Novel Biodegradable Poly(disulfide amine)s for Gene Delivery with High Efficiency and Low Cytotoxicity

Abstract

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.

RAFT Polymerization and Thiol Chemistry: A Complementary Pairing for Implementing Modern Macromolecular Design

“…[97][98][99][100][101] reduced glutathione, [106,107] offering an approach to the release of proteins/siRNA, [97,[108][109][110] drugs, [111] imaging agents, [112] or the synthesis of biodegradable polymers. [113] There are two types of disulfides: symmetric and asymmetric. Asymmetric disulfides are essentially obtained using thiol-disulfide exchange chemistry with an electrophilic (activated) sulfur reagent (Scheme 6), while a symmetric disulfide is obtained by oxidation of a thiol with an oxidizing agent, such as iodine, oxygen, or FeCl 3 .…”

Characteristic as a Gene Delivery System of Water Soluble Chitosan Conjugated with Cationic Peptide

“…현재 유전자 전달에 많이 사용하고 양이 온 고분자는 poly(ethyleneimine)(PEI), poly(L-lysine), poly (amidoamine), chitosan, dendrimer등이 있다. 그러나 이러한 고분자들은 높은 농도로 사용할 경우 독성이 나타난다는 문 제점과 낮은 유전자 전이 효율을 가지고 있어서 그 응용성에 제한을 받고 있다 [13][14][15][16]. 이러한 문제점을 극복하기 위하여 유전자 전이 효율을 높이고, 독성 문제를 해결한 유전자전달 체의 개발이 이루어지고 있다 [17,18].…”

Reductive Degradation Behavior of Bioreducible Poly(disulfide amine) for Enhancing SiRNA Efficiency

“…In the recent works, we have designed and synthesized different types of bioreducible poly(disulfide amine)s containing multiple disulfide bonds for non-viral gene delivery systems that performed cell transfection quite efficiently with very low cellular toxicity, presumably resulted from the unique structural characteristics. [13][14][15] Thus, it was hypothesized that the reductive degradation behavior could be more favorable for intracellular delivery of siRNA. In this study, we reported physico-chemical characteristics and reduction degradation behavior of the bioreducible cationic polymer/siRNA polyelectrolyte complexes (polyplexes).…”