纳米颗粒表面蛋白冠的形成机制和表征技术研究进展

张永杰 黄彬铜 翟月明

引用本文: 张永杰, 黄彬铜, 翟月明. 纳米颗粒表面蛋白冠的形成机制和表征技术研究进展[J]. 无机化学学报, 2024, 40(12): 2318-2334. doi: 10.11862/CJIC.20240247 shu
Citation:  Yongjie ZHANG, Bintong HUANG, Yueming ZHAI. Research progress of formation mechanism and characterization techniques of protein corona on the surface of nanoparticles[J]. Chinese Journal of Inorganic Chemistry, 2024, 40(12): 2318-2334. doi: 10.11862/CJIC.20240247 shu

纳米颗粒表面蛋白冠的形成机制和表征技术研究进展

    通讯作者: 黄彬铜, E-mail: bthuang@whu.edu.cn; 翟月明, E-mail: yueming@whu.edu.cn
  • 基金项目:

    国家重点研发计划 2021YFA0909900

    中央高校基本科研业务费 2042024kf0012

    中国博士后科学基金 2023M742689

    中国博士后科学基金 GZB20230545

摘要: 在生物体液环境中,纳米颗粒表面会快速结合蛋白质,形成蛋白冠,这会极大地改变其理化性质,并影响与生物系统的相互作用。理解蛋白冠的形成机制和动态变化有助于优化纳米颗粒的设计,提高纳米药物的靶向性和有效性,减少副作用。在这篇综述中,我们首先回顾了蛋白冠的研究进展,详细介绍了蛋白冠的形成机制、影响因素以及调控方法和现阶段表征技术,最后讨论了现阶段蛋白冠研究面临的挑战,并基于此提出展望,以期不断深化对蛋白冠的认识,推动纳米医学和生物技术的发展,拓展其应用范围。

English

  • 纳米颗粒已经成为现代医学诸多方面不可或缺的一部分,其独特的光、电、磁学性质,在药物运输、医学成像、疾病诊断与治疗等领域得到广泛应用[1-3]。当纳米颗粒进入生物体内,由于其表面势能高于其他生物分子,表面会迅速吸附蛋白质、核酸、磷脂膜等生物分子,形成一系列“纳米-生物界面”。在界面处,生物分子与纳米颗粒发生动态物理化学相互作用、动力学和热力学交换[4]。实验表明,这些生物分子最终会在纳米颗粒表面形成具有生物特性的“分子冠”,并且决定着纳米颗粒在生物体内的分布行为、代谢过程以及最终的命运[5-6]。现阶段,对于生物分子冠形成机制和组成结构等问题,主要都以蛋白质吸附于纳米颗粒表面形成的蛋白冠为研究模型[7],因此本综述重点聚焦在蛋白冠。

    “蛋白冠”概念的发展改变了研究人员对纳米医学中生物系统与纳米颗粒相互作用的理解。“蛋白冠”一词于2007年由Cedervall等[8]提出,用于描述蛋白质在纳米颗粒表面上的竞争吸附并定义颗粒的生物学特性。然而在19世纪50和60年代Bangham等[9]和Vroman[10]已经开始进行蛋白质吸附研究。他们证明蛋白质吸附在整个生物相互作用以及与纳米材料表面的反应中发挥重要作用。在随后的20世纪80和90年代,研究主要集中在脂质体和聚合纳米颗粒与蛋白质吸附。旨在开发抗吸附表面和纳米材料,以防止其被细胞识别和吸附,并延长静脉注射的纳米颗粒血液循环半衰期。其中最大的突破是开发了亲水性聚合物聚乙二醇,它的涂覆和修饰可以稳定纳米颗粒并减少其与血液蛋白的相互作用[11]。过去十几年里,蛋白质吸附研究发生显著的变化,研究者主要利用质谱(mass spectrometry,MS)、透射电子显微镜(transmission electron microscope,TEM)以及多种光谱手段研究纳米颗粒表面的蛋白冠形成机制与组成成分[12]

    蛋白冠主要是由静电相互作用、范德瓦耳斯力、疏水相互作用、氢键和蛋白质与蛋白质的相互作用等形成[13]。当蛋白质开始与纳米颗粒相互作用时,高迁移率、低亲和力的蛋白质借助弱相互作用(如氢键和疏水作用)吸附在纳米颗粒表面,形成高度动态且松散的蛋白质“软冠”。随着时间的推移,这些低亲和力的蛋白会被亲和力更强的蛋白质取代,进而形成与纳米颗粒紧密结合的内层蛋白质“硬冠”。一般情况下,软冠和硬冠的形成不是截然分开的2个步骤,而是一个动态的、相互影响的过程[7, 14-15]。因此,对纳米颗粒蛋白冠的硬冠和软冠的表征存在极大的挑战。另一方面,纳米颗粒在体内形成的蛋白冠是由内源性配体组成,这些配体会掩盖纳米颗粒表面固有的反应活性,改变纳米材料的本征理化特性,并赋予其新的化学生物学特性,进而改变纳米颗粒的体内生物学行为,包括细胞摄取、免疫反应、血液循环、靶向效率、生物分布以及毒性等[13]

    除此之外,蛋白冠也为体外诊断和个性化纳米医疗创造了新的机遇[2]。一些疾病生物标志物,特别是罕见蛋白或糖蛋白可以富集到纳米颗粒上,形成蛋白冠独特的“指纹”,这可以用于个性化检测,有助于疾病预防、早期诊断和风险分层管理[16-17]。癌症或者某些疾病可能导致特定的蛋白表达上调或者下调,同时疾病微环境也改变蛋白质的物理化学性质,进而影响蛋白质与纳米颗粒的结合能力,最终导致蛋白冠组成结构和功能发生显著变化。利用这些蛋白组学差异,结合生物信息学手段,可以实现不同肿瘤患者以及不同肿瘤亚型和健康人群的区分[16, 18-19]。在个性化纳米医疗中,主要也是通过蛋白冠中组学个体化差异进行分析,设计更精准的治疗策略,优化给药方案以及药物递送系统,减小特定药物反应和潜在毒性,并提高治疗的效果[20-22]

    鉴于近年来纳米颗粒在生物系统中的应用越来越多,深入地理解蛋白冠的特性及其对纳米材料体内命运的影响是调控纳米材料有效性和提高安全性的重要科学基础。在这篇综述中,我们对纳米颗粒中蛋白冠的形成及影响因素和表征手段进行了深入讨论,总结现阶段蛋白冠研究面临的挑战和机遇。

    蛋白冠的形成是一个复杂的过程,受到纳米颗粒组成及其尺寸、形状、表面电荷和化学修饰等本征理化特性、生物流体性质以及环境因素的多方面影响。这些因素共同作用决定了蛋白冠的最终组成和性质,进而影响纳米颗粒在生物体中的行为和功能。

    蛋白冠的形成及其与纳米颗粒的结合强度在很大程度上取决于纳米颗粒的组成及其结构特性,如大小、形状、形态等[1, 23]。不同种类纳米颗粒的蛋白冠成分的差别较大。Weiss等[24]发现介孔二氧化硅纳米颗粒在人血清中会形成以补体蛋白、糖蛋白、血清淀粉样蛋白、凝血因子、凝胶蛋白等为主要成分的蛋白冠。Pinals等[25]通过MS表征了人血清中DNA功能化的单壁碳纳米管上蛋白冠的组成,其主要成分为血清白蛋白、富含组氨酸糖蛋白和载脂蛋白A-Ⅰ、补体蛋白C3、补体蛋白C4等。Ashby等[26]采用场流分离法和超离心分离法研究了人血清中超顺磁性氧化铁纳米颗粒上形成的蛋白冠,其主要成分为白蛋白、α-1-抗胰蛋白酶、免疫球蛋白A、触球蛋白、巨球蛋白等。以上3种不同纳米颗粒与人血清相互作用吸附了不同成分的蛋白,表明材料种类对蛋白冠组成具有较大影响。

    纳米颗粒具有较大的表面积,这有利于它们与蛋白的非特异性相互作用,其尺寸大小影响蛋白质吸附量,并且决定蛋白冠的结构。一般而言,较大的纳米颗粒比较小的纳米颗粒吸附的蛋白质更多。Piella等[27]研究了粒径为3.5~150 nm柠檬酸盐稳定的金纳米颗粒在含有10%胎牛血清细胞培养液中的蛋白冠形成过程,发现蛋白冠的厚度和密度取决于金纳米颗粒的尺寸(图 1a)。粒径小于12 nm的金纳米颗粒表面的蛋白冠层不完整,12~80 nm的金纳米颗粒可以形成完整的单层蛋白冠,而大于80 nm时,蛋白冠表现为多层结构。但是颗粒粒径越小,其结合蛋白达到平衡的时间也越快。此外,纳米颗粒尺寸会影响吸附蛋白质的种类。Tenzer等[28]研究了粒径为20、30和100 nm的3种不同大小的二氧化硅纳米颗粒在人血清中蛋白冠的成分。他们发现即使粒径仅有10 nm的差异也会显著影响蛋白冠的成分,脂蛋白与较小的纳米颗粒结合能力增强,而凝血酶原或肌动蛋白优先与较大的纳米颗粒结合。

    图 1

    图 1.  (a) 不同尺寸金纳米颗粒的蛋白冠形成过程示意图[27]; (b) 球形和棒状的介孔二氧化硅纳米颗粒在血液中的蛋白冠形成过程示意图[23]
    Figure 1.  (a) Schematic diagram of protein corona formation process of gold nanoparticles with different sizes[27]; (b) Schematic diagram of protein corona formation process of spherical and rod-shaped mesoporous silica nanoparticles in blood[23]

    不同形状的纳米颗粒提供了不同的物理环境,这将影响蛋白质与纳米颗粒相互作用区域和空间位阻。例如,与球形纳米颗粒相比,片状和棒状纳米颗粒可提供更多的平面区域,有利于特定蛋白质的吸附[23, 29]。Visalakshan等[23]研究了球形和棒状介孔二氧化硅纳米颗粒在血清和血浆中形成的蛋白冠(图 1b)。这2种介孔二氧化硅纳米颗粒具有相同的化学性质、孔隙度、表面电位和γ-维尺寸,只存在形状上的差异。与球体相比,棒状颗粒上附着的蛋白质数量更多,并且在球形和棒状介孔二氧化硅纳米颗粒上吸附的白蛋白和纤维蛋白原等蛋白含量不同,呈现出形状依赖性差异。其根本原因是这2种纳米颗粒的晶体不同,球形纳米颗粒为无定形结构,而棒状纳米颗粒主要以六方晶体结构排列,导致其具有不同的表面能,使其蛋白冠成分和数量产生差异。

    不同形貌的金纳米颗粒,例如球形、棒状、星形、片状、立方体等,因其表面结构和表面能的差异,与蛋白质的相互作用方式各不相同,吸附蛋白质的种类和数量也相差较大[30-32],从而导致不同的蛋白冠特征,如图 2a所示。星形纳米颗粒具有复杂形貌,不同部位的曲率差异会导致吸附蛋白种类和数量差异较大[33]。Diloknawarit等[30]发现在短支和长支的金纳米星结构上具有不同的蛋白冠分布(图 2b),且较长分支的金纳米星结构与癌细胞细胞膜上的人表皮生长因子受体2的结合效果更好。金纳米星的长支链高曲率尖端处吸附的小蛋白质促进了DNA适配体与人表皮生长因子受体2的结合。同时,具有高曲率尖端的颗粒在细胞实验和体内实验中表现出不同的生物学行为,如细胞摄取效率和循环时间等[34]。García-Álvarez等[35]研究了小鼠静脉注射各向异性金纳米颗粒(纳米棒和纳米星),经过血液循环后体内蛋白冠形成的情况。结果表明在注射后10 min就已形成了复杂的蛋白冠,不同颗粒表面吸附蛋白质总量和蛋白冠组成差异较大。上述研究表明,在生物体内颗粒的种类、大小和形状对吸附蛋白质总量和蛋白冠组成都有影响,通过调控金纳米颗粒的形貌和大小,可以实现对蛋白冠组成和结构的精细控制,从而优化纳米颗粒在生物医学领域的应用。关于不同纳米材料形成的蛋白冠归纳如表 1所示。

    图 2

    图 2.  (a) 不同形状金纳米颗粒的TEM图像以及蛋白冠中的蛋白含量[31]; (b) 不同分支长度的金纳米星形成蛋白冠的TEM图[30]
    Figure 2.  (a) TEM images of the differently shaped gold nanoparticles and the total number of identified corona proteins[31]; (b) TEM images of short and long gold nanostars with protein coronas[30]

    表 1

    表 1  典型纳米颗粒的蛋白冠组成
    Table 1.  Protein corona composition of typical nanoparticles
    下载: 导出CSV
    Structure Type of nanoparticles Protein corona component Ref.
    Nanosphere SiO2 Complement protein, glycoprotein, serum amyloid protein, coagulation factor, gel protein, serum albumin, immunoglobulin G, complement protein C1q [24]
    Fe2O3 Albumin, α-1-antitrypsin, immunoglobulin A, haptoglobulin, macroglobulin, transferrin, bovine serum albumin, α-2-HS glycoprotein, apolipoprotein A-Ⅰ [26, 36]
    Ag Apolipoprotein, lactoferrin, human serum albumin, phospholipid-binding protein A1/A2, hemoglobin, metallothionein 1, plasma ceruloplasmin [37-38]
    Au Complement protein C3, apolipoprotein A-Ⅰ, complement factor H, α-2-HS glycoprotein, viral spike protein S1, concanavalin A, bovine serum albumin [39-41]
    CeO2 Bovine serum protein, immunoglobulin [42]
    C60 Bovine serum protein, fibrinogen, hemoglobin [43]
    L-Au Human serum albumin, complement protein C3, fibronectin 1, epididymal luminal protein 214, complement protein C4-A, serum transferrin, complement factor H, apolipoprotein A-Ⅰ, platelet factor 4, immunoglobulin [44]
    D-Au Human serum albumin, complement protein C3, complement protein C4-A, complement factor H, fibronectin, epididymal luminal protein 214, adiponectin B, platelet factor 4, apolipoprotein C-Ⅲ, and complement protein C1q
    Polystyrene Apolipoprotein A-Ⅰ, fibrinogen [45-46]
    Nanostar Au Fibrinogen, trypsin, complement protein C3, complement factor H, human serum albumin, α-2-macroglobulin, plasminogen [30, 32]
    Nanocube Au Human serum albumin, histidine-rich glycoprotein, complement component C9, apolipoprotein B-100, coagulation factor Ⅻ [31]
    Nanorod Au Fibrinogen, trypsin, bovine serum albumin, human serum albumin, complement protein C3, complement protein C1q, apolipoprotein A-Ⅰ, apolipoprotein A-Ⅱ [31-32, 47]
    Nanotube Single-walled carbon nanotube Human serum albumin, histidine-rich glycoprotein, apolipoprotein A-Ⅰ, complement protein C3, complement protein C4 [25]
    Quantum dot CdSe@ZnS Human serum albumin, lysozyme [48]
    Nanosheet Graphene oxide Fibrinogen α chain, human serum albumin, apolipoprotein B-100, apolipoprotein E, complement protein C3 [49]
    Black phosphorus Human serum albumin, α-2-macroglobulin, complement protein C3, transferrin, heme binding protein, apolipoprotein B [50-51]

    纳米颗粒的表面性质包括表面手性、电荷、亲疏水性以及表面化学修饰等,也是影响蛋白冠组分的重要因素。手性纳米颗粒具有原子级的表面手性,在蛋白冠形成过程中表现出与普通纳米颗粒显著不同的动力学、热力学和吸附取向行为。这些差异主要体现在手性纳米颗粒与蛋白质的相互作用中表现出的立体选择性和功能性,使其在生物医学应用中具有更大的潜力和优势。Xu等[52]利用手性纳米结构的不对称性调控免疫应答,发现左手性纳米颗粒与抗原呈递细胞表面受体蛋白亲和性高于右手性颗粒(图 3a)。在跨膜蛋白作用下,左手性纳米颗粒进入细胞的速率为右手性颗粒的2倍。Baimanov等[44]研究了具有不同手性的金纳米颗粒形成的蛋白冠的成分。他们发现手性金纳米颗粒对蛋白冠成分表现出表面手性特异性识别,最终导致体内不同的细胞摄取和组织积累。左手性纳米颗粒蛋白冠中白蛋白、血清转铁蛋白、纤维蛋白原和纤维连接蛋白含量相对较高,而大多数补体和脂蛋白家族蛋白在右手性表面的吸附增强(图 3b)。除此之外,纳米颗粒在吸附蛋白后,形成的复合物会诱导纳米颗粒产生手性[47]。纳米颗粒的表面电荷会影响蛋白质的吸附行为[53]、蛋白冠的组成以及分布代谢情况[54-55]。由于血液中大多数蛋白质分子带负电荷,带正电的纳米颗粒表面通过静电吸引蛋白质。这种静电吸引力通常较强,可以导致蛋白质牢固吸附,引起蛋白质的构象变化甚至变性。表面配体和溶液pH都可以对纳米颗粒的表面带电情况进行调控。Wang等[56]采用十六烷基三甲基溴化铵、聚乙二醇和柠檬酸盐3种不同配体修饰的金纳米颗粒对牛血清白蛋白吸附的过程进行了分析(图 3c),发现带负电的金纳米粒子倾向于在其表面形成蛋白冠,而带正电的纳米粒子则倾向于聚集。此外,一些带正电荷的纳米颗粒很容易被调理素识别而被网状内皮系统和单核吞噬细胞系统吞噬,导致这些纳米颗粒清除速度加快,有效浓度显著下降[57]。表面修饰除了可以改变表面电荷,还可以改变其亲疏水性、稳定性和提供特异性结合位点等[58-59]。利用二氧化硅的介电特性以及表面的硅烷醇官能团,可改性超顺磁性氧化铁纳米颗粒亲疏水性和稳定性,防止团聚并给蛋白质提供结合位点[60]。在纳米颗粒的表面可以修饰一些合适的聚合物,例如聚乙二醇,以减少非特异性结合,抑制调理素作用,提高纳米颗粒表面基团与细胞表面的受体特异性结合[61]。近期Dridi等[62]使用一系列基于二氢硫辛酸(dihydrolipoic acid,DHLA)修饰的配体研究金纳米颗粒和血清蛋白之间的相互作用(图 3d),研究发现使用小配体(例如DHLA或柠檬酸盐)能够通过静电斥力稳定金纳米颗粒,并促进蛋白质的非特异性吸附,形成蛋白冠。相比之下,给DHLA上附加亲水基,如聚乙二醇或两性离子基团,基本上消除了颗粒与蛋白的非特异性相互作用,避免了蛋白冠的形成。除此之外,纳米颗粒表面粗糙度和弹性都会导致蛋白冠的组成差异[63-64]。因此,纳米颗粒的表面特性与化学修饰在蛋白冠形成中起到关键作用,为纳米颗粒的生物应用设计提供了有价值的信息。

    图 3

    图 3.  (a) 左旋和右旋金纳米颗粒的SEM图(左)以及手性纳米颗粒与细胞受体的相互作用示意图(右)[52]; (b) 三肽修饰的手性金纳米颗粒的示意图(左)以及在人血清中手性金纳米颗粒蛋白冠成分鉴定的热图(右)[44]; (c) 不同表面电荷的金纳米颗粒形成蛋白冠的示意图[56]; (d) DHLA修饰的金纳米颗粒(上)和聚乙二醇修饰的金纳米颗粒(下)与蛋白相互作用的示意图[62]
    Figure 3.  (a) SEM images of L-P+ and D-P- gold nanoparticles (left) and a diagram of the interaction between chiral nanoparticles and cell receptors (right)[52]; (b) Schematic diagram of chiral gold nanoparticles modified with tripeptides (left) and heatmap for identification of protein corona components of chiral gold nanoparticles in human serum (right)[44]; (c) Schematic diagram of protein corona formed by negatively charged gold nanoparticles and positively charged gold nanoparticles[56]; (d) Schematic diagram of the interaction of DHLA-coated modified gold nanoparticles (top) and polyethylene glycol-coated modified gold nanoparticles (bottom) with proteins[62]

    纳米颗粒所处的环境也会对蛋白冠的形成产生影响,环境介质的pH、组成、温度都会影响纳米颗粒与蛋白质的相互作用,改变其与生物体的亲和力,从而影响其在生物体内的分布、代谢和毒性等。此外,肿瘤的缺氧和酸性微环境、炎症和发热等疾病状态下会改变生物体的介质环境,且不同器官的组织液中含有的有机物和无机盐等代谢物质也不同,对体内蛋白冠的形成和组成产生巨大影响[12, 65-66]。甚至在某些疾病(如神经退行性疾病)中,特定蛋白质如β-淀粉样蛋白、Tau蛋白和α-突触核蛋白等异常聚集可能主导蛋白冠的组成,反映出疾病的特征性变化[67-68]

    多个体外实验和计算模拟研究表明,不同pH下蛋白质与纳米颗粒的吸附作用力不同[69-72]。当pH接近蛋白质的等电点(protein isoelectric point,pI)时,蛋白质的净电荷为零,静电排斥力减弱,蛋白质更容易通过其他相互作用(如疏水作用、范德瓦耳斯力)吸附在纳米颗粒表面。当pH远离蛋白质的pI时,蛋白质带有显著的净电荷,静电相互作用增强[69]。极端的pH可能导致蛋白质变性,使其结构展开,从而改变其吸附行为和在纳米颗粒表面的排列方式(图 4a)[71]

    图 4

    图 4.  (a) 不同pH下TiO2颗粒表面蛋白冠的示意图[71]; (b) 不同温度下聚苯乙烯纳米颗粒的蛋白冠的组成[45]; (c) 正常和高胆固醇血症小鼠体内纳米颗粒生物分布的实验过程示意图(上)、正常和高胆固醇血症小鼠的血清胆固醇含量(左下)、正常和高胆固醇血症小鼠血清中形成的蛋白冠中载脂蛋白和补体蛋白的百分比(右下)[12]
    Figure 4.  (a) Formation of protein corona of TiO2 particles at different pH values[71]; (b) Composition of the protein corona of polystyrene nanoparticles at different temperatures[45]; (c) Scheme of the experiments for in vivo bio-distribution of nanoparticles in normal and hypercholesterolemic mice (top), the serum cholesterol content in normal and hypercholesterolemic mice (bottom left), the percentage of apolipoproteins and complement proteins identified in protein corona formed in serums from normal and hypercholesterolemic mice (bottom right)[12]

    环境温度的变化主要会影响蛋白质的扩散和对纳米颗粒的表面亲和力。Oberländer等[45]研究了在人血浆中温度对聚苯乙烯纳米颗粒蛋白冠的形成和组成的影响,他们观察到低温(4 ℃)下形成的蛋白冠成分与生理温度(37 ℃)下形成的成分不同(图 4b)。蛋白冠中的簇蛋白和血清白蛋白数量随着温度的升高而增加,而载脂蛋白A-Ⅰ和纤维蛋白原的数量随着温度的升高而减少。在4 ℃时,凝固蛋白是蛋白冠中的优势蛋白组,而在37 ℃时,脂蛋白和玻璃结合蛋白是蛋白冠中的优势蛋白组。

    介质环境中,特别是体内与疾病相关的小分子对于调节蛋白冠的组成和纳米颗粒在体内的命运具有重要作用。近期有研究表明环境中胆固醇浓度的改变可以调控蛋白冠的组成成分[12]。高水平的胆固醇通过改变蛋白质与纳米颗粒的结合亲和力,导致形成载脂蛋白富集和补体蛋白减少的蛋白冠(图 4c)。胆固醇介导的蛋白冠可以诱导更强的巨噬细胞对纳米颗粒的炎症反应,并促进肝细胞摄取。体内的结果表明,与健康小鼠相比,高胆固醇血症小鼠体内的蛋白冠主要被输送到肝脏、脾脏和大脑,极少被输送到肺部。随着对体内介质环境深入理解和控制,可以更有效地设计和开发疾病诊断和治疗工具,实现精准医疗的目标。

    通过对蛋白冠系统的表征,有助于全面理解蛋白冠的组成、结构和功能,可以优化纳米颗粒的设计,为纳米颗粒在生物医学中的应用提供重要依据。现阶段,大多数研究依然采用非原位检测技术进行蛋白冠分析,这类技术需要通过分离纯化将带有蛋白冠的纳米颗粒从未吸附的蛋白质中分离。利用蛋白冠的尺寸、密度、电荷以及磁性等性质差异,通过离心法[46, 73-75]、层析色谱法[76-77]、磁分离法[78]和场流分离法[79-80]等方法有效地分离并纯化蛋白冠纳米颗粒。蛋白冠的分析表征主要包括对其形貌结构、成分定性和定量分析、形成过程和吸附中亲和力和动力学以及构象变化等分析,目前已发展了多种针对蛋白冠的测量方法和技术手段。本节总结了可用于蛋白质-纳米颗粒复合物表征的主要技术,并对目前一些原位检测技术进行介绍。

    TEM[28, 46]、SEM[81-82]和原子力显微镜(atomic force microscopy,AFM)[83-84]是蛋白冠研究中强大的形态学和结构分析工具。虽然单独的TEM成像难以直接观察到蛋白冠,但是通过结合负染色、电子显微镜断层扫描和免疫金标记等技术,研究人员可以深入了解蛋白冠的形成机制、结构特征和在纳米粒子表面的分布情况。Sheibani等[46]采用负染色和冷冻电子透射显微镜研究了单颗粒水平上羧基化的聚苯乙烯纳米颗粒暴露于人血浆后其表面的生物分子的积累和分布(图 5a)。研究发现颗粒表面的分子冠呈簇状分布,且分子簇可能在分子冠形成前已经存在,同时在多次洗涤后,分子簇仍然很明显。Kelly等[85]使用抗体来标记金纳米颗粒,采用微分离心沉降和各种成像技术,识别蛋白质的空间位置、功能基序和结合位点(图 5b)。他们发现,对于转铁蛋白包被的聚苯乙烯纳米颗粒,只有少数吸附的蛋白质表现出功能基序,并且空间排列呈现随机性。尽管SEM和TEM能够提供高分辨率的结构信息,但是其样品制备复杂,染色和干燥过程以及高真空环境可能导致蛋白冠结构改变。AFM能够在液体环境和空气中操作,不需要高真空环境,也能在纳米级分辨率情况下实时监测蛋白冠的形成过程和动态变化,但是扫描速度较慢,在接触模式下AFM探针与蛋白冠表面之间会存在相互作用。

    图 5

    图 5.  (a) 聚苯乙烯纳米颗粒表面生物分子簇洗涤前(上)和洗涤3次后(下)的TEM图[46]; (b) 不同浓度的免疫金标记的聚苯乙烯纳米颗粒-蛋白复合物的TEM图[85]; (c) 美国17个不同测试机构及其对相同蛋白冠样品分析流程的示意图[97]; (d) 同步加速器源的SAXS原理(上)及SiO2纳米颗粒形成蛋白冠和聚集体的示意图(下)[110]
    Figure 5.  (a) TEM images of biomolecule clusters on the surface of polystyrene nanoparticles before washing (top) and after washing three times (bottom)[46]; (b) TEM images of immunogold-labelled polystyrene nanoparticle corona complexes with different concentrations of immunogold labels[85]; (c) Schematic diagram of 17 proteomics core facilities in the USA and their analysis processes for the same protein corona sample[97]; (d) Schematic diagram of SAXS of synchrotron source (top), and SiO2 nanoparticles forming protein corona and aggregates (bottom)[110]

    通过动态光散射(dynamic light scattering,DLS)[86-87]和纳米颗粒实时跟踪分析(nanoparticle tracking analysis,NTA)[88-89]可以表征纳米颗粒和蛋白复合物的水合尺寸、ζ电位以及分散性。DLS样品制备简单,对样品的破坏性小,但是对多分散体系的分辨能力有限。NTA可以在单颗粒水平对粒径和浓度进行分析,但是样品需要良好的透明度。除此之外,多种基于X射线的技术已用于表征纳米颗粒的结构、成分、大小或聚集状态[90-91]

    对吸附在纳米颗粒上的蛋白冠的组成和定量研究,可以为纳米颗粒与蛋白质的相互作用机制及其生物学效应提供线索。目前大多数蛋白质定量和定性技术都需要将蛋白冠与游离蛋白分离,然后将蛋白冠组成成分从纳米颗粒上洗脱下来,并通过紫外可见吸收光谱(UV visible absorption spectrum)[31, 92]、二辛可宁酸(bicinchoninic acid,BCA)测定法[93]、酶联免疫法(enzyme-linked immunosorbent assay,ELISA)[94]和MS[6, 39, 95]等方法进行定量。具有芳香族侧链氨基酸(色氨酸、酪氨酸等)的蛋白质在280 nm处具有独特的紫外吸光度[96],因此紫外吸收光谱比较适用于单一蛋白质的测定,并且样品中一些非蛋白质成分可能会干扰吸光度测量。BCA是比色检测总蛋白质定量的常用方法,适用于大多数蛋白质的测定。ELISA具有高特异性、高灵敏度优势,但是测试过程中依赖于特异性抗体,成本较高,特别是对于血液等生物体液中蛋白冠组成复杂的样本。目前,MS是大多数蛋白质组学实验的基础,具有高通量、免标记等优势,特别是液相色谱与MS联用(liquid chromatography coupled to mass spectroscopy,LC-MS/MS)是表征蛋白冠中的蛋白数量和相对丰度的主要方法。但是标准的样品制备和测试分析流程对LC-MS/MS分析蛋白冠至关重要。Ashkarran等[97]采用17个不同测试机构对相同样品分析发现,4 022种蛋白中只有73种蛋白(1.8%)鉴定一致,数据差异巨大(图 5c)。在统一参数变量和标准化实验流程后,5家机构鉴定样本中有253种(35.3%)一致,11家测试机构样本中有16.2%的蛋白种类一致[98]。因此,发展蛋白冠研究标准化样品处理过程,减少实验样品制备和测试误差,确保可重复性,这对加速纳米技术在诊断和治疗上的临床应用有巨大意义。此外,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacryla-mide gel electrophoresis,SDS-PAGE)可以用于蛋白的分离和结合染色定性分析蛋白冠中蛋白组分[50, 99]。然而,SDS-PAGE不能提供精确的蛋白质信息,并且存在通量低和灵敏度低等问题。因此,SDS-PAGE也常常与LC-MS/MS技术联用以同时鉴定和定量蛋白冠[28, 39]。除了上述的一些定量分析方法,石英晶体微天平(quartz crystal micro-balance,QCM)、热重分析(thermogravimetric analysis,TGA)也可用于监测吸附蛋白质的质量变化[93, 100]

    蛋白冠的形成是一个复杂的动力学过程,受到多种因素的影响。当纳米颗粒进入生物体液时,纳米颗粒表面首先与溶液中的蛋白质分子发生相互作用,之后蛋白质分子的吸附和解吸过程会继续进行,达到动态平衡,形成相对稳定的蛋白冠。已经有多项研究通过等温滴定量热法(isothermal titration calorimetry,ITC)[101-102]、表面等离子体共振(surface plasmon resonance,SPR)[103-104]、QCM[105]、微尺度热泳法(microscale thermophoresis,MST)[106-107]、生物层干涉法(biolayer interferometry,BLI)[108-109]等测量亲和力常数,深入分析了蛋白冠形成过程。ITC通过测量在固定温度下纳米颗粒与蛋白分子相互作用过程中产生的热量变化来获得热力学参数,但是测试过程中需要的样本量较大,对于弱相互作用灵敏度不足。SPR通过检测电磁波在金属表面传播时发生的共振条件变化,实时监测蛋白质与纳米颗粒的结合和解离过程。蛋白冠形成过程中伴随着颗粒的无规则聚集,导致结果出现偏差,而且上述测量技术大多数得到的是平均信息,因此无法了解纳米颗粒的异质性。一些基于X射线光子相关光谱(X-ray photon correlation spectroscopy,XPCS)的技术可以直接探测并区分纳米颗粒和生物分子信号,部分地解决了上述问题。在大多数情况下,纳米颗粒的X射线散射截面较小,因此需要利用同步辐射的技术。Cardoso等[110]基于同步加速器的小角X射线散射(small-angle X-ray scattering,SAXS)研究了二氧化硅颗粒表面蛋白冠的形成过程(图 5d),通过该技术可以清楚区分蛋白冠和颗粒的聚集。Wang等[111]利用X射线吸收近边结构(X-ray absorption near edge structure,XANES)直接观察到金纳米棒表面牛血清白蛋白冠中的Au—S键。此外,一些基于光谱的技术被发展用于单颗粒水平原位观察蛋白冠的形成过程[112-113]和颗粒表面蛋白质的分布[114-115],避免了分离带来的影响。Dolci等[88]利用散射显微镜,在单颗粒水平原位实时跟踪全血清中不同形状的金属纳米颗粒和介电纳米颗粒表面蛋白吸附形成蛋白冠的全过程(图 6),并且无需任何标记,得到了单颗粒表面吸附蛋白的质量、亲和力和动力学等参数。

    图 6

    图 6.  介电纳米颗粒和等离激元纳米颗粒蛋白冠形成示意图(左), 以及全内反射激发散射显微镜装置(右)[88]
    Figure 6.  Schematic diagram of the formation of protein corona for dielectric nanoparticles and plasmonic nanoparticles (left), and the setup of total internal reflection excited scattering microscope (right)[88]

    蛋白冠的形成会改变纳米颗粒的生物相容性和功能,而蛋白质在吸附到纳米颗粒表面时可能发生结构变化,影响其功能。X射线晶体衍射(X-ray crystal diffraction,XRD)[116]或冷冻电子显微镜(cryo-electron microscopy,cryo-EM)[117]具有出色的空间分辨率,为蛋白质静态结构解析提供了大量信息,但是蛋白质构象动力学和纳米颗粒相互作用的动态过程研究存在极大挑战。核磁共振波谱(nuclear magnetic resonance spectroscopy,NMR)[118]、SAXS[119]和小角中子散射(small-angle neutron scattering,SANS)[120]等谱学技术可以用于获取蛋白质三维结构以及动力学信息,但是得到的都是整个系统的平均信息,还需要较高浓度的样品进行长时间的数据采集。圆二色光谱(circular dichroism,CD)[121]、同步辐射圆二色光谱(synchrotron radiation CD,SR-CD)[122-123]、傅里叶变换红外光谱(Fourier transform infrared,FTIR)[32, 81]以及表面增强拉曼光谱(surface enhanced Raman spectroscopy,SERS)[124]都被用来提供蛋白质的二级结构信息。CD对于监测环境因素导致的蛋白冠结构改变非常有效,可以获得α-螺旋、β-折叠和无规则卷曲等二级结构的相对比例。FTIR利用分子结构特有的模式和频率振动,从纳米颗粒表面获取蛋白质的信息,对蛋白质质子化状态、氢键和不同基团的构象(包括肽骨架、氨基酸侧链、内部水分子或辅因子)都很敏感[125]。其中蛋白质和肽最重要的红外特征是周期性肽基团的集体振荡,这会产生酰胺Ⅰ区(主要是C=O拉伸,波数约为1 650 cm-1)和酰胺Ⅱ区(N—H弯曲和C—N拉伸,波数约为1 550 cm-1)[126]。蛋白质分子吸附在金纳米颗粒表面,能够显著增强红外光谱信号,增强区域大概在表面10 nm左右,这个长度与蛋白质的直径(1~20 nm)非常吻合[127],因此,利用红外增强研究蛋白冠形成的相关过程是一种有吸引力的方法。SERS同样也可以获得蛋白质分子的结构信息,具有极高的表面检测灵敏度,而且大多数拉曼谱峰窄,分辨率高,适合溶液环境多组分分析[128-129]。蛋白质中的一些特定氨基酸,如苯丙氨酸、络氨酸和组氨酸等,以及酰胺Ⅰ区α-螺旋(1 655 cm-1附近)、β-折叠(1 665 cm-1附近)和无规则卷曲(1 670 cm-1附近)以及酰胺Ⅱ区α-螺旋(1 280 cm-1附近)、β-折叠(1 235 cm-1附近)和无规则卷曲(1 245 cm-1附近)等二级结构都具有特定的SERS信号[82, 124, 130]。Szekeres等[128]利用SERS对金纳米颗粒在细胞中形成蛋白冠的过程以及蛋白质和金纳米颗粒相互作用进行了表征。与正常细胞质相比,由纳米颗粒孵育后的活细胞中纳米颗粒与蛋白质多肽骨架相互作用减少,而溶酶体中变性蛋白质片段与金纳米颗粒的作用增加。

    蛋白冠研究深化了对纳米颗粒在生物体系中行为的理解,为开发更安全、高效的纳米药物和诊断工具提供了科学依据。目前为止,绝大多数的蛋白冠研究都是在体外利用血浆等体液进行,只有极少数的体内研究。体外实验已揭示部分蛋白冠形成的不同因素及其与纳米颗粒的化学组成和理化性质的相关性,但是精准调控蛋白冠的形成,以及基于纳米颗粒类型和其性质进行蛋白冠组成预测等基本问题还没有得到解决[7]。因此,可以进一步通过表面化学修饰精准调控蛋白质吸附,从而设计出具有特定功能的纳米颗粒,并开发对环境变化(如pH、温度、光照等)敏感的纳米材料,以动态调节蛋白冠的组成和特性。同时发展理论模型和计算模拟,与实验研究相结合,预测和验证蛋白冠的形成和功能。此外,对纳米颗粒在体内生理环境中的蛋白冠形成机制还缺乏足够的了解和认识,直接通过体外的研究去推测纳米颗粒在体内的命运以及免疫毒性等性质需要更谨慎的考虑。相对于体外蛋白冠研究,体内具有复杂的内环境,比如分子种类更多、不同的血流动力学、与内环境相互作用和免疫反应等会导致蛋白冠差异。因此,为了更全面地理解蛋白冠在生物体内形成机制、动态变化和功能作用等,需要发展并完善表征方法,进行广泛和系统的体内模型研究。值得注意的是蛋白冠在不同疾病状态下的组成和变化规律与正常状态差异巨大,因此在疾病诊断和治疗中的潜在应用也需要进一步探索。

    蛋白冠表征过程中离心纯化过程可能会破坏蛋白冠的组成,引入多种误差,影响实验结果的准确性和可靠性。同时,纳米颗粒也会对其周围的蛋白质造成一系列的影响,如:蛋白质构象的改变、新结合位点的暴露、功能被干扰(由于结构效应或局部高浓度)或由相同蛋白质的紧密堆叠引起的亲和效应等。要深入了解纳米颗粒的生物效应,需要了解与纳米颗粒相关的蛋白质的平衡和动力学结合特性。因此,发展蛋白冠的原位表征手段,在不破坏蛋白冠的自然状态下实时动态监测蛋白冠的形成过程,以及分析吸附过程中构象变化等,对深入理解蛋白冠的机制和功能具有重要意义。一些新的单分子表征手段如纳米孔传感有望被引入用于蛋白冠的原位表征。固态纳米孔经过二十多年的发展,由于其灵敏快捷、免标记、通用性强等优势而受到广泛的关注,成为目前极具前景的单颗粒、单分子检测技术[131-132]。Yusko等[133]在氮化硅纳米孔上涂上一层脂质双分子层,以防止或减少纳米孔对蛋白的非特异性黏附。基于此,他们同时测定了多种蛋白质在水溶液中的体积、形状、偶极矩、旋转扩散系数和表面电荷等参数。Niedzwiecki等[134]利用固态纳米孔研究了单分子水平上蛋白质与无机界面相互作用过程,以及蛋白质吸附在硅基上的方向性。除此之外,还可以利用纳米孔对颗粒的捕获方式[135-136],实现蛋白的构型实时变化研究,以及结合动力学和亲和力差异等性质。

    总的来说,纳米颗粒形成蛋白冠是一把双刃剑。它既能提高纳米颗粒在生物系统中的稳定性和功能性,也可能带来不可预测的生物学效应和潜在的副作用。因此,在纳米医药和纳米技术的应用中,理解和控制蛋白冠的形成和影响是关键。现阶段,蛋白冠研究面临着诸多技术挑战,如生物安全性评估的复杂性,伦理法规以及个体差异的影响等问题,但是通过合理的设计和调控,并逐步解决上述问题,将促进更可预测的纳米治疗设计和个性化纳米医学的发展。


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  • 图 1  (a) 不同尺寸金纳米颗粒的蛋白冠形成过程示意图[27]; (b) 球形和棒状的介孔二氧化硅纳米颗粒在血液中的蛋白冠形成过程示意图[23]

    Figure 1  (a) Schematic diagram of protein corona formation process of gold nanoparticles with different sizes[27]; (b) Schematic diagram of protein corona formation process of spherical and rod-shaped mesoporous silica nanoparticles in blood[23]

    图 2  (a) 不同形状金纳米颗粒的TEM图像以及蛋白冠中的蛋白含量[31]; (b) 不同分支长度的金纳米星形成蛋白冠的TEM图[30]

    Figure 2  (a) TEM images of the differently shaped gold nanoparticles and the total number of identified corona proteins[31]; (b) TEM images of short and long gold nanostars with protein coronas[30]

    图 3  (a) 左旋和右旋金纳米颗粒的SEM图(左)以及手性纳米颗粒与细胞受体的相互作用示意图(右)[52]; (b) 三肽修饰的手性金纳米颗粒的示意图(左)以及在人血清中手性金纳米颗粒蛋白冠成分鉴定的热图(右)[44]; (c) 不同表面电荷的金纳米颗粒形成蛋白冠的示意图[56]; (d) DHLA修饰的金纳米颗粒(上)和聚乙二醇修饰的金纳米颗粒(下)与蛋白相互作用的示意图[62]

    Figure 3  (a) SEM images of L-P+ and D-P- gold nanoparticles (left) and a diagram of the interaction between chiral nanoparticles and cell receptors (right)[52]; (b) Schematic diagram of chiral gold nanoparticles modified with tripeptides (left) and heatmap for identification of protein corona components of chiral gold nanoparticles in human serum (right)[44]; (c) Schematic diagram of protein corona formed by negatively charged gold nanoparticles and positively charged gold nanoparticles[56]; (d) Schematic diagram of the interaction of DHLA-coated modified gold nanoparticles (top) and polyethylene glycol-coated modified gold nanoparticles (bottom) with proteins[62]

    图 4  (a) 不同pH下TiO2颗粒表面蛋白冠的示意图[71]; (b) 不同温度下聚苯乙烯纳米颗粒的蛋白冠的组成[45]; (c) 正常和高胆固醇血症小鼠体内纳米颗粒生物分布的实验过程示意图(上)、正常和高胆固醇血症小鼠的血清胆固醇含量(左下)、正常和高胆固醇血症小鼠血清中形成的蛋白冠中载脂蛋白和补体蛋白的百分比(右下)[12]

    Figure 4  (a) Formation of protein corona of TiO2 particles at different pH values[71]; (b) Composition of the protein corona of polystyrene nanoparticles at different temperatures[45]; (c) Scheme of the experiments for in vivo bio-distribution of nanoparticles in normal and hypercholesterolemic mice (top), the serum cholesterol content in normal and hypercholesterolemic mice (bottom left), the percentage of apolipoproteins and complement proteins identified in protein corona formed in serums from normal and hypercholesterolemic mice (bottom right)[12]

    图 5  (a) 聚苯乙烯纳米颗粒表面生物分子簇洗涤前(上)和洗涤3次后(下)的TEM图[46]; (b) 不同浓度的免疫金标记的聚苯乙烯纳米颗粒-蛋白复合物的TEM图[85]; (c) 美国17个不同测试机构及其对相同蛋白冠样品分析流程的示意图[97]; (d) 同步加速器源的SAXS原理(上)及SiO2纳米颗粒形成蛋白冠和聚集体的示意图(下)[110]

    Figure 5  (a) TEM images of biomolecule clusters on the surface of polystyrene nanoparticles before washing (top) and after washing three times (bottom)[46]; (b) TEM images of immunogold-labelled polystyrene nanoparticle corona complexes with different concentrations of immunogold labels[85]; (c) Schematic diagram of 17 proteomics core facilities in the USA and their analysis processes for the same protein corona sample[97]; (d) Schematic diagram of SAXS of synchrotron source (top), and SiO2 nanoparticles forming protein corona and aggregates (bottom)[110]

    图 6  介电纳米颗粒和等离激元纳米颗粒蛋白冠形成示意图(左), 以及全内反射激发散射显微镜装置(右)[88]

    Figure 6  Schematic diagram of the formation of protein corona for dielectric nanoparticles and plasmonic nanoparticles (left), and the setup of total internal reflection excited scattering microscope (right)[88]

    表 1  典型纳米颗粒的蛋白冠组成

    Table 1.  Protein corona composition of typical nanoparticles

    Structure Type of nanoparticles Protein corona component Ref.
    Nanosphere SiO2 Complement protein, glycoprotein, serum amyloid protein, coagulation factor, gel protein, serum albumin, immunoglobulin G, complement protein C1q [24]
    Fe2O3 Albumin, α-1-antitrypsin, immunoglobulin A, haptoglobulin, macroglobulin, transferrin, bovine serum albumin, α-2-HS glycoprotein, apolipoprotein A-Ⅰ [26, 36]
    Ag Apolipoprotein, lactoferrin, human serum albumin, phospholipid-binding protein A1/A2, hemoglobin, metallothionein 1, plasma ceruloplasmin [37-38]
    Au Complement protein C3, apolipoprotein A-Ⅰ, complement factor H, α-2-HS glycoprotein, viral spike protein S1, concanavalin A, bovine serum albumin [39-41]
    CeO2 Bovine serum protein, immunoglobulin [42]
    C60 Bovine serum protein, fibrinogen, hemoglobin [43]
    L-Au Human serum albumin, complement protein C3, fibronectin 1, epididymal luminal protein 214, complement protein C4-A, serum transferrin, complement factor H, apolipoprotein A-Ⅰ, platelet factor 4, immunoglobulin [44]
    D-Au Human serum albumin, complement protein C3, complement protein C4-A, complement factor H, fibronectin, epididymal luminal protein 214, adiponectin B, platelet factor 4, apolipoprotein C-Ⅲ, and complement protein C1q
    Polystyrene Apolipoprotein A-Ⅰ, fibrinogen [45-46]
    Nanostar Au Fibrinogen, trypsin, complement protein C3, complement factor H, human serum albumin, α-2-macroglobulin, plasminogen [30, 32]
    Nanocube Au Human serum albumin, histidine-rich glycoprotein, complement component C9, apolipoprotein B-100, coagulation factor Ⅻ [31]
    Nanorod Au Fibrinogen, trypsin, bovine serum albumin, human serum albumin, complement protein C3, complement protein C1q, apolipoprotein A-Ⅰ, apolipoprotein A-Ⅱ [31-32, 47]
    Nanotube Single-walled carbon nanotube Human serum albumin, histidine-rich glycoprotein, apolipoprotein A-Ⅰ, complement protein C3, complement protein C4 [25]
    Quantum dot CdSe@ZnS Human serum albumin, lysozyme [48]
    Nanosheet Graphene oxide Fibrinogen α chain, human serum albumin, apolipoprotein B-100, apolipoprotein E, complement protein C3 [49]
    Black phosphorus Human serum albumin, α-2-macroglobulin, complement protein C3, transferrin, heme binding protein, apolipoprotein B [50-51]
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  • 发布日期:  2024-12-10
  • 收稿日期:  2024-06-30
  • 修回日期:  2024-10-23
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