Preparation And Antitumor Activity Of Aptamer-mediated Targeting Gene Delivery System For Human Hepatocellular Carcinoma In Vitro And In Vivo

Objective:To develop a novel targeted gene delivery system of RNA aptamer EpDT3-modified recombinant adenovirus carrying tumor suppressor gene PTEN(Ad5-PTEN)to form EpDT3-PEG-Ad5-PTEN(EPAP)and overcome the immunogenicity,low targeting of Ad5-PTEN and the poor stability of RNA aptamer EpDT3,prolong the circulation time of the drug and enhance its targeting,and further confirm preliminarily the antitumor effect on hepatocellular carcinoma.Moreover,the present study is to investigate whether EPAP could improve the serum stability and gene transfection rate,change the morphology of HepG2 cells,inhibit the proliferation and migration of HepG2 cell and its tumor growth,and reduce the toxic and side effects of EPAP.Methods:In this study,EPAP was prepared using a homobifunctional polyethylene glycol(PEG)linker to conjugate epithelial cell adhesion molecule(EpCAM)aptamer EpDT3 and Ad5-PTEN by amide reaction to form PEG-Ad5-PTEN that was confirmed by SDS-PAGE electrophoresis.The formation of EPAP was identified by fluorescence intensity detection,the particle size and zeta potential determination and TBE-PAGE electrophoresis,respectively.Afterwards,the stability of EPAP in human serum was measured in human embryonic kidney(HEK293)cells.EpCAM-positive HepG2 cells and normal human liver cells L-02 were treated respectively with saline and EPAL(EpDT3-PEG-Ad5-LacZ)prepared as the same method as EPAP with a recombinant adenovirus carrying report gene LacZ(Ad5-LacZ)to investigate the gene transfection efficiency and cellular uptake of EPAP.The cells were separately administrated with EpDT3,5-fluorouracil(5-FU),Ad5-PTEN and three different doses of EPAP to observe the changes of cell morphology of HepG2 cells,detect the cell viability of HepG2 cells,EpCAM-negative HEK293 and L-02 cells.The migration ability of HepG2 cells was also detected.Subsequently,the in vivo side effects of EPAP were assessed by acute toxicity trial in normal mice.Finally,the nude mouse HepG2 xenograft tumor model was constructed to evaluate the uptake in tumor tissues and anticancer activity of EPAP.Results:The experimental results showed that the PEGylation of Ad5-PTEN is 95.2±4.58%and the yield of EPAP is about 80-90%.The particle size and zeta potential of EPAP are 98.26±2.85nm and-21.56±9.75 mV,respectively.The findings from the in vitro assays suggested that the serum stability and transfection efficiency of EPAP were both greatly improved.EPAP could specifically target EpCAM-positive HepG2 cells and tumor tissues but not to the normal liver cells,resulting in much more uptake of EPAP by HepG2 cells than L-02 cells.EPAP significantly influenced the morphology and reduced the activity and migration ability of HepG2 cells,but it almost had no effect against HEK293and normal liver cells.The in vivo acute toxicity test indicated that the EPAP at high dose of 1.73×10~100 PFU/mL showed mild liver toxicity while there were no noteworthy side effects for both low-and medium-dose of EPAP.EPAP could suppress the growth and differentiation of liver cancer tumor in nude mice.Conclusions:In the present study,we have successfully constructed the targeted gene delivery system EPAP.It could specifically target EpCAM-positive hepatoma carcinoma cells both in vitro and in vivo,inhibit cellular proliferation and migration,exhibit antitumor activity and restrain the growth and differentiation of hepatocellular carcinoma.Both the low-and medium-dose of EPAP was suitable for the treatment of liver cancer.