| Malignant tumor seriously affect human life and health,and incidence rate and mortality rate has been rising.However,due to the complex process of tumor,monotherapy could not achieve the ideal therapeutic efficacy,and combination therapy has gradually become a hot spot.Combination of chemotherapy and immunotherapy(chemoimmunotherapy)has been a promising strategy for cancer therapy.Different immunotherapeutic drugs focusing on these immune cells,such as immune checkpoint inhibitors,cytokines,have been widely combined with chemotherapeutic drugs to achieve chemoimmunotherapeutic effect.However,the administrations of different drugs simultaneously were faced with challenges,such as the distinct distribution in vivo of different drugs,the short half-lives,the insufficient tumor accumulation,the unpredictable ratio of different drugs in tumor,and the serious systemic adverse effects,resulting in the limited synergistic antitumor efficacy.Therefore,a novel delivery strategy is urgently needed to response these challenges and achieve the optimal chemoimmunotherapeutic antitumor efficacy.Recently,cell therapy has emerged as a novel immunotherapeutic approach for cancer treatment,which directly transports therapeutic immune cells,such as T cells,NK cells and macrophages,to eliminate cancer cells.Compared with traditional drug molecules,cells with exquisite sensitivity and specificity can sense diverse signals,move to specific sites in the body,make decisions and execute complex response behaviors.Based on these characteristics,cell therapy shows the advantages of higher specificity and lower side effects.Some studies have developed smarter generations of cell therapy based combination therapy strategies,such as the fourth-generation CAR-T were engineered to express pro-inflammatory cytokines,combining CAR-T therapy with immune checkpoint inhibitors and combining CAR-T therapy with vaccines.Macrophages are the major tumor-infiltrating immune cells population with a critical role in regulating tumor progression,educated by the tumor microenvironments to differentiate into M1-type macrophages(M1)and M2-type macrophages(M2).M1-type macrophages secreting immunogenic cytokines,such as IL-12 and TNF-?,improved the immune response to exert inhibitory effects on tumor growth,and M2-type macrophages secreting immunosuppressive cytokines,such as IL-10 and TGF-?,impaired antitumor immunity to enhance tumor growth.Therefore,M1-type macrophages-based cell therapy strategies show enormous potential in tumor immunotherapy.Cell-mediated drug delivery systems,such as stem cells,neutrophil or macrophages,have become novel and beneficial drug delivery strategies.Cell-mediated drug delivery systems employ specific cells as drug carriers to transport drugs to the lesion by the natural targeting functions of cells,which have the advantages of increasing drug accumulation in tumor tissues,evading reticuloendothelial system(RES)clearance,prolonging circulation time and improving biocompatibility.Macrophages could be recruited to tumor tissues by some chemokine,such as CCL2.Moreover,macrophages are major phagocytes with innate phagocytotic capability.Consequently,macrophages might be ideal delivery vessels to target deliver chemotherapeutic drugs to tumor tissues and enhance therapeutic efficacy.Herein,we hypothesized a "cell-drug" strategy,which refers to using M1-type macrophages with both of tumor targeting ability and immunotherapeutic efficacy as chemotherapeutic drugs delivery vessel to develope a M1-type macrophages-based targeting delivery and combination therapy system.On the one hand,M1-type macrophages were used as therapeutic tool to provide immunotherapeutic effect;on the other hand,M1-type macrophages were used as chemotherapeutic drugs delivery vessels to target deliver chemotherapeutic drugs to tumor tissue and enhance chemotherapy effect.In this study,a polarized macrophages-based treatment and drug delivery system involving M1-type macrophages carrying sorafenib(SF)loaded lipid nanoparticles(M1/SLNP)was designed for enhanced chemoimmunotherapy of hepatocellular carcinoma(HCC).SF is a multityrosine kinase inhibitor and the first-line chemotherapy drug for HCC and approved by FDA in 2007.CCL2 required for recruitment of macrophages was overexpressed in HCC and macrophages could be recruited to the tumor tissues effectively.SF loaded lipid nanoparticles(SLNP)were formed by loading SF into lipid nanoparticles,and then loaded into M1-type macrophages.Lipid nanoparticles with favorable biocompatibility and excellent safety were widely used as drug delivery vessels,could avoide the damage of SF to macrophages.The Main methods and results were listed as follows:Chapter 1.Determination method establishment of SFHigh performance liquid chromatography was used to establish the determination method of SF.The method was characterized by good linear relationships and high precisions,and could be used for the determination of SF.Chapter 2 Preparation and characterization of Ml/SLNPSLNP was prepared by nanoprecipitation method.The formulations were optimized,and 12:100 was determined as the optimal SF/soya lecithin mass ratio,7.5 mg/mL was determined as the optimal soya lecithin concentration.SLNP were successfully prepared with particle size of 67.63 ± 5.02 nm,and drug-loading efficiency(DL%)of 5.58 ± 0.41%.The transmission electronic microscopy(TEM)image of SLNP showed that SLNP were nearly spherical particles and with good dispersibility.M1/SLNP was obtained by incubating macrophages with SLNP.The drug-loading(?g/10 6 cells)of M1/SLNP was 38.18 ± 0.80?g/106 cells.The macrophages phenotype was evaluated,and the results indicated that the percentage of M1-type macrophages in M1/SLNP group was increased significantly.The levels of cytokines(IL-12?TNF-?)were analyzed,and the results indicated that the levels of IL-12 and TNF-? were increased in M1/SLNP group.The release profiles of M1/SLNP were evaluated.The cumulative release of total SF from M1/SLNP at 72 h was 48.4%,indicating that SF could be released from M1/SLNP.SF would be released from M/SLNP as the form of SF or SLNP.Chapter 3 The tumor targeting ability and deep tumor-penetrating ability evaluation of M1/SLNPThe tumor targeting ability of M/SLNP and M1/SLNP was evaluated in vitro and in vivo,proving M/SLNP and M1/SLNP could reach the tumor tissues and enhance the tumor targeting delivery.The deep tumor-penetrating ability of M1/SLNP were evaluated in vitro and in vivo,proving M1/SLNP exhibited deep tumor-penetrating ability.Chapter 4 The chemoimmunotherapeutic antitumor efficacy evaluation of M1/SLNPThe in vitro cytotoxicity was investigated by methylthiazol tetrazolium(MTT)assay,suggesting that M1/SLNP could improve the cytotoxicity of SLNP.The therapy effect of M1/SLNP was evaluated in vivo.Tumor volumes of M1/SLNP group were significantly decreased compared with SLNP group(p<0.01),indicating M1/SLNP exhibited enhanced antitumor efficacy.The anti-proliferation ability were evaluated by H&E staining and Ki-67 staining.The resuts of haemolysis assay in vivo showed that M1/SLNP could increase the percentage of M1-type macrophages,CD3+CD4+T cell,CD3+CD8+T cell in tumor tissues,reduce the percentage of Treg in tumor tissues,promote the level of IL-12 and TNF-?,indicating that M1/SLNP could relieve the immunosuppressive tumor microenvironments.The primary safety of M1/SLNP was good.In summary,we developed a M1-type macrophages-based drug delivery system.M1-type macrophages with both of tumor targeting ability and immunotherapeutic efficacy were used as drug delivery vessel,enhancing drug accumulation in tumor sites and enhancing the therapeutic effect of chemotherapy.Meanwhile,M1-type macrophages were used as therapeutic tool displayed immunotherapeutic antitumor effect,relieving the immunosuppressive tumor microenvironments,achieving the enhanced chemoimmunotherapeutic antitumor efficacy. |
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