A method of preparing a dry powder of milk extracellular vesicles (MEVS) and MEV dry powder obtained thereof

Abstract

The invention relates to a method for the preparation of a dry powder of milk extracellular vesicles (mEVs), wherein the method essentially comprises spray-drying a suspension of mEVs isolated from milk in the presence of a stabilising agent consisting of the combination of mannitol and leucine. The invention also relates to the mEV dry powder obtained with the method of the invention, and therapeutic applications thereof.

Description

[0001] A METHOD OF PREPARING A DRY POWDER OF MILK EXTRACELLULAR VESICLES (MEVS) AND MEV DRY POWDER OBTAINED THEREOF

[0002] Technical Field

[0003] The present invention relates to a method for the preparation of milk extracellular vesicles (mEVs) stabilized in the form of dry powders, as well as the mEV dry powders thus obtained and their uses in the medical field. Background art

[0004] Despite the great progress made in recent years in studies exploring the potential of extracellular vesicles (EVs) for early disease diagnosis, therapeutic application and drug administration, the identification of favourable storage conditions for EVs is still an open chal- lenge.

[0005] The essential need in all the clinical and research areas that study and use EVs, in fact, is to find a system for stabilising EV preparations which allows long-term shelf life without any change in their physical / functional characteristics. EVs, especially milk EVs, have well- known therapeutic effects but also significant critical issues. EVs, in fact, are structures that can undergo varying degrees of modification during storage, resulting in loss of size, shape, function, and content. According to current knowledge, the preservation of the morphological and functional characteristics of EVs is partially possible only at low temperatures (- 80°C), but this methodology is obviously not practicable if the EVs are intended for clinical and medical applications.

[0006] Freeze-drying techniques for the preservation of EVs have recently been tested, with or without the addition of cryoprotectants. These techniques have been shown to be promising in ensuring EV preservation only for a rather limited period of time [Drug Deliv. 2021;28(l): 1501-1509. doi: 10.1080 / 10717544.202 E 1951896],

[0007] International patent application WO 2023 / 067490 describes the use of the spray-drying technology for the preparation of milk EV (mEV) formulations, without the use of any type of excipient. However, WO 2023 / 067490 does not demonstrate the effectiveness and longterm stability of the product obtained.

[0008] Summary of the invention

[0009] Considering the prior art, there is still a need to provide a method for stabilising milk extracellular vesicles (mEVs), which allows their long-term preservation without any change in their function and composition, thus making the use of mEVs feasible in various fields, particularly in the medical field.

[0010] These and other needs are now met by the present invention, which refers to a method for the preparation of a dry powder of milk extracellular vesicles (mEVs), as defined in the appended claim 1.

[0011] The scope of the invention also includes a dry powder of mEVs that is stable in the long term under ambient conditions, as well as therapeutic uses thereof.

[0012] Further features and advantages of the invention are defined in the appended dependent claims.

[0013] The appended claims form an integral part of the present specification.

[0014] Detailed description

[0015] The scope of the invention includes a method for the preparation of a stable dry powder of milk extracellular vesicles (mEVs), and the resulting stable dry powder of EVs.

[0016] The method of the invention comprises the following steps: a) Providing a suspension of isolated milk extracellular vesicles (mEVs). mEVs are preferably from milk, preferably bovine milk. The milk used may be raw or may have been previously pasteurized. Optionally, the milk used may also be ultrafiltered. In a preferred embodiment, prior to isolation of the mEVs, the milk is treated with enzymes, salts, or acidic solutions such as chymosin, ethylenediaminetetraacetic acid (EDTA), acetic acid, hydrochloric acid, or sodium citrate to remove the proteins in solution more effectively during the isolation process.

[0017] Differential centrifugation associated with ultracentrifugation is advantageously used for the isolation of the mEVs from milk, for example using the following protocol: centrifugation of the milk at 300-5000* g for 5-30 min at room temperature or 4°C; one to three successive centrifugations of the supernatant at 10,000 - 35,000* g for 30-90 min at 4°C, after adding an equal volume of 50-500 mM EDTA (pH 7.4) to the supernatant and incubating for 0-30 min on ice; a final ultracentrifugation between 100,000 and 200,000* g for 60-90 minutes at 4°C.

[0018] The mEV suspension is preferably prepared by collecting the mEV pellet obtained by differential centrifugation and ultracentrifugation and resuspending this pellet in water, distilled water, or isotonic solution, such as, for example, PBS. The water or isotonic solution used is preferably sterile. b) Quantification of the protein content

[0019] The total protein content of the mEVs in the suspension is measured on an aliquot of the suspension by any known methodology, e.g. by absorption assay or fluorimetry. To this end, a preferred methodology is the Bradford assay. The Bradford protein assay is a colorimetric method used to determine the concentration of proteins in a solution or suspension. This assay is based on the ability of the Coomassie Brilliant Blue G-250 dye to bind to proteins, causing a change in colour that can be measured spectrophotometrically. The amount of protein contained in the mEVs in the suspension or solution is proportional to the intensity of the colour developed. c) Addition of a stabilising agent

[0020] A stabilising agent, namely a combination of mannitol and leucine (D- or L-leucine), is added to the mEV suspension.

[0021] The weight ratio of the stabilising agent to the protein content of the mEVs in the suspension ranges from 20: 1 to 1 :20, preferably from 10: 1 to 1 : 1. A particularly preferred ratio of the stabilising agent to the protein content in the mEV suspension is 2: 1, or more preferably 1 : 1.

[0022] The combination of mannitol and leucine used as the stabilising agent is intended to improve the properties of the dry powder obtained as a result of the method, especially in terms of stability. In particular, leucine helps to form a protective barrier on the surface of the dried particles, improving their stability, and prevents the aggregation of the particles (doi: 10.1016 / j.xphs.2024.06.018; doi: j.powtec.2021.02.031). Mannitol helps to reduce the degradation of the mEVs due to heat or moisture during drying and enhances the ease of handling (doi: j.powtec.2021.02.031). These properties are particularly valuable for preserving the activity of the biologically active substances contained in the mEVs. This is demonstrated by the fact that the dry mEV powder obtained using the method of the invention has been shown to maintain long-term biological efficacy for at least 18 months after its preparation. d) Spray-drying

[0023] The mEV suspension with the stabilising agent is spray-dried to obtain a dry mEV powder.

[0024] In a preferred embodiment, the solute concentration in the suspension of mEVs and stabilising agent that is subjected to the spray-drying step is between 1% and 20% w / v, more preferably between 1.5 and 5% w / v.

[0025] In the spray-drying step, the prepared suspension of mEVs and stabilising agent is atomized into fine droplets using a laboratory- or industrial-scale spray dryer. The spray dryer has, for example, a pneumatic or rotary disc atomizer and a co-current or counter-current configuration. Atomization increases the surface of the liquid, facilitating rapid drying. The fine droplets then enter the drying chamber, where they come into contact with a hot gas (usually air) at an inlet temperature typically between 120°C and 160°C (a temperature of 140°C is preferred). The hot gas causes moisture to evaporate rapidly from the droplets, resulting in dry particles, i.e., mEV dry powder. Process parameters, such as air flow rate, feed rate, and vacuum speed, can be adjusted as needed.

[0026] The resulting dry powders are stable under ambient or refrigerated conditions for at least 18 months, while maintaining the biological activity of the mEVs.

[0027] This spray-drying step is essential to transform the mEV suspension into a stable dry powder form, which can be easily stored, handled and used for therapeutic applications.

[0028] The main advantage of the spray-drying process is that a product is obtained which exhibits greater chemical-physical stability than the suspension of the mEVs in a liquid medium. Furthermore, compared to other drying processes such as freeze-drying, spray-drying requires very short time and is less expensive because it does not need low process temperatures. During the spray-drying process, the mEVs are exposed to high temperatures for a very short period of time; for this reason, and due to the presence of the selected stabilisers, the active components of the mEVs do not degrade. Spray-drying is also a highly adjustable technique that allows the properties of the particles to be controlled and can therefore allow the development of specific therapeutic applications.

[0029] The scope of the invention also includes a dry powder of mEVs resulting from the method described above.

[0030] The dry mEV powder includes the combination of mannitol and leucine as the stabilising agent.

[0031] The weight ratio of the stabilising agent to the proteins of the mEVs is between 20: 1 and 1 :20, even more preferably between 10: 1 and 1 : 10, e.g., 2: 1 or 1 : 1. The particle size distribution of the dry powder preferably ranges from 0.1 pm to 5 mm, more preferably from 0.1 pm to 990 pm, even more preferably from 1 to 60 pm.

[0032] A particularly advantageous feature of the mEV dry powder of the invention is its stability under ambient or refrigerated conditions for at least 18 months, while maintaining the biological activity of the mEVs.

[0033] These features ensure that the mEV dry powder of the invention is stable, easy to handle, and maintains its therapeutic properties for long periods, making it suitable for various medical and nutraceutical applications.

[0034] The mEV dry powder of the invention can be reconstituted in a pharmaceutically acceptable vehicle to form a suspension of reconstituted mEVs. A pharmaceutically acceptable vehicle is, for example, a saline solution.

[0035] The mEV dry powder of the invention can be used in pharmaceutical compositions, for example, for oral, inhalation or topical administration, in particular for the treatment of inflammatory diseases such as chronic inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis).

[0036] The molecular load of mEVs isolated from milk of various species has in fact been widely characterized, highlighting the presence of proteins, metabolites, and RNA species that play a role in mammary gland physiology, milk production, immunity and immune response, cell homeostasis, and intestinal mucosal maturation. It is also known that mEVs exhibit antiinflammatory and immunomodulatory properties, as demonstrated in vitro in intestinal inflammation models and in immune cells. The beneficial effects of the mEVs on the intestine have also been demonstrated in experimental models of necrotising colitis. The preferred route of administration for the mEVs is the oral route, which is optimal since mEVs are known to be capable of passing intact through the gastric barrier.

[0037] The mEV dry powder of the invention can also be used in nutraceutical products, such as food supplements or functional foods. A nutraceutical product containing the mEV dry powder of the invention may, for example, be a dietary supplement or food supplement in tablets or capsules, or a functional food such as an additive to liquid food products (milk and yogurt) or solid food products such as bars or snacks.

[0038] The examples that follow are provided for illustration purposes only and do not limit the scope of the invention as defined in the appended claims.

[0039] Example 1

[0040] Materials and Methods

[0041] 1. Isolation of extracellular vesicles from milk

[0042] Raw milk was collected from a single livestock farm under the supervision of the Department of Veterinary Medicine of Perugia, Italy. To account for individual variability, samples were collected from bulk (pooled) milk directly from the collection tank. Mid-lactation Holstein Friesian cows, managed according to a standard intensive farming system, were selected for sampling. Several aliquots (1-2 litres) of bulk milk were collected from the tank to characterize the extracellular vesicles derived from fresh milk (mEVs) and to produce batches of mEV powder. The milk was processed immediately to avoid cryopreservation and minimize potential artifacts.

[0043] The mEVs were isolated by subjecting the bulk raw milk to two consecutive centrifugations at 3000*g (Eppendorf® Centrifuge 5810R with F34-6-38 rotor) for 10 minutes at room temperature to remove cells and cell debris (in the pellet) as well as the milk fat component (top layer). Once the supernatant was recovered, an equal volume of 0.25 M EDTA (pH 7.4) was added to promote protein precipitation, incubated for 15 min on ice and centrifuged at 10,000*g for 1 hour at 4°C. The supernatant was then ultracentrifuged at 35,000*g (Beckman Coulter Optima L-100 XP ultracentrifuge with Type 45 Ti rotor) for 1 hour at 4°C, which allowed most of the remaining proteins (mainly caseins) to be removed. The supernatant was recovered and a final ultracentrifugation was carried out at 200,000 / g for 90 minutes at 4°C and the mEVs were collected in the form of a pellet. The pellet was then resuspended in sterile phosphate buffered saline (PBS) and filtered through a 0.22 pm filter.

[0044] 2, Dry Powder Production

[0045] The isolated mEVs were transformed into dry powders by spray-drying to obtain a formulation that is stable over time and therefore allow long-term use of the mEVs. The technique involves drying mEV suspensions in a single step by atomization-drying and collection by means of a cyclone system. In detail, the protein concentration of the mEV suspensions in PBS was measured by the Bradford assay. A solution of excipients in double-distilled water was added to the suspensions to a final concentration twice the previously measured protein concentration in the mEV sample. The excipients used were mannitol and D-leucine in a 1 : 1 ratio. The prepared mEV / excipient suspensions were concentrated, in terms of total solute, between 1.5 and 5%. These were processed through a Mini Spray -Dry er model B-290 (Biichi, Milan, Italy) to obtain a dry microparticulate formulation by setting the following process parameters: inlet temperature 140°C, air flow rate 357 L / h, feed flow rate 2.5 mL / min, vacuum flow rate 20 m3 / h. These parameters are compatible with the type of instrument used. The Mini Spray -Dry er model used has a nozzle diameter between 0.5 and 2 mm to obtain a particle size distribution of the dry powder in the range of 1-60 pm.

[0046] 3, Morphological and functional characterization

[0047] Morphology

[0048] Dry mEV powders were observed under a FEG-LEO 1525 scanning electron microscope (LEO Electron Microscopy Inc., NY). The source voltage was maintained at 2 keV. The samples were placed on a double-sided carbon tape attached to an aluminium stub. The stubs were coated with chromium at 20 mA for 25 seconds.

[0049] The size distribution of the powders was measured using the Accusizer C770 instrument (PSS, Santa Barbara, CA) equipped with an auto-dilution system. The powders were dispersed in acetonitrile and immediately analysed in the same solvent in gravity mode. The dimensions were expressed as the volumetric mean diameter.

[0050] In addition, both the mEV powders resuspended in ultrapure water and the fresh mEVs from which the powders were obtained were analysed using the Malvern Panalytical NanoSight NS300 NTA system (Malvern, Worcestershire, U.K.) to assess the size range and concentration of fresh mEVs in the preparations.

[0051] Protein and Nucleic Acid Quantification

[0052] Protein and nucleic acid concentration associated with the fresh mEVs and the dry mEV powder resuspended in aqueous solvent was evaluated for any changes following the spraydrying process.

[0053] The Bradford assay consists of adding Coomassie Brilliant Blue G-250 to the protein solution. Coomassie Blue dye is associated with basic and aromatic amino acids, causing a shift in absorbance during protein determination. The absorbance is proportional to the proteins present. The Bradford assay was performed in a microplate by mixing 1 part of the sample with 50 parts of Bradford reagent. The absorbance was measured at 595 nm and protein concentration was determined by comparison with a standard curve obtained by measuring bovine serum albumin. Nucleic acid content was measured by fluorimetry (Qubit 4.0 Thermo Fisher). Fluorescence-based quantification methods use dyes that bind specifically to dsDNA, ssDNA, or RNA, causing a conformational shift that produces fluorescence upon excitation. The concentration was determined by comparison with a standard curve.

[0054] Toxicity assessment

[0055] For the toxicity assay, THP1 cells were seeded at a concentration of 3xlOA4 / well in a 96 well plate in medium containing 16 pM Phorbol 12-Myristate 13-Acetate (PMA) to induce differentiation. After 24 hours, the cells were washed in PBS and treated with scalar concentrations of mEVs (10A8 particles / mL to 10A14 particles / mL corresponding to a protein amount of 0.00025 pg / pl to 250 pg / pl) isolated from bovine milk, either processed or not processed into dry powders.

[0056] MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to the cells one hour before the end of the 24-hour treatment. The assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, or MTT) into purple formazan crystals by metabolically active cells as a result of the fact that viable cells contain NAD(P)H-dependent reductase enzymes that reduce MTT into formazan. The insoluble formazan crystals are then dissolved in DMSO before spectrophotome- teric reading at 590 nm to determine viability.

[0057] Gene Expression Assessment

[0058] To determine gene expression, THP1 cells were seeded at a concentration of 0.2xl0A6 / / well in a 12 well plate in medium containing 16 pM PMA to induce the differentiation thereof. After 24 hours, the cells were washed in PBS and stimulated with Interferon-gamma (IFNy, 10 mg / ml) and lipopolysaccharide (LPS, 1 pg / ml) to induce an inflammatory state, and treated with mEVs (5x 10A10 parti cles / ml - approximately 125 pg / pl of protein) isolated from bovine milk, either processed or not processed into dry powders. After 6 and 18 hours, the medium was recovered and the cells were lysed in the RLT lysis solution of the Qiagen RNA extraction kit (74104-RNeasy Mini Kit). The RT-qPCR reaction was performed with 5 pL of 1 : 10 diluted cDNA and SsoFast™ Eva-Green® Supermix (BioRad, Hercules, CA, USA). The amplification was performed with a CFX96™ Real-Time PCR Detection System (BioRad, Hercules, CA, USA) under the following conditions: 95°C for 1 minute, 40 cycles of 95°C for 10 seconds followed by 60°C for 15 seconds. The fluorescence was measured at the end of the second step, and the melting curve was produced at the end of the reaction by increasing the temperature by 0.5°C every 0.05 seconds until reaching 95°C, to assess the specificity of the signal. Gene expression was then assessed for target genes such as interleukin 1 beta (IL1B), IL6, interleukin 8 (CAULS), and tumor necrosis factor (TNFA), using genes encoding ribosomal proteins 37A and 0 (RPL37A and RPLO) as reference genes. The primers were designed using the Primer3 on-line platform (https: / / primer3.ut.ee), placing the two primers for the same gene (sense and antisense) in different exons or at exon-exon junctions to prevent signalling due to amplification of contaminating genomic DNA (Table 1). The data was normalized based on the expression levels of the two reference genes, after assessing their stability under the two different experimental conditions, using the Norm algorithm included in the Bio-Rad CFX Maestro software (ver. 4.1 BioRad, Hercules, CA, USA). The relative normalized expression was assessed using the 2-AACTstatistical method. After normalization to the expression of the reference genes (ACT), the difference between the two ACT values (AACT) of the samples from the groups treated with fresh mEVs or with mEVs transformed into dry powders was calculated compared to that of the control group (inflamed cells). Table 1- Sequences of the primers used to test gene expression in RT-qPCR. Statistical analysis was performed in the R environment by applying a one-way ANOVA variance analysis and Student’s t-test for pairwise comparisons. Statistical significance was tested by comparing the various treatments with the group subjected to inflammation alone (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). In particular, for the experiment on the assessment of the anti-inflammatory power, samples from the untreated group (Control), inflamed cells treated with fresh mEVs (mEVs), inflamed cells treated with mEVs transformed into dry powder stored for 6 months (powder_mEVs_M6), and inflamed cells treated with mEVs transformed into dry powder stored for 18 months (powder_mEVs_M18) were compared with the inflamed cells (Inflamed).

[0059] The effect of the excipients alone on the same inflamed cells was also assessed to verify that the reduction in gene expression caused by the powders was actually induced by the mEVs. Therefore, the expression of the tested genes in the inflamed cells (Inflamed) was compared with that of naive cells (Control) and inflamed cells subsequently treated with excipients (Inf exc).

[0060] Comparison of LPS content

[0061] Lipopolysaccharide (LPS) is considered to be a potential contaminant with inflammatory properties in mEVs extracted from milk. Therefore, the possible presence of LPS in the suspension of fresh mEVs and in the dry powder of mEVs was assessed with the ToxinSensor Chromogenic LAL Endotoxin Assay kit (GenScript, USA).

[0062] Results

[0063] The dry powders were subjected to morphological and dimensional characterization. The results are shown in Figure 1, which represents the morphology and the corresponding volumetric size distributions of mEV powders from bovine milk, a-c represent three replicates produced under the same conditions and stored for different times. Specifically, mEV powder stored for 22 months (a), mEV powder stored for 9 months (b), and freshly produced mEV powder (c). The graph in Figure 1 shows the dimensional analysis evaluated using the Accusizer C770 particle counter. The dimensions were expressed as the volumetric mean diameter.

[0064] Figure 2 shows the SEM image of fresh mEVs prior to transformation into dry powders. The volumetric mean diameters of the transformed mEVs range from 11 to 20 pm.

[0065] The protein content and particle number were assessed by the Bradford assay and NTA on dry powders, respectively (Table 2).

[0066] Table 2- Characterization of the content of the dry powders

[0067] For the assessment of protein and nucleic acid content, the samples were normalized by NTA establishing a number of mEVs of 2.2 xlOA12 for both fresh mEVs and dry powders derived therefrom. The mean and standard deviation values of the two distributions (“fresh mEVs” and “resuspended dry powders”) are given in Table 3, including the statistical significance of the difference between the means by applying Student’ s t-test.

[0068] Table 3- Protein and nucleic acid quantifications before and after drying the mEVs

[0069] The toxicity of the fresh mEV suspension and of the mEVs transformed into dry powder was then tested on human macrophage cell lines (THP-1) by assessing cell viability with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Figure 3). The two preparations show very similar toxicity and, based on this analysis, we selected a quantity of 5x10A10 particles / ml as the treatment for the following in vitro functional studies. The graphs in Figure 3 show the results of the viability test on the THP1 macrophage cell line after administration of scalar concentrations of mEVs freshly isolated from bovine milk (fresh bovine mEVs) and of the microparticulate powder (dry-powder bovine mEVs).

[0070] The effect of mEVs stored as dry powders for 6 and 18 months after production was then assessed on inflammatory processes by testing the gene expression in the same THP1 cell line pretreated with LPS and fFNy and subjected to two treatment times, 6 hours (6h) (Figure 4) and overnight (ON) (Figure 5). This made it possible to assess the functional stability of the dry formulation of mEVs.

[0071] The graphs in Figure 4 show the gene expression in untreated cells (Control), cells treated with a pro-inflammatory stimulus (Inflamed), inflamed cells treated for 6h with fresh mEVs (mEVs), dry mEV powder stored for 6 months (powder_mEVs_M6) and 18 months (pow- der_mEVs_M18). Statistical analysis was performed in the R environment by applying a one-way ANOVA variance analysis and Student’ s t-test for pairwise comparisons. Statistical significance was tested by comparing the control group (Control) and the various treatments (mEVs, powder_mEVs_M6 and powder_mEVs_M18) with the group subjected to inflammation alone (Inflamed) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0072] The graphs in Figure 5 show the gene expression in untreated cells (Control), cells treated with a pro-inflammatory stimulus (Inflamed), inflamed cells treated ON with fresh mEVs (mEVs), dry mEV powder stored for 6 months (powder_mEVs_M6) and 18 months (pow- der_mEVs_M18). Statistical analysis was performed in the R environment by applying a one-way ANOVA variance analysis and Student’ s t-test for pairwise comparisons. Statistical significance was tested by comparing the control group (Control) and the various treatments (mEVs, powder_mEVs_M6 and powder_mEVs_M18) with the group subjected to inflammation alone (Inflamed) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0073] The effect of excipients alone on inflamed cells was then assessed (Figure 6).

[0074] The graphs in Figure 6 show the gene expression in untreated cells (Control), cells treated with a pro-inflammatory stimulus (Inflamed), inflamed cells treated for 6h (A) and overnight (B) with excipients (Inf exc). Statistical analysis was performed in the R environment by applying a one-way ANOVA variance analysis and Student’s t-test for pairwise comparisons. Statistical significance was tested by comparing the control group (Control) and the excipient treatment (Inf exc) to the group subjected to inflammation alone (Inflamed) (*p<0.05, **p<0.01, ****p<0.0001).

[0075] As mentioned above, the presence of LPS was tested in both powder and fresh mEVs as it is considered a contaminant that could potentially impair the natural anti-inflammatory effect of the preparations. LPS range for the two preparations was found to be 10-50 pg per IO10particles, i.e., a residual amount that does not produce inflammatory effects in naive cells (Figure 7).

[0076] The graphs in Figure 7 show the results relating to the effect of excipients alone, freshly isolated mEVs, and EV powder at 6 months of storage on naive cells for A) 6h treatment, B) overnight. Statistical analysis was performed in the R environment by applying a one-way ANOVA variance analysis and Student’s t-test for pairwise comparisons (ns: not statistically significant), comparing cells treated with excipients alone (Exc), fresh mEVs, or mEV powder stored for 6 months (powder_mEVs_M6) with untreated naive cells (Control).

[0077] Discussion

[0078] The experimental part of the present description illustrates, by way of example, the transformation of milk extracellular vesicles (mEVs) into a dry microparticulate powder using the spray-drying technology.

[0079] The morphological transformation of the mEVs is evident (Figure 1 a-c and Figure 2), with the conversion of the mEVs into collapsed particles which are typical of the applied drying process. The volumetric mean diameters of the transformed mEVs range from 11 to 20 pm.

[0080] The protein and nucleic acid content, with the same number of particles in solution, remained almost unchanged between the preparation of fresh mEVs and the dry powders (Table 3), showing that there were no molecular changes due to the drying process.

[0081] Also from a functional point of view, the dry powders did not undergo changes compared to the fresh mEVs (Figure 3). In fact, the study showed a considerable stability of the mEVs stored as dry powders at room temperature for 6 and 18 months.

[0082] The product obtained was shown to maintain the same anti-inflammatory effect as the fresh mEVs in vitro, reducing the gene expression of the tested pro-inflammatory cytokines (Figures 4 and 5). It was also shown that the anti-inflammatory effect is strictly induced by the mEVs of the powder formulation and not by the excipients, which did not change gene expression either on inflamed cells (Figure 6) or on naive cells (Figure 7).

[0083] Lastly, the presence of lipopolysaccharides (LPS) was tested in both the dry powder and the fresh mEVs as they are potential contaminants that could impair the natural anti-inflammatory effect of the dry mEV powders, and it was found that the residual amount of LPS does not produce inflammatory effects in naive cells.

[0084] Example 2

[0085] Comparison of formulations with mannitol or lactose

[0086] Preparation of dry powders containing mEVs

[0087] The mEV-containing comparison formulations were obtained by spray-drying and collection by means of a cyclone system. The protein concentration of the mEV suspensions in PBS was measured by the Bradford assay. In one case mannitol / leucine in a 1 : 1 w / w ratio, and in the other case lactose / leucine in a 2.5: 1 w / w ratio, were added to the ultrapure water suspensions of mEVs, as per the literature (Prior art DI), to obtain a total concentration of 1.5% w / v. In both cases, the excipients were dissolved to a final concentration twice the previously measured protein concentration in the mEV sample. Both preparations were processed with a Mini Spray -Dry er model B-290 (Buchi, Milan, Italy) under the same conditions: inlet temperature 140°C, air flow rate 357 L / h, feed flow rate 2.5 mL / min, vacuum flow rate 20 m3 / h. The resulting powders were collected and stored under ambient conditions.

[0088] Morphological and dimensional characterization

[0089] Morphological analyses were performed with a FEG-LEO 1525 scanning electron microscope (LEO Electron Microscopy Inc., NY). The source voltage was maintained at 2 keV. The samples were placed on a double-sided carbon tape attached to an aluminium stub. The stubs were coated using a high-resolution sputter coating system with chromium (Quorum Technologies, East Essex, UK). The coating was carried out at 20 mA for 30 s. Dimensional analysis: Accusizer C770 particle counter (PSS, Santa Barbara, CA) equipped with an autodilution system. The powders were dispersed in hexane and immediately analysed in the same solvent in gravity mode. The dimensions were expressed as the volumetric mean diameter.

[0090] Nanoparticle Quantification and Size Range

[0091] The mEVs contained in the dry powders obtained with mannitol / leucine and lactose / leucine were extracted by resuspending the powders in ultrapure water and were analysed using the Malvern Panalytical NanoSight NS300 NTA system (Malvern, Worcestershire, U.K.) to assess the size range and concentration of fresh mEVs in the preparations (Table 4). The results are reported as the mean + / - standard deviation of 5 measurements for each preparation.

[0092] Protein and Nucleic Acid Quantification

[0093] Protein and nucleic acid content was measured by fluorimetry (Qubit 4.0 Thermo Fisher). Fluorescence-based quantification methods use dyes that bind specifically to proteins, dsDNA, ssDNA, or RNA, causing a conformational shift that produces fluorescence upon excitation. The concentration was determined by comparison with a standard curve. For the assessment of protein and nucleic acid content, the samples were normalized by NTA establishing a number of mEVs of 2.2 xlOA12 after extraction from the dry powder formulations obtained with mannitol / leucine and lactose / leucine (Figure 10). The statistical significance of the difference between the means was tested by applying Student’s t-test. Results

[0094] The two dry powder formulations obtained with lactose in one case and mannitol in the other showed similar characteristics in terms of size and morphology. Figure 8 shows the morphology of the mEV-loaded dry powders obtained using the excipients: a. lactose / leucine or b. mannitol / leucine; the graph in the figure shows the relative size distributions of the powders expressed as volumetric diameter.

[0095] The particles with lactose / leucine were found to be slightly larger, with a volumetric mean diameter of approximately 16 pm compared to 13 pm for those with mannitol / leucine. The morphology is very similar since it is related to the identical processing conditions, where the only difference is the type of sugar and the ratio to leucine.

[0096] The mEVs extracted from the powders were also similar in terms of size (Figure 9 and Table 4) even though the powder with lactose / leucine showed a slightly higher mEV content in terms of numbers (Table 4). Panel a. of Figure 9 refers to the mannitol / leucine combination, panel b. refers to the lactose / leucine combination.

[0097] Table 4. Number of particles / mg dry powder and mean diameter measured by Nanoparticle Tracking Analysis after extraction from the formulations obtained with mannitol or lactose. Results are expressed as mean + / - standard deviation (SD) (n=5).

[0098] Despite this, analysis of protein and nucleic acid content in mEVs extracted from powders showed a significant reduction of all parameters in the preparation with lactose / leucine compared to that with mannitol (Figure 10). Figure 10 shows the analysis of protein, RNA and DNA content in mEVs after extraction from the dry powder formulations obtained with mannitol / leucine and lactose / leucine. *p<0.00001, **p< 0.05, ***p< 0.01 (n=3). This result suggests a greater stabilising ability of mannitol compared to lactose during the drying process. This result could also be related to the reducing nature of lactose, which can promote Maillard reactions with peptides, leading to denaturation of proteins in particular, especially at the high temperatures required for the drying process.

Claims

CLAIMS1. A method of preparing a dry powder of milk extracellular vesicles (mEVs) comprising the steps of: a) providing a suspension of isolated milk extracellular vesicles (mEVs) in water or in an isotonic solution; b) quantifying the protein content of the mEVs in the suspension; c) adding to the mEV suspension a stabilising agent consisting of the combination of mannitol and leucine, wherein the weight ratio of the stabilising agent to the total protein content of the mEVs in the suspension is between 20:1 and 1:20; d) spray-drying the mEV suspension added with the stabilising agent, thereby obtaining a dry powder of mEVs.

2. The method according to claim 1, wherein the stabilising agent consists of the combination of mannitol and D-leucine.

3. The method according to claim 2, wherein the weight ratio of mannitol to D-leucine is between 10:1 and 1:10.

4. The method according to any one of claims 1 to 3, wherein the mEV suspension added with the stabilising agent to be spray-dried according to step d) has a total solute concentration of between 1% and 20% w / v.

5. The method according to any one of claims 1 to 4, wherein the dry powder obtained in step d) is stored under ambient conditions or in a refrigerator.

6. The method according to any one of claims 1 to 5, which comprises the further step of reconstituting the dry powder of mEVs in a pharmaceutically acceptable vehicle to form a suspension of mEVs reconstituted in the pharmaceutically acceptable vehicle.

7. A dry powder of milk extracellular vesicles (mEVs) spray-dried with a stabilising agent consisting of the combination of mannitol and leucine.

8. The dry powder of mEVs according to claim 7, wherein the stabilising agent consists of the combination of mannitol and D-leucine.

9. The dry powder of mEVs according to claim 7 or 8, having a particle size distribution ranging from 0.1 pm to 5 mm, preferably ranging from 0.1 pm to 990 pm.

10. The dry powder of mEVs according to any one of claims 7 to 9, wherein the weight ratio of the stabilising agent to the total protein content of the mEVs is between 20: 1 and 1 :20.

11. The dry powder of mEVs according to any one of claims 7 to 10, which is stable under ambient conditions for at least 18 months after its preparation.

12. A pharmaceutical composition comprising a dry powder of mEVs according to any one of claims 7 to 11 and at least one pharmaceutically acceptable vehicle, diluent and / or excipient.

13. A pharmaceutical composition comprising a dry powder of mEVs according to any one of claims 7 to 11, which is reconstituted in a pharmaceutically acceptable vehicle.

14. The pharmaceutical composition according to claim 12, which is formulated for oral administration, administration by inhalation or topical administration.

15. The pharmaceutical composition according to any one of claims 12 to 14 or the dry powder according to any one of claims 7 to 11, for use as a medicament.

16. The pharmaceutical composition or dry powder for use according to claim 15, in the therapeutic treatment of an inflammatory disease.

17. The pharmaceutical composition or dry powder for use according to claim 16, wherein the inflammatory disease is a chronic inflammatory bowel disease.

18. The pharmaceutical composition or dry powder for use according to claim 17, wherein the chronic inflammatory bowel disease is Crohn’s disease or ulcerative colitis.

19. A nutraceutical product comprising a dry powder of mEVs as defined in any one of claims 7 to 11, which is optionally reconstituted in a pharmaceutically acceptable vehicle.

20. The nutraceutical product according to claim 19, which is a food supplement or a functional food.

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