da Costa Marques Richard, Hüppe Natkritta, Speth Kai R, Oberländer Jennifer, Lieberwirth Ingo, Landfester Katharina, Mailänder Volker
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany; Dermatology Clinic, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany.
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.
Acta Biomater. 2023 Dec;172:355-368. doi: 10.1016/j.actbio.2023.10.010. Epub 2023 Oct 13.
The intracellular protein corona has not been fully investigated in the field of nanotechnology-biology (nano-bio) interactions. To effectively understand intracellular protein corona formation and dynamics, we established a workflow to isolate the intracellular protein corona at different uptake times of two nanoparticles - magnetic hydroxyethyl starch nanoparticles (HES-NPs) and magnetic human serum albumin nanocapsules (HSA-NCs). We performed label-free quantitative LC-MS proteomics to analyze the composition of the intracellular protein corona and correlated our findings with results from conventional methods for intracellular trafficking of nanocarriers, such as flow cytometry, transmission electron microscopy (TEM), and confocal microscopy (cLSM). We determined the evolution of the intracellular protein corona. At different time stages the protein corona of the HES-NPs with a slower uptake changed, but there were fewer changes in that of the HSA-NCs with a more rapid uptake. We identified proteins that are involved in macropinocytosis (RAC1, ASAP2) as well as caveolin. This was confirmed by blocking experiments and by TEM studies. The investigated nanocarrier predominantly trafficked from early endosomes as determined by RAB5 identification in proteomics and in cLSM to late endosomes/lysosomes (RAB7, LAMP1, cathepsin K and HSP 90-beta) We further demonstrated differences between nanoparticles with slower and faster uptake kinetics and determined the associated proteome at different time points. Analysis of the intracellular protein corona provides us with effective data to examine the intracellular trafficking of nanocarriers used in efficient drug delivery and intracellular applications. STATEMENT OF SIGNIFICANCE: Many research papers focus on the protein corona on nanoparticles formed in biological fluids, but there are hardly any articles dealing with proteins that come in contact with nanoparticles inside cells. The "intracellular protein corona" studied here is a far more complex and highly demanding field. Most nanocarriers are designed to be taken up into cells. Given this, we chose two different nanocarriers to reveal changes in the proteins in dendritic cells during contact at specific times. Further studies will allow us to examine molecular target proteins using these methods. Our research is a significant addition towards the goal of understanding and thus improving the efficacy of drug nanocarriers.
在纳米技术与生物学（纳米 - 生物）相互作用领域，细胞内蛋白质冠层尚未得到充分研究。为了有效理解细胞内蛋白质冠层的形成和动态变化，我们建立了一种工作流程，用于在两种纳米颗粒——磁性羟乙基淀粉纳米颗粒（HES - NPs）和磁性人血清白蛋白纳米囊（HSA - NCs）的不同摄取时间分离细胞内蛋白质冠层。我们进行了无标记定量液相色谱 - 质谱蛋白质组学分析，以分析细胞内蛋白质冠层的组成，并将我们的研究结果与纳米载体细胞内运输的传统方法（如流式细胞术、透射电子显微镜（TEM）和共聚焦显微镜（cLSM））的结果相关联。我们确定了细胞内蛋白质冠层的演变。在不同时间阶段，摄取较慢的HES - NPs的蛋白质冠层发生了变化，但摄取较快的HSA - NCs的蛋白质冠层变化较少。我们鉴定出参与巨胞饮作用的蛋白质（RAC1、ASAP2）以及小窝蛋白。这通过阻断实验和TEM研究得到了证实。通过蛋白质组学和cLSM中RAB5的鉴定确定，所研究的纳米载体主要从早期内体运输到晚期内体/溶酶体（RAB7、LAMP1、组织蛋白酶K和热休克蛋白90 - β）。我们进一步证明了摄取动力学较慢和较快的纳米颗粒之间的差异，并确定了不同时间点的相关蛋白质组。对细胞内蛋白质冠层的分析为我们提供了有效数据，以研究用于高效药物递送和细胞内应用的纳米载体的细胞内运输。重要性声明：许多研究论文关注生物流体中形成的纳米颗粒上的蛋白质冠层，但几乎没有任何文章涉及与细胞内纳米颗粒接触的蛋白质。这里研究的“细胞内蛋白质冠层”是一个更加复杂且要求很高的领域。大多数纳米载体被设计用于被细胞摄取。鉴于此，我们选择了两种不同的纳米载体来揭示树突状细胞在特定时间接触过程中蛋白质的变化。进一步的研究将使我们能够使用这些方法研究分子靶蛋白。我们的研究对于理解并因此提高药物纳米载体的功效这一目标具有重要意义。
