Preparation And Evaluation Of Ultrasound-sensitive Smart Nano-micelles Targeted To Hepatocellular Carcinoma

Hepatocellular carcinoma（HCC）is one of the most serious diseases that threaten human life.Chemotherapy is the most extensive and effective treatment for liver cancer.But for traditional small-molecule drugs,there are many disadvantages such as low utilization,side toxic effects,toleranceand and the insufficient concentration of drugs will induce cancer metastasis.Here,we designed a multifunctional nano-carrier with liver targeting and ultrasonic-sensitive drug release function to treat HCC.The liver active-targeted function of nano-micelles can enhance the accumulation of nano-carriers in the tumor tissues.Ultrasonic-sensitive nano-micelles release drug rapidly under ultrasonic stimulation can increase the accumulation of anticancer drug in the HCC cells in a short period of time.Therefore,the combination of liver targeting group and ultrasonic responsive linker can make up for the deficiency of traditional anticancer drugs.The main contents are as follows:（1）The synthesis and characterization of polymer（p LAMA-OC）In this thesis,a new class of liver targeted and ultrasonic-sensitive glycopolymer（poly（lactobionamidoethyl methacrylate）-b-（Hydroxy tetramethyl piperidinooxy）stearate,p LAMA-OC）,was synthesized by single electron transfer-nitroxide radical coupling（SET-NRC）.The liver targeted hydrophilic polymer of pLAMA-OC with different length(poly（lactobionamidoethyl methacrylate）,pLAMA:p LAMA₃₀;p LAMA₄₀;pLAMA₅₀)consisted of lactobionamidoethyl methacrylate.The hydrophobic compound of pLAMA-OC is（4-Hydroxy-2,2,6,6-tetramethyl-piperidinooxy）stearate（TEPMO-OC）.The structure of the polymers was characterized by ¹H NMR,HRMS and MALDI-TOF MS methods.（2）The characterization of nano-micellesThe nano-micelles with different length of hydrophilic polymer named pLAMA₃₀-OC;p LAMA₄₀-OC and p LAMA₅₀-OC respectively were obtained by dialysis method.The dynamic light scattering（DLS）and transmission electron microscopy（TEM）results showed that the nano-micelles were uniformly distributed with a particle size of about 200 nm.The surface of nano-micelles were negatively charged,which guaranted the stability of nano-micelles in storage and blood circulation.The nano-micelles had excellent stability and ultrasonic-sensitive property in vitro.Hemolysis test and cytotoxicity test proved the low toxicity and biocompatibility of the nano-micelles.（3）The characterization of the drug-loading nano-micellesDoxorubicin（DOX）was used as a model drug,drug-loading nano-micelles were obtained by dialysis method.pLAMA₄₀-OC nano-micelles and pLAMA₅₀-OC nano-micelles possessed higher drug loading content and entrapment efficiency.pLAMA₄₀-OC nano-micelles are more stable in storage and blood circulation.The drug loading of pLAMA₄₀-OC/DOX nano-micelles with drug/vector ratio 1/10 was 5.66±0.18%and the entrapment efficiency was 60.01±2.04%.the pLAMA₄₀-OC/DOX nano-micelles were uniform dispersed spherical structure with the size of 265.5±7.8 nm.The pLAMA₄₀-OC/DOX nano-micelles exhibited an obvious ultrasonic-sensitive drug release behavior,and in PBS solution（pH 5.0）,ultrasonic stimulation increased the cumulative release of DOX by 27.8%.（4）Liver targeting capacity and cytotoxicity of the drug-loading nano-micelles in vitroThe effect of liver targeting and the ultrasonic responsiveness in vitro of p LAMA₄₀-OC/DOX nano-micelles was analyzed by flow cytometry（FCM）and laser confocal microscope（CLSM）.The results showed the affinity of drug-loading micelles for HCC cells,and p LAMA40-OC/DOX nano-micelles could be internalized by recognizing asialoglycoprotein receptor（ASGP-R）on Hep-G2 cells.Furthermore,ultrasound can stimulate anticancer drug DOX to release from the nanoparticles.The average half inhibitory concentration(IC₅₀)of p LAMA₄₀-OC/DOX for ultrasound-treatedgroupwas 6.61μg/m L,while 38.80μg/m L for non-ultrasonic treated-groupis.These results demonstrated that the pLAMA40-OC/DOX nano-micelles could release DOX in response to ultrasonic stimulation and improve its anti-cancer activity.（5）The ability to inhibit cancer of the drug-loading nano-micelles in vivoThe mouse breast cancer cell 4T1 was inoculated to the Balb/c mice to build the model mice.The therapeutic outcome displayed that the tumor proliferation rate（T/C%）of PBS/US treatment group was 122.90%（>60%,ineffective cure）,but the T/C%of p LAMA₄₀-OC/DOX/US treatment group was 28.58%（<60%,effective cure）.These results showed that the drug-loading nano-micelles can effectively cure cancer after ultrasonic stimulation in vivo.The HE photographs also proved that the drug-loading nano-micelles can ablate and kill cancer cells.Hence,the p LAMA₄₀-OC/DOX in this thesis can cure the HCC after ultrasonic stimulation,offering a new way and platform for the liver cancer therapy.