| Background and objective:Gene therapy refers to the treatment of diseases by inserting,removing or correcting faulty genes.As a new treatment strategy,gene therapy has brought the possibility of curing cancer,genetic diseases,viral infections and other incurable diseases.However,nucleic acid molecules themselves are less capable of penetrating cell membranes,targeting the organ and they are extremely unstable in the physiological environment.At present,gene delivery systems are mainly divided into viral vectors and non-viral vectors.Liposomes are the most widely used non-viral vectors in clinical trials.Compared with viral vectors,the formulation of liposomes is simple and safe,and it is easy to alter the properties of liposomes by changing the surface modification and thus have a variety of functions.It has been proved that pegylated liposomes can improve the stability of the complex in serum and significantly prolong the half-life of the carrier.However,the effect of PEG modification on gene delivery efficiency of liposome vector in vivo has not been discussed.As a target organ for DNA transfection,skeletal muscle has many physiological advantages.Therefore,this topic explores the effects of different PEG surface modifications on the efficiency of cationic lipid nanomaterials delivery system targeting muscle cells to deliver plasmid DNA.It will provide a theoretical basis for the application of PEG modified cationic liposome vectors in gene therapy and a feasible strategy for realizing stable long-term expression of target genes in vivo.Methods:A cationic liposome containing DNA was synthesized by thin-film hydration method and then modified with Polyethylene glycol（PEG）or malimide（MAL-PEG）respectively.Nanoscale characterization was performed by Malvern particle size analyzer for in vitro and in vivo experiments.In vitro experiments were conducted by utilizing those vectors to transfect RFP DNA into 293 FT cells,and the transfection results were detected by flow cytometry.We investigated the effects of different modifications on the efficiency of such vector system for the delivery of reporter and functional genes targeting muscle cells in vivo.Reporter gene expression was detected by fluorescence staining in frozen sections of muscle tissue at injection site and imaging of small animals.The expression of functional genes was detected by ELISA,CBA and flow cytometry.In addition,the toxicity of modified cationic liposome vectors was detected by HE staining of muscle tissue at the injection site and organs.Results:1.Uniform and stable cationic liposome nanocarriers were successfully synthesized by thin-film hydration method for gene delivery.2.In vitro experiments,the transfection efficiency of the liposomes with Mal surface modification was significantly higher than those with PEG surface modification,even though the overall transfection efficiency in both cases was low.It was confirmed that different PEG surface modification was an important factor affecting gene delivery efficiency.3.The cationic liposome drug delivery system can achieve long-term expression of plasmid DNA in muscle cells.4.PEG surface modification has significant influence on the delivery efficiency of lipid nanomedicine delivery system.Liposomes with PEG surface modification are more efficient in reporter genes delivery while Mal surface modification on the nanoparticles appears to improve the delivery of functional genes.We considered that it might be related to molecular weight of nucleic acid and other factors,nevertheless the specific mechanism needs to be further studied.5.The cationic liposome drug delivery system has good biocompatibility.Conclusion:Our study confirmed that the cationic liposome drug delivery system can achieve long-term expression of plasmid DNA in muscle cells.It was confirmed that PEG surface modification had a significant effect on the delivery efficiency of lipid nanomedicine delivery system. |
