Preclinical Safety Evaluation And Hepatotoxicity Mechanism Study Of Copper Sulfide Nanoparticle-based Photothermal Agents

Photothermal therapy(PTT)utilizes photothermal agents(PAs)to convert external light into heat energy to increase the temperature surrounding tumor cells,aiming to trigger tumor cell death or increase the sensitivity of other treatment types.Copper sulfide nanoparticles(Cu2-xS NPs,0≤x≤1)are strong near-infrared(NIR)absorbing agents widely recognized in PTT and photoacoustic imaging(PAI).Compared with the photothermal agents such as small molecular organic dyes(such as ICG)and Au NPs or carbon NPs,Cu2-x S NPs has better biocompatibility and photostability.Therefore,Cu2-xS NPs is expected to become the next photothermal agent to enter clinical research besides gold nanoshells and ICG.However,the road to clinical translation of Cu2-xS NPs using green controllable preparation methods is still lengthy,mainly limited by detailed preclinical safety evaluation.Currently,there are limited preclinical studies on the systematic safety evaluation and toxicity related mechanisms of Cu2-xS NPs.In this study,two kinds of BSA@Cu2-xS NPs with different sizes were synthesized using bovine serum albumin(BSA)as template by biomeralization method,and a series of preclinical studies on their physicochemical properties,PTT and PAI effects in vivo and in vitro,distribution,metabolism and pharmacokinetics(DMPK)in vivo,safety evaluation and related toxicity mechanism were evaluated.The main findings are as follows:Chapter 1:Firstly,the research progress of PTT,PAI,photothermal agents and imaging agents is briefly reviewed.Then,the application and advantages of Cu2-xS NPs in various diagnostic and therapeutic platforms for diseases are summarized.At last,the basis of the topic selection,design ideas and research contents of the doctoral project are specified.Chapter 2:A green synthesis method of BSA@Cu2-xS NPs was established.Two sizes BSA@Cu2-xS NPs——L-BSA@Cu2-xS NPs and S-BSA@Cu2-xS NPs were successfully synthesized by biomineralization,the average particle sizes were 17.8 nm and 2.8 nm respectively under electron microscopy.The results show that L-and SBSA@Cu2-xS NPs have excellent dispersibility,chemical stability,photothermal stability,strong near-infrared light absorption ability,as well as good photothermal heating and photoacoustic imaging effect.The photothermal conversion efficiency of L-BSA@Cu2-xS NPs and S-BSA@Cu2-xS NPs was 42.7%and 44.9%respectively.In vivo experiments demonstrated that L-and S-BSA@Cu2-xS NPs mediated PTT could effectively trigger tumor cells death and prevent malignant metastasis of tumor cells.Chapter 3:The absorption,distribution,metabolism and excretion of L-and SBSA@Cu2-xS NPs in rats showed that S-BSA@Cu2-xS NPs were distributed more slowly and cleared faster than L-BSA@Cu2-xS NPs.Moreover,the two kinds of NPs were mainly distributed in the liver,and were mainly eliminated from feces by hepatobiliary elimination,the clearance rate of S-BSA@Cu2-xS NPs in feces and urine was higher than that of L-BSA@Cu2-xS NPs.S-BSA@Cu2-xS NPs was basically metabolized completely in rats within 7 days after single administration,while LBSA@Cu2-xS NPs still accumulated a lot in the liver.L-and S-BSA@Cu2-xS NPs were confirmed the relatively good biocompatibility in the safety evaluation of the 14day consecutive administration,but there was a potential risk of hepatotoxicity.A large number of NPs was observed in the pathological sections of the liver.The hepatotoxicity of L-BSA@Cu2-xS NPs was characterized by a large amount of inflammatory cell infiltration,while that of S-BSA@Cu2-xS NPs were characterized a small amount of inflammatory cell infiltration and local fibrous tissue formation in the pathological sections.However,after 28 days of recovery period,the hepatotoxicity symptoms of the rats in S-BSA@Cu2-xS NPs group basically recovered,while those in the L-BSA@Cu2-xS NPs group did not recover,and the inflammatory infiltration was more serious.Chapter 4:In vitro cell experiments proved that the difference of hepatotoxicity between the two sizes BSA@Cu2-xS NPs,S-BSA@Cu2-xS NPs were more easily accumulated in hepatocytes and rapidly discharged from the body through the pathway of hepatobiliary elimination,so the hepatotoxicity produced by S-BSA@Cu2XS NPs could be quickly recovered after drug cessation.However,L-BSA@Cu2-xS NPs were more easily accumulated in Kupffer cells and difficultly metabolized in the liver,resulting in long-term and irreversible hepatotoxicity.Chapter 5:The hepatotoxicity of two sizes BSA@Cu2-xS NPs and their differences were further demonstrated from the perspective of molecular biology through transcriptomic,proteomics and RT-qPCR methods.After 14 consecutive days of administration,the expression levels of genes related to the inflammatory pathway,lipid metabolism pathway,bile acid and cholesterol metabolism pathway,CYP450 pathway of drug metabolism and copper ion transport pathway were significantly altered.Moreover,the expression of genes significantly altered in S-BSA@Cu2-xS NPs group returned to normal levels during the 28-day recovery period,whereas the significantly altered genes of the L-BSA@Cu2-xS NPs-administered rats can not be recovered.In this study,L-and S-BSA@Cu2-xS NPs prepared by green synthesis method have good stability,biocompatibility,high photothermal conversion efficiency,good PTT and PAI effect,which are NIR-Ⅱ PAs with potential clinical transformation ability.Safety evaluation experiments suggested that L-and S-BSA@Cu2-xS NPs might have potential hepatotoxicity.However,the hepatotoxicity caused by SBSA@Cu2-xS NPs could basically recover to normal level within 28 days after drug cessation,but the hepatotoxicity caused by L-BSA@Cu2-xS NPs could not be recovered.Thereby,the results of this study demonstrate that S-BSA@Cu2-xS NPs with size of 2.8 nm has more clinical transformation value as a diagnostic nanoagent for PTT and PAI of tumor.