Therapeutic Anti-Methamphetamine Antibody Fragment-Nanoparticle Conjugates: Synthesis and in Vitro Characterization
Therapeutic Anti-Methamphetamine Antibody Fragment-Nanoparticle Conjugates: Synthesis and in Vitro Characterization
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DOI：
10.1021/bc300204n
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发表时间：
2012-09-01
影响因子：
4.7
通讯作者：
Peterson, Eric C.
中科院分区：
文献类型：
--
作者：
Nanaware-Kharade, Nisha;Gonzalez, Guillermo A., III;Peterson, Eric C.
非常需要针对甲基苯丙胺（METH）滥用引起的医疗问题的治疗。为了实现这一目标，我们正在开发具有可定制药代动力学特性的新型多价抗METH抗体片段-纳米颗粒缀合物。我们设计了一种新型的抗METH单链抗体片段，其末端带有一个工程化的半胱氨酸（scFv 6 H4 Cys）。第3代（G3）聚酰胺-胺树状聚合物纳米颗粒由于其单分散性和多个胺官能团而被选择用于缀合。ScFv 6 H4 Cys通过异双官能PEG交联剂与G3树枝状聚合物缀合，所述异双官能PEG交联剂对一端上的游离胺和另一端上的硫醇基团具有反应性。通过使PEG交联剂与树枝状聚合物以11：1的化学计量比反应来合成PEG修饰的树枝状聚合物，其进一步与3倍摩尔过量的抗METH scFv 6 H4 Cys反应。该反应产生G3-PEG-scFv 6 H4 Cys缀合物（树枝状体）的异质混合物，其中三至六个scFv 6 H4 Cys缀合至每个树枝状聚合物。使用亲和色谱将树枝状体与未反应的PEG修饰的树枝状聚合物和scFv 6 H4 Cys分离。对PEG修饰的树枝状聚合物和树枝状体进行详细的体外表征，以确定大小、纯度和METH结合功能。在饱和结合测定中，发现树枝状体对METH具有与未缀合的scFv 6 H4 Cys相同的亲和力，而PEG修饰的树枝状聚合物对METH没有亲和力。这些数据表明，抗METH scFv可以成功地缀合至PEG修饰的树枝状聚合物纳米颗粒，而对METH结合性质没有不利影响。这项研究是临床前表征和开发用于治疗METH滥用的新型纳米药物的关键一步。
Treatments specific to the medical problems caused by methamphetamine (METH) abuse are greatly needed. Toward this goal, we are developing new multivalent anti-METH antibody fragment-nanoparticle conjugates with customizable pharmacokinetic properties. We have designed a novel anti-METH single chain antibody fragment with an engineered terminal cysteine (scFv6H4Cys). Generation 3 (G3) polyamidoamine dendrimer nanoparticles were chosen for conjugation due to their monodisperse properties and multiple amine functional groups. ScFv6H4Cys was conjugated to G3 dendrimers via a heterobifunctional PEG cross-linker that is reactive to a free amine on one end and a thiol group on the other. PEG modified dendrimers were synthesized by reacting the PEG cross-linker with dendrimers in a stoichiometric ratio of 11:1, which were further reacted with 3-fold molar excess of anti-METH scFv6H4Cys. This reaction resulted in a heterogeneous mix of G3-PEG-scFv6H4Cys conjugates (dendribodies) with three to six scFv6H4Cys conjugated to each dendrimer. The dendribodies were separated from the unreacted PEG modified dendrimers and scFv6H4Cys using affinity chromatography. A detailed in vitro characterization of the PEG modified dendrimers and the dendribodies was performed to determine size, purity, and METH binding function. The dendribodies were found to have affinity for METH identical to that of the unconjugated scFv6H4Cys in saturation binding assays, whereas the PEG modified dendrimers had no affinity for METH. These data suggest that an anti-METH scFv can be successfully conjugated to a PEG modified dendrimer nanoparticle with no adverse effects on METH binding properties. This study is a critical step toward preclinical characterization and development of a novel nanomedicine for the treatment of METH abuse.