Method for the production of extracellular vesicles derived from plant

Abstract

Method for the production of extracellular vesicles derived from plant raw material (PDEVs) obtained from biological agriculture, comprising the following steps: a. inspecting visually the plant raw material; b. transferring the plant raw material into plastic food containers and store it for short periods in the refrigerator at a temperature of +4°C, c. washing the plant raw material, d. carrying out an initial processing of the plant raw material for extraction, e. extracting the first semi-finished product using automatic extractors, f. storing the first semi-finished product in plastic bottles, g. storing the first semi-finished product in the refrigerator, h. carrying out an initial filtration step of the first semi-finished product in a filter with a mesh size between 0.2 mm and 1 mm, i. storing the filtered product not immediately used for a short period in the refrigerator at a temperature of +4°C, l. performing an initial centrifugation of the filtered product in centrifuge bottles at a speed of 2,000 x g for 30 minutes, m. filtering the supernatant with a nylon filter with a mesh size of up to 100 µm to obtain a mixture of extracellular vesicles and discarding the pellet, n. storing the mixture of extracellular vesicles in a refrigerator for a short time at a temperature of +4°C in plastic or borosilicate glass bottles, o. adding to the mixture of extracellular vesicles a quantity of maltodextrin between 4% and 35% by weight, p. drying the mixture of extracellular vesicles by atomization, q. collecting and storing the powdered product at room temperature in amber borosilicate glass containers.

Description

[0001] METHOD FOR THE PRODUCTION OF EXTRACELLULAR VESICLES DERIVED FROM PLANT

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The present invention relates to an innovative method for the production of extracellular vesicles from plant raw materials of biological origin, i.e. obtained from biological agriculture. In particular, the extracellular vesicles are extracted from organic fruits and / or vegetables and the method, which is the object of the present invention, allows the production of extracellular vesicles suitable for use in the cosmetic sector, in particular as a dermocosmetic and in the pharmaceutical sector.

[0005] Background art

[0006] As is known, research has only begun to shed light on the mechanisms of action of extracellular vesicles (EVs) in recent years, but it is already clear that they could offer a platform for the development of innovative therapies. EVs are released by all cell types in the body and, consequently, are present in all body fluids. They can be divided into three types: exosomes, the smallest vesicles that are released from inside the cell and follow the endosomal route; microvesicles, which are also released from inside the cell, but are produced using the cell membrane itself that incorporates the cargo to be transported outwards; and apoptotic bodies, characteristic of dying cells.

[0007] EVs are, by their very nature, transporters. Consequently, it is easy to imagine that scientists have thought of exploiting their ability to cross cellular barriers without problems to transfer small payloads (drugs, proteins, different types of RNA). Loading various molecules into these vesicles, however, requires the manipulation of the vesicles themselves (exogenous loading, i.e. small molecules are incorporated into isolated EVs) or of the cells from which they come (endogenous loading, in which the cell receives the means necessary to incorporate small molecules into EVs during the formation process). The strong interest in extracellular vesicles has led to a greater knowledge of them and the awareness that they could be a useful and applicable tool in various fields of research and experimentation.

[0008] Furthermore, the industrial use of extracellular vesicles of vegetal origin, i.e. derived from plants, fruits and vegetables, is bio-renewable and sustainable. This suggests that the use of plants as industrial sources (nanofactories) for the production of extracellular vesicles for preventive and therapeutic use could represent a new approach in nanomedicine. It is expected that the promotion of research and development of extracellular vesicles for the transport of therapeutic molecules will significantly contribute to the development of natural nanomedicines.

[0009] In particular, extracellular vesicles of plant origin and from biological agriculture have all the characteristics to allow an adequate transport of therapeutic molecules with a low level of toxicity and last but not least with a very low level of environmental pollution.

[0010] These assumptions are leading and will increasingly lead to a greater demand for raw materials, i.e. to a greater demand for production availability of extracellular vesicles, especially of plant origin from raw material from organic farming. The extraction of extracellular vesicles from plants, to date, is based on standard methods (for example using “gold standard” technology). The known methodologies through which extracellular vesicles are obtained from various fruits and / or vegetables include repeated cycles of centrifugation and ultracentrifugation, as required by internationally shared standard procedures (ISEV).

[0011] However, as described above, to meet the increased demand for extracellular vesicles, continuous improvements in the technical field related to the production of such vesicles are necessary.

[0012] Therefore, the need arises to obtain an innovative method, which differs from standard methodologies, extremely efficient to extract extracellular vesicles of plant origin quickly and economically.

[0013] Summary of the invention

[0014] The subject of the present invention is therefore a method of producing extracellular vesicles from plant raw material, in particular of biological origin, i.e. from biological agriculture.

[0015] The method, the subject of the present invention, allows to obtain extracellular vesicles both from dried petals, leaves, berries and roots derived from biological plants and from fresh fruits and / or vegetables also derived from biological agriculture, in particular from Italy.

[0016] Advantageously, the production of extracellular vesicles through the method according to the present invention achieves an optimization in terms of extract volume, i.e. the number of vesicles obtained with respect to the initial quantity of raw material used.

[0017] Advantageously, the extracellular vesicles obtained through the method according to the present invention are better in terms of purity and stability.

[0018] Advantageously, the production of extracellular vesicles through the method according to the present invention ensures a considerable facilitation in the storage of raw materials.

[0019] Advantageously, the production of extracellular vesicles is obtained through a considerable reduction in production times.

[0020] Advantageously, the production of extracellular vesicles is obtained through a considerable saving of water for washing and electricity.

[0021] Therefore, according to a first aspect of the present invention, an innovative method of production of extracellular vesicles from vegetal raw material from biological agriculture is defined, as specified in the independent method claim annexed.

[0022] In accordance with a second aspect of the present invention, a new use of the extracellular vesicles directly obtained from the production method for the cosmetic and pharmaceutical sectors is defined.

[0023] The dependent claims outline particular and further advantageous aspects of the invention.

[0024] Brief description of the drawings

[0025] These and other advantages of the invention will now be described in detail, with reference to the attached drawings, which represent an exemplary embodiment of the invention, in which:

[0026] - Figure 1 illustrates the quality control graph of the concentration and size distribution of extracellular vesicles obtained from roots (e.g. liquorice) by Nanoparticle Tracking Analysis (NTA); Figure 2 illustrates the quality control plot of the concentration and size distribution of extracellular vesicles obtained from petals (e.g. rose) by Nanoparticle Tracking Analysis (NTA);

[0027] - Figure 3 illustrates the Quality Control plot of concentration and size distribution of extracellular vesicles obtained from leaves (e.g. green tea) by Nanoparticle Tracking Analysis (NTA);

[0028] - Figure 4 graphically illustrates the results relating to the evaluation of the elasticity of the skin treated with a serum comprising the extracellular vesicles extracted according to the method of the present invention and the skin treated with a control serum;

[0029] - Figure 5 graphically illustrates the results relating to the evaluation of the reduction of wrinkles of the skin treated with the serum comprising the extracellular vesicles extracted according to the method of the present invention and the skin treated with a control serum.

[0030] Detailed description

[0031] According to the present invention, the plant-derived extracellular vesicles (PDEVs) produced by means of the method object of the present invention, are obtained from plants from biological agriculture (plant raw material).

[0032] Biological agriculture means agriculture that uses a cultivation technique and a way of producing food that respects natural life cycles. That is, without the use of chemical pesticides, synthetic fertilizers, antibiotics and other substances that are subject to severe restrictions. In addition, the crops are rotated so that the resources on site are used efficiently; the resources on site are exploited, such as manure for fertilizer or feed produced on the farm. Furthermore, by definition, biological agriculture does not use genetically modified organisms (OGMs). Instead, plant and animal species that are resistant to disease and adapted to the environment are used. To this end, techniques such as protecting beneficial insects, which are antagonists of parasites, are used; rustic, more resistant plants are chosen; mulching is practiced, which consists of covering the soil with hay or fresh grass to protect it from temperature changes and hinder the growth of weeds; green manure is used, that is, the sowing of certain plants (clover, vetch, watercress, lamb's lettuce, spinach, rapeseed, and so on) which, once in flower, are buried to fertilize the soil and protect it from erosion; crop rotation is practiced, which consists of alternating the cultivation of plants that improve the fertility of the soil, for example by enriching it with nitrogen, with plants that impoverish it, by removing nutrients; manure and organic fertilizers such as compost, a mixture of soil, plant remains, wood ash and anything else that is biodegradable and non-polluting is used on the farm.

[0033] In particular, the method of the present invention applies to both dried biological petals, leaves, berries and roots and to biological fresh fruits and / or vegetables, obtained through: a) research and selection of certified Italian biological farms; b) research, selection and obtaining of vegetable raw material: dried petals, leaves, berries and roots and / or fruits and / or vegetables certified from Italian biological farming.

[0034] The method for the production of extracellular vesicles from vegetable raw material from biological agriculture, includes the following phases: a. inspecting petals, leaves, berries and dried roots, fruits and / or vegetables from Italian organic crops (vegetable raw material) by means of documents and visual inspection, b. transferring the plant raw material into plastic food containers and storing it for 4 to 8 hours maximum in the refrigerator (preferably at a temperature of +4°C) while waiting for the extraction phase, c. washing the plant raw material, d. carrying out an initial processing of the plant raw material for extraction: in the case of dried basic material, carrying out an infusion phase in ultrafiltered water for 24 hours; in the case of fresh basic material, peeling and cutting the fresh basic material into pieces, e. extracting the first semi-finished product, i.e. the first infusion or extraction juice, using professional automatic extractors, f. storing the first semi-finished product in plastic bottles of the type High Density Polyethylene (HDPE), Polyethylene Terephthalate (PET), Polypropylene (PP), Polycarbonate (PC), to avoid the release of toxic substances. g. storing the first semi-finished product in the refrigerator at a temperature of +4°C if processed immediately, h. performing an initial filtration phase of the first semi-finished product in a filter with a mesh size between 0.2 mm and 1 mm. i. storing the filtered product not immediately used in the refrigerator (preferably at a temperature of +4°C), for a maximum of 1 hour, l. performing an initial centrifugation of the filtered product in polypropylene or PPCO centrifuge bottles at a speed of 2,000 x g for 30 minutes, m. filtering the supernatant containing the extracellular vesicles of plant origin with a very fine mesh nylon filter, preferably with a mesh size of up to 100 pm, and discard the pellet to obtain a mixture of PDEVs. In particular, for pharmaceutical preparations (medical devices), filter with a nylon filter with a mesh size of 0.45 pm and subsequently with a 0.22 filter to ensure the removal of microorganisms such as yeasts, fungi and bacteria (to comply with the limits set by the pharmacopoeia (reference Ph.Eur.), n. storing the PDEVs mixture in plastic bottles (PET, HDPE, PC) or borosilicate glass 3.3 in the refrigerator for a short period, approximately 1 hour (preferably at +4°C), o. adding a quantity of maltodextrin between 4% and 35% by weight to the PDEVs mixture, p. drying the PDEVs mixture by atomization (spray drying or “SPRAYDRYING”), q. collecting and storing the powdered product at room temperature (preferably between 20°C and 25°C) in tightly closed amber borosilicate glass containers, away from heat sources, not exposed to light and humidity.

[0035] Phase a) includes the registration of the incoming plant raw material: petals, leaves, berries and roots or dried or dehydrated fruits / vegetables (registration of goods flows) and the related quality control: transport conditions, packaging conditions, physical appearance of the basic products received. Generally, dried basic products (e.g. petals, leaves, berries and roots) arrive already washed. In addition, quality control also includes physical / chemical analysis, heavy metals, polycyclic aromatic hydrocarbons, mycotoxins, microbiology, pesticides, microelements of the extract.

[0036] Phase d) includes the grinding of some types of dried plant raw material (mainly roots), while petals, leaves and berries are directly infused. The infusion process is performed with ultrafiltered water. From 1 to 1000 grams of dried raw material infused with water (from 2 to 10000 ml infusion). The infusion of dried / dehydrated plants, such as roots and leaves, is a phase that presents particular technical challenges, since the secretion of extracellular vesicles in these matrices requires specific conditions, such as temperature control, infusion time and type of solvent. In particular, the extraction of extracellular vesicles from plants with a low liquid content requires specific optimization, achieved by means of the method. The method according to the invention aims to use a wide range of plant derivatives, including leaves, roots and fruits with low liquid content, for the extraction of extracellular vesicles.

[0037] Phase g) of conservation of the first semi-finished product, if the latter is not immediately processed, is also possible at a temperature of -20°C for up to three months, at a temperature of -40°C for up to six months and at a temperature of - 80°C for up to one year. In these cases, before filtration, phase g1) of defrosting in the refrigerator at a temperature of +4°C of the first semi-finished product previously stored at a temperature of -20°C, -40°C, -80°C is necessary.

[0038] Phase h) of filtration of the first semi-finished product is carried out by means of filters with specific degrees of porosity. The optimal filter meshes between 0.2 mm and 1 mm were obtained through numerous laboratory tests in order to effectively isolate the extracellular vesicles, without compromising their integrity. Further experiments were also conducted to optimize the quality and quantity of the extracted vesicles, since each type of plant requires specific treatment parameters. Phase p) of drying is carried out according to the spray technique or "Spray Drying" which is one of the methods of choice for the production of powders starting from aqueous (and / or organic-aqueous) solutions or suspensions. This technique involves the atomization of a liquid in a chamber in which a hot gas recirculates. To ensure thorough cleaning of the incoming air of the atomizer “Spray Drying” follows three types of filters: 1) filter and regulator (5 microns), 2) filter (0.01 microns) and 3) air dryer.

[0039] According to the method of the present invention, the drying phase q) is performed according to optimized process parameters, namely:

[0040] - “Drying Gas” Drying gas flow rate (m3 / h) = 30 - 35

[0041] - ’’Inlet T” Inlet temperature (°C) = 115 - 140

[0042] - ’’Spray Gas” Atomized gas flow rate (l / h) = 1000 - 1500

[0043] - ’’Pump 1” Pump flow rate (ml / min): 6 -15

[0044] - “Outlet T” outlet temperatures (°C) = 60 - 75

[0045] Advantageously, the raw material used in the drying phase is a mixture of noble derivatives extracted by infusion from different plants. The starting material is therefore richer in extracellular vesicles, which can be isolated more easily through the process, reducing waste and improving the quality of the final product.

[0046] Advantageously, the use of a blend of Italian and biological plants and fruits, specifically selected for their ability to produce high-quality extracellular vesicles, and the use of the “Spray Drying” phase to ensure industrial scalability, imply the optimization of various technical parameters (e.g., drying temperature, air flow, and raw material concentration) through specific experiments and adjustments to obtain the desired product. Advantageously, at the end of the process a sampling of the product is carried out for subsequent quality controls.

[0047] The attached figures 1-3 illustrate the results of the control tests carried out on the extracellular vesicles, PDEVs, obtained through the innovative method object of the present invention.

[0048] In particular, the quality controls were performed on the PDEVs purified by spray drying and tested by means of the “Nanoparticle Tracking Analysis” (NTA), using the Nanosight. The results on three samples extracted from roots (e.g. licorice - Fig. 1), petals (e.g. rose - Fig. 2) or leaves (e.g. green tea - Fig. 3) demonstrated that the size distribution and concentration of the PDEVs produced by spray drying is equivalent to that of the PDEVs produced by the “gold standard” technology, i.e. by ultracentrifugation. The resulting % variability = < 10%, therefore not significant.

[0049] Further quality controls are also carried out through:

[0050] - analysis of the moisture content (%MC) of the product,

[0051] - analysis of the size, particle charge (zeta potential) and molecular weight of the product by “Dynamic Light Scattering” (DLS),

[0052] - analysis of the pH of the product by pH-meter,

[0053] - antioxidant analysis of the product by colorimetric kits,

[0054] - in vitro toxicity tests on fibroblasts,

[0055] - physical / chemical tests, heavy metals, polycyclic aromatic hydrocarbons, mycotoxins, microbiology, pesticides, microelements at an external laboratory (test report) for the purposes of the certificate of analysis (COA) of the product, - stability tests of the product.

[0056] Advantageously, the PDEVs purified by spray drying are accompanied by a technical data sheet, safety data sheet, certificate of analysis (COA).

[0057] Advantageously, the PDEVs purified by spray-drying are controlled for pH by pH-meter. The pH of PDEVs obtained from roots, petals and leaves by the infusion-extraction-spray drying processes is: pH roots: 3 - 5.5; pH petals: 3 - 5.5; pH leaves: 3 - 5.5.

[0058] Advantageously, the PDEVs purified by spray drying are checked for their antioxidant power by a commercially available colorimetric kit. The absorbance is read by spectrophotometer. The antioxidant power of PDEVs obtained from roots, petals and leaves by the infusion-extraction-spraydrying processes was found to be consistent with the same measured in PDEVs preparations obtained by gold standard methodology (ultracentrifugation): (% variability = < 10%, not significant). Therefore, the spray drying process does not compromise in any way the antioxidant content of PDEVs.

[0059] Advantageously, the PDEVs purified by spray drying are checked for their functionality by in vitro tests on human fibroblasts following treatment for 24h and 48h. Toxicity analysis by cell proliferation and mortality tests showed that PDEVs obtained from roots, petals and leaves by infusion-extraction-spraydrying processes do not induce any toxicity in vitro on human fibroblasts. Furthermore, following in vitro scratch tests on human fibroblasts, treatment with PDEVs (isolated from petals, leaves and roots) induced wound closure after 48 hours of treatment, demonstrating their complete functionality. Advantageously, stability tests on purified PDEVs by spray-drying are obtained through an accelerated stability analysis, comprising the following steps:

[0060] - sampling at stability points: months to, t1 , t3, t6 at end points,

[0061] - product: compatibility with primary packaging,

[0062] - organoleptic evaluation of the product: appearance, color and odor; pH at 25 ± 2°C (on the product diluted 1 to 9 in water),

[0063] - total count of mesophilic aerobic bacteria (TAMC), yeasts and molds (TYMC) according to the European Pharmacopoeia,

[0064] - detection of two pathogenic microorganisms (S. Aureus, P. Aeruginosa) method according to the European Pharmacopoeia.

[0065] The results obtained from the infusion process, according to the method of the present invention, showed a high concentration of PDEVs from a small amount of raw material.

[0066] Table 1 shows the results obtained from the infusion process and production by spray-drying of PDEVs obtained from dried leaves, petals and roots. The results are normalized on a gram (1 g) of dried product. The parameters taken into consideration are: volume (ml) of initial infusion, volume (ml) of infusion used for Spray Drying, number of PDEVs, size (nm) of PDEVs, number of PDEVs / ml of initial infusion, number of PDEVs / g of dried raw material, % of support used for the spray drying process (maltodextrin), grams of powder I gram dried raw material obtained from the spray drying process, zeta potential (mV) and conductivity (mS / cm) in water. Infusion / Extraction Process and Production by Spray Drying of Extracellular Vesicles from Dried Plant Raw Material (PDEVs) from Italian Agriculture

[0067] PARAMETERS CONSIDERED PDEVs from PDEVs from PDEVs from

[0068] Dried leaves Dried Petals Dried Roots (e.g. Rosa

[0069] (e.g. green tea) (e.g. Licorice) canina)

[0070] Dried Raw Material (grams) 1 1 1

[0071] Vol (ml) of Initial Infusion 6-14 6-14 6-14

[0072] Vol (ml) of Infusion Used for 4 - 12 4 - 12 4-9 Spray Drying

[0073] Number of PDEVs 0,9E+13 - 2,4E+13 3,7E+12 - 8,7E+12 l,0E+12 -

[0074] 2,4E+12

[0075] Dimensions (nm) of PDEVs 160 - 300 140 - 300 140 - 300

[0076] PDEVs / ml of initial infusion 0,9E+12 - 2,4E+12 3,7E+11 — 8,7E+11 l,0E+ll-

[0077] 2,4E+11

[0078] PDEVs / g of dried raw 0,9E+13 - 2,4E+13 3,7E+12 - 8,7E+12 l,0E+12- material 2,4E+12

[0079] % Carrier (maltodextrin) 4 - 35 4-35 8-35

[0080] Final grams of powder / 0,3 - 2,5 0,4 - 3,5 0,2-2, 5 gram of dried raw material

[0081] Zeta potential (mV) da -20 a -40 da -15 a -35 da -9 a -30

[0082] Conductivity (mS / cm) in 0,001 - 20,000 0,001 - 5,000 0,001 - 5,000 water pH 3 - 5.5 3 - 5.5 3 - 5.5

[0083] Antioxidant capacity Yes Yes Yes

[0084] In vitro functionality Yes Yes Yes

[0085] In vitro toxicity No No No

[0086] Table 1

[0087] The results of comparative tests between the method for the production of extracellular vesicles (PDEVs) from plant raw material from organic agriculture object of the present invention and the commonly used method for the extraction of extracellular vesicles (“Gold standard”) are shown in Table 2 and Table 3.

[0088] Table 2

[0089]

[0090] Table 3

[0091] As can be seen from the results in the tables, the advantages of the method object of the present invention are multiple, among these the most important are:

[0092] - reduction of the initial quantity of raw material (greater efficiency of the method),

[0093] - reduction of water consumption for washing, - facilitation of the storage of raw materials.

[0094] Furthermore, the phase q) of drying of the PDEVs mixture by atomization brings the further advantages:

[0095] - reduction of production times,

[0096] - increase in production capacity, - greater scalability. Advantageously, the method for the production of raw material based on extracellular vesicles from plant raw material from biological agriculture is suitable for use in the cosmetic sector, in particular dermocosmetics and in the pharmaceutical sector.

[0097] An example of application for dermocosmetics, entirely exemplary and not limiting, of the method object of the present invention, is described below, for the production of PDEVs from a mix of fresh Italian biological fruits and / or vegetables: ginger (Zingiber officinale), avocado (Persea americana) and nettle (llrtica dioica).

[0098] The method of the present invention is applied to ginger, avocado and nettle according to the following phases:

[0099] - searching and selecting certified Italian biological farms;

[0100] - researching, selecting and obtaining ginger (Zingiber officinale), avocado (Persea americana) and nettle (llrtica dioica) certified as Italian biological farming,

[0101] - inspecting with documents and visually ginger (Zingiber officinale), avocado (Persea americana) and nettle (Urtica dioica) from Italian biological agriculture,

[0102] - transferring the fruits / vegetables into plastic food containers and storing in the refrigerator (+ 4°C) for short periods,

[0103] - washing the fruits / vegetables: washing with tap water, washing with tap water (or distilled) and sodium bicarbonate, washing with ultrafiltered distilled water,

[0104] - peeling the fruits / vegetables with the peel and cutting them into pieces,

[0105] - extracting the juice, using professional automatic juice extractors,

[0106] - carrying out an initial quality control of the juice: physical / chemical analysis, heavy metals, polycyclic aromatic hydrocarbons, mycotoxins, microbiology, pesticides, microelements of fruit / vegetable juices, - storing the juice in plastic bottles such as High Density Polyethylene (HDPE), Polyethylene Terephthalate (PET), Polypropylene (PP), Polycarbonate (PC),

[0107] - storing the juice in the refrigerator at a temperature of +4°C if processed immediately, at a temperature of -20°C for up to three months, at a temperature of -40°C for up to six months and at a temperature of -80°C for up to one year,

[0108] - defrosting the juice in the refrigerator at a temperature of +4°C if stored at -20, - 40, -80°C and preparing the mixture of juice extracted from ginger, avocado and nettle,

[0109] - carrying out an initial phase of filtering the juice in a filter with a mesh size between 0.2 mm and 1 mm,

[0110] - storing the filtered product, which is not used immediately, for a short period in the refrigerator (preferably at +4°C),

[0111] - carrying out an initial centrifugation of the filtered product in polypropylene or PPCO type centrifuge bottles at a speed of 2,000 x g for a period of 30 minutes,

[0112] - filtering with a very fine mesh nylon filter (preferably with a mesh size up to 100 pm) the supernatant comprising the extracellular vesicles of plant origin (PDEVs), discard the pellet and obtaining the mixture of PDEVs,

[0113] - storing for a short time in the refrigerator (preferably at +4°C) the mixture of PDEVs in plastic bottles (PET, HDPE, PC) or borosilicate glass 3.3,

[0114] - adding to the mixture of PDEVs a quantity of maltodextrin between 4% and 35% by weight,

[0115] - drying the mixture of PDEVs by atomization (spray drying or “SPRAY-DRYING”), - collect and store at room temperature (T = 20 - 25°C) the powdered product in well-closed amber borosilicate glass containers, away from heat sources, not exposed to light and humidity.

[0116] Advantageously, the production of PDEVs through the method according to the present invention achieves an optimization in terms of extract volume, number of vesicles (up to 40% optimization) and reduction of production times (up to 90% optimization).

[0117] Further tests were conducted to evaluate the industrial scalability of two products (Product 1 and Product 2), composed of mixtures of Italian biologic fruit extracts (Table 4). The mixtures are obtained from a plurality of plants and allow to obtain extracellular vesicles with differentiated characteristics, based on the type of plant used. The specificity of the method that includes infusion, filtration and conservation is therefore developed on these particular conditions. The filtration step in the method, according to the invention, is supported by studies that highlight the importance of optimizing centrifugation conditions and filtration compositions to obtain high-quality vesicles.

[0118] Table 4

[0119] The results showed a significant increase in process capacity, from 400-600 ml to 2.8-3.2 liters per hour (=6 times more than the laboratory scale). Furthermore, the powder yield improved from 30-40% in the laboratory up to 80- 97% in industrial scale, maintaining product quality and efficacy.

[0120] In conclusion, the specific steps of the method such as drying carried out according to the “Spray Drying” technique and the optimization of the extraction and filtration conditions, are essential to obtain high-quality extracellular vesicles and to ensure the industrial scalability of the process.

[0121] Clinical tests, as illustrated in Figures 4 and 5, have demonstrated that the extracellular vesicles obtained, through the method of the present invention, from a mix of fresh Italian biological fruits and / or vegetables: ginger (Zingiber officinale), avocado (Persea americana) and nettle (llrtica dioica) are suitable for use in the dermocosmetics sector.

[0122] Advantageously, extracellular vesicles alone in a formulation with sterile water and glutamannan are able to pass the transdermal barrier without the use of “enhanchers” i.e. permeabilizers that break down the keratin barrier to allow the passage of bioactives and to have an efficacy on wrinkles and skin elasticity.

[0123] Figures 4 and 5 show the comparison of the results obtained on the facial skin of 40 volunteers for a period of 45 days following the application of a serum comprising PDEVs extracted from ginger, avocado and nettle and other ingredients such as: water, Sorbitol, Propanediol, Benzyl, Sorbitol, Propanediol, Benzyl alcohol, Polyglyceryl-4 caprate, Glucomannan, Silybum marianum ethyl ester, Tetrasodium glutamate diacetate, Potassium sorbate and a control serum (first of PDEVs).

[0124] The graphs relating to the evaluation of skin elasticity (Figure 4) and wrinkle reduction (Figure 5) at the beginning (TO), after twenty days (T20) and after forty- five days (T45) between the skin treated with the serum comprising the extracellular vesicles and the skin treated with the control serum show a notable increase in both elasticity and wrinkle reduction in the first case. Therefore, the extracellular vesicles obtained, through the method object of the present invention, are suitable for their use in products in the dermocosmetics sector.

[0125] Although at least one exemplary embodiment has been presented in the summary and detailed descriptions, it should be understood that there are a large number of variations that fall within the scope of the invention. Furthermore, it should be understood that the embodiment or embodiments presented are merely examples that are not intended to limit in any way the scope of the invention or its application or configurations. Rather, the summary and detailed descriptions provide the person skilled in the art with a convenient guide to implement at least one exemplary embodiment, it being understood that numerous variations may be made in the function and assembly of the elements described therein, without departing from the scope of the invention as set forth in the appended claims and their technical-legal equivalents.

Claims

AMENDED CLAIMS received by the International Bureau on July 28, 2025 (28.07.2025)CLAIMS1. Method for the production of extracellular vesicles derived from plant raw material (PDEVs) obtained from biological agriculture, comprising the following steps: a. inspecting visually the plant raw material; b. transferring the plant raw material into plastic food containers and store it for short periods in the refrigerator at a temperature of +4°C, c. washing the plant raw material, d. carrying out an initial processing of the plant raw material for extraction, e. extracting the first semi-finished product using automatic extractors, f. storing the first semi-finished product in plastic bottles, g. storing the first semi-finished product in the refrigerator, h. carrying out an initial filtration step of the first semi-finished product in a filter with a mesh size between 0.2 mm and 1 mm, i. storing the filtered product not immediately used for a short period in the refrigerator at a temperature of +4°C, l. performing an initial centrifugation of the filtered product in centrifuge bottles at a speed of 2,000 x g for 30 minutes, m. filtering the supernatant with a nylon filter with a mesh size of up to 100 pm to obtain a mixture of extracellular vesicles and discarding the pellet, n. storing the mixture of extracellular vesicles in a refrigerator for a short time at a temperature of +4°C in plastic or borosilicate glass bottles, o. adding to the mixture of extracellular vesicles a quantity of maltodextrin between 4% and 35% by weight, p. drying the mixture of extracellular vesicles by atomization, q. collecting and storing the powdered product at room temperature in amber borosilicate glass containers.

2. Method according to claim 1 wherein the phase d) of preparation of the plant raw material comprises an infusion step in ultrafiltered water for 24 hours in the case of dried or dehydrated plant raw material.

3. Method according to claim 1 wherein the phase d) of preparation of the plant raw material comprises a phase of peeling and cutting into pieces in the case of fresh plant raw material.

4. Method according to one of claims 1 to 3 where the plant raw material comprises at least one of petals, leaves, berries, roots, fruits and vegetables.

5. Method according to any of claims from 1 to 4 wherein the phase g) is carried out at a temperature of +4°C, if the first semi-finished product is processed immediately, at a temperature of -20°C if the first semi-finished product is processed within three months, at a temperature of -40°C if the first semi-finished product is processed within six months and at a temperature of -80°C if the first semi-finished product is processed within one year.

6. Method according to claim 5 wherein phase g) is followed by a phase g1) of defrosting the first semi-finished product in the refrigerator at a temperature of +4°C if previously the first semi-finished product was stored at a temperature of -20°C, -40°C, -80°C.

7. Method according to any of the preceding claims where phase m) of filtration of the supernatant, for pharmaceutical preparations, is to be carried out with a nylon filter with a mesh size of 0.45 pm.

8. Method according to any of the preceding claims wherein the drying phase p) is carried out according to the spray technique or “Spray Drying” in accordance with the following process parameters:- Drying gas flow rate (m3 / h) = 30 - 35- Inlet temperature (°C) = 115 - 140- Atomized gas flow rate (l / h) = 1000 - 1500- Pump flow rate (ml / min): 6 -15- Outlet temperatures (°C) = 60 - 75.

9. Extracellular vesicles derived from plant raw material (PDEVs) obtained from biological agriculture according to the method as in any of the preceding claims.

10. Use of extracellular vesicles from plant raw material (PDEVs) according to claim 9 as a dermocosmetic or cosmetic.

Images