Characterization of Extracellular Vesicles from Human Saliva: Effects of Age and Isolation Techniques

Abstract

Salivary extracellular vesicles (EVs) represent an attractive source of biomarkers due to the accessibility of saliva and its non-invasive sampling methods. However, the lack of comparative studies assessing the efficacy of different EV isolation techniques hampers the use of salivary EVs in clinical settings. Moreover, the effects of age on salivary EVs are largely unknown, hindering the identification of salivary EV-associated biomarkers across the lifespan. To address these questions, we compared salivary EV concentration, size mode, protein concentration, and purity using eight EV isolation techniques before and after magnetic bead immunocapture with antibodies against CD9, CD63, and CD81. The effects of age on salivary EVs obtained with each isolation technique were further investigated. Results showed higher expression of CD63 on isolated salivary EVs compared to the expression of CD81 and flotillin-1. Overall, magnetic bead immunocapture was more efficient in recovering salivary EVs with Norgen's Saliva Exosome Purification Kit and ExoQuick-TC ULTRA at the cost of EV yield. Regardless of age, Invitrogen Total Exosome Isolation Solution showed the highest level of protein concentration, whereas Izon qEVOriginal-70nm columns revealed the highest purity. This study provides the first comprehensive comparison of salivary EVs in younger and older adults using different EV isolation techniques, which represents a step forward for assessing salivary EVs as a source of potential biomarkers of tissue-specific diseases throughout the life cycle.

Keywords: TEM; aging; exosomes; extracellular vesicles; magnetic bead immunocapture; saliva; tetraspanins.

Figure 1

Schematic overview of the protocol followed with each technique for the isolation of salivary EVs. Salivary EV isolation was performed in sextuplicate with eight techniques in younger and older adults, respectively. Protein concentration and purity of salivary EV samples were only assessed before magnetic bead immunocapture with anti-CD9, -CD63, and -CD81 antibodies. TEIS: Total Exosome Isolation Solution; RT: room temperature; UC: differential ultracentrifugation; BCA: Bicinchoninic acid protein assay; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy.

Figure 2

Salivary EV yield obtained with each technique (N = 6 replicates) before and after magnetic bead immunocapture with anti-CD9, -CD63, and -CD81 antibodies (left and middle columns, respectively) in younger and older adults (top and bottom rows, respectively). The right column shows the percentage of recovered salivary EVs obtained with each technique after magnetic bead immunocapture. Due to the lack of significant age × technique interaction on the percentage of recovered salivary EVs, the results of the post hoc analysis for the EV isolation techniques were identical in the two age groups. Statistical differences in EV yield between techniques resulted from post hoc comparisons as long as a significant main or interaction effect was previously obtained with aligned ranks transformation ANOVAs. The p-values were adjusted by a Bonferroni correction for multiple comparisons. Statistical significance is expressed as ^† (p ≤ 0.05); # (p ≤ 0.001); * (p ≤ 0.0001).

Figure 3

Mean salivary EV size mode obtained with each technique (N = 6 replicates), before and after magnetic bead immunocapture with anti-CD9, -CD63, and -CD81 antibodies (left and right columns, respectively) in younger and older adults (top and bottom rows, respectively). Note that the EV size mode significantly varied with age and magnetic bead immunocapture, but not with EV isolation techniques. nm: nanometers.

Figure 4

Protein concentration and purity of salivary EVs (left and right columns, respectively) obtained with each technique (N = 6 replicates) before magnetic bead immunocapture with anti-CD9, -CD63, and -CD81 antibodies in younger and older adults (top and bottom rows, respectively). Statistical differences in protein concentration or purity between techniques resulted from post hoc comparisons as long as a significant main or interaction effect was previously obtained with aligned ranks transformation ANOVAs. The p-values were adjusted by a Bonferroni correction for multiple comparisons. Statistical significance is expressed as ^† (p ≤ 0.05); # (p ≤ 0.001); * (p ≤ 0.0001).

Figure 5

Western blot analyses of salivary EVs obtained with the eight techniques in younger and older adults (top and bottom row, respectively), before and after magnetic bead immunocapture with anti-CD9, -CD63, and -CD81 antibodies (left and right columns, respectively). Antibodies against CD63, CD81, and flotillin-1 (FLOT-1) were used as positive markers of exosomes. As negative markers of exosomes, we used antibodies against Glucose-regulated protein 78 (GRP78) and Calnexin, two endoplasmic reticulum proteins. Levels of lipoprotein and albumin contamination were further assessed by using antibodies against APOB and albumin, respectively. The SH-SY5Y human neuroblastoma cell line was used as a control.

Figure 6

TEM imaging analysis of salivary EVs obtained with each technique after magnetic bead immunocapture with anti-CD9, -CD63, and -CD81 antibodies in younger (A) and older adults (B). For each technique, we showed a representative negative-stained TEM image at 80,000× magnification together with a histogram displaying the relative frequency distribution of salivary EV size obtained from 30 TEM images (6 replicates × 5 images/technique) acquired at 10,000× magnification. The number of salivary EVs detected with each technique was also included. nm: nanometers.

Figure 7

Age effects on salivary EVs obtained with Norgen’s Saliva Exosome Purification Kit comparing 10 younger vs. 10 older adults. (A) NTA characterization of salivary EVs before and after magnetic bead immunocapture with anti-CD9, CD63, and CD81 antibodies in younger and older adults. Age differences in salivary EV concentration (B), size mode (C), percentage of recovered EVs after magnetic bead immunocapture (D), level of protein concentration estimated with BCA before magnetic bead immunocapture (E), salivary EV purity estimated with the particle-to-protein ratio before magnetic bead immunocapture (F), and western blot analyses of salivary EVs obtained before (left panel) and after magnetic bead immunocapture (right panel) in 5 younger and 5 older adults (G). Statistical significance is expressed as ^† (p ≤ 0.05); # (p ≤ 0.01); * (p ≤ 0.001). IC: immunocapture; nm: nanometers.