Prepartion Of Multifunctionall Polymer Nano-carriers And Their Antitumor Efficacy Study

Cancer has always been one of the major diseases that threaten human life.How to save the lives of cancer patients has become a major challenge that we have to face today.Chemotherapy is one of the most important methods of cancer treatment in clinical cancer treatment,but it will inevitably produce severe side effects.In addition,the poor water solubility and low bioavailability of some chemotherapeutic drugs also affect their anticancer effects.At present,although the drug delivery system based on nanocarriers has gradually demonstrated its unique advantages in solving the defects and improving the therapeutic effect of drugs,there are still some problems to be solved.In view of the current low delivery efficiency of nanoparticles,unsatisfactory therapeutic effect of monotherapy with only a single-drug and difficulty of treating multidrug resistant tumors,this dissertation aims to build different multi-functional drug nanocarriers based on different strategies to significantly improve the antitumor effect of chemotherapeutic drugs.In the chapter 2,we successfully synthesized and prepared tumor vascular-targeted multifunctional hybrid polymer micelle,PLEPMPss-c RGD,which can selectively expand tumor vessels by producing NO in situ to enhance EPR effect of nanoparticles.The morphology and stability of the hybrid micelles were characterized by TEM and DLS.The results showed that the hybrid micelles had a uniform core-shell structure and good kinetic stability.Subsequently,the ability of hybrid micelles to catalyze GSNO decomposition to generate NO and endothelial cell targeting were investigated by in vitro experiments.The results indicated that hybrid micelles could remove m PEG-CDM protective layer in mildly acidic conditions and promote NO generation through a catalytic reaction between copper ions and RSNO and have a good targeting ability of endothelial cells.DOX was selected as the model drug to be loaded into hybrid micelles,and DOX release ability and cytotoxicity were investigated.The results showed that high concentration of GSH could accelerate the release of DOX and achieve better antitumor effect.The morphological changes of tumor blood vessels confirmed that the hybrid micelle could effectively expand tumor blood vessels in vivo.In addition,the results of drug distribution in vivo confirmed that the hybrid micelle could improve the enrichment of DOX in tumor tissues and reduce its side effects.In vivo antitumor experiments indicated that the design and preparation of hybrid micelles had good antitumor effect and biological safety,extending the survival time of mice.Additionally,the analysis results of H&E,TUNEL and Ki67 staining of tumor tissues on day 16 showed that the hybrid micelles induced the highest level of tumor cell apoptosis under light irradiation.In chapter 3,we successfully synthesized and prepared a novel multifunctional micelle(termed HCCT)with multiple stimulus-responsive ability(hyaluronidase-,GHS-,and light-responsiveness),and cell membrane-and mitochondrion-dual-targeting functions.This micelle can encapsulate antineoplastic cisplatin-prodrug(designated as PCT@HCCT)during the self-assembly process.The chemical structures of copolymer and cisplatin prodrug were confirmed by ~1H NMR.The core-shell structure of micelles was confirmed by ~1H NMR.TEM and DLS confirmed the uniform distribution and dynamic stability of micelles.In vitro response results showed that HAase and GSH could accelerate the destruction of HCCT micelle structure and thus accelerate the release of cisplatin prodrug.In vitro cell experiments demonstrated that the PCT@HCCT micelle could be internalized by B16F10tumor cells through CD44 receptor-mediated targeting,successfully escape lysosomes,release PCT into the cytoplasm rapidly,and target the mitochondria via the TPP function and allow conversion to cisplatin-prodrug to free cisplatin through redox-responsiveness.At the same time,the membrane permeability of mitochondria would be improved by the generated ROS with light irradiation,thus allowing the entry of cisplatin into the mitochondria that causes mitochondrial damage,ultimately resulting in mitochondria-mediated apoptosis.The B16F10 cells was stained by the Annexin V-FITC/PI Apoptosis Detection kit after treated with different methods,and the apoptotic cells were enumerated by FACS.It was found that PCT@HCCT micelles could induce more apoptosis of B16F10 cells under light.The results of drug distribution in vivo confirmed that PCT@HCCT micelles could effectively enrich in tumor tissues.The results of antitumor experiments in vivo confirmed that PCT@HCCT micelles had better inhibition of tumor growth with light irradiation than that without light irradiation,and the survival rate of mice was highter.In addition,by analyzing the H&E and TUNEL staining of tumors on day 15,it was found that PCT@HCCT micelles caused the highest level of apoptosis of tumor cells under light irradiation,which further confirmed its antitumor effect.In the last chapter,we synthesized a new FA-modified polydopamine nanoparticle(FAPPs)for the treatment of multidrug-resistant tumors.The nanoparticle could be used to loading hydrophobic NO donor BNN6 and chemotherapy drug DOX on the surface of FAPPs respectively.Uv-vis detected the LC of BNN6@FAPPs and DOX@FAPPs,respectively.and confirmed that the two nanoparticles maintained good stability in PBS and medium(containing 10%FBS).In vitro experiments confirmed that BNN6@FAPPs could produce NO under NIR irradiation,and the produced NO could reduce the expression level of P-gp in MCF-7/ADR cells,and inhibit cellular respiration to reduce the content of ATP in cells,thereby reversing MDR and increasing the cytotoxicity of DOX@FAPPs.The frozen section of tumor tissue was observed via CLSM and the results showed that the amount of accumulation of free DOX in MCF-7/ADR tumor was very little,while pre-injection BNN6@FAPPs with NIR irradiation could significantly increase the accumulation of DOX in tumor,moreover most drugs were located in the nucleus.In vivo antitumor experiments confirmed that pre-injection of BNN6@FAPPs with NIR irradiation could improve the tumor inhibition effect of DOX@FAPPs,and combinaion with the photothermal treatment effect of FAPPs under NIR irradiation could significantly inhibit the growth rate of MCF-7/ADR tumor volume,indicating that this combination treatment strategy has the best antitumor effect.H&E and TUNEL staining were used to analyze tumor cells in mice treated on day 18.It was found that a large number of cell nuclei were broken and cell apoptosis occured,which further confirmed the antitumor effect at the histological level.Western blot showed that treatment strategy of combining BNN6@FAPPs and DOX@FAPPs together with NIR irradiation could down-regulate the expression of anti-apoptotic protein Bcl-2 and up-regulate the expression of pro-apoptotic protein Caspase-3.