Dataset Information

Combining confocal microscopy, dSTORM, and mass spectroscopy to unveil the evolution of the protein corona associated with nanostructured lipid nanocarriers during blood-brain barrier crossing

ABSTRACT: Upon entering in contact with the biological environment, nanostructures are immediately covered by biomolecules, and in particular proteins forming the so-called “protein corona” (PC). The phenomenon of PC formation has gained great attention in recent years due to its implication for the use of nanostructures in biomedicine. In fact, it has been shown that the formation of the PC can impact the performance of nanostructures by reducing their stability, causing aggregation, increasing their toxicity, and causing unexpected and undesired nanostructure-cell interactions. In this work, we decided to study for the first time the formation and the evolution of PC on the surface of nanostructured lipid carriers loaded with superparamagnetic iron oxide nanoparticles (SPIONs), before and after the crossing of an in vitro model of the blood-brain barrier. Combining confocal microscopy, direct STochastic Optical Reconstruction Microscopy (dSTORM), and proteomic analysis, we were able to provide a complete analysis of the PC formation and evolution. In particular, we evidence that PC formation is a fast process, being formed around particles even after just 1 min of exposure to fetal bovine serum. Moreover, PC formed around particles is extremely heterogeneous: while some particles have no associated PC at all, others are completely covered by proteins. Lastly, the interaction with an in vitro blood-brain barrier model strongly affects the PC composition: in particular, a large amount of the proteins forming the initial PC is lost after the BBB passage and is partially replaced by new proteins derived from both the brain endothelial cells and from the cell culture medium. Altogether, the presented data could potentially provide new insights into the design and fabrication of lipid nanostructures for the treatment of central nervous system disorders.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Martina Bartolucci

LAB HEAD: Andrea Petretto

PROVIDER: PXD031034 | Pride | 2022-10-14

REPOSITORIES: Pride

ACCESS DATA

Publications

Combining confocal microscopy, dSTORM, and mass spectroscopy to unveil the evolution of the protein corona associated with nanostructured lipid carriers during blood-brain barrier crossing.

Battaglini Matteo M Feiner Natalia N Tapeinos Christos C De Pasquale Daniele D Pucci Carlotta C Marino Attilio A Bartolucci Martina M Petretto Andrea A Albertazzi Lorenzo L Ciofani Gianni G

Nanoscale 20220922 36

Upon coming into contact with the biological environment, nanostructures are immediately covered by biomolecules, particularly by proteins forming the so-called "protein corona" (PC). The phenomenon of PC formation has gained great attention in recent years due to its implication in the use of nanostructures in biomedicine. In fact, it has been shown that the formation of the PC can impact the performance of nanostructures by reducing their stability, causing aggregation, increasing their toxici ...[more]

PMID: 36063033

Similar Datasets

Project description:Background: Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we provide initial evidence for potential roles of AKR1C3 in PCa progression. Methods: Spatial distribution of AKR1C3 was analyzed using immunohistochemical staining in prostate adenocarcinoma tissue array. Human PCa PC-3 cells were stably transfected with AKR1C3 cDNA to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analyses were performed to identify pathways that are activated by elevated AKR1C3 expression in PCa cells. Functional confirmation of microarray and bioinformatics results was performed by immunoblot analysis and an in vitro Matrigel angiogenesis assay. Results: Elevated AKR1C3 expression was specifically limited to human prostate adenocarcinoma. Microarray and bioinformatics analysis suggested that elevated AKR1C3 expression in PC-3 cells modulates estradiol and androgen metabolism and activates insulin growth factor (IGF)-1 and Akt signaling pathways. Immunoblots confirmed that phosphorylated levels of IGF-1 receptor (IGF-1R) and Akt are significantly up-regulated in PC3-AKR1C3 as compared to mock transfectants. PC3-AKR1C3 transfectants promoted endothelial cell tube formation in Matrigel as compared to parental PC-3 cells and mock transfectants. Conclusion: Microarray and bioinformatics data followed by biological analyses suggest that elevated AKR1C3 expression in PC-3 cells promotes PCa angiogenesis and aggressiveness. These results suggest AKR1C3 can promote the aggressiveness of PCa through modulating estrogen and androgen metabolism with subsequent activation of growth factor IGF-1 and cytoplasmic Akt signaling pathways. Total RNA from mock- and ACR1C3 transfected PC-3 cells was isolated, with 2 or 3 biological replicates each. Gene expression data from AKR1C3 transfected PC-3 cells were compared with mock-transfected data.

Project description:Objective: Production of pathogenic autoantibodies by self-reactive plasma cells (PC) is a hallmark of autoimmune diseases. Investigating the prevalence of PC in autoimmune disease and their relationship with known pathogenic pathways may increase our understanding of the role of PC in disease progression and treatment response. Methods: We developed a sensitive gene expression based method to overcome the challenges of measuring PC using flow cytometry. Whole genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA, IGJ, IGKC, IGKV, and TNFRSF17, expressed predominantly in PC. The sensitivity of the PC signature score created from the combined expression levels of these genes was assessed through ex vivo experiments with sorted cells. This PC gene expression signature was used for monitoring changes in PC levels following anti-CD19 therapy; evaluating the relationship between PC and other autoimmune disease-related genes; and estimating PC levels in affected blood and tissue from multiple autoimmune diseases. Results: The PC signature was highly sensitive and capable of detecting as few as 300 PCs. The PC signature was reduced over 90% in scleroderma patients following anti-CD19 treatment and this reduction was highly correlated (r = 0.77) with inhibition of collagen gene expression. Evaluation of multiple autoimmune diseases revealed 30-35% of lupus, rheumatoid arthritis, and scleroderma patients with increased PC levels. Conclusion: This newly developed PC signature provides a robust and accurate method to measure PC levels in the clinic. Our results highlight subsets of patients across multiple autoimmune diseases that may benefit from PC depleting therapy. To examine gene expression in purified cellular fractions, normal human blood was collected from 2 donors as per institutional policy. The granulocyte (CD15+), monocyte (CD14+), T cell (CD3+), B cell (Non-PC gated, CD19+), and PC (CD27++CD38++) fractions from peripheral blood were separated. White blood cells were washed with FACS buffer (PBS + 0.5%BSA + 2mM EDTA (Gibco)) and incubated with 20% heat-inactivated FBS for 10-15 minutes on ice. The following mAbs were added directly to the cells: CD15 (HI98); CD14 (M5E2), CD3 (UCHT1), CD27 (M-T271), CD38 (HB7), and DAPI (Molecular probes). Cells were sorted on a Becton Dickinson FACS Aria II flow cytometer. All sorted fractions were collected in FACS buffer, centrifuged, and the resulting cell pellet was suspended in RNA lysis buffer (Ambion).

Dataset Information

Combining confocal microscopy, dSTORM, and mass spectroscopy to unveil the evolution of the protein corona associated with nanostructured lipid nanocarriers during blood-brain barrier crossing

Publications

Combining confocal microscopy, dSTORM, and mass spectroscopy to unveil the evolution of the protein corona associated with nanostructured lipid carriers during blood-brain barrier crossing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure