Construction Of Bifunctional Complex Vectors For Gene Delivery And Bioimaging

Gene therapy is generally considered as one of the most promising approaches in the treatment of cancer diseases,of which the key technique is the development of gene vectors.Among various gene vectors,multifunctional vector could provide solution for both of gene delivery and biological imaging.Rare-earth ion doped upconversion nanomaterials are a new type of fluorescent probes,which are extensively applied in the biotechnology field.Therefore,a cationic tri-peptide lipid was used to coat upconversion nanoparticles(UCNPs)for constructing the gene vectors with dual functions of gene delivery and biological imaging.The high-temperature solution phase reaction process was used to prepare Yb3+ and Er3+co-doped hexagonal phase NaYF4 nanoparticles.The structure and morphology of UCNPs were characterized through X-ray diffraction(XRD),scanning electron microscope(SEM)and transmission electron microscope(TEM).The results show that optimal products could be given,when the mole ratios of rare earth chloride hexahydrate,oleic acid(OA),ammonium hydrogen fluoride and sodium hydroxide were 1:19:2.5:2.5 in octadecene at 300? for 3 h.CSLNs were prepared by thin-film dispersing and ultrasonic dispersing method.They were characterized for their morphology and Zeta potential by using SEM,TEM and laser light scattering particle size analyzer.The results showed that the CSLNs appeared to be spherical,with the average sizes of 50 nm and PDI of 0.2,and Zeta potential was +80 mV.The electrostatic binding interactions between the CSLNs and Luc-siRNA at varying weight ratios were measured by the gel retardation assay.The results indicated that CSLNs completely inhibited Luc-siRNA at the ratios of 4/1 and above.The cell uptake and gene silencing activity were evaluated in vitro transfection.The cell uptake rates of CSLNs were over 75%against A549,HeLa,NCI-H460 and Hep-2,higher than DOTAP and Lipofectamine 2000 at the CSLNs/Luc-siRNA ratio of 3/1.It was found that CSLNs effectively entered the cytoplasm of A549 cells after transfection of 30 min,and the hexagonal UCNPs were clearly observed after transfection of 4 h by TEM.CSLNs were tested for the ability to deliver Luc-siRNA to A549 lung cells which can stably express firefly luciferase.The maximum silencing efficiency of 60%could be obtained in A549 cells.The results indicated that they had comparable transfection efficiency with that of DOTAP and Lipofectamine 2000.The upconversion fluorescence intensity of the CSLNs was evaluated,after they transferred various tumor cells.Fluorescence microscopy detection demonstrates that CSLNs could emit bright yellow-green fluorescence from the cell under laser excitation of 980 nm,therefore,they could be used as a fluorescent marker of tumor cells.The cell lysis liquids were used to detect luminescent intensity by fluorescence spectrometer.The results showed green characteristic peak of rare-earth upconversion nanomaterials,consistent with the results of cellular imaging.BALB/c-nude mice were planted A549 cells to establish a mouse model.CSLNs were injected into mice to investigate the upconversion luminescence imaging in vivo.Yellow-green fluorescence was observed at the tumor site of mice under laser excitation of 980 nm,indicating upconversion fluorescence signal.At the same time,yellow-green fluorescence was observed for tumor,liver and lung through dissection of mice to show CSLNs had the potential as bioprobes.Cytotoxicity of the CSLNs was measured by the MTT method against A549,HeLa,NCI-H460 and Hep-2 cells.The results indicated that all the cells could tolerate CSLNs with the survival rates over 90%.It was superior to that of the two commercial transfection reagents DOTAP and Lipofectamine 2000.The CSLNs showed no obvious toxicity to mice,as the body weights of the mice were stably increased similar to the controls at the dosage of 20 mg kg-1.