Autologous Fixed Tumor Vaccine: a Formulation with Cytokine-microparticles for Protective Immunity Against Recurrence of Human Hepatocellular Carcinoma

Japanese Journal of Cancer Research : Gann. Apr, 2002  |  Pubmed ID: 11985784

We developed a tumor vaccine consisting of fixed hepatocellular carcinoma (HCC) cells/tissue fragments, biodegradable microparticles encapsulating granulocyte-macrophage-colony stimulating factor and interleukin-2, and an adjuvant. The vaccine protected 33% of syngeneic mice from HCC cell challenge. The vaccine containing human autologous HCC fragments showed essentially no adverse effect in a phase I/IIa clinical trial and 8/12 patients developed a delayed-type hypersensitivity (DTH) response against the fragments. Although 2 of 4 DTH-response-negative patients had recurrence after curative resection, the DTH-response-positive patients had no recurrence. The time before the first recurrence in the vaccinated patients was significantly longer than that in 24 historical control patients operated in the same department (P < 0.05). This formulation is a promising candidate to prevent recurrence of human HCC.

Polyphosphoester Microspheres for Sustained Release of Biologically Active Nerve Growth Factor

Biomaterials. Sep, 2002  |  Pubmed ID: 12109702

Controlled delivery of neurotrophic proteins to a target tissue by biodegradable polymer microspheres has been explored widely for its potential applications in the treatment of various disorders in the nervous system. We investigated in this study the potential of polyphosphoester microspheres as carriers for the sustained release of nerve growth factor (NGF), a water-soluble neurotrophic protein. Two polyphosphoesters (PPEs), P(BHET-EOP/TC) and P(DAPG-EOP), as well as poly(lactide/glycolic acid) (PLGA), were used to fabricate microspheres by a W/O/W emulsion and solvent evaporation/extraction method. With bovine serum albumin as a model protein to optimize processing parameters. P(DAPG-EOP) microspheres exhibited a lower burst effect but similar protein entrapment levels and efficiencies when compared with those made of PLGA. Bioactive NGF could be released for at least 10 weeks from the P(DAPG-EOP) microspheres, as confirmed by a neurite outgrowth assay of the PC12 cells. These NGF containing microspheres were incorporated into the nerve guide conduits that were implanted to bridge a 10 mm gap in a rat sciatic nerve model. Two weeks after implantation, immunostaining with an antibody against the neurofilament protein confirmed the presence of axons at the distal end of regenerated cables within the NGF microsphere-loaded conduits. These results demonstrated the feasibility of using biodegradable PPEs for microencapsulation of NGF and provided a basis for future therapeutic application of the microspheres.

Intranasal Gene Transfer by Chitosan-DNA Nanospheres Protects BALB/c Mice Against Acute Respiratory Syncytial Virus Infection

Human Gene Therapy. Aug, 2002  |  Pubmed ID: 12215263

Respiratory syncytial virus (RSV) infection is often associated in infancy with life-threatening bronchiolitis, which is also a major risk factor for the development of asthma. At present, no effective prophylaxis is available against RSV infection. Herein, we describe an effective prophylactic intranasal gene transfer strategy utilizing chitosan-DNA nanospheres (IGT), containing a cocktail of plasmid DNAs encoding all RSV antigens, except L. A single administration of IGT (25 microg/mouse) induces expression of the mRNA and proteins of all antigens in the lung and results in a significant reduction of viral titers and viral antigen load after acute RSV infection of these mice. IGT-administered mice show no significant change in airway reactivity to methacholine and no apparent pulmonary inflammation. Furthermore, IGT results in significant induction of RSV-specific IgG antibodies, nasal IgA antibodies, cytotoxic T lymphocytes, and interferon-gamma production in the lung and splenocytes compared with controls. Together, these results demonstrate the potential of IGT against acute RSV infection.

New Polyphosphoramidate with a Spermidine Side Chain As a Gene Carrier

Journal of Controlled Release : Official Journal of the Controlled Release Society. Sep, 2002  |  Pubmed ID: 12220847

A new cationic polymer (PPA-SP), polyphosphoramidate bearing spermidine side chain, was prepared as a non-viral vector for gene delivery. PPA-SP was synthesized from poly(1,2-propylene H-phosphonate) by the Atherton-Todd reaction. The weight average molecular weight of PPA-SP was 3.44x10(4) with a number average degree of polymerization of 90, as determined by GPC/LS/RI method. The average net positive charge per polymer chain was 102. PPA-SP was able to condense plasmid DNA efficiently and formed complexes at an N/P ratio (free amino groups in polymer to phosphate groups in DNA) of 2 and above, as determined by agarose gel electrophoresis. This new gene carrier offered significant protection to DNA against nuclease degradation at N/P ratios above 2, and showed lower cytotoxicity than PLL and PEI in cell culture. The LD(50) of PPA-SP was 85 microg/ml in COS-7 cells, in contrast to 20 and 42 microg/ml for PLL and PEI, respectively. The complexes prepared in saline at N/P ratios of 5 approximately 10 had an average size of 250 nm and zeta-potential of 26 mV. PPA-SP mediated efficient gene transfection in a number of cell lines, and the transfection protocol was optimized in HEK293 cells using a luciferase plasmid as a marker gene. Gene expression mediated by PPA-SP was greatly enhanced when chloroquine was used in conjunction at a concentration of 100 microM. Under the optimized condition, PPA-SP/DNA complexes yield a luciferase expression level closed to PEI/DNA complexes or Transfast mediated transfection. In a non-invasive CNS gene delivery model, PPA-SP/DNA complexes yielded comparable bcl-2 expression as PEI/DNA complexes in mouse brain stem following injection of the complexes in the tongue.

Adhesion Contact Dynamics of HepG2 Cells on Galactose-immobilized Substrates

Biomaterials. Feb, 2003  |  Pubmed ID: 12485802

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.

Preparation and Characterization of Polypseudorotaxanes Based on Block-selected Inclusion Complexation Between Poly(propylene Oxide)-poly(ethylene Oxide)-poly(propylene Oxide) Triblock Copolymers and Alpha-cyclodextrin

Journal of the American Chemical Society. Feb, 2003  |  Pubmed ID: 12580604

A series of new polypseudorotaxanes were synthesized in high yields when the middle poly(ethylene oxide) (PEO) block of poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEO-PPO) triblock copolymers was selectively recognized and included by alpha-cyclodextrin (alpha-CD) to form crystalline inclusion complexes (ICs), although the middle PEO block was flanked by two thicker PPO blocks, and a PPO chain had been previously thought to be impenetrable to alpha-CD. X-ray diffraction studies demonstrated that the IC domains of the polypseudorotaxanes assumed a channel-type structure similar to the necklace-like ICs formed by alpha-CD and PEO homopolymers. Solid-state CP/MAS (13)C NMR studies showed that the alpha-CD molecules in the polypseudorotaxanes adopted a symmetrical conformation due to the formation of ICs. The compositions and stoichiometry of the polypseudorotaxanes were studied using (1)H NMR, and a 2:1 (ethylene oxide unit to alpha-CD) stoichiometry was found for all polypseudorotaxanes although the PPO-PEO-PPO triblock copolymers had different compositions and block lengths, suggesting that only the PEO block was closely included by alpha-CD molecules, whereas the PPO blocks were uncovered. The hypothesis was further supported by the differential scanning calorimetry (DSC) studies of the polypseudorotaxanes. The glass transitions of the PPO blocks in the polypseudorotaxanes were clearly observed because they were uncovered by alpha-CD and remained amorphous, whereas the glass-transition temperatures increased, because the molecular motion of the PPO blocks was restricted by the hard crystalline phases of the IC domains formed by alpha-CD and the PEO blocks. The thermogravimetric analysis (TGA) revealed that the polypseudorotaxanes had better thermal stability than their free components due to the inclusion complexation. Finally, the kinetics of the threading process of alpha-CD onto the copolymers was also studied. The findings reported in this article suggested interesting possibilities in designing other cyclodextrin ICs and polypseudorotaxanes with block structures.

Microcapsules with Improved Mechanical Stability for Hepatocyte Culture

Biomaterials. May, 2003  |  Pubmed ID: 12593959

Packed-bed or fluidized-bed bioreactor filled with microencapsulated hepatocytes has been proposed as one of the promising designs for bioartificial liver assist device (BLAD) because of potential advantages of high mass transport rate and optimal microenvironment for hepatocyte culture. Recently, we have developed a microcapsule system for the encapsulation of hepatocytes. The microcapsules consist of an inner core of modified collagen and an outer shell of terpolymer of methyl methacrylate, methacrylate and hydroxyethyl methacrylate. Cells encapsulated in these microcapsules exhibit enhanced cellular functions. Improving the mechanical stability of the microcapsules to withstand the shear stress induced by high perfusion rate would be crucial to the success of BLAD applications. In this study, we investigated the effects of terpolymer molecular weight (M(w)) on the mechanical property of these microcapsules and the differentiated functions of encapsulated hepatocytes. Six terpolymers with different M(w) were synthesized using radical polymerization in solution by adjusting the reaction temperature and the initiator concentration. All the terpolymers formed microcapsules with the methylated collagen. While the terpolymer M(w) had little effect on the capsule membrane thickness and permeability of serum albumin, the mechanical property of the microcapsules was significantly improved by the higher M(w) of the terpolymer. Differentiated functions of the hepatocytes cultured in the microcapsules, including urea synthesis, albumin synthesis and cytochrome P450 metabolic activity, were not significantly affected by the terpolymer M(w).

Peripheral Nerve Regeneration with Sustained Release of Poly(phosphoester) Microencapsulated Nerve Growth Factor Within Nerve Guide Conduits

Biomaterials. Jun, 2003  |  Pubmed ID: 12699678

Prolonged delivery of neurotrophic proteins to the target tissue is valuable in the treatment of various disorders of the nervous system. We have tested in this study whether sustained release of nerve growth factor (NGF) within nerve guide conduits (NGCs), a device used to repair injured nerves, would augment peripheral nerve regeneration. NGF-containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site-specific delivery of NGF. The conduits were used to bridge a 10 mm gap in a rat sciatic nerve model. Three months after implantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower G-ratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, as compared with those from control conduits loaded with either saline alone, BSA microspheres, or NGF protein without microencapsulation. Beneficial effects on fiber diameter, G-ratio and fiber density were also observed in the permeable PPE NGCs. Thus, the results confirm a long-term promoting effect of exogenous NGF on morphological regeneration of peripheral nerves. The tissue-engineering approach reported in this study of incorporation of a microsphere protein release system into NGCs holds potential for improved functional recovery in patients whose injured nerves are reconstructed by entubulation.

Polyphosphoesters in Drug and Gene Delivery

Advanced Drug Delivery Reviews. Apr, 2003  |  Pubmed ID: 12706047

Polymers with repeating phosphoester bonds in the backbone are structurally versatile, and biodegradable through hydrolysis, and possibly enzymatic digestion at the phosphoester linkages under physiological conditions. These biodegradable polyphosphoesters are appealing for biological and pharmaceutical applications because of their potential biocompatibility and similarity to bio-macromolecules such as nucleic acids. In the first part of this review, we will focus on one particular structure synthesized by extending oligomeric lactide prepolymers with ethylphosphate groups. This amorphous to semi-crystalline polymer is promising in delivering anti-cancer therapeutics in the form of microspheres. In the second half, we will discuss the conjugation of charged groups to the side chain of the phosphate, constituting one of the few biodegradable cationic polymers in the field for non-viral gene delivery. Capable of delivering exogenous genes to a cell nucleus or providing an extracellular sustained release of DNA, these cationic polyphosphoesters also serve as a valuable model to understand the important characteristics that render a polymer an effective gene carrier.

Injectable Drug-delivery Systems Based on Supramolecular Hydrogels Formed by Poly(ethylene Oxide)s and Alpha-cyclodextrin

Journal of Biomedical Materials Research. Part A. May, 2003  |  Pubmed ID: 12734812

Polymeric hydrogels long have attracted interest for biomaterials applications because of their generally favorable biocompatibility. High in water content, they are particularly attractive for delivery of delicate bioactive agents, such as proteins. However, because they require covalent crosslinking for gelation, many hydrogels can be applied only as implantables, and incorporation of drugs by sorption may be time-consuming and limiting with regard to the loading level. Therefore a delivery formulation where gelation and drug loading can be achieved simultaneously, taking place in an aqueous environment and without covalent crosslinking, would be attractive. Herein is described a new class of injectable and bioabsorbable supramolecular hydrogels formed from poly(ethylene oxide)s (PEOs) and alpha-cyclodextrin (alpha-CD) for controlled drug delivery. The hydrogel formation is based on physical crosslinking induced by supramolecular self-assembling with no chemical crosslinking reagent involved. The supramolecular structure of the hydrogels was confirmed with wide-angle X-ray diffraction studies. The gelation kinetics was found to be dependent on the concentrations of the polymer and alpha-CD as well as on the molecular weight of the PEO used. The rheologic studies of the hydrogels showed that the hydrogels were thixotropic and reversible and that they could be injected through fine needles. The components of the supramolecular hydrogels potentially are biocompatible and nontoxic. Drugs can be encapsulated directly into the hydrogels in situ at room temperature without any contact with organic solvents. The supramolecular hydrogels were evaluated in terms of their in vitro release kinetics. The rate-controlling mechanism of macromolecular drug release might be the erosion of the hydrogels.

Chitosan Nanoparticles Containing Plasmid DNA Encoding House Dust Mite Allergen, Der P 1 for Oral Vaccination in Mice

Vaccine. Jun, 2003  |  Pubmed ID: 12798609

Our previous studies indicated that intramuscular (i.m.) immunisation with full length Der p 1 cDNA induced significant humoral response to the left domain (approximately corresponding to amino acids 1-116) but not to the right domain (approximately corresponding to amino acids 117-222) of Der p 1 allergen. This study explored the use of chitosan-DNA nanoparticles for oral immunisation to induce immune responses specific to both the left and right domains of Der p 1. DNA constructs pDer p 1 (1-222) and pDer p 1 (114-222) were complexed with chitosan and delivered orally followed by an i.m. injection of pDer p 1 (1-222) 13 weeks later. Such approach has successfully primed Th1-skewed immune responses against both domains of Der p 1. This strategy could be further optimised for more efficacious gene vaccination for full length Der p 1.

In Vivo US Monitoring of Catheter-based Vascular Delivery of Gene Microspheres in Pigs: Feasibility

Radiology. Aug, 2003  |  Pubmed ID: 12893906

In this study, the authors tested the feasibility of using ultrasonography (US) to monitor catheter-based vascular gene microsphere delivery. Polymeric biodegradable microspheres (mean diameter, 5 microm) were prepared by using a double-emulsion technique to encapsulate DNA-plasmid-encoding green fluorescent protein (GFP) genes. With use of gene-delivery catheters, GFP microspheres were locally delivered into the left femoral arterial walls of six pigs; the contralateral arteries were not infused with microspheres and thus served as negative control vessels. The delivery procedures were monitored with high-frequency (8-15-MHz) transducer US. The effectiveness of monitoring with US was compared with the effectiveness of monitoring with immunohistochemical anti-GFP staining. A highly echogenic "star burst" sign around the entire vessel wall was seen at US and correlated with immunohistochemical findings that showed the destination of the gene microspheres. Study results demonstrate the potential of US for monitoring catheter-based vascular gene microsphere delivery in vivo.

Multifunctional Nanorods for Gene Delivery

Nature Materials. Oct, 2003  |  Pubmed ID: 12970757

The goal of gene therapy is to introduce foreign genes into somatic cells to supplement defective genes or provide additional biological functions, and can be achieved using either viral or synthetic non-viral delivery systems. Compared with viral vectors, synthetic gene-delivery systems, such as liposomes and polymers, offer several advantages including ease of production and reduced risk of cytotoxicity and immunogenicity, but their use has been limited by the relatively low transfection efficiency. This problem mainly stems from the difficulty in controlling their properties at the nanoscale. Synthetic inorganic gene carriers have received limited attention in the gene-therapy community, the only notable example being gold nanoparticles with surface-immobilized DNA applied to intradermal genetic immunization by particle bombardment. Here we present a non-viral gene-delivery system based on multisegment bimetallic nanorods that can simultaneously bind compacted DNA plasmids and targeting ligands in a spatially defined manner. This approach allows precise control of composition, size and multifunctionality of the gene-delivery system. Transfection experiments performed in vitro and in vivo provide promising results that suggest potential in genetic vaccination applications.

Poly(D,L-lactide-co-ethyl Ethylene Phosphate)s As New Drug Carriers

Journal of Controlled Release : Official Journal of the Controlled Release Society. Sep, 2003  |  Pubmed ID: 14499184

Many biodegradable polymers have been developed for controlled drug delivery. The plethora of drug therapies and types of drugs demand different formulations, fabrications conditions and release kinetics. No one single polymer can satisfy all the requirements. To extend the properties of poly(D,L-lactide) (PLA), we synthesized copolymers of PLA and poly(ethylethylene phosphate) (PEEP) by ring-opening polymerization using Al(Oipr)3 as the initiator. The copolymers were structurally characterized by IR and 1H NMR. DSC data confirmed the formation of random microphase structure in all the copolymers, and showed a decrease of Tg from 43.2 to -22.6 degrees C when the molar content of ethylethylene phosphate (EEP) increased from 5 to 40%. The hydrophilicity of the copolymers increased with EEP content. In contrast to the degradation behavior of PLA, disc samples made of PLAEEP90 showed a linear weight loss profile in PBS (pH 7.4) at 37 degrees C. BSA microspheres using PLAEEP90 were prepared by double-emulsion method, yielding a loading level of 4.3% and a loading efficiency of 75%. The BSA release profile consisted of an initial burst (9%) on the first day, followed by a daily 4% release for the following 40 days, resulting in 91% of the BSA release in a near linear manner. The released BSA remained intact according to SDS-PAGE data. Cytotoxicity and histopathology studies showed low toxicity in HeLa cells and good tissue biocompatibility in mouse brain, respectively. PLAEEP is a promising biodegradable polymer for controlled drug delivery.

Use of Ultrathin Shell Microcapsules of Hepatocytes in Bioartificial Liver-assist Device

Tissue Engineering. 2003  |  Pubmed ID: 14511471

We previously encapsulated hepatocytes in ultrathin shell microcapsules and showed them to have enhanced differentiated functions over cells cultured in monolayer. Here we have used these microencapsulated hepatocytes in a bioartificial liver-assisted device (BLAD) with a rat hepatectomy model. Primary rat hepatocytes were encapsulated in 150- to 200-microm microcapsules, using an electrostatic droplet generator. The microencapsulated hepatocytes exhibited good in vitro urea synthesis activity in plasma from rats with fulminant hepatic failure (FHF). The ex vivo hemoperfusion was conducted in FHF rats by perfusing plasma at a rate of 1-2 mL/min through 1.5-2 x 10(8) encapsulated hepatocytes packed into a packed-bed bioreactor. Hemoperfusion with the bioreactor was initiated 5 h after operative induction of liver failure and continued for 7 h. The BLAD-treated rats showed improvements over the control groups in survival time and metabolic indicators, including ammonia and total bilirubin levels. Furthermore, expanded bed adsorption (EBA) detoxification technology using Streamline-SP resin was explored to complement the bioreactor with microencapsulated hepatocytes. In vitro experiments indicated that serum ammonia could be specifically removed in dose-dependent manner, whereas the total serum proteins were unaffected by the resin. In ex vivo experiments, hemoperfusion with the resin was initiated 3 h after operative induction of liver failure and continued for 7 h. The resin-treated rats showed obvious serum ammonia removal with no observable total blood protein and blood cell adsorption. Therefore, Streamline-SP resin can potentially be integrated into a BLAD for improved efficacy.

Effect of Side-chain Structures on Gene Transfer Efficiency of Biodegradable Cationic Polyphosphoesters

International Journal of Pharmaceutics. Oct, 2003  |  Pubmed ID: 14522120

Cationic polyphosphoesters (PPEs) with different side-chain charge groups were designed and synthesized as biodegradable gene carriers. Poly(N-methyl-2-aminoethyl propylene phosphate) (PPE-MEA), with a secondary amino group (-CH(2)CH(2)NHCH3) side chain released DNA in several hours at N/P (amino group of polymer to phosphate group of DNA) ratios from 0.5 to 5; whereas PPE-HA, bearing -CH(2)(CH2)(4)CH(2)NH(2) groups in the side chain, did not release DNA at the same ratio range for 30 days. Hydrolytic degradation and DNA binding results suggested that side chain cleavage, besides the polymer degradation, was the predominant factor affected the DNA release and transfection efficiencies. The side chain of PPE-MEA was cleaved faster than that of PPE-HA, resulting poor cellular uptake and no transgene expression for PPE-MEA/DNA complexes in COS-7 cells at charge ratios from 4 to 12. In contrast, PPE-HA/DNA complexes were stable enough to be internalized by cells and effected gene transfection (3400 folds higher than background at a charge ratio of 12). Interestingly, gene expression levels mediated by PPE-MEA and PPE-HA in mouse muscle following intramuscular injection of complexes showed a reversed order: PPE-MEA/DNA complexes mediated a 1.5-2-fold higher luciferase expression in mouse muscle as compared with naked DNA injection, while PPE-HA/DNA complexes induced delayed and lowered luciferase expression than naked DNA. These results suggested that the side chain structure is a crucial factor determining the mechanism and kinetics of hydrolytic degradation of PPE carriers, which in turn influenced the kinetics of DNA release from PPE/DNA complexes and their transfection abilities in vitro and in vivo.

Thermally Responsive Polymeric Micellar Nanoparticles Self-assembled from Cholesteryl End-capped Random Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide): Synthesis, Temperature-sensitivity, and Morphologies

Journal of Colloid and Interface Science. Oct, 2003  |  Pubmed ID: 14527452

Cholesteryl end-capped thermally responsive amphiphilic polymers with two different hydrophobic/hydrophilic chain-length ratios were synthesized from the hydroxyl-terminated random poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) and cholesteryl chloroformate. The hydroxyl-terminated precursor polymers with narrow molecular weight distributions were synthesized by free-radical polymerization using 2-hydroxyethanethiol as a chain-transfer agent. The aqueous solutions of the cholesteryl end-capped copolymers exhibited reversible phase transitions at temperatures slightly above human body temperature, with the lower critical solution temperature values being 37.7 and 38.2 degrees C, respectively. The critical micelle concentration values of the two cholesteryl end-capped polymers were 9 and 25 mg/L, respectively. Polymeric micellar nanoparticles were prepared from the amphiphilic polymers using a dialysis method as well as a direct dissolution method. Transmission electron microscope studies showed that the micellar nanoparticles existed in different morphologies, including spherical, star-like, and cuboid shapes. Pyrene as a model hydrophobic compound could be readily encapsulated in these polymeric nanoparticles, at loading levels of 1.0 and 0.8 mg/g for the two cholesteryl end-capped polymers, respectively. The temperature sensitivity and unusual morphology of these novel polymeric nanoparticles would make an interesting drug delivery system.

Galactosylated PVDF Membrane Promotes Hepatocyte Attachment and Functional Maintenance

Biomaterials. Dec, 2003  |  Pubmed ID: 14559002

One of the major challenges in BLAD design is to develop functional substrates suitable for hepatocyte attachment and functional maintenance. In the present study, we designed a poly(vinylidene difluoride) (PVDF) surface coated with galactose-tethered Pluronic polymer. The galactose-derived Pluronic F68 (F68-Gal) was adsorbed on PVDF membrane through hydrophobic-hydrophobic interaction between PVDF and the polypropylene oxide segment in Pluronic. The galactose density on the modified PVDF surface increased with the concentration of the F68-Gal solution, reaching 15.4 nmol galactosyl groups per cm2 when a 1 mg/ml of F68-Gal solution was used. The adsorbed F68-Gal remained relatively stable in culture medium. Rat hepatocytes attachment efficiency on F68-Gal modified PVDF membrane was similar to that on collagen-coated surface. The attached hepatocytes on PVDF/F68-Gal membrane self-assembled into multi-cellular spheroids after 1 day of culture. These attached hepatocytes in spheroids exhibited higher cell functions such as albumin synthesis and P450 1A1 detoxification function compared to unmodified PVDF membrane and collagen-coated surface. These results suggest the potential of this galactose-immobilized PVDF membrane as a suitable substrate for hepatocyte culture.

High Density of Immobilized Galactose Ligand Enhances Hepatocyte Attachment and Function

Journal of Biomedical Materials Research. Part A. Dec, 2003  |  Pubmed ID: 14624494

Galactosylated surface is an attractive substrate for hepatocyte culture because of the specific interaction between the galactose ligand and the asialoglycoprotein receptor on hepatocytes. In this study, we described a scheme to achieve high density of immobilized galactose ligands on polyethylene terephthalate (PET) surface by first surface-grafting polyacrylic acid on plasma-pretreated PET film under UV irradiation, followed by conjugation of a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the grafted polyacrylic acid chains. A high galactose density of 513 nmol/cm(2) on the PET surface was used in this study to investigate the behavior of cultured hepatocyte. This engineered substrate showed high affinity to fluorescein isothiocyanate-lectin binding. Primary rat hepatocytes, when seeded at a density of 2 x 10(5) cells/cm(2), attached to the galactosylated PET substrate at a similar efficiency compared with collagen-coated substrate. The hepatocytes spontaneously formed aggregates 1 day after cell seeding and showed better maintenance of albumin secretion and urea synthesis functions than those cultured on collagen-coated surface.

Peripheral Nerve Regeneration by Microbraided Poly(L-lactide-co-glycolide) Biodegradable Polymer Fibers

Journal of Biomedical Materials Research. Part A. Feb, 2004  |  Pubmed ID: 14704970

Tiny tubes with fiber architecture were developed by a novel method of fabrication upon introducing some modification to the microbraiding technique, to function as nerve guide conduit and the feasibility of in vivo nerve regeneration was investigated through several of these conduits. Poly(L-lactide-co-glycolide) (10:90) polymer fibers being biocompatible and biodegradable were used for the fabrication of the conduits. The microbraided nerve guide conduits (MNGCs) were characterized using scanning electron microscopy to study the surface morphology and fiber arrangement. Degradation tests were performed and the micrographs of the conduit showed that the degradation of the conduit is by fiber breakage indicating bulk hydrolysis of the polymer. Biological performances of the conduits were examined in the rat sciatic nerve model with a 12-mm gap. After implantation of the MNGC to the right sciatic nerve of the rat, there was no inflammatory response. One week after implantation, a thin tissue capsule was formed on the outer surface of the conduit, indicating good biological response of the conduit. Fibrin matrix cable formation was seen inside the MNGC after 1 week implantation. One month after implantation, 9 of 10 rats showed successful nerve regeneration. None of the implanted tubes showed tube breakage. The MNGCs were flexible, permeable, and showed no swelling apart from its other advantages. Thus, these new poly(L-lactide-co-glycolide) microbraided conduits can be effective aids for nerve regeneration and repair and may lead to clinical applications.

A Nonlinear Hyperelastic Mixture Theory Model for Anisotropy, Transport, and Swelling of Annulus Fibrosus

Annals of Biomedical Engineering. Jan, 2004  |  Pubmed ID: 14964725

A precise knowledge of the local mechanical and chemical environment around the nerve endings and disc cells in the annulus fibrosus will shed insight on understanding the mechanism of low-back pain and disc degeneration. It would also present an effective tool for the studies of the intervertebral disc structure-function relationship and provide guidance to disc tissue engineering. Experimental difficulties preclude the direct and simultaneous measurement of many of the important physical quantities, such as annulus pressurization, nutrient and electrolyte transport, and mechanical and swelling deformation. Considering that many of these quantities are coupled and that the annulus is highly anisotropic, interpretation of the results would be extremely challenging without an appropriate theoretical framework. In this study, we develop a nonlinear hyperelastic fiber-reinforced continuum mixture theory model for the annulus fibrosus. Special attention is given to the anisotropic nature of the annulus. On the basis of the lamella structure of annulus, and derived from a Helmholtz energy function, a locally transversely isotropic stress-strain relation is adopted for explicit representation of the collagen fiber orientations in general finite deformation situation. The exponential form of the Helmholtz energy function naturally reduces to the infinitesimal deformation form, and the equivalence between the current model coefficients and engineering elastic constants is established under the infinitesimal deformation. This model is able to describe the anisotropic finite and infinitesimal deformation, tension-compression nonlinearity, osmotic swelling, pressurization, electrical potential and current, and water and ion transports as well as the electroneutral nutrient (or growth factor) transport within the annulus.

Water-soluble and Nonionic Polyphosphoester: Synthesis, Degradation, Biocompatibility and Enhancement of Gene Expression in Mouse Muscle

Biomacromolecules. Mar-Apr, 2004  |  Pubmed ID: 15002988

A nonionic and water-soluble polyphosphoester, poly(2-hydroxyethyl propylene phosphate) (PPE3), was synthesized by chlorination of poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane), followed by esterification with 2-benzyloxyethanol and deprotection of the hydroxyl group by catalytic hydrogenation in the presence of Pd-C. PPE3 degraded rapidly in PBS 7.4 at 37 degrees C. The cytotoxicity and tissue compatibility assays suggested good biocompatibility of PPE3 in vitro and in vivo. The expression of pVR1255 Luc plasmid in mouse muscle after intramuscular (i.m.) injection of DNA formulated with PPE3 solution in saline was enhanced up to 4-fold compared with that of naked DNA. These results suggest the potential of this polyphosphoester for naked DNA-based gene therapy. The advantages of this polymer design include the biodegradability of the polyphosphoester and its structural versatility, which allows the fine-tuning of the physicochemical properties to optimize the enhancement of gene expression in muscle.

Phase II Randomized Trial of Autologous Formalin-fixed Tumor Vaccine for Postsurgical Recurrence of Hepatocellular Carcinoma

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Mar, 2004  |  Pubmed ID: 15014006

We conducted a Phase II clinical trial with randomized patients to determine whether autologous formalin-fixed tumor vaccine (AFTV) protects against postsurgical recurrence of hepatocellular carcinoma (HCC).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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

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