Engineering peptide-lipoplexes and recombinant proteins for non-viral gene deliver

Modular gene delivery vectors were developed to enhance transfection by both bolus and surface-mediated gene delivery in vitro by altering the intracellular trafficking of exogenous DNA. For lipid/DNA complexes, termed lipoplexes, and polymer/DNA complexes, termed polyplexes, the efficiency of gene delivery by surface immobilization was investigated, and this efficiency was compared to bolus delivery. For polyplexes, the efficiency of trafficking of cellassociated polyplexes to the nucleus for surface delivery was similar or less than bolus delivery, while the efficiency of nuclear association for cell-associated lipoplexes was similar or greater for surface delivery relative to bolus, suggesting that strategies to enhance surface-meditaed gene delivery for polyplexes should target the vector design to maintain polyplex potency, whereas enhancing lipoplex delivery should target the material design to increase internalization. In addition, confocal microscopy studies indicate that intracellular trafficking of exogenous DNA is dependent on substrate properties and gene delivery reagent; recombinant fibronectin fragmentcoated substrates resulted in increased intracellular trafficking efficiency and caveolar DNA for lipoplexes compared to full-length fibronectin and protein polymer without bioactive sequences, potentially due to the increased amount of cell-adhesive bioactive sequences on the substrate.;Peptides can potentiate lipid-mediated gene delivery by modifying lipoplex physiochemical properties to overcome rate-limiting steps to gene transfer. Inclusion of peptides in lipoplexes resulted in a significant increase in percentage of cells transfected and protein expressed when delivered as a bolus. Relative to lipid alone, peptide-modified lipoplexes enhanced cellular association, which has been reported as a rate-limiting step for bolus transfection with lipoplexes. For substrate-mediated gene delivery, the presence of the peptide in the lipoplex increased transfection, internalization efficiency, decreased the percentage of lysosomal DNA and increased the efficiency of nuclear accumulation relative to lipid alone. These results demonstrate peptide-induced enhancement of gene transfer by surface immobilization due to increased intracellular trafficking efficiency. Finally, peptide-lipoplexes were covalently tethered to PEG hydrogels using tissue transglutaminase. Covalent tethering of lipoplexes can alter DNA release from a biomaterial for sustained and controlled gene expression, which is especially desired for in vivo applications such as regenerative medicine.