Studies On The Folate Receptor Targeted And PH Sensitive Decoy-ODN Loaded Nanoparticles

Signal transducer and activator of transcription3(STAT3), a kind of cytoplasmic transcription factor, is a number of the Janus kinase (JAK)/STAT signaling pathway, which is involved in the occurrence and development of tumor. Previous studies have confirmed that blocking the signaling pathway of STAT3can restrain the cancer cell proliferation and increase its apoptosis.The "Decoy" oligonucleotides (Decoy-ODN) will compete with the endogenous cis-element within the regulatory regions of target genes to inhibit its binding with STAT3; prevent the transcription and activation of the oncogene, and subsequent result in cell apoptosis and inhibition of tumor cell proliferation. However, naked nuclenic acids are usually prone to degradation in vivo. In order to improve its stability and transfection efficiency, we developed a kind of safe and effective gene carrier which are multifunctional including cellular targeting, long circulation time and endosomal escape.Water solubility trimethyl chitosan chloride (TMC) and folate acid (FA) coupling polyethylene glycol (PEG) derivatives TMC (FA-PEG-TMC) was synthesized, their structure were identified by nuclear magnetic resonce and infrared spectrum. The degree of quaternary of TMC and the percentage of FA in FA-PEG-TMC was45%and8.626%, respectively. It was noteworthy that, compared with TMC, the cytotoxicity of FA-PEG-TMC was greatly reduced, but slightly higher than PEG-TMC at higher concentration.Folate receptor (FR) targeted and pH sensitive Decoy-ODN loaded nanoparticles (FPT-MAA-ODN) were prepared via electrostatic combination. These nanoparticles were spherically shaped, the average particle size was about130nm, the zeta potential was12mV, and the entrapment efficiency was above90%. When the Decoy-ODN/MAA (molar ratio) was10:1, the nanoparticles were pH sensitive. Moreover, the nanoparticles were able to protect Decoy-ODN from nuclease degradation and have good stability in human blood plasma.Most importantly, compared with naked Decoy-ODN, the condensed nanoparticles showed good transfection efficiency which was appropriate with Lipofectamine2000and much higher than PT-MAA-ODN nanoparticles in KB, Hela and HepG2cell, and folate binding study confirmed that the significant difference was mediated by FR. Besides, the addition of MAA did not affect the transfection efficiency of FPT-MAA-ODN nanoparticles. Moreover, FPT-MAA-ODN nanoparticles could inhibit cell proliferation better than FPT-ODN and PT-MAA-ODN nanoparticles (p<0.05) with low cytotoxicity. Apoptosis assay showed that FPT-MAA-ODN nanoparticles could induce nearly20%cell apoptosis, which was superior to Lipofectamine2000, FPT-ODN and PT-MAA-ODN nanoparticles.The study on anti-tumor in vivo showed that FPT-MAA-ODN nanoparticles could inhibite hepatocellular carcinoma (HCC) efficicently both via intratumor injection and tail injection, which was better than that of FPT-ODN and FPT-MAA-ODN nanoparticles. Owing to the poor stability and non-targeted in vivo, the suppression effect of Lipo2000-ODN by tail injection was significantly lower than that by intratumor injection.In conclusion, FPT-MAA-ODN nanoparticles were safe, efficient, FR targeted and pH sensitve gene delivery system.