In JoVE (1)

Other Publications (199)

Articles by Kam W. Leong in JoVE

Other articles by Kam W. Leong on PubMed

Photo-crosslinkable Microcapsules Formed by Polyelectrolyte Copolymer and Modified Collagen for Rat Hepatocyte Encapsulation

Biomaterials. Aug, 2004  |  Pubmed ID: 15020127

New anionic polyelectrolyte tetra-copolymers with photo-crosslinkable 4-(4-methoxycinnamoyl)phenyl methacrylate monomer in addition to a HEMA-MMA-MAA ter-copolymer system were synthesized. The tetra-copolymers were used to form photo-crosslinkable microcapsules with modified collagen by complex coacervation for rat hepatocytes encapsulation. The hepatocytes were encapsulated within a two-layered membrane comprising of modified collagen as the inner core and an outer photo-crosslinkable copolymer shell. Upon photo-crosslinking of the microcapsules with UV-Vis light irradiation, the mechanical strength and chemical stability of the microcapsules, and the cellular functions of the encapsulated hepatocytes were enhanced. Particularly, the mechanical stability of the microcapsules was dramatically strengthened. The new photo-crosslinkable tetra-copolymer formulation described in this article has opened a way to the development of hepatocyte microencapsulation technology for bioartifical liver assist device.

Bioadhesive Characterization of Poly(methylidene Malonate 2.12) Microparticle on Model Extracellular Matrix

Biomaterials. Aug, 2004  |  Pubmed ID: 15046923

The efficacy of a drug delivery system is predicated on its retention in the target tissue. Microparticle is one of the most popular and effective drug delivery configurations. Recently, it has been shown that the interaction between drug-loaded microparticles and tissues is related to the effectiveness of paclitaxel delivery to the bladder wall of mice for treating superficial bladder cancer. In this study, the adhesive interaction between poly(methylidene malonate 2.12) or PMM 2.1.2 microparticles and collagen, which serves as the model extracellular matrix for bladder wall, was probed with confocal reflectance interference contrast microscopy (C-RICM), single-particle compressive force measurement and contact mechanics theory. Young's modulus of single PMM 2.1.2 microparticle was determined as 1.56 +/- 0.25 x 10(4)N/m(2). For plain PMM 2.1.2 microparticle in water (pH 5.5), the degree of deformation (a/R) on collagen coated substrate decreased from 0.77 to 0.26 against the increase of mid-plane diameter from 2 to 18 microm. The adhesion energy of PMM 2.1.2 microparticle was determined from Maguis-JKR theory and remained at around 1.5 mJ/m(2) against the increase of particle diameter. At pH 4, the average degree of particle deformation and adhesion energy was increased by 11% and 32%, respectively, in comparison with that at pH 5.5. The loading of paclitaxel in PMM 2.1.2 microspheres enhanced the deformation and adhesion of microspheres at pH 5.5. It is hypothesized that the electrostatic repulsion between paclitaxel and collagen at pH 4 reduces the adhesion energy of PMM 2.1.2-paclitaxel microsphere. This study may offer insight for design of future microparticulate delivery systems by providing the experimental and theoretical tools to study the bioadhesive interaction between drug-loaded microparticles and model extracellular matrices.

The Effect of the Degree of Chitosan Deacetylation on the Efficiency of Gene Transfection

Biomaterials. Oct, 2004  |  Pubmed ID: 15110480

Chitosans with various degrees of deacetylation were synthesized by acetylation with acetic anhydride. These chitosans were evaluated for efficacy of nanoparticle formation, DNA binding efficiency, morphology, and in vitro and in vivo gene transfection efficiency. DNA binding efficacy was reduced as degree of deacetylation was decreased, therefore requiring an increased +/-ratio to effect complete DNA complexation. For chitosan with a molecular weight of 390 kDa, the +/-ratio to achieve complete DNA complexation for degrees of deacetylation of 90%, 70% and 62% was 3.3:1, 5.0:1, and 9.0:1, respectively. The size and morphology of these nanoparticle formulations were not significantly different. The decreased degree of deacetylation results in a decrease in overall luciferase expression levels in HEK293, HeLa, and SW756 cells due to particle destabilization in the presence of serum proteins. However, intramuscular luciferase expression levels increased with decreasing deacetylation over the time points tested. Degree of chitosan deacetylation is an important factor in chitosan-DNA nanoparticle formulation as it affects DNA binding, release and gene transfection efficiency in vitro and in vivo.

In Vitro Release of Vascular Endothelial Growth Factor from Gadolinium-doped Biodegradable Microspheres

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Jun, 2004  |  Pubmed ID: 15170848

A drug delivery vehicle was constructed that could be visualized noninvasively with MRI. The biodegradable polymer poly(DL-lactic-co-glycolic acid) (PLGA) was used to fabricate microspheres containing vascular endothelial growth factor (VEGF) and the MRI contrast agent gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). The microspheres were characterized in terms of size, drug and contrast agent encapsulation, and degradation rate. The PLGA microspheres had a mean diameter of 48 +/- 18 microm. The gadolinium loading was 17 +/- 3 microg/mg polymer and the VEGF loading was 163 +/- 22 ng/mg polymer. Electron microscopy revealed that the Gd was dispersed throughout the microspheres and it was confirmed that the Gd loading was sufficient to visualize the microspheres under MRI. VEGF and Gd-DTPA were released from the microspheres in vitro over a period of approximately 6 weeks in three phases: a burst, followed by a slow steady-state, then a rapid steady-state. Biodegradable Gd-doped microspheres can be effectively used to deliver drugs in a sustained manner, while being monitored noninvasively with MRI.

Poly(phosphoester) Ionomers As Tissue-engineering Scaffolds

Journal of Biomedical Materials Research. Part B, Applied Biomaterials. Jul, 2004  |  Pubmed ID: 15199588

Regenerative medicine requires scaffolds of divergent physicochemical properties for different tissue-engineering applications. To this end, a series of biodegradable poly(phosphoester) ionomers of the general composition [p(BHET-EOP-HOP/TC)] was synthesized, with BHET(bis-hydroxyl ethylene phosphate):EOP(ethylene phosphate):HOP(free phosphate) ratios of 60:20:20, 70:10:20, and 75:5:20, respectively. The 60/20/20 ionomer possessed the best tensile properties, exhibiting an average tensile modulus of 68 MPa and strain at break of 31%. Calcium treatment of the ionomer films led to significantly higher hardness and elastic moduli as measured by indentation. Calcium binding was evident from the increase in glass transition and melting temperatures, and a shift in the P-->O absorption in the FTIR spectrum. Stable N-hydroxysuccinimide ester (NHS) of the ionomers could be synthesized to facilitate derivatization, as demonstrated by conjugation of GRGDS in this study. The polymers conjugated with NHS were hydrolyzed in a biphasic mode, with a fast initial phase occurring in the first few hours, followed by a slower phase in the next few days. These ionomers represent a novel class of biomaterials with readily controllable physical and chemical attributes for tissue engineering.

Evaluation of Collagen and Methylated Collagen As Gene Carriers

International Journal of Pharmaceutics. Jul, 2004  |  Pubmed ID: 15234800

This study explores the potential of DNA complexes prepared with methylated collagen (MC) and unmodified native collagen (NC) to deliver genes into cells. The physicochemical properties and transfection abilities of these two types of complexes are studied in parallel. MC was prepared by methylation of the carboxyl groups of collagen, rendering the collagen net positively charged at neutral pH. NC/DNA complexes were prepared at pH approximately 3, but aggregated rapidly at neutral pH. These complexes did not confer significant protection to DNA due to its poor stability in serum. MC carried a positive charge at neutral pH and formed complexes with DNA in PBS; therefore MC improved DNA binding ability and the stability of the complexes at physiological conditions. MC/DNA complexes were smaller and more stable than NC/DNA complexes in PBS, and sustained released of DNA from MC/DNA complexes was observed for up to 3 weeks in PBS at 37 degrees C. In contrast, NC/DNA complexes released almost all the DNA within 6h under the same condition. In vitro gene transfection experiments revealed that MC mediated a higher gene expression than NC, although the level of gene expression was still much lower than that achieved with polyethyleneimine/DNA complexes. In contrast to in vitro results, NC/DNA complexes yielded a 3.8-fold higher gene expression than naked DNA and MC/DNA complexes (P < 0.05) at week 2 following intramuscular injection at a DNA dose of 3 microg per muscle and a weight ratio of 1. Higher weight ratios resulted in significant decrease of transfection efficiency, particularly for MC/DNA complexes. The results suggested that gene delivery via the intramuscular route followed a different mechanism that demands a different set of physiochemical properties of the carrier from other parental routes. The potential of these collagen-based gene carriers for other administration routes remain to be further investigated.

Biodegradable Polyphosphoester Micelles for Gene Delivery

Journal of Pharmaceutical Sciences. Aug, 2004  |  Pubmed ID: 15236461

A new biodegradable polyphosphoester, poly[[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide] ethyl phosphate] (PCEP) was synthesized and investigated for gene delivery. Carrying a positive charge in its backbone and a lipophilic cholesterol structure in the side chain, PCEP self-assembled into micelles in aqueous buffer at room temperature with an average size of 60-100 nm. It could bind and protect plasmid DNA from nuclease digestion. Cell proliferation assay indicated a lower cytotoxicity for PCEP than for poly-L-lysine and Lipofectamine. The IC50 determined by the WST-1 assay was 69.8, 51.6, and 12.1 microg/mL for PCEP, Lipofectamine, and poly-L-lysine, respectively. PCEP efficiently delivered DNA to several cell lines such as HEK293, Caco-2, and HeLa. The highest efficiency was achieved when PCEP/DNA complex was prepared in Opti-MEM with a +/- charge ratio of 1.5-2. The transfection efficiency did not change significantly when the complex was used 3 days after preparation. The addition of chloroquine to the formulation increased transfection efficiency 10- to 50-fold compared to the complex alone. In vivo studies showed a luciferase expression by PCEP/DNA complexes in muscle increasing with time during 3 months, although the expression level was lower than that by direct injection of naked DNA. In addition to biodegradability and lower toxicity, the PCEP micelle carrier offers structural versatility. The backbone charge density and the side chain lipophilicity are two parameters that can be varied through copolymerization and monomer variation to optimize the transfection efficiency.

Polyelectrolyte Complex Films Derived from Polyethyleneoxide-maleic Acid Copolymer and Chitosan: Preparation and Characterization

Macromolecular Bioscience. May, 2004  |  Pubmed ID: 15468245

Polyelectrolyte complex films were prepared with polyethyleneoxide-maleic acid copolymer and chitosan using a casting/solvent evaporation method. The films were examined in terms of their IR spectra, surface and cross-section morphologies, cytotoxicity, and swelling behavior at different pH levels. To assess the potential of these films as a biomedical device, the profiles of the release of model drug from the CS/PEOMA films were examined at pH 4.8. The surface morphology of the films was quite smooth and uniform, and the cross-sectional morphology was dense and homogeneous. The swelling behaviors of CS/PEOMA films were found to depend on the pH of the solution as well as on the CS/PEOMA composition. Drug release from different CS/PEOMA films at pH 4.8 was found to be dependent on film composition. The results showed the potential applicability of CS/PEOMA film as a drug delivery vehicle.

Encapsulation of Biologics in Self-assembled Fibers As Biostructural Units for Tissue Engineering

Journal of Biomedical Materials Research. Part A. Dec, 2004  |  Pubmed ID: 15499568

The concept of a "biostructural unit" is presented as the combination of biological and structural building blocks to create scaffolds or constructs via a bottom-up approach. Three types of biostructural units were constructed using the process of fiber formation by interfacial polyelectrolyte complexation: protein-encapsulated fiber, ligand-immobilized fiber, and cell-encapsulated fiber units. Water-soluble chitin (WSC) and alginate were used as the polyelectrolyte combination to form fiber. Encapsulation and sustained release of bovine serum albumin from the fiber could be achieved, release profiles being dependent on the WSC/alginate concentration ratio. Released nerve growth factor (NGF) retained its bioactivity, as demonstrated on PC12 cells. Biotinylated fiber could be fabricated by biotinylating alginate before drawing fiber with WSC, enabling biotinylated NGF to be immobilized to fiber via an avidin bridge. The immobilized NGF induced the differentiation of PC12 cells seeded on the fiber. Bovine pulmonary endothelial cells, human dermal fibroblasts, and human mesenchymal stem cells were encapsulated, demonstrating good viability as determined by Live/Dead and WST-1 assays. The assembly of biostructural units into constructs was illustrated by using human mesenchymal stem cell-encapsulated fiber units. Cells in the resulting constructs could be induced to differentiate along chondrogenic and osteogenic lineages.

Coculture of Mesenchymal Stem Cells and Respiratory Epithelial Cells to Engineer a Human Composite Respiratory Mucosa

Tissue Engineering. Sep-Oct, 2004  |  Pubmed ID: 15588402

In this study, we describe a novel in vitro reconstitution system for tracheal epithelium that could be useful for investigating the cellular and molecular interaction of epithelial and mesenchymal cells. In this system, a Transwell insert was used as a basement membrane on which adult bone marrow mesenchymal stem cells (MSCs) were cultured on the lower side whereas normal human bronchial epithelial (NHBE) cells were cultured on the opposite upper side. Under air-liquid interface conditions, the epithelial cells maintained their capacity to progressively differentiate and form a functional epithelium, leading to the differentiation of mucin-producing cells between days 14 and 21. Analysis of apical secretions showed that mucin production increased over time, with peak secretion on day 21 for NHBE cells alone, whereas mucin secretion by NHBE cells cocultured with MSCs remained constant between days 18 and day 25. This in vitro model of respiratory epithelium, which exhibited morphologic, histologic, and functional features of a tracheal mucosa, could help to understand interactions between mesenchymal and epithelial cells and mechanisms involved in mucus production, inflammation, and airway repair. It might also play an important role in the design of an composite prosthesis for tracheal replacement.

Formulation of Chitosan-DNA Nanoparticles with Poly(propyl Acrylic Acid) Enhances Gene Expression

Journal of Biomaterials Science. Polymer Edition. 2004  |  Pubmed ID: 15648571

Poly(propyl acrylic acid) (PPAA) is a polymer specifically designed to disrupt lipid bilayer membranes within a sharply defined pH range. The pH sensitivity can be used to enhance the release of endocytosed drugs into the cytoplasmic compartment of the cell. By incorporating this polymer in a polymeric gene carrier, chitosan, the release of plasmid DNA from the endosomal compartment was enhanced. In vitro transfection studies confirmed that the incorporation of PPAA into the chitosan-DNA nanoparticles enhanced gene expression in both HEK293 and HeLa cells compared to chitosan nanoparticles alone. The dose and time at which PPAA was incorporated during the complex formation affected the release of DNA and transfection efficiency. The optimal dose of PPAA incorporated into the chitosan nanoparticles was determined to be 10 microg, corresponding to a PPAA/DNA weight ratio of 1:1. At this dose, the ternary complexes are approx. 400 nm in size with a net negative surface charge of -17.4 mV. Intracellular trafficking studies confirmed the association of PPAA, DNA and chitosan at 24 h post-transfection and the subsequent release of DNA and PPAA from the chitosan at 48 h. The diffuse appearance of the majority of the DNA and the PPAA at later time points suggests that the PPAA triggered membrane disruption resulting in the release of DNA from the endosomal compartment. Finally, the lack of colocalization between PPAA and Lysotracker indicated that the PPAA-loaded nanoparticles were not trafficked through a lysosomal pathway. This study suggests the promising strategy of including PPAA in the formulation of polymer-DNA complexes for non-viral gene delivery.

Adhesion Contact Dynamics of Primary Hepatocytes on Poly(ethylene Terephthalate) Surface

Biomaterials. Mar, 2005  |  Pubmed ID: 15353200

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R proportional to t0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R proportional to t0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.

Stable Immobilization of Rat Hepatocyte Spheroids on Galactosylated Nanofiber Scaffold

Biomaterials. May, 2005  |  Pubmed ID: 15585256

Primary rat hepatocytes self-assemble into multi-cellular spheroids and maintain differentiated functions when cultured on a two-dimensional (2-D) substrate conjugated with galactose ligand. The aim of this study is to investigate how a functional nanofiber scaffold with surface-galactose ligand influences the attachment, spheroid formation and functional maintenance of rat hepatocytes in culture, as compared with the functional 2-D substrate. Highly porous nanofiber scaffolds comprising of fibers with an average diameter of 760 nm were prepared by electrospinning of poly(epsilon-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a novel biodegradable copolymer. Galactose ligand with a density of 66 nmol/cm(2) was achieved on the nanofiber scaffold via covalent conjugation to a poly(acrylic acid) spacer UV-grafted onto the fiber surface. Hepatocytes cultured on the galactosylated PCLEEP nanofiber scaffold exhibited similar functional profiles in terms of cell attachment, ammonia metabolism, albumin secretion and cytochrome P450 enzymatic activity as those on the functional 2-D substrate, although their morphologies are different. Hepatocytes cultured on galactosylated PCLEEP film formed 50-300 microm spheroids that easily detached from surface upon agitation, whereas hepatocytes cultured on galactosylated nanofiber scaffold formed smaller aggregates of 20-100 microm that engulfed the functional nanofibers, resulting in an integrated spheroid-nanofiber construct.

Ternary Complexes Comprising Polyphosphoramidate Gene Carriers with Different Types of Charge Groups Improve Transfection Efficiency

Biomacromolecules. Jan-Feb, 2005  |  Pubmed ID: 15638504

To understand the influence of charge groups on transfection mediated by polymer complexes, we have synthesized a series of biodegradable and cationic polyphosphoramidates (PPAs) with an identical backbone but different side chains. Our previous study showed that PPA with a spermidine side chain (PPA-SP) showed high transfection efficiency in culture, whereas PPAs with secondary, tertiary, and quaternary amino groups were significantly less efficient. To investigate whether the coexistence of 1 degrees amino charge groups with 3 degrees and 2 degrees amino charge groups in the DNA/polymer complexes would enhance their transfection efficiency, we evaluated a ternary complex system containing DNA and PPAs with 1 degrees amino groups (PPA-SP) and 3 degrees amino groups (PPA-DMA) and a quaternary complex system containing DNA and PPAs with 1 degrees and 2 degrees and 3 degrees amino groups (PPA-EA/PPA-MEA/PPA-DMA), respectively. Ternary complexes mediated 20 and 160 times higher transfection efficiency in COS-7 cells than complexes of DNA with PPA-SP or PPA-DMA alone, respectively. Similarly, quaternary complexes exhibited 8-fold higher transfection efficiency than PPA-EA/DNA complexes. The mechanism of enhancement in transfection efficiency by the mixture carriers appears to be unrelated to the particle size, zeta potential, or DNA uptake. The titration characterization and the transfection experiments using a proton pump inhibitor suggest that the enhancement effect is unlikely due to the slightly improved buffering capacity of the mixture over PPA-SP. This approach represents a simple strategy of developing polymeric gene carriers and understanding the mechanisms of polymer-mediated gene transfer.

In Vitro Gene Delivery Using Polyamidoamine Dendrimers with a Trimesyl Core

Biomacromolecules. Jan-Feb, 2005  |  Pubmed ID: 15638538

Polyamidoamine (PAMAM) dendrimer represents one of the most efficient polymeric gene carriers. To investigate the effect of the core structure and generation of dendrimers on the complex formation and transfection efficiency, a series of PAMAM dendrimers with a trimesyl core (DT) at different generations (DT4 to DT8) were developed as gene carriers and compared with the PAMAM dendrimers derived from pentaerythritol (DP) and inositol (DI). The minimal generation number of DTs at which the dendrimer has enough amino group density to effectively condense DNA was higher (generation 6) than those of DPs and DIs (generation 5). DTs of generation 6 or higher condensed DNA into complexes with an average diameter ranging from 100 to 300 nm, but the 4th and 5th generations of DT (DT4 and DT5) formed only a severe aggregate with DNA. Interestingly, the DT6/pDNA complex was determined to be much smaller (100-300 nm) than those prepared with DP5 or DI5 (>600 nm) at N/P ratios higher than 15. The optimal generation numbers at which the dendrimers showed the highest transgene expression in COS-7 cells were 5 for DPs and DIs but 6 for DTs. The DT6/pDNAcomplex with smaller size mediated higher transgene expression in COS-7 cells than those prepared with DP5 or DI5. The in vitro transfection efficiency of the DT dendrimers as evaluated in HeLa cells, COS-7 cells, and primary hepatocytes decreased in the order of DT6 > DT7 > DT8 > DT5 > DT4. The transfection mediated by DT6 was significantly inhibited by bafilomycin A1. The acid-base titration curve for DT6 showed high buffer capacity in the pH range from 5.5 to 6.4 (pK(a) approximately 6). This permits dendrimers to buffer the pH change in the endosomal compartment. However, the transfection efficiency mediated by DT6 decreased significantly in the presence of serum in both HeLa cells and COS-7 cells. The cytotoxicity of DTs evaluated in HeLa cells using the 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay showed a trend of increasing toxicity with the polymer generations. The LD50 values of DT4 through DT8 were 628, 236, 79, 82, and 77 microg/mL, respectively, which were higher than that of poly(ethyleneimide) (18 microg/mL) and poly(L-lysine) (28 microg/mL) in the same assay. With a lower cytotoxicity and versatility for chemical conjugation, these PAMAM dendrimers with a DT core warrant further investigation for nonviral gene delivery.

Galactosylated Ternary DNA/polyphosphoramidate Nanoparticles Mediate High Gene Transfection Efficiency in Hepatocytes

Journal of Controlled Release : Official Journal of the Controlled Release Society. Feb, 2005  |  Pubmed ID: 15681095

Galactosylated polyphosphoramidates (Gal-PPAs) with different ligand substitution degrees (6.5%, 12.5% and 21.8%, respectively) were synthesized and evaluated as hepatocyte-targeted gene carriers. The in vitro cytotoxicity of Gal-PPA decreased significantly with an increase in galactose substitution degree. The affinity of Gal-PPA/DNA nanoparticles to galactose-recognizing lectin increased with galactose substitution degree. However, decreased transfection efficiency was observed for these galactosylated PPAs in HepG2 cells. Based on the results of gel retardation and polyanion competition assays, we hypothesized that the reduced transfection efficiency of Gal-PPA/DNA nanoparticles was due to their decreased DNA-binding capacity and decreased particle stability. We therefore prepared nanoparticles by precondensing DNA with PPA at a charge ratio of 0.5, yielding nanoparticles with negative surface charge, followed by coating with Gal-PPA, resulting in a Gal-PPA/ DNA/PPA ternary complex. Such a ternary nanoparticle formulation led to significant size reduction in comparison with binary nanoparticles, particularly at low N/P ratios (2 to 5). In HepG2 cells and primary rat hepatocytes, and at low N/P ratios (2 to 5), transfection efficiency mediated by ternary nanoparticles prepared with 6.5% Gal-PPA was 6-7200 times higher than PPA-DPA/DNA nanoparticles. Transgene expression increased slightly at higher N/P ratios in HepG2 cells and reached a plateau at N/P ratios between 5 and 10 for primary rat hepatocytes. Such an enhancement effect was not observed in HeLa cells that lack of asialoglycoprotein receptor (ASGPR). Nevertheless, transfection efficiency of ternary particles decreased dramatically, presumably due to the decreased DNA binding capacity and particle stability, as PPA galactosylation degree increased. This highlights the importance of optimizing ligand conjugation degree for PPA gene carrier.

MR Imaging of Biodegradable Polymeric Microparticles: a Potential Method of Monitoring Local Drug Delivery

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Mar, 2005  |  Pubmed ID: 15723408

Gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) was encapsulated into biodegradable, bioadhesive polymeric microparticles to enable noninvasive monitoring of their local intravesical delivery with MRI. The microparticles were characterized by contrast agent encapsulation and release kinetics, T(1) relaxation rates, and contrast enhancement in vivo. The level of Gd-DTPA loading into microparticles was 14.3 +/- 0.6 mug/mg polymer. The measured T(1) relaxation rates of the microparticles showed a direct dependence on Gd-DPTA content. Both 1.5T and 4.7T MR scanners were used to image murine bladders instilled intravesically with Gd-DTPA-loaded particles in vivo. MR images showed ring-shaped regions of enhancement inscribing the bladder wall, which were attributed to the microparticles that were preferentially adherent to the mucosa lining the urothelium. The images of controls exhibited no such enhancement. The normalized signal intensities measured from post-instillation images were significantly greater (P < 0.05) than those in the pre-instillation images. Contrast enhancement was observed for at least 5 days after the initial instillation, although the enhancement decreased due to microparticle degradation or mucosa renewal. The localized distribution of biodegradable, bioadhesive microparticles encapsulating Gd-DTPA was successfully visualized with MRI in vivo, allowing particle-mediated delivery to be temporally and spatially monitored noninvasively.

Biodegradable Poly(terephthalate-co-phosphate)s: Synthesis, Characterization and Drug-release Properties

Journal of Biomaterials Science. Polymer Edition. 2005  |  Pubmed ID: 15794482

To develop biodegradable polymers with favorable physicochemical and biological properties, we have synthesized a series of poly(terephthalate-co-phosphate)s using a two-step poly-condensation. The diol 1,4-bis(2-hydroxyethyl) terephthalate was first reacted with ethylphosphorodichloridate (EOP), and then chain-extended with terephthaloyl chloride (TC). Incorporation of phosphate into the poly(ethylene terephthalate) backbone rendered the co-polymers soluble in chloroform and biodegradable, lowered the Tg, decreased the crystallinity and increased the hydrophilicity. With an EOP/TC molar feed ratio of 80: 20, the polymer exhibited good film-forming property, yielding at 86.6 +/- 1.6% elongation with an elastic modulus of 13.76 +/- 2.66 MPa. This polymer showed a favorable toxicity profile in vitro and good tissue biocompatibility in the muscular tissue of mice. In vitro the polymer lost 21% of mass in 21 days, but only 20% for up to 4 months in vivo. It showed no deterioration of properties after sterilization by gamma-irradiation at 2.5 Mrad on solid CO2. Release of FITC-BSA from the microspheres was diffusion-controlled and exceeded 80% completion in two days. Release of the hydrophobic cyclosporine-A from microspheres was however much more sustained and near zero-ordered, discharging 60% in 70 days. A limited structure-property relationship has been established for this co-polymer series. The co-polymers became more hydrolytically labile as the phosphate component (EOP) was increased and the side chains were switched from the ethoxy to the methoxy structure. Converting the methoxy group to a sodium salt further increased the degradation rate significantly. The chain rigidity as reflected in the Tg values of the co-polymers decreased according to the following diol structure in the backbone: ethylene glycol > 2-methylpropylene diol > 2,2-dimethylpropylene diol. The wide range of physicochemical properties obtainable from this co-polymer series should help the design of degradable biomaterials for specific biomedical applications.

Nanopattern-induced Changes in Morphology and Motility of Smooth Muscle Cells

Biomaterials. Sep, 2005  |  Pubmed ID: 15814139

Cells are known to be surrounded by nanoscale topography in their natural extracellular environment. The cell behavior, including morphology, proliferation, and motility of bovine pulmonary artery smooth muscle cells (SMC) were studied on poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces comprising nanopatterned gratings with 350 nm linewidth, 700 nm pitch, and 350 nm depth. More than 90% of the cells aligned to the gratings, and were significantly elongated compared to the SMC cultured on non-patterned surfaces. The nuclei were also elongated and aligned. Proliferation of the cells was significantly reduced on the nanopatterned surfaces. The polarization of microtubule organizing centers (MTOC), which are associated with cell migration, of SMC cultured on nanopatterned surfaces showed a preference towards the axis of cell alignment in an in vitro wound healing assay. In contrast, the MTOC of SMC on non-patterned surfaces preferentially polarized towards the wound edge. It is proposed that this nanoimprinting technology will provide a valuable platform for studies in cell-substrate interactions and for development of medical devices with nanoscale features.

PH Responsive Adhesion of Phospholipid Vesicle on Poly(acrylic Acid) Cushion Grafted to Poly(ethylene Terephthalate) Surface

Colloids and Surfaces. B, Biointerfaces. May, 2005  |  Pubmed ID: 15893225

Polymer-supported lipid bilayer is a key enabling technology for the design and fabrication of novel biomimetic devices. To date, the physical driving force underlying the formation of polymer-supported lipid bilayer remains to be determined. In this study, the interaction between dipalmitoylphosphocholine (DPPC) vesicle and poly(ethylene terephthalate) [PET] surface with or without grafted poly(acrylic acid) [PAA] layer is examined with several biophysical techniques. First, vesicle deformation analysis shows that the geometry of adherent vesicle on either plain PET or PAA-grafted PET surface is best described by a truncated sphere model. At neutral pH, the degree of deformation and adhesion energy are unaltered by the grafted polymerization of acrylic acid on PET surface. Interestingly, the average magnitude of adhesion energy is increased by 185% and -43% on PAA-grated PET and plain PET surface, respectively, towards an increase of pH at room temperature. Our results demonstrate the possibility of tuning the adhesive interaction between vesicle and polymer cushion through the control of polyelectrolyte ionization on the solid support.

Controlled Release from Fibers of Polyelectrolyte Complexes

Journal of Controlled Release : Official Journal of the Controlled Release Society. May, 2005  |  Pubmed ID: 15907585

Controlled release systems for delicate compounds, such as proteins, often suffer the drawbacks of decreased bioactivity and low encapsulation efficiency. This study introduces the concept of producing drug-loaded fibers from interfacial polyelectrolyte complexation. Chitosan-alginate fibers were produced by pulling from the interface between two polyelectrolyte solutions at room temperature. Depending on the component properties, the release time of encapsulated components from these fibers can range from hours to weeks. Dexamethasone was completely released within 2 h, whereas charged compounds such as BSA, PDGF-bb, and avidin showed sustained release for 3 weeks. The fibers were able to release PDGF-bb in a steady fashion for over 3 weeks without an initial burst. Furthermore, the bioactivity of PDGF-bb was retained over this period. Release kinetics could be controlled by the inclusion of heparin, which contains specific binding sites for various growth factors. By varying the alginate/heparin ratios in the anionic polyelectrolyte solution, the release of PDGF-bb could be significantly altered. In this study, interfacial polyelectrolyte complexation has been demonstrated to be a promising technique for producing drug-loaded fibers with high encapsulation efficiency, sustained release kinetics, and capacity to retain the bioactivity of the encapsulants.

Polyethylenimine-grafted Multiwalled Carbon Nanotubes for Secure Noncovalent Immobilization and Efficient Delivery of DNA

Angewandte Chemie (International Ed. in English). Jul, 2005  |  Pubmed ID: 15995988

Sustained Release of Proteins from Electrospun Biodegradable Fibers

Biomacromolecules. Jul-Aug, 2005  |  Pubmed ID: 16004440

Electrospinning is a simple and versatile technique of producing polymeric fibers ranging from submicron to micron in diameter. Incorporation of bioactive agents into the fibers could make a biofunctional tissue engineering scaffold. In this study, we investigated the feasibility of encapsulating human beta-nerve growth factor (NGF), which was stabilized in a carrier protein, bovine serum albumin (BSA) in a copolymer of epsilon-caprolactone and ethyl ethylene phosphate (PCLEEP) by electrospinning. Partially aligned protein encapsulated fibers were obtained and the protein was found to be randomly dispersed throughout the electrospun fibrous mesh in aggregate form. A sustained release of NGF via diffusion process was obtained for at least 3 months. PC12 neurite outgrowth assay confirmed that the bioactivity of electrospun NGF was retained, at least partially, throughout the period of sustained release, thus clearly demonstrating the feasibility of encapsulating proteins via electrospinning to produce biofunctional tissue scaffolds.

Engineering Microenvironment for Expansion of Sensitive Anchorage-dependent Mammalian Cells

Journal of Biotechnology. Sep, 2005  |  Pubmed ID: 16026880

Tissue engineering involves ex vivo seeding of anchorage-dependent mammalian cells onto scaffolds, or transplanting cells in vivo. The cell expansion currently requires repeated cell detachment from solid substrata by enzymatic, chemical or mechanical means. The report here presents a high yield three-dimensional culture and harvest system circumventing the conventional detachment requirements. Cells mixed with dilute cationic collagen were microencapsulated within an ultra-thin shell of synthetic polymers. The cationic collagen could rapidly form a conformal layer of collagen fibers around cells to support cell proliferation and functions. The collagen could be readily removed from cells with a buffer rinse after harvesting from the fragile microcapsules. The cells harvested from this system demonstrate improved attachment, morphology and functions over conventionally cultured cells, upon binding to ligand-conjugated polymer surfaces. The harvested cells can be re-encapsulated and allowed to proliferate again, or used immediately in applications.

Micellization Phenomena of Biodegradable Amphiphilic Triblock Copolymers Consisting of Poly(beta-hydroxyalkanoic Acid) and Poly(ethylene Oxide)

Langmuir : the ACS Journal of Surfaces and Colloids. Sep, 2005  |  Pubmed ID: 16142948

This paper reports the studies on micelle formation of new biodegradable amphiphilic poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) triblock copolymer with various PHB and PEO block lengths in aqueous solution. Transmission electron microscopy showed that the micelles took an approximately spherical shape with the surrounding diffuse outer shell formed by hydrophilic PEO blocks. The size distribution of the micelles formed by one triblock copolymer was demonstrated by dynamic light scattering technique. The critical micellization phenomena of the copolymers were extensively studied using the pyrene fluorescence dye absorption technique, and the (0,0) band changes of pyrene excitation spectra were used as a probe for the studies. For the copolymers studied in this report, the critical micelle concentrations ranged from 1.3 x 10(-5) to 1.1 x 10(-3) g/mL. For the same PEO block length of 5000, the critical micelle concentrations decreased with an increase in PHB block length, and the change was more significant in the short PHB range. It was found that the micelle formation of the biodegradable amphiphilic triblock copolymers consisting of poly(beta-hydroxyalkanoic acid) and PEO was relatively temperature-insensitive, which is quite different from their counterparts consisting of poly(alpha-hydroxyalkanoic acid) and PEO.

Design of Polyphosphoester-DNA Nanoparticles for Non-viral Gene Delivery

Advances in Genetics. 2005  |  Pubmed ID: 16240998

Development of safe and effective non-viral gene carriers is still critical to the ultimate success of gene therapy. This review highlights our attempt to design the gene carriers in a systematic manner. We have synthesized a series of polymers with a phosphoester backbone containing different charge groups in the sidechain connected to the backbone through a phosphate (P-O) or a phosphoramide (P-N) bond. These gene carriers have different charge groups, sidechain lengths, and branching structures, but they are structurally related to allow a systematic investigation of the structure-property relationship, including DNA binding capacity, cytotoxicity, DNA protection, biodegradability, DNA release kinetics, and transfection efficiency.

Design of Polyphosphoester-DNA Nanoparticles for Non-Viral Gene Delivery

Advances in Genetics. 2005  |  Pubmed ID: 16243068

Development of safe and effective non-viral gene carriers is still critical to the ultimate success of gene therapy. This review highlights our attempt to design the gene carriers in a systematic manner. We have synthesized a series of polymers with a phosphoester backbone containing different charge groups in the sidechain connected to the backbone through a phosphate (PO) or a phosphoramide (PN) bond. These gene carriers have different charge groups, sidechain lengths, and branching structures, but they are structurally related to allow a systematic investigation of the structure-property relationship, including DNA binding capacity, cytotoxicity, DNA protection, biodegradability, DNA release kinetics, and transfection efficiency.

Proliferation and Differentiation of Human Embryonic Germ Cell Derivatives in Bioactive Polymeric Fibrous Scaffold

Journal of Biomaterials Science. Polymer Edition. 2005  |  Pubmed ID: 16268248

Human embryonic germ cell derivatives, a heterogeneous population of uncommitted embryoid body derived (EBD) cells, were studied in a bioactive three-dimensional (3D) fibrous culture. Their proliferation, morphology, gene expression and differentiation were investigated to gain insights on development of 3D bioactive scaffold for pluripotent stem cells. The expansion of the EBD cells in 3D environment was significantly higher than their two-dimensional controls after 21 days. No apparent differentiation of the EBD cells cultured in the 3D environment, as indicated by histology and gene expression profile analysis, was evident. Extracellular matrix production was weak in the long-term 3D culture, and the EBD cells maintained their multilineage gene expressions for the period studied. When nerve growth factor (NGF) was surface-immobilized on the fibrous scaffold via chemically-modified Pluronic, the EBD cells cultured in this scaffold showed evidence of entering the neural pathway. An upregulation of tyrosine hydroxylase mRNA expression was observed when EBD cells were cultured in the NGF-immobilized fibrous scaffold, as demonstrated by real-time PCR and immunofluorescence staining. The study suggests the value of such fibrous 3D culture in manipulating stem cell proliferation/differentiation and as a model for developing a bioactive scaffold.

Evaluation of Hyperbranched Poly(amino Ester)s of Amine Constitutions Similar to Polyethylenimine for DNA Delivery

Biomacromolecules. Nov-Dec, 2005  |  Pubmed ID: 16283742

New hyperbranched poly(amino ester)s were synthesized via A3 + 2BB'B' ' approach, represented by the Michael addition polymerization of trimethylol-propane triacrylate (TMPTA) (A3-type monomers) with a double molar 1-(2-aminoethyl)piperazine (AEPZ) (BB'B''-type monomer) performed in chloroform at ambient temperature. The results obtained by in situ monitoring the polymerization using NMR and MS indicated that hyperbranched poly(TMPTA1-AEPZ2) was formed via a A(B'B'')2 intermediate, and the B' ' (the formed 2 degrees amine) was kept intact in the reaction. Therefore, poly(TMPTA1-AEPZ2) contained secondary and tertiary amines in the core and primary amines in the periphery similar to polyethylenimine (PEI). The chemistry of protonated poly(TMPTA1-AEPZ2) was further confirmed by 13C NMR, and the molecular weight, the radius of gyration (Rg), and the hydrodynamic radius (Rh) were determined using GPC, small-angle X-ray scattering (SAXS), and laser dynamic light scattering (LDLS), respectively. The ratio of Rg/Rh of ca. 1.1 verified the hyperbranched structure. Protonated hyperbranched poly(TMPTA1-AEPZ2) is degradable and less cytotoxic as compared with PEI (25 K). Gel electrophoresis reflected that stable complexes could be formed from protonated hyperbranched poly(TMPTA1-AEPZ2) and DNA, and the size and xi-potential of the complexes were characterized. Remarkably, protonated hyperbranched poly(TMPTA1-AEPZ2) showed transfection efficiency comparable to PEI (25 k) for in vitro DNA delivery.

Galactosylated Poly(vinylidene Difluoride) Hollow Fiber Bioreactor for Hepatocyte Culture

Tissue Engineering. Nov-Dec, 2005  |  Pubmed ID: 16411812

To overcome the limitations of long-term expression of highly differentiated hepatocyte functions, we have developed a novel bioreactor in which hepatocytes are seeded in a ligand-immobilized hollow fiber cartridge. Galactosylated Pluronic polymer is immobilized on poly(vinylidene difluoride) (PVDF) hollow fiber surface through an adsorption scheme yielding a substrate with hepatocyte-specific ligand and a hydrophilic surface layer, which can resist nonspecific protein adsorption and facilitate cell binding to the galactose ligand. Interestingly, the galactosylated PVDF hollow fiber shows enhanced serum albumin diffusion across the membrane. Freshly isolated rat hepatocytes were seeded and cultured in the extralumenal space of the hollow fiber cartridge for 18 days in a continuously circulated system. Albumin secretion function of the seeded hepatocytes was monitored by analyzing circulating medium by enzyme-linked immunosorbent assay. Urea synthesis and P-450 function (7-ethoxycoumarin dealkylase activity) were measured periodically by doping the circulating medium with NH4Cl and 7-ethoxycoumarin, respectively. Hepatocytes cultured on galactosylated PVDF hollow fibers maintained better albumin secretion and P-450 functions than on unmodified and serum-coated PVDF hollow fibers when cultured in serum-containing medium. Morphological examination by scanning electron microscopy showed that hepatocytes cultured on galactosylated PVDF hollow fibers developed significant aggregation, in contrast to those cultured on unmodified PVDF fibers or on serum-coated PVDF fibers. Transmission electron microscopy images revealed that tight junctions and canaliculus-like structures formed in these aggregates. These results suggest the potential application of this galactosylated PVDF hollow fiber cartridge for the design of a bioartificial liver assist device.

Three-dimensional Co-culture of Rat Hepatocyte Spheroids and NIH/3T3 Fibroblasts Enhances Hepatocyte Functional Maintenance

Acta Biomaterialia. Jul, 2005  |  Pubmed ID: 16701821

Functional maintenance of primary hepatocytes in culture can be improved by several distinct approaches involving optimization of the extracellular matrix microenvironment, media composition and cell-cell interactions, both homotypic and heterotypic. Using a galactose-decorated surface, we have developed a method to combine these two approaches by co-culturing rat primary hepatocyte spheroids with NIH/3T3 mouse fibroblast cells. Spheroids were performed by culturing hepatocytes for 3 days on galactosylated poly(vinylidene difluoride) membrane; NIH/3T3 cells were subsequently seeded and co-cultured with the spheroids. Results showed that although NIH/3T3 cells alone responded poorly to the galactosylated PVDF surface and displayed limited attachment, NIH/3T3 fibroblasts attached to the periphery of the hepatocyte spheroids and proliferated around them. Co-cultured hepatocyte spheroids exhibited significantly higher liver-specific functions as compared to spheroids cultured alone. Albumin secretion level in this co-culture system peaked on day 11, which was 1.8- and 2.9-times higher than the peak expression level in spheroid homo-culture control in serum-free (day 3) and serum-containing media (day 4), respectively. The albumin secretion function was maintained for at least two weeks; it was 5.1 (in serum-free medium) and 17.8 (in serum-containing medium) times higher than spheroid homo-culture on day 13. Similarly, the co-culture system also expressed approximately 5.5- and 3.1-times higher 3-methylcholanthrene-induced cytochrome P450 enzymatic activity on day 14 as compared to the homo-culture control in serum-free and serum-containing medium, respectively. In conclusion, this unique co-culture system demonstrated the synergistic roles of homotypic cell-cell interaction, heterotypic cell-cell interaction, cell-substrate interaction and soluble stimuli in hepatocyte functional maintenance.

Significance of Synthetic Nanostructures in Dictating Cellular Response

Nanomedicine : Nanotechnology, Biology, and Medicine. Mar, 2005  |  Pubmed ID: 17292053

Cell-substratum interaction is influenced by topographical in addition to chemical cues. The majority of patterning studies on cellular response have been conducted on micropatterned surfaces. Cells clearly respond to the topography of substrates in the micron range in terms of adhesion, proliferation, migration, and gene expression. However, cells in their natural environment also interact with extracellular matrix components in the nanometer scale. This review will cover recent studies that show mammalian cells responding to nanoscale features on a synthetic surface. An important and exciting direction of research in nanomedicine would be to gain a better understanding of the interaction between cells and nanostructures. This will facilitate the creation of the next generation of biomaterials with well-defined nanotopography that can elicit the desired cellular and tissue response.

Temperature-responsive Hydroxybutyl Chitosan for the Culture of Mesenchymal Stem Cells and Intervertebral Disk Cells

Biomaterials. Jan, 2006  |  Pubmed ID: 16115680

Temperature-responsive polymers are attractive candidates for applications related to injectable delivery of biologically active therapeutics, such as stem cells. In this study, we evaluate the potential of thermosensitive hydroxybutyl chitosan (HBC) as a biomaterial for the culture of human mesenchymal stem cells (hMSC) and cells derived from the intervertebral disk, with the eventual goal of using the HBC polymer as an injectable matrix/cell therapeutic. Conjugation of hydroxybutyl groups to chitosan renders the polymer water soluble and thermally responsive. Below its lower critical solution temperature, a solution of HBC can be maintained indefinitely in its solvated state. Upon exposure to a 37 degrees C environment, within 60 s, a 3.8 wt% HBC solution rapidly forms a gel that can be maneuvered with forceps. Upon cooling, the gel once again is able to revert to its solvated state. The gel exhibits a dramatic increase in both G' and G'' with increasing temperature, signifying a temperature-dependent enhancement of gel mechanical properties. Although a solid structure upon gelation, due to its physical nature of polymer interaction and gel formation, the gel exhibits a fluid-like viscoelastic behavior when exposed to shear stresses of up to 10% strain, with both G' and G'' approaching zero with increasing shear stress. Formulations of HBC gels presented in this study have gelation temperatures ranging from 13.0 to 34.6 degrees C and water contents of 67-95%. Minimal cytotoxicity in MSC and disk cell cultures was observed with these polymers up to a concentration of 5 wt%. Detection of metabolic activity, genetic analysis of synthesized mRNA, and histological staining of MSC and disk cell cultures in these gels collectively indicate cell proliferation without a loss in metabolic activity and extracellular matrix production. This study suggests the potential of HBC gel as an injectable carrier for future applications of delivering therapeutics to encourage a biologically relevant reconstruction of the degenerated disk.

Biodegradable and Photocrosslinkable Polyphosphoester Hydrogel

Biomaterials. Mar, 2006  |  Pubmed ID: 16125222

A new biodegradable, photocrosslinkable and multifunctional macromer, poly(6-aminohexyl propylene phosphate) (PPE-HA)-ACRL, was synthesized by conjugation of acrylate groups to the side chains of PPE-HA. By controlling the synthetic conditions, different weight fractions of acrylate in the macromers were achieved as confirmed by 1H NMR. The hydrogels obtained from PPE-HA-ACRL through photocrosslinking were dominantly elastic. With increasing acrylate contents in the macromers, the hydrogels exhibited a lower swelling ratio and higher mechanical strength. The hydrogels with different crosslinking densities lost between 4.3% and 37.4% of their mass in 84 days when incubated in phosphate-buffered saline at 37 degrees C. No significant cytotoxicity of the macromers against bone marrow-derived mesenchymal stem cells from goat (GMSC) was observed at a concentration up to 10mg/ml. Finally, GMSCs encapsulated in the photopolymerized gel maintained their viability when cultured in osteogenic medium for three weeks. Clear mineralization in the hydrogel scaffold was revealed by Von Kossa staining. This study suggests the potential of these biodegradable and photocrosslinkable as injectable tissue engineering scaffolds.

Targeted Tumor Cell Death Induced by Autologous Tumor-specific T Lymphocyte Recognition of Wild-type P53-derived Peptides

Journal of Neuro-oncology. Jan, 2006  |  Pubmed ID: 16132498

Autologous tumor-specific T lymphocyte (ATTL) lines were derived from the peripheral blood mononuclear cells (PBMC) of a healthy volunteer with human leukocyte antigen (HLA) -A*0201. These lines were achieved using interleukins -1beta, -2, -4, and -6 and the p53-based peptide from the 264-272 sequence of the wild-type p53 protein with a strong affinity against HLA-A*0201.;The frequencies of CD3+, CD4+, and CD8+ lymphocytes were 94-96%, 30-34%, and 69-74%, respectively. ATTLs killed most of the T2 cells pulsed with p53-derived peptide, but not against the T2 cells non-pulsed or pulsed with an irrelevant peptide. ATTLs also killed TKB-14 cells, which have been derived from human glioblastoma multiforme, and exhibited HLA-A*0201 molecule and immunohistochemical accumulation of p53 protein. These cytotoxic activities were inhibited by anti-CD3, anti-CD8, and anti-class I antibodies. These findings suggested that these ATTL lines might include CTL populations, which could recognize p53-derived peptide on HLA-A*0201 and the p53-based peptide may play as an antigen on HLA-A*0201. When tumor antigens would be more analyzed in the future, ATTL could be induced without the primary-cultured cells from tumor tissue and could be applied for cancer therapy.

Controlled Release of Heparin from Poly(epsilon-caprolactone) Electrospun Fibers

Biomaterials. Mar, 2006  |  Pubmed ID: 16305806

Sustained delivery of heparin to the localized adventitial surface of grafted blood vessels has been shown to prevent the vascular smooth muscle cell (VSMC) proliferation that can lead to graft occlusion and failure. In this study heparin was incorporated into electrospun poly(epsilon-caprolactone) (PCL) fiber mats for assessment as a controlled delivery device. Fibers with smooth surfaces and no bead defects could be spun from polymer solutions with 8%w/v PCL in 7:3 dichloromethane:methanol. A significant decrease in fiber diameter was observed with increasing heparin concentration. Assessment of drug loading, and imaging of fluorescently labeled heparin showed homogenous distribution of heparin throughout the fiber mats. A total of approximately half of the encapsulated heparin was released by diffusional control from the heparin/PCL fibers after 14 days. The fibers did not induce an inflammatory response in macrophage cells in vitro and the released heparin was effective in preventing the proliferation of VSMCs in culture. These results suggest that electrospun PCL fibers are a promising candidate for delivery of heparin to the site of vascular injury.

Surface-immobilization of Adhesion Peptides on Substrate for Ex Vivo Expansion of Cryopreserved Umbilical Cord Blood CD34+ Cells

Biomaterials. May, 2006  |  Pubmed ID: 16376984

The interaction between integrins and extracellular matrix proteins play an important role in the regulation of hematopoiesis. Human hematopoietic progenitor cells express very late antigen-4 (VLA-4) and VLA-5, which mediate their interaction with fibronectin by recognizing the connecting segment-1 (CS-1 and RGD motifs, respectively. In this study, we investigated the ex vivo expansion of human umbilical cord blood (UCB) CD34+ cells on synthetic substrates surface-immobilized with peptides containing the CS-1 binding motif (EILDVPST) and the RGD motif (GRGDSPC). These peptides were covalently conjugated to poly(ethylene terephthalate) (PET) film at a surface density of 2.0-2.3 nmol/cm2. UCB CD34+ cells were cultured for 10 days in serum-free medium supplemented with recombinant human thrombopoietin, stem cell factor, flt3-ligand and interleukin 3. The highest cell expansion fold was observed on the CS-1 peptide-modified surface, where total nucleated cells, total colony forming unit, and long-term culture initiating cells were expanded by 589.6+/-58.6 (mean+/-s.d.), 76.5+/-8.8, and 3.2+/-0.9-fold, respectively, compared to unexpanded cells. All substrates surface-immobilized with peptides, including the control peptides, were more efficient in supporting the expansion of CD34+, CFU-GEMM and LTC-ICs than tissue culture polystyrene surface. Nevertheless, after 10-days of ex vivo expansion from 600 CD34+ cells, only cells cultured on CS-1-immobilized surface yielded positive engraftment, even though the frequency was low. PET surface immobilized with RGD peptide was less efficient than that with CS-1 peptide. Our results suggest that covalently immobilized adhesion peptides can significantly influence the proliferation characteristics of cultured UCB CD34+ cells.

Chitosan-g-PEG/DNA Complexes Deliver Gene to the Rat Liver Via Intrabiliary and Intraportal Infusions

The Journal of Gene Medicine. Apr, 2006  |  Pubmed ID: 16389625

Chitosan has been shown to be a non-toxic and efficient vector for in vitro gene transfection and in vivo gene delivery through pulmonary and oral administrations. Recently, we have shown that chitosan/DNA nanoparticles could mediate high levels of gene expression following intrabiliary infusion 1. In this study, we have examined the possibility of using polyethylene glycol (PEG)-grafted chitosan/DNA complexes to deliver genes to the liver through bile duct and portal vein infusions.

PEI-g-chitosan, a Novel Gene Delivery System with Transfection Efficiency Comparable to Polyethylenimine in Vitro and After Liver Administration in Vivo

Bioconjugate Chemistry. Jan-Feb, 2006  |  Pubmed ID: 16417264

Polyethylenimine-graft-chitosan (PEI-g-chitosan) was synthesized by performing cationic polymerization of aziridine in the presence of water-soluble oligo-chitosan (M(n) = 3400). The absolute molecular weight and chemistry of the PEI-g-chitosan obtained were characterized using GPC, 13C and 1H NMR, respectively. The results indicated that all the amines of chitosan were grafted with oligo-PEI, and the average length of the oligo-PEI side chains was determined by the feed molar ratio of aziridne/amine in chitosan. PEI-g-chitosan of M(n) = 7400 with a polydispersity index (PDI) of 1.50, and PEI side chains of M(n) = 206 was prepared for gene delivery. Gel electrophoresis showed that DNA migration was retarded completely at a N/P ratio of 2.5/1, indicating good DNA condensation capability of PEI-g-chitosan. The sizes and the zeta-potentials of the complexes of PEI-g-chitosan/DNA were characterized. The cytotoxicity of PEI-g-chiotsan was evaluated, and the results reflected that PEI-g-chitosan had a lower cytotoxicity than PEI (25 K). Gene transfection efficiency of PEI-g-chitosan in HepG2, HeLa, and primary hepatocytes cells and after administration in the common bile duct of rat liver was determined. Remarkably, PEI-g-chitosan showed a higher transfection efficiency than that of PEI (25 K) both in vitro and in vivo. The systematic distribution and the distribution in liver of the gene expression of the complexes of PEI-chitosan/DNA were determined as well.

Nonviral Gene Delivery from Nonwoven Fibrous Scaffolds Fabricated by Interfacial Complexation of Polyelectrolytes

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2006  |  Pubmed ID: 16497560

We investigated a novel nonwoven fibrous scaffold as a vehicle for delivery of DNA. Fibers were formed by polyelectrolyte complexation of water-soluble chitin and alginate, and PEI-DNA nanoparticles were encapsulated during the fiber drawing process. Nanoparticles released from the fibers over time retained their bioactivity and successfully transfected cells seeded on the scaffold in a sustained manner. Transgene expression in HEK293 cells and human dermal fibroblasts seeded on the transfecting scaffolds was significant even after 2 weeks of culture compared to 3-day expression in two-dimensional controls. Fibroblasts seeded on scaffolds containing DNA encoding basic fibroblast growth factor (bFGF) demonstrated prolonged secretion of bFGF at levels significantly higher than baseline. This work establishes the potential of this fibrous scaffold as a matrix capable of delivering genes to direct and support cellular development in tissue engineering.

Dynamic and Static Light Scattering Studies on Self-aggregation Behavior of Biodegradable Amphiphilic Poly(ethylene Oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene Oxide) Triblock Copolymers in Aqueous Solution

The Journal of Physical Chemistry. B. Mar, 2006  |  Pubmed ID: 16553399

The self-aggregation behavior of two amphiphilic poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) triblock copolymer samples with nearly identical PHB block lengths but different PEO block lengths, PEO-PHB-PEO(2000-810-2000) and PEO-PHB-PEO(5000-780-5000), was studied with dynamic and static light scattering (DLS and SLS), in combination with fluorescence spectroscopy and transmission electron microscopy (TEM). The formation of polymeric micelles by the two PEO-PHB-PEO triblock copolymers was confirmed with fluorescence technique and TEM. DLS analysis showed that the hydrodynamic radius (R(h)) of the monodistributed polymeric micelles increased with an increase in PEO block length. The relative thermostability of the triblock copolymer micelles was studied by SLS and DLS at different temperatures. The aggregation number and the ratio of the radius of gyration over hydrodynamic radius were found to be independent of temperature, probably due to the strong hydrophobicity of the PHB block. The combination of DLS and SLS studies indicated that the polymeric micelles were composed of a densely packed core of hydrophobic PHB blocks and a corona shell formed by hydrophilic PEO blocks. The aggregation numbers were found to be approximately 53 for PEO-PHB-PEO(2000-810-2000) micelles and approximately 37 for PEO-PHB-PEO(5000-780-5000) micelles. The morphology of PEO-PHB-PEO spherical micelles determined by DLS and SLS measurements was further confirmed by TEM.

Self-assembled Supramolecular Hydrogels Formed by Biodegradable PEO-PHB-PEO Triblock Copolymers and Alpha-cyclodextrin for Controlled Drug Delivery

Biomaterials. Aug, 2006  |  Pubmed ID: 16584769

A materials design of a new supramolecular hydrogel self-assembled between alpha-cyclodextrin and a biodegradable poly(ethylene oxide)-poly[(r)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) triblock copolymer was demonstrated. The cooperation effect of complexation of PEO segments with alpha-cyclodextrin and the hydrophobic interaction between PHB blocks resulted in the formation of the supramolecular hydrogel with a strong macromolecular network. The in vitro release kinetics studies of fluorescein isothiocyanate labeled dextran (dextran-FITC) model drug from the hydrogel showed that the hydrogel was suitable for relatively long-term sustained controlled release of macromolecular drugs, which many simple triblock copolymer hydrogel systems could not achieve. The hydrogel was found to be thixotropic and reversible, and can be applied as a promising injectable drug delivery system.

Inducing Hepatic Differentiation of Human Mesenchymal Stem Cells in Pellet Culture

Biomaterials. Aug, 2006  |  Pubmed ID: 16616366

Extensive cell-cell or cell-matrix interaction in three-dimensional (3D) culture is important for the maintenance of adult hepatocyte function and the maturation of hepatic progenitors. However, although there is significant interest in inducing the transdifferentiation of adult stem cells into the hepatic lineage, very few studies have been conducted in a 3D culture configuration. The aim of this study is to investigate the differentiation of mesenchymal stem cells (MSC) into hepatocytes in a pellet configuration, with or without the presence of small intestinal submucosa (SIS). After 4 weeks of differentiation with growth factors bFGF, HGF, and OsM, we obtained hepatocyte-like cells that expressed a subset of hepatic genes, secreted albumin and urea, stored glycogen, and showed inducible CYP3A4 mRNA levels. When these cells were implanted into livers of hepatectomized rats, they secreted human albumin into the bloodstream. The hepatic differentiation of MSC was faster in cell pellets without SIS. The plausible explanations for this finding may be related to the mass transport issues of the two different pellets and the role of cell-cell contact over cell-matrix interactions. The findings of this study should help in the design of optimal culture configurations for efficient hepatic differentiation of adult stem cells.

Enhanced Extracellular Matrix Production and Differentiation of Human Embryonic Germ Cell Derivatives in Biodegradable Poly(epsilon-caprolactone-co-ethyl Ethylene Phosphate) Scaffold

Acta Biomaterialia. Jul, 2006  |  Pubmed ID: 16709468

Extracellular environment regulates cell behavior and also influences the differentiation of stem cells. Two cell lines of pluriopotent human embryonic germ cell derivatives (EBD cells) were cultured on a biodegradable poly(epsilon-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) and non-degradable cellulose acetate scaffold. Their cell behaviors including proliferation, differentiation, cell distribution and extracellular matrix production were studied for 4 weeks and 10 months. The proliferation of the EBD cells was enhanced in both of the three-dimensional scaffolds in the first 5 weeks of culture, regardless of the material difference, compared to monolayer culture. While the gene expression profile remained multilineage for the EBD cells cultured in the cellulose acetate fibrous scaffold, much of the neuronal lineage markers were down-regulated in EBD cells cultured in the PCLEEP scaffold. On the other hand, extracellular matrix production was significantly enhanced in the PCLEEP scaffold. The study showed that the polymer substrate could influence the differentiation and growth of pluripotent stem cells in the absence of exogenous biochemical signals.

Enhancing Efficacy of HIV Gag DNA Vaccine by Local Delivery of GM-CSF in Murine and Macaque Models

Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research. Jun, 2006  |  Pubmed ID: 16734558

Controlled release of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein by albumin-heparin microparticles administered via intramuscular vaccination in conjunction with HIV DNA vaccines stimulated HIV Gag-specific immune responses. In the murine model, Gag-specific cytotoxic T lymphocyte (CTL) and T helper (Th) responses were significantly enhanced by administration of murine GM-CSF microparticles. This effect was comparable to a GM-CSF encoded plasmid. In three of four rhesus monkeys, enhancement of Gag-specific antibody (Ab), Th, and CTL responses was observed 1 month after the first immunization with coadministration of human GM-CSF microparticles and HIV Gag plasmid. The second, third, and fourth booster immunizations, however, did not increase the Gag-specific immune responses. Subsequent application of Gag protein in complete Freund's adjuvant (CFA) significantly enhanced Ab and Th, but not CTL. However, Gag-specific CTL response was triggered by cytokine and Gag p55-encapsulated microparticles in all animals. The strategy of priming immune responses by coadministration of cytokine microparticles and DNA vaccines, followed by boosting with cytokine and antigen protein-encapsulated microparticles, may prove effective in improving an HIV DNA vaccine design.

Expansion of Engrafting Human Hematopoietic Stem/progenitor Cells in Three-dimensional Scaffolds with Surface-immobilized Fibronectin

Journal of Biomedical Materials Research. Part A. Sep, 2006  |  Pubmed ID: 16739181

An efficient and practical ex vivo expansion methodology for human hematopoietic stem/progenitor cells (HSPCs) is critical in realizing the potential of HSPC transplantation in treating a variety of hematologic disorders and as a supportive therapy for malignant diseases. We report here an expansion strategy using a three-dimensional (3D) scaffold conjugated with an extracellular matrix molecule, fibronectin (FN), to partially mimic the hematopoietic stem cell niche. FN-immobilized 3D polyethylene terephthalate (PET) scaffold was synthesized and evaluated for HSPC expansion efficiency, in comparison with a FN-immobilized 2D PET substrate and a 3D scaffold with FN supplemented in the medium. Covalent conjugation of FN produced substrate and scaffold with higher cell expansion efficiency than that on their unmodified counterparts. After 10 days of culture in serum-free medium, human umbilical cord blood CD34+ cells cultured in FN-conjugated scaffold yielded the highest expansion of CD34+ cells (approximately 100 fold) and long-term culture initiating cells (approximately 47-fold). The expanded human CD34+ cells successfully reconstituted hematopoiesis in NOD/SCID mice. This study demonstrated the synergistic effect between the three-dimensionality of the scaffold and surface-conjugated FN, and the potential of this FN-conjugated 3D scaffold for ex vivo expansion of HSPCs.

Natural Polymers for Gene Delivery and Tissue Engineering

Advanced Drug Delivery Reviews. Jul, 2006  |  Pubmed ID: 16762443

Although the field of gene delivery is dominated by viral vectors and synthetic polymeric or lipid gene carriers, natural polymers offer distinct advantages and may help advance the field of non-viral gene therapy. Natural polymers, such as chitosan, have been successful in oral and nasal delivery due to their mucoadhesive properties. Collagen has broad utility as gene activated matrices, capable of delivering large quantities of DNA in a direct, localized manner. Most natural polymers contain reactive sites amenable for ligand conjugation, cross-linking, and other modifications that can render the polymer tailored for a range of clinical applications. Natural polymers also often possess good cytocompatibility, making them popular choices for tissue engineering scaffolding applications. The marriage of gene therapy and tissue engineering exploits the power of genetic cell engineering to provide the biochemical signals to influence proliferation or differentiation of cells. Natural polymers with their ability to serve as gene carriers and tissue engineering scaffolds are poised to play an important role in the field of regenerative medicine. This review highlights the past and present research on various applications of natural polymers as particulate and matrix delivery vehicles for gene delivery.

Hyperbranched Poly(amino Ester)s with Different Terminal Amine Groups for DNA Delivery

Biomacromolecules. Jun, 2006  |  Pubmed ID: 16768410

Hyperbranched poly(amino ester)s containing tertiary amines in the core and primary, secondary, and tertiary amines in the periphery, respectively, were evaluated for DNA delivery in vitro. The same core structure facilitated the investigation on the effects of the terminal amine type on the properties of hyperbranched poly(amino ester)s for DNA delivery. The hydrolysis of the poly(amino ester)s was monitored using (1)H NMR. The results reflected that the terminal amine type had negligible effects on the hydrolysis rate but was much slower than that of linear poly(amino ester)s, probably due to the compact hyperbranched spatial structure preventing the accessibility of water. In comparison with PEI 25 K, the hyperbranched poly(amino ester)s showed much lower cytotoxicity in Cos7, HEK293, and HepG2 cells. Gel electrophoresis indicated that poly(amino ester)s could condense DNA efficiently, and the zeta potentials and sizes of the complexes formed with different weight ratios of hyperbranched poly(amino ester)s and DNA were measured. Remarkably, all the hyperbranched poly(amino ester)s showed DNA transfection efficiency comparable to PEI 25 K in Cos7, HEK293, and HepG2 cells regardless of the terminal amine type. Therefore, the terminal amine type had insignificant effects on the hydrolysis rate, cytotoxicity, DNA condensation capability, and in vitro DNA transfection efficiency of the hyperbranched poly(amino ester)s.

Surface-aminated Electrospun Nanofibers Enhance Adhesion and Expansion of Human Umbilical Cord Blood Hematopoietic Stem/progenitor Cells

Biomaterials. Dec, 2006  |  Pubmed ID: 16854459

Interaction between hematopoietic stem/progenitor cells (HSPCs) and their extra cellular matrix components is an integral part of the signaling control for HSPC survival, proliferation and differentiation. We hypothesized that both substrate topographical cues and biochemical cues could act synergistically with cytokine supplementation to improve ex vivo expansion of HSPCs. In this study, we compared the ex vivo expansion of human umbilical cord blood CD34(+) cells on unmodified, hydroxylated, carboxylated and aminated nanofibers and films. Results from 10-day expansion cultures showed that aminated nanofiber mesh and film were most efficient in supporting the expansion of the CD34(+)CD45(+) cells (195-fold and 178-fold, respectively), as compared to tissue culture polystyrene (50-fold, p<0.05). In particular, aminated nanofiber meshes supported a higher degree of cell adhesion and percentage of HSPCs, as compared to aminated films. SEM imaging revealed the discrete colonies of cells proliferating and interacting with the aminated nanofibers. This study highlights the potential of a biomaterials approach to influence the proliferation and differentiation of HSPCs ex vivo.

Interaction of Human Mesenchymal Stem Cells with Disc Cells: Changes in Extracellular Matrix Biosynthesis

Spine. Aug, 2006  |  Pubmed ID: 16915085

To evaluate the in vitro interactions between human mesenchymal stem cells (MSCs) and degenerative disc cells.

Proliferation and Differentiation of Human Mesenchymal Stem Cell Encapsulated in Polyelectrolyte Complexation Fibrous Scaffold

Biomaterials. Dec, 2006  |  Pubmed ID: 16919722

A biofunctional scaffold was constructed with human mesenchymal stem cells (hMSCs) encapsulated in polyelectrolyte complexation (PEC) fibers. Human MSCs were either encapsulated in PEC fibers and constructed into a fibrous scaffold or seeded on PEC fibrous scaffolds. The proliferation, chondrogenic and osteogenic differentiation of the encapsulated and seeded hMSCs were compared for a culture period of 5.5 weeks. Gene expression and extracellular matrix production showed evidences of chondrogenesis and osteogenesis in the cell-encapsulated scaffolds and cell-seeded scaffolds when the samples were cultured in the chondrogenic and osteogenic differentiation media, respectively. However, better cell proliferation and differentiation were observed on the hMSC-encapsulated scaffolds compared to the hMSC-seeded scaffolds. The study demonstrated that the cell-encapsulated PEC fibers could support proliferation and chondrogenic and osteogenic differentiation of the encapsulated-hMSCs. Together with our previous works, which demonstrated the feasibility of PEC fiber in controlled release of drug, protein and gene delivery, the reported PEC fibrous scaffold system will have the potential in composing a multi-component system for various tissue-engineering applications.

In Vivo Evaluation of Plasmid DNA Encoding OP-1 Protein for Spine Fusion

Spine. Sep, 2006  |  Pubmed ID: 16946649

A posterolateral lumbar interbody arthrodesis animal model was selected to evaluate the percutaneous delivery of OP-1 plasmid DNA. OBJECTIVE.: To evaluate the feasibility of achieving ectopic bone formation using nonviral gene delivery with a minimally invasive technique, by coinjecting plasmid DNA encoding OP-1 with collagen into the paraspinal muscle.

Co-culture of Umbilical Cord Blood CD34+ Cells with Human Mesenchymal Stem Cells

Tissue Engineering. Aug, 2006  |  Pubmed ID: 16968157

Insufficient numbers of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) sometimes limit allogenic transplantation of umbilical cord blood (UCB). Ex vivo expansion may overcome this limitation. Mesenchymal stem cells (MSCs), as non-hematopoietic, well-characterized skeletal and connective-tissue progenitor cells within the bone marrow stroma, have been investigated as support cells for the culture of HSCs/HPCs. MSCs are attractive for the rich environmental signals that they provide and for immunological compatibility in transplantation. Thus far, HSC/MSC co-cultures have mainly been performed in 2-dimensional (2D) configuration. We postulate that a 3-dimensional (3D) culture environment that resembles the natural in vivo hematopoietic compartment might be more conducive for regulating HSC expansion. In this study, we compared the co-culture of HSCs and MSCs in 2D and 3D configurations. The results demonstrated the benefit of MSC inclusion in HSC expansion ex vivo. Direct contact between MSCs and HSCs in 3D cultures led to statistically significantly higher expansion of cord blood CD34+ cells than in 2D cultures (891- versus 545-fold increase in total cells, 96- versus 48-fold increase of CD34+ cells, and 230- versus 150-fold increase in colony-forming cell assay [CFC]). Engraftment assays in non-obese diabetic/severe combined immunodeficiency mice also indicated a high success rate of hematopoiesis reconstruction with these expanded cells.

Small Intestinal Submucosa As a Potential Bioscaffold for Intervertebral Disc Regeneration

Spine. Oct, 2006  |  Pubmed ID: 17023850

To evaluate the capacity of porcine small intestine submucosa to support the in vitro proliferation of human disc cells and the synthesis of extracellular matrix that could restore the biochemical properties of the disc.

Evaluating the Intracellular Stability and Unpacking of DNA Nanocomplexes by Quantum Dots-FRET

Journal of Controlled Release : Official Journal of the Controlled Release Society. Nov, 2006  |  Pubmed ID: 17081642

We demonstrate a highly sensitive method to characterize the structural composition and intracellular fate of polymeric DNA nanocomplexes, formed by condensing plasmid DNA with cationic polymers through electrostatic interactions. Rational design of more efficient polymeric gene carriers will be possible only with mechanistic insights of the rate-limiting steps in the non-viral gene transfer process. To characterize the composition and binding dynamics of nanocomplexes, plasmid and its polymer carrier within nanocomplexes were labeled with quantum dots (QDs) and fluorescent organic dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled precise detection of discrete changes in nanocomplex state at the single-particle level, against various intracellular microenvironments. The distribution and unpacking of individual nanocomplexes within cells could thus be unambiguously followed by fluorescence microscopy. QD-FRET is a highly sensitive and quantitative method to determine the composition and dynamic stability of nanocomplexes during intracellular transport, where barriers to gene delivery may be identified to facilitate gene carrier optimization.

Chitosan-DNA Nanoparticles Delivered by Intrabiliary Infusion Enhance Liver-targeted Gene Delivery

International Journal of Nanomedicine. 2006  |  Pubmed ID: 17369870

The goal of this study was to examine the efficacy of liver-targeted gene delivery by chitosan-DNA nanoparticles through retrograde intrabiliary infusion (RII). The transfection efficiency of chitosan-DNA nanoparticles, as compared with PEI-DNA nanoparticles or naked DNA, was evaluated in Wistar rats by infusion into the common bile duct, portal vein, or tail vein. Chitosan-DNA nanoparticles administrated through the portal vein or tail vein did not produce detectable luciferase expression. In contrast, rats that received chitosan-DNA nanoparticles showed more than 500 times higher luciferase expression in the liver 3 days after RII; and transgene expression levels decreased gradually over 14 days. Luciferase expression in the kidney, lung, spleen, and heart was negligible compared with that in the liver. RII of chitosan-DNA nanoparticles did not yield significant toxicity and damage to the liver and biliary tree as evidenced by liver function analysis and histopathological examination. Luciferase expression by RII of PEI-DNA nanoparticles was 17-fold lower than that of chitosan-DNA nanoparticles on day 3, but it increased slightly over time. These results suggest that RII is a promising routine to achieve liver-targeted gene delivery by non-viral nanoparticles; and both gene carrier characteristics and mode of administration significantly influence gene delivery efficiency.

Hepatic Differentiation Potential of Commercially Available Human Mesenchymal Stem Cells

Tissue Engineering. Dec, 2006  |  Pubmed ID: 17518684

The ready availability and low immunogenicity of commercially available mesenchymal stem cells (MSC) render them a potential cell source for the development of therapeutic products. With cell source a major bottleneck in hepatic tissue engineering, we investigated whether commercially available human MSC (hMSC) can transdifferentiate into the hepatic lineage. Based on previous studies that find rapid gain of hepatic genes in bone marrow-derived stem cells cocultured with liver tissue, we used a similar approach to drive hepatic differentiation by coculturing the hMSC with rat livers treated or untreated with gadolinium chloride (GdCl(3)). After a 24-hour coculture period with liver tissue injured by GdCl(3) in a Transwell configuration, approximately 34% of the cells differentiated into albumin-expressing cells. Cocultured cells were subsequently maintained with growth factors to complete the hepatic differentiation. Cocultured cells expressed more hepatic gene markers, and had higher metabolic functions and P450 activity than cells that were only differentiated with growth factors. In conclusion, commercially available hMSC do show hepatic differentiation potential, and a liver microenvironment in culture can provide potent cues to accelerate and deepen the differentiation. The ability to generate hepatocyte-like cells from a commercially available cell source would find interesting applications in liver tissue engineering.

Quantum-dots-FRET Nanosensors for Detecting Unamplified Nucleic Acids by Single Molecule Detection

Nanomedicine (London, England). Jun, 2006  |  Pubmed ID: 17716215

Chitosan Nanoparticles for Oral Drug and Gene Delivery

International Journal of Nanomedicine. 2006  |  Pubmed ID: 17722528

Chitosan is a widely available, mucoadhesive polymer that is able to increase cellular permeability and improve the bioavailability of orally administered protein drugs. It can also be readily formed into nanoparticles able to entrap drugs or condense plasmid DNA. Studies on the formulation and oral delivery of such chitosan nanoparticles have demonstrated their efficacy in enhancing drug uptake and promoting gene expression. This review summarizes some of these findings and highlights the potential of chitosan as a component of oral delivery systems.

Mechanical Properties of Single Electrospun Drug-encapsulated Nanofibres

Nanotechnology. Aug, 2006  |  Pubmed ID: 19079553

The mechanical and structural properties of a surface play an important role in determining the morphology of attached cells, and ultimately their cellular functions. As such, mechanical and structural integrity are important design parameters for a tissue scaffold. Electrospun fibrous meshes are widely used in tissue engineering. When in contact with electrospun scaffolds, cells see the individual micro- or nanofibres as their immediate microenvironment. In this study, tensile testing of single electrospun nanofibres composed of poly(ε-caprolactone) (PCL), and its copolymer, poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP), revealed a size effect in the Young's modulus, E, and tensile strength, σ(T). Both strength and stiffness increase as the fibre diameter decreases from bulk (∼5 μm) into the nanometre region (200-300 nm). In particular, E and σ(T) of individual PCL nanofibres were at least two-fold and an order of magnitude higher than that of PCL film, respectively. PCL films were observed to have more pronounced crystallographic texture than the nanofibres; however no difference in crystalline fraction, perfection, or texture was detected among the various fibres. When drugs were encapsulated into single PCLEEP fibres, mechanical properties were enhanced with 1-20 wt% of loaded retinoic acid, but weakened by 10-20 wt% of encapsulated bovine serum albumin. This understanding of the effect of size and drug and protein encapsulation on the mechanical properties of electrospun fibres may help in the optimization of tissue scaffold design that combines biochemical and biomechanical cues for tissue regeneration.

Radio-responsive Gene Therapy for Malignant Glioma Cells Without the Radiosensitive Promoter: Caspase-3 Gene Therapy Combined with Radiation

Cancer Letters. Feb, 2007  |  Pubmed ID: 16644107

Caspase-3 plays a critical role as an executioner of apoptosis. The aim of this study is to evaluate the potential of the combination of caspase-3 gene therapy and radiation treatment. We prepared a plasmid (pCI-CSP3) that contained the human caspase-3 gene and the cytomegalovirus promoter. We introduced this plasmid into U251 and U87 human glioma cells and subjected the cells to radiation treatment. The degree of cell death and apoptosis were evaluated. None of the cell lines underwent apoptosis by the overexpression of caspase-3 alone, but the degree of cell death and apoptosis were markedly enhanced by the addition of radiation treatment. Next, we prepared another plasmid (EGR-CSP3) that contained the caspase-3 gene and a radiation-sensitive promoter. Each treatment system using either pCI-CSP3 or EGR-CSP3 showed radio response. The treatment system using pCI-CSP3 more effectively induced apoptosis than that using EGR-CSP3. Caspase-3 gene therapy in combination with radiation treatment has the potential to serve as a radio-responsive gene therapy without any radiation-sensitive promoter.

Dynamics of Smooth Muscle Cell Deadhesion from Thermosensitive Hydroxybutyl Chitosan

Biomaterials. Mar, 2007  |  Pubmed ID: 17157377

Thermoresponsive polymer (TRP) enables the enzyme-free harvesting of cells through an acute increase in surface hydrophilicity of TRP across its lower critical solution temperature (LCST), rendering feasible the generation of polymer-free cell sheets for regenerative medicine applications. To date, the intricate mechanisms of cell deadhesion/detachment on TRP surface remain obscure. Elucidation of such biophysical responses would be valuable for the cell sheet technology. In this study, integrative biophysical techniques are applied to probe the thermal-induced deadhesion kinetics of smooth muscle cell (SMC) on thermoresponsive hydroxybutyl chitosan (HBC29) against different periods of pre-culture time at 37 degrees C. Atomic force microscopy demonstrates that both the surface topography and mechanical property of HBC29 film in water are acutely modulated across its LCST. Firstly, cells show negligible changes in adhesion contact area during low-temperature incubation on unmodified tissue culture polystyrene (TCPS). Secondly, the recession of adhesion contact and retraction of cell body for cells with different pre-culture times are triggered by HBC29 coating on TCPS. Interestingly, the initial rate of reduction in the normalized adhesion contact area of SMC is negatively correlated with the pre-culture time. Thirdly, the degree of cell deformation and average adhesion energy are reducing functions of time only for SMCs with the lowest pre-culture time. In contrast, adhesion energy per cell is a reducing function of time irrespective of the change of pre-culture time. Lastly, the temporal dynamics of cytoskeleton organization and beta-actin/smoothelin-B mRNA expression for SMCs is strongly dependent on the pre-culture time. Overall, this study demonstrates that the thermal-induced deadhesion of SMC on TRP is characterized by the evolution of its contractile phenotypes.

Biomaterials Approach to Expand and Direct Differentiation of Stem Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. Mar, 2007  |  Pubmed ID: 17264853

Stem cells play increasingly prominent roles in tissue engineering and regenerative medicine. Pluripotent embryonic stem (ES) cells theoretically allow every cell type in the body to be regenerated. Adult stem cells have also been identified and isolated from every major tissue and organ, some possessing apparent pluripotency comparable to that of ES cells. However, a major limitation in the translation of stem cell technologies to clinical applications is the supply of cells. Advances in biomaterials engineering and scaffold fabrication enable the development of ex vivo cell expansion systems to address this limitation. Progress in biomaterial design has also allowed directed differentiation of stem cells into specific lineages. In addition to delivering biochemical cues, various technologies have been developed to introduce micro- and nano-scale features onto culture surfaces to enable the study of stem cell responses to topographical cues. Knowledge gained from these studies portends the alteration of stem cell fate in the absence of biological factors, which would be valuable in the engineering of complex organs comprising multiple cell types. Biomaterials may also play an immunoprotective role by minimizing host immunoreactivity toward transplanted cells or engineered grafts.

Effects of MIP-1 Alpha, MIP-3 Alpha, and MIP-3 Beta on the Induction of HIV Gag-specific Immune Response with DNA Vaccines

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2007  |  Pubmed ID: 17356539

Transfection of DNA vaccines with chemokines may recruit dendritic cells (DCs) locally to capture the antigenic genes and their gene products to generate enhanced CD8(+) cytotoxic T lymphocytes (CTLs). In this study, we investigated the effects of macrophage inflammatory protein (MIP)-1 alpha, MIP-3 alpha, and MIP-3beta on human immunodeficiency virus (HIV) Gag DNA vaccination. The chemokine plasmids markedly enhanced the local infiltration of inflammatory cells and increased the presence of CD11c(+) B7.2(+)-activated DCs. MIP-1 alpha and MIP-3 alpha were potent adjuvants in augmenting CTLs and afforded strong protection to immunized animals against challenge with vaccinia virus expressing Gag (vv-Gag). However, decreased humoral response was observed. MIP-3beta plasmid did not dramatically alter immunity. The chemokine inoculation time with respect to DNA vaccine priming was also investigated. The injection of pMIP-3 alpha three days before Gag plasmid (pGag) vaccination markedly increased specific CTLs compared with simultaneous injection and led to higher protection against vv-Gag. Immunity was also shifted toward a T-helper type-1 (Th1) response. In contrast, inoculation with pMIP-3 alpha three days after pGag vaccination shifted immunity toward a Th2 response. Our data suggest that administration of a chemokine with DNA vaccines offers a valuable strategy to modulate the efficacy and polarization of specific immunity and that chemokine-antigen timing is critical in determining overall biological effects.

Synthetic Nanostructures Inducing Differentiation of Human Mesenchymal Stem Cells into Neuronal Lineage

Experimental Cell Research. May, 2007  |  Pubmed ID: 17428465

Human mesenchymal stem cells (hMSCs) have been shown to trans-differentiate into neuronal-like cells by culture in neuronal induction media, although the mechanism is not well understood. Topography can also influence cellular responses including enhanced differentiation of progenitor cells. As extracellular matrix (ECM) in vivo comprises topography in the nanoscale, we hypothesize that nanotopography could influence stem cell differentiation into specific non-default pathways, such as transdifferentiation of hMSCs. Differentiation and proliferation of hMSCs were studied on nanogratings of 350 nm width. Cytoskeleton and nuclei of hMSCs were aligned and elongated along the nanogratings. Gene profiling and immunostaining showed significant up-regulation of neuronal markers such as microtubule-associated protein 2 (MAP2) compared to unpatterned and micropatterned controls. The combination of nanotopography and biochemical cues such as retinoic acid further enhanced the up-regulation of neuronal marker expressions, but nanotopography showed a stronger effect compared to retinoic acid alone on unpatterned surface. This study demonstrated the significance of nanotopography in directing differentiation of adult stem cells.

Tissue-engineered Bone Formation with Gene Transfer and Mesenchymal Stem Cells in a Minimally Invasive Technique

The Laryngoscope. Jul, 2007  |  Pubmed ID: 17507830

The objective of this study was to use a chitosan-alginate gel to implant bone marrow-derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote bone formation by the simultaneously transferred osteogenic protein (OP)-1 (bone morphogenic protein-7) gene.

Tissue Compatibility of Interfacial Polyelectrolyte Complexation Fibrous Scaffold: Evaluation of Blood Compatibility and Biocompatibility

Tissue Engineering. Feb, 2007  |  Pubmed ID: 17518574

Interfacial polyelectrolyte complexation (PEC) fiber has been proposed as a biostructural unit and biological construct for tissue engineering applications, with its ability to incorporate proteins, drug molecules, DNA nanoparticles, and cells. In this study, we evaluated the biocompatibility and blood compatibility of PEC fiber in order to assess its potential for in vivo applications in tissue engineering. Although chitosan-alginate PEC fibrous scaffold was found to be thrombogenic, the blood compatibility of the scaffold could be significantly improved by incorporating a small amount of heparin in the polyelectrolyte solution during fiber formation. The platelet microparticle production and platelet adhesion on the chitosan-alginate-heparin fibrous scaffold were comparable to those on the resting control. In vitro cytotoxicity test showed that the scaffold was not toxic to human mesenchymal stem cells (hMSCs). In the in vivo biocompatibility test in rats, no acute inflammation was observed in the subcutaneously or intramuscularly implanted specimens. Good cell infiltration and vascularization were observed after 2 months of implantations. Enhanced extracellular matrix (ECM) deposition was observed when hMSCs were cultured in the transforming growth factor-beta3 (TGF-beta3)-encapsulated PEC fibrous scaffold in vitro, or when the TGF-beta3-encapsulated PEC was implanted intramuscularly in vivo. The results showed that this versatile PEC fibrous scaffold could be used in various tissue engineering applications for its good biocompatible and blood compatible properties.

A Dual-functional Fibrous Scaffold Enhances P450 Activity of Cultured Primary Rat Hepatocytes

Acta Biomaterialia. Sep, 2007  |  Pubmed ID: 17532276

We have designed a novel dual-functional electrospun fibrous scaffold comprising two fiber mesh layers that were modified differently to induce two separate biological responses from hepatocytes. The first fiber layer was galactosylated on the surface to mediate hepatocyte attachment, while the second layer was loaded with 3-methylcholanthrene (3-Mc) to enhance cytochrome P450 activity of hepatocytes. Primary rat hepatocytes cultured on the galactosylated fibrous scaffolds loaded with different concentrations of 3-Mc were compared for their cell attachment efficiency, albumin secretion activity and cytochrome P450-dependent 7-ethoxycoumarin O-deethylase activity. This hybrid fibrous scaffold mediated hepatocyte attachment with slightly lower efficiency (76+/-2.3%) than a single-layer galactosylated fibrous scaffold (84+/-3.5%). More importantly, the cytochrome P450 activity of the hepatocytes cultured on the hybrid scaffold correlated well with the 3-Mc loading level. The results also showed that transfer of 3-Mc to hepatocytes through direct cell-fiber contact was the dominant transport route, with the induced cytochrome P450 activity being 1.9- to 4.8-fold higher than that of transfer of 3-Mc to hepatocytes via dissolution from fibers to medium. This study demonstrates the feasibility of creating multi-functional fibrous scaffolds that serve both as an adhesive substrate and as a delivery vehicle for bioactive molecules.

Functional Nanofiber Scaffolds with Different Spacers Modulate Adhesion and Expansion of Cryopreserved Umbilical Cord Blood Hematopoietic Stem/progenitor Cells

Experimental Hematology. May, 2007  |  Pubmed ID: 17577926

Nanofiber scaffolds with amino groups conjugated to fiber surface through different spacers (ethylene, butylenes, and hexylene groups, respectively) were prepared and the effect of spacer length on adhesion and expansion of umbilical cord blood hematopoietic stem/ progenitor cells (HSPCs) was investigated.

Myogenic Induction of Aligned Mesenchymal Stem Cell Sheets by Culture on Thermally Responsive Electrospun Nanofibers

Advanced Materials (Deerfield Beach, Fla.). 2007  |  Pubmed ID: 18584057

Aligned Protein-Polymer Composite Fibers Enhance Nerve Regeneration: A Potential Tissue-Engineering Platform

Advanced Functional Materials. 2007  |  Pubmed ID: 18618021

Sustained release of proteins from aligned polymeric fibers holds great potential in tissue-engineering applications. These protein-polymer composite fibers possess high surface-area-to-volume ratios for cell attachment, and can provide biochemical and topographic cues to enhance tissue regeneration. Aligned biodegradable polymeric fibers that encapsulate human glial cell-derived neurotrophic factor (GDNF, 0.13 wt%) were fabricated via electrospinning a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP) with GDNF. The protein was randomly dispersed throughout the polymer matrix in aggregate form, and released in a sustained manner for up to two months. The efficacy of these composite fibers was tested in a rat model for peripheral nerve-injury treatment. Rats were divided into four groups, receiving either empty PCLEEP tubes (control); tubes with plain PCLEEP electrospun fibers aligned longitudinally (EF-L) or circumferentially (EF-C); or tubes with aligned GDNF-PCLEEP fibers (EF-L-GDNF). After three months, bridging of a 15 mm critical defect gap by the regenerated nerve was observed in all the rats that received nerve conduits with electrospun fibers, as opposed to 50% in the control group. Electrophysiological recovery was seen in 20%, 33%, and 44% of the rats in the EF-C, EF-L, and EF-L-GDNF groups respectively, whilst none was observed in the controls. This study has demonstrated that, without further modification, plain electrospun fibers can help in peripheral nerve regeneration; however, the synergistic effect of an encapsulated growth factor facilitated a more significant recovery. This study also demonstrated the novel use of electrospinning to incorporate biochemical and topographical cues into a single implant for in vivo tissue-engineering applications.

In Vitro Chondrogenesis of Mesenchymal Stem Cells in Recombinant Silk-elastinlike Hydrogels

Pharmaceutical Research. Mar, 2008  |  Pubmed ID: 17404809

In this study the chondrocytic differentiation and cartilage matrix accumulation of human mesenchymal stem cells (hMSCs) were investigated after encapsulation in a genetically engineered silk-elastinlike protein polymer SELP-47 K as an injectable matrix for delivery of cell-based therapeutics.

The Effect of the Alignment of Electrospun Fibrous Scaffolds on Schwann Cell Maturation

Biomaterials. Feb, 2008  |  Pubmed ID: 17983651

Peripheral nerve regeneration can be enhanced by the stimulation of formation of bands of Büngner prior to implantation. Aligned electrospun poly(epsilon-caprolactone) (PCL) fibers were fabricated to test their potential to provide contact guidance to human Schwann cells. After 7 days of culture, cell cytoskeleton and nuclei were observed to align and elongate along the fiber axes, emulating the structure of bands of Büngner. Microarray analysis revealed a general down-regulation in expression of neurotrophin and neurotrophic receptors in aligned cells as compared to cells seeded on two-dimensional PCL film. Real-time-PCR analyses confirmed the up-regulation of early myelination marker, myelin-associated glycoprotein (MAG), and the down-regulation of NCAM-1, a marker of immature Schwann cells. Similar gene expression changes were also observed on cells cultured on randomly oriented PCL electrospun fibers. However, up-regulation of the myelin-specific gene, P0, was observed only on aligned electrospun fibers, suggesting the propensity of aligned fibers in promoting Schwann cell maturation.

In Vivo Wound Healing of Diabetic Ulcers Using Electrospun Nanofibers Immobilized with Human Epidermal Growth Factor (EGF)

Biomaterials. Feb, 2008  |  Pubmed ID: 17997153

Biodegradable polymers were electrospun and recombinant human epidermal growth factor (EGF) was immobilized on the electrospun nanofibers for the purpose of treating diabetic ulcers. Amine-terminated block copolymers composed of poly(epsilon-caprolactone) [PCL] and poly(ethyleneglycol) [PEG] and PCL were electrospun to biocompatible nanofibers with functional amine groups on the surface via PEG linkers. EGF was chemically conjugated to the surface of the nanofibers. The conjugation amount of EGF on the nanofibers was quantitated by X-ray photoelectron scattering. Human primary keratinocytes were cultivated on EGF-conjugated nanofibers in order to investigate the effect of EGF nanofibers on the differentiation of keratinocytes. Wound healing effects of the EGF nanofibers were confirmed in diabetic animals with dorsal wounds. The expression of keratinocyte-specific genes significantly increased with application of EGF-conjugated nanofibers. The EGF-nanofibers exerted superior in vivo wound healing activities compared to control groups or EGF solutions. Furthermore, immunohistochemical-staining results showed that EGF-receptor (EGFR) was highly expressed in the EGF nanofiber group. This study showed that EGF-conjugated nanofiber could potentially be employed as a novel wound healing material by increasing proliferation and phenotypic expression of keratinocytes.

Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed from Quantum Dot-FRET

Molecular Therapy : the Journal of the American Society of Gene Therapy. Feb, 2008  |  Pubmed ID: 18180773

A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.

Label-free, High-throughput Measurements of Dynamic Changes in Cell Nuclei Using Angle-resolved Low Coherence Interferometry

Biophysical Journal. Jun, 2008  |  Pubmed ID: 18326642

Accurate measurements of nuclear deformation, i.e., structural changes of the nucleus in response to environmental stimuli, are important for signal transduction studies. Traditionally, these measurements require labeling and imaging, and then nuclear measurement using image analysis. This approach is time-consuming, invasive, and unavoidably perturbs cellular systems. Light scattering, an emerging biophotonics technique for probing physical characteristics of living systems, offers a promising alternative. Angle-resolved low-coherence interferometry (a/LCI), a novel light scattering technique, was developed to quantify nuclear morphology for early cancer detection. In this study, a/LCI is used for the first time to noninvasively measure small changes in nuclear morphology in response to environmental stimuli. With this new application, we broaden the potential uses of a/LCI by demonstrating high-throughput measurements and by probing aspherical nuclei. To demonstrate the versatility of this approach, two distinct models relevant to current investigations in cell and tissue engineering research are used. Structural changes in cell nuclei due to subtle environmental stimuli, including substrate topography and osmotic pressure, are profiled rapidly without disrupting the cells or introducing artifacts associated with traditional measurements. Accuracy > or = 3% is obtained for the range of nuclear geometries examined here, with the greatest deviations occurring for the more complex geometries. Given the high-throughput nature of the measurements, this deviation may be acceptable for many biological applications that seek to establish connections between morphology and function.

Radiation-inducible Caspase-8 Gene Therapy for Malignant Brain Tumors

International Journal of Radiation Oncology, Biology, Physics. Jun, 2008  |  Pubmed ID: 18407431

Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue.

Gene Transfer to Hemophilia A Mice Via Oral Delivery of FVIII-chitosan Nanoparticles

Journal of Controlled Release : Official Journal of the Controlled Release Society. Dec, 2008  |  Pubmed ID: 18634839

Effective oral delivery of a non-viral gene carrier would represent a novel and attractive strategy for therapeutic gene transfer. To evaluate the potential of this approach, we studied the oral gene delivery efficacy of DNA polyplexes composed of chitosan and Factor VIII DNA. Transgene DNA was detected in both local and systemic tissues following oral administration of the chitosan nanoparticles to hemophilia A mice. Functional factor VIII protein was detected in plasma by chromogenic and thrombin generation assays, reaching a peak level of 2-4% FVIII at day 22 after delivery. In addition, a bleeding challenge one month after DNA administration resulted in phenotypic correction in 13/20 mice given 250-600 microg of FVIII DNA in chitosan nanoparticles, compared to 1/13 mice given naked FVIII DNA and 0/6 untreated mice. While further optimization would be required to render this type of delivery system practical for hemophilia A gene therapy, the findings suggest the feasibility of oral, non-viral delivery for gene medicine applications.

Viscoelastic Behaviour of Human Mesenchymal Stem Cells

BMC Cell Biology. 2008  |  Pubmed ID: 18644160

In this study, we have investigated the viscoelastic behaviour of individual human adult bone marrow-derived mesenchymal stem cells (hMSCs) and the role of F-actin filaments in maintaining these properties, using micropipette aspiration technique together with a standard linear viscoelastic solid model.

Engineering Strategies to Enhance Nanoparticle-mediated Oral Delivery

Journal of Biomaterials Science. Polymer Edition. 2008  |  Pubmed ID: 19017470

Oral delivery is the most preferred route of drug administration due to convenience, patient compliance and cost-effectiveness. Despite these advantages it remains difficult to achieve satisfactory bioavailability levels via oral administration due to the harsh environment of the gastrointestinal (GI) tract, particularly for biomacromolecules. One promising method to increase the bioavailability of macromolecular drugs such as proteins and nucleic acids is to encapsulate them in nanoparticles before oral administration. This review describes innovative strategies for increasing the efficacy of nanoparticle-mediated delivery to the GI tract. Approaches to optimize nanoparticle formulation by exploiting mucoadhesion, environmental responsiveness and external delivery control mechanisms are discussed. The application of recent advances in nanoparticle synthesis using supercritical fluids, microfluidics and imprint lithography to oral delivery are also presented, as well as possible strategies for incorporating nanoparticles into micro- and macroscale oral delivery devices.

Effect of Electromechanical Stimulation on the Maturation of Myotubes on Aligned Electrospun Fibers

Cellular and Molecular Bioengineering. Sep, 2008  |  Pubmed ID: 19774099

Tissue engineering may provide an alternative to cell injection as a therapeutic solution for myocardial infarction. A tissue-engineered muscle patch may offer better host integration and higher functional performance. This study examined the differentiation of skeletal myoblasts on aligned electrospun polyurethane (PU) fibers and in the presence of electromechanical stimulation. Skeletal myoblasts cultured on aligned PU fibers showed more pronounced elongation, better alignment, higher level of transient receptor potential cation channel-1 (TRPC-1) expression, upregulation of contractile proteins and higher percentage of striated myotubes compared to those cultured on random PU fibers and film. The resulting tissue constructs generated tetanus forces of 1.1 mN with a 10-ms time to tetanus. Additional mechanical, electrical, or synchronized electromechanical stimuli applied to myoblasts cultured on PU fibers increased the percentage of striated myotubes from 70 to 85% under optimal stimulation conditions, which was accompanied by an upregulation of contractile proteins such as α-actinin and myosin heavy chain. In describing how electromechanical cues can be combined with topographical cue, this study helped move towards the goal of generating a biomimetic microenvironment for engineering of functional skeletal muscle.

Poly(ethylene Imine)-g-chitosan Using EX-810 As a Spacer for Nonviral Gene Delivery Vectors

Journal of Biomedical Materials Research. Part A. Mar, 2009  |  Pubmed ID: 18404706

Polyelectrolyte complexes have been widely studied as gene carriers in recent years. In this study, poly (ethylene imine) was grafted onto chitosan (PEI-g-CHI) as a nonviral gene carrier in order to improve the water solubility as well as the inherent transfection efficiency of chitosan. We present a novel method to conjugate the amine or hydroxyl groups of chitosan (CHI) and the amine groups of PEI through opening the epoxide rings of ethylene glycol diglycidyl ether (EX-810), which also brings the merits as mentioned in PEGylation chemistry. The degree of substitution of PEI onto CHI was characterized by NMR. The preliminarily cellular mechanisms, from the cellular entry to the endosomal release, were investigated by the correlations among the physicochemical properties of the DNA-polymer complexes, the buffering capacity of the modified polymer, the cytotoxicity, and the efficiency of the transgene expression. The cytotoxicity assayed by MTT shows that cell viability of PEI-g-CHI is higher than CHI especially noticeable at high concentrations using human kidney 293T cells. The efficiency of transgene expression and the amount of intracellular plasmid were monitored using green fluorescent protein (GFP) and visualized by fluorescence microscopy. The transfection efficiency of PEI-g-CHI/DNA polyplex is significantly better than CHI/DNA polyplex when using the weight ratios higher than 2.5.

The Convergence of Quantum-dot-mediated Fluorescence Resonance Energy Transfer and Microfluidics for Monitoring DNA Polyplex Self-assembly in Real Time

Nanotechnology. Mar, 2009  |  Pubmed ID: 19417478

We present a novel convergence of quantum-dot-mediated fluorescence resonance energy transfer (QD-FRET) and microfluidics, through which molecular interactions were precisely controlled and monitored using highly sensitive quantum-dot-mediated FRET. We demonstrate its potential in studying the kinetics of self-assembly of DNA polyplexes under laminar flow in real time with millisecond resolution. The integration of nanophotonics and microfluidics offers a powerful tool for elucidating the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Sustained Viral Gene Delivery Through Core-shell Fibers

Journal of Controlled Release : Official Journal of the Controlled Release Society. Oct, 2009  |  Pubmed ID: 19539680

Although viral gene transfer is efficient in achieving transgene expression for tissue engineering, drawbacks of virus dissemination, toxicity and transient gene expression due to immune response have hindered its widespread application. Many tissue engineering studies thus opt to genetically engineer cells in vitro prior to their introduction in vivo. However, it would be attractive to obviate the need for in vitro manipulation by transducing the infiltrating progenitor cells in situ. This study introduces the fabrication of a virus-encapsulated electrospun fibrous scaffold to achieve sustained and localized transduction. Adenovirus encoding the gene for green fluorescent protein was efficiently encapsulated into the core of poly(epsilon-caprolactone) fibers through co-axial electrospinning and was subsequently released via a porogen-mediated process. HEK 293 cells seeded on the scaffolds expressed high level of transgene expression over a month, while cells inoculated by scaffold supernatant showed only transient expression for a week. RAW 264.7 cells cultured on the virus-encapsulated fibers produced a lower level of IL-1 beta, TNF-alpha and IFN-alpha, suggesting that the activation of macrophage cells by the viral vector was reduced when encapsulated in the core-shell PCL fibers. In demonstrating sustained and localized cell transduction, this study presents an attractive alternative mode of applying viral gene transfer for regenerative medicine.

Electrohydrodynamics: A Facile Technique to Fabricate Drug Delivery Systems

Advanced Drug Delivery Reviews. Oct, 2009  |  Pubmed ID: 19651167

Electrospinning and electrospraying are facile electrohydrodynamic fabrication methods that can generate drug delivery systems (DDS) through a one-step process. The nanostructured fiber and particle morphologies produced by these techniques offer tunable release kinetics applicable to diverse biomedical applications. Coaxial electrospinning/electrospraying, a relatively new technique of fabricating core-shell fibers/particles have added to the versatility of these DDS by affording a near zero-order drug release kinetics, dampening of burst release, and applicability to a wider range of bioactive agents. Controllable electrospinning/spraying of fibers and particles and subsequent drug release from these chiefly polymeric vehicles depends on well-defined solution and process parameters. The additional drug delivery capability from electrospun fibers can further enhance the material's functionality in tissue engineering applications. This review discusses the state-of-the-art of using electrohydrodynamic technique to generate nanofiber/particles as drug delivery devices.

Development of Universal Antidotes to Control Aptamer Activity

Nature Medicine. Oct, 2009  |  Pubmed ID: 19801990

With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

Mast Cell-derived Particles Deliver Peripheral Signals to Remote Lymph Nodes

The Journal of Experimental Medicine. Oct, 2009  |  Pubmed ID: 19808250

During infection, signals from the periphery are known to reach draining lymph nodes (DLNs), but how these molecules, such as inflammatory cytokines, traverse the significant distances involved without dilution or degradation remains unclear. We show that peripheral mast cells, upon activation, release stable submicrometer heparin-based particles containing tumor necrosis factor and other proteins. These complexes enter lymphatic vessels and rapidly traffic to the DLNs. This physiological drug delivery system facilitates communication between peripheral sites of inflammation and remote secondary lymphoid tissues.

Simultaneous Non-invasive Analysis of DNA Condensation and Stability by Two-step QD-FRET

Nano Today. Apr, 2009  |  Pubmed ID: 20161048

Nanoscale vectors comprised of cationic polymers that condense DNA to form nanocomplexes are promising options for gene transfer. The rational design of more efficient nonviral gene carriers will be possible only with better mechanistic understanding of the critical rate-limiting steps, such as nanocomplex unpacking to release DNA and degradation by nucleases. We present a two-step quantum dot fluorescence resonance energy transfer (two-step QD-FRET) approach to simultaneously and non-invasively analyze DNA condensation and stability. Plasmid DNA, double-labeled with QD (525 nm emission) and nucleic acid dyes, were complexed with Cy5-labeled cationic gene carriers. The QD donor drives energy transfer stepwise through the intermediate nucleic acid dye to the final acceptor Cy5. At least three distinct states of DNA condensation and integrity were distinguished in single particle manner and within cells by quantitative ratiometric analysis of energy transfer efficiencies. This novel two-step QD-FRET method allows for more detailed assessment of the onset of DNA release and degradation simultaneously.

Balancing Protection and Release of DNA: Tools to Address a Bottleneck of Non-viral Gene Delivery

Journal of the Royal Society, Interface / the Royal Society. Feb, 2010  |  Pubmed ID: 19734186

Engineering polymeric gene-delivery vectors to release an intact DNA payload at the optimal time and subcellular compartment remains a formidable challenge. An ideal vector would provide total protection of complexed DNA from degradation prior to releasing it efficiently near or within the nucleus of a target cell. While optimization of polymer properties, such as molecular weight and charge density, has proved largely inadequate in addressing this challenge, applying polymeric carriers that respond to temperature, light, pH and redox environment to trigger a switch from a tight, protective complex to a more relaxed interaction favouring release at the appropriate time and place has shown promise. Currently, a paucity of gene carriers able to satisfy the contrary requirements of adequate DNA protection and efficient release contributes to the slow progression of non-viral gene therapy towards clinical translation. This review highlights the promising carrier designs that may achieve an optimal balance of DNA protection and release. It also discusses the imaging techniques and three-dimensional in vitro models that can help study these two barriers in the non-viral gene transfer process. Ultimately, efficacious non-viral gene therapy will depend on the combination of intelligent material design, innovative imaging techniques and sophisticated in vitro model systems to facilitate the rational design of polymeric gene-delivery vectors.

Nanotopography-induced Changes in Focal Adhesions, Cytoskeletal Organization, and Mechanical Properties of Human Mesenchymal Stem Cells

Biomaterials. Feb, 2010  |  Pubmed ID: 19879643

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Transport of Chitosan-DNA Nanoparticles in Human Intestinal M-cell Model Versus Normal Intestinal Enterocytes

European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences. Jan, 2010  |  Pubmed ID: 19913612

Oral vaccination is one of the most promising applications of polymeric nanoparticles. Using two different in vitro cellular models to partially reproduce the characteristics of intestinal enterocytes and M-cells, this study demonstrates that nanoparticle transport through the M-cell co-culture model is 5-fold that of the intestinal epithelial monolayer, with at least 80% of the chitosan-DNA nanoparticles uptaken in the first 30 min. Among the properties of nanoparticles studied, ligand decoration has the most dramatic effect on the transcytosis rate: transferrin modification enhances transport through both models by 3- to 5-fold. The stability of the nanoparticles also affects transport kinetics. Factors which de-stabilize the nanoparticles, such as low charge (N/P) ratio and addition of serum, result in aggregation and in turn decreases transport efficiency. Of these stability factors, luminal pH is of great interest as an increase in pH from 5.5 to 6.4 and 7.4 leads to a 3- and 10-fold drop in nanoparticle transport, respectively. Since soluble chitosan can act as an enhancer to increase paracellular transport by up to 60%, this decrease is partially attributed to the soluble chitosan precipitating near neutral pH. The implication that chitosan-DNA nanoparticles are more stable in the upper regions of the small intestine suggests that higher uptake rates may occur in the duodenum compared to the ileum and the colon.

Characterization of Topographical Effects on Macrophage Behavior in a Foreign Body Response Model

Biomaterials. May, 2010  |  Pubmed ID: 20138663

Current strategies to limit macrophage adhesion, fusion and fibrous capsule formation in the foreign body response have focused on modulating material surface properties. We hypothesize that topography close to biological scale, in the micron and nanometric range, provides a passive approach without bioactive agents to modulate macrophage behavior. In our study, topography-induced changes in macrophage behavior was examined using parallel gratings (250 nm-2 mum line width) imprinted on poly(epsilon-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(dimethyl siloxane) (PDMS). RAW 264.7 cell adhesion and elongation occurred maximally on 500 nm gratings compared to planar controls over 48 h. TNF-alpha and VEGF secretion levels by RAW 264.7 cells showed greatest sensitivity to topographical effects, with reduced levels observed on larger grating sizes at 48 h. In vivo studies at 21 days showed reduced macrophage adhesion density and degree of high cell fusion on 2 mum gratings compared to planar controls. It was concluded that topography affects macrophage behavior in the foreign body response on all polymer surfaces examined. Topography-induced changes, independent of surface chemistry, did not reveal distinctive patterns but do affect cell morphology and cytokine secretion in vitro, and cell adhesion in vivo particularly on larger size topography compared to planar controls.

Microscale Oral Delivery Devices Incorporating Nanoparticles

Nanomedicine (London, England). Feb, 2010  |  Pubmed ID: 20148626

Low Oxygen Tension and Synthetic Nanogratings Improve the Uniformity and Stemness of Human Mesenchymal Stem Cell Layer

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2010  |  Pubmed ID: 20179678

A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. As a first step toward this goal, a confluent, uniform hMSC layer with a high degree of alignment and stemness maintenance needs to be created. Hypothesizing that topographical cue and a physiologically relevant low-oxygen condition could promote the formation of such an hMSC layer, we studied the culture of hMSCs on synthetic nanogratings (350 nm width and 700 nm pitch) and either under 2 or 20% O(2). Culturing hMSCs on the nanogratings highly aligned the cells, but it tended to create patchy layers and accentuate the hMSC differentiation. The 2% O(2) improved the alignment and uniformity of hMSCs, and reduced their differentiation. Over a 14-day culture period, hMSCs in 2% O(2) showed uniform connexon distribution, secreted abundant extracellular matrix (ECM) proteins, and displayed a high progenicity. After 21-day culture on nanogratings, hMSCs exposed to 2% O(2) maintained a higher viability and differentiation capacity. This study established that a 2% O(2) culture condition could restrict the differentiation of hMSCs cultured on nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications.

Dual-sensitive Micellar Nanoparticles Regulate DNA Unpacking and Enhance Gene-delivery Efficiency

Advanced Materials (Deerfield Beach, Fla.). Jun, 2010  |  Pubmed ID: 20440698

Electrosprayed Core-shell Microspheres for Protein Delivery

Chemical Communications (Cambridge, England). Jul, 2010  |  Pubmed ID: 20485844

This communication describes a single-step electrospraying technique that generates core-shell microspheres (CSMs) with encapsulated protein as the core and an amphiphilic biodegradable polymer as the shell. The protein release profiles of the electrosprayed CSMs showed steady release kinetics over 3 weeks without a significant initial burst.

Polymer Hydrogels: Chaperoning Vaccines

Nature Materials. Jul, 2010  |  Pubmed ID: 20571480

Implantation of Mouse Embryonic Stem Cell-derived Cardiac Progenitor Cells Preserves Function of Infarcted Murine Hearts

PloS One. 2010  |  Pubmed ID: 20634944

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Quantum Dot-based Theranostics

Nanoscale. Jan, 2010  |  Pubmed ID: 20648364

Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), have advanced the fields of molecular diagnostics and nanotherapeutics. Much of the initial progress for QDs in biology and medicine has focused on developing new biosensing formats to push the limit of detection sensitivity. Nevertheless, QDs can be more than passive bio-probes or labels for biological imaging and cellular studies. The high surface-to-volume ratio of QDs enables the construction of a "smart" multifunctional nanoplatform, where the QDs serve not only as an imaging agent but also a nanoscaffold catering for therapeutic and diagnostic (theranostic) modalities. This mini review highlights the emerging applications of functionalized QDs as fluorescence contrast agents for imaging or as nanoscale vehicles for delivery of therapeutics, with special attention paid to the promise and challenges towards QD-based theranostics.

Nanoscale Surfacing for Regenerative Medicine

Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology. Sep-Oct, 2010  |  Pubmed ID: 20803682

Cells in most tissues reside in microenvironment surrounded with specific three-dimensional features. The extracellular matrix or substratum with which cells interact often includes topography at the nanoscale. For example, the basement membrane of many tissues displays features of pores, fibers and ridges in the nanometer range. The nanoscale topography has significant effects on cellular behavior. Knowledge of the cell-substratum interactions is crucial to the understanding of many fundamental biological questions and to regenerative medicine. Rapid advances in nanotechnology enable cellular study on engineered nanoscale surfaces. Recent findings underscore the phenomenon that mammalian cells do respond to nanosized features on a synthetic surface. This review covers the commonly used techniques of engineering nanoscale surface and the techniques which have not been adapted but are of great potential in regenerative medicine, surveys the applications of nanoscale surface in regenerative medicine including vascular, bone, neural and stem cell tissue engineering, and discusses the possible mechanisms of cellular responses to nanoscale surface. A better understanding of the interactions between cells and nanoscale surfacing will help advance the field of regenerative medicine.

Deformation of Stem Cell Nuclei by Nanotopographical Cues

Soft Matter. Apr, 2010  |  Pubmed ID: 21297875

Cells sense cues in their surrounding microenvironment. These cues are converted into intracellular signals and transduced to the nucleus in order for the cell to respond and adapt its function. Within the nucleus, structural changes occur that ultimately lead to changes in the gene expression. In this study, we explore the structural changes of the nucleus of human mesenchymal stem cells as an effect of topographical cues. We use a controlled nanotopography to drive shape changes to the cell nucleus, and measure the changes with both fluorescence microscopy and a novel light scattering technique. The nucleus changes shape dramatically in response to the nanotopography, and in a manner dependent on the mechanical properties of the substrate. The kinetics of the nuclear deformation follows an unexpected trajectory. As opposed to a gradual shape change in response to the topography, once the cytoskeleton attains an aligned and elongation morphology on the time scale of several hours, the nucleus changes shape rapidly and intensely.

Emerging Links Between Surface Nanotechnology and Endocytosis: Impact on Nonviral Gene Delivery

Nano Today. Dec, 2010  |  Pubmed ID: 21383869

Significant effort continues to be exerted toward the improvement of transfection mediated by nonviral vectors. These endeavors are often focused on the design of particulate carriers with properties that encourage efficient accumulation at the membrane surface, particle uptake, and endosomal escape. Despite its demonstrated importance in successful nonviral transfection, relatively little investigation has been done to understand the pressures driving internalized vectors into favorable nondegradative endocytic pathways. Improvements in transfection efficiency have been noted for complexes delivered with a substrate-mediated approach, but the reasons behind such enhancements remain unclear. The phenotypic changes exhibited by cells interacting with nano- and micro-featured substrates offer hints that may explain these effects. This review describes nanoscale particulate and substrate parameters that influence both the uptake of nonviral gene carriers and the endocytic phenotype of interacting cells, and explores the molecular links that may mediate these interactions. Substrate-mediated control of endocytosis represents an exciting new design parameter that will guide the creation of efficient transgene carriers.

Bioavailability of Metalloporphyrin-based SOD Mimics is Greatly Influenced by a Single Charge Residing on a Mn Site

Free Radical Research. Feb, 2011  |  Pubmed ID: 20942564

In the cell Mn porphyrins (MnPs) likely couple with cellular reductants which results in a drop of total charge from 5+ to 4+ and dramatically increases their lipophilicity by up to three orders of magnitude depending upon the length of alkylpyridyl chains and type of isomer. The effects result from the interplay of solvation, lipophilicit and stericity. Impact of ascorbate on accumulation of MnPs was measured in E. coli and in Balb/C mouse tumours and muscle; for the latter measurements, the LC/ESI-MS/MS method was developed. Accumulation was significantly enhanced when MnPs were co-administered with ascorbate in both prokaryotic and eukaryotic systems. Further, MnTnHex-2-PyP(5+) accumulates 5-fold more in the tumour than in a muscle. Such data increase our understanding of MnPs cellular and sub-cellular accumulation and remarkable in vivo effects. The work is in progress to understand how coupling of MnPs with ascorbate affects their mechanism of action, in particular with respect to cancer therapy.

Efficacy of Engineered FVIII-producing Skeletal Muscle Enhanced by Growth Factor-releasing Co-axial Electrospun Fibers

Biomaterials. Feb, 2011  |  Pubmed ID: 21084118

Co-axial electrospun fibers can offer both topographical and biochemical cues for tissue engineering applications. In this study, we demonstrate the sustained treatment of hemophilia through a non-viral, tissue engineering approach facilitated by growth factor-releasing co-axial electrospun fibers. FVIII-producing skeletal myotubes were first engineered on aligned electrospun fibers in vitro, followed by implantation in hemophilic mice with or without a layer of core-shell electrospun fibers designed to provide sustained delivery of angiogenic or lymphangiogenic growth factors, which serves to stimulate the lymphatic or vascular systems to enhance the FVIII transport from the implant site into systemic circulation. Upon subcutaneous implantation into hemophilic mice, the construct seamlessly integrated with the host tissue within one month, and specifically induced either vascular or lymphatic network infiltration in accordance with the growth factors released from the electrospun fibers. Engineered constructs that induced angiogenesis resulted in sustained elevation of plasma FVIII and significantly reduced blood coagulation time for at least 2-months. Biomaterials-assisted functional tissue engineering was shown in this study to offer protein replacement therapy for a genetic disorder such as hemophilia.

Transient Depletion of Kupffer Cells Leads to Enhanced Transgene Expression in Rat Liver Following Retrograde Intrabiliary Infusion of Plasmid DNA and DNA Nanoparticles

Human Gene Therapy. Jul, 2011  |  Pubmed ID: 21091274

In this report, we have demonstrated that by temporarily removing Kupffer cells (KCs), the transgene expression levels mediated by retrograde intrabiliary infusion (RII) of plasmid DNA, polyethylenimine-DNA, and chitosan nanoparticles were enhanced by 1,927-, 131-, and 23,450-fold, respectively, in comparison with the respective groups without KC removal. KC removal also led to significantly prolonged transgene expression in the liver that received all three carriers. This increased transgene expression was correlated with significantly reduced serum tumor necrosis factor-α level as an indicator for KC activation. These results suggest that KC activation is a significant contributing factor to the lowered transgene expression by polycation-DNA nanoparticles delivered by RII. More importantly, the combination of RII and transient removal of KCs may be adopted as an effective approach to achieving high and persistent transgene expression in the liver mediated by nonviral nanoparticles.

Simultaneous Delivery of SiRNA and Paclitaxel Via a "two-in-one" Micelleplex Promotes Synergistic Tumor Suppression

ACS Nano. Feb, 2011  |  Pubmed ID: 21204585

Combination of two or more therapeutic strategies with different mechanisms can cooperatively prohibit cancer development. Combination of chemotherapy and small interfering RNA (siRNA)-based therapy represents an example of this approach. Hypothesizing that the chemotherapeutic drug and the siRNA should be simultaneously delivered to the same tumoral cell to exert their synergistic effect, the development of delivery systems that can efficiently encapsulate two drugs and successfully deliver payloads to targeted sites via systemic administration has proven to be challenging. Here, we demonstrate an innovative "two-in-one" micelleplex approach based on micellar nanoparticles of a biodegradable triblock copolymer poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(2-aminoethyl ethylene phosphate) to systemically deliver the siRNA and chemotherapeutic drug. We show clear evidence that the micelleplex is capable of delivering siRNA and paclitaxel simultaneously to the same tumoral cells both in vitro and in vivo. We further demonstrate that systemic administration of the micelleplex carrying polo-like kinase 1 (Plk1) specific siRNA and paclitaxel can induce a synergistic tumor suppression effect in the MDA-MB-435s xenograft murine model, requiring a thousand-fold less paclitaxel than needed for paclitaxel monotherapy delivered by the micelleplex and without activation of the innate immune response or generation of carrier-associated toxicity.

High-throughput Screening of Microscale Pitted Substrate Topographies for Enhanced Nonviral Transfection Efficiency in Primary Human Fibroblasts

Biomaterials. May, 2011  |  Pubmed ID: 21334062

Optimization of nonviral gene delivery typically focuses on the design of particulate carriers that are endowed with desirable membrane targeting, internalization, and endosomal escape properties. Topographical control of cell transfectability, however, remains a largely unexplored parameter. Emerging literature has highlighted the influence of cell-topography interactions on modulation of many cell phenotypes, including protein expression and cytoskeletal behaviors implicated in endocytosis. Using high-throughput screening of primary human dermal fibroblasts cultured on a combinatorial library of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP(+) cells was observed independent of proliferation rate, accompanied by SEM and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems.

Engineering of a Microfluidic Cell Culture Platform Embedded with Nanoscale Features

Lab on a Chip. May, 2011  |  Pubmed ID: 21442110

Cells residing in a microenvironment interact with the extracellular matrix (ECM) and neighboring cells. The ECM built from biomacromolecules often includes nanotopography. Through the ECM, interstitial flows facilitate transport of nutrients and play an important role in tissue maintenance and pathobiology. To create a microenvironment that can incorporate both nanotopography and flow for studies of cell-matrix interactions, we fabricated microfluidic channels endowed with nanopatterns suitable for dynamic culture. Using polymer thin film technology, we developed a versatile stitching technique to generate a large area of nanopatterned surface and a simple microtransfer assembly technique to assemble polydimethylsiloxane-based microfluidics. The cellular study showed that both nanotopography and fluid shear stress played a significant role in adhesion, spreading, and migration of human mesenchymal stem cells. The orientation and deformation of cytoskeleton and nuclei were regulated through the interplay of these two cues. The nanostructured microfluidic platform provides a useful tool to promote the fundamental understanding of cell-matrix interactions and may be used to regulate the fate of stem cells.

Tuning Physical Properties of Nanocomplexes Through Microfluidics-assisted Confinement

Nano Letters. May, 2011  |  Pubmed ID: 21506589

The future of genetic medicine hinges on successful intracellular delivery of nucleic acid-based therapeutics. While significant effort has concentrated on developing nanocarriers to improve the delivery aspects, scant attention has been paid to the synthetic process of poorly controlled nanocomplex formation. Proposed here is a reliable system to better control the complexation process, and thus the physical properties of the nanocomplexes, through microfluidics-assisted confinement (MAC) in picoliter droplets. We show that these homogeneous MAC-synthesized nanocomplexes exhibit narrower size distribution, lower cytotoxicity, and higher transfection efficiency compared to their bulk-synthesized counterparts. MAC represents a physical approach to control the energetic self-assembly of polyelectrolytes, thereby complementing the chemical innovations in nanocarrier design to optimize nucleic acid and peptide delivery.

Diverse Functions of Cationic Mn(III) N-substituted Pyridylporphyrins, Recognized As SOD Mimics

Free Radical Biology & Medicine. Sep, 2011  |  Pubmed ID: 21616142

Oxidative stress, a redox imbalance between the endogenous reactive species and antioxidant systems, is common to numerous pathological conditions such as cancer, central nervous system injuries, radiation injury, diabetes etc. Therefore, compounds able to reduce oxidative stress have been actively sought for over 3 decades. Superoxide is the major species involved in oxidative stress either in its own right or through its progeny, such as ONOO⁻, H₂O₂, •OH, CO₃•⁻, and •NO₂. Hence, the very first compounds developed in the late 1970-ies were the superoxide dismutase (SOD) mimics. Thus far the most potent mimics have been the cationic meso Mn(III) N-substituted pyridylporphyrins and N,N'-disubstituted imidazolylporphyrins (MnPs), some of them with k(cat)(O₂·⁻) similar to the k(cat) of SOD enzymes. Most frequently studied are ortho isomers MnTE-2-PyP⁵⁺, MnTnHex-2-PyP⁵⁺, and MnTDE-2-ImP⁵⁺. The ability to disproportionate O₂·⁻ parallels their ability to remove the other major oxidizing species, peroxynitrite, ONOO⁻. The same structural feature that gives rise to the high k(cat)(O₂·⁻) and k(red)(ONOO⁻), allows MnPs to strongly impact the activation of the redox-sensitive transcription factors, HIF-1α, NF-κB, AP-1, and SP-1, and therefore modify the excessive inflammatory and immune responses. Coupling with cellular reductants and other redox-active endogenous proteins seems to be involved in the actions of Mn porphyrins. While hydrophilic analogues, such as MnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺ are potent in numerous animal models of diseases, the lipophilic analogues, such as MnTnHex-2-PyP⁵⁺, were developed to cross blood brain barrier and target central nervous system and critical cellular compartments, mitochondria. The modification of its structure, aimed to preserve the SOD-like potency and lipophilicity, and diminish the toxicity, has presently been pursued. The pulmonary radioprotection by MnTnHex-2-PyP⁵⁺ was the first efficacy study performed successfully with non-human primates. The Phase I toxicity clinical trials were done on amyotrophic lateral sclerosis patients with N,N'-diethylimidazolium analogue, MnTDE-2-ImP⁵⁺ (AEOL10150). Its aggressive development as a wide spectrum radioprotector by Aeolus Pharmaceuticals has been supported by USA Federal government. The latest generation of compounds, bearing oxygens in pyridyl substituents is presently under aggressive development for cancer and CNS injuries at Duke University and is supported by Duke Translational Research Institute, The Wallace H. Coulter Translational Partners Grant Program, Preston Robert Tisch Brain Tumor Center at Duke, and National Institute of Allergy and Infectious Diseases. Metal center of cationic MnPs easily accepts and donates electrons as exemplified in the catalysis of O₂·⁻ dismutation. Thus such compounds may be equally good anti- and pro-oxidants; in either case the beneficial therapeutic effects may be observed. Moreover, while the in vivo effects may appear antioxidative, the mechanism of action of MnPs that produced such effects may be pro-oxidative; the most obvious example being the inhibition of NF-κB. The experimental data therefore teach us that we need to distinguish between the mechanism/s of action/s of MnPs and the effects we observe. A number of factors impact the type of action of MnPs leading to favorable therapeutic effects: levels of reactive species and oxygen, levels of endogenous antioxidants (enzymes and low-molecular compounds), levels of MnPs, their site of accumulation, and the mutual encounters of all of those species. The complexity of in vivo redox systems and the complex redox chemistry of MnPs challenge and motivate us to further our understanding of the physiology of the normal and diseased cell with ultimate goal to successfully treat human diseases.

Dynamic Topographical Control of Mesenchymal Stem Cells by Culture on Responsive Poly(ε-caprolactone) Surfaces

Advanced Materials (Deerfield Beach, Fla.). Aug, 2011  |  Pubmed ID: 21626577

Stem Cell Differentiation Indicated by Noninvasive Photonic Characterization and Fractal Analysis of Subcellular Architecture

Integrative Biology : Quantitative Biosciences from Nano to Macro. Aug, 2011  |  Pubmed ID: 21695342

We hypothesised that global structural changes in stem cells would manifest with differentiation, and that these changes would be observable with light scattering microscopy. Analysed with a fractal dimension formalism, we observed significant structural changes in differentiating human mesenchymal stem cells within one day after induction, earlier than could be detected by gene expression profiling. Moreover, light scattering microscopy is entirely non-perturbative, so the same sample could be monitored throughout the differentiation process. We explored one possible mechanism, chromatin remodelling, to account for the changes we observed. Correlating with the staining of HP1α, a heterochromatin protein, we applied novel microscopy methods and fractal analysis to monitor the plastic dynamics of chromatin within stem cell nuclei. We showed that the level of chromatin condensation changed during differentiation, and provide one possible explanation for the changes seen with the light scattering method. These results lend physical insight into stem cell differentiation while providing physics-based methods for non-invasive detection of the differentiation process.

Uptake and Intracellular Fate of Multifunctional Nanoparticles: a Comparison Between Lipoplexes and Polyplexes Via Quantum Dot Mediated Förster Resonance Energy Transfer

Molecular Pharmaceutics. Oct, 2011  |  Pubmed ID: 21740056

Lipoplexes and polyplexes represent the two major nanocarrier systems for nucleic acid delivery. Previous studies examining their uptake and intracellular unpacking rely on organic fluorophores fraught with low signal intensity and photobleaching. In this work quantum dot mediated Förster resonance energy transfer (QD-FRET) was first used to study and compare the cellular uptake and the intracellular fate of oligodeoxynucelotide (ODN)-based lipoplexes and polyplexes. QD605-amine and Cy5-labeled ODN (Cy5-GTI2040) were chosen as the FRET pair. By adjusting the lipid/ODN ratio of lipoplexes and the nitrogen/phosphate (N/P) ratio of polyplexes, lipoplexes and polyplexes with comparable physical properties were produced. The biological activities of dual-labeled lipoplexes and polyplexes remained unaltered compared to their unlabeled counterparts as evidenced by their comparable antisense activities against protein R2 in KB cells. Flow cytometry and confocal microscopy revealed similar pattern of uptake for these two types of nanoparticles, although polyplexes had a higher dissociation rate than lipoplexes in KB cells. We demonstrate that QD-FRET is a sensitive tool to study the uptake and intracellular unpacking of lipoplexes and polyplexes, which may help optimize their formulations for various theranostics applications.

Three-dimensional Culture of Rabbit Nucleus Pulposus Cells in Collagen Microspheres

The Spine Journal : Official Journal of the North American Spine Society. Oct, 2011  |  Pubmed ID: 21843975

Degenerative disc disease poses an increasing threat to our quality of life as we age. Existing treatments have limitations. New treatment modalities focusing on biologic rather than surgical approach would be appealing.

Nucleic Acid-binding Polymers As Anti-inflammatory Agents

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2011  |  Pubmed ID: 21844380

Dead and dying cells release nucleic acids. These extracellular RNAs and DNAs can be taken up by inflammatory cells and activate multiple nucleic acid-sensing toll-like receptors (TLR3, 7, 8, and 9). The inappropriate activation of these TLRs can engender a variety of inflammatory and autoimmune diseases. The redundancy of the TLR family encouraged us to seek materials that can neutralize the proinflammatory effects of any nucleic acid regardless of its sequence, structure or chemistry. Herein we demonstrate that certain nucleic acid-binding polymers can inhibit activation of all nucleic acid-sensing TLRs irrespective of whether they recognize ssRNA, dsRNA or hypomethylated DNA. Furthermore, systemic administration of such polymers can prevent fatal liver injury engendered by proinflammatory nucleic acids in an acute toxic shock model in mice. Therefore these polymers represent a novel class of anti-inflammatory agent that can act as molecular scavengers to neutralize the proinflammatory effects of various nucleic acids.

Mechanical Behavior of Human Embryonic Stem Cell Pellet Under Unconfined Compression

Biomechanics and Modeling in Mechanobiology. Aug, 2011  |  Pubmed ID: 21858691

As a prelude to the understanding of mechanotransduction in human embryonic stem cell (hESC) differentiation, the mechanical behavior of hESCs in the form of cell pellet is studied. The pellets were tested after 3 or 5 weeks of cell culture in order to demonstrate the effect of the duration of cell culture on the mechanical properties of the pellets. A micromechanical tester was used to conduct unconfined compression on hESC pellet, and experimental, numerical, and analytical methods were combined to determine the mechanical properties of hESC pellet. It is assumed that the mechanical behavior of hESC pellets can be described by an isotropic, linear viscoelastic model consisting of a spring and two Maxwell units in parallel, and the Poisson's ratio of the hESC pellet is constant based on pellet deformation in the direction perpendicular to the compression direction. Finite element method (FEM) simulation was adopted to determine the values of Poisson's ratio and the five parameters contained in the viscoelastic model. The variations of Poisson's ratio and the initial elastic modulus are found to be larger compared with those of the four other parameters. Results show that longer duration of cell culture leads to higher modulus of hESC pellet. The effect of pellet size error on the values of mechanical parameters determined is studied using FEM simulation, and it is found that the effect of size error on Poisson's ratio and initial elastic modulus is much larger than that on the other parameters.

Cytotoxic Effects of Mn(III) N-alkylpyridylporphyrins in the Presence of Cellular Reductant, Ascorbate

Free Radical Research. Nov, 2011  |  Pubmed ID: 21859376

Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O(2)(·-), reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP(5+), MnTnHex-2-PyP(5+), and a meta isomer MnTnHex-3-PyP(5+), which differ greatly with regard to their metal-centered reduction potential, E(1/2) (Mn(III)P/Mn(II)P) and lipophilicity, were explored. Employing Mn(III)P/Mn(II)P redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP(5+) was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP(5+) is most prone to oxidative degradation with H(2) , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.

Pluripotent Stem Cell-derived Cardiac Tissue Patch with Advanced Structure and Function

Biomaterials. Dec, 2011  |  Pubmed ID: 21906802

Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell-derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25 cm/s, and 3) significant contractile forces of up to 2 mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease.

Detection of Single Enzymatic Events in Rare or Single Cells Using Microfluidics

ACS Nano. Oct, 2011  |  Pubmed ID: 21936557

In the present study we demonstrate highly sensitive detection of rare, aberrant cells in a population of wild-type human cells by combining a rolling-circle-enhanced enzyme activity single-molecule detection assay with a custom-designed microfluidic device. Besides reliable detection of low concentrations of aberrant cells, the integrated system allowed multiplexed detection of individual enzymatic events at the single cell level. The single cell sensitivity of the presented setup relies on the combination of single-molecule rolling-circle-enhanced enzyme activity detection with the fast reaction kinetics provided by a picoliter droplet reaction volume and subsequent concentration of signals in a customized drop-trap device. This setup allows the fast reliable analyses of enzyme activities in a vast number of single cells, thereby offering a valuable tool for basic research as well as theranostics.

Microfluidics-mediated Isothermal Detection of Enzyme Activity at the Single Molecule Level

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. Aug, 2011  |  Pubmed ID: 22255034

Conventional analysis of enzymatic activity, often carried out on pools of cells, is blind to heterogeneity in the population. Here, we combine microfluidics with a previously developed isothermal rolling circle amplification-based assay to investigate multiple enzymatic activities in down to single cells. This microfluidics-meditated assay performs at very high sensitivity in picoliter incubators with small quantities of biological materials. Furthermore, we demonstrate the assay's capability of multiplexed detection of at least three enzyme activities at the single molecule level.

Nucleic Acid-based Nanoengineering: Novel Structures for Biomedical Applications

Interface Focus. Oct, 2011  |  Pubmed ID: 23050076

Nanoengineering exploits the interactions of materials at the nanometre scale to create functional nanostructures. It relies on the precise organization of nanomaterials to achieve unique functionality. There are no interactions more elegant than those governing nucleic acids via Watson-Crick base-pairing rules. The infinite combinations of DNA/RNA base pairs and their remarkable molecular recognition capability can give rise to interesting nanostructures that are only limited by our imagination. Over the past years, creative assembly of nucleic acids has fashioned a plethora of two-dimensional and three-dimensional nanostructures with precisely controlled size, shape and spatial functionalization. These nanostructures have been precisely patterned with molecules, proteins and gold nanoparticles for the observation of chemical reactions at the single molecule level, activation of enzymatic cascade and novel modality of photonic detection, respectively. Recently, they have also been engineered to encapsulate and release bioactive agents in a stimulus-responsive manner for therapeutic applications. The future of nucleic acid-based nanoengineering is bright and exciting. In this review, we will discuss the strategies to control the assembly of nucleic acids and highlight the recent efforts to build functional nucleic acid nanodevices for nanomedicine.

Nucleic Acid-binding Polymers As Anti-inflammatory Agents: Reducing the Danger of Nuclear Attack

Expert Review of Clinical Immunology. Jan, 2012  |  Pubmed ID: 22149331

Synthetic Mast-cell Granules As Adjuvants to Promote and Polarize Immunity in Lymph Nodes

Nature Materials. Jan, 2012  |  Pubmed ID: 22266469

Granules of mast cells (MCs) enhance adaptive immunity when, on activation, they are released as stable particles. Here we show that submicrometre particles modelled after MC granules augment immunity when used as adjuvants in vaccines. The synthetic particles, which consist of a carbohydrate backbone with encapsulated inflammatory mediators such as tumour necrosis factor, replicate attributes of MCs in vivo including the targeting of draining lymph nodes and the timed release of the encapsulated mediators. When used as an adjuvant during vaccination of mice with haemagglutinin from the influenza virus, the particles enhanced adaptive immune responses and increased survival of mice on lethal challenge. Furthermore, differential loading of the particles with the cytokine IL-12 directed the character of the response towards Th1 lymphocytes. The synthetic MC adjuvants replicate and enhance the functions of MCs during vaccination, and can be extended to polarize the resulting immunity.

Comparative Study of Nanoparticle-mediated Transfection in Different GI Epithelium Co-culture Models

Journal of Controlled Release : Official Journal of the Controlled Release Society. Feb, 2012  |  Pubmed ID: 22326811

Oral nonviral gene delivery is the most attractive and arguably the most challenging route of administration. To identify a suitable carrier, we studied the transport of different classes (natural polymer, synthetic polymer and synthetic lipid-polymer) of DNA nanoparticles through three well-characterized cellular models of intestinal epithelium (Caco2, Caco2-HT29MTX and Caco2-Raji). Poly(phosphoramidate-dipropylamine) (PPA) and Lipid-Protamine-DNA (LPD) nanoparticles consistently showed the highest level of human insulin mRNA expression and luciferase protein expression in these models, typically at least three orders of magnitude above background. All of the nanoparticles increased tight junction permeability, with PPA and PEI having the most dramatic transepithelial electrical resistance (TEER) decreases of (35.3±8.5%) and (37.5±1.5%) respectively in the first hour. The magnitude of TEER decrease correlated with nanoparticle surface charge, implicating electrostatic interactions with the tight junction proteins. However, confocal microscopy revealed that the nanoparticles were mostly uptaken by the enterocytes. Quantitative uptake and transport experiments showed that the endocytosed, quantum dot (QD)-labeled PPA-DNA nanoparticles remained in the intestinal cells even after 24h. Negligible amount of quantum dot labeled DNA was detected in the basolateral chamber, with the exception of the Caco2-Raji co-cultures, which internalized nanoparticles 2 to 3 times more readily compared to Caco2 and Caco2-HT29MTX cultures. PEGylation decreased the transfection efficacy by at least an order of magnitude, lowered the magnitude of TEER decrease and halved the uptake of PPA-DNA nanoparticles. A key finding was insulin mRNA being detected in the underlying HepG2 cells, signifying that some of the plasmid was transported across the intestinal epithelial layer while retaining at least partial bioactivity. However, the inefficient transport suggests that transcytosis alone would not engender a significant therapeutic effect, and this transport modality must be augmented by other means in vivo to render nonviral oral gene delivery practical.

Understanding Nonviral Nucleic Acid Delivery with Quantum Dot-FRET Nanosensors

Nanomedicine (London, England). Apr, 2012  |  Pubmed ID: 22471720

Nonviral delivery of nucleic acids is a potentially safe and viable therapeutic modality for inherited and acquired diseases. However, current systems have proven too inefficient for widespread clinical translation. The rational design of improved carriers depends on a quantitative, mechanistic understanding of the rate-limiting barriers to efficient intracellular delivery. Separation of the nucleic acid from the carrier is one of the barriers, which may be analyzed by Förster resonance energy transfer (FRET), a mechanism used to detect interactions between fluorescently labeled molecules. When applied to the molecular components of polymer or lipid-based nanocomplexes, FRET provides information on their complexation status, uptake, release and degradation. Recently, the design of FRET systems incorporating quantum dots as energy donors has led to improved signal stability, allowing prolonged measurements, as well as increased sensitivity, enabling direct detection and the potential for multiplexing. The union of quantum dots and FRET is providing new insights into the mechanisms of nonviral nucleic acid delivery through convergent characterization of delivery barriers, and has the potential to accelerate the design of improved carriers to realize the potential of nucleic acid therapeutics and gene medicine.

Nanotopography As Modulator of Human Mesenchymal Stem Cell Function

Biomaterials. Jul, 2012  |  Pubmed ID: 22516607

Nanotopography changes human mesenchymal stem cells (hMSC) from their shape to their differentiation potential; however little is known about the underlying molecular mechanisms. Here we study the culture of hMSC on polydimethylsiloxane substrates with 350 nm grating topography and investigate the focal adhesion composition and dynamics using biochemical and imaging techniques. Our results show that zyxin protein plays a key role in the hMSC response to nanotopography. Zyxin expression is downregulated on 350 nm gratings, leading to smaller and more dynamic focal adhesion. Since the association of zyxin with focal adhesions is force-dependent, smaller zyxin-positive adhesion as well as its higher turnover rate suggests that the traction force in focal adhesion on 350 nm topography is decreased. These changes lead to faster and more directional migration on 350 nm gratings. These findings demonstrate that nanotopography decreases the mechanical forces acting on focal adhesions in hMSC and suggest that force-dependent changes in zyxin protein expression and kinetics underlie the focal adhesion remodeling in response to 350 nm grating topography, resulting in modulation of hMSC function.

Microfluidic Synthesis of Multifunctional Janus Particles for Biomedical Applications

Lab on a Chip. May, 2012  |  Pubmed ID: 22584998

Multifunctional Janus particles have a variety of applications in a wide range of fields. However, to achieve many of these applications, high-throughput, low-cost techniques are needed to synthesize these particles with precise control of the various structural/physical/chemical properties. Microfluidics provides a unique platform to fabricate Janus particles using carefully controlled liquid flow in microfluidic channels to form Janus droplets and various types of solidification methods to solidify them into Janus particles. In this Focus article, we summarize the most recent representative works on Janus particle fabrication in microfluidics. The applications of Janus particles in biomedical areas are emphasized. We believe that microfluidics-enabled multifunctional Janus particles could resolve multiple prevalent issues in biomedicine (e.g., disease monitoring at an early stage, high-throughput bioassays, therapeutic delivery) if persistent effort and collaboration are devoted to this direction.

Guidance of Stem Cell Fate on 2D Patterned Surfaces

Biomaterials. Oct, 2012  |  Pubmed ID: 22748769

Stem cells possess unique abilities as they can renew themselves for extended periods of time and have the capacity to differentiate into a variety of lineages. They hold promise for treating a plethora of diseases ranging from musculoskeletal defects to myocardial infarction and to neural disorders. Understanding how to control the fate decision of these cells to self-renew or differentiate is paramount in stem cell tissue engineering. Recently, significant progress has been made in guiding stem cell differentiation in vitro, and we are beginning to understand the complex interplay of factors that control their fate. Here, we highlight the recent approaches for guidance of stem cells through patterning of surfaces at the micro- and nanoscale. Particular attention is given to chemical patterning of substrates with adhesion ligands and physical patterning with a variety of topographical features. These surface-mediated biochemical and mechanical cues have proven influential in altering a wide range of stem cell phenotypes. This approach to guide or ultimately control stem cells by surface patterning has enormous potential implications in cell therapies and regenerative medicine.

The Inhibition of Anti-DNA Binding to DNA by Nucleic Acid Binding Polymers

PloS One. 2012  |  Pubmed ID: 22808279

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs) on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3), hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

Nucleic Acid Scavengers Inhibit Thrombosis Without Increasing Bleeding

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2012  |  Pubmed ID: 22837404

Development of effective, yet safe, antithrombotic agents has been challenging because such agents increase the propensity of patients to bleed. Recently, naturally occurring polyphosphates such as extracellular DNA, RNA, and inorganic polyphosphates have been shown to activate blood coagulation. In this report, we evaluate the anticoagulant and antithrombotic activity of nucleic acid-binding polymers in vitro and in vivo. Such polymers bind to DNA, RNA, and inorganic polyphosphate molecules with high affinity and inhibit RNA- and polyphosphate-induced clotting and the activation of the intrinsic pathway of coagulation in vitro. Moreover, [NH(2)(CH(2))(2)NH(2)](G = 3);dendri PAMAM(NH(2))(32) (PAMAM G-3) prevents thrombosis following carotid artery injury and pulmonary thromboembolism in mice without significantly increasing blood loss from surgically challenged animals. These studies indicate that nucleic acid-binding polymers are able to scavenge effectively prothrombotic nucleic acids and other polyphosphates in vivo and represent a new and potentially safer class of antithrombotic agents.

Effects of Topographical and Mechanical Property Alterations Induced by Oxygen Plasma Modification on Stem Cell Behavior

ACS Nano. Oct, 2012  |  Pubmed ID: 22970773

Polymeric substrates intended for cell culture and tissue engineering are often surface-modified to facilitate cell attachment of most anchorage-dependent cell types. The modification alters the surface chemistry and possibly topography. However, scant attention has been paid to other surface property alterations. In studying oxygen plasma treatment of polydimethylsiloxane (PDMS), we show that oxygen plasma treatment alters the surface chemistry and, consequently, the topography and elasticity of PDMS at the nanoscale level. The elasticity factor has the predominant effect, compared with the chemical and topographical factors, on cell adhesions of human mesenchymal stem cells (hMSCs). The enhanced focal adhesions favor cell spreading and osteogenesis of hMSCs. Given the prevalent use of PDMS in biomedical device construction and cell culture experiments, this study highlights the importance of understanding how oxygen plasma treatment would impact subsequent cell-substrate interactions. It helps explain inconsistency in the literature and guides preparation of PDMS-based biomedical devices in the future.

Cartilage Tissue Engineering Using Differentiated and Purified Induced Pluripotent Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2012  |  Pubmed ID: 23115336

The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP- cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.

Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-causing Plasmodium Parasites Based on Enzyme Activity Measurement

ACS Nano. Dec, 2012  |  Pubmed ID: 23121492

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.

Nonviral Direct Conversion of Primary Mouse Embryonic Fibroblasts to Neuronal Cells

Molecular Therapy. Nucleic Acids. Jul, 2012  |  Pubmed ID: 23344148

Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5 µg cm(-2) doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1(+) (neuron-specific class III β-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP(+) cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes.

Advanced Materials and Processing for Drug Delivery: the Past and the Future

Advanced Drug Delivery Reviews. Jan, 2013  |  Pubmed ID: 23088863

Design and synthesis of efficient drug delivery systems are of vital importance for medicine and healthcare. Materials innovation and nanotechnology have synergistically fueled the advancement of drug delivery. Innovation in material chemistry allows the generation of biodegradable, biocompatible, environment-responsive, and targeted delivery systems. Nanotechnology enables control over size, shape and multi-functionality of particulate drug delivery systems. In this review, we focus on the materials innovation and processing of drug delivery systems and how these advances have shaped the past and may influence the future of drug delivery.

Mechanism of Oral Tolerance Induction to Therapeutic Proteins

Advanced Drug Delivery Reviews. Jun, 2013  |  Pubmed ID: 23123293

Oral tolerance is defined as the specific suppression of humoral and/or cellular immune responses to an antigen by administration of the same antigen through the oral route. Due to its absence of toxicity, easy administration, and antigen specificity, oral tolerance is a very attractive approach to prevent unwanted immune responses that cause a variety of diseases or that complicate treatment of a disease. Many researchers have induced oral tolerance to efficiently treat autoimmune and inflammatory diseases in different animal models. However, clinical trials yielded limited success. Thus, understanding the mechanisms of oral tolerance induction to therapeutic proteins is critical for paving the way for clinical development of oral tolerance protocols. This review will summarize progress on understanding the major underlying tolerance mechanisms and contributors, including antigen presenting cells, regulatory T cells, cytokines, and signaling pathways. Potential applications, examples for therapeutic proteins and disease targets, and recent developments in delivery methods are discussed.

Comparison of Mixed and Lamellar Coculture Spatial Arrangements for Tissue Engineering Capillary Networks in Vitro

Tissue Engineering. Part A. Mar, 2013  |  Pubmed ID: 23171167

Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent monolayers or EC networks. Factors influencing this transition are not known. In this study, we examined whether the spatial arrangement of EC-SMC cocultures affected EC migration, network morphology, and angiogenic protein secretion. Human umbilical cord blood-derived ECs (hCB-ECs) were grown in coculture with human aortic SMCs in either a mixed or lamellar spatial geometry and analyzed over a culture period of 12 days. The hCB-ECs cultured on SMCs in a mixed system had higher cell speeds, shorter persistence times, and lower random motility coefficients than ECs in a lamellar system. By day 12 of coculture, mixed systems demonstrated greater anastomoses and capillary loop formation than lamellar systems as evidenced by a higher number of branch points, angle of curvature between branch points, and percentage of imaged area covered by networks. The network morphology was more uniform in the mixed systems than the lamellar systems with fewer EC clusters present after several days in culture. Proliferation of hCB-ECs was higher for mixed cocultures during the first 24 h of coculture, and then declined dramatically suggesting that proliferation only contributed to network formation during the early stages of coculture. Proteome assay results show reduced solution levels, but no change in intracellular levels of angiogenic proteins in lamellar systems compared to mixed systems. These data suggest that mixing ECs and SMCs together favors the formation of EC networks to a greater extent than a lamellar arrangement in which ECs form a cell layer above a confluent, quiescent layer of SMCs.

Transfection Efficiency and Transgene Expression Kinetics of MRNA Delivered in Naked and Nanoparticle Format

Journal of Controlled Release : Official Journal of the Controlled Release Society. Mar, 2013  |  Pubmed ID: 23306021

Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

In Vitro and in Vivo Models for the Study of Oral Delivery of Nanoparticles

Advanced Drug Delivery Reviews. Jun, 2013  |  Pubmed ID: 23415952

Oral delivery is an attractive route to deliver therapeutics via nanoparticles due to its ease of administration and patient compliance. This review discusses laboratory techniques for studying oral delivery of nanoparticles, which offer protection of cargo through the gastrointestinal tract. Some of the difficulties in modeling oral delivery include the harsh acidic environment, variable pH, and the tight monolayer of endothelial cells present throughout the gastrointestinal tract. The use of in vitro techniques including the Transwell ® system, simulated gastric/intestinal fluid, and diffusion chambers addresses these challenges. When studying effects after oral delivery in vivo, bioimaging of nanoparticle biodistribution using radioactive markers has been popular. Functional assays such as immune response and systemic protein concentration analysis can further define the merits of the oral delivery systems. As biologics become increasingly more important in chronic therapies, nanoparticle-mediated oral delivery will assume greater prominence, and more sophisticated in vitro and in vivo models will be required.

Nanoparticle- and Biomaterials-mediated Oral Delivery for Drug, Gene, and Immunotherapy

Advanced Drug Delivery Reviews. Jun, 2013  |  Pubmed ID: 23434918

A Programmable Microenvironment for Cellular Studies Via Microfluidics-generated Double Emulsions

Biomaterials. Jun, 2013  |  Pubmed ID: 23522800

High throughput cellular studies require small sample volume to reduce costs and enhance sensitivity. Microfluidics-generated water-in-oil (W/O) single emulsion droplet systems, in particular, provide uniform, well defined and discrete microenvironment for cell culture, screening, and sorting. However, these single emulsion droplets are incapable of continuous supply of nutrient molecule and are not compatible with aqueous phase-based analysis. A solution is to entrap W/O droplets in another aqueous phase, forming water-in-oil-in-water (W/O/W) double emulsions. The external aqueous phase efficiently prevents desiccation and reduces the amount of organic component, and yet retaining the advantages of compartmentalization. The internal environment can also be programmed dynamically without the need of rupturing the droplets. In this study, we explore the potential application of W/O/W double emulsion droplets for cell cultivation, genetic activation and study of more complicated biological events such as bacteria quorum-sensing as an example. This study demonstrates the advantages and potential application of double emulsion for the study of complex biological processes.

Transcription Factors MYOCD, SRF, Mesp1 and SMARCD3 Enhance the Cardio-inducing Effect of GATA4, TBX5, and MEF2C During Direct Cellular Reprogramming

PloS One. 2013  |  Pubmed ID: 23704920

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca(2+) transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

A Robust Strategy for Negative Selection of Cre-loxP Recombination-based Excision of Transgenes in Induced Pluripotent Stem Cells

PloS One. 2013  |  Pubmed ID: 23717601

Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1α promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. A reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.

Induced Pluripotent Stem Cell-derived Cardiac Progenitors Differentiate to Cardiomyocytes and Form Biosynthetic Tissues

PloS One. 2013  |  Pubmed ID: 23785459

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

RNA-guided Gene Activation by CRISPR-Cas9-based Transcription Factors

Nature Methods. Oct, 2013  |  Pubmed ID: 23892895

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

Differentiation of Mouse Induced Pluripotent Stem Cells (iPSCs) into Nucleus Pulposus-like Cells in Vitro

PloS One. 2013  |  Pubmed ID: 24086564

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

Multifunctional Nanorods Serving As Nanobridges to Modulate T Cell-mediated Immunity

ACS Nano. Nov, 2013  |  Pubmed ID: 24088178

Electrodeposited nanorods serving as multivalent bridges were fabricated and surface-decorated with ligands for immune cells. Gold and nickel solutions were sequentially electrodeposited on nanoporous anodized disc templates and the template was dissolved to retrieve bisegmented nanorods with different lengths. Gold and nickel segmented nanorods were surface-immobilized with mannose and RGD peptides to prepare immune-cell recruiting nanorods. Surface-functionalization of nanorods were confirmed by fluorescence-labeling of each ligands and confocal microscopy. Dendritic cells and T cells were co-incubated with the surface-functionalized nanorods, and the proximity between the nanorods and the immune cells was visualized by variable pressure scanning electron microscopy and confocal microscopy. The long nanorods were associated with the immune cells, whereas the shorter nanorods were rather endocytosed by cells, suggesting a feasibility of the longer nanorods as bridging for the cells. Cytokine releases from the immune cells were monitored by cultivating lipopolysaccharide-activated dendritic cells with T cells. Interleukine-2 and interferon-γ release profiles showed a strong correlation with the length of the nanorod, where the 4 μm nanorods induced the highest levels of cytokine release compared to 1 or 2 μm nanorods. Thus, we concluded that the proximity of the immune cells increased by bridging the immune cells with the nanobridging system, which subsequently increased cytokine release by facilitating the antigen presentation process.

Temperature-controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-assembled DNA Nanocage

ACS Nano. Nov, 2013  |  Pubmed ID: 24168393

We demonstrate temperature-controlled encapsulation and release of the enzyme horseradish peroxidase using a preassembled and covalently closed three-dimensional DNA cage structure as a controllable encapsulation device. The utilized cage structure was covalently closed and composed of 12 double-stranded B-DNA helices that constituted the edges of the structure. The double stranded helices were interrupted by short single-stranded thymidine linkers constituting the cage corners except for one, which was composed by four 32 nucleotide long stretches of DNA with a sequence that allowed them to fold into hairpin structures. As demonstrated by gel-electrophoretic and fluorophore-quenching experiments this design imposed a temperature-controlled conformational transition capability to the structure, which allowed entrance or release of an enzyme cargo at 37 °C while ensuring retainment of the cargo in the central cavity of the cage at 4 °C. The entrapped enzyme was catalytically active inside the DNA cage and was able to convert substrate molecules penetrating the apertures in the DNA lattice that surrounded the central cavity of the cage.

Microfluidic Preparation of Polymer-nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

Scientific Reports. Nov, 2013  |  Pubmed ID: 24193511

As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

Rapid Formation of Multicellular Spheroids in Double-emulsion Droplets with Controllable Microenvironment

Scientific Reports. Dec, 2013  |  Pubmed ID: 24322507

An attractive option for tissue engineering is to use of multicellular spheroids as microtissues, particularly with stem cell spheroids. Conventional approaches of fabricating spheroids suffer from low throughput and polydispersity in size, and fail to supplement cues from extracellular matrix (ECM) for enhanced differentiation. In this study, we report the application of microfluidics-generated water-in-oil-in-water (w/o/w) double-emulsion (DE) droplets as pico-liter sized bioreactor for rapid cell assembly and well-controlled microenvironment for spheroid culture. Cells aggregated to form size-controllable (30-80 μm) spheroids in DE droplets within 150 min and could be retrieved via a droplet-releasing agent. Moreover, precursor hydrogel solution can be adopted as the inner phase to produce spheroid-encapsulated microgels after spheroid formation. As an example, the encapsulation of human mesenchymal stem cells (hMSC) spheroids in alginate and alginate-arginine-glycine-aspartic acid (-RGD) microgel was demonstrated, with enhanced osteogenic differentiation further exhibited in the latter case.

Materials Innovation for Co-delivery of Diverse Therapeutic Cargos

RSC Advances. Dec, 2013  |  Pubmed ID: 24818000

Co-delivery is a rapidly growing sector of drug delivery that aspires to enhance therapeutic efficacy through controlled delivery of diverse therapeutic cargoes with synergistic activities. It requires the design of carriers capable of simultaneously transporting to and releasing multiple therapeutics at a disease site. Co-delivery has arisen from the emerging trend of combination therapy, where treatment with two or more therapeutics at the same time can succeed where single therapeutics fail. However, conventional combination therapy offers little control over achieving an optimized therapeutic ratio at the target site. Co-delivery via inclusion of multiple therapeutic cargos within the same carrier addresses this issue by not only ensuring delivery of both therapeutics to the same cell, but also offering a platform for control of the delivery process, from loading to release. Co-delivery systems have been formulated using a number of carriers previously developed for single-therapeutic delivery. Liposomes, polymeric micelles, PLGA nanoparticles, and dendrimers have all been adapted for co-delivery. Much of the effort focuses on dealing with drugs having dissimilar properties, increasing loading efficiencies, and controlling loading and release ratios. In this review, we highlight the innovations in carrier designs and formulations to deliver combination cargoes of drug/drug, drug/siRNA, and drug/pDNA toward disease therapy. With rapid advances in mechanistic understanding of interrelating molecular pathways and development of molecular medicine, the future of co-delivery will become increasingly promising and prominent.

SOD Therapeutics: Latest Insights into Their Structure-activity Relationships and Impact on the Cellular Redox-based Signaling Pathways

Antioxidants & Redox Signaling. May, 2014  |  Pubmed ID: 23875805

Superoxide dismutase (SOD) enzymes are indispensable and ubiquitous antioxidant defenses maintaining the steady-state levels of O2·(-); no wonder, thus, that their mimics are remarkably efficacious in essentially any animal model of oxidative stress injuries thus far explored.

Magnetoactive Sponges for Dynamic Control of Microfluidic Flow Patterns in Microphysiological Systems

Lab on a Chip. Feb, 2014  |  Pubmed ID: 24310854

We developed a microfluidic flow-control system capable of dynamically generating various flow patterns on demand. The flow-control system is based on novel magnetoactive sponges embedded in microfluidic flow channels. Applying a non-uniform magnetic field compresses the magnetoactive sponge, significantly reducing porosity and hydraulic conductivity. Tuning the applied magnetic field can dynamically vary the flow rate in the microfluidic channel. Pulsatile and physiological flow patterns with frequency between 1 and 3 Hz, flow rates between 0.5 and 10 μL min(-1) and duration over 3 weeks have been achieved. Smooth muscle cells in engineered blood vessels perfused for 7 days aligned perpendicular to the flow direction under pulsatile but not steady flow, similar to the in vivo orientation. Owing to its various advantages over traditional flow-control methods, the new system potentially has important applications in microfluidic-based microphysiological systems to simulate the physiological nature of blood flow.

Harnessing Localized Ridges for High-aspect-ratio Hierarchical Patterns with Dynamic Tunability and Multifunctionality

Advanced Materials (Deerfield Beach, Fla.). Mar, 2014  |  Pubmed ID: 24339233

A simple method for fabricating high-aspect-ratio, hierarchical, and dynamically tunable surface patterns is invented by harnessing localized-ridge instabilities in gold nanofilms coated on elastomer substrates (a); a theoretical model to calculate the critical parameters (e.g., wavelength and amplitude) for designing the new patterns is developed (b); and novel applications of the patterns as super-hydrophobic coatings (c) and biomimetic cell-culture substrates (d) capable of on-demand tunability are demonstrated.

Whole Blood Cells Loaded with Messenger RNA As an Anti-tumor Vaccine

Advanced Healthcare Materials. Jun, 2014  |  Pubmed ID: 24339387

The use of a cell-based vaccine composed of autologous whole blood cells loaded with mRNA is described. Mice immunized with whole blood cells loaded with mRNA encoding antigen develop anti-tumor immunity comparable to DC-RNA immunization. This approach offers a simple and affordable alternative to RNA-based cellular therapy by circumventing complex, laborious and expensive ex vivo manipulations required for DC-based immunizations.

Three-dimensional Hydrodynamic Focusing Method for Polyplex Synthesis

ACS Nano. Jan, 2014  |  Pubmed ID: 24341632

Successful intracellular delivery of nucleic acid therapeutics relies on multiaspect optimization, one of which is formulation. While there has been ample innovation on chemical design of polymeric gene carriers, the same cannot be said for physical processing of polymer-DNA nanocomplexes (polyplexes). Conventional synthesis of polyplexes by bulk mixing depends on the operators' experience. The poorly controlled bulk mixing process may also lead to batch-to-batch variation and consequent irreproducibility. Here, we synthesize polyplexes by using a three-dimensional hydrodynamic focusing (3D-HF) technique in a single-layered, planar microfluidic device. Without any additional chemical treatment or postprocessing, the polyplexes prepared by the 3D-HF method show smaller size, slower aggregation rate, and higher transfection efficiency, while exhibiting reduced cytotoxicity compared to the ones synthesized by conventional bulk mixing. In addition, by introducing external acoustic perturbation, mixing can be further enhanced, leading to even smaller nanocomplexes. The 3D-HF method provides a simple and reproducible process for synthesizing high-quality polyplexes, addressing a critical barrier in the eventual translation of nucleic acid therapeutics.

Synthesis of Fluorosurfactants for Emulsion-based Biological Applications

ACS Nano. Apr, 2014  |  Pubmed ID: 24646088

Microemulsion represents an attractive platform for fundamental and applied biomedical research because the emulsified droplets can serve as millions of compartmentalized micrometer-sized reactors amenable to high-throughput screening or online monitoring. However, establishing stable emulsions with surfactants that are compatible with biological applications remains a significant challenge. Motivated by the lack of commercially available surfactants suitable for microemulsion-based biological assays, this study describes the facile synthesis of a biocompatible fluorosurfactant with nonionic tris(hydroxymethyl)methyl (Tris) polar head groups. We have further demonstrated compatibility of the developed surfactant with diverse emulsion-based applications, including DNA polymeric nanoparticle synthesis, enzymatic activity assay, and bacterial or mammalian cell culture, in the setup of both double- and multiphases of emulsions.

The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

Biomaterials. Jul, 2014  |  Pubmed ID: 24709523

Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2(+) neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming.

Intranasal MRNA Nanoparticle Vaccination Induces Prophylactic and Therapeutic Anti-tumor Immunity

Scientific Reports. Jun, 2014  |  Pubmed ID: 24894817

Direct in vivo administration of messenger RNA (mRNA) delivered in both naked and nanoparticle formats are actively investigated because the use of dendritic cells transfected ex vivo with mRNA for cancer therapy is expensive and needs significant infrastructure. Notably, intravenous and subcutaneous injections are the only routes of administration tested for mRNA nanoparticle tumor vaccination. In this report, we demonstrate that tumor immunity can be achieved via nasal administration of mRNA. Mice nasally immunized with mRNA delivered in nanoparticle format demonstrate delayed tumor progression in both prophylactic and therapeutic immunization models. The observed tumor immunity correlates with splenic antigen-specific CD8+ T cells and is achieved only when mRNA is delivered in nanoparticle but not in naked format. In conclusion, we demonstrate, as a proof-of-concept, a non-invasive approach to mRNA tumor vaccination, increasing its potential as a broadly applicable and off-the-shelf therapy for cancer treatment.

Messenger RNA (mRNA) Nanoparticle Tumour Vaccination

Nanoscale. Jul, 2014  |  Pubmed ID: 24904987

Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

Recent Advances in Nanoparticle-mediated SiRNA Delivery

Annual Review of Biomedical Engineering. Jul, 2014  |  Pubmed ID: 24905873

Inhibiting specific gene expression by short interfering RNA (siRNA) offers a new therapeutic strategy to tackle many diseases, including cancer, metabolic disorders, and viral infections, at the molecular level. The macromolecular and polar nature of siRNA hinders its cellular access to exert its effect. Nanoparticulate delivery systems can promote efficient intracellular delivery. Despite showing promise in many preclinical studies and potential in some clinical trials, siRNA has poor delivery efficiency, which continues to demand innovations, from carrier design to formulation, in order to overcome transport barriers. Previous findings for optimal plasmid DNA delivery cannot be generalized to siRNA delivery owing to significant discrepancy in size and subtle differences in chain flexibility between the two types of nucleic acids. In this review, we highlight the recent advances in improving the stability of siRNA nanoparticles, understanding their intracellular trafficking and release mechanisms, and applying judiciously the promising formulations to disease models.

Use of Cartilage Derived from Murine Induced Pluripotent Stem Cells for Osteoarthritis Drug Screening

Arthritis & Rheumatology (Hoboken, N.J.). Nov, 2014  |  Pubmed ID: 25047145

The discovery of novel disease-modifying drugs for osteoarthritis (OA) is limited by the lack of adequate genetically defined cartilage tissues for application in high-throughput screening systems. We addressed this need by synthesizing cartilage from induced pluripotent stem cells (iPSCs) to establish and validate an in vitro model of OA.

Shape-controlled Synthesis of Hybrid Nanomaterials Via Three-dimensional Hydrodynamic Focusing

ACS Nano. Oct, 2014  |  Pubmed ID: 25268035

Shape-controlled synthesis of nanomaterials through a simple, continuous, and low-cost method is essential to nanomaterials research toward practical applications. Hydrodynamic focusing, with its advantages of simplicity, low-cost, and precise control over reaction conditions, has been used for nanomaterial synthesis. While most studies have focused on improving the uniformity and size control, few have addressed the potential of tuning the shape of the synthesized nanomaterials. Here we demonstrate a facile method to synthesize hybrid materials by three-dimensional hydrodynamic focusing (3D-HF). While keeping the flow rates of the reagents constant and changing only the flow rate of the buffer solution, the molar ratio of two reactants (i.e., tetrathiafulvalene (TTF) and HAuCl4) within the reaction zone varies. The synthesized TTF-Au hybrid materials possess very different and predictable morphologies. The reaction conditions at different buffer flow rates are studied through computational simulation, and the formation mechanisms of different structures are discussed. This simple one-step method to achieve continuous shape-tunable synthesis highlights the potential of 3D-HF in nanomaterials research.

A Novel Immune Competent Murine Hypertrophic Scar Contracture Model: a Tool to Elucidate Disease Mechanism and Develop New Therapies

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Nov-Dec, 2014  |  Pubmed ID: 25327261

Hypertrophic scar (HSc) contraction following burn injury causes contractures. Contractures are painful and disfiguring. Current therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune-competent murine HSc model. A third-degree burn was created on dorsum of C57BL/6 mice. Three days postburn, tissue was excised and grafted with ear skin. Graft contraction was analyzed and tissue harvested on different time points. Outcomes were compared with human condition to validate the model. To confirm graft survival, green fluorescent protein (GFP) mice were used, and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus in contraction was analyzed. Cellularity was assessed with 4',6-diamidino-2-phenylindole. Collagen maturation was assessed with Picro-sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Elastic moduli of skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ∼45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild-type mice, and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed that panniculus carnosus doesn't contribute to contraction. Microscopic analyses showed that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT-PCR showed up-regulation of transforming growth factor beta, alpha smooth muscle actin, and rho-associated protein kinase 2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.

A CRISPR/Cas9-based System for Reprogramming Cell Lineage Specification

Stem Cell Reports. Dec, 2014  |  Pubmed ID: 25448066

Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. We show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

Highly Aligned Nanofibrous Scaffold Derived from Decellularized Human Fibroblasts

Advanced Functional Materials. May, 2014  |  Pubmed ID: 25484849

Native tissues are endowed with a highly organized nanofibrous extracellular matrix (ECM) that directs cellular distribution and function. The objective of this study is to create a purely natural, uniform, and highly aligned nanofibrous ECM scaffold for potential tissue engineering applications. Synthetic nanogratings (130 nm in depth) were used to direct the growth of human dermal fibroblasts for up to 8 weeks, resulting in a uniform 70 μm-thick fibroblast cell sheet with highly aligned cells and ECM nanofibers. A natural ECM scaffold with uniformly aligned nanofibers of 78 ± 9 nm in diameter was generated after removing the cellular components from the detached fibroblast sheet. The elastic modulus of the scaffold was well maintained after the decellularization process because of the preservation of elastin fibers. Reseeding human mesenchymal stem cells (hMSCs) showed the excellent capacity of the scaffold in directing and supporting cell alignment and proliferation along the underlying fibers. The scaffold's biocompatibility was further examined by an in vitro inflammation assay with seeded macrophages. The aligned ECM scaffold induced a significantly lower immune response compared to its unaligned counterpart, as detected by the pro-inflammatory cytokines secreted from macrophages. The aligned nanofibrous ECM scaffold holds great potential in engineering organized tissues.

Vector Modifications to Eliminate Transposase Expression Following PiggyBac-mediated Transgenesis

Scientific Reports. Dec, 2014  |  Pubmed ID: 25492703

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.

Nanotopography Alters Nuclear Protein Expression, Proliferation and Differentiation of Human Mesenchymal Stem/stromal Cells

PloS One. 2014  |  Pubmed ID: 25521962

Mesenchymal stem/stromal cells respond to physical cues present in their microenvironment such as substrate elasticity, geometry, or topography with respect to morphology, proliferation, and differentiation. Although studies have demonstrated the role of focal adhesions in topography-mediated changes of gene expression, information linking substrate topography to the nucleus remains scarce. Here we show by two-dimensional gel electrophoresis and western blotting that A-type lamins and retinoblastoma protein are downregulated in mesenchymal stem/stromal cells cultured on 350 nm gratings compared to planar substrates; these changes lead to a decrease in proliferation and changes in differentiation potential.

Nanograting Structure Promotes Lamellipodia-based Cell Collective Migration and Wound Healing

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference. 2014  |  Pubmed ID: 25570601

Wound healing is a dynamic and complex process of replacing missing or dead cell structures and tissue layers. The aim of this research is to discover biocompatible materials and drugs that can promote cell migration in the wound area and thus enhance desirable wound healing effects. In this paper, we report that PDMS nanogratings could accelerate the migration of epithelial cells along the grating axis, and the addition of Imatinib could further increase the epithelial cell wound healing speed to 1.6 times the speed of control cells. We also demonstrate that this migration is mediated by lamellipodia protrusion, and is Rac1-GTPase activity dependent. Lastly, we discuss the potential application and prospect of different nanostructured biomaterials for wound healing studies.

Abstract 166: Electrospun Synthetic Scaffolds: a Biomimetic Approach to Prevent Hypertrophic Scar Contraction

Plastic and Reconstructive Surgery. Mar, 2014  |  Pubmed ID: 25942276


Biomaterials. Jan, 2015  |  Pubmed ID: 25443403

Knockdown of the Cell Cycle Inhibitor P21 Enhances Cartilage Formation by Induced Pluripotent Stem Cells

Tissue Engineering. Part A. Apr, 2015  |  Pubmed ID: 25517798

The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded ∼150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering.

Mitigation of Hypertrophic Scar Contraction Via an Elastomeric Biodegradable Scaffold

Biomaterials. Mar, 2015  |  Pubmed ID: 25591962

Hypertrophic scar (HSc) occurs in 40-70% of patients treated for third degree burn injuries. Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc contraction. HSc contraction leads to formation of a fixed, inelastic skin deformity. We propose that BSEs should maintain their architecture in the wound bed throughout the remodeling phase of repair to prevent HSc contraction. In this work we study a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using human dermal fibroblasts demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using a validated immune-competent murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d30 in vivo. Together, these data suggest that scaffolds which persist throughout the remodeling phase of repair may represent a clinically translatable method to prevent HSc contraction.

Plant-based Oral Tolerance to Hemophilia Therapy Employs a Complex Immune Regulatory Response Including LAP+CD4+ T Cells

Blood. Apr, 2015  |  Pubmed ID: 25700434

Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80(+) cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103(+) and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4(+) regulatory T cells (CD4(+)CD25(-)LAP(+) cells with upregulated IL-10 and transforming growth factor-β (TGF-β) expression) as well as conventional CD4(+)CD25(+) regulatory T cells systemically suppressed anti-FIX responses.

Aptamer Nanomedicine for Cancer Therapeutics: Barriers and Potential for Translation

ACS Nano. Mar, 2015  |  Pubmed ID: 25731717

Aptamer nanomedicine, including therapeutic aptamers and aptamer nanocomplexes, is beginning to fulfill its potential in both clinical trials and preclinical studies. Especially in oncology, aptamer nanomedicine may perform better than conventional or antibody-based chemotherapeutics due to specificity compared to the former and stability compared to the latter. Many proof-of-concept studies on applying aptamers to drug delivery, gene therapy, and cancer imaging have shown promising efficacy and impressive safety in vivo toward translation. Yet, there remains ample room for improvement and critical barriers to be addressed. In this review, we will first introduce the recent progress in clinical trials of aptamer nanomedicine, followed by a discussion of the barriers at the design and in vivo application stages. We will then highlight recent advances and engineering strategies proposed to tackle these barriers. Aptamer cancer nanomedicine has the potential to address one of the most important healthcare issues of the society.

Engineering Mesenchymal Stem Cells for Regenerative Medicine and Drug Delivery

Methods (San Diego, Calif.). Aug, 2015  |  Pubmed ID: 25770356

Researchers have applied mesenchymal stem cells (MSC) to a variety of therapeutic scenarios by harnessing their multipotent, regenerative, and immunosuppressive properties with tropisms toward inflamed, hypoxic, and cancerous sites. Although MSC-based therapies have been shown to be safe and effective to a certain degree, the efficacy remains low in most cases when MSC are applied alone. To enhance their therapeutic efficacy, researchers have equipped MSC with targeted delivery functions using genetic engineering, therapeutic agent incorporation, and cell surface modification. MSC can be genetically modified virally or non-virally to overexpress therapeutic proteins that complement their innate properties. MSC can also be primed with non-peptidic drugs or magnetic nanoparticles for enhanced efficacy and externally regulated targeting, respectively. Furthermore, MSC can be functionalized with targeting moieties to augment their homing toward therapeutic sites using enzymatic modification, chemical conjugation, or non-covalent interactions. These engineering techniques are still works in progress, requiring optimization to improve the therapeutic efficacy and targeting effectiveness while minimizing any loss of MSC function. In this review, we will highlight the advanced techniques of engineering MSC, describe their promise and the challenges of translation into clinical settings, and suggest future perspectives on realizing their full potential for MSC-based therapy.

Integration of Drug, Protein, and Gene Delivery Systems with Regenerative Medicine

Drug Delivery and Translational Research. Apr, 2015  |  Pubmed ID: 25787742

Regenerative medicine has the potential to drastically change the field of health care from reactive to preventative and restorative. Exciting advances in stem cell biology and cellular reprogramming have fueled the progress of this field. Biochemical cues in the form of small molecule drugs, growth factors, zinc finger protein transcription factors and nucleases, transcription activator-like effector nucleases, monoclonal antibodies, plasmid DNA, aptamers, or RNA interference agents can play an important role to influence stem cell differentiation and the outcome of tissue regeneration. Many of these biochemical factors are fragile and must act intracellularly at the molecular level. They require an effective delivery system, which can take the form of a scaffold (e.g., hydrogels and electrospun fibers), carrier (viral and nonviral), nano- and microparticle, or genetically modified cell. In this review, we will discuss the history and current technologies of drug, protein, and gene delivery in the context of regenerative medicine. Next, we will present case examples of how delivery technologies are being applied to promote angiogenesis in nonhealing wounds or prevent angiogenesis in age related macular degeneration. Finally, we will conclude with a brief discussion of the regulatory pathway from bench to bedside for the clinical translation of these novel therapeutics.

NanoCluster Beacons As Reporter Probes in Rolling Circle Enhanced Enzyme Activity Detection

Nanoscale. May, 2015  |  Pubmed ID: 25901841

As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.

Dynamic Control and Quantification of Bacterial Population Dynamics in Droplets

Biomaterials. Aug, 2015  |  Pubmed ID: 26005763

Culturing and measuring bacterial population dynamics are critical to develop insights into gene regulation or bacterial physiology. Traditional methods, based on bulk culture to obtain such quantification, have the limitations of higher cost/volume of reagents, non-amendable to small size of population and more laborious manipulation. To this end, droplet-based microfluidics represents a promising alternative that is cost-effective and high-throughput. However, difficulties in manipulating the droplet environment and monitoring encapsulated bacterial population for long-term experiments limit its utilization. To overcome these limitations, we used an electrode-free injection technology to modulate the chemical environment in droplets. This ability is critical for precise control of bacterial dynamics in droplets. Moreover, we developed a trapping device for long-term monitoring of population dynamics in individual droplets for at least 240 h. We demonstrated the utility of this new microfluidic system by quantifying population dynamics of natural and engineered bacteria. Our approach can further improve the analysis for systems and synthetic biology in terms of manipulability and high temporal resolution.

MicroRNA Delivery for Regenerative Medicine

Advanced Drug Delivery Reviews. Jul, 2015  |  Pubmed ID: 26024978

MicroRNA (miRNA) directs post-transcriptional regulation of a network of genes by targeting mRNA. Although relatively recent in development, many miRNAs direct differentiation of various stem cells including induced pluripotent stem cells (iPSCs), a major player in regenerative medicine. An effective and safe delivery of miRNA holds the key to translating miRNA technologies. Both viral and nonviral delivery systems have seen success in miRNA delivery, and each approach possesses advantages and disadvantages. A number of studies have demonstrated success in augmenting osteogenesis, improving cardiogenesis, and reducing fibrosis among many other tissue engineering applications. A scaffold-based approach with the possibility of local and sustained delivery of miRNA is particularly attractive since the physical cues provided by the scaffold may synergize with the biochemical cues induced by miRNA therapy. Herein, we first briefly cover the application of miRNA to direct stem cell fate via replacement and inhibition therapies, followed by the discussion of the promising viral and nonviral delivery systems. Next we present the unique advantages of a scaffold-based delivery in achieving lineage-specific differentiation and tissue development.

3D Printing of Highly Stretchable and Tough Hydrogels into Complex, Cellularized Structures

Advanced Materials (Deerfield Beach, Fla.). Jul, 2015  |  Pubmed ID: 26033288

A 3D printable and highly stretchable tough hydrogel is developed by combining poly(ethylene glycol) and sodium alginate, which synergize to form a hydrogel tougher than natural cartilage. Encapsulated cells maintain high viability over a 7 d culture period and are highly deformed together with the hydrogel. By adding biocompatible nanoclay, the tough hydrogel is 3D printed in various shapes without requiring support material.

3D Printing: 3D Printing of Highly Stretchable and Tough Hydrogels into Complex, Cellularized Structures

Advanced Materials (Deerfield Beach, Fla.). Jul, 2015  |  Pubmed ID: 26172844

X. Zhao and co-workers develop on page 4035 a new biocompatible hydrogel system that is extremely tough and stretchable and can be 3D printed into complex structures, such as the multilayer mesh shown. Cells encapsulated in the tough and printable hydrogel maintain high viability. 3D-printed structures of the tough hydrogel can sustain high mechanical loads and deformations.

Smart Multifunctional Drug Delivery Towards Anticancer Therapy Harmonized in Mesoporous Nanoparticles

Nanoscale. Sep, 2015  |  Pubmed ID: 26260245

Nanomedicine seeks to apply nanoscale materials for the therapy and diagnosis of diseased and damaged tissues. Recent advances in nanotechnology have made a major contribution to the development of multifunctional nanomaterials, which represents a paradigm shift from single purpose to multipurpose materials. Multifunctional nanomaterials have been proposed to enable simultaneous target imaging and on-demand delivery of therapeutic agents only to the specific site. Most advanced systems are also responsive to internal or external stimuli. This approach is particularly important for highly potent drugs (e.g. chemotherapeutics), which should be delivered in a discreet manner and interact with cells/tissues only locally. Both advances in imaging and precisely controlled and localized delivery are critically important in cancer treatment, and the use of such systems - theranostics - holds great promise to minimise side effects and boost therapeutic effectiveness of the treatment. Among others, mesoporous silica nanoparticles (MSNPs) are considered one of the most promising nanomaterials for drug delivery. Due to their unique intrinsic features, including tunable porosity and size, large surface area, structural diversity, easily modifiable chemistry and suitability for functionalization, and biocompatibility, MSNPs have been extensively utilized as multifunctional nanocarrier systems. The combination or hybridization with biomolecules, drugs, and other nanoparticles potentiated the ability of MSNPs towards multifunctionality, and even smart actions stimulated by specified signals, including pH, optical signal, redox reaction, electricity and magnetism. This paper provides a comprehensive review of the state-of-the-art of multifunctional, smart drug delivery systems centered on advanced MSNPs, with special emphasis on cancer related applications.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels

Scientific Reports. Oct, 2015  |  Pubmed ID: 26456074

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Immobilization of Nucleic Acid Binding Polymers As Anti-inflammatory Agent in Autoimmunity

Journal of Controlled Release : Official Journal of the Controlled Release Society. Sep, 2015  |  Pubmed ID: 27005091

Intranasal MRNA Nanoparticle Vaccination Induces Prophylactic and Therapeutic Anti-tumor Immunity

Journal of Controlled Release : Official Journal of the Controlled Release Society. Sep, 2015  |  Pubmed ID: 27005208

Biostable Electrospun Microfibrous Scaffolds Mitigate Hypertrophic Scar Contraction in an Immune-competent Murine Model

Acta Biomaterialia. Mar, 2016  |  Pubmed ID: 26708709

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). In this study, we evaluate the impact of an elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In vitro, fibroblast-seeded PU scaffolds contracted significantly less and demonstrated fewer αSMA(+) myofibroblasts compared to fibroblast-seeded collagen lattices. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. Our data suggest that electrospun ccPU scaffolds meet the requirements to reduce HSc contraction including reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, our data suggest that a more long-term scaffold may possess merit in reducing HSc.

Transdifferentiation of Human Endothelial Progenitors into Smooth Muscle Cells

Biomaterials. Apr, 2016  |  Pubmed ID: 26874281

Access to smooth muscle cells (SMC) would create opportunities for tissue engineering, drug testing, and disease modeling. Herein we report the direct conversion of human endothelial progenitor cells (EPC) to induced smooth muscle cells (iSMC) by induced expression of MYOCD. The EPC undergo a cytoskeletal rearrangement resembling that of mesenchymal cells within 3 days post initiation of MYOCD expression. By day 7, the reprogrammed cells show upregulation of smooth muscle markers ACTA2, MYH11, and TAGLN by qRT-PCR and ACTA2 and MYH11 expression by immunofluorescence. By two weeks, they resemble umbilical artery SMC in microarray gene expression analysis. The iSMC, in contrast to EPC control, show calcium transients in response to phenylephrine stimulation and a contractility an order of magnitude higher than that of EPC as determined by traction force microscopy. Tissue-engineered blood vessels constructed using iSMC show functionality with respect to flow- and drug-mediated vasodilation and vasoconstriction.

Coupling Spatial Segregation with Synthetic Circuits to Control Bacterial Survival

Molecular Systems Biology. Feb, 2016  |  Pubmed ID: 26925805

Engineered bacteria have great potential for medical and environmental applications. Fulfilling this potential requires controllability over engineered behaviors and scalability of the engineered systems. Here, we present a platform technology, microbial swarmbot, which employs spatial arrangement to control the growth dynamics of engineered bacteria. As a proof of principle, we demonstrated a safeguard strategy to prevent unintended bacterial proliferation. In particular, we adopted several synthetic gene circuits to program collective survival in Escherichia coli: the engineered bacteria could only survive when present at sufficiently high population densities. When encapsulated by permeable membranes, these bacteria can sense the local environment and respond accordingly. The cells inside the microbial swarmbot capsules will survive due to their high densities. Those escaping from a capsule, however, will be killed due to a decrease in their densities. We demonstrate that this design concept is modular and readily generalizable. Our work lays the foundation for engineering integrated and programmable control of hybrid biological-material systems for diverse applications.

Nanografted Substrata and Triculture of Human Pericytes, Fibroblasts, and Endothelial Cells for Studying the Effects on Angiogenesis

Tissue Engineering. Part A. Apr, 2016  |  Pubmed ID: 27019156

For the successful treatment of severe wounds, angiogenesis must be induced to provide a sufficient blood supply to the wound site. There have been many studies on various structural and biochemical factors regulating angiogenesis, such as surface topography, surface modifications, angiogenic factors, and various cell types. However, there is a paucity of research on the effects of micro- and nanoscale topography and the application of pericytes in angiogenesis. In this study, we utilized nanoscale topography combined with a triculture system consisting of human pericytes, fibroblasts, and endothelial cells. We investigated the effects of the nanografted substrata and a triculture system on proangiogenic therapies in vitro. Human dermal fibroblasts and human umbilical vein endothelial cells were seeded with human pericytes from the placenta directly onto nanografted substrata composed of polydimethylsiloxane. We demonstrated that key elements of angiogenesis, including segment and capillary-like tubular structure formation, as well as the secretion of extracellular matrix, were increased by interaction between the nanografted substrata and the coculture containing pericytes. Thus, nanografted surfaces and triculture systems containing human pericytes, fibroblasts, and endothelial cells promote angiogenesis.

Efficient One-Step Production of Microencapsulated Hepatocyte Spheroids with Enhanced Functions

Small (Weinheim an Der Bergstrasse, Germany). May, 2016  |  Pubmed ID: 27038291

Hepatocyte spheroids microencapsulated in hydrogels can contribute to liver research in various capacities. The conventional approach of microencapsulating spheroids produces a variable number of spheroids per microgel and requires an extra step of spheroid loading into the gel. Here, a microfluidics technology bypassing the step of spheroid loading and controlling the spheroid characteristics is reported. Double-emulsion droplets are used to generate microencapsulated homotypic or heterotypic hepatocyte spheroids (all as single spheroids <200 μm in diameter) with enhanced functions in 4 h. The composition of the microgel is tunable as demonstrated by improved hepatocyte functions during 24 d culture (albumin secretion, urea secretion, and cytochrome P450 activity) when alginate-collagen composite hydrogel is used instead of alginate. Hepatocyte spheroids in alginate-collagen also perform better than hepatocytes cultured in collagen-sandwich configuration. Moreover, hepatocyte functions are significantly enhanced when hepatocytes and endothelial progenitor cells (used as a novel supporting cell source) are co-cultured to form composite spheroids at an optimal ratio of 5:1, which could be further boosted when encapsulated in alginate-collagen. This microencapsulated-spheroid formation technology with high yield, versatility, and uniformity is envisioned to be an enabling technology for liver tissue engineering as well as biomanufacturing.

Poly(ethylene Glycol) Hydrogel Scaffolds Containing Cell-Adhesive and Protease-Sensitive Peptides Support Microvessel Formation by Endothelial Progenitor Cells

Cellular and Molecular Bioengineering. Mar, 2016  |  Pubmed ID: 27042236

The development of stable, functional microvessels remains an important obstacle to overcome for tissue engineered organs and treatment of ischemia. Endothelial progenitor cells (EPCs) are a promising cell source for vascular tissue engineering as they are readily obtainable and carry the potential to differentiate towards all endothelial phenotypes. The aim of this study was to investigate the ability of human umbilical cord blood-derived EPCs to form vessel-like structures within a tissue engineering scaffold material, a cell-adhesive and proteolytically degradable poly(ethylene glycol) (PEG) hydrogel. EPCs in co-culture with angiogenic mural cells were encapsulated in hydrogel scaffolds by mixing with polymeric precursors and using a mild photocrosslinking process to form hydrogels with homogeneously dispersed cells. EPCs formed 3D microvessels networks that were stable for at least 30 days in culture, without the need for supplemental angiogenic growth factors. These 3D EPC microvessels displayed aspects of physiological microvasculature with lumen formation, expression of endothelial cell proteins (connexin 32, VE-cadherin, eNOS), basement membrane formation with collagen IV and laminin, perivascular investment of PDGFR-β and α-SMA positive cells, and EPC quiescence (<1% proliferating cells) by 2 weeks of co-culture. Our findings demonstrate the development of a novel, reductionist system that is well-defined and reproducible for studying progenitor cell-driven microvessel formation.

Can Microfluidics Address Biomanufacturing Challenges in Drug/gene/cell Therapies?

Regenerative Biomaterials. Jun, 2016  |  Pubmed ID: 27047674

Translation of any inventions into products requires manufacturing. Development of drug/gene/cell delivery systems will eventually face manufacturing challenges, which require the establishment of standardized processes to produce biologically-relevant products of high quality without incurring prohibitive cost. Microfluidicu technologies present many advantages to improve the quality of drug/gene/cell delivery systems. They also offer the benefits of automation. What remains unclear is whether they can meet the scale-up requirement. In this perspective, we discuss the advantages of microfluidic-assisted synthesis of nanoscale drug/gene delivery systems, formation of microscale drug/cell-encapsulated particles, generation of genetically engineered cells and fabrication of macroscale drug/cell-loaded micro-/nano-fibers. We also highlight the scale-up challenges one would face in adopting microfluidic technologies for the manufacturing of these therapeutic delivery systems.

Nanoparticle-mediated Inhibition of Survivin to Overcome Drug Resistance in Cancer Therapy

Journal of Controlled Release : Official Journal of the Controlled Release Society. Oct, 2016  |  Pubmed ID: 27091696

The acquired resistance of human cancer cells to apoptosis is one of the defining hallmarks of cancer. Upregulated expression of inhibitors of apoptosis proteins (IAP) has been implicated in drug resistance in several cancers. Survivin (encoded by BIRC5), the smallest member of the IAP family, has been correlated with both the control of cell apoptosis and regulation of cell mitosis in cancer. Owing to its critical role in regulation of cell survival and development of cancer resistance, as well as its distinguishingly high level of expression in many types of cancer, survivin has long been regarded as a promising therapeutic target for cancer therapy. This review first presents an overview of the mechanism by which survivin regulates cell function, followed by a discussion of the current state of survivin-targeted therapies. We focus on the application of nanoparticulate systems to deliver survivin inhibitors, co-delivery of survivin inhibitors with chemotherapeutic agents, synchronous targeting of survivin, other drug resistant molecules, and survivin regulators. We conclude by highlighting the current limitations associated with survivin-targeted therapies and speculating on the future strategies to surmount these impediments.

Signal-on Protein Detection Via Dye Translocation Between Aptamer and Quantum Dot

ACS Applied Materials & Interfaces. May, 2016  |  Pubmed ID: 27101438

A unique interaction between the cyanine dye and negatively charged quantum dot is used to construct a signal-on biaptameric quantum dot (QD) Förster resonance energy transfer (FRET) beacon for protein detection and distinct aptamer characterization. The beacon comprises a pair of aptamers, one intercalated with the cyanine dye (YOYO-3) and the other conjugated to a negatively charged, carboxyl-QD. When the target protein is present, structural folding and sandwich association of the two aptamers take place. As a consequence, YOYO-3 is displaced from the folded aptamer and transferred to the unblocked QD surface to yield a target concentration-dependent FRET signal. As a proof-of-principle, we demonstrate the detection of thrombin ranging from nanomolar to submicromolar concentrations and confirm the dye translocation using cylindrical illumination confocal spectroscopy (CICS). The proposed beacon provides a simple, rapid, signal-on FRET detection for protein as well as a potential platform for distinct aptamer screening.

Inducing Enhanced Immunogenic Cell Death with Nanocarrier-based Drug Delivery Systems for Pancreatic Cancer Therapy

Biomaterials. Sep, 2016  |  Pubmed ID: 27343466

Immunogenic cell death (ICD) occurs when apoptotic tumor cell elicits a specific immune response, which may trigger an anti-tumor effect, via the release of immunostimulatory damage-associated molecular patterns (DAMPs). Hypothesizing that nanomedicines may impact ICD due to their proven advantages in delivery of chemotherapeutics, we encapsulated oxaliplatin (OXA) or gemcitabine (GEM), an ICD and a non-ICD inducer respectively, into the amphiphilic diblock copolymer nanoparticles. Neither GEM nor nanoparticle-encapsulated GEM (NP-GEM) induced ICD, while both OXA and nanoparticle-encapsulated OXA (NP-OXA) induced ICD. Interestingly, NP-OXA treated tumor cells released more DAMPs and induced stronger immune responses of dendritic cells and T lymphocytes than OXA treatment in vitro. Furthermore, OXA and NP-OXA exhibited stronger therapeutic effects in immunocompetent mice than in immunodeficient mice, and the enhancement of therapeutic efficacy was significantly higher in the NP-OXA group than the OXA group. Moreover, NP-OXA treatment induced a higher proportion of tumor infiltrating activated cytotoxic T-lymphocytes than OXA treatment. This general trend of enhanced ICD by nanoparticle delivery was corroborated in evaluating another pair of ICD inducer and non-ICD inducer, doxorubicin and 5-fluorouracil. In conclusion, although nanoparticle encapsulation did not endow a non-ICD inducer with ICD-mediated anti-tumor capacity, treatment with a nanoparticle-encapsulated ICD inducer led to significantly enhanced ICD and consequently improved anti-tumor effects than the free ICD inducer. The proposed nanomedicine approach may impact cancer immunotherapy via the novel cell death mechanism of ICD.

Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells

Cell Stem Cell. Sep, 2016  |  Pubmed ID: 27524438

Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Polycationic Nanofibers for Nucleic Acid Scavenging

Biomacromolecules. Oct, 2016  |  Pubmed ID: 27741396

Dying cells release nucleic acids (NA) and NA-containing complexes that activate inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation frequently encountered in autoimmune and inflammatory diseases. In this study, grafting of cationic polymers onto a nanofibrous mesh enabled local scavenging of negatively charged pro-inflammatory molecules in the extracellular space. Nucleic acid scavenging nanofibers (NASFs) formed from poly(styrene-alt-maleic anhydride) conjugated with 1.8 kDa bPEI resulted in nanofibers of diameters 486 ± 9 nm. NASFs inhibited the NF-κB response stimulated by the negatively charged agonists, CpG and poly(I:C), in Ramos-blue cells but not Pam3CSK4, a nonanionic agonist. Moreover, NASFs significantly impeded NF-κB activation in cells stimulated with damage-associated molecular pattern molecules (DAMPs) released from doxorubicin killed cancer cells. In vivo application of NASFs to open wounds demonstrated nucleic acid scavenging in wounds of diabetic mice infected with Pseudomonas aeruginosa, suggesting the in vivo efficacy of NASFs. This simple technique of generating NASF results in effective localized anti-inflammation in vitro and local nucleic acid scavenging in vivo.

Cell-laden Microfluidic Microgels for Tissue Regeneration

Lab on a Chip. Nov, 2016  |  Pubmed ID: 27797383

Regeneration of diseased tissue is one of the foremost concerns for millions of patients who suffer from tissue damage each year. Local delivery of cell-laden hydrogels offers an attractive approach for tissue repair. However, due to the typical macroscopic size of these cell constructs, the encapsulated cells often suffer from poor nutrient exchange. These issues can be mitigated by incorporating cells into microscopic hydrogels, or microgels, whose large surface-to-volume ratio promotes efficient mass transport and enhanced cell-matrix interactions. Using microfluidic technology, monodisperse cell-laden microgels with tunable sizes can be generated in a high-throughput manner, making them useful building blocks that can be assembled into tissue constructs with spatially controlled physicochemical properties. In this review, we examine microfluidics-generated cell-laden microgels for tissue regeneration applications. We provide a brief overview of the common biomaterials, gelation mechanisms, and microfluidic device designs that are used to generate these microgels, and summarize the most recent works on how they are applied to tissue regeneration. Finally, we discuss future applications of microfluidic cell-laden microgels as well as existing challenges that should be resolved to stimulate their clinical application.

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