title: Enhanced Performance of Plasmid DNA Polyplexes Stabilized by a Combination of Core Hydrophobicity and Surface PEGylation.
abstract: Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cellsin vitrofollowing incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability, endosomal escape, and resultant high transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))-pDNA polyplexes underscores their potential utility both for local delivery from scaffolds as well as systemic, intravenous delivery.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Adolph
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Nelson
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Werfel
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN.
authors.forename: Ruijing
authors.initials: R
authors.lastname: Guo
authors.affiliation: Department of Pathology, Vanderbilt University Medical Center, Nashville, TN ; Research Service, VA Tennessee Valley Healthcare System, Nashville, TN.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Davidson
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN ; Department of Biomedical Engineering, Vanderbilt University, Nashville, TN ; Center for Bone Biology, Vanderbilt University Medical Center, Nashville, TN.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Guelcher
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN.
authors.forename: Craig L
authors.initials: CL
authors.lastname: Duvall