Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 1;12(10):1117.
doi: 10.3390/v12101117.

Electrostatic Surface Properties of Blood and Semen Extracellular Vesicles: Implications of Sialylation and HIV-Induced Changes on EV Internalization

Hussein Kaddour  1 , Tyler D Panzner  1 , Jennifer L Welch  2   3   4 , Nadia Shouman  1 , Mahesh Mohan  5 , Jack T Stapleton  2   3   4 , Chioma M Okeoma  1
Affiliations

Electrostatic Surface Properties of Blood and Semen Extracellular Vesicles: Implications of Sialylation and HIV-Induced Changes on EV Internalization

Hussein Kaddour et al. Viruses. .

Abstract

Although extracellular vesicle (EV) surface electrostatic properties (measured as zeta potential, ζ-potential) have been reported by many investigators, the biophysical implications of charge and EV origin remains uncertain. Here, we compared the ζ-potential of human blood EVs (BEVs) and semen EVs (SEVs) from 26 donors that were HIV-infected (HIV+, n = 13) or HIV uninfected (HIV-, n = 13). We found that, compared to BEVs that bear neutral surface charge, SEVs were significantly more negatively charged, even when BEVs and SEVs were from the same individual. Comparison of BEVs and SEVs from HIV- and HIV+ groups revealed subtle HIV-induced alteration in the ζ-potential of EVs, with the effect being more significant in SEVs (∆ζ-potential = -8.82 mV, p-value = 0.0062) than BEVs (∆ζ-potential = -1.4 mV, p-value = 0.0462). These observations were validated by differences in the isoelectric point (IEP) of EVs, which was in the order of HIV + SEV ≤ HIV-SEV ≪ HIV + BEV ≤ HIV-BEV. Functionally, the rate and efficiency of SEV internalization by the human cervical epithelial cell line, primary peripheral blood lymphocytes, and primary blood-derived monocytes were significantly higher than those of BEVs. Mechanistically, removal of sialic acids from the surface of EVs using neuraminidase treatment significantly decreased SEV's surface charge, concomitant with a substantial reduction in SEV's internalization. The neuraminidase effect was independent of HIV infection and insignificant for BEVs. Finally, these results were corroborated by enrichment of glycoproteins in SEVs versus BEVs. Taken together, these findings uncover fundamental tissue-specific differences in surface electrostatic properties of EVs and highlight the critical role of surface charge in EV/target cell interactions.

Keywords: HIV-1; biological membranes; blood; extracellular vesicles; glycocalyx; semen; zeta potential.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 5
Figure 5
Sialic acids affect SEV ζ-potential and internalization efficiency. (a) Schematic of EV surface membrane showing a membrane glycoprotein and glycosphingolipid decorated with sialic acids. (b) ζ-potential of HIV- (blue) and HIV+ (red) BEVs and SEVs before (full bars) and after (pattern bars) neuraminidase treatment. (c) Representative 10× images of EV internalization. Scale bar is 20 µm. Primary and secondary masks were set in the DAPI and GFP channels, respectively. Min and Max object size for the primary mask were 5 and 55 µm, respectively. GFP intensity for each cell was measured by reducing the primary mask with 4 µm and expanding the secondary mask with 8 µm. (d) Relative EV internalization at 24 h of intact and neuraminidase-treated HIV- and HIV+ BEVs and SEVs. Represented data are an average of 16 fields of view per well and 6 wells per treatment. (e) Venn diagram between the differentially present proteins (DPPs) enriched in SEVs as compared to BEV from the HIV- and HIV+ groups, as analyzed in [51]. (f) Heatmap clustering analysis of the 21 glycocalyx-related SEV-enriched DPPs (rows). Columns correspond to the analyzed EV samples. Spearman rank correlation single linkage was applied to rows and columns. Heatmap was generated using heatmapper, an online data visualization application [53]. Ordinary one-way ANOVA test was performed to determine the differences between the treatments and the labelled PBS. Unpaired t-test with Welch’s correction was used to determine the differences between the two-group comparisons. Error bars indicate S.D. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, non-significant.
Figure 1
Figure 1
ζ-potential measurements using Nano Tracking Analysis (NTA). (a) Schematic showing that the ζ-potential corresponds to the electrokinetic potential at the slipping plane between the diffuse layer of the vesicle and the diluent. (b) Graph showing ζ-potential ranges of EVs. (c) Graph showing 10 repeat measurements of the same sample (pooled, n = 10, HIV- or HIV+, BEV or SEV). (d) Graph of ζ-potential measurements of the same sample before and after dilution.
Figure 2
Figure 2
ζ-potential measurements of HIV- and HIV+ BEVs and SEVs. (a,b) Mean ζ-potential of quintuplet measurements of HIV- BEVs and HIV- SEVs from 10 unmatched donor samples (a) and 3 matched donor samples (b). Error bars correspond to S.D. (c) A graph showing the mean ζ-potential for all 13 HIV- BEV and HIV- SEV samples. Numbers below and above the data are, respectively, the mean of the means ± S.D and the p-value of an unpaired t-test with Welch’s correction. (d) Mean ζ-potential of quintuplet measurements of HIV+ BEVs and SEVs, from 13 matched donors. Error bars correspond to S.D. (e) Mean ζ-potential for all 13 HIV+ BEV and SEV samples. Numbers below the data are the mean of the means ± S.D. The number on top of the data corresponds to the p-value of an unpaired t-test with Welch correction. (f) Effects of HIV-1 on the ζ-potential of BEVs and SEVs. The numbers on top of the data represent the p-values of unpaired t-tests with Welch correction between the HIV- and HIV+ groups. (g) ζ-potential of pools (n = 10) of HIV- and HIV+ BEVs and SEVs as a function of pH. Blue and red dashed vertical lines correspond to the IEP of HIV- and HIV+ BEVs, respectively. Error bars are S.D. of quintuplet measurements.
Figure 3
Figure 3
Internalization rate and efficiency of HIV- and HIV+ BEVs and SEVs by epithelial cells. (a) Representative images of TZM-bl cells 24 h post-internalization experiment. Scale bar is 50 µm. (b) Quantification strategy for EV internalization as described in Materials and Methods. (c) Kinetics of EV internalization. Error bars represent S.D. of 4 wells, each with stitched 4 fields of view. Experiment was repeated three times with similar results. (d) Luciferase assay post-internalization showing no LTR promoter activation beyond the basal level of cells, which was set to 100. “ns” indicates not significant based on one-way ANOVA, compared to PBS control. (e) Linear regression between relative internalization and ζ-potential of EVs.
Figure 4
Figure 4
Internalization rate and efficiency of HIV- BEVs and SEVs by primary peripheral blood lymphocytes (PBLs) and monocytes. (a,b) Kinetics of EV internalization by (a) PBLs and (b) monocytes. Fluorescence intensity from the green-fluorescence protein (GFP) channel for each sample was normalized to that of labelled PBS which was set to 1. Error bars represent SD of 6 wells. Experiment was done three times with three different donors (D1-D3). (c,d) MTT viability assay post- internalization for (c) PBLs and (d) monocytes showing no difference in cell viability compared to labelled PBS which was set to 100. (e) Comparison of internalization efficiency amongst cell types at 24 h. 2-way ANOVA test (Sidak’s correction) was performed to determine the differences. Error bars indicate S.E.M. of 3 independent experiments. * p < 0.05; **** p < 0.0001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Vance J.E., Steenbergen R. Metabolism and functions of phosphatidylserine. Prog. Lipid Res. 2005;44:207–234. doi: 10.1016/j.plipres.2005.05.001. - DOI - PubMed
    1. Chen B., Le W., Wang Y., Li Z., Wang D., Ren L., Lin L., Cui S., Hu J.J., Hu Y. Targeting negative surface charges of cancer cells by multifunctional nanoprobes. Theranostics. 2016;6:1887. doi: 10.7150/thno.16358. - DOI - PMC - PubMed
    1. Fröhlich E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int. J. Nanomed. 2012;7:5577–5591. doi: 10.2147/IJN.S36111. - DOI - PMC - PubMed
    1. Goldenberg N.M., Steinberg B.E. Surface charge: A key determinant of protein localization and function. Cancer Res. 2010;70:1277–1280. doi: 10.1158/0008-5472.CAN-09-2905. - DOI - PubMed
    1. Bondar O.V., Saifullina D.V., Shakhmaeva I.I., Mavlyutova I.I., Abdullin T.I. Monitoring of the Zeta Potential of Human Cells upon Reduction in Their Viability and Interaction with Polymers. Acta Nat. 2012;4:78–81. doi: 10.32607/20758251-2012-4-1-78-81. - DOI - PMC - PubMed

Publication types