Aliyandi Aldy, Reker-Smit Catharina, Zuhorn Inge S, Salvati Anna
Department of Nanomedicine & Drug Targeting, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713AV Groningen, the Netherlands.
Department of Biomedical Engineering, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, the Netherlands.
Acta Biomater. 2023 Jan 1;155:507-520. doi: 10.1016/j.actbio.2022.11.010. Epub 2022 Nov 9.
Targeted drug delivery requires -among others- specific interaction of nanocarriers with cell surface receptors enabling efficient internalization into the targeted cells. Thus, identification of receptors allowing efficient nanocarrier uptake is essential to improve the design of targeted nanomedicines. Here we used methods based on cell surface biotinylation to identify cell surface receptors mediating nanoparticle uptake by cells. We used human brain and liver endothelial cells as representative examples of cells typically showing very low and very high nanoparticle uptake, respectively. Amino-modified and carboxylated silica were used as model nanoparticles usually associated with high and low uptake into cells, respectively, and carrying different coronas after exposure in full human plasma. Using cell surface biotinylation of live cells and receptor pull-down assays, we compared the receptors internalized in control untreated cells and those internalized upon exposure to nanoparticles. In this way, we identified receptors associated with (high) nanoparticle uptake. The candidate receptors were further validated by decorating the nanoparticles with an artificial corona consisting of the respective receptor ligands. We found that a vitronectin corona can be used to target integrin receptors and strongly enhances nanoparticle uptake in brain and liver endothelial cells. The increased uptake was maintained in the presence of serum, suggesting that the vitronectin-corona could resist interaction and competition with serum. Furthermore, plasminogen-coated nanoparticles promoted uptake in endothelial cells of the liver, but not of the brain. The presented approach using reversible biotinylation of cell surface receptors in live cells allows for receptor-based targeting of nanocarriers that are instrumental in nanoparticle uptake, which can be exploited for targeted drug delivery. STATEMENT OF SIGNIFICANCE: In order to deliver drugs to their site of action, drug-loaded nanocarriers can be targeted to cell receptors enabling efficient uptake into target cells. Thus, methods to identify nanocarrier receptors are invaluable. Here we used reversible biotinylation of live cells and receptor pull-down approaches for receptor identification. By comparative analysis of the individual receptors internalized in untreated cells and cells exposed to nanoparticles, we identified receptors enabling high nanoparticle uptake into liver and brain endothelial cells. Their role was confirmed by decorating nanoparticles with an artificial corona composed of the receptor ligands. In conclusion, live cell reversible biotinylation of cell surface proteins is a powerful tool for the identification of potential receptors for receptor-based targeting of nanocarriers.
靶向药物递送尤其需要纳米载体与细胞表面受体进行特异性相互作用，从而实现高效内化进入靶细胞。因此，识别能够使纳米载体有效摄取的受体对于改进靶向纳米药物的设计至关重要。在此，我们使用基于细胞表面生物素化的方法来识别介导细胞摄取纳米颗粒的细胞表面受体。我们分别使用人脑内皮细胞和肝内皮细胞作为通常显示出极低和极高纳米颗粒摄取的细胞的代表实例。氨基修饰的二氧化硅和羧基化的二氧化硅用作模型纳米颗粒，通常分别与细胞内的高摄取和低摄取相关，并且在全人血浆中暴露后携带不同的冠层。通过活细胞的细胞表面生物素化和受体下拉分析，我们比较了未处理的对照细胞内化的受体和暴露于纳米颗粒后内化的受体。通过这种方式，我们鉴定出与（高）纳米颗粒摄取相关的受体。通过用由各自受体配体组成的人工冠层修饰纳米颗粒，进一步验证了候选受体。我们发现玻连蛋白冠层可用于靶向整合素受体，并强烈增强纳米颗粒在脑和肝内皮细胞中的摄取。在血清存在的情况下，摄取增加得以维持，这表明玻连蛋白冠层可以抵抗与血清的相互作用和竞争。此外，纤溶酶原包被的纳米颗粒促进了肝内皮细胞而非脑内皮细胞的摄取。本文提出的在活细胞中使用细胞表面受体的可逆生物素化的方法允许基于受体的纳米载体靶向，这在纳米颗粒摄取中起重要作用，可用于靶向药物递送。重要性声明：为了将药物递送至其作用部位，载药纳米载体可以靶向细胞受体，从而实现高效摄取进入靶细胞。因此，识别纳米载体受体的方法非常宝贵。在此，我们使用活细胞的可逆生物素化和受体下拉方法进行受体识别。通过对未处理细胞和暴露于纳米颗粒的细胞内化的各个受体的比较分析，我们鉴定出能够使纳米颗粒高效摄取进入肝和脑内皮细胞的受体。通过用由受体配体组成的人工冠层修饰纳米颗粒，证实了它们的作用。总之，细胞表面蛋白的活细胞可逆生物素化是识别基于受体的纳米载体靶向潜在受体的有力工具。
