Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes
Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes
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DOI：
10.1016/j.biomaterials.2004.07.047
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发表时间：
2005-05-01
期刊：
影响因子：
14
通讯作者：
Langer, K
中科院分区：
文献类型：
--
作者：
Balthasar, S;Michaelis, K;Langer, K
已建立的蛋白质化学方法可用于将药物靶向配体有效附着到基于蛋白质的纳米颗粒的表面。在本工作中，明胶纳米颗粒用于附着生物素化的抗CD 3抗体的抗生物素蛋白-生物素复合物的形成。这些抗体修饰的纳米颗粒代表了一种有前途的载体系统，用于靶向T淋巴细胞的特异性药物。这项工作的目的是在这些细胞特异性纳米颗粒的制备过程中的每个化学反应步骤的全面定量。明胶纳米颗粒通过两步去溶剂化过程形成。第一次去溶剂化步骤后，通过尺寸排阻色谱法（SEC）分析剩余沉淀物和上清液的分子量分布。然后使用高分子明胶级分形成纳米颗粒，随后通过戊二醛交联进行稳定。颗粒表面上的可检测的氨基的一部分与2-亚氨基硫杂环戊烷反应，以引入反应性巯基。将硫醇化的纳米颗粒偶联至NeutrAvidin（TM）（NAv），其先前用异双官能交联剂磺基-MBS活化。所有这些反应步骤都通过光度法或重量法定量。通过生物素-4-荧光素测定证实共价缀合后NAv的功能性。然后将NAv修饰的纳米颗粒用于通过抗生物素蛋白-生物素复合物形成来结合生物素化的抗CD 3抗体。通过不同的间接方法：免疫印迹法和荧光测定法，确定了配体的高效附着。因此，建立了具有药物靶向配体修饰的定义明确的纳米颗粒系统，其有望用于进一步有效的临床前测试。(C)2004 Elsevier Ltd.保留所有权利。
Established methods of protein chemistry can be used for the effective attachment of drug targeting ligands to the surface of protein-based nanoparticles. In the present work gelatin nanoparticles were used for the attachment of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. These antibody modified nanoparticles represent a promising carrier system for the specific drug targeting to T-lymphocytes. The objective of this work was the comprehensive quantification of every chemical reaction step during the preparation procedure of these cell specific nanoparticles. Gelatin nanoparticles were formed by a two-step desolvation process. After the first desolvation step the remaining sediment and the supernatant were analysed for molecular weight distribution by size exclusion chromatography (SEC). Nanoparticles then were formed using the high molecular gelatin fraction and subsequently were stabilised by glutaraldehyde crosslinking. A part of the detectable amino groups on the particle surface was reacted with 2-iminothiolane in order to introduce reactive sulfhydryl groups. The thiolated nanoparticles were coupled to NeutrAvidin((TM)) (NAv) which previously was activated with the heterobifunctional crosslinker sulfo-MBS. All these reaction steps were quantified by photometry or gravimetry. The functionality of NAv after covalent conjugation was confirmed by a biotin-4-fluorescein assay. The NAv-modified nanoparticles then were used for the binding of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. A highly effective attachment of the ligand was ascertained by different, indirect methods: immunoblotting and fluorimetry. Therefore, a well-defined nanoparticle system with drug targeting ligand modification was established that holds promise for further effective preclinical testing. (C) 2004 Elsevier Ltd. All rights reserved.