Multistage Targeted Nanoparticles In Cancer Chemo/Chemodynamic Synergistic Therapy

Cancer is known as one of the diseases that has seriously endangered the human health.Currently,the available single-mode treatments,such as surgery,radiotherapy and chemotherapy are not able to meet people’s needs.Nanoparticles with dual-mode synergistic therapeutic effects,especially those combined with chemotherapy（CT）and chemodynamic therapy（CDT）,the effect is extremely prominent in the treatment of cancer.However,the accuracy,selectivity,efficiency and safety of most CT/CDT nanoparticles in cancer treatment still needs to be further improved.Based on these mentioned concerns,this thesis aims to design and develop a series of nanoparticles for cancer treatment,which combines the functions of both CT and CDT,multistage-targeting,spatiotemporally controlled release of chemotherapy drug and magnetic resonance imaging（MRI）,so as to enhance the antitumor efficiency.The pH-responsive prodrug(PEG_2K-NH-N-DOX),ROS-responsive prodrug(PEG_2K-S-S-CPT-ROS),organic CDT agents(TPP-PEG_2K-LND,TPP-PEG_2K-TOS),and MRI contrast agents（Gd-DTPA-N16-16）were designed and synthesized.The targeted mitochondria,pH/ROS dual responsive nanoparticle（TLDCAG）with endogenous ROS burst and spatiotemporally controlled multiple drug release ability was prepared by the above-mentioned molecular assembly and encapsulation of the small anti-tumor angiogenic drug combrestatin A4（CA4）.The average particle size of TLDCAG is less than 200 nm,and has outstanding storage stability,serum stability and pH/ROS dual responsiveness.The results of drug release showed that TLDCAG has the ability to control multi-drug release in time and space under slightly acidic and high ROS level.Confocal laser and flow cytometry results demonstrated that the uptake of TLDCAG by MCF-7 cells was time-dependent.Besides,confocal laser results confirmed that TLDCAG has the targeting ability to MCF-7cells mitochondria,and also TLDCAG can cause the outbreak of endogenous ROS in MCF-7cells and reduce its mitochondrial membrane potential.Western blotting confirmed that TLDCAG induced apoptosis of MCF-7 cells through mitochondrial endogenous routes.The relaxation efficiency of TLDCAG is 3.89 times that of the clinical contrast agent Gd-DTPA,and the imaging effect is significant in vivo and in vitro.Both cytotoxicity and in vivo anti-cancer activity test results showed that TLDCAG has excellent anti-tumor activity.Therefore,TLDCAG provides an effective strategy for the synergistic effect of CT/CDT.The GSH-responsive and breast cancer targeting prodrug(Bio-PEG_2K-S-S-CPT)was designed and synthesized.The multifunctional nanoparticle（BDTLAG）with breast cancer tissue-cells-mitochondria targeting,spatiotemporally controlled multiple drugs release to the corresponding active sites,GSH depletion,and endogenous ROS burst induced ability was successfully prepared by the Bio-PEG_2K-S-S-CPT,PEG_2K-NH-N-DOX,TPP-PEG_2K-TOS,TPP-PEG_2K-LND,Gd-DTPA-N16-16 assembly and encapsulation of CA4.The average particle size of BDTLAG was determined as≈140 nm,and has outstanding storage stability,serum stability,dilution stability and pH/GSH dual responsiveness.The results of drug release showed that BDTLAG has the ability to control multi-drug release in time and space.Confocal laser and flow cytometry results demonstrated that BDTLAG has the multistage-targeting ability to breast cancer.Besides,confocal laser results confirmed that BDTLAG could obviously cause mitochondrial superoxide generation to guarantee the formation of much more amount of ROS species in mitochondria.Western blotting confirmed the BDTLAG induced apoptosis of MCF-7 cells through mitochondrial endogenous routes.In vitro and in vivo MRI results showed that BDTLAG has high relaxation efficiency and remarkable MRI ability.Cytotoxicity,anticancer activity and toxicity experiments confirmed that BDTLAG has outstanding antitumor activity and negligible toxicity in vivo.Therefore,such a single nanoparticle that integrates CT,CDT,and MRI functions along with GSH depletion reflects the“all in one”concept and improves the accuracy,selectivity,efficiency and safety of nanoparticles in the treatment of cancer.The GSH-responsive and liver cancer targeting prodrug(GA-PEG_2K-S-S-CPT)was designed and synthesized.The multifunctional nanoparticle（CDTLG）with cancer tissue-cells-mitochondria targeting,spatiotemporally controlled multiple drugs release to the corresponding active sites,MRI and endogenous ROS burst induced ability was prepared by the GA-PEG_2K-S-S-CPT,PEG_2K-NH-N-DOX,TPP-PEG_2K-TOS,TPP-PEG_2K-LND and Gd-DTPA-N16-16 self-assembly.The average particle size of CDTLG was determined as≈147 nm,and has outstanding storage stability,serum stability,dilution stability and pH/GSH dual responsiveness.The results of confocal laser,flow cytometer,in vivo MRI confirmed that CDTLG has the multistage-targeting ability to liver cancer.The confocal laser images confirmed that CDTLG can cause the outbreak of endogenous ROS in HepG2 cells and reduce the mitochondrial membrane potential.Cytotoxicity and anti-tumor activity experiments in vivo confirmed that CDTLG realized the synergistic effect of CT/CDT and has strong antitumor activity.After treatment,the weight,hematology,serum biochemical and H&E staining of various organs of tumor-bearing mice showed that CDTLG has high biological safety.The multifunctional nanoparticle（CDTLG）provides a strategy to improve the treatment efficiency of liver cancer.