Preparation, Characterization And Transfection Efficiency Of Cationic PEGylated PLA Nanoparticles As Gene Delivery Systems

Recently, the methods of gene delivery are gradually developing and have made great progresses. So the gene therapy of cancer is a hot spot of the biology and clinic medical currently.The gene delivery vectors and vectors associated immune response, cell toxicity and safety are the bottleneck of research and clinic application of gene therapy. The two main systems for DNA delivery are viral and non-viral vectors. Although, the efficiency of gene delivery by non-viral-based vectors is less than that by the viral vectors, non-viral systems are preferred in terms of safety, stability, the relative ease of large-scale production and characterization, and the lack of immunogenicity Nanoparticle is developing as a new non-viral gene delivery vector. Gene therapeutical molecules including DNA, RNA, and so on are encapsulated into or loaded on the surface of nanoparticles. At the same time, specific ligands and monoclonal antibodies are linked onto the surface of nanoparticles. The nanoparticles/DNA complexes can be taken into target cells by receptor-mediated endocytosis, so the effective target gene therapy is achieved.It is vital for nanoparticle-mediated gene delivery and gene therapy to select proper materials. The nanoparticle materials must have very good biodegradability and biocompatibility and be non-immunogenic. The biodegradable polymer nanoparticles have some advantages, such as stabilization, non-toxicity, non-immunogenicity and good biocompatibility. In addition, they can protect the loaded DNA from degradation. New progress proved that the binding between nanoparticles and DNA was accomplished by electrostatic attraction. The negatively charged phosphate backbone of DNA could bind the positively charged stuffs. Therefore, modification of the nanoparticles with biomolecules yields surface properties that make the nanoparticles effective DNA carriers for gene delivery.Objective: To construct an idea gene delivery system with high efficacy of gene transfer, targeting ability, effect of protection to pDNA from DNase degradation and good biocompatibility, especially high stability and the ability to prolong gene transfer.Methods: We used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), and chitosan (CS) to prepare MePEG-PLA-CS NPs by a diafiltration method. The morphology and particle size of the NPs were surveyed by the atomic force microscope (AFM) and zeta potentials were determined with the laser grain analyzer.The cytotoxicities of the MePEG-PLA-CS NPs to liver cancer cell lines (HepG2 cells) and normal liver cell line (L-02 cells) were examined by the MTT assay. DNase I was used to study the gene protection effect of NPs to pDNA. pEGFP Cl carried by MePEG-PLA-CS nanoparticles was used as the reporter plasmid to assay the transfection efficiency in COS7 cells in vitro. Lipofectamine was used as a positive control according to the manufacturer's procedures. Naked DNA was used as a negative control. Furthermore, EGFP gene carried by MePEG-PLA-CS nanoparticles was used to assay the transfection efficiency in BALB/C tumor-bearing mice.Results: They are biodegradable, biocompatible and non-immunogenic. The surface of MePEG-PLA-CS NPs was positive that make the nanoparticles could bind DNA. At the same time, we found that MePEG-PLA-CS/DNA complexes could protect the DNA from degradation of DNasel. In our research, we found that MePEG-PLA-CS NPs had no obvious cell toxicity to L-02 cells at proper concentration, but cell toxicity could be showed at high concentration. The EGFP expression plasmid could be transfected effectively into COS7 cells by MePEG-PLA-CS NPs in vitro. The transfection efficiency was about 43%, which was higher than that of liposome. Compared with lipofectamine, MePEG-PLA-CS NPs had higher transfection efficiency while with much lower toxicity in vivo. The fact that in MePEG-PLA-CS NPs treated mice group the expression level of EGFP in tumor was significant higher than other tested organs indicated PEG can reduce the opsonization, prolong the circulation time of the nanoparticles and enhance the targeted property of gene carriers since coatings of PEG can create a cloud of chains on the particle surface which will repel plasma proteins.Conclusion: In conclusion, chitosan coated PEGylated PLA nanoparticles yielded positively charged nanoparticles and uniform morphology with higher transfection efficiency through in vitro and in vivo experiments. MePEG-PLA-CS NPs is a versatile gene carrier with minimal cytotoxicity and has great potential for gene therapy.