Extracellular vesicles for therapy

Abstract

The present invention relates to algal extracellular vesicles (EVs) for use in therapy and recovery or maintenance of health in a subject. In particular, the present invention relates to the use of algal EVs in therapy wherein the EVs themselves have an advantageous effect on the health of subjects administered them. In particular, algal EVs are used to treat conditions or disorders associated with cell proliferation and / or cell migration and / or non-physiological ECM production and deposition. The disorders also particularly include inflammation, neoplastic disorders, fibroproliferative diseases, and the like. The invention also relates to methods of treatment of subjects as well as pharmaceutical and nutraceutical compositions. The invention also relates to a cosmetic method in which algae EVs are applied to the affected skin.

Description

Technical Field

[0001] The present invention relates to algal extracellular vesicles (EVs) for use in therapy of a subject and in restoring or maintaining the health of a subject.

[0002] In particular, the present invention relates to the use of algal EVs in therapy, wherein the EVs themselves have a beneficial effect on the health of the subject to which they are administered.

[0003] In particular, algal EVs are used to treat conditions or disorders associated with cell proliferation and / or cell migration and / or non-physiological ECM production and deposition, including inflammation, neoplastic disorders, and fibroproliferative diseases.

[0004] The invention also relates to methods of treatment of a subject and to pharmaceutical and nutraceutical compositions. The invention also relates to cosmetic methods, wherein algal EVs are applied to the affected skin. Background Art

[0005] Kuruvinashetti et al. (Algal Extracellular Vesicles for Therapeutic Applications. 2020 IEEE 20th International Conference on Nanotechnology (IEEE-NANO) July 29-31 2020, Virtual Conference) envisioned the use of algae-derived EVs as drug delivery constructs in cancer therapy, although there was no experimental evidence or direct reference.

[0006] CN110448696A describes a method for preparing EVs derived from seaweed (Dunaliella salina) and the use of EVs derived from seaweed (Dunaliella salina) as drug delivery constructs. CN111543637A, CN111567798A and WO2021032794A1 also describe the use of (brown) algae-derived EVs as drug delivery constructs. WO2011090731 describes microalgae that produce heterologous proteins and EVs derived from such algae as delivery constructs for heterologous proteins.

[0007] CN113209140 and CN113462645 describe EVs derived from Phaeophyceae (multicellular brown algae) that activate NK cells by increasing the expression of, for example, TNFα. However, a closer look at the methods described in these patent applications reveals that the first step in preparing EVs is to obtain fucoidan from these seaweeds. In other embodiments, powdered fucoidan is used to prepare EVs. Fucoidan is a sulfated polysaccharide that has been widely studied for its biological activity and possible therapeutic applications (Luthuli et al., Therapeutic Effects of Fucoidan: A Review on Recent Studies. Mar Drugs. 2019; 17 (9): 487, published on August 21, 2019, doi: 10.3390 / md17090487). Thus, neither CN113209140 nor CN113462645 appear to provide evidence of actual isolation of EVs, or specifically teach the use of algae-derived EVs as active agents in therapy. If such use is not taught in either CN113209140 or CN113462645, this would be used as a delivery construct for fucoidan, similar to that described in the above applications. It is worth noting that fucoidan is typical of brown algae, not unicellular or green algae.

[0008] Algae are widely used in food and food supplements and have various health benefits. However, the various compounds that make up algal cells can have different or even opposite effects, or the effects of certain compounds (or extracts) can be masked by other compounds (or extracts).

[0009] The inventors have discovered that well-defined algal extracellular vesicles are useful in therapy. Summary of the invention

[0010] 1. The present invention relates to algal extracellular vesicles (EVs) for use in therapy, preferably in the therapy of disorders.

[0011] Preferably, the present invention relates to algal EVs for use in treating a disorder in a subject. The subject is an animal. In particular, the subject is a vertebrate, such as a fish, an amphibian or a reptile, preferably a mammal or a bird, very preferably a mammal, in particular a human subject.

[0012] As used herein, algae are photosynthetic prokaryotic or eukaryotic organisms that are not embryophytes (land plants).

[0013] As used herein, algae are preferably organisms that live in water (fresh or sea water).

[0014] The prokaryotic algae used herein are preferably prokaryotic photoautotrophic organisms.

[0015] In other embodiments, the prokaryotic algae used herein are heterotrophic organisms.

[0016] As used herein, prokaryotic algae are organisms preferably belonging to the phylum Cyanobacteriota, preferably to the class Cyanophyceae.

[0017] As used herein, eukaryotic algae are preferably eukaryotic photoautotrophic organisms that are not embryophytes.

[0018] The eukaryotic algae used herein are preferably photosynthetic and / or heterotrophic organisms that do not have the matrix tissue characteristic of plants (parenchyma, collenchyma and sclerenchyma) nor have special reproductive organs.

[0019] As used herein, eukaryotic algae are organisms preferably selected from the group consisting of Chlorophyta, Streptophyta, Rhodophyta (red algae), Ochrophyta (brown algae) and Cyanobacteriota phylum.

[0020] Preferably, the algae used herein are as defined in the detailed description.

[0021] In a highly preferred embodiment, the algal EVs are derived from species selected from

[0022] Chlorella sorokiniana, Parachlorella kessleri, Chlorella vulgaris, Chlorella pyrenoidosa (Auxenochlorella pyrenoidosa), Chlamydomonas reinhardtii, Hormidiospora verrucosa, Haematococcus lacustris, Tetraselmis chui, Zygnema peliosporum, Klebsormidium nitens, Spirogyra sp., Vischeria polyphem, Ascophyllum nodosum, Palmaria palmata, Microcystis aeruginosa aeruginosa, Nostoc linckia, Spirulina platensis (Arthrospira platensis), Spirulina maxima (Limnospira maxima), Arthronema africanum, Synechococcus sp., Aphanizomenon flos-aquae or other plants of the same genus.

[0023] Algal EVs according to the present invention are preferably derived from eukaryotic algae.

[0024] Algal EVs according to the present invention are preferably derived from prokaryotic algae.

[0025] The algal EVs according to the present invention are preferably derived from microalgae present alone or in the form of chains or groups.

[0026] The algal EVs according to the present invention are preferably derived from macroalgae, either alone or in the form of chains or groups.

[0027] Algal EVs according to the present invention are derived from freshwater algae.

[0028] The algae according to the invention are derived from saltwater and / or marine algae.

[0029] Preferably, the algal EVs according to the present invention are derived from eukaryotic freshwater algae, preferred variants of which are different from brown algae and / or different from red algae.

[0030] In a particularly preferred embodiment, the algae is a green algae, preferably a freshwater green algae.

[0031] In particularly preferred embodiments, the algae is green algae, preferably saltwater green algae.

[0032] In particularly preferred embodiments, the algae is food grade algae, preferably dried algae.

[0033] In particularly preferred embodiments, EVs are provided in the form of medical devices, pharmaceuticals, cosmetics or nutraceutical compositions comprising one or more pharmaceutically or nutritionally acceptable excipients.

[0034] In particularly preferred embodiments, EVs are provided in the form of pharmaceutical or nutraceutical compositions comprising one or more pharmaceutically or nutritionally acceptable excipients.

[0035] In a preferred embodiment, EV types from multiple sources are combined.

[0036] In a preferred embodiment, the EVs used as active agents are combined with drugs or other active agents for the same disease. 2. Preferably, algal EVs are used to treat disorders associated with cell proliferation, in particular cell hyperproliferation and / or cell migration, in particular pathological cell migration and / or ECM-formation disorders, in particular defects in the regulation of ECM-formation, more particularly the production of non-physiological (abnormal, non-healthy) ECM and / or the production or deposition of physiological ECM, in particular the regulation of ECM production and / or deposition. Cell proliferation, migration and ECM production and / or deposition are often related and in particular interrelated processes in many pathological processes (see Figure 108). Preferably, the disorder manifests itself due to defects in cell proliferation and / or migration and / or ECM formation, such as ECM production and / or deposition, in particular cell hyperproliferation and / or pathological cell migration and / or non-physiological ECM production / deposition.

[0037] In a particular embodiment, algal EVs are used for the therapy of fibrosis, in particular fibroproliferative disorders (preferably as defined in paragraph 4). In particular fibrosis, in particular fibroproliferative disorders are associated with defects in cell migration and / or cell proliferation and / or ECM-formation.

[0038] In a particular embodiment, algal EVs are used for the therapy of a neoplastic, preferably tumorogenic disorder (preferably as defined in paragraph 5). In particular, the neoplastic disorder is associated with defects in cell migration and / or cell proliferation and / or ECM-formation.

[0039] In a specific embodiment, algal EVs are used for the treatment of an immune disorder, preferably as described in paragraph 6. In particular, the immune disorder is associated with defects in cell migration and / or cell proliferation and / or ECM-formation. 3. The present invention preferably relates to algal EVs for use in a therapy according to any of paragraphs 1 or 2, wherein the disorder is a proliferation / migration disorder.

[0040] Proliferation / migration disorders are disorders in which both cell proliferation and migration play an important role in the development of a condition (particularly an unhealthy condition) (e.g., in the etiology and / or manifestation of a disease) and in certain stages of the condition, non-physiological ECM is produced or deposited. Typically, such proliferation / migration disorders are progressive fibrosis or scarring of tissue, neoplastic conditions or cancer, particularly solid tumors, inflammation, and conditions and stages that lead or may lead to their manifestation.

[0041] In certain embodiments, the proliferative / migratory disorder is progressive fibrosis or tissue scarring, a neoplastic condition, or cancer.

[0042] In certain embodiments, the algal EVs used according to the invention have an anti-proliferative effect.

[0043] 4. The present invention relates to algal EVs for use in a therapy according to any of the above paragraphs (for example, according to paragraph 3), wherein the disorder (such as a proliferation / migration disorder) is fibrosis including progressive fibrosis, or a fibroproliferative disease, in particular a fibroproliferative disease involving progressive fibrosis.

[0044] In a particular embodiment, it is skin fibrosis or fibrosis of gastrointestinal organs, in particular fibrosis of the intestine, peritoneum or abdominal organs or the kidneys or lungs or heart.

[0045] In certain embodiments, the fibrosis is fibrosis of the lung.In certain embodiments, the fibrosis is fibrosis of the peritoneum.

[0046] Fibrosis as defined herein (eg, progressive) can occur in virtually any part of a subject's body that is capable of developing fibrosis, particularly progressive fibrosis.

[0047] In embodiments, EVs inhibit the production of ECM.

[0048] In other embodiments, fibrosis is considered herein as a dynamic process of ECM production or degradation. Abnormal processes (such as damage on the one hand and progressive fibrosis on the other hand) shift the balance of such dynamic processes to extremes or out of balance. In variants, the production / degradation of ECM becomes irregular (irregulated). In a preferred embodiment, the EVs or compositions of the present invention improve the regulation of ECM production, preferably maintaining its dynamic properties or maintaining normal balance.

[0049] 5. Algal EVs for use in a therapy according to any of the above paragraphs (e.g., according to paragraph 3), wherein the disorder (e.g., a proliferation / migration disorder) is a neoplastic disease, preferably cancer, preferably tumor formation (tumourgenesis), in particular a solid tumor.

[0050] 6. Algal EVs for use in a therapy according to paragraph 3, wherein the proliferative / migratory disorder is inflammation, in particular acute or chronic inflammation and / or can be classified as migratory / proliferative inflammation.

[0051] The disorder is preferably an inflammatory disorder, particularly an autoimmune disorder. In an embodiment, the inflammation occurs in the form of a chronic disorder. In an embodiment, the inflammation occurs in an autoimmune disorder. Preferably, algal EVs are used as anti-inflammatory agents.

[0052] Inflammation can be induced by many agents or causes and may be of toxicological origin (e.g., induced by chemical agents, mechanical origin such as injury, oxidative stress), biological origin (e.g., autoimmune disease or infection), etc.

[0053] In embodiments, EVs inhibit migration or immune cell migration in inflammation. In embodiments, EVs inhibit immune cell recruitment.

[0054] In embodiments, EVs inhibit proliferation in inflammation or proliferative inflammation.

[0055] In other preferred embodiments, the inflammation is selected from:

[0056] - Inflammation associated with fibrosis, especially progressive fibrosis;

[0057] - Cancer-related inflammation,

[0058] - Inflammation associated with tissue deterioration, such as joint inflammation

[0059] -Inflammation associated with autoimmune diseases.

[0060] 7. In an embodiment, the disorder according to any one of paragraphs 1 to 6 is an advanced glycation end product (AGE)-related disease as defined herein.

[0061] Advanced glycation end products (AGEs) are proteins, lipids or nucleic acids that become glycosylated due to exposure to sugars and are involved in the development or exacerbation of diseases associated with oxidative stress and inflammation, such as metabolic syndrome, diabetes, inflammatory bowel disease, atherosclerosis, chronic kidney disease, Alzheimer's disease. For example, an AGE-related disease is selected from the group consisting of these diseases and related conditions. As an example, an AGE-related disease is a diabetic ulcer characterized by ECM disintegration, which can also be treated with EVs that can increase the production / deposition of ECM.

[0062] Preferably, EVs are used as anti-aging AGE agents. Preferably, EVs are used as inhibitors of AGE formation. Preferably, EVs are used as inhibitors of activation of AGE receptors.

[0063] 8. Algal EVs for use in treating a disorder, preferably a disorder in a vertebrate subject, according to any of paragraphs 1 to 6, wherein the disorder is:

[0064] - Skin (preferred)

[0065] - Gastrointestinal organs, especially the intestine (preferably) (colon)

[0066] - abdominal organs (liver),

[0067] - Cardiovascular, especially heart

[0068] -peritoneum,

[0069] - Urinary system, especially the kidneys,

[0070] - The respiratory system, especially the lungs.

[0071] In certain embodiments, EVs penetrate into cells of an animal, in particular cells of a vertebrate (eg, as defined in paragraph 1) subject, preferably cells of any organ as defined herein.

[0072] Among them, the proliferation and migration of fibroblasts preferably contribute to the manifestation of the disease.

[0073] EVs are able to pass through (or cross) barriers in the body, such as the blood-brain barrier, blood-ocular barrier, epithelial layer, endothelial cell layer, etc. Therefore, there may be fibrosis, inflammation or cancer in the brain or eye, etc.

[0074] 9. Algal EVs for use in a therapy according to any one of paragraphs 1 to 8

[0075] Wherein, EVs are used to inhibit the activity of a signaling pathway selected from the group consisting of

[0076] -PDE

[0077] -PDGF receptor α and / or PDGF receptor β signaling pathway, preferably PDGF-induced signaling, preferably PDGF-BB-induced signaling pathway, wherein, if the PDGF-BB-induced signaling pathway is mentioned herein, it can be understood in a broader manner,

[0078] - a signaling pathway induced by TGFβ, preferably TGFβ-1, wherein if TGFβ-1 or TGFβ-induced signaling pathway is mentioned herein, it can be understood in a broader manner,

[0079] -EGF-induced signaling pathway.

[0080] In certain embodiments,

[0081] -EVs are used to inhibit cell migration, preferably EGF-induced cell migration,

[0082] - EVs are used to inhibit cell proliferation, preferably PDGF-BB-induced cell proliferation, and / or

[0083] -EVs are used to inhibit ECM production, preferably TGFβ-induced ECM production / deposition.

[0084] - EVs are used to inhibit a condition selected from cell migration, cell proliferation and / or inflammation and / or ECM production, preferably a growth factor cocktail-induced or in particular a PDE-induced condition.

[0085] In certain embodiments, EVs are used to inhibit cell migration induced by PDE, EGF, PDGF-BB and / or TGFβ.

[0086] In certain embodiments, EVs are used to inhibit cell proliferation induced by PDE, EGF, PDGF-BB and / or TGFβ.

[0087] In certain embodiments, EVs are used to inhibit PDE, EGF, PDGF-BB and / or TGFβ-induced inflammation.

[0088] In certain embodiments, EVs are used to inhibit PDE, EGF, PDGF-BB and / or TGFβ-induced ECM production / deposition.

[0089] In certain embodiments, EVs are used to inhibit cell migration, cell proliferation, inflammation, and / or ECM production induced by a formulation comprising multiple growth factors or a mixture of growth factors (e.g., a mixture of EGF, PDGF-BB, and / or TGFβ, such as PDE).

[0090] In certain embodiments:

[0091] Algal EVs are used to treat disorders caused by cell migration, wherein cell migration is induced by activation of the PDE EGF signaling pathway. Preferably, algal EVs are administered to reduce cell migration.

[0092] Algal EVs are used to treat disorders caused by cell proliferation, wherein the cell proliferation is induced by PDE, the PDGF-BB signaling pathway is activated to induce cell proliferation, and wherein the algal EVs are administered to reduce cell proliferation.

[0093] Algal EVs are used to treat disorders caused by ECM-production, wherein ECM-production is induced by activation of the PDE TGFβ signaling pathway. Preferably, algal EVs are administered to reduce / alter ECM production. Preferably, algal EVs are administered to modulate ECM production. Preferably, algal EVs are administered to increase physiological ECM production and reduce non-physiological ECM production.

[0094] Signaling pathway activation involves activation of part of the pathway (partial activation) as long as the activation leads to activation of receptors for PDGF-BB and / or TGF-β and / or EGF (i.e., PDGF-BB and / or TGF-β and / or EGF receptors as used herein).

[0095] Algal EVs for use in the therapy according to any one of paragraphs 1 to 9, wherein the EVs are used to inhibit the activity of a signaling pathway selected from the group consisting of

[0096] -Signaling pathways induced by multiple growth factors or mixtures of growth factors such as PDE

[0097] -PDGF-BB-induced signaling pathway,

[0098] -TGFβ-induced signaling pathways,

[0099] -EGF-induced signaling pathway, in which

[0100] In such disorders, one or more PDGF-BB and / or TGF-β and / or EGF receptors are activated.

[0101] Alternatively, EVs are used to inhibit the activity of the pathway, particularly the activation of any one or more of the receptors.

[0102] The present invention relates to a method as defined herein, wherein the level or phosphorylation or localization or activation of one or more of the following factors and or signaling molecules (preferably at least 2, 3, 5, 7, 10 or 15 factors) is altered, i.e. different from the normal range defined for a healthy population, e.g. a population with common features associated with:

[0103] AKT1 AKT serine / threonine kinase 1 EGF Epidermal Growth Factor-Level FOS Fos proto-oncogene, AP-1 transcription factor subunit GRB2 Growth factor receptor binding protein 2 HRAS HRas proto-oncogene, GTPase JAK1 Janus kinase 1 JUN Jun proto-oncogene, AP-1 transcription factor subunit MAP2K1 Mitogen-activated protein kinase 1-phosphorylation MAP2K2 Mitogen-activated protein kinase 2 MAPK1 Mitogen-activated protein kinase 1 MAPK3 Mitogen-activated protein kinase 3 MTOR Mechanistic target of rapamycin kinase STAT1 signal transducer and activator of transcription 1 STAT3 Signal transducer and activator of transcription 3

[0104] In alternative embodiments, the levels of the factors are listed in any one of Tables 4-6.

[0105] 10. The present invention relates to algal EVs for use in a therapy according to any one of paragraphs 1 to 9, wherein the disorder is selected from the group consisting of

[0106] - Fibroproliferative disorders,

[0107] - Neoplastic disorders,

[0108] -Inflammatory disorders.

[0109] 11. The present invention relates to algal EVs for use in the therapy according to any of paragraphs 1 to 10, wherein the EVs are derived from green algae, brown-red algae or cyanobacterial algae, preferably from green algae (e.g., Chlorophyta or Streptophytes), brown algae (e.g., Chlorophyta), red algae (e.g., Rhodophyta) - cyanobacteria (e.g., Cyanobacteria).

[0110] The algae are preferably microalgae or macroalgae.

[0111] In a specific embodiment, algae are used in the therapy according to any of the preceding paragraphs, wherein the EVs are derived from Chlorophyta, Rhodophyta, Paleophyta and Streptophyta, preferably from Chlorella sorokinensis, Pseudochlorella kessleri, Chlorella vulgaris, Chlorella pyrenoidosa (Chlorella pyrenoidosa), Chlamydomonas reinhardtii, Homiella verrucosa, Haematococcus lacustris, Platymonas zhouei, Indigosporum, Klebsiella, Spirogyra, Boweishi Algae, Ascophyllum, and Drosophila palmatum.

[0112] In a specific embodiment, algae are used in the therapy according to any of the preceding paragraphs, wherein the EVs are derived from Cyanobacteria, preferably from Microcystis aeruginosa, Nostoc lindneri, Spirulina platensis (Arthrospira platensis), Spirulina maxima (Arthrospira maxima), Annelidae, Synechococcus, Aphanizomenon flos-aquae.

[0113] In particular, EVs are preferably derived from green algae. EVs are preferably derived from sweet and saltwater phyla including Chlorophyta, Streptophyta, Rhodophyta, Paleophyta and Cyanobacteria.

[0114] In a highly preferred embodiment, the algae is selected from Chlorella sorokinensis, Pseudomonas keslerii, Chlorella vulgaris, Chlorella pyrenoidosa (Chlorella pyrenoidosa), Chlamydomonas reinhardtii, Homiella verrucosus, Haematococcus lacustrine, Platymonas zhouei, Indigosporus diastereois, Klebsiella, Spirogyra, Bowei's algae, Ascophyllum nodosum, Drosophila palmatum, Microcystis aeruginosa, Nostoc lindenii, Spirulina platensis (Arthrospira platensis), Spirulina maxima (Arthrospira maxima), Annelidae africana, Synechococcus, and Aphanizomenon.

[0115] In a preferred embodiment, the algae are selected from those given in the definitions.

[0116] In a preferred embodiment, the algae is selected from the algae given in the detailed description, in particular in the section on algae useful in the present invention.

[0117] 12. The present invention relates to algal EVs for use in a therapy according to any one of paragraphs 1 to 11, wherein the algal EVs are prepared by a method comprising the following steps

[0118] - providing isolated algal cells; in particular an algal cell culture, preferably wherein the algal cells are propagated in a medium or dried, preferably rehydrated freeze-dried or spray-dried algal cells,

[0119] - Obtaining EVs from the algal cells

[0120] -Preferably, the EVs are formulated into an EV preparation.

[0121] Herein, isolation is understood as a process in which the natural environment of EVs is artificially altered, i.e., by human action as a technical step. Preferably, the EV population is also altered by selecting EVs according to their size and removing cellular material and / or debris, and / or other bioactive molecules.

[0122] The present invention preferably relates to a method for preparing algal EVs or a composition comprising said EVs.

[0123] EVs and / or EV compositions (or preparations) prepared by the technology according to the present invention can be used for any of the above-mentioned indications (or therapies or methods of treatment or treatment of disorders), for example, in any of paragraphs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and / or 11 or a combination thereof.

[0124] Preferably, EVs from any algae as defined in either or both of paragraphs 1 and 11 can be used.

[0125] In certain embodiments, the algae from which EVs are isolated are dried, preferably rehydrated freeze-dried or spray-dried algal cells.

[0126] In certain embodiments, EVs are freeze-dried or spray-dried.

[0127] 13. Specifically, the present invention relates to algal EVs used in the therapy according to paragraph 12, wherein the algal EVs are obtained by

[0128] - Culture supernatant of algal cells, by isolating EVs from the culture supernatant,

[0129] - dried algal cells, by rehydrating said algal cells with a culture medium from which the cells and cell debris are removed, in particular pelleted, and EVs are separated from the supernatant culture medium,

[0130] - Suspended algal cells, in particular cultured algal cells or dried and rehydrated algal cells, in particular rehydrated pelleted algal cells, are preferably applied by mechanical or physical effect.

[0131] 14. In particular, the present invention relates to algal EVs for use in a therapy according to paragraph 12 or 13, wherein the algal EVs are obtained by a method comprising the following steps

[0132] - Removal of algal cells and / or cellular components constituting fragments of algal cells by a technique selected from the group consisting of

[0133] Ultracentrifugation, centrifugation, filtration, tangential flow filtration (TFF), size exclusion chromatography (SEC), dialysis of the supernatant of algal cells.

[0134] Preferably, the cell supernatant is also removed.

[0135] 15. In particular, the present invention relates to algal EVs for use in a therapy according to any of paragraphs 12 to 14, wherein

[0136] Obtaining EVs from algal cells comprises filtration, preferably ultrafiltration (UF).

[0137] 16. In particular, the present invention relates to algal EVs for use in therapy according to any of paragraphs 12 to 15, wherein

[0138] Obtaining EVs from algal cells comprises size exclusion chromatography (SEC), preferably SEC on a SEC chromatography matrix, preferably having a pore size of 10 to 1000 nm, preferably 10 to 500 nm, or 35 to 500 nm, or 35 to 350 nm, in particular 70-1000 nm, or 70 to 500 nm. Highly preferably, the medium pore size is 60-80 nm, including about 70 nm.

[0139] Optionally, the method comprises preparing a SEC column, optionally passing liposomes. Preferably, the size (average diameter) of the liposomes is defined as the pore size of the EVs as described above.

[0140] In other preferred embodiments, the SEC column is prepared from EVs. Preferably, the EVs are the same or similar to the EVs to be prepared on the SEC column.

[0141] 17. In particular, the present invention relates to algal EVs for use in a therapy according to any of paragraphs 12 to 16, wherein

[0142] Isolating EVs from algal cells includes concentrating EVs. Preferably, the concentration is performed by filtration, such as UF. In particular, there is a cutoff point that allows proteins and nucleic acids to flow through into the filtrate. The cutoff point is 20 to 500 kDa, 20 to 400 kDa, 20 to 300 kDa or 50 to 400 kDa, 50 to 300 kDa. Alternatively, the cutoff point is, for example, 20 to 200 kDa, preferably 50 to 200 kDa or 50 to 150 kDa, and highly preferably about 100 kDa. In an embodiment, a variety of membranes with different cutoff points are applied.

[0143] In certain embodiments, cells are removed through a cell filter (eg, a bacterial filter having a pore size of about 0.2 microns).

[0144] 18. In particular, the present invention relates to algal EVs for use in a therapy according to any one of paragraphs 1 to 17, wherein the size intensity distribution of the algal EVs is characterized by an intensity distribution of 10 or 10 2 Up to 10 3 nm, or 10 to 10 2 nm, especially 1.8×10 2 to 1.8×10 3 The intensity distribution is on the order of nm.

[0145] Highly preferably, the majority of EVs have a size (or diameter) of 70 to 500 nm. In various embodiments, the diameter of EVs is 10 to 1000 nm, preferably 10 to 500 nm, or 35 to 500 nm, or 35 to 350 nm, in particular 70-1000 nm. Alternatively, at least 70%, 80%, 90%, or at least 95%, or at least 98% of EVs have a diameter within the range given herein.

[0146] 19. In particular, the present invention relates to algal EVs for use in a therapy according to any of paragraphs 1 to 18, wherein

[0147] EVs are used as active agents for a condition defined in any one of paragraphs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and / or 11 or a combination thereof, and

[0148] - the EVs comprise an additional active agent for treating the same disorder as defined in any of paragraphs 1 to 10; or

[0149] - wherein, in said therapy, EVs are administered together with another active agent for treating the same disorder as defined in any of paragraphs 1 to 10.

[0150] Preferably, EVs are defined in any of the preceding paragraphs, in particular in paragraph 1 and / or paragraph 11 and or by the method defined in any of paragraphs 12 to 18.

[0151] Preferably, a disorder as defined in any of paragraphs 1 to 10 or 11,

[0152] Wherein, the other active agent is an anti-fibrotic agent.

[0153] Wherein, the other active agent is an anti-tumor agent.

[0154] Wherein, the other active agent is an anti-inflammatory agent.

[0155] In certain embodiments, EVs or a combination or composition thereof include as other active agents compounds selected from vitamins, trace elements, tonics, etc.

[0156] In certain embodiments, the algae are transgenic algae to produce a protein or nucleic acid active agent.

[0157] 20. Algal EVs for use in a therapy according to any one of paragraphs 1 to 19, wherein the EVs are administered in the therapy

[0158] - orally, preferably in the form of a composition for oral administration,

[0159] - topically, preferably in the form of a composition for topical administration,

[0160] - intraperitoneally, preferably in the form of a composition for intraperitoneal administration,

[0161] - intranasally, preferably in the form of a composition for nasal administration,

[0162] - subcutaneous / intradermal, preferably in the form of a composition for subcutaneous administration,

[0163] - intramuscularly, preferably in the form of a composition for intramuscular administration, or

[0164] - Intravenously, preferably in the form of a composition for intravenous administration.

[0165] - In a specific embodiment, the administration is intratumoral administration.

[0166] - In a specific embodiment, the administration is intraocular administration.

[0167] In certain embodiments, the administration is oral administration.

[0168] In certain embodiments, the administration is intraperitoneal administration.

[0169] In certain embodiments, the administration is intravenous.

[0170] In certain embodiments, the administration is intradermal administration.

[0171] 21. The present invention also relates (among other aspects) to a composition for treating a disorder as defined in any of the preceding paragraphs, in particular in paragraphs 1 to 10, comprising algal EVs as an active agent and / or a biologically acceptable carrier for a mammalian subject.

[0172] Preferably, the EVs are EVs as defined in any of paragraphs 1 and / or 11.

[0173] 22. The present invention also relates to a composition according to paragraph 21, comprising algal EVs as defined in any of paragraphs 11 to 20.

[0174] In certain embodiments, the number of algal EVs in a dosage unit of the composition is at least 10 3 , preferably at least 10 5 , or at least 10 6 or at least 10 7 or at least 10 8 or at least 10 9 or at least 10 10 In an embodiment, the number of algal EVs in a dosage unit of the composition is at most 10 20 , preferably at most 10 15, or up to 10 12 or up to 10 11 In particular, the number of algal EVs in a dosage unit of the composition is at least or at least 3×10 6 or at least 3×10 7 or at least 3×10 8 or at least 3×10 9 Preferably, the number of algal EVs in the composition is at least 3×10 7 or at least 3×10 8 or at least 3×1×10 9 or at least 3×10 10 .

[0175] In an embodiment, the above data relate to concentrations and 1 ml dosage units.

[0176] In a specific example, the dose used to treat an animal is at least 3×10 7 EVs or 3×10 8 or 3×10 9 EVs / treatment, for example in mice as a model animal. In mice, the recommended dose is equivalent to at least 1.5x10 EVs / kg body weight (kgbw). 9 Ev or at least 1.5x10 10 EV. In larger animals such as humans, the dose per kg bw may be smaller, for example, 1.5 × 10 8 EVs or at least 1.5×10 9 EVs.

[0177] In a specific embodiment, in the case of mammals (e.g., in humans), the dosage range of the dosage unit of the composition is at least 10 5 And at most 10 8 EVs, or at least 10 per dose 6 And at most 10 9 EV, or at least 10 per dose 7 And at most 10 10 EV, or at least 10 per dose 8 And at most 10 11 EVs. In embodiments, this is a daily dose.

[0178] 23. The present invention also relates to a composition according to any one of paragraphs 21 or 22, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0179] 24. The present invention also relates to a composition according to any one of paragraphs 21 to 23, which is formulated as

[0180] For oral administration,

[0181] For topical application,

[0182] For intraperitoneal administration,

[0183] For nasal administration,

[0184] For subcutaneous administration,

[0185] For intramuscular administration, or

[0186] For intravenous administration.

[0187] For intratumoral administration.

[0188] For intraocular administration;

[0189] In certain embodiments

[0190] - for oral administration, and / or

[0191] - for intraperitoneal administration, and / or

[0192] - for intravenous administration, and / or

[0193] - for topical application, and / or

[0194] - for intradermal administration,

[0195] - administration as defined in paragraph 20.

[0196] 25. The present invention also relates to a composition according to any one of paragraphs 21 to 24, which further comprises an additional active agent for treating a disorder as defined in any one of paragraphs 1 to 10.

[0197] Preferably, EVs are defined in any of the preceding paragraphs, in particular in any of paragraphs 1 and / or 11, or by a method according to any of claims 12 to 18.

[0198] Preferably, a disorder as defined in any of paragraphs 1 to 10 or 11,

[0199] Wherein, the other active agent is an anti-fibrotic agent, more preferably nintedanib;

[0200] Wherein, the other active agent is an anti-tumor agent, more preferably axitinib or ponatinib;

[0201] Wherein, the other active agent is an anti-inflammatory agent; and / or

[0202] Wherein, the other active agent is an anti-AGE agent.

[0203] 26. The present invention also relates to a composition according to any one of paragraphs 21 or 22, which is a nutraceutical composition, comprising a nutritionally acceptable carrier.

[0204] Preferably selected from the group consisting of

[0205] Food (dietary) supplements, fortified foods, medicinal foods, functional foods, dietary formulas or compositions related to health.

[0206] Thus, a nutraceutical is defined herein to include any composition of food that has a health-related purpose or use and / or is provided for that purpose or use.

[0207] 27. The present invention also relates to a composition according to any one of paragraphs 21 to 26, which is a dermatological cosmetic composition comprising a dermatologically tolerable excipient.

[0208] 28. The present invention also relates to a composition according to paragraph 27, wherein the composition is a cosmetic composition comprising a dermatologically tolerable excipient.

[0209] 29. The present invention also relates to a composition according to any one of paragraphs 27 to 28, further comprising an agent that resists ECM deposition, preferably non-physiological ECM production or deposition.

[0210] 30. The present invention also relates to a composition according to any one of paragraphs 21 to 29, which is configured for storage, preferably the carrier comprises a medium for storing algal EVs.

[0211] Preferably, the medium comprises a stabilizer.

[0212] Preferably, the medium includes substances for preserving the integrity of EVs, such as salts or sugars.

[0213] Preferably, EVs are concentrated.

[0214] Optionally, the EVs are lyophilized or freeze-dried or spray-dried, preferably spray-dried.

[0215] 31. The present invention also relates to the use of EVs as defined herein in the manufacture of a composition for therapy, preferably therapy of a disorder, preferably therapy of a subject's disorder or a method of treatment, each as defined herein. In particular, the disorder is a disorder as defined in any of the preceding paragraphs, in particular paragraphs 1 to 10 or paragraph 11. Specifically, the composition is as defined in any of paragraphs 21 to 30, in particular a pharmaceutical composition or a nutraceutical composition as defined herein. In particular, algal EVs are as defined in any of the preceding paragraphs, in particular as defined in any of paragraphs 1 and / or 11 or by a process according to any of paragraphs 12 to 18.

[0216] 32. In other aspects, the invention relates to a method for treating a mammalian subject having a disorder as defined in any one of paragraphs 1 to 11, the method comprising administering the algal EVs to the mammalian subject.

[0217] 33. The present invention also relates to a method for treating a mammalian subject according to paragraph 32, the method comprising - orally administering the composition of any one of paragraphs 21 to 26 to the mammalian subject,

[0218] - topically administering the composition of any one of paragraphs 27 to 29 to said mammalian subject.

[0219] - administering the composition of any one of paragraphs 21 to 23 intraperitoneally to the mammalian subject.

[0220] - administering the composition of any one of paragraphs 21 to 23 intranasally to the mammalian subject.

[0221] - administering the composition of any one of paragraphs 21 to 23 intramuscularly to said mammalian subject.

[0222] - administering the composition of any one of paragraphs 21 to 23 intravenously to the mammalian subject.

[0223] 34. The present invention also relates to a method for treating a mammalian subject according to paragraph 32 or 33, the method comprising

[0224] The composition is administered routinely, preferably monthly, weekly or every two days, or preferably daily.

[0225] 35. The present invention also relates to a method for treating a mammalian subject according to paragraph 32 or 33, the method comprising

[0226] The administration continues for one or two weeks, and preferably the composition is administered for at least one or two months.

[0227] 36. In other aspects, the present invention relates to a cosmetic method for treating a mammal to modulate / inhibit / induce extracellular matrix (ECM) production, the method comprising administering a cosmetic composition comprising algal EVs as an active agent.

[0228] In certain embodiments, ECM production / deposition is induced, for example, in the treatment of ulcers.

[0229] In certain embodiments, such as when treating keloids, ECM production / deposition is inhibited.

[0230] The cosmetic composition is a composition as defined herein comprising a dermatologically tolerable or useful excipient.

[0231] Preferably, the subject is in need of inhibiting scarring of skin tissue or production of extracellular matrix (ECM).

[0232] definition

[0233] In short, "progressive fibrosis" is characterized by the process when ECM remodeling shifts toward the accumulation and / or nonphysiological direction of ECM-producing cells (such as fibroblasts or myofibroblasts), for example, excessive deposition of ECM components leads to tissue architecture damage or destruction and / or gradual decline in organ function, which is a nonphysiological ECM production / deposition.

[0234] Progressive fibrosis can lead to the formation of permanent scar tissue, can cause tissue or organ failure, and may result in death. In "progressive fibrosis," ECM components and ECM-producing cells, particularly fibrous ECM components (such as type I and III collagen and fibronectin) and the cells that produce them, continue to accumulate even after the homeostatic / regenerative phase of ECM remodeling.

[0235] The process by which excess ECM replaces normal parenchyma or ECM, which is a typical feature of tissues affected by progressive fibrosis, can also be considered "progressive fibrosis." The process is characterized by excessive proliferation of ECM-producing cells (e.g., fibroblasts), as well as excessive, unregulated or dysregulated deposition of ECM components in various tissues after injury and / or abnormal repair processes.

[0236] Collagen accumulation can be measured by the Sirius Red assay.

[0237] A "fibroproliferative disorder" is particularly a disorder characterized by the presence of progressive fibrosis, particularly wherein at least part of the ECM remodeling is shifted towards the accumulation of ECM-producing cells (e.g., fibroblasts) and / or to excessive deposition of ECM components, leading to impaired or destroyed tissue architecture and / or a gradual decline in organ function.

[0238] A "neoplasm" is a type of abnormal and excessive growth of tissue. (The process by which a neoplasm forms or develops is called "neoplasia.") The growth of a neoplasm is out of coordination with the growth of surrounding normal tissue and may continue to grow abnormally even if the original cause is removed. This abnormal growth often forms a mass, at which point it may be called a tumor. Tumor cells often migrate to various organs.

[0239] "Inflammation" as defined herein refers to the biological response of the animal body, particularly the vertebrate or mammalian body, and particularly its immune system, to one or more stimuli recognized by the body, particularly the immune system, as harmful, typically involving injured or damaged cells, in which inflammatory cells (macrophages, dendritic cells, granulocytes, lymphocytes, fibroblasts, endothelial cells and epithelial cells) and mediators, particularly cytokines, chemoattractants, complement, growth and angiogenic factors, are produced at levels higher than the level in the same body without inflammation.

[0240] In the present context, "ECM deposition" is understood as a process that results in an increase in the amount of ECM components in the space between / in between tissue cells (ie, outside of tissue cells).

[0241] "Physiological" or "regulated" or "normally regulated" deposition of ECM occurs when the deposition of ECM components serves to restore tissue architecture and / or tissue function itself. Physiological deposition of ECM components is regulated or maintained under control to avoid non-physiological ECM deposition or production. Preferably, the regulatory process of the surrounding healthy tissue to the adverse deposition is set in the body to reverse or at least prevent such deposition.

[0242] "Non-physiological" or "dysregulated" deposition of ECM occurs when the deposition of ECM components results in damage (i.e., disruption of tissue architecture and / or tissue function itself). Unregulated or dysregulated deposition of ECM components is a specific hallmark of non-physiological ECM deposition or production. Preferably, deposition of ECM components is considered "non-physiological" (in the case of excessive fibrosis) when there is no evidence that regulatory processes surrounding healthy tissue that counteract deposition are capable of reversing or at least preventing such deposition.

[0243] As used herein, a "subject" is an individual of an animal species, preferably a vertebrate, more preferably a mammalian or avian species, particularly a mammalian species, very preferably the individual is a primate, hominid or human.

[0244] The term "mammal" is well known in the art and refers to species of animals in which females feed their young with milk from their own bodies, and exemplary mammals include humans, primates, livestock animals (including cattle, pigs, goats, sheep, horses, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats), all or any of which are contemplated herein.

[0245] "Patient" means a subject who is undergoing or is intended to undergo medical or veterinary observation, supervision, diagnosis, or treatment.

[0246] "Treatment" refers to any process, action, application, or therapy, etc., in which a subject or patient receives assistance, especially medical or veterinary assistance, with the aim of directly or indirectly improving the condition of the subject or patient. Improving the condition of a subject may include improving the aesthetic condition (cosmetic treatment) and / or may include, in particular, restoring or maintaining the normal function of an organ or tissue, preferably at least partially restoring or maintaining health (medical or veterinary treatment). Treatment generally refers to the administration of an effective amount of a compound or composition as described herein. Treatment may involve or include medical or veterinary treatment, and cosmetic treatment, in particular medical or veterinary treatment.

[0247] "Preventing" or "prevention" of the development of a disease or condition means at least reducing the likelihood of risk or susceptibility to developing a disease or disorder, or preferably reducing the likelihood that a patient who may be exposed to or susceptible to the disease but does not yet experience or display symptoms of the disease will not develop at least one clinical symptom of the disease or disorder.

[0248] The term "effective amount" or "therapeutically effective amount" is intended to define the amount of a therapeutic agent required to alleviate to some extent the symptoms of one or more conditions, diseases or disorders, including but not limited to: 1) reducing the number of fibroblasts or myofibroblasts; 2) reducing the synthesis of ECM components, and / or increasing the degradation of ECM components; 3) reducing the size of fibrous tissue; 4) improving the physiological function of the tissue at least to some extent due to any of 1) to 3); 5) reducing the size of tumor tissue; 6) inhibiting the formation of tumor cell metastasis; 7) inhibiting the proliferation or activation of immune cells, including the production of cytokines, growth factors or antibodies.

[0249] The compounds of the present invention also have pharmaceutical (drug), nutritional and cosmetic uses.

[0250] A "composition" relates to a composition of matter for in vivo administration in humans or animals, comprising an active agent (particularly algal EVs) and one or more additional substances used as carriers. The term "carrier" refers to a diluent, adjuvant, filler, excipient, stabilizer or vehicle with which an agent is formulated for administration.

[0251] A "pharmaceutical composition" relates to a composition for use in treating the human or animal body to restore or maintain health, said composition comprising algal EVs as active agent and one or more additional substances used as carriers.

[0252] "Nutrient" refers to a food that provides health benefits in addition to basic nutritional value. Nutrients have physiological benefits or provide protection against physiological disorders or discomfort. A "nutritive composition" is a nutritive that includes a composition of the present invention and at least an additional substance, such as a nutraceutical carrier or food component.

[0253] The term "dietary supplement" refers to a nutraceutical, such as a nutraceutical composition, that is intended to provide nutrients that may not otherwise be consumed in adequate amounts.

[0254] "Functional food" is also a nutraceutical (eg, a nutraceutical composition) and refers to any modified food or food ingredient that can provide a benefit beyond the traditional nutrients it contains or provide protection against a physiological disorder or discomfort.

[0255] A "health claim" defines the health benefit of a nutraceutical and has regulatory approval under national or equivalent law (similar to the indications for drugs). "Health claims" are used as food labels and in food marketing.

[0256] As used herein, "algae" are photosynthetic or heterotrophic prokaryotes or eukaryotes that are not embryophytes (land plants).

[0257] As used herein, algae are preferably organisms that live in water (fresh or sea water).

[0258] The prokaryotic algae used herein are preferably prokaryotic photoautotrophs.

[0259] As used herein, prokaryotic algae are organisms preferably belonging to the phylum Cyanobacteriota, preferably to the class Cyanophyceae.

[0260] The eukaryotic algae used herein are preferably eukaryotic photoautotrophic organisms that do not belong to embryophytes. The eukaryotic algae used herein are preferably photosynthetic organisms that do not have the matrix tissue characteristics of plants (thin wall tissue, collenchyma tissue and sclerenchyma tissue) nor have special reproductive organs.

[0261] The eukaryotic algae used herein preferably belong to the following groups: Chlorophyta, Catenella, Rhodophyta, Paleophyta and Cyanobacteria

[0262] The singular forms "a," "an," and "the," or at least "a," include plural referents unless the context clearly dictates otherwise.

[0263] The term "comprises" or "comprising" or "including" is interpreted herein as having a non-exhaustive meaning and allows for the addition or reference of further features or method steps or components to anything that includes the listed features or method steps or components. If practice of a given language variant requires, "comprising" can be replaced with "including", or can be limited to "consisting essentially of" if other members or components are not essential to putting the invention into practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0264] Figure 1 Representative immunofluorescence staining of α-smooth muscle actin (α-SMA, green) and cytokeratin (CK) 18 (red) in human primary peritoneal fibroblasts from patient A (phPFB / A). Nuclei were counterstained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0265] Figure 2 Representative immunofluorescence staining of α-smooth muscle actin (α-SMA, green) and cytokeratin (CK) 18 (red) in human primary peritoneal mesothelial cells (HPMC). Nuclei were counterstained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0266] Figure 3 Representative immunofluorescence staining of α-smooth muscle actin (α-SMA, green) and cytokeratin (CK) 18 (red) in human primary colon fibroblasts from patient A (phCFB / A). Nuclei were counterstained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0267] Figure 4 Representative immunofluorescence staining of α-smooth muscle actin (α-SMA, green) and cytokeratin (CK) 18 (red) in human primary colon fibroblasts from patient B (phCFB / B). Nuclei were counterstained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0268] Figure 5 Representative immunofluorescence staining of α-smooth muscle actin (α-SMA, green) and cytokeratin (CK) 18 (red) in human primary skin fibroblasts from patient A (phSFB / A). Nuclei were counterstained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0269] Figure 6 Representative immunofluorescence staining of α-smooth muscle actin (α-SMA, green) and cytokeratin (CK) 18 (red) in human primary skin fibroblasts from patient B (phSFB / B). Nuclei were counterstained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0270] Figure 7. EVs from Chlorella MACC-360 were observed by transmission electron microscopy (TEM) after negative staining with uranyl acetate (-1) (red arrows). The average particle size / particle size distribution and particle number of EVs were measured by dynamic light scattering (DLS), and the data are as follows:

[0271] C360 fraction 2_WO dissolved in PBS

[0272]

[0273] and multiple resistive pulse sensing (MRPS).

[0274] Run Details Box Category: TS400 Mold ID: P12.4.2 Packing date: 210303 Sample Description: 02AC360 1 10xdil Calibrated particle size (nm): 1,0 Calibration particle concentration ( / mL): l,00E+00 analyze Minimum size range (nm): 65,0 Maximum size range (nm): 400,0 Total concentration ( / mL): 1,05E+09 Number of particles detected: 1990 Statistical error of concentration (%): 2.5％ D10(nm): 68,4 D50(nm): 81,8 D90(nm): 128,7 Data processing username: mrps Measurement parameters Measurement date: 20210528 Measurement Username: mrps Get time(s): 490,0l,90E+00 Measuring volume (nL) 1,000 Diameter Scale Factor 1,000 Concentration scaling factor

[0275] Figure 8Representative Western blot analysis of CTGF, PDGF-B, and TGF-β levels in peritoneal dialysis effluent (PDE) from patients with different underlying renal diseases.

[0276] Fig. 9 Penetration of primary human peritoneal fibroblasts from patient A (phPFB / A) by EVs derived from Chlorella MACC-360. phPFB / A cells were treated with cell culture medium containing 3x10^8 particles / ml of DiI-labeled EVs (red) for 24 h. Images were taken with a 20x objective.

[0277] Fig.10 .Permeation of primary human peritoneal fibroblasts from patient B (phPFB / B) by EVs derived from Chlorella MACC-360. phPFB / B cells were treated with cell culture medium containing 3x 10^8 particles / ml of DiI-labeled EVs (red) for 24 h. DiI-labeled PBS (in the absence of EVs) was used as a negative control. Cell nuclei were stained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0278] Fig.11 .Permeation of primary human peritoneal mesothelial cells (HPMC) by EVs derived from Chlorella MACC-360. HPMC were treated with cell culture medium containing 3×10^8 particles / ml of DiI-labeled EVs (red) for 24 h. DiI-labeled PBS (in the absence of EVs) was used as a negative control. Cell nuclei were stained with Hoechst 33342 (blue). Images were taken with a 20x objective.

[0279] Fig.12 Penetration of primary human colonic fibroblasts from patient A (phCFB / A) by EVs derived from Chlorella MACC-360. phCFB / A cells were treated with cell culture medium containing 3x 10^8 particles / ml of DiI-labeled EVs (red) for 24 h. Images were taken with a 20x objective.

[0280] Fig.13 Penetration of primary human dermal fibroblasts from patient A (phSFB / A) by EVs derived from Chlorella MACC-360. phSFB / A cells were treated with cell culture medium containing 3x 10^8 particles / ml of DiI-labeled EVs (red) for 24 h. Images were taken with a 20x objective.

[0281] Fig.14.Effect of EVs derived from Chlorella MACC-360 on the proliferation of primary human peritoneal fibroblasts (phPFB / A) from patient A induced by peritoneal dialysis effluent (PDE). phPFB / A cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test. **p<0.01 3x 10 relative to C (PBS), *p<0.05 3x 10 relative to C (PBS), ##p<0.01 relative to C (PDF), $$3x 10 relative to PDE.

[0282] Fig.15 Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of primary human peritoneal fibroblasts from patient A (phPFB / A). phPFB / A cells were treated with PDGF-B and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 h. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. EV concentrations in the figures refer to the concentration of EV stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test. **p<0.01 3x 10 vs. C(PBS), *p<0.05 3×10 vs. C(PBS), ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. 3x 10PDGF.

[0283] Fig.16 .Effect of EVs derived from Chlorella MACC-360 on PDE-induced proliferation of primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with peritoneal dialysis effluent (PDE) and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 relative to PDF, $$p<0.01 3x 10 relative to 3x10PDE.

[0284] Fig.17 .Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with PDGF-B and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. 3x 10PDGF.

[0285] Fig.18 .Effect of EVs derived from Chlorella MACC-360 on the proliferation of primary human colonic fibroblasts (phCFB / B) from patient B induced by peritoneal dialysis effluent (PDE). phCFB / B cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 relative to PDF, $$p<0.01 3x 10 relative to 3x10PDE.

[0286] Fig.19 .Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of primary human colonic fibroblasts from patient B (phCFB / B). phCFB / B cells were treated with PDGF-B and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. 3x 10PDGF.

[0287] Fig. 20Effect of EVs derived from Chlorella MACC-360 on the proliferation of primary human skin fibroblasts (phSFB / A) from patient A induced by peritoneal dialysis effluent (PDE). phSFB / A cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 3x 10 vs. C (PBS), *p<0.05 3×10 vs. C (PBS), ##p<0.01 vs. PDF, $$p<0.01 3x 10 vs. PDE.

[0288] Fig.21 Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 h. PBS and HCl were used as vehicle controls. Proliferation induced by PDGF-B treatment was determined by MTT assay. The concentration of EVs in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 3x10 vs. C(PBS), *p<0.05 3×10 vs. C(PBS), ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. 3x 10PDGF.

[0289] Fig. 22.Effect of Chlorella MACC-360-derived EVs on peritoneal dialysis effluent (PDE)-induced proliferation of primary human skin fibroblasts from patient B (phSFB / B). phSFB / B cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of Chlorella MACC-360-derived EVs for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 relative to PDF, $$p<0.01 3x 10 relative to 3x10PDE.

[0290] Fig.23 .Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of primary human skin fibroblasts from patient B (phSFB / B). phSFB / B cells were treated with PDGF-B and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0291] Fig.24 .Effect of EVs derived from Chlorella MACC-360 on the proliferation of human lung fibroblast cell line (MRC-5) induced by peritoneal dialysis effluent (PDE). MRC-5 cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##P<0.01 relative to PDF, $$p<0.01 3x 10 relative to PDE, $p<0.3x 1005 relative to PDE.

[0292] Fig.25.Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of human lung fibroblast cell line (MRC-5). MRC-5 cells were treated with PDGF-B and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 relative to C(HCl), $$p<0.01 3x 10 relative to PDGF.

[0293] Fig.26 .Effect of EVs derived from Chlorella MACC-360 on collagen production in primary human peritoneal fibroblasts (phPFB / A) from patient A induced by peritoneal dialysis effluent (PDE). phPFB / A cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. The accumulation of collagen was determined by Sirius Red assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test. ##p<0.01 relative to PDF, $$p<0.01 3x 10 relative to PDE.

[0294] Fig. 27 .Effect of Chlorella MACC-360-derived EVs on TGF-β-induced collagen production in primary human peritoneal fibroblasts from patient A (phPFB / A). phPFB were treated with TGF-β and 3x 10^7 or 3x 10^8 particles / ml of Chlorella MACC-360-derived EVs for 72 hours. PBS and HCl were used as vehicle controls. Collagen accumulation was determined by Sirius Red assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test. ##p<0.01 vs. C(HCl), $p<0.053x 10 vs. TGF-β.

[0295] Fig.28.Effect of EVs derived from Chlorella MACC-360 on collagen production in primary human colonic fibroblasts (phCFB / A) from patient A induced by peritoneal dialysis effluent (PDE). phCFB / A cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. The accumulation of collagen was determined by Sirius Red assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 relative to PDF, $$p<0.01 3x 10 relative to PDE.

[0296] Fig.29 .Effect of EVs derived from Chlorella MACC-360 on TGF-β-induced collagen production in primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A were treated with TGF-β and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and HCl were used as vehicle controls. Collagen accumulation was determined by Sirius Red assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.013x10 vs. 3x10 TGF-β.

[0297] Fig.30 .Effect of EVs derived from Chlorella MACC-360 on collagen production in primary human colonic fibroblasts (phCFB / A) from patient A induced by peritoneal dialysis effluent (PDE). phCFB / A cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. The accumulation of collagen was determined by Sirius Red assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 relative to PDF, $$p<0.01 3x 10 relative to PDE.

[0298] Fig.31.Effect of EVs derived from Chlorella MACC-360 on TGF-β-induced collagen production in primary human colonic fibroblasts from patient B (phCFB / B). phCFB / B were treated with TGF-β and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and HCl were used as vehicle controls. Collagen accumulation was determined by Sirius Red assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.013x10 vs. 3x10 TGF-β.

[0299] Fig.32 .Effect of EVs derived from Chlorella MACC-360 on collagen production in primary human skin fibroblasts (phSFB / A) of patient A induced by peritoneal dialysis effluent (PDE). phSFB / A cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. The accumulation of collagen was determined by the Sirius Red assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. €€p<0.01 vs. C(PBS), *p<0.05 3x 10 vs. C(PBS), ##p<0.01 vs. PDF, $$p<0.01 3x 10 vs. 3x 10PDE.

[0300] Fig.33 Effect of EVs derived from Chlorella MACC-360 on TGF-β-induced collagen production in primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with TGF-β and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and HCl were used as vehicle controls. Collagen accumulation was determined by Sirius Red assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. *p<0.05 vs. C (PBS), ##p<0.01 vs. C (HCl), $$p<0.013x10 vs. 3x10 TGF-β.

[0301] Fig.34 .Effect of EVs derived from Chlorella MACC-360 on collagen production in primary human skin fibroblasts (phSFB / B) of patient B induced by peritoneal dialysis effluent (PDE). phSFB / B cells were treated with PDE and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. The accumulation of collagen was determined by Sirius Red assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t test or Mann-Whitney U test. €€p<0.01 relative to C(PBS), *p<0.05 3x 10 relative to C(PBS), **p<0.01 3x 10v relative to C(PBS), ##p<0.01 relative to PDF, $$p<0.01 3x 103x 10 relative to PDE.

[0302] Fig.35 .Effect of EVs derived from Chlorella MACC-360 on TGF-β-induced collagen production in primary human skin fibroblasts from patient B (phSFB / B). phSFB / B were treated with TGF-β and 3x 10^7 or 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and HCl were used as vehicle controls. Collagen accumulation was determined by Sirius Red assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. *p<0.05 3×10 versus C(PbS), **p<0.01 3×10 versus C(PBS), ##p<0.01 versus C(HCl), $$p<0.01 3×10 versus 3×10 TGF-β.

[0303] Fig.36.Effect of EVs derived from Chlorella MACC-360 on collagen production in TGF-β-induced lung fibroblasts (MRC-5). MRC-5 cells were treated with TGF-β and 3x 10^7 particles / ml of EVs derived from Chlorella MACC-360 for 72 hours. PBS and HCl were used as vehicle controls. The accumulation of collagen was determined by Sirius Red assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 relative to C (PBS), ##p<0.01 3x10 relative to C (HCl), $p<0.05 3×10 relative to TGF-β

[0304] Fig.37 .Effect of EVs derived from Chlorella MACC-360 on IL10 gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with EVs derived from Chlorella MACC-360 at a concentration of 3x 10^8 particles / ml for 24 hours. PBS was used as a vehicle control. The mRNA expression of IL10 was measured by real-time RT-PCR and determined as the ratio of RPLP0 as an internal control. The EVs concentration in the figure refers to the concentration of the EV stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by Mann-Whitney U test. *p<0.05 3x 10 relative to C (PBS).

[0305] Fig.38 .Effect of EVs derived from Chlorella MACC-360 on IL13 gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with EVs derived from Chlorella MACC-360 at a concentration of 3x 10^8 particles / ml for 24 hours. PBS was used as a vehicle control. The mRNA expression of IL13 was measured by real-time RT-PCR and determined as the ratio of RPLP0 as an internal control. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test p = NS 3x 10 relative to C (PBS).

[0306] Fig.39.Effect of EVs derived from Chlorella MACC-360 on PDGFB gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with EVs derived from Chlorella MACC-360 at a concentration of 3x10^8 particles / ml for 24 hours. PBS was used as a vehicle control. The mRNA expression of PDGFB was measured by real-time RT-PCR and determined as the ratio of RPLP0 as an internal control. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test **p<0.01 3x 10 relative to C (PBS).

[0307] Fig.40 .Effect of EVs derived from Chlorella MACC-360 on TGFB gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with EVs derived from Chlorella MACC-360 at a concentration of 3x10^8 particles / ml for 24 hours. PBS was used as a vehicle control. The mRNA expression of TGFB was measured by real-time RT-PCR and determined as the ratio of RPLP0 as an internal control. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test U test. p = NS 3x 10 relative to C (PBS).

[0308] Fig.41 .Effect of EVs derived from Chlorella MACC-360 on IL6 gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. The mRNA expression of IL6 was measured by real-time RT-PCR and determined as the ratio of RPLP0 as an internal control. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). After normality test, significance analysis was performed by unpaired t test. **p < 0.01 relative to C (PBS).

[0309] Fig.42.Effect of EVs derived from Chlorella MACC-360 on IL8 gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. The mRNA expression of IL8 was measured by real-time RT-PCR and determined as the ratio of RPLP0 as an internal control. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). After normality test, significance analysis was performed by unpaired t test. **p < 0.01 relative to C (PBS).

[0310] Fig.43 .Effect of EVs derived from Chlorella MACC-360 on MCP1 gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with 3x10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. The mRNA expression of MCP1 was measured by real-time RT-PCR and determined as the ratio of RPLP0 as an internal control. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). After normality test, significance analysis was performed by unpaired t test. **p < 0.01 relative to C (PBS).

[0311] Fig.44 .Effect of EVs derived from Chlorella MACC-360 on IL10 gene expression in peripheral blood mononuclear cells (PBMCs) under physiological and PHA (phytohemagglutinin) stimulation conditions. PBMCs were treated with PHA and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. The mRNA expression of IL10 was measured by real-time RT-PCR and determined as the ratio of RN18S as an internal control. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). After normality test, significance analysis was performed by unpaired t test. **p<0.01 relative to C (PBS), $$p<0.01 relative to PHA.

[0312] Fig.45.Effect of EVs derived from Chlorella MACC-360 on TGFB gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with PHA and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. The mRNA expression of TGFB was measured by real-time RT-PCR and determined as the ratio of RN18S as an internal control. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). After the normality test, the significance analysis was performed by the Mann-Whitney U test. **p<0.01 relative to C (PBS), $$p<0.01 relative to PHA.

[0313] Fig.46 .Effect of EVs derived from Chlorella MACC-360 on IL13 gene expression in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with PHA and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. The mRNA expression of IL13 was measured by real-time RT-PCR and determined as the ratio of RN18S as an internal control. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). After the normality test, the significance analysis was performed by the Mann-Whitney U test. p = NS.

[0314] Fig.47 Effect of EVs derived from Chlorella MACC-360 on IL-6 protein levels in adenocarcinoma human alveolar basal epithelial cells (A549). A549 cells were treated with lipopolysaccharide (LPS), phytohemagglutinin (PHA), polyinosilic:polycytidilic acid [poly(I:C)] and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. The protein level of IL-6 was determined by ELISA. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 4 / group). After normality test, significance analysis was performed by unpaired t test. **p < 0.01 relative to C (PBS), ##p < 0.01 relative to C (PBS), $$p < 0.01 relative to PHA or poly (I:C).

[0315] Fig.48Effect of EVs derived from Chlorella MACC-360 on IL-6 protein levels in human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with lipopolysaccharide (LPS), phytohemagglutinin (PHA), polyinosinic:polycytidylic acid [poly(I:C)], and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. IL-6 protein levels were determined by ELISA. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 4 / group). Significance analysis was performed by unpaired t-test after normality test. **p<0.01 vs. C(PBS), ##p<0.01 vs. C(PBS), $$p<0.01 vs. PHA or poly(I:C).

[0316] Fig.49 .Effect of EVs derived from Chlorella MACC-360 on methylglyoxal (MGO)-induced cytotoxicity in primary human omental mesothelial cells (HPMC). HPMC were treated with 600 μM and 700 μM MGO and 3x10^7 or 3x 10^8 particles / ml Chlorella MACC-360-derived EVs for 24 hours. PBS was used as a vehicle control. Cell viability was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by Mann-Whitney U3×103×103×10-test. *##p<0.01 relative to C (PBS), $$p<0.01 relative to the corresponding MGO.

[0317] Fig.50.Effect of EVs derived from Chlorella MACC-360 on methylglyoxal (MGO)-induced cytotoxicity in primary human omental mesothelial cells (HPMC). HPMC were treated with 700 μM MGO and 3x 10^7 or 3x10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS was used as a vehicle control. Cytotoxicity was determined by LDH assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by Mann-Whitney U test. **p<0.01EV 3x 10 vs. C (PBS), ##p<0.01 vs. C (PBS), $$p<0.01 3x 10 vs. 700 μM MGO.

[0318] Fig.51 .Effect of Chlorella MACC-360-derived EVs on methylglyoxal (MGO)-induced toxicity in primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with 100 μM MGO and 3x 10^8 particles / ml of MACC-360-derived EVs for 24 h. PBS was used as a vehicle control. Cytotoxicity was determined by LDH assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). After normality test, significance analysis was performed by Mann-Whitney U test. #p<0.05 relative to C (PBS), $$p<0.01 relative to 100 μM MGO.

[0319] Fig.52 Effect of EVs derived from Chlorella vulgaris MACC-360 on 3,4-dideoxyglucosone-3-ene (DGE)-induced cytotoxicity in primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with 75 μM DGE and 3x 10^8 particles / ml MACC-360-derived EVs for 24 h. PBS was used as vehicle control. Cytotoxicity was determined by LDH assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). After normality test, significance analysis was performed by Mann-Whitney U test. #p<0.01 vs. C (PBS), $$p<0.01 vs. 75 μM DGE.

[0320] Fig.53 Effect of EVs derived from Chlorella vulgaris MACC-360 on 5-Hydroxymethyl-2-furaldehyde (HMF)-induced cytotoxicity in primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with 10 mM HMF and 3 x 10^8 particles / ml MACC-360-derived EVs for 24 h. PBS was used as vehicle control. Cytotoxicity was determined by LDH assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). After normality test, significance analysis was performed by Mann-Whitney U test. ##p<0.01 vs. C (PBS), $$p<0.01 vs. 10 mM HMF.

[0321] Fig.54 Effect of Chlorella MACC-360-derived EVs on methylglyoxal (MGO)-induced cytotoxicity in primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with 300 μM MGO and 3x 10^8 particles / ml of MACC-360-derived EVs for 24 h. PBS was used as a vehicle control. Cell viability was determined by MTT assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). After normality test, significance analysis was performed by Mann-Whitney U test. **p<0.01 vs. C (PBS), ##p<0.01 vs. C (PBS), $$p<0.01 vs. 300 μM MGO.

[0322] Fig.55 Effect of Chlorella MACC-360-derived EVs on methylglyoxal (MGO)-induced cytotoxicity in primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with 300 μM MGO and 3x 10^8 particles / ml of MACC-360-derived EVs for 24 h. PBS was used as a vehicle control. Cytotoxicity was determined by LDH assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). After normality test, significance analysis was performed by Mann-Whitney U test. *p<0.05 vs. C (PBS), ##p<0.01 vs. C (PBS), $$p<0.01 vs. 300 μM MGO.

[0323] Fig.56.Effect of EVs derived from Chlorella MACC-360 on 5-Hydroxymethyl-2-furaldehyde (HMF)-induced cytotoxicity in primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with 5 mM HMF and 3 x 10^8 particles / ml MACC-360-derived EVs for 24 h. PBS was used as a vehicle control. Cell viability was determined by MTT assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). After normality test, significance analysis was performed by Mann-Whitney U test. **p<0.01 relative to C (PBS), ##p<0.01 relative to C (PBS), $p<0.05 relative to 5 mM HMF.

[0324] Fig.57 .Effect of EVs derived from Chlorella MACC-360 on methylglyoxal (MGO)-induced cytotoxicity in primary human umbilical cord endothelial cells (HUVEC). HUVEC cells were treated with 500 μM MGO and 3x 10^8 particles / ml EVs derived from Chlorella MACC-360 for 24 hours. PBS was used as a vehicle control. Cell viability was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by Mann-Whitney U test. ##p<0.01 relative to C (PBS), $$p<0.01 3x 10 relative to MGO.

[0325] Fig.58 .Effect of EVs derived from Chlorella MACC-360 on methylglyoxal (MGO)-induced cytotoxicity of primary human umbilical cord endothelial cells (HUVEC). HUVEC cells were treated with 500 μM MGO and 3x 10^8 particles / ml Chlorella MACC-360-derived EVs for 24 hours. PBS was used as a vehicle control. Cytotoxicity was determined by LDH assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by Mann-Whitney U test. ##p<0.01 relative to C (PBS), $$p<0.01 3x 10 relative to MGO.

[0326] Fig.59.Effect of EVs derived from Chlorella MACC-360 on the migration of primary human colonic fibroblasts (phCFB / A) from patient A. phCFB / A cells were treated with 10 ng / ml EGF and 3x 10^8 particles of EVs derived from Chlorella MACC-360. PBS was used as vehicle control. Cell migration was determined by TAS assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean ± SD (n = 6 / group). Significance analysis was performed by two-way ANOVA. $$p<0.01EGF versus EGF+EV 3x10, at relevant times.

[0327] Fig.60 .Effect of EVs derived from Chlorella MACC-360 on the migration of primary human skin fibroblasts (phSFB / A) from patient A. phSFB / A cells were treated with 10ng / ml EGF and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360. PBS was used as a vehicle control. Cell migration was determined by TAS assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean ± SD (n = 6 / group). Significance analysis was performed by two-way ANOVA. $$p<0.01EGF versus EGF+EV 3x10, at relevant times.

[0328] Fig.61 .Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of adenocarcinoma human alveolar basal epithelial cells (A549). A549 cells were treated with PDGF-B and 3x 10^8 or 3x 10^9 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 3x 10 relative to C (PBS), ##p<0.01 3×10 relative to C (HCl), $$p<0.01 3x 103x 10 relative to PDGF.

[0329] Fig.62.Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of human colorectal adenocarcinoma cell line (HT-29). HT-29 cells were treated with PDGF-B and 3x 10^8 or 3x 10^9 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 3x 10 relative to C (PBS), ##p<0.01 3×10 relative to C (HCl), $$p<0.01 3x 103x 10 relative to PDGF.

[0330] Fig.63 .Effect of EVs derived from Chlorella MACC-360 on PDGF-B-induced proliferation of colon carcinoma cell line (Caco-2). Caco-2 cells were treated with PDGF-B and 3x 10^8 or 3x 10^9 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 3x 10 relative to C (PBS), ##p<0.01 3x 10 relative to C (HCl), $$p<0.01 3x 103x 10 relative to PDGF.

[0331] Fig.64 .Effect of EVs derived from Chlorella MACC-360 on cell migration of colon cancer cell line (Caco-2). phSFB / A cells were treated with EGF or peritoneal dialysis effluent (PDE) and 3x 10^8 particles / ml EVs derived from Chlorella MACC-360. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell migration was determined by TAS assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean ± SD (n = 6 / group). Significance analysis was performed by two-way ANOVA. $$P<0.01PDE relative to PDE+EV 3x 103x 10, at relevant times.

[0332] Fig.65.Effect of EVs derived from Chlorella MACC-360 on the proliferation of human breast cancer cell line (Hs578-T) induced by peritoneal dialysis effluent (PDE). Hs578-T cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test. **p<0.01, ##p<0.01 relative to C (PDF), $$p<0.01 relative to PDE.

[0333] Fig.66 .Effect of EVs derived from Chlorella MACC-360 on cell death of human breast cancer cell line (Hs578-T) treated with peritoneal dialysis effluent (PDE). Hs578-T cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cytotoxicity was determined by LDH assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t test. p = NS.

[0334] Fig.67 .Effect of EVs derived from Chlorella MACC-360 on the proliferation of human large cell lung cancer cell line (LCLC-103H) induced by peritoneal dialysis effluent (PDE). LCLC-103H cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test and Mann-Whitney test. **p<0.01 relative to C (PBS), ##p<0.01 relative to C (PDF), $$p<0.01 relative to PDE.

[0335] Fig.68.Effect of EVs derived from Chlorella MACC-360 on cell death of human large cell lung cancer cell line (LCLC-103H) treated with peritoneal dialysis effluent (PDE). LCLC-103H cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cytotoxicity was determined by LDH assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t test. p = NS.

[0336] Fig.69 .Effect of EVs derived from Chlorella MACC-360 on the proliferation of human hepatocellular carcinoma cell line (HEPG2) induced by peritoneal dialysis effluent (PDE). HEPG2 cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed using an unpaired t-test. **p<0.01 relative to C (PBS), ##p<0.01 relative to C (PDF), $$p<0.01 relative to PDE.

[0337] Fig.70 .Effect of EVs derived from Chlorella MACC-360 on cell death of human hepatocellular carcinoma cell line (HEPG2) treated with peritoneal dialysis effluent (PDE). HEPG2 cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cytotoxicity was determined by LDH assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test and Mann-Whitney test. **p<0.01 relative to C (PBS)

[0338] Fig.71Effects of EVs derived from Chlorella MACC-360 on peritoneal thickness in a chlorhexidin-gluconate (CG)-induced peritoneal fibrosis model in mice. Peritoneal fibrosis was induced by daily intraperitoneal injection (ip) of CG for 7 consecutive days. 1*10^9 particles of EVs from Chlorella MACC-360 were injected ip twice a week. The extent of fibrosis in peritoneal sections was observed microscopically (blue) after Masson trichrome staining (-1), and the submesothelial thickness (-2) was quantified graphically. Images were taken with a 20x objective. The EVs concentrations in the figures refer to the concentrations of the EVs stock solution used to treat mice. Results are presented as mean + SD, and points represent individual values. Significance analysis was performed by Mann-Whitney U test. ##p<0.01 vs. C (PBS), $$p<0.01 3x 10 vs. CG.

[0339] Fig.72 The average particle size / size distribution of EVs isolated from Chlorella MACC-360 using different separation methods (-1: high-speed centrifugation; -2: Amicon and SEC) was measured by dynamic light scattering (DLS):

[0340] UCF_10 times dissolved in PBS

[0341]

[0342] Amsecfr1_10 times dissolved in PBS

[0343]

[0344] UCF_10 times dissolved in PBS

[0345]

[0346] Fig.73.Effect of Chlorella MACC-360-derived EVs (TFF EVs) isolated by tangential flow filtration ultrafiltration on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^8 EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0347] Fig.74 .Effect of Chlorella MACC-360-derived EVs separated by tangential flow filtration and size exclusion chromatography (TFF+SEC EVs) on PDGF-B-induced proliferation of primary human dermal fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x10^8 particles / ml of Chlorella MACC-360-derived EVs for 24 h. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentrations in the figures refer to the concentrations of the EVs stock solution used for cell treatment. Results are presented as mean+SD, and points represent individual values ​​(n=5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0348] Fig.75 .Effect of Chlorella MACC-360-derived EVs (UCF EVs) isolated by ultracentrifugation on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml of Chlorella MACC-360-derived EVs for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0349] Fig.76.Effect of Chlorella MACC-360-derived EVs (AMICON EVs) isolated by ultrafiltration using Amicon filters on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml of Chlorella MACC-360-derived EVs for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01PDGF vs. C(HCl), $$p<0.01PDGF+3x 10^8 particles / ml AMICON EV vs. PDGF.

[0350] Fig.77 .Effect of Chlorella MACC-360-derived EVs (AM+SEC EVs) separated by Amicon filter and size exclusion chromatography ultrafiltration on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml of Chlorella MACC-360-derived EVs for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $p<0.05 3x 10 vs. PDGF.

[0351] Fig.78 .Effect of EVs derived from Chlorella MACC-360 isolated by size exclusion chromatography on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml Chlorella MACC-360-derived EVs for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0352] Fig.79 .Effect of EVs derived from Chlorella MACC-360 (EXTR EVs) isolated by ultrasound / extraction on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0353] Fig.80 .Effect of Chlorella MACC-360-derived EVs (EXTR+SECEV) separated by ultrasound / extraction and size exclusion chromatography on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x10^8 particles / ml of Chlorella MACC-360-derived EVs for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean+SD, and points represent individual values ​​(n=5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0354] Figure 81. Transmission electron microscopy (TEM) of EVs from Pseudomonas kesleri after negative staining with uranyl acetate (-1) (arrows). The average particle size / particle size distribution and particle number of EVs from Pseudomonas kesleri were measured by dynamic light scattering (DLS) C38 Amicon SECFR2

[0355]

[0356] and multi-resistance pulse sensing (MRPS).

[0357] Run Details Box Category: TS400 Mold ID: P12.4.2 Packing date: 210303 Sample Description: 05_BC360_2_10xdil Calibrated particle size (nm): 1,0 Calibration particle concentration ( / mL): 1,00E+00 analyze Minimum size range (nm): 65,0 Maximum size range (nm): 400,0 Total concentration ( / mL): 7,94E+08 Number of particles detected: 2247 Statistical error of concentration (%): 2,4％ D10(nm): 65,0 D50(nm): 75,1 D90(nm): 108,6 Data processing username: mrps Measurement parameters Measurement date: 20210528 Measurement Username: mrps Get time(s): 620,0 Measuring volume (nL) 2,83E+00 Diameter Scale Factor 1,000 Concentration scaling factor 1,000

[0358] Fig.82.Effect of EVs derived from Pseudochlorella kassleri on PDGF-B-induced proliferation of human colonic fibroblasts (phCFB / A). phCFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml of EVs derived from Pseudochlorella kassleri for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 3x 10 relative to C (PBS), ##p<0.01 relative to C (HCl), $$p<0.01 3x 10 relative to PDGF.

[0359] Fig.83 .Effect of Chlorella vulgaris-derived EVs on TGF-β-induced collagen production in primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with TGF-β and 3x 10^8 particles / ml of Chlorella vulgaris-derived EVs for 72 h. PBS and HCl were used as vehicle controls. Collagen accumulation was determined by Sirius Red assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. TGF-β.

[0360] Fig.84 .Effect of EVs derived from Pseudomonas kesleri on TGF-β-induced collagen production in primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with TGF-β and 3x 10^8 particles / ml of EVs derived from Pseudomonas kesleri for 72 hours. PBS and HCl were used as vehicle controls. The accumulation of collagen was determined by the Sirius Red assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. TGF-β.

[0361] Figure 85. EVs (arrows) from Spirulina platensis (-1, -2) and Chlorella pyrenoidosa (-3, -4) were observed by transmission electron microscopy (TEM) after negative staining with uranyl acetate (-1, -3). Average size / size distribution of EVs measured by dynamic light scattering (DLS) (-2, -4): SP lio FR1_10 times dissolved in PBS

[0362]

[0363] CV lio FR1_10 times dissolved in PBS

[0364]

[0365] The protein and lipid contents and ratios were analyzed by Fourier-transform infrared spectroscopy (FTIR) (-5).

[0366] Fig.86 .Effect of EVs derived from Spirulina platensis (AMICON EVs) isolated by ultrafiltration using an Amonic filter on PDGF-B-induced proliferation of primary human dermal fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x10^8 particles / ml of EVs derived from Spirulina platensis for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 relative to C(HCl), $$p<0.3x 10 relative to PDGF.

[0367] Fig.87 .Effect of Spirulina-derived EVs (AM+SECEVs) isolated using Amicon filters and size exclusion chromatography ultrafiltration on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml of Spirulina-derived EVs for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentrations in the figures refer to the concentrations of the EVs stock solution used for cell treatment. Results are expressed as mean+SD, and points represent individual values ​​(n=5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x10 vs. PDGF.

[0368] Fig.88.Effect of EVs derived from Chlorella vulgaris (AMICON EVs) isolated by ultrafiltration using an Amonic filter on PDGF-B-induced proliferation of primary human dermal fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x10^8 particles / ml of EVs derived from Chlorella vulgaris for 24 hours. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentrations in the figures refer to the concentrations of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0369] Fig.89 .Effect of EVs derived from Chlorella vulgaris (AM+SEC EVs) isolated using Amicon filters and size exclusion chromatography ultrafiltration on PDGF-B-induced proliferation of primary human skin fibroblasts from patient A (phSFB / A). phSFB / A cells were treated with PDGF-B and 3x10^8 particles / ml of Chlorella vulgaris-derived EVs for 24 h. PBS and HCl were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figures refers to the concentration of the EVs stock solution used for cell treatment. Results are expressed as mean+SD, and points represent individual values ​​(n=5 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. ##p<0.01 vs. C(HCl), $$p<0.01 3x 10 vs. PDGF.

[0370] Fig.90 EVs derived from Chlorella MACC-360 are internalized into the liver in vivo. 10^9 DiI-labeled EVs (red) were intravenously injected into the tail of C57 / BL6J mice. Mice were sacrificed 24 hours later and the liver was harvested. Images were taken with a 100x objective.

[0371] Figure 91-1 .MACC-1 (Chlorella vulgaris)-derived EVs are internalized into the visceral peritoneum in vivo. 10^9 DiI-labeled EVs (red) were intraperitoneally administered to C57 / BL6J mice. Mice were sacrificed 24 hours later, and the visceral peritoneum was obtained. Cell nuclei were stained with DAPI (blue). Images were taken with a 20x objective.

[0372] Figure 91-2.MACC-3 (Chlorella vulgaris)-derived EVs are internalized into the visceral peritoneum in vivo. 10^9 DiI-labeled EVs (red) were intraperitoneally administered to C57 / BL6J mice. Mice were sacrificed 24 hours later, and the visceral peritoneum was obtained. Nuclei were stained with DAPI (blue). Images were taken with a 20x objective.

[0373] Fig.92 EVs derived from MACC-1 (Chlorella vulgaris), MACC-3 (Chlorella pyrenoidosa), and MACC-1023 (Homiella verrucosa) are internalized into peritoneal lavage cells in vivo. 10^9 DiI-labeled EVs (red) were administered intraperitoneally to C57 / BL6J mice. Mice were killed 24 hours later, and peritoneal lavage fluid was collected. Cell nuclei were stained with DAPI (blue). Fig.93 EVs derived from Chlorella MACC-360 are internalized into the skin in vivo. 10^9 DiI-labeled EVs (red) were intradermally administered to C57 / BL6J mice. After 24 hours, the mice were killed and the skin was harvested. Images were taken with a 20x objective.

[0374] Figure 94. Particle number of Chlorella MACC-360-derived EVs was measured by nanoparticle tracking analysis (NTA) (-1) and multi-resistance pulse sensing (MRPS) (-2) to determine the exact dose for oral administration (gavage) to C57 / BL6 mice.

[0375] Graph 94-1 shows PARTICLEMETRIX measurements, ie, Electrophoresis & Brownian Motion Video Analysis Laser Scattering Microscopy at Electrolyte: PBS, Temperature: 25.04°C sensed, Input pH 7.0, Conductivity: 15000.00 pS / cm sensed.

[0376] Results (size in nm)

[0377]

[0378] Chart 94-2

[0379]

[0380] Fig.95.Effect of EVs derived from Chlorella MACC-360 on the disease activity index (DAI) of the mouse colitis model induced by dextrane sodium sulfate (DSS). In brief, C57BL / 6J mice received 2.5% DSS in drinking water for 7 days (DSS). In addition, mice were treated with 10^9 EVs of Chlorella MACC-360 by oral gavage every day (DSS+MACC-360). Changes in DAI were monitored daily throughout the experiment. Results are expressed as mean + SD. Significance analysis was performed by two-way ANOVA. *p<0.05DSS+MACC-360EV versus DSS, on the relevant day.

[0381] Fig.96 .Effect of EVs derived from Chlorella MACC-360 cultured with autotrophs (auto) and heterotrophs (het) on the proliferation of human hepatocellular carcinoma cell line (HEPG2) induced by peritoneal dialysis effluent (PDE). HEPG2 cells were treated with PDE and 3x10^8 particles / ml of Chlorella MACC-360-derived EVs (from autotrophic and heterotrophic cultures) for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t test. **p<0.01 relative to C (PBS), ##p<0.01 relative to C (PDF), $$p<0.01 relative to PDE, $p<0.05 relative to PDE.

[0382] Fig.97 .Effect of autotrophic (auto) and heterotrophic (het) cultures of Chlorella MACC-360-derived EVs on cell death in human hepatocellular carcinoma cell line (HEPG2) treated with peritoneal dialysis effluent (PDE). HEPG2 cells were treated with PDE and 3x 10^8 particles / ml of Chlorella MACC-360-derived EVs (from autotrophic and heterotrophic cultures) for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cytotoxicity was determined by LDH assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test and Mann-Whitney U test. **p < 0.01 relative to C (PBS).

[0383] Fig.98.Effect of EVs derived from Chlorella MACC-360 (using freeze-dried (lio) and fresh samples) on the proliferation of primary human colonic fibroblasts (phCFB / A) of patient A induced by peritoneal dialysis effluent (PDE). phCFB / A cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 (from freeze-dried (lio) and fresh samples) for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t test. **p<0.01 relative to C (PBS), ##p<0.01 relative to C (PDF), $$p<0.01 relative to PDE.

[0384] Fig.99 .Effect of Chlorella MACC-360-derived EVs on cell death of primary human colonic fibroblasts (phCFB / A) treated with peritoneal dialysis effluent (PDE) of patient A using freeze-dried (lio) and fresh samples. phCFB / A cells were treated with PDE and 3x 10^8 particles / ml of Chlorella MACC-360-derived EVs (from freeze-dried (lio) and fresh samples) for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cytotoxicity was determined by LDH assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test and Mann-Whitney U test. **p<0.01 relative to C (PBS).

[0385] Fig.100.Effect of EVs derived from Chlorella MACC-360 - treated with fresh hydrochloric acid (HCl) (37°C, 1h, pH=4.6) and 0.25% trypsin-EDTA (TFF:trypsin-EDTA=2:1, 37°C, 1h) samples - on the proliferation of primary human colonic fibroblasts (phCFB / A) from patient A induced by peritoneal dialysis effluent (PDE). phCFB / A cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from Chlorella MACC-360 for 24 hours, and samples were treated with fresh HCl (37°C, 1h) and trypsin (37°C, 1h). PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. The results are expressed as mean + SD, and the points represent individual values ​​(n=6 / group). Significance analysis was performed by unpaired t test. **P<0.01 relative to C(PBS), ##P<0.01, $$P<0.01 relative to PDE.

[0386] Fig.101 Effect of Chlorella MACC-360-derived EVs on platelet-derived growth factor B (PDGF-B)-induced proliferation of primary human colonic fibroblasts from patient A (phCFB / A) using fresh, heat-denatured (denat), hydrochloric acid (HCl)-treated, heat-denatured and HCl-treated (denat+HCl), and proteinase K-treated (protK) samples. phCFB / A cells were treated with PDGF-B and 3x 10^8 particles / ml Chlorella MACC-360-derived EVs for 24 hours. PBS was used as a vehicle control. Cell proliferation was determined by MTT assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test. ##p<0.01 vs. C (PBS), $$p<0.01 vs. PDGF-B.

[0387] Fig.102.Effect of EVs derived from Chlorella MACC-360 in combination with MACC-1 (Chlorella vulgaris) and MACC-908 (Spirulina platensis) on peritoneal dialysis effluent (PDE)-induced proliferation of primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with PDE and 3x 10^8 particles / ml of EVs derived from MACC-360, MACC-1 and MACC-908 or their combination for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. The EVs concentrations in the figures refer to the final concentration of the EVs stock solution used for cell treatment. Results are expressed as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by two-way ANOVA. **p<0.01 relative to PBS, ##p<0.01 relative to PDF, $$p<0.01 relative to PBS; PDE.

[0388] Fig.103 Effect of EVs derived from Chlorella MACC-360 in combination with the platelet-derived growth factor receptor alpha (PDGFR-α) inhibitor Ponatinib on platelet-derived growth factor B (PDGF-B)-induced proliferation of primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with PDGF-B and Ponatinib for 24 h in the absence or presence of 3x 10^8 particles / ml MACC-360-derived EVs. PBS was used as a vehicle control. Cell proliferation was determined by MTT assay. The EVs concentration in the figure refers to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by one-way ANOVA. ##p<0.01 vs. C (PBS), $$p<0.01 vs. PDGF-B, €€p<0.01 vs. PDGF-B, ΩΩp<0.01 vs. PDGF-B.

[0389] Fig.104Effect of EVs derived from Chlorella sp. MACC-360 in combination with the platelet-derived growth factor receptor β (PDGFR-β) inhibitor Axitinib on platelet-derived growth factor B (PDGF-B)-induced proliferation of primary human colonic fibroblasts from patient A (phCFB / A). phCFB / A cells were treated with PDGF-B and Axitinib for 24 h in the absence or presence of 3 x 10^8 particles / ml MACC-360-derived EVs. PBS was used as vehicle control. Cell proliferation was determined by MTT assay. The concentration of EVs in the figures refers to the final concentration of the EVs stock solution used for cell treatment. Results are presented as mean + SD, and points represent individual values ​​(n = 5 / group). Significance analysis was performed by one-way ANOVA. ##p<0.01 vs. C (PBS), $$p<0.01 vs. PDGF-B, €€p<0.01 vs. PDGF-B, ΩΩp<0.01 vs. PDGF-B.

[0390] Figure 105. Effect of EVs derived from Chlorella MACC-360 on protein kinase phosphorylation in primary human colonic fibroblasts (phCFB / A) from patient A induced by recombinant protein mixture (recMix) (-1, -2) and peritoneal dialysis effluent (PDE) (-3, -4). Protein kinase phosphorylation was determined by the Proteome Profiler Human Phospho-Kinase Array Kit. Analytes and their membrane locations on the array membrane (Figure 105-1). Representative images of the array membrane of different treatment groups in each experiment (-1, -2). In each experiment, phCFB / A cells were treated for 30 minutes. The average pixel density of the analyte was quantified by densitometry. In each experiment, the fold change values ​​of the optical density of a given group were compared ( Figure 105-2 , Chart 105-3) or normalized and expressed as a ratio of its control value (-3, -4).

[0391] Chart 105-1

[0392]

[0393]

[0394] Chart 105-2

[0395]

[0396]

[0397] Chart 105-3

[0398]

[0399]

[0400] Fig.106 .Effect of EVs derived from Spirulina platensis powder isolated by extraction (SP extr) on the proliferation of primary human colonic fibroblasts (phCFB / A) from patient A induced by peritoneal dialysis effluent (PDE). phCFB / A cells were treated with PDE and 18 μg of EVs from Spirulina platensis powder for 24 h. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay. Results are expressed as mean + SD, points represent individual values ​​(n = 6 / group). Significance analysis was performed by unpaired t-test or Mann-Whitney U test. **p<0.01 relative to C (PBS), $$p<0.01 relative to PDE.

[0401] Fig.107 . Schematic representation of the interactions between inflammation, fibrosis, and cancer in human disease. These pathological processes intersect at multiple levels and through multiple pathways and are thought to be causal for the development of many diseases and important host factors that affect the effectiveness of drug therapies. CAF: cancer-associated fibroblasts; DAMP: danger-associated molecular patterns; ECM: extracellular matrix; IL: interleukin; PAMP: pathogen-associated molecular patterns; ROS: reactive oxygen species; TAM: tumor-associated macrophages; TAN: tumor-associated neutrophils; TGF: transforming growth factor [Dong, J., & Ma, Q. (2019). Integration of inflammation, fibrosis, and cancer induced by carbon nanotubes. Nanotoxicology, 13(9), 1244-1274.]. DETAILED DESCRIPTION

[0402] It is known in the art that extracellular vesicles (EVs) are potential vehicles for delivering drugs in therapeutic treatments.

[0403] The present invention relates to the use of algal EVs in therapy, wherein the EVs themselves have a beneficial effect on the health of the subject to which they are administered.

[0404] In particular, the inventors surprisingly found that EVs isolated from algae, including prokaryotic and eukaryotic algae as defined herein, exert complex beneficial effects in a number of fibrosis and tumor formation and inflammation models, which involve inhibiting the production of extracellular matrix (ECM), cell proliferation and cell migration, and also increasing the production of anti-inflammatory cytokines including IL-6, IL-10 and TGFb.

[0405] These processes (i.e., ECM production, cell proliferation, cell migration, and inflammation) help maintain or restore the health of a subject (e.g., after injury or infection), however, when excessive or uncontrolled, they can lead to various disorders. These processes can indicate the stages of development of various disease conditions and also play a role in them. They are not independent, but rather overlap as stages of symptom development. Necessarily, they are tailored by a complex network of interconnected biochemical and signaling pathways, but they can still be considered details or fragments of a larger picture. Therefore, these processes (e.g., inflammatory stages, tissue scarring or progressive fibrosis, cell hyperproliferation, migration, or non-physiological ECM production and / or deposition) can be considered to be conditions to be treated to restore or prevent impairment of a subject's health. ( Fig.107 ) The etiology of some symptomatically defined disorders involves these (excessive or uncontrolled) processes, which should be treated either on their own or within the therapeutic framework of a given disease. While specific molecules used as active substances in medicine typically act on several off-target sites of drugs and various biochemical pathways, typically one or two of which are considered the main "entry points", the inventors provide a wealth of experimental evidence showing that algal EVs as active substances act on different off-targets, which is consistent with their complex, still well-defined nature.

[0406] Surprisingly, these EVs have effects at several points in the matrix of such conditions, including broadly speaking inflammatory processes, cell hyperproliferation, migration, and ECM production and deposition, with factors associated with inflammation, fibrosis, tumors, etc., among others, as shown in the exemplary model experiments taught herein. In the example, EVs lead to increased production of anti-inflammatory IL-10.

[0407] In certain embodiments, EVs affect the production of IL-6, IL-10, and / or TGFβ, for example, having the following effects: IL-6- inhibits the production of TNF and IL-1 by macrophages

[0408] IL-10 - inhibits the production of monocyte / macrophage and neutrophil cytokines and suppresses TH1 lymphocyte responses; has humoral and cellular effects,

[0409] TGFb-inhibits monocyte / macrophage MHC class II expression and the synthesis of proinflammatory cytokines; however, the anti-inflammatory properties promote the production of proinflammatory cytokines.

[0410] Thus, typically, the cytokines affected by the EVs of the present invention are Janus-faced molecules with anti-inflammatory aspects.

[0411] Disorders according to the present invention typically induce inflammation or are associated with inflammation. For example, inflammation and fibrosis are interrelated. Due to the regulation of cytokines, the regulation of EVs on immune responses can be multiple and ultimately depends on the specific situation. However, the results of the regulation of immune responses are beneficial in this article, which is reasonable. On the one hand, EVs do not seem to directly and strongly cause a proinflammatory response. The animal experiments provided herein support this well. For example, in the DSS-induced inflammation model, the DAI scores of animals were reduced and the clinical status improved. In another experiment, the antifibrotic effect of EVs was also tested in vivo in a peritoneal fibrosis model induced by chlorhexidine gluconate (CG). EVs significantly inhibited the increase in CG-induced submesothelial thickness. Submesothelial thickness is positively correlated with the severity of peritoneal fibrosis, and therefore, inflammation does not lead to progressive fibrosis ( Fig.71 ).

[0412] Therefore, regulation of inflammation by EVs contributes to their beneficial effects.

[0413] In other embodiments, fibrosis is considered herein as a dynamic process of ECM production or degradation. Abnormal processes (such as damage on the one hand and progressive fibrosis on the other hand) shift the balance of this dynamic process to extremes or out of balance. In variants, the production / degradation of ECM becomes irregular. In preferred embodiments, the EV or composition of the present invention improves the regulation of ECM production, preferably maintaining its dynamic properties or maintaining normal balance.

[0414] For example, in certain experiments, the inventors found that EVs shifted ECM production at controlled levels, which could indicate a regulatory role and shift in ECM production / degradation dynamics.

[0415] As an example of diabetic ulcers, induction of ECM is useful.

[0416] These characteristics of EVs can be used to treat a mammalian subject whose health condition is to be restored or to use EVs for prevention. In some embodiments, EVs can be used to improve the performance of a subject.

[0417] In a sense, ECM production, fibrosis, cell migration and even inflammation are natural processes that should be controlled within limits and thus gently mitigated. An example is wound healing, where fibrotic processes are necessary for wound healing, however, under physiological conditions, such processes (such as ECM deposition) are controlled and restore tissue architecture and lead to restoration of function.

[0418] As another example of aging, fibrosis can be considered an inevitable occurrence.

[0419] However, often in otherwise healthy elderly people, the kidneys become affected by a progressive fibrotic process and thus become impaired in their functioning.

[0420] Thus, in one aspect, the compositions of the invention can be part of the diet and used as a dietary supplement, nutraceutical, functional food, nutritional food (including beverages) or, in some cases, as a medical food or beverage.

[0421] In other variations, if applied to the skin, the cosmetic treatment may have a similar effect of maintaining or achieving a healthy state of skin in which tissue scarring is no longer evident.

[0422] In addition to restoring health, treating and preventing disease conditions, the algal EVs of the present invention help control these processes or modulate these processes and thereby improve performance in otherwise healthy subjects.

[0423] In their experiments, the inventors found a general anti-fibrotic effect from a very wide range of algae types, wherein the pro-fibrotic inducer was applied to lung, peritoneal, colon and skin fibroblasts, and essentially every algae type tested caused a decrease in fibroblast proliferation, such as shown by the MTT assay. Nevertheless, the yield of ECM increased significantly rather than decreased in a few algae species / strains, particularly on skin fibroblasts, while it decreased in other algae species / strains. These data collectively indicate the regulatory role of algae EVs in fibroblasts, wherein, in addition to significant anti-fibrotic effects, the regulation of ECM is part of the overall benefit. The inventors also show the preferred effect of algae strain combinations (e.g., see Figure X102). It is reasonable that the anti-fibrotic effect can be optimized by the combination of algae strains, and the careful selection of strains will result in significant anti-fibrotic effects and balanced ECM production. This can have advantages in any anti-fibrotic (anti-fibroblast) application, particularly in the lungs, peritoneum, colon and skin, fibroblasts (particularly preferably skin fibroblasts).

[0424] Algae useful in the present invention

[0425] Algae that can be used in the present invention include prokaryotic algae and eukaryotic algae, for example, as listed in the definitions.

[0426] In particular, algae as used herein are micro- and macroalgae.

[0427] In particular, algae as used herein are prokaryotic or eukaryotic algae.

[0428] In an embodiment, the eukaryotic algae used herein are preferably organisms belonging to the following groups: The eukaryotic algae of Chlorophyta, Streptomyces, Rhodophyta and Palaeophyta as used herein are preferably organisms belonging to the following groups: Chlorophyta (green algae),

[0429] Preferably, it is Ulvophyceae, Chlorophyceae, Trebouxiophyceae, Chlorodendrophyceae, Pyramimonadales, Mamiellophyceae, Pycnococcaceae, Nephroselmidaceae, Prasinococcales or Palmophyllales, more preferably Ulvophyceae, Chlorophyceae or Trebouxiophyceae;

[0430] Chain plants (charophyta, green algae),

[0431] Preferred are Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Charophyceae, Zygnematophyceae, and Coleochaetephyceae, more preferably Klebsormidiophyceae or Chlorokybophyceae;

[0432] Phaeophyceae (brown algae)

[0433] Paleophyceae (Paleophyceae)

[0434] Rhodophyta (red algae), preferably Cyanidiophyceae, Rhodellophyceae, Compsopogonophyceae, Bangiophyceae, Florideophyceae, Porphyridiophyceae, Stylonematophyceae, more preferably Florideophyceae.

[0435] Glaucophyta, preferably Cyanophoraceae (Cyanophorales), Gloeochaetaceae (Gloeochaetales), Glauccystidaceae (Glaucocystales).

[0436] As used herein, eukaryotic algae are organisms that may belong to the following groups:

[0437] Euglenozoa (Euglenophyceae),

[0438] Stramenopiles

[0439] Dinoflagellata,

[0440] Haptophyta (Haptophytina, Prymnesiophyta, Haptophyceae, Prymnesiophyceae),

[0441] Cryptophyta (Cryptophyceae, Cryptomonada),

[0442] Chlorarachniophyceae.

[0443] As used herein, eukaryotic algae are organisms preferably belonging to the following groups: Chlorophyta, Catenella (Charophyta), Pleurophyta, Rhodophyta.

[0444] The eukaryotic algae used herein preferably belong to the following group: Ulvae, Chlorophyceae, Symphycophyceae, Chlorophyceae, Pyramiales, Dinoflagellates, Pycnococcaceae, Nephrolepsis, Cyanophyceae, Palmophyllales, Mesophyceae, Chlorokybophyceae, Cremorales, Charophyceae, Diplophyceae, Chaetophyceae, Phaeophyceae, Paleophyceae (Paleophyceae), Rhodophyceae, Haematococcus, Flexicoccus, Rhodophyceae, Euryophyceae, Porphyrococcales, Stenophyceae,

[0445] Cyanobacteriaceae (Cyanobacteriales), Glaucophyceae (Glaucophyceales), Glaucophyceae (Glaucophyceales), more preferably Ulvae, Chlorophyceae, Coccolophyceae, Cremophyceales, Diplophyceae, Phaeophyceae, and Paleophyceae (Phylum Paleophyceae).

[0446] The eukaryotic algae used herein preferably belong to the following groups: Ulvae, Chlorophyceae, Symglobaceae, Cremophyceales, Diplophyceae, Phaeophyceae, Palaeophyceae (Phylum), Euryophyceae.

[0447] In certain embodiments, the algae may be selected from the group consisting of the following algae species:

[0448]

[0449]

[0450]

[0451]

[0452] In a particular embodiment, EVs are preferably derived from Chlorophyta, preferably Chlorellaceae, more preferably Chlorella and / or Chlorella-like and / or Spirulina, highly preferably derived from

[0453] Chlorella Sorokinensis, Chlorella kessleri, Chlorella vulgaris, Chlorella protothecoides, Chlamydomonas reinhardtii, Homi spores, Haematococcus lacustris, Zhou's flat algae

[0454] In a particular embodiment, the EVs are preferably derived from catenella plants, preferably Dinocampus indigosporum, Klebsiella pneumoniae, Spirogyra.

[0455] In a particular embodiment, the EVs are preferably derived from the phylum Paleophyta, preferably from Boehmeria and Ascophyllum.

[0456] In a particular embodiment, the EVs are preferably derived from Rhodophyta, preferably from Rhodophyta palmatum.

[0457] In another embodiment, the EVs are derived from a species belonging to the phylum Cyanobacteria, preferably to the subclass Oscillatoria, more preferably to the family Microcoelenteriae, more preferably to Arthrospira, highly preferably to Spirulina platensis (Arthrospira platensis).

[0458] In fact, we tested the following: Microcystis aeruginosa, Nostoc lindneri, Spirulina platensis (Arthrospira platensis)

[0459] Spirulina maxima (Arthrospira maxima), Annelidium africanum, Synechococcus sp., Aphanizomenon flos-aquae

[0460] In a specific embodiment, EVs are derived from Chlorophyta, Rhodophyta, Paleophyta and Streptophytes, preferably derived from Chlorella sorokinensis, Pseudochlorella kessleri, Chlorella vulgaris, Chlorella pyrenoidosa (Chlorella pyrenoidosa), Chlamydomonas reinhardtii, Homiella verrucosa, Haematococcus lacustris, Platymonas zhouei, Indigosporum, Klebsiella, Spirogyra, Boweishi Algae, Ascophyllum, and Drosophila palmatum.

[0461] In a particular embodiment, the EVs are from the phylum Cyanobacteria, preferably from Microcystis aeruginosa, Nostoc lindneri, Spirulina platensis (Arthrospira platensis), Spirulina maxima (Arthrospira maxima), Annelidae, Synechococcus, Aphanizomenon flos-aquae.

[0462] In certain embodiments, the algae from which the EVs are isolated belong to any of the taxonomic groups listed in the "Algae Table" below.

[0463] The results are described herein, for example, in Tables 1-1 to 1-4 and 2-1 to 2-4.

[0464] In a particularly preferred embodiment, the algae species of the invention belongs to:

[0465] Chlorophyta (phylum), core green algae (clade), preferably Trebouxiophyceae (class), preferably Chlorella (order), preferably Chlorellaceae (family), preferably Chlorella clade, preferably Chlorella genus -

[0466] The Cyanobacteria / Melainabacteria group, preferably Cyanobacteria (phylum), preferably Oscillatoria (subclass), preferably Oscillatorias (order), preferably Microcoelenteriae (family), preferably Arthrospira (genus).

[0467] As described herein, the inventors examined MACC360 in a very broad range of assays.

[0468] After understanding its several beneficial effects on disease conditions, several algae from different species have also been studied to exemplify these effects.

[0469] Specifically, the following algae strains were tested: MACC-215 (Chlamydomonas reinhardtii), MACC-1 (Chlorella vulgaris), MACC-3 (Chlorella pyrenoidosa), MACC-1023 (Homias verrucosus), MACC-21 (Dinocampus indigosporum), MACC-1042 (Klebsiella), MACC-918 (Spigorodactyla), MACC-1022 (Bowei's algae), MACC-888 (Microcystis aeruginosa), MACC-612 (Nostoc lindl.), MACC-908 (Spirulina platensis), MACC-909 (Spirulina maxima), MACC-90 (Haematococcus lagenbergii), MACC-42 (Annella africana), MACC-115 (Synechococcus), Ascophyllum nodosum, Drosophila palmatum, Platymonas zhouii, and Aphanizomenon aquatica.

[0470] Algae separation

[0471] According to the present invention, algal EVs can be isolated in a variety of ways.

[0472] For example, algae can be separated by

[0473] - Culture supernatant of algal cells, by isolating EVs from the culture supernatant,

[0474] - dried algal cells, by rehydrating said algal cells with a culture medium from which the cells and cell debris are removed, in particular pelleted, and EVs are separated from the supernatant culture medium,

[0475] - Suspended algal cells, in particular cultured algal cells or dried and rehydrated algal cells, in particular rehydrated pelleted algal cells, are preferably applied by mechanical or physical effect.

[0476] In particular embodiments, the algae can be separated from an algal cell suspension or from dried and subsequently rehydrated algal cells by disruption of the algal cells, in particular by mechanical disruption or ultrasound.

[0477] In embodiments, the method comprises pelleting cells and cell fragments and isolating EVs from the pellets. In one method, cells and cell fragments are pelleted from a culture or dried algae after rehydration, and the pellets are mechanically processed (e.g., extruded) to produce artificial EVs.

[0478] The present invention preferably relates to a method for preparing algal EVs or a composition comprising said EVs, in particular for use in any of the indications defined herein,

[0479] Specifically, after obtaining EVs

[0480] - granulation of algae cells,

[0481] - Filter the pellet supernatant, preferably

[0482] - Filter out cells

[0483] - Preparation of EV fractions by filtration, wherein biomolecules are allowed to transfer into the filtrate,

[0484] -Subject the EV fraction to SEC.

[0485] Cell proliferation and migration in various disorders

[0486] Cell proliferation and migration play a very important role in the disease conditions contemplated in the present invention and indicated herein, which are, in accordance with the literature in this field, referred to as proliferation / migration disorders.

[0487] Proliferation and migration work by increasing the number of cells in a part of the body.

[0488] To see an example of a proliferation / migration disorder, fibroproliferative disorders include scarring of tissues in response to chronic damage to mammalian tissues and organs. In addition, inflammation typically occurs in tissue damage. Tissue damage increases the synthesis of inflammatory mediators, activates tissue fibroblasts, increases their division and migration activity, and the production of extracellular matrix (ECM) components.

[0489] In terms of their molecular markers and / or functions, fibroblasts form a rather heterogeneous cell population and may be derived from specific cells, such as mesenchymal cells and mesenchymal stromal cells (MSCs), endothelial cells, epithelial cells, adipocytes, bone marrow cells and mesoderm-derived pericytes (Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 2009 Jun; 119(6): 1429-37. doi: 10.1172 / JCI36183. Epub 2009 Jun 1. PMID: 19487819; PMCID: PMC2689132; Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer. 2016 Aug 23;16(9):582-98.doi:10.1038 / nrc.2016.73.PMID:27550820; LeBleu VS, Kalluri R. Apeek into cancer-associated fibroblasts: origins, functions and translational impact. Dis Model Mech. 2018Apr 19;11(4):dmm029447.doi:10.1242 / dmm.029447.PMID:29686035;PMCID:PMC5963854.2018;11:dmm029447;Lynch MD,Watt FM.Fibroblast heterogeneity:implications for humandisease.J Clin Invest.2018Jan 2; 128(1): 26-35. doi: 10.1172 / JCI93555. Epub 2018 Jan 2. PMID: 29293096; PMCID: PMC5749540). There are many hypotheses about the origin of fibroblasts. The common assumption is that fibroblasts in different tissues are formed by differentiation and dedifferentiation of cells originating from the mesoderm.

[0490] Fibroblasts are characterized by their strong proliferation, migration and increased production of ECM during activation, which provides a special example of the interrelationship of these three processes. In addition, fibrosis and chronic inflammation are closely related. Cytokines, growth factors, etc. produced during the inflammatory response activate fibroblasts, that is, increase their proliferation, migration and production of ECM.

[0491] Different PDGF isoforms, including PDGF-BB, as well as other factors, such as TGFβ or EGF, play important roles in the activation of fibroblasts, such as their proliferation, migration and ECM production.

[0492] These growth factors and fibroblasts also play a role in the occurrence and migration of different tumors. The pathophysiological role of tumor-associated fibroblasts (CAF) in heterogeneous tumor microenvironments has attracted more and more attention. CAF plays a key role in tumor progression and response to chemotherapy. Some cytokines and chemokines are involved in the activation of CAF, and some of them form a feedback loop between cancer cells and CAF. In addition, the physical force between tumor cells and CAF promotes the synergistic invasion or co-migration of two types of cells (Go J.Yoshida Regulation of heterogeneous cancer-associated fibroblasts: the molecular pathology of activated signalling pathways, Experimental & Clinical Cancer Research volume 39, Article number: 112 (2020)).

[0493] In our study, proliferation, migration, and ECM production of different fibroblasts, including MRC5 and primary fibroblasts derived from human skin, colon, or peritoneum, were induced in vitro by peritoneal dialysis (PD) effluent (PDE). In addition, proliferation, migration, or ECM production of skin, colon, and peritoneal primary fibroblasts were induced by PDGF-B, EGF, and TGF-β, respectively.

[0494] During PD, the amount of glucose, AGE, inflammatory factors, and profibrotic growth factors in the peritoneal cavity increases (all of which can induce peritoneal thickening), which is characterized by increased fibroblast activation and excessive ECM deposition. Therefore, the PDE released from the peritoneal cavity at the end of dialysis contains a large amount of these proinflammatory and fibrotic factors. In fact, the inventors have shown that the peritoneal dialysis effluent used contains CTGF (connective tissue growth factor), PDGF-B and TGF-β, which are all involved in the initiation of fibrosis (Lipson, KE, Wong, C., Teng, Y. et al., CTGF is a central mediator of tissue remodelling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenesis Tissue Repair 5, S24 (2012); Ying HZ, Chen Q, Zhang WY et al., PDGF signalling pathway in hepatic fibrosis pathogenesis and therapeutics (Review). Mol Med Rep. 2017; 16 (6): 7879-7889. doi: 10.3892 / mmr.2017.7641; Gallini R, Lindblom P, Bondjers C, Betsholtz C, Andrae J. PDGF-A and PDGF-B induces cardiac fibrosis in transgenic mice.Exp CellRes.2016Dec 10;349(2):282-290; Piotr Czochra, Borut Klopcic, Erik Meyer, JohannesHerkel, Jose Francisco Garcia-Lazaro, Florian Thieringer, Peter Schirmacher, Stefan Biesterfeld, Peter R.Galle, Ansgar W.Lohse, Stephan Kanzler, Liverfibrosis induced by hepatic overexpression of PDGF-B in transgenic mice, Journal of Hepatology, Volume 45, Issue 3, 2006, Pages 419-428; Meng, Xm., Nikolic-Paterson, D. & Lan, H. TGF-β: the master regulator of fibrosis. Nat Rev Nephrol 12,325–338(2016). Biernacka A, Dobaczewski M, Frangogiannis NG. TGF-β signaling in fibrosis. Growth Factors. 2011; 29(5): 196-202. doi: 10.3109 / 08977194.2011.595714; Dirk Pohlers, Julia Brenmoehl, Ivonne. Cornelia K.Müller,Carola Leipner,Stefan Schultze-Mosgau,Andreas Stallmach,Raimund W.Kinne,Gunter Wolf,TGF-βandfibrosis in different organs—molecular pathway imprints,Biochimica etBiophysica Acta(BBA)-Molecular Basis of Disease,Volume 1792,Issue 8,2009,Pages 746-756).

[0495] Therefore, the inventors used PDE (see below) to simulate the complex molecular environment of fibrotic tissue / organ in vitro (Yu, F., Chen, J., Wang, X. et al., Establishment of a novel mouse peritonealdialysis-associated peritoneal injury model. Clin Exp Nephrol (2022). PMID: 35353282). This in vitro model (more complex fluid) provides an environment very close to the in vivo environment.

[0496] Effects of EVs from different algae sources on proliferation, cytotoxicity, collagen production and migration of primary human colon fibroblasts (phCFB / A), primary human skin fibroblasts (phSFB / A), human hepatocellular carcinoma cell lines (HEPG2) and human colon cancer cell lines (Caco-2) induced by peritoneal dialysis effluent (PDE) of patient A. The results are shown in Tables 1-1 to 1-5 and Tables 2-1 to 2-5. All cell types were treated with PDE and 3×10^8 EVs from different algae sources for 24 hours. PBS and peritoneal dialysis fluid (PDF) were used as vehicle controls. Cell proliferation was determined by MTT assay, cytotoxicity was determined by LDH assay, collagen deposition was determined by Sirius Red assay, and cell migration was determined by TAS assay. Significance analysis was performed using unpaired t test, Mann-Whitney test or two-way ANOVA. p<0.05↑ means that the proliferation, cytotoxicity, collagen production or migration of the EV, PDE or PDE+EV treated groups are enhanced compared with the respective appropriate control groups (control group or PDE treated groups, respectively); p<0.05↓ means that the proliferation, cytotoxicity, collagen production or migration of the EV, PDE or PDE+EV treated groups are decreased compared with the respective appropriate control groups (control group or PDE treated groups, respectively); p=NS means that there is no significant difference in proliferation, cytotoxicity, collagen production or migration between the EV, PDE or PDE+EV treated groups and the respective appropriate control groups (control group or PDE treated groups, respectively).

[0497] Brief information of the algae strains examined is provided in the table of algae:

[0498] Algae Table

[0499]

[0500] Tables 1-1 to 1-4 and Tables 2-1 to 2-4 provide the results for the algae strains examined herein:

[0501] Table 1-1

[0502]

[0503]

[0504] Table 1-2

[0505]

[0506]

[0507] Table 1-3

[0508]

[0509]

[0510] Table 1-4

[0511]

[0512]

[0513] cancer

[0514] Table 2-1

[0515]

[0516] Table 2-2

[0517]

[0518] Table 2-3

[0519]

[0520]

[0521] Table 2-4

[0522]

[0523] The mechanisms of fibrosis are identical or very similar in virtually any tissue or organ. Thus, taken together, experiments with multiple types of fibroblasts treated with profibrotic growth factors or PDEs and in vivo models of peritoneal fibrosis support the potential of algal EVs to treat fibrotic or fibroproliferative diseases in virtually any tissue or organ in a subject.

[0524] Below, supporting experiments and specific embodiments of the present invention are discussed.

[0525] The ability of EVs to penetrate (mammalian) cells

[0526] Primary cells were characterized by immunofluorescence staining ( Figure 1-6 ). The fibroblast cytoplasm stained positively for the specific fibroblast marker α-SMA and was negative for the mesothelial marker CK18, while the mesothelial cells showed CK18 immunopositivity.

[0527] EVs were found to penetrate into different cells ( Figure 9-13 ), making it possible for it to act in and / or on cells.

[0528] Furthermore, EVs administered into the tail vein of (C57 / BL6J) mice were targeted and internalized into the liver ( Fig.90). This result suggests that EVs could be particularly useful in treating diseases such as fibroproliferative diseases or liver cancer.

[0529] EVs have been administered intraperitoneally (Figures 91-92) or intradermally ( Fig.93 ) to the skin.

[0530] Effects of EVs on fibroblast migration

[0531] EVs were found to inhibit the migration of primary colon and skin fibroblasts. In the TAS migration assay, the effect of EVs on cell migration was shown on different cell types. The migration of various primary fibroblasts was induced by EGF ( Fig.59 and Fig.46 ). These experiments showed that the addition of EVs could inhibit cell migration, especially EGF-induced cell migration ( Fig.59 , Fig.60 ).

[0532] In a further set of experiments, the migration of primary colon and skin fibroblasts was initiated with PDE and measured by the TAS assay. These experiments are shown in Tables 1-1 to 1-4. As can be seen, there was a significant reduction in the number of EVs migrated from several algae, and no increase in EVs. Effect of EVs on fibroblast proliferation

[0533] EVs were found to inhibit cell proliferation. Fig.14 , Fig.16 , Fig.18 , Fig. 20 , Fig. 22 , Fig.24 ) or PDGF-BB( Fig.15 , Fig.17 , Fig.19 , Fig.21 , Fig.23 , Fig.25 ) induced the proliferation of various primary fibroblasts, and the Chlorella MACC-360 Chlorella (Chlorella vulgaris) ( Figures 88-89 )、Spirulina (Spirulina platensis) Figures 86-87 ) and Chlorella (Pseudochlorella kessleri) ( Figures 58-60 ) EVs were able to inhibit this effect, as measured by MTT assay.

[0534] In a further set of experiments, PDE was used to initiate the proliferation of fibroblasts on primary colon and skin fibroblasts, and the proliferation was measured by MTT assay. These experiments are shown in Tables 1-1 to 1-5. It can be seen that EVs from each algae significantly reduced the proliferation of fibroblasts and thus provided an anti-fibrotic effect.

[0535] Effects of EVs on ECM production in fibroblasts

[0536] We found that EVs from different sources have a strong effect on ECM production in various primary fibroblasts. Fig.26 , Fig.28 , Fig.30 , Fig.32 and Fig.34 ) or TGF-β( Fig. 27 , Fig.29 , Fig.31 , Fig.33 , Fig.35 , Fig.36 , Fig.63 , Fig.64 ) induced collagen production. Different concentrations of EVs inhibited the increase in collagen production induced by PDE or TGF-β.

[0537] In a further set of experiments, the accumulation of collagen on primary colonic and skin fibroblasts was stimulated with PDE. These experiments are shown in Tables 1-1 to 1-5. As can be seen, EVs from each algae significantly reduced collagen production, providing a clear and general anti-fibrotic effect.

[0538] Effects of EVs on the production of profibrotic factors by immune cells.

[0539] EVs inhibited PDGF-B gene expression in peripheral blood mononuclear cells (PBMCs) Fig.39 ), which suggests their antifibrotic potential.

[0540] In addition, the anti-fibrotic effect of EVs in vivo was also studied in a chlorhexidine gluconate (CG)-induced peritoneal fibrosis model in C57Bl / 6 mice. MACC-360-derived EVs significantly inhibited the increase in submesothelial thickness induced by CG. Submesothelial thickness was positively correlated with the severity of peritoneal fibrosis ( Fig.71 )

[0541] In vivo effects of EVs in the DSS-induced colitis model.

[0542] The anti-inflammatory effect of EVs in vivo was also tested in a DSS-induced colitis model in C57Bl / 6 mice. Changes in the disease activity index (DAI) were monitored throughout the experiment. Mice were killed after the end of treatment (7 days), and the disease activity index was measured and recorded. EVs-treated mice showed significantly improved DAI ( Figure X 95)

[0543] Effects of EVs on cancer cell proliferation.

[0544] It was found that EVs can inhibit the proliferation of cancer cells. PDGF-BB induced the proliferation of various cancer cells (A549, HT29, CACO-2, Hs578-T, LCLC-103H, HepG2) ( Fig.47 , Fig.48 , Fig.49 ), and EVs from Chlorella MACC-360 were able to inhibit this effect, as measured in the MTT assay ( Figure 61-63 , Fig.65 , Fig.67 , Fig.69 ) and did not induce cell death ( Fig.66 , Fig.68 , Fig.70 ).

[0545] In a further set of experiments, the proliferation of HepG2 and Caco-2 cells was initiated with PDE. These experiments are shown in Tables 1-1 to 1-4. As can be seen, EVs from each algae significantly reduced the proliferation of these cells, providing a clear and general anti-cancer effect. Effects of EVs on cancer cell migration.

[0546] In the TAS migration assay, it was found that EVs derived from Chlorella MACC-360 inhibited the migration of Caco-2 colon cancer cells ( Fig.50 ). EVs inhibited PDF and PDE induced the migration of Caco-2 cells.

[0547] Anti-inflammatory effects of EVs

[0548] The anti-inflammatory effect of MACC-360-derived EVs was manifested by the large induction of IL-6 ( Fig.41 )、IL-10( Fig.37 ) gene expression. In addition, the IL-10 and TGFB gene expressions of PBMCs were evaluated after co-treatment with lectins and EVs ( Figure 44-45 ). In addition, in primary skin fibroblasts co-treated with LPS, PHA, poly(I:C), and EVs ( Fig.48 ) and adenocarcinoma human alveolar basal epithelial cells co-treated with PHA, poly(I:C) and EVs ( Fig.47 ), the IL-6 protein level was even more prominent.

[0549] In vivo anti-inflammatory effects of EVs in a DSS-induced colitis model.

[0550] The in vivo anti-inflammatory effects of MACC-360-derived EVs were also tested in a DSS-induced colitis model in C57Bl / 6 mice. Changes in the disease activity index (DAI) were monitored throughout the experiment. At the end of treatment (after 7 days), the mice were killed and the disease activity index was measured and recorded. EVs-treated mice showed significantly improved DAI ( Fig.95 )

[0551] Cytoprotective effects of EVs

[0552] The cytoprotective effect of EVs was confirmed in methylglyoxal (MGO)-induced cytotoxicity experiments in primary mesothelial cells, primary colonic and skin fibroblasts, and human umbilical vein endothelial cell lines, as well as in primary colonic and skin fibroblasts induced by 3,4-dideoxyglucose ketone-3-ene (DGE) and 5-hydroxymethyl-2-furfural (HMF). Fig.49 , Fig.54 , Fig.56 , Fig.57 ) and LDH release (LDH assay, Figure 50-53 , Fig.55 , Fig.58 ) showed that EVs have cytoprotective effects.

[0553] Combinatorial use of algal EVs

[0554] Furthermore, EVs may be used in combination, ie algal EVs may be preferably isolated from various algal sources and used together, for example in the form of a mixture or composition.

[0555] For example, in the MTT assay, MACC-360 and other algal EVs in combination inhibited PDE-induced fibroblast proliferation more effectively than single EV types alone (see Fig.102 ).

[0556] In another embodiment, algal EVs are combined with other active agents. Surprisingly, ponatinib and axitinib used together with EVs reduced PDGF-B-induced proliferation of primary human colonic fibroblasts compared to the drugs used alone ( Fig.103 and 104 ). The effects of EVs remain unchanged after harsh environmental impacts.

[0557] The inventors also found that EVs are surprisingly resistant to a variety of environmental influences and preparation methods.

[0558] For example, samples treated with denaturation, hydrochloric acid (HCl), denaturation and HCl, and proteinase K still had significant effects on platelet-derived growth factor B (PDGF-B)-induced proliferation of primary human colonic fibroblasts, as shown by MTT assay ( Fig.101 )

[0559] Similarly, HCl-treated and trypsin-EDTA-treated samples still had a significant effect on platelet-derived growth factor B (PDGF-B)-induced proliferation of primary human colonic fibroblasts, as shown by MTT assay ( Fig.100 ).

[0560] The culture method (autotrophic vs heterotrophic) can affect the efficacy of algal EVs, however, het culture ( Fig.97 ). Cytotoxicity measurements did not refute this finding.

[0561] Isolation method has no effect on the biological effects of EVs

[0562] EVs were isolated from the supernatant of algal cultures and rehydrated algae, and also from the algae themselves. Figures 73-80 It was shown that the isolation method had no effect on the biological effects of EVs.

[0563] Furthermore, the antiproliferative effects of EVs remained largely unchanged after freeze-drying, as evidenced by the cytotoxicity of peritoneal dialysis effluent (PDE)-induced primary human colonic fibroblasts ( Fig.99 ) as shown.

[0564] Effects of EVs on the kinase panel

[0565] The effects of Chlorella MACC-360 derived EVs on recombinant protein mixture (recMix) and peritoneal dialysis slipout (PDE) induced primary human colonic fibroblast protein kinase phosphorylation were also investigated. Representative images of array membranes are shown in Figure 105. Although different kinase groups have been affected, as discussed above, it is clearly shown that EVs are effective on various kinase pathways activated by growth factors, as demonstrated in other experiments herein.

[0566] Therefore, in the present invention, EVs can affect

[0567] - PDGF receptor α and / or PDGF receptor β signaling pathway, preferably PDGF-induced signaling, preferably PDGF-BB-induced signaling pathway,

[0568] - signaling pathways induced by TGFβ, preferably TGFβ-1,

[0569] -EGF-induced signaling pathway.

[0570] PDEs include multiple profibrotic growth factors.

[0571]

[0572]

[0573] EVs are suitable for preventing or treating fibrosis

[0574] Our results directly indicate that EVs are suitable for preventing or treating conditions, disorders or diseases associated with or characterized by fibrosis, such as fibroproliferative conditions, disorders or diseases or proliferative / migratory conditions, disorders or diseases, including the production / deposition of non-physiological ECM, for example for preventing or treating the following conditions, disorders or diseases.

[0575] Examples of renal diseases associated with or characterized by fibrosis: diabetic nephropathy, hypertensive nephropathy, glomerular diseases including proliferative glomerulonephritis (mesangial proliferative, membranous proliferative, focal proliferative, diffuse proliferative, crescentic), lupus nephritis with glomerulonephritis, bacterial endocarditis, vasculitis, chronic hepatitis, infections (e.g., hantavirus), non-inflammatory glomerular diseases (minimal change nephritis, focal glomerulosclerosis, membranous nephropathy, fibrotic glomerular disease), glomerular diseases associated with Hodgkin's disease, antibiotics, drugs (aspirin, ibuprofen, acetaminophen, tacrolimus, cyclosporine, contrast media, chemotherapy or heroin toxicity), HIV infection. Hereditary nephritis (Alport syndrome), vascular diseases including renal artery stenosis, sickle cell disease, hemolytic uremic syndrome, atypical hemolytic uremic syndrome. Tubulointerstitial diseases including pyelonephritis, analgesic nephritis, allergic interstitial nephritis, granulomatous interstitial nephritis, autoimmune interstitial nephritis, noninflammatory diseases (eg, reflux nephropathy), obstructive uropathy (anatomical abnormalities such as posterior urethral valves, or calculi, or malignancy or prostatic hyperplasia), myeloma kidney, and transplant disease (eg, chronic rejection, drug toxicity, recurrent disease, allograft glomerulopathy).

[0576] Examples of lung diseases associated with or characterized by fibrosis: bronchitis, asthma, idiopathic pulmonary fibrosis, usual interstitial pneumonia, pulmonary fibrosis induced by gas or ionizing radiation, nitrofurantoin, tobacco smoke-induced pulmonary fibrosis, emphysema, chronic obstructive pulmonary disease, tuberculosis, pulmonary fibrosis induced by rheumatoid arthritis, pulmonary fibrosis induced by systemic lupus erythematosus, sarcoidosis, Wegener's granulomatosis, nonspecific interstitial pneumonia, Hamman-Rich syndrome Syndrome), diffuse fibrosing alveolitis, inhalation of environmental and occupational pollutants (fumed silica, asbestos, nitrogen and sulfur gases, smoke, detergent vapors, cleaning agents, hydrochloric acid, herbicides, hair sprays), drug-induced pulmonary fibrosis (bleomycin, amiodarone, busulfan, methotrexate, apotropine, nitrofurantoin, phenytoin) and radiation therapy, Torquetenovirus, pneumoconiosis, etc.

[0577] Examples of pancreatic diseases associated with or characterized by fibrosis: alcoholic chronic pancreatitis, hereditary pancreatitis, autoimmune pancreatitis, obstructive chronic pancreatitis, tropical calcific pancreatitis, fibrolithic pancreatic diabetes, chronic nonalcoholic pancreatitis, chronic atrophic pancreatitis, groove pancreatitis,

[0578] Examples of intestinal diseases associated with or characterized by fibrosis: ulcerative colitis, Crohn's disease, collagen colitis, microscopic colitis, diversion colitis, necrotizing enterocolitis, chemical colitis, ischemic enteritis, Helicobacter pylori-induced gastritis, chronic gastritis, subepithelial esophageal fibrosis, Barrett's esophagus, gastroesophageal reflux disease, oral submucous fibrosis, esophageal atresia,

[0579] Examples of liver diseases associated with or characterized by fibrosis: nonalcoholic steatohepatitis, autoimmune hepatitis, viral hepatitis (hepatitis A, B, C, D), alcoholic hepatitis, toxic and drug-induced hepatitis, nonalcoholic fatty liver disease, cirrhosis, hepatic fascioliasis, schistosomiasis, liver fluke-induced fibrosis, primary sclerosing cholangitis, Budd-Chiari syndrome, biliary atresia, Alagille syndrome, progressive familial intrahepatic cholestasis, serotonergic agonist drugs: diet pills (fenfluramine, chlorphentermine, aminorex), anti-migraine drugs (ergotamine, dimethylergonovine), anti-Parkinson's disease drugs (pergolide, cabergoline), recreational drugs (MDA, MDMA, DOI, mCPP),

[0580] Examples of eye diseases associated with or characterized by fibrosis: diabetic retinopathy, corneal fibrosis, neovascular glaucoma, retinopathy of prematurity, age-related macular degeneration, premyeloid fibrosis, herpetic keratitis, palpebral fissures, capsular fibrosis, posterior capsule fibrosis, retinal fibrovascular scarring, retinal gliosis, complications of retinal detachment surgery, viral infection of the cornea, retinal damage caused by hypoxia or inflammation, trachoma, congenital fibrosing syndrome, levator fibrosis, congenital ocular muscle fibrosis, congenital extraocular muscle fibrosis, proliferative retinopathy, macular fibrosis, talc retinopathy, subretinal fibrosis, syndrome, subconjunctival fibrosis,

[0581] Examples of metabolic diseases associated with or characterized by fibrosis: atherosclerosis, complications of type 2 diabetes, arteriosclerosis, diabetic foot, metabolic syndrome, hyperlipidemia, hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, galactosemia, glycogen storage diseases I-IV, VI, IX, XI, urate nephropathy, hyperlipoproteinemia IV, common hypercholesterolemia, mucopolysaccharidosis types I-VII, mucolipidosis III-IV, Fabry disease (diffuse angiokeratoma corporis), pseudoxanthoma elasticum,

[0582] Examples of autoimmune diseases associated with or characterized by fibrosis: Complications of type 1 diabetes, rheumatoid arthritis, ankylosing spondylitis (Bechterew's disease), systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome ( syndrome), CREST syndrome, polymyositis, dermatomyositis, primary biliary cirrhosis, primary sclerosing cholangitis, vasculitis: giant cell arteritis, Takayasu arteritis, polyarteritis nodosa, granulomatous vasculitis, thromboangiitis obliterans, sarcoidosis, Goodpasture's syndrome, mixed connective tissue disease, hypersensitivity vasculitis-pneumonia syndrome,

[0583] Skin diseases associated with or characterized by fibrosis: related to trauma, surgery, piercing, acne, chickenpox, infection, incision, hematoma, spontaneous (spontaneusly), granuloma, tick granuloma, solaris atrophia, burns, pseudocyst (pseudocatrix stellat), ulcers associated with anthrax, gonorrhea, ulcers and anthrax, tularemia, bedsores, diabetic foot syndrome, diabetic skin, diabetic lipid necrosis, varicose veins, thrombophlebitis, infections: fasciitis necrosis, ecthyma simplex, ecthyma gangrene, pheromone abscesses, furuncle, carbuncle, anthrax, granuloma, lymphogranuloma, tularemia, tbc (lupus vulgaris, scrofulous tuberculosis of the skin), leprosy, Lyme borreliosis, lymphadenopathy (tick-bone lymph nodes), syphilis, actinomycosis, fungal infections, secondary infection of scar tissue, herpes simplex virus (HSV), varicella zoster virus (varicella zoster virus) virus, VZV), erythema multiforme, dermatitis herpetiformis; scars associated with prurigo (infection, allergy, irritation, paraneoplastic, eclampsia, diabetes), acne: ecthyma simplex, acne inverse, comedones, rosacea, rhinoplasty, dermatitis, seborrhea, Cushing syndrome), surgery, postoperative side effects (e.g., infection, sponging, splintering),

[0584] Examples of urogenital diseases associated with or characterized by fibrosis: Menstrual disorders: endometriosis, polycystic ovary syndrome (PCOS), adrenal diseases (congenital adrenal hyperplasia (CAH), Cushing's disease, virilization, acne, seborrheic dermatitis), Asherman's syndrome (-iatrogen), endometritis, intrauterine contraceptive devices (IUDs), device, IUD)), infections: perinephritis, perinephritis, pyelonephritis, pyelitis and chronic pyelonephritis, pyelonephritis, chronic urethritis (gonorrhea, Escherichia coli, Proteus, HSV), retroperitoneal fibroma, chronic cystitis, post-radiation cystitis, simple ulcer (Hunner), trichomoniasis, tuberculosis (kidney, bladder, epididymitis, prostate), actinomycosis, pelvic peritonitis, vulvovaginitis, genital herpes, genital human papillomavirus (human papillomavirus, HPV), chronic cervicitis, endometritis, salpingitis, abscess, tubo-ovarian abscess, syphilis, gonorrhea, chlamydia, trichomoniasis, HPV, chancroid, human immunodeficiency virus (Human Immunodeficiency Virus, HIV), tuberculosis,

[0585] Examples of pathological pregnancy-related fibroproliferative diseases associated with or characterized by fibrosis: pruritus and hyperemesis, bullous pemphigoid, impetigo herpetiformis, cesarean section (or other procedures), uterine rupture, postpartum ulcer, endometritis, postpartum myometritis, postpartum adnexitis, postpartum pelvic inflammatory disease, postpartum parametritis, thrombophlebitis, postpartum mastitis. Male: penis, prostate, testicles: cavernitis, corpus cavernosum induration, prostatitis, abscess, chronic orchitis, epididymitis. Obstructive uropathy with anatomical abnormalities of posterior urethral valves, subvesical obstruction, vesicoureteral reflux urolithiasis, inflammation, gouty arthritis, hyperthyroidism, hypercalcemia, hyperoxalate, cystic aciduria, aceketonuria.

[0586] Examples of cardiovascular diseases associated with or characterized by fibrosis:

[0587] Dilated and hypertrophic cardiomyopathy, myocardial infarction, valvular disease, arrhythmias, cardiac hypertrophy, hypertension-induced cardiac fibrosis, Marfan syndrome, left ventricular fibrosis, myocardial necrosis and apoptosis-induced cardiac fibrosis, vascular fibrosis, arteriosclerosis, atherosclerosis, venous sclerosis

[0588] Examples of musculoskeletal system disorders associated with or characterized by fibrosis: myelofibrosis, muscular fibrosis.

[0589] Examples of CNS diseases associated with or characterized by fibrosis: stroke and tissue injury-induced glial fibrosis, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease.

[0590] The role of PDGFR inhibitors in the treatment of fibrotic diseases (e.g., pulmonary fibrosis, myelofibrosis, systemic sclerosis, nephrogenic systemic fibrosis) has been clinically demonstrated. Our results suggest that EVs can be used similarly to PDGFR inhibitors to treat fibrotic diseases. For example, pegpleranib (an anti-PDGF aptamer) is involved in the treatment of subretinal fibrosis and subfoveal fibrosis, and E10030 (another anti-PDGF aptamer) is used to treat macular degeneration. Our results suggest that EVs can be used similarly to anti-PDGF aptamer drugs in the treatment of fibrotic diseases.

[0591] TGF-β neutralizing antibodies have been tested in clinical trials for the treatment of fibrotic diseases (e.g., fresolimumab in systemic sclerosis). Our results suggest that EVs could be used similarly to TGF-β neutralizing antibodies in the treatment of fibrotic diseases.

[0592] EGFR neutralizing antibodies are clinically tested for the treatment of fibrotic diseases, for example, nimotuzumab for the treatment of COVID-19-induced pulmonary fibrosis. Our results suggest that EVs could be used similarly to EGFR neutralizing antibodies in the treatment of fibrotic diseases.

[0593] PDGF neutralization? .....

[0594] Methylglyoxal (MGO), a potent reactive carbonyl species, is a precursor to the formation of advanced glycation end products (AGEs) by reacting with free amino groups of lysine and arginine. Therefore, experiments with MGO provide a model for the effects of AGEs and their possible reversal. [Leone, Alessia et al., The Dual-Role of Methylglyoxal in Tumour Progression–Novel Therapeutic Approaches” Frontiers in Oncology 2021(11) DOI=10.3389 / fonc.2021.645686]. AGEs induce ECM overaccumulation and expression of inflammatory and profibrotic cytokines. In addition, studies on the receptor for advanced glycation end-products (RAGE) have shown that ligand-RAGE interactions activate several intracellular signaling cascades associated with several fibrotic diseases. AGEs induce ECM overdeposition and enhance the expression of profibrotic cytokines, including TGF-β (Kyung SY, Byun KH, Yoon JY, Kim YJ, Lee SP, Park JW, Lee BH, Park JS, Jang AS, Park CS, Jeong SH. Advanced glycation end-products and receptor for advanced glycation end-products expression in patients withidiopathic pulmonary fibrosis and NSIP. Int J Clin Exp Pathol. 2013 Dec 15; 7(1): 221-8. PMID: 24427342; PMCID: PMC3885476.). In our experiments, we found that EVs had a protective effect against MGO-induced cytotoxicity, therefore, EVs are good candidates for the treatment of AGEs-induced / related diseases, including fibrotic diseases.

[0595] Inflammation is an important cause of fibrosis and many other diseases (including infection / inflammatory / autoimmune diseases) (Mack: Inflammation and fibrosis. Matrix Biol. (2018) 68-69, 106-121). The anti-inflammatory effect shown in our experiments also supports that EVs are suitable for preventing or treating fibrosis and inflammation (in vivo DSS model)?

[0596] These experiments show that the effects of AGEs can be reversed by algal EVs, and their use can benefit diseases where AGEs develop via AGE receptors (such as fibrosis, cancer, and inflammation), and can be used for autoimmune diseases, diabetes and atherosclerosis, chronic degenerative diseases (such as Alzheimer's disease, Parkinson's disease, and alcoholic brain damage). [Byunab, Kyunghee et al., Advanced glycation end-products produced systemically and by macrophages: A common contributor to inflammation and degenerative diseases" Pharmacology & Therapeutics Volume 177, September 2017, Pages 44-55].

[0597] Anti-tumor effects of EVs

[0598] We found that EVs inhibit cell proliferation and cell migration, suggesting that EVs can be used for anticancer therapy. EVs can be used to prevent, treat / inhibit tumor growth or metastasis, and by reducing CAF activation and thus lowering intratumoral pressure, they can also facilitate the delivery of anticancer drugs to tumor cells, thereby enhancing the efficacy of anticancer therapy.

[0599] EVs can be used in a method for preventing or treating cancer, preferably, wherein the cancer is selected from: acoustic neuroma, acute lymphoblastic leukemia, acute myeloid leukemia, cancer, melanoma, prostate cancer, anal cancer, anaplastic large cell lymphoma, astructural thyroid cancer, angioimmunoblastoma, T-cell lymphoma, anorectal melanoma, astrocytoma, bile duct cancer (cholangiocarcinoma), bladder cancer, blood cancer, bone cancer, intestinal cancer, brain cancer, breast cancer, male breast cancer, Burkitt's lymphoma, unknown primary cervical cancer, chondrosarcoma, chordoma, chronic lymphocytic leukemia , chronic myeloid leukemia, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, ductal carcinoma in situ, early (localized) prostate cancer, ependymoma, essential thrombocythemia, Ewing's sarcoma, eye cancer (ocular melanoma), fallopian tube cancer, follicular lymphoma, gallbladder cancer, gastrointestinal stromal tumors, germ cell ovarian cancer, glioma, hemangioblastoma, head and neck cancer, Hodgkin lymphoma, inflammatory breast cancer, laryngeal (throat) cancer, leiomyosarcoma, leukemia, liver cancer, localized prostate cancer, lung cancer, lymph node cancer, secondary lymphoblastic lymphoma, lymphoma, MALT lymphoma, mantle cell lymphoma, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, mesothelioma, oral cancer, myelodysplasia, myelofibrosis, myeloma, paranasal sinus cancer, nasopharyngeal cancer, neuroendocrine tumors, intranodal marginal zone B-cell lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, esophageal cancer, oligodendroglioma, oropharyngeal cancer, osteosarcoma, ovarian cancer, Paget's disease of the breast, pancreatic cancer, papillary and follicular thyroid cancer, parathyroid cancer, penile cancer, peripheral T-cell lymphoma, phyllodes tumor, pineal Body tumors, pituitary tumors, polycythemia vera, primary central nervous system lymphoma, primary mediastinal large B-cell lymphoma, primary peritoneal cancer, prostate cancer, pseudomyxoma peritonei, rare cancers, rectal cancer, recurrent melanoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, secondary bone cancer, secondary breast cancer, secondary cancer, secondary liver cancer, secondary lung cancer, skin cancer, small intestine cancer, small cell lung cancer, small lymphocytic lymphoma, soft tissue sarcoma, spinal cord tumor, splenic marginal zone lymphoma, gastric cancer, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, tongue cancer, tracheal cancer. Triple negative breast cancer, ureter and renal pelvis cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia ( macroglobulinaemia), uterine cancer.

[0600] By inhibiting PDGFR activation-induced tumor cell proliferation and migration, EVs can suppress both tumor growth and metastasis formation.

[0601] The role of PDGFR inhibitors in cancer therapy is well documented in the art. Our results suggest that EVs can be used similarly to PDGFR inhibitor anticancer drugs, i.e., in the treatment or prevention of cancer. Examples of cancer types for which PDFR inhibitor drugs have been clinically tested are: glioblastoma multiforme, chordoma, meningioma, dermatofibrosarcoma protuberans, gastrointestinal stromal tumor, soft tissue sarcoma, osteosarcoma, chronic myeloproliferative disorder, hypereosinophilic syndrome, prostate cancer, non-small cell lung cancer, neuroblastoma, liver cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, stromal cell tumor. Known PDGFR targeted (inhibitor) drugs include Nilotinib, Dasatinib, Ponatinib, Sunitinib, Axitinib, Midostaurin, Pazopanib, Regorafenib, Sorafenib, Nintedanib, Lenvatinib, Masitinib, and Crenolanib.

[0602] EVs may also be used to enhance the effects of other anticancer drugs by inhibiting PDGF-induced activation (e.g., proliferation, migration, ECM production, tissue contraction) of fibroblasts present in the tumour stroma (tumour-associated fibroblasts).

[0603] PDGF induces contraction of fibroblasts present in the tumor stroma, leading to shrinkage of tumor tissue in solid tumors. As a result, intratumoral pressure increases, reducing capillary flow and thus limiting the amount of anticancer drugs that reach tumor cells. EVs can be used to treat solid tumors selected from the group consisting of acoustic neuroma, melanoma, prostate cancer, anal cancer, astructural thyroid cancer, anorectal melanoma, astrocytoma, bile duct cancer (cholangiocarcinoma), bladder cancer, bone cancer, intestinal cancer, brain cancer, breast cancer, male breast cancer, solid cancer of unknown primary, cervical cancer, chondrosarcoma, chordoma, colon cancer, craniopharyngioma, ductal carcinoma in situ, early (localized) prostate cancer, ependymoma, Ewing's sarcoma, eye cancer (ocular melanoma), fallopian tube cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell ovarian cancer, glioma, hemangioblastoma, head and neck cancer, inflammatory breast cancer, laryngeal (larynx) cancer, leiomyosarcoma, liver cancer, local Prostate cancer, lung cancer, secondary medullary thyroid cancer, medulloblastoma, meningioma, mesothelioma, oral cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroendocrine tumors, non-small cell lung cancer, esophageal cancer, oligodendroglioma, oropharyngeal cancer, osteosarcoma, ovarian cancer, Page's disease of the breast, pancreatic cancer, papillary and follicular thyroid cancer, parathyroid cancer, penile cancer, phyllodes tumors, pineal region tumors, pituitary tumors, primary peritoneal cancer, prostate cancer, pseudomyxoma peritonei, rectal cancer, recurrent melanoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, secondary bone cancer, secondary breast cancer, secondary solid cancer, skin cancer, small intestine cancer, small cell lung cancer, soft tissue sarcoma, spinal cord tumors, stomach cancer, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, tongue cancer, trachea cancer, triple-negative breast cancer, ureter and renal pelvis cancer, vaginal cancer, vulvar cancer, uterine cancer.

[0604] Our finding that EVs inhibited TGF-induced signaling pathway activation and PDGFB gene expression (see above) also suggests that EVs could be used in anticancer therapy. The role of TGFβ signaling pathway inhibitors in cancer therapy is well documented in the art. Our results suggest that EVs could be used similarly to TGFβ inhibitor anticancer drugs, i.e., in the treatment or prevention of cancer.

[0605] The clinical application of TGFβ inhibitor signaling pathway has been tested for, for example, gastric cancer, non-small cell lung cancer, ovarian cancer, colon cancer, Ewing's sarcoma, hepatocellular carcinoma, prostate cancer, and uterine cancer. Known TGFβ-targeted drugs include Vactosertib, Galunisertib, Fresolimumab, Bintrafusp alfa, Trabedersen, Belagenpumatucel-L, Luspatercept, Tasisulam, AVID200, LY3200882, A83-01, LY2109761, and SB-431542.

[0606] Our results suggest that EVs could be used similarly to EGFR inhibitor anticancer drugs, i.e., in the treatment or prevention of cancer.

[0607] The clinical application of EGFR inhibitors has been tested for, for example, acute myeloid leukemia, colorectal cancer, head and neck cancer, lung cancer, breast cancer and pancreatic cancer. Known EGFR inhibitor drugs include erlotinib, osimetinib, neratinib, gefitinib, cetuximab, panitumumab, dacomitinib, lapatinib, necitumumab, mobocertinib, vandetanib, almonertinib, brigatinib, olmutinib, pyrotinib, afatinib, acomitinib, icotinib, simotinib.

[0608] Our results indicate that EVs can be used to modulate, preferably inhibit, the activity of the PDGF signaling pathway. Preferably, EVs are used to modulate the activity of any of the signaling molecules associated with PDGFR in Table 1. Preferably, EVs are used to prevent or treat conditions, disorders or diseases associated with the activity of any of the signaling molecules in Table 1.

[0609] Table 1 shows the signal transduction molecules that may be affected by EVs.

[0610]

[0611]

[0612]

[0613]

[0614] Our results indicate that EVs can be used to modulate, preferably inhibit, the activity of EGFR-related signaling pathways. Preferably, EVs are used to modulate the activity of any of the signal transduction molecules in Table 2. Preferably, EVs are used to prevent or treat conditions, disorders or diseases associated with the activity of any of the signal transduction molecules in Table 2.

[0615] Table 2 shows the signal transduction molecules that may be affected by EVs.

[0616] Table 2

[0617]

[0618]

[0619]

[0620]

[0621]

[0622] Our results indicate that EVs can be used to modulate, preferably inhibit, the activity of TGFR-related signaling pathways. Preferably, EVs are used to modulate the activity of any of the signal transduction molecules in Table 3. Preferably, EVs are used to prevent or treat conditions, disorders or diseases associated with the activity of any of the signal transduction molecules in Table 3.

[0623] Table 3 shows the signal transduction molecules that may be affected by EVs.

[0624] Table 3

[0625]

[0626]

[0627]

[0628]

[0629] Our results indicate that EVs can be used to modulate, preferably inhibit, the activity of common elements of the PDGFR and EGFR signaling pathways. Preferably, EVs are used to modulate the activity of any of the signaling molecules in Table 4. Preferably, EVs are used to prevent or treat conditions, disorders or diseases associated with the activity of any of the signaling molecules in Table 4.

[0630] Table 4 shows the signal transduction molecules that may be affected by EVs.

[0631] Table 4

[0632]

[0633]

[0634] Our results indicate that EVs can be used to modulate, preferably inhibit, the activity of common elements of the PDGFR and EGFR signaling pathways. Preferably, EVs are used to modulate the activity of any of the signaling molecules in Table 5. Preferably, EVs are used to prevent or treat conditions, disorders or diseases associated with the activity of any of the signaling molecules in Table 5.

[0635] Table 5 shows the signal transduction molecules that may be affected by EVs.

[0636] Table 5

[0637]

[0638]

[0639] Our results indicate that EVs can be used to modulate, preferably inhibit, the activity of common elements of the TGF and EGF signaling pathways. Preferably, EVs are used to modulate the activity of any of the signaling molecules in Table 6. Preferably, EVs are used to prevent or treat conditions, disorders or diseases associated with the activity of any of the signaling molecules in Table 6.

[0640] Table 6 shows the signal transduction molecules that may be affected by EVs.

[0641] Table 6

[0642]

[0643]

[0644] Our results indicate that EVs can be used to modulate, preferably inhibit, the activity of TGF, EGFR and PDGF-induced signaling pathways, preferably inhibit the activity of common elements of TGF, EGFR and PDGF-induced signaling pathways. Preferably, EVs are used to modulate the activity of any of the signal transduction molecules in Table 7. Preferably, EVs are used to prevent or treat conditions, disorders or diseases associated with the activity of any of the signal transduction molecules in Table 7.

[0645] Table 7 shows the signal transduction molecules that may be affected by EVs.

[0646] Table 7

[0647] AKT1 AKT serine / threonine kinase 1 EGF Epidermal Growth Factor FOS Fos proto-oncogene, AP-1 transcription factor subunit GRB2 Growth factor receptor binding protein 2 HRAS HRas proto-oncogene, GTPase JAK1 Janus kinase 1 JUN Jun proto-oncogene, AP-1 transcription factor subunit MAP2K1 Mitogen-activated protein kinase kinase 1 MAP2K2 Mitogen-activated protein kinase kinase 2 MAPK1 Mitogen-activated protein kinase 1 MAPK3 Mitogen-activated protein kinase 3 MTOR Mechanistic target of rapamycin kinase STAT1 Signal transducer and activator of transcription 1 STAT3 Signal transducer and activator of transcription 3

[0648] Our results suggest that EVs can modulate IL-10 signaling through a receptor complex consisting of two IL-10 receptor-1 and two IL-10 receptor-2 proteins (Berman RM, Suzuki T, Tahara H, Robbins PD, Narula SK, Lotze MT (July 1996). "Systemic administration of cellular IL-10 induces an effective, specific, and long-lived immune response against established tumours in mice". Journal of Immunology. 157(1):231–8. PMID 8683120). IL-10 has pleiotropic effects in immunomodulation and inflammation. In fact, it has been shown to downregulate the expression of Th1 cytokines, MHC class II antigens and co-stimulatory molecules on macrophages. In addition, it enhances B cell proliferation, survival and antibody production. IL-10 is able to block the activity of NF-κB and participates in the regulation of the JAK-STAT signaling pathway.IL-10 also leads to control of primary tumor growth and reduces metastatic burden (Zheng LM, Ojcius DM, Garaud F, Roth C, Maxwell E, Li Z, Rong H, Chen J, Wang XY, Catino JJ, King I (August 1996). "Interleukin-10 inhibits tumour metastasis through an NK cell-dependent mechanism". The Journal of Experimental Medicine. 184(2):579–84. doi:10.1084 / jem.184.2.579.PMC 2192723. PMID 8760811; Fujii S, Shimizu K, Shimizu T, Lotze MT (October 2001). "Interleukin-10 promotes the maintenance of antitumour CD8(+)T-cell effector function in "Systemic administration of cellular IL-10 induces an effective, specific, and long-lived immune response against established tumors in mice". Journal of Immunology. 157(1):231–8. PMID8683120).

[0649] Endogenous metabolites generated by oxidation of glucose and other sugars (such as MGO, glyoxal, 3,4DGE, HMF, etc.) react with proteins, lipids or nucleic acids to form advanced glycation end products (AGEs). The accumulation of AGEs and their binding to their receptors promote oxidative stress and inflammation. Oxidative stress can interfere with intracellular signaling, turning it into a pathological state, especially insulin-mediated metabolic reactions and insulin resistance (Shen CY, Lu CH, Wu CH, Li KJ, Kuo YM, Hsieh SC, Yu CL. The Development of Maillard Reaction, and Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signalling Inhibitors as Novel Therapeutic Strategies for Patients with AGE-Related Diseases. Molecules. 2020 Nov 27; 25(23): 5591. doi: 10.3390 / molecules25235591. PMID: 33261212; PMCID: PMC7729569). Protein glycation and the formation of AGEs play an important role in the pathogenesis of diabetic complications (e.g., retinopathy, nephropathy, neuropathy, cardiomyopathy) (Singh VP, Bali A, Singh N, JaggiAS. Advanced glycation end products and diabetic complications. Korean JP Hysiol Pharmacol. 2014; 18(1): 1-14. doi: 10.4196 / kjpp.2014.18.1.1). AGEs play a pathogenic role in the development and progression of different oxidative diseases including diabetes.(Vistoli G, De Maddis D, Cipak A, Zarkovic N, Carini M, Aldini G. Advanced glycoxidation and lipoxidation endproducts (AGEs and ALEs): an overview of their mechanisms of formation. Free Radic Res. 2013 Aug; 47 Suppl 1: 3-27. doi: 10.3109 / 10715762.2013.815348. PMID: 23767955.). According to our results (. Figure 41-44 ), EVs are suitable for preventing or treating disorders or diseases associated with impaired glucose metabolism, such as insulin resistance, diabetes, diabetic complications or disorders or diseases associated with diabetes, and preventing or treating conditions, disorders or diseases associated with MGO formation, such as insulin resistance, type I diabetes, type II diabetes, diabetic nephropathy, diabetic ulcers, diabetic retinopathy, macular degeneration, myocardial infarction, hypertension, arteriosclerosis, atherosclerosis, venous sclerosis, irritable bowel syndrome, rheumatoid arthritis, osteoarthritis, Parkinson's disease, Alzheimer's disease, dementia, depression, anxiety disorders, schizophrenia and various cancers.

[0650] Our results suggest that EVs can be used in therapies similar to PDGFR, EGFR or TGFR inhibitors or agents that inhibit any signaling pathway activated by PDGF, EGF or TGF, or preferably inhibit the activity of common elements of signaling pathways induced by TGF and EGFR, or induced by TGF and PDGF, or induced by EGFR and PDGF, preferably TGF and EGFR and PDGF. Thus, EVs can be used to treat conditions, disorders or diseases in which PDGFR, EGFR or TGFR inhibitors or agents that inhibit any signaling pathway mediated by PDGF, EGF or TGF are useful.

[0651] Our results also suggest that EVs can be used for non-medical (e.g., cosmetic) treatment of fibroproliferative or fibrotic conditions (such as age-related tissue fibrosis). Thus, also provided are food or cosmetic compositions comprising or consisting essentially of algal EVs for treating or inhibiting fibroproliferative or fibrotic conditions.

[0652] EVs can be formulated into compositions as disclosed herein when preparing EVs. In fact, the examples describe providing EVs in appropriate amounts and concentrations and in any appropriate medium. EVs are generally stable, and descriptions of methods for their cultivation, concentration, pelleting, and changing media are provided. In addition, providing EVs in the form of compositions and developing them into drugs is well known in the art.

[0653] The following publications provide examples of the development of EVs as formulations. The skilled artisan will appreciate that formulation methods and compositions developed for EVs can also be applied to algal EVs, if necessary.

[0654] Karamanidou, T.; Tsouknidas, A. Plant-Derived Extracellular Vesicles asTherapeutic Nanocarriers.Int.J.Mol.Sci.2022,23,191.https: / / doi.org / 10.3390 / ijms23010191

[0655] Klyachko, Natalia L et al. "Extracellular Vesicle-Based Therapeutics: Preclinical and Clinical Investigations." Pharmaceutics vol.12,121171.1Dec.2020,doi:10.3390 / pharmaceutics12121171

[0656] Wiklander, Oscar PB et al. “Advances in therapeutic applications of extracellular vesicles.” Science translational medicine vol.11,492(2019):eaav8521.doi:10.1126 / scitranslmed.aav8521

[0657] Claridge B et al.Development of Extracellular Vesicle Therapeutics:Challenges,Considerations,and OpportunitiesCell Dev.Biol.,20September2021https: / / doi.org / 10.3389 / fcell.2021.734720Ana Rita Marques PereiraTrindade,FORMULATION STRATEGIES FOR EXTRACELLULAR VESICLE DELIVERY"PhDthesis,2018.

[0658] Example - Method:

[0659] Algae culture: Chlorella species MACC-360 and Pseudocerulina kessleri were grown in TAP medium (UTEX, USA), MACC-215 (Chlamydomonas reinhardtii), MACC-1 (Chlorella vulgaris), MACC-3 (Chlorella pyrenoidosa), MACC-1023 (Homicystis verrucosum), MACC-21 (Diplocystis indigosporum), MACC-1022 (Boweizia), MACC-888 (Microcystis aeruginosa) and MACC-90 (Haematococcus lacustris) were grown in Bristol medium (UTEX, USA), MACC-612 (Lin Nian) was grown in Bristol medium (UTEX, USA). MACC-918 (Spigodium) and MACC-42 (Annella africana) were grown in Z-8 medium (http: / / www.sccap.dk / media / freshwater / 7.asp), MACC908 (Spirulina platensis), MACC909 (Spirulina maxima), MACC115 (Synechococcus) and MACC1042 (Klebsiella) were grown in BG-11 (N) medium (UTEX, USA), and 250 ml conical flasks were placed on an orbital shaker at 150 rpm at room temperature. Unless otherwise stated, autotrophic culture was provided by optimized conditions, using white light bulbs to ensure a 12-hour day / 12-hour night photoperiod until the cell count reached about 7x10^7 cells / ml. In the case of heterotrophic culture, Chlorella MACC-360 was cultured in flasks under dark conditions, using RPMI 1640 medium (ATCC, USA) to ensure adequate nutrient sources. Algal strains were provided by Molnár Zoltán (Mosonmagyaróvár Algal Culture Collection, Mosonmagyaróvár, Hungary).

[0660] Chlorella pyrenoidosa (Hangzhou Natural Food Co., Ltd., China), Spirulina platensis (Hangzhou Natural Food Co., Ltd., China), Ascophyllum nodosum (Giant Algae, https: / / www.futunatura.hu), Dulse digitata (Dulse digitata, https: / / www.futunatura.hu), Tetraselmis chui (https: / / www.allmashop.com / produto / tetraselmis-chui-em-po), Aphanizomenon flos-aquae (https: / / www.futunatura.hu) were purchased as edible powders and rehydrated in phosphate buffered saline (PBS) at a concentration of 20 mg / ml for 1 hour at room temperature with gentle stirring.

[0661] Extracellular vesicle (EV) isolation: To remove microbial contaminants and cell debris, samples were centrifuged at 2000 rpm for 20 min and then filtered through a 0.22 μm pore size filter (Millipore The supernatant was filtered using the ELISA PLUS (Merck, Germany). Subsequently, the EVs content of a given sample was concentrated and / or EVs were isolated by several methods (summarized in Table 8).

[0662] (1) Samples were ultrafiltered and concentrated by tangential flow filtration using TFF-easy filters (Hansa BioMed Life Sciences, BioCentral Laboratory Suppliers Ltd., Hungary).

[0663] (2) Samples Ultra-15 (100 kDa MWCO filter, Millipore PLUS, Merck, Germany) ultrafiltration and concentration.

[0664] (3) The samples were ultracentrifuged at 4°C for 1 hour using a Beckman L7-55 ultracentrifuge, a 70.1 Ti fixed-angle titanium rotor (Beckman Coulter, USA) and 10 ml open-top thick-walled polycarbonate tubing (Beckman Coulter, USA).

[0665] (4) Sample SnakeSkin TM The cells were dialyzed for 24 h using dialysis tubing (3.5K MWCO, Thermo Fisher Scientific, USA) and 20% polyethylene glycol solution (20K MW, Merck, Germany).

[0666] (5) Samples were homogenized in a multi-step sonication process (Kerry Steel Probe Sonicator, immersed in liquid) or in a 2 ml Potter Elvehjem tissue grinder (in the case of Spirulina platensis powder) and then The extruder (Evonic Industries, Germany) was used with a polycarbonate membrane filter (Whatman, UK) at 60°C and subjected to freeze-thaw cycles (-80-60°C) before extrusion.

[0667] (6) The partially separated EV samples from steps (1), (2), (4), and (5) were chromatographically analyzed on an IZON qEVoriginal / 70 nm pore size column (IZON,

[0668] The product was further purified by size exclusion chromatography (SEC) at 4 °C in the USA.

[0669] Unless otherwise stated, EVs isolated from Chlorella MACC-360 by TFF (1) + SEC (6) were used during in vitro experiments.

[0670] Table 8 Methods for isolating EVs from different algal sources.

[0671]

[0672]

[0673] According to the recommendations of ISEV (International Society for Extracellular Vesicles), each fraction was collected and analyzed by the following method to demonstrate and validate the EV content of these samples.

[0674] Protein content, spectrophotometric method: The protein content of the samples was determined by a NanoDrop ND-100 spectrophotometer (BCM, USA) based on the absorbance of the samples at 280 nm. Protein concentrations above 100 μg / ml indicate successful EV isolation. In an alternative method, the protein content of EV samples was determined by the QubitTM Protein Assay Kit (Q33211, Thermo Fisher Scientific, USA) according to the manufacturer's instructions.

[0675] Microfluidic resistive pulse sensing (MRPS): Microfluidic resistive pulse sensing (MRPS) is a novel non-optical method for measuring the size and concentration of various nano- and micro-particles using the Coulter principle. MRPS measurements were performed using an nCS1 instrument (Spectradyne LLC, USA). Samples were diluted 10 times with 1 mg / ml bovine serum albumin (BSA, Merck Kft, Hungary) solution in PBS buffer (Merck Kft, Hungary) according to the manufacturer's instructions. Factory-calibrated TS-400, TS-900, and TS-2000 cartridges were used for measurements with a measurement range of 65 nm to 2000 nm.

[0676] Nanoparticle tracking analysis (NTA): The size distribution, median size, and particle concentration of EVs were determined by NTA. The measurements were performed using ZetaView PMX-120 (Particle Metrix GmbH, Meerbusch, Germany) with ZetaVIEW software.

[0677] Dynamic light scattering (DLS): The hydrodynamic diameter of EVs was measured using a W130i dynamic light scattering (DLS) instrument (AvidNano, UK). DLS measurements were performed using small-volume disposable plastic cuvettes (UVette; Eppendorf, Austria) and data were evaluated using iSize 3.0 software (AvidNano).

[0678] Fourier-transform infrared spectroscopy (FTIR): The protein and lipid content of EVs was characterized by Fourier transform infrared spectroscopy (FTIR). FTIR measurements were performed using a Varian 2000 spectrometer (Scimitar Series, USA) equipped with a diamond attenuated total reflectance unit ('Golden Gate' single reflection ATR unit, Specac, UK). About 5 μl of sample was pipetted onto the diamond ATR surface, and a thin dry film was obtained by slowly evaporating the solvent under ambient conditions (about .10 min). Typically, 2 cm -1 64 scans were collected at a nominal resolution of 1.3 Å. ATR correction, buffer background spectrum subtraction and other spectral evaluations were performed using the Grams / 32 software package (Galactic Inc., USA).

[0679] Freeze-fracture combined transmission electron microscopy (FF-TEM): The morphology of EVs was demonstrated using freeze-fracture combined transmission electron microscopy (FF-TEM). EV samples were mixed with glycerol (Sigma-Aldrich, Hungary) as a cryoprotectant at a sample:glycerol volume ratio of 3:1. About 2 μl of vesicle sample was pipetted onto a gold sample holder and immediately frozen by placing it in partially solidified Freon for 20 s. Fracture was performed in a Balzers freeze-fracture apparatus (Balzers BAF 400D, Balzers AG, Liechtenstein) at −100 °C. Replicas of the fracture surfaces were made using the platinum-carbon evaporation method and then washed with an aqueous solution of a surfactant and with distilled water. The platinum-carbon replica was placed on a 200-mesh copper grid and examined under a MORGAGNI268D (FEI, The Netherlands) transmission electron microscope.

[0680] Lyophilization: Peritoneal dialysis (PD) effluent (PDE) and EV samples were flash frozen onto the wall of a 50 ml Falcon centrifuge tube (PDE) or 2 ml glass vial (EV samples) by spinning in liquid nitrogen to increase the drying surface. Freeze drying and setting of the lyophilization protocol were performed using a ScanVacCoolSafe Touch Superior device (LaboGen A / S, Allerød, Denmark).

[0681] Human samples: Peritoneal dialysis (PD) effluent (PDE) was obtained from patients undergoing peritoneal dialysis (PD) at the 1st Department of Paediatrics, Semmelweis University, Budapest, Hungary.

[0682] Peritoneal samples were collected at the first insertion of a Tenckhoff peritoneal catheter and at the time of removal of the peritoneal dialysis catheter (loss of peritoneal ultrafiltration capacity) at the First Department of Pediatrics, Szentmevis University, Budapest, Hungary (31224-5 / 2017 / EKU).

[0683] Colonic samples were collected during routine endoscopy at the First Department of Pediatrics, Semmelweis University, Budapest, Hungary, and the remnants of these samples were used in our work (19048-4 / 2018 / EKU).

[0684] Skin samples were provided by the Department of Dermatology, Venereology and Dermatooncology, Semmelweis University (Kende PhD(IV / 1707-6 / 2020 / EKU)

[0685] Primary cells: primary human peritoneal fibroblasts (phPFB), primary human colonic fibroblasts (phCFB), primary human skin fibroblasts (phSFB) were prepared by enzymatic digestion of relevant tissues with 1 mg / ml collagenase type II (LifeTechnologies Kft, Hungary), and primary human peritoneal mesothelial cells (HPMC) were prepared by enzymatic digestion of relevant tissues using 0.25% trypsin-EDTA (Life Technologies Kft, Hungary).

[0686] Primary fibroblasts (phFB) were cultured at 37°C in a 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium / nutrient mixture F-12 (DMEM-F12, Life Technologies Kft, Hungary) supplemented with 10% heat-inactivated fetal calf serum (FCS, Life Technologies Kft, Hungary), 100 μg / ml streptomycin and 100 U / ml penicillin (Life Technologies Kft, Hungary) at 37°C in a humidified atmosphere of air.

[0687] Primary HPMCs were cultured in M199 medium supplemented with 10% FCS, 400 nM hydrocortisone (Life Technologies Kft, Hungary), 870 nM insulin (Life Technologies Kft, Hungary), 20 mM HEPES (Life Technologies Kft, Hungary), 3.3 nM epithelial growth factor (EGF, R&D, UK) and 100 μg / ml streptomycin and 100 U / ml penicillin. Monolayers were identified as phFB or HPMC by their morphology and as α-SMA (phFB) or cytokeratin 18 positive (HPMC) by immunofluorescence staining.

[0688] HUVEC (#CRL-1730) human umbilical vein endothelial cells (ATCC, USA) were cultured at 37°C in a 5% CO 2The cells were cultured in a humidified atmosphere of air in Vascular Cell Basal Medium (ATCC, USA) supplemented with Endothelial Cell Growth Kit-VEGF (ATCC, USA), 10% FCS, 100 μg / ml streptomycin and 100 U / ml penicillin.

[0689] Peripheral blood mononuclear cells (PBMCs) from control patients were isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, Hungary). After isolation, the cells were placed in RPMI 1640 medium (ATCC, USA) and incubated at 37°C with 5% CO 2 The cells were maintained in a humidified atmosphere of air supplemented with L-glutamine (Life Technologies Kft, Hungary), 10% FCS, 100 μg / ml streptomycin and 100 U / ml penicillin.

[0690] Cell lines: MRC-5 (#CCL-171) human lung fibroblasts, A549 (#CRM-CCL-185) human lung epithelial adenocarcinoma cells and Caco-2 (#HTB-37) human colon cancer cells (American Type Culture Collection (ATCC), USA) were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, USA), HEPG2 human hepatocellular carcinoma cell line (American Type Culture Collection (ATCC), USA) was grown in Dulbecco's modified Eagle's medium / nutrient mixture F-12 (DMEM-F12) medium. Hs578-T human breast cancer cell line (American Type Culture Collection (ATCC), USA) and LCLC-103H human large cell lung cancer cell line (German Collection of Microorganisms and Cell Cultures, Germany) were grown in RPMI1640 medium (ATCC, USA). HT-29 (#HTB-38), a human colon cancer cell line (ATCC), was cultured in McCoy's 5A medium (Thermo Fisher Scientific, USA) supplemented with 10% FCS, 100 μg / ml streptomycin, and 100 U / ml penicillin at 37°C in a humidified atmosphere containing 5% CO. 2 Cultivate in a humidified atmosphere of air.

[0691] Immunofluorescence staining: To characterize primary human cells isolated from tissue samples, immunofluorescence staining was performed. Monolayers were identified as phFB or HPMC based on their morphology and α-SMA (phFB) or CK18 immunopositivity (HPMC). 4phPFB, phCFB, phSFB and HPMC cells were seeded at 80% confluence in 4-well cell culture slides (CorningCostar, Sigma-Aldrich, Hungary). After a methanol fixation step, the chambers were first incubated for 2 h at room temperature with primary antibodies against the fibroblast marker smooth muscle actin α (α-SMA, Sigma-Aldrich, Hungary) and the mesothelial marker cytokeratin 18 (CK18, Santa Cruz Biotechnology, Hungary) (1:500, Santa Cruz, USA). After multiple washes with PBS, the slides were incubated with the corresponding Alexa Fluor 400 antibody. The coupled secondary antibodies (1:1000, Life Technologies Kft, Hungary) were incubated for 1 hour at room temperature. Appropriate controls were performed, omitting the primary antibodies to avoid autofluorescence. Cell nuclei were stained with Hoechst 33342 (1:1000, Sigma-Aldrich, Hungary) for 10 minutes at room temperature. Finally, the slides were covered with ProLongTM Gold antifade mounting medium (Invitgen, Hungary). Cell morphology, α-SMA and CK18 immunopositivity were visualized using an Olympus IX-81 fluorescence microscope system (Olympus Corporation, Japan).

[0692] EVs in vitro and in vivo uptake experiments: To test the in vitro internalization of EVs, samples were labeled with the lipophilic fluorescent dye DiI (DiIC18(3), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate, Molecular Probes, UK) according to the manufacturer's instructions. Briefly, 100 μl of EVs were incubated with 2 μl of 1 mg / ml DiI dye for 30 min at 37°C. After washing the labeled EVs with 400 μl PBS, unbound dye was eliminated by SEC. In parallel, the same volume of dye was incubated in PBS (in the absence of EVs) and subjected to the same incubation period, and then SEC was performed as before after the washing steps. The latter sample was used as a negative control.

[0693] 6×10 4 phPFB, phCFB, phSFB, and HPMC cells were seeded at 80% confluence in 4-well cell culture slides (Corning Costar, Sigma-Aldrich, Hungary). The cells were then incubated with cell culture medium containing 109 particles / ml of DiI-labeled EVs at 37°C, 5% CO 2The cells were incubated in a humidified atmosphere for 24 h. Subsequently, the nuclei were stained with Hoechst 33342 (1:1000, Merck Kft, Hungary) for 10 min at room temperature. Finally, the slides were covered with ProLongTM Gold antifade mounting medium (Invitgen, Hungary). The internalized EVs were visualized using an Olympus IX-81 fluorescence microscope system (Olympus Corporation, Japan).

[0694] In order to test the in vivo internalization of enterovirus of Chlorella MACC-360 in liver and skin, and the internalization of MACC-1, MACC-3 and MACC-1023 in visceral peritoneum and peritoneal lavage cells, 10^9 DiI-labeled enterovirus was injected intravenously or intraperitoneally or intradermally into the tail vein of C57 / BL6J mice. Mice were killed after 24 hours, and then liver, visceral peritoneal samples, peritoneal lavage cells and skin samples were collected, 3 μm thick sections were prepared (in the case of liver and peritoneal samples), and finally ProLongTM Gold anti-fading mounting medium DAPI (Invitrogen, Hungary) coverslips were used. The internalized EVs were visualized with an Olympus IX-81 fluorescence microscope system.

[0695] Western blotting: PDE samples were freeze-dried, dissolved in water to the same protein concentration, and then denatured by adding standard Leammli buffer and boiling for 5 minutes. The samples were separated by electrophoresis on a 4-20% gradient SDS-polyacrylamide gel (BioRad, USA) and transferred to a nitrocellulose membrane (BioRad, USA). To verify the transfer, the membrane was stained with Ponceau S (Sigma-Aldrich, Hungary), then washed and blocked with 5% skim milk in TRIS buffered saline (TBS) at room temperature for 1 hour. Thereafter, the membrane was incubated with specific primary antibodies (1:1000) against anti-fibrotic factors (PDGF-BB, TGF-β1 / 2 / 3 and CTGF, Santa Cruz, USA) at 4°C overnight. After repeated washing with TBS containing 0.05% Tween-20 and 1% skim milk, the membrane was incubated with the corresponding HRP-conjugated antibody (1:2000, Merck Kft, Hungary, Life Technologies Kft, Hungary) at room temperature for 1 hour. The immunoreactive bands of interest were detected using enhanced chemiluminescence detection (Western Blotting Luminol reagent, GE Healthcare, USA).

[0696] MTT (cell proliferation and viability) assay: MTT assay was performed on phPFB, phCFB, phSFB, MRC-5, Caco2, HEPG2, Hs578-T, LCLC-103H, HT-29 and A549 cells treated with platelet-derived growth factor B (PDGF-B, 10 ng / ml, R&D Systems, USA) or peritoneal dialysis effluent (PDE, 30%), HPMC HUVEC and phSFB / A cells treated with methylglyoxal (MGO, 300-700 μM, Merck Kft, Hungary), and phSFB / A cells treated with 5 mM hydroxymethylfurfural (HMF) in the presence or absence of 10^7 or 10^8 particles / ml EV. Vehicle-treated cells (4 mM HCL in the case of PDGF, 30% Fresenius 1.5% glucose-containing peritoneal dialysis fluid (PDF) in the case of PDE, and an equal volume of PBS in the case of MGO and EVs) served as controls. 24 h after treatment, cell proliferation / viability was determined by colorimetric assay based on intracellular mitochondrial dehydrogenase activity of attached cells. Briefly, 10 μl of 5 mg / ml thiazolyl blue tetrazolium bromide (in sterile H 2 MTT reagent (diluted in 1% MgCl2O) was added to each well, along with cells and 100 μl of supernatant, and then incubated at 37°C for 4 hours. Thereafter, the supernatant was removed from the cells using a pipette, and the intracellular MTT crystals were dissolved by adding 100 μl of a 1:1 mixture of DMSO and ethanol (all reagents were purchased from Merck, Germany). The absorbance at 570 nm and 690 nm was recorded as background in a SPECTROstar nanoplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, Germany). The results were normalized and determined as a percentage of the control group's values.

[0697] LDH (cytotoxicity) assay: LDH assay was performed on Hs578-T, LCLC-103H, HepG2, phCFB / A and MGO (100-700 μM) treated HPMC, HUVEC and phCFB / A cells and phCFB / A cells treated with HMF (10 mM) (Merck Kft, Hungary) or 3,4-dideoxyglucosidone-3-ene (DGE) (75 μM) (Biosynth Carbosynth, UK) in the presence or absence of 10^7 or 10^8 particles / ml EV. Vehicle-treated cells (equal volume of PBS) served as controls. The extent of cell death was determined by colorimetry at 24 h after treatment based on lactate dehydrogenase (LDH) enzyme activity in the supernatant released by damaged cells. An equal volume (40 μl) of the desired culture medium was mixed with LDH reagent in a sterile 96-well plate, containing 109 mM lactate, 3.3 mM β-nicotinamide-adenine-dinucleotide hydrate, 2.2 U / ml diaphorase, 3 mM TRIS, 30 mM HEPES, 10 mM NaCl, 350 μM thiazolyl blue tetrazolium bromide (all reagents purchased from Merck Kft, Hungary), and then incubated at 37°C for 1 hour. The absorbance at 570 nm and 690 nm was recorded as background in a SPECTROstar nanoplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, Germany). The results were normalized and determined as a percentage of the control group's values.

[0698] Sirius Red (Collagen Detection) Assay: Sirius Red assay was performed on phPFB, phCFB, phSFB and MRC-5 lung cells treated with transforming growth factor β (TGF-β, 1 nM, R&D Systems, USA) or PDE (30%) in the presence or absence of 10^7 or 10^8 particles / ml EV. Vehicle-treated cells (4 mM HCL in the case of TGF-β, 30% PDF in the case of PDE, PBS in the case of EV) were used as controls. After 48 hours of treatment, collagen deposition was determined based on Sirius Red, a basic histological dye incorporated into triple-helical collagen molecules. After removing the supernatant, the cells were incubated in a fixative solution containing 26% EtOH, 3.7% formaldehyde, and 2% glacial acetic acid for 15 minutes at room temperature. Samples were stained at room temperature with 0.1% Sirius red (DirectRed80) solution dissolved in 1% acetic acid for 1 hour, then washed three times with 200 μl 0.1M HCl, and finally added 100 μl 0.1M NaOH to dissolve the bound dye (all reagents are purchased from Merck, Germany). Use SPECTROstar Nano MARS v3.32 software (BMG Labtech, Germany) to record the absorbance at 544nm and 690nm as background in a SPECTROstar nano-microplate reader. The results were normalized and determined as the percentage of the control group value.

[0699] TAS (cell migration) assay: According to the study of Apor Veres Székely et al., TAS assay was performed on phCFB, phSFB treated with epidermal growth factor (EGF, 10 ng / ml, R&D Systems, USA) and Caco-2 cells treated with EGF or PDE (30%) in the presence or absence of 10^8 particles / ml EV. Vehicle-treated cells (PBS in the case of EGF and EV, 30% PDF in the case of PDE) served as controls. In brief, cells were seeded at a density close to full density into 96-well plates containing a non-toxic gel barrier to create a cell-free area. After incubation for 24 hours, the barrier was removed, the wells were washed with PBS, and the cells were treated. At 0, 24, 48, and 72 hours after treatment, bright field images of each well were taken using an Olympus IX-81 microscope system (Olympus, Japan). The cell-free gap area was measured using ImageJ1.48v software and determined as the ratio of the initial gap area at 0 hours.

[0700] Real-time reverse transcription polymerase chain reaction (qRT-PCR): Total RNA was isolated from PBMCs treated with EV (10^8 particles / ml, 24 hours of treatment) or 1μg / ml phytohemagglutinin (PHA) (Merck Kft, Hungary) and EV by Geneaid Total RNA Mini Kit (Geneaid Biotech, Taiwan, China). The RNA was reversely transcribed using the Maxima First-Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific, USA) to produce the first-strand cDNA. The mRNA expression of inflammation or fibrosis-related markers (IL10, IL13, PDGFB, TGFβ, IL6, MCP1) was determined by real-time PCR using the LightCycler 480SYBR GreenI Master enzyme mixture on the LightCycler 96 system (Roche Diagnostics, USA). The results were analyzed using LightCycler 96 software v1.1.0.1320 (Roche Diagnostics). The corresponding mRNA expression was determined by the ΔΔCt method using ribosomal protein stalk subunit P0 (RPRP0) or 18s ribosomal RNA (RN18S) as internal controls. The data were normalized and expressed as the ratio of their control values.

[0701] IL-6 protein level (ELISA method): 10 4 A549 and phSFB / A cells were seeded into 96-well plates and treated with 100 ng / ml lipopolysaccharide (LPS, Merck Kft, Hungary), 1 μg / ml PHA, Merck Kft, Hungary) and 10 μg / ml polyinositol:polycytidylic acid [poly (I:C), Merck Kft, Hungary] for 24 h in the presence or absence of 10^8 particles / ml of Chlorella MACC-360-derived EVs. Vehicle-treated cells (PBS) served as controls. Protein levels of IL-6 were measured by human IL-6 DuoSet ELISA (#DY206, R&D Systems, Inc, USA) according to the manufacturer's instructions.

[0702] Kinase array: 5 × 10 5phCFB / A cells were seeded into 6-well plates and treated with peritoneal dialysis fluid (PDE, 30%) or recombinant mixture (PDGF-B 10 ng / ml, TGF-β 0.5 nM, EGF 10 ng / ml, CTGF 100 ng / ml, R&D Systems, Inc, USA) for 30 minutes in the presence or absence of 10^8 particles / ml EVs from Chlorella MACC-360. Vehicle-treated cells [Fresenius 1.5% peritoneal dialysis fluid containing glucose (PDF), PBS] served as controls. The corresponding levels of human protein kinase phosphorylation were determined using the Proteom profiler human phosphokinase array kit (#ARY003C, R&D Systems, Inc, USA) according to the manufacturer's instructions.

[0703] Testing EV stability: Chlorella MACC-360 samples ultrafiltered and concentrated by TFF simple filter were (1) incubated with hydrochloric acid (HCl, Merck Kft, Hungary) at pH 4.6 for 1 hour at room temperature, (2) treated with 0.25% trypsin-EDTA (TFF: trypsin-EDTA = 2:1 ratio) at room temperature for 1 hour, (3) incubated with HCl at pH 2-3 at room temperature, (4) or incubated at 95°C for 1 hour and then neutralized with sodium hydroxide (Merck Kft, Hungary); (5) incubated at 95°C for 1 hour; (6) incubated with 20 μg / ml proteinase K (Life Technologies Kft, Hungary) at 37°C for 1 hour. After the above treatments, all groups were subjected to SEC treatment to separate uniform EV fractions. MTT assay was performed on phCFB / A cells treated with platelet-derived growth factor B (PDGF-B, 10 ng / ml) or peritoneal dialysate (PDE, 30%) according to the above protocol.

[0704] Testing the effect of EV-EV and EV-platelet-derived growth factor receptor alpha and beta (PDGFR-α, PDGFR-β) inhibitor combinations on proliferation: The effect of EVs derived from Chlorella MACC-360 in combination with MACC-1 and MACC-908 and the PDGFR-α inhibitors ponatinib (Merck Kft, Hungary) and PDGFR-β inhibitors axitinib (Merck Kft, Hungary) on peritoneal dialysis effluent (PDE)-induced proliferation of primary human colonic fibroblasts (phCFB / A) from patient A was determined. Briefly, phCFB / A cells were treated with PDE and 3x 10^8 particles / ml MACC-360, MACC-1 and MACC-908 derived EVs and their 1μM ponatinib or axitinib combinations for 24 hours in the presence or absence of 3x 10^8 particles / ml Chlorella MACC-360 derived EVs. Proliferation was determined by MTT assay according to the above protocol (see MTT paragraph).

[0705] Effects of EV in vivo treatment on peritoneal fibrosis and DSS-induced colitis models in mice: Animal experiments were performed on 6-8 week old male C57Bl / 6J mice (Charles River Laboratories, Germany). Animals were housed in a temperature-controlled (22 ± 1 °C) room with alternating light and dark cycles with free access to standard laboratory rodent chow and water. The Institutional Animal Welfare Committee approved all experiments (PEI / 001 / 1731-9 / 2015). Mice were randomly divided into four groups:

[0706] (A) Vehicle-treated (PBS) mice served as controls (n=6);

[0707] (B) On days 2 and 5, mice were intraperitoneally injected (ip) with 300 μl of 10^9 particles / ml EV in PBS (n=6).

[0708] (C) Mice were intraperitoneally injected daily with 300 μl of 0.1% chlorhexidine digluconate (CG) and 15% ethanol in PBS (n=6);

[0709] (D) Mice received the same EV and CG treatments as groups B and C (4 h interval) (n = 8);

[0710] In the DSS-induced colitis model, C57BL / 6J mice received 2.5% DSS in drinking water for 7 days. During DSS treatment, mice were orally administered 100 μl of approximately 1.5 10^10 particles / ml EV of Chlorella MACC-360 or vehicle (PBS) only (DSS or DSS+MACC-360 group (n=8) respectively) every day. Changes in the disease activity index (DAI) were monitored throughout the experiment. At the end of treatment (after 7 days), mice were killed, and parietal peritoneal samples were collected and fixed in 4% buffered formaldehyde or quickly frozen and stored at -80°C until further molecular biological measurements.

[0711] Masson trichrome staining: Formalin-fixed peritoneum was embedded in paraffin and prepared into 3 μm thick sections. To evaluate the extent of peritoneal fibrosis, standard Masson trichrome histological staining was performed, and then tissue sections were photographed using an Olympus IX-81 microscope system (Olympus, Japan). Collagen-rich tissue areas (blue) were quantitatively measured using ImageJ 1.48v software. In the case of each section, the thickness of the submesothelial tissue was determined as the average of 10 independent measurements of non-overlapping areas.

[0712] Statistical analysis: Data were analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc., USA). Significance analysis was performed by unpaired t-test or Mann-Whitney test after testing normality by Kolmogorov-Smirnov test. In case of EV-EV and EV combination tests, two-way ANOVA was used to analyze differences. Results are presented as mean + SD of the corresponding groups. Applied tests, significance and number of elements (n) are indicated in each figure legend.

Claims

1. An algal extracellular vesicle (EVs) for use as an agent in the treatment of disorders.

2. Algal EVs for use in therapy according to claim 1, wherein the algal EVs are used in the treatment of disorders associated with excessive cell proliferation and / or migration and / or dysregulated extracellular matrix (ECM) production or deposition.

3. The algae EVs used in the therapy according to claim 2, wherein: The disorder is a proliferative / migratory disorder.

4. Algal EVs for use in the therapy according to any one of claims 1 to 3, wherein The disorder is a fibroproliferative disorder and / or the disorder is a dysregulation of ECM production / deposition.

5. Algal EVs for use in the therapy according to any one of claims 1 to 3, wherein The disorder is a neoplastic disorder, preferably a solid tumor.

6. Algal EVs for use in the therapy according to any one of claims 1 to 3, wherein The disorder is inflammation, Wherein, preferably, the inflammation is selected from the group consisting of - chemically induced inflammation, preferably oxidative stress induced inflammation, - Biologically induced inflammation - Mechanical stress-induced inflammation, -Inflammation as an advanced glycation endproduct (AGE)-related disorder, and EVs serve as anti-AGE agents.

7. Algal EVs for use in the therapy according to any one of claims 1 to 6, wherein The disorder is an advanced glycation end product (AGE)-related disorder, and the EVs are used as anti-AGE agents.

8. Algal EVs for use in the therapy according to any one of claims 1 to 7, wherein The EVs are used to inhibit the activity of a signaling pathway selected from the group consisting of -PDGF-BB-induced signaling pathway, -TGFβ-induced signaling pathways, - EGF-induced signaling pathways, and / or - a signaling pathway induced by a mixture of growth factors, including in particular PDGF-BB, TGFβ and EGF, -Signaling pathways induced by peritoneal dialysis effluent (PDE).

9. Algal EVs for use in the therapy according to any one of claims 1 to 8, wherein - the EVs are used to inhibit cell migration, preferably EGF or PDE-induced cell migration, and / or -The EVs are used to inhibit cell proliferation, preferably PDGF-BB or PDE-induced cell proliferation.

10. Algal EVs for use in the therapy according to any one of claims 1 to 9, wherein The EVs are used to regulate the production / deposition of ECM, preferably inhibit the production / deposition of ECM, preferably the production of ECM induced by TGFβ or PDE.

11. Algal EVs for use in the therapy according to any one of claims 1 to 10, wherein The EVs are used to regulate inflammation, preferably inhibit inflammation.

12. Algal EVs for use in a therapy according to any one of the preceding claims, wherein: The EVs are microalgae or macroalgae, particularly green algae.

13. Algal EVs for use in a therapy according to any one of the preceding claims, wherein: The EVs were derived from Chlorophyta, Streptophyta, Rhodophyta, Paleophyta, and Cyanobacteria.

14. Algal EVs for use in the therapy according to any one of claims 1 to 13, wherein The EVs are derived from Chlorella sorokinensis, Pseudomonas keslerii, Chlorella vulgaris, Chlorella pyrenoidosa (Chlorella pyrenoidosa), Chlamydomonas reinhardtii, Homiella verrucosa, Haematococcus lacustrine, Platymonas zhouli, Indigosporus diastereois, Klebsiella, Spirogyra, Bowei's algae, Ascophyllum nodosum, Drosophila palmatum, Microcystis aeruginosa, Nostoc lindenii, Spirulina platensis (Arthrospira platensis), Spirulina maxima (Arthrospira maxima), Annelidae africana, Synechococcus, and Aphanizomenon.

15. Algal EVs for use in a therapy according to any one of claims 2 to 14, wherein the disorder is a disorder selected from the group consisting of gastrointestinal disorders, skin disorders, lung disorders, kidney disorders, liver disorders and cardiovascular disorders and peritoneal disorders, In particular, neoplastic disorders, proliferative disorders, fibroproliferative disorders, inflammatory disorders and / or ECM production / deposition disorders.

16. A composition comprising algal extracellular vesicles (EVs) as an active agent for use in a method for treating a subject as defined in any one of claims 1 to 15.

17. A pharmaceutical composition comprising algal EVs as an active agent and a pharmaceutically acceptable excipient.

18. A nutraceutical composition comprising algal EVs as an active agent and a nutritionally acceptable excipient.

19. A composition according to any one of claims 16 to 18 comprising algal extracellular vesicles (EVs) as an active agent for use as an active agent in a method of treating a subject, wherein The composition includes a combination of EVs from multiple algae.

20. A composition according to any one of claims 16 to 19 comprising algal extracellular vesicles (EVs) as an active agent for use in a method of treating a subject, wherein: The composition comprises an additional non-EV active agent for treating a disorder associated with excessive cell proliferation and / or migration and / or dysregulated extracellular matrix (ECM) production or deposition, preferably a proliferation / migration disorder.

21. A cosmetic method comprising administering algal EVs as an active agent to the skin of a subject in need of inhibiting scarring of skin tissue or extracellular matrix (ECM) production, or Increases ECM production.

Images