Construction and Performance of Pullulan-based Nano-carrier for Tumor Drug/Gene Combination Therapy

Author:Chen Li Li

Supervisor:wang jing yun


Degree Year:2019





Tumor is one of the malignant diseases that threaten human health seriously and overcoming cancer has become a worldwide problem and challenge.However,in some case.treating malignant tumor with single therapy including surgery.radiotherapy.gene therapy and chemotherapy could not achieve satisfactory curative effect because the occurrence and development of tumor are regulated by complex signaling networks in the body.Therefore.integrating two or more different cancer treatments together.that is the so called combination therapy,has emerged as an important strategy for effective cancer treatments nowadays.It has been reported that chemotherapy combined with gene therapy could inhibit tumor growth from different pathways or distinctive mechanism so as to achieve the enhanced therapeutic effect against tumors.However.The key of drug/gene combination therapy is how to co-deliver hydrophobic antitumor drug and negatively charged genes(DNA or RNA)to tumor cells that drug and gene can synergistically play a role in tumor suppression.In this paper.three novel amphiphilic nanocarriers were designed and synthesized based on the biocompatible pullulan,and the ability of the nanomicelles to co-delivery drug/gene to targeted tumor site and their application for combination therapy of tumor were studied.1.Preparation of bifunctional nanocarrier PDP for co-delivery of drug/gene.In the second chapter,the cationic amphiphilic polymer PDP was successfully prepared by modifying pullulan polysaccharide(P)with hydrophobic deoxycholic acid(DA)and cationic polyethyleneimine(PEI)to simultaneously deliver hydrophobic chemotherapeutic drug doxorubicin(DOX)and tumor suppressor gene(p53)to tumor site for the combination treatment of tumor.The biocompatibility of PDP was investigated by evaluating cytotoxicity and erythrocyte hemolysis rate,showing that PDP was safe and non-toxic for biological application.In the self-assembly process of polymer PDP into micelles,hydrophobic drug DOX was encapsulated via hydrophobic interaction to obtain drug-loaded nanomicelles PDP/DOX with high drug loading(7.64%)and encapsulation efficiency(84.05%).In addition.drug-loaded micelles PDP/DOX exhibited better sustained drug release profile than the free DOX in the drug release evaluation.Gel electrophoresis experiments showed that PDP could bind genes via electrostatic interaction,protect genes from being degraded and carry genes effectively into the cancer cells.In vitro apoptosis analysis and MTT assay both indicated that the co-loaded drug/gene nanomicelles PDP/DOX/p53 could effectually inhibit the tumor cell growth.More importantly,the tumor volume of mice treated with PDP/DOX/p53 was only 1/10 in ratio of that in the PBS treatment group after 14 days,indicating that PDP/DOX/p53 could inhibit the tumor growth in nude mice effectively.Therefore,PDP,as a good biocompatible nanocarrier,can efficiently co-deliver doxorubicin and anti-oncogene p53 to tumor cells simultaneously,presenting good clinical application prospects in the combined treatment of tumor.2.Preparation of folate receptor-targeted co-delivery drug/gene multifunctional nanomicelle FPDP.In the third chapter,the folate molecule was conjugated to the PDP polymer synthesized previously to obtain a novel nanocarrier FPDP for targeted co-delivery of DOX and the interfering RNA of cellular autophagy factor Beclinl(shBeclinl)to the tumor site.Cell uptake assay results showed that nanomicelles FPDP could efficiently delivery DOX to the tumor cells that overexpressed folate receptor.vitro apoptosis analysis showed that FPDP/DOX/shBeclin1 micelles could significantly inhibit the tumor cell with marked elevated apoptosis rate against cancer cells when compared with other three micelles including FPDP/DOX,FPDP/shBeclinl and PDP/DOX/shBeclinl.In vivo tumor inhibition assays demonstrated that the tumor volume of the mice after the treatment of FPDP/DOX/shBeclinl for 21 days has reduced to 1/14 in ratio of that of the mice treated with merely PBS.The interfering RNA of autophagy factor Beclinl could interfere with the initiation of the autophagy process of tumor cells induced by DOX,which endowed the FPDP/DOX/shBeclinl micelles with the enhanced sensitivity of tumor cell against DOX so as to achieve an efficient combined therapeutic effect against tumors.3.An intelligent multi-functional nanocarrier HP-ss-DP with CD44 targeting ability and tumor microenvironment responsiveness.In the fourth chapter of this paper,disulfide bond was used as the reduction sensitive group to connect pullulan polysaccharide and hydrophobic small molecule deoxycholic acid.Subsequently,PEI was grafted onto the polysaccharide skeleton for cationic modification.After the polymer was prepared into micelles,hyaluronic acid(HA)was modified into micelles by electrostatic action so as to obtain the multifunctional nanomicelles named HP-ss-DP.It was found that HP-ss-DP nanomicelles had an appreciable response to the reduction environment in terms of both the change of particle size and the drug release behavior in vitro under the reduction environment.Cell uptake experiments showed that HP-ss-DP nanomicelles could efficiently target CD44-overexpressed cancer cells.The results of cell scratch test and Transwell experiment showed that HP-ss-DP/DOX/shSNAI1 micelles could effectively inhibit the metastasis,invasion and growth of cancer cells,validating that HP-ss-DP/DOX/shSNAIl micelles possess a better combined therapeutic effect against lung cancer.In summary,in this paper,a series of malfunctional nanocarriers with good biocompatibility was developed for efficient co-delivery of chemotherapeutic drugs and therapeutic genes to achieve combination therapy against tumors.Three therapeutic genes were respectively combined with DOX to achieve drug/gene therapy by inducing apoptosis,inhibiting multidrug resistance and preventing metastasis and invasion of cancer cells.The results of this paper fully demonstrated that the multifunctional nanocarriers based on pullulan polysaccharide present good application prospects in anti-cancer combined therapy.