(462c) Assessment of Biotin-Streptavidin Surface Stability In the Presence of Various Proteases for Controlled Release of DNA Polyplexes

Tokatlian, T. - Presenter, University of California, Los Angeles

Non-viral gene delivery is an ideal approach to guide tissue regeneration through the delivery of genes that encode for bioactive signals. However, gene delivery efficiency and controlled release of DNA from tissue engineering scaffolds have proven to be major limitations in their widespread application. One possible solution to address both of these issues is to immobilize and release DNA near the proximity of the targeted cells. Immobilization of polyplexes to cell binding surfaces has been shown to enhance non-viral gene transfer, and through the modulation of the tether degradation rate, one could control the polyplex release rate resulting in controlled transgene expression. Our group has proposed that through the immobilization of DNA/PEI complexes to biomaterials via protease sensitive tethers, release can be targeted to cells that express these proteases. In our proposed approach the cationic polymer used to complex DNA is modified with protease sensitive peptide tethers. These tethers also contain a functional group (e.g. biotin) that can be used to immobilize the polyplexes to a cell-binding surface that has an affinity to the functional group (e.g. streptavidin). Biotin-streptavidin systems have been used widely for immobilization and purification of proteins and have been previously used for the immobilization of biotinylated polyplexes. However, to date there have been no studies conducted regarding the stability of streptavidin surfaces in the presence of various buffers and proteases. We conducted a thorough study to analyze the robustness of streptavidin surfaces and in preventing release of fluorescently labeled biotinylated nanoparticles in the presence of phosphate buffered saline (PBS), conditioned cell media, matrix metalloproteinase 2 (MMP-2), collagenase I and trypsin. Particles were immobilized to a streptavidin surface and incubated at 37oC for up to 128 hours with the solution of interest. Surface fluorescence was quantified using a Typhoon scanner and particle release was directly correlated to biotin-streptavidin stability. After an initial burst release of loosely bound particles over the first 20 hours, the surface proved to be stable in PBS, mesenchymal stem cell (MSC) conditioned media, 50ng/ml MMP-2, and 1.25mg/ml collagenase I, averaging only 10%, 8%, 8%, and 15% particle release from the surface over the following 100 hours, respectively. Only trypsin proved to be detrimental to the surface stability with a sharp burst release followed by steady continued release. Overall, incubation in trypsin resulted in 78% of the particles being released from the surface over 120 hours. However, surfaces incubated in trypsin for 48 hours post 120 hours of incubation in PBS showed only 19% release indicating that prolonged incubation in PBS may have aided in further stabilizing the biotin-streptavidin bond. To ensure that the biotin-streptavidin bond was also stable in the presence of cells, multiple cell types (HeLa, MSC, M202, and PAE) were incubated on top of the bound particles and particle internalization was qualitatively assessed for up to 5 days using a fluorescence microscope. Extensive particle internalization would indicate the surface is not stable for cell culture. No extensive particle internalization was observed for any cell type even when the cells were found to express high levels of proteases enzymes. The immobilization of DNA/PEI polyplexes via a biotinylated peptide tether to a protease resistant streptavidin surface would ensure that release kinetics would be dominated by tether sensitivity to the proteases and not surface instability. We have shown here that the biotin-streptavidin surface is an ideal 2D system to study DNA/PEI polyplex release and cell internalization kinetics.