(44d) Carbon Nanotubes-Poly(lactide-co-glycolide) Conjugates for the Delivery of Pro-Apoptotic Transcription Factors in Osteosarcoma Cells

Gene therapy approaches have offered a new avenue to treat diseases. Such approaches rely on transferring proteins or genes to program cells into desired fate. Traditionally, liposomes, synthetic polymers and viral vectors have been used as gene carriers. However, the enthusiasm to use liposomes and synthetic polymers has been hampered by the low specificity and transfection efficiency. The application of viral vectors has also been limited due to host immune response. Recently, carbon nanotubes have received significant attention for their promising potential in penetrating cell membranes and transfecting cells effectively. However, major concerns still remain with the toxicity side-effects of carbon nanotubes. The present study introduces a new generation of CNT-based gene vectors based on the use of PLGA to reduce the intrinsic toxicity of carbon nanotubes, and to control the temporal release of genes within cells. Carbon nanotube-PLGA conjugates were fabricated through an atom transfer radical polymerization process. Briefly, oxalyl chloride activated carboxyl carbon nanotubes were linked to PLGA. Caspase-3 (CP3), as a pro-apoptosis protein, was conjugated to PLGA by carbodiimide crosslinking chemistry that included activation of carboxyl group in PLGA by N-hydroxysuccinimide (NHS) and ethyl-dimethylaminopropyl carbodiimide (EDC). Our chemical characterization results demonstrated PLGA wrapping over individual carbon nanotube as confirmed by TEM, UV and XRD. Cellular uptake of CNT-PLGA-protein conjugates was examined through fluorescent microscopy. Images showed CNT-PLGA conjugates were able to penetrate osteosarcoma cell membrane and release caspase-3 with a transfection efficiency of close to 100%. Strikingly, upon the introduction of CNT-PLGA-CP3 conjugates, cell survival significantly diminished to ~20% on day 3, and remained suppressed till day 7. Our results confirmed this effect on cell survival was solely due to the release of CP3 and not CNT-PLGA conjugates (negative control). In contrast, liposomes (positive control) demonstrated an inhibition of only ~50% on day 1 with cells continuing to grow back to fully confluent on day 3. Our results all together demonstrate the successful development and application of CNT-PLGA-protein conjugates to deliver transcription factors within cells. This approach has a broad applicability to a number of research areas including cancer therapeutics, regenerative medicine, cellular imaging and tissue engineering.