Construction And Evaluation Of The Effectiveness Of Nanoparticles With Molecular Targeted Drugs And Radioiodine Co-loaded For Anaplastic Thyroid Cancer Cells

Background: Thyroid cancer is the most common endocrine malignancy.Some patients with differentiated thyroid cancer have poor prognosis because of occurrence of resistance to radioiodine therapy.Moreover,molecular targeted drugs have obvious side effects and might be prone to drug resistance.The prototypical HSP90 inhibitor 17-AAG and m TOR inhibitor Torin2 could restrain the proliferation of anaplastic thyroid cancer（ATC）cells and induce apoptosis.Mesoporous silica nanoparticles（MSNs）could offer an effective nanotechnology platform for solubilization,co-loaded and targeted therapy of multiple drugs.Objective: We plan to construct a new 3-in-1 nanoparticles,and investigated the function of the nanoparticles,which loaded dual drugs and labeled with radioactive nuclide,in vitro of anaplastic thyroid cancer.Methods: First we constructed the nanoparticles（NPs）,which co-loading dual molecular targeted drugs and labeled with radioiodine and Anti-VEGFR2 antibody.We labeled the MSNs with ¹³¹I by chloramine-T method and analysed its labeling efficiency and radiochemical yield.We also evaluated the dimensions,the level of drug loading,targeting,drug release kinetics.Then anti-proliferative effects of 17-AAG and Torin2,alone and in combination,on growth of FRO human anaplastic thyroid cancer cells were assessed by MTS.The combination index（CI）was analysed according to the Chou-Talalay method.The target binding and cellular uptake of the NPs in FRO cell line were observed by fluorescence confocal microscopy and flow cytometry.The time-dependent cellular uptake of ¹³¹I-labeled NPs was also analysed.In addition,cell experiments MTS assay confirmed that drug-loaded and ¹³¹I labeled NPs have a killing effect on tumor cells.Results:（1）Successfully prepared the new 3-in-1 NPs.The labeling efficiency of ¹³¹I-NPs was 66-78%,the radiochemical yield was up to 98.8-99.4%.The released studies showed that after 180 min,maximum release rate of the loaded 17-AAG and Torin2 were 15%、50%.Maximum release rate of free 17-AAG and Torin2 were 40% and 20%.（2）MTS assay showed that 17-AAG and Torin2,alone and in combination all could kill tumor cells.Synergistic effects were generated（CI < 1）in FRO cell lines when total concentrations of the two drugs > 0.52μM（the molar rate of 17-AAG and Torin2 is 1:1）.（3）Fluorescence confocal microscopy and flow cytometry experiments confirmed that FRO cells can uptake NPs quickly,the ability of cellular uptake of VEGFR2 targeted NPs was much higher than non-targeted NPs.After incubation,the rapid radioiodine uptake of ¹³¹I-labeled NPs reached maximum levels at 3h.Cells could uptake much more（17-AAG+Torin2）@MSNs-(Anti-VEGFR2 ab+BSA-¹³¹I)than Na¹³¹I（P<0.001）.（4）MTS assay demonstrated that there was an obvious killing effect of（17-AAG+Torin2）@MSNs-(Anti-VEGFR2 ab+BSA-¹³¹I)on ATC cells.The cell inhibitions of（17-AAG+Torin2）@MSNs-（Anti-VEGFR2 ab+BSA）was higher than（17-AAG+Torin2）,（t= 0.55-5.75,P<0.05）（except concentration 5μM）;（17-AAG+Torin2）@MSNs-(Anti-VEGFR2 ab+BSA-¹³¹I)was higher than（17-AAG+Torin2）@MSNs-（Anti-VEGFR2 ab+BSA）,（t= 0.33-6.64,P<0.05）（except concentration 2μM、5μM）.MSNs-(Anti-VEGFR2 ab+BSA-¹³¹I)was higher than Na¹³¹I（t= 3.93-26.08,P<0.01）;（17-AAG+Torin2）@MSNs-(Anti-VEGFR2 ab+BSA-¹³¹I)was higher than MSNs-(Anti-VEGFR2 ab+BSA-¹³¹I)（P<0.001）。Conclusion: FRO cell lines of ATC could uptake the new 3-in-1 ¹³¹I-labeled nanoparticles quickly,and VEGFR2 targeted NPs could be uptake more effectively.The NPs could resident in tumor cells smoothly and have a killing effect on them.