Category: Manufacturing and Bioprocessing
Purpose: Non-viral vectors exhibit numerous significant advantages over their viral counterparts e.g. lower immunogenicity, low acute toxicity, ease of manipulation, higher stability and cost-effective. While the conventional preparation method, bulk mixing (BM) is straightforward, the formation of the DNA/Polymer complexes (known as Polyplexes) is not well controlled. Therefore, it is essential to develop a better method that can produce complexes with uniform size distribution for more effective and reliable in-vitro and in-vivo gene transfection. In this work, as proof of concept, we have developed a novel method [i.e. the simultaneous spray (SS) technique-shown in Fig-1] for the polyplexes production at a high throughput scale. Secondly, to overcome the issues of PEI cytotoxicity as a transfecting agent, we have developed a non-toxic derivative of PEI i.e. “L-arginine modified oligo (alkylaminosiloxanes)-graft- polyethyleneimine” called as P(SiDAAr)5P3 for the development of non-viral polyplexes formulation.
Methods: In this work, as proof of concept, we have examined the simultaneous spray (SS) technique suitability for the production of polyplexes as an alternate way to produce polyplexes at a larger scale (High throughput method). 1H NMR (D2O) and mass spectroscopy was used to characterize the P(SiDAAr)5P3 polymer. The pDNA-P(SiDAAr)5P3 polyplexes were prepared by different methods i.e. standard bulk mixing (BM) and simultaneous mixing (SS) and characterized for size, shape, charge, hydrodynamic stress profiling, polyplexes stability, effect of N/P ratios, the effect of distance, angle of spray, nozzle size of spraying nozzle and the ratio of pDNA to P(SiDAAr)5P3 for the polyplexes formation. Polyplexes were also compared for cytotoxicity and transfection efficiency using neuronal cell line and primary neurons.
Results: The 1H NMR (D2O) results confirmed the presence of PEI, arginine, siloxane, and PEG groups on the synthesized polymer and confirm the formation of P(SiDAAr)5P3 polymer. Polyplexes prepared using BM and SS were compared and results showed that the polyplexes prepared by SS are more uniform & smaller (150 nm compared to 250 nm for BM), more stable and better transfection efficiency (78 % compared to 60% for BM). Also, P(SiDAAr)5P3 Polyplexes showed significantly higher cell viability ( > 80%) compared to jetPEI polyplex (< 55%) after 48 hrs of treatment in primary neurons and neuronal cell lines (SKNMC & SHSY5Y). Thus, signifying that polyplex preparation using P(SiDAAr)5P3 via SS method is a feasible method for the preparation of polyplexes without inducing any damage to delicate biological molecules (pDNA) and can be used as an alternate method for upscaling of polyplexes production for non-viral gene delivery.
Conclusion: In conclusion, we showed that lab developed transfecting polymer [P(SiDAAr)5P3] is non-toxic in nature when compared against marketed PEI (jetPEI). Also, showed that SS technique is better compared to standard lab bulk mixing (BM) protocol and can be used as a scale-up method for the production of non-viral gene delivery complexes. Currently, we are exploring the lab developed nontoxic derivative of PEI [i.e. P(SiDAAr)5P3] and SS method for the development of oxytocin (OT) encoding CRISPR/Cas-9 plasmid formaultion for the treatment of drug abuse.
Rahul Dev Jayant– Amarillo, Texas