Method for the preparation of nanometric phytovesicles and uses thereof

Abstract

The present invention relates to the field of plant-derived cellular vesicles, and in particular of nanovesicles obtained from plant protoplasts. A method for their preparation and uses thereof in the medical, cosmetic and nutraceutical fields are described.

Description

[0001] METHOD FOR THE PREPARATION OF NANOMETRIC PHYTOVESICLES AND USES THEREOF ★★★★★★

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of plant-derived cellular vesicles, and in particular of nanovesicles obtained from plant protoplasts called nanometric PhytoVesicles (Phyto-NanoVes). A method for their preparation and uses thereof in the medical, cosmetic, food, and nutraceutical fields are described.

[0005] STATE OF THE ART

[0006] In recent decades, numerous nanomaterials have been exploited and applied from diagnostics to pharmacology as drugs and / or as carriers, in the latter case with the aim of encapsulating drugs or phytochemicals and delivering them to specific organs and cells, without degradation or negative side effects.

[0007] In particular, the terms micro and nano refer to how small the size of the object is. Today, nanomedicine is a medical application resulting from nanotechnology and nano-science. It therefore deals with all the nanoscale knowledge and technologies that have a medical use.

[0008] The transition from nano to micro is not only a matter of scale, but it implies changes in the system properties that can open new routes for the development of increasingly high-performance strategies.

[0009] Nano- and microstructures have profoundly different chemical-physical characteristics, such as: size, load capacity, zeta potential, high surface area / volume ratio. Furthermore, nanostructures and microstructures interact in profoundly different ways with biological systems, for example, in crossing biological barriers, being absorbed at the cellular level, in distributing themselves to the various tissues and organs, in the elimination processes at the renal level, in the accumulation processes, in the ability to be recognized by the immune system, in the processes of genotoxicity and tumorigenesis, in the rate of propagation and reaching the target site, and in the choice of the administration route.

[0010] Furthermore, through nano-encapsulation, the solubility, stability and bioavailability of drugs and bioactive substances, such as phytomolecules, are greatly improved.

[0011] The above-mentioned differences due to the different systems’ sizes modify the therapeutic, diagnostic, cosmetic, and nutraceutical properties of the systems.

[0012] Nanocarriers currently used could be roughly classified into two classes, artificial and natural ones. The ongoing concern for biosafety of using artificially synthesized nanomaterials for the delivery of drugs or natural products has accelerated the discovery and use of cell-derived nanovesicles (CDNs). In the field of plant-derived vesicles, there are naturally secreted nanovesicles called extracellular vesicles (EVs), nanovesicles obtained from the destruction of plant tissue (Plant-derived nanovesicles PDNs), and microvesicles derived from protoplasts (plant cells without wall).

[0013] Extracellular Vesicles (EVs)

[0014] Extracellular vesicles (EVs) are secreted by various cell types and deliver specific cargo to target cells, thereby mediating paracrine effects in physiological and pathological mechanisms. It is well known that there is no single method suitable for the isolation of EVs from various sources. Several isolation methods have been introduced, including differential ultracentrifugation, density gradient ultracentrifugation, gel filtration chromatography, ultrafiltration, polymer precipitation, and immunoaffinity separation.

[0015] Of these methods, separation by differential centrifugation is considered the gold standard for EV isolation. Since plant EVs are mainly present in the apoplastic space, the most critical step in EV isolation is the isolation of clean apoplastic washing fluid (AWF) obtained by a filtration-centrifugation method. Subsequent purification of plant EVs involves differential centrifugation of AWF. It has to be noted that, in distinct protocols for isolation of plant EVs, differences lie not only in the centrifugation speed for final EV sedimentation, but also in AWF collection. So far, there is no standard protocol for isolation of EVs from AWF from different plant species. Extraction of apoplastic fluid by vacuum infiltration yields only a few EVs due to the barrier effect of the cell wall, which limits the passage of EVs. This low extraction efficiency results in waste of a large amount of plant raw material and the method does not meet the requirements of bulk preparation.

[0016] Plant-Derived Nanovesicles (NDPs)

[0017] Disruption of plant tissue (fruits, leaves, roots, seeds, etc.) is the most commonly used method that yields large amounts of NDPs. After homogenization, five approaches are applicable for isolating plant nanovesicles, namely differential density and gradient ultracentrifugation, precipitation using highly hydrophilic polymers (e.g. protamine, dextran, polyethylene glycol), size exclusion chromatography and immunoprecipitation, microfiltration. Typically, the homogenized plant, which is a very complex matrix, forms the starting material for the isolation of NDPs, thus making the isolation process very challenging, and the obtained product is highly variable. In fact, since these vesicles derive from whole plant tissues subjected to cell disruption, a mixture of vesicles of inhomogeneous size and activity is extracted. NDPs contain a complex set of membrane-bound intracellular vesicles (transport vesicles, secretory vesicles, vesicles) deriving from the breakdown of plant cells, as well as EVs, and any impurities that are formed due to the homogenization process. The inhomogeneity of the composition influences the final results, and the potential applications thereof.

[0018] Cellular Protoplast-derived Microvesicles

[0019] Protoplast-derived microvesicles are obtained from a bacterial, fungal, or plant cell, or the like, from which a cell wall is removed. The protoplast (cell without a cell wall) is destroyed by different techniques: cytolysis by osmosis, electroporation, sonication, homogenization, detergent treatment, freezethaw, extrusion, mechanical degradation or chemical treatment, and reassembled to form microvesicles.

[0020] The aim of the present invention is therefore to provide a new class of plant protoplast-derived vesicles having a size in the order of nanometers, with an innovative method that overcomes the disadvantages related to the micrometer size for the same application.

[0021] SUMMARY OF THE INVENTION

[0022] The invention therefore concerns a new method for producing plant nanovesicles, in particular protoplast nanovesicles, having the innovative characteristics of being of smaller dimensions compared to known ones.

[0023] Nano- and microstructures have profoundly different chemical-physical characteristics (size, load capacity, zeta potential, high surface area / volume ratio) and interact with a biological system in profoundly different ways.

[0024] Nanostructures deriving from various biological sources, including mammals, plants, fungi and bacteria, have emerged as a new category of carriers. In particular, plant-derived nanovesicles are gaining increasing interest as eco- friendly, sustainable and biocompatible materials for the development of nextgeneration transport carriers. Furthermore, these nanovesicles intrinsically contain plant-derived bioactive compounds (phytocompounds), which are widely used for therapeutic, cosmetic and nutraceutical purposes.

[0025] In a first aspect, the present invention therefore relates to a process for the preparation of plant protoplast nanovesicles having the steps of: a. isolating protoplasts from plant tissue; b. purifying the isolated protoplasts of step a.; c. expanding the purified protoplasts of step b.; and d. preparing nanovesicles from the expanded protoplasts of step c., wherein said nanovesicles have a diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and a polydispersity index PDI 0.25±0.15 (< 0.5), as measured with a Zetasizer instrument (Malvern Panalytical).

[0026] The inventors have developed an innovative process for producing nanovesicles produced starting from protoplasts obtained from plant cells (isolated and maintained in culture through an expansion process) for applications ranging from food to nutraceuticals, cosmetics and pharmaceuticals.

[0027] In a second aspect, a plant protoplast nanovesicle having a diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and having a polydispersity index PDI 0.25±0.15 (< 0.5), as measured with a Zetasizer instrument (Malvern Panalytical), is described herein.

[0028] The invention concerns, in a third aspect, a composition comprising a plant protoplast nanovesicle and excipients suitable for use.

[0029] In a fourth aspect, the use of the plant protoplast nanovesicle, or the composition comprising it, as a medicinal product is described.

[0030] In a fifth aspect, the cosmetic use of the plant protoplast nanovesicle according to the invention, or the composition comprising it, is claimed.

[0031] In a further aspect, the invention relates to the food use of the plant protoplast nanovesicle, or the composition comprising it, in particular the use as a nutraceutical.

[0032] The invention concerns, in a seventh aspect, the plant protoplast nanovesicle obtainable from the process described above, said nanovesicle having a nanometric diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and a polydispersity index PDI 0.25±0.15 (< 0.5), as measured with a Zetasizer instrument (Malvern Panalytical).

[0033] The dependent claims describe particular embodiments of the invention.

[0034] DESCRIPTION OF THE FIGURES

[0035] The invention will now be described in detail and with reference to the attached Figures.

[0036] Figure 1 Scheme of the plant tissue incubation procedure.

[0037] Figure 2 Scheme of purification of protoplasts that are arranged on a sugar gradient.

[0038] Figure 3 Scheme of protoplast counting and culture.

[0039] Figure 4 Scheme of the extrusion process.

[0040] Figure 5 Visualization of protoplasts in culture (A), protoplast counting in a Burker chamber (B) and fluorescence analysis and measurement (C).

[0041] Figure 6 Mini-syringe extruder and related components (A). Schematic illustration of the mini-syringe extruder parts (B).

[0042] Figure 7 Nanovesicle characterization: size and concentration analysis (A), fluorescence analysis (B), fluorescence intensity analysis histogram (C).

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] Plant tissue disruption is the most commonly used method to produce large amounts of plant-derived nanovesicles. However, since these vesicles derive from whole plant tissues subjected to cell disruption, such vesicles are made of a mixture of vesicles of different origin, including EVs, with non-uniform size and activity, and highly variable batches.

[0045] Therefore, the development of a new method leading to the production of vesicles with a size in the order of nanometers (nanovesicles) starting from plant cell protoplast is not only fundamental for the preparation of a new typology of plant-derived vesicles but, since such vesicles are obtained from expandable and engineerable protoplasts, this process presents itself as a scalable, sustainable and reproducible platform for the creation and generation of nanovesicles with unique and peculiar characteristics.

[0046] In a first aspect, the present invention therefore relates to a process for the preparation of plant protoplast nanovesicles having the steps of: a. isolating protoplasts from plant tissue; b. purifying the isolated protoplasts of step a.; c. expanding the purified protoplasts of step b.; and d. preparing nanovesicles from the expanded protoplasts of step c., wherein said nanovesicles have a diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and a polydispersity index PDI 0.25±0.15 (< 0.5), as measured with a Zetasizer instrument (Malvern Panalytical). In a preferred embodiment, the isolation of protoplasts of step a. is mediated by an enzymatic treatment wherein enzymatic degradation of the plant cell wall occurs.

[0047] Preferably, in the method of the invention said plant tissue is selected from the group consisting of leaves, stem, roots, fruits, corns, embryonic tissue and cotyledons, preferably from leaves, corns and cotyledons.

[0048] More preferably said plant tissue is a leaf, preferably a leaf of Citrus limon, basil (Ocimum basilicum), Diplotaxis, strawberry and pyrethrum (Tanacetum cinerariifolium). In the process for protoplast extraction, the plant and tissue having the most useful characteristics for the final purpose of nanovesicles, and richest in the components and molecules of interest, are selected.

[0049] Advantageously, in the method described herein, the purification of step b. is performed by centrifugation, preferably centrifugation on a gradient of 25% sucrose and 13% mannitol.

[0050] After purification, the protoplasts can be placed in liquid culture under suitable conditions that allow their multiplication, or expansion, as in step c. of the present method.

[0051] Culturing protoplasts is essential because it allows us to have a constantly available (well characterized) pool from which nanovesicles can be obtained, thus making the process scalable, sustainable and reproducible. Furthermore, protoplasts could be engineered or simply grown in conditioned media rich in active ingredients, in order to then produce vesicles enriched or loaded with molecules of interest.

[0052] The characteristics of plant protoplast nanovesicles depend not only on the physiological state of the cell but also on the cell type from which they are derived, as well as on the extraction protocol. The process shown herein allows us to have uniform nanovesicles since they are obtained from the same tissue; furthermore, with the growth in culture of protoplasts it is possible to obtain well standardized and reproducible protoplast clones from which nanovesicles are produced. In one embodiment, the nanovesicles preparation of step d. occurs through one or more processes selected from the group consisting of extrusion, sonication, cavitation and homogenization, or a combination thereof.

[0053] Compared to nanovesicles obtained by tissue destruction, that may contain not only EVs but also contaminants deriving from all the plant tissue components (wall, elements of the extracellular matrix) resulting in a significant variability of the vesicles produced, the process for the preparation of plant protoplast nanovesicles can be standardized in terms of the type of nanovesicles produced and is reproducible.

[0054] All the steps of the process described in the present invention and aimed at obtaining plant protoplast nanovesicles are preferably performed under sterile conditions.

[0055] The nanovesicles of the present invention differ from the known ones in that they are not extracellular vesicles and are not vesicles produced by destruction and homogenization of the plant tissue, and are not microvesicles.

[0056] In a second aspect, a plant protoplast nanovesicle having a diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and a polydispersity index PDI 0.25±0.15 (< 0.5), as measured with a Zetasizer instrument (Malvern Panalytical), is described herein.

[0057] In a preferred embodiment, the plant protoplast nanovesicle according to the invention contains phytocompounds and bioactive molecules of plant origin, preferably said bioactive molecules are selected from the group consisting of polyphenols, carotenoids and glucosinolates with antioxidant, antiinflammatory, anti-tumor, anti-neurodegenerative activity. In fact, the nanovesicles contain active ingredients present in the cell that are encapsulated during the production process and are not present in EVs, and are not identified or characterized in the protoplast-derived microvesicles. Protoplasts can be grown in media conditioned with active ingredients or be stimulated to produce a greater amount of bioactive molecules (phytocompounds) that are normally produced by them, or engineered, thus obtaining vesicles with unique properties. Plant protoplast nanovesicles can be loaded with a substance, for example a drug, a contrast agent, a fluorescent probe, a cosmetic substance, a food substance or a nutraceutical substance.

[0058] The invention concerns, in a third aspect, a composition comprising a plant protoplast nanovesicle and excipients suitable for use.

[0059] In a fourth aspect, the use of the plant protoplast nanovesicle, or the composition comprising it, as a medicament is described.

[0060] In the pharmaceutical field, doxorubicin (an antitumor drug) has been encapsulated in liposomes thus improving its therapeutic effect, and liposomes have been used for the production of vaccines. Vesicles of natural origin, such as the plant protoplasts nanovesicles described herein, are considered valid carriers for the therapies of the future. In fact, due to their origin, compared to synthetic nanocarriers (liposomes), natural vesicles are able to easily penetrate biological barriers (including the blood-brain barrier), be internalized by specialized recipient cells with different, more or less specific, mechanisms, not be accumulated in tissues in a non-specific manner, be less recognized by phagocytic cells as foreign material to be eliminated, in addition to being biocompatible; thanks to the presence of specific proteins and membrane receptors on their surface, they can be endowed with an intrinsic ability to target certain cells and tissues.

[0061] In a fifth aspect, the cosmetic use of the plant protoplast nanovesicle according to the invention, or the composition that includes it, is claimed.

[0062] The plant protoplast nanovesicles described herein may be of great interest for the cosmetics industry. In fact, cosmetic applications may include skin care products such as creams, lotions, sunscreens, tanning agents or products intended for make-up, such as lipsticks, mascaras, foundations and powders. In a further aspect, the invention relates to the food use of the plant protoplast nanovesicle, or the composition comprising it, in particular the use as a nutraceutical.

[0063] The invention concerns, in a seventh aspect, the plant protoplast nanovesicle obtainable from the process described above, said nanovesicle having a nanometric diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and a polydispersity index PDI 0.25±0.15 (< 0.5), as measured with a Zetasizer instrument (Malvern Panalytical). In a preferred embodiment, said protoplast nanovesicles contain phytocompounds and / or bioactive molecules of plant origin, preferably said bioactive molecules are selected from the group consisting of polyphenols, carotenoids and glucosinolates.

[0064] The plant protoplast nanovesicles of the present invention are produced by a renewable, sustainable and scalable process leading to the production of environmentally sustainable nanomaterials by minimizing the use of toxic chemicals, reducing waste, and generating less greenhouse gases.

[0065] Examples of embodiments of the present invention, provided for illustrative purposes, are reported below.

[0066] EXAMPLES

[0067] Example 1 : Isolation of protoplasts from plant material

[0068] For the isolation of protoplasts, different types of explants (leaves, corn, embryos, cotyledons, etc.) can be used.

[0069] To optimize the release of protoplasts from lemon leaves (Citrus limon (L.) Osbeck) the enzyme solution (Table 1 ) and the BH3 culture medium (Table 2) were used).

[0070] The solutions were sterilized with a 0.22 pm filter.

[0071] Table 1 : Enzyme solution composition

[0072] Table 2: BH3 solution composition

[0073] About 100 mg of leaf tissue were minced with a scalpel blade in 7 mL of BH3, to which 3 mL of enzyme solution were added. After preparation, the sample was incubated with gentle shaking at 24°C for 18h in the dark (Figure 1 ), the quality of the yield was checked by inverted microscope in transmitted light. The material was filtered through 40 pm filters to eliminate the coarser residues.

[0074] Example 2: Purification of protoplasts on gradient

[0075] The filtered solution was centrifuged, the supernatant removed, the pellet obtained was resuspended in 5 mL of 25% sucrose solution and 2 mL of 13% mannitol solution were gently layered on the suspension.

[0076] The sample was centrifuged again, intact protoplasts were identified at the interface between 25% sucrose and 13% mannitol. The gradient-purified protoplasts arranged in a ring at the interface between the two sugars (Figure 2) were collected with a Pasteur pipette and resuspended in 5 mL of the culture medium.

[0077] At this point, the protoplasts were further centrifuged, and the pellet was resuspended in 1 mL of medium for determining the concentration with the hematocytometer (Burker Chamber) to bring the final concentration of the protoplast suspension necessary for the subsequent steps.

[0078] Example 3: Nanovesicle production

[0079] In order to produce the nanovesicles, the protoplasts obtained, for example through gradient centrifugation, are subjected to one or more of the following processes or combinations thereof:

[0080] 1. Extrusion,

[0081] 2. Sonication,

[0082] 3. Cavitation,

[0083] 4. Homogenization Extrusion example

[0084] An extruder is used to prepare vesicles by using the mechanical force imposed by the extrusion process to disrupt the cell membrane structure and allow it to reform into nanoscale vesicles. Specifically, the extrusion method takes advantage of the fact that when cells are forced through the pores of a filter with pores smaller than their diameter, they break down into smaller vesicles closer to the pore size (Figure 4).

[0085] Step 1 : Preparation of protoplasts for extrusion.

[0086] Protoplasts are centrifuged at 2,000 rpm to remove growth medium and washed twice ith saline or PBS (Figure 5A). Protoplasts in saline are counted using a Burker chamber until a concentration of 2 to 5 million per mL is obtained (Figure 5B). As a first step, a fluorescence analysis at different excitation wavelengths (523, 555, 647 nm) of protoplasts compared to animal cells (intestinal epithelial cells CaCo2) was performed, in order to highlight the presence in protoplasts of some phytomolecules (polyphenols, phycoerythrins, phycocyanins, carotenoids, glucosinolates etc.) that are not present in animal cells in vitro (Figure 5C).

[0087] Step 2. Assembly of the extruder apparatus

[0088] In Figure 6, an example of a mini-syringe extruder and its schematic illustration are depicted. Before assembling the extruder, all parts (except for polycarbonate membranes and filter supports) are thoroughly cleaned with a mild detergent solution, followed by rinsing with hot tap water and deionized or distilled water. All parts are allowed to dry and UV sterilized before assembling the apparatus.

[0089] Step 3. Protoplast extrusion

[0090] 500 uL of protoplasts (about 2 million) are inserted into the syringe of the extrusion apparatus and extruded through polycarbonate membranes of different sizes, 1 ,000 nm, 400 nm, 200 nm and 100 nm, respectively. A total of 10 passages are performed for each type of membrane. In order to purify the vesicles produced after the extrusion process, the latter are subjected to two washes with saline or PBS by ultracentrifugation at 100,000g for 70 minutes at 4°C.

[0091] Example 4: Nanovesicle characterization

[0092] After purification, the nanovesicles, resuspended in PBS, were analyzed by Nanosight which allows a rapid and automated analysis of the size distribution and concentration of all types of nanovesicles (Figure 7A). As can be seen, the size of the nanovesicles is in a range of 30 to 200 nm with a predominant species at about 100 nm and with a concentration of 1.40 x 1010nanovesicles / mL with a PDI 0,25±0,1 and a zeta potential between -10 and - 50 mV. Furthermore, from scanner (Figure 7B) and fluorimeter (Figure 7C) fluorescence analyses, at different excitation and emission wavelengths, it is apparent that the vesicles produced contain phytocomplexes (polyphenols, carotenoids, glucosinolates, etc.) like the protoplasts from which they originate.

[0093] From the detailed description and the Examples reported above, the advantages achieved by the method for the production of plant protoplast nanovesicles of the invention are apparent.

Claims

CLAIMS1 . A process for the preparation of plant protoplast nanovesicles having the steps of: a. isolating protoplasts from plant tissue; b. purifying the isolated protoplasts of step a.; c. multiplying the purified protoplasts of step b.; and d. preparing nanovesicles from the multiplied protoplasts of step c., wherein said nanovesicles have a diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and a polydispersity index PDI of 0.25±0.15, and said nanovesicle containing phytocompounds and bioactive molecules of plant origin chosen from the group consisting of polyphenols, carotenoids, and glucosinolates.

2. The process according to claim 1 , wherein the isolation of protoplasts of step a. is mediated by enzymatic treatment.

3. The process according to any one of claims 1 or 2, wherein said plant tissue is selected from the group consisting of leaves, corns, cotyledons, embryos, stem, roots, and fruits.

4. The process according to any one of claims 1 to 3, wherein said plant tissue is a leaf, preferably a leaf of Citrus limon.

5. The process according to any one of claims 1 to 4, wherein the purification of step b. is carried out by centrifugation, preferably centrifugation on a gradient of 25% sucrose and 13% mannitol.

6. The process according to any one of claims 1 to 5, wherein the preparation of the nanovesicles of step d. is carried out by one or more processes selected from the group consisting of extrusion, sonication, cavitation, and homogenization, or combinations thereof.

7. A plant protoplast nanovesicle having a nanometer diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and having a polydispersity index PDI of 0.25±0.15 as measured with a Zetasizer instrument, said nanovesicle containing phytocompounds and bioactive molecules of plant origin selected from the group consisting of polyphenols, carotenoids, and glucosinolates.

8. The plant protoplast nanovesicle according to claim 7, loaded with a substance.

9. The plant protoplast nanovesicle according to claim 8, wherein said substance is a drug, a contrast agent, a fluorescent probe, a cosmetic substance, or a nutraceutical substance.

10. A composition comprising one or more plant protoplast nanovesicles according to any one of claims 7 to 9, and excipients suitable for use.1 1 . The plant protoplast nanovesicle according to any one of claims 7 to 9, or the composition according to claim 10, for use as a medicament.

12. Cosmetic use of the plant protoplast nanovesicle according to any one of claims 7 to 10, or the composition according to claim 10.

13. Dietary use of the plant protoplast nanovesicle according to any one of claims 7 to 10, or the composition according to claim 10.

14. The use according to claim 13, wherein said nanovesicle is a nutraceutical.

15. A plant protoplast nanovesicle obtainable by the process according to any one of claims 1 to 6, said nanovesicle having a diameter in the range of 30 to 200 nm, a negative zeta potential in the range from -10 to -50 mV, and apolydispersity index PDI of 0.25±0.15 as measured with a Zetasizer, and said nanovesicle containing phytocompounds and bioactive molecules of plant origin chosen from the group consisting of polyphenols, carotenoids, and glucosinolates.

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