| At present, breast cancer is mostly treated in combination with multiple methods such as operations, chemotherapy, radiation therapy, hormone and targeted therapy in clinical practices. As one of means for treating breast cancer, chemotherapy has been unavoidable all the time. Concerning chemotherapy, drugs are intravenously administered all over body. Except for some anatomical barriers like blood brain barriers, tumor tissues, most tissues and organs are impacted by routine chemotherapy drugs, which thereby damage normal and malignant cells of the body without any choice. After several cycles of chemotherapy, toxicity and side effects would be accumulated for patients. As a result, patients’ tolerance to chemotherapy and sensitivity to chemotherapy drugs are weakened. Hence, medical workers have made constant efforts to explore treatment of breast cancer, in order to seek more effective treatment mode.Mesoporous silica-coated Au nanorods(Au NR@Si O2-HA) modified by hyaluronic acid(HA), studied in this paper, are expected to solve the above problem and become ideal drug carriers. With a diameter of about 70 nm, Au NR@Si O2 can enter cells through endocytosis of cell membranes. Being structurally stable, it can be discharged via human metabolism, so it is only a little toxic to cells. With characteristics of mesoporous silica, Au NR@Si O2 is characterized by high specific surface area. Once loaded with drugs, it can release them to different extent. It may be also modified by HA on its surface to block drugs, in order that drugs aren’t exposed under normal physiological conditions. HA may specifically bind with CD44 receptors over expressed on breast cancer cells and enter the cells through endocytosis, so as to intelligently control drug release. Furthermore, the Au nanorods inside this system can produce photothermal effects under near infrared light.In view of above reasons, HA-modified mesoporous silica-coated Au nanorods(Au NR@Si O2-HA), as nanoparticles drug delivery systems, are designed in this paper. Au nanorods are coated by mesoporous silica and amino-modified on their surface after they are loaded with Dox, so as to block drugs by binding amide bonds with HA. In reaching target cells, HA specifically binds with CD44 receptors over expressed on the surface and has numerous nano-carriers or particles centralized inside cells. Then, HA is degraded by hyaluronidase inside cells and Dox(a chemotherapy drug) is released for the purpose of targeted drug delivery. With photothermal effects, HA-modified mesoporous silica-coated Au nanorods absorb light and transform it into heat under near infrared light. Tumor cells are killed by increasing local temperature in combination with drugs and chemotherapy. Methods and ResultsIn this experiment, drug carrier of HA-modified mesoporous silica-coated Au nanorods was prepared to test their properties. It was experimentally demonstrated to be effective for strengthening Dox-mediated apoptosis of breast cancer cells(MDA-MB-231) under dual effects of chemotherapy and thermal therapy. I. Preparation of Au NR@Si O2-HA and Test of Its Properties(1)CTAB-coated gold nano seeds were further prepared after HAu Cl4 was reduced by Na BH4. Protected by CTAB, they grew into nanorods, on the surface of which CTAB was used as template for wrapping mesoporous silica(through sol-gel) that was amino-modified on the surface. It was proven to be successfully prepared by transmission electron microscope, and its photothermal effects were demonstrated by ultraviolet absorption spectroscopy.(2)Hyaluronic acid(HA) were grafted to the surface of mesoporous silica. The surface modification process was verified by infrared spectroscopy, Zeta potential and gain diameter distribution map.(3)Photothermal effects of the nano-carrier were observed under 808 nm near infrared light(1.8W/cm2, 8min). It was discovered that the temperature of Au NR@Si O2-HA solution at 0.1mg/m L increased from 19 oC to 30.3oC and its photothermal effects increased with the increase in concentration. In addition, the photothermal effects were further verified by images of infrared cameras. II. Drug Release Testing after Loading Dox on Au NR@Si O2-HA(1)Dox was proven to be successfully loaded on Dox-Au NR@Si O2-HA with the appearance of Dox adsorption peak in ultraviolet-visible absorption spectrums. Effects of Dox-Au NR@Si O2-HA for drug release(PBSp H 7.4, PBSp H 4.5 and Hyal-1) were investigated under different conditions. Research shows Dox was proven to be released under control as triggered by hyaluronidase in that system.(2)After Au NR@Si O2-HA was marked by fluorescence, it was then incubated with MDA-MB-231 cell rich in CD44 on the surface. The confocal fluorescence imaging and flow cytometry suggested that nano-particles marked by FITC appeared inside MDA-MB-231 cells in large quantities. This proved that Au NR@Si O2-HA could effectively and selectively enter targeted tumor cells.(3)Au NR@FITC-Si O2 and Au NR@FITC-Si O2-HA were incubated with MDA-MB-231 cells and NIH-3T3 cells(fibroblasts) of the control group respectively. There was a lack of CD44 receptors on the surface of NIH-3T3. According to test results of flow cytometry, Au NR@FITC-Si O2 didn’t differentiate between two groups of cells. After Au NR@FITC-Si O2-HA was modified by HA, there was a significant increase in the amount of such nanorods entering MDA-MB-231 cells, which proved the nanorods targeted MDA-MB-231 cells. III. Cytotoxicity Test of Dox-Au NR@Si O2-HA(1)According to the results of an MTT experiment, Au NR@Si O2-HA wasn’t toxic to MDA-MB-231 and NIH-3T3 cells. Dox-Au NR@Si O2-HA was more toxic to MDA-MB-231 cells than separate Dox, while Dox-Au NR@Si O2-HA was less toxic to NIH-3T3 cells than separate Dox.(2)The synergetic effects of chemotherapy and thermal therapy in apoptosis were evaluated by an MTT experiment as follows: Dox-Au NR@Si O2-HA+NIR Group > Dox-Au NR@Si O2-HA > Au NR@Si O2-HA+NIR Group ≈ Dox Group. In this case, it was more effective for killing tumor cells under the synergetic effects of drug therapy and excessively high-temperature treatment, because the illumination of 80 nm near-infrared light was strengthened after its entrance to cells.(3)According to the test of Annexin V-FITC/PI staining, the apoptosis of MDA-MB-231 cells were as follows; the nano-carrier acted upon such cells through chemotherapy and thermal therapy: Dox-Au NR@Si O2-HA+NIR Group > Dox-Au NR@Si O2-HA Group > Dox Group > Control Group IV. Evaluating Tumor-suppressing Effects of Dox-Au NR@Si O2-HA for Tumor-bearing Mice Inoculated with MDA-MB-231 Cells.(1)After tumor growth curves and final tumor volume were measured and calculated, the tumor-suppressing effects of the drug-loading system were determined as follows: Dox-Au NR@Si O2-HA+NIR Group > Dox-Au NR@Si O2-HA Group > Dox Group > Control Group.(2)The Tunel assay proved that, Dox-Au NR@Si O2-HA+NIR showed a more evident trend of apoptosis than tumor tissues treated in the Dox-Au NR@Si O2-HA Group and Dox Group.(3)After apoptosis factors were tested by Western blot, it was confirmed that after Dox-Au NR@Si O2-HA+NIR, Dox-Au NR@Si O2-HA and separate Dox acted upon MDA-MB-231 cells, the expressions of p53 and Bax in the former group were higher than those in the latter two groups, whereas the expression of bcl-2 was in the former were lower than those in the latter two groups. ConclusionsIn this paper, the nanoparticles-based load delivery system known as Au NR@Si O2 were firstly integrated with HA and pertinently applied in MDA-MB-231 cells(breast cancer cells) rich in CD44 on the surface by loading Dox on mesopores of the system. This system was verified to have relatively great advantages in specifically killing cancer cells and reducing toxicity and side effects of chemotherapy. Additionally, it was further observed to produce synergetic effects with photothermal therapies to significantly increase the apoptosis efficiency of cancer cells, in hope of solving problems about resistance to chemotherapy drugs after multiple cycles of chemotherapy. |
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