Extracellular vesicles from oranges and their use

Abstract

Extracellular vesicles (PDEVs) for reaching the blood-brain barrier characterized by: be obtained from oranges from organic farming and to contain within the lipid membrane a ratio between the concentration of Phosphatidylethanolamine (PE) and the concentration of Phosphatidic Acid (PA) between 8 and 15.

Description

[0001] EXTRACELLULAR VESICLES FROM ORANGES AND THEIR USE

[0002] DESCRIPTION

[0003] Technical sector of the invention

[0004] The present invention relates to extracellular vesicles derived from oranges of biological origin and their use as natural transporters of bioactives, in particular for reaching anatomical districts that are difficult to reach .

[0005] Known art

[0006] As is known, one of the greatest discoveries of recent decades has been extracellular vesicles and in particular those of nanometric dimensions called exosomes. Exosomes are considered the connectors between cells not only within the same organ or compartment, but also at a distance. Exosomes of plant origin have aroused great interest in a wide range of applications in the field of health protection as they are able to convey their content (cargo) to target cells. EVs are, therefore, important intercellular mediators and are involved in a multitude of processes, both physiological (coagulation, immune surveillance, etc.) and pathological (tumorigenesis, formation of metastases, etc.). It is also known that human EVs are able to cross biological barriers, in particular to reach the central nervous system , to be internalized in the cell with high specificity.

[0007] What is not known or is only partially known is the level of biodistribution of plant-derived extracellular vesicles (“plant-derived extracellular vesicles”; PDEVs) within the organism of living beings, including humans.

[0008] It is also not known whether PDEVs from fruits and vegetables differ in their ability to reach various anatomical districts (“homing”), in particular whether these PDEVs are able to reach the central nervous system and cross the blood-brain barrier. This information is of fundamental importance for the use of extracellular vesicles obtained from plants both as a new category of nutraceuticals and as a vehicle for drugs intended for the treatment of various pathologies.

[0009] There is therefore a need to obtain extracellular vesicles derived from fruits from Italian biological farming, capable of reaching anatomical districts that are difficult to reach.

[0010] Summary of the invention

[0011] The object of the present invention are therefore extracellular vesicles purified from plants originating from Italian biological farming containing within the lipid membrane at least one bioavailable substance and their use as natural transporters of bioactive compounds, in particular for reaching anatomical districts that are difficult to reach, such as the blood-brain barrier.

[0012] The present invention uses a selected population of extracellular vesicles (EVs) purified from biologically derived oranges.

[0013] According to the present invention, the biodistribution of PDEVs from different fruits (e.g., orange and grape) from Italian biological farming was verified in time course, compared to artificial nanoparticles (i.e. , liposomes).

[0014] According to the present invention, extracellular vesicles isolated from biological oranges have been characterized on the basis of their “ drug delivery ", i.e. their ability to transport drugs and bioactives into the central nervous system that are normally incapable of doing so. Therefore, according to a first aspect of the present invention, extracellular vesicles isolated from biological farming oranges are defined, as specified in the attached independent claim.

[0015] According to a further aspect, the use of such extracellular vesicles for a nasal spray formulation and for injectable solutions is defined, as specified in the independent use claims.

[0016] The dependent claims outline particular and further advantageous aspects of the invention.

[0017] Brief description of the drawings

[0018] 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:

[0019] Figure 1 illustrates the graph of the dimensional characterization of the extracellular vesicles isolated from orange , according to the present invention, Figure 2 illustrates the graph of the dimensional characterization of the extracellular vesicles isolated from grapes, according to the present invention, Figure 3 illustrates the graph of the size characterization of liposomes,

[0020] Figure 4 illustrates the in vivo analysis of the 6-hour biodistribution of the PDEVs purified from orange, according to the present invention,

[0021] Figure 5 illustrates the in vivo analysis of the 6-hour biodistribution of PDEVs purified from grapes, according to the present invention,

[0022] Figure 6 illustrates the in vivo analysis of the 6-hour biodistribution of the liposomes, according to the present invention,

[0023] Figure 7 illustrates the in vivo analysis of the 24-hour biodistribution of the PDEVs purified from orange, according to the present invention,

[0024] Figure 8 illustrates the in vivo analysis of the 24-hour biodistribution of PDEVs purified from grapes, according to the present invention,

[0025] Figure 9 illustrates the in vivo analysis of the 24-hour biodistribution of liposomes, according to the present invention,

[0026] Figure 10 illustrates the in vivo analysis of the 72-hour biodistribution of the

[0027] PDEVs purified from orange, according to the present invention,

[0028] Figure 11 illustrates the in vivo analysis of the 72-hour biodistribution of PDEVs purified from grapes, according to the present invention,

[0029] Figure 12 illustrates the in vivo analysis of the 72-hour biodistribution of the liposomes, according to the present invention,

[0030] Figure 13 illustrates the in vivo analysis of the 7-day biodistribution of the PDEVs purified from orange, according to the present invention,

[0031] Figure 14 illustrates the in vivo analysis of the 7-day biodistribution of the PDEVs purified from grapes, according to the present invention,

[0032] Figure 15 illustrates the in vivo analysis of the 7-day biodistribution of the liposomes , according to the present invention,

[0033] Figure 16 illustrates the 6-hour distribution in organs of PDEVs purified from orange, according to the present invention,

[0034] Figure 17 illustrates the 6-hour distribution in organs of PDEVs purified from grapes, according to the present invention,

[0035] Figure 18 illustrates the distribution of liposomes according to the present invention at 6 hours in the organs,

[0036] Figure 19 illustrates the 24-hour distribution in organs of PDEVs purified from orange, according to the present invention,

[0037] Figure 20 illustrates the 24-hour distribution in organs of PDEVs purified from grapes, according to the present invention,

[0038] Figure 21 illustrates the 24-hour distribution in organs of the liposomes, according to the present invention,

[0039] Figure 22 illustrates the 72-hour distribution in organs of PDEVs purified from orange, according to the present invention,

[0040] Figure 23 illustrates the 72-hour distribution in organs of PDEVs purified from grapes, according to the present invention,

[0041] Figure 24 illustrates the 72-hour distribution in organs of the liposomes according to the present invention,

[0042] Figure 25 illustrates the 1-week distribution in the organs of the PDEVs purified from orange, according to the present invention,

[0043] Figure 26 illustrates the 1-week distribution in the organs of PDEVs purified from grapes, according to the present invention,

[0044] Figure 27 illustrates the 1-week distribution in the organs of liposomes according to the present invention,

[0045] Figure 28 illustrates the distribution in the organism of PDEVs, isolated from orange (A), labeled with fluorescent probe DI Led and inoculated intranasally in Balb / c mice.

[0046] Figure 29 illustrates the distribution in the organism of liposomes (B) labeled with DIL fluorescent probe and inoculated intranasally in Balb / c mice.

[0047] Detailed description According to the present invention and based on the attached figures, extracellular vesicles (EVs) have been purified from orange and grape from organic farming.

[0048] Organic farming is defined as 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 strict restrictions. In addition, crops are rotated so that on-site resources are used efficiently; on-site resources are exploited, such as manure for fertilizer or feed produced on the farm. Furthermore, by definition, organic farming does not use genetically modified organisms (GMOs). On the contrary, plant and animal species that are resistant to disease and adapted to the environment are used. To this end, techniques such as the protection of useful insects, 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 some plants (clover, vetch, cress, lamb's lettuce, spinach, rapeseed and so on) that once they have flowered 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 enriching it with nitrogen, with plants that impoverish it, subtracting nutrients ; manure and organic fertilizers such as compost are used, a mixture of soil, plant remains, wood ash and anything else that exists on the farm that is biodegradable and nonpolluted.

[0049] The methodology through which the extracellular vesicles EVs were obtained is obtained through standard methodologies and in particular includes the following additional phases:

[0050] - Cold extraction: performed with professional extractors equipped with augers, which operate at a speed of 40-100 rotations per minute, simulating the human chewing process. This method preserves the integrity of the PDEVs;

[0051] - Purification: carried out through a specific sequence of filtrations (e.g. with nylon filters, polypropylene, stainless steel, borosilicate glass microfiber filters, etc.) and centrifugations, which allows the separation and purification of PDEVs from fibers, cellular organelles, cellular debris, cellulose and pectins;

[0052] - Concentration, achieved through: a. tangential flow filtration, b. ultrafiltration;

[0053] - Sterilization: performed with a 0.22 micron filter (e.g. with polyethersulfone (PES) membrane, polyvinylidenefluoride (PVDF), nylon, cellulose acetate (CA), polypropylene (PP) membrane, etc.), in compliance with the Pharmacopoeia standards,

[0054] - Dilution to the optimal concentration: carried out using sterile and pyrogen-free ultrafiltered water.

[0055] The process of extraction, concentration, purification and sterilization of plant vesicles is innovative and exclusive. It is based on physical separation methods, avoiding the use of chemical solvents.

[0056] A series of preliminary in vitro experiments in normal human cell cultures was performed in order to select the PDEVs to be compared in the in vivo experiments. In the in vivo experimentation, PDEVs selected in vitro, from orange and grape from organic farming, were used in comparison with the liposomes available on the market.

[0057] As illustrated in Figures 1 - 3, orange, grape and liposome PDEVs, respectively, were characterized for their size distribution, membrane potential and phospholipid concentration.

[0058] The average size (according to “Nanoparticle Tracking Analysis”, NTA) of the Orange PDEVs is 142.1 ± 3.2 nm and the zeta potential is (-)21.8 ± 1.4 mV.

[0059] The average size (according to “Nanoparticle Tracking Analysis”, NTA) of llva PDEVs is 175.0 ± 3.9 nm, the zeta potential is (-) 16.69 ± 0.87 mV and the phospholipids quantified in 1O10PDEVs are 102.06 ± 0.02 pM.

[0060] The average size (according to “Nanoparticle Tracking Analysis”, NTA) of liposomes is 199.8 ± 3.2 nm, the zeta potential is (-) 3.23 ± 0.8 mV.

[0061] Different PDEVs and liposomes were administered intraperitoneally to immunocompetent BALB / C mice. Subsequently, at various time points (6 h, 24 h, 72 h and 1 week) the biodistribution of PDEVs and liposomes administered intraperitoneally was evaluated using in vivo imaging technology (“In Vivo Imaging Systems”, IVIS), which allows to analyze the distribution of both molecules and nanoparticles in a non-invasive manner. This set of experiments aims to verify the biodistribution of labeled PDEVs compared to labeled liposomes. It is of crucial importance to demonstrate not only the ability of PDEVs to distribute within the organism, and therefore to be identifiable within the main organs, but also their ability to cross the blood-brain barrier (BBB) and to persist within the central nervous system. At each time point, the distribution of the various extracellular vesicles by IVIS was measured both in vivo and ex vivo in the organs explanted at the time of sacrifice. The data were collected and analyzed to verify the statistical significance.

[0062] With the single-handed scruff restraint technique, the animal was immobilized to have the other hand free and to be able to work quickly and in succession, having all the material ready. The mouse was held with its head down, in order to have the organs move towards the head and perform the injection (27-30G insulin syringe) without the risk of perforating the intestine, bladder and diaphragm. The division of the abdomen into 4 quadrants was taken into consideration, taking as reference the lower quadrant of the abdomen inside the cavity of the left or right hind leg (better the right of the animal due to the presence of the cecum on the left side).

[0063] Again, a good grip and the speed of the procedure decrease the discomfort to the animal of the grip and contact with the operator. A firm containment does not allow the animal any movement avoiding unnecessary state of agitation and is then placed in its social context immediately after the procedure.

[0064] I VIS (In vivo imaging system) uses the principle of bioluminescence for in vivo applications. In the murine model it allows in-depth analysis in experimental studies, which provide qualitative and quantitative analysis data.

[0065] I VIS applied to preclinical studies is characterized as a Refinement procedure as it minimizes possible states of stress in the animal, and as a Reduction procedure, as by observing the same animal over time, the number of experimental animals is reduced, providing, at the same time, valuable data for the in vivo study. It is possible to define that with bioluminescence studies the right balance is found between experimental data and the use of the animal in the experimental procedure.

[0066] Bioluminescence assessment was performed once a week using the IVIS optical imaging system (Spectrum). Before each imaging under the IVIS, 1 mg / kg luciferin (substrate) was inoculated subcutaneously and then the animals anesthetized with 1-4 % isoflurane were immediately placed inside a heated instrument chamber under continuous infusion of 1- 2% isoflurane.

[0067] Each measurement took from a few seconds to a maximum of 5 minutes for image acquisition (this depends on the intensity of the emitted signal).

[0068] As shown in figures 4-6, in vivo analysis at 6 hours showed an early brain concentration of purified orange PDEVs (figure 4). In contrast, in mice treated with both grape PDEVs (figure 5) and liposomes (figure 6), no positivity was detected in the brain or in other organs. Ex vivo analysis of organs confirmed positivity in the brain only in mice treated with orange PDEVs. In particular, orange PDEVs (figure 16) were distributed in all organs examined (brain, heart, lung, spleen, liver and kidneys). In mice treated with grape PDEVs (figure 17) and liposomes (figure 18), instead, the distribution was mainly confined to the liver and lungs, with a minor presence in the spleen and kidneys.

[0069] As clearly illustrated in figures 7-9, the in vivo examination at 24 hours revealed that the brain localization of purified orange PDEVs (figure 7) remained constant, while no brain positivity emerged in mice treated with grape PDEVs (figure 8) and liposomes (figure 9). The analysis of the organs after explantation substantially confirmed the 6-hour data, with a high positivity in all the examined organs (brain, heart, lung, spleen, liver and kidneys) in mice treated with orange PDEVs (figure 19). In mice treated with grape PDEVs (figure 20), the distribution was mainly concentrated in the liver, followed by lungs and spleen, with a slight presence also in the kidneys. Liposomes (figure 21) being localized only in the liver.

[0070] As highlighted in figures 10-12, the in vivo examination confirmed, even 3 days after inoculation, the brain localization of the PDEVs purified from orange (figure 10), while negativity was confirmed both at the brain level and in all other organs in the mice treated with the PDEVs obtained from grapes (figure 11) and with liposomes (figure 12). The analysis of the organs after explantation substantially confirmed the data from 6 and 24 hours, with positivity at the brain level (although significantly decreasing) only in the mice inoculated with the orange PDEVs, while the grape PDEVs continued not to show positivity at the brain level. The rest of the results were confirmed: the orange PDEVs (figure 22) showed clear positivity at the liver, lungs, kidneys, heart and spleen, with the signal remaining high especially in the liver and lungs, while it significantly decreased in the other organs. Grape PDEVs (figure 23) remained confined mainly to the liver, spleen, kidneys (with little signal) and lungs (with very weak signal) . Liposomes accumulated mainly in the liver (although the signal was decreasing over 24 hours) and to a small extent in the kidneys, spleen and to a very small extent in the lungs (figure 24).

[0071] After 7 days, a significant and uniform change in the distribution of positivity was observed both in vivo and ex vivo . Figures 13-15 show a substantial negativity for both orange (figure 13) and grape PDEVs (figure 14), as well as for liposomes (figure 15). Post-organ explant analysis showed that orange PDEVs

[0072] (figure 25) were no longer localized in the brain, but were still present mainly in the liver, and to a lesser extent in the spleen, lungs, kidneys and heart. Grape PDEVs (figure 26) were predominantly localized in the liver, with a minor presence in the kidneys, lungs and spleen. Liposomes (figure 27) were accumulated in the liver (predominantly) and spleen.

[0073] Experimental tests have clearly demonstrated that purified and concentrated PDEVs from plants, administered systemically (intraperitoneally), are able to distribute early in the organism and cross a barrier often considered insurmountable for most drugs in use: the blood-brain barrier (BBB). However, this is not true for all PDEVs. As demonstrated in the tests performed, PDEVs from oranges are able to cross the blood-brain barrier, but not those from grapes. Furthermore, artificial nanoparticles are not able to cross the BBB and tend to accumulate in the filter organs (liver and spleen). PDEVs from oranges not only remain in the brain of treated mice up to 72 hours after inoculation, but are distributed in practically all the organs analyzed: heart, lungs, spleen, liver and kidneys. Supporting tests were performed both in vivo (IVIS technology) and ex vivo in organs explanted after sacrifice at the end of the experiment.

[0074] The fact that PDEVs are able to enter the central nervous system and remain there opens new perspectives for health management based on the use of PDEVs as therapeutic tools, also capable of transporting drugs to districts that are difficult to reach, such as the central nervous system.

[0075] Further tests highlighted the pulmonary biodistribution of PDEVs purified from orange. In figures 28-29 the distribution in the organism of isolated orange vegetal extracellular vesicles (PDEVs,) (A) and liposomes (B) both labeled with a fluorescent probe DIL, and inoculated intranasally in Balb / c mice, were compared. The results were analyzed with I VIS technique in the organs explanted at the time of sacrifice, after 18 hours from the treatment. The figure shows the results of two different mice (mouse 1 and mouse 2), compared to a mouse left without any treatment (naive CTR).

[0076] Advantageously, these data support the use of purified PDEVs from biological oranges in certain nasal spray and injectable solutions formulations.

[0077] It is clear that only in mice that had been administered orange PDEVs intranasally there was positivity at the pulmonary level, while in other organs no fluorescence was detected. On the contrary, in mice inoculated with labeled liposomes there were no fluorescence signals detectable with I VIS in all the organs examined.

[0078] These results represent the definitive confirmation that intranasally administered plant extracellular vesicles, in particular those isolated from orange, are able to circulate within the organism and stop in the lung. Artificial nanoparticles (i.e. liposomes) administered intranasally, on the other hand, have shown a tendency not to circulate freely in the organism. The permanence of PDEVs at the lung level, even if administered intranasally, confirms the extreme bioavailability of PDEVs.

[0079] Finally, the phospholipid composition of PDEVs was evaluated.

[0080] For liposomes it is as follows:

[0081] 50% Phosphatidylcholine (PC) 20% Phosphatidylglycerol (PG)

[0082] 10% Phosphatidylethanolamine (PE)

[0083] 10% Cholesterol.

[0084] For Orange PDEVs it is as follows:

[0085] 25% Phosphatidylcholine (PC)

[0086] 40% Phosphatidylethanolamine (PE) 5% Phosphatidic Acid (PA) 12 of Phosphatidylinositol (PI) For llva's PDEVs it is as follows:

[0087] 16% Phosphatidylcholine (PC)

[0088] 26% Phosphatidylethanolamine (PE)

[0089] 53% Phosphatidic Acid (PA)

[0090] 5% Phosphatidylinositol (PI)

[0091] The ratio of PE to PA is a substantial difference for the vesicles to reach a target organ. In particular, a high ratio, for example in a PE / PA range between 8 and 15 with PE between 40% and 45% and PA between 3% and 5%, which characterizes the composition of orange PDEVs, determines the brain (ability to permeate the blood-brain barrier) and lung (ability to permeate the blood-air barrier) distribution. On the contrary, in grape PDEVs, the PE / PA ratio is low i.e. PE between 25% and 5% and PA between 50% and 60%, preventing them from permeating the blood-brain barrier. Liposomes contain exclusively PE at a low concentration i.e. 10%, which makes it difficult for them to reach the specific target organ (e.g. lung, brain). An example of a pharmaceutical composition as a nasal spray comprises a concentration of orange PDEVs between 1% and 20 % with the lipid composition in favor of PE (phosphatidylethanolamine) and comprising at least one of the following components: preservatives : benzalkonium chloride 0.01% stabilisers : sodium edetate 0.1% solvents : purified water qs 80% and 100%.

[0092] A composition per 5 ml of nasal spray includes in volumetric percentages:

[0093] Orange PDEVs: 10% (0.5 ml), concentrated in favor of phosphatidylethanolamine (PE) ,

[0094] - Benzalkonium chloride (preservative): 0.01% (0.0005 ml),

[0095] - Sodium edetate (Stabilizer): 0.1% (0.005 ml),

[0096] - Purified water (Solvent): qs up to 100% (4.4945 ml).

[0097] Advantageously, intranasal administration is functional for reaching the lungs.

[0098] An example of a pharmaceutical composition as an injection solution comprises a concentration of orange PDEVs between 1% and 4% with the lipid composition in favor of PE (phosphatidylethanolamine) and comprising at least one of the following components:

[0099] - solvent: water for injections commonly used as a solvent. In some cases, saline solutions (NaCI 0.9%) or other specific solutions at 94%-95% may be used;

[0100] - excipients: additional ingredients that help stabilize the solution, improve the solubility of the active ingredient, or extend shelf life. Common examples include: sodium chloride (used to make the solution isotonic with blood), sodium hydroxide or hydrochloric acid (used to adjust the pH of the solution);

[0101] - preservatives: such as benzalkonium chloride, to prevent microbial contamination;

[0102] - stabilizers: some drugs may require specific stabilizers to maintain their effectiveness during storage and use in a concentration between 1%-2% .

[0103] A composition for 5 ml of injection solution includes in volumetric percentages :

[0104] • Orange PDEVs: 2% (0.1 ml), with lipid composition in favor of phosphatidylethanolamine (PE)

[0105] • Sodium chloride (NaCI) (excipient): 0.9% (0.045 ml), used to make the solution isotonic with blood

[0106] • Benzalkonium chloride (preservative): 0.01% (0.0005 ml)

[0107] • Stabilizers: 1.5% (0.075 ml), to ensure the stability of the solution

[0108] • Water for injections (solvent): qs up to 100% (4.7795 ml)

[0109] 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 30 December 2025 (30.12.2025)1. Extracellular vesicles (PDEVs) : obtained from oranges from organic farming and containing within the lipid membrane a ratio between the concentration of Phosphatidylethanolamine (PE) and the concentration of Phosphatidic acid (PA) between 8 and 15; characterized by an average size of 142.1 ± 3.2 nm and a zeta potential of (-) 21.8 ± 1.4 mV.

2. Extracellular vesicles (PDEVs) according to claim 1 , where the concentration of Phosphatidylethanolamine (PE) is between 40% and 45% and the concentration of Phosphatidic acid (PA) is between 3% and 5%.

3. Pharmaceutical composition as a nasal spray comprising a volumetric concentration, with respect to the total volume of said pharmaceutical composition, of orange extracellular vesicles (PDEVs), according to any of the preceding claims, of between 1 % and 20% and at least one of the following compounds in concentration, with respect to the total volume of said pharmaceutical composition, of:- 0.01% benzalkonium chloride- 0.1 % sodium edetate- purified water qs 80% and 100%.

4. Pharmaceutical composition as an injection solution comprising a volumetric concentration, with respect to the total volume of said pharmaceutical composition, of orange extracellular vesicles (PDEVs), according to one ofthe preceding claims, between 1% and 4% and at least one of the following compounds: solvent: water for injections or NaCI in volumetric concentration, with respect to the total volume of said pharmaceutical composition, of 0,9% or other specific solutions in a volumetric concentration, with respect to the total volume of said pharmaceutical composition, between 94% and 95%; excipients: sodium chloride or sodium hydroxide or hydrochloric acid; preservatives: benzalkonium chloride; stabilizers in a volumetric concentration, with respect to the total volume of said pharmaceutical composition, between 1%-2%.

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