College of Pharmacy, Dali University, No. 2 Hongsheng Road, Dali 671003, China.
Yunnan Key Laboratory of Screening and Research on Anti-Pathogenic Plant Resources from Western Yunnan, Dali University, Xueren Road, Dali 671003, China.
Molecules. 2024 Oct 11;29(20):4818. doi: 10.3390/molecules29204818.
Lipid nanoparticles (LNPs), widely used in disease diagnosis and drug delivery, face the challenge of being surrounded by biological macromolecules such as proteins upon entering the human body. These molecules compete for binding sites on the nanoparticle surfaces, forming a protein corona. The impact of different types of protein coronas on LNP delivery remains unclear. In this study, we employed a newly developed, highly sensitive LNP labeling platform and analyzed the endocytosis of HeLa cells under different nutritional conditions using proteomics to address this critical issue. Our research found that under conditions of complete medium and amino acid starvation, most DNA-FITC vesicles in HeLa cells were located in the perinuclear region 4 h after transfection. In contrast, under serum starvation conditions, only a small portion of DNA-FITC vesicles were in the perinuclear region. On the other hand, through proteomics, we discovered that cells that were enriched in amino acids and complete medium contained more proteins, whereas those under serum starvation had relatively fewer enriched proteins. Through KEGG pathway enrichment analysis, we identified the phagosome and endocytosis pathways as particularly important. Lastly, differential analysis of proteins in these pathways revealed that proteins such as F-actin, Coronin, vATPase, TUBA, TUBB, MHCII, and TSP may have significant impacts on cellular endocytosis. Our research findings indicate that it is necessary to regulate cellular endocytosis based on different protein coronas to achieve optimal cytoplasmic release.
脂质纳米颗粒（LNPs）广泛应用于疾病诊断和药物递送，但在进入人体后，它们面临着被蛋白质等生物大分子包围的挑战。这些分子会竞争纳米粒子表面的结合位点，形成蛋白质冠。不同类型的蛋白质冠对 LNP 传递的影响尚不清楚。在这项研究中，我们采用了一种新开发的、高度敏感的 LNP 标记平台，并通过蛋白质组学分析在不同营养条件下 HeLa 细胞的内吞作用，解决了这一关键问题。我们的研究发现，在完全培养基和氨基酸饥饿条件下，转染后 4 小时，HeLa 细胞中大多数 DNA-FITC 囊泡位于核周区域。相比之下，在血清饥饿条件下，只有一小部分 DNA-FITC 囊泡位于核周区域。另一方面，通过蛋白质组学，我们发现富含氨基酸和完全培养基的细胞含有更多的蛋白质，而血清饥饿的细胞相对较少。通过 KEGG 途径富集分析，我们确定吞噬体和内吞作用途径尤为重要。最后，对这些途径中的蛋白质进行差异分析表明，F-肌动蛋白、冠蛋白、vATP 酶、TUBA、TUBB、MHCII 和 TSP 等蛋白质可能对细胞内吞作用有重大影响。我们的研究结果表明，有必要根据不同的蛋白质冠来调节细胞内吞作用，以实现最佳的细胞质释放。
