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.

Sulfhydryl cross-linking poly(ethylene glycol) (PEG)-peptides and glycopeptides were prepared and tested for spontaneous polymerization by disulfide bond formation when bound to plasmid DNA, resulting in stable PEG-peptide and glycopeptide DNA condensates. A 20 amino acid synthetic peptide possessing a single sulfhydryl group on the N-terminal cysteine, with two or five internal acetamidomethyl (Acm)-protected cysteine residues, was reacted with either PEG vinyl sulfone or iodoacetamide tyrosinamide triantennary N-glycan. Following RP-HPLC purification, Acm groups were removed by silver tetrafluoroborate to generate sulfhydryl cross-linking PEG-peptides and glycopeptide that were characterized by either (1)H NMR or LC-MS. Sulfhydryl cross-linking PEG-peptides and glycopeptides were found to bind to plasmid DNA and undergo disulfide cross-linking resulting in stable DNA condensates with potential utility for in vivo gene delivery.

The biodistribution, metabolism, cellular targeting, and gene expression of a nonviral peptide DNA gene delivery system was examined. 125 I-labeled plasmid DNA was condensed with low molecular weight peptide conjugates and dosed i.v. in mice to determine the influence of peptide DNA formulation parameters on specific gene targeting to hepatocytes. Optimal targeting to hepatocytes required the combined use of a triantennary glycopeptide (Tri-CWK 18) and a polyethylene glycol-peptide (PEG-CWK 18) to mediate specific recognition by the asialoglycoprotein receptor and to reduce nonspecific uptake by Kupffer cells. Tri-CWK 18 /PEG-CWK 18 DNA co-condensates were stabilized and protected from metabolism by glutaraldehyde crosslinking. An optimized formulation targeted 60% of the dose to the liver with 80% of the liver targeted DNA localized to hepatocytes. Glutaraldehyde crosslinking of DNA condensates reduced the liver elimination rate from a t 1 /2 of 0.8 to 3.6 h. An optimized gene delivery formulation produced detectable levels of human ␣1-antitrypsin in mouse serum which peaked at day 7 compared to no expression using control formulations. The results demonstrate the application of formulation optimization to improve the targeting selectivity and gene expression of a peptide DNA delivery system.

Recent interest in sulfhydryl cross-linked nonviral gene delivery systems, designed to trigger the intracellular release of DNA, has inspired studies to establish their utility in vitro. To determine if this concept can be extrapolated to in vivo gene delivery, sulfhydryl cross-linking peptides (dp 20), derivatized with either an N-glycan or polyethylene glycol (PEG), were used to generate sulfhydryl cross-linked gene formulations. The biodistribution, metabolism, cell-type targeting, and gene expression of sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates were examined following i.v. dosing in mice. Optimal targeting to hepatocytes was achieved by condensing (125)I-DNA with an add-mixture of 10 mol % triantennary glycopeptide, 5 mol % PEG-peptide, and 85 mol % backbone peptide. Four backbone peptides were substituted into the formulation to examine the influence of peptide metabolism and disulfide bond strength on the rate of DNA metabolism and the level of gene expression in vivo. The half-life of DNA in liver was extended from 1 to 3 h using a backbone peptide composed of d-amino acids, whereas substituting penicillamine for cysteine failed to further increase the metabolic stability of DNA. Optimized gene delivery formulations transiently expressed secreted alkaline phosphatase in mouse serum for 12 days. The results suggest that disulfide bond reduction in liver hepatocytes proceeds rapidly, followed by peptide metabolism, ultimately limiting the metabolic half-life of sulfhydryl cross-linked DNA condensates in vivo.

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