Block Catiomer Polyplexes with Regulated Densities of Charge and Disulfide Cross-Linking Directed To Enhance Gene Expression

Abstract

A block catiomer polyplex, showing a high stability in the extracellular medium and an efficient release of plasmid DNA (pDNA) in the intracellular compartment, was developed by controlling both the cationic charge and disulfide cross-linking densities of the backbone polycations. Poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) was thiolated using either of two thiolation reagents, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane (Traut's reagent), to investigate the effects of both the charge and disulfide cross-linking densities on the properties of the polyplexes. The introduction of thiol groups by SPDP proceeded through the formation of amide linkages to concomitantly decrease the cationic charge density of PLL segment, whereas Traut's reagent promoted the thiolation with the introduction of cationic imino groups to keep the charge density constant. These thiolated PEG-PLLs were complexed with pDNA to form the disulfide cross-linked block catiomer polyplexes, which had the size of approximately 100 nm. Both thiolation methods were similarly effective in introducing disulfide cross-links to prevent the polyplex from the dissociation through a counter polyanion exchange in the extracellular oxidative condition. On the other hand, the efficient release of pDNA responding to the reductive condition mimicking the intracellular environment was only achieved for the polyplex thiolated with SPDP, a system compensating for the decrease in the charge density with the disulfide cross-linking. This distinctive sensitivity toward oxidative and reductive environments was nicely correlated with the remarkable difference in the transfection efficiency between these two types of thiolated polyplexes (SPDP and Traut's reagent types): the former revealed approximately 50 times higher transfection efficiency toward 293T cells than the latter. Obviously, the balance between the densities of the cationic charge and disulfide cross-linking in the thiolated polyplex played a crucial role in the delivery and controlled release of entrapped pDNA into the microenvironment of intracellular compartment to achieve the high transfection efficiency.

Study on Development of Polymeric Micellar Gene Carrier and Evaluation of Its Functionality

“…The incorporated gene in the polyplex micelle needs to be strongly protected from nucleases and serum proteins in the extracellular milieu such as in the blood, while it must be released from the micelle only under the appropriate environment. In order to satisfy these requirements, disulfide crosslinks were introduced into the core of polyplex micelles 13,14) (Fig. 2).…”

Smart polymeric gels: Redefining the limits of biomedical devices

“…Similarly, a block polycationic complex, demonstrating high stability in the extracellular medium efficiently released plasmid DNA (pDNA) in the intracellular compartment. Obviously, engineering an optimum balance between the densities of the cationic charge and those of the disulfide linkages played a crucial role in the delivery and controlled release of entrapped pDNA into the intracellular compartment, enhancing transfection efficiency [241].…”

A review of RGD-functionalized nonviral gene delivery vectors for cancer therapy

“…To refine their stability and transfection efficiency, disulfide cross-linkages have been introduced, and the resulting polyplex micelles have shown improved transfection efficiencies both in vitro and in vivo mouse models. 75,76 For the targeted delivery of therapeutic genes into tumor sites, cyclic RGD-installed polyplex micelles were introduced. [77][78][79] Thiolated cyclo(RGDfK)-poly(ethylene glycol)-block-poly(L-lysine), (c(RGDfK)-PEG-P(Lys-SH)) (Figure 3a) underwent spontaneous association with pDNA to form targetable disulfide cross-linked polyplex micelles in aqueous solution.…”