| ObjectiveFirstly,doxorubicin-loaded copper sulfide nanoparticles(DOX@BSA-CuS NPs)were synthesized for local injection into hepatic artery.Secondly,the multifunctional therapeutic effect of DOX@BSA-CuS NPs on hepatoma cells was explored in vitro.Finally,the efficacy and safety of local injection of DOX@BSA-CuS NPs in the treatment of hepatocellular carcinoma in rats were evaluated.Methods1.Characterization of copper sulfide nanoparticles:size,shape and surface structure of nanoparticles were observed by scanning electron microscopy(SEM);the hydrodynamic radius and optical absorption properties of nanoparticles were measured by dynamic light scattering and ultraviolet-visible spectrophotometer;the photothermal conversion of nanoparticles was measured by near infrared emitter and thermal imager.2.In vitro experiments:Cell uptake of DOX@BSA-CuS NPs was observed by fluorescence microscopy and fluorophotometer,and the efficacy of multifunctional DOX@BSA-CuS NPs in treating hepatoma cells was tested by flow cytometry.3.Establishment and imaging examination of rat primary hepatocellular carcinoma model:Rat orthotopic hepatocellular carcinoma model was established by intrahepatic injection of N1-S1 cells,meanwhile,three techniques were used to detect rat hepatocellular carcinoma,including enhanced computed tomography(CT),ultrasound and magnetic resonance(MRI).4.In vivo experiments:DOX@BSA-CuS NPs were administered in the hepatic artery via retrograde intubation of the gastroduodenum in rats.The temperature change of tumor lesions within 5 min after near-infrared irradiation were detected by thermal imaging;the distribution of DOX@BSA-CuS NPs in the tumor tissue was observed by transmission electron microscopy(TEM);the drug release properties of nanocomposite particles was detected by immunofluorescence;the effectiveness of treatment was evaluated by MRI monitoring the size of tumors and pathological examination.The safety of treatment was evaluated by blood biochemical detection of aspartate transaminase,alanine transaminase,creatinine and urea nitrogen.Results1.X-ray diffraction(XRD)showed that the characteristic peaks of nanoparticles were consistent with CuS;SEM demonstrated that the CuS NPs and BSA-CuS NPs were round and well dispersed.Meanwhile,HRTEM displayed the surface stripes of CuS NPs and BSA-CuS NPs were unidirectionally distributed and both of them were single crystal structures;Hydrodynamic radius exhibited that the diameter of CuS NPs was 54.6nm±5.3nm.When CuS NPs surround BSA,the diameter of BSA-CuS NPs increased to 65.4nm±10.6nm;Ultraviolet-visible spectrophotometer showed that the absorptive capacity of DOX@BSA-CuS NPs was the sum of the free DOX and CuS NPs;Zata potential exhibited that the surface potential of CuS NPs was-17,BSA-CuS NPs was-30.However,DOX@BSA-CuS NPs was+5.2.Fluorescence microscopy showed that the N1-S1 cells in the DOX@BSA-CuS NPs group took more drugs than the free DOX group.However,after NIR irradiation,the uptake of DOX@BSA-CuS NPs was significantly increased;In addition,the results of the microplate reader demonstrated that the absorbance(IOD)of the Control group,Control+NIR group,DOX group,DOX+NIR group,DOX@BSA-CuS group and DOX@BSA-CuS+NIR group were 0.025±0.001,0.025±0.003,0.245 ±0.024,0.259±0.030,0.353±0.065,0.494±0.115 respectively;Flow cytometry showed that the necrosis rate(%)were Control group:8.10±0.60,Control+NIR group:9.67±0.61,DOX group:23.20±3.48,DOX+NIR group:24.30±6.06,DOX@BSA-CuS group:27.43±6.37 and DOX@BSA-CuS+NIR group:52.73±10.91.3.N1-S1 cells were suspended cells and grew in aggregation,and the N1-S1 hepatocellular carcinoma was nodular;MRI showed the N1-S1 hepatocellular carcinoma in was hypointensity on T1 and hyperintensity in T2,the maximum transverse diameter of the tumor was 7.98+2.03 mm.In arterial phase,high signal intensity at the edge of the tumors,low signal intensity in internal,and clearance in venous phase were observed in enhanced CT,showing "fast-in and fast-out" characteristics.Ultrasound examination showed a strong echo at the edge of the tumor lesion and a low echo inside.4.After 4 hours of hepatic artery injection of the suspension of DOX@BSA-CuS NPs and lipiodol,transmission electron microscopy observed that DOX@BSA-CuS NPs accumulated inside the tumor tissue mostly;Thermal imager showed the temperature of the tumor was significantly increased under NIR irradiation,reaching 60? within 5 min;Fluorescence microscopy showed that most DOX raleased from DOX@BSA-CuS NPs into tumor tissue after 24 hours;MRI showed that the tumor volume of Control group,DOX+Lipiodol group,DOX@BSA-CuS+Lipiodol group and DOX@BSA-CuS+Lipiodol+NIR group were 513.11mm3 ± 196.77mm3,214.35mm3 ± 101.25mm3,152.30mm3±85.33mm3,89.43mm3 ± 32.57mm3(P<0.001);Histopathological examination demonstrated that the tumor cell necrosis rate were 8.11%±3.64%,16.89%±4.45%,17.90%±4.13%and 27.30%±7.20%(P=0.005<0.05).Tumor cell proliferation rates were 26.97%±9.55%,18.70%±5.87%,17.99%± 4.43%and 10.34%±6.11%,respectively(P=0.024<0.05).ConclusionsThis study demonstrates the local injection of multifunctional DOX@BSA-CuS NPs via the hepatic artery,achieving a local combination therapy strategy(TACE combined with PTA)for HCC,simplifying the operation steps and improving the effectiveness of treatment It provides a new drug system and treatment strategy for interventional treatment and combination therapy of HCC,and provides reference for related research. |
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