Intramuscular gene delivery materials are of great importance in plasmid-based gene therapy system, but there is limited information so far on how to design and synthesize them. A previous study showed that the peptide dendron-based triblock copolymer with its components arranged in a reversed biomembrane architecture could significantly increase intramuscular gene delivery and expression. Herein, we wonder whether copolymers with biomembrane-mimicking arrangement may have similar function on intramuscular gene delivery. Meanwhile, it is of great significance to uncover the influence of electric charge and molecular structure on the function of the copolymers. To address the issues, amphiphilic triblock copolymers arranged in hydrophilic–hydrophobic–hydrophilic structure were constructed despite the paradoxical characteristics and difficulties in synthesizing such hydrophilic but electroneutral molecules. The as-prepared two copolymers, dendronG2(l-lysine-OH)-poly propylene glycol2k(PPG2k)-dendronG2(l-lysine-OH) (rL2PL2) and dendronG3(l-lysine-OH)-PPG2k-dendronG3(l-lysine-OH) (rL3PL3), were in similar structure but had different hydrophilic components and surface charges, thus leading to different capabilities in gene delivery and expression in skeletal muscle. rL2PL2 was more efficient than Pluronic L64 and rL3PL3 when mediating luciferase, β-galactosidase, and fluorescent protein expressions. Furthermore, rL2PL2-mediated growth-hormone-releasing hormone expression could significantly induce mouse body weight increase in the first 21 days after injection. In addition, both rL2PL2 and rL3PL3 showed good in vivo biosafety in local and systemic administration. Altogether, rL2PL2-mediated gene expression in skeletal muscle exhibited applicable potential for gene therapy. The study revealed that the molecular structure and electric charge were critical factors governing the function of the copolymers for intramuscular gene delivery. It can be concluded that, combined with the previous study, both structural arrangements either reverse or similar to the biomembrane are effective in designing such copolymers. It also provides an innovative way in designing and synthesizing new electroneutralized triblock copolymers, which could be used safely and efficiently for intramuscular gene delivery.

For efficient transgene delivery and expression, internalized nucleic acids should quickly escape from cellular endosomes and lysosomes to avoid enzymatic destruction and degradation. Here, we report a novel strategy for safe and efficient endosomal/lysosomal escape of transgenes mediated by Pluronic L64, a neutral amphiphilic triblock copolymer. L64 enhanced the permeability of biomembranes by structural disturbance and pore formation in a concentration- and time-dependent manner. When applied at optimal concentration, it rapidly reached the endosome/lysosome compartments, where it facilitated escape of the transfection complex from the compartments and dissociation of the complex. Therefore, when applied properly, L64 not only significantly increased polyethylenimine- and liposome-mediated transgene expression, but also decreased the cytotoxicity occasioned by transfection process. Our studies revealed the function and mechanism of neutral amphiphilic triblock copolymer as potent mediator for safe and efficient gene delivery.Keywords: amphiphilic triblock copolymer; biomembrane; endocytosis; endosomal/lysosomal escape; gene delivery; PluronicL64

Hydrophilic–hydrophobic–hydrophilic triblock copolymers, such as Pluronic L64, P85, and P105, have attracted more attention due to their enhancement in muscular gene delivery. In the present study, a new kind of electroneutralized triblock copolymer, LPL, dendron G2(l-lysine-Boc)-PEG2k-dendron G2(l-lysine-Boc), was designed and investigated. This hydrophobic–hydrophilic–hydrophobic copolymer is composed of a structure reverse to that of L64, one of the most effective materials for intramuscular gene delivery so far. Our results showed that LPL exhibited good in vivo biocompatibility after intramuscular and intravenous administration. LPL mediated higher reporter gene expression than L64 in assays of β-galactosidase (LacZ), luciferase, and fluorescent protein E2-Crimson. Furthermore, LPL-mediated mouse growth hormone expression significantly accelerated mouse growth within the first 10 days. Altogether, LPL-mediated gene expression in skeletal muscle exhibits the potential of successful gene therapy. The current study also presented an innovative way to design and construct new electroneutralized triblock copolymers for safe and effective intramuscular gene delivery.Keywords: amphiphilic; electroneutralization; gene delivery; pluronics; triblock copolymer

Efficient DNA electrotransfer into muscles can be achieved by combining two types of electronic pulses sequentially: short high-voltage (HV) pulse for the cell electropermeabilization and long low-voltage (LV) pulse for the DNA electrophoresis into cells. However, the voltages currently applied can still induce histological and functional damages to tissues. Pluronic L64 has been considered as a molecule possessing cell membrane-disturbing ability. For these reasons, we hope that L64 can be used as a substitute for the HV pulse in cell membrane permeabilization, and a safe LV pulse may still keep the ability to drive plasmid DNA across the permeabilized membrane. In this work, we optimized the electrotransfer parameters to establish a safe and efficient procedure using a clinically applied instrument, and found out that the critical condition for a successful combination of electrotransfer with L64 was that the injection of plasmid/L64 mixture should be applied 1 h before the electrotransfer. In addition, we revealed that the combined procedure could not efficiently transfer plasmid into solid tumor because the uncompressed plasmid may rapidly permeate the leaky tumor vessels and flow away. Altogether, the results demonstrate that the combined procedure has the potential for plasmid-based gene therapy through safe and efficient local gene delivery into skeletal muscles.