Colostrum Extracellular Vesicle Isolation, Characterization, and Function

1. Introduction

“First milk,” known as colostrum, is a concentrated nutrient-rich breast milk that a mother produces in pregnancy and after giving birth [1,2]. It is a rich source of protein, fat, carbohydrates, vitamins, minerals, growth factors, immunoglobulins, proline polypeptides, and antioxidants. Each supports neonatal development and immunity [3,4,5]. Commercialized bovine colostrum is a “super food,” with nearly twice the fat and four times the protein, 250 times immunoglobulins, vitamins B₂, B₁₂, E, and D, and minerals (calcium, copper, iron, zinc, magnesium, manganese, and phosphorus) compared with mature milk. [6,7,8]. Colostrum is rich in bioactive oligosaccharides and probiotics, which benefit the gut microbiota [4]. Colostrum supplements are heavily marketed for gut health, immune support, and athletic performance. However, to date, there is scant evidence to support any of its broad nutritional claims. Nonetheless, attention was drawn to the fact that colostrum can serve as a natural therapeutic with regenerative, wound-healing, anti-inflammatory, and immunomodulatory functions [9]. Bovine colostrum can reduce intestinal inflammation by regulating IL-8. This is achieved by affecting epithelial pathogen immunity. There is also evidence for neuroprotective activities in rat models of cerebral ischemia. Reduced neuroinflammation is associated with lower levels of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, and improved neurological outcomes [10].

Our research supports these claims for colostrum extracellular vesicles (C-EVs) [11]. EVs are the active components of colostrum, as they facilitate cell-to-cell communication by transferring bioactive cargoes. These include proteins, lipids, and nucleic acids [12]. While EVs demonstrate significant therapeutic potential for intestinal and hepatic health, their bioactivities are the active colostrum product. Like crude colostrum, they can reduce gut inflammation, protect against liver injury [13], and improve gut barrier function [14]. Notably, EVs can treat ulcerative colitis by suppressing inflammatory responses and restoring regulatory T cell (Treg)/Th17 immune balance, including control of TLR4/NF-κB and NLRP3 signaling [15]. Additional studies report that EVs are protective against mastitis [16], steatohepatitis [17], oxidative stress, ferroptosis [18], and rheumatoid arthritis [19]. Our previous work supports these observations by demonstrating that C-EVs are both neuroprotective and anti-inflammatory when tested in a model of Parkinson’s disease (PD) [11]. While preclinical research offers translational pathways, bulk EV isolation is limited by recovery techniques. These include size-exclusion chromatography and serial ultracentrifugation. Both provide pure exosome preparations but are often limited by low sample capacity and time [20]. Higher-yield approaches such as polymer precipitation (PEG) and tangential flow filtration are more efficient but yield lower purity and have time constraints [21,22]. Ultrasonic Nanofiltration (UNF) provides an ultrafast isolation alternative (Exodus) [23,24]. UNF employs an automated dual-membrane nanofiltration system with periodic negative-pressure oscillation (NPO) and double-coupled harmonic oscillations (HO) [25]. Comparative EV isolation techniques showed that all preserve the physical and functional properties of EVs. However, the best recovery was made by UNF, which showed equivalent vesicle size distribution with improved recoveries. Nanoparticle Tracking Analysis (NTA) showed that the UNF approach yielded optimal morphology when examined by transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo-EM). Critical anti-inflammatory EV properties were preserved. Western blotting analysis for EV markers demonstrated high purity of the isolated EVs. These findings demonstrated that the UNF system enabled rapid isolation with short processing times while achieving high yields with minimal contamination by colostrum cell components.

2. Methods

2.1. EV Isolation

As a control, C-EVs were isolated by a conventional Opti-Prep method (OPT) [11]. Briefly, 50 mL of colostrum was centrifuged at 4700 × g for 20 min at 4 °C to remove fat globules and debris, followed by centrifugation at 2000 × g for 20 min at 4 °C to remove smaller fat globules. The supernatant was then filtered through Whatman grade 1 filter paper. After filtration, an equal volume of 0.25 M EDTA (pH 7) was added, and the mixture was incubated on ice for 20 min. The pH was then adjusted to 4.6 using 6 N acetic acid to precipitate the casein. Next, the samples were ultracentrifuged at 65,000 × g for 1.5 h, followed by filtration through grade 1 filter paper and 0.45- and 0.22-micron filters. 10 mL of the filtrate was diluted with phosphate-buffered saline (PBS) and centrifuged at 110000 × g for 2 h. The resulting pellets were then resuspended in PBS. Gradient ultracentrifugation was performed using 40, 30, 20, 10, and 5% Opti prep density gradients at 186,000 × g for 18 h at 10 °C using an SW 41 Ti swinging bucket rotor (Beckman Coulter, IN, USA). Each fraction was collected and washed with PBS at 110,000 × g for 90 min at 4 °C. The resulting pellets were resuspended in 1X PBS.

2.2. Ultrasonic Nanofiltration

C-EV were isolated by UNF as a comparator (Exodus system, MA, USA) [25]. Fifty ml of colostrum was centrifuged at 2,000 × g for 30 min at 4 °C to remove cells and debris. The supernatant pH was adjusted to 4.6 with 2 M HCl to reach the isoelectric point of κ-casein, then incubated on ice for 10-15 min. The samples were further centrifuged 2- times at 10,000 × g for 60 min, followed by 10,000 × g for 30 min at 4 °C to remove any remaining casein. The clarified supernatant was subsequently diluted with PBS (1:5) and filtered through 0.45 and 0.22-micron filters. Finally, the processed sample was loaded into the Exodus system for EV isolation, according to the manufacturer’s protocol.

2.3. Nanoparticle Tracking

Concentration and size distribution of C-EVs were determined by NTA using a NanoSight NS300 (Malvern Panalytical Ltd., MA, USA) at 25 °C. Nanoparticle tracking analysis [26,27,28] was performed using an NS300 Nanosight (Malvern Panalytical Ltd., MA, USA) with a green 532 nm laser. NTA 3.4 Build 3.4.4 software was used to acquire and analyze all the measurements. The system was set to the following specifications: frame rate, 24,9825 fps; Slider Gain, 219; Shutter Speed, 30.8 ms; Screen Gain, 1.8; Camera Level, 14; and Detection Limit, 5. The samples were diluted 1:1000 in particle-free PBS. The measurement chamber was flushed with 1 mL of PBS before the analysis and between samples to prevent carryover. For each run, 500 µL of the sample was loaded and videos were recorded in triplicate (3 × 60s). Independent experiments were performed in quadruplicate. Data were processed using the NTA software suite, and the C-EV concentration was expressed as EVs/mL.

2.4. Electron Microscopy

EVs were initially fixed using 2% glutaraldehyde and then deposited onto 200-mesh Formvar-coated copper grids for 5 min at room temperature. After incubation, the grids were immersed in an uranyl acetate solution for negative staining. Excess stain was removed by rinsing with PBS, and the grids were air-dried at room temperature before imaging. Transmission electron microscopy was performed using a Hitachi H-7500 microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 200 kV. Cryo-electron microscopy analysis was conducted at the contract facilities of the Hormel Institute, University of Minnesota Medical Research Center, USA.

2.5. Dynamic Light Scattering

C-EVs were diluted in PBS (1:1000) prior to characterization with Dynamic light scattering (DLS). DLS measurements were performed in triplicate for each sample using a Zetasizer Nano (Malvern Panalytical Ltd., MA, USA). The measurement parameters were set as follows: dispersant, PBS; temperature, 25 °C; viscosity, 1 cP; material refractive index (polystyrene latex), 1.590; and absorption, 0.010.

2.6. Cell Culture

The microglial cell line (BV2 cells) was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO₂ atmosphere, using 75 cm² culture flasks for routine maintenance. For different experiments, 2.0 × 10⁵ (western blotting) or 1.0 × 10⁵ (qPCR) cells were seeded in 6-well or 24-well plates and allowed to adhere for 12 h. The experimental protocol consisted of treating cells with C-EVs (500 EVs/cell) for 22 h, followed by stimulation with 100 ng/mL lipopolysaccharide (LPS) to induce inflammation. For qPCR analysis, cells were incubated with LPS for 2h prior to RNA extraction to evaluate mRNA expression levels. For Western blot analysis, cells were incubated with LPS for 24 h before protein extraction to assess protein expression.

2.7. Western Blot

After EV isolation, an equal number of EVs (1 × 10¹⁰ EVs) were mixed with 5X Laemmli buffer. The samples were then incubated at 99 °C for 15 min. For cellular protein analysis, BV2 cells treated with EVs and LPS were lysed in RIPA buffer, followed by sonication and centrifugation, and the total protein concentration was quantified using the Pierce BCA Protein Assay kit (23227, Thermo Fisher Scientific, MA, USA). Samples were prepared and loaded at equal concentrations and electrophoresed under reducing conditions in 10–15% polyacrylamide gels. After electrophoresis, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Cat. No. IPVH00010 (Millipore Sigma, MO, USA) and blocked with 5% non-fat dry milk prepared in 1X Tris-buffered saline containing 0.1% Tween-20. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies. After primary antibody incubation, the membranes were treated with secondary antibodies for 1h at RT. Chemiluminescence detection was performed using SuperSignal West Pico, Dura, Femto, and Atto substrates (Cat. No. No. Nos. 34580, 34076, 34096, A38556; Thermo Fisher Scientific, MA, USA) using an iBright750 Imager (Thermo Fisher Scientific, MA, USA). Images were quantified using ImageJ Launcher software (v1.4.3.67, NIH, MD, USA), and fold changes in protein expression were normalized to β-actin as an internal control.

2.8. RNA Isolation

Total RNA was extracted using 500 µL of TRIzol™ Reagent (Thermo Fisher Scientific, MA, USA). Subsequently, 100 µL of chloroform was added, and the samples were vigorously shaken and incubated for 3 min at room temperature, followed by centrifugation at 12,000 × g for 15 min at 4 °C to achieve phase separation. The upper aqueous phase containing the RNA was carefully transferred to a new tube. RNA was precipitated by adding 250 µL of isopropanol and incubated at room temperature for 10 min. Samples were centrifuged at 12,000 × g for 10 min at 4 °C to pellet the RNA. The RNA pellet was washed with 1 mL of 75% ethanol, vortexed briefly, and centrifuged at 7,500 × g for 4 min at 4 °C. The supernatant was discarded, and the pellet was air-dried for approximately 8 min. The RNA pellet was resuspended in RNase-free water and incubated at 55 °C for 10 min to facilitate dissolution. RNA concentration and purity were measured using NanoDrop. RNA purity was confirmed by A260/A280 (1.8-2.0) and A260/A230 (2.0-2.2).

2.9. Real-Time qPCR

Purified, isolated RNA was reverse transcribed into cDNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. The primers (Thermo Fisher Scientific, MA, USA) used included TNF-alpha (Mm00443258_m1), IL-1β (Mm00434228_m1), and IL-6 (Mm00446190_m1). Five microliters of the generated cDNA were used in qPCR, along with 10 μL of TaqMan 2× Universal PCR Master Mix (4364337, Thermo Fisher Scientific, MA, USA), 2 μL of nuclease-free water, and 1 μL of the respective 20× TM primer. mRNA expression was quantified by normalizing to GAPDH (Mm99999915_g1), and the fold change was calculated. The specificity of RT-qPCR was verified using non-template controls. Quantitative real-time PCR was performed using a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, MA, USA).

2.10. Immunocytochemistry

BV2 microglial cells were seeded at a density of 0.1 × 10⁶ cells per well in a 24-well plate containing 1 mL of complete medium with coverslips and allowed to adhere for 24 h. The cells were then treated EVs followed by stimulation with LPS (100 ng/mL). Following the treatments, immunocytochemistry was performed using standard protocols [26]. Briefly, the cells were gently washed with PBS, then replaced with fresh PBS and incubated on a rocker for 10-15 min at room temperature. Cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at room temperature. After fixation, the cells were washed three times with PBS for 5 min each on a rocker. Cells were then incubated with blocking buffer for 1h at room temperature to minimize non-specific binding. Cells were permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific, MA, USA). Next, the cells were incubated with IBA1 antibody (ab283342, Abcam, MA, USA) overnight at 4 °C. The next day, the cells were incubated with 488-fluorophore-tagged secondary antibody (Alexa Fluor 488; A-11001, Invitrogen, CA, USA) for 2h at room temperature. Next, the cells were washed with PBS for 15 min at room temperature and mounted with DAPI (P36935, Invitrogen, CA, USA). Imaging was performed using a Z1 inverted microscope (Carl Zeiss, Thornwood, NY, USA), and the analysis was performed using AxioVision software (version 4.8.0.0; Carl Zeiss Microimaging GmbH). The mean fluorescence intensity was quantified using ImageJ software (v1.4.3.67; NIH, Bethesda, MD, USA).

2.11. Statistical Analysis

All grouped data are presented as mean ± standard error of the mean (SEM). A t-test for two groups and a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons were used to assess statistical significance across groups using GraphPad Prism software (version 10), and a p-value < 0.05 was considered statistically significant.

3. Results

3.1. Characterization

C-EVs were isolated by OPT and UNF (Figure 1). Isolated UNF EVs showed a significantly higher particle concentration than OPT EVs (Figure 2A). Nanoparticle tracking analysis showed that UNF EVs had a broader particle size distribution (50 to 210 nm) with a peak around 110 nm, whereas OPT EVs had lower particle counts across all size ranges, with a peak at 90 nm (Figure 2B). Dynamic Light Scattering Analysis showed a polydispersity index (PDI) of ~0.3-0.4 for both methods, indicating heterogeneous EV size (Figure 2B). Western blot analysis confirmed exosomal markers, including ALG-2 interacting protein X (Alix), cluster of differentiation (CD) - CD63, Tumor susceptibility gene 101 (TSG101), and flotillin, in EVs isolated by both methods (Figure 2D). In contrast, the ER marker calnexin (a negative EV marker) was absent in the isolated EV samples, indicating minimal contamination by cellular debris or intracellular organelles. To ensure that residual casein did not interfere with anti-inflammatory responses, western blotting using a casein antibody (ab166596, Abcam, MA, USA) was performed, and the results showed the absence of casein in the isolated EVs. TEM revealed that EVs isolated by both methods had characteristic spherical and cup-shaped morphologies with intact membrane structures (Figure 2E). Cryo-EM further confirmed the presence of well-preserved lipid bilayer vesicles in all samples (Figure 2F).

3.2. C-EV and Innate Immunity

Cytokines play a vital role in initiating innate cell inflammatory responses [29,30,31,32]. In this study, we used a microglial cell line (BV2 cells) pretreated with isolated colostrum EVs (from both methods) for 22 h, followed by LPS (100 ng/mL) for 2h to induce an inflammatory response. We then performed qPCR analysis of key proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, in the different groups. Quantitative PCR (qPCR) analysis revealed that LPS stimulation significantly upregulated (p < 0.0001) TNF-α, IL 1β, and IL6 expression compared with the control group. EV pre-treatment significantly (p<0.0001) attenuated LPS-induced upregulation of IL-1β, IL-6, and TNF-α, indicating a pronounced anti-inflammatory effect of UNF and OPT colostrum EVs. Notably, EVs administered alone did not increase inflammatory gene expression, indicating that neither EV preparation elicited a pro-inflammatory effect under basal conditions (Figure 3A–C).

3.3. C-EVs Attenuate Inflammasome Activation

Next, C-EVs were assessed using western blotting to modulate inflammasome expression. LPS stimulation significantly upregulated NLRP3 expression compared to that in the control group (p<0.0001). EVs isolated by either method significantly suppressed the LPS-induced increase in NLRP3 expression (p < 0.0001) (Figure 4A). Similar results were observed for the other components of the NLRP3-signaling pathway. As shown in Figure 4B–D, LPS induced a significant increase (p<0.0001) in the expression of mature caspase-1, lL1β, and IL18, whereas C-EVs pre-treatment suppressed (p<0.0001) LPS-induced mature caspase-1, lL1β, and IL18. In contrast, treatment with EVs alone did not significantly alter NLRP3 and mature forms of caspase-1, IL-1β, or IL-18 expression compared to the control group, suggesting that EVs were not cytotoxic or pro-inflammatory.

3.4. C-EV Controls Microglial Activation

Next, to assess the comparative role of C-EVs isolated by both methods in microglial activation, we performed immunocytochemistry for the microglial activation marker Iba1 across the different groups. As shown in Figure 5A, LPS induced increased (p<0.0001) Iba1 expression, demonstrating microglial activation compared to the control, while pre-treatment with EVs significantly suppressed (p<0.0001) LPS-induced microglial activation. Furthermore, quantification data demonstrated that the mean fluorescence intensity of Iba-1 was significantly decreased (p<0.0001) upon pre-treatment with either isolation technique compared to LPS alone. In addition, treatment with EVs did not induce microglial activation (Figure 5B)

3.5. C-EV Anti-Inflammatory Activities

Lipopolysaccharide (LPS) stimulation activates the NF-κB signaling pathway and the formation of the NLRP3-ASC inflammasome complex in microglia. This activation increased transcription of pro-inflammatory precursors, including pro-caspase-1, pro-IL-1β, and pro-IL-18, which were subsequently cleaved by caspase-1 into their mature active forms. As a result, elevated levels of inflammatory cytokines, including IL-1β, IL-18, TNF-α, and IL-6, were observed, contributing to neuroinflammation. Pre-treatment with colostrum milk-derived extracellular vesicles (EVs), isolated from both methods, attenuated these inflammatory responses (Figure 6).

4. Discussion

The deployment of the EV isolation technique by an automated, ultrasonic nanofiltration system was a significant advantage for time, recovery, and preservation of functional immune-based anti-inflammatory properties [33]. While ultracentrifugation was similarly capable of eliciting functional responses, concerns remained about capturing pure exosomes and removing contaminants that can trigger adverse immune responses [24]. The reductions in isolation times relative to conventional 30-hour protocols led to higher yields and reduced batch-to-batch variance, which is required for clinical translational studies. Future studies will best assess protein removal and free-pure recoveries. Perhaps the most notable advance is the recovery of exosomes using a label-free approach, with no reliance on antibodies, which could compromise the biological integrity of the recovered materials [34]. The absence of particle aggregation during the extraction procedure and the potential to recover functionally intact particles all highlighted significant advantages over more commonly used techniques [24].

EVs show great therapeutic potential across a broad range of diseases [35]. Morphologically, they are characterized by a lipid bilayer membrane [36] and are reflective of the cell biology from which they were produced [37]. EVs range in size from approximately 30 nm to 10 µm [38] and are categorized into subtypes. These include exomeres, exosomes, small EVs, and apoptotic bodies [39]. EVs are released by all living cells and can be isolated from a wide range of biological fluids, including blood, saliva, urine, cerebrospinal fluid, breast milk, and bovine milk [40]. However, their biological characteristics and functions largely depend on their biogenesis pathways [12]. Among EV sources, milk-derived bovine EVs have attracted interest for their biocompatibility, low immunogenicity, and disease-fighting therapeutic potential [41]. EVs are now regarded as critical components in maintaining cellular homeostasis under normal physiological conditions due to the unique characteristics and functions of their bioactive cargo [42]. It acts as an intercellular communication vehicle, mediating paracrine, endocrine, and autocrine signaling either by cell surface interactions without cargo release, or by membrane fusion with target cells or endocytosis, whereby cargo is released into the cytoplasm or endosomal compartment, respectively; the latter necessitating escape from the endosomal compartment to exert a biological effect [43,44,45]. The variety of biological effects of EVs is broad [46,47], including modulation of pro- and anti-inflammatory immune responses [48], tissue regeneration and repair [48], and control of neurological function, particularly synaptic plasticity, myelination, and neuroprotection [11,49,50,51]. EVs have demonstrated therapeutic potential in various neurodegenerative diseases [52,53], including PD [11,54,55,56] and AD [41,57,58,59,60]. Functionally, bovine C-EVs significantly suppress the expression of apoptosis-related genes, including Bax, p53, and caspase-3, as well as pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, in intestinal epithelial cells [61]. In contrast, mature milk-derived EVs showed limited effects on apoptosis-related genes and primarily reduced the expression of inflammatory mediators, including TNF-α and IL-6, in intestinal epithelial cells [61], suggesting that C-EVs possess superior bioactive and therapeutic potential. Supporting this, C-EVs accelerate wound healing by promoting fibroblast proliferation and migration, enhancing angiogenesis, regulating extracellular matrix remodeling, and suppressing inflammation [62]. In addition, C-EVs exhibit anti-microbial, anti-inflammatory, and immunomodulatory activities in neonatal calf diarrhea models through reduced bacterial adherence and modulation of immune-related gene expression [63]. C-EVs also protect intestinal epithelial cells from LPS-induced injury by improving barrier integrity, enhancing cell proliferation, and reducing apoptosis and inflammatory responses, exerting stronger protective effects than mature EVs [61]. Recently, bovine colostrum EVs were reported to alleviate atopic dermatitis by modulating the gut-skin axis, restoring gut microbiota balance, regulating immune responses, and improving intestinal metabolite profiles [64]. Collectively, these findings highlight the therapeutic and regenerative potential of bovine colostrum-derived EVs as promising natural nanotherapeutics for inflammatory and immune-related disorders.

Our own work demonstrated that bovine C-EVs have anti-inflammatory and neuroprotective effects in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD [11]. We showed that treatment with C-EVs rescued dopaminergic neurons, reduced the number of reactive microglia, and diminished inflammasome cascade expression, thereby decreasing neuroinflammation. Based on this background, we employed an upgraded UNF to isolate C-EVs using exosome detection by UNF which employs an automated dual-membrane nanofiltration system integrated by periodic negative NPO and double-coupled HO (Exodus, [25], and compared with OPT-isolated EVs for structural and functional integrity.

We accept that, as of today, clinical adoption of C-EVs remains restricted [53]. EVs have generally been avoided in human treatments due to production challenges, safety concerns, and poor targeted delivery [65]. One key limitation that has stopped widespread clinical use includes, but is not limited to, EV isolation and standardization. Simply put, it has been difficult to separate pure, uniform preparations of EVs from other cellular materials that contain proteins and lipids [66]. These challenges, amongst others, must be overcome to guarantee that each batch of vesicles isolated from diverse materials, including cells and body fluids, meets the requirements of any regulatory human drug. Without question, isolating pure, uniform EVs is technically challenging. Recurrent concerns stem from the fact that EVs are commonly derived from living cells and, as such, there remains a risk of viral, endotoxic, and other cellular contaminants [67]. Because of these contamination risks, EVs could trigger adverse immune reactions or promote cancer. There is also the need to overcome poor tissue targeting. When EVs are injected systemically, most are rapidly cleared by the liver and spleen before they even reach target tissues of interest, which requires high dosing and carries an additional potential for long-term toxicities. We also understand that no approved EV-based therapies currently exist, which means such therapies remain highly experimental. C-EVs have a strong safety profile and rarely cause systemic toxicities, primarily due to their natural origin and structural biocompatibility. This high safety margin is notable compared with other EVs derived from natural cells. First, as naturally occurring lipid-bound nanoparticles designed to cross mammals, C-EVs bypass immune clearance and prevent unwanted inflammatory or toxic responses when circulating in the body [11]. Second, C-EV payloads, which consist of beneficial microRNAs, proteins, and lipids, act as gene-expression modulators rather than toxins, allowing them to selectively alter cellular machinery without inducing notable cytotoxicity [11,64]. Third, C-EVs are engineered to withstand harsh environments, such as the gastrointestinal tract, enabling them to cross biological barriers safely without triggering systemic toxicity or off-target adverse effects [68,69]. C-EVs naturally carry high concentrations of growth- and immune-related proteins that actively suppress inflammation and reduce oxidative stress, further protecting the body from unintended toxicities. Because of these inherent protective mechanisms, C-EVs are widely studied as highly biocompatible vehicles for drug delivery and therapeutics.

To best evaluate the feasibility, efficiency, and scalability of ultrasonic nanofiltration for isolating C-EVs, we compared it with our previously optimized EV isolation gradient method. Characterization of C-EVs confirmed successful isolation using both methods, with conventional ultracentrifugation requiring extended time, while UNF isolation was completed in just a few hours. The yield of UNF C-EV was 2-3-fold higher than that of OPT-EV isolations. Western blot analysis demonstrated the presence of canonical exosomal markers (ALIX, CD63, TSG101, Flotillin) in isolation methods, while the absence of calnexin confirmed minimal contamination from intracellular organelles. In addition, the absence of casein further validated the purity of the EV preparations, indicating the effective removal of milk protein contaminants. Morphological analysis by EM confirmed the characteristic cup-shaped, membrane-bound vesicular structure with intact lipid bilayers, supporting the structural integrity of EVs isolated using both methods. Functionally, both EV preparations significantly suppressed LPS-induced neuroinflammatory responses by modulating the key pathways involved in microglial activation and inflammasome signaling. Inflammasomes. Notably, NLRP3, drive chronic inflammation by activating caspase-1, which processes IL-1β/IL-18 and triggers pyroptotic cell death [70,71]. The induction of NLRP3, along with the activation of downstream signaling molecules, included cleaved caspase-1, mature IL-1β, and IL-18. Both UNF and OPT EVs significantly suppressed NLRP3 inflammasome priming and activation. The reduction in caspase-1 activation and downstream cytokine maturation suggests that C-EVs act at an upstream regulatory level affecting inflammasome assembly and NF-Κβ-dependent priming signals [72]. The anti-inflammatory role of C-EVs was demonstrated by pre-treatment with C-EVs, which robustly attenuated this response. The results demonstrated a broad anti-inflammatory response by the C-EVs. Microglia support neural homeostasis [73]. Chronic activation drives the release of pro-inflammatory cytokines, disrupting neuron-microglia communication, impairing synaptic structure and plasticity, promoting excitotoxicity, and promoting neurodegeneration [76].

Overall, the combined results of EV characterization, cytokine profiling, inflammasome signaling, and Iba1 immunostaining provide strong, consistent evidence that bovine colostrum-derived EVs possess potent immunomodulatory properties. Their ability to suppress both microglial activation and NLRP3 inflammasome signaling highlights their therapeutic potential as natural nanomedicine candidates for treating neuroinflammatory and neurodegenerative diseases. Given the central role of chronic microglial activation [75,77,78], these findings suggest that C-EVs may represent a promising strategy for mitigating a broad range of diseases both within and outside the nervous system. Furthermore, UNF Exodus EVs yielded higher recovery and reduced isolation time, suggesting that EVs can represent an efficient approach for clinical translation.

5. Conclusions

C-EVs were isolated in bulk within hours while preserving vesicle integrity and function. The procedures effectively suppressed neuroinflammatory responses. Overall, these findings suggest that the Exodus UNF-based isolation method is a rapid, scalable, safe, and efficient platform for producing biologically active C-EVs with promising therapeutic potential. Long-term stability, storage conditions, biodistribution, and pharmacokinetic properties of isolated EVs will be determined before clinical translation is initiated.