Cationic Polymer Modified Halloysite Nanotubes As Gene Vectors For Treatment Of Cancer And Their Toxicity Evaluation In Vivo And In Vitro

In recent years,utilizing the unique properties of nanomaterials to design non-viral drug carriers for cancer research is one of the hotspots for cancer therapy.In this paper,natural occurred halloysite nanotubes(HNTs)were modified with polyethyleneimine(PEI)and polyamidoamine(PAMAM),and then designed as non-viral vectors for the treatment of breast cancer.At the same time,the in vivo and in vitro toxicity of HNTs was also studied using cells and zebrafish models systematically.(1)Here,natural occurred halloysite nanotubes(HNTs)were developed as a novel non-viral gene vector.To increase the efficiency of endocytosis,HNTs were firstly shortened into an appropriate size(~200 nm).Then PEI was grafted onto HNTs to bind green fluorescence protein(GFP)labeled pDNA.The structure and physical-chemical properties of PEI grafted HNTs(PEI-g-HNTs)were characterized by various methods.PEI-g-HNTs show lower cytotoxicity than PEI.PEI-g-HNTs are positively charged and can bind DNA tightly at designed N/P ratio from 5:1 to 40:1.PEI-g-HNTs/pDNA complexes show much higher transfection efficiency towards both 293 T and HeLa cells compared with PEI/pDNA complexes at the equivalent N/P ratio.The transfection efficiencies of PEI-g-HNTs/pDNA complex towards HeLa cell can reach to 44.4% at N/P ratio of 20.PEI-g-HNTs/pDNA complexes possess a higher GFP protein expression than PEI/pDNA from simple western immunoblots.So,PEI-g-HNTs are potential gene vectors with good biocompatibility and high transfection efficiency,which have promising applications in cancer gene therapy.(2)PAMAM grafted HNTs(PAMAM-g-HNTs)were synthesized for loading of siRNA in order to intracellular delivery of siRNA and treat of breast cancer via gene therapy.PAMAM-g-HNTs were characterized with morphological and chemical analyses methods,and all results suggested the successful grating of PAMAM on HNTs.The size,zeta potential,and grafting ratio of PAMAM-g-HNTs is ~206.2 nm,+19.8 mV,and 3.02%,respectively.PAMAM-g-HNTs showed good cytocompatibility towards HUVECs(84.7%)and MCF-7 cells(82.3%)even at high concentration of 100 μg/mL.Flow cytometry results showed that the cellular uptake efficiency of PAMAM-g-HNTs/siRNA achieved cellular uptake efficacy of 94.3%,which was higher than that of Lipofectamine 2000(Lipo 2000)/siRNA complexes(83.6%).PAMAM-g-HNTs/small interfering vascular endothelial growth factor(siVEGF),led to 78.0% knockdown of cellular VEGF mRNA and induced 33.6% apoptosis in the MCF-7 cells,which is also much higher than that of Lipo 2000.In vivo anti-cancer result demonstrated that PAMAM-g-HNTs/si VEGF treated 4T1-bearing mice showed enhanced anticancer efficacy than Lipo2000 group.Also,the nanocarrier system did not show any obviously toxic effects to major organs.In vivo fluorescence imaging studies showed that there is slight decrease in the fluorescence signal of PAMAM-g-HNTs/cy5-siVEGF after post-injection for 72 h.Therefore,PAMAM-g-HNTs show promising application as novel nanovectors for siRNA delivery and gene therapy of cancer.(3)The impact of HNTs on the environment and human health should be taken into consideration.In order to clearly show the cell uptake and the distribution of HNTs in zebrafish,HNTs are labeled with fluorescein isothiocyanate(FITC),coded as FITC-HNTs.The cytotoxicity of HNTs towards human umbilical vein endothelial cells(HUVECs)and human breast adenocarcinoma(MCF-7)cells shows that the cell viability has been above 85% even at a concentration of 200 μg/mL.Confocal laser scanning microscope results show the uptake of HNTs by the HUVECs and MCF-7 cells.The in vivo toxicity of HNTs was then investigated in early developing zebrafish embryos.The survival rate of zebrafish embryos and larvae shows no significant changes at specific end points(24,48,72,96,and 120 hpf)treated with different HNTs concentrations(0.25~10 mg/mL).Besides,HNTs can promote the hatching of zebrafish embryos and do not affect the morphological development of zebrafish at the concentration of ≤ 25 mg/mL.HNTs are enriched on the surface of zebrafish chorion with a dose-dependence.HNTs also can be absorbed and majorly accumulated in the gastrointestinal tract.Furthermore,the fluorescence intensity of FITC-HNTs decreases gradually with time,which suggests that HNTs can be excreted out of the zebrafish larvae through the gastrointestinal metabolism.Therefore,it can be concluded that HNTs are eco-friendly nanomaterials for its rapid development in industrial applications.