Isolation and Functional Characterization of Carob-Derived Nanovesicles Reveals Anti-Inflammatory and Regenerative Potential

Article type: Research Article

Keywords: carob (, extracellular vesicles, inflammation, size-exclusion chromatography, tangential flow filtration, wound-healing

Authors: Mari Cruz Manzaneque-López ⚬, Christian M. Sánchez-López ⚬, Antonio Marcilla ⚬, Pedro Pérez-Bermúdez ⚬, Carla Soler ⚬

Affiliations: Parasites & Health Research Group, Àrea de Parasitologia, Departament Farmàcia i Tecnologia Farmacèutica i Parasitologia, Facultat de Farmacia i Ciències de l’Alimentació, Universitat de València, 46100 Valencia, Spain; mari.c.manzaneque@uv.es (M.C.M.-L.); christian.sanchez@uv.es (C.M.S.-L.); antonio.marcilla@uv.es (A.M.); Joint Research Unit on Endocrinology, Nutrition and Clinical Dietetics UV-IIS La Fe, 46012 Valencia, Spain; Departament Biologia Vegetal, Facultat de Farmacia i Ciències de l’Alimentació, Universitat de València, 46100 Valencia, Spain; Departament Medicina Preventiva i Salut Pública, Facultat de Farmacia i Ciències de l’Alimentació, Universitat de València, 46100 Valencia, Spain

License: © 2026 by the authors. CC BY 4.0 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.

Article links: DOI: 10.3390/plants15040590  |  PubMed: 41754297  |  PMC: PMC12944457

Relevance: Relevant: mentioned in keywords or abstract

Full text: PDF (1.8 MB)

1. Introduction

The carob tree (Ceratonia siliqua L.) is an evergreen woody tree belonging to the Fabaceae family. It is predominantly cultivated in Mediterranean basin countries contributing to an annual production of over 135,000 tons in 2019 [ref. 1]. The carob tree is a species of high ecological value since its adaptation to arid climates and its remarkable drought tolerance and ability to thrive in nutrient-poor soil makes it a strategic species against desertification in Mediterranean environments. Furthermore, its notable capacity for carbon dioxide sequestration enhances its relevance as a climate-mitigating crop [ref. 2]. In several Mediterranean regions, particularly in Spain, Italy, and Portugal, renewed efforts are being made to revitalize this crop, driven by increasing interest in its food applications and environmental sustainability [ref. 3].

Among the principal industrial applications of carob fruits, the production of carob bean gum (CBG) stands out. The CBG, extracted from the endosperm of the seeds, is a high molecular weight polysaccharide composed of galactomannans. This food additive (E-410) is widely used for its thickening, stabilizing, and gelling properties and is incorporated into various formulations such as ice creams, desserts, sauces, baby foods, and gluten-free products, where it improves texture and stability [ref. 3,ref. 4]. The CBG extraction process yields the carob pod (fruit pulp) as a by-product, which accounts for approximately 80–90% of the fruit’s total weight. Although rich in natural sugars, dietary fiber, minerals, D-pinitol and phenolic compounds [ref. 5,ref. 6], this fraction has traditionally been undervalued and primarily used for animal feed or biomass production. In recent years, however, increasing attention has been directed toward its valorization through the development of sweet flours, fermented beverages, healthy bakery products, and as a source of functional ingredients with antioxidant, anti-inflammatory, and/or prebiotic potential. This approach not only promotes more sustainable resource utilization but also creates new opportunities within the food, nutraceutical, and cosmetic industries.

A growing body of evidence has documented the antioxidant, antidiabetic, hypolipidemic, anti-inflammatory, and antimicrobial activities of compounds present in the carob pod, suggesting that it may represent a valuable source of functional ingredients with nutraceutical and therapeutic applications. Nevertheless, substantial knowledge gaps remain concerning the mechanisms underlying the transport, stability, and bioavailability of these bioactive compounds.

In this regard, the study of plant-derived extracellular vesicles (EVs) has recently gained prominence due to their involvement in intercellular communication and the transport of bioactive molecules (proteins, nucleic acids, and secondary metabolites). These lipid bilayer-enclosed nanovesicles are secreted by plant cells and, according to numerous studies, may exert key physiological functions within plants, but also affect mammalian systems, modulating inflammatory, oxidative, and metabolic processes with associated therapeutic effects such as anticancer, antioxidant and anti-inflammatory [ref. 7,ref. 8].

However, a lack of standardization exists in the isolation protocols for plant EVs, which results in diverse nomenclatures for denoting plant nanovesicles (NVs) isolated from different plant materials. Usually, the NVs isolated, without cellular disruption, from the apoplast, from phloem sap or from in vitro plant cultures are regarded as authentic extracellular vesicles and named PDEVs (plant-derived extracellular vesicles). The term PDNVs (plant-derived nanovesicles) is applied more accurately to vesicles obtained by squeezing fruits (juices) or homogenizing plant tissues, since these procedures involve cellular disruption and the probable generation of membranous structures resembling natural EVs. The absence of standardized terminology and isolation protocols complicates inter-study comparisons and data reproducibility. Moreover, unlike mammalian extracellular vesicles, those of plant origin lack universally accepted molecular markers for the confirmation of identity and purity, representing an additional obstacle for their reliable characterization and for progress in this emerging field [ref. 9,ref. 10].

Given the positive biological effects attributed to the carob pod and the current uncertainty regarding the compounds responsible for these activities, it is hypothesized that some of its functional properties may be mediated, at least in part, by the action of PDEVs or PDNVs.

The isolation of NVs from agri-food waste and by-products remains largely unexplored, with only two reports to date. Potato peels were used to extract NVs, via size-exclusion chromatography, which were enriched in proteins linked to plant defense [ref. 11], and NVs isolated by density gradient separation from olive vegetation water, which contained molecules with well-documented antioxidant and anti-inflammatory properties [ref. 12]. Comparative studies addressing diverse sources of agri-food wastes and by-products and NV isolation methods are still lacking, as well as the characterization of their molecular cargo and biological functions [ref. 13]. In this context, carob by-products represent a promising and sustainable source of PDNVs with potential biological activity.

This study aims to isolate EVs from the apoplast of carob pulp, the dried pericarp of the fruit, using the combination of two isolation techniques, tangential flow filtration (TFF) coupled with size exclusion chromatography (SEC). Our research includes the characterization of the carob nanovesicles (CbNVs) and the evaluation of their functional properties in vitro.

2. Results

2.1. Isolation and Characterization of Carob Nanovesicles

CbNVs were successfully isolated from the Carob apoplastic washing fluid (CbAWF) through a sequential strategy combining TFF and SEC (Figure 1a) following the protocol of Sánchez-López et al. [ref. 14]. Transmission electron microscopy (TEM) images confirmed the presence of NVs in the CbAWF of carob pods, displaying both spherical and oval morphologies (Figure 1b). Nanoparticle tracking analysis (NTA) determined a modal particle size of 167.7 nm (Figure 1c) and a concentration of 5.31 ± 0.22 × 10⁸ particles/mL. Based on the average protein content (84.9 μg/mL) determined from three independent samples, the particle-to-protein ratio was calculated, yielding a mean value of 6.25 × 10⁶ particles/μg protein (Figure 1d).

2.2. Proteome Analysis of Carob Nanovesicles

To characterize the protein composition of CbNVs and to identify potential EV markers, three independent biological replicates of CbNVs were analyzed using LC-MS/MS. From a total of 261 proteins detected, those presenting at least one peptide with an identification probability greater than 95% were selected, resulting in the identification of 197 proteins. Among these, we detected several proteins previously reported in plant NVs [ref. 10] including annexins, elongation factors, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat shock proteins (HSP70), malate dehydrogenase, and tetraspanin 8 (TET8).

In order to further characterize the protein content of CbNVs, Gene Ontology (GO) analysis was performed to classify the identified proteins according to biological processes, molecular function, and cellular component (Figure 2a). Based on biological processes, most proteins were associated with metabolic functions, followed by response to stimulus and cellular processes. Similar to observations reported for other NVs derived from apoplastic fluid [ref. 11], we identified enzymes involved in cell wall modification, such as pectin acetylesterase and pectinesterase.

With respect to molecular function, CbNVs proteins were mainly associated with GO terms binding, hydrolase activity, and oxidoreductase activity. In the analysis of GO terms related to cellular component, proteins linked to the cytoplasm and nucleus were identified, which may reflect the origin of the NVs. In addition, the extracellular region was also represented, suggesting a distinctive feature of these nanoparticles.

The CbNVs proteome was also analyzed using the STRING v.10 software to evaluate potential protein–protein interaction networks. Of the 197 proteins, only 23 could be examined because the database does not include Ceratonia siliqua; therefore, Vigna angularis, a species from the same family, was used as a proxy. Nevertheless, the analysis revealed an interaction network connecting the majority of these proteins (Figure 2b). An interaction was detected between pectinesterase (A0A0L9TEU4) and HSP70 (A0A0L9TGV7), suggesting a potential link between cell wall remodeling and NV release, particularly under stress.

2.3. Carob Nanovesicles Are Not Cytotoxic

Plant-derived nanovesicles are generally non-cytotoxic. To assess whether CbNVs compromise cell viability, we performed MTT assays on differentiated THP-1 macrophages, Caco-2 intestinal cells, and HaCaT keratinocytes. These cells were exposed to 5, 10, or 20 μg/mL of CbNVs, doses that are equivalent to 3.13 × 10⁷, 6.25 × 10⁷, or 1.25 × 10⁸ particles/mL, respectively. These concentrations of CbNVs were selected for our experiments based on the results obtained in a preliminary trial. Dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS) served as positive and negative controls, respectively. CbNVs did not reduce cell viability after 24 or 48 h, indicating a high level of cellular tolerance or even a slight proliferative effect of CbNVs (Figure 3).

2.4. Carob Nanovesicles Have Anti-Inflammatory and Wound-Healing Activities

Several studies have reported anti-inflammatory effects of carob extracts, which have been attributed to their bioactive compounds [ref. 15,ref. 16,ref. 17,ref. 18]. Among these, D-pinitol, a cyclic polyalcohol abundant in carob pulp, emerges as a key candidate, as it exhibits multiple therapeutic activities, including anti-inflammatory and wound-healing properties [ref. 19,ref. 20].

Once it has been demonstrated that CbNVs do not exhibit cytotoxicity, we evaluated whether these NVs have therapeutic properties similar to those of carob extracts.

Experiments to assess their effects on NF-κB expression in human macrophages were carried out using CbNVs at the lowest (5 μg/mL) and highest (20 μg/mL) non-toxic concentrations (Figure 4). In contrast to the pronounced increase induced by bacterial lipopolysaccharide (LPS), none of the CbNVs used concentrations significantly altered NF-κB levels. Similarly, D-pinitol at 6 or 32 μM did not affect NF-κB expression (Figure 4a).

To evaluate the anti-inflammatory potential of CbNVs and D-pinitol, THP-1 macrophages were pre-treated with LPS for 1 h, followed by treatment with CbNVs, D-pinitol, or PBS. NF-κB levels were measured 24 h post-treatment (Figure 4). Notably, only the highest concentrations of CbNVs (20 μg/mL) and D-pinitol (32 μM) significantly inhibited NF-κB production (Figure 4b).

In addition, wound-healing potential was evaluated using HaCaT keratinocytes, a standard model for epidermal repair assays [ref. 21]. Based on their superior anti-inflammatory activity CbNVs (20 μg/mL) and D-pinitol (32 μM) were selected for testing. A scratch assay was performed in growth medium containing 10% FBS, and cells were treated with CbNVs, D-pinitol, CbTE (20 μg/mL total extract from carob apoplastic fluid), or PBS. Dexamethasone served as a negative control. After 24 h, wound closure reached 67.24 ± 2.22% under treatment with CbNVs, compared to 24.61 ± 2.53% with D-pinitol, which was significantly lower than the dexamethasone-treated cells (45.11 ± 1.28%). After 48 h, CbNVs treated cells achieved 98.06 ± 1.75% closure, markedly higher than D-pinitol (34.23 ± 2.89%) and dexamethasone (73.99 ± 1.45%) (Figure 4c). Interestingly, CbTE promoted wound closure more effectively than D-pinitol but less than CbNVs, suggesting that vesicle isolation enhances the activity of bioactive components compared to the crude extract (Figure 4c).

3. Discussion

Our findings demonstrate that carob pods contain NVs within the apoplast of the pericarp, a food industry byproduct, which adds further value from a circular economy perspective. Through proteomic analysis, we identified protein markers related to plants EVs. We also showed that CbNVs were not cytotoxic in three cell lines assayed, decreased NF-κB activity in THP-1 cells and promoted the wound closure rate in HaCaT cell cultures.

The apoplast, a space external to the plasma membrane of plant cells, encompasses the plant cell wall and the intercellular air spaces within plant tissues. It is involved in a diverse range of physiological processes: water and nutrient transport, plant defense mechanisms, cell interactions, the maintenance of cell walls, and the transmission of environmental and developmental signals [ref. 22]. NVs isolated from apoplastic fluid are considered as “natural bona fide” EVs due to the absence of any destructive methods in the isolation process of nanovesicles from plant tissues or organs [ref. 10]. The most commonly used technique for collecting the apoplastic liquid is vacuum infiltration centrifugation, where the tissue is subjected to buffer infiltration under vacuum, and the resulting intercellular fluid is subsequently gathered through centrifugation [ref. 22]. Regente et al. [ref. 23] first described the extraction of NVs from the apoplastic compartment employing peeled sunflower seeds in their research. Based on this study, Rutter and Innes [ref. 24] implemented changes in the infiltration buffer that led to a new buffer formulation, widely used by others [ref. 25,ref. 26,ref. 27]. In our study, PBS (1×, pH 8) has been used as an infiltration buffer. This buffer was selected due to its hypothesized capacity for mitigating adverse effects on the structural integrity of the plasma membrane.

Differential ultracentrifugation is the most widely used technique for EV separation and concentration [ref. 10]. However, it is associated with low specificity, as it can lead to disruption of EVs, aggregation or co-precipitation with contaminant particles, and also is time consuming and has limited scalability [ref. 28,ref. 29]. For this reason, alternative methods emerge with the aim of improving time, yield, purity, or scalability. Tangential flow filtration (TFF) is a membrane separation technology in which a feed stream flows tangentially to a membrane, preventing the accumulation of molecules [ref. 30]. It can be used for concentration or diafiltration of NVs from large volumes [ref. 31]. It has been reported that the use of TFF in conjunction with size exclusion chromatography (SEC) is a more efficient, specific, and reproducible method than ultracentrifugation with SEC in mammal EVs [ref. 32]. In the field of plant studies, our group has been the first to employ this combination for the isolation of NVs from pomegranate juice [ref. 14] and from phloem sap [ref. 33]. In this study, we replicated that protocol and confirmed by TEM the presence of NVs in the apoplast of carob pericarp. In relation to yield and purity, CbNVs exhibit a ratio of less than 2 × 10⁹ particles/mL.

As usually observed with NVs isolated from fruit juices, the yield of apoplastic fluid-derived NVs is also strongly influenced by both the plant source and the isolation method; for instance, concentrations of 2.9 × 10⁷ to 2.3 × 10⁸ particles/mL from hemp leaf apoplastic fluid were reported using ultracentrifugation, whereas NVs from sorghum leaf apoplast, isolated via iodixanol density-gradient centrifugation, reached 1.20 × 10⁹ ± 4.17 × 10⁷ particles/mL [ref. 25,ref. 27]. These differences underscore the critical role of both starting material and purification strategy in determining NVs yield, which may in turn affect their biological activity and potential applications.

CbNVs cargo contains proteins such as annexins, elongation factors, G3PDH, HSP70, malate dehydrogenase and TET8, identified in the proteomes of NVs isolated from apoplastic fluids of diverse plant species, although these are not universally accepted or validated reference markers for NVs [ref. 9].

The MISEV guidelines [ref. 29] recommend the identification of molecular markers for the characterization of EVs/NVs, but plant NVs diverge markedly from animal exosomes. The high diversity of plant species, together with the heterogeneity of isolation methods, contributes to poor reproducibility of results, further exacerbated by the limited number of comparative experimental studies.

For these reasons, a multi-parameter approach was adopted in the present study as there is a lack of bona fide universally accepted reference markers that allow for their unequivocal identification, and of well-validated antibodies targeting plant EV/NV-associated proteins (i.e., TET8 or PEN1). Thus, the complete characterization of CbNVs involves negative staining and TEM, NTA, BCA, and proteomic analysis based on LC-MS/MS.

In relation to the above, it is worth noting a recent study by De la Cuesta and colleagues where the authors presented a list of several protein families as potential markers for genuine plant EVs, standing out among them aquaporins, vacuolar-typeATPase complex subunits, fasciclin-like arabinogalactan proteins FLA 10 and FLA13, syntaxins other than PEN1, germin-like proteins and calreticulins [ref. 9]. Furthermore, a Plant Task Force group from the International Society for EVs (ISEV) is dealing with the identification of universal markers.

Interestingly, our results suggest the association between pectin esterase and HSP70, a chaperone that protects cells from various forms of stress. Cell wall remodeling is a critical component of plant adaptation to abiotic stress, including heat stress. In this process, enzymes such as pectin methylesterases play a central role by modulating cell wall properties in concert with HSPs [ref. 34]. Thus, the association between pectin esterase and HSP70 may support the hypothesis proposed by Ambrosone et al. [ref. 35], suggesting that pectin modification in the cell wall contributes to NVs secretion in plants under stress conditions.

CbNVs, likely owing to their protein and metabolite cargo, showed no cytotoxicity in three cell types and displayed both anti-inflammatory and wound-healing activities. These findings are consistent with previous studies reporting that PDNVs generally show no cytotoxicity in diverse cell lines. For example, Beta vulgaris–derived NVs had no toxic effects on cancer cell lines such as HeLa, MCF-7 and N2A at 10 and 100 µg/mL [ref. 36]. Similarly, HepG2 viability was unaffected by blueberry-derived NVs at 50–200 µg/mL [ref. 37] and HaCaT, HDF and RAW264.7 cells remained viable after exposure to high concentrations of cabbage and red cabbage NVs (1 × 10¹¹ and 2 × 10¹¹ particles/mL) [ref. 38]. However, cytotoxic responses can differ between cancerous and non-cancerous cells. Garlic-derived NVs (5–50 μg/mL) induced cytotoxicity in A498 and A549 cancer cell lines but not in HDFs [ref. 39]. Likewise, edible tea flower NVs (0.5–64 µg/mL) decreased viability in MCF-7, 4T1, A549, and HeLa cells, while sparing HUVEC and HEK293T cells [ref. 40]. Comparable results were reported for non-edible plants such as Cannabis sativa, where NVs reduced viability in HepG2 and Huh-7 cells (25–200 µg/mL) but had no effect on HUVECs [ref. 41].

These observations suggest that PDNVs are generally well tolerated by normal cells, while in some cases NVs display selective cytotoxicity toward cancer cells. In addition to their selective cytotoxic activity, PDNVs have also been related to other biological processes relevant to tissue homeostasis and repair, such as inflammation and wound healing.

Wound-healing is a complex physiological process comprising multiple stages: hemostasis, inflammation, proliferation, and remodeling [ref. 42]. During the inflammatory phase, immune cells such as macrophages are recruited to the site and evolve from a pro-inflammatory to an anti-inflammatory state, thereby supporting tissue repair. Consequently, anti-inflammatory properties are often sought to prevent dysregulated inflammation and irreversible tissue damage. Studies on PDNVs have demonstrated their capacity to exert both anti-inflammatory and wound-healing effects [ref. 43,ref. 44,ref. 45]. Indeed, pomegranate NVs have been shown to reduce NF-κB expression in macrophages and accelerate wound closure in Caco-2 cells [ref. 14]. These findings are further supported by studies linking NV-induced anti-inflammatory responses or enhanced cellular proliferation to improved tissue regeneration and wound-healing [ref. 44,ref. 46,ref. 47].

Several studies have reported that carob extracts exhibit anti-inflammatory effects, which have been attributed to their bioactive compounds [ref. 15,ref. 16,ref. 17,ref. 18]. Among these, D-pinitol possesses multiple therapeutic properties, including anti-inflammatory and wound-healing activities [ref. 19,ref. 20]. In light of our results, CbNVs demonstrated both anti-inflammatory effects and the ability to promote wound healing. Comparison with D-pinitol revealed that while this compound exerts anti-inflammatory activity comparable to that of the NVs, it lacks the wound-healing capacity observed with CbNVs.

4. Materials and Methods

4.1. Plant Material

Pods of Ceratonia siliqua L., cv. SDC, employed in this study were supplied by the Pedro Pérez Martínez S.L.U. company (Bugarra, Valencia, Spain). Once collected in the field, mature and dried carob fruits were subjected to an industrial seed removal process, which resulted in seeds separated from the pericarp (pod). These dried and chopped pods, supplied by the agricultural company as “carob kibbles”, were the starting plant material for our research.

4.2. Carob Apoplastic Washing Fluid Preparation

For analysis, homogeneous carob pod fragments (ca. 4–8 mm), free from visible damage, external contamination, or signs of deterioration were selected. Carob kibbles were thoroughly washed to avoid contamination, first rinsed twice in distilled water and then with 1× phosphate-buffered saline (PBS; pH 8). To infiltrate the apoplast of the pericarp with the buffer, 100 g of carob pods were put into a Kitasato flask with 250 mL PBS for 10 min and samples were vacuumed 3 times, 15 s each, with rest periods of 30 s. Buffer-embedded carob kibbles were separated with a stainless-steel sieve (1 mm) and distributed in several 10 mL syringes, without the plungers, which were placed into 50 mL conical tubes. Then, the tubes with the samples were centrifuged at 2000× g for 30 min at 4 °C to collect ca. 30 mL of the carob apoplastic washing fluid (CbAWF). To remove debris, the CbAWF was sequentially centrifuged, at 1000× g for 15 min and then at 3000× g for 15 min, always at 4 °C. The supernatants were transferred to a new tube with protease inhibitors (Complete™ Mini without EDTA, Roche, Madrid, Spain) and centrifuged at 16,000× g for 20 min at 4 °C. The final CbAWF was concentrated by tangential flow filtration (TFF-Easy 20 nm pores, HansaBioMed, Tallinn, Estonia) to obtain 3–5 mL of total extract (CbTE).

4.3. NVs Isolation

The isolation of nanovesicles was performed by size-exclusion chromatography (SEC) as described previously by Sánchez-López et al. [ref. 14]. Briefly, 10 mL Sepharose-CL2B (Sigma-Aldrich, St. Louis, MO, USA) were stacked in a 12 mL syringe (Sigma-Aldrich) and washed with filtered PBS. 1 mL of CbTE was loaded onto the column, and a total of 20 fractions of 0.5 mL each were collected using filtered PBS as an elution buffer. Carob NVs (CbNVs) were eluted in the fractions 6 to 10, whereas soluble proteins were collected in fraction 20 (F20). Finally, the fractions 6–10 were centrifuged at 3000× g for 15 min at 4 °C using centrifugal filter devices (MilliporeSigma™ Amicon™ Ultra-2, Billerica, MA, USA) to concentrate CbNVs.

4.4. CbNVs Characterization

4.4.1. Protein Quantification

Protein quantification was performed by Micro Bicinchoninic Acid (BCA) Protein Assay kit (Thermo Fisher Scientific, Barcelona, Spain). Samples were lysed with Tritón-X-100 (0.05%) and sodium dodecyl sulfate (0.01%) in Milli-Q water. The absorbance was measured at 595 nm on an iMarkTM microplate absorbance reader (Bio-Rad, Hercules, CA, USA). A standard linear curve was set up using bovine serum albumin (BSA) (Thermo Fisher Scientific).

4.4.2. Nanoparticle Tracking Analysis (NTA)

For calculating the concentration and size distribution of CbNVs by NTA we used a NanoSight LM10 system (Malvern Instrument Ltd., Malvern, UK) with 405 nm laser and a scientific complementary metal-oxide-semiconductor camera. Data analysis was performed with NTA software version 3.3 (Dev Build 3.3.104) with these parameters: camera level of 15, a detection threshold of 5, and three readings of 30 s. Automatic settings were selected for maximum jump distance, blur and minimum track length. Samples were diluted in filtered PBS and were injected into the sample chamber using a sterile 1 mL syringe.

4.4.3. Transmission Electron Microscopy (TEM)

CbNVs morphology was determined using a HITACHI HT7800 transmission electron microscope (Central Service for Experimental Research (SCSIE) at the Universitat de València) operating at 100 kV. Samples were prepared as described previously by Sánchez-López et al. [ref. 14]. Briefly, 6 μL of NVs samples were fixed 1:1 with 2% paraformaldehyde for 30 min. Fixed samples were loaded on formvar-carbon coated EM grids for 15 min. Afterwards, samples underwent a PBS rinse followed by a 5 min postfixation in 1% glutaraldehyde. Subsequently, they were washed with distilled water and subjected to contrast staining using a blend of uranyl acetate (1%) and methyl cellulose (0.5%).

4.5. Proteomic Analysis of CbNVs

4.5.1. Sample Preparation

CbNVs were dried in a rotary evaporator at 50 °C. The dried residues were reduced and alkylated. Protein reduction was performed for 20 min at 60 °C with 2 mM dithiothreitol. Subsequent alkylation was conducted in the dark for 30 min with 5.5 mM iodoacetamide. Finally, 800 ng of trypsin were added and incubated overnight at 37 °C. Digestion was stopped with 10% trifluoroacetic acid at a final concentration of 1%. Digested peptides were diluted with formic acid and loaded in an Evotip pure tip (Evosep, Odense, Denmark).

4.5.2. LC-MS/MS and Data Analysis

Liquid chromatography-tandem mass spectrometry analysis (LC–MS/MS) was performed in a Tims TOF fleX mass spectrometer (Bruker, Ettlingen, Germany). The sample loaded in the Evotip pure was eluted to an analytical column by the Evosep One system and resolved with the 100 SPD chromatographic method. The eluted peptides were ionized in a CaptiveSpray and analyzed in a ddaPASEF mode. The PASER system (Bruker) was used to search the MS and MSMS data with the Sequest algorithm (Prolucid, Mississauga, ON, Canada) with the following parameters: SwissProt 23.03.10 database; Trypsin specificity; IAM cys-alkylation and taxonomy not restricted. A database was generated by Uniprot_Fabaceae and NCBI_CeratoniaSiliqua. The uncharacterized identifications were subjected to a supplementary search on the NCBI databases (https://www.ncbi.nlm.nih.gov/) through the utilization of the basic local alignment search tool (BlastP). The proteomic analysis was performed in the proteomics facility of the SCSIE- Universitat de València.

4.6. Cell Culture Conditions

To study distinct functional properties of CbNVs we have employed different cell types/lines. The THP-1-XBlue™-CD14 cell line (InvivoGen, Toulouse, France) was generated from the monocytic THP-1 lineage by transfection with a genetic construct encoding the secreted embryonic alkaline phosphatase reporter gene, driven by a promoter responsive to the transcription factors NF-κB and AP1. Cells were maintained in RPMI 1640 medium supplemented with L-glutamine (Gibco, Waltham, MA, USA).

Immortalized human keratinocytes (HaCaT) (ATCC, Manassas, VA, USA) and human colon epithelial (Caco-2(ATCC) cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing high glucose and L-glutamine (Gibco). All media were supplemented with streptomycin (100 μg/mL), penicillin (100 U/mL) (Sigma-Aldrich), and 10% fetal bovine serum (FBS) (Gibco). Cultures were maintained in 75-cm² flasks at 37 °C in a humidified atmosphere of 5% CO₂.

For macrophage differentiation, THP-1-XBlue™-CD14 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) at 100 ng/mL for 72 h. Following differentiation, the medium was replaced with fresh culture medium, and cells were incubated for an additional 72 h prior to treatment. The cell cultures were performed in the cell culture facility of the SCSIE-Universitat de València.

4.7. Cell Cytotoxicity Assays

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to assess cell viability. Caco-2, HaCaT, and THP-1-XBlue™-CD14 macrophage-differentiated cells were seeded in 96-well plates at a density of 3 × 10⁵ cells/mL and incubated at 37 °C. Cells were exposed for 24 or 48 h to: (a) increasing concentrations of CbNVs (5, 10, and 20 μg/mL; protein content determined by the Micro BCA Protein Assay Kit (Thermo Fisher Scientific), (b) the NV-depleted soluble protein fraction derived from SEC (F20), or (c) CbTE. Controls included 20 μL PBS and 20% dimethyl sulfoxide (DMSO) as a cytotoxicity reference.

After the initial incubation, cells were refreshed with serum-free medium, supplemented with 10 μL MTT, and incubated for an additional 4 h. Subsequently, 180 μL DMSO was added, and absorbance was recorded at 570 nm (signal) and 695 nm (background) using an iMark™ microplate absorbance reader (Bio-Rad). Final values were obtained by subtracting background from signal absorbance. Results are expressed as the percentage of ΔA relative to untreated control cells (defined as 100% MTT reduction or viability).

4.8. NF-ĸB Reporter Assay

To assess NF-κB activation, the Quanti-Blue™ colorimetric reagent (InvivoGen) was employed to monitor SEAP production according to the manufacturer’s instructions. THP-1-XBlue™-CD14 monocytes differentiated into macrophages were treated with PBS, CbNVs (5 or 20 μg/mL), or with 300 ng/mL lipopolysaccharide (LPS) from Escherichia coli K12 (InvivoGen) as a pro-inflammatory positive control. In parallel, to evaluate potential anti-inflammatory effects of these treatments and D-pinitol (6 and 32 μM), macrophages were pre-stimulated with 300 ng/mL LPS for 1 h prior to treatment in serum-free medium. Following 24 h of incubation at 37 °C, 5% CO₂, and 100% humidity, 20 μL of cell culture supernatant was collected and mixed with 180 μL Quanti-Blue™ reagent. Plates were incubated for 5 h at 37 °C, and optical density was measured at 655 nm using an iMark™ microplate absorbance reader (Bio-Rad).

4.9. Scratch Assay

Wound healing is a critical process that, on the one hand, promotes tissue repair following injury or surgery, but on the other, can result in persistent infections and increased risk of severe complications. In this study, the wound-healing potential of CbNVs was assessed in keratinocytes (HaCaT).

The wound-healing assay was performed as described previously [ref. 14]. Briefly, cells were seeded in 24-well plates at a density of 3 × 10⁵ cells/mL and cultured until confluence was reached. Then, the medium was removed, and a scratch was made using a 500 μL pipette tip to generate a wound. Cells were then washed with PBS and incubated with fresh medium supplemented with 10% FBS. HaCaT cells were treated with PBS, 20 μg/mL CbNVs, 32 μM D-pinitol or with 10 μM dexamethasone (Sigma-Aldrich) as a negative control of wound closure. Wound closure (confluence) was documented at 40× magnification using an Eclipse TE2000-S inverted microscope (Nikon, Amstelveen, The Netherlands) equipped with a Nikon F-601 camera (SCSIE-UV at the Universitat de València). Results were monitored over 0–48 h. The migration area was quantified by measuring the wound with ImageJ2 software, and using the following formula [ref. 8]:

where A0 represents the initial wound area at day 0, and At is the remaining wound area at the corresponding time point.

4.10. Statistical Analyses

The analyses were performed using GraphPad Prism 8 software (San Diego, CA, USA). Data are presented as mean ± SEM. Statistical significance was assessed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test when appropriate. For parameters that did not exhibit a normal distribution, the Kruskal–Wallis non-parametric test was used. A p value < 0.05 was considered statistically significant.

5. Conclusions

In the current study we demonstrate that carob pods contain NVs which can be isolated, intact and functional, from the apoplast of the pericarp by vacuum infiltration, TFF, and SEC. Future research should focus on the scalability of the vesicle isolation process.

The complete molecular characterization of carob nanovesicles (including transcriptomics, lipidomics and metabolomics) should pave the way in elucidating the molecular mechanisms responsible for their biological activities observed in vitro.

Our findings suggest that CbNVs may encapsulate bioactive compounds that act synergistically to enhance both their anti-inflammatory and wound-healing effects and open new opportunities for the valorization of the pericarp of carob pods, a fraction commonly considered an agricultural by-product, as a natural source of NVs with potential therapeutic or cosmetic applications. Research on agricultural by-products aligns with the principles of circular bioeconomy, reinforcing the commitment to the comprehensive utilization of traditional crops with high ecological, economic, and cultural values in vulnerable Mediterranean regions.

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