| Breast cancer, occurred in the breast epithelial tissue, is the commonest malignancy in women and comprises nearly about 10% of all female cancers. Breast cancer susceptibility is generally inherited and seriously impact on women’s health and even life. Presently, with changing and updating of the treatment concept, the treatment for the breast cancer enters into the age of comprehensive therapy modalities with both local and systemic treatment. According to tumor stage and condition of the patients, appropriately use of surgery, radiotherapy, chemo-, hormonal, biological therapy and targeted adjuvant treatment with traditional Chinese medicine, and so on, has been performed. Usually, in order to "prevent future trouble", the patients would face a final mastectomy treatment, which will lead to a loss of women’s sexuality. Moreover, the side effects of the radio- and chemo- therapy bring harm to their body and make them fall into fear and worries of life. This greatly endures mental pain and pressure for the patients. So it is very necessary to find out new and more efficiency treatments. Recently, the nano-materials investigated for medical application has got more and more attention. Of them, Gold nanorods (GNR) has a strong spectra absorption near infrared (NIR) light, causing that subsequent release of energy via heat, which can be used for hyperthermia therapy. In this work, we will study the preparation and characterization of curcumin and gold nanorods-loaded albumin nanoparticles (Cur/GNRs-BSA-NPs) and investigate its treatment for MCF-7 human breast cancer cells in vitro. The main contents are as follows:Part I Preparation and Characterization of GNRs and GNRs-BSA nanoparticles and Its Thermal-dynamic Effect[Objective] This part aims to study the preparation and characterization of Gold nanorods (GNRs) and albumin modified GNRs nanoparticles (GNRs-BSA-NPs). While, thermodynamic test of the GNRs-BSA-NPs was also studied.[Methods] ① GNRs were prepared by seedless method, and GNRs-BSA were prepared using a desolvation method, followed by cross-linking with Bovine serum albumin (BSA) to modify the surface of the GNRs. ② After preparation, the characteristics of GNRs and GNRs-BSA were detected by transmission electron microscope (TEM), ultraviolet photometer, and ZETA SIZER3000 laser particle size analyzer. ③ Thermodynamic test was performed to observe the temperature change of various doses of GNRs-BSA-NPs at the near-infrared (NIR) light exposure with the wavelength of 808 nm and the output power of 2.5 W.[Results] The TEM images show that GNRs were rodlike with stable rectangular and round-ended morphology. They have uniform sizes and good dispersion. The modified GNRs-BSA-NPs were nearly spherical with uniform sizes and loose/gathered distribution. While the analysis results of ZETA SIZER3000 analyzer demonstrated that the UV-Vis absorption spectra of the GNR have two bands:a strong long-wavelength band at 814 nm and a weak short-wavelength band around 508 nm. GNRs-BSA-NPs have just one band at about 149.7 nm. In a neutral environment of PH 7.4, the free GNRs displayed positive zeta potential of 42.43±0.74 mV, as well as that GNRs-BSA-NPs displayed negative zeta potential of -39.83±0.29 mV. ② The thermodynamic test results showed that the prepared GNRs-BSA-NPs could rise to a steady temperature ranging from 39.1℃ to 47.8 ℃ with different concentrations. Moreover, In the process of heating, the temperature rose rapidly within 15 min, and stabilized up to a certain level after 40 min.[Conclusion] ①The GNR was successfully prepared by seedless method, while the prepared GNRs were successfully wrapped into BSA, forming GNRs-BSA nanoparticles (GNRs-BSA-NPs). ② The prepared GNRs demonstrated good optical properties. The modified GNRs-BSA-NPs also have specific characteristics of hyperthermia, which is very suitable for cancer NIR therapy.Part II Preparation and Characterization of Cur/GNRs-BSA-NPs[Objective] ① This part aims to study the preparation and characterization of Cur-BSA nanoparticles and Cur/GNRs-BSA nanoparticles. ② Also, the thermodynamic effect and biocompatibility of Cur/GNRs-BSA-NPs were studied.[Methods] ① Cur-BSA-NPs and Cur/GNRs-BSA-NPs were prepared using a desolvation and cross-linking method. ② Their characteristics were detected by TEM and ZETA SIZER3000 laser particle size analyzer. ③ The prepared Cur/GNRs-BSA-NPs were placed at the exposure of NIR light to observe their temperature changes in vitro with different concentrations. ④ The cytotoxicity of prepared GNRs-BSA-NPs, Cur-BSA-NPs, and Cur/GNRs-BSA-NPs in vitro were determined by CCK-8 assay.[Results] ① TEM images show that the prepared Cur-BSA-NPs are spherical with stable morphology, uniform size, and well dispersion. And the prepared Cur/GNRs-BSA-NPs are approximately spherical with consistent sizes and loose/gathered distribution. The size analyzer results demonstrated that Cur-BSA-NPs have just one UV-Vis absorption spectra band with average diameter of 139.7 nm and Cur/GNR-BSA-NPs also have one band with average diameter of 190.1 nm. In a neutral environment of PH 7.4, Cur-BSA-NPs have zeta potential of -44.87±1.12 mV, while Cur/GNRs-BSA-NPs have zeta potential of -37.27±0.52 mV. The thermodynamic test results showed that the prepared GNRs-BSA-NPs could rise to a steady temperature ranging from 38℃ to 49℃ with different concentrations. The temperature rising level is positive related to the concentration of Cur/GNRs-BSA-NPs. In the process of heating, the temperature rose rapidly within 15 min, and stabilized up to a certain level after 40 min. ③ CCK-8 test results indicated that, coating with albumin molecules, the Cur-BSA-NPs, GNR-BSA-NPs, and Cur/GNRs-BSA-NPs did not show cytotoxicity for L929 cells in vitro. The toxicity is just determined with Grade 1.[Conclusion] ① The Cur-BSA nanoparticles and Cur/GNRs-BSA nanoparticles were successfully prepared. ② The temperature of Cur/GNRs-BSA-NPs solution could rise to higher level with higher concentration at the exposure of NIR light. This characteristic is very suitable for cancer hyperthermia treatment.③ Good biocompatibility was found for the prepared Cur-BSA-NPs, GNRs-BSA-NPs and Cur/GNRs-BSA-NPs due to albumin modification.Part Ⅲ Therapeutic and mechanism experiment of Cur/GNRs-BSA-NPs for human breast cancer MCF-7 cells[Objective] This part aims to study anti-proliferation and apoptosis effect of Cur/GNRs-BSA-NPs on breast cancer MCF-7 cells at the exposure of NIR light.[Methods] CCK-8 assay was employed to examine the biological effects of the prepared Cur/GNRs-BSA-NPs on the MCF-7 cancer cells in terms of cell proliferation inhibition rate. In order to preliminarily analysis and discuss the anti-tumor mechanism, the Western blotting assay was carried out.[Results] The therapeutic results of the CCK-8 assay showed that anti-tumor proliferation efficiency of Cur/GNRs-BSA-NPs group is higher than that of either Cur-BSA-NPs group or GNRs-BSA-NPs group. The Western blotting experiment results indicated that the anti-tumor mechanism of the Cur/GNRs-BSA-NPs is related with the increasing expression of protein P53, cytochrome C, and caspase 3 in the apoptotic cancer cells, due to the help of both curcumin and hyperthermia effect.[Conclusion] Therapeutic effect of Cur/GNRs-BSA-NPs is better than that of either just hyperthermia group of GNRs-BSA-NPs or just drug group of Cur-BSA-NPs, due to the combined drug treatment and hyperthermia therapy irradiated by NIR light. |
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