Cationic, amphiphilic copolymer micelles as nucleic acid carriers for enhanced transfection in rat spinal cord

Abstract

Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1 %, and 8.1 %, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration.

RhoA knockdown by cationic amphiphilic copolymer/siRhoA polyplexes enhances axonal regeneration in rat spinal cord injury model

“…Complete gel retardation was observed for PgP/siRhoA polyplexes formed at N/P ratio of 10 or above (Fig 1). This result is consistent with the stability of PgP/pDNA polyplexes observed in our previous study [39]. …”

“…Currently, only a few studies have investigated non-viral delivery of siRNA in spinal cord injury models [51-53], non-viral delivery of siRNA in spinal cord injury models [51-53]. Previously, we have reported the synthesis of PgP, a micelle forming block copolymer designed for combinatorial drug/nucleic acid delivery and demonstrated its capability for in vitro transfection of siRNA and pDNA in the presence of serum and pDNA in the uninjured rat spinal cord [39]. Here, we investigated the ability of PgP to deliver siRNA targeting RhoA in a rat spinal cord compression injury model.…”

“…Cationic, amphiphilic copolymer PgP (poly(lactide-co-glycolide)–g-polyethylenimine) was synthesized using PLGA (4 kDa, 50:50) with a carboxylic end group and branched polyethylenimine (bPEI, 25 kDa) as previously described [39]. The formation of stable polyplexes was evaluated by gel retardation assay.…”

“…Toward this end, we have previously synthesized and characterized a cationic, amphiphilic block co-polymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) [39]. PgP micelles offer a hydrophobic core for solubilization of neuroprotective or neurogenic drugs, while the cationic shell can form polyelectrolyte complexes with therapeutic nucleic acids.…”

“…), specificity, and the ability to design sequences for different targets with minimal change in overall physicochemical properties that might affect carrier interactions and delivery properties. Previously, we have shown that PgP can efficiently transfect a variety of neural cell lines in vitro in the presence of 10 % serum as well as deliver pDNA to the normal rat spinal cord [39]. Using RhoA as a well-established therapeutic target, here we investigate the ability of PgP to deliver siRNA (siRhoA) in B35 cells and in a rat compression spinal cord injury model.…”