Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, United States.
Division of Hematology/Oncology, University of North Carolina at Chapel Hill, United States.
Acta Biomater. 2017 Nov;63:181-189. doi: 10.1016/j.actbio.2017.08.049. Epub 2017 Sep 9.
Pretargeting represents a promising strategy to enhance delivery of nanoparticles. The strategy involves first introducing bispecific antibodies or fusion proteins (BFP) that can bind specific epitopes on target cells with one arm, and use the other arm to capture subsequently administered effector molecules, such as radionuclides or drug-loaded nanoparticles. Nevertheless, it remains unclear whether BFP that bind slowly- or non-internalizing epitopes on target cells can facilitate efficient intracellular delivery. Here, we investigated the cellular uptake of biotin-functionalized nanoparticles with streptavidin-scFv against TAG-72, a membrane protein on Jurkat T-cell leukemia cells. Unlike conventional active-targeted nanoparticles, we found that pretargeting resulted in preferential retention of ∼100nm nanoparticles at the plasma membrane rather than internalization into cells. We found no improvement in nanoparticle internalization by simply reducing nanoparticle concentration or surface biotin density. Interestingly, by adding both the BFP and a monoclonal antibody against TAG-72, we observed a twofold improvement in internalization of pretargeted nanoparticles. Our work illustrates that the cellular fate of pretargeted nanoparticles can be controlled by carefully tuning the interactions between pretargeting molecules and nanoparticles on the cell surface.
Pretargeting is a multi-step strategy that utilizes bispecific proteins that recognize both cellular epitopes and subsequently administered therapeutic molecules. This approach has been extensively studied for radiotherapy of blood cancers; however, pretargeting remains largely underexplored for nanoparticle targeting, including whether pretargeting can facilitate efficient intracellular delivery. Here, we found that high density of targeting proteins on the cell surface can effectively limit internalization of pretargeted nanoparticles. Our work underscores the need to carefully assess specific cell-pretargeting molecule pairs for applications requiring intracellular delivery, and the key design requirements for such bispecific pretargeting molecules.
前靶向代表了一种增强纳米颗粒递送的有前途的策略。该策略涉及首先引入能够与靶细胞上的特定表位结合的双特异性抗体或融合蛋白（BFP），其一条臂与靶细胞结合，另一条臂用于捕获随后给予的效应分子，如放射性核素或载药纳米颗粒。然而，目前尚不清楚结合靶细胞上缓慢内化或非内化表位的 BFP 是否能够促进有效的细胞内递送。在这里，我们研究了链霉亲和素-scFv 与 TAG-72（Jurkat T 细胞白血病细胞上的一种膜蛋白）结合的生物素化纳米颗粒的细胞摄取。与传统的主动靶向纳米颗粒不同，我们发现前靶向导致约 100nm 纳米颗粒优先保留在质膜上，而不是内吞到细胞内。我们发现简单地降低纳米颗粒浓度或表面生物素密度并不能改善纳米颗粒的内化。有趣的是，通过添加 BFP 和针对 TAG-72 的单克隆抗体，我们观察到前靶向纳米颗粒内化提高了两倍。我们的工作表明，可以通过仔细调整前靶向分子与细胞表面上纳米颗粒之间的相互作用来控制前靶向纳米颗粒的细胞命运。
前靶向是一种利用同时识别细胞表位和随后给予的治疗分子的双特异性蛋白的多步策略。这种方法已被广泛研究用于血液癌的放射治疗；然而，前靶向在纳米颗粒靶向中仍在很大程度上未得到探索，包括前靶向是否可以促进有效的细胞内递送。在这里，我们发现细胞表面上高浓度的靶向蛋白可以有效地限制前靶向纳米颗粒的内化。我们的工作强调了需要仔细评估需要细胞内递送的应用所需的特定细胞-前靶向分子对，以及此类双特异性前靶向分子的关键设计要求。
