J. LU1, Z. Liang1, Q. Yao1, C. Chen1 1Baylor College Of Medicine,Surgical Research/Surgery,Houston, TX, USA
Introduction: Although gene therapy holds great promise, the progress has been slow because the current gene delivery systems are less successful for clinical applications due to their low efficiency and high toxicity. The objective of this study was to develop a better gene delivery system from two biocompatible polymers, poly(lactic-co-glycolic acid) (PLGA) and polyethylenimine (PEI), potentially for clinical applications.
Methods: The PLGA-PEI copolymer was prepared by directly mixing PLGA and PEI in organic solvent. Self-assembly nanoparticles (NPs) of PLGA-PEI copolymer and DNA were prepared by adding the DNA solution to the water solution of PLGA-PEI copolymer. The size and morphology of PLGA-PEI/DNA NPs were determined with dynamic light scattering and scanning electronic microscopy. The cytotoxicity of PLGA-PEI/DNA NPs at different copolymer to DNA ratios was performed in pancreatic cancer cell line (PANC-1) with an MTT assay. For the gene transfection assay in PANC-1 cells, plasmid DNA containing a green or red fluorescence protein (GFP or RFP) gene was used. In vivo toxicity and transfection efficiency of PLGA-PEI PLGA-PEI/DNA NPs were carried in mouse models with the tail vein administration.
Results: PLGA-PEI copolymer was produced in one-step by mixing PEI and PLGA in the tetrahydrofuran solution and PLGA to PEI ratio (0.5:1). Through the analysis of primary amines of PEI before and after its chemical reaction with PLGA, the chemical structure of the PLGA-PEI copolymer was demonstrated. PLGA was broken down to LGA single units, which were covalently linked to the primary amine groups of PEI; while PEI was intact. PLGA-PEI copolymers spontaneously formed NPs (~100 nm in diameter) with plasmid DNA at the 1.5:1 ratio due to change of the surface charge, achieving 100% DNA loading. The particle size can be controlled by justifying copolymer and DNA ratios. PLGA-PEI significantly reduced the toxicity of PEI in both PANC-1 cells and mouse models. PLGA-PEI copolymer more efficiently delivered GFP or RFP plasmid into PANC-1 cells compared with commercially available transfection reagents with additional advantages of less toxicity, serum independency and long duration of transgene expression. More importantly, PLGA-PEI/DNA NPs were tested in the mouse model and showed an effective gene delivery to liver, spleen, and pancreas. Direct intratumor administration of PLGA-PEI/DNA NPs also showed a high transfection rate in the nude mouse model.
Conclusions: PLGA-PEI copolymer is a new gene delivery material, which has high DNA loading capacity and low toxicity in vitro and in vivo; and it condenses DNA into small sized NPs. The PLGA-PEI/DNA NPs have a high transfection efficiency in cell cultures and mouse models. The current study demostrates a better gene delivery system, which may have board clinical applications.