Study On The Antitumor Application Of Photothermal-responsive Double-loading Erythrocyte Membrane Nanovesicles

In recent years,integrating chemotherapeutic drugs and photosensitizers into multi-functional nanocarriers to achieve better antitumor efficacy and reduce the toxicity of chemotherapeutic drugs normal tissues has become a hot topic in the field of tumor therapy.In this study,relying on the excellent biological characteristics of the natural erythrocyte membrane,a novel photothermally responsive erythrocyte membrane nanovesicle double loading delivery system?DIRNPs?with a long cycle and tumor targeting was constructed utilizing nano-co-extrusion process and ligand modification strategy.The DIRNPs can locate the tumor tissue specifically,release chemotherapeutic drugs and quickly respond to the near-infrared stimulation,so as to realize the combination of the advantages of chemotherapy and hyperthermia for the tumor and achieve the purpose of improving the synergistic effect.The main research contents of this paper are as follows:Polylactic acid-glycolic acid copolymer?PLGA?nanoparticles loaded with dual drugs?doxorubicin and indocyanine green?were prepared by nano-deposition technology,and the natural erythrocyte membrane was coated on the outer layer of the nanoparticles and the targeting ligand?DSPE-PEG2000-FA?and long-cycle ligand?DSPE-PEG2000?was modified on the surface,the nano-co-extrusion technology was used to finally construct photothermally responsive erythrocyte membrane nanovesicle double loading delivery system?DIRNPs?.The average particle size of the nano-drug loading system was 158.4±2.6 nm,the surface potential was-5.79 mV,and the drug encapsulation efficiency of doxorubicin and indocyanine green was 61.78±1.37%and 62.4±3.73%,respectively.The research results show that the DIRNPs system has good light-to-heat conversion efficiency,after being irradiated with near-infrared laser?1.5 W/cm²,808 nm?,the maximum temperature can reach76.7°C.The in vitro release results showed that within 120 h,the cumulative release rates of DIRNPs in different PBS media?pH=5.0,6.0,7.4?were 99.53%,75.28%,and 54.21%,respectively.When the near-infrared laser irradiation is given at the same time,the release amount of the system under the condition of pH7.4 can be increased to 90.32%,indicating that its drug release has pH-dependent and photo-promoting properties.The results of the stability experiments showed that during the test period?4°C,30 d?,the particle size of the DIRNPs system did not change significantly.Compared with 0 h,the encapsulation efficiency of doxorubicin was greater than 90%,and the fluorescence degradation rate was only 6.56±1.32%.At the same time,the system can also significantly improve the light and thermal stability of indocyanine green.The cytotoxicity test results showed that DIRNPs can significantly inhibit the growth of tumor cells in vitro compared with the free doxorubicin control group.When DIRNPs were incubated with HepG2cells for 48 h,the incubation concentration?as doxorubicin?was increased to 1.0?g/mL,the cell survival rate decreased to 46.80%,and the IC₅₀ was0.77?g/mL.When DIRNPs combined with near-infrared laser irradiation for 180 s,the survival rate of HepG2 cells was only 10.11%,and the IC₅₀decreased to 0.57?g/mL.The uptake of DIRNPs by HepG2 cells is an energy-dependent active transport process,which is regulated by caveolin,clathrin,and folate receptor-mediated endocytosis.DIRNPs can significantly increase the level of reactive oxygen species in Hep G2 cells.When combined with near-infrared laser irradiation,it can further promote apoptosis and necrosis of HepG2 cells,and inhibit cell migration simultaneously,suggesting its potential application for tumor metastasis treatment.The results of pharmacokinetic studies in vivo show that compared with the free drug doxorubicin,DIRNPs can significantly increase the concentration of doxorubicin in the plasma and prolong the circulation time of the drug in the body.Its C_max,AUC_0-t,MRT,and t_1/2 were 7.71±0.40?g/mL,60.22±6.51 mg/mL·h,13.81±0.78 h,and 17.61±1.87 h,respectively.DIRNPs are mostly distributed in the liver and spleen tissues,and the drug is less distributed in the heart,suggesting that it reduces doxorubicin-related side effects.Antitumor effects of DIRNPs combined with chemotherapy and hyperthermia in vivo were investigated in tumor-bearing mice.The tumor growth dynamics of tumor-bearing mice were monitored for 17 days,the tumor volume of the PBS group and the free doxorubicin group was3354.03 mm³ and 1167.72 mm³,respectively,while the tumor volume of the DIRNPs combined laser treatment group was only 472.86 mm³.The tumor weight,the tumor burden was 0.26 g,0.94%,and the tumor growth inhibition rate of H22 tumor-bearing mice is as high as 88.38%,suggesting that the dual therapy of DIRNPs combined with chemotherapy and hyperthermia has a significant inhibitory effect on H22 liver cancer cells,and the treatment effect is much higher than chemotherapy or hyperthermia alone?p<0.001?.Histological analysis showed that DIRNPs combined with laser can cause severe tumor tissue necrosis,showing typical heat loss characteristics of coagulative necrosis and nuclear dissolution,showing a large number of sparse and hollow cell distributions,and H&E staining images of normal tissues showed that DIRNPs can be significantly reduced Myocardial tissue toxicity caused by doxorubicin is of great significance for its clinical application.After injecting DIRNPs into the tail vein of mice,laser irradiation can rapidly increase the local temperature of the tumor area,and it can reach 47.3?for 3 minutes,which can cause severe thermal damage to tumor cells.In summary,the DIRNPs drug-loading system can effectively co-deliver anti-tumor drugs and photosensitizers and specifically target to tumor tissues.The weakly acidic microenvironment of the tumor and near-infrared laser can promote the rapid release of drugs at the tumor site.The efficient photothermal conversion of photosensitizers can rapidly increase the local temperature of tumor tissues to promote the apoptosis and necrosis of tumor cells,and synergistically improve the anti-tumor treatment effect.It shows great potential in anti-tumor combination therapy.