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In Vitro Targeted Gene Delivery of Pegylated Adenoviral Vectors from PLGA Microspheres

Author: YeMing
Tutor: WangZhiGang
School: Chongqing Medical University
Course: Medical Imaging and Nuclear Medicine
Keywords: Recombinant adenoviruses PEGylated Ultrasound microbubbles Contrast media Covalent binding PLGA-COOH ultrasound contrast agents Targeted
CLC: R445.1
Type: Master's thesis
Year: 2011
Downloads: 22
Quote: 0
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Abstract


Objective To prepare PEGylated recombinant adenovirus(Adv) for microencapsulation within poly(lactic-co-glycolic acid)(PLGA) ultrasound contrast agents.Then the basic physical properties, the release test and the bioactivity of viruses incorporated in vitro, the relative extent of the immune respone for Adv and PEG-Adv encapsulated within PLGA microspheres were detected.Methods PEGylated Adv(PEG-Adv) was constructed by PEGylated adenoviral capsid proteins with activated methoxypolyethylene glycols, encapsulate the recombinant adenovirus in PLGA using a water-in-oil-in-water(W/O/W) double emulsion and solvent evaporation method. 2. The diameter was determined. The release test and the bioactivity of viruses incorporated in vitro were studied, and the relative extent of the immune respone for Adv and PEG-Adv encapsulated within PLGA Results 1. The amounts of mPEG-SPA at 1:1 molar ratios of mPEG/ADV surface amine group were added to the solution. It was previously reported that the amount of primary amine groups on the surface of ADV was determined by the fluorescamine assay. After reacting overnight at 4oC, PEGylated ADV was dialyzed against 3%(w/v) sucrose/PBS solution at 4oC and kept at -20oC until use. 2. The diameter of PLGA carrying PEG-Adv was 6.23±1.2μm. More than 17.23% of adenovirus was encapsulated in PLGA. Adv and PEG-Adv were both released from PLGA similarly in a sustained fashion. However, PEG-Adv microspheres demonstrated a higher GFP gene transfection efficiency than Adv microspheres into HUVEC. The PEG-Adv micropheres also exhibited a reduced extent of innate immune response for macrophage cell.Conclusions PEGylated recombinant adenovirus can be formulated within PLGA increasing gene transfection efficiency and safety, which can be ideal gene therapy vectors for cardiovascular disease in future. Objective To prepare a kind of targeted PEG-ADV-loaded PLGA-COOH microspheres covalently binding with monoclonal antibody and investigate its affinity for Human umbilical vein endothelial cell (HUVEC) in vitro.Methods Targeted PLGA mirospheres were prepared by combining PLGA-COOH microspheres with anti-E-selectin monoclonal antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide hydrochloride(EDC) and N-hydroxysuccinimide(NHS) mediated cross linking agents. The characteristic of targeting to HUVEC with light microscope and served non-targeted PLGA microspheres as the control group were valued.Results In the targeting study in vitro, it was shown that many targeted high molecular polymer microspheres conjugated with HUVEC tightly.Conclusion Targeted PEG-ADV-loaded PLGA-COOH microspheres were prepared successfully and can bind to HUVEC effectively in vitro.

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