Dataset Information

Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

ABSTRACT: Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (?40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability.

SUBMITTER: Werfel TA

PROVIDER: S-EPMC5517028 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

Werfel Thomas A TA Swain Corban C Nelson Christopher E CE Kilchrist Kameron V KV Evans Brian C BC Miteva Martina M Duvall Craig L CL

Journal of biomedical materials research. Part A 20160111 4

Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporate ...[more]

PMID: 26691570

Similar Datasets

Project description:Stimuli-responsive polycations have been developed for improved nucleic acid transfection and enhanced therapeutic efficacy. The most reported mechanisms for controlled release of siRNA are based on polyelectrolyte exchange reactions in the cytoplasm and the degradation of polycations initiated by specific triggers. However, the degradation strategy has not always been sufficient due to unsatisfactory kinetics and binding of cationic fragments to siRNA, which limits the gene silencing effect. In this study, a new strategy that combines degradation and charge reversal is proposed. Methods: We prepared a polycation (CrossPPA) by crosslinking of phenylboronic acid (PBA)-grafted 1.8k PEI with alginate. It was compared with 25k PEI, 1.8k PEI and 1.8k PEI-PBA on siRNA encapsulation, ATP-responsive behavior and mechanism, cytotoxicity, cell uptake, siRNA transfection, in vivo biodistribution and in vivo anti-tumor efficacy. The in vitro and in vivo experiments were performed on 4T1 murine breast cancer cells and 4T1 tumor model separately. Results: The crosslinking strategy obviously improve the siRNA loading ability of 1.8k PEI. We validated that intracellular levels of ATP could trigger CrossPPA disassembly and charge reversal, which resulted in efficient and rapid siRNA release due to electrostatic repulsion. Besides, CrossPPA/siRNA showed strong cell uptake in 4T1 cells compared with 1.8k PEI/siRNA. Notably, the cytotoxicity of CrossPPA was pretty low, which was owing to its biodegradability. Furthermore, the crosslinked polyplexes significantly enhanced siRNA transfection and improved tumor accumulation. The high gene silencing ability of CrossPPA polyplex led to strong anti-tumor efficacy when using Bcl2-targeted siRNA. Conclusion: These results indicated that the ATP-triggered disassembly and charge reversal strategy provided a new way for developing stimuli-responsive siRNA carriers and showed potential for nucleic acid delivery in the treatment of cancer.

Project description:The effective delivery of DNA locally could increase the applicability of gene therapy in tissue regeneration and therapeutic angiogenesis. One promising approach is through use of porous hydrogel scaffolds that incorporate and deliver DNA in the form of nanoparticles to the affected sites. While we have previously reported on caged nanoparticle encapsulation (CnE) to load DNA polyplexes within hydrogels at high concentrations without aggregation, frequent issues with limited polyplex release following CnE have been encountered. In this study, we report two alternative approaches to polyplex presentation for decreasing aggregation in porous hydrogels. The first approach reduces polyplex aggregation by utilizing polyethylene glycol modification of the gene carrier polymer polyethyleneimine (sPEG-PEI) to mitigate charge-charge interactions between polyplexes and the scaffold during gelation. The second approach electrostatically presents polyplexes on the surfaces of scaffold pores as opposed to an encapsulated presentation. The sPEG-PEI polymer formed a smaller, less toxic, and more stable polyplex that exhibited less aggregation within HA gels when compared to the traditionally used linear PEI (LPEI) polymer. Surface-coated polyplexes also resulted in a more homogenous distribution of polyplexes in hydrogels. Furthermore, sPEG-PEI polyplexes retained transfection abilities comparable to LPEI in 3D surface-coated transfections. These results demonstrate a significant improvement in scaffold-mediated gene delivery and show promise in applications to multi-gene delivery systems.A promising gene delivery approach for regenerative medicine is implanting porous hydrogel scaffolds loaded with DNA nanoparticles for delivery to affected sites. However, loading DNA polyplexes at high concentrations within hydrogels results in significant aggregation. Here, we describe two methods for decreasing aggregation of DNA polyplexes in porous gels. First, the gene carrier polymer polyethyleneimine (PEI) was modified with polyethylene glycol (sPEG-PEI) to mitigate the electrostatic interactions between polyplexes and scaffold polymer to in turn decrease aggregation. Second, polyplexes were presented along the surfaces of the pores of the hydrogel instead of being encapsulated within the gel. These methods allow for highly tunable and sustained transgene expression from scaffold-mediated gene delivery while avoiding polyplex aggregation.

Dataset Information

Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

Publications

Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets