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Differential Recognition of Nanoparticle Protein Corona and Modified Low Density Lipoprotein by Macrophage Receptor with Collagenous Structure Sandra Lara, André Perez-Potti, Luciana M. Herda, Laurent Adumeau, Kenneth A. Dawson, and Yan Yan ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02014 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Differential Recognition of Nanoparticle Protein Corona and Modified Low Density Lipoprotein by Macrophage Receptor with Collagenous Structure Sandra Lara1, André Perez-Potti1, Luciana M. Herda1, Laurent Adumeau1, Kenneth A. Dawson1*, Yan Yan1,2* 1

Centre for BioNano Interactions, School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland.

2

UCD Conway Institute of Biomolecular and Biomedical Research, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland

*Corresponding Authors Email: [email protected], [email protected]

KEYWORDS: nanoparticles, biomolecular corona, scavenger receptors, protein unfolding, MARCO, modified lipoproteins.

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ABSTRACT Key practical challenges such as to understand the immunological processes at the nanoscale, and to control the targeting and accumulation of nano-objects in vivo now further stimulate efforts to underpin phenomenological knowledge of the nanoscale with more mechanistic and molecular insight. Thus, the question as to what constitutes nanoscale biological identity continues to evolve. Certainly nanoparticles in contact with complex biological milieu develop a biological identity, differing from the original nanomaterial, now referred to as the ‘biomolecular corona’. However, this surface-adsorbed layer of biomolecules may in some circumstance lead to different forms of receptor-particle interactions not evident only from the identity of the surface adsorbed biomolecules, and hard to predict or detect by current physiochemical methods. Here we show that scavenger receptors may recognise complex as yet unidentified biomolecular surface layer motifs, even when no current physiochemical analysis is capable of doing so. For instance, fluorescently-labelled SiO2 nanoparticles, in biological milieu are strongly recognized by the macrophage receptor with collagenous structure (MARCO) scavenger receptor in even dense biological media (human serum) apparently using a form of binding with which most of MARCO’s known ligands (e.g. LPS, modified LDL) fail to compete. Such observations may suggest the need for a much stronger emphasis on nanoscale receptor-corona and other biomolecular interaction studies, if one wishes to unravel how biomolecular recognition drives outcomes in the nanoscale-biological domain.


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Nanostructures (NPs) in contact with a biological environment form a ‘biomolecular corona’ on their surface that contains biological materials derived that environment. In situ, the biomolecular corona composition may continue to slowly evolve via contact with evolving environments. However, at each stage, it is now widely accepted that significant aspects of the overall biological identity of nanoparticles arise from biological recognition of the particle-environment (corona) interface.1-20 We seek to connect the microscopic details of particles in realistic milieu to their interaction and (for example) accumulation in organs, such as the liver. Forging such a connection will certainly be challenging, and we envisage a number of important factors being involved.21-25 Still, the most basic question is whether the particles are recognized by the key receptors presented by the relevant (e.g. Kupffer) cells charged with sweeping the bloodstream free of pathogenic, endogenous debris, particulate and other foreign matter.26 There are significant technical challenges in answering even this question, but they can be overcome. While our understanding of the nanoscale biomolecule-based interface processing and recognition at cell level will need to deepen, we know that significant aspects of ‘biological identity’ involve suitably presented ‘biomolecular recognition motifs’ at the nanoparticle surface, for durations that exceed the cellular ‘recognition time’.2 Thus, while we cannot yet classify the modes of particle surface-presented biomolecular-receptor-driven corona recognition some obvious categories are becoming understood. For instance, corona’s containing biomolecules which themselves have known receptor binding sites, if appropriately presented on the particle surface, can lead to obvious nanoparticle-receptor interactions.27 One should be aware that the mere presence of a biomolecule on the particle surface in no way guarantees that it presents a suitably disposed recognition motif.28 However, it is now possible to map out likely binding sites, on a particle-by-particle basis,


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using a variety of techniques, and this (the simplest) class of corona recognition phenomena is likely to be mastered.29-31 Examples of specific nanoparticle presentation of ligands include apolipoprotein B-100, immunoglobulins, complement proteins, and TGF-b1 recognized respectively by low-density lipoprotein receptor (LDLR), Fc-gamma R1, Mac-1 receptor, and TGF receptors.27,32-34 Other forms of nanoparticle biological recognition derive from more complex biological recognition domains presented at the nanoparticle surface. The simplest examples are based on analogies with more developed arenas of scavenger receptor research.35,36 Thus, it is expected that endogenous proteins that have been in some way disrupted offer de novo receptor recognition patterns, resulting in interaction with scavenger receptors, which are abundantly represented in various clearance systems in vivo. However, more generally, pattern recognition, scavenger and other such receptors can bind to complex (often poorly understood) and highly variable recognition domains, absent in native endogenous biomolecules. While the investigations to be reported here do not permit comprehensive answers to broader questions, they suggest the need for a more complete understanding of particle-cell-scavenger-receptor recognition. Scavenger receptors belong to a super group comprising a diverse range of structurally unrelated receptors that recognise and bind a wide spectrum of ligands, including modified low-density lipoproteins (e.g. acetylated LDL, acLDL) apoptotic cells, altered proteins, bacteria, viruses and other pathogens. Thereby they play critical roles in nanoparticle liver clearance, secretion of pro-inflammatory cytokines,8,34,37 lipid metabolism, inflammation, cell adhesion and antigen presentation.36,38 The microscopic basis of the poly-specificity of scavenger receptors has long been a surprisingly difficult question to resolve, and the existing limited structural information on scavenger receptors does not always lead to unifying insights. One of the best studied, macrophage receptor with collagenous structure (MARCO)


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is a trimeric membrane glycoprotein with five domains, including a small N-terminal intracellular domain (Domain I), transmembrane region (Domain II), a short spacer domain (Domain III), a long triple-helical collagenous domain (Domain IV), and a C-terminal cysteine-rich domain (SRCR, Domain V) (Figure 1a). Based on the crystal structure of the purified SRCR domain, site-directed mutagenesis, and phage display screening, a complex ligand-binding model has been put forward in which the SRCR domain is crucial for ligand binding. In particular, a β-sheet region in this domain contains several arginines, forming an electrostatic cluster, provides key binding sites for acLDL and other polyanionic ligands.39 Furthermore, the identification of hydrophobic peptides that bind to the SRCR domain has suggested multiple binding interfaces co-existing in the SRCR domain, which may have partial overlapping with acLDL binding sites.40 Having previously concluded that the identification of nanoparticle-scavenger interactions based on usual silencing and knock out approaches is prone to confounding crosscouplings,41 we instead transfected HEK-293T cells (themselves having undetectable levels of human MARCO expression) with a plasmid coding an affinity tag, and human MARCO fusion protein (Figure 1b). The fact that fusion receptors are inserted without matched downstream cellular uptake and processing machinery means that cells essentially act as functional receptor ‘host’ membrane surfaces, without the complicating aspects of intracellular accumulation. Then, for cell populations, the fusion label allows for simultaneous tracking and correlation of the expression levels of the receptor (itself correlated to membrane-expression) and the binding of the biomolecular corona-nanoparticle complexes using flow cytometry (Figure 1c), all in complex media.

Such correlations

between receptor expression and particle association can extend beyond across several orders of magnitude allowing for a semi-quantitative interpretation of the outcome. We report that


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this MARCO model faithfully recapitulates interactions with all of the expected ligands (e.g. LPS, modified LDL etc., here used as controls). Our studies establish that the interactions are driven by the relatively fixed strongly associated ‘hard corona’, which may be isolated and studied in different complex media. The latter may modulate the interactions with MARCO (as indeed observed with all receptors interactions studied so far-Transferrin, etc.), but the basic observation remains intact. Thus, while, by conventional and state-of-the-art methods, silica particles have long been reported to produce insignificant surface disruption of the proteins (as also they do in this case),42 we find a distinctive interaction with MARCO, under many different conditions. It is striking that this nanoparticle-corona-MARCO interaction fails to compete with most of the knowns ligands (even at their saturation concentrations), except for aggregated protein particles (which are able to displace the nanoparticles), suggesting a distinctive mode of binding to the receptor. We are thereby lead to either question the conclusion that proteins at the silica nanoparticle surface are not disrupted. Other possibilities however cannot be ignored, and there has been no in-depth investigation of the potentially emergent nanoscale-biocorona surface recognition sites for MARCO (and other such receptors).

RESULTS AND DISCUSSION Physiochemical Characterization of 100 nm SiO2 NPs in Biological Media. We present results from labelled amorphous SiO2 NPs 100 nm in diameter (Figure 2a), (other sizes are reported in Figure S1) synthesized in a clean environment, characterized as described previously and tested to ensure they are free from contaminants (such as LPS) that are known to be ligands for the receptor (Figure 2b).36 The particles were dispersed in different concentrations of human serum, consistent with the choice of human derived MARCO receptor. The dispersions determined by Differential Centrifugal Sedimentation (DCS) in


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situ. The DCS data showed that corona-NP complexes in 30%, 50%, 80%, and 100% human serum had similar size and colloidal stability compared to the pristine SiO2 NPs dispersed in phosphate-buffered saline (PBS) (Figure 2c). Notable aggregation was observed in the 10% human serum dispersion, but all other dispersions are lead to nearly perfectly dispersed single particles, with well developed corona layers. Corona-NP complexes can either be studied in situ in these dispersions, or isolated by centrifugation, washed with PBS extensively to remove unbound proteins, and re-dispersed. For comparison, the hard corona proteins were derived from equivalent number of corona-NPs, and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 2d). As expected, the 10% serum corona showed the lowest amount of proteins compared with other coronas, consistent with a limited surface coverage of proteins, and dispersion instability in agreement with previous studies.43 In Situ Interactions of Human Serum Corona-NPs with MARCO. Next, we examine the uptake of stable corona-NP dispersions by MARCO in media containing increasing concentration of human serum. HEK-293T cells were transfected with plasmids coding either HaloTag®-MARCO fusion protein (MARCO) or HaloTag® protein (Empty vector) and expression in cell populations confirmed both by RT-qPCR and western blot (Figure S2), and dye-labeling of the tag, and flow cytometry. Firstly, we examined the in situ interaction of corona-NPs by MARCO. The cells were incubated with NPs in human serum at various concentrations for 4 h, followed by extensive washes to remove unbound NPs. The use of populations with different expression levels of MARCO allows us to correlate the level of nanoparticle interaction, supporting the role of the receptor in cell-particle association. As shown in Figure 3a, although corona-NPs-MARCO interaction was decreased with the increase of serum concentration, in 100% human serum the uptake in MARCO expressing


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cells remained 3 times as high as in the empty vector transfected cells. The recognition of the corona by MARCO in high concentrations of serum in our experience suggests that MARCO at least partially binds to the corona via sites different from the targets of endogenous binding ligands present in serum. Therefore, the recognition of corona cannot be completely abolished by the competition binding of endogenous ligands. We further investigated the interaction of isolated hard corona complexes of SiO2 NPs formed at 100% serum (isolated from excess serum) with MARCO. 100 nm SiO2 NPs were dispersed in 100% human serum to form hard corona and then re-dispersed in different media including different concentrations of serum. DCS analysis confirmed that all dispersions were stable (Figure S3). MARCO-mediated uptake of NP-corona complexes occurred at all serum concentrations tested (i.e. 10-80%) (Figure 3b-d). Similar effects are observed with other particles sizes (see results for 30 nm SiO2 NPs, Figure S4). Human Serum corona−SiO2 NP Uptake in Competitive Media. Next, we probed the binding sites of MARCO with the 100% hard corona by a set of competition binding experiments. Known MARCO ligands in 80% human serum (including LPS, acLDL,44 oxLDL up to considerable excess) fail to compete with particle binding (Figure 4a and Figure S5). However, unfolded BSA inhibited the corona-NP-MARCO interaction (Figure 4). Indeed, when acLDL or oxLDL were combined with unfolded BSA corona-NPs-MARCO interaction was not further decreased compared with unfolded BSA alone (Figure 4a). Competitive interaction of unfolded BSA was further confirmed using fluorescently labelled unfolded BSA and simultaneous measurement of corona-NPs and unfolded BSA MARCO interaction. A transition from predominant NP uptake to predominant unfolded BSA uptake occurred at the concentration of 5 mg mL-1 unfolded BSA (Figure 4b). Such competition is absent with modified LDL (Figure S5). This finding suggests that existence of distinct binding sites on MARCO for corona from the ones for modified LDL. To identify key


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domains of MARCO for corona recognition, further structure-function relationship investigations, such as directed-mutagenesis, would be required.

CONCLUSIONS In broad terms this study confirms that receptor-nanoparticle interactions in situ can be studied using host-cell scavenger receptor models. This could provide significant molecularly relevant information in our understanding of the role of biomolecular-corona interactions in organ-level clearance. While simpler examples of adsorbed environmental proteins may confer to the particle the known receptor-protein site recognition27 the origins of more subtle scavenger-particle interactions may not be evident from current physiochemical nanoparticle measurements. However, one is also alerted to the possibility that nanoscale surface recognition could reside in much more complex organizational properties of the adsorbed biomolecular layer than hitherto foreseen. The structural tools to investigate these more complex recognition scenarios are largely absent, leaving the scavenger, pattern-recognition and related receptors themselves the best signal of such interactions.

METHODS Materials. 100 nm SiO2 NPs fluorescein isothiocyanate (FITC)-labelled with a plain surface were synthesized at the CBNI following the protocol previously described.45 PierceTM Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit (88282) was purchased from ThermoFisher. Alexa Fluor™ 488 acLDL (L23380), acLDL (L35354), and oxLDL (L34357) were purchased from ThermoFisher. Bovine serum albumin (BSA) lowendotoxin (CAS 9048-46-8) was purchased from Santa Cruz Biotechnology. Alexa Fluor™ 647 NHS Ester (Succinimidyl Ester) (A37573) and Alexa Fluor™ 488 5-SDP Ester (Alexa Fluor™ 488 Sulfodichlorophenol Ester) (A30052) were purchased from ThermoFisher. 9 ACS Paragon Plus Environment

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Human serum was purchased from BIOCHROM (collected off the clot from healthy humans (pool of donors with mixed gender and age)). The plasmid vector for expression of MARCO scavenger receptor (FHC10062) was purchased from KAZUSA DNA Research Institute. PureYield™ Plasmid Midiprep System (A2492), FuGENE® 6 Transfection Reagent (E2692), and Anti-HaloTag® antibody (G9211) were purchased from Promega. Rabbit antimouse IgG H+L (HRP) (ab6728) was purchased from Abcam. Color plus prestained protein ladder broad range (10−230 kDa) (P7711S) and blue loading buffer for SDS-PAGE were purchased from New England Bio-Laboratories (B77035). BCA protein assay kit (23227) and RIPA buffer (89901) were purchased from ThermoFisher. Propan-2-ol (P/7507/17) was ordered from Fisher Chemical. Ampicillin (A9393), select agar (A5054), and reagents used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Sigma-Aldrich, and they were used as received. PVC calibration standard 0.483 µm used for DCS measurements was purchased from Analytik Ltd. NP Characterization. SiO2 NPs were characterized by DCS to verify the stability of the dispersions. For corona preparation, 1 mg mL-1 of 100 nm SiO2 NPs were incubated with 1 mL of 10% human serum (corresponding to 5 mg mL−1 of total protein concentration), 30% human serum (15 mg mL−1 total protein), 50% human serum (25 mg mL−1 total protein), 80% human serum (40 mg mL-1), or 100% human serum (50 mg mL-1), depending on the experiment, at 37 °C for 1 h on a shaker followed by centrifugation at 16 000 rcf for 20 min. After centrifugation, the pellets were re-dispersed and washed with phosphate-buffered saline (PBS, pH 7.4), this step was repeated three times. The protein corona was analyzed by SDSPAGE. The NP dispersions used in this study were characterized by DCS using a CPS disc centrifuge DC24000. A sucrose density gradient of 8−24% prepared in PBS was used at 20 000 rpm (disk speed). Measurements were performed between 0.001 and 0.5 µm. Prior to each


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measurement, the instrument was calibrated with a PVC standard with a nominal size of 483 nm (Analytic Ltd.). LAL assay. LPS or endotoxin content of the NP dispersions were tested at the working concentration used in the cell experiments by Pierce™ Limulus Amebocyte lysate (LAL) Chromogenic Endotoxin Quantitation Kit (88282). 50 µL of each NP dispersion were tested in duplicates following the LAL chromogenic assay manufacturer’s indications. Cell Culture. Human embryonic kidney (HEK-293T) cells (from female human foetus) purchased from and authenticated by ATCC (ATCC CRL-3216) were cultured in DMEM, high glucose, Glutamax (GIBCO), supplemented with 10% foetal bovine serum (FBS, GIBCO) in a humidified chamber at 37 °C under 5% CO2. Cells were grown in their preferred environment and passaged three times a week (cells were subcultured up to passage 20), as they approached 70−80% surface coverage. The cells were monthly tested for mycoplasma contamination by MycoAlert assay kit (Cambrex Bio Science, Nottingham, UK). Transfection of HEK-293T Cells. HEK-293T cells at a seeding density of 1 × 105 per well in 1 mL of complete DMEM Glutamax (GIBCO) medium supplemented with 10% FBS were plated into 12-well plate (Cellstar Greiner bio-one) for 24 h prior to transfection plate. Cells were transfected at a 3.5:1 FuGENE 6 (Promega)-to-DNA (0.02 µg µL−1). Plasmid DNA used for the expression of MARCO scavenger receptor was added into a sterile tube containing opti-MEM reduced serum media (GIBCO). FuGENE 6 was added to the plasmid DNA solution and mixed by pipetting 15 times. The solution was incubated for 10 min at room temperature. Next, 50 µL of FuGENE 6 transfection reagent/plasmid DNA mixture were added to each well and incubated for 24 h at 37 °C and 5% CO2. Cellular Uptake of NPs. After 24h of transfection, prior to NP exposure, cells were washed for 30 min in serum-free DMEM and replaced subsequently by the freshly prepared NP


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dispersions used in the study. Different NP dispersions were added to the cells at 0.1 mg mL−1 at 37 °C and 5% CO2 for 4 h. Following the incubation, cells were washed with 1 mL of completed DMEM. One-fifth of the volume in each well was replaced with TMR HaloTag® ligand at a concentration of 200 nM in completed DMEM and incubated at 37 °C, 5% CO2 for 15 min. Prior to harvesting the cells by trypsinization, cells were washed with completed DMEM once and twice with PBS. Cells were spun down, re-dispersed in fresh completed DMEM and placed on ice. Cell fluorescence intensity was measured using a Beckman Coulter CyAn ADP flow cytometer equipped with 3 lasers (488, 561, and 643 nm). Results are reported as the median of cell fluorescence intensity of transfected cells (high TMR subpopulation). At least 15 000 cells were analyzed in each repeat.

ACKNOWLEDGMENTS This work was supported by the Science Foundation Ireland (SFI) Principal Investigator Award (Agreement No. 12/IA/1422), Starting Investigator Researcher Grant (Agreement No. 15/SIRG/3423), SFI MAGneTISe Grant (Grant No. 16/ENM-ERA/3457), and Science Foundation Ireland (SFI) and the National Natural Science Foundation Of China (NSFC) under the SFI-NSFC Partnership Programme (Grant No: 17/NSFC/4898). This publication acknowledges the support of the EU FP7 FutureNanoNeeds project (Agreement No. 604602). The authors acknowledge A. Blanco’s assistance in the Conway Institute Flow Cytometry facility. D. Garry and F. Bertoli are also acknowledged for their assistance. T. Miclaus is acknowledged for acquiring TEM micrographs of the nanoparticles under study.

AUTHOR CONTRIBUTIONS S.L. performed all cell biology experiments, analyzed and interpreted the data, A.P.P. contributed with experimental method development for LPS testing, L.M.H. contributed with


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the synthesis of 100 nm SiO2 NPs, L.A. contributed with the synthesis of 30 nm SiO2 NPs. K.A.D. and Y.Y. conceived the idea and designed the experiments. All authors discussed and contributed to writing the manuscript.

COMPETING INTEREST The authors declare no competing financial interest.

ASSOCIATED CONTENT Supporting information. Physicochemical characterisation of 30 nm SiO2 nanoparticles; Assessment of MARCO scavenger receptor expression by Western Blot and RT-qPCR in HEK-293T cells; Physicochemical characterization of human serum-corona 100 nm SiO2 NPs re-dispersed in several concentrations of human serum; Human serum corona-30 nm SiO2 NPs uptake in several concentrations of human serum; LPS and oxLDL failed to compete the uptake of human serum corona-100 nm SiO2 NPs by MARCO in 80% human serum. The Supporting Information is available free of charge on the ACS Publications website.

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FIGURES

Figure 1. Scheme representation of MARCO scavenger receptor transfected HEK-293T cells. (a) Schematic representation MARCO scavenger receptor with its proposed domains. (b) Scheme of the receptor fused with a HaloTag® protein at its N-terminus. The HaloTag® protein forms a covalent bond with a fluorescent HaloTag® ligand, TMR. (c) Scatter blot representation of two color flow cytometry determination of SiO2 NPs uptake in MARCOtransfected HEK-293T cells stained with TMR HaloTag® Ligand (Y-axis) versus SiO2 NPs fluorescence intensity (X-axis) in 10% human serum. Different populations of receptor expression are resolved via TMR receptor staining intensity, with high level receptor expression, which is the population with highest NPs uptake, and low level receptor expression with lower NPs uptake.


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Figure 2. Physicochemical characterization of 100 nm SiO2 NPs. (a) TEM micrograph showing 100 nm SiO2 NPs. Scale bar: 100 nm (inset). (b) Detection of endotoxin in 100 nm SiO2 NPs by Limulus Amebocyte Lysate (LAL) assay. (c) Physicochemical characterization of 100 nm SiO2 NPs dispersed in cell culture media supplemented with various human serum concentrations by Differential Centrifugal Sedimentation (DCS). (d) 100 nm SiO2 NPs were dispersed in 10, 30, 50, 80 and 100% human serum. SDS-PAGE of protein coronas recovered from 100 nm SiO2 NPs incubated at various human serum concentrations. 100 nm SiO2 NPs were incubated in 10, 30, 50, 80, and 100% human serum for 1 h at 37 °C, after centrifugation the pellets were re-dispersed in PBS, this step was repeated three times prior loading the samples in 1D SDS-PAGE.


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Figure 3. Uptake of 100 nm SiO2 NPs in several concentrations of human serum measured by flow cytometry. (a) In site uptake of 100 nm SiO2 NPs dispersed in 30, 50, 80, and 100% human serum by MARCO-transfected or empty vector transfected cells at 37 °C for 4 h. (b) Uptake of human serum corona-SiO2 NP complexes derived from 100% human serum by MARCO-transfected or empty vector transfected cells in 10, 30, 50, and 80% human serum at 37 °C for 4 h. Data represent the median fluorescence intensity of transfected cells performed in duplicates. At least 15 000 cells were analyzed in each repeat by flow cytometry Beckman Coulter CyAn ADP Cytometers. (c and d) Examples of a 2D plot graph of flow cytometry data showing correlation between MARCO expression (TMR intensity) and uptake of human serum corona-SiO2 NP complexes in 10% (c) and 80% (d) (NPs intensity). Blue: MARCO-transfected cells. Red: Empty vector transfected cells. UT: Untreated cells.


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Figure 4. Competitive uptake of human serum corona-SiO2 NPs re-dispersed in 80% human serum measured by flow cytometry. (a) Competitive uptake of human serum corona-SiO2 NPs by MARCO-transfected cells in 80% human serum in presence of 15 µg mL-1 of acLDL (NP+acLDL), or 15 µg mL-1 of oxLDL (NP+oxLDL), or 1 EU mL-1 of LPS (NP+LPS), or 15 mg mL-1 of unfolded BSA (NP+Unf. BSA), or 15 mg mL-1 of unfolded BSA and 15 µg mL-1 of acLDL (NP+acLDL+unf. BSA), or 15 mg mL-1 of unfolded BSA and 15 µg mL-1 of oxLDL (NP+oxLDL+unf. BSA) at 37 °C for 4 h. (b and c) Uptake of human serum corona-SiO2 NPs competed with Alexa Fluor 647 labelled unfolded BSA (1, 5, 10, and 15 mg mL-1) in 80% human serum. Data represent the median fluorescence intensity of transfected cells performed in duplicates. At least 15 000 cells were analyzed in each repeat by flow cytometry Beckman Coulter CyAn ADP Cytometers. Blue: MARCO-transfected cells. Red: Empty vector transfected cells. UT: Untreated cells.


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