Construction Of Dually PH/redox Responsive Nano Drug Delivery System And Antitumor Efficacy Evaluation

In this study,one kind of polymer with p H/reduction response to specific tumor microenvironment was synethesized with rational molecular design.The stimuli-responsive nanoparticles based on the self-assembly of polymer was studied for efficient delivery of small-molecullar chemotherapy drug to inhibit subcutaneous tumor growth via enhanced cellular internalization and on-demand intracellular durg release.The multifuncaitonal polymer was then further modified to coat cationic liposomes to form smart co-delivery system for combine chemotherapy and gene therapy to synergistically inhibit proliferation and metastasis of orthotopic NSCLC,which was expected to provide a promising strategy for precise and effective cancer therapy.Chapter 1: The process and character of tumorigenesis and development was summarized.The characteristic and application of smart nano drug delivery system in response to tumor microenvironment in targeted tumor therapy was then expounded.Besides,the combination of chemotherapy and gene therapy for synergistic antitumor effect was introduced to put forward this study.The idea and proposal of this paper were then presented.Chapter 2: An amphilic p H/reduction-responsive polymer based on chitosan with histidine and lipoic acid graft(HCSL)was synethesized and characterizated via FT-IR and 1H NMR spectrum.HCSL micelles were prepared via the probe ultrasonic method.The size and zeta potential in different p H or reduction condition was investigated.HCSL micelles showed particle size of 106 nm and zeta potential of-25.0 m V at p H 7.4.When exposed to p H 6.5,zeta potential was reversed to 4.4 m V and 20.0 m V at p H 5.3.Broader size distribution was triggered by p H 5.3 and 10 m M GSH.DOX loaded micells(DOX/HCSL)was prepared via the dialysis method with diameter of 126.2 nm,EE % of 92.71 % and DL % of 23.22 %.In vitro drug release of DOX/HCSL exhibited p H/reduction responsed,which showed slow and little DOX release at p H 7.4 while sharp and almost complete durg release under p H 5.3 and 10 m M GSH incubation.Cellular uptake and intracellular distribution studied by confocal laser scanning microscope(CLSM),flow cytometry and the live cell station indicated that extracellular p H 6.5 significantly enhanced cell internalization of DOX/HCSL.Besides,HCSL encapsulation facilitated the sharp and on-demand intracellular drug release.MTT assay suggested that the cytotoxicity of DOX micelles against tumor cells obviously increased under p H 6.5 incubation.In vivo targeting and biodistribution study demonstrated that HCSL micelles could be effectively targeted in tumor sites and greatly lower the accumulation in normal tissues.In vivo antitumor activity study presented that compared with DOX·HCl and DOX/CSL micelles,DOX/HCSL micelles significantly inhibit the growth of subcutaneous tumor with lower systemic toxicity.These results indicated that the environment-responsive HCSL nanoparticles could deliver chemotherapy agents effectively and lower the side effect of chemodrug and finally greatly enhanced antitumor activity.Chapter 3: In this study,we modified p H/reduction sensitive polymer HCSL with PEGylation for prolonged circulation to obtain multifunctionally amphilic PHCL polymer.Cationic liposomes was used to load si VEGF and ETO via positive charge and phospholipid bilayer,followed by PHCL coating to construct multistimuli-responsive co-delivery system(PHCL-Lip/ETO-si VEGF).The co-delivery nanoparticles with particle size of 176.5 nm and zeta potential of-12.06 m V exhibited round shape and uniform distribution,which possessed high ecapsulating ability with EE % of 89.88% and DL % of 15.24 % and absolute adsorption of si RNA when N/P up to 6.PHCL-Lip/ETO-si VEGF perfectly inherited the charge-reversal and reduction sensivity of PHCL,which exhibited sharp and complete drug release responding to p H 5.3 and 10 m M stimuli.Besides,it was found that nanopartilces showed obviously higher cellular uptake under extracellular p H condition and could escape from endo-lysosomes followed by sharp payloads release.PHCL-Lip/si VEGF effectively downregulated the expression of VEGF,resulted in tumor cell molibility and invasion ability decrease.In vivo study demonstrated that PHCL-Lip effectively accumulated in orthotopic lung tumor.The combination of si VEGF and ETO co-delivred by PHCL-Lip presented significant inhibition of tumor proliferation and metastasis with minimal systemic side effects.This study confirmed that the dually p H/reduction polymer could improve the drawback of cationic liposomes to construct a effective and safe co-delivery system,and that synergitic suppress the growth and metastasis via combination of si VEGF and ETO would be promising for effective NSCLC treatment.