Homogenous And Different-target Hybrid Nanoparticles Loading With Sorafenib And IMD-0354 For Hepatocellular Carcinoma Chemo-immunotherapy

Hepatocellular carcinoma is the fifth most frequent malignancy,and the third most leading cause of cancer-related deaths worldwide.The main treatments for HCC include radical hepatectomy,liver transplant,local ablation,chemotherapy and immunotherapy.Most HCC patients experience complications due to advanced disease and underlying liver dysfunction.chemotherapy is regarded as the essential strategy for cases with unresectable HCC or those who are not transplant candidates.Sorafenib(SF)as an multi-tyrosine kinase inhibitor for systemic pharmacotherapy,has multiple mechanisms on tumor such as anti-proliferation and anti-angiogenesis,which acts by inhibiting the RAF/MEK/ERK signaling pathway and prevents angiogenesis via inhibiting vascular endothelial growth factor and platelet-derived growth factor receptors.However,it was reported that treating with SF may cause tumor immunosuppression on account of anti-angiogenesis effects.Therefore,new drugs and new treatment regimens are expected to enhance the therapeutic effect of hepatocellular carcinoma.Recently,the rapid development of immunotherapy leads to major changes in tumor therapy.Tumor associated macrophages(TAM)have increasingly gained interest as a therapeutic target since they represent an integral component of the tumor microenvironment.TAMs can be polarized into M1-type and M2-type.M1-type macrophages mediate resistance to intracellular pathogens and tumors,whereas M2-type macrophages mediate resistance to immunoregulation and tissue repair and remodeling.In fact,M2-type macrophage accumulation in solid tumors is associated with poor prognosis and therapy failure.TAM targeted treatment show enormous potential in HCC immunotherapy.Combining chemotherapy and immunotherapy is one of the strategies currently under investigation to solve above problems.Activated immune system and improved tumor immunosuppression microenvironment could be achieved by immunotherapy.Chemotherapy combinations of TAM targeted treatment could effectively enhance the therapeutic effect of HCC,which reprogramming M2-type macrophages towards an M1-type with anti-tumor activity by gene therapy represents a promising therapeutic approach.NF-?B signaling is the central mechanism that maintains the alternative phenotype of TAMs.IMD-0354 is a kind of selective IKK? inhibitor,which could effectively restrain the NF-?B pathway and reprogram M2-type macrophages towards an M1-type.Co-administration of SF and IMD-0354 were supposed to overcome sorafenib immunosuppressive effect and enhance the efficacy of treatment.The differences of physical and chemical properties,in vivo distribution and targeting cells between two drugs were supposed to be consideration when co-administration of SF and IMD-0354.Established two kinds of drug-loading nanocarriers,respectively and administrated at the same time could realize the goal of delivery to different cells.In order to overcome the different in vivo distributions,homogeneous nanocarriers were come into focus,which have the similar physical and chemical properties and lead to similar distributions.Hybrid nanoparticles which were made from two or more materials with different physical and/or chemical natures exhibited superior advantages to load drugs with different physicochemical properties and achieve combination therapy.Meanwhile,hierarchy structure could be observed in hybrid nanoparticles,which were able to combine homogeneity and different target cell delivery.Herein,two homogeneous hybid nanoparticles were designed loading SF and IMD-0354,respectively(CMCS/SF-cLNS and CMCS/M-IMD-cLNS).The same pH sensitive biomacromolecule materials CMCS were used as shell to achieve homogenization.SF and IMD-0354 were loading in lipid-based cationic cores(SF-cLNS and IMD-cLNS).Mannose was chosen as the M2-type TAM targeting ligand to modified the cationic core loading with IMD-0354(M-IMD-cLNS).When co-administration,CMCS could be disassembled in response to tumor acidic microenvironment and then M-IMD-cLNS and SF-cLNS could deliver to M2-type TAM and tumor cells,respectively.Combination efficacy and enhanced effects could be achievedMain methods and results were as follows:.Chapter 1.Determination method of SF and IMD-0354 in nanoparticlesDetermination method of SF and IMD-0354 were established by HPLC.Both drugs showed good linear relationships under determinated concentrations,and the precisions and recoveries could meet the requirements.Chapter 2 Preparation and characterization of CMCS/SF-cLNSCMCS/SF-cLNS was prepared by nanoprecipitation and electrostatic absorbtion method.The formulations were optimized by the single-factor research.The optimized molar ratio of drug to lipid was 1:6,molar ratio of DOTAP to soylecithin was 1:5,and concentration of CMCS was 0.6 mg/mL.The transmission electron micrograph of CMCS/SF-cLNS showed that the nanoparticles were nearly spherical particles and well dispersed.Core-shell structure of CMCS/SF-cLNS was visible,indicating that CMCS was absorbed on the surface of SF-cLNS.The particle size and zeta potential of CMCS/SF-cLNS were 117.9±3.4 nm and-21.1±2.5 mV,DL%was 5.22%±0.25%.The shell of CMCS/SF-cLNS could come off at weak acid pH.The cumulative release percentage of SF from CMCS/SF-cLNS at pH 6.5 was higher than that at pH 7.4(p<0.01).In HepG2 and Hepal-6 cell lines,the cellular uptake of CMCS/cLNS at pH 6.5 was much higher than that at pH 7 4(p<0.05 in HepG2 cells,p<0.001 in Hepal-6 cells).Meanwhile,the cytotoxicity of CMCS/SF-cLNS was better than SF solution in both HepG2 and Hepal-6 cell lines.Chapter 3 Preparation and characterization of CMCS/M-IMD-cLNSMannose-DOPE(M-DOPE)was synthesized as the targeting material for M2-type TAM,and it was confirmed by 1H NMR and IR spectrums.CMCS/M-IMD-cLNS was prepared by nanoprecipitation and electrostatic absorbtion method.The formulations were optimized by the single-factor research.The optimized molar ratio of drug to lipid was 2:9,molar ratio of DOTAP was 10%,and concentration of CMCS was 0.6 mg/mL.The transmission electron micrograph of CMCS/M-IMD-cLNS showed that the nanoparticles were nearly spherical particles with core-shell structure and were well dispersed.The particle size and zeta potential of CMCS/M-IMD-cLNS were 129.4±6.8 nm and-25.5±0.8 mV,DL%was 7.43%±0.51%.CMCS/M-IMD-cLNS showed the similar physicochemical property as CMCS/SF-cLNS,which indicated their homogenous property.The shell of CMCS/M-IMD-cLNS could come off at weak acid pH.The cumulative release percentage of IMD-0354 from CMCS/M-IMD-cLNS at pH 6.5 was higher than that at pH 7.4(p<0.01).Results of cell uptake showed that the cellular uptake of CMCS/M-cLNS at pH 6.5 was much higher than that at pH 7.4 in RAW264.7 cells.Results of the competitive binding assay showed that the cellular uptake of M-cLNS was lower after incubating with free mannose for 1 h.It was suggested that M-cLNS were internalized through CD206-mediated endocytosis leading to efficient delivery to M2-type TAM.The results of cell phenotype conversion showed that the TAM could be reprogramed from M2-type to Ml-type after treated with CMCS/M-IMD-cLNS.Chapter 4 In vivo evaluation of two homogenous nanoparticle coadministeredNear-infrared fluorescence imaging showed that the distribution of CMCS/cLNS and CMCS/M-cLNS were similar,and they could both increase the accumulation of drugs in the tumor site.In vivo antu-tumor activity experiments were investigated in Hepal-6 tumor bearing mice and showed that CMCS/SF-cLNS and CMCS/M-IMD-cLNS co-administration could significantly enhance the anti-tumor activity compared with CMCS/SF-cLNS alone.The immunological evaluation showed that co-administration could promote TAM repolarize from M2-type to Ml-type.Therefore,co-administration could reverse the immunosuppressive microenviroment which were caused by CMCS/SF-cLNS and enhance the therapeutic effect of liver cancer.Histological analysis illustrated that there were no pathological change of organs of mice which were co-administrated with CMCS/SF-cLNS and CMCS/M-IMD-cLNS.The primary safety of the nanoparticles was good.In conclusion,two homogeneous hybid nanoparticles were designed loading SF and IMD-0354,respectively for chemo-immunotherapy.The immunosuppression caused by sorafenib could be reversed and the anti-tumor efficacy was improved.A new idea for chemo-immunotherapy was obtained.