Font Size: a A A

Investigation Of Pulluan-based Nanoparticle System For Drug Delivery Against Hepatma

Posted on:2016-05-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y Y LiuFull Text:PDF
GTID:1224330503952038Subject:Oncology
Abstract/Summary:PDF Full Text Request
Objective:Hepatocellular carcinoma(HCC) is one of the most common malignancies worldwide and its incidence is rising year by year. Chemotherapy is a common method to treat patients with advanced-stage in clinical treatment on HCC, but most HCC can easily produce multi-drug resistance against chemotherapeutic drugs. Therefore, the explorations of efficient therapeutic methods for HCC are of great significance. Pullulan is a water-soluble mucopolysaccharide that is produced from starch by the fungus Aureobasidium pullulans. Pullulan has many good biological properties and is a natural ligand for asialoglycoprotein receptor(ASGPR), which is often over-expressed on HCC cells, thus it can be used as a drug carrier material targeting to HCC. In this project, two kinds of pullulan-based nanoparticle systems were designed for the co-carrying and targeted delivery of different antitumor drugs or gene and antitumor drug, thus realize the effective treatment on HCC through the combination therapies.Methods:Section 1: Urocanyl pullulan(URPA), a p H-sensitive pullulan derivative, was synthesized by grafting urocanic acid to pullulan backbone. Chemotherapeutic drug methotrexate(MTX) was conjugated to UPRA via ester bond to form polymeric prodrug. The chemical structures of URPA and MTX-URPA were characterized by the analysis methods of infrared spectroscopy(IR) and proton nuclear magnetic resonance(1H NMR). Prodrug nanoparticles were prepared from MTX-URPA using self-assembly technique. Anti-angiogenic drug, combretastatin A4(CA4), was physically encapsulated into MTX-URPA nanoparticles, thus realized co-loading of two anti-tumor drugs with different mechanisms. The morphology of CA4-loaded MTX-URPA(CA4/MTX-URPA) nanoparticles was observed using transmission electron microscope(TEM). The size and size distribution of these nanoparticles were measured by dynamic laser scattering method, and their surface charge character was also assessed by zeta potential detector. The loading contents of MTX and CA4 in this nanoparticle system were detected using ultraviolet(UV)spectrophotometric method. The cytotoxicity of CA4/MTX-URPA nanoparticles was evaluated in HCC Hep G2 cells using MTT assay. The uptakes of CA4/MTX-URPA nanoparticles in HCC Hep G2 cells were detected by the flow cytometry method. The nude mice model with transplanted HCC was established; the blood concentrations and tissue distributions of MTX and CA4 were measured using high-performance liquid chromatography(HPLC) method after intravenous injection of CA4/MTX-URPA nanoparticles. Moreover, the anti-tumor activity of CA4/MTX-URPA nanoparticles was also evaluated by assessing the tumor growth in Hep G2-bearing nude mice and the blood vessel density in tumor tissues.Section 2: A cationic biodegradable polymer, poly(beta-amino ester)(PBAE) was synthesized using Michael addition reaction. Plasmid DNA expressing green fluorescent protein(p EGFP), as a model gene, was fully condensed with cationic PBAE to form PBAE/p EGFP nanocomplex and the efficiency of this complex system was assessed by agarose gel electrophoresis, thus the optimal PBAE/p EGFP weight ratio could be obtained. Anti-tumor drug, MTX, was conjugated to pullulan to form polymeric prodrug of MTX-PL. The chemical structure of MTX-PL was confirmed by the IR and 1H NMR, and MTX content was determinated using UV method. Next, MTX-PL was coated on the surface of PBAE/p EGFP nanocomplex via adsorption to form hydrophilic polysaccharide shell, thus realized the co-loading of gene and chemotherapeutic drug in a same nanoparticle system. The morphology of MTX-PL/PBAE/p EGFP nanoparticles was observed using TEM technique. The size and size distribution of these nanoparticles were measured by dynamic laser scattering method, and their surface charge character was also assessed by zeta potential detector. The confocal microscopy and flow cytometry were used to measure the affinity of MTX-PL/PBAE/p EGFP nanoparticles for Hep G2 cells and the cellular transfection efficiency of p EGFP. The cytotoxicity of MTX-PL/PBAE/p EGFP nanoparticles was assessed by Cell Counting Kit-8(CCK8) assay. The nude mice model with transplanted HCC was established and tissue distribution of MTX-PL/PBAE/p EGFP nanoparticles was observed using in vivo imaging system.Results:Section 1: URPA was successfully synthesized and the degree of substitution(DS) of urocanyl group in URPA was about 5.2%. URPA exhibited obvious p H-sensitivity and its sensitive p H value was about 6.5. MTX was efficiently conjugated to URPA to form polymeric prodrug of MTX-URPA, in which MTX content was about 17.8%. MTX-URPA nanoparticles had spherical shapes and their mean size was about 187.1 nm. MTX-URPA nanoparticles showed a high affinity for HCC Hep G2 cells, on which ASGPR was over-expressed. CA4 was physically loaded into MTX-URPA nanoparticles and exhibited p H-sensitive in vitro release property. CA4 and MTX showed orderly releasing property both in vitro and in vivo, thus was favor to exert their combination effects. After intravenous injection to nude mice bearing human HCC PLC/PRF/5 cells, CA4 loaded MTX-URPA(CA4/MTX-URPA) nanoparticles achieved the enhanced antitumor and antiangiogenic effects, the prolonged circulation time in blood, and the increased distributions both in the liver and the tumor.Section 2: PBAE and MTX-PL were successfully synthesized and their chemical structures were confirmed by IR and 1H NMR spectra. MTX content in MTX-PL was about 15.2%. PBAE exhibited relatively high condensation capability for p EGFP and the PBAE/p EGFP weight ratio of 50/1 was believed as an optimal weight ratio. MTX-PL/PBAE/p EGFP nanoparticles had a spherical shape with classic “core-shell” structure, and their size was 172.9 nm with a relatively narrow distribution. Compared with PBAE/p EGFP nanocomplex, MTX-PL/PBAE/p EGFP nanoparticles showed the neutral surface charge property, which was favor to decrease the biological toxicity of PBAE. In addition, MTX-PL/PBAE/p EGFP nanoparticles realized the efficient transfection of p EGFP in Hep G2 cells and exhibited significant inhibitory effect on the cell proliferation. In Hep G2 tumor-bearing nude mice, MTX-PL/PBAE/p EGFP nanoparticles were mainly distributed in the tumor after 24 h post intravenous injection.Conclusions:In this study, two kinds of pullulan-based nanoparticle systems were designedand prepared for the co-loading and targeted delivery of different antitumor drugs or gene and antitumor drug. One is a nanocarrier system with co-loading of angiogenesis inhibitor and chemotherapeutic drug. This nanocarrier system realized HCC-targeted delivery of two anti-tumor drugs with different effect mechanisms and exhibited significant enhanced synergistic effect on HCC. The other nanocarrier system realized the efficient co-loading of model gene and chemotherapeutic drug, their synergistic effects at cellular level, and their HCC-targeted co-delivery in mice. Taken together, the designs of these pullulan-based nanoparticle systems are favorable for the combination therapy on HCC and provide new strategies for solving the clinical challenge of HCC treatment.
Keywords/Search Tags:nanocarrier, pullulan, anti-angiogenesis, chemotherapy gene, combination, therapy
PDF Full Text Request
Related items