Polymer Nanoparticles for Optimized Gene Delivery to Mucosal Tissues

Date of Award

Fall 10-1-2021

Document Type


Degree Name

Doctor of Philosophy (PhD)


Engineering and Applied Science

First Advisor

Saltzman, Mark


After decades of pre-clinical and clinical research, gene therapies are finally achieving clinical success. There is rapidly growing interest in using the potential of nucleic acids to develop new treatment options in diverse fields of medicine, but effective gene therapies are still limited by effective delivery systems. Localized gene delivery to mucosal tissues—which include the eyes, respiratory system, digestive system, and female reproductive tract—are being developed to treat disorders and diseases ranging from cystic fibrosis to HIV. However, agent penetration through the mucus barrier that is common to all of these tissues poses a significant challenge to delivery vector design. Many groups have reported that coating delivery vehicles with polyethylene glycol (PEG) can improve diffusion through mucus, but PEG coatings can also inhibit gene delivery.In this dissertation, we examined the effect of PEGylation on gene delivery with nanoparticles made of poly(amine-co-ester) (PACE). PACEs are family of cationic polymers that can effectively deliver a variety of nucleic acids, such as DNA plasmids, mRNA and siRNA. By tuning the lactone content, PACE can be synthesized as a liquid polymer that forms polyplexes by electrostatic attraction to nucleic acids—or as a solid polymer that forms nanoparticles with sustained nucleic acid release. We developed a method of producing PACE polyplexes with tunable PEG content by blending different ratios of PACE and PEG-conjugated PACE. PACE polyplexes with PACE-PEG content as low as 0.25% by weight were more stable than non-PEGylated polyplexes over 3 days, but similar percentages of PACE-PEG inhibited transfection of pDNA, mRNA, and siRNA in vitro. By contrast, incorporation of up to 5% PACE-PEG significantly improved intratracheal mRNA transfection to the lung compared to non-PEGylated polyplexes, highlighting the potential benefits of optimized PEGylation for gene delivery. Next, we tested PACE polymers with modified end group chemistry to enhance endosomal escape of mRNA and improve protein expression. We demonstrated that intratracheal delivery of mRNA polyplexes with end-capped PACE (1,3-diamino-2-hydroxypropane) produced 100-fold higher protein expression in the lung compared to unmodified polymer. Blending end group-modified PACE with PACE-PEG further improved polyplex distribution and mRNA transfection in the lung. Our results indicate that PACE end-capped with 1,3-diamino-2-hydroxypropane and blended with 10% PACE-PEG is a promising delivery vehicle for inhaled mRNA therapies. Finally, we evaluated the safety and kinetics of bioadhesive nanoparticles (BNPs) when administered to the vaginal mucosa of non-human primates. We administered BNPs formed from a block copolymer of poly(lactic acid) and hyperbranched polyglycerol—a system previously shown to be effective in mice for intravaginal delivery of anti-retroviral drugs. We used PET imaging to examine the retention and distribution of 89Zr-labeled BNPs after vaginal application and found that 1.7% of BNPs were retained in the vaginal canal for 24 hours, and BNPs were still detectable after at least 5 days. Additionally, we tracked inflammatory vaginal biomarkers before and after BNP administration to confirm vehicle safety. We observed no significant increase in TNFα, IL-6, MIP-1α, and IL-1RA, suggesting that BNPs are non-reactive and biocompatible, even after multiple vaginal doses. Stealthy nanoparticles—with coatings of PEG or HPG—are promising vehicles for the delivery of genes or drugs to mucosal tissues. This dissertation work concludes that optimized PEGylation can significantly improve the stability and pulmonary transfection efficiency of PACE polyplexes. We also highlight the promise of bioadhesive HPG coatings for safe and prolonged delivery of vaginal therapeutics.

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