Light Localized Controlled Release Nano-macrophage Drug Delivery System Enhanced HCC Cell-chemotherapy

Hepatocellular carcinoma is one of the most common malignant tumors.Chemotherapy as a systemic therapy has become the main treatment for patients with advanced hepatocellular carcinoma.However,only a few of the chemotherapeutic drugs can reach the target tissue after administration,which limits their efficacy.Therefore,in order to achieve the targeted delivery and accumulation of drugs at the tumor site,the construction of a targeted delivery system is very important.Macrophages have attracted much attention because of their many advantages,such as tumor homing ability,antitumor drug tolerance,unique phagocytosis ability and ability to cross biological barrier.In addition,macrophages also have potential immunotherapeutic capabilities,and the M1-type macrophage have a favorable antitumor effect.Many studies have used macrophages as cell carriers and achieved good antitumor therapeutic effects.However,how to control the localization and release of drugs in macrophages at the tumor site while exerting the immunotherapy effect of macrophages is still a difficulty in current design.To address the above problems,we propose a light localized controlled release strategy,selecting light as the external stimulus to control drug release.O-nitrobenzyl is a common photo-responsive group.The photo-responsive micelles constructed by amphiphilic block copolymers containing o-nitrobenzyl can be cleaved under the irradiation of 365 nm ultraviolet light(Ultraviolet,UV),triggering drug release.In addition,a certain degree of UV can promote the production of exosomes by macrophages and promote drug efflux.However,the weak penetration ability of UV limits its application,and the near infrared(NIR)with strong tissue penetration and high safety can just make up for its shortcomings.Therefore,UCNP are used to convert NIR to UV,and the two are combined to achieve the localized release of drugs in macrophages at the tumor site.Based on the above strategy,this thesis selects polyethylene oxide and nitrobenzyl methacrylate to synthesize amphiphilic block copolymers containing o-nitrobenzyl groups to prepare photoresponsive drug-loaded nanoparticles for the construction of light localized controlled release nano-macrophage drug delivery system,which can trigger drug release under the stimulation of strong penetrating NIR,and then promote drug efflux by UV-promoted exosomes produced by macrophages.Macrophages have strong plasticity and are easily acclimated to M2-type by the tumor immunosuppressive microenvironment.In addition,tumor-associated macrophages at tumor sites are mostly M2-type,which play a role in promoting tumor development.IMD 0354 is a hydrophobic IKKβ inhibitor that can effectively inhibit the NF-κB pathway,polarize carrier macrophages to maintain the M1 phenotype and repolarize tumor-associated macrophages to the M1 phenotype,and play an anti-tumor immune response,improve the tumor immunosuppressive microenvironment and inhibit tumor growth.Sorafenib is the first first-line chemotherapy drug approved for patients with advanced liver cancer.It can not only inhibit tumor cell proliferation,but also target vascular endothelial growth factor receptors to inhibit tumor angiogenesis and achieve dual anti-tumor effects.Therefore,in this paper,IMD 0354 was selected to polarize macrophages and tumor-associated macrophages,maintain the activity of macrophages while exerting its cell therapy effect,and synergize with the chemotherapeutic drug Sorafenib to effectively kill tumors.In summary,based on the light localized controlled release strategy,amphiphilic block copolymers were synthesized by atom transfer radical polymerization,and co-loaded with sorafenib,IMD 0354 and upconversion nanoparticles.After co-incubation with macrophages,it was taken up by macrophages,and a photolocalized controlled-release nano-macrophage drug delivery system was constructed for the treatment of hepatocellular carcinoma.Utilize the natural targeting ability of macrophages to achieve targeted delivery of drugs.After the NIR is converted into UV by the UCNP,the drug release is controlled while maintaining the activity of macrophages,and the drug efflux is promoted.The released IMD 0354 simultaneously polarized carrier macrophages and tumor-associated macrophages to an anti-tumor M1 phenotype,enhanced T cell immunity,and combined with the tumor-killing effect of sorafenib to effectively inhibit tumor growth.Main methods and results were as follows:Chapter 1.Determination method of SF and IMD 0354A method for the determination of SF and IMD 0354 was established by HPLC.Its specificity is good,the linear relationship between SF and IMD 0354 is good in the concentration range of 1-30 μg/mL and 0.5-20 μg/mL,and the intra day and inter day precision and method recovery meet the content determination requirements.It can be used for the in vitro content determination of SF and IMD 0354.Chapter 2.Preparation and characterization of USIP and USIP@MFirstly,the UV responsive amphiphilic block copolymer PNB was synthesized,and the near-infrared light responsive drug loaded nanoparticles usip co loaded with ucnp,SF and IMD 0354 were prepared.The successful synthesis of PNB was verified by 1H-NMR and GPC.Nanoparticles usip have uniform particle size,round shape and good dispersion.They were prepared by co-incubation in vitro USIP@M and the optimum incubation concentration of USIP was SF 200 μg/mL and the best incubation time was 2 h.The phototoxicity was verified by living and dead cell experiment.The results showed that near-infrared light would not affect the USIP@M cause obvious damage.The investigation of release form shows that some drugs will be released in the form of exosomes,and the light response release of drugs can promote the polarization of macrophage cells to the pro-inflammatory M1 phenotype.Chapter 3.The tumor targeting ability and in vitro uptake evaluation of USIP@MThe effect of H22 cells on near infrared irradiation was investigated UCP@M The results showed that UCP@M+L The release solution can better enter the cells and significantly promote the uptake of H22 Tumor cells.It is proved by Trans well experiment and small animal imaging experiment USIP@M It has good tumor targeting ability in vivo and in vitro,and can recruit and infiltrate well in tumor tissues.The investigation of tumor deep permeability showed that near-infrared light irradiation could promote UCP@M deep penetration of the tumor.Chapter 4.The study on antitumor efficacy and safety of USIP@MUsing H22 as a cell model,the in vitro antitumor effect of USIP@M was investigated by cytotoxicity experiments.Using H22-bearing Balb/c mice as an animal model to investigate the in vivo antitumor effect of USIP@M.Cytotoxicity test and pharmacodynamics test in vivo showed that USIP@M+L It can combine the tumor killing effect of SF,the immune activation effect of IMD 0354 and the immunotherapeutic effect of macrophages,significantly inhibit tumor growth and have significant antitumor effect.The number of TAM,CTL,Treg and T cells in the tumor and the levels of pro-inflammatory and anti-inflammatory factors in serum were measured.The results showed that USIP@M+L can effectively polarize TAM into M1 type and effectively improve the tumor immunosuppressive microenvironment.Hemolysis test and H&E staining of main organs of mice proved that USIP@M+L The preliminary safety is good.