Study On The Application Of Phototherapy And Its Synergistic Effect With Chemotherapy In The Treatment Of Thyroid Cancer

Background:In recent years,the incidence of thyroid cancer has continued to rise,and it has become the seventh most common malignant tumor in China.Most patients have good prognosis after surgery,radioactive 131 or endocrine therapy,but the treatment of advanced differentiated thyroid cancer(DTC)and anaplastic thyroid cancer(ATC)has always been a clinical difficulty.These patients have high mortality rate,short survival,and lack of effective treatment methods.Even with the application of molecular targeted therapy and immunotherapy,the effect is still very poor.Therefore,research and development of new treatment methods for thyroid cancer is imminent.As a non-invasive treatment method,photothermal(PTT)or photodynamic therapy(PDT)mediated by photothermal conversion reagents or photosensitizers is widely used in malignant tumors,skin disease,and rheumatism due to its non-invasive,low toxicity,reproducible and non-resistant characteristics.The anatomical location of thyroid cancer is superficial,and light does not need to penetrate deeply,so it is very suitable for phototherapy.However,on account of the poor solubility,instability under physiological environment,and less accumulation in tumor sites of photothermal conversion reagents and photosensitizers,the final therapeutic effect of phototherapy is not ideal.With the development of material chemistry and bioengineering technology,antitumor nanodrugs formed by nano-sized nanocarriers and active drugs have attracted great attention.Compared with traditional anticancer drugs,they have the advantages of good water solubility,strong targeting,less side effects,and easy modification.So far,about 2000 nanodrugs have been approved for marketing or clinical trials.If a photothermal conversion reagent or photosensitizer is prepared into a nano-preparation,its circulation time in the body,tumor targeting as well as the therapeutic effect can be improved.The delivery of the first-generation nanomedicine has problems such as non-specific uptake,early drug leakage,and uncontrollable drug release.In order to better achieve the anticancer goal of high curative effect and low toxicity,people have developed tumor microenvironment-responsive smart nanomedicine systems.They can sense and respond to specific triggers in tumor which are absent in normal tissues,such as hypoxia,weak acidity,excessive glutathione,hydrogen peroxide or certain enzymes,to achieve tumor-specific drug release.Such nanomedicines have huge application potential due to their maximized anticancer effect and minimized system toxicity.If multiple therapies are combined organically with the help of a responsive drug platform,they can complement each other and inhibit tumor development by different mechanisms to exert a better anticancer effect.Purpose:(1)Synthesize the high-efficiency photothermal conversion material polyethylene glycol(PEG)gold nanostars(AuNS)and nanomicelles(Ce6 NP)encapsulating photosensitizer chlorin e6(Ce6),and explore the therapeutic effects of PTT and PDT on thyroid cancer,respectively,to investigate the feasibility of applying phototherapy to treat thyroid cancer.(2)Design and construct the light-promoted hypoxia-responsive paclitaxel dimer prodrug nanosystem(Ce6/PTX2-Azo NP),and explore the effect of its photodynamic-chemotherapy synergistic therapy in the treatment of thyroid cancer.Furthermore,we would like to verify whether it can enhance the antitumor efficacy,improve the drug loading and drug release controllability,and reduce the side effects caused by drug off-target at the same time,to obtain a win-win situation of high efficacy and low toxicity.Methods:(1)Use tetrachloroauric acid and sulfhydryl modified PEG as raw materials to synthesize the nano-sized photothermal conversion material AuNS.Its physical and chemical characteristics and stability were evaluated by transmission electron microscopy(TEM),dynamic light scattering(DLS)and ultraviolet-visible light spectrophotometry(UV-vis).In the in vitro environment,the temperature real-time recorder and infrared thermal imager were used to detect the heating ability of AuNS.Human-derived DTC cell TPC-1 was selected to explore the killing mode and killing effect of AuNS-mediated PTT on thyroid cancer cells by means of cytotoxic MTT detection,live-dead cell staining and flow cytometric detection.Finally,a nude mouse thyroid cancer xenograft model was constructed to explore the biological safety and anti-tumor effect of AuNS.(2)Using paclitaxel(PTX)and Ce6 as raw materials,the hypoxia-responsive dimer prodrug PTX2-Azo and Ce6 grafted polymer mPEG-Poly(Ser-Ce6)were synthesized,respectively.Proton nuclear magnetic resonance and linear ion trap mass spectrometry were used to detect the structure and purity of these synthesized products.After that,the hypoxic conditions of tumors were simulated in vitro to verify the responsive lysis of PTX2-Azo by high performance liquid chromatography.The Ce6/PTX2-Azo NP was prepared by solvent exchange method.Its physical and chemical properties as well as stability were characterized and tested by TEM,UV-vis,fluorescence spectrometer,DLS and other equipment.9,10-Anthradiiyl-bis(methylene)dimalonic acid was employed to detect the production of reactive oxygen species(ROS).At the cellular level,the laser confocal microscope was used to investigate the endocytosis of drugs.Subsequently,the ROS/hypoxia detection probe was used to verify that Ce6/PTX2-Azo NP could generate ROS under light and consume oxygen which leading to aggravation of hypoxia.To compare the sensitivity of different thyroid cancer types to the nanomedicine,DTC cells TPC-1 and ATC cells C643 were selected for cytotoxicity testing.Single PDT Ce6 NP,single chemotherapy PTX2-Azo NP and the PTX injection Taxol were set as controls.Live and dead cell staining experiment was conducted to verify MTT results.At the level of animal experiments,real-time detection of drug distribution in the body and tumor accumulation after system administration was done through the in vivo fluorescence imager.Finally,the antitumor experiment was performed to evaluate the anticancer efficacy and biological safety.Results:(1)The PEGylated gold nanostar AuNS with branched morphology was synthesized.The method is simple and the batch difference is small.Different from traditional photothermal materials gold nanorods and gold nanoshells,AuNS has both nano spherical shells and nano-tip enhanced plasmon resonance.Due to its irregular shape and large specific surface area,it has much red shift in the absorption spectrum and better ability to generate heat.Through in vitro experiments,we verified the stability under physiological conditions and photothermal conversion ability of AuNS.It could rapidly heat up under 808 nm laser irradiation,which leading to the necrosis of TPC-1 cells,thereby effectively killing cancer cells.The nanoparticle has good biocompatibility.After intravenous injection of the drug into the nude mouse subcutaneous thyroid cancer model,AuNS could accumulate at the tumor site.Through local irradiation,the tumor tissue was significantly warmed up to inhibit further growth.According to the calculation of tumor volume,the tumor inhibition rate is as high as 95%.During the experiment,AuNS had no side effects on the systemic system.No obvious organ damage was observed.(2)First,the hypoxia-sensitive azobenzene bond was used to connect the chemotherapeutic drug PTX to prepare the dimer prodrug PTX2-Azo.By using sodium dithionite to simulate azo reductase in vitro,we proved that the responsive prodrug can break and release PTX monomer under the hypoxic conditon.Then we encapsulated the prodrug in polymer mPEG-Poly(Ser-Ce6)to obtain nanoparticle Ce6/PTX2-Azo NP.This nanoparticle has small size,good stability,and high drug loading.It could be taken up and endocytosed by cells.Under light irradiation,Ce6 generated singlet oxygen,which not only resulted in cell death through oxidative stress,but also consumed oxygen to aggravate the hypoxic degree in tumor and then promoted the cleavage of dimer prodrug to release active PTX and exert the chemotherapeutic effect.In C643 and TPC-1 cells,we observed enhanced cytotoxicity caused by Ce6/PTX2-Azo NP-mediated synergy therapy.The IC50 value of C643 cells is lower,indicating that the nanodrug system is more effective for ATC.Through real-time in vivo fluorescence imaging monitoring,we found that compared with small molecule drugs,Ce6/PTX2-Azo NP had a significantly longer circulation time and increased tumor accumulation.Compared with the control group or the monotherapy group,the photodynamic-chemotherapy synergy group had the best treatment effect.No side effects on liver and kidney function were observed.Conclusion:(1)It has been confirmed that the synthesized AuNS can effectively inhibit thyroid cancer at the molecular,cellular,and animal level,which provides a new option for the non-invasive treatment of early microthyroid cancer or advanced thyroid cancer that cannot be operated on.(2)The prepared photo-promoted PTX dimer prodrug nanosystem can realize the controlled release of drugs and reduce the systemic toxicity caused by off-target effect.Meanwhile,the photodynamic-chemotherapy synergistic pattern can solve the defect of monotherapy treatment and enhance antitumor efficacy.