An Experimental Study On Treatment Of Liver Cancer By Sorafenib Loaded Prussian Blue Nanoparticles Combined With Anti-PD-L1 Monoclonal Antibody

BackgroundLiver cancer is a common and malignant tumor with poor prognosis in China.It's prone to recurrence and metastasis and has few effective treatment.Studies have found that hypoxia and immunosuppression in the tumor microenvironment are important causes of recurrence and metastasis.Recently,nanoprobes and immunotherapy have emerged as promising cancer treatment strategies.Nanoprobes with photothermal properties and antitumor drug delivery capabilities can accurately and effectively kill tumors,and reduce local tumor recurrence.Nanoenzyme with catalase-like ability decompose H2O2 to produce(O2),and relieve tumor hypoxia.Immunotherapy helps the immune system to better act against cancer.Studies have demonstrated that hypoxia relief and photothermal effects can promote antitumor immunity and alleviate immunosuppression.However,a nanoprobe that combined antitumor drug-photothermal therpary and hypoxia relief has never been reported.Therefore,we designed a liver cancer targeting multifunctional nanoprobe that incorporate above functions.We hope our nanoprobe can reduce local recurrence,combined with immunotherapy,inhibit metastasis and recurrence in liver cancer.Objectives1.To prepare sorafenib(SF)loaded prussian blue nanoparticles(PBNPs)and conjugate it with SP94(liver cancer specific peptide)and cyanine(Cy)5.5.2.To explore the efficacy of SP94-PB-SF-Cy5.5 alone and SP94-PB-SF-Cy5.5 combined with anti-PD-L1 monoclonal antibody(mAb)in the treatment of liver cancer.Methods1.Preparation and characterization of nanoprobe:PBNPs were prepared via one-step method.1-tetradecanol was coloaded with SF for controlled drug release.PBNPs were further modified by SP94 and Cy5.5,and finally SP94-PB-SF-Cy5.5 NPs was synthesized.The features of SP94-PB-SF-Cy5.5 NPs were observed by transmission electron microscopy(TEM),fourier transform infrared spectrometer(FTIR),dynamic light scattering(DLS),high performance liquid chromatography(HPLC)and so on.2.Function verification of nanoprobe:cytotoxicity was evaluated by CCK-8 assay and Calcein-AM and propidium iodide staining.Fluorescence imaging,magnetic resonance imaging and photoacoustic imaging were used for tumor imaging and NPs biodistribution monitoring.Immunofluorescence staining,chemiluminescence,enzyme-linked immunosorbent assay and flow cytometry were used to analyze the effects of SP94-PB-SF-Cy5.5 NPs on hypoxia relief and immune promoting in the tumor microenvironment.3.In vivo antitumor efficacy evaluation:HepG2 tumor-bearing BALB/c nude mice were used and treated with nanoprobe.The tumor volume,tumor weight,body weight and survival was monitored.Serum samples were collected for liver and renal function assay.Major organs were collected for histopathological analysis.Hepa1-6 tumor-bearing C57BL/6N mice were used for tumor metastasis and recurrence model.The mice were treated with SP94-PB-SF-Cy5.5 NPs and anti-PD-L1 mAb.The growth of tumors was monitored by measuring tumor volume,tumor weight and bioluminescent imaging.The mechanism of the synergistic antitumor effects was analyzed by flow cytometry.Results1.By TEM,we observed quadrate and homogeneous NPs with diameters ranging from 90 to 110 nm.FTIR and UV-Vis-NIR spectroscopy demonstrated the successful conjugation of SP94 and Cy5.5 to the NPs.SF loading content was 5%.At 42?,?74%of the SF was released.After 5 min of irradiation,the maximum temperatures of SP94-PB-SF-Cy5.5 NPs were 48.1 7±0.95?.There was almost no difference in temperature increase across five rounds of irradiation.NPs retained excellent catalytic activity after multiple additions of H2O2.2.CCK-8 assay showed that the viability of cells incubated with SP94-PB-SF-Cy5.5 NPs was>90%.After the NIR irradiation was applied,the viability decreased to 20.86±5.67%.The treatment of SP94-PB-SF-Cy5.5+NIR induced immunogenic cell death,stimulated dendritic cell maturation,promoted infiltration of cytotoxic T lymphocytes,alleviated the tumor hypoxia and reduced M2 macrophages.Multimodality imaging showed that signals of the tumors in the SP94-PB-SF-Cy5.5 NP group were obviously stronger.3.In the SP94-PB-SF-Cy5.5+NIR group,tumors were completely eradicated.No obvious side effects were observed during the observation period and the survival rate was obviously longer than mice in other groups.In the SP94-PB-SF-Cy5,5 NPs+NIR+anti-PD-L1 mAb treatment group,the growth of metastatic and recurrent tumors were inhibited.In metastatic tumors,flow cytometry showed that the percentages of cytotoxic T lymphocytes and T helper cells increased while regulatory T cells and myeloid-derived suppressor cells decreased.In spleens,an increase in the percentage of effector memory T cells was revealed by flow cytometry.ConclusionWe for the first time developed a safe and multifunctional SP94-PB-SF-Cy5.5 NP,which realized targeted and controlled SF release.The combination of SF and photothermal therapy effectively eliminated tumor cells in the tumor site.Moreover,the catalase-like ability and photothermal effect of SP94-PB-SF-Cy5.5 NP effectively alleviated tumor hypoxia and immunosuppression.When combined with anti-PD-L1 mAb treatment,the NPs also displayed promsing inhibitory effects on tumor metastasis and recurrence.