| Objective:Cancer has become one of the diseases that threaten human health and life.Nowadays,the main methods of cancer therapy are surgery,chemotherapy and radiotherapy.However,the traditional treatment methods have many side effects,such as decreased chemotherapy effect due to the resistance of the drug,the damage of radiotherapy and chemotherapy on the immune system of human body,and etc.With continuous development of nanotechnology,the application of novel metal nanomaterials provides a new opportunity to overcome the side effects of traditional treatment methods.In this paper,Au NRs@Si O2-G3 nanocomposites were prepared by coating mesoporous silica and dendrimer polymer onto the surface of Au nanorods?Au NRs?and used as nanoplatforms for deliverying the chemotherapeutic drug DOX and Bcl-2 targeted si RNA into the tumor site for chemotherapy and gene therapy.The photothermal therapy induced by NIR laser irradiation was performed to treat tumors at the same time.Combined with the gene therapy,chemotherapy and photothermal therapy,the multi-functional cancer treatment system was formed to improve the therapeutic effect on cancer.Contents:The research in this paper is divided into two parts.Part 1:Preparation of Au NRs@Si O2-G3 nanocomposites,characterization of their morphologies and structures and investigation of their photothermal conversion performance,gene loading performance,drug loading and release performance.Part 2:In vitro investigation of Au NRs@Si O2-G3 nanocomposites,including photothermal treatment performance,cell uptake,inhibition to protein expression and cytotoxicity,etc.Methods:1.Preparation and characterization of Au NRs@Si O2-G3 nanocomposites:Au NRs with a certain length-diameter ratio were synthesized via the classical seed-mediated growth method.Then mesoporous silica was used to cover the surface of Au NRs using the sol-gel method followed by further introducing amino groups from APS.Finally,methyl acrylate and ethanediamine were used to grow PAMAM polymers onto the surface of mesoporous silica coated Au NRs via three-generation Michael additions and amidation reactions.Thus the Au NRs@Si O2-G3nanocomposites were obtained.Next,the products obtained in each step were characterized.The morphology,length-diameter ratio and dispersion state of Au NRs covered with different surface layers were characterized via transmission electron microscope?TEM?.Fourier translation infrared?FT-IR?spectra and UV-vis-NIR absorption spectra were used to characterize the structures of the related products to comfirm the successful coating of mesoporous Si O2 and PAMAM on the surface of Au NRs.The photothermal effect of Au NRs@Si O2-G3 nanocomposites was detected via real-time temperature monitor under the NIR laser irradiation.The gene loading capacity of the Au NRs@Si O2-G3 nanocomposites was investigated via agarose gel electrophoresis.The drug loading and release of the Au NRs@Si O2-G3nanocomposites were investigated under different conditions using DOX as a drug model.2.In vitro experiment:The cellular uptake and distribution of Au NRs@Si O2-G3nanocomposite in cell were detected via laser scanning confocal fluorescence microscope.The effect of photothermal therapy was confirmed via living-dead cell staining experiment.The expression of specific proteins and their gene silencing efficiency were detected via Western blot?WB?assay.The cytotoxicity of Au NRs@Si O2-G3 nanocomposites were investigated via MTT assay and the effect of combined treatment were evaluated preliminarily.Results:1.The Au NRs@Si O2-G3 nanocomposites with a certain length-diameter ratio were successfully synthesized from the results of UV-vis-NIR spectra,FT-IR spectra and TEM images.Au NRs showed the homogeneous structure and good dispersity from the observation of TEM images.The average thickness of Si O2 layer was 20 nm.After being coated with PAMAM polymer,the shell thickness of Au NRs was changed from 20 nm to 26 nm.The zeta potential of Au NRs changed from+49.7 m V to-11.7m V after being coated with Si O2,and to+34.6 m V after being coated with PAMAM further,suggesting the successful preparation of Au NRs@Si O2-G3 nanocomposites.The Au content in Au NRs@Si O2-G3 nanocomposites detected by inductively coupled plasma-mass spectrometry?ICP-MS?was 25.8%.The results of gel electrophoresis showed that Au NRs@Si O2-G3 and plasmid DNA could be fully complexed when the mass ratio was 30:1.The temperature of the Au NRs@Si O2-G3 nanocomposites solution at Au concentration of 25?g m L-1 could be increased to more than 42°C under NIR laser irradiation at 2 w cm-2 for 10 minutes,indicating the good photothermal conversion effect.The results of living-dead cell assay showed that a large number of cells died under the irradiation site after incubation with Au NRs@Si O2-G3 nanocomposites,verifying the photothermal therapy effect of nanocomposites.2.The drug loading of the Au NRs@Si O2-G3 nanocomposites was increased with the increase of initial concentration of DOX solution in a certain range and level off at33.0%,with the corresponding encapsulation rate of 35.0%.The results of drug release experiment showed that the release rate of DOX in Au NRs@Si O2-G3nanocomposites was increased with the decrease of p H of release medium,indicating the characteristics of p H-sensitive drug release.The release rate of DOX in the composites under the NIR laser irradiation was increased compared with that in the experimental group without irradiation,suggesting the good photothermal-stimulated drug release in Au NRs@Si O2-G3 nanocomposites.3.From the observation of confocal fluorescence microscopy,Au NRs@Si O2-G3nanocomposites could co-delivery DOX and FAM-DNA into tumor cells and localized in cytoplasm.Western-blotting results confirmed that Au NRs@Si O2-G3nanocomposites could carry Bcl-2 targeted si RNA into cells and reduce the expression of Bcl-2 protein effectively,indicating good gene transfection efficiency.4.The results of MTT assay showed that compared to free DOX,free si RNA,free Au NRs@Si O2-G3 with laser and DOX or si RNA single loaded Au NRs@Si O2-G3nanocomposites with laser,the combination nanoplatform of DOX and si RNA co-loaded Au NRs@Si O2-G3 nanocomposites with laser showed the best therapeutic effect toward the MCF-7 cells in vitro,which confirmed the synergistic treatment was superior to the single function.Conclusions:Au NRs@Si O2-G3 nanocomposites with the uniform size and stable structure were successfully prepared in this study.They can delivery the chemotherapeutic drugs DOX and anti-Bcl-2 protein expression gene into the cells effectively and show good photothermal therapy,drug therapy and anti-protein expression effects.The Au NRs@Si O2-G3 nanocomposites with low cytotoxicity as multifunctional nanoplatforms by combining photothermal therapy,gene silencing and chemotherapy are expected to become a new generation of material for the synergistic therapy of cancer. |
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