The Fabrication and Synergistic Anticancer Effects in Vitro of Self-assembled Folate-biotin-quaternized Starch Nanoparticles as Co-carrier of Doxorubicin and siRNA

Author:Li Liang Ping

Supervisor:gong ren min


Degree Year:2019





Tumor has become one of the diseases with the highest mortality rate in China.Due to it’s complexity of pathogenic factors,interactivity and variability of disease progression,and the various uncertainties caused by individual characteristics of patients,the difficulty of cancer treatment has increased.Existing mainstream clinical therapies such as surgery,chemotherapy and radiotherapy,whether used separately or in combination,are difficult to achieve ideal efficacy,and unable to effectively curb the deterioration and high mortality of tumors.Facing the severe treatment situation,the research of new and efficient treatment methods and drug preparation has always been the development direction of cancer treatment.Chemotherapy is one of the main treatment methods for cancer and is used in the formation and development stage of most tumors.Chemotherapeutics can inhibit the proliferation of cancer cells or effectively kill them by interfering with the anabolism of nucleic acid,suppressing cell mitosis and protein synthesis.However,the anti-apoptosis mechanism and multidrug resistance of cancer cells lead to the failure of chemotherapy.In addition,the systemic distribution of chemotherapeutics often causes severe side effects to other normal tissues and organs at the same time of tumor inhibition,bringing many unbearable damages to patients.Gene therapy is often used for the knockout(silencing)of oncogenes and the introduction or activation of anti-oncogenes,so as to achieve the therapeutic purpose of inhibiting tumor development.But gene drugs are easy to be degraded and deactivated in vivo,and also without tumor target specificity and accumulation ability in tumor tissues.So the application of tumor-targeting nanocarrier system encapsulating chemotherapeutics and gene drugs for the co-delivery of the two drugs to target cancer tissues is a hopeful method and research focus of cancer treatment that likely achieve the synergistic suppression of cancer by chemotherapy and gene therapy.This treatment method can avoid the systemic distribution of chemotherapeutics and gene drugs,reduce the side effects and a large number of drugs losses,improve the drug efficacy and accumulation to tumor target sites.Based on the enhanced permeability and retention effect(EPR effect)of tumor tissue and the affinity interaction between folate and its specific receptors,a novel tumor-targeted co-carrier of anti-cancer drugs and siRNAs,folate-biotin-quaternized starch nanoparticles(FBqS NPs),was designed and prepared.The amphiphilic grafted polymer(FBqS)can be self-assembled into core-shell spherical nanoparticles in aqueous solution.Their core can encapsulate chemotherapeutics through hydrophobic interaction,and the cationic hydrophilic shell can load siRNA by electrostatic adsorption,which can achieve the targeted co-delivery of chemotherapeutics and siRNAs.The hydrophobic doxorubicin(DOX)was used as the model of chemotherapeutics encapsulated into the core of FBqS NPs.The siRNAIGF1R degrading the mRNA of type 1 insulin-like growth factor receptor(IGF1R) was used as the gene drug,which was loaded into the positively charged hydrophilic shell of FBqS NPs by electrostatic adsorption.As a result,the siRNAIGF1R/DOX FBqS NPs were prepared.The physicochemical properties of siRNAIGF1R/DOX/FBqS NPs and the combined inhibitory effect of DOX and siRNAIGF1R on cancer cells in vitro were observed and evaluated by a series of experiment.The main research contents and results are as follows:1)The starch was firstly modified by quaternary reagent to obtain cationic starch.Then amphiphilic grafted polymer(FBqS)was prepared by a one-pot synthesis via DCC/NHS/DMAP-mediated esterification reaction and self-assembled folate-biotin-quaternized starch nanoparticles were fabricated by ultrasonication.The physicochemical properties of the prepared FBqS NPs were characterized.The FBqS NPs were used as co-carrier of siRNA and DOX with satisfactory loading capacity and encapsulation efficiency.The siRNA encapsulated in FBqS NPs exhibited excellent serum stability.The release characteristics of DOX and siRNA from FBqS NPs were studied in different pH environment and the release behaviors of two drugs were all pH-sensitive.The FBqS NPs as a potential co-carrier of anticancer agents and gene drugs was expected to achieve future practical application in vitro and in vivo.2)The FBqS NPs were used as carrier of DOX and siRNAIGF1R for codelivery of both into A549 human lung adenocarcinoma cell lines in vitro.The cytotoxicity,targeted ligand competition,cell proliferation inhibition,cellular uptake,endocytosis mechanism and target protein suppression of drug-loaded FBqS NPs were evaluated in detail.Compared with several other drug formulations under same condition,siRNAIGF1R/DOX/FBqS NPs exhibited the greatest cytotoxicity to A549 cells and the cytotoxicity was competitively inhibited by free folate in dose dependent manner.The A549 cells treated by siRNAIGF1R/DOX/FBqS NPs showed the lowest cell proliferation capacity.The energy-dependent clathrin-and caveolae-mediated endocytosis might be the primary cellular uptake mechanism of drug-loaded FBqS NPs.The expression of IGF1R protein in A549 cells treated by siRNAIGF1R/FBqS NPs declined dramatically.So the FBqS NPs were expected as the co-carrier system of chemotherapeutants and siRNAs for future clinical application.