(70e) DNA Delivery from Enzymatically Degradable Synthetic Hydrogels to Invading Cells Results In Sustained Transgene Expression

We are interested in engineering artificial extracellular matrixes (aECM) that are capable of delivering nucleic acid-based bioactive signals to promote tissue regeneration when implanted at diseased or injured sites. The general idea is to design a scaffold that allows and supports cellular infiltration while also delivers genes, encoding for bioactive signals, to the infiltrating cells. Here the delivery of DNA/polyethylene imine (PEI) polyplexes from matrix metalloproteinase (MMP) degradable polyethylene glycol (PEG) hydrogels to the invading cells is demonstrated. Michael addition chemistry of PEG-vinyl sulfone (PEG-VS) with protease degradable bis-cystine consisting peptides was used for crosslinking. To allow cellular attachment, PEG-VS was modified with Arg-Gly-Asp (RGD) prior to gelation. Although the DNA/PEI polyplexes were physically encapsulated into the hydrogel scaffold during hydrogel gelation, the DNA release profiles showed limited diffusion (< 15%) of the polyplexes out of the gel in PBS and mouse cloned mesenchymal stem cell (D1) conditioned media, indicating that the majority of the polyplexes remain entrapped in the gel and are available for internalization by the invading cells. No changes in mechanical properties were observed due to the encapsulation of polyplexes. Cells were seeded inside the hydrogels using two approaches, homogeneously throughout the hydrogel or inside a fibrin cluster. The fibrin cluster cell plating approach was designed to mimic cells infiltrating the hydrogel post implantation in vivo. Low toxicity to cells seeded inside the polyplex containing hydrogels was observed using the LIVE/DEAD assay. The reporter gene expression pattern of invading cells was found to be different from that of cells homogenously suspended inside the hydrogel. Invading cells showed multiple surges in reporter gene expression throughout the 21-day incubation, which resulted in a sustained cumulative transgene expression and suggest that the cells are transfected several times as they infiltrate the scaffold. In contrast, cells seeded homogeneously only showed one surge in transgene expression with cumulative transgene expression leveling off after 7-days, indicating that the cells were transfected only once during the 21-day incubation. Importantly, we found that using green fluorescent protein as a reporter gene, which is widely used by the other research groups to study gene transfer from hydrogel scaffolds, often generates false positives, with cells that were not transfected or transfected by a different reporter plasmid showing green fluorescence. The ability to repeatedly transfect cells invading DNA containing aECMs maybe result in prolonged expression of bioactive factors in vivo which may have implications in tissue regeneration applications.