Parallel Synthesis and Biophysical Characterization of a Degradable Polymer Library for Gene Delivery

Journal of the American Chemical Society. May, 2003  |  Pubmed ID: 12720443

We recently reported the parallel synthesis of 140 degradable poly(beta-amino esters) via the conjugate addition of 20 primary or secondary amine monomers to seven different diacrylate monomers. To explore possible structure/function relationships and further characterize this class of materials, we investigated the ability of each DNA-complexing polymer to overcome important cellular barriers to gene transfer. The majority of vectors were found to be uptake-limited, but complexes formed from polymers B14 and G5 displayed high levels of internalization relative to "naked" DNA (18x and 32x, respectively). Effective diameter and zeta potential measurements indicated that, in general, small particle size and positive surface charge led to higher internalization rates. Of the 10 DNA/polymer complexes with the highest uptake levels, all had effective diameters less than 250 nm and nine had positive zeta potentials. Lysosomal trafficking was investigated by measuring the pH environment of delivered DNA. Complexes prepared with polymers G5, G10, A13, B13, A14, and B14 were found to have near neutral pH measurements, suggesting that they were able to successfully avoid trafficking to acidic lysosomes. This work highlights the value of parallel synthesis and screening approaches for the discovery of new polymers for gene delivery and the elucidation of structure/function relationships for this important class of materials.

Semi-automated Synthesis and Screening of a Large Library of Degradable Cationic Polymers for Gene Delivery

Angewandte Chemie (International Ed. in English). Jul, 2003  |  Pubmed ID: 12866105

Synthesis of Poly(beta-amino Ester)s Optimized for Highly Effective Gene Delivery

Bioconjugate Chemistry. Sep-Oct, 2003  |  Pubmed ID: 13129402

Several families of synthetic polymers, including degradable poly(beta-amino ester)s, have been previously shown to effectively mediate gene transfer. However, the combined impact of potentially significant factors-such as polymer molecular weight, polymer chain end-group, and polymer/DNA ratio-on different gene transfer properties has yet to be systematically investigated. The elucidation of these relationships may aid in the design of nonviral vectors with greatly enhanced transfection properties. To examine these factors, two distinct poly(beta-amino ester) structures, Poly-1 and Poly-2, were generated by adding 1,4-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively, to 1-aminobutanol. Twelve unique versions of each structure were synthesized by varying amine/diacrylate stoichiometric ratios, resulting in polymers with either amine or acrylate end-groups and with molecular weights ranging from 3350 to 18000. Using high throughput methods, all polymers were tested in quadruplicate at nine different polymer/DNA ratios ranging from 10:1 w/w to 150:1 w/w. Through the optimization of molecular weight, polymer chain end-group, and polymer/DNA ratio, these polymers successfully mediated gene transfer at levels that surpassed both PEI and Lipofectamine 2000 in vitro.

Degradable Poly(amino Alcohol Esters) As Potential DNA Vectors with Low Cytotoxicity

Biomacromolecules. Nov-Dec, 2003  |  Pubmed ID: 14606906

The synthesis of a new degradable polymer system, poly(amino alcohol esters) and the resulting polymers' potential for use in gene transfection vectors are reported. The polymerization proceeded in a one step reaction from commercially available bis(secondary amines) monomers (N,N'-dimethyl-1,3-propanediamine and N,N'-dimethyl-1,6-hexanediamine, respectively) through nucleophilic addition to the diglycidyl ester of dicarboxylic acid (diglycidyl adipate). Poly(amino alcohol ester) 1 and 2 were synthesized with a yield of 89% and 91% with Mn = 24,800 and Mn = 36,400, respectively. Poly(amino alcohol ester) 1 degraded hydrolytically in phosphate buffer at pH 7.4 with a half-life of approximately 5 days. Both polymers readily self-assembled with plasmid DNA into nanometer-sized DNA/polymer complexes less than 180 nm diameter and are significantly less cytotoxic than the commonly used DNA delivery polymer, poly(ethylene imine) (PEI).

PH-triggered Release of Macromolecules from Spray-dried Polymethacrylate Microparticles

Pharmaceutical Research. Oct, 2003  |  Pubmed ID: 14620503

pH-triggered microparticles release their therapeutic payloads at acidic pH (e.g., in the phagosome), making intracellular drug delivery more efficient. Here we modify lipid-based microparticles that are safe and efficacious in nerve and brain and are potentially inhalable, making them pH-triggerable by incorporating an acid-soluble polymethacrylate, Eudragit E100 (E100).

Nanoliter-scale Synthesis of Arrayed Biomaterials and Application to Human Embryonic Stem Cells

Nature Biotechnology. Jul, 2004  |  Pubmed ID: 15195101

Identification of biomaterials that support appropriate cellular attachment, proliferation and gene expression patterns is critical for tissue engineering and cell therapy. Here we describe an approach for rapid, nanoliter-scale synthesis of biomaterials and characterization of their interactions with cells. We simultaneously characterize over 1,700 human embryonic stem cell-material interactions and identify a host of unexpected materials effects that offer new levels of control over human embryonic stem cell behavior.

Poly-beta Amino Ester-containing Microparticles Enhance the Activity of Nonviral Genetic Vaccines

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2004  |  Pubmed ID: 15210954

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-beta amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.

PH-triggered Microparticles for Peptide Vaccination

Journal of Immunology (Baltimore, Md. : 1950). Aug, 2004  |  Pubmed ID: 15294974

Improving vaccine delivery to human APCs is a way to increase the CTL response to vaccines. We report the use of a novel pH-triggered microparticle that exploits the ability of APCs to cross-present MHC I-restricted Ags that have been engulfed in the low pH environment of the phagosome. A model MHC class I-restricted peptide Ag from the influenza A matrix protein was encapsulated in spray-dried microparticles composed of dipalmitoylphosphatidylcholine and the pH-sensitive polymethacrylate Eudragit E100. Release of the peptide from the particle was triggered by a drop in pH to the acidity normally found in the phagosome. The particles were efficiently phagocytosed by human monocytes and dendritic cells with minimal cellular toxicity and no functional impairment. Encapsulation of the peptide in the microparticles resulted in efficient presentation of the peptide to CD8(+) T cells by human dendritic cells in vitro, and was superior to unencapsulated peptide or peptide encapsulated in an analogous pH-insensitive particle. Vaccination of human HLA-A*0201 transgenic mice with peptide encapsulated in pH-triggering microparticles resulted in priming of CTL responses. These microparticles can be modified to coencapsulate a range of adjuvants along with the Ag of interest. Encapsulation of MHC I epitopes in pH-triggered microparticles increases Ag presentation and may improve CD8(+) T cell priming to peptide vaccines against viruses and cancer.

Materials Science. Smart Biomaterials

Science (New York, N.Y.). Sep, 2004  |  Pubmed ID: 15448260

A Polymer Library Approach to Suicide Gene Therapy for Cancer

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2004  |  Pubmed ID: 15520369

Optimal gene therapy for cancer must (i) deliver DNA to tumor cells with high efficiency, (ii) induce minimal toxicity, and (iii) avoid gene expression in healthy tissues. To this end, we generated a library of >500 degradable, poly(beta-amino esters) for potential use as nonviral DNA vectors. Using high-throughput methods, we screened this library in vitro for transfection efficiency and cytotoxicity. We tested the best performing polymer, C32, in mice for toxicity and DNA delivery after intratumor and i.m. injection. C32 delivered DNA intratumorally approximately 4-fold better than one of the best commercially available reagents, jetPEI (polyethyleneimine), and 26-fold better than naked DNA. Conversely, the highest transfection levels after i.m. administration were achieved with naked DNA, followed by polyethyleneimine; transfection was rarely observed with C32. Additionally, polyethyleneimine induced significant local toxicity after i.m. injection, whereas C32 demonstrated no toxicity. Finally, we used C32 to deliver a DNA construct encoding the A chain of diphtheria toxin (DT-A) to xenografts derived from LNCaP human prostate cancer cells. This construct regulates toxin expression both at the transcriptional level by the use of a chimeric-modified enhancer/promoter sequence of the human prostate-specific antigen gene and by DNA recombination mediated by Flp recombinase. C32 delivery of the A chain of diphtheria toxin DNA to LNCaP xenografts suppressed tumor growth and even caused 40% of tumors to regress in size. Because C32 transfects tumors locally at high levels, transfects healthy muscle poorly, and displays no toxicity, it may provide a vehicle for the local treatment of cancer.

Structure/property Studies of Polymeric Gene Delivery Using a Library of Poly(beta-amino Esters)

Molecular Therapy : the Journal of the American Society of Gene Therapy. Mar, 2005  |  Pubmed ID: 15727939

Here we describe the synthesis and characterization of a library of 486 second-generation poly(beta-amino esters). To understand better the structure/property relationships governing polymeric gene delivery, we synthesized polymers with 70 different primary structures, at 6 to 12 different molecular weights, using monomers previously identified as common to effective gene delivery polymers. This library was characterized by (1) molecular weight, (2) particle size upon complexation with DNA, (3) surface charge upon complexation with DNA, (4) optimal polymer/DNA ratio, and (5) transfection efficiency. In this library, polymers with 20 of the 70 primary structures possess transfection efficiencies as good as or better than one of the best commercially available lipid reagents, Lipofectamine 2000. In general, the most effective polymers condense DNA into sub-150-nm complexes with positive surface charge. Among this group, the 2 most effective polymers condensed DNA to the smallest particle sizes (71 and 79 nm). Interestingly, the top 9 polymers were all formed from amino alcohols, and the structure of the 3 top performing polymers differs by only one carbon. This convergence in structure of the top performing polymers suggests a common mode of action and provides a framework with which future polymers can be designed.

Biomaterial Microarrays: Rapid, Microscale Screening of Polymer-cell Interaction

Biomaterials. Aug, 2005  |  Pubmed ID: 15763269

The identification of biomaterials that induce optimal gene expression patterns and allow for appropriate levels of cellular attachment is of central importance in tissue engineering and cell therapy. Herein, we describe the creation of cell-compatible, biomaterial microarrays, that allow rapid, microscale testing of biomaterial interactions with cells. As proof of principle, we simultaneously characterized over 3456 human mesenchymal stem cell (hMSC)-biomaterial composite interactions, and describe preliminary studies on the utility of these arrays with a neural stem cell line (NSC), and primary articular chondrocytes.

Beta-amino Ester Polymers Facilitate in Vivo DNA Transfection and Adjuvant Plasmid DNA Immunization

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jul, 2005  |  Pubmed ID: 15963932

Increased in vivo expression of intramuscularly delivered plasmid DNA will be essential for clinical success in gene therapy and plasmid DNA vaccination. We screened polymers from a library of beta-amino esters for their ability to augment transgene expression as measured by beta-galactosidase activity and cellular immune responses. Among the candidates identified in this screen, poly[(1,6-di(acryloxyethoxy)hexane)-co-(4-aminobutanol)] enhanced plasmid DNA transgene expression by sevenfold (P=0.0001) and its immunogenicity by 70% (P=0.03). We found that polymers with moderately hydrophobic backbones and terminal alcohol groups facilitated transfection most effectively in vivo. We also observed a log-linear correlation (R2=0.93) between peak cellular immune responses and transgene activity in all evaluated polymer-plasmid DNA formulations, clarifying the relationship between immunogenicity and the quantity of expressed antigen.

Direct Patterning of Mammalian Cells Onto Porous Tissue Engineering Substrates Using Agarose Stamps

Biomaterials. Dec, 2005  |  Pubmed ID: 15979701

This paper describes simple, inexpensive, and potentially generic methodology for generating patterns of mammalian cells on porous scaffolds for tissue engineering using replica printing. Circular patterns (diameter: 200, 700, and 1000 microm) of human osteoblasts were transferred directly from topographically patterned agarose stamps onto porous hydroxyapatite scaffolds or onto fibronectin-coated glass slides. The use of hydrogel stamps provided a "wet", biocompatible surface and maintained the viability of cells adsorbed on stamps during the patterning process. Stamps inked once with suspensions of cells allowed the repeated patterning of substrates. Direct stamping of human osteoblasts (and, potentially other mammalian cells) can be used to control the size, spacing, and geometry of patterns of cells printed on porous tissue engineering substrates. This approach may find use in controlling the spatial invasion of scaffolds, promoting the hierarchical organization of cells, and in controlling cell-cell interactions as a step in preservation of phenotypes of cells.

Biodegradable Polymeric Vectors for Gene Delivery to Human Endothelial Cells

Bioconjugate Chemistry. Sep-Oct, 2006  |  Pubmed ID: 16984124

Endothelial cells are an important cell type to both cardiovascular disease and cancer, as they play critical roles in vascular function and angiogenesis. However, effective and safe gene delivery to primary endothelial cells in the presence of serum proteins is known to be particularly challenging. A library of biodegradable poly(beta-amino esters) was synthesized for use as potential vectors. Promising vectors were optimized for high efficacy and low cytotoxicity to human umbilical vein endothelial cells (HUVECs) in serum. Vector parameters including polymer type, polymer weight, and DNA loading were varied, and biophysical properties including particle size, zeta potential, and particle stability over time were studied. While many of the poly(beta-amino ester) vectors have similar biophysical properties in the presence of buffer, their biophysical properties changed differentially in the presence of serum proteins, and the properties of these serum-interacting particles correlated to transfection efficacy. Leading poly(beta-amino ester) vectors were found to transfect HUVECs in the presence of serum significantly higher (47 +/- 9% positive, n = 10) than the best commercially available transfection reagents including jetPEI (p < 0.001) and Lipofectamine 2000 (p < 0.01). These results demonstrate the potential of a new class of biomaterials, poly(beta-amino esters), for effective human endothelial cell gene therapy.

Synthesis of Poly(beta-amino Ester)s with Thiol-reactive Side Chains for DNA Delivery

Journal of the American Chemical Society. Oct, 2006  |  Pubmed ID: 17002366

The safe and efficient delivery of DNA remains the major barrier to the clinical application of non-viral gene therapy. Here, we present novel, biodegradable polymers for gene delivery that are capable of simple graft modification and demonstrate the ability to respond to intracellular conditions. We synthesized poly(beta-amino ester)s using a new amine monomer, 2-(pyridyldithio)-ethylamine (PDA). These cationic, degradable polymers contain pyridyldithio functionalities in the side chains that react with high specificity toward thiol ligands. This reactivity is demonstrated using both mercaptoethylamine (MEA) and the thiol peptide RGDC, a ligand that binds with high affinity to certain integrin receptors. These two polymer derivatives displayed strong DNA binding as determined using electrophoresis and dye exclusion assays. In addition, the MEA-based polymer and plasmid DNA were shown to self-assemble into cationic complexes with effective diameters as low as 100 nm. Furthermore, this DNA binding ability was substantially reduced in response to intracellular glutathione concentrations, which may aid in DNA unpackaging inside the cell. These complexes also displayed low cellular toxicity and were able to mediate transfection at levels comparable to PEI in human hepatocellular carcinoma cells. These results suggest that PDA-based poly(beta-amino ester)s may serve as a modular platform for polymer-mediated gene delivery.

Electrostatic Ligand Coatings of Nanoparticles Enable Ligand-specific Gene Delivery to Human Primary Cells

Nano Letters. Apr, 2007  |  Pubmed ID: 17362046

A general method of coating polymer/DNA nanoparticles was developed. Peptide coated nanoparticles were found to have favorable biophysical characteristics including small particle size, near-neutral zeta potential, and stability in serum. At appropriate formulation conditions including near-neutral charge ratio, the coated nanoparticles enabled effective ligand-specific gene delivery to human primary endothelial cells in serum-containing media. As this nanoparticulate drug delivery system has high efficacy, ligand-based specificity, biodegradability, and low cytotoxicity, it may be potentially useful in several clinical applications.

Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(beta-amino Ester)s

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jul, 2007  |  Pubmed ID: 17375071

Poly(beta-amino ester)s are cationic degradable polymers that have significant potential as gene delivery vectors. Here we present a generalized method to modify poly(beta-amino ester)s at the chain ends to improve their delivery performance. End-chain coupling reactions were developed so that polymers could be synthesized and tested in a high-throughput manner, without the need for purification. In this way, many structural variations at the polymer terminus could be rapidly evaluated. End-modification of the terminal amine structure of a previously optimized poly(beta-amino ester), C32, significantly enhanced its in vitro transfection efficiency. In vivo, intraperitoneal (IP) gene delivery using end-modified C32 polymers resulted in expression levels over one order of magnitude higher than unmodified C32 and jet-polyethylenimine (jet-PEI) levels in several abdominal organs. The rapid end-modification strategy presented here has led to the discovery of many effective polymers for gene delivery and may be a useful method to develop and optimize cationic polymers for gene therapy.

Nanoparticulate Delivery of Suicide DNA to Murine Prostate and Prostate Tumors

The Prostate. Jun, 2007  |  Pubmed ID: 17427200

Currently available treatments for benign prostatic hyperplasia (BPH) and localized prostate cancer are generally effective but are often attended by serious side effects that impact on the quality of life. In particular, most current therapies are non-specific, with surgery, radiation, and chemical ablation having the potential to cause damage to surrounding tissue. Here, we demonstrate the effectiveness of a prostate-specific, locally delivered gene therapy for the targeted killing of prostate cells.

Effective RNAi-mediated Gene Silencing Without Interruption of the Endogenous MicroRNA Pathway

Nature. Oct, 2007  |  Pubmed ID: 17898712

Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipoprotein B and factor VII) that achieved efficient (approximately 80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA.

Gene Delivery Properties of End-modified Poly(beta-amino Ester)s

Bioconjugate Chemistry. Nov-Dec, 2007  |  Pubmed ID: 17929884

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.

TOF-SIMS Analysis of a 576 Micropatterned Copolymer Array to Reveal Surface Moieties That Control Wettability

Analytical Chemistry. Jan, 2008  |  Pubmed ID: 18044847

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used in a high-throughput fashion to obtain mass spectra from the surfaces of 576 novel acrylate-based polymers, synthesized using a combinatorial approach and in a micropatterned format. To identify variations in surface chemistry within the library, principal component analysis (PCA) was used. PCA clearly identified surface chemical commonality and differences within the library. The TOF-SIMS spectra were also used to determine the relationship between water contact angle (WCA) and the surface chemistry of the polymer library using partial least-squares regression (PLS). A good correlation between the TOF-SIMS data from the novel polymers and water contact angle was obtained. Examination of the PLS regression vector allowed surface moieties that correlate with high and low WCA to be identified. This in turn provided an insight into molecular structures that significantly influence wettability. This study demonstrates that multivariate analysis can be successfully applied to TOF-SIMS data from a large library of samples and highlights the potential of these techniques for building complex surface property/chemistry models.

Nanomechanical Control of Cell Rolling in Two Dimensions Through Surface Patterning of Receptors

Nano Letters. Apr, 2008  |  Pubmed ID: 18321075

We envisioned that label-free control of the transport of cells in two dimensions through receptor-ligand interactions would enable simple separation systems that are easy to implement yet retain the specificity of receptor-ligand interactions. Here we demonstrate nanomechanical control of cell transport in two dimensions via transient receptor-ligand adhesive bonds by patterning of receptors that direct cell rolling through an edge effect. HL-60 cells rolling on P-selectin receptor patterns were deflected at angles of 5-10 degrees with respect to their direction of travel. Absence of this effect in the case of rigid microsphere models of cell rolling suggests that this two-dimensional motion depends on nanomechanical properties of the rolling cell. This work suggests the feasibility of simple continuous-flow microfluidic cell separation systems that minimize processing steps and yet retain the specificity of receptor-ligand interactions.

A Combinatorial Library of Lipid-like Materials for Delivery of RNAi Therapeutics

Nature Biotechnology. May, 2008  |  Pubmed ID: 18438401

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

Heme Oxygenase-1 is an Anti-inflammatory Host Factor That Promotes Murine Plasmodium Liver Infection

Cell Host & Microbe. May, 2008  |  Pubmed ID: 18474360

The clinically silent Plasmodium liver stage is an obligatory step in the establishment of malaria infection and disease. We report here that expression of heme oxygenase-1 (HO-1, encoded by Hmox1) is upregulated in the liver following infection by Plasmodium berghei and Plasmodium yoelii sporozoites. HO-1 overexpression in the liver leads to a proportional increase in parasite liver load, and treatment of mice with carbon monoxide and with biliverdin, each an enzymatic product of HO-1, also increases parasite liver load. Conversely, mice lacking Hmox1 completely resolve the infection. In the absence of HO-1, the levels of inflammatory cytokines involved in the control of liver infection are increased. These findings suggest that, while stimulating inflammation, the liver stage of Plasmodium also induces HO-1 expression, which modulates the host inflammatory response, protecting the infected hepatocytes and promoting the liver stage of infection.

A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery

Accounts of Chemical Research. May, 2008  |  Pubmed ID: 18507402

The potential of gene therapy to benefit human health is tremendous because almost all human diseases have a genetic component, from untreatable monogenic disorders to cancer and heart disease. Unfortunately, a method for gene therapy that is both effective and safe has remained elusive. It has been said that "there are only three problems in gene therapy - delivery, delivery, and delivery." (quote from I. M. Verma in Jaroff, L. TIME, 1999; Jan 11). This Account describes an alternative strategy to viral gene delivery: the design of biodegradable polymers that are able to deliver DNA like a synthetic virus. Using high-throughput synthesis and screening techniques, we have created libraries of over 2000 structurally unique poly(beta-amino esters) (PBAEs). PBAEs are formed by the conjugate addition of amines to diacrylates. These biomaterials are promising for nonviral gene delivery due to their ability to condense plasmid DNA into small and stable nanoparticles and their ability to promote cellular uptake and endosomal escape. Our laboratory has iteratively improved PBAE nanoparticles through polymer end modifications and nanoparticle coatings. Lead PBAEs have high gene delivery efficacy and low cytotoxicity both in vitro and in vivo. Certain polymer structural characteristics are important for effective gene delivery. The best PBAEs are linear polymers of approximately 10 kDa that contain hydroxyl side chains and primary amine end groups. These polymers bind DNA to form nanoparticles that are small (<200 nm) and stable and have near-neutral zeta potential in the presence of serum-containing media. Lead PBAEs also contain tertiary amines that can buffer the low pH environment of endosomes and facilitate escape of polymer/DNA particles into the cytoplasm. Diamine end-modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers (C32) bind DNA more tightly and form smaller nanoparticles than other PBAEs. These nanoparticles also have higher cellular uptake and the best gene expression of all gene delivery polymers in the library. These polymers are more effective for gene delivery than top commercially available nonviral vectors including jet-PEI and Lipofectamine 2000 and are comparable to adenovirus for in vitro gene delivery to human primary cells. In vivo, these PBAE/DNA particles are promising as cancer therapeutics. This Account summarizes the results of our laboratory in using a combinatorial polymer library approach to elucidate polymer structure/function relationships and enable the development of polymeric gene delivery nanoparticles with viral-like efficacy.

Nanoparticle Delivery of Suicide DNA for Epithelial Ovarian Cancer Therapy

Advances in Experimental Medicine and Biology. 2008  |  Pubmed ID: 18546630

Intraperitoneal administration of polymeric nanoparticles to deliver DNA encoding suicide genes holds much promise as an effective therapy for advanced epithelial ovarian cancer. Poly(beta-amino ester)s, a class of cationic, biodegradable polymers complex to DNA to form nanoparticles that deliver DNA to cells in ovarian tumors. Modifications to poly(beta-amino ester)s can improve both the efficiency and specificity with which DNA is delivered to tumor cells. Preclinical studies to test therapeutic efficacy of gene therapy strategies that are under development make use of mouse models for epithelial ovarian cancer and new imaging technologies.

Therapeutic RNAi Targeting PCSK9 Acutely Lowers Plasma Cholesterol in Rodents and LDL Cholesterol in Nonhuman Primates

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2008  |  Pubmed ID: 18695239

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

Nanoparticles for Gene Transfer to Human Embryonic Stem Cell Colonies

Nano Letters. Oct, 2008  |  Pubmed ID: 18754690

We develop biodegradable polymeric nanoparticles to facilitate nonviral gene transfer to human embryonic stem cells (hESCs). Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA. By varying the end group of the polymer, we can tune the biophysical properties of the resulting nanoparticles and their gene-delivery efficacy. We created an OCT4-driven GFP hES cell line to allow the rapid identification of nanoparticles that facilitate gene transfer while maintaining an hESC undifferentiated state. Using this cell system, we synthesized nanoparticles that have gene delivery efficacy that is up to 4 times higher than that of the leading commercially available transfection agent, Lipofectamine 2000. Importantly, these materials have minimal toxicity and do not adversely affect hESC colony morphology or cause nonspecific differentiation.

Delivery of Small Interfering RNA for Inhibition of Endothelial Cell Apoptosis by Hypoxia and Serum Deprivation

Biochemical and Biophysical Research Communications. Nov, 2008  |  Pubmed ID: 18765230

RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). siRNA was designed and synthesized targeting tumor necrosis factor-alpha receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-alpha expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-alpha expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia.

Assessing SiRNA Pharmacodynamics in a Luciferase-expressing Mouse

Molecular Therapy : the Journal of the American Society of Gene Therapy. Dec, 2008  |  Pubmed ID: 18781145

A significant barrier to the successful general development of small-interfering RNA (siRNA) therapeutics is the ability to deliver them systemically to target organs and cell types. In this study, we have developed a mouse strain that will facilitate the evaluation of the efficacy of siRNA delivery strategies. This strain contains robust ubiquitous expression of firefly luciferase from germ line Cre-mediated recombination of the ROSA26-LSL-Luc allele. We show that luciferase is highly and uniformly expressed in all tissues examined. Using this mouse model, we describe a facile assay that enables the assessment of the pharmacodynamics of a systemically delivered siRNA formulation. These mice can also be used as universal donors, enabling the efficient and sensitive monitoring of cell trafficking or tissue transplantation. The primary advantage of this approach is that siRNA efficacy against a nonessential target can be easily evaluated in any tissue. This strain should generally enhance the ability to rapidly screen, compare and optimize various siRNA formulations for tissue-targeted or -enhanced systemic delivery in a preclinical development setting.

Cell-compatible, Multicomponent Protein Arrays with Subcellular Feature Resolution

Small (Weinheim an Der Bergstrasse, Germany). Oct, 2008  |  Pubmed ID: 18844310

Nanoparticulate Delivery of Diphtheria Toxin DNA Effectively Kills Mesothelin Expressing Pancreatic Cancer Cells

Cancer Biology & Therapy. Oct, 2008  |  Pubmed ID: 19039293

Pancreatic cancer is the fourth leading cause of cancer-related deaths in this country, and there is currently no effective targeted treatment for this deadly disease. A dire need exists to rapidly translate our molecular understanding of this devastating disease into effective, novel therapeutic options. Mesothelin is a candidate target protein shown by a number of laboratories to be specifically overexpressed in pancreatic cancers and not in the adjacent normal tissue. Translational investigations have shown promising results using this molecule as a therapeutic target (e.g., vaccine strategies). In addition, the mesothelin promoter has been cloned and dissected and can therefore be used as a vehicle for regulating expression of DNA sequences. Using a novel, proven, biodegradable nanoparticulate system, we sought to target mesothelin-expressing pancreatic cancer cells with a potent suicide gene, diphtheria toxin-A (DT-A). We first confirmed reports that a majority of pancreatic cancer cell lines and resected pancreatic ductal adenocarcinoma specimens overexpressed mesothelin at the mRNA and protein levels. High mesothelin-expressing pancreatic cancer cell lines produced more luciferase than cell lines with undetectable mesothelin expression when transfected with a luciferase sequence under the regulation of the mesothelin promoter. We achieved dramatic inhibition of protein translation (>95%) in mesothelin-expressing pancreatic cancer cell lines when DT-A DNA, driven by the mesothelin promoter, was delivered to pancreatic cancer cells. We show that this inhibition effectively targets the death of pancreatic cancer cells that overexpress mesothelin. The work presented here provides evidence that this strategy will work in pre-clinical mouse pancreatic cancer models, and suggests that such a strategy will work in the clinical setting against the majority of pancreatic tumors, most of which overexpress mesothelin.

Poly(beta-amino Esters): Procedures for Synthesis and Gene Delivery

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19085119

Non-viral gene delivery systems are promising as they avoid many problems of viral gene therapy by having increased design flexibility, high safety, large DNA cargo capacity, and ease of manufacture. Here, we describe the use of polymeric vectors, in particular biodegradable poly(beta-amino esters) (PBAEs), for non-viral gene delivery. These polymers are able to self-assemble with DNA and form positively charged gene delivery nanoparticles. Methods for synthesis of these polymers, particle self-assembly, and transfection using these particles are delineated. A standard protocol is presented as well as a high-throughput screening technique that can be used to more quickly optimize transfection parameters for efficient delivery.

Knocking Down Barriers: Advances in SiRNA Delivery

Nature Reviews. Drug Discovery. Feb, 2009  |  Pubmed ID: 19180106

In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids.

Claudin-3 Gene Silencing with SiRNA Suppresses Ovarian Tumor Growth and Metastasis

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2009  |  Pubmed ID: 19208807

Claudin-3 (CLDN3) is a tight junction protein that is overexpressed in 90% of ovarian tumors. Previous in vitro studies have indicated that CLDN3 overexpression promotes the migration, invasion, and survival of ovarian cancer cells. Here, we investigated the efficacy of lipidoid-formulated CLDN3 siRNA in 3 different ovarian cancer models. Intratumoral injection of lipidoid/CLDN3 siRNA into OVCAR-3 xenografts resulted in dramatic silencing of CLDN3, significant reduction in cell proliferation, reduction in tumor growth, and a significant increase in the number of apoptotic cells. Intraperitoneal injection of lipidoid-formulated CLDN3 siRNA resulted in a substantial reduction in tumor burden in MISIIR/TAg transgenic mice and mice bearing tumors derived from mouse ovarian surface epithelial cells. Ascites development was reduced in CLDN3 siRNA-treated mice, suggesting the treatment effectively suppressed metastasis. Toxicity was not observed after multiple i.p. injections. Importantly, treatment of mice with nonimmunostimulatory 2'-OMe modified CLDN3 siRNA was as effective in suppressing tumor growth as unmodifed siRNA. These results suggest that lipidoid-formulated CLDN3 siRNA has potential as a therapeutic for ovarian cancer.

Development of Lipidoid-siRNA Formulations for Systemic Delivery to the Liver

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2009  |  Pubmed ID: 19259063

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

Preparation of Monodisperse Biodegradable Polymer Microparticles Using a Microfluidic Flow-focusing Device for Controlled Drug Delivery

Small (Weinheim an Der Bergstrasse, Germany). Jul, 2009  |  Pubmed ID: 19296563

Degradable microparticles have broad utility as vehicles for drug delivery and form the basis of several therapies approved by the US Food and Drug Administration. Conventional emulsion-based methods of manufacturing produce particles with a wide range of diameters (and thus kinetics of release) in each batch. This paper describes the fabrication of monodisperse, drug-loaded microparticles from biodegradable polymers using the microfluidic flow-focusing (FF) devices and the drug-delivery properties of those particles. Particles are engineered with defined sizes, ranging from 10 microm to 50 microm. These particles are nearly monodisperse (polydispersity index = 3.9%). A model amphiphilic drug (bupivacaine) is incorporated within the biodegradable matrix of the particles. Kinetic analysis shows that the release of the drug from these monodisperse particles is slower than that from conventional methods of the same average size but a broader distribution of sizes and, most importantly, exhibit a significantly lower initial burst than that observed with conventional particles. The difference in the initial kinetics of drug release is attributed to the uniform distribution of the drug inside the particles generated using the microfluidic methods. These results demonstrate the utility of microfluidic FF for the generation of homogenous systems of particles for the delivery of drugs.

A Novel High-throughput Cell-based Method for Integrated Quantification of Type I Interferons and in Vitro Screening of Immunostimulatory RNA Drug Delivery

Biotechnology and Bioengineering. Jul, 2009  |  Pubmed ID: 19338049

A hallmark of immune activation by certain RNA sequences is the generation of interferon responses. However, the study of immunostimulatory RNA (isRNA) has been hindered by costly and slow methods, particularly in vitro. We have developed a cell-based assay to detect human type I interferon (IFN) that reliably senses both IFN-alpha and IFN-beta simultaneously. The human 293T cell line was stably transfected with a fusion gene of monomeric red fluorescent protein (mRFP) under the transcriptional control of an interferon-stimulated response element (ISRE). High levels of mRFP are expressed following activation of the type I IFN receptor (IFNAR). Using this method, detection limits for IFN similar to that of ELISA can be achieved but with a greater dynamic range and in a high-throughput manner. As a proof of concept, we utilized this method to screen a library of cationic lipid-like materials that form nanoparticle complexes with RNA for induction of innate immune responses in vitro. We expect the screening and detection methods described herein may provide a useful tool in elucidating mechanisms that govern the delivery of RNA molecules to effector cells and receptors of the innate immune system. We apply this tool to investigate isRNA drug delivery, but it may also find use in other applications for which high-throughput detection of type 1 IFN is desired.

Gold, Poly(beta-amino Ester) Nanoparticles for Small Interfering RNA Delivery

Nano Letters. Jun, 2009  |  Pubmed ID: 19422265

The safe and effective delivery of RNA therapeutics remains the major barrier to their broad clinical application. Here we develop a new nanoparticulate delivery system based on inorganic particles and biodegradable polycations. First, gold nanoparticles were modified with the hydrophilic polymer poly(ethylene glycol) (PEG), and then small interfering RNA (siRNA) was conjugated to the nanoparticles via biodegradable disulfide linkages, with approximately 30 strands of siRNA per nanoparticle. The particles were then coated with a library of end-modified poly(beta-amino ester)s (PBAEs), previously identified as capable of facilitating intracellular DNA delivery. Nanoparticulate formulations developed here facilitate high levels of in vitro siRNA delivery, facilitating delivery as good or better than the commercially available lipid reagent, Lipofectamine 2000.

Locally Delivered Growth Factor Enhances the Angiogenic Efficacy of Adipose-derived Stromal Cells Transplanted to Ischemic Limbs

Stem Cells (Dayton, Ohio). Aug, 2009  |  Pubmed ID: 19544425

Ischemia is a potentially fatal medical event that is associated with as many as 30% of all deaths. Stem cell therapy offers significant therapeutic promise, but poor survival following transplantation to ischemic tissue limits its efficacy. Here we demonstrate that nanosphere-mediated growth factor delivery can enhance the survival of transplanted human adipose-derived stromal cells (hADSCs) and secretion of human angiogenic growth factors per cell, and substantially improve therapeutic efficacy of hADSCs. In vitro, in hypoxic (1% oxygen) and serum-deprived conditions that simulate in vivo ischemia, fibroblast growth factor-2 (FGF2) significantly reduced hADSC apoptosis and enhanced angiogenic growth factor secretion. In vivo, hADSCs delivered intramuscularly into ischemic hind limbs in combination with FGF2 resulted in significant improvements in limb survival and blood perfusion, as well as survival of the transplanted hADSCs and secretion of human angiogenic growth factors (i.e., vascular endothelial growth factor, hepatocyte growth factor, and FGF2). Interestingly, the majority of transplanted hADSCs were localized adjacent to the microvessels rather than being incorporated into them, suggesting that their major contribution to angiogenesis might be to increase paracrine secretion of angiogenic growth factors. This study demonstrates the potential of hADSCs in combination with growth factors for use in the treatment of ischemia.

High-throughput Membrane Surface Modification to Control NOM Fouling

Environmental Science & Technology. May, 2009  |  Pubmed ID: 19544900

A novel method for synthesis and screening of fouling-resistant membrane surfaces was developed by combining a high-throughput platform (HTP) approach together with photoinduced graft polymerization (PGP)forfacile modification of commercial poly(aryl sulfone) membranes. This method is an inexpensive, fast, simple, reproducible, and scalable approach to identify fouling-resistant surfaces appropriate for a specific feed. In this research, natural organic matter (NOM)-resistant surfaces were synthesized and indentified from a library of 66 monomers. Surfaces were prepared via graft polymerization onto poly(ether sulfone) (PES) membranes and were evaluated using an assay involving NOM adsorption, followed by pressure-driven-filtration. In this work new and previously tested low-fouling surfaces for NOM are identified, and their ability to mitigate NOM and protein (bovine serum albumin)fouling is compared. The best-performing monomers were the zwitterion [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, and diacetone acrylamide, a neutral monomer containing an amide group. Other excellent surfaces were synthesized from amides, amines, basic monomers, and long-chain poly(ethylene) glycols. Bench-scale studies conducted for selected monomers verified the scalability of HTP-PGP results. The results and the synthesis and screening method presented here offer new opportunities for choosing new membrane chemistries that minimize NOM fouling.

Drug Delivery-mediated Control of RNA Immunostimulation

Molecular Therapy : the Journal of the American Society of Gene Therapy. Sep, 2009  |  Pubmed ID: 19584813

RNA interference (RNAi) has generated significant interest as a strategy to suppress viral infection, but in some cases antiviral activity of unmodified short-interfering RNA (siRNA) has been attributed to activation of innate immune responses. We hypothesized that immunostimulation by unmodified siRNA could mediate both RNAi as well as innate immune stimulation depending on the mode of drug delivery. We investigated the potential of immunostimulatory RNAs (isRNAs) to suppress influenza A virus in vivo in the mouse lung. Lipidoid 98N12-5(1) formulated with unmodified siRNA targeting the influenza nucleoprotein gene exhibited antiviral activity. Formulations were optimized to increase antiviral activity, but the antiviral activity of lipidoid-delivered siRNA did not depend on sequence homology to the influenza genome as siRNA directed against unrelated targets also suppressed influenza replication in vivo. This activity was primarily attributed to enhancement of innate immune stimulation by lipidoid-mediated delivery, which indicates increased toll-like receptor (TLR) activation by siRNA. Certain chemical modifications to the siRNA backbone, which block TLR7/8 activation but retain in vitro RNAi activity, prevented siRNA-mediated antiviral activity despite enhanced lipidoid-mediated delivery. Here, we demonstrate that innate immune activation caused by unmodified siRNA can have therapeutically relevant effects, and that these non-RNAi effects can be controlled through chemical modifications and drug delivery.

Minicells Overcome Tumor Drug-resistance

Nature Biotechnology. Jul, 2009  |  Pubmed ID: 19587665

High Throughput Optimization of Stem Cell Microenvironments

Combinatorial Chemistry & High Throughput Screening. Jul, 2009  |  Pubmed ID: 19601753

Stem cells have great potential as cell sources for regenerative medicine due to both their self-renewal and multi-lineage differentiation capacity. Despite advances in the field of stem cell biology, major challenges remain before stem cells can be widely used for therapeutic purposes. One challenge is to develop reproducible methods to control stem cell growth and differentiation. The niche in which stem cells reside is a complex, multi-factorial environment. In contrast to using cells alone, biomaterials can provide initial structural support, and allow cells to adhere, proliferate and differentiate in a three-dimensional environment. Researchers have incorporated signals into the biomaterials that can promote desired cell functions in a spatially and temporally controlled manner. Despite progress in biomaterial design and methods to modulate cellular behavior, many of the complex signal networks that regulate cell-material interactions remain unclear. Due to the vast numbers of material properties to be explored and the complexity of cell-surface interactions, it is often difficult to optimize stem cell microenvironments using conventional, iterative approaches. To address these challenges, high throughput screening of combinatorial libraries has emerged as a novel approach to achieve rapid screening with reduced materials and costs. In this review, we discuss recent research in the area of high throughput approaches for characterization and optimization of cellular interactions with their microenvironments. In contrast to conventional approaches, screening combinatorial libraries can result in the discovery of unexpected material solutions to these complex problems.

Nanoparticle-delivered Suicide Gene Therapy Effectively Reduces Ovarian Tumor Burden in Mice

Cancer Research. Aug, 2009  |  Pubmed ID: 19643734

There is currently no effective therapy for patients with advanced ovarian cancer. To address the need for a more effective treatment for this deadly disease, we conducted preclinical tests in ovarian tumor-bearing mice to evaluate the therapeutic efficacy of using a cationic biodegradable poly(beta-amino ester) polymer as a vector for nanoparticulate delivery of DNA encoding a diphtheria toxin suicide protein (DT-A). The promoter sequences of two genes that are highly active in ovarian tumor cells, MSLN and HE4, were used to target DT-A expression to tumor cells. Administration of DT-A nanoparticles directly to s.c. xenograft tumors and to the peritoneal cavity of mice bearing primary and metastatic ovarian tumors resulted in a significant reduction in tumor mass and a prolonged life span compared to control mice. Minimal nonspecific tissue and blood chemistry toxicity was observed following extended treatment with nanoparticles. DT-A nanoparticle therapy suppressed tumor growth more effectively than treatment with clinically relevant doses of cisplatin and paclitaxel. Our findings suggest that i.p. administration of polymeric nanoparticles to deliver DT-A encoding DNA, combined with transcriptional regulation to target gene expression to ovarian tumor cells, holds promise as an effective therapy for advanced-stage ovarian cancer.

Microfabrication of Homogenous, Asymmetric Cell-laden Hydrogel Capsules

Biomaterials. Dec, 2009  |  Pubmed ID: 19800116

Cell encapsulation has been broadly investigated as a technology to provide immunoprotection for transplanted endocrine cells. Here we develop a new fabrication method that allows for rapid, homogenous microencapsulation of insulin-secreting cells with varying microscale geometries and asymmetrically modified surfaces. Micromolding systems were developed using polypropylene mesh, and the material/surface properties associated with efficient encapsulation were identified. Cells encapsulated using these methods maintain desirable viability and preserve their ability to proliferate and secrete insulin in a glucose-responsive manner. This new cell encapsulation approach enables a practical route to an inexpensive and convenient process for the generation of cell-laden microcapsules without requiring any specialized equipment or microfabrication process.

Nanoparticle-delivered Multimeric Soluble CD40L DNA Combined with Toll-Like Receptor Agonists As a Treatment for Melanoma

PloS One. 2009  |  Pubmed ID: 19812695

Stimulation of CD40 or Toll-Like Receptors (TLR) has potential for tumor immunotherapy. Combinations of CD40 and TLR stimulation can be synergistic, resulting in even stronger dendritic cell (DC) and CD8+ T cell responses. To evaluate such combinations, established B16F10 melanoma tumors were injected every other day X 5 with plasmid DNA encoding a multimeric, soluble form of CD40L (pSP-D-CD40L) either alone or combined with an agonist for TLR1/2 (Pam(3)CSK(4) ), TLR2/6 (FSL-1 and MALP2), TLR3 (polyinosinic-polycytidylic acid, poly(I:C)), TLR4 ( monophosphoryl lipid A, MPL), TLR7 (imiquimod), or TLR9 (Class B CpG phosphorothioate oligodeoxynucleotide, CpG). When used by itself, pSP-D-CD40L slowed tumor growth and prolonged survival, but did not lead to cure. Of the TLR agonists, CpG and poly(I:C) also slowed tumor growth, and the combination of these two TLR agonists was more effective than either agent alone. The triple combination of intratumoral pSP-D-CD40L + CpG + poly(I:C) markedly slowed tumor growth and prolonged survival. This treatment was associated with a reduction in intratumoral CD11c+ dendritic cells and an influx of CD8+ T cells. Since intratumoral injection of plasmid DNA does not lead to efficient transgene expression, pSP-D-CD40L was also tested with cationic polymers that form DNA-containing nanoparticles which lead to enhanced intratumoral gene expression. Intratumoral injections of pSP-D-CD40L-containing nanoparticles formed from polyethylenimine (PEI) or C32 (a novel biodegradable poly(B-amino esters) polymer) in combination with CpG + poly(I:C) had dramatic antitumor effects and frequently cured mice of B16F10 tumors. These data confirm and extend previous reports that CD40 and TLR agonists are synergistic and demonstrate that this combination of immunostimulants can significantly suppress tumor growth in mice. In addition, the enhanced effectiveness of nanoparticle formulations of DNA encoding immunostimulatory molecules such as multimeric, soluble CD40L supports the further study of this technology for tumor immunotherapy.

High Throughput Methods Applied in Biomaterial Development and Discovery

Biomaterials. Jan, 2010  |  Pubmed ID: 19815273

The high throughput discovery of new bio materials can be achieved by rapidly screening many different materials synthesised by a combinatorial approach to identify the optimal composition that fulfils a particular biomedical application. Here we review the literature in this area and conclude that for polymers this process is best achieved in a microarray format, which enable thousands of cell-material interactions to be monitored on a single chip. Polymer microarrays can be formed by printing pre-synthesised polymers or by printing monomers onto the chip where on-slide polymerisation is initiated. The surface properties of the material can be analysed and correlated to the biological performance using high throughput surface analysis, including time-of-flight secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements. This approach enables the surface properties responsible for the success of a material to be understood, which in turn provides the foundations of future material design. The high throughput discovery of materials using polymer microarrays has been explored for many cell-based applications including the isolation of specific cells from heterogeneous populations, the attachment and differentiation of stem cells and the controlled transfection of cells. Further development of polymerisation techniques and high throughput biological assays amenable to the polymer microarray format will broaden the combinatorial space and biological phenomenon that polymer microarrays can explore, and increase their efficacy. This will, in turn, facilitate the discovery of optimised polymeric materials for many biomaterial applications.

Tissue-specific Gene Delivery Via Nanoparticle Coating

Biomaterials. Feb, 2010  |  Pubmed ID: 19850333

The use of biomaterials for gene delivery can potentially avoid many of the safety concerns with viral gene delivery. However, the efficacy of polymeric gene delivery methods is low, particularly in vivo. One significant concern is that the interior and exterior composition of polymeric gene delivery nanoparticles are often coupled, with a single polymer backbone governing all functions from biophysical properties of the polymer/DNA particle to DNA condensation and release. In this work we develop electrostatically adsorbed poly(glutamic acid)-based peptide coatings to alter the exterior composition of a core gene delivery particle and thereby affect tissue-specificity of gene delivery function in vivo. We find that with all coating formulations tested, the coatings reduce potential toxicity associated with uncoated cationic gene delivery nanoparticles following systemic injection. Particles coated with a low 2.5:1 peptide:DNA weight ratio (w/w) form large 2 micro sized particles in the presence of serum that can facilitate specific gene delivery to the liver. The same particles coated at a higher 20:1w/w form small 200nm particles in the presence of serum that can facilitate specific gene delivery to the spleen and bone marrow. Thus, variations in nanoparticle peptide coating density can alter the tissue-specificity of gene delivery in vivo.

Lipid-like Materials for Low-dose, in Vivo Gene Silencing

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2010  |  Pubmed ID: 20080679

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.

Nanoparticulate Cellular Patches for Cell-mediated Tumoritropic Delivery

ACS Nano. Feb, 2010  |  Pubmed ID: 20121215

The targeted delivery of therapeutics to tumors remains an important challenge in cancer nanomedicine. Attaching nanoparticles to cells that have tumoritropic migratory properties is a promising modality to address this challenge. Here we describe a technique to create nanoparticulate cellular patches that remain attached to the membrane of cells for up to 2 days. NeutrAvidin-coated nanoparticles were anchored on cells possessing biotinylated plasma membrane. Human bone marrow derived mesenchymal stem cells with nanoparticulate patches retained their inherent tumoritropic properties as shown using a tumor model in a 3D extracellular matrix. Additionally, human umbilical vein endothelial cells with nanoparticulate patches were able to retain their functional properties and form multicellular structures as rapidly as unmodified endothelial cells. These results provide a novel strategy to actively deliver nanostructures and therapeutics to tumors utilizing stem cells as carriers and also suggest that nanoparticulate cellular patches may have applications in tissue regeneration.

Electrostatic Surface Modifications to Improve Gene Delivery

Expert Opinion on Drug Delivery. Apr, 2010  |  Pubmed ID: 20201712

Gene therapy has the potential to treat a wide variety of diseases, including genetic diseases and cancer.

Rapid Biocompatibility Analysis of Materials Via in Vivo Fluorescence Imaging of Mouse Models

PloS One. 2010  |  Pubmed ID: 20386609

Many materials are unsuitable for medical use because of poor biocompatibility. Recently, advances in the high throughput synthesis of biomaterials has significantly increased the number of potential biomaterials, however current biocompatibility analysis methods are slow and require histological analysis.

Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D

Biomacromolecules. Aug, 2010  |  Pubmed ID: 20614932

Embryonic stem cells (ESCs) are promising cell sources for tissue engineering and regenerative medicine. Scaffolds for ESC-based tissue regeneration should provide not only structural support, but also signals capable of supporting appropriate cell differentiation and tissue development. Extracellular matrix (ECM) is a key component of the stem cell niche in vivo and can influence stem cell fate via mediating cell attachment and migration, presenting chemical and physical cues, as well as binding soluble factors. Here we investigated the effects of combinatorial extracellular matrix proteins on controlled human ESC (hESC) differentiation. Varying ECM compositions in 3D markedly affects cell behavior, and optimal compositions of ECM hydrogels are identified that facilitate specific-lineage differentiation of stem cells. To our knowledge, this is the first combinatorial analysis of ECM hydrogels for their effects on hESC differentiation in 3D. The 3D matrices described herein may provide a useful platform for studying the interactive ECM signaling in influencing stem cell differentiation.

Combinatorial Approach to Determine Functional Group Effects on Lipidoid-mediated SiRNA Delivery

Bioconjugate Chemistry. Aug, 2010  |  Pubmed ID: 20715849

The application of RNA interference (RNAi), either in the clinic or in the laboratory, requires safe and effective delivery methods. Here, we develop a combinatorial approach to synthesize a library of delivery vectors based on two lipid-like substrates with known siRNA delivery capabilities. Members of this library have a mixture of lipid-like tails and feature appendages containing hydroxyl, carbamate, ether, or amine functional groups as well as variations in alkyl chain length and branching. Using a luciferase reporter system in HeLa cells, we studied the relationship between lipid chemical modification and delivery performance in vitro. The impact of the functional group was shown to vary depending on the overall amine content and tail number of the delivery vector. Additionally, in vivo performance was evaluated using a Factor VII knockdown assay. Two library members, each containing ether groups, were found to knock down the target protein at levels comparable to those of the parent delivery vector. These results demonstrate that small chemical changes to the delivery vector impact knockdown efficiency and cell viability both in vitro and in vivo. The work described here identifies new materials for siRNA delivery and provides new insight into the parameters for optimized chemical makeup of lipid-like siRNA delivery materials.

Combinatorial Development of Biomaterials for Clonal Growth of Human Pluripotent Stem Cells

Nature Materials. Sep, 2010  |  Pubmed ID: 20729850

Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content, have a moderate wettability and employ integrin alpha(v)beta(3) and alpha(v)beta(5) engagement with adsorbed vitronectin to promote colony formation. The structure-function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.

CanScript, an 18-Base Pair DNA Sequence, Boosts Tumor Cell-specific Promoter Activity: Implications for Targeted Gene Therapy

Cancer Biology & Therapy. Nov, 2010  |  Pubmed ID: 20798601

Gene therapy protocols for the treatment of cancer often employ gene promoter sequences that are known to be over-expressed in specific tumor cell types relative to normal cells. These promoters, while specific, are often weakly active. It would be desirable to increase the activity of such promoters, while at the same time retain specificity, so that the therapeutic gene is more robustly expressed. Using a luciferase reporter DNA construct in both in vitro cell transfection assays and in vivo mouse tumor models, we have determined that in the absence of any other DNA sequence, a previously identified 18-base pair enhancer sequence called CanScript, lying upstream of the MSLN gene, has ~25% of the promoter activity of CAG, a very strong non-specific promoter/enhancer, in tumor cells in which MSLN is highly expressed. Furthermore, tandem repeat copies of CanScript enhance transcription in a dose-dependent manner and, when coupled with promoter sequences that are active in tumor cells, increase promoter activity. These findings suggest that the incorporation of CanScript into gene constructs may have application in enhancing activity of promoters used in cancer-targeting gene therapy strategies, thereby improving therapeutic efficacy.

A High Throughput Micro-array System of Polymer Surfaces for the Manipulation of Primary Pancreatic Islet Cells

Biomaterials. Dec, 2010  |  Pubmed ID: 20828808

We developed a high throughput micro-arrayed polymer system for the study of polymer surfaces for islet cell culture. A micro-arrayed library with 496 different polymers was synthesized and used to examine attachment and insulin expression of islet cells. While most polymers were not supportive, several related polymers were identified as suitable ("hit's"). The "hit" arrays composed of "hit" polymers with 36 replicates were fabricated to confirm their capacities to support the attachment of islet cells, and these capacities were further validated in large surfaces. Notably, the attachment of islet cells on these synthetic polymeric films has been found to be as supportive as 804G supernatant coated tissue culture polystyrene dishes, one of the most extensively used substrates for the islet cell attachment. Interestingly, the polymeric surfaces optimal for a different cell type, hES derived cells, were distinct, highlighting the utility of these approaches for identifying cell type specific surfaces.

Polymer Surface Functionalities That Control Human Embryoid Body Cell Adhesion Revealed by High Throughput Surface Characterization of Combinatorial Material Microarrays

Biomaterials. Dec, 2010  |  Pubmed ID: 20832108

High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterization (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates the ability to identify surface moieties that control protein adsorption and subsequent cell adhesion using ToF SIMS and multivariate analysis.

High Throughput Surface Characterization: A Review of a New Tool for Screening Prospective Biomedical Material Arrays

Journal of Drug Targeting. Dec, 2010  |  Pubmed ID: 20945971

The application of high throughput surface characterization (HTSC) to the analysis of polymeric biomaterial libraries is an important advancement for the discovery and development of new biomedical materials and is the focus of this review. The potential for HTSC to identify structure/activity relationships for large libraries of materials can be utilized to accelerate materials discovery as well as providing insight into the underlying biological-material interactions. Furthermore, the correlations identified between surface chemical structure and cellular behavior could not have been predicted by a rational design approach based simply on review of bulk structure, which demonstrates the importance of HTSC in the assessment of cell-material and cell-biomolecular interactions that are dependent on surface properties.

DNA-controlled Assembly of a NaTl Lattice Structure from Gold Nanoparticles and Protein Nanoparticles

Nature Materials. Nov, 2010  |  Pubmed ID: 20953184

The formation of diamond structures from tailorable building blocks is an important goal in colloidal crystallization because the non-compact diamond lattice is an essential component of photonic crystals for the visible-light range. However, designing nanoparticle systems that self-assemble into non-compact structures has proved difficult. Although several methods have been proposed, single-component nanoparticle assembly of a diamond structure has not been reported. Binary systems, in which at least one component is arranged in a diamond lattice, provide alternatives, but control of interparticle interactions is critical to this approach. DNA has been used for this purpose in a number of systems. Here we show the creation of a non-compact lattice by DNA-programmed crystallization using surface-modified Qβ phage capsid particles and gold nanoparticles, engineered to have similar effective radii. When combined with the proper connecting oligonucleotides, these components form NaTl-type colloidal crystalline structures containing interpenetrating organic and inorganic diamond lattices, as determined by small-angle X-ray scattering. DNA control of assembly is therefore shown to be compatible with particles possessing very different properties, as long as they are amenable to surface modification.

Genetic Engineering of Human Stem Cells for Enhanced Angiogenesis Using Biodegradable Polymeric Nanoparticles

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2010  |  Pubmed ID: 19805054

Stem cells hold great potential as cell-based therapies to promote vascularization and tissue regeneration. However, the use of stem cells alone to promote angiogenesis remains limited because of insufficient expression of angiogenic factors and low cell viability after transplantation. Here, we have developed vascular endothelial growth factor (VEGF) high-expressing, transiently modified stem cells for the purposes of promoting angiogenesis. Nonviral, biodegradable polymeric nanoparticles were developed to deliver hVEGF gene to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs). Treated stem cells demonstrated markedly enhanced hVEGF production, cell viability, and engraftment into target tissues. S.c. implantation of scaffolds seeded with VEGF-expressing stem cells (hMSCs and hESdCs) led to 2- to 4-fold-higher vessel densities 2 weeks after implantation, compared with control cells or cells transfected with VEGF by using Lipofectamine 2000, a leading commercial reagent. Four weeks after intramuscular injection into mouse ischemic hindlimbs, genetically modified hMSCs substantially enhanced angiogenesis and limb salvage while reducing muscle degeneration and tissue fibrosis. These results indicate that stem cells engineered with biodegradable polymer nanoparticles may be therapeutic tools for vascularizing tissue constructs and treating ischemic disease.

Real-time in Vivo Detection of Biomaterial-induced Reactive Oxygen Species

Biomaterials. Mar, 2011  |  Pubmed ID: 21146868

The non-specific host response to implanted biomaterials is often a key challenge of medical device design. To evaluate biocompatibility, measuring the release of reactive oxygen species (ROS) produced by inflammatory cells in response to biomaterial surfaces is a well-established method. However, the detection of ROS in response to materials implanted in vivo has not yet been demonstrated. Here, we develop a bioluminescence whole animal imaging approach to observe ROS released in response to subcutaneously-implanted materials in live animals. We compared the real-time generation of ROS in response to two representative materials, polystyrene and alginate, over the course of 28 days. High levels of ROS were observed near polystyrene, but not alginate implants, and persisted throughout the course of 28 days. Histological analysis revealed that high levels of ROS correlated not only with the presence of phagocytic cells at early timepoints, but also fibrosis at later timepoints, suggesting that ROS may be involved in both the acute and chronic phase of the foreign body response. These data are the first in vivo demonstration of ROS generation in response to implanted materials, and describe a novel technique to evaluate the host response.

A Novel Family of Biodegradable Poly(ester Amide) Elastomers

Advanced Materials (Deerfield Beach, Fla.). Mar, 2011  |  Pubmed ID: 21394790

Spatiotemporal Effects of a Controlled-release Anti-inflammatory Drug on the Cellular Dynamics of Host Response

Biomaterials. Jul, 2011  |  Pubmed ID: 21429573

In general, biomaterials induce a non-specific host response when implanted in the body. This reaction has the potential to interfere with the function of the implanted materials. One method for controlling the host response is through local, controlled-release of anti-inflammatory agents. Herein, we investigate the spatial and temporal effects of an anti-inflammatory drug on the cellular dynamics of the innate immune response to subcutaneously implanted poly(lactic-co-glycolic) microparticles. Noninvasive fluorescence imaging was used to investigate the influence of dexamethasone drug loading and release kinetics on the local and systemic inhibition of inflammatory cellular activities. Temporal monitoring of host response showed that inhibition of inflammatory proteases in the early phase was correlated with decreased cellular infiltration in the later phase of the foreign body response. We believe that using controlled-release anti-inflammatory platforms to modulate early cellular dynamics will be useful in reducing the foreign body response to implanted biomaterials and medical devices.

Chondrogenic Priming Adipose-mesenchymal Stem Cells for Cartilage Tissue Regeneration

Pharmaceutical Research. Jun, 2011  |  Pubmed ID: 21494923

Chondrocytes lose their ability to produce cartilaginous matrix during multiplication in culture through repeated passages, resulting in inferior tissue phenotype. To overcome the limited amount of primary chondrocytes, we aimed to determine the optimal culture condition for in vitro/in vivo cartilage regeneration using human adipose-derived mesenchymal stem cells (AMSCs).

Development of Cationic Polymer Coatings to Regulate Foreign-body Responses

Advanced Materials (Deerfield Beach, Fla.). Jun, 2011  |  Pubmed ID: 21567481

Degradable Polyelectrolyte Multilayers That Promote the Release of SiRNA

Langmuir : the ACS Journal of Surfaces and Colloids. Jun, 2011  |  Pubmed ID: 21574582

We report an approach to the design of degradable polyelectrolyte-based films for the controlled release of siRNA from surfaces. Our approach is based on stepwise, layer-by-layer assembly of multilayered polyelectrolyte films (or "polyelectrolyte multilayers", PEMs) using siRNA and a hydrolytically degradable poly(β-amino ester) (polymer 1). Fabrication of films using siRNA sequences for green fluorescent protein (GFP) or firefly luciferase resulted in linear growth of ultrathin films (∼50 nm thick) that promoted the surface-mediated release of siRNA upon incubation in physiologically relevant media. Physicochemical characterization of these siRNA-containing films revealed large differences in film growth profiles, physical erosion profiles, and siRNA release profiles as compared to PEMs fabricated using polymer 1 and larger plasmid DNA constructs. For example, whereas films fabricated using plasmid DNA erode gradually and release DNA over a period of ∼48 h, films fabricated using siRNA released ∼65% of incorporated siRNA within the first hour of incubation, prior to the onset of any observed film erosion. This initial burst of release was followed by a second, slower phase of release (accompanied by gradual film erosion) over the next 23 h. These differences in release profiles and other behaviors likely result, at least in part, from large differences in the sizes of siRNA and plasmid DNA. Finally, we demonstrate that the siRNA in these films is released in a form that remains intact, functional, and able to silence targeted protein expression upon administration to mammalian cells in vitro. The results of this investigation provide a platform for the design of thin films and coatings that could be used to localize the release of siRNA from surfaces in a variety of fundamental and applied contexts (e.g., for development of new research tools or approaches to delivery from film-coated implants and other devices).

Five Years of SiRNA Delivery: Spotlight on Gold Nanoparticles

Small (Weinheim an Der Bergstrasse, Germany). Jul, 2011  |  Pubmed ID: 21681985

Gold nanoparticles have become widely used in scientific research due to their unique physical and chemical properties. In the last several years their use as siRNA delivery agents has been investigated. Here, progress made using gold nanoparticles for siRNA delivery is described and the different strategies employed are compared.

Poly(β-amino Ester)-DNA Complexes: Time-resolved Fluorescence and Cellular Transfection Studies

Journal of Controlled Release : Official Journal of the Controlled Release Society. Sep, 2011  |  Pubmed ID: 21699928

A large number of different polymers have been developed and studied for application as DNA carriers for non-viral gene delivery, but the DNA binding properties are not understood. This study describes the efficiency of nanoparticle formation by time-resolved fluorescence measurements for poly(β-amino esters), cationic biodegradable polymers with DNA complexation and transfection capability. From the large library of poly(β-amino esters) ten polymers with different transfection efficacies were chosen for this study. The binding constants for nanoparticle formation were determined and compared to with the same method. Although the DNA binding efficiency of the amine groups are similar for both types of polymers, the overall binding constants are an order of magnitude smaller for poly(β-amino esters) than for 25 kDa polyethylenimines, yet poly(β-amino esters) show comparable DNA transfection efficacy with polyethylenimines. Within this series of polymers the transfection efficacy showed increasing trend in association with relative efficiency of nanoparticle formation.

Synergistic Silencing: Combinations of Lipid-like Materials for Efficacious SiRNA Delivery

Molecular Therapy : the Journal of the American Society of Gene Therapy. Sep, 2011  |  Pubmed ID: 21750531

Despite the promise of RNA interference (RNAi) therapeutics, progress toward the clinic has been slowed by the difficulty of delivering short interfering RNA (siRNA) into cellular targets within the body. Nearly all siRNA delivery vehicles developed to date employ a single cationic or ionizable material. In order to increase the material space available for development of siRNA delivery therapeutics, this study examined the possibility of using binary combinations of ionizable lipid-like materials to synergistically achieve gene silencing. Interestingly, it was found that ineffective single lipid-like materials could be formulated together in a single delivery vehicle to induce near-complete knockdown of firefly luciferase and factor VII in HeLa cells and in mice, respectively. Microscopy experiments suggested that synergistic action resulted when combining materials that respectively mediated cellular uptake and endosomal escape, two important steps in the delivery process. Together, the data indicate that formulating lipid-like materials in combination can significantly improve siRNA delivery outcomes while increasing the material space available for therapeutic development. It is anticipated that this binary formulation strategy could be applicable to any siRNA delivery material in any target cell population that utilizes the two-step endosomal delivery pathway.

Combinatorial Synthesis of Chemically Diverse Core-shell Nanoparticles for Intracellular Delivery

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2011  |  Pubmed ID: 21784981

Analogous to an assembly line, we employed a modular design for the high-throughput study of 1,536 structurally distinct nanoparticles with cationic cores and variable shells. This enabled elucidation of complexation, internalization, and delivery trends that could only be learned through evaluation of a large library. Using robotic automation, epoxide-functionalized block polymers were combinatorially cross-linked with a diverse library of amines, followed by measurement of molecular weight, diameter, RNA complexation, cellular internalization, and in vitro siRNA and pDNA delivery. Analysis revealed structure-function relationships and beneficial design guidelines, including a higher reactive block weight fraction, stoichiometric equivalence between epoxides and amines, and thin hydrophilic shells. Cross-linkers optimally possessed tertiary dimethylamine or piperazine groups and potential buffering capacity. Covalent cholesterol attachment allowed for transfection in vivo to liver hepatocytes in mice. The ability to tune the chemical nature of the core and shell may afford utility of these materials in additional applications.

Directing Human Embryonic Stem Cell Differentiation by Non-viral Delivery of SiRNA in 3D Culture

Biomaterials. Nov, 2011  |  Pubmed ID: 21835461

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.

Nanoparticles Targeting the Infarcted Heart

Nano Letters. Oct, 2011  |  Pubmed ID: 21899318

We report a nanoparticulate system capable of targeting the heart after myocardial infarction (MI). Targeting is based on overexpression of angiotensin II type 1 (AT1) receptor in the infarcted heart. Liposomes 142 nm in diameter were conjugated with a ligand specific to AT1. The nanoparticles were able to specifically target cardiac cells in vitro, and in the infarcted heart after intravenous injection in vivo. This system may be useful for delivering therapeutic agents specifically to the infarcted heart.

Islets Transplanted in Immunoisolation Devices: a Review of the Progress and the Challenges That Remain

Endocrine Reviews. Dec, 2011  |  Pubmed ID: 21951347

The concept of using an immunoisolation device to facilitate the transplantation of islets without the need for immunosuppression has been around for more than 50 yr. Significant progress has been made in developing suitable materials that satisfy the need for biocompatibility, durability, and permselectivity. However, the search is ongoing for a device that allows sufficient oxygen transfer while maintaining a barrier to immune cells and preventing rejection of the transplanted tissue. Separating the islets from the rich blood supply in the native pancreas takes its toll. The immunoisolated islets commonly suffer from hypoxia and necrosis, which in turn triggers a host immune response. Efforts have been made to improve the supply of nutrients by using proangiogenic factors to augment the development of a vascular supply in the transplant site, by using small islet cell aggregates to reduce the barrier to diffusion of oxygen, or by creating scaffolds that are in close proximity to a vascular network such as the omental blood supply. Even if these efforts are successful, the shortage of donor islet tissue available for transplantation remains a major problem. To this end, a search for a renewable source of insulin-producing cells is ongoing; whether these will come from adult or embryonic stem cells or xenogeneic sources remains to be seen. Herein we will review the above issues and chart the progress made with various immunoisolation devices in small and large animal models and the small number of clinical trials carried out to date.

The Influence of Scaffold Elasticity on Germ Layer Specification of Human Embryonic Stem Cells

Biomaterials. Dec, 2011  |  Pubmed ID: 21963156

Mechanical forces are critical to embryogenesis, specifically, in the lineage-specification gastrulation phase, whereupon the embryo is transformed from a simple spherical ball of cells to a multi-layered organism, containing properly organized endoderm, mesoderm, and ectoderm germ layers. Several reports have proposed that such directed and coordinated movements of large cell collectives are driven by cellular responses to cell deformations and cell-generated forces. To better understand these environmental-induced cell changes, we have modeled the germ layer formation process by culturing human embryonic stem cells (hESCs) on three dimensional (3D) scaffolds with stiffness engineered to model that found in specific germ layers. We show that differentiation to each germ layer was promoted by a different stiffness threshold of the scaffolds, reminiscent of the forces exerted during the gastrulation process. The overall results suggest that three dimensional (3D) scaffolds can recapitulate the mechanical stimuli required for directing hESC differentiation and that these stimuli can play a significant role in determining hESC fate.

Therapeutic SiRNA Silencing in Inflammatory Monocytes in Mice

Nature Biotechnology. Nov, 2011  |  Pubmed ID: 21983520

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.

Surface-engineered Substrates for Improved Human Pluripotent Stem Cell Culture Under Fully Defined Conditions

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2011  |  Pubmed ID: 22065768

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

An Injectable Thiol-acrylate Poly(ethylene Glycol) Hydrogel for Sustained Release of Methylprednisolone Sodium Succinate

Biomaterials. Jan, 2011  |  Pubmed ID: 20880573

Clinically available injectable hydrogels face technical challenges associated with swelling after injection and toxicity from unreacted constituents that impede their performance as surgical biomaterials. To overcome these challenges, we developed a system where chemical gelation was controlled by a conjugate Michael addition between thiol and acrylate in aqueous media, with 97% monomer conversion and 6 wt.% sol fraction. The hydrogel exhibited syneresis on equilibration, reducing to 59.7% of its initial volume. It had mechanical properties similar to soft human tissue with an elastic modulus of 189.8 kPa. Furthermore, a mesh size of 6.9 nm resulted in sustained release of methylprednisolone sodium succinate with a loading efficiency of 2 mg/mL. Functionalization with 50 μg/mL of an oligolysine peptide resulted in attachment of freshly isolated murine mesenchymal stem cells. The rational design of the physical, chemical and biological properties of the hydrogel makes it a potentially promising candidate for injectable applications.

Alkane-modified Short Polyethyleneimine for SiRNA Delivery

Journal of Controlled Release : Official Journal of the Controlled Release Society. Dec, 2011  |  Pubmed ID: 22155553

RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.

Treating Metastatic Cancer with Nanotechnology

Nature Reviews. Cancer. Jan, 2012  |  Pubmed ID: 22193407

Metastasis accounts for the vast majority of cancer deaths. The unique challenges for treating metastases include their small size, high multiplicity and dispersion to diverse organ environments. Nanoparticles have many potential benefits for diagnosing and treating metastatic cancer, including the ability to transport complex molecular cargoes to the major sites of metastasis, such as the lungs, liver and lymph nodes, as well as targeting to specific cell populations within these organs. This Review highlights the research, opportunities and challenges for integrating engineering sciences with cancer biology and medicine to develop nanotechnology-based tools for treating metastatic disease.

Action and Reaction: The Biological Response to SiRNA and Its Delivery Vehicles

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2012  |  Pubmed ID: 22252451

RNA interference (RNAi)-based therapeutics have significant potential for the treatment of human disease. Safe and effective delivery of RNA to target tissues remains a major barrier to realizing its clinical potential. Several factors can affect the in vivo performance of short interfering RNA (siRNA) delivery formulations, including siRNA sequence, structure, chemical modification, and delivery formulation. This review provides an introduction to the principles underlying the pharmacokinetics and pharmacodynamics of systemically administered siRNA and its delivery formulations, including the factors that lead to its degradation, clearance, and tissue uptake, as well as its potential for immunogenicity, toxicity, and off-target effects within the body.

Origins of Tumor-associated Macrophages and Neutrophils

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2012  |  Pubmed ID: 22308361

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b(+) Ly-6C(hi) monocytic and CD11b(+) Ly-6G(hi) granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

Combinatorial Library of Lipidoids for in Vitro DNA Delivery

Bioconjugate Chemistry. Jan, 2012  |  Pubmed ID: 22148515

A combinatorial library of lipidoids was constructed and studied for in vitro gene delivery. The library of lipidoids was synthesized by reacting commercially available amines with lipophilic acrylates, acrylamides, or epoxides. Lipidoids derived from amine 86 (N,N-bis(2-hydroxyethyl)ethylene diamine) and amine 87 (N-(3-aminopropyl)diethaneamine) showed high efficiency in DNA delivery, some with a higher transfection efficiency than Lipofectamine 2000, a commonly used commercial gold standard for in vitro gene delivery. The structure-activity relationship between the lipidoids was further studied with respect to small variations in chemical structures and the resulting efficiency in DNA delivery in vitro. Since these lipidoids are easy to synthesize and do not require a colipid for efficient DNA delivery, they could offer an inexpensive but effective alternative to other commonly used commercial gene delivery carriers.