Preparation And Preliminary Study On The Anticancer Activities Of Camptothecin And 3-MA Conjugated Gold Nanoparticles (CPT-Au-3MA NPs)

Objective:Camptothecin has a very good therapeutic effect on most tumors.However,unexpected factors such as poor water solubility and systemic toxicity have restricted its clinical use.In addition,the drug resistance has emerged with the extensive use of drugs,which become one of the main limitations of current cancer treatments.At present,nanoparticles used as drug carriers have many obvious advantages,which can deliver the drugs to tumor sites and improve the solubility of drugs.It was reported that inhibiting autophagy can make cancer cells sensitive to drugs again.This study aims to use gold nanoparticles as a drug delivery vehicle deliver CPT and 3-MA to the tumor site,and use the synergistic effect of the two drugs to improve the anti-tumor effect.Methods:In this study,ethylene glycol was used as the main chain,one end was connected to lipoic acid（LA）,and the other end was connected to drugs（CPT or 3-MA）.they were connected by covalent bonds to form the Linker.Then linkers and gold nanoparticles were connected through the Au-S bonds.In the experiment,18 Linkers were synthesized by changing the length and functional groups of the main chain.And 9 kinds of drug-loaded gold nanoparticles were prepared.The structures of the synthesized Linkers were determined by Mass Spectrometer,Nuclear Magnetic Resonance Spectrometer,FT-IR Spectrometer.UV-Visible spectrophotometer,Malvern particle size analyzer,and Transmission electron microscope were used to characterize the drug-loaded gold nanoparticles.The drug loading capacity of the nanoparticles was determined by UV-Visible spectrophotometer and High performance liquid chromatography.The growth inhibitory effects of prototype drugs and drug-loaded nanoparticles on MCF-7 and K150 cells were tested by MTT assay.Compu Syn software was used to calculate the combined index（CI）.Finally,Flow cytometry was used to test the apoptosis rate of the drug-loaded gold nanoparticles LC-Au-LM NPs on MCF-7cells.Results:1.In this experiment,a total of 18 compounds were synthesized,and the structures were confirmed by FT-IR spectroscopy,ESI-MS,¹H-NMR,and ¹³C-NMR.9 kinds of drug-loaded gold nanoparticles were prepared,and the characterization of the nanoparticles were determined by UV-Visible spectrophotometer,Malvern particle size analyzer,and Transmission electron microscope.The drug loading of LC-Au-LM NPs was measured,and the results showed that the drug loading of CPT was 4.3 wt%and the drug loading of 3-MA was 9.8 wt%.The cumulative release rates of 3-MA and CPT were 45.08%and 14.47%respectively after 24 h in p H 5.4 buffer solution.2.MTT results showed that the IC₅₀ of CPT to MCF-7 cells was 23.96±3.68μM.The IC₅₀of 3-MA on MCF-7 cells was 14.34±2.14 m M.Using different concentrations of CPT and3-MA for combined use,the CI values of the 10 groups were all less than 1,which indicated that the combined use of CPT and 3-MA has a synergistic effect on inhibiting the proliferation of tumor cells.3.The inhibitory effects of the prepared 9 drug-loaded gold nanoparticles on the MCF-7cells were screened by the MTT assay,and the results showed that LC-Au-LM NPs exhibited better inhibitory effect on of MCF-7 cells under the same drug concentration,which was higher than that of the combined use group of the prototype drug CPT and 3-MA at the same concentration.LC-Au-LM NPs also showed similar results on K150 cells.The results of flow cytometry showed that the apoptotic rate of the LC-Au-LM NPs group was 27.36%,which was higher than the 11.84%of the prototype drug combination.Conclusion:In this study,18 Linkers were synthesized by connecting CPT,3-MA and LA through main chains of different lengths and functional groups such as ethylene glycol.And Linkers were connected to gold nanoparticles through Au-S bonds,and 9 kinds of drug-loaded gold nanoparticles were prepared.The drug loading of LC-Au-LM NP was tested.According to the calculation principle of Chou Talaly,the combined use of CPT and 3-MA showed a synergistic effect on MCF-7 cells.Among the 9 drug-loaded gold nanoparticles,LC-Au-LM NPs showed the strongest proliferation inhibitory activity against MCF-7.The Flow cytometry results also showed that the apoptosis rate of MCF-7 cells in the same concentration of drug-loaded gold nanoparticles group were higher than that of the prototype drug combination group.