Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size.

Covalent fluorescent labels are important tools for monitoring the in vitro and in vivo localization of plasmid DNA nanoparticles, but must meet several criteria including high DNA labeling efficiencies and minimal impact on nanoparticle size. We developed a novel fluorescent labeling strategy utilizing an aryl azide photolabel conjugated to a short cationic peptide to label plasmid DNA with Cyanine 5 and sulfo-Cyanine 5. Using a simple camera flash apparatus, photolabel-peptide-dyes can be conjugated to DNA in minutes with preservation of DNA structure and minimal dye photobleaching. The addition of two anionic sulfonates to the Cyanine 5 core greatly improved labeling efficiencies from ~13 to ~53% and mitigated PEGylated polyacridine peptide-DNA nanoparticle size increases over a range of labeling densities. Comparison of our sulfo-Cyanine 5 peptide label to the Mirus Bio Label IT-Cy5 kit revealed that while both did not affect nanoparticle sizes appreciably, labeling efficiencies with our conjugate were higher, possibly due to the higher positive charge density on the peptide linker. The results from this work provide important considerations for choosing fluorophore tags to track DNA nanoparticles.

Gene transfection of primary mouse hepatocytes in 384-well plates.

We report the development of an improved in vitro transfection assay to test the efficiency of non-viral vector DNA nanoparticle transfection of primary hepatocytes. The protocol describes the isolation of viable hepatocytes from a mouse by collagenous perfusion. Primary mouse hepatocytes are plated in 384-well plates and cultured for 24 h prior to transfection with polyethylenimine (PEI) or peptide DNA nanoparticles. Luciferase expression is measured after 24 h following the addition of ONE-Glo substrate. The gene transfer assay for primary hepatocytes was optimized for cell plating number, DNA dose, and PEI to DNA ratio. The assay was applied to compare the expression mediated by mRNA relative to two plasmids possessing different promoters. The reported assay provides reliable in vitro expression results that allow direct comparison of the efficiency of different non-viral gene delivery vectors.

The reduced-charge melittin analogue MelP5 improves the transfection of non-viral DNA nanoparticles.

Melittin is a 26-amino acid amphiphilic alpha-helical peptide derived from honeybee venom. Prior studies have incorporated melittin into non-viral delivery systems to effect endosomal escape of DNA nanoparticles and improve transfection efficiency. Recent advances have led to the development of two newer melittin analogues, MelP5 and Macrolittin 70, with improved pore formation in lipid bilayers while possessing fewer positive charges relative to natural melittin. Consequently, MelP5 and Macrolittin 70 were conjugated through a disulfide bond to a DNA binding polyacridine peptide. The resulting peptide conjugates were used to prepare DNA nanoparticles to compare their relative endosomolytic potency by transfection of HepG2 cells. Melittin and MelP5 conjugates were equally potent at mediating in vitro gene transfer, whereas PEGylation of DNA nanoparticles revealed improved transfection with MelP5 relative to melittin. The results demonstrate the ability to substitute a potent, reduced-charge analogue of melittin to improve overall DNA nanoparticle biocompatibility needed for in vivo testing.

Heat-shrinking DNA nanoparticles for in vivo gene delivery.

The particle size of a PEG-peptide DNA nanoparticle is a key determinant of biodistribution following i.v. dosing. DNA nanoparticles of <100 nm in diameter are sufficiently small to cross through fenestrated endothelial cells to target hepatocytes in the liver. In addition, DNA nanoparticles must be close to charge-neutral to avoid recognition and binding to scavenger receptors found on Kupffer cells and endothelial cells in the liver. In the present study, we demonstrate an approach to heat shrink DNA nanoparticles to reduce their size to <100 nm to target hepatocytes. An optimized protocol heated plasmid DNA at 100 °C for 10 min resulting in partial denaturation. The immediate addition of a polyacridine PEG-peptide followed by cooling to room temperature resulted in heat-shrunken DNA nanoparticles that were ~70 nm in diameter compared with 170 nm when heating was omitted. Heat shrinking resulted in the conversion of supercoiled DNA into open circular to remove strain during compaction. Heat-shrunken DNA nanoparticles were stable to freeze-drying and reconstitution in saline. Hydrodynamic dosing established that 70 nm heat-shrunken DNA nanoparticles efficiently expressed luciferase in mouse liver. Biodistribution studies revealed that 70 nm DNA nanoparticles are rapidly and transiently taken up by liver whereas 170 nm DNA nanoparticles avoid liver uptake due to their larger size. The results provide a new approach to decrease the size of polyacridine PEG-peptide DNA nanoparticles to allow penetration of the fenestrated endothelium of the liver for the purpose of transfecting hepatocytes in vivo.

Metabolically stabilized double-stranded mRNA polyplexes.

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a fully complementary dsmRNA polyplex. The results established dsmRNA as a novel metabolically stable and transfection-competent form of mRNA.

PEG-Peptide Inhibition of Scavenger Receptor Uptake of Nanoparticles by the Liver.

PEGylated polylysine peptides represent a new class of scavenger receptor inhibitors that may find utility at inhibiting DNA nanoparticle uptake by Kupffer cells in the liver. PEG-peptides inhibit scavenger receptors in the liver by a novel mechanism involving in situ formation of albumin nanoparticles. The present study developed a new in vivo assay used to explore the structure-activity-relationships of PEG-peptides to find potent scavenger receptor inhibitors. Radio-iodinated PEG-peptides were dosed i.v. in mice and shown to saturate liver uptake in a dose-dependent fashion. The inhibition potency (IC50) was dependent on both the length of a polylysine repeat and PEG molecular weight. PEG30kda-Cys-Tyr-Lys25 was confirmed to be a high molecular weight (33.5 kDa) scavenger receptor inhibitor with an IC50 of 18 µM. Incorporation of multiple Leu residues improved potency, allowing a decrease in PEG MW and Lys repeat, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys that inhibited scavenger receptors with an IC50 = 20 µM. A further decrease in PEG MW to 2 kDa increased potency further, resulting in a low molecular weight (4403 g/mol) PEG-peptide with an IC50 of 3 µM. Optimized low molecular weight PEG-peptides also demonstrated potency when inhibiting the uptake of radio-iodinated DNA nanoparticles by the liver. This study demonstrates an approach to discover low molecular weight PEG-peptides that serve as potent scavenger receptor inhibitors to block nanoparticle uptake by the liver.

Miniaturization of gene transfection assays in 384- and 1536-well microplates.

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35µl and in 1536-well plates in a total assay volume of 8µl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.

Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery.

PEGylated polylysine peptides of the general structure PEG30 kDa-Cys-Trp-LysN (N = 10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per µg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of (125)I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased 10-fold when comparing PEG30 kDa-Cys-Trp-Lys10 (IC50 of 20.2 µM) with PEG30 kDa-Cys-Trp-Lys25 (IC50 of 2.1 µM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes, resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.

Nanoparticle ligand presentation for targeting solid tumors.

Among the many scientific advances to come from the study of nanoscience, the development of ligand-targeted nanoparticles to eliminate solid tumors is predicted to have a major impact on human health. There are many reports describing novel designs and testing of targeted nanoparticles to treat cancer. While the principles of the technology are well demonstrated in controlled lab experiments, there are still many hurdles to overcome for the science to mature into truly efficacious targeted nanoparticles that join the arsenal of agents currently used to treat cancer in humans. One of these hurdles is overcoming unwanted biodistribution to the liver while maximizing delivery to the tumor. This almost certainly requires advances in both nanoparticle stealth technology and targeting. Currently, it continues to be a challenge to control the loading of ligands onto polyethylene glycol (PEG) to achieve maximal targeting. Nanoparticle cellular uptake and subcellular targeting of genes and siRNA also remain a challenge. This review examines the types of ligands that have been most often used to target nanoparticles to solid tumors. As the science matures over the coming decade, careful control over ligand presentation on nanoparticles of precise size, shape, and charge will likely play a major role in achieving success.

A Convergent Synthesis of Homogeneous Reducible Polypeptides.

The convergent syntheses of homogeneous disulfide cross-linked polypeptides are reported. Reducible polypeptides were synthesized containing four and eight dodecapeptides in two and three linear conjugation steps. Critical for the convergent methodology was the use of orthogonally protected cysteines as either acetamidomethyl (Acm) or Fmoc-thiazolidine (Thz). Both groups could be selectively deprotected with silver trifluoromethanesulfonate in the presence of internal disulfide bonds using TFA and aqueous conditions, respectively. This approach allows for large, reducible polypeptides to be synthesized in efficient yields and minimizes the number of conjugation steps, allowing the development and optimization of gene delivery polypeptides containing multiple peptide components necessary to overcome the numerous in vivo barriers for efficacious gene delivery.

Iterative Reducible Ligation to form Homogeneous Penicillamine Cross-linked Polypeptides.

The syntheses of homogeneous penicillamine disulfide cross-linked polypeptides are reported. Dodecapeptides containing N-terminal, C-terminal, or N- and C-terminal Pen were serially ligated into 36 amino acid polypeptides linked through Cys-Pen, Pen-Cys or Pen-Pen disulfide bonds. Critical to the syntheses was the incorporation of thiazolidine masked Cys and Pen as the N-terminal residues and selective hydrolysis with silver trifluoromethanesulfonate in acidic aqueous conditions to generate a free thiol for subsequent ligation. This approach allows the synthesis of homogeneous disulfide cross-linked polypeptides that have different reductive stabilities and have application in gene delivery by undergoing a tempered reductive triggered release of DNA.

In silico prediction of the 3D structure of trimeric asialoglycoprotein receptor bound to triantennary oligosaccharide.

In this study, we present a general-purpose methodology for deriving the three-dimensional (3D) arrangement of multivalent transmembrane complexes in the presence of their ligands. Specifically, we predict the most likely families of structures of the experimentally intractable trimeric asialoglycoprotein receptor (ASGP-R), which consists of human hepatic subunits (two subunits of H1 and one subunit of H2), bound to a triantennary oligosaccharide (TA). Because of the complex nature of this multivalent type-II transmembrane hetero-oligomeric receptor, structural studies have to date been unable to provide the 3D arrangement of these subunits. Our approach is based on using the three-pronged ligand of ASGP-R as a computational probe to derive the 3D conformation of the complex and then using this information to predict the relative arrangement of the protein subunits on the cell surface. Because of interprotein subunit clashes, only a few families of TA conformers are compatible with the trimeric structure of ASGP-R. We find that TA displays significant flexibility, matching that detected previously in FRET experiments, and that the predicted complexes derived from the viable TA structures are asymmetric. Significant variation exists with respect to TA presentation to the receptor complex. In summary, this study provides detailed information about TA-ASGP-R interactions and the symmetry of the complex.

N-glycan targeted gene delivery to the dendritic cell SIGN receptor.

A novel nonviral gene delivery vector composed of a high-mannose N-glycan conjugated to a polyacridine peptide was prepared. The glycopeptide was designed to bind to plasmid DNA by a combination of polyintercalation and ionic binding, and to the DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin) receptor expressed on CHO cells by recognition of the high-mannose N-glycan. The glycopeptide conjugate was prepared by purification of a high-mannose N-glycan from affinity fractionated soybean agglutinin (SBA). The SBA was proteolyzed to release the N-glycan which was then modified on its N-terminus with Tyr and a propionate maleimide. A DNA binding polyacridine peptide, Cys-(Acr-Lys)(4), was prepared by solid-phase peptide synthesis using Fmoc-Lys(Acr), then conjugated to the maleimide on the N-glycan to produce a glycopeptide. The glycopeptide bound to DNA with high affinity as determined by fluorophore displacement assay and DNA band shift on agarose gel. When bound to Cy5 labeled DNA, the glycopeptide mediated specific uptake in DC-SIGN CHO (+) cells as determined by FACS analysis. In vitro gene transfer studies established that the glycopeptide increased the specificity of gene transfer in DC-SIGN CHO (+) cells 100-fold relative to CHO (-) cells. These studies suggest that a high-mannose N-glycan conjugated to a polyacridine peptide may also facilitate receptor mediated gene delivery in dendritic cells and thereby find utility in the delivery of DNA vaccines.

Synthesis and in vitro testing of new potent polyacridine-melittin gene delivery peptides.

The combination of a polyacridine peptide modified with a melittin fusogenic peptide results in a potent gene transfer agent. Polyacridine peptides of the general formula (Acr-X)(n)-Cys were prepared by solid-phase peptide synthesis, where Acr is Lys modified on its epsilon-amine with acridine, X is Arg, Leu, or Lys and n is 2, 3, or 4 repeats. The Cys residue was modified by either a maleimide-melittin or a thiolpyridine-Cys-melittin fusogenic peptide resulting in reducible or non-reducible polyacridine-melittin peptides. Hemolysis assays established that polyacridine-melittin peptides retained their membrane lytic potency relative to melittin at pH 7.4 and 5. When combined with plasmid DNA, the membrane lytic potency of polyacridine-melittin peptides was neutralized. Gene transfer experiments in multiple cell lines established that polyacridine-melittin peptides mediate expression as efficiently as PEI. The expression was very dependent upon a disulfide bond linking polyacridine to melittin. The gene transfer was most efficient when X is Arg and n is 3 or 4 repeats. These studies establish polyacridine peptides as a novel DNA binding anchor peptide.

Discovery of metabolically stabilized electronegative polyacridine-PEG peptide DNA open polyplexes.

Cationic condensing peptides and polymers bind electrostatically to DNA to form cationic polyplexes. While many cationic polyplexes are able to achieve in vitro transfection mediated through electrostatic interactions, few have been able to mediate gene transfer in vivo. The present study describes the development and testing of polyacridine PEG-peptides that bind to plasmid DNA by intercalation resulting in electronegative open polyplex DNA. Polyacridine PEG-peptides were prepared by chemically conjugating 6-(9-acridinylamino) hexanoic acid onto side chains of Lys in PEG-Cys-Trp-(Lys)(3, 4, or 5). The resulting PEG-Cys-Trp-(Lys-(Acr))(3, 4, or 5) peptides bound tightly to DNA by polyintercalation, rather than electrostatic binding. Unlike polycationic polyplexes, polyacridine PEG-peptide polyplexes were anionic and open coiled, as revealed by zeta potential and atomic force microscopy. PEG-Cys-Trp-(Lys-(Acr))(5) showed the highest DNA binding affinity and the greatest ability to protect DNA from metabolism by DNase. Polyacridine PEG-peptide DNA open polyplexes were dosed intramuscularly and electroporated in mice to demonstrate their functional activity in gene transfer. These results establish polyacridine PEG-peptide DNA open polyplexes as a novel gene delivery method for in vivo use.

Potency of siRNA versus shRNA mediated knockdown in vivo.

The intracellular delivery of small interfering RNA (siRNA) is a therapeutic strategy to transiently block gene expression. Two silencing RNA strategies utilize either synthetic double stranded RNA or plasmid DNA encoding a short hairpin RNA (shRNA). In the present study, we have quantitatively compared the potency of siRNA (siLuc1) and shRNA (pShagLuc) mediated knockdown of luciferase expression in vivo using hydrodynamic dosing and bioluminescence imaging (BLI). Following hydrodynamic coadministration of siLuc1 or pShagLuc with a plasmid encoding luciferase (pGL3), mice were analyzed for transgene expression by BLI. The knockdown of luciferase expression by siLuc1 or pShagLuc was observed at 3 h and persisted for 3 days. The potency of siLuc1 and pShagLuc was equivalent with maximal effect at 10 microg coadministered with 1 microg of pGL3 resulting in >80% knockdown. Combined dosing of siLuc1 and pShagluc (5 microg each) with 1 microg of pGL3 resulted in >99% knockdown. Analysis of the data established that shRNA was significantly more potent than siRNA at mediating knockdown when compared on a mole basis. The combination of hydrodynamic dosing and BLI to measure siRNA or shRNA mediated knockdown of luciferase provide an attractive in vivo quantitative method to test formulations that target the liver.

Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration.

UNLABELLED: Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions. INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation. MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT. RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition. CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics.

Long-term in vivo gene expression via delivery of PEI-DNA condensates from porous polymer scaffolds.

Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding beta-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for beta-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55-60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of beta-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI-condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.

"Evolving nanoparticle gene delivery vectors for the liver: What has been learned in 30 years".

Nonviral gene delivery to the liver has been under evolution for nearly 30years. Early demonstrations established relatively simple nonviral vectors could mediate gene expression in HepG2 cells which understandably led to speculation that these same vectors would be immediately successful at transfecting primary hepatocytes in vivo. However, it was soon recognized that the properties of a nonviral vector resulting in efficient transfection in vitro were uncorrelated with those needed to achieve efficient nonviral transfection in vivo. The discovery of major barriers to liver gene transfer has set the field on a course to design biocompatible vectors that demonstrate increased DNA stability in the circulation with correlating expression in liver. The improved understanding of what limits nonviral vector gene transfer efficiency in vivo has resulted in more sophisticated, low molecular weight vectors that allow systematic optimization of nanoparticle size, charge and ligand presentation. While the field has evolved DNA nanoparticles that are stable in the circulation, target hepatocytes, and deliver DNA to the cytosol, breaching the nucleus remains the last major barrier to a fully successful nonviral gene transfer system for the liver. The lessons learned along the way are fundamentally important to the design of all systemically delivered nanoparticle nonviral gene delivery systems.

Fabrication and in vitro testing of polymeric delivery system for condensed DNA.

Polyethylenimine (PEI) was combined with plasmid DNA and freeze dried following the addition of sucrose as a lyoprotectant and pore-forming agent. Freeze-dried PEI DNA condensates were dry mixed with granular polylactideglycolic acid (PLGA) then compression molded and sponged to encapsulated PEI DNA. A measurement of the elastic modulus indicated that 91 wt% sucrose substituted for 95 wt% sodium chloride as a porogen, resulting in PLGA sponges with a mechanical modulus of 100 kPa. The PEI DNA was retained (80%) within PLGA sponges prepared with sucrose during the leaching and subsequent 2-week release studies, whereas sodium chloride PLGA sponges caused the premature release (100%) of PEI DNA within 2 days. In vitro gene transfer studies with PEI DNA PLGA sponges established that adherent and infiltrating fibroblasts expressed reporter gene for 15 days compared with the short, 3-day expression mediated by direct gene of PEI DNA on cells in culture. The results demonstrate an approach to encapsulate condensed DNA in a PLGA sponge for the purpose of retaining DNA within the matrices and creating efficient gene transfer during tissue engineering.