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.
Highlights
Spinal cord injury (SCI) leads to complex pathological changes that include neuronal and glial cell death and axonal demyelination and degeneration
We demonstrate that hydrophobic dye-loaded poly (lactide-co-glycolide)-graft-polyethylenimine (PgP)/pDNA polyplexes are retained in injured spinal cord for up to 5 days after intraspinal injection and that PgP can efficiently deliver pβ-Gal in a rat compression SCI model
We have shown that PgP/pDNA forms stable polyelectrolyte complexes at N/P ratios of 10/1 and greater and achieves the highest transfection efficiency without significantly increased cytotoxicity at N/P of 30/117
Summary
Spinal cord injury (SCI) leads to complex pathological changes that include neuronal and glial cell death and axonal demyelination and degeneration. The ultimate goal of our work is to develop cationic, amphiphilic polymeric micelle nanoparticles for simultaneous delivery of drugs and therapeutic nucleic acids (pDNAs, siRNAs, ODNs, and miRNAs) to promote axonal regeneration and plasticity. Toward this end, we recently reported the synthesis and characterization of poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) and its ability to efficiently transfect pGFP in various neural cell lines and primary chick forebrain neurons in 10% serum condition in vitro, as well as in the normal rat spinal cord[17]. We demonstrate that hydrophobic dye-loaded PgP/pDNA polyplexes are retained in injured spinal cord for up to 5 days after intraspinal injection and that PgP can efficiently deliver pβ-Gal in a rat compression SCI model
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