Chitosan-zein nano-in-microparticles capable of mediating in vivo transgene expression following oral delivery

Abstract

The oral route is an attractive delivery route for the administration of DNA-based therapeutics, specifically for applications in gene therapy and DNA vaccination. However, oral DNA delivery is complicated by the harsh and variable conditions encountered throughout gastrointestinal (GI) transit, leading to degradation of the delivery vector and DNA cargo, and subsequent inefficient delivery to target cells. In this work, we demonstrate the development and optimization of a hybrid-dual particulate delivery system consisting of two natural biomaterials, zein (ZN) and chitosan (CS), to mediate oral DNA delivery. Chitosan-Zein Nano-in-Microparticles (CS-ZN-NIMs), consisting of core Chitosan/DNA nanoparticles (CS/DNA NPs) prepared by ionic gelation with sodium tripolyphosphate (TPP), further encapsulated in ZN microparticles, were formulated using a water-in-oil emulsion (W/O). The resulting particles exhibited high CS/DNA NP loading and encapsulation within ZN microparticles. DNA release profiles in simulated gastric fluid (SGF) were improved compared to un-encapsulated CS/DNA NPs. Further, site-specific degradation of the outer ZN matrix and release of transfection competent CS/DNA NPs occurred in simulated intestinal conditions with CS/DNA NP cores successfully mediating transfection in vitro. Finally, CS-ZN-NIMs encoding GFP delivered by oral gavage in vivo induced the production of anti-GFP IgA antibodies, demonstrating in vivo transfection and expression. Together, these results demonstrate the successful formulation of CS-ZN-NIMs and their potential to improve oral gene delivery through improved protection and controlled release of DNA cargo.

Development of a ZHER3‐Affibody‐Targeted Nano‐Vector for Gene Delivery to HER3‐Overexpressed Breast Cancer Cells

“…Fluorescence was evaluated by a VICTOR Multilabel Plate Reader (PerkinElmer, USA). The encapsulation efficiency of the RALA‐HER3 nanoparticles was evaluated by calculating the difference between the amount of evaluated free DNA and the primary amount of DNA which was used in the nanoparticle formation as described previously …”

“…RALA‐HER3/pDNA complex with N:P ratio of 10 was formed as described earlier and then was oven dried before scanning electron microscopy (SEM) (ZEISS, SIGMA VP) imaging. The nanoparticles were mounted to double‐sided carbon tape on brass stubs, and sputter covered by gold under argon atmosphere prior to evaluation …”

Oral non-viral gene delivery for applications in DNA vaccination and gene therapy

“…Our group has investigated the use of the natural material, zein (ZN), in the development of a dual material particulate oral gene delivery system [21]. ZN, a prolamine from corn that is amphiphilic in nature, has good film forming capabilities, is resistant to degradation in acidic conditions, and is degraded in an intestinal enzyme-mediated manner, was used to encapsulate inner core CS/DNA NPs [21].…”

“…Our group has investigated the use of the natural material, zein (ZN), in the development of a dual material particulate oral gene delivery system [21]. ZN, a prolamine from corn that is amphiphilic in nature, has good film forming capabilities, is resistant to degradation in acidic conditions, and is degraded in an intestinal enzyme-mediated manner, was used to encapsulate inner core CS/DNA NPs [21]. Oral delivery of the CS-ZN nano-in-microparticle system (encapsulating a reporter transgene) resulted in significantly higher production of mucosal IgA antibodies against the delivered transgene compared to naked CS/DNA NPs that lacked the ZN coating.…”

“…Oral delivery of the CS-ZN nano-in-microparticle system (encapsulating a reporter transgene) resulted in significantly higher production of mucosal IgA antibodies against the delivered transgene compared to naked CS/DNA NPs that lacked the ZN coating. These findings suggest that ZN plays a significant role in protecting the encapsulated CS/DNA cores from gastric transit, while still allowing for CS/DNA NP release and transfection of intestinal cells to generate mucosal immune responses [21]. This platform shows promise as an oral vaccine platform due to its ability to protect genetic cargo in the stomach, and allow for intestinal release of CS/DNA NPs that are: 1) stable in the intestine, 2) have reproducible transfection efficiencies, and 3) have been shown to have adjuvant properties [22].…”