PHARMACEUTICAL COMPOSITION IN THE FORM OF A HYDROGEL COMPRISING ORANGE-DERIVED EXTRACELLULAR VESICLES

Abstract

A method of promoting angiogenesis and cell proliferation in a subject in need of tissue repair and/or regenerative therapy involves administering to the subject a pharmaceutical composition in the form of a hydrogel that includes orange-derived extracellular vesicles (EVs) having a diameter ranging from 10 to 500 nm and showing pro-angiogenic activity. The orange-derived EVs are dispersed in a hydrogel matrix and are releasable from the hydrogel matrix. A method for preparing the pharmaceutical composition in the form of a hydrogel is also provided.

Claims

1. A method of promoting angiogenesis and cell proliferation in a subject in need of tissue repair and/or regenerative therapy, said method comprising administering to the subject a pharmaceutical composition in the form of a hydrogel, comprising: (i) orange-derived extracellular vesicles (EVs); (ii) a polymer gelling agent; (iii) water in an amount of at least 10% by weight of the total weight of the pharmaceutical composition; and (iv) optionally, pharmaceutically acceptable vehicles, excipients, and/or diluents, wherein the orange-derived extracellular vesicles (EVs) are enclosed by a lipid bilayer membrane, have a diameter ranging from 10 to 500 nm as measured by light scattering-based nanoparticle tracking analysis (NTA), and show pro-angiogenic activity, and wherein the polymer gelling agent and the water form a hydrogel matrix in which the orange-derived EVs are dispersed, said orange-derived EVs being releasable from the hydrogel matrix.

2. The method of claim 1, wherein the orange-derived EVs are derived from one or more Citrus sinensis plants and/or one or more fruits of said one or more Citrus sinensis plants.

3. The method of claim 1, wherein the amount of the orange-derived EVs is in the range of from 10.sup.10 to 10.sup.12 EVs/ml on the total volume of the pharmaceutical composition.

4. The method of claim 1, wherein the amount of orange-derived EVs proteins is in the range of from 5 μg to 500 μg/ml on the total volume of the pharmaceutical composition.

5. The method of claim 1, wherein the protein content of the orange-derived EVs is in the range of from 1 to 1×10.sup.3 ng/10.sup.9 EVs.

6. The method of claim 1, wherein the RNA content of the orange-derived EVs is in the range of from 10 to 60 ng/10.sup.10 EVs.

7. The method of claim 1, wherein the polymer gelling agent is selected from the group consisting of cellulose and cellulose-based polymers, including carboxymethylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, cellulose gum, collagen, hydrolyzed collagen, chitosan, gelatin, hyaluronic acid (HA), glycerol, polyethylene glycol (PEG), polysulfated glycosaminoglycan (PSGAG), glycerine, propylene glycol, calcium alginate, and any combination thereof.

8. The method of claim 1, wherein the pharmaceutical composition further comprises a therapeutic agent selected from the group consisting of antibiotics, antibacterial agents, antifungal agents, anti-inflammatory agents, growth factors, pro-regenerative agents, and any combination thereof.

9. The method of claim 1, wherein the pharmaceutical composition has a pH comprised between 3.5 and 7.0.

10. The method of claim 1, wherein the tissue repair and/or regenerative therapy is for closing a tissue lesion.

11. The method of claim 1, wherein the subject is affected by a disease selected from the group consisting of ischemic ulcers, optionally the ischemic ulcers being pressure ulcers, arterial ulcers, venous ulcers, diabetic ulcers, exudative ulcers, dysmetabolic ulcers, traumatic ulcers, mucosal lesions, optionally the mucosal lesions being diabetic lesions, burns, fistulae, psoriasis, keratosis, keratitis, fissures, traumatic ulcers, mucosal lesions including traumatic lesions due to prothesis and mouth, decubital, genital mucosal lesions, dermatitis including acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, dermatitis herpetiformis, and cellular damage induced by pro-apoptotic drugs aimed to treat pre-cancerous lesions including actinic keratosis.

12. The method of claim 1, wherein the pharmaceutical composition is suitable for topical administration or for administration by injection.

13. The method of claim 12, wherein treatment comprises release of the orange-derived EVs from the hydrogel matrix over a period of at least 1 day.

14. The method of claim 13, wherein the release of the orange-derived EVs from the hydrogel matrix is performed over a period of at least 7 days.

15. A method for preparing a pharmaceutical composition in the form of a hydrogel, comprising: (i) orange-derived extracellular vesicles (EVs); (ii) a polymer gelling agent; (iii) water in an amount of at least 10% by weight of the total weight of the pharmaceutical composition; and (iv) optionally, pharmaceutically acceptable vehicles, excipients, and/or diluents, wherein the orange-derived extracellular vesicles (EVs) are enclosed by a lipid bilayer membrane, have a diameter ranging from 10 to 500 nm as measured by light scattering-based nanoparticle tracking analysis (NTA), and show pro-angiogenic activity, and wherein the polymer gelling agent and the water form a hydrogel matrix in which the orange-derived EVs are dispersed, said orange-derived EVs being releasable from the hydrogel matrix, the method comprising the steps of: (i) dissolving the polymer gelling agent in a predetermined amount of water to obtain the hydrogel matrix, wherein the pre-determined amount of water represents at least 10% by weight of the total weight of the pharmaceutical composition; (ii) adding the orange-derived extracellular vesicles (EVs) to the hydrogel matrix; and (iii) mixing said orange-derived EVs with the hydrogel matrix, thereby obtaining the pharmaceutical composition in the form of a hydrogel in which the EVs are dispersed in the hydrogel matrix.

Description

EXAMPLES

[0073] The following experimental section is provided purely by way of illustration and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section, reference is made to the appended drawings, wherein:

[0074] FIG. 1 shows the characterization of orange-derived EVs in experimental example 1. (A) Representative image of NanoSight analysis and transmission electron microscopy photographs, (at original magnifications of ×60,000, scale bar 500 nm (B) and ×150,000, scale bar 200 nm (E)), showed a size in diameter typical of orange-derived EVs. The bar graphs show (C) the mean protein content of EVs expressed as nanograms (ng) of protein in 10.sup.9 EVs, and (D) the RNA content expressed as nanograms (ng) of RNA in 10.sup.10 EVs, as measured on several preparations of orange-derived EVs.

[0075] FIG. 2 shows the release of orange-derived EVs from different hydrogels in experimental example 2. The release of EVs from hydrogels (carboxymethylcellulose and collagen) was evaluated as number of EVs (A for carboxymethylcellulose, C for collagen) and protein amount (B for carboxymethylcellulose, D for collagen) released over time. To perform the experiment, the hydrogel or the pharmaceutical composition, comprising hydrogel and EVs at different doses (10.sup.8, 10.sup.9 and 10.sup.10 EVs), were kept in a 24 well plate in direct contact with saline solution. The saline solution was analyzed for the presence of EVs at time 0 (0) and after 30 minutes (30 min), 1 hour (1 h), 3 hours (3 h), 6 hours (6 h), 24 hours (24 h), 48 hours (48 h), 72 hours (72 h). EVs and proteins were quantified in the saline solution at each timepoint by using the NanoSight instrument and the BCA kit, respectively. The amount of EVs released by hydrogel was expressed as number of EV/ml, whereas the amount of released protein was expressed as μg/ml. N=3 experiments were performed for each data set.

[0076] FIG. 3 shows the incorporation of the orange-derived EVs of the pharmaceutical composition in target cells in experimental example 3. HMEC target cells were treated with mixtures of fluorescently labelled EVs (fluorochrome PKH26) and hydrogel (carboxymethylcellulose, collagen, ethylcellulose, chitosan, gelatin, PEG). EVs uptake in target cells was measured by Citofluorimetry at different time-points: 6 hours (6 h), 24 hours (24 h), 48 hours (48 h) and 72 hours (72 h). The EVs uptake is shown as percentage of signal intensity compared to target cells alone (negative control, CTR−). N=3 experiments were performed for each data set. CTR−: unstimulated endothelial cells. 10.sup.8, 10.sup.9 or 10.sup.10 EV: dose of orange-derived EVs used with or without hydrogel. The statistical significance was calculated comparing vesicle uptake of EVs alone or in combination with hydrogel. p: ***<0.005; ****<0.001.

[0077] FIG. 4 shows the ability of the pharmaceutical composition of the invention to promote angiogenesis on endothelial cells in vitro in experimental example 4. Tube formation assay was performed by stimulating endothelial cells with orange-derived EVs alone or hydrogel alone or the combination of EVs and hydrogel, for 24, 48 and 72 hours. The histograms show the total length of tube structure formed on Matrigel (mean±SEM) upon stimulation carried out as above described. N=3 experiments were performed for each data set. CTR−: unstimulated endothelial cells. CTR+: medium supplemented with Fetal Bovine Serum and Growth Factors. EV: 10.sup.9 orange-derived EVs. H1: carboxymethylcellulose hydrogel alone. H1+EV: carboxymethylcellulose hydrogel with 10.sup.9 orange-derived EVs. H2: collagen hydrogel alone. H2+EV: collagen hydrogel with 10.sup.9 orange-derived EVs. The statistical significance was calculated comparing each condition with every other condition. p: ****<0.0001 vs. CTR−; #<0.0001 vs. H1; § <0.0001 vs. H2; α<0.05 vs EV.

[0078] FIG. 5 shows the ability of the pharmaceutical composition of the invention to promote endothelial cell migration in vitro in experimental example 4. Scratch test assay was performed by stimulating endothelial cells with orange-derived EVs alone or with hydrogel alone or the combination of EVs and hydrogel, for 24, 48 and 72 hours. The histograms show the percentage of wound closure (mean±SEM) upon stimulation carried out as above described. N=3 experiments were performed for each data set. CTR−: unstimulated endothelial cells. CTR+: medium supplemented with Fetal Bovine Serum and Growth Factors. EV: 10.sup.9 orange-derived EVs. H1: carboxymethylcellulose hydrogel alone. H1+EV: carboxymethylcellulose hydrogel with 10.sup.9 orange-derived EVs. H2: collagen hydrogel alone. H2+EV: collagen hydrogel with 10.sup.9 orange-derived EVs. The statistical significance was calculated comparing each condition with every other condition. p: ****<0.0001 vs. CTR−; **<0.01 vs. CTR−; *<0.05 vs. CTR−; #<0.0001 vs. H1; § <0.0001 vs. H2; α<0.05 vs EV.

[0079] FIG. 6 shows the ability of the pharmaceutical composition of the invention to promote fibroblast cell migration in vitro in experimental example 4. Scratch test assay was performed by stimulating fibroblast cells with orange-derived EVs alone or with hydrogel alone or the combination of EVs and hydrogel, for 24, 48 and 72 hours. The histograms show the percentage of wound closure (mean±SEM) upon stimulation carried out as above described. N=4 experiments were performed for each data set. CTR−: unstimulated fibroblasts. CTR+: medium supplemented with 10% Fetal Bovine Serum. EV: 10.sup.9 orange-derived EVs. H1: carboxymethylcellulose hydrogel alone. H1+EV: carboxymethylcellulose hydrogel with 10.sup.9 orange-derived EVs. H2: collagen hydrogel alone. H2+EV: collagen hydrogel with 10.sup.9 orange-derived EVs. The statistical significance was calculated comparing each condition with every other condition. p: ****<0.0001 vs. CTR−; ***<0.001 vs. CTR−; #<0.0001 vs. H1; § <0.0001 vs. H2; α<0.05 vs EV.

[0080] FIG. 7 shows the absence of toxicity of different types of hydrogel on endothelial cells and fibroblasts in vitro in experimental example 4. Endothelial cells and fibroblasts were stimulated with carboxymethylcellulose hydrogel (H1) or collagen hydrogel (H2) and toxicity was measured as reduction of the proliferation rate assessed by BrdU proliferation assay, in comparison to the negative control. The graphs show the percentage of proliferation (mean±SEM) for fibroblasts (upper-left) and endothelial cells (upper-right). N=3 experiments were performed for each data set. CTR−: unstimulated cells. CTR+: medium supplemented with 10% of Fetal Bovine Serum for fibroblasts, or medium supplemented with Fetal Bovine Serum and Growth Factors (Endogro) for endothelial cells. H1: carboxymethylcellulose hydrogel alone. H2: collagen hydrogel alone. The statistical significance was calculated comparing each condition with CTR−. p: ****<0.0001; **<0.01.

[0081] Moreover, FIG. 7 shows the ability of to promote endothelial cell proliferation in vitro in experimental example 4. Endothelial cells were stimulated with orange-derived EVs alone or hydrogel alone, or the combination of EVs and hydrogel, for 24, 48 and 72 hours. The graph in the lower part of the figure shows endothelial cells proliferation expressed as percentage (mean±SEM) upon stimulation carried out for 24 hours as above described. N=3 experiments were performed for each data set. CTR−: unstimulated cells. CTR+: medium supplemented with 10% of Fetal Bovine Serum for fibroblasts, or medium supplemented with Fetal Bovine Serum and Growth Factors (Endogro) for endothelial cells. EV: 10.sup.9 orange-derived EVs. H1: carboxymethylcellulose hydrogel alone. H1+EV: carboxymethylcellulose hydrogel with 10.sup.9 orange-derived EVs. H2: collagen hydrogel alone. H2+EV: collagen hydrogel with 10.sup.9 orange-derived EVs. The statistical significance was calculated comparing each condition with every other condition. p: ***<0.001 vs. CTR−; #<0.05 vs. H1; § <0.001 vs. H2; α<0.05 vs EV.

[0082] FIG. 8 shows that the pharmaceutical composition of the invention accelerates tissue regeneration in an in vivo model of ulcers in experimental example 5. Ulcers were induced in mice and treated with carboxymethylcellulose (H1), carboxymethylcellulose with 10.sup.9 EVs (H1+EV), chitosan (H2), chitosan with 10.sup.9 EVs (H2+EV). As controls, untreated ulcers were used (CTR−). Wound area, i.e. the area of the wound still open, measured after 6 days of treatment is reported as percentage, compared to the original wound area at time 0. Representative images of treated wounds are shown below the graph. N=5 animals were used for each data set. CTR−: untreated wounds. The statistical significance was calculated comparing the effect of the different treatments with the untreated group (CTR−): p: *<0.05; ****<0.001. The statistical significance was calculated by comparing the effect of each hydrogel with the same with EVs (H1 versus H1+EV, or H2 versus H2+EV): p: $<0.05.

[0083] FIG. 9 shows that the pharmaceutical composition of the invention accelerates tissue regeneration in a diabetic in vivo model of ulcers in experimental example 5. Ulcers were induced in mice and treated with carboxymethylcellulose (Hydrogel), carboxymethylcellulose with 10.sup.9 EVs (Hydrogel+EV). As controls, untreated ulcers were used (CTR−). (A) Wound area, i.e. the area of the wound still open, measured after 3, 6, 10 and 14 days of treatment is reported as percentage, compared to the original wound area at time 0. N=5 animals were used for each data set. CTR−: untreated wounds. The statistical significance was calculated comparing the effect of different treatments: p: *<0.05; **<0.01; ***<0.005; ****<0.001. (B, C, D) Results of histological analysis of wound tissues after 14 days of treatment. Hematoxylin and eosin staining was used to measure (B) the wound width intended as wound size (μm), (C) the percentage of re-epithelization as the percentage of newly formed epithelium in the wound in comparison to the original wound size, (D) the epithelial thickness of the wound (μm). The treatment with the combination of hydrogel and EVs was compared with the hydrogel alone (hydrogel) and untreated wounds (CTR−). N=5 animals were used for each data set. CTR−: untreated wounds. The statistical significance was calculated comparing the effect of different treatments: p: ***<0.005; ****<0.001.

MATERIALS AND METHODS

Extracellular Vesicles Isolation

[0084] Extracellular vesicles were isolated from orange juice. Fruits of Citrus sinensis plants were squeezed and the juice was sequentially filtered using decreasing order of pores to remove fibers. EVs were then purified with differential ultracentrifugation or tangential flow filtration. For differential ultracentrifugation, the juice was centrifuged at 1,500 g for 30 minutes to remove debris and other contaminants. Then, EVs were purified by a first ultracentrifugation at 10,000 g followed by ultracentrifugation at 100,000 g for 1 hour at 4° C. (Beckman Coulter Optima L-90K, Fullerton, CA, USA). The final pellet was resuspended with phosphate buffered saline added with 1% DMSO and filtered with 0.22 micrometer filters to sterilize.

[0085] Extracellular vesicles were used or stored at −80° C. for long time. For tangential flow filtration, at first the juice was clarified by filtration with depth filter sheet discs Supracap 50 (Pall) to exclude fibers and debris. Then, the filtered juice was purified by concentration and diafiltration using a tangential flow filtration cassette TFF Omega (Pall Cadence). Finally, the retentate from tangential flow filtration was sterilized by filtration with a 0.2 nm filter.

Nanoparticle Tracking Analysis (NTA)

[0086] Nanoparticle tracking analysis (NTA) was used to define the EV dimension in diameter and profile using the NanoSight LM10 system (NanoSight Ltd., Amesbury, UK), equipped with a 405 nm laser and with the NTA 3.1 analytic software. The Brownian movements of EVs present in the sample subjected to a laser light source were recorded by a camera and converted into size and concentration parameters by NTA through the Stokes-Einstein equation. Camera levels were for all the acquisition at 16 and for each sample, five videos of 30 s duration were recorded. Briefly, purified EVs were diluted 1:2000 in 1 ml vesicle-free saline solution (Fresenius Kabi, Runcorn, UK). NTA post-acquisition settings were optimized and maintained constant among all samples, and each video was then analyzed to measure EV mean, mode and concentration.

Transmission Electron Microscopy (TEM)

[0087] Transmission electron microscopy of EVs was performed by loading EVs onto 200 mesh nickel formvar carbon coated grids (Electron Microscopy Science, Hatfield, PA) for 20 minutes. EVs were then fixed with a solution containing 2.5% glutaraldehyde and 2% sucrose and after repeated washings in distilled water, samples were negatively stained with NanoVan (Nanoprobes, Yaphank, NK, USA) and examined by Jeol JEM 1010 electron microscope.

Protein Extraction and Quantification

[0088] Proteins were extracted from EVs by RIPA buffer (150 nM NaCl, 20 nM Tris-HCl, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 1% Triton X-100, pH 7.8) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, Missouri, USA). The protein content was quantified by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) following manufacturer's protocol. Briefly, 10 μl of sample were dispensed into wells of a 96-well plate and total protein concentrations were determined using a linear standard curve established with bovine serum albumin (BSA).

RNA Extraction and Quantification

[0089] Total RNA was isolated from EVs and cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA concentration of samples was quantified using spectrophotometer (mySPEC, VWR, Radnor, PA, USA).

Hydrogel Preparation

[0090] Hydrogels were produced by resuspending the hydrogel in water or saline solution. Hydrogels used included carboxymethylcellulose, collagen, ethylcellulose, chitosan, gelatin and PEG (Sigma-Aldrich, St. Louis, Missouri, USA). After extensive mixing the hydrogel was sterilized by filtering with filter 0.22 μm and ready to use. In order to combine hydrogel with orange-derived EVs, hydrogel was mixed with different doses of EVs (10.sup.8, 10.sup.9, 10.sup.10 EVs).

Cell Culture

[0091] Human microvascular endothelial cells (HMEC) were obtained by immortalization with simian virus 40 of primary human dermal microvascular endothelial cells. HMEC were cultured in Endothelial Basal Medium supplemented with bullet kit (EBM, Lonza, Basel, Switzerland) and 1 ml Mycozap CL (Lonza). Normal Human Dermal Fibroblasts (NHDF) are primary adult fibroblasts (Lonza). NHDF were cultured in FGM-2 Growth Media (Lonza) supplemented with bullet kit and 1 ml Mycozap PR (Lonza).

Extracellular Vesicles Uptake

[0092] In order to evaluate the uptake into cells of orange-derived EVs, EVs were labeled with PKH-26 dye (Sigma-Aldrich, St. Louis, Missouri, USA) for 30 min at 37° C. and washed by ultracentrifugation at 100,000 g for 2 h at 4° C. using a 10 mL polycarbonate tube (SW 90 Ti rotor, Beckman Coulter Optima L-90 K ultracentrifuge). For uptake experiments, 25,000 HMEC cells/well were plated in 24-well plates and cultured with a transwell with pores of 0.4 μm containing saline solution (CTR−), the hydrogel or the combination of hydrogel and EVs. After each time point (6, 24, 48 and 72 hours) the transwell was removed, cells were extensively washed, detached with trypsin and their fluorescence was measured by FACS using the CytoFLEX flow cytometer with CytExpert software (Beckman Coulter Optima L-90K, Fullerton, CA, USA).

In Vitro Tube Formation Assay

[0093] In vitro formation of tubes or capillary-like structures was studied on growth factor-reduced Matrigel (BD Bioscience, Franklin Lakes, NJ, USA) in 24-well plates. HMEC (25,000 cells/well) were seeded onto Matrigel-coated wells in DMEM alone (untreated control) or EndoGRO MV-VEGF medium (positive control), or in DMEM in the presence of hydrogel alone or hydrogel containing EVs (dose 10.sup.9 EVs). Each condition was performed in triplicate. After incubation for 24 hours, five random-field phase-contrast images (magnification, ×10) were recorded for each well. The total length of the network of tube structures was measured using ImageJ software. Results are expressed as the mean of the total length per each condition. The test was repeated with conditioned medium collected at 24, 48 and 72 hours.

In Vitro Scratch-Test Assay

[0094] Endothelial cells (HMEC) and fibroblasts (NHDF) were seeded at a density of ˜50×10.sup.3 cells/well in 24-well plates in the appropriate culture medium. When the cells reached complete confluence, scratch were created with a sterile tip to mimic a wound. Prior to stimulation (t=0), micrographs of the well were obtained using a Leica camera (Leica, Wetzlar, Germany). The cells were then stimulated with DMEM alone (negative control), or Endogro (Lonza) for HMEC or DMEM with 10% FBS for NHDF as positive control or DMEM containing EVs (dose 10.sup.9 EVs) or DMEM in the presence of hydrogel alone or hydrogel containing EVs (dose 10.sup.9 EVs). The ‘wound closure’ phenomenon was monitored for 24 hours, taking pictures using the Leica camera. Images were analyzed by ImageJ software (Bethesda, MD, USA) measuring the wound area. Results are expressed as a percentage of wound closure considering the wound area at t0 as 100% and calculating the corresponding area occupied by cells at 24 hours. The test was repeated with conditioned medium collected at 24, 48 and 72 hours.

In Vitro Proliferation Assay

[0095] Endothelial cells (HMEC) and fibroblasts (NHDF) were seeded in a 96 well plate at a density of 2,000 cells/well and left to adhere. The culture medium was replaced with DMEM to leave overnight. Then, cells were stimulated with DMEM alone (negative control), or Endogro (Lonza) for HMEC or DMEM with 10% FBS for NHDF as positive control or DMEM in the presence of hydrogel alone or hydrogel containing EVs (dose 10.sup.9 EVs) for 72 hours (. Each condition was performed in quadruplicate. Then 10 μl of BrdU labeling solution (BrdU colorimetric assay, Roche) were added to each well and the plate was incubated overnight. The effects of the stimuli were analyzed after 24 hours of incubation. The development of the assay was performed according with manufacturer's instruction. Absorbance was measured by an ELISA reader at 370 nm. The mean absorbance for each condition was calculated. Absorbance is directly proportional to proliferation rate. All mean absorbances were normalized for the mean of untreated control (CTR−), used as reference samples. The results show the relative proliferation rate compared to negative control, which is equal to 1.

In Vivo Experiments

[0096] Normal male (BALB) or diabetic NOD.CB17-Prkdcscid/NCrCrl (NGS) male mice at 9 weeks old were anesthetized and back cutaneous hair was removed by electrical shaving. Diabetes was induced on NOD.CB17-Prkdcscid/NCrCrl (NGS) mice by intraperitoneal injection of streptozotocin (40 mg/kg) for 4 days and animals were treated after about 20 days of diabetes, measured as glycemia>200. Four 6-mm diameter full-thickness skin wounds were created on each side of the midline using a disposable Biopsy Punch (PMD Medical). Each animal received different treatments on each wound. The day of the wounding was counted as day 0. Animals were monitored at different times (day 0, 3, 6 normal mice; and 0, 3, 6, 10, and 14 for diabetic mice) by wound measurement. At each time, the wounds received a fresh application of the treatments. At the last timepoint, mice were sacrificed and back skin wounds were collected for histological analysis. Histological analysis was performed using hematoxylin and eosin staining and was used to measure the wound width, the percentage of re-epithelization as the percentage of newly formed epithelium in the wound, the epithelial thickness of the wounds, using Imagej software.

Statistical Analysis

[0097] Data analysis was carried out with the software Graph Pad 8, demo version. Results are expressed as mean±standard error (SEM). One-way analysis of variance (ANOVA) was used to substantiate statistical differences between groups, while Student's t-test was used for comparison between two samples. We used p<0.05 as a minimal level of significance.

RESULTS/EXAMPLES

Example 1

[0098] For their experiments, the inventors used orange-derived EVs. EVs were isolated by differential ultracentrifugation or tangential flow filtration. Results from the characterization of EVs obtained with the two methods were identical. Orange-derived EVs displayed a size in diameter in the range of 25-400 nm by Nanosight analysis, with a mean size in diameter of approximately 200 nm (FIG. 1A). Orange-derived EVs showed a round morphology delimited by an electrondense membrane as demonstrated by transmission electron microscopy (TEM) analysis (FIGS. 1B and 1E). TEM analysis confirmed the mean size of EVs in diameter is approximately 200 nm.

[0099] In order to examine the content of orange-derived EVs, the protein and RNA content of EVs was measured. The protein concentration is shown in FIG. 1C and demonstrate a heterogeneous protein content with a mean of 600 ng of proteins for 10.sup.9 EVs. The RNA concentration is shown in FIG. 1D and demonstrate a variable RNA content with a mean of 22 ng of RNA for 10.sup.10 EVs.

[0100] Further analysis demonstrated that orange-derived EVs contain proteins characteristic of vesicles, such as HSP70, HSP80, glyceraldehyde-3-phosphate dehydrogenase (G3PD) and fructose-bisphosphate aldolase 6 (FBA6); and plant proteins, such as Patellin-3-like and clathrin heavy chain.

[0101] In addition, the lipid content of orange-derived EVs revealed a cargo of lipids, including 24-Propylidene cholesterol, Beta sitosterol, Glycidol stearate, Dipalmitin, Campesterol, Eicosanol, Eicosane, Hexadecane, Hexadecanol, Octadecane, Octadecanol, Tetradecane, Tetradecene, Valencene and Stearate.

Example 2

[0102] The present inventors carried out experiments in order to assess that hydrogel preserves and releases orange-derived EVs in a controlled manner. For this purpose, the amount of EVs and the EV protein released in the microenvironment around the pharmaceutical composition was measured at increasing times (FIG. 2). Briefly, the pharmaceutical composition of the invention was put in contact with saline solution and the release of EVs was measured in the saline solution at increasing time points. This analysis clearly demonstrated that hydrogels composed of either carboxymethylcellulose or collagen allow the release of EVs as particles already after 30 minutes (FIG. 2 A, C). Moreover, the quantification of proteins confirmed that EVs are released from hydrogels already after 30 minutes (FIG. 2 B, D). EVs release was already detectable after 30 minutes, increased over time, from 24 to 48 hours, and was still present after 72 hours. Similar data were obtained also with other hydrogels (ethylcellulose, chitosan, gelatin, PEG). Taken together, these results demonstrated that all hydrogels are able to preserve orange-derived EVs and allow their controlled release at increasing timings when in contact with cell or skin surface, starting already after 30 minutes of treatment and at least up to 3 days.

Example 3

[0103] To perform their biological actions, orange-derived EVs have to be released by hydrogels and enter into target cells. In order to evaluate the functionality of EVs incorporated in the pharmaceutical composition, experiments were conducted to analyze the ability of these vesicles to enter (uptake) into target cells at different time points (6, 24, 48, and 72 hours) (FIG. 3). For this type of experiment, increasing doses of orange-derived EVs (10.sup.8 EVs, 10.sup.9 EVs, 10.sup.10 EVs) labelled with a fluorochrome were included in the pharmaceutical composition.

[0104] The compositions were put into contact with target cells and EVs uptake in target cells (endothelial cells) was quantified by cytofluorimetric analysis. The uptake rate was evaluated by comparing the pharmaceutical composition of the invention with EVs alone (without hydrogel) and target cells alone (negative control, CTR−). The results shown in FIG. 3 demonstrates that both the EVs included in the pharmaceutical composition and the EVs alone enter into target cells. Interestingly, the uptake of EVs alone was significantly higher compared to EVs included in the hydrogel after 6 and 24 hours of treatment. However, at increased time, i.e. at 48 and 72 hours, the trend was inverted and the combination of EVs with hydrogel allowed a significantly higher uptake in target cells. This effect increased with time, reaching the maximum peak at 72 hours. Taken together these data demonstrate that hydrogel can preserve EVs from degradation and allows a more efficient and prolonged release of these vesicles as well as their uptake into target cells, compared to EVs alone, already after 24 hours. Similar data were obtained with different hydrogels (carboxymethylcellulose, collagen, ethylcellulose, chitosan, gelatin, PEG).

Example 4

[0105] In order to investigate the therapeutic potential of the pharmaceutical composition of the invention, in vitro functional tests were performed. In particular, the ability of the pharmaceutical composition including orange-derived EVs and hydrogel to promote endothelial cell angiogenesis and migration, as well as fibroblasts migration was tested in vitro.

[0106] The pharmaceutical composition containing hydrogel and orange-derived EVs was used to stimulate cells in vitro for 24, 48 and 72 hours. The results showed herein are obtained employing two kinds of hydrogels, carboxymethylcellulose hydrogel (H1) and collagen hydrogel (H2). Similar results were obtained with other types of hydrogels, including gelatin, chitosan, PEG and ethylcellulose.

[0107] EVs pro-angiogenic ability was tested by means of a tube formation assay, seeding endothelial cells on Matrigel (FIG. 4) with the pharmaceutical composition containing hydrogel and orange-derived EVs or hydrogel alone or EVs alone, and incubating for 24, 48 and 72 hours. Endothelial cells were stimulated with orange-derived EVs alone (EV), hydrogel alone (H1, H2), or with the pharmaceutical composition comprising hydrogel and orange-derived EVs (H1+EV, H2+EV). All conditions were tested after EVs release for 24, 48, or 72 hours. A significant increase in total tube length was observed with EVs alone (EV) compared to untreated control (CTR−), and with the composition comprising hydrogel and EVs (H1+EV, H2+EV) compared to hydrogel alone (H1 and H2). Moreover, the composition comprising hydrogel and EVs (H1+EV, H2+EV) was shown to be significantly more effective in promoting angiogenesis than EVs alone (EV) and hydrogel alone (H1, H2). Such result was observed at each time point (24, 48 and 72 hours). This demonstrates that a composition comprising orange-derived EVs and hydrogel has a synergistic effect in promoting angiogenesis on endothelial cells and such pharmaceutical composition is significantly more effective than hydrogel alone or orange-derived EVs alone already after one day (24 hours) of treatment. Said effect is maintained over at least 3 days of treatment.

[0108] The effect of the pharmaceutical composition of the invention on endothelial cells and fibroblasts migration was assessed by performing a scratch on a monolayer of cells and measuring the migration of cells after 24 hours. The pharmaceutical composition containing hydrogel and orange-derived EVs was used to stimulate cells in vitro for 24, 48 and 72 hours.

[0109] The results shown are obtained using two kinds of hydrogels, i.e. carboxymethylcellulose hydrogel (H1) and collagen hydrogel (H2). Similar results were obtained with other types of hydrogels, including gelatin, chitosan, PEG and ethylcellulose.

[0110] The present inventors observed a significantly higher migration rate of endothelial cells (FIG. 5) and fibroblasts (FIG. 6) using the combination of orange-derived EVs and hydrogel (H1+EV and H2+EV) compared to EVs alone (EV), which are also effective in promoting cell migration but at a statistically significant lower extent, and hydrogel alone (H1 and H2), which is not effective and comparable to the untreated control. All conditions were tested after EVs release for 24, 48 or 72 hours and on both kind of cells. The experiments demonstrate that orange-derived EVs and hydrogel have a synergistic effect not only in the formation of new blood vessels but also in promoting migration of endothelial cells and such pharmaceutical composition is significantly more effective than hydrogel alone or orange-derived EVs alone already after one day (24 hours) of treatment. Said effect is maintained over at least 3 days of treatment (FIG. 5). In fact, the migration of endothelial cells contributes to the formation of new blood vessels (angiogenesis) and this data support the pro-angiogenic activity of the pharmaceutical composition of the invention.

[0111] Similar results were obtained on fibroblast cells (FIG. 6), demonstrating that the pharmaceutical composition comprising orange-derived EVs and hydrogel exploits a therapeutic effect also by promoting other cell types present in the lesion. Fibroblast, in fact, are involved in the regenerative process and the promotion of their migration contributes to accelerate the wound closure. The fact that the pharmaceutical composition of the invention can act on multiple cells in the lesion site supports its beneficial effect on accelerating tissue regeneration and wound closure.

[0112] In addition, the absence of toxicity of different types of hydrogel on endothelial cells and fibroblasts was assessed in vitro by a proliferation assay (FIG. 7). The present inventors observed that carboxymethylcellulose hydrogel (H1) or collagen hydrogel (H2) do not affect and do not reduce the proliferation rate of fibroblasts (Figure, upper panel on the right) and endothelial cells (FIG. 7, upper panel on the left). Similar results were also obtained with different types of hydrogel, including gelatin, chitosan, ethylcellulose and PEG. The toxicity of a compound is revealed by the reduction of proliferation of cells. The percentage of proliferation of fibroblasts and endothelial cells stimulated with H1 or H2 is comparable to the basal condition (unstimulated control, CTR−). This data demonstrate that hydrogels are not toxic and can be used in combination with orange-derived EVs for therapeutic purposes.

[0113] In addition, the present inventors observed that orange-derived EVs released from hydrogel (H1+EV and H2+EV) significantly increased proliferation of endothelial cells compared to orange-derived EVs alone (EV) or hydrogel alone (H1 and H2) (FIG. 7, down panel). In the composition of the invention, orange-derived EVs and hydrogel have a synergistic effect in promoting proliferation of endothelial cells compared to untreated control (CTR−), and said composition is significantly more effective than hydrogel alone or orange-derived EVs alone. The results shown are obtained by employing two kinds of hydrogels, carboxymethylcellulose hydrogel (H1) and collagen hydrogel (H2). Similar results were obtained with other types of hydrogels, including gelatin, chitosan and ethylcellulose.

[0114] All experiments illustrated in example 4 confirm that EVs can be gradually released from hydrogel and maintain their biological effect at each time point (24, 48, and 72 hours). The pharmaceutical composition of the invention improves both hydrogel and EV biological activity already after few hours (6 hours) of treatment. Said effect is maintained over at least 3 days of treatment. In fact, the pharmaceutical composition containing orange-derived EVs and hydrogel is effective already after few hours of treatment and maintains the effectiveness for several days and guarantees a gradual distribution of EVs to recipient cells.

[0115] All results shown in experimental example 4 demonstrate that the therapeutic activity of the pharmaceutical composition comprising orange-derived EVs and hydrogel is mainly mediated by its pro-angiogenic activity. In fact, the combination of EVs and hydrogel promote the proliferation and migration of endothelial cells, and induce a higher formation of new blood vessels. Moreover, the combination of EVs and hydrogel acts also on other cell types involved in the lesion regeneration and present in the lesion, such as fibroblast cells. By promoting the migration of fibroblast cells, the combination of EVs and hydrogel allows fibroblast to migrate to the lesion site and deposit collagen, contributing to the promotion of wound closure.

Example 5

[0116] To evaluate the therapeutic effect of the pharmaceutical composition, different hydrogels were combined with orange-derived EVs and their activity was tested on an in vivo model of ulcers (FIG. 8). Ulcers were induced in a mice model and treated with carboxymethylcellulose (H1), carboxymethylcellulose and EVs (H1+EV), chitosan (H2), chitosan and EVs (H2+EV). After 3 days the medication was changed and the therapeutic effect was evaluated after 6 days of treatment. The effect was measured as percentage of wound area in comparison to the initial wound area at time 0 before treatment. Data clearly demonstrated that both hydrogels alone (carboxymethylcellulose and chitosan) were able to slightly reduce the wound area in comparison to untreated wound (CTR−) (FIG. 8). Importantly, the combination of each hydrogel with orange-derived EVs significantly increased their therapeutic effect (H1+EV versus H1, and H2+EV versus H2). The results confirm the therapeutic efficacy of the pharmaceutical composition comprising hydrogel and orange-derived EVs. More specifically, this experiment demonstrates that the combination of hydrogel with EVs is more efficient in accelerating wound closure than hydrogel alone. For this reason, the pharmaceutical composition of the invention is particularly useful for the treatment of several kinds of lesions where the angiogenesis favors and accelerates the healing process, such as burns, fistulae, psoriasis, keratosis, keratitis, fissures, traumatic ulcers, mucosal lesions (such as traumatic lesions due to prothesis and such, mouth, decubital, genital mucosal lesions), dermatitis (including acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, dermatitis herpetiformis), cellular damage induced by pro-apoptotic drugs aimed to treat pre-cancerous lesions (e.g. actinic keratosis).

[0117] With the aim to further analyze the therapeutic activity of the pharmaceutical composition of the invention, the regenerative effect of said composition was evaluated on in vivo mice model of diabetic ulcers (FIG. 9). Diabetic ulcers, in fact, are more difficult to close than normal ulcers because of the diabetic condition that impairs tissue regeneration ability. The wound area was expressed as percentage of wound area in respect with the original area at time 0 before treatment (FIG. 9A). The wounds were performed on diabetic mice and medications were changed every three days. The wound area measurement was performed at time 0 and after 3, 6, 10 and 14 days. The analysis clearly demonstrated that the treatment with the pharmaceutical composition of the invention was the most effective in accelerating wound closure in comparison to untreated wound (CTR−) and hydrogel alone (hydrogel) at each timepoint (FIG. 9A).

[0118] Moreover, the treatment with the combination of hydrogel and EVs was the only able to induce a significant reduction of wound area already after 3 days of treatment (FIG. 9A). The histological analysis of the tissues confirmed the higher therapeutic effect of the composition of the invention in comparison with hydrogel alone and untreated wounds (CTR−) (FIG. 9 B, C, D). Tissue sections stained with hematoxylin and eosin were used to measure wound width (FIG. 9B), the percentage of re-epithelization (FIG. 9C) and the epithelial thickness (FIG. 9D).

[0119] The wound width was significantly reduced upon treatment with the combination of hydrogel and EVs compared with the treatment with hydrogel alone or untreated control (FIG. 9B).

[0120] The analysis of the percentage of re-epithelization demonstrated that the treatment with the combination of hydrogel and EVs induced a significantly higher formation of new epithelium in the wound compared with the treatment with hydrogel alone or untreated control (FIG. 9C). The new epithelium is essential to the wound closure.

[0121] Finally, the tissue section subjected to the treatment with the combination of hydrogel and EVs presented a higher epithelial thickness in comparison to the treatment with hydrogel alone or untreated control (FIG. 9D), showing that the regeneration is more efficient and allows the restoration of a normal higher epithelial thickness.

[0122] Taken together, all the data confirm the higher therapeutic effect of the pharmaceutical composition of the invention in accelerating wound closure of diabetic ulcers in comparison to the treatment with hydrogel alone, already after 3 days of treatment. This demonstrates that such pharmaceutical composition is particularly useful in the treatment of several kinds of lesions where the natural angiogenetic processes are impaired or compromised, as in case of diabetic foot ulcers, diabetic mucosal ulcers, dysmetabolic ulcers, or in presence of ischemic damages, including ischemic ulcers, such as pressure ulcers, arterial ulcers, venous ulcers, ischemic ulcers, exudative ulcers, traumatic ulcers, and other mucosal lesions.