Multifunctional Drug Delivery Systems For Tumor Targeting Therapy

The purpose of the thesis is to develope multi-functional drug delivery systems to solid tomor. (1) The over-expression of ASGPR in HepG2 cells facilitated the receptor-ligand mediated tumor targeting. Novel multi-functional polymeric nanoparticles consist of methoxy-polyethylene glycols-b-poly (D-galactopyranose) (MPEG-b-PMaIPG) were prepared with effective ability of hepatoma targeting and pH triggered drug release. (2) Glucose conjugation to the delivery system confers tumor-targeting property through facilitative glucose metabolism by the glucose transporters in the tumor. Glucose transporters such as GLUT-1 are also known to be over-expressed on many tumors. For this reseason, N-acetyl-D-glucosamine (NAG) as a ligand has received significant attention in the development of drug targeting systems. A novel targeting drug delivery system containing poly (styrene-alt-maleic anhydride)58-b-polystyrene130 (P(St-alt-MA)58-b-PSt130) as a copolymer backbone, N-acetyl glucosamine (NAG) as a targeting moiety was designed and synthesized.An amphiphilic block copolymers containing D-galactopyranose and MPEG was prepared by atom transfer radical polymerization (ATRP) with the polydispersity of 1.21. The chemical structure and composition of the resultant copolymers were characterized by 1H NMR. The molecular weight and molecular weight distribution was also evaluated by gel permeation chromatography (GPC). Thus, the formation of amphiphilic block copolymer can be expressed as MPEG42-b-PMaIPG2o. The self-assembly behavior of the polymer were characterized by fluorescence spectrum, dynamic laser light scattering instrument (DLS), Zeta potential and transmission electron microscopy (TEM). The critical micelle concentration (CMC) of the amphiphilic block copolymer was about 0.005 mg/mL; the particle size was about 100±4.43 nm, zeta potential was approximately-32.8 mV at pH 7.4. DOX as a model drug was easily entrapped by the nanoparticles with high DL% and EE% of 24.77±2.68% and 66.12±9.44%, respectively. TEM images showed that the morphology of the non-loaded nanoparticles and doxorubicin (DOX) loaded nanoparticles were spheres with diameters of approximately 50 nm and 60 nm, respectively. Drug release studies were conducted using a dialysis method to investigate the pH triggered-release behavior of MPEG42-b-PMaIPG2o nanoparticles. After 72 h in the different environment, the cumulative release of DOX loaded nanoparticles about 37% of pH 5.0, whereas in pH 6.5 and pH 5.0 the corresponding values were 65% and 37%, respectively. The results suggested that the nanoparticles may be of suitable properties for pH-sensitive drug delivery application. The in vitro hemolysis rates of the nanoparticles (up to 1.0 mg/mL) to human red blood cells were found to be minimal and less than 5% which is considered safe for iv injection. MTT assay showed that the nanoparticles were nontoxic and biocompatible to L929 cells and HepG2 cells. Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to verify the targeting efficiency of D-galactopyranose modified nanoparticles. Both the microscopy image and quantitative determination showed that at the same condition DOX-NPs was easier to be endocytosed by HepG2 cells than A549 cells. The anticancer activity of DOX-NPs was investigated in HepG2 cells and A549 cells by the MTT assay. DOX-NPs exhibited superior anticancer activity to HepG2 than A549 cells in the experimental concentration range at any certain time. The in vitro experiment of the research indicated that the MPEG42-b-PMaIPG20 nanoparticles may serve as an effective drug delivery system for hepatoma cancer therapy.Another polymeric nanocarrier for targeting drug delivery was prepared by self-assemble of NAG grafted copolymer (NAG-P(St-alt-MA)58-b-PSt130), which was characterized by FTIR and 'H NMR. The CMC of nanoparticles was 0.028 mg/mL, determined by fluorescence spectroscopy using pyrene as a fluorescence probe. The nanoparticle was determined using a zetasizer (DLS, Malvern Instruments Ltd., UK). The zeta potential of NAG-NPs was -41.46±0.99 mV at pH 7.4, with the average size of 56.27±0.43 nm, PDI=0.099. A model hydrophobic anticancer drug, DOX, was efficiently entrapped in the NAG-NPs. The loading capacity and encapsulation efficiency of NAG-NPs-DOX was 25.83±1.09% and 69.69±3.98%, with the average size of 64.21±0.96 nm, PDI=0.10. The morphology of NAG-NPs and NAG-NPs-DOX were observed by TEM, and showed to be monospheres. Drug release of NAG-NPs-DOX and NPs-DOX was evaluated by the dynamic dialysis method. The results indicated that the drug loaded nanoparticles exhibited sustained release behavior in PBS buffer solution. MTT assay showed that NPs and NAG-NPs were nontoxic and biocompatible to AT II cells. CLSM and FCM results showed that the NAG targeting moiety enhanced the internalization and targeting ability of NAG-NPs. Anticancer activity toward MCF-7 cells and HT29 cells showed that DOX-loaded NAG-P(St-alt-MA)58-b-PSt130 nanoparticles exhibited a higher antitumor activity compare to DOX-loaded P(St-alt-MA)58-b-PSt130 nanoparticles, which could attribute to NAG receptor-mediated endocytosis. These results suggested that the biocompatible and non-toxic NAG-P(St-alt-MA)58-b-PSt130 nanoparticles which containing glucose transporter protein 1 (GLUT 1) ligand N-acetyl-D-amino glucose molecules may be used as an effective targeting drug delivery system for cancer therapy.In summary, this work has investgated two kinds of receptor-ligand mediated tumor active targeting technology and evaluated the biocompatibility and targeting of the nanocarrier materials. These results showed that the nanocarriers are promising for targeted cancer therapy and expected to be sustained controlled release and tumor targeting in the integration of anticancer drug delivery systems.