Antioxidant Effect of a Plant‐Derived Extracellular Vesicles’ Mix on Human Skin Fibroblasts. Induction of a Reparative Process

1. Introduction

Skin is the largest organ in our body representing the most important barrier against external stimuli, such as pollution and UV radiation [1,2]. The most important function of stratum corneum is the protection against dryness; in fact, impairments of skin integrity led to increasing of trans-epidermal water loss and consequent insufficient skin moisture [3,4,5]. As widely demonstrated, UV can induce oxidative stress in human skin cells, generating an increased production of reactive oxidative species that can in turn affect lipid integrity, reach the genetic material in cell nuclei and trigger DNA damages, with consequent impair of cellular function and cell death. Skin aging is a complex mechanism due to both intrinsic and extrinsic factors. However, many evidences support a key role of redox imbalance as the prime cause of skin aging. Many environmental factors, take part in the skin aging, including UV-exposure and pollution [6,7,8].

Mitochondria are fundamental in the skin protection against inflammation, and local ROS, mostly through the collagen and elastin production. Mitochondria are considered the powerhouse of cells. In fact, they generate energy in form of ATP [9], which involves a series of enzyme complexes and transporters. Mitochondrial Membrane Potential derives from redox transformation with the activity of Krebs cycle and serves as an intermediate form of energy storage [10]. Mitochondrial Membrane Potential is considered a valuable indicator of mitochondria’s health, inasmuch as it maintains an active control of the ions’ transport [10,11,12]. Actually, Mitochondrial Membrane Potential is not a stable value, and the oxidative stress represents one of the most important perturbing factors leading to a reduction of Mitochondrial Membrane Potential [10,13,14]. Mitochondria generate 90% of cellular reactive oxygen species and the imbalance between mitochondrial ROS and antioxidant defense results in oxidative stress, with consequent damages to mitochondrial components that, without therapeutic intervention, inevitably leads to cellular degenerative processes [15,16]. In particular, Mitochondrial Superoxide Anion is normally converted in oxygen and water by cellular antioxidant systems, but in pathological conditions it may overcome the antioxidant apparatus leading to different levels of cellular damages [17,18]. During the oxidative stress and inflammation, passing through a mitochondrial dysfunction, different proteins are involved, including Sirtuin 1, the most extensively studied member of Sirtuins’ family. Sirtuin 1 is a NAD⁺-dependent deacetylase that regulates some intracellular pathways often leading to chromatin silencing and a general decrease of the energetic state of the cell [19]. Sirtuin, participates also to the regulation of several proteins involved in age-related processes at the transcriptional level [20]. It was also shown that the age-related decrease of Sirtuin 1 content is associated to a significant inhibition of cellular proliferation [21,22,23]. Natural compounds, such as resveratrol, exert beneficial effects in skin-derived cells by increasing Sirtuin 1 expression [24,25,26,27,28,29,30].

There is clear evidence that plants contain different antioxidants that have been used in treating different diseases caused by oxidative damages, including chronic wounds, carcinogenesis, and skin aging [31,32,33,34,35,36,37]. More recently, the beneficial effect on skin health of extracellular vesicles isolated from plant extracts (PDEVs) has been widely reported. In fact, PDEVs contain a variety of antioxidants, with both enzymatic that non-enzymatic activity, that efficiently reduce damages induced by oxidative stress and help wound healing in treated cells. In this regard, increased ROS at the skin level, leads to decreased proliferation of both Fibroblasts and Keratinocytes, with a reduced epidermal turnover and decreased production of elastin [8,38,39].

Wound healing is a dynamic skin process that involves different events such as inflammation, proliferation and migration of different cell types such as Fibroblasts [40,41,42]. In this process are involved some proteins, such as Matrix Metalloproteinase and Vimentin, that drive cellular proliferation and migration to the wound site [43,44,45,46,47,48,49,50].

PDEVs can mediate wound healing by modulating gene expression and/or activating signaling pathways involved in this process. For example, exosomes from wheat and grapefruit increase collagen production along with Fibroblasts proliferation and migration to the wound site. Moreover, exosomes promote the formation of tube-like structures involved in the angiogenetic processes [51,52,53].

All in all the antioxidants constitutively contained in PDEVs can efficiently contrast damages related to skin aging, reducing intracellular ROS levels and stimulating collagen production [39,54,55,56,57,58,59].

With the above background we decided to test the effect of a “PDEVs’ magic formula”, as represented by a mix fruits, on human Skin Fibroblasts in different culture conditions, in order to assess the possible use of this PDEVs’ mix as a skin-aging reversing agent.

2. Materials and Methods

2.1. Fruit Material

Tangerine (Citrus Reticulata), Blood Orange (Citrus sinensis ‘Blood Orange’), Papaya (Carica papaya L.), Pomegranate (Punica granatum) and Grape (Vitis vinifera) were purchased from several Italian farms with organic farming certification. The fruits were washed with water and bicarbonate, peeled, and extracted with a fruit juice extractor. Fruit juices were stored at −80 °C.

2.2. Nanovesicles Isolation

Fruit juices were centrifuged at 500× g × 10 min; the supernatants were filtered with 100 µm filters and serially centrifugated at 2000× g for 20 min to eliminate cell debris and then at 15,000× g for 30 min to collect the fraction enriched in microvesicles. The supernatants were subsequently ultracentrifuged in a Sorvall WX Ultracentrifuge Series (Thermo Fisher Scientific, USA) at 110,000× g for 1 h 30 min to collect the nanovesicles. The pellet was resuspended in an appropriate buffer for downstream analyses.

2.3. Total Antioxidant Activity Assay

The detection and quantification of Total Antioxidant Capacity were performed in PDEVs using a colorimetric assay, the Antioxidant Assay kit (MAK334, Sigma-Aldrich, USA). Antioxidant Assay Kit measures total antioxidant capacity in which Cu²⁺ is reduced by an antioxidant to Cu⁺. The resulting Cu⁺ specifically forms a colored complex with a dye reagent. The optical densities were read at 570 nm and the color intensity is proportional to TAC in the sample.

2.4. Ascorbic Acid Assay

Detection and quantification of Ascorbic Acid in PDEVs were performed using a fluorometric Ascorbic Acid Assay Kit (Sigma-Aldrich, USA). Samples were diluted in ascorbic acid buffer in a 96-well plate and subsequently to each well was added a catalyst and then reaction mix (the reaction mix is composed of an ascorbic acid buffer, ascorbic acid probe and ascorbic acid enzyme mix). After 5 min of incubation, fluorescence was read in a microplate reader at Ex/Em = 535/587 nm.

2.5. ATP Assay Kit

Total ATP was measured in PDEVs with the ATP Assay Kit (Colorimetric) (Abcam, Cambridge, UK). The assay was based on the phosphorylation of glycerol in order to generate a product that was quantified colorimetrically. After plating standard wells, sample wells, and sample background control wells at the optimal dilution, the reaction mix was added to each standard and sample well, and the background reaction mix was added to the background control sample wells. The samples were incubated at room temperature for 30 min, protected from light, and the absorbance was measured using a microplate reader at OD 570 nm.

2.6. Catalase Activity Assay

For the Catalase Activity Assay (Abcam, Cambridge, UK), a fluorometric kit was used for detection and quantification of the Catalase activity in fruit-derived nanovesicles. Briefly, samples resuspended in H₂O were loaded in a 96-well plate; a stop solution was added in the control samples and incubated for 5 min at 25 °C to inhibit the Catalase activity. Catalase reaction mix (with H₂O₂) was added to both the control and high control samples for 30 min at 25 °C. The reaction in the high control samples and standard samples was stopped with the stop solution, the developer was added to all wells and after 10 min the fluorescence was read at Ex/Em = 535/587 nm on a microplate reader (Promega, Madison, WI, USA). Data were analyzed using the manufacturer’s instructions. One unit of Catalase corresponds to the amount of Catalase that will decompose 1 µmol of H₂O₂ per minute at pH 4.5 at 25 °C.

2.7. Citric Acid Assay

The Detection and quantification of Citric Acid were performed in Skin Cells Regeneration using a colorimetric assay kit, Citric Acid (CA) Colorimetric Assay Kit (MBS2563963, MyBioSource, USA).

The samples were incubated with the reaction reagents for 30 minutes at room temperature, and then the reaction product was analysed at a wavelength of 545 nm.

2.8. Reduced Glutathione (GSH) Detection and Quantification Assay

The Glutathione Colorimetric Detection Kit (Thermo Fisher, USA), a colorimetric assay, was used for detection and quantification of reduced glutathione (GSH) levels in PDEVs. Detection reagent and reaction mixture (NADPH and glutathione reductase) were added to samples and after 20 min of incubation at room temperature, the optical densities were recorded at 405 nm.

2.9. Superoxide Dismutase (SOD) Activity Assay

The Superoxide Dismutase Activity kit (Thermo Fisher, USA), a colorimetric assay, was used for detection and quantification of the superoxide dismutase activity in PDEVs’ preparations. Samples were incubated for 20 min at room temperature, after the addition of substrate and chromogenic detection reagent. The optical densities were recorded at 450 nm.

2.10. Nanoparticle Tracking Analysis

Nanoparticle Tracking Analysis (NTA) from Malvern (NanoSight NS300, UK) was used for the measurement of size distribution and concentration of extracellular vesicle samples in the liquid suspension. Five videos of typically 60 s duration were taken. Data were analyzed using the NTA 3.4 software (Malvern Instruments), which was optimized to first identify and then track each particle on a frame-by-frame basis. The Brownian motion of each particle was tracked using the Stokes–Einstein equation: D° = kT/6πηr, where D° is the diffusion coefficient, kT/6πηr = f0 is the frictional coefficient of the particle, for the special case of a spherical particle of radius r moving at a uniform velocity in a continuous fluid of viscosity η, k is Boltzmann’s constant, and T is the absolute temperature.

2.11. Dynamic Light Scattering

Dynamic Light Scattering (DLS) (Malvern Instrument, UK) was used for the evaluation of zeta potential of Plant-Derived Extracellular Vesicles. Evaluation of zeta potential allows prediction of the stability or electrostatic interactions of extracellular vesicles in dispersions.

Data from were analyzed using ZS XPLORER software (Malvern Instrument, UK).

2.12. Transmission Electron Microscopy (TEM)

The isolated PDEVs were resuspended in PBS and after a proper dilution, placed on 200 mesh carbon-coated copper grids. PDEVs were let adhere to the grids, and fixed with glutaraldehyde 2% (Electron Microscopy Sciences, USA) in PBS. Then, 3 washing steps with milliQ water were performed, followed by negative staining with phosphotungstic acid 2% solution. TEM images were captured using a Philips CM 100 Electron Microscope [60,61,62,63].

2.13. Cell line

Normal human dermal fibroblast cells (NHDFs) (Sigma-Aldrich, USA) isolated from the dermis of adult skin where cultured in Low-serum cell culture medium (Fibroblast Growth Medium 2, Sigma-Aldrich, USA) supplemented with antibiotics and antimycotics in incubator with an atmosphere of 5% CO₂.

2.14. Staining Protocol of PDEVs with a fluorescent probe (DIl 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)

To study cellular uptake of PDEVs, vesicles were marked with Dil, a fluorescent lipophilic cationic indocarbocyanine dye widely used to label membrane lipid bilayer (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 42364, Sigma-Aldrich, USA).

A stock solution of Dil was prepared dissolving the power in DMSO and then incubating the extracellular vesicles with a 5 µM dye solution for 30 minutes at 37°C. After incubation, the vesicles were ultracentrifugated at 110,000 rpm for one hour to remove the unlabeled dye and labelled-PDEVs were used for cellular treatment.

Fibroblasts (NHDF) were treated with Dil-labeled PDEVs and after 24 h, 48 h and 72 h of treatment, cells were fixed with paraformaldehyde (4%). The slides were counterstained with DAPI (DAPI/Antifade solution, Sigma-Aldrich, USA) and images were acquired with the Optika microscope (IM-5FLD, Optika Microscopes Italy). Mean Intensity of Fluorescence was measured using ImageJ software (National Institutes of Health of the United States).

2.15. Mitochondrial Membrane Potential Measurement

Mitochondrial membrane potential using MitoTracker^® Dyes for Mitochondria Labeling (Thermo Fisher, USA), a green-fluorescent mitochondrial stain, which appears to localize to mitochondria regardless of mitochondrial membrane potential. To label mitochondria, live cells were incubated with 100 nM of MitoTracker^® probes, which passively diffused across the plasma membrane and accumulated in active mitochondria. The reduced probes do not fluoresce until they enter live cells, where they are oxidized to the corresponding fluorescent mitochondrion-selective probe and then sequestered in the mitochondria. After the induction of oxidative stress (500 µM H₂O₂), Fibroblasts were incubated with prewarmed MitoTracker^® probe staining solution at 37 °C and 5% CO₂ for 30 min. After incubation cells were centrifugated and resuspended in a fresh prewarmed medium or buffer. Green fluorescence was read at Ex/Em = 490/516 using a Fluorescence Microplate Reader. Data are expressed as M.I.F. (a.u.).

2.16. Mitochondrial Superoxide Assay

To evaluate the antioxidant effect of PDEVs, NHDF were treated with 500 µM H₂O₂ for 24 hours to induce oxidative damages, and then with PDEVs. After 24-hour treatment, Mitochondrial superoxide was measured in cells treated with PDEVs and control untreated cells using Mitochondrial Superoxide Detection Kit (Abcam, UK). This is a sensitive fluorometric one-step assay based on MitoROS 580 dye to detect intracellular superoxide radical in live cells. The dye is cell-permeable and selectively reacts with mitochondrial superoxide present in live cells to generate a red fluorescence signal at Ex/Em = 540/590 nm. Mean Intensity of Fluorescence was measured using a Fluorescence Microplate Reader. Data are expressed as M.I.F. (a.u.).

2.17. Sirtuin quantification

Detection and quantification of Sirtuin in PDEVs were performed using an ELISA kit: SIRT1(Sirtuin1) Human SimpleStep ELISA^® Kit (ab171573, Abcam, UK). To perform the assay, samples or standards are added to the wells, followed by the addiction of antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm microplate reader.

2.18. Wound healing Assay

Fibroblast cells (NHDF) were seeded in order to have a monolayer. Once the monolayer was formed, we made a scratch using a micropipette tip to mimic a wound. The cells were then treated with PDEVs and images were acquired at time 0, after 24-hour and 48-hour treatment using the Optika microscope (IM-5FLD, Optika Microscopes Italy). Wound gaps were measured using the PROview software (Optika Microscopes Italy).

2.19. Matrix Metalloproteinase-9 evaluation

As described above, we induced a scratch in the monolayer of Fibroblasts seeded on coverslips. After 24-hours treatment, cells were fixed with paraformaldehyde (4%) and stained with Monoclonal Anti-Mmp9 - FITC (SAB5200306, Sigma-Aldrich, USA). The slides were counterstained with DAPI (DAPI/Antifade solution, Sigma-Aldrich, USA) and images were acquired with the Optika microscope (IM-5FLD, Optika Microscopes Italy). Mean Intensity of Fluo-rescence was measured using ImageJ software (National Institutes of Health of the United States).

2.20. Vimentin quantification

Extracellular Vimentin was measured using VIM (Human) ELISA Kit (KA3127, Novus Biologicals, UK). This kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. The purified anti-Vimentin antibody was pre-coated onto 96-well plates, then standards, test samples and anti-Vimentin HRP conjugated antibody were added to the wells subsequently, mixed and incubated. Then, unbound conjugates were washed away with wash buffer. TMB substrates (A & B) were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding an acidic stop solution. The density of yellow is proportional to the Vimentin amount of sample captured in plate. The O.D. absorbance were read at 450 nm in a microplate reader.

2.21. Statistical analysis

Results are reported as the means ± standard error (SE), and calculations were done using GraphPad Prism software (USA). An unpaired t-test (Student’s t-test) and one-way ANOVA Bonferroni were applied to analyze the results. Statistical significance was set at p < 0.05.

3. Results

3.1. Characterization of the PDEVs mix.

3.1.1. Bioactives’ content

We have first characterized the content in bioactives of a mix of different fruits. In our experiment we exploited a combination of PDEVs isolated from grape (Vitis vinifera), pomegranate (Punica granatum), orange (Citrus sinensis), tangerine (Citrus reticulata) and papaya (Carica papaya). The mix of these fruits has shown a very high level of bioactive compounds. PDEVs were characterized through different Colorimetric kit to detect and quantify their antioxidant cargo. In Table 1 are summarized the results obtained from the analysis of 10⁹ PDEVs.

3.1.2. Size distribution and zeta potential analysis

Then we characterized through Nanoparticle Tracking Analysis the PDEVs’ mix in order to establish their concentration and size distribution. As reported in Figure 1a, PDEVs isolated from the above-mentioned mix of fruit extracts, have the typical size distribution of extracellular vesicles, with size equal to 189,5 ± 2,1 nm. Moreover, in order to further characterize PDEVs isolated from fruits, we used the Zetasizer to analyze the zeta potential in a liquid suspension. The zeta potential measured was -33,68 ± 1,3 mV and the results are reported in Figure 1b.

3.1.3. Morphological Characterization

PDEVs were also characterized by Transmission Electron Microscopy to better evaluate their PDEVs-like morphology [64]. In fact, this analysis, due its nanometer resolution, can efficiently distinguish Extracellular Vesicles from non-EV-particles. TEM analysis clearly showed the presence of numerous typically rounded, whole, and undamaged vesicles with a size ranging from 50-80 nm (Figure 2a) to 150-200 nm (Figure 2b) suggesting their EV-like nature. Moreover, TEM analysis demonstrated the presence of an unbroken bilayer membrane, visible as a thin white filament surrounding the electron-dense exosome content (Figure 2). Thus, our PDEVs contained a heterogeneous population in size, round in shape, and enclosed by a membrane.

3.2. PDEVs uploading into Human Skin Fibroblasts

In order to track the uptake of extracellular vesicles over time, we labelled extracellular vesicles with Dil, an orange-red lipophilic dye. This dye is usually weakly fluorescent until incorporated into membranes. The stain with lipophilic dye is usually used to label extracellular vesicles and trace their distribution in in vitro and in vivo cells [65,66,67,68,69,70].

In our experiment we stained PDEVs with Dil for 20 minutes at 37°C, then we washed unincorporated dye and used PDEVs-Dil in cells treatment for different time points. Data reported in Figure 3, showed that PDEVs were efficiently internalized in cells after 24 h (Figure 3a) and that fluorescent signal was directly proportional to treatment time. Moreover, results demonstrated that PDEVs can efficiently reach cell nucleus where they may affect the genome (Figure 3c).

3.3. Effect on mitochondrial metabolism

For the reasons reported in the introduction section, we wanted to investigate how PDEVs can mitigate progressive aging using a model of Fibroblasts treated with H₂O₂ [71,72]. To evaluate the effect of PDEVs in cellular metabolism, we induced an oxidative stress in in vitro Fibroblasts through H₂O₂ treatment and then we treated cells with the PDEVs’ mix. As shown in Figure 4a, PDEVs can restore unbalanced Mitochondrial Membrane Potential induced by oxidative stress. H₂O₂ treatment significantly reduced Mitochondrial Membrane Potential (447 ± 3 M.I.F.; p<0,001) compared to control cells (3434 ± 56 M.I.F.), whereas cells subjected to oxidation and then treated with PDEVs have an increased potential level (2626 ± 27 M.I.F.; p<0,01) compared to the H₂O₂-treated fibroblasts.

In the following series of experiments, we evaluated also the Mitochondrial Superoxide Anion levels in untreated cells, either cells undergone H₂O₂ or cells treated with PDEVs following H₂O₂. In the H₂O₂-treated cells we measured a 13% increase of Mitochondrial Superoxide Anion compared to control cells (p<0,05), with values equal to 578 ± 11 M.I.F. (a.u.) of untreated cells and 654 ± 6 M.I.F. (a.u.) of oxidated cells. The most significant result was observed in Fibroblasts subjected to oxidation and then treated with Extracellular Vesicles. Indeed, as shown in Figure 4b, a significant reduction (p<0,001) in superoxide anion levels was observed in the samples treated with PDEVs’ mix (65 ± 2 M.I.F.) compared to oxidated cells; in particular the anion value is 10-fold lower in PDEVs treated samples compared to human fibroblast undergone H₂O₂ treatment.

3.4. Effects of the PDEVs’ mix on some aging-related molecules

Sirtuin 1 is the most extensively studied member of Sirtuins family that is involved in oxidative stress response, inflammation and mitochondrial functions, as well as in the transcriptional regulation of several proteins involved in age-related processes [20]. We thus quantified the concentration of Sirtuin 1 in cell culture supernatants of human Fibroblasts treated with either H₂O₂ or H₂O₂ + PDEVs or left untreated. As reported in Figure 5, extracellular Sirtuin 1 concentration in significantly reduced after oxidation (2,32 ± 0,03 ng/ml; CTR 3,91 ± 0,07 ng/ml; p<0,0001); while the treatment of oxidated skin fibroblasts with the PDEVs’ mix efficiently increased Sirtuin 1 levels as compared to cells treated with H₂O₂ only (2,84 ± 0,02 ng/ml; p<0,0001).

These data showed that the PDEVs’ mix efficiently restored Sirtuin 1 levels in skin-derived Human Fibroblasts after oxidative stress, supporting the beneficial effect of PDEVs’ mix in mitigating progressive skin aging.

3.5. Skin repair: Wound healing

Fibroblasts play a crucial role in skin repair. We thus used this cellular model to investigate the effects of our PDEVs’ mix on the wound healing after the induction of a scratch in the Fibroblasts’ monolayer. Fibroblasts were seeded in 24-cell plate in order to have a uniform monolayer of cells, then we induced an injury in the monolayer and treated then with PDEVs. Images of untreated control and treated Fibroblasts were acquired after 24 and 48 hours. For in vitro evaluation of wound closure, we measured the distance between the two cell fronts. The results are summarized in Figure 6. Our experiments have shown that the PDEVs’ mix significantly speeded up wound healing at both 24 (p<0,0001) and 48 hours (p<0,0001), by reducing the distances between cells’ fronts. As showed in Figure 6, wound gap at 24 h in control cells was 412 ± 21 µm, while after the PDEVs’ mix treatment it was reduced to 165 ± 14 µm. Similarly, after 48 hours the distance between the two cells’ fronts was 148 ± 10 µm in untreated control cells compared to 16 ± 3 µm in PDEV-treated cells with an almost complete closure of the scratch after PDEVs’ mix treatment.

Wound healing repair is a complex process involving different events including mesenchymal cell differentiation, proliferation and migration to the wound site and ri-epithelization [41,73,74]. In this process Matrix Metalloproteinases (MMPs), a family of zinc-containing enzymes, play an important role in the decomposition of Extracellular Matrix after injury. Among these enzyme, MMP-9 is a fundamental protein involved in cell migration and recruitment at wound site, together with the regulation of angiogenesis process [43,44,45,46,75]. In order to investigate PDEVs’ mechanism of action in Skin Cells, we evaluated MMP-9 expression in Skin Fibroblast after the induction of an injury. Briefly, cells were seeded on coverslips in order to have a uniform monolayer, then we made a scratch and treated cells for 24 hours with PDEVs. After treatment, we evaluated MMP-9 expression through Immunofluorescence Microscopy. In Figure 7 shows images of the wound site, since it has been widely demonstrated that MMP-9 is highly overexpressed in epithelial cells at the front of the migrating epithelial sheet [43,76]. Notably, we measured a 6-fold increase of MMP-9 expression (p<0,01) after 24-hours treatment with PDEVs (4,73 ± 1,15 M.I.F.) compared to untreated control cells (0,73 ± 0,03 M.I.F).

Vimentin is another key protein involved in cellular proliferation and wound repair. In fact, Vimentin is an intermediate filament protein that participates in numerous processes including cell adhesion, migration and invasion, signaling, differentiation. In tissue repair, Vimentin regulates the migration after an injury [47,48,49,50] and its concentration is directly related to the ability of cells to close a wound [77,78,79]. We thus measured Vimentin concentration in cell culture supernatants after treatment with the PDEVs’ mix. Briefly, Fibroblasts were treated with the PDEVs’ mix for 24 hours and the concentration of Vimentin was measured in the cell culture supernatant. The results showed a significant increase of Vimentin in the supernatant of the PDEVs’ mix-treated cells compared to control cell cultures (Figure 8). Notably we found a strong increase of Vimentin in the PDEVs’ mix treated human skin fibroblasts cell cultures (CTR: 56 ± 2,4 ng/ml, PDEVs: 71 ± 1,3 ng/ml; p<0,0001).

These results highly supported the results obtained in the wound healing experiments.

4. Discussion

PDEVs naturally contain a wide array of molecules, including proteins, lipids and nucleic acids, that are all involved in the intercellular communication within the same species and between the different species and nature kingdoms. In fact, PDEVs contain antioxidant molecules that can moderate oxidative stress damages, scavenging ROS and enhancing antioxidant defense systems of cells [80]. PDEVs can efficiently influence the expression of genes involved in skin health, such as regeneration, skin barrier, moisturization and aging-related genes [81]. Taking into account these considerations, we combine different fruit extracts widely characterized to exert beneficial effect in skin regeneration and mitigation of oxidative stress damages [82,83,84,85,86,87,88,89,90,91]

First of all, we characterized PDEVs bioactive molecules, detecting a huge amount of antioxidants; including both enzymatic bioactives such Superoxide Dismutase and Catalase and non-enzymatic ones such as Glutathione and Ascorbic Acid. Moreover, ATP was detected in Extracellular Vesicles and it demonstrated not only its role as energy source but also as signaling molecule that mediates interactions between organs and systems [92]. Consistently with other works showing that ATP positively influence skin cell proliferation, DNA repair and collagen synthesis [79,93], we assume that ATP in PDEVs could positively exert beneficial regenerative action, in combination with antioxidant naturally contained into the PDEVs. Moreover, skin cells have a high turnover paying the price of a high energy consumption [93,94,95]. Thus, we hypothesized that the ATP supplementation from PDEVs could be helpful in maintaining physiological skin cell turnover, keeping cells proliferating and highly producing molecules involved in regenerative processes.

In this study we set up a PDEVs’ mix that, while combining a heterogeneous population of extracellular vesicles in term of size distribution, confirmed by NTA and TEM analysis, has proven highly stable in liquid suspension, also confirming previous reports [34,35,96]. Moreover, the PDEVs used in this study had a very negative zeta potential. Notably, the zeta potential value characterized the fusogenic properties of PDEVs with plasma membrane of target/recipient cells, and the more negative is the zeta potential of the PDEVs the more fusogenic they are. Based on this background, we analyzed the cellular uptake of our PDEVs mix by human skin fibroblasts. To this purpose we labelled PDEVs with a fluorescent probe that specifically bound the membrane phospholipids of the extracellular vesicles. The labelled PDEVs were detectable in the target Fibroblasts up to 24 hours after treatment, with an increase of the fluorescence values in the following 48 and 72 hours. Furthermore after 72 hours of treatment the signal of the PDEVs overlaps with that of the nuclei, suggesting that PDEVs are able to effectively reach the genome of the target cells, possibly influencing the gene expression of recipient cells. This set of results witnessed the ability of PDEVs to be entirely uploaded into the target cells where they fully exert their action.

Damages induced by oxidative stress can affect virtually all the cellular pathways, in turn inducing a heavy unbalance of the cellular homeostasis, of course including mitochondrial activities as well. To reproduce this condition, we treated Human Skin Cell Fibroblasts with hydrogen peroxide, that induces a redox imbalance into the cells; following the H₂O₂ treatment we first tested the ability of our PDEVs mix to revert the imbalanced Mitochondria Membrane Potential, that is the most important indicator of Mitochondria health. The results showed that PDEVs can efficiently restore the physiological mitochondrial membrane potential after oxidation stimuli, from the first 24 hours after treatment. Supporting this result, PDEVs reduce the production of Mitochondrial Superoxide Anion in treated cells, further demonstrating their beneficial effect on mitochondria metabolism. Also, the increase of oxidative stress damages is related to the decreased production of molecules involved in age-related processes, such as Sirtuin family of proteins. Thus, we compared the levels of extracellular Sirtuin 1 before and after treatment of oxidated cells with PDEVs, showing that that PDEVs increased the extracellular sirtuin levels in treated cultures, thus further supporting their skin anti-aging effect.

In a further series of experiments, we wanted to directly show the ability of our PDEVs mix to influence cellular mechanisms involved in the skin regenerative processes. To this purpose we used the model of wound healing in cultures of human Skin Fibroblasts. We first showed the ability of our PDEVs mix to lead to a full closure of the wound produced into the human skin fibroblasts’ monolayer. Then we showed in the same model the wound repair was associated to the increase of MMP-9 concentrations, a protein actively involved in cellular repair mechanism. Of interest, in our experiments, MMP-9 was more expressed in the fibroblasts’ cultures treated with PDEVs, particularly at the wound site, where the wound closure actually started. In the same cultures we found a higher concentration of extracellular Vimentin in Fibroblasts treated with the PDEVs mix as compared to untreated Fibroblasts. Consistently with previous reports [97] we assumed that the polyphenols contained in our PDEVs mix could well contribute to the wound healing we showed in our experiments.

All in all the results of our study provided a scientific evidence that a mix of PDEVs from different fruits from organic farming may have a clear beneficial effect on skin aging processes. This effect passes through a clear anti-oxidant action due to the high levels of anti-oxidant molecules contained into the PDEVs mix. Here we powerfully show that the PDEVs effect was due to a full uploading of the extracellular vesicles into the target cells. Our results are also supported by previous reports showing that Plant-derived Extracellular Vesicles can efficiently penetrate to recipient cells, decrease levels of oxidants species and positively regulate the regenerative processes [52,53,80,98,99,100,101,102].

5. Conclusions

Here we show that PDEVs isolated and concentrated from a mix of fruit extracts (Grape, Blood Orange, Tangerine, Papaya and Pomegranate) contain different detectable antioxidants (Superoxide Dismutase, Catalase, Glutathione and Ascorbic Acid). These vesicles are efficiently uptaken by human Skin Fibroblasts, where they mitigate aging processes related to oxidative damages, restoring the physiological mitochondrial function and increasing the concentration of Sirtuin 1, whose principal role is the regulation of age-related processes. Moreover, here we show that PDEVs are actively involved in maintaining the skin's barrier function, assisting wound repair by also modulating the expression of MMP-9 and Vimentin, essential proteins involved in tissue repair processes.

We believe that our results will contribute in positively changing the approach of local treatment of skin aging through the set-up of entirely new products, but implementing the local treatments of skin wounds. We can’t exclude that the local anti-oxidant effect of our PDEVs mixes may be improved by the systemic treatment with the same mixes, that have shown to burst an antioxidant reaction in the whole body [34].