The formation of the biomolecule corona on the surface of nanoparticles upon exposure to biological fluids critically influences nanocarrier performance in drug delivery. It has been shown that in some cases corona proteins can mediate specific nanoparticle interactions with cell receptors. Within this context, in order to identify corona proteins affecting nanoparticle uptake, in this work, correlation analysis is performed between the corona composition of a panel of silica nanoparticles of different sizes and surface functionalities and their uptake in four endothelial cell types derived from different organs. In this way, proteins that correlate with increased or decreased uptake were identified, and their effects were validated by studying the uptake of nanoparticles coated with a single protein corona and competition studies in brain and liver endothelium. The results showed that precoating nanoparticles with histidine-rich glycoprotein (HRG) alone strongly decreased uptake in both liver and brain endothelium. Furthermore, our results suggested the involvement of the transferrin receptor in nanoparticle uptake in liver endothelium and redirection of the nanoparticles to other receptors with higher uptake efficiency when the transferrin receptor was blocked by free transferrin. These data suggested that changes in the cell microenvironment can also affect nanoparticle uptake and may lead to a different interaction site with nanoparticles, affecting their uptake efficiency. Overall, correlating the composition of the protein corona and nanoparticle uptake by cells allows for the identification of corona molecules that can be used to increase as well as to reduce nanoparticle uptake by cells.
In order to improve the current success of nanomedicine, a better understanding of how nano-sized materials interact with and are processed by cells is required. Typical in vitro nanoparticle-cell interaction studies often make use of cells cultured at different cell densities. However, in vivo, for their successful delivery to the target tissue, nanomedicines need to overcome several barriers, such as endothelial and epithelial cell barriers. Unlike sub-confluent or confluent cell cultures, cell barriers are tight cell monolayers, expressing a series of specialized tight junction proteins between adjacent cells to limit paracellular transport and ensure close cell-to-cell interactions. A clear understanding on how the development of cells into a cell barrier may affect the uptake of nano-sized drug carriers is still missing. To this aim, here, human primary umbilical vein endothelial cells (HUVEC) are used as a model cell line to form endothelial cell barriers. Then, nanoparticle uptake is assessed in the developed endothelial barriers and compared to the uptake in sub-confluent or confluent HUVEC cultures. The results clearly show that the organization of cells into a cell barrier leads to a differential gene expression of endocytic markers, and - interestingly - this is accompanied by reduced nanoparticle uptake levels. Transport inhibitors are used to characterise the mechanisms involved in the uptake. However, we show that some of them can strongly compromise barrier integrity, thus impairing the interpretation of the outcomes, and overall, only a partial inhibition of nanoparticle uptake could be obtained.
Nanoparticles are promising tools for nanomedicine in a wide array of therapeutic and diagnostic applications. Yet, despite the advances in the biomedical applications of nanomaterials, relatively few nanomedicines made it to the clinics. The formation of the biomolecular corona on the surface of nanoparticles has been known as one of the challenges toward successful targeting of nanomedicines. This adsorbed protein layer can mask targeting moieties and creates a new biological identity that critically affects the subsequent biological interactions of nanomedicines with cells. Extensive studies have been directed toward understanding the characteristics of this layer of biomolecules and its implications for nanomedicine outcomes at cell and organism levels, yet several aspects are still poorly understood. One aspect that still requires further insights is how the biomolecular corona interacts with and is “read” by the cellular machinery. Within this context, this review is focused on the current understanding of the interactions of the biomolecular corona with cell receptors. First, we address the importance and the role of receptors in the uptake of nanoparticles. Second, we discuss the recent advances and techniques in characterizing and identifying biomolecular corona-receptor interactions. Additionally, we present how we can exploit the knowledge of corona-cell receptor interactions to discover novel receptors for targeting of nanocarriers. Finally, we conclude this review with an outlook on possible future perspectives in the field. A better understanding of the first interactions of nanomaterials with cells, and -in particular -the receptors interacting with the biomolecular corona and involved in nanoparticle uptake, will help for the successful design of nanomedicines for targeted delivery.
Cell-derived extracellular vesicles (EVs) are effectors of cell-to-cell communication that are in the spotlight as promising candidates for in vivo drug delivery because of their ability to enter cells and deliver cargo. For example, proteins of interest can be loaded into EVs to mediate protein transfer into target cells. To determine causality between EV content and function, which is also important to assess the clinical safety of EVs, it is crucial to comprehensively characterize their complete molecular composition. Here, we investigated EVs loaded with the chaperone protein DNAJB6. Chaperone proteins assist in protein folding and have been suggested to alleviate protein aggregation diseases, such as Alzheimer’s disease and Huntington’s disease. We analyzed and compared the proteome of EVs isolated from wildtype HEK293T cells with that of EVs from HEK 293T cells overexpressing DNAJB6-WT or loss-of-function mutant DNAJB6-M3. Comprehensive analysis of proteomics data showed enhanced levels of DNAJB6 as well as protein-folding-related proteins in EVs derived from DNAJB6-overexpression cells. Interestingly, upregulation of a chaperone and its protein-folding-related proteins resulted in downregulation of another chaperone plus its related proteins, and vice versa. This implies the presence of compensatory mechanisms in the cellular expression of chaperones. Collectively, we provide the proteomic EV signatures underlying EV mediated DNAJB6 transmission by HEK293T cells, with the aim of establishing a causal relationship between EV protein content and EV function.
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