Study On The Treatment Of Radioiodine-refractory Differentiated Thyroid Cancer With Silica Nanoparticles Loaded With Adriamycin

Differentiated thyroid cancer（DTC）can be divided into papillary thyroid cancer（PTC）and follicular thyroid cancer（FTC）,which make up95%of all thyroid malignant diseases.The incidence growth of thyroid cancer is boosting in recent years,and thyroid cancer has become the fastest growing malignant carcinoma among women.DTC is an indolent cancer,and has good treatment outcome and prognosis.The 5-year survival rate of DTC is 98.1%.The combination of surgery,radioactive iodine treatment,endocrine suppression treatment is the most common clinical treatments for DTC,and can stabilize and relieve the condition of most DTCs.But for a few DTC patients,their DTC may progress clinically,showing loss of differentiation and iodine-taking ability,which leads to RAI treatment failure.This is called radioiodine-refractory differentiated thyroid cancer（RAIR-DTC）,and has a 15%-20%10-year survival rate.Chemical treatment based on Adriamycin（Dox）used to be the only choice for RAIR-DTC patients,but due to its poor spread in thyroid gland and severe blood and cardiac toxicity,its clinical application is restricted.Targeted therapy using kinase inhibitor was introduced to the treatment of RAIR-DTC 10 years ago,but most researches have proven it can only prolong the progression-free survival,but not the overall survival rate.Therefore,the treatment of RAIR-DTC is now a key and difficult research point.In recent years,the application of nanoparticles loaded drugs has shown its particular potential in treating tumors.In fact,a series of nanoparticles anti-tumor drugs has already been approved into clinical application or trials.To improve RAIR-DTC treatment and decrease side effects of chemotherapy,our research uses silica（SiO₂）as carrier to load Adriamycin,aims to kill RAIR-DTC cells,which can selectively accumulate in tumor lesions via EPR effect that solid tumors particularly have.Thyrotropin Receptor（TSHR）is still highly specific to RAIR-DTC or low-differentiated lesions and metastasis,which can be used as a highly specific targeting point.The combination of TSH and nanoparticles increasing density and anti-tumor effect has been reported.The specific bonding of TSHR and TSH can be applied in the targeted nanoparticle delivery system.And to further decrease toxic effect and improve treatment,acid-triggered anti-tumor drug release is a more advanced method.Objective:To construct a targeted delivered,targeted released nanoparticle anti-tumor drug,providing a novel treatment for RAIR-DTC.Methods:（1）ConstructingTSHRtargetedandacid-triggered TSH-SiO₂@Dox nanoparticles.（2）Measure the shape and diameters of nanoparticles with TEM and DLS.（3）Releasing experiment in different PH conditions and active targeting experiment to test the acid-respond drug releasing effect and TSHR-active targeting activity.（4）Selecting undifferentiated FTC-133 by phenotypic function test,examining FTC-133 cells internalize TSH-Si O₂@Dox by confocal laser scanning microscopy（CLSM）and flow cytometry,observing cytotoxicity by MTT and colony-forming test,and measure tumor cell apoptosis by flow cytometry.（5）Establish subcutaneous thyroid tumor transplanted model in vivo,treat tumor with TSH-SiO₂@Dox nanoparticles and evaluate tumor suppressing effect,estimate expression of apoptosis related genes and systematic safety.Results:1.TSH-SiO₂@Dox nanoparticles are successfully compounded.The diameter and shape of the nanoparticles are qualified by TEM and DLS.The drug-loaded nanoparticles can accumulate selectively in tumor via EPR effect which is particular in solid tumors.2.Adriamycin is linked to nanoparticles via acid-reactive bond.It makes the drug can only be released inside tumor,in which has a much more acid environment compared to normal tissues.3.After bonding TSH to its surface,the nanoparticle has active-targeting effect against RAIR-DTC cells.4.TSH-SiO₂@Dox can specifically bind to the surface of FTC-133,and can be taken into tumor cells by endocytosis.The amount of intake is increased with time.5.TSH-SiO₂@Doxhasstrongercytotoxiceffect,andis dose-dependent.6.TSH-SiO₂@Dox can enhance FTC-133 apoptosis induced by Adriamycin.7.TSH-SiO₂@Dox can reduce the growth of subcutaneous transplanted thyroid tumor（FTC-133）significantly,indicating a preferable anti-tumor effect.8.TSH-SiO₂@Dox has enhanced the expression of apoptosis-inducing Gene caspase-3 and caspase-9,while reduced the expression of apoptosis-reducing Gene Bcl-2.This enhanced the apoptosis effect of DOX.9.TSH-SiO₂@Dox does not change the body weight of mice,and does not cause any organ damage.The side effects of anti-tumor drugs are decreased.Conclusions:1.TSH-SiO₂@Dox nanoparticles are successfully compounded.The nanoparticles have passive targeting function by EPR effect,and have active targeting function by bonded TSH.The anti-tumor drug can be released specifically inside tumor cells as result of the acid-reactive bond between Adriamycin and nanoparticle.2.TSH-SiO₂@Dox can be taken specifically by FTC-133 cells in vito,and has stronger cytotoxic effect compared to free Dox,and can enhance the ability of Dox inducing apoptosis.3.In vivo cell experiment shows that TSH-SiO₂@Dox can be taken by retained differentiation thyroid cancer cell line（FTC-133）,and it can release Adriamycin specifically and rapidly in the acid environment that tumor cells have and has a significant tumor-killing effect against thyroid cancer cells.Animal antitumor experiments have proven its anti-growth effect of tumor cells,in which the tumor almost stopped growing,cell necrosis is observed,the expression of intracellular anti-apoptotic gene Bcl-2 is significantly reduced,while expression of apoptotic genes such as Caspse-3 and Caspase-9 are significantly enhanced,prompting the drug-loaded functionally modified silica nanoparticles can induce apoptosis of large number of thyroid cancer cells,showing excellent anti-tumor effects.4.System safety assessment indicates that the nanoparticle has high tumor distribution and releasing specificity and has low systemic toxic side effects.The result shows that TSH-Si O₂@Dox can be used as a novel treatment for RAIR-DTCs in the future.