Synergistic Effects Of Gold Nanocages In Hyperthermia And Radiotherapy Treatment Under The Monitor Of Infrared Thermal Imaging

BackgroundAt present, the general diagnosis and treatment of breast cancer in China is not satisfactory. The diagnosis of breast cancer is mainly dependent on traditional imaging techniques, such as mammography, ultrasound, MRI, etc.. However, these techniques have some difficulties in the diagnosis of early lesions and atypical lesions. In terms of treatment, although many advanced treatment methods have been used for early treatment of breast cancer, recurrence of the tumor and treatment related adverse reactions have been a difficult clinical problem, especially in triple negative breast cancer (TNBC). TNBC is a lack of estrogen receptor, progesterone receptor and proto oncogene Her2 receptor expression of breast cancer. At present, the global treatment of breast cancer has reached a consensus:the disease is a systemic disease, we must pay attention to the combination of various treatment modalities.However, in the actual diagnosis and treatment of breast cancer, due to the diagnosis, chemotherapy, radiotherapy, surgery are independent departments, multidisciplinary joint diagnosis and treatment is facing great challenges. In recent years, with the development and integration of nanomaterials and molecular biology techniques, multi-functional nanoparticles with "diagnosis" and "treatment" provide a feasible way to solve the above problems, and it has shown great potential and value in the modern diagnosis and treatment of malignant tumors.In a variety of nano-materials, gold nanoparticles because of its unique physical and chemical characteristics have attracted much attention. Its advantages are as follows: ① simple preparation, good biocompatibility and safety, and easy modification. ② Fine hyperthermia because of SPR effect of the gold shell and strong radiation sensitizing effect because of its high atomic number, which may results in doubled treatment effect if this synergistic treatment is used. ③ Real-time monitoring for the treatment to achieve the integration of "diagnosis and treatment" by used of the infrared thermal imaging.In this study, the high expression of CD44 receptor in breast cancer had been used as a specific target of TNBC of biological imaging and therapy. The CD44-PEG-GNC molecular probe was constructed. Animal models of breast cancer were used for treatment under the monitoring of infrared thermal imaging to achieve the real-time image monitoring of diagnosis and therapeutics. The aim of the study was to explore the comprehensive diagnosis and treatment of breast cancer, and provide a feasible method.ObjectiveThe aim of the study was to construct a novel CD44-PEG-GNC molecular probe, to study the effects of this gold nanocages in hyperthermia and radiotherapy treatment under real-time monitoring of infrared thermal imaging, and to find a feasible method to improve the disgnosis and treatment of breast cancer.MethodsCytology test:HAuCl4 solution was used to synthesize the GNCs, then aqueous PEG solution was added to the GNCs and maintained at 4℃. After the PEG-GNCs were prepared, the monoclonal anti-CD44 antibody was added to conjugate the PEG-GNCs. TEM was used to determine the morphology and size of GNCs. UV-vis spectroscopy was used to evaluate whether CD44-antibody bound to GNPs successfully. Immunohistochemistry was employed to verify the expression of CD44 receptors on 4T1 cells. Inductively coupled plasma mass spectrometry (ICP-MS) and TEM were employed to analyze the GNCs concentration uptaken by 4T1 cells, and the CCK-8 assay was also used to detect the impact of the immuno-probe on the activity of cells. Near infrared laser (NIR) was employed to irradiate the tumor cells, and the treatment effect of GNCs was detect by CCK-8 assay and Hoechst/PI double staining. The 4T1 cells were irradiated by 6MV X-ray, and then the colony assay and Hoechst/PI were used to detect the activity of tumor cells.Animal experiment:4T1 cells were injected to the subcutaneous of four week old Balb/c mice. After tumor formation, thermal infrared imager to take pictures of the tumor-bearing mice was used, then the tumor site was injected with 100 μ 1 GNCs or PBS. The tumor site was irradiated with NIR for 5 minutes, and then the infrared thermal imaging instrument was used to take pictures and to detect the temperature. The tumor bearing mice were divided into 12 groups, which were injected with GNCs or PBS, and then irradiated with NIR or 6MV X-ray. The tumor specimen was scliced and HE staining was performed to evaluate the effects of different treatments on tumor. We utilized intravenous (i.v.) and intratumoral (i.t.) injections.Results1. According to TEM, the mean diameter of our GNCs was about 58.14±4 nm, and the UV-vis extinction spectrum showed that the plasma resonance peak for GNCs was 780 nm, while the peak for CD44-PEG-GNCs occurred at 813 nm. A characteristic red-shift was observed in the localized plasma resonance peak, demonstrating the conjugation of the anti-CD44 antibody on the GNCs.2. The immunohistochemistry showed that many CD44 receptors were expressed on the 4T1 cell membrane, while few receptors were also expressed in the cytoplasm. ICP-MS and TEM showed many GNCs were uptaken by 4T1 cells, and the amount of CD44-PEG-GNCs uptake by 4T1 was more than that of PEGylated GNCs. Moreover, the TEM showed that GNCs aggregate around the membrane of 4T1 cell firstly, and then enter the cell through endocytosis. Subsequently, many GNCs were dispersed in the cytoplasm and localized around the organelle membranes. CCK-8 assay showed that the cell viability of tumor cells was less affected by GNCs, and after 48 hours, the cell viability was 81.2％.3. After NIR laser irradiation and radiotherapy, CCK-8 assay, Hoechst/PI double staining and the colony assay showed the apoptosis of a great deal of 4T1 cells with immuno GNCs in the groups of phototherml therapy, radiotherapy and combined treatment, and the treatment effect was much better than that in control group.4. The infrared thermal imaging showed that the temperature of breast cancer was significantly higher than that of the normal tissue. When the tumor site was injected with PBS and irradiated by NIR, the temperature difference was 4℃. However, when injected with the GNCs, the temperature difference was14℃.5. In PBS injection group, loosely arranged cells, the cell morphology did not change significantly; In PEG injection groups, the nuclear fragmentation, nuclear fusion and inflammatory cell infiltration were observed; In CD44-PEG-GNC injection group, the tumor tissue had obvious necrosis cells, disintegration of extracellular matrix and the increase of cytoplasmic eosinophilic, and the obvious nuclear fragmentation, nuclear dissolution and inflammatory cell infiltration were also observed. The tumor necrosis in synergistic treatment group was significantly more serious than that in other groups.ConclusionThe immuno-probe has less cytotoxicity but good targeting to 4T1 cells. Moreover, the GNCs molecular probes can be used in photothermal therapy and radiotherapy sensitization, and the multiplication effect of synergistic therapeutic can be achieved. Infrared thermal imaging can be used as a real-time monitoring method for the treatment to achieve the synchronous diagnosis and treatment, which provides a feasible technique for the comprehensive diagnosis and treatment of breast cancer.