PLANT-DERIVED EXTRACELLULAR VESICLE (EVS) COMPOSITIONS AND USES THEREOF
20220142938 · 2022-05-12
Inventors
- Giovanni CAMUSSI (TORINO, IT)
- Chiara GAI (Bagnasco (Cuneo), IT)
- Margherita Alba Carlotta POMATTO (Piossasco (Torino), IT)
Cpc classification
International classification
Abstract
A composition comprising a population of plant-derived extracellular vesicles (EVs) having a diameter ranging from 10 to 500 nm and showing pro-angiogenic, and anti-bacterial activity, for use in therapeutic applications is provided. A method for loading one or more negatively-charged biologically-active molecules into the population of plant-derived extracellular vesicles (EVs) is also provided.
Claims
1. A method of promoting angiogenesis and inhibiting bacterial growth in a subject in need thereof, said method comprising administering to said subject a composition comprising a population of plant-derived extracellular vesicles (EVs), the plant-derived extracellular vesicles (EVs) in said population being delimited by a lipid bilayer membrane and having a diameter ranging from 10 to 500 nm, a protein content in the range of from 1 to 55 ng/10.sup.9 EVs, and an RNA content in the range of from 10 to 60 ng/10.sup.10 EVs, wherein said subject is affected by a disease or condition selected from the group consisting of ulcers, dermatites, corneal damages, eye diseases, mucosal lesions and infective lesions.
2. The method of claim 1, wherein the ulcers are selected from the group consisting of pressure ulcers, arterial ulcers, venous ulcers, diabetic ulcers, ischemic ulcers, exudative ulcers, dysmetabolic ulcers, traumatic ulcers, burns, fistulae, fissures and traumatic ulcers.
3. The method of claim 1, wherein the dermatites are selected from the group consisting of acne, eczema, seborrheic dermatitis, atopic dermatitis, contact dermatitis, dyshidrotic eczema, neurodermatitis, dermatitis herpetiformis, keratosis, keratitis and psoriasis.
4. The method of claim 1, wherein the corneal damages and eye diseases are selected from the group consisting of ulcers, traumatic injuries, degeneration injuries, abrasions, chemical injuries, contact lens problems, ultraviolet injuries and/or keratitis, conjunctivitis and dry eye.
5. The method of claim 1, wherein the mucosal lesions are selected from the group consisting of traumatic lesions due to prosthesis, diabetic, mouth, decubital or genital mucosal lesions.
6. The method of claim 1, wherein the infective lesions are selected from the group consisting of virus infections, herpes infections and bacterial infections.
7. The method of claim 1, wherein the EVs have a diameter in the range of from 20 to 400 nm.
8. The method of claim 1, wherein the EVs population is derived from one or more plants selected from the group consisting of: the family Rutaceae, the family Rosaceae, the family Vitaceae, the family Brassicaceae, the family Selaginellaceae, the family Asteraceae, the family Oleaceae, the family Xanthorrhoeaceae, the family Nelumbonaceae, the family Araliaceae, the family Lamiaceae, the family Hypericaceae, the family Pedaliaceae, the family Ginkgoaceae, the family Piperaceae, and the family Rubiaceae.
9. The method of claim 8, wherein the EVs population is derived from one or more plants selected from the group consisting of: the genus Citrus, including lemon, orange, tangerine, clementine, bergamot, pompia; Malus pumila; Vitis vinifera; Anastatica hierochuntica; Selaginella lepidophylla; Calendula officinalis; Olea europaea; Aloe vera; Nelumbo; the subgenus Panax; Lavandula; Hypericum perforatum; Harpagophytum procumbens; Ginkgo biloba; Piper kadsura, Piper futokadsura; and Hedyotis diffusa.
10. The method of claim 9, wherein the EVs are native EVs or EVs loaded with one or more negatively-charged biologically-active molecules selected from the group consisting of drugs, nucleic acid molecules and lipophilic molecules, including lipophilic vitamins, wherein the nucleic acid molecules are selected from the group consisting of miRNA, mRNA, tRNA, rRNA, siRNA, regulating RNA, non-coding and coding RNA, DNA fragments and DNA plasmids.
11. (canceled)
12. The method of claim 1, wherein the composition is formulated as a pharmaceutical composition for topic application, local injection or oral administration, or is formulated as a food supplement preparation.
13. A method for loading one or more negatively-charged biologically active molecules into a population of plant-derived extracellular vesicles (EVs), comprising the steps of: (i) contacting and co-incubating a population of plant-derived extracellular vesicle (EVs) as defined in claim 1, with a polycationic substance and one or more negatively-charged biologically active molecules; and (ii) purifying the loaded EVs obtained in step (i) from the polycationic substance and the remaining one or more free negatively-charged active molecules.
14. The method of 13, wherein the polycationic substance is selected from the group consisting of protamine, polylisine, cationic dextrans and combinations thereof.
15. The method of claim 13, wherein the one or more negatively-charged biologically active molecules are selected from the group consisting of drugs, nucleic acid molecules and lipophilic molecules, including lipophilic vitamins, wherein the nucleic acid molecules comprise miRNA, mRNA, tRNA, rRNA, siRNA, regulating RNA, non-coding and coding RNA, DNA fragments and DNA plasmids.
Description
EXAMPLES
[0054] 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:
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MATERIALS AND METHODS
Extracellular Vesicles Isolation
[0074] Extracellular vesicles were isolated from plant juice. Fruits were squeezed and the juice was sequentially filtered using decreasing order of pores to remove fibers. EVs were then purified with ultracentrifugation. For differential ultracentrifugation the juice was first centrifuged at 1,500 g for 30 minutes to remove debris and other contaminants. Then, EVs were purified by a first centrifugation at 10,000 g followed by ultracentrifugation at 100,000 g for 1 hour at 4° C. (Beckman Coulter Optima L-90K, Fullerton, Calif., USA). The final pellet was resuspended with phosphate buffered saline added with 1% DMSO and filtered with 0.22 micrometer filters to sterilize. Extracellular vesicles were used or stored at −80° C. for long time. Purified EVs were characterized by nanoparticle tracking analysis and electron microscopy.
Nanoparticle Tracking Analysis (NTA)
[0075] Nanoparticle tracking analysis (NTA) was used to define the EV dimension 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 and engineered EVs were diluted (1:1000 and 1:200, respectively) 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
[0076] 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 min. 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.
Cell Culture
[0077] 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, Basel, Switzerland).
[0078] Immortalized human keratinocytes (HaCat) were cultured with DMEM (Lonza, Basel, Switzerland) supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, Waltham, Mass., USA) at 37° C. with 5% CO2. The cells were seeded at density 3.5×102 cell/cm2, using 1 ml of medium/cm2 and subcultured when cell confluence was 70-80%. Briefly, flasks were washed with HEPES buffer saline solution, incubated with trypsin solution for 6 min and then trypsin was neutralized with medium containing 10% FBS. If the cells were not completely detached within 7 min, incubation with trypsin was repeated.
[0079] Endothelial cells derived from human renal carcinoma (TECs) were isolated from specimens of clear-cell type renal cell carcinomas using anti-CD105 Ab coupled to magnetic beads by magnetic cell sorting using the MACS system (Miltenyi Biotec, Auburn, Calif., USA). TEC cell lines were established and maintained in culture in Endogro basal complete medium (Merck Millipore, Billerica, Mass., USA). TEC were previously characterized as endothelial cells by morphology, positive staining for vWF antigen, CD105, CD146, and vascular endothelial-cadherin and negative staining for cytokeratin and desmin.
Protein Analysis
[0080] 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, Mo., USA). The protein content was quantified by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Mass., 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).
In Vitro Scratch-Test Assay
[0081] Keratinocyes (HaCaT) and endothelial cells (HMEC) were seeded at a density of ˜50×10.sup.3 cells/well in 24-well plates in DMEM supplemented with 10% FCS. When the cells reached complete confluence, they were starved with medium without FCS overnight. The following day, scratch wounds were created with a sterile tip. Prior to stimulation (t=0), micrographs of the well were obtained using a Leica microscope (Leica, Wetzlar, Germany). The cells were then stimulated with DMEM with 10% FBS or EGF as positive controls (CTR+) or EVs (10,000 (10 k) or 50,000 (50 k) or 100,000 (100 k) EVs/target cells). The ‘wound closure’ phenomenon was monitored for 48 hr using the Leica microscope and images were analyzed by ImageJ software (Bethesda, Md., USA) observing the decrease of the wound area in cells stimulated with EVs in comparison to cells not stimulated with EVs.
In Vitro Angiogenic Assay
[0082] In vitro formation of capillary-like structures was studied on growth factor-reduced Matrigel (BD Bioscience, Franklin Lakes, N.J., USA) in 24-well plates. HMEC or TECs (25,000 cells/well) were seeded onto Matrigel-coated wells in DMEM or EndoGRO MV-VEGF medium, respectively, containing EVs (50,000 or 30,000 EVs/target cells). Treatments were performed in triplicate. Cell organization onto Matrigel was imaged with a Nikon Eclipse TE200. After incubation for 24 h, phase-contrast images (magnification, ×10) were recorded and the total length of the network structures was measured using ImageJ software. The total length per field was calculated in five random fields and expressed as a percentage to the respective control.
In Vitro Proliferation Assay
[0083] HMEC were plated in a 96 well plate at a density of 2,000 cells/well and left to adhere.
[0084] The culture medium was replaced with DMEM to leave overnight. Then, the plate was closed in a hypoxic chamber filled with the following mixture of gas: 5% CO2, 1% 02, 94% N. The hypoxic chamber was placed in CO2 incubator for 24 h. Then the plate was removed from hypoxic chamber, cells were treated with DMEM alone (CTR), positive control (10 ng/ml of EGF, CTR+), increasing doses of native plant derived EVs (10,000, 30,000, 50,000, and 100,000 EVs/cell). Each condition is 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 following procedure was performed according with BrdU assay 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 CTR, which is equal to 1.
Measurement of EV Charge
[0085] The analysis was performed by Zeta-sizer nanoinstrument (Malvern Instruments, Malvern, UK). All samples were analyzed at 25° C. in filtered (cutoff=200 nm) saline solution. Zeta-potential (slipping plane) is generated at x distance from the particle indicating the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion. Negative Zeta-potential indicates a high grade of dispersion across the particles.
Engineering of EVs with Protamine
[0086] EVs were mixed with protamine (1.0 μg/ml) (Sigma-Aldrich, St. Louis, Mo.) and co-incubated at 37° C. for 5-30 minutes to allow the binding to EV surface. Various doses of protamine (1.0 ng/ml, 0.1 ng/ml, 0.01 ng/ml) was used. Next, synthetic miRNA molecules (100 pmol/ml) (miRNA mimics or antimiR, Qiagen, Hilden, Germany), negative-charged, were added to the mixture and co-incubated at 37° C. for 3 hours. The mixture was diluted with saline solution and stored at 4° C. overnight. Ultracentrifugation with 100,000 g for 2 hours at 4° C. (Beckman Coulter Optima L-90K, Fullerton, Calif., USA) allowed the elimination of free miRNA and protamine molecules, and the pellet was resuspended with phosphate buffered saline added with 1% DMSO and filtered with 0.22 micrometer filters to sterilize.
RNAse Treatment
[0087] EVs were treated with RNAse A (Thermo Fisher Scientific, Waltham, Mass., USA), using a concentration of 0.2 μg/ml, for 30 minutes at 37° C. The RNAse inhibitor (Thermo Fisher Scientific, Waltham, Mass., USA) was used to stop the reaction as described by the manufacturer's protocol and EVs were washed by ultracentrifugation at 100,000 g for 1 hour at 4° C. (Beckman Coulter Optima L-90K, Fullerton, Calif., USA).
Confocal Microscopy
[0088] For EV incorporation, EVs were labeled with a red membrane fluorescent dye for membranes, PKH26 (Sigma-Aldrich, St. Louis, Mo.) and engineered with a green fluorescent (FITC) labeled siRNA (Qiagen, Hilden, Germany). Labeled-EVs were used to treat TEC plated in 24-well plates (30,000 cells/well) for different timing (30 min, 1 h, 3 h, 6 h, 18 h, 24 h). The uptake of EVs was analyzed using confocal microscopy (Zeiss LSM 5 Pascal, Carl Zeiss, Oberkochen, Germany).
RNA Extraction
[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).
MiRNA and mRNA Analysis by qRT-PCR
[0090] For miRNA analysis, miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) was used. Briefly, RNA samples were reverse transcribed using the miScript Reverse Transcription Kit and the cDNA was then used to detect and quantify miRNAs of interest. Experiments were run in triplicate using 3 ng of cDNA for each reaction as described by the manufacturer's protocol (Qiagen). For mRNA analysis, cDNA was obtained using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Five nanograms of cDNA were added to SYBR GREEN PCR Master Mix (Applied Biosystems) and run on a 96-well QuantStudio 12K Flex Real-Time PCR (qRT-PCR) system (Thermo Fisher Scientific, Waltham, Mass., USA). GAPDH was used as a housekeeping gene. Fold change (Rq) in miRNA expression among all samples was calculated as 2.sup.−ΔΔCt compared to control samples.
Extracellular Vesicle Electroporation
[0091] Electroporation was performed on a Neon Transfection System (Thermo Fisher Scientific, Waltham, Mass., USA) following manufacturer's protocol. For every electroporation, the sample volume was fixed at 200 μL.
In Vivo Experiments
[0092] Ingenol mebutate (ingenol-3-angelate, Picato) was used for the topical treatment of pre-cancerous lesions induced by actinic keratosis. The drug was applicated for 3 days on actinic keratosis lesions removing the pre-cancerous lesion but inducing the formation of tissue apoptotic lesions. After the treatment, native orange-derived EVs were topically administered on one tissue lesion, whereas one untreated lesion on the same patient was used as control. The effect of plant-derived EVs was evaluated after 3-7 days of treatment.
Statistical Analysis
[0093] Data analysis was carried out with the software package Graph Pad Demo version 6.01. 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. p: *<0.05, **<0.01, ***<0.005, ****<0.001.
RESULTS/EXAMPLES
Example 1
[0094] To investigate the feasibility of the method of the present invention, the inventors used native EVs purified from different plants, including lemon, orange, grape, Anastatica hierochuntica and Selaginella lepidophylla. EVs were isolated by microfiltration and differential ultracentrifugation or tangential flow filtration and they displayed a size in the range of 25-350 nm by Nanosight analysis (
[0095] In order to examine the content of plant-derived EVs, the protein content of native EVs isolated from apple, orange, lemon, grape, Anastatica hierochuntica and Selaginella lepidophylla was measured by using the BCA assay. The results are shown in the
TABLE-US-00001 TABLE 1 EV protein content. mean protein ng/E+08 EV SD Apple EV 12.06 0.71 Grape EV 47.93 2.17 Lemon EV 63.13 4.55 Orange EV 143.74 49.21 AH EV 210.58 11.05 SL EV 504.58 13.06
[0096] Moreover, deeper analysis demonstrated that native plant-derived EVs contain proteins characteristic of vesicle, 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.
[0097] In addition, the lipid content of native plant-derived EVs revealed a cargo of lipids variable in amount depending on the plant, including 24-Propylidene cholesterol, Beta sitosterol, Glycidol stearate, Dipalmitin, Campesterol, Eicosanol, Eicosane, Hexadecane, Hexadecanol, Octadecane, Octadecanol, Tetradecane, Tetradecene, Valencene and Stearate.
Example 2
[0098] The ability of native plant-derived EVs to promote endothelial cell migration and angiogenesis was tested. By performing a scratch on a monolayer of endothelial cells, the present inventors observed a significantly higher migration rate of endothelial cells using different doses of native plant-derived EVs compared to negative control (CTR) (
[0099] In addition, the capacity of native plant-derived EVs to promote vessel formation was evaluated by angiogenesis assay to verify their effect on vessel formation in vitro. Results showed in
[0100] Moreover, the ability of native plant-derived EVs to promote cell proliferation on endothelial cells was tested after hypoxic damage in vitro. Different doses of native orange-derived EVs significantly promoted cell proliferation rate compared to negative control (CTR), demonstrating the beneficial effect of EVs on endothelial cells (
Example 3
[0101] Native plant-derived EVs were analyzed for their anti-microbial activity. Most of the pathogenic bacteria associated with infected lesions in humans need a pH value>6 and their growth is inhibited by lower pH values. Native plant-derived EVs show a low pH ranging from 4 to 5. Applying native plant-derived EVs to the lesion surface creates an acidic environment unfavorable for the growth and the multiplication of bacterial pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella spp., Proteus spp., Citrobacter spp., S. epidermidis, S. pyogenes, streptococci, and enterococci.
[0102] The application of plant-derived EVs is effective in clearing bacterial pathogens from contaminated or infected lesions by lowering the pH. In fact, the treatment with native plant-derived EVs restored the average surface pH of the skin (normally ranging from about 4.2 to 5.6) controlling the topic infection increasing the natural antimicrobial activity of the skin. Moreover, the decrease of pH has been demonstrated to enhances the antibacterial activity of other drugs against both gram-positive and gram-negative bacteria.
Example 4
[0103] Native plant-derived EVs were analyzed for their therapeutic effect in vivo. Native plant-derived EVs were used to treat a skin damage induced by Ingenol mebutate (ingenol-3-angelate, Picato) in a human volunteer. This substance is an inducer of cell death and was used for the topical treatment of a pre-cancerous lesion, the actinic keratosis. Results illustrated in
Example 5
[0104] In order to modify plant-derived EVs with a method based on charge interaction, native plant-derived EVs were analyzed for their surface charge. The analysis of Zeta potential was performed on different preparations showing that native EVs derived from orange display a negative charge of −13.59±1.83 mV (
[0105] To investigate the method to load negatively-charged molecules on EV surface using a charged positive linker, loaded plant-derived EVs modified using protamine were mixed with miRNA molecules as illustrated in
Example 6
[0106] In order to understand whether loaded molecules could be efficiently transferred to target cells, the transfer of loaded molecules mediated by loaded plant-derived EVs to target cells was analyzed. Firstly, orange derived EVs were labeled with PKH26 (red fluorescent dye) and engineered with FITC fluorescent labeled synthetic siRNA. Loaded EVs were co-incubated with human endothelial cells derived from renal carcinoma (TECs) at different time points (30 min, 1 h, 3 h, 6 h, 18 h, 24 h). Analysis by confocal microscopy revealed that small nucleic acids present on EV's surface did not alter their uptake by target cells.
Example 7
[0107] In order to deeper investigate the use of a positive charged linker, different doses of protamine were evaluated to load plant-derived EVs. Positively-charged molecules, such as protamine, can form micelles around negatively-charged molecules, such as miRNAs. Then, orange derived EVs were co-incubated with decreasing doses of protamine and a representative negatively-charged molecule, the miRNA miR-221-3p. The size analysis of EVs performed by Nanosight measured the mean and mode size of loaded EVs. Results showed that the initial amount of protamine (1.0 μg/ml) induced an increase in both mean and mode size, with a significantly difference in mean (
Example 8
[0108] As a representative example of the modification method to further improve the native activity of plant-derived EVs, the modification of plant-derived EVs was used to improve their native activity in promoting wound closure of keratinocytes. In these experiments, loaded orange derived EVs were engineered with miRNA miR-21 using protamine as positively-charged linker. Human keratinocytes were treated with three different doses of native EVs, loaded EVs incubated with protamine alone (EV+P) as control, and loaded EVs with protamine and miR-21 (EV+miR-21). EV+P and EV+miR-21 was used at the intermediate dose (50 k). The measurement of the wound closure in each condition was used as parameter of EV activity. The graph in the
Example 9
[0109] The modification method can also be used to change the biological activity of native plant-derived EVs. Plant-derived EVs can be engineered with negatively-charged molecules that provide different or new biological effects. As a representative example, orange derived EVs were modified with different anti-angiogenic miRNAs and their ability to inhibit angiogenesis was evaluated by angiogenesis assay in vitro on TEC cells. In particular, loaded EVs were engineered with mimics for anti-angiogenic miRNAs (miR-221, miR-223, miR-145) and anti-miRNAs for pro-angiogenic miRNAs (miR-29, miR-126, miR-31) and their effect on endothelial cell vessel formation was evaluated after 24 hours of treatment (
Example 10
[0110] The therapeutic effect of loaded plant-derived EVs was evaluated using different doses of a positively-charged linker. As a representative example, loaded orange derived EVs were engineered with two different doses of protamine (1 μg/ml, 1 ng/ml) and three different antiangiogenic miRNAs (antimir-29, miR-145 and miR-221-3p). Loaded EVs were used to stimulate endothelial cells (TEC) for 24 hours and their activity was evaluated by angiogenesis assay. The results showed in
Example 11
[0111] The modification method of plant-derived EVs with negatively-charged molecules can be further improved by transfection protocols. In fact, the bound of negatively-charged molecules (e.g. miRNAs) to EV surface through a positively-charged linker (e.g. protamine) could facilitate their entrance inside EVs. The closeness of negatively-charged molecules to EVs could increase their loading after transfection protocols such as electroporation, as illustrated in
[0112] To test this hypothesis, orange-derived EVs were modified using protamine and the miR-221-3p and their capacity to inhibit vessel formation was evaluated. The