Method for production of pharmaceutical composition of exosomes comprising lipophilic and / or hydrophilic active substance

Abstract

The present invention is referred to a method for production of a pharmaceutical composition, said composition comprises: A) naturally secreted exosomes from normal human embryonic lung fibroblast cells (MRC-5 cell line), wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method comprises the following steps in the following order: a) separation of exosomes by ultrafiltration, b) collection of exosomes by membrane-based affinity binding method, c) loading of exosomes with active substance, or B) artificial exosomes, wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method is a microfluidic method and said method comprises the following steps in the following order: a) extraction of lipids from a cell line of normal human embryonic lung fibroblast cells (MRC-5 cell line) to obtain a lipid film, b) resuspending of the lipid film in an organic solvent and mixing with the aqueous phase in a microfluidics device to obtain exosomes, wherein the aqueous phase comprises Tween 20 and wherein in the microfluidics device I) the flow rate ratio of the organic phase to the flow rate of the aqueous phase (flow rate ratio, FRR) is set from 1:5 to 1:3, ii) the organic phase flow rate (OPFR) is preferably set at 200 pL / min or 400 pL / min and ill) the aqueous phase flow rate (APFR) is preferably set at 100 pL / min or 1000 pL / min, c) purification of the obtained exosomes using size exclusion chromatography, d) loading of the exosomes with active substance, e) purification of the obtained exosomes using size exclusion chromatography to remove the unloaded drug.

Description

[0001] Method for production of pharmaceutical composition of exosomes comprising lipophilic and / or hydrophilic active substance

[0002] Description of the invention

[0003] The present invention is referred to a method for production of a pharmaceutical composition, said composition comprises:

[0004] A) naturally secreted exosomes from normal human embryonic lung fibroblast cells (MRC-5 cell line), wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method comprises the following steps in the following order: a) separation of exosomes by ultrafiltration, b) collection of exosomes by membrane-based affinity binding method, c) loading of exosomes with active substance, or B) artificial exosomes, wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method is a microfluidic method and said method comprises the following steps in the following order: a) extraction of lipids from a cell line of normal human embryonic lung fibroblast cells (MRC-5 cell line) to obtain a lipid film, b) resuspending of the lipid film in an organic solvent and mixing with the aqueous phase in a microfluidics device to obtain exosomes, wherein the aqueous phase comprises Tween 20 and wherein in the microfluidics device i) the flow rate ratio of the organic phase to the flow rate of the aqueous phase (flow rate ratio, FRR) is set from 1:5 to 1:3, ii) the organic phase flow rate (OPFR) is preferably set at 200 pL / min or 400 pL / min and iii) the aqueous phase flow rate (APFR) is preferably set at 100 pL / min or 1000 pL / min, c) purification of the obtained exosomes using size exclusion chromatography, d) loading of the exosomes with active substance, e) purification of the obtained exosomes using size exclusion chromatography to remove the unloaded drug.

[0005] Exosomes are nano-scaled, round-shaped lipid biovesicles naturally released by all cells in both physiological and pathological conditions. Whilst they were initially considered as molecular waste carriers, soon their unique properties as key mediators of cellular communication were clarified [Pan, B.-T., & Johnstone, R. M. (1983). Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective exte realization of the receptor. Cell, 33(3), 967-978.; Raposo, G., Nijman, H. W., Stoorvogel, W., Liejendekker, R., Harding, C. V, Melief, C. J., & Geuze, H. J, (1996). B lymphocytes secrete antigen-presenting vesicles. Journal of Experimental Medicine, 183(3), 1161-1172] and led many scientists to indulge in exosome-based therapeutics for many diseases spanning the field of neurodegenerative medicine to oncology [Rezaie, J,, Feghhi, M., & Etemadi, T. (2022). A review on exosomes application in clinical trials: perspective, questions, and challenges. Cell Communication and Signaling, 20(1), 145].

[0006] The exploitation of exosomes in oncology is based on the potential selective delivery of the encapsulated conventional or advanced biological drugs at the target malignant cells, minimizing off-target effects on normal tissues. Advanced biotechnology-based drugs and pharmaceutical molecules have been incorporated into exosomes, constituting innovative systems for targeting cancer cells and showing enhanced stability, bioavailability, and better therapeutic potential of the loaded agent [Srivastava, A., Amreddy, N., Razaq, M., Towner, R., Zhao, Y. D., Ahmed, R. A., Munshi, A., & Ramesh, R. (2018). Exosomes as Theranostics for Lung Cancer (pp. 1- 33).; Wang, J., Zheng, Y., & Zhao, M. (2017). Exosome-Based Cancer Therapy: Implication for Targeting Cancer Stem Cells. Frontiers in Pharmacology, 7],

[0007] Also, exosomes, as biological vesicles that are naturally released by all cell types, exhibit advantageous biopharmaceutical properties as drug delivery systems in cancer therapy, compared to synthetic nanoparticles. Human fetal lung fibroblast MRC-5 cells are commonly used as a sustainable industrial cellular factory for the production of vaccines, approved by regulatory systems as safe biological materials with no further tests required.

[0008] Despite the prominent biopharmaceutical properties of exosomes as drug delivery "bio- shuttles" for oncotherapy, the required multistage exosome isolation procedures, the lack of standardized isolation and characterization methodologies, the innate heterogeneity and "batch-to-batch" variation in exosomal formulations, as well as the non-clarified exosomes intrinsic targetability towards specific cancer cells, hinder the "translational process" of exosome-based drug delivery systems towards clinical application for cancer management [Meng, Y., Asghari, M., Aslan, M. K., Yilmaz, A<, Mateescu, B., Stavrakis, S., & deMello, A. J. (2021). Microfluidics for extracellular vesicle separation and mimetic synthesis: Recent advances and future perspectives. Chemical Engineering Journal, 404, 126110]. So far, many isolation methods have been studied, employing traditional, widely applied approaches as well as more advanced emerging technologies. However, the established exosome isolation methods cannot yet guarantee high yield and purity in the final exosome formulation, Besides, some methodologies require specialized staff and costly equipment, leaving the field of exosomes in its infancy [Gao, J., Li, A., Hu, J,, Feng, L., Liu, L., & Shen, Z. (2023). Recent developments in isolating methods for exosomes. Frontiers in Bioengineering and Biotechnology, 10].

[0009] The difficulty of isolating large quantities hinders their subsequent exploitation in clinical practice. Furthermore, they are characterized by the insufficient capacity to entrap large amounts of drug, the difficulty of loading with methods that disrupt their physicochemical properties, and the inefficient separation of the non-entrapped-free drug from the entrapped drug in the exosomes.

[0010] Due to the increasing quality and safety requirements and therefore the high demands when preparing pharmaceutical exosome compositions with high efficiency and purity in the final composition, there is a need for an Improved production method. However, in the prior art, there are no mentions yet of a method for the production of pharmaceutical composition, said composition comprises:

[0011] A) naturaliy secreted exosomes from normal human embryonic lung fibroblast cells (MRC-5 cell line), wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method comprises the following steps in the following order: a) separation of exosomes by ultrafiltration, b) collection of exosomes by membrane-based affinity binding method, c) loading of exosomes with active substance, or

[0012] B) artificial exosomes, wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method is a microfluidic method and said method comprises the following steps in the following order: a) extraction of lipids from a cell line of normal human embryonic lung fibroblast cells (MRC-5 cell line) to obtain a lipid film, b) resuspending of the lipid film in an organic solvent and mixing with the aqueous phase in a microfluidics device to obtain exosomes, wherein the aqueous phase comprises Tween 20 and wherein in the microfluidics device i) the flow rate ratio of the organic phase to the flow rate of the aqueous phase (flow rate ratio, FRR) is set from 1:5 to 1:3, ii) the organic phase flow rate (OPFR) is preferably set at 200 pl / min or 400 pL / min and iii) the aqueous phase flow rate (APFR) is preferably set at 100 pL / min or 1000 pL / min, c) purification of the obtained exosomes using size exclusion chromatography, d) loading of the exosomes with active substance, e) purification of the obtained exosomes using size exclusion chromatography to remove the unloaded drug. More specifically, the present invention is defined by the following: Definition 1, Method for production of a pharmaceutical composition, said composition comprises:

[0013] A) naturally secreted exosomes from normal human embryonic lung fibroblast ceils (MRC-5 cell line), wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method comprises the foliowing steps in the following order: a) separation of exosomes by ultrafiltration, b) collection of exosomes by membrane-based affinity binding method, c) loading of exosomes with active substance, or

[0014] B) artificial exosomes, wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method is a microfluidic method and said method comprises the following steps in the following order: a) extraction of lipids from a cell line of normal human embryonic lung fibroblast cells (MRC-5 cell line) to obtain a lipid film, b) resuspending of the lipid film in an organic solvent and mixing with the aqueous phase in a microfluidics device to obtain exosomes, wherein the aqueous phase comprises Tween 20 and wherein in the microfluidics device i) the flow rate ratio of the organic phase to the flow rate of the aqueous phase (flow rate ratio, FRR) is set from 1:5 to 1:3, ii) the organic phase flow rate (OPFR) is preferably set at 200 pL / min or 400 pL / min and iii) the aqueous phase flow rate (APFR) is preferably set at 100 pL / mln or 1000 pL / min, c) purification of the obtained exosomes using size exclusion chromatography, d) loading of the exosomes with active substance, e) purification of the obtained exosomes using size exclusion chromatography to remove the unloaded drug. Definition 2. Method for production of a pharmaceutical composition according to definition 1, wherein the active substance is preferably curcumin and the loading of curcumin into the exosomes is preferably in a curcumin: exosomes ratio of 5:1, said method comprises the following steps in the following order: a) mixing the exosomes with curcumin dissolved in phosphate buffered saline (PBS) and ethanol, preferably to a concentration of ethanol less than or equal to 1%, b) incubation preferably for 3 hours at room temperature away from light and c) ultracentrifugation to remove the unloaded curcumin preferably for 2 hours at 135,000xg.

[0015] Definition 3. Method for production of a pharmaceutical composition according to definition 1, wherein the active substance is preferably doxorubicin and the loading of doxorubicin into the exosomes is preferably in a 1:1 ratio, said method comprises the following steps in the following order: a) either ultrasonic treatment of the exosomes in a solution of doxorubicin hydrochloride preferably with more than one cycles with intermediate cooling periods, preferably of 2 minutes, followed by incubation preferably at 37°C for 90 minutes and then ultracentrifugation to remove the unloaded doxorubicin preferably for 2 hours at 135,000xg, b) either resuspension of the previously lyophilized exosomes in a solution of doxorubicin hydrochloride, followed by incubation preferably at 37°C for 90 minutes and then ultracentrifugation to remove the unloaded doxorubicin preferably for 2 hours at 135,000xg.

[0016] Definition 4. Method for production of a pharmaceutical composition according to any one of the definitions 1 to 3, wherein the exosomes exhibit zeta potential values generally ranging from -100 mV to +100 mV, preferably from -100 mV to 0 mV, more preferably from -35 mV to -5 mV. Definition 5. Method for production of a pharmaceutical composition according to any one of the definitions 1 to 4, wherein the exosomes present the CD9 protein (CD9 antigen) and the CD63 protein (CD63 antigen) on their surface and comprise at least 39 other proteins.

[0017] Definition 6. Method for production of a pharmaceutical composition according to any one of the definitions 1 to 5, wherein the exosomes comprise at least 92 miRNAs, preferably the mature miRNAs hsa-miR-10400-5p (Homo sapiens-miRNA-10400-Sp), hsa-miR-365b~5p (Homo sapiens-miRNA-365b-5p), hsa-miR-1469 (Homo sapiens- miRNA-1469), hsa-miR-1268a (Homo sapiens-mJRIMA-1268a) and mature miRNAs of the let-7 family.

[0018] Definition 7. Method for production of a pharmaceutical composition according to any one of the definitions 1 to 2 and 4 to 6, wherein the exosomes are curcumin nanocarriers with a loading rate from 2,5 to 25%, preferably at 9%.

[0019] Definition 8. Method for production of a pharmaceutical composition according to any one of the definitions 1 and 3 to 6, wherein the exosomes are doxorubicin nanocarriers with a loading rate from 2,5 to 25%, preferably at 16,5%.

[0020] Definition 9. Method for production of a pharmaceutical composition according to any one of the definitions 1 and 3 to 6 and 8, wherein the exosomes are in lyophilized form.

[0021] Definition 10. Method for production of a pharmaceutical composition according to any one of the definitions 1 to 9, said composition is formulated with suitable pharmaceutically acceptable excipients and administered intravenously or subcutaneously or intraperitoneally or orally. Definition 11. Microfluidics method for production of a pharmaceutical composition according to the definition 1, wherein the exosomes comprise lipids of the MRC-5 cell line, cholesterol and Tween 20 in a molar ratio from (55 to 75) :(25 to 35);(2 to 8), preferably at 65:30:5.

[0022] Definition 12. Microfluidics method for production of a pharmaceutical composition according to any one of the definitions 1 and 11, wherein the total concentration of lipids in the exosomes ranges from 3 mg / mL to 15 mg / mL, preferably is 11 mg / mL.

[0023] Definition 13. Microfluidics method for production of a pharmaceutical composition according to any one of the definitions 1 and from 11 to 12, wherein the active substance is preferably doxorubicin and the loading of doxorubicin into the exosomes is preferably carried out by the remote loading method, wherein: a) exosomes are prepared using an ammonium sulfate solution with Tween 2.0 (pH=5,48) as the aqueous phase in the microfluidics apparatus, b) purification of the obtained exosomes using size exclusion chromatography at pH =7,4, so that the exosomes present size of up to 150 nm, c) addition of doxorubicin solution to create a transmembrane pH gradient and stirring for 1 hour at 60°C, d) re-purification of the obtained exosomes using size exclusion chromatography at pH =7,4 to remove the unloaded doxorubicin, so that the exosomes present size of up to 150 nm.

[0024] Definition 14. Microfluidics method for production of a pharmaceutical composition according to any one of the definitions 1 and from 11 to 13, wherein the exosomes exhibit a size of from 100 nm to 150 nm.

[0025] Definition 15. Microfluidics method for production of a pharmaceutical composition according to any one of the definitions 1 and from 11 to 14, wherein the exosomes are in lyophilized form. Definition 16. Microfluidics method for production of a pharmaceutical composition according to any one of the definitions 1 and from 11 to 15, said composition is formulated with suitable pharmaceutically acceptable excipients and administered intravenously or subcutaneously or intraperitoneally or orally.

[0026] I) Method of production of naturally secreted exosomes

[0027] The present invention, taking into account all the aforementioned data, solves all the above problems of the state of the art, and indeed with unexpectedly advantages and better results in terms of improved purity in the exosomes sample obtained.

[0028] It was surprisingly found that the method of the present invention provides exosomal samples comprising unexpectedly fewer protein impurities, i.e. unexpectedly higher purity, as confirmed by protein quantification with the colorimetric method BCA (Bicinchoninic acid assay), as well as by the zeta potential of the exosomal membrane, for example with a value of -30,9 mV,

[0029] It was surprisingly found that the method of the present invention provides exosomal samples with unexpectedly better results, in terms of maintaining the size of the exosomes with a diameter of for example 30-90 nm (typical exosomal size 30-150 nm).

[0030] It was surprisingly found that the method of the present invention provides exosomal samples with unexpectedly better results, in terms of reducing the aggregation of the exosomes, for example to sizes of 40 nm.

[0031] It was surprisingly found that the method of the present invention provides exosome compositions that exhibit selective uptake by "heterologous” A549 cells (Adenocarcinomic human alveolar basal epithelial cells) and even more particularly by HSC-3 cells (Human Tongue Squamous Carcinoma Ceil Line). Considering the regulator / framework, exosome-based drug delivery systems fall into the category of biological drugs (biopharmaceuticals) which are accompanied by intrinsic biological variability and a high degree of complexity in the development of the final formulation. It was found that the present methodology contributes to the standardization and simplification of the development of exosomal nanocarriers, resulting in a reduction in the heterogeneity of the biopharmaceutical characteristics of the final formulation. Finally, it was surprisingly found that the more is restricted the scope of the claims, the more intense are the effects and the advantages of the present invention.

[0032] When lipophilic and hydrophilic active substances are simultaneously comprised, we simultaneously have the advantages of both active substances. Notably, when no active substance is comprised in the exosomes, the cytotoxicity of the exosomes is zero.

[0033] II) Method for production ©f artificial exosomes via microfluidics

[0034] The present invention, taking into account all the aforementioned data, solves all the above problems of the state of the art, and indeed with unexpectedly advantages and better results in terms of providing unexpectedly improved physicochemical characteristics of the received exosomes, as well as increased drug loading rate and controlled drug release.

[0035] It was surprisingly found that the method of the present invention enables the production of large quantities of exosomes ready for exploitation. In particular, after the lipids are isolated from the cells and the aqueous and organic phases of the fluid mechanics system are prepared, for example, it is possible to produce an exosome suspension at a rate of 1.2 mL / min. It was surprisingly found that the method of the present invention provides exosome samples with unexpectedly better results, in terms of maintaining the size of the exosomes with a diameter of 100 nm (typical size of exosomes 30-150 nm), as well as a similar lipid composition to naturally secreted exosomes, It was surprisingly found that the method of the present invention provides exosome samples with unexpectedly better results, in terms of the highest drug loading percentage, above 30%, e.g. 60%, preferably 48%.

[0036] It was surprisingly found that the method of the present invention provides exosome compositions with a sustained drug release profile, which profile exploits cell killing through apoptosis mechanisms.

[0037] It was surprisingly found that the method of the present invention provides exosome compositions which maintain their size and homogeneity (monodispersity) at constant levels after the first 2 weeks. Considering the regulatory framework, exosome-based drug delivery systems fall into the category of biological drugs (biopharmaceuticals) which are accompanied by intrinsic biological variability and a high degree of complexity in the development of the final formulation. It was found that the present methodology contributes to the standardization and simplification of the development of exosomal nanocarriers, resulting in a reduction in the heterogeneity of the biopharmaceutical characteristics of the final formulation.

[0038] Finally, it was surprisingly found that the more is restricted the scope of the claims, the more intense are the effects and the advantages of the present invention.

[0039] When lipophilic and hydrophilic active substances are simultaneously comprised, we simultaneously have the advantages of both active substances.

[0040] Notably, when no active substance is comprised in the exosomes, the cytotoxicity of the exosomes is zero.

[0041] Ill) Caparison of the two methodologies

[0042] It is noted that the method of naturally secreted exosomes is preferable in cases where, for example, we need immediate release of the drug, or in cases of cell lines where we want the exosomes to comprise more proteins and / or receptors for more selective uptake by the cells, as naturally secreted exosomes resemble the exosomes of the cell even more. Also, naturally secreted exosomes may be able to be involved in more biochemical pathways of the cell.

[0043] Of course, artificial exosomes prepared by the microfluidics method are advantageous over naturally secreted exosomes when we target other characteristics, such as loading rate, size homogeneity and controlled release.

[0044] The present invention is further described by the following indicative, but non-limiting examples.

[0045] IV) Method for production of naturally secreted exosomes

[0046] Example 1: Morphological and physicochemical characterization of exosomes isolated using three different isolation methods

[0047] The method for production of a pharmaceutical composition, said composition comprises exosomes, as described in the present invention, was compared with two other methods reported in the prior art, (a) ultracentrifugation and (b) membraneaffinity binding, Exosomes isolated by each methodology were then subjected to physicochemical and morphological evaluation by of using techniques Dynamic Light Scattering (DLS) and Cryogenic Transmission Electron Microscopy (cryo-TEM).

[0048] According to the DLS results, in the exosomes isolated by the method of the naturally secreted exosomes of the current invention, a size distribution peak was observed at approximately 30 nm (with a size distribution range of 30-90 nm). According to the cryo-TEM results, spherical-shaped exosome vesicles were observed within the range obtained by DLS. In addition, no aggregation phenomena were observed.

[0049] The ^-potential value was -30,9 mV, indicating good colloidal stability of particles in suspension. The negative value of the surface potential of the exosomes verifies the removal of free protein "contaminants" originating from the donor cell secretion or from fetal bovine serum supplement during ultrafiltration, which enhances the purity of the exosomal samples.

[0050] Notably, the method of the naturally secreted exosomes of the present invention resulted in the absence of aggregates, which is a very significant advantage compared to the ultracentrifugation method (Figure 1), as well as a low percentage of total proteins, lower than that of the membrane-affinity binding method, which is a significant advantage in terms of the purity of the exosome sample obtained (Figure 2). Example 2: Loading of curcumin in exosomes

[0051] Curcumin loading into exosomes was conducted by mixing a fixed proportion (5:1) of MRC5~derived exosomal protein (PBS IX pH 7.4) with curcumin (dissolved in ethanol and diluted with phosphate buffer saline (PBS) to achieve a final ethanol concentration less than or equal to 1%). The mixture was incubated for 3 hours at room temperature protected from light to achieve efficient binding of lipophilic curcumin with exosomal lipid membrane. To remove the unbound free drug, the preparation was ultracentrifuged for 2 hours at 135.000xg. The estimation of the amount of curcumin loaded in exosomes was achieved with the direct lysing method. Briefly, the curcumin- loaded exosomal pellet was lysed with Radioimmunoprecipitation Assay buffer / RIPA IX, followed by bath-sonication to facilitate the release of curcumin from exosomal vesicles. The lysed exosomal suspension was then diluted with Phosphate buffered saiine / PBS IX pH 7.4 and the amount of curcumin loaded in exosomes was estimated via Fluorescence spectroscopy with 42.0nm excitation and 550nm emission.

[0052] Cellular uptake assays were also conducted by using Confocal Laser Scanning Microscopy / CLSM) and cell viability assays upon incubation of empty or drug-loaded exosomes derived from MRC-5 cells, with "autologous" normal MRC-5 cells and "heterologous" A549 (Adenocarcinomic human alveolar basal epithelial cells) and HSC- 3 (Human Tongue Squamous Carcinoma Cell Line).

[0053] Curcumin-loaded exosomes accumulate to a greater extent in A549 and HSC-3 cancer cells than in normal MRC-5 cells.

[0054] Curcumin-loaded exosomes display selectivity and enhanced antiproliferative activity in A549 and HSC-3 cancer cells compared to normal MRC-5 cells (Figure 3).

[0055] Therefore, curcumin-loaded exosomes exhibit increased "endogenous" targeting in A549 and HSC-3 tumor cells. Example 3: Doxorubicin loading in exosomes

[0056] For doxorubicin HCI loading in exosomes, the doxorubicin HCI salt was diluted in PBS IX pH 7 A and two different loading methods were investigated: (a) ultrasonication of exosomes in doxorubicin HCI solution, and, (b) resuspension of previously freeze-dried exosomes in doxorubicin HCL solution, For both methods, a fixed proportion (1:1) of

[0057] MRC-5 cell-derived exosomal protein (intact exosomes), as estimated via BCA, with doxorubicin HCL was added. For the sonication method, the doxorubicin -exosome mixture was sonicated using a 130-watt Ultrasonic Processor; CV18 Converter and keeping the flowing standard settings: 20% amplitude, 3 cycles of 10s on / off for 1 minute. Between each cycle, a constant two-minute cooling interval was mediated to avoid exosomal membrane disintegration due to extensive heating. For the lyophilization method, the exosomes were freeze-dried overnight and then resuspended in doxorubicin HCL solution, After the completion of each loading procedure, the exosomes-doxorubicin mixtures were incubated at 37°C for 90 minutes to facilitate the recovery of the exosomal membrane, followed by ultracentrifuged for 2 hours at 135,000xg to remove the excess-free unincorporated doxorubicin and isolate doxorubicin -loaded exosomes.

[0058] The same amount of doxorubicin HCI previously added into exosomal suspensions was also ultra centrifuged with the same conditions to enable the quantification of doxorubicin loading in exosomes. The doxorubicin loaded in exosomes was quantified via the indirect supernatant-based method. A standard calibration curve was constructed and the intrinsic fluorescence of doxorubicin was detected via Fluorescence spectroscopy with 488nm excitation wavelength and 590nm emission wavelength.

[0059] Cellular uptake studies using Confocal Laser Scanning Microscopy (CLSM) and cell viability assays were also performed upon incubation of empty or drug-loaded exosomes derived from MRC-5 cells with "autologous" normal MRC-5 cells and "heterologous" tumorigenic A549 and HSC-3 cells. Doxorubicin hydrochloride-loaded exosomes accumulate to a greater extent in A549 and HSC-3 tumor cells than in normal MRC-5 cells.

[0060] Doxorubicin hydrochloride-loaded exosomes exhibit higher antiproliferative activity in both normal MRC-5 and A549 and HSC-3 tumor cells than free doxorubicin hydrochloride (Figure 4).

[0061] Therefore,, doxorubicin hydrochloride- loaded exosomes exhibit increased

[0062] "endogenous"' targeting to A549 and HSC-3 cancer cells.

[0063] Dual co-administration of curcumin-loaded exosomes in combination with doxorubicin hydrochloride-loaded exosomes enhances the observed growth inhibition of HSC-3 cancer cells (Figure 5).

[0064] The above results are better understood by referring to the attached Figures 1 to 5 where:

[0065] Figure 1: Comparative morphological evaluation via cryo-TEM of MRC-5 cell-derived exosomes concentrated via ultrafiltration (a, 3) and collected via collected via membrane-based binding affinity method (y, 6). Spherical vesicles (pointed by white arrows) with diameter of about 30-60 nm represent the isolated exosomes. The bright white circles 'which surround the exosomal lumen are the lipid bilayer of the exosomal vesicles. Exosomes aggregates (pointed by red arrows) generated by the high-speed centrifugation are also presented.

[0066] Figure 2: Comparison of various exosome isolation methodologies in terms of exosome yield as determined by total protein estimation via BCA assay. ** p<0;005, **** p<0,0001. Figure 3: In vitro viability of "autologous" lung normal MRC-5 ceils (a) and '’heterologous" lung tumor A549 (3) and tongue tumor HSC-3 cells (y) after incubation for 48 hours with control medium (control), IpM free CUR, IpM CUR-loaded in exosomes (4.9ppg exosomal protein), 3pM free CUR and 3uM (CUR as curcumin). CUR-loaded in exosomes (I4.75pug exosomal protein), 96-well plates were used and the viability of the cells was assessed by performing CCK-8 assays. The graph was created in GraphPad Prism, One-way ANOVA with Tukey's multiple comparison test was applied. *p<0.05, **p<0.01, ***p<0.005

[0067] Figure 4: In vitro viability of "autologous" lung normal MRC-5 ceils (a) and "heterologous" lung tumor A549 (3) and tongue tumor HSC-3 cells (y) after incubation for 48 hours with control medium (control), 0.3pM free DOX, 0,3pM DOX-loaded in exosomes (3.1ppg exosomal protein), 0.7pM free DOX and 0.7pM DOX-loaded in exosomes (7,2ppg exosomal protein) (DOX as doxorubicin). 96-well plates were used and the viability of the ceils was assessed by performing CCK-8 assays. The graph was created in GraphPad Prism. One-way ANOVA with Tukey's multiple comparison test was applied, *p<0.05

[0068] Figure 5: In vitro viability of "autologous" lung normal MRC-5 cells (a) and "heterologous" lung tumor A549 (p) and tongue tumor HSC-3 cells (y) after coincubation for 48 hours with 0.7pM DOX loaded in exosomes (7.2ppg exosomal protein) and IpM CUR loaded in exosomes (4.9ppg exosomal protein). 96-well plates were used and the viability of the cells was assessed by performing CCK-8 assays. The graph was created in GraphPad Prism. One-way ANOVA with Tukey's multiple comparison test was applied. *p<0.05. V) Method of production of artificial exosomes via microfluidics

[0069] Example 4; Morphological and physicochemical characterization of exosomes

[0070] The microfluidics method for preparing a pharmaceutical composition comprising exosomes ("artificial exosomes" based on MRC-5 cell lipids) was compared with the method for obtaining naturally secreted exosomes from MRC-5 cells.

[0071] The exosomes isolated by each methodology were then subjected to physicochemical and morphological evaluation using Dynamic Light Scattering (DLS) and Cryogenic Transmission Electron Microscopy (cryo-TEM) techniques.

[0072] According to the DLS results, naturally secreted exosomes maintain a size of 30-90 nm (Figure 6a) and the "artificial exosomes", isolated by the microfluidics method, maintain a size of 40-110 nm (Figure 6y).

[0073] The peak size distribution of naturally secreted exosomes is located at 30 nm, while the peak size distribution of artificial exosomes is located at .100 nm. According to the literature, vesicles with a diameter in the range of 100-150 nm favor cellular uptake and are able to escape from the capillaries of blood vessels in diseased tissues (such as kidneys, heart and lungs) and enter through the vessels into the tumor microenvironment. In addition, the larger size of "artificial exosomes" compared to naturally secreted exosomes favors a greater drug loading capacity (both hydrophilic and lipophilic) inside them.

[0074] Hydrophilic drugs can be more easily entrapped in the aqueous lumen of vesicles due to their larger internal aqueous volume and lower surface-area-to-volume ratio. Hydrophobic-lipophilic drugs can also be more easily incorporated into their lipid membrane due to the larger surface area of the lipid bilayer that can accommodate more lipophilic drug molecules. According to DLS size distribution analysis, naturally secreted exosomes exhibit heterogeneity in size. According to the literature, cells secrete vesicles with different sizes and different molecular contents, which makes it difficult to standardize the isolation and formulation process. In addition, the size distribution of exosomes can vary depending on variations in the culture process of the exosome donor cells (fluctuations in the nutrient medium, oxygen-temperature conditions) and is also altered by alterations in cell physiology due to aging or stress.

[0075] In contrast, as demonstrated by the size distribution analysis of "artificial exosomes", these vesicles maintain a narrow size distribution with significant homogeneity without the presence of vesicles of variable size. Therefore, the development of artificial vesicles utilizing cell lipids can be more easily standardized, reducing batch-to-batch variations, while complying with the regulatory framework. Furthermore, the artificial exosomes prepared by the microfluidics method exhibit satisfactory stability at 4°C, as observed from their analysis after 2 and 4 weeks via the DLS method. More specifically, they maintain their size and homogeneity (monodispersity) at constant levels after the first 2 weeks. As shown by cryo-TEM analyses (Figure 6p & Figure 66), both naturally secreted exosomes and artificial exosomes appear to have the same morphology, i.e. they are spherical vesicles surrounded by a lipid bilayer.

[0076] The membrane integrity of the artificial exosomes prepared by the microfluidics method was evaluated by the Caicein Release Assay and proved to be particularly satisfactory especially for the form developed by the microfluidics device with a total flow rate of 1200pL / min. More specifically, it was observed that at 25 hours only 20% of the entrapped caicein was released. Example 5: Doxorubicin loading in exosomes

[0077] The loading capacity of doxorubicin in the "artificial exosomes" produced by the microfluidics method was compared with that of naturally secreted exosomes from MRC-5 cells.:

[0078] The study of loading of vesicles with doxorubicin hydrochloride showed that naturally secreted exosomes exhibit a significantly reduced capacity to load hydrophilic drug compared to artificial exosomes prepared by the microfluidics method. Specifically: Naturally secreted exosomes are isolated from the supernatant of MRC-5 cell culture. The exosomes were lyophilized and then resuspended with doxorubicin hydrochloride solution, as well as the use of ultrasound on the exosomes in doxorubicin hydrochloride solution, in order to create pores In the exosomal membrane and allow the drug to enter.

[0079] In the case of the development of artificial exosomes using the microfluidics method, the possibility of utilizing different drug loading methods is given. Lipophilic molecules can be solubilized directly in the ethanolic phase of the microfluidic chip, together with the lipids of the cells, and then after entering the chip, be incorporated into the lipid layer of the liposomal vesicles. On the contrary, for hydrophilic molecules, different methods can be utilized depending on their physicochemical and acid-base properties,

[0080] Also an important role in the microfluidics method is played by the method of remote loading of doxorubicin hydrochloride into exosomal vesicles. Doxorubicin is a weak base and its degree of ionization can change depending on its environment. This method allows loading of a large amount of doxorubicin through the process of transmembrane pH-gradient.

[0081] Therefore, through this method, the interior of the exosome is acidic, while the external pH value is adjusted to physiological conditions. The uncharged doxorubicin that is incubated with the exosomes diffuses into the vesicles and is protonated inside them. The more positively charged doxorubicin cannot cross the bilayer and is trapped inside the exosomes.

[0082] The loading rate of doxorubicin in naturally secreted exosomes, through the methods of lyophilization and sonication, was approximately 6% and 15% respectively, while the loading rate in artificial exosomes through remote loading with the microfluidics method reached 48% (Figure 7).

[0083] This study therefore shows that while artificial exosomes mimic the morphological and physicochemical characteristics of exosomes, with the fluid mechanics method we are given the opportunity to exploit loading methods that give almost three times the loading rate.

[0084] Example 6: Cytotoxicity study of target cells incubated with "artificial exosomes" produced from MRC-5 hpids by the microfluidics method in comparison with naturally secreted exosomes of the same cells

[0085] The cytotoxicity study showed that both naturally secreted exosomes loaded with doxorubicin and artificial exosomes produced by the microfluidics method loaded with the drug can reduce the viability of target cancer cells (Figure 8). Specifically:

[0086] Naturally secreted exosomes from MRC-5 cells, when loaded with doxorubicin and incubated with MRC-5 and A549 cells, showed a decrease in cell viability that was proportional to the amount / concentration of drugs entrapped. It was observed that naturally secreted exosomes exhibited a significantly similar cytotoxicity profile to free doxorubicin and no statistically significant difference was noted in most comparisons.

[0087] The similar cytotoxicity profile of exosomal and free doxorubicin is likely due to the rapid release of doxorubicin from the lumen of the exosomes (both extracellularly and after their incorporation into the cells) without evidence of sustained release. This observation leads us to the conclusion that naturally secreted exosomes do not exhibit sufficient stability inside cells, resulting in rapid drug release, leading to rapid necrosis and reduced apoptosis activation potential, resulting in the potential release of cellular debris and inflammatory molecules, the development of chemotherapy-resistant cancer cells, and the favored dissemination of cancer cells with increased invasiveness and metastatic potential.

[0088] In contrast, in the "artificial exosomes" prepared by the microfluidics method, a difference in the cytotoxicity1' profile between vesicle-entrapped and free doxorubicin is presented. While both forms show a decrease in the viability of both cell lines, free doxorubicin shows a statistically significant greater decrease in viability compared to doxorubicin entrapped In artificial exosomes.

[0089] Therefore, doxorubicin entrapped in artificial exosomes is released gradually, showing a prolonged release profile. In this way, the cells are driven to death in a more smooth and organized manner and exploit the apoptotic cell killing mechanism, resulting in a more prolonged anticancer effect with a longer exposure time to the chemotherapeutic agent, a possible reduction in the resistance of cancer cells to the drug and ultimately a more desirable anticancer effect profile.

[0090] The different mechanism of target cell killing between free and doxorubicin encapsulated in artificial exosomes was also demonstrated by apoptosis induction studies using flow cytometry as well as by expression studies of the apoptosis -related genes BAX and Bcl-2. The increased Bax / Bcl-2 ratio reduces cellular resistance to apoptotic stimuli, leading to increased cell death through apoptosis pathways. It was observed that especially in A549 cells, during their Incubation with the "artificial exosomes" loaded with doxorubicin, the Bax / Bcl-2 ratio increased (increase in Bax and decrease in Bc!-2) statistically significantly compared to ceils grown under physiological conditions. The above results are better understood by referring to the attached Figures 6 to 8 where:

[0091] Figure 6: Morphological characterization of naturally secreted exosomes from MRC-5 cells and of artificial exosomes grown via the microfluidics device from the lipid composition of MRC-5 cells, by means of the Dynamic Light Scattering (DLS) and Transmission Electron Microscopy or Cryogenic Transmission Electron Microscopy (TEM and cryo-TEM):

[0092] (a) Size distribution of naturally secreted exosomes isolated by the methodology combining ultrafiltration and membrane-based binding affinity method.

[0093] (p) Morphological evaluation by cryo-TEM of naturally secreted exosomes isolated from MRC-5 cells by the combined method.

[0094] (y) Size distribution of vesicles grown from the lipid composition of MRC-5 cells by the microfiuidics method.

[0095] (8) TEM analysis of vesicles developed from the lipid composition of MRC-5 cells via the microfluidics device,

[0096] Figure 7: Comparative analysis of the percentage (%) loading of doxorubicin in naturally secreted exosomes and "artificial exosomes":

[0097] (a) Percentage (%) loading diagram of doxorubicin in naturally secreted exosomes that were lyophilized and resuspended with doxorubicin hydrochloride solution or by sonication of exosomes in doxorubicin hydrochloride solution.

[0098] (P) Percentage (%) loading diagram of doxorubicin in "artificial exosomes" MRC-5 lipid- based through the remote loading procedure using microfluidics method. Figure 8: Cytotoxicity study of MRC-5 and A549 cells incubated with naturally secreted exosomes from MRC-5 cells (a, p) and with "artificial exosomes" developed via the microfluidics device from the lipid composition of MRC-5 cells (y, 6).

Claims

Claims1. Method for production of a pharmaceutical composition, said composition comprises:A) naturally secreted exosomes from normal human embryonic lung fibroblast cells (MRC-5 cell line), wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method comprises the following steps in the following order: a) separation of exosomes by ultrafiltration, b) collection of exosomes by membrane- based affinity binding method, c) loading of exosomes with active substance, orB) artificial exosomes, wherein said exosomes comprise a lipophilic and / or hydrophilic active substance, said method is a microfluidic method and said method comprises the following steps in the following order: a) extraction of lipids from a cell line of normal human embryonic lung fibroblast cells (MRC-5 cell line) to obtain a lipid film, b) resuspending of the lipid film in an organic solvent and mixing with the aqueous phase in a microfluidics device to obtain exosomes, wherein the aqueous phase comprises Tween 20 and wherein in the microfluidics device i) the flow rate ratio of the organic phase to the flow rate of the aqueous phase (flow rate ratio, FRR) is set from 1:5 to 1:3, ii) the organic phase flow rate (OPFR) is preferably set at 200 pL / min or 400 pL / min and ill) the aqueous phase flow rate (APFR) is preferably set at 100 pL / min or 1000 pL / min, c) purification of the obtained exosomes using size exclusion chromatography, d) loading of the exosomes with active substance, e) purification of the obtained exosomes using size exclusion chromatography to remove the unloaded drug,2. Method for production of a pharmaceutical composition according to claim 1, wherein the active substance is preferably curcumin and the loading of curcumin into the exosomes is preferably in a curcumin:exosomes ratio of 5:1, said method comprises the following steps in the. following order: a) mixing the exosomes with curcumin dissolved in phosphate buffered saline (PBS) and ethanol, preferably to a concentration of ethanol less than or equal to 1%, b) incubation preferably for 3 hours at room temperature away from light and c) ultracentrifugation to remove the unloaded curcumin preferably for 2 hours at 135,000xg,3. Method for production of a pharmaceutical composition according to claim 1, wherein the active substance is preferably doxorubicin and the loading of doxorubicin into the exosomes is preferably in a 1:1 ratio, said method comprises the following steps in the following order: a) either ultrasonic treatment of the exosomes in a solution of doxorubicin hydrochloride preferably with more than one cycles with intermediate cooling periods, preferably of 2 minutes, followed by incubation preferably at 37°C for 90 minutes and then ultracentrifugation to remove the unloaded doxorubicin preferably for 2 hours at 135,000xg, b) either resuspension of the previously lyophilized exosomes in a solution of doxorubicin hydrochloride, followed by incubation preferably at 37°C for 90 minutes and then ultracentrifugation to remove the unloaded doxorubicin preferably for 2 hours at 135,000xg.

4. Method for production of a pharmaceutical composition according to any one of the claims 1 to 3, wherein the exosomes exhibit zeta potential values generally ranging from -100 mV to +100 mV, preferably from -100 mV to 0 mV, more preferably from -35 mV to -5 mV,5. Method for production of a pharmaceutical composition according to any one of the claims 1 to 4, wherein the exosomes present the CD9 protein (CD9 antigen) and the CD63 protein (CD63 antigen) on their surface and comprise at least 39 other proteins.

6. Method for production of a pharmaceutical composition according to any one of the claims 1 to 5, wherein the exosomes comprise at least 92 miRNAs. preferably the mature miRNAs hsa-miR-10400-5p (Homo sapiens-miRNA-10400~5p), hsa-miR-365b- 5p (Homo sapiens-miRNA-365b-5p), hsa-miR-1469 (Homo sapiens-miRNA-1469), hsa- miR-1268a (Homo sapiens-miRNA-1268a) and mature miRNAs of the let-7 family.

7. Method for production of a pharmaceutical composition according to any one of the claims 1 to 2 and 4 to 6, wherein the exosomes are curcumin nanocarriers with a loading rate from 2,5 to 25%, preferably at 9%.

8. Method for production of a pharmaceutical composition according to any one of the claims 1 and 3 to 6, wherein the exosomes are doxorubicin nanocarriers with a loading rate from 2,5 to 25%, preferably at 16,5%.

9. Method for production of a pharmaceutical composition according to any one of the claims 1 and 3 to 6 and 8, wherein the exosomes are in lyophilized form.

10. Method for production of a pharmaceutical composition according to any one of the claims 1 to 9, said composition is formulated with suitable pharmaceutically acceptable excipients and administered intravenously or subcutaneously or intraperitoneally or orally.

11. Microfluidics method for production of a pharmaceutical composition according to the claim 1, wherein the exosomes comprise lipids of the MRC-5 cell line, cholesterol and Tween 20 in a molar ratio from (55 to 75):(25 to 35):(2 to 8), preferably at 65:30:5.

12. Microfluidics method for production of a pharmaceutical composition according to any one of the claims 1 and 11, wherein the total concentration of lipids in the exosomes ranges from 3 mg / mL to 15 mg / mL, preferably is 11 mg / mL.

13. Microfluidics method for production of a pharmaceutical composition according to any one of the claims 1 and from 11 to 12, wherein the active substance is preferably doxorubicin and the loading of doxorubicin into the exosomes is preferably carried out by the remote loading method, wherein: a) exosomes are prepared using an ammonium sulfate solution with Tween 20 (pH=5,48) as the aqueous phase in the microfluidics apparatus, b) purification of the obtained exosomes using size exclusion chromatography at pH-7,4, so that the exosomes present size of up to 150 nm, c) addition of doxorubicin solution to create a transmembrane pH gradient and stirring for 1 hour at 60°C, d) re- purification of the obtained exosomes using size exclusion chromatography at pH=7,4 to remove the unloaded doxorubicin, so that the exosomes present size of up to 150 nm.

14. Microfluidics method for production of a pharmaceutical composition according to any one of the claims 1 and from 11 to 13, wherein the exosomes exhibit a size of from 100 nm to 150 nm.

15. Microfluidics method for production of a pharmaceutical composition according to any one of the claims 1 and from 11 to 14, wherein the exosomes are in lyophilized form.

16. Microfluidics method for production of a pharmaceutical composition according to any one of the claims 1 and from 11 to 15, said composition is formulated with suitablepharmaceutically acceptable excipients and administered intravenously or subcutaneously or intraperitoneally or orally.

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