Physicochemical stability and transfection efficiency of cationic amphiphilic copolymer/pDNA polyplexes for spinal cord injury repair

Abstract

Multiple age-related and injury-induced characteristics of the adult central nervous system (CNS) pose barriers to axonal regeneration and functional recovery following injury. In situ gene therapy is a promising approach to address the limited availability of growth-promoting biomolecules at CNS injury sites. The ultimate goal of our work is to develop, a cationic amphiphilic copolymer for simultaneous delivery of drug and therapeutic nucleic acids to promote axonal regeneration and plasticity after spinal cord injury. Previously, we reported the synthesis and characterization of a cationic amphiphilic copolymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) and its ability to efficiently transfect cells with pDNA in the presence of serum. We also demonstrated the efficacy of PgP as a therapeutic siRhoA carrier in a rat compression spinal cord injury model. In this work, we show that PgP/pDNA polyplexes provide improved stability in the presence of competing polyanions and nuclease protection in serum relative to conventional branched polyethylenimine control. PgP/pDNA polyplexes maintain bioactivity for transfection after lyophilization/reconstitution and during storage at 4 °C for up to 5 months, important features for commercial and clinical application. We also demonstrate that PgP/pDNA polyplexes loaded with a hydrophobic fluorescent dye are retained in local neural tissue for up to 5 days and that PgP can efficiently deliver pβ-Gal in a rat compression SCI model.

Suicide Gene Therapy By Amphiphilic Copolymer Nanocarrier for Spinal Cord Tumor

“…We also observed that PgP can efficiently protect pDNA from nucleases in serum for up to 24 h, whereas pDNA without PgP was completely degraded by serum nucleases within 3 h. These results show that PgP may be a promising non-viral gene carrier for systemic gene therapy in vivo . In the long-term storage stability study, we observed that the transfection efficiency of PgP/pGFP polyplexes in C6 (rat glioma) cells was maintained up to four months at 4 °C and this result is consistent with the long term stability of PgP/pGFP and PgP/siRhoA observed in B35 (neuroblastoma) cells in our laboratory [21,24]. This demonstrates that PgP can form a stable complex with both pDNA and siRNA and preserve the bioactivity of nucleic acids during long-term storage, an important challenge for the clinical translation of non-viral vectors.…”

“…A cationic, amphiphilic copolymer, PgP, has been developed by our group and has been shown to perform as an efficient drug and non-viral gene carrier in vitro as well as in in vivo animal models [21,22,24]. As a block copolymer that forms polymeric micelles in aqueous solution with a hydrophobic core and hydrophilic, cationic shell, PgP has the potential to serve as a combinatorial carrier for the simultaneous delivery of hydrophobic drugs and anionic therapeutic nucleic acids.…”

Influence of Alkyl Chains of Modified Polysuccinimide‐Based Polycationic Polymers on Polyplex Formation and Transfection

“…These results suggest that the hydrophobic alkyl moieties present in the side chains of PSI contributed to the increase of polymer/DNA polyplexes interaction via Van der Waal's interactions/modulation, which have been reported to facilitate interactions between the polymer backbones and DNA in previous studies . Although an efficient gene delivery can be observed in polyplexes with strong pDNA binding via hydrogen bonding, the presence of an alkyl chain can modulate the integrity of polyplexes by hydrophobic interactions, increasing the binding between the polymer and the DNA.…”

“…To evaluate the binding strength of PSI derivatives and polyplex integrity, heparin displacement assay was performed. In this experiment, the amount of uncomplexed plasmid is accessed once free double‐stranded DNA can be detected after displacement of DNA from polyplexes by different concentrations of heparin using a commonly described method . For bPEI 25, the optimal complexation ratio is N:P 5, and pEGFP‐N3 was displaced by heparin from concentrations above 0.6 IU per sample (Figure S7, Supporting Information).…”

Polycaprolactone electrospun fiber scaffold loaded with iPSCs-NSCs and ASCs as a novel tissue engineering scaffold for the treatment of spinal cord injury

“…SCI is a common central nervous system disorder characterized by destruction or dysfunction of spinal cord caused by trauma or other causes. [42][43][44] SCI can cause a variety of complications that lead to irreversible loss of sensory and motor functions. Recently, the incidence of SCI showed a significant upward trend especially in developing countries, and this is a heavy burden for the patients' family and society.…”