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In the present work, B. jararaca EVs (Bj-EVs) were purified from fresh crude venom. Several conserved EV markers and many types of processing enzymes were identified by proteomic analysis. Using fluorescence and electron microscopy, we demonstrated that mammalian cells uptake Bj-EVs, which establishes a basis for discovering new mechanisms of interaction between venoms and recipient cells, further indicating the potential of cross-organism communication. Based on these findings, we hypothesize that venom-EVs may play a role in toxin processing and stability in the venom gland.
3. DiscussionDespite the extensive studies of B. jararaca snake venom in recent decades, which revealed several intraspecific differences in terms of composition and biological activities [13,14], this is the first report of extracellular vesicles in B. jararaca snake venom.Previous works using size exclusion chromatography, one-step ultracentrifugation, or magnetic beads for EV isolation reported a single fraction of EVs from other snake venoms [29,30,31,32,33]. In this study, two EV populations were isolated from B. jararaca venom by differential ultracentrifugation (Figure S1). Together, morphologic (Figure 1) and proteomic data (Figure 2, Figure 3 and Figure S4) support the existence of two different EV populations, P20K and P100K. Such heterogeneity is expected in EVs isolated from biological fluids [35]. Many EV types have been discovered recently, such as ARMMS, migrasomes, and mitovesicles [69,70,71], as well as extracellular nanoparticles (which are membranelles), such as exomeres and supermeres [72,73]. Among them, microvesicles and exosomes are well described in the literature and classified according to their cellular origin. Microvesicles are released directly from the plasma membrane and have a size range of 100–10,000 nm. Multivesicular bodies (MVBs) are members of the endosomal system and have many intraluminal vesicles (ILVs) ranging from ~30–150 nm. When MVBs fuse with the plasma membrane, exosomes are released into the extracellular space [34,38].Souza-Imberg et al. observed small vesicles in the lumen of the venom gland of Crotalus durissus terrificus, budding directly from the plasma membrane of secretory cells and in the collected fresh venom after one-step centrifugation [31]. Although exosomes could not be excluded from the EV pool, the observation of budding vesicles in the C. d. terrificus venom gland strongly indicates the presence of microvesicles in this venom. In the case of Bj-EVs, the biogenesis was hypothesized based on the identification of some EV markers in these fractions (Table S1). EV markers are recurrently identified proteins enriched in certain types of EVs compared with their parental cells. The EV markers can be either membrane or cytosolic proteins that are specifically loaded in EVs during their biogenesis [34].Among the EV markers identified in Bj-EVs, syntenin-1 is a previously demonstrated exosome or small EV biomarker [74,75,76], which plays an important role in EV cargo sorting and membrane budding by interacting with syndecans and ALIX [76,77]. The chromatin modifier protein 5 (CHMP5)/Vsp60 is responsible for membrane scission events together with Vsp4 and ALIX ESCRT-III-associated proteins. Syntenin-1 and ALIX were identified in both Bj-EV fractions, while CHMP5 was detected only in P100K. In parallel, the microvesicle-enriched protein Annexin A1 was identified in the P20K fraction [38,78]. The proteomic identification of EV markers and the morphological characterization presented here indicate that P100K is likely a small EV-enriched fraction, while P20K is a more heterogeneous EV population, composed, at least in part, by microvesicles.In the present work, we classified some EV-enriched proteins found in B. jararaca and other snake venoms as venom-related EV markers, such as 5′-nucleotidase (5NTD), dipeptidyl-peptidase IV, phosphodiesterase, and aminopeptidases (Table 2). These proteins were already known as low abundant toxins in snake venoms and were previously described in crude venom proteomes and venom gland transcriptomes [79,80,81,82]. In addition, most of them have a membrane-protein topology [58,83] and were reported in other snake venom EVs [30,31,33]. The biological function of these toxins is controversial. For instance, a few 5NTDs and PDEs were isolated from snake venoms and showed blood clotting and platelet aggregation inhibitory activity [84,85,86,87,88,89]. Hypotheses supporting their hypotensive effects were also published [90,91,92,93]. On the other hand, orthologous proteins of the dipeptidases, aminopeptidases, dipeptidyl peptidase, and serine carboxypeptidase (Table 2) have a consolidated biological role as processing enzymes [94,95,96,97].Based on the identified venom-processing EV markers, we hypothesize that Bj-EVs may be involved in processing venom components and/or processing prey proteins. Ogawa et al. isolated EVs from Gloydius b. blomhoffi snake venom and demonstrated that those EVs had processing activity on physiological oligopeptides, i.e., angiotensin II, glucose-dependent insulinotropic peptide, and glucagon-like peptide-1 [30]. These results indicated the possible involvement of EVs in disturbing physiological processes after envenomation, such as blood pressure and glucose homeostasis imbalance [30]. Additionally, snake venom EVs may be involved in toxin processing and maturation inside the venom gland.In viperid snakes, venom is stored for long periods in the extracellular space, in the large venom gland basal lumen [98,99]. Several toxins lose their prodomain and undergo maturation and post-translational modifications after reaching the lumen of the venom gland. Typically, SVSPs and SVMPs are secreted as zymogens [100,101,102]. The bradykinin-potentiating and C-type natriuretic peptides (BPP-CNP) are toxins expressed as a large precursor cleaved in many mature peptides [103,104,105]. Other toxins, such as B. jararaca disintegrins, have proteoforms identified in the crude venom in which the N or C-terminus amino acids are missing, with the unprocessed and processed forms coexisting in the toxin pool [14,106,107]. Portes-Junior et al. observed that the toxin jararhagin, an SVMP from B. jararaca venom, is secreted as a zymogen. Its prodomain is significantly cleaved only when the toxin reaches the lumen of the venom gland [108]. In another study with primary cultures of secretory cells, SVMPs were identified in their zymogen form in the culture medium, in opposition to what is observed in natural venom. These findings indicated that the processing molecules are not supplied by the same toxin-producing cells, supporting the assumption that processing occurs after protein secretion in the lumen of the venom gland [109]. However, the underlying mechanisms and the enzymes involved are not clear. Thus, based on the proteins found enriched in Bj-EVs (Table 2), i.e., aminopeptidases, dipeptidases, and carboxypeptidases, we propose that Bj-EV proteins may be involved in toxin processing (post-translational modification, zymogen cleaving, and toxin maturation) and overall venom stability (presence of isomerases and PLA2 inhibitor).The 5NTD is the most abundant protein family in Bj-EVs. Many 5NTDs from snake venoms are homologous to the ecto-5′-nucleotidases, also known as CD73, which carry a GPI-anchor that attaches proteins to membranes [88,110,111,112]. The first report of EVs in the literature described high 5′-nucleotidase activity, and the authors hypothesized a role in the dephosphorylation of membrane constituents of recipient cells [113]. Since then, CD73 has been described as enriched in many other EVs, for example, the EVs from immune and tumor cells that play a role in immune suppression [114,115,116,117].In many types of cells from diverse organisms, CD73 works with other ecto-nucleotidases, including CD39 (NTPdases) and PDEs (NPPs), in extracellular purinergic signaling. CD39 and PDEs release AMP from ATP and ADP; subsequently, CD73 releases free adenosine (Ado) from 5′-AMP [115,116]. In the immune system of mammals, Ado acts through adenosine receptors, inducing immune suppression by blocking the activation and proliferation of lymphocytes and neutrophils while impairing cytokine secretion in macrophages [117,118,119,120]. Furthermore, the super expression of CD39 and CD73 in cancer cells promotes immune escape, tumor survival, and metastasis [114,116,121,122,123,124]. In this context, these nucleotidases have been considered pharmacological targets for cancer treatment [112,115,120,125].In the case of snake venoms, 5NTD may be acting synergistically with another enriched Bj-EV protein, the PDE, generating Ado in the venom and/or in the blood circulation of the envenomed organism. Some authors proposed the existence of purinergic hypotension caused by snake venoms [90,91,92,93]. In this perspective, Ado would act as an acute hypotensive molecule. However, these mechanisms were not demonstrated experimentally or with the involvement of EVs.The Ado pharmacological effects differ based on the purinergic receptors type and in which cell lineage they occur. Ado can cause vasodilation via endothelial A2A and A2B receptors, increase vascular permeability by mast cell activation (A3), provoke central sedative effects via neuronal A1 receptors, and bradycardia through A1 receptor, which together can contribute to hypotension and paralysis [90,126,127]. Considering the aspect of vascular permeability, Bj-EVs carrying 5NTD could contribute to venom spreading, since soluble 5NTD from snake venom causes vascular leakage in vivo [128]. On the other hand, Ado is immunosuppressive when immune cells express P1 receptors [117,118,119,120,129], which may be related to the long-term effects of snakebites [24,25,26,27,28]. In accordance with this, a strong immune suppressive function is well characterized in EVs from wasp venoms [48,54,55].5NTD is ubiquitous in snake venoms [79,91], is a minor toxin fraction observed in venom proteomes [14,130,131], is enriched in snake venom EVs (as demonstrated here and in previous works [30,31,33]), and is usually predicted to have a GPI-anchor. Based on this knowledge and in our results, we suggest that a fraction of 5NTD is structurally related to EVs. Furthermore, we speculate that EVs may be part of all snake venoms. The presence of free nucleosides and nucleotides in high abundance in snake venoms (which can exceed 8% of their dry weight) corroborates these claims [3,90,91,132]. EVs have previously been isolated from venoms of other taxons, such as wasp and spider venoms, indicating their presence in this type of biological fluid.Lipophilic dyes have been extensively used for EV labeling for in vitro and in vivo uptake assays [133,134,135]. In this study, fluorescence microscopy and flow cytometry revealed that P20K vesicles are internalized by muscle cells and macrophages. Interestingly, the pre-incubation of P20K with ABS reduced the cellular uptake of EVs (Figure 6I). The ABS is a polyclonal serum obtained after immunizing horses with a mixture of crude venoms collected from five Bothrops snakes: B. jararaca (50%), B. jararacussu (12.5%), B. moojeni (12.5%), B. alternatus (12.5%), and B. neuwiedi (12.5%). ABS was shown to react and neutralize the main Bothrops snake venom toxins [14,118,119]. Our results suggest that specific EV surface proteins are important for internalization and that the ABS may partially protect the cells from EVs. As expected, Western blotting showed that the ABS recognized the entire CV protein profile. In contrast, Bj-EV proteins were partially recognized (Figure S8A), which may be associated with the low abundance of Bj-EVs in the venom. In addition, the ABS recognized differentially the Bj-EVs obtained from distinct pools of animals (Figure S8B). Similarly to what is known for crude snake venoms, which show individual intraspecific variability, especially in B. jararaca [14,136,137,138], these results indicate that the protein composition of Bj-EVs varies with each animal/venom sampling.Even though macrophages were expected to uptake more vesicles due to their phagocytosis function, the ABS treatment blocked P20K uptake by muscle cells more efficiently than macrophages, inhibiting 72% and 49% of the EV internalization, respectively. This result indicates that distinct EV surface proteins could be required for EV uptake by different cell lines.
In electron microscopy, after 24 h of cell treatment with both P20K and P100K, cytosol vacuolization and disorganization, possible mitochondrial alterations, and abnormal chromatin condensation were observed. In addition, using fluorescence microscopy, we observed a change in the actin filament distribution, mainly in macrophages. Similarly, HUVEC cells treated with spider venom EVs also exhibited actin disorganization [50]. Whether these alterations contribute to the development of envenoming clinical effects is still an open question.Macrophages are cells with high phagocytic ability. In most cases, these cells uptake circulating vesicles when administered in vivo [134,139,140]. The parasitoid wasp venom suppresses the Drosophila immune system, facilitating wasp offspring development and survival [48,53,141,142]. Notably, the macrophage-like immune cells of Drosophila internalize the wasp venom EVs that circulate in the hemolymph, which leads to the destruction of the phagolysosomal compartments and cause their death by apoptosis [54,55]. Based on the CD73+PDE+ phenotype found in Bj-EVs, we expect that the vesicles can modulate macrophages and other immune cells in vivo. The same may happen for muscle cells, as the cellular alterations observed in the A7R5 aortic smooth muscle cell line may also represent a possible participation of this tissue in the pathophysiology of the envenoming. Viperid venoms affect the endothelial cells and the integrity of blood vessels causing edema, hemorrhage, and extravasation of plasma to the extravascular space [16,17,143,144]. These effects are due to direct cellular damage, increased vascular permeability, and basement membrane degradation. Alongside this, myotoxicity and myonecrosis are relevant aspects of a viperid snakebite [17,22]. Accordingly, the skeletal muscle tissue may also be a target of Bj-EVs. These results, together with the insights regarding the interaction of Bj-EVs with mammalian targets in vivo, will certainly provide a more complete picture of animal response to envenoming.Snakebite envenoming poses a threat to life as it can cause an acute inflammatory response, ranging from debilitation to death. The possible effects of snake venom EVs on the prey/victim immune system and other tissues may be linked to the less frequent cases of late effects and chronic manifestations. The enrichment of RNA-binding proteins in Bj-EVs, such as Y-box-binding protein 1, polyadenylate-binding protein, regulator of nonsense transcripts 1, and the heterogeneous nuclear ribonucleoprotein Q (Tables S1, Supplementary Data S1 and S2), supports the assumption that they also contain nucleic acids [145,146]. In fact, mRNAs have been unexpectedly found in snake venoms [147,148,149]. The occurrence of mRNA in EVs could explain how such labile molecules remain undegraded in venoms. Since EV RNAs from other organisms have been shown to reprogram the recipient cell phenotype [150,151], we will investigate Bj-EV RNAs and how they may contribute to the understanding of envenoming.We have proposed some varied possible biological functions related to Bj-EVs. They are all reasonable, as venoms have evolved to be multifunctional and are able to have several molecular targets at the same time [152]. Additionally, snake venom EVs may be important to protect their cargo from proteases, which requires further investigation. The present results lay a foundation for further studies on the biological effects of snake venom EVs. 5. Materials and Methods 5.1. Bothrops jararaca VenomAdult specimens of Bothrops jararaca maintained at the serpentarium of Instituto Vital Brazil (Niterói, Rio de Janeiro, Brazil) were previously anesthetized by CO2 inhalation. Fresh venom was manually extracted and pooled. Each extraction group contained at least 11 specimens including males and females, all native to the southeastern region of Brazil. Fresh venom pools were kept at 4 °C until extracellular vesicle isolation.
5.2. Extracellular Vesicles IsolationAfter being centrifuged at 8,000× g for 25 min for cell debris removal, the venom was diluted in sterile 0.1 mM citrate buffer pH 5.0 containing 100 U/mL penicillin and 100 µg/mL streptomycin and was further centrifuged at 20,000× g for 25 min. The first EV pellet was collected (P20K) and the supernatant was centrifuged at 100,000× g for 2 h. The second EV pellet (P100K) was obtained and the P100K supernatant (SP100K) corresponded to the venom depleted of vesicles. The P20K and P100K samples were then washed in citrate buffer using the respective centrifugation speeds. The samples were considered clean from extravesicular proteins when the buffer absorbances at 280 nm reached near 0 values. After washing, EVs were resuspended in a new buffer, immediately frozen in nitrogen, and stored at −20 °C until use. The protein contents of the crude venom and venom fractions were quantified using a Qubit protein assay kit (Thermo Fisher Scientific, Waltham, MA USA ) (Figure S1). 5.3. Transmission Electron Microscopy (TEM)P20K and P100K were washed twice with PBS pH 7.2 and fixed in 2.5% glutaraldehyde, 0.1 M sodium cacodylate buffer pH 7.2 for at least 1 h. After fixation, vesicles were washed in cacodylate buffer pH 7.2 and postfixed in a solution containing 1% osmium tetroxide, 1.25% potassium ferrocyanide, and 5 mM calcium chloride in 0.1 M cacodylate buffer for 30 min at room temperature in the dark. Next, pellets were washed in the same buffer, dehydrated in increasing acetone concentrations, and embedded in Epoxy resin. The first steps of fixation, post-fixation, and washes of P100K were conducted in a micro ultracentrifuge (Optima MAX-XP, TLA 55 rotor, Beckman Coulter) at 100,000× g due to the low density of the vesicle population. Next, ultrathin sections were obtained in an ultramicrotome (RMC Boeckeler, Tucson, AZ, USA), stained with 5% uranyl acetate and lead citrate solution.
For negative staining, isolated vesicles were directly resuspended in the fixation solution (2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2), absorbed for 2 min in formvar/carbon-coated copper grids, and stained in 5% uranyl acetate for 30 s. Both samples were visualized in a FEI Tecnai Spirit microscope at 120 kV.
5.4. EV Size AnalysisThe size of venom vesicles was determined by nanoparticle tracking analysis using ZetaView® (Particle Metrix, Meerbusch, Germany). Samples were diluted in citrate buffer and analyzed in size distribution mode with 85% sensitivity and 55 shutter pre-acquisition. Post-acquisition parameters were set to a minimum brightness of 50 and a minimum and maximum size of 5 nm and 500 nm, respectively, using ZetaView 8.02.31 software. Exported data were normalized for initial particle concentration and plotted in GraphPad Prism 8.
Due to the wide size distribution with the remarkable dynamic range within vesicle populations, morphometric analysis was conducted in images of negative staining using Image J 1.50i (NIH, USA), dimensioning n = 705 and 174 particles in P20K and P100K, respectively.
5.5. SDS-PAGE and Western Blot15 µg of B. jararaca venom fractions were loaded on a discontinuous system of 12% polyacrylamide gels under reducing conditions, stained with Coomassie Brilliant Blue G-250 (MP Biomedicals), and visualized in a ChemiDoc MP Image System (Bio Rad) in the EPI-fluorescent mode. For Western blotting, 10 µg from each fraction were submitted to the same electrophoretic conditions and transferred to 0.45 µm PVDF membrane (GE Healthcare) at 200 V for 2 h in a mini wet Trans-Blot system (Bio-Rad). Membranes were blocked with 5% BSA for at least 2 h. The first antibody was the polyclonal antibothropic antivenom (ABS) with dilutions of 1:4000 or 1:5000 from a 32 µg/µL solution for 2 h at room temperature (Instituto Vital Brazil, http://www.vitalbrazil.rj.gov.br, accessed on 10 September 2021). The second antibody was the anti-Horse IgG—Peroxidase (Sigma, SAB3700130) incubated for 1 h with a 1:5000 dilution. Membranes were revealed using Clarity Max ECL reagent (Bio-Rad) in the ChemiDoc MP Image System (Bio-Rad). 5.6. Proteomics 5.6.1. In-Gel Protein Digestion and Mass SpectrometryProtein bands of interest were sliced from a Coomassie-G250 stained SDS-PAGE gel and subjected to in-gel reduction (10 mM dithiothreitol), alkylation (55 mM iodacetamide), and overnight trypsin digestion at 37 °C (Promega). Tryptic digests were dried, redissolved in 3% acetonitrile and 0.1% formic acid solution, and submitted to LC-MS/MS using Q-TOF micro mass equipment (Waters Corporation, Milford, MA, USA).
Electrospray voltage was set at 3500 V, source temperature at 80 °C, and cone voltage at 30 V. Instrument control and data acquisition were conducted using a MassLynx data system (Version 4.1, Waters), and experiments were performed by scanning from a mass-to-charge ratio (m/z) of 400–2000 using a scan time of 1 s, applied during the whole chromatographic process. Data-dependent MS/MS acquisitions (DDA) were performed on precursors with charge states of 2, 3, or 4 over a range of 50–2000 m/z and under a 2 m/z window. A maximum of 3 ions were selected for MS/MS from a single MS survey obtained by collision-induced dissociation (CID). Exported MS data were analyzed in Mascot MS/MS Ions Search, with parameters described in Figure S3. 5.6.2. Protein Digestion and Mass Spectrometry for Shotgun ProteomicsProtein samples (30 µg) were precipitated in cold acetone overnight at −20 °C. Then, samples were resuspended in 50 µL of 8 M urea, 2 M thiourea, and 2% sodium deoxycholate (DOC) in 25 mM ammonium bicarbonate pH 8.0. Incubation steps were followed at room temperature (RT) as described: protein reduction (10 mM DTT for 60 min), alkylation (55 mM iodoacetamide—IAA for 30 min), and IAA quenching (55 mM of DTT for 15 min). The last two steps were undertaken in the dark. Finally, urea was diluted to <1 M with 25 mM ammonium bicarbonate, and trypsin digestion (Sequencing grade, Promega) proceeded overnight at a ratio of 1:20 at 37 °C.
DOC was precipitated by solution acidification with 1% trifluoroacetic acid (TFA), repeating 2 centrifugations steps at 12,000× g for 30 min at 4 °C. Tryptic peptides were desalted in a homemade C18 column using POROS® 50 R2 reversed-phase packing matrix (Applied Biosystems) and eluted peptides were dried under vacuum.
For LC-MS/MS analyses, samples were resuspended in 0.1% formic acid (FA), quantified by Qubit® Protein Assay Kit (Thermo Fisher Scientific) and injected into a nano-HPLC system Easy-nLC 1000 (Thermo Scientific). First, at reverse-phase trap column (2 cm × 150 µm i.d., ReproSil-Pur C18 AQ, 5 µm, 120 Å, Dr. Maisch GmbH) followed by the analytical column (15 cm × 75 µm i.d. ReproSil-Pur C18 AQ, 3 µm, 120 Å, Dr. Maisch GmbH). The flow rate was set to 250 nL/min with 0.1% FA + 5% acetonitrile (solution A) and 0.1% FA + 95% acetonitrile (solution B). A linear gradient was developed with a flush time of 10 min with 100% A, followed by 5–40% B for 107 min, 40–95% B for 5 min, and isocratically 90% B for 8 min. Eluting peptides were analyzed online by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) using a LTQ Velos Orbitrap (Thermo Fisher Scientific, San Jose, CA) controlled by Tune 2.7.0.1103 SP1 and Xcalibur 2.2 SP1.48.
A full scan was performed in high resolution (60,000) in the Orbitrap with 500 ms accumulation time and 350–2000 m/z range. In DDA mode, the 10 most abundant ions were selected for Higher Energy Collision Dissociation (HCD) with an isolation width of 2.5 m/z, normalized collision energy of 35, and 10 ms activation time. MS2 analyses were performed in the Orbitrap (7500 resolution) with 100 ms accumulation time and 45 s excluding time.
Data Analysis-Peptide Spectrum Matching (PSM)
Data were analyzed with PatternLab for proteomics software v4.1.1.22, which uses the comet algorithm [153]. A database was created merging “Uniprot serpentes” downloaded on 22 March 2019, B. jararaca accessory gland proteome [154] and B. jararaca tissues 454-sequecing [155], resulting in 227,747 sequences. B. jararaca translated databases were kindly provided by the authors. Carbamidomethylation of cysteines was set as a fixed modification, while methionine oxidation was set as a variable modification. For the peptide search, the parameters were 3 trypsin missed cleavages, mass error tolerance of 40 ppm (for precursor ions), and 1.0005 bin (for fragment ions). PSM-generated data were processed and filtered using the Search Engine Processor (SEPro) [153,156]. The pre-processing quality filters included a Delta mass of 30 ppm and a Delta CN of 0.001. Proteins were assigned with a cutoff of 2 peptides and a minimum of 3 spectral counts, accepted based on a 1% false-discovery rate (FDR) at protein level. The final protein list was obtained by grouping proteins with maximum parsimony.-Quantitative analysis
For a descriptive protein list and relative protein abundances in each sample, NSAF was used with a cutoff of 2 peptides and a minimum of 3 spectral counts. For relative percent abundances, contaminants were manually removed. Protein family clustering was also manually assembled (Supplementary Data S2). For quantitative comparisons between samples, XIC data were used to calculate the principal component analysis (PCA) (linear Kernel PCA) and to analyze differentially expressed proteins by the T-Fold algorithm [153,157]. Exported PCA coordinates, protein ratios, and quantitative t-test p-values were used to construct PCA and volcano plots in GraphPad Prism 8. 5.7. Immunofluorescence MicroscopyP20K vesicles were labeled using the lipophilic membrane marker Dil (Invitrogen®, #V22885) with 0.25 µL of Dil equivalent to 1 µg of total P20K proteins, for 30 min at 37 °C. Vesicles were washed 3 times at 20,000× g, for 10 min in Hepes buffer for excess dye removal. RAW 264.7 macrophages and A7R5 smooth muscle cell lines were seeded at a density of 5 × 104 cells/mL in 24-well plates and cultivated for 24 h to the adherence. To synchronize the interactions and internalization of vesicles by cells, they were first maintained at 4 °C for 5 min. Then, labeled P20K (equivalent to 5 µg of total proteins) per well were added on ice, and plates were kept for 10 min at 4 °C, followed by incubation at 37 °C for 4 or 24 h.
After incubation with the labeled EVs, the cells were 3× washed with PHEM buffer pH 7.2 (25 mM MgCl2, 35 mM KCl, 5 mM EGTA, 10 mM HEPES, 30 mM PIPES). Then, they were fixed in a freshly prepared solution of 4% formaldehyde in 0.1 M PHEM for 4h at room temperature, washed 3 times, and subsequently permeabilized by incubation in acetone for 5 min at −20 °C. Next, cells were incubated in 50 mM ammonium chloride for 30 min, and then blocked in PBS buffer containing 3% BSA and 0.025% fish gelatin, pH 7.2, twice for 30 min or overnight at 4 °C. After blocking non-specific antigenic sites, cells were incubated with the Alexa Fluor 488® phalloidin marker at a final concentration of 25 µg/mL for 1 h. Alexa Fluor 488® phalloidin is a fluorescent marker conjugated to phalloidin with a high affinity for actin filaments. Finally, cells were incubated with Hoechst at 5 µg/mL for nucleus labeling, mounted on glass slides with n-propyl gallate, and sealed. Samples were observed using a Leica DMI 6000B fluorescence microscope, where optical cuts were made on the Z-axis followed by 3D deconvolution processing using LAS X software version v. 3.2.1.9702.
5.8. Bj-EV Uptake QuantificationTo quantify the cellular uptake of vesicles, flow cytometry was performed for positive fluorescent events count. RAW 264.7 macrophages and A7R5 smooth muscle cell lines were seeded at a 5 × 104 cells/mL density in 6-well plates. After 24 h of cultivation, cells were either treated with Dil-labeled P20K (equivalent to 5 µg of total proteins) or with vesicles preincubated with antibothropic serum (ABS) for 24 h. ABS in a 32 mg/mL solution was diluted at 1:1000, incubated with pre-stained P20K for 90 min at 37 °C, and then washed before cell treatment.
After incubation, cells were washed 3 times with serum-free medium and removed from the plates using a solution of 2% trypsin, washed in PBS pH 7.2 buffer, and resuspended in 500 µL of PBS pH 7.2 for flow cytometry analysis. In an Accuri C6 cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), 15,000 events were evaluated, and the fluorescent population was measured compared to control cells without Dil-P20K treatment. Data were plotted and submitted to 2-way ANOVA statistical analysis using the GraphPad Prism 8.0.2 software (USA).
5.9. Transmission Electron Microscopy in Scanning Electron Microscopy (STEM-IN-SEM)RAW 264.7 macrophages and A7R5 smooth muscle cell lines were cultivated in 25 cm3 culture flasks. Before cell confluence, they were treated with P20K or P100K (equivalent to 10 µg of total proteins) for 4 or 24 h. Control cultures received vesicle vehicles instead. Fixation, post-fixation, embedding, and ultramicrotomy were conducted as described above for TEM. Cells were visualized by transmission electron microscopy in scanning electron microscopy (STEM-IN-SEM) using a VEGA 3 LMU (Tescan Brno, Czech Republic) microscope, at 30 kV in STEM mode.
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