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. 2024 Oct 27;31(3):879–889. doi: 10.1111/odi.15171

Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation‐Based Protocols

Castillejos‐García Itzel 1, Martínez‐Martínez Eduardo 2, Ramírez‐Amador Velia 3, Cisneros‐Villanueva Mireya 4, Hidalgo‐Miranda Alfredo 4, Ramos‐Godínez María del Pilar 5, Anaya‐Saavedra Gabriela 3,✉
PMCID: PMC12021316  PMID: 39462790

ABSTRACT

Introduction

The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer‐based precipitation and UC (PBP + UC).

Methods

Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT‐PCR. Statistical analyses comparing groups were performed using ANOVA.

Results

All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR‐16, miR‐27a, and miR‐99a was successfully detected using all methods.

Conclusions

The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time‐sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses.

Keywords: extracellular vesicles, miRNAs, PEG, saliva, ultracentrifugation

1. Introduction

Extracellular vesicles (EVs) are lipid‐bilayer particles ranging from nano‐ to micro‐size (30 to 1000 nm) that are unable to replicate on their own. They are released by various cell types and found in diverse biofluids (Welsh et al. 2024). EVs carry selectively packaged cargo, including proteins, lipids, and nucleic acids, reflecting the composition of the originating cell (Dixson et al. 2023; Théry et al. 2018).

EV‐associated proteins serve as reliable markers for identification, shedding light on the biogenesis process (Welsh et al. 2024; Théry et al. 2018; Yáñez‐Mó et al. 2015). Similarly, nucleic acids, particularly non‐coding RNAs such as microRNAs (miRNAs) and long non‐coding RNAs (lncRNAs), exhibit specific sorting patterns indicative of their cellular origin, making them potential biomarkers (Dixson et al. 2023; O'Brien et al. 2020; Hessvik and Llorente 2018). The transfer of EV cargo to nearby and distant target cells modulates multiple signaling pathways, leading to phenotypic and functional changes (Miron and Zhang 2023; Joshi et al. 2020). Consequently, EVs play a pivotal role in intercellular communication by regulating various physiological and pathological processes (Joshi et al. 2020; Kalluri and LeBleu 2020; Van Niel, D'Angelo, and Raposo 2018; Yáñez‐Mó et al. 2015). Additionally, EVs hold therapeutic potential for certain diseases, continually revealing novel biological functions (Wiklander et al. 2019).

The functional roles of EVs and their potential as biomarkers in biofluids have attracted significant interest in EV research aimed at identifying markers for various diseases through liquid biopsy (Yao et al. 2023; Yu, Li, Wang, et al. 2022; Yu, Li, Zhang, et al. 2022; Liu et al. 2021; Valencia and Montuenga 2021; Sun et al. 2018). While most EV research has focused on plasma and serum (Martins, Vaz, and Henriques 2023), saliva has emerged as an interesting source of EVs due to its easy accessibility and non‐invasive sampling collection (Dawes and Wong 2019; Sullivan et al. 2020).

Saliva is a complex fluid that contains EVs from various sources, including the salivary glands, the oral microbiome, and the epithelial oral cells. Additionally, some EVs found in saliva are believed to originate from the bloodstream, crossing epithelial barriers (Yu, Li, Zhang, et al. 2022). Numerous methods for separating and analyzing EVs have been documented, including ultracentrifugation (UC), size exclusion chromatography (SEC), immunoaffinity capture‐based methods (IA), ultrafiltration (UF), and polymer‐based precipitation (PBP) (Akbar et al. 2022; Cheng et al. 2020; García‐Romero et al. 2019; Monguió‐Tortajada et al. 2019; Théry et al. 2018; Helwa et al. 2017; Kim et al. 2015). Each method has inherent limitations, and sample characteristics can influence purity and separation efficiency (Martins, Vaz, and Henriques 2023; Théry et al. 2018). Combining methods for EVs separation has demonstrated improved yield and purity among diverse biological samples, with UC + UF and UC + SEC approaches being the most employed (Stam et al. 2021; Koh et al. 2018).

In the case of saliva, ultracentrifugation, PBP, and SEC are the main techniques for separating EVs (Hofmann et al. 2022; Gallo et al. 2012; Lässer et al. 2011). Studies comparing EV separation methods for saliva are limited, and outcomes regarding efficiency and purity are varying. However, overall data suggest that UC provides the highest yield of salivary EVs, even compared to some commercial kits, followed by SEC and PBP methods (Reseco et al. 2024; Tengler et al. 2024; Boulestreau et al. 2024; Jangholi et al. 2023; Li et al. 2021; Han et al. 2020; Deregibus et al. 2016; Sun et al. 2018). Nevertheless, UC has concerns, such as the lack of a unified protocol with adjusting speeds, times, and centrifugation cycles and its low scalability for clinical applications (Konoshenko et al. 2018). The selection of the appropriate protocol should be based on the characteristics of the biological sample type (Konoshenko et al. 2018).

Some studies have demonstrated that combining PBP with UC enhances the separation of EVs from cell culture media and blood‐derived mesenchymal stem cells, resulting in a quality sufficient to carry out proteomic and transcriptomic analysis (Tsiapalis et al. 2023; Ludwig et al. 2018; Rider, Hurwitz, and Meckes 2016). However, the efficacy of this combined approach (PBP + UC) in EVs from saliva has not been evaluated.

Thus, the present study compares three EV separation strategies for whole saliva: ultracentrifugation (UC), UC with purification step (UC + PS), and combined polymer‐based precipitation with UC (PBP + UC).

2. Materials and Methods

Five healthy donors with no prior medical history participated in this research. Clinical and demographical data were obtained through a customized questionnaire. All the participants gave their written informed consent. Figure 1 depicts the protocol flowchart from collecting and preprocessing saliva samples to EV characterization.

FIGURE 1.

FIGURE 1

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Workflow of salivary extracellular vesicle separation. Unstimulated whole saliva was collected from five healthy individuals, and preprocessing for EV separation by Ultracentrifugation (UC) (red box), ultracentrifugation + purification step (UC + PS) (yellow box), and polymer‐based precipitation + UC (PBP + UC) (blue box). The EV characterization was performed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western blotting (WB), and total protein and RNA quantification. Additionally, an RT‐qPCR was performed to assess miRNA expression.

2.1. Saliva Sample Collection and Preprocessing

Unstimulated whole saliva was collected from the healthy donors. Each donor provided three saliva samples from successive days and abstained from eating or drinking for an hour before sample collection. Each sample of 7 mL of saliva was collected for 10–12 min using the drooling method, between 9:00 and 11:00 am, in polypropylene DNase‐ and RNase‐free conical tubes (Thermo Fisher Scientific, MA, USA) and placed on ice. Within 30 min of collection, the samples underwent a centrifugation protocol of 400 g for 10 min and 2000 g for 20 min at 4°C to remove contaminating cells and debris (Figure 1). From each donor, three free‐cell saliva samples were pooled to avoid day‐to‐day variations. The resulting pooled samples, with a final volume of 20–21 mL, were divided into equal aliquots and preserved at −80°C for downstream assays.

2.2. Extracellular Vesicles Separation

Salivary EVs were separated from cell‐free saliva diluted at a 1:1 ratio with filtered PBS (0.22‐μm pore‐size polyethersulfone filter, Merck Millipore, MA, USA) through three ultracentrifugation‐based protocols. The first protocol involved a single UC at 118,000 g for 90 min. The second approach incorporated an additional purification step (UC + PS), whereas the third integrated a preliminary polymer‐based precipitation step before ultracentrifugation (PBP + UC) (Figure 1).

2.3. Ultracentrifugation (UC)

In this method, cell‐free saliva diluted with filtered PBS was ultracentrifuged at 118,000 g for 90 min at 4°C in thick‐walled polycarbonate ultracentrifuge tubes (70 Ti rotor/k‐factor 44, Centrifuge Optima L‐100 XP, Beckman Coulter, Brea, CA, EUA). The resulting pellet was resuspended for downstream assays in 250 μL of PBS or 400 μL of TRIzol (Invitrogen, MA, USA).

2.4. Ultracentrifugation with purification step (UC + PS)

This protocol involves the ultracentrifugation of cell‐free saliva diluted with filtered PBS at 118,000 g for 90 min at 4°C in thick‐walled polycarbonate ultracentrifuge tubes (70 Ti rotor/k‐factor 44, Centrifuge Optima L‐100 XP, Beckman Coulter, Brea, CA, USA). The resulting pellet was resuspended in 2.5 mL of PBS, and the ultracentrifugation step was repeated at 118,000 g for 90 min at 4°C (SW 55 Ti rotor/k‐factor 48, Centrifuge Optima L‐100 XP, Beckman Coulter, Brea, CA, USA). For downstream assays, the pellet was resuspended in 250 μL of PBS or 400 μL of TRIzol (Invitrogen, MA, USA).

2.5. Polymer‐Based Precipitation and Ultracentrifugation (PBP + UC)

A 50% (w/v) solution of PEG 8000 (Sigma Aldrich) was used to precipitate total EVs. The reagent was added to each sample of diluted cell‐free saliva in a 1:5 ratio (polymer/fluid) and incubated overnight at 4°C. The following day, the samples were centrifuged at 1500 g for 30 min. The supernatant was decanted, and the residual liquid was centrifuged at 1500 g for 5 min to remove the remaining liquid. The resulting pellet was resuspended in 2.5 mL of PBS into thick‐walled polycarbonate tubes (Beckman Coulter) and ultracentrifuged at 118,000 g (SW 55 Ti rotor/k‐factor 48, Centrifuge Optima L‐100 XP, Beckman Coulter, Brea, CA, USA) for 90 min. The final pellet was resuspended in 250 μL of PBS or 400 μL of TRIzol for downstream assays.

2.6. Transmission Electron Microscopy (TEM)

EVs in 250 μL of PBS were fixed with 400 μL of 2.5% glutaraldehyde and 2% formaldehyde (Electron Microscopy Sciences [EMS]). The PBS was exchanged for a fixative solution using an Amicon‐0.5 mL filter (3 kDa cut‐off) until it reached a final concentration of 200 μL. After 45 min, EVs were adsorbed onto 200‐mesh formvar and carbon‐coated grids (Electron Microscopy Sciences [EMS]). Each grid was washed by transfer seven times to drops of water for 2 min. The sample on the grid was stained with 4% uranyl acetate for 5 min. Finally, the image was captured using a JEOL JEM‐1010 electron microscope with an AMT digital camera.

2.7. Nanoparticle Tracking Analysis (NTA)

Particle size distribution and EV quantification were analyzed by NTA using a NanoSight NS300 (Malvern Panalytical, UK) equipped with a blue laser (488 nm). The analysis parameters were set with camera sCOMOS at level 12 and a detection threshold 5. EV samples were diluted at 1:10,000 in PBS and loaded into the chamber using a constant‐flow injection syringe. Two 60‐s videos were captured for each sample, and subsequent data analysis was performed using NTA 3.2 software (Malvern Panalytical, UK).

2.8. Protein Quantification

EV samples were processed with TRIzol reagent (Invitrogen, MA, USA), and protein was recovered from the organic phase following the manufacturer's instructions. Protein concentration was determined using a BCA assay (Pierce BCA Protein Assay, Thermo Scientific, MA, USA), following the manufacturer's guidelines, with bovine serum albumin (BSA) as a standard. Absorbance was measured at 562 nm using a Nanodrop 2000 spectrophotometer (Thermo Scientific, MA, USA).

2.9. Western Blotting

An equal amount of protein (5 μg) from the EV sample mixed with the loading buffer was heated to 95°C for 5 min and separated by SDS‐PAGE (7.5% and 10% gel, 200 V, 30 min) using the TGX FastCast Acrylamide Kit (Bio‐Rad, CA, USA). Protein was transferred to a PVDF membrane using a Trans‐Blot Turbo Transfer System (Bio‐Rad, CA, USA). Cell lysate and EVs from the MDA‐MB‐468 cell line were positive controls for intracellular contamination and EV markers, respectively. After washing with Tween Tris‐Buffered Saline (TTBS), membranes were incubated overnight at 4°C with primary antibodies against CD63 (1:2000, #sc‐81178 Santa Cruz Biotechnology), CD9 (1:1000, #10626D Thermo Fisher Scientific), GAPDH (1:10,000, #ab181602 Abcam), and calnexin (1:1000, #sc‐6465 Santa Cruz Biotechnology) diluted with blocking solution (5% non‐fat milk in TBST). Gels for CD9 and CD63 were run under non‐reducing conditions. Subsequently, membranes were washed three times with TTBS for 5 min, followed by incubation with secondary detection reagents (m‐IgG Fc BP‐HRP, #sc‐525409, and anti‐goat‐HRP #sc2020, Santa Cruz Biotechnology) at room temperature, for 1.5 h. Detection of antibodies was performed with a luminol‐based enhanced chemiluminescence (SuperSignal West Pico PLUS, ThermoFisher Scientific, MA, USA) substrate for detecting horseradish peroxidase (HRP) coupled to the secondary reagent according to the manufacturer's instructions. The intensity of bands was quantified with ImageLab software (Bio‐Rad), and densitometric data normalization was carried out with housekeeping protein loading control (GAPDH) of EV samples.

2.10. RNA Extraction Protocol

RNA extraction was performed using the TRIzol reagent (Invitrogen, MA, USA). The EV pellet was resuspended in 400 μL of TRIzol, and subsequent steps were performed following the manufacturer's guidelines. The RNA pellet was solubilized in nuclease‐free water, and all procedures were conducted in an RNase‐free environment.

2.11. Quality and Quantity Assessment

RNA yield and purity were determined using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). Representative samples from each experimental group, selected based on the highest total amount of RNA, were analyzed with capillary electrophoresis by the Agilent 2100 Bioanalyzer (Agilent Technologies Sweden AB, Kista, Sweden).

2.12. Quantitative RT‐PCR

To analyze the miRNA expression in EVs separated by all methods, three miRNAs (miR‐16, miR‐27a, and miR‐99a) were selected, as they have previously been identified in EVs from biofluids (Burdiel et al. 2023; Han et al. 2020; Gai et al. 2018; Gallo et al. 2012). Specifically, miR‐16 has been used as an endogenous control to miRNA expression assays of EVs (Burdiel et al. 2023; Coon, Kingsley & Howard, 2020). The assay involves reverse transcription (RT) and quantitative polymerase chain reaction (qPCR). First, cDNA was synthesized using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA). Briefly, 2.5 ng of RNA from each sample was mixed with 1.5 μL of 100 nM stem‐loop RT primer, 0.8 μL of 1x RT buffer, 0.1 μL of 100 mM of dNTPs, 0.5 of 50 U/μL MultiScribe Reverse Transcriptase, 0.1 of 20 U/μL of RNase inhibitor, and 2 μL of nuclease‐free water. The resulting 7.5 μL mixture was incubated in an Applied Biosystems 9700 thermal cycler at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min, followed by cooling to 4°C.

For real‐time PCR, the TaqMan PCR Kit protocol (Thermo Fisher Scientific, MA, USA) was performed as per the manufacturer's instructions. Briefly, 1 μL of RT product was mixed with 5.0 μL of TaqMan Universal PCR Master Mix, 0.5 μL of TaqMan‐specific probe (miR‐16, miR‐99a, and miR‐27a) and 3.5 μL of nuclease‐free water. The PCR reaction mixture (10 μL) was incubated in a 96‐well plate of QuantStudio 5 Real‐Time PCR System thermocycler (Applied Biosystems Life Technologies, Carlsbad, USA) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed in triplicate.

2.13. Statistical Analysis

Experimental data were analyzed using GraphPad Prism 9 (GraphPad Software, USA). Analysis of variance (ANOVA) and post hoc tests were applied to compare multiple groups, with a significance level of p < 0.05.

3. Results

All healthy donors were female, with a median age of 34 (Q1–Q3: 25–40) years. They were non‐smokers and had no history of strenuous physical exercise in the last 6 months. Regarding alcohol consumption, all participants reported being current drinkers with an average of 14.5 years of consumption and a median intake of 125 (Q1–Q3: 100–412.5) mL per month. At the sampling time, no donors had any oral lesions and presented adequate oral hygiene.

3.1. UC + PS and PBP + UC Methods Yielded the Highest Total EVs With High Purity

All three protocols successfully separated extracellular vesicles from cell‐free saliva samples. TEM confirmed the presence of EVs using all methods. Salivary EVs appeared scattered throughout the sample, exhibiting their characteristic morphology and a range of sizes. EVs obtained through UC protocol presented the larger vesicles (Figure 2a–c).

FIGURE 2.

FIGURE 2

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Characterization of salivary EVs by NTA. Representative transmission electron microscopy (TEM) images of EVs separated by UC (a), UC + PS (b), and PBP + UC (c) protocols exhibit characteristic morphology and differing sizes. Magnified morphology (100,000×) of typical EVs can be observed within the small square for each method (a–c). Histogram of separated EVs depicts distinct patterns of particle size distribution and concentration among the three methods, with the broadest EV distribution in UC method (d). NTA analysis demonstrated that UC + PS (yellow) and PBP + UC (blue) protocols yielded higher EV recovery than UC (red) (e). Regarding size, all methods showed greater concentrations of vesicles under 200 nm than those over 200 nm; nonetheless, UC methods exhibited the highest concentration of EVs exceeding 200 nm in comparison to UC + PS and PBP + UC (f). In agreement, UC method (red) separated the largest EVs (mean and median), while PBP + UC yielded the smallest (blue) (g). The total protein amount measured by the BCA assay displays a similar total amount among all methods (h). Similarly, the EV purity (particle number/protein ratio) was comparable across all methods (i). Data are presented as mean ± SEM and median with interquartile intervals (n = 5 per method), ****p < 0.0001, ***p = 0.0004, **p = 0.001, and ns = not significant.

The particle size distribution revealed differences in the concentration of specific sizes separated by UC + PS compared with PBP + UC (p = 0.004) and UC (p < 0.001). EVs separated through UC + PS showed a prominent peak at 115–150 nm, while PBP + UC EVs presented a significant peak at 97–130 nm. The UC method showed the broadest EV distribution, with two nanoparticle peaks (102–135 nm and 157–196 nm) (Figure 2d).

The average concentrations of salivary EVs separated by UC + PS and PBP + UC [1.36 × 1010 ± 9.0 × 108 and 1.38 × 1010 ± 8.5 × 108 particles/mL, respectively (p = 0.89)] were similar, but significantly higher than UC (1.31 × 1010 ± 6.3 × 108 particles/mL, p < 0.0001) (Figure 2e). According to NTA, all methods mainly recovered EVs smaller than 200 nm (p < 0.0001), showing comparable concentrations among all methods (p = 0.99). However, UC demonstrated significantly higher concentrations of EVs larger than 200 nm (7.9 × 109± 3.7 × 108 particles/mL) compared to UC + PS and PBP + UC [(6.1 × 109 ± 4.0 × 108 and 6.6 × 109 ± 4.7 × 108 particles/mL, respectively) (p < 0.001)] (Figure 2f).

The mean size of EVs separated by UC + PS and PBP + UC was comparable, measuring 193.7 ± 0.74 nm and 183.9 ± 2.08 nm (p = 0.66), respectively. These sizes were significantly smaller than those obtained through UC (237.1 ± 13.46 nm, p = 0.001). Furthermore, the median also demonstrated resembling measurement, with UC + PS and PBP + UC showing 176.3 nm and 173.0 nm, respectively, while UC had a median size of 226.3 nm (Figure 2g).

The total amount of protein tends to be higher in PBP + UC‐separated EVs than in UC + PS and UC (98.9 ± 26.2 μg vs. 61.5 ± 4.4 μg and 68.1 ± 4.9 μg, respectively). Still, no significant difference was found (p = 0.78) (Figure 2h). Additionally, when assessing the ratio of total particle count to total protein amount, all three methods exhibited similar purity, with UC + PS and PBP + UC being slightly higher than UC (Figure 2i).

3.2. All Methods Recover CD9 + and CD63 + Extracellular Vesicles

Western blotting analysis was performed to detect EV‐associated proteins CD9 and CD63, demonstrating the expression of both markers in EVs separated by all methods (Figure 3a), with comparable relative levels among all (p = 0.20) (Figure 3b). Interestingly, EV samples from all methods used showed more abundance of CD63 than CD9. Furthermore, the endoplasmic reticulum marker calnexin was evaluated in salivary EVs alongside a positive control (cell lysate from MDA‐MB‐468 cells) to exclude the possibility of co‐isolation of intracellular contaminants. As shown in Figure 3a, calnexin was not detected in the salivary EVs.

FIGURE 3.

FIGURE 3

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Western blotting. The expression of CD9 and CD63 markers was confirmed on salivary EVs separated by all protocols and verified with positive control of EVs from the cell line (MDA‐MB‐468). The absence of a calnexin signal was observed in EV samples compared to cell lysate control (a). The relative expression of tetraspanins in relation to GAPDH of EV samples was not significantly different between all methods (b). Data are presented as mean ± SEM (n = 3 per method). All experiments and quantification were conducted in triplicate, ns = not significant.

3.3. All EV Separation Methods Obtained RNA With Classical EV‐Like Patterns and High Quality to Amplify miRNAs

The RNA concentration was measured to assess the influence of EVs separation methods on RNA yield. The UC + PS protocol yielded the highest amount of RNA with a mean of 22.68 ± 3.9 ng/μL, followed by UC (15.66 ± 3.2 ng/μL) and PBP + UC (11.90 ± 1.6 ng/μL). However, this difference was not statistically significant (Figure 4a).

FIGURE 4.

FIGURE 4

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Total RNA and miRNA expression in salivary EVs. RNA concentration varied for each EV separation protocol without statistical significance (a). Representative bioanalyzer electropherograms of EVs show the total RNA size distribution and fluorescence intensity (FU). The absence of ribosomal RNA molecules is evident, and the peak observed at short RNA represents the typical RNA pattern of EVs (b–d). This distinctive pattern differentiates EVs from control cell lines (e). The qPCR results show C q values of three miRNAs from EV‐derived preparations obtained by UC (red), UC + PS (yellow), and PBP + UC (blue) (f). Data are presented as mean ± SEM (n = 5 per method), ns = not significant.

As shown in Figure 4b–d, the RNA electropherograms of salivary EVs showed short nucleotide sequences with variations in RNA quantity and quality between the different separation methods. Furthermore, we confirm the absence of 18S and 28S ribosomal RNA molecules, unlike the pattern observed in the cell line control, which exhibited high levels of ribosomal RNA (Figure 4e).

To demonstrate the utility of salivary EVs separated in assessing miRNA expression levels by RT‐qPCR and explore potential differences between the three protocols, we examined three miRNAs (miR‐16, miR‐27a, and miR‐99a) previously detected in EVs (Burdiel et al. 2023; Han et al. 2020; Gai et al. 2018; Gallo et al. 2012). As shown in Figure 4f, all protocols successfully amplify all miRNAs. As expected, miR‐16 showed higher levels among all samples with a mean C q value of 23.1, and the UC + PS protocol recovered higher quantities of miR‐16. The miR‐27a and miR‐99a were amplified afterward with a mean C q values of 27.4 and 33.7, respectively. Both miRNAs show similar quantities in EVs separated by UC + PS and PBP + UC (Figure 4f).

4. Discussion

This study evaluated three methods for separating EVs from whole saliva to determine their effectiveness in recovery and purity. All three methods successfully separated CD9+ and CD63+ extracellular vesicles, enriched in sizes < 200 nm. The UP+PS and PBP + UC protocols yielded the highest EV recovery with high purity, and they were found to be effective strategies for separating salivary EVs, resulting in high‐quality RNA for qPCR.

Despite few studies have evaluated the EV yield in the whole saliva of healthy donors, the concentrations observed in our study are consistent with the reported range of 6.1 × 108 to 6.6 × 1010 particles/mL (Reseco et al. 2024; Sjoqvist and Otake 2023; Jangholi et al. 2023; Hofmann et al. 2022; Li et al. 2021; Chaparro Padilla et al. 2020; Han et al. 2020; Aqrawi et al. 2017; Deregibus et al. 2016). Individual variations may account for the differences in concentration observed in different studies. Additionally, factors such as sampling method, preprocessing, and separation protocols also impact the yield and purity of EVs (Jangholi et al. 2023; Li et al. 2021; Deregibus et al. 2016). However, a standard limitation is the lack of detailed methodological information that hinders the reproducibility of salivary EV separation protocols.

Our study showed that the UC + PS and PBP + UC protocols yielded the highest EV recovery. The enhanced EV recovery obtained through UC + PS is contrary to the EV loss documented after the purification step (Lee et al. 2019; Webber and Clayton 2013). However, our findings are consistent with a recent study that demonstrated higher recovery and purity of salivary EVs using UC + PS compared to the UC protocol (Tengler et al. 2024). Interestingly, this approach has demonstrated even more efficacy than SEC, which showed a 67% lower yield for salivary EVs (Tengler et al. 2024). Although SEC has been reported as an effective separation method, particularly for plasma‐EVs, in saliva there are varying results and currently no consensus regarding the EV‐enriched fractions (Tengler et al. 2024; Jangholi et al. 2023).

On the other hand, PEG‐precipitation improves EV separation by reducing the solubility of EVs in the solution (Akbar et al. 2022). This can also co‐precipitate non‐vesicular extracellular particles (NEVPs), potentially leading to an overestimation of the EV concentration (Akbar et al. 2022; Li et al. 2021; García‐Romero et al. 2019; Rider, Hurwitz, and Meckes 2016). However, our study shows that applying UC after PBP leads to a higher yield of EVs from saliva with high purity (> 3 × 1010 particles/μg) (Webber and Clayton 2013), confirming the findings previously observed in other samples (Ludwig et al. 2018; Rider, Hurwitz, and Meckes 2016). These results suggest that, despite the ubiquitous impurities in PEG isolates, the PBP + UC protocol effectively reduces NEVPs that could affect downstream applications.

Although all three methods in this study demonstrated significant enrichment in EVs smaller than 200 nm, they also recovered larger vesicles. However, EV separation by UC protocol had the highest concentration of vesicles larger than 200 nm. These findings are in agreement with previous studies that reported salivary EVs with diameters ranging from 45 to 309 nm (Jangholi et al. 2023; Sjoqvist and Otake 2023; Hofmann et al. 2022; Li et al. 2021; Chaparro Padilla et al. 2020; Aqrawi et al. 2017; Deregibus et al. 2016), indicating the presence of diverse EVs and both small and large EV populations in saliva (Ogawa et al. 2024, 2011).

The analysis of the specific EV markers CD9+ and CD63 showed successful EV recovery across all three methods. These tetraspanins are known to be involved in protein trafficking (Dixson et al. 2023; Hessvik and Llorente 2018; Théry et al. 2018; Hurwitz et al. 2016; Yáñez‐Mó et al. 2015). All samples displayed prominent CD63 bands, which aligns with previous findings on salivary EVs from both healthy donors and individuals with diverse health conditions (Hofmann et al. 2022; Han et al. 2020; Zlotogorski‐Hurvitz et al. 2015; Gallo et al. 2012). The levels of these markers may vary owing to the biochemical individuality of salivary composition; however, some authors have proposed that the abundance of CD63 in salivary EVs indicates an immune‐associated function in the oral mucosa (Reseco et al. 2024; Ogawa, Tsujimoto, and Yanoshita 2016). Additionally, the effectiveness of EV purification depends on proper decontamination during preprocessing and separation protocol (Li et al. 2021). The absence of calnexin protein in salivary EV samples indicates the successful removal of contaminants, such as apoptotic bodies and microsomes, during saliva sample collection. Calnexin is a membrane‐bound protein of the endoplasmic reticulum (ER), highly expressed in cell lysate, and a recommended marker of ER content in EVs (Welsh et al. 2024). Even though calnexin can be slightly present in large EVs, its absence is used to confirm the removal of contaminants because its presence in EVs may primarily result from cell destruction prior to centrifugation (Mebarek et al. 2023; Lischnig et al. 2022; Mathieu et al. 2021). According to this, recent research on quantitative proteomics of small and large EVs showed slightly detectable amounts of calnexin. Still, its presence is not a differential marker to distinguish large VEs (Lischnig et al. 2022).

While our study achieved an average RNA concentration above the reported range for EVs in saliva (1.0–4.0 ng/μL) and serum (2.4–12 ng/μL) of healthy individuals (Cross et al. 2023; Kumar et al. 2020), the variation in methodological RNA extraction across studies poses challenges for comparative analysis. In agreement with previous studies, our salivary EVs separated using all methods were devoid of whole‐cell ribosomal RNA and displayed peaks of short RNA species on the electropherogram (Ogawa, Tsujimoto, and Yanoshita 2016; Lässer et al. 2011).

In our research, salivary EV separated by all methods exhibited successful amplification of three selected miRNAs, indicating the high‐quality RNA for assessing miRNA expression through qPCR. Specifically, we observed a robust amplification of miR‐16 with C q mean values below 25 indicating its high abundance in all samples. Although there is limited information about miRNA expression in saliva samples from healthy donors, miR‐16 is one of the most reported in EVs. It has even been used as an endogenous control for miRNA expression assays of EVs, including saliva (Burdiel et al. 2023; Coon, Kingsley, and Howard 2020). Conversely, miR‐27a and miR‐99a showed slightly higher C q values, with mean around 29.5 and 31.1, respectively, consistent with their reported expression levels in salivary EVs (Gai et al. 2018). These variations in C q values may reflect differences in the abundance of specific RNA sequences among samples.

In conclusion, our findings demonstrate that both UC + PS and PBP + UC protocols achieve similar efficiency in separating highly pure EVs from saliva, which make them suitable for downstream RT‐qPCR analysis. However, the PBP + UC protocol represents a cost‐effective option with a significant advantage: the initial phase is readily applicable in clinical settings, where rapid sample processing is crucial, and precipitation does not require sophisticated equipment. This approach could optimize UC protocol and reduce the time needed for UC + PS. Nevertheless, it is essential to emphasize that further validation is necessary to confirm the protocol's efficacy for transcriptomic or proteomic analyses, highlighting the need for future research in this area.

Author Contributions

Castillejos‐García Itzel: conceptualization, investigation, methodology, visualization, writing – original draft, writing – review and editing, formal analysis, funding acquisition. Martínez‐Martínez Eduardo: investigation, funding acquisition, writing – original draft, writing – review and editing, validation, methodology, formal analysis. Ramírez‐Amador Velia: conceptualization, investigation, funding acquisition, writing – review and editing, formal analysis. Cisneros‐Villanueva Mireya: writing – original draft, methodology, validation, visualization, writing – review and editing. Hidalgo‐Miranda Alfredo: conceptualization, investigation, methodology, validation, funding acquisition, writing – original draft, writing – review and editing, project administration, supervision, resources. Ramos‐Godínez María del Pilar: methodology, validation, visualization, writing – review and editing, supervision. Anaya‐Saavedra Gabriela: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, methodology, formal analysis, project administration, supervision, resources, data curation.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

During the study, ICG received a fellowship from CONACYT‐Mexico (517377) as a Ph.D. student of the Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Mexico.

Funding: This research was funded by Consejo Nacional de Humanidades, Ciencia y Tecnología (64382; FORDECYT‐PRONACES), Secretaria de Salud (FPIS2024‐INMEGEN‐6940) to E.M.‐M. and the Academic Support for Research Strengthening from the Rector of Universidad Autónoma Metropolitana, Campus Xochimilco.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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