Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona dependent

Extracellular vesicles (EVs) derived from mesenchymal stem cells are promising nanotherapeutics in liver diseases due to their regenerative and immunomodulatory properties. Nevertheless, a concern has been raised regarding the rapid clearance of exogenous EVs by phagocytic cells. Here we explore the impact of protein corona on EVs derived from two culturing conditions in which specific proteins acquired from media were simultaneously adsorbed on the EV surface. Additionally, by incubating EVs with serum, simulating protein corona formation upon systemic delivery, further resolved protein corona–EV complex patterns were investigated. Our findings reveal the potential influences of corona composition on EVs under in vitro conditions and their in vivo kinetics. Our data suggest that bound albumin creates an EV signature that can retarget EVs from hepatic macrophages. This results in markedly improved cellular uptake by hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells. This phenomenon can be applied as a camouflage strategy by precoating EVs with albumin to fabricate the albumin-enriched protein corona–EV complex, enhancing non-phagocytic uptake in the liver. This work addresses a critical challenge facing intravenously administered EVs for liver therapy by tailoring the protein corona–EV complex for liver cell targeting and immune evasion.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Dot blot data were collected by Image Lab software V4.1 (Bio-Rad, UK).Size and concentration of EV data were collected by Nanosight NTA 3.2 software (Malvern Instruments, UK).Zeta potential of EV data were collected by Zetasizer version 7.12 (Malvern Instruments, UK).Data for microBCA were collected by Omega (BMG LABTECH).Flow cytometry data were collected by BD FACStation™ software V6.0 and BD FACSDiva Software V9.2 (BD Biosciences, USA).In vivo and ex vivo imaging data were collected by Living Image® V4.7.3 Software (PerkinElmer, USA).

Data analysis
Microplate reader data analysis was performed through MARS software V2.40.Flow cytometry data analysis was performed by FlowJo v.10.7.2 (TreeStar/BD Bioscience).Proteomic analysis was performed by MassLynx 4.1.Progenesis® QI for Proteomics Software Version 2.0 (using human and bovine UniProt database).Multivariate analysis was performed by Minitab V20 and The Unscrambler X. Bioinformatic analysis was performed by FunRich 3.1.3.Statistical analyses of the data were performed using Prism 9.4.1.MATLAB 9.11 was used to generate heatmaps of data.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.

Validation
Mouse anti-human CD81 antibody has been validated by the manufacturer (BioLegend) to be specific to human species and was quality control tested by immunofluorescent staining with flow cytometric analysis.Application in WB and IP are reported in the literature (PMID: 14966136) Mouse anti-human CD9 antibody has been validated by the manufacturer (BioLegend) to be specific to human species and was quality control tested by immunofluorescent staining with flow cytometric analysis.Application in dot blot is reported in the literature (PMID: 33368666) Rabbit anti-human CD63 antibody has been validated by the manufacturer (Abcam) to be specific to human species and was verified for application in WB and IHC-P.
TSG101 polyclonal antibody has been validated by the manufacturer (Proteintech) to be specific to human, mouse, and rat species and was verified for application in WB, RIP, IP, IHC, IF, FC, and ELISA.HRP-goat anti-rabbit antibody and HRP-goat anti-mouse antibody were thoroughly validated by the manufacturer (Cell Signalling Technologies) with CST primary antibodies and will work optimally with the CST western immunoblotting protocol, ensuring accurate and reproducible results.
Anti-mouse CD45 antibody (PerCP), Anti-mouse CD45 antibody (PE), Anti-mouse F4/80 antibody (FITC), Anti-mouse CD11b antibody, and Anti-mouse CD31 antibody (PE) has been validated by the manufacturer (BioLegend) to be specific to mouse species and were quality control tested by immunofluorescent staining with flow cytometric analysis.
ASGPR1 antibody has been validated by the manufacturer (Santa Cruz Biotechnology) to be used for detecting ASGPR1 of mouse, rat and human origin by WB, IP, IF and FCM (cited in 12 publications) GFAP antibody has been validated by the manufacturer (Miltenyi Biote) to be used for detecting GFAP of human/ mouse/rat by ICFC, MICS, 3D-IF, IF, and IHC.CD146-FITC antibody has been validated by the manufacturer (Miltenyi Biote) to be used for detecting CD146 of mouse by FC, MICS, IF, and IHC.SPARC Alexa Fluor® 647-conjugated Antibody has been validated by the manufacturer (R&D systems) to be used for detecting SPARC/ Osteonectin of mouse by intracellular staining by flow cytometry.

Eukaryotic cell lines
Policy information about cell lines and Sex and Gender in Research Cell line source(s) HepG2 (human hepatocellular carcinoma, ATCC® HB-8065) and J774A.1 (BALB/c mouse macrophage, ATCC®TIB-67™) were purchased from American Type Culture Collection (ATCC).

Authentication
The cell lines used in the study were not authenticated.

Mycoplasma contamination
All the cell lines were tested negative for mycoplasma contamination.