Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, 4301 West Markham Street, #611, Little Rock, Arkansas 72205, USA.
Bioconjug Chem. 2012 Sep 19;23(9):1864-72. doi: 10.1021/bc300204n. Epub 2012 Aug 28.
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.
非常需要针对冰毒（METH）滥用引起的医学问题的特定治疗方法。为此，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。我们设计了一种具有工程化末端半胱氨酸（scFv6H4Cys）的新型抗 METH 单链抗体片段。由于其单分散性质和多个胺官能团，我们选择了第三代（G3）聚酰胺胺树枝状纳米粒子进行缀合。scFv6H4Cys 通过异双官能 PEG 交联剂与 G3 树枝状大分子缀合，该交联剂的一端对游离胺反应，另一端对硫醇基反应。通过将 PEG 交联剂与树枝状大分子以 11:1 的化学计量比反应合成 PEG 修饰的树枝状大分子，然后将其与 3 倍摩尔过量的抗 METH scFv6H4Cys 进一步反应。该反应导致 G3-PEG-scFv6H4Cys 缀合物（树状抗体）的混合物不均匀，每个树枝状大分子上有三到六个 scFv6H4Cys 缀合。使用亲和色谱法将树状抗体与未反应的 PEG 修饰的树枝状大分子和 scFv6H4Cys 分离。对 PEG 修饰的树枝状大分子和树状抗体进行了详细的体外表征，以确定其大小、纯度和 METH 结合功能。在饱和结合测定中，发现树状抗体对 METH 的亲和力与未缀合的 scFv6H4Cys 相同，而 PEG 修饰的树枝状大分子对 METH 没有亲和力。这些数据表明，抗 METH scFv 可以成功地与 PEG 修饰的树枝状大分子纳米颗粒缀合，而对 METH 结合特性没有不利影响。这项研究是朝着开发用于治疗 METH 滥用的新型纳米医学的临床前表征和开发迈出的关键一步。
