Silver Nanoparticle Protein Corona Composition in Cell Culture Media

The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial’s surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index), the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic and hydrophobic interactions in the formation of the PC which may have broad biological and toxicological implications.

纳米材料作为药物递送系统及其他产品的潜在应用正持续拓展。当被引入生理环境并受能量驱动时，纳米材料会迅速与蛋白质结合，在其表面形成蛋白冠（protein corona, PC）。该蛋白冠会改变纳米材料的表面特性，并可能影响其与细胞的相互作用。为明确纳米材料暴露的生物学影响以及纳米治疗应用，理解蛋白冠的形成机制十分必要。本研究采用无标记质谱蛋白质组学方法，对四种银纳米颗粒（silver nanoparticles, AgNPs）的蛋白冠组成进行了分析，包括柠檬酸盐稳定和聚乙烯吡咯烷酮（polyvinylpyrrolidone, PVP）稳定的胶体银，粒径分别为20 nm或110 nm。为模拟细胞培养条件，将AgNPs在添加10%胎牛血清的杜氏改良伊格尔培养基（Dulbecco’s Modified Eagle Medium, DMEM）中孵育1小时，随后进行洗涤，溶解冠层蛋白，并通过无标记液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）对蛋白进行鉴定与定量。为明确哪些属性会影响蛋白冠的形成，我们分别在纯水和添加10%胎牛血清的细胞培养基中对AgNPs进行了表征。所有AgNPs均结合了11种蛋白的共同子集，包括白蛋白、载脂蛋白、角蛋白及其他血清蛋白。相较于20 nm柠檬酸盐稳定和PVP稳定的AgNPs（分别结合45种和48种蛋白），110 nm柠檬酸盐稳定和PVP稳定的AgNPs结合的蛋白数量最多（分别为79种和85种），这表明蛋白冠的形成存在基于表面曲率的差异。虽然未发现其他蛋白参数（等电点或脂肪族指数）与蛋白冠形成存在关联，但20 nm AgNPs（PVP和柠檬酸盐稳定）表面的蛋白冠所含疏水蛋白多于110 nm AgNPs，这意味着此类蛋白更易受曲率诱导的折叠和拥挤作用影响，以换取水环境中更高的水合程度。这些观察结果证实了静电和疏水相互作用在蛋白冠形成过程中的重要性，这可能具有广泛的生物学和毒理学意义。