Cell culture medium (RPMI-1640), fetal bovine serum, and antibiotics [antibiotic-antimycotic solution] were purchased from Biosera (Nuaille, France). Phorbol 12-myristate 13-acetate (PMA), ascorbic acid, and polyethylene glycol 6,000 and 4-pyridyl-1-oxide-N-tert-butylnitrone (POBN) were obtained from Sigma Aldrich (St. Louis, Missouri, United States of America). Rabbit polyclonal anti-malondialdehyde (MDA) antibody and anti-NADPH oxidase 4 (anti-NOX4), were purchased from Abcam (Cambridge, United Kingdom) and HRP-conjugated goat anti-rabbit antibody was purchased from BioRad. CD63 monoclonal antibodies, NOX2 polyclonal antibody, and secondary antibody (HRP-conjugated goat anti-mouse) were obtained from Proteintech (GmbH Germany). Protease and phosphatase inhibitors were purchased from Roche (Mannheim, Germany) and Protein A-Agarose (sc-2001) was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Please refer to the Supplementary Material for a catalog of reagents and clones of antibodies.

U-937 and THP-1 cells were purchased from the American Type Culture Collection (ATCC; Rockville, Maryland, United States) and cultured in RPMI 1640 medium containing 1% antibiotic v/v and 10% fetal bovine serum (FBS). To avoid contamination of EVs present in FBS, EV-depleted FBS was used. Using a 0.2 μm polyether sulfone membrane syringe filter (VWR International, Puerto Rico, United States of America), particles larger than 0.2 μm were removed (Shelke et al., 2014), and the filtrate was collected and measured (Shu et al., 2021) (Figure 1). Furthermore, to get rid of the smaller component <0.2 μm (Figure 1A), centrifugation was performed at 21,000 × g for 3 h and the supernatant was collected. Following this step, the obtained FBS was used for further cell culture.

From the media containing cultured cells (U-937) in the log phase (5 days after cell passage) and with viability ∼90–95%, the cell products were harvested and subjected to centrifugation at 300 g for 10 min at 4°C for removal of cells from the harvested media. The supernatant was collected and filtered through a 0.2 µm polyether sulfone membrane syringe filter and then centrifuged at 21,000 × g for 3 h at 4°C. After this, the supernatants were discarded, and the pellet was washed again at 21,000 × g (2X, 3 h) with ice-cold phosphate buffer saline (PBS, 1X). Subsequently, the pellet was suspended in 250 µL of HPLC grade water. The isolated EVs were stored at -20°C until further processing. The same procedure was followed for THP-1 cells.

The medium containing cultured cells and cell products was harvested and then centrifugation was carried out at 300 × g for 10 min at 4°C for cell removal from the harvested medium. The supernatant was collected and filtered through 0.2 µm polyether sulfone membrane syringe filter. 50% of the PEG6000 solution was prepared in d/w, after which the filtrate was mixed with the PEG: filtrate (1: 2.5) followed by a short vortex and incubated at 4°C overnight to form aggregates. After overnight incubation, the PEG mixture was centrifuged at 13,000 g for 60 min at 4°C. The pellet was then washed twice with ice-cold PBS at 13,000×g. The collected pellet was stored at -20°C until further processing.

To determine the size distribution of isolated particles in the suspension, DLS measurements were performed using a Malvern Zetasizer RED Pro using a detector angle of 173° (backscatter) at a temperature of 25°C. For sampling, 25 μL of isolated vesicles were diluted in 975 mL of d/w and particle size analysis was carried out for both EVs isolated using centrifugation (as in Section 2.3) and PEG enrichment (as in Section 2.4). Control measurements were performed using EV -free FBS. To confirm that our isolate contains an EV, we used a transmission electron microscope (TEM) to visualize the particles (data not shown).

Based on the results obtained from the basic characterization of the isolated EVs, since potential aggregation was observed in EVs prepared using PEG enrichment (Figure 1C), further studies were carried out using EVs isolated using centrifugation. For protein isolation, EVs (in d/w) were sonicated (40% amplitude; 1 cycle for 30 s) in ice-cold Radioimmunoprecipitation Assay (RIPA) buffer [150 mM NaCl, 50 mM Tris (pH 8.0), 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40] containing 1% protease inhibitor and phosphatase inhibitor. Sonication was repeated 6 times with a 30 s gap before the next sonication. EVs were kept on ice after each cycle of sonication. Following this step, centrifugation at 18,400 × g was done for 30 min at 4°C to remove cell debris and to obtain proteins in the supernatant.

Estimation of protein was done using Pierce bicinchoninic (BCA) protein estimation kit (Thermo Fisher Scientific, Paisley, United Kingdom). The measurement procedure used is as outlined in the manufacturer’s guidelines with minor modifications as described in our previous study (Manoharan et al., 2023; Prasad et al., 2023).

Immunoblotting was performed with CD63 surface markers on EVs. The samples were prepared with 5 × Laemmli sample buffer along with 100 mM Dithiothreitol (DTT) and a protein concentration of 10 μg was used for electrophoresis. Prior to loading the samples, the protein samples were incubated at 70°C for 10 min in a dry bath. Subsequently, the sample was loaded onto 10% Tricine SDS-PAGE. The proteins were then transferred to either PVDF membrane (Bio-Rad, California, United States of America) which was charged prior with methanol or nitrocellulose membrane. The protein transfer to the membrane was achieved using the Trans-Blot Turbo transfer system (Bio-Rad, California, United States). Following the transfer, to ensure that the loading of protein samples is uniform (loading control), Ponceau staining (0.1% Ponceau S: 1% acetic acid, w: v; diluted to 100 mL d/w) was done. After washing, the membrane was blocked with 5% BSA in Tris-buffered saline (TBS) (pH 7.4) and 0.1% Tween 20 (referred to as TBST) for 90 min at room temperature (RT). After blocking, washing with TBST (3x) was done for 10 min each. The blocked membranes were then incubated for 90 min at RT with anti-CD63 mouse monoclonal antibody (dilution 1: 5000) followed by 3x washing (10 min each) with TBST. Subsequently, the membrane was incubated for another 90 min at RT with HRP-conjugated goat anti-mouse secondary antibody (dilution 1:10,000) and washed with TBST (3x, 10 min each). Immunocomplexes were imaged using the Amersham imager 600 (GE Healthcare, United Kingdom) and Immobilon Western Chemiluminescent HRP Substrate (Sigma Aldrich, GmbH, Germany).

Co-culture experimental setups were made. For the setting of the plates, U-937 and THP-1 controls with differentiating agents [250 nM or 150 nM PMA and ascorbic acid (5 μΜ and 10 μΜ)] were prepared. For the experimental sample, cells (1×10⁵/mL) under the above conditions (PMA or ascorbic acid) were cultured in the presence of exogenous EVs (20 μL, the same isolates were used for all experiments within the setup). Cells were treated with PMA/ascorbic acid for 72 h (for monocyte to macrophage differentiation) in a 6 well plate and isolated EVs (section 2.3) were added at the start of the treatment. Cell viability at 24h and 72 h were monitored using the trypan blue test to follow cell proliferation and effect of EVs and/or differentiating agents. For protein isolation, 5 mL of non-treated and treated cells were used, and quantification was done based on the method described in Section 2.6. Following quantification, 10 μg protein sample/lane was used for immunoblotting.

Immunoblotting was performed against NOX4 and NOX2. Until the blocking of membranes, the procedure as described in Section 2.8 was followed. The blocked membranes were then incubated for 90 min at room temperature (RT) with either anti-NOX4 rabbit monoclonal antibody (dilution 1:5000) or anti-NOX2 mice polyclonal antibody followed by 3x washing (10 min each) with TBST. Subsequently, the membranes were incubated for another 90 min at RT with HRP-conjugated goat anti-rabbit secondary antibody and HRP-conjugated goat anti-mice secondary antibody, respectively (dilution 1:10,000) and washed with TBST (3x, 10 min each). Immunocomplexes were imaged using the procedures described in Section 2.7.

To confirm MDA-protein adduct formation, nitrocellulose membranes transferred with MDA adduct proteins were incubated with rabbit polyclonal anti-malondialdehyde antibody prepared at a dilution of 1:5000 in TBST and incubated for 90 min at RT. Following three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibody (1:10,000) for 90 min at RT. The blots were imaged as described above.

Chemically generated HO^• using Fenton reagent was detected by spin-trapping with 10 mM POBN (4-pyridyl-1-oxide-N-tert-butylnitrone) containing 170 mM ethanol. The measurements were performed using an electron paramagnetic resonance spectrometer (MiniScope MS400, Magnettech GmbH, Berlin, Germany) (Pou et al., 1994). Experiments were carried out in the absence and presence of different concentrations of EVs isolated from U-937 cells resuspended in HPLC grade water. EPR spectra were recorded under the following parameters: microwave power (10 mW), modulation amplitude (1 G), modulation frequency (100 kHz), sweep width (100 G), scan rate (1.62 G s^–1).

Using the dynamic light scattering method, we measured the mean Z-average (mean hydrodynamic size) of the sample obtained by centrifugation from U-937 cells. For the control (filtered and centrifuged FBS), the Z-average was recorded to be 15.7 nm. More than one peak with a polydispersity index of 0.43 was observed (Figure 1A; Table 1). It can be seen that in the sample isolated by centrifugation based on the method described by Shelke and co-workers (with minor modifications) (Shelke et al., 2014), a Z-average of ∼154.8 nm was observed with a standard deviation of about 15.2 nm and a polydispersity index of 0.30 (Figure 1B; Table 1). The presence of multiple peaks in the case of PEG samples can be attributed to the centrifugation, aggregation, and orientation of the vesicle (Figure 1C) (Genneback et al., 2013). As the sample acquired by the centrifugation method includes EVs falling within the 30–200 nm range and exhibits the presence of the CD63 marker, it is a likelihood that the isolates may be classified as exosome particles. However, we are uncertain due to a lack of understanding about their origin.

In order to confirm the presence of EVs in the isolated fractions, Western blotting was performed to identify the expression of exosomal protein markers. CD63 is a 30–60 kDa lysosomal membrane protein that is composed of four alpha-helical transmembrane domains with two extracellular loops (Jung et al., 2006; Gonzalez et al., 2014). Both the N- and the C-terminus point toward the inside of EVs. In EVs, several tetraspanins, especially CD63, CD81, and CD9, have been used as markers of EVs for the last 2 decades. In our study, endogenous CD63 was monitored as a surface marker for EVs (Kim et al., 2019; Mathieu et al., 2021). Proteins were extracted using the procedure outlined in Section 2.6 from EVs obtained through centrifugation. Our immunoblotting analysis revealed a single band with a molecular weight of approximately 60 kDa (Figure 2 and Supplementary Data S2) in U-937 and THP-1 cells. The band at 60 kDa is most likely due to post-translational modifications (glycosylation of CD63 protein) (Engering et al., 2003). Bands at different positions in the range 30 kDa–85 kDa have been reported which can be due to multiple reasons such as but not limited to post-translational modifications and proteolytic cleavage, and other experimental factors (Engering et al., 2003).

Cells were treated with differentiation inducers in the absence and presence of EVs (Figure 3). In order to evaluate whether the addition of differentiation inducers led to a change in cell viability of U-937 cells, the trypan blue test was performed in all experimental samples at 24 h and 72 h, including those treated with EVs. In the control (no differentiation agent + no EVs), a viability percentage of 90% and 80% were observed at 24 h and 72 h, respectively. In PMA-differentiated specimens, the recorded viability was 86.5% at 24 h and increased to 95% at 72 h. On the contrary, in samples treated with ascorbic acid, viability remained consistently in the same range, exceeding 80% at various concentrations (5 μM and 10 μM). In EVs treated samples differentiated using PMA, 88% viability was observed at 24 h and 94% at 72 h was recorded. Similarly, in EVs treated samples differentiated using ascorbic acid, it was recorded to be in the same range and maintained above 80% at different concentrations (5 μM and 10 μM); the details have been presented in the form of Table (Supplementary Data S3). On the basis of this dataset, it can be concluded that neither exogenous addition of differentiation agents (either PMA or ascorbic acid) nor supplementation of EVs led to any stress leading to alterations in cell proliferation and viability.

Based on immunoblotting using NOX4 antibody, we monitored its expression at 67 kDa. In a non-differentiated control, a visible expression of NOX4 can be observed, which is typical for monocytes, as monocytes are well known to express this isoform. In differentiated controls [either PMA or ascorbic acid (5 μM and 10 μM)], the expression of NOX4 is slightly suppressed in all cases except for 10 µM ascorbic acid, which can be hypothesized based on the fact that NOX4 expression is not a characteristic of macrophages. However, under the condition where the EVs are present along with cells, no influence on NOX4 expression is evident compared to the control. Interestingly, in differentiated cells supplemented with EVs for 72 h, a strong suppression of NOX4 expression is evident (especially in ascorbic acid differentiated cells and U-937 cells), which can be hypothesized that when monocytes are transformed to macrophages, NOX4 expression might have been regulated by microRNA (miRNA) (Figure 4). Interestingly, NOX2 expression was found to be only suppressed in macrophages co-cultured with EVs, predominantly in U-937, while in THP-1 cells, no significant suppression was observed (Figure 5). In monocytes co-cultured with EV, an increase in NOX2 expression was observed [Figure 5A (lane c) and Figure 5B (lane c)].

In THP-1 cells, we validated our findings using immunoprecipitation of NOX2 using NOX2 antibody and protein A agarose beads. NOX2 expression was found to be significantly suppressed in EVs co-cultured macrophages (Supplementary Data S7). We also observed a decrease in overall MDA formation in EVs treated macrophages (Figure 6), which agrees with previous reports (Song et al., 2015; Song et al., 2021).

We measured the suppression of ROS under the exogenous addition of EVs on chemically generated HO^•. The intensity of the EPR signal was observed in the control samples (Figure 7A). It can be seen that in the sample that did not contain a Fenton reagent (negative control), a relative signal intensity of about ∼800 was observed. Chemically generated HO^• using Fenton reagent (0.1 μM FeSO₄ and 2 mΜ H₂O₂) led to a high signal intensity reflecting the formation of α-hydroxyethyl radical adduct of POBN [POBN-CH(CH₃)OH adduct] (Figure 7A). The intensity of the EPR signal was found to be significantly and linearly suppressed with the exogenous addition of EVs in a dose-dependent manner (Figures 7B, C). The addition of EVs suppressed the signal by up to almost 90%, indicating the antioxidant capacity of the EVs. EVs specifically exosomes mitigate oxidative stress in recipient cells by directly delivering the enzymatic antioxidant [GSH, superoxidedismutase1 (SOD1), thioredoxinreductase1 (TrxR1), methioredoxin reductase (TrxR2) and glutathione peroxidase, among others] or antioxidative enzyme mRNA that later translates aiding to prevention of oxidative stress (Yan et al., 2017; Lin et al., 2022). EVs therapy therefore can be contemplated as an emerging and promising area of regenerative medicine (Muthu et al., 2021; Thakur et al., 2022). EVs through the transfer of cargo to recipient cells can influence cellular functions and therefore, exosomes isolated from different sources can specifically be chosen to target different therapeutic applications. It has been applied in regenerative medicine to promote tissue repairs, in neurological disorders such as Alzheimer’s and Parkinson’s disease, in cancer therapy, among others (Gao et al., 2021; Zhang et al., 2023).