Localized Surface Plasmon Resonance Based On Black Phosphorus@AuNPs Nanosheet To Enhance The Photodynamic/Thermal Synergistic Therapy Of Hepatocellular Carcinoma

Background:Hepatocellular carcinoma（HCC）is one of the most frequent malignant tumor.Currently,the anti-cancer methods mainly include surgery,radiotherapy and chemotherapy in clinical,but all of them have their limitations,Some patients cannot receive timely surgical treatment due to physical weakness or poor tumor location,and radiotherapy or chemotherapy often lead to systemic side effects.Moreover,photothermal therapy（PTT）and photodynamic therapy（PDT）are new types of tumor treatment methods,both of which are light-dependent treatment methods with advantages of minimally invasive and high selectivity.The PDT therapy is heavily depending on three essential components,including the content of oxygen,photosensitizer（PS）,and appropriate wavelength of laser irradiation.Photosensitizers are stimulated to generate highly oxidizing singlet oxygen（¹O₂）under the irradiation of light with specific wavelength,which can destroy DNA,lipids and other biological active substances in cells and induce cell apoptosis.especially ¹O₂ through absorbing the electromagnetic wave energy to destroy the intracellular DNA,lipids and other bioactive substances to induce cell apoptosis under light irradiation.However,the wavelength of some PS excitation light sources is too short to effectively penetrate biological tissue.Moreover,some of the biocompatibility of PS is poor,which together with the hypoxic microenvironment of tumors,leads to the decline of ¹O₂ production.Therefore,photodynamic therapy alone cannot effectively inhibit tumor growth.In recent years,the multi-mode cooperative treatment strategy based on nanotechnology that combinate different treatment methods to form complementary advantages,which can effectively improve the anti-tumor effect.However,most combined therapies require multiple light sources of different wavelengths to stimulate or hybridize multiple nano-complexes,which limits the further application of multi-mode cooperative treatment strategy.Herein,the black phosphorus（BP）nanosheet is integrated with gold nanoparticles（AuNPs）to simultaneously enhance the singlet oxygen generation and the photothermal conversion efficiency by localized surface plasmon resonance（LSPR）,to achieve the photothermal/photodynamic synergistic treatment of hepatocellular carcinoma under the single light source irradiation.Purposes:Synthesizing two-dimensional nanosheets,uniform size of BP-PEI/Au and studying the inhibition of HCC growth by the photothermal-photodynamic synergistic effect of BP-PEI/Au in a single light source,to provide new ideas for the treatment of hepatocellular carcinomaMethods:（1）Firstly,BP,Polyethyleneimine（PEI）and HAuCl₄·H₂O were used as raw materials to prepare BP-PEI/Au by nano-precipitation method,and the morphology,structure,size distribution,potential,material composition and spectroscopy of BP-PEI/Au were characterized by transmission electron microscope（TEM）,DSL dynamic light scattering,Raman spectra,Fourier infrared spectrometer,UV-NIR absorption spectroscopy and fluorescence spectrometer.Secondly,9,10-anracenediyl-bis（methylene）-dimalonic Acid（ABDA）was used as ¹O₂ probe to evaluate the photodynamic performance of BP-PEI/Au.Lastly,the photothermal performance of BP-PEI/Au were evaluated by the thermal heating curve,photothermal conversion efficiency,photothermal stability and thermal imager.（2）The dark toxicity of BP-PEI/Au was evaluated by the Cell Counting Kit-8（CCK-8）assay.2,7-Dichlorodihydrofluorescein diacetate（DCFH-DA）was used to determinate the intracellular ¹O₂ production.the thermal imager was used to evaluate thermal effect of cells treated with BP-PEI/Au.In addition,the CCK-8 assay,Calcein-AM/PI cells LIVE/DEAD double staining assay and Annexin V-FITC/Propidium Iodide double staining flow cytometry were used to detect killed HepG2 cells by the joint effect of photothermal and photodynamic.（3）Creating subcutaneous in BALB/c nude mice（SPF）model of HCC with HepG2 cells.The PTT effects of BP-PEI/Au was evaluated through the thermal image of the temperature of tumors in mice.The change of tumor size and pathological biopsy of tumor tissue to further evaluate the photothermal-photodynamic synergy effect of inhibiting tumor cell growth in mice.In addition,the biocompatibility of the materials was evaluated by the changes in body weight of the mice treated with BP-PEI/Au and the pathological results of the main organs（heart,liver,spleen,lung and kidney）in mice.Results:（1）The two-dimensional and uniform of BP-PEI/Au were proved by TEM,DSL dynamic light scattering,Raman spectra,Fourier infrared spectrometer,UV-NIR absorption spectroscopy and fluorescence spectrometer.The BP-PEI/Au showed lamellar structure with an average diameter of 343±58.6 nm and the zeta potential of BP-PEI was about+21.4 mV.The temperature elevation of BP-PEI/Au was significantly increased from 28.5°C to 55.3°C.The photothermal conversion efficiency of BP-PEI/Au was 65.70%.（2）The CCK-8 assay showed that the survival rate of HepG2 cells treated with BP-PEI/Au reached more than 85%.The ¹O₂ production of BP-PEI/AuNPs treated cells were much higher than that of BP-PEI treated cells by ABDA probe.The temperature of infrared radiation thermal image that BP-PEI/Au treated HepG2 cells with 670nm laser irradiation after centrifugation was 46.3℃.The mass production of intracellular ¹O₂ detected by 2,7-dichlorodi-hydrofluorescein diacetrate（DCFH-DA）.In addition,the CCK-8 assay and Calcein-AM/PI cells LIVE/DEAD double staining assay showed that the HepG2 cells treated with BP-PEI/Au were killed more than 80%,and Annexin V-FITC/Propidium Iodide double staining flow cytometry showed that the cell dead or apoptosis rate of BP-PEI/Au group was 97.8%.（3）The temperature reached to 52.3°C within 5 min could be observed at the tumor site of mice treated with BP-PEI/AuNPs.After 2 weeks,the tumor volume of mice treated with BP-PEI/AuNPs decreased significantly,and the pathological sections showed that BP-PEI/AuNPs had a significant inhibitory effect on the proliferation of tumor cells.Moreover,there was no significant change in the body weight of the mice treated with BP-PEI/Au for 2 weeks,and no significant damage was found in the pathological results of the main organs（heart,liver,spleen,lung and kidney）of the mice.Conclusions:The nanosheets of BP-PEI/Au can effectively inhibit the proliferation of HCC with the synergistic effect of photothermal-photodynamic therapy in a single light source.