Modified membrane vesicles as active agents in cell proliferative diseases

Abstract

The invention relates to modified membrane vesicles (mMVs) for use in inhibiting the activity of a growth fac- tor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway, wherein the mMVs are capable of binding the growth factor when the mMVs are administered to a subject, and wherein the mMVs are produced by a process comprising at least one non-naturally occurring step.

Description

[0001] MODIFIED MEMBRANE VESICLES AS ACTIVE AGENTS IN CELL PROLIFERATIVE DISEASES

[0002] FIELD OF THE INVENTION

[0003] The invention relates to modified membrane vesicles (rnMVs) for use in inhibiting the activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway, wherein the rnMVs are capable of binding the growth factor when the rnMVs are administered to a subject, and wherein the rnMVs are produced by a process comprising at least one non-naturally occurring step.

[0004] BACKGROUND OF THE INVENTION

[0005] Health-related applications of extracellular vesicles has become an intensively searched and quickly developing field. Extracellular vesicles (EVs) together with similar membrane compartments are often used as carriers for various active agents. Bacterial membrane vesicles (BMVs) have emerged as novel and promising platforms for the development of vaccines and immunotherapeutic strategies against infectious and noninfectious diseases. Besides being able to deliver active agents against infectious diseases, tumour antigens and other drug cargos, BMVs just like bacteria can activate trained immunity and may have an adjuvant effect in vaccines. (Qiong Long, Peng Zheng, Xiao Zheng, Weiran Li, Liangqun Hua, Zhongqian Yang, Weiwei Huang, Yanbing Ma, Engineered bacterial membrane vesicles are promising carriers for vaccine design and tumor immunotherapy, Advanced Drug Delivery Reviews, Volume 186, 2022, 114321)

[0006] K Lv et al. ((2022) Extracellular vesicles as advanced therapeutics for the resolution of organ fibrosis: Current progress and future perspectives. Front Immunol. 2022, 13 1042983) in their summary article on EVs derived from different cells of human origin discuss the development of engineered EVs may enhance therapeutic potency of native EVs). The authors suggest that native EVs can be re-engineered by biological methods and serve as advanced nanomedicines for the resolution of organ fibrosis.

[0007] The authors list three types of engineered EVs. i) Genetic engineering of parental cells is a common strategy to produce functional EVs. This idea supports the approach that the features of the parental cell are transmitted to EVs. For example, engineered hucMSC-EVs with antagomir-21a-5p showed higher potential in inhibiting fibroblast activation in vitro than native EVs. ii) Due to their bilayer structure and cargo transferring capacity, EVs can serve as natural carriers of therapeutic agents and protect them against in vivo degradation. In this regard mammalian MSC-EV-s are offered. iii) A third option is the use of EVs from preconditioned cell, like those preconditioned with hypoxia or cytokines to acquire and retain phenotypes relevant for therapeutic applications. This is a concept which further supports that EVs are viewed as carriers of effective molecule from their parent cells.

[0008] Taken together, in the prior art, engineered membrane vesicles are generally mentioned as promising carriers, the production of which is more manageable than that of naturally occurring vesicles. When the medical effect of the EV themselves are mentioned, in general a skilled person concludes that the effect of such EVs is due to the biomolecules they carry.

[0009] SUMMARY OF THE INVENTION

[0010] The invention relates to modified membrane vesicles (artificial (or engineered) membrane vesicles) for use in the treatment of a disease as defined herein, in particular a cell proliferative disease or preferably a fibroproliferative disease or preferably neoplasm. Modified membrane vesicles (mMVs) are provided for use in inhibiting the effect or activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway.

[0011] A method for inhibiting the effect or activity of a growth factor, a receptor of a growth factor or modulating, preferably inhibiting the activity or effect of a growth factor mediated signaling pathway (s) is provided, the method comprising administering m Vs to a subject in need thereof.

[0012] Modified membrane vesicles (mMVs) are provided for use in the treatment of a cell proliferative disorder.

[0013] A method for the treatment of a cell proliferative disorder is provided, the method comprising administering mMVs to a subject in need thereof.

[0014] A composition comprising mMVs is provided. Preferably the composition is for use in inhibiting the activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway(s). Preferably the composition is for use in the treatment of a cell proliferative disorder.

[0015] The invention relates to modified membrane vesicles obtainable by mixing isolated lipids of a human cell and isolated lipids of an algal cell, a bacterial cell or a yeast cell (hybrid lipid mMV) under conditions allowing the formation of vesicles. Preferably the hybrid lipid mMV is for use in inhibiting the effect or activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway or for use in the treatment of a cell proliferative disease.

[0016] The invention relates to modified membrane vesicles obtainable by combining membrane components of a first cell and membrane components a second cell, wherein the first and the second cells are from different organisms, preferably from organisms belonging to different taxonomical units (e.g. species), e.g. the first cell is a human cell and the second cell is an algal cell or the first cell is from a human subject and the second cell is from a different human subject.

[0017] The invention relates to modified membrane vesicles obtainable by co-extruding extrudates of a human cell and extrudates of an algal cell, a bacterial cell or a yeast cell (hybrid extrudate mMV). Preferably the hybrid extrudate mMV is for use in inhibiting the effect or activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway or for use in the treatment of a cell proliferative disease.

[0018] The invention relates to lipid mMVs comprising a protein corona. Preferably the lipids are of non-human (e.g. alga) origin and the corona is of human origin (e.g. human serum albumin or of human serum origin).

[0019] The invention relates to human, algal yeast, or bacterial EVs comprising a human protein corona or a protein corona comprising a human protein, preferably algal, yeast or bacterial EVs comprising a human protein corona or a protein corona comprising a human protein.

[0020] EVs are provided for use in the treatment of a cell proliferative disease or preferably neoplasm or preferably a fibroprotiferative disease or for use in inhibiting the effect or activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway, wherein the EVs are derived from human MSCs, IPSs, yeast cells, preferably Kluyveromyces sp. cells, preferably Kluyve- romyces lactis cells, preferably Saccharomyces sp. cells, preferably Saccharomyces cerevisiae cells, in particular fromBY4741 cells, preferably Saccharomyces pombe cells, preferably Saccharomyces pastorianus cells, prefera- bly Yarrowia sp. cells, preferably Yarrowia lipolytica cells, bacterial cells, in particular Escherichia coli or Lactobacillus sp. cells (preferably Lactobacillus bulgaricus) or Lactococcus sp. cells (preferably Lactococcus lactis) or Cyanobacteria cells, preferably Spirulina cells, in particular MACC-908 cells.

[0021] Preferably the EVs are from yeast cells, preferably Saccharomyces cerevisiae cells, preferably from BY4741 cells, preferably Saccharomyces pombe cells, preferably Saccharomyces pastorianus cells, preferably Kluyveromyces lactis cells preferably Yarrowia sp. cells, preferably Yarrowia lipolytica cells.

[0022] Preferably the EVs are from bacterial cells, in particular Escherichia coli or Lactobacillus sp. cells (preferably Lactobacillus bulgaricus) or Lactococcus sp. cells (preferably Lactococcus lactis).

[0023] Preferably the EVs are from Cyanobacteria cells, preferably Spirulina cells, in particular MACC-908 cells. Preferably the EVs are from algal cells, preferably Chlorella sp. cells, preferably Chlorella vulgaris cells. Preferably the rnMVs are capable of binding the growth factor. Preferably the rnMVs are capable of binding the growth factor present in the extracellular space when the rnMVs are administered to a subject.

[0024] Preferably the rnMVs are produced by a process comprising at least one non-naturally occurring step.

[0025] Preferably the rnMVs are selected from the group consisting of i) a synthetic MV, wherein the synthetic MV is an MV synthesized de novo from molecular components, ii) an artificial, cell derived MV wherein the artificial, cell derived MV is an MV produced by a process comprising a non-naturally occurring step of disrupting a cell, iii) a lipid MV, wherein the lipid MV is an MV produced by a process comprising a non-naturally occurring step of lipid isolation, iv) an impaired MV, wherein the impaired MV is an MV produced from a cell or a native extracellular vesicle (nEV) by a process comprising at least one non-naturally occurring step that results in

[0026] - a decrease in the diversity of components or a decreased amount of a component of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, or

[0027] - a decrease in the diversity of biologically active components or a decreased amount of a biologically active component of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, or

[0028] - the loss of a biological function of a component of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, v) an induced MV, wherein the induced MV is an MV obtained from a cell by a process comprising at least one non-naturally occurring step that results in

[0029] - a decrease in the diversity of components or a decreased amount of a component of the mMV compared to the cell, or

[0030] - a decrease in the diversity of biologically active components or a decreased amount of a biologically active component of the mMV compared to the cell, or

[0031] - the loss of a biological function of a component of the mMV compared to the cell, or vi) a supplemented MV, wherein the supplemented MV is an MV obtained from an nEV or a cell by a process comprising at least one non-naturally occurring step that results in

[0032] - an increase in the diversity of components or an increased amount of a component of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, or - an increase in the diversity of biologically active components or an increased amount of a biologically active component of the mMV compared to the nEV obtained by the same process lacking the at least one non-naturally occurring step, or

[0033] - gain of a biological function of a component of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, vii) a hybrid of nEVs derived from different species, viii) a combination of rn Vs or a combination of nEV(s) and rnMV(s).

[0034] Preferably the rnMVs are selected from ii) an artificial, cell derived MV, wherein the artificial, cell derived MV is an MV produced by the extrusion of the cell, iii) a lipid MV, wherein the lipid MV is produced by a process comprising a non-naturally occurring step of lipid isolation from a cell, followed by vesicle formation, iv) an impaired MV, wherein the impaired MV is depleted in functional proteins, v) an induced MV, wherein the induced MV is a giant plasma membrane vesicle, vii) a hybrid or combination of any one of ii)-v).

[0035] Preferably the rnMVs are selected from a lipid mMV, an impaired MV, an artificial, cell derived MV, an induced MV and hybrids / combinations thereof, preferably selected from a lipid mMV, an impaired MV and any hy- brids / combinations thereof, preferably a lipid MV and an impaired MV depleted in functional proteins and any hybrids / combinations thereof, preferably a lipid MV depleted in functional proteins and an impaired MV depleted in functional proteins and any hybrids / combinations thereof.

[0036] Preferably the rnMVs are selected from a lipid MV, wherein the membrane of the lipid MV essentially lacks functional protein components, an impaired MV, wherein the membrane of the impaired MV essentially lacks functional protein components, a supplemented MV, which is preferably a lipid MV with a membrane essentially lacks functional protein components and which is supplemented with a human polypeptid, lipid or polysaccharide, wherein the human polypeptide is not a receptor of the growth factor, a hybrid or combination thereof.

[0037] Preferably the rnMVs are lipid MVs.

[0038] Preferably the membrane of the rnMVs essentially lacks functional protein components.

[0039] Preferably the rnMVs essentially lack functional (active) protein components.

[0040] Preferably the rnMVs are artificial, cell derived MVs, preferably MVs obtained by the extrusion of cells, preferably MVs designated exMVs herein

[0041] Preferably the rnMVs are induced MVs, preferably giant plasma membrane vesicles, preferably MVs designated as gMVs herein.

[0042] Preferably the cell proliferative disorder is selected from a neoplasm or a fibroproliferative disease.

[0043] Preferably the cell proliferative disorder is a fibroproliferative disease.

[0044] Preferably the rnMVs are for use in inhibiting ECM production, preferably collagen production.

[0045] Preferably the rnMVs are for use in inhibiting PDGF, CTGF, TGF, EGF, FGF or PDE induced collagen production, preferably TGF or PDE induced collagen production.

[0046] Preferably the rnMVs are for use in inhibiting cell migration, preferably EGF or PDE induced cell migration. Preferably the mMVs are for use in the treatment of a disease associated with cell proliferation, cell migration and / or dysregulated extracellular matrix (ECM) formation.

[0047] Preferably the mMV s are for use in the therapy of a fibroproliferative disease by the inhibition of ECM production. Preferably the mMVs are for use in the therapy of a fibroproliferative disease by the inhibition of cell proliferation and migration.

[0048] Preferably the growth factor is selected from PDGF, TGF, EGF, FGF, preferably the growth factor is selected fromPDGF and TGF, preferably the growth factor is PDGF -B, preferably PDGF-BB. Preferably the growth factor is TGF-B. Preferably the growth factor is EGF. Preferably the growth factor is FGF-1.

[0049] Preferably the mMVs are free of an added active agent for use in inhibiting the activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway.

[0050] Preferably the mMVs are for use in inhibiting cell proliferation, preferably PDGF, CTGF, TGF or PDE induced cell proliferation, preferably PDGF-B (PDGF-BB) or TGF-B induced cell proliferation.

[0051] Preferably the mMVs are free of an added active agent for use in the treatment of a cell proliferative disorder.

[0052] Preferably the mMVs are free of an added active agent for use in the treatment of a cell migration / ECM production disorder.

[0053] Preferably the mMV is of mammalian, preferably human, algal, bacterial or of yeast origin. Preferably the mMV is of algal, bacterial or of yeast origin. Preferably the MV is of human or algal origin.

[0054] Preferably the composition is a pharmaceutical composition and comprises at least one pharmaceutically acceptable excipient.

[0055] Preferably the mMVs are comprised in a composition consisting essentially of the mMVs as active agents and one or more additional substance useful as carrier.

[0056] Pharmaceutical combination comprising the mMVs or the composition consisting essentially of the mMVs as active agents and at least one pharmaceutically acceptable excipient. A kit comprising the mMVs, the pharmaceutical combination or the composition consisting essentially of the mMVs as active agents and a further active agent. Preferably the further active agent is selected from the group consisting of antifibrotic agents, anticancer agents, anti-inflammatory agents, inhibitors of a PDGFR, inhibitors of EGF, inhibitors of TGF (preferably TGFP), and inhibitors of FGF (preferably FGF-1). Preferably the pharmaceutical combination, the composition consisting essentially of the mMVs as active agents or the kit comprises more than one type of mMV.

[0057] Preferably the mMV is free of added cargo. Preferably the term cargo refers to compounds present inside of the mMV.

[0058] Preferably the innate polypeptides / proteins of the mMV are impaired. Preferably the mMV comprises a low amount of functional polypeptides / proteins or essentially lacks functional polypeptides / proteins. Preferably the mMV comprises a low amount of active polypeptides / proteins or essentially lacks active polypeptides / proteins. Preferably the protein content of the mMV is decreased, not detectable by a protein assay. Preferably the innate proteins or the proteins in the mMV are inactivated or essentially inactivated, heat treatment, heat-thaw cycles, high-shear force, e.g. by grinding, extrusion or homogenization, by chemical means, by peptidase, e.g. protease digestion or treatment, in a preferred embodiment by trypsin treatment. Preferably the polypeptides / proteins are impaired by the manufacturing process, preferably by heat treatment or peptidase or protease treatment or by the at least one non-naturally occuring (artificial manipulation step). Preferably the mMVs are not for use as a drug delivery device.

[0059] BRIEF DESCRIPTION OF THE FIGURE

[0060] Figure 1. Protein content of the different type of EVs. Protein analysis of nanoparticle-samples by (a) silver staining, (b) UV-spectrophotometry, (c) protein assay. Sample IDs: 0 - Molecular weight marker; 1 and 3 - lipid extract (IMVs) of primary human peritoneal fibroblast (pFB); 2 and 4 - trypsin treated IMVs (t lMVS) of pFB; 5 - IMVs of MACC-360; 6 - tJMVs of MACC-360; 7 - native EV (nEV) of MACC-360 origin; 8 - nEV of mesenchymal stem cells (MSCs) of the peritoneal dialisate; 9 - giant plasma membrane vesicle giant MVs oof pFB; 10 - nEVs isolated from the peritoneal effluent of a patient with peritoneal dyalisis; 11 - MACC-360 extrudate (exMV of MACC-360).

[0061] DEFINITIONS

[0062] The term “membrane vesicles” („MV”-s) refers to objects which cannot replicate on their own and are delimited from their surroundings by a lipid layer.

[0063] „Native extracellular vesicles” (nEVs) are particles naturally released from cells, are delimited by a lipid bilayer and are generated by a process that comprises only naturally occuring steps, apart from the step of isolation of the native extracellular vesicles from their natural environment. The term „native extracellular vesicles” may refer to native extracellular vesicles as defined in DOI: 10.1002 / jev2.12404. The term „native extracellular vesicles” may refer to particles that are naturally released as exosomes or microvesicles from cells.

[0064] „Modified membrane vesicles” (mMVs) are membrane vesicles obtained by a process that comprises at least one non-naturally occuring step, i.e. an artificial manipulation step, wherein the at least one non-naturally occuring step is not a step of isolating a membrane vesicle from the medium it was generated in. Preferably the non-naturally occurring step is not a step of adding a component capable of binding a protein, in particular a growth factor to the MV. Preferably modified membrane vesicles are obtained from a cell. Preferably the process of obtaining the modified membrane vesicle does not comprise steps before the provision of a cell from which the modified membrane vesicle is obtained.

[0065] The term modified membrane vesicles may refer to EV mimetics as defined in DOI: 10.1002 / jev2.12404 page 5 2.2; table 2 (EV mimetic): An EV mimetic is an EV-like particle that is produced through direct artificial manipulation.

[0066] “Biological membranes” are lipid bilayer membranes composed of amphiphilic phospholipids, i.e. amphiphilic phospholipids are decisive components in forming a phospholipid bilayer structure whereas in addition to the phospholipid bilayer structure other essential membrane constituents like membrane proteins and sugars, or carbohydrates, glycolipids, glycoproteins ant / or sterols are also present.

[0067] “Membrane polypeptides”, e.g. proteins play a vital role in biological membranes, as they help to maintain the structural integrity, organization and flow of material through membranes. Sugars are found on the outer side of the biological membrane bilayers only and may be attached by covalent bonds to certain lipids and proteins. It is generally believed that membrane polypeptides and possible sugars play an essential part in MV activity.

[0068] The terms “polypeptide” and “protein” may be used interchangeably herein.

[0069] “Extracellular vesicles” (EVs) are membrane vesicles released from a cell. In a wording EVs are membrane vesicles of natural origin produced by natural organisms. “Exosomes” are extracellular vesicles which are membranous vesicles of endocytic origin (about 30-200 nm), however, are present outside the cell. “Exosomes” are EVs from internal compartments of the cell that are released via the multivesicular body.

[0070] “Microvesicles” are vesicles of various size (100-1000 nm in diameter) that bud directly from the plasma membrane and are shed to the extracellular space.

[0071] “Plasma membrane vesicles” are membrane vesicles which are formed from cellular plasma membranes. In a particular embodiment plasma membrane vesicles are engineered membrane vesicles (engineered plasma membrane vesicles). In another embodiment plasma membrane vesicles are extracellular vesicles.

[0072] “Artificial membrane vesicles” are membrane vesicles which are formed by human interaction. “Artificial membrane vesicles” are preferably formed according to the invention from biological membranes.

[0073] “Engineered membrane vesicles” as used herein are formed according to descriprion, in particular the examples from biological membranes by engineering method. In an embodiment engineered membrane vesicles are preferably formed from biological membranes, preferably cellular membranes. In a preferred embodiment engineered membrane vesicles are manufactured from biological membranes (“manufactured membrane vesicles”). Also engineered membrane vesicles may be prepared from natural membranes vesicles by processing such vesicles (“processed membrane vesicles”). Examples for engineered membrane vesicles of biomembrane origin are giant plasma membrane vesicles, extruded membrane vesicles, membrane vesicles obtained by lipid extract and peptidase- treated variants thereof (peptidase treated artificial membrane vesicle of biomembrane origin), e.g. peptidase- treated lipid extract. “Engineering method” refers to a method comprising at least one step that does not occur in nature (i.e. without human intervention) during the formation of a membrane vesicle. The result of an engineering method is a membrane vesicle that does not occur in nature (i.e. without human intervention). “Nature” in some cases includes an in vitro cell culture. “Engineered membrane vesicles” and “modified membrane vesicles” are used interchangeably herein. In some cases “modified membrane vesicles” do not include fully artificial vesicles, ie. synthetic vesicles.

[0074] “Extrusion” as used herein is broadly understood herein as grinding or cutting soft substance (i.e. a substance which is suitable for such cutting, grinding, homogenization or sonication) into small pieces and / or forcing soft substance through an opening or multiple openings. Optionally further steps like sonication, freeze-thaw, optionally in multiple cycles, ultrafiltration and concentration are applied.

[0075] Extruded membrane vesicles may be prepared from various sources like algae or plants or any other soft substance. “Giant plasma membrane vesicles (GPMVs)” are engineered membrane vesicles obtained from cells by incubating them in a buffer containing PF A, and DTT, particularly preferably containing, as an example, HEPES, NaCl, CaC12, pH 7.4 PF A, and DTT. GPMVs may be isolated by any alternative method resulting in such vesicles. GPMVs may be harvested by centrifugation the supernatant of the cells

[0076] A “lipid extract” is an engineered membrane vesicle obtained by a lipid isolation method from a biomembrane and comprising enriched level of lipids; preferably obtained by treatment of the cells with alcohol, preferably isopropanol; and preferably by harvesting the vesicles, pelleting and resuspending them in alcohol, preferably an isopro- panol-methanol mixture or chloroform, then lysing the cells, e.g. by ultrasound, then obtaining the lipid extracts from the supernatant and optionally by selecting the so obtained lipid extracts by size.

[0077] Taken together from a biological membrane the engineered membrane vesicle, optionally manufactured or processed membrane vesicle can be prepared by physical methods, like extrusion as defined herein, lipid isolation, or, optionally additionally, heat treatment by chemical methods, like preparing GPMVs, and / or additionally biochemical method, like enzymatic treatment, e.g. protease (or peptidase) treatment or RNase treatment or glucosidase treatment, in particular protease (or peptidase) treatment.

[0078] A peptidase-treated or peptidase-digested lipid extract is obtained by treating the lipid extract by a peptidase. Preferably a peptidase-treated or peptidase-digested lipid extract is a trypsin-treated lipid extract.

[0079] Definition of disease conditions

[0080] A “cell proliferation-related disorder” is a disorder characterized by unwanted cell proliferation and / or excessive cell proliferation.

[0081] In one aspect of the embodiment, the cell proliferation disorder is a neoplastic disorder, e.g. a pre-cancer or cancer. In another aspect of the embodiment, the cell proliferation disorder is a hyperproliferative disorder.

[0082] In another embodiment, prevention or treatment of the cell proliferation disorder, cancer or hyperproliferative disorder occurs through the binding of a growth factor by the membrane vesicle according to the invention in the extracellular space.

[0083] In a preferred embodiment the disease is one related to the growth factor receptor signalling. In an embodiment the growth factor is selected from PDGF and TGF, preferably PDGF-beta and TGF-beta, in particular PDGF-beta. A “neoplasm” is a type of abnormal and excessive growth of tissue / cells. (The process that occurs to form or produce a neoplasm is called “neoplasia”.) The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists in growing abnormally, even if the original trigger is removed. This abnormal growth usually forms a mass, when it may be called a tumour. Tumour cells often metastasize to various organs. “Progressive fibrosis” in short is characterized by a process, wherein fibroblast proliferate, migrate and produce extracellular matrix, ECM remodeling is shifted towards accumulation of ECM producing cells, like fibroblasts or myofibroblasts, and / or towards non-physiological, e.g. excessive deposition of ECM components leading to impairment or destruction of tissue architecture and / or to gradual decline of organ function, which is a kind of non- physiological ECM production / deposition.

[0084] Progressive fibrosis may lead to the formation of permanent scar tissue, may cause tissue or organ failure and might lead to death. In “progressive fibrosis” ECM components and ECM producing cells, in particular fibrillar ECM components like type I and III collagen and fibronectin, as well as the cells producing them continue to accumulate even beyond the homeostatic / regenerative phase of ECM remodeling.

[0085] The process in which an excessive amount of ECM replaces normal parenchyma or the ECM which is typical to the tissue affected by progressive fibrosis may also be considered “progressive fibrosis”. This process is characterized by overproliferation of ECM producing cells, e.g. fibroblasts, and excessive, unregulated or dysregulated deposition of ECM components and / or abnormal repair processes in different tissues upon injury.

[0086] Collagen accumulation can be measured by Sirius Red assay.

[0087] “Fibroproliferative disorder” is a disorder which is characterized by inter alia the presence of progressive fibrosis, in particular wherein at least partially ECM remodeling is shifted towards accumulation of ECM producing cells, like fibroblasts, and / or towards excessive deposition of ECM components leading to impairment or destruction of tissue architecture and / or to gradual decline of organ function.

[0088] “Deposition of ECM” is understood herein as a process leading to an increase in the amount of ECM components in a space among / between (i.e. outside) the cells of a tissue. A “physiological” or “regulated” or “normally regulated” deposition of ECM occurs when deposition of ECM components serves to restore tissue architecture and / or tissue function itself. The physiological deposition of ECM components is regulated or maintained under control to avoid non-physiological ECM deposition or production. Preferably, in in vivo setting the regulatory processes of the surrounding healthy tissue in question counter-acting deposition are capable of reversing, or at least arresting such deposition.

[0089] A “non-physiological” or “dysregulated” deposition of ECM occurs when deposition of ECM components leads to impairment, i.e. destruction of tissue architecture and / or tissue function itself. The unregulated or abnormally regulated deposition of ECM components is a particular hallmark of non-physiological ECM deposition or production. Preferably, deposition of ECM components is considered as “non-physiological” (in case of fibrosis excessive) when there are no signs that regulatory processes of the surrounding healthy tissue in question counteracting deposition are capable of reversing, or at least arresting such deposition.

[0090] A “treatment” refers to any process, action, application, therapy, or the like, wherein the subject or patient is under aid, in particular medical or veterinarian aid with the object of improving the subjects’s or patient’s condition, either directly or indirectly. Improving the subjects’s condition may include improving an aesthetic condition (cosmetic treatment) and / or may include, in particular, restoring or maintaining normal lunction of an organ or tissue, preferably at least partly restoring or maintaining health (medical or veterinarian treatment). Treatment typically refers to the administration of an effective amount of a compound or composition described herein. Treatment may relate to or include medical or veterinarian treatment and cosmetic treatment, in particular medical or veterinarian treatment. Treatment may be prophylactic.

[0091] “Preventing” or “prevention” of the development of a disease or condition refers to at least the reduction of likelihood of the risk of or susceptibility to acquiring a disease or disorder, or preferably causing at least one of the clinical symptoms of the disease or disorder not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease.

[0092] The terms “effective amount” or “therapeutically effective amount” are intended to qualify the amount of a therapeutic agent required to relieve to some extent one or more of the symptoms of a condition, disease or disorder, including but not limited to: 1) reducing the number of fibroblasts or myofibroblasts ; 2) reducing the synthesis of the ECM components, and / or increasing the degradation of the ECM component; 3) reducing the size of the fibrous tissue; 4) improving to at least some extent the physiological function of the tissue due to any of 1) to 3); 5) reducing the size of a tumour tissue; 6) inhibits the formation of tumour cell metastases; 7) inhibits immune cell proliferation or activation, including production of cytokines, growth fators or antibodies.

[0093] The provided modified MVs have pharmaceutical (medicinal), nutritional, and cosmetic uses as well.

[0094] The term “comprises” or “comprising” or “including” are to be construed here as having a non-exhaustive meaning and allow the addition or involvement of further features or method steps or components to anything which comprises the listed features or method steps or components. “Comprising” can be substituted by “including” if the practice of a given language variant so requires or can be limited to “consisting essentially of’ if other members or components are not essential to reduce the invention to practice. Optionally “consisting essentially of’ can be replaced and limited to “consisting of’.

[0095] ABBREVIATIONS

[0096] A549 Human lung carcinoma

[0097] DETAILED DESCRIPTION OF THE INVENTION

[0098] Anti-proliferative effect of modified membrane vesicles Surprisingly, the present inventors have observed that different MVs, including exMV, gMV, 1MV, nEV, tJMV, t nEV, Co exMV, CoJMV, Ch lMV, HASJMV of MSC, IPS, pFB, cFB, RBC, A549, HCT-116, HK-2, MACC- 1, MACC-3, MACC-360, MACC-908, Chlorella vulgaris powder, Spirulina platensis powder, BY4741, K. lactis, S. pastorianus, S. pombe, Y. lipolytica, L. bulgaricus, L. lactis, E. coli, Pseudomonas aeruginosa, and wheat grass origin altered the PDGF-BB induced proliferation of pFB, cFBs, sFBs and IFBs.

[0099] Modified membrane vesicles from different sources, namely lipid extract of MACC-360, trypsin treated lipid extract of MACC-360, lipid extract of pFB, trypsin treated lipid extract of pFB significantly enhanced the effect of nintedanib to reduce PDGF-B induced proliferation (MTT assay) of pFB. Nintedanib, which also acts on PDGF- receptor, is used as an antiproliferative and antifibrotic agent. The results show that the modified membrane vesicles have an antiproliferative effect additional to and perhaps similar to nintedanib.

[0100] Modified membrane vesicles, namely lipid extract of pFB significantly enhanced the effect of Axitinib and Ponatinib to reduce PDGF-B induced proliferation.

[0101] Binding of PDGF-B and TGF-B to modified membrane vesicles

[0102] Fluorescence resonance energy transfer (FRET) revealed that native EVs from different sources, namely from MSCs, MACC-360 EV, Spirulina platensis powder, BY4741 EV, E.coli, and MVs from pFBs (1MV, t lMV, gMV) and MACC-360 (1MV) bound PDGF-B, and native EVs from Spirulina platensis powder, BY4741 EV, E.coli bound TGF-B.

[0103] Optical waveguide lightmode spectroscopy (OWLS) revealed that nEVs originated from MSCs, MACC-360 and IMVs originated from MACC-360 bound EGF, FGF-1, HSA, PDGF-BB, Human serum, and TGF-B. It was also demonstrated that 1_MV of MSC, Chlorella vulgaris and Spirulina platesnis powder bound EGF, FGF-1, HSA, and PDGF-BB and that 1_MV and HSA 1MV originated from Chlorella vulgaris powder bound TGF-B mMVs inhibit collagen production

[0104] Different MVs, including exMV, gMV, 1MV, nEV, t_nEV, CoJMV of MSC, IPS, pFB, cFB, RBC, A549, HCT- 116, HK-2, MACC-360, MACC-908, Chlorella vulgaris powder, Spirulina platensis powder, BY4741, K. lactis, S. pastorianus, S. pastorianus, S. pombe, Y. lipolytica, E. coli origin altered the TGF-P induced collagen production of cFBs and sFBs. mMVs inhibit EGF or PDE induced migration

[0105] IMVs of MACC-360, Spirulina platensis powder, K. lactis, Y. lipolytica, L. lactis, L. bulgaricus origin altered EGF or PDE induced migration of cFBs.

[0106] Lipid extracts (IMVs) contain no detectable protein

[0107] Independent multiple analysis showed that modified membrane vesicles from lipid extracts contain no detectable protein (protein analysis of nanoparticle-samples by (a) silver staining, (b) UV-spectrophotometry, (c) protein assay). Analysis was carried out by lipid extract of primary human peritoneal fibroblast, trypsin treated lipid extract of the same primary human peritoneal fibroblast, as well as lipid extract from algae (MACC-360), trypsin treated lipid extract of the same algae; and, MACC-360 extrudate. This shows that modified membrane vesicles do not have the same cargo content as native extracellular vesicles. In particular, it was found that protein content and / or quality of the original biological membranes is impaired by the engineering process.

[0108] Internalization of modified membrane vesicles of pFB and cFB cells

[0109] Lipid extract of pFB were successfully internalized by pFB and primary human colon fibroblast (cFB), as well. Traditionally it is assumed that the favorable effects of native EVs are due to their cargo and there is a general assumption in the art that polypeptide, in particular protein cargo of the EV plays an essential part. The cargo of a native (naturally occuring) EV is a product of the natural processing an EV goes through in the organism and the characteristics (e.g. type of protein) of the cargo are determined by e.g. the cell of origin.

[0110] The present inventors have surprisingly found that modified MVs (artificial membrane vesicles, in particular engineered membrane vesicles from membranes of biological origin) are capable of binding growth factors PDGF- beta and / or TGF-beta (as shown among others by FRET and OWLS measurements) and thereby reduce the level of such factors in the intercellular space.

[0111] The reduction of the levels of PDGF-beta and / or TGF-beta results in a neutralization effect, i.e. a reduced binding of the growth factors to their respective receptors.

[0112] The PDGF-beta and / or TGF-beta binding activity of the modified membrane vesicles neutralizes the effect of PDGF-beta and / or TGF-beta which, i.e. their effect on their respective membrane receptors are reduced. Thus, this neutralization effect is, as to its result, is inhibitory, i.e. it is equivalent with or at least similar to the effect of an inhibitor acting directly on the respective receptor.

[0113] Reduced binding of PDGF-beta and / or TGF-beta on their respective receptors results in an adjustment of the downregulation of the PDGF-beta and / or TGF-beta signalling pathways. By this “clean-up” process wherein these growth factors are sequestered and removed from the intercellular space, their effect on their receptors is lowered. Thereby for example the cell proliferation-inducing, fibrosis-inducing property of a growth factor such as PDGF- beta or TGF-beta is reduced when bound to the modified membrane vesicles. The same is true for other effects of these factors. These effects have been shown by the inventors by experiments for cell proliferation (MTT assays), cell migration (TAS) and ECM deposition.

[0114] Reasonably other growth factors including PDGF-A, PDGF-C, PDGF-D, VEGF-A, VEGF-B, VEGF-C, VEGF- D, and PIGF containinc growth factor core domain, in particular PDGF-alpha having a similar structure to PDGF- beta (in particular its mature form) will show the same phenomenon. PDGF-alpha and PDGF-beta form both homodimers and heterodimers with each other. Therefore, through its presence in the heterodimer, the effect of PDGF-alpha (PDGFA) may also be affected via the lowered level of PDGF-beta (PDGFB). Moreover, PDGF-BB acts on both PDGFR-A and PDGFR-B. In particular, modified membrane vesicles bind growth factors mediating a cell proliferative disease or preferably a fibroproliferative disease. By said growth factor binding the level of said growth factor(s) are lowered in the extracelluar space which results in a lowered activity on their respective receptors. For example an increased binding of PDGF-beta by the modified membrane vesicles results in a lowered effect on the PDGF-beta receptor and thereby an improvement in the PDGF-beta-mediated condition, e.g. disease. PDGF-beta, TGF-beta, FGF-1, EGF like other growth factors, exert their effects via their receptors in cell membranes. According to this aspect, the invention is related to the surprising discovery that membrane vesicles of biomembrane origin (or modified membrane vesicles), even if artificially processed or manufactured are capable of binding growth factors, in particular PDGF-beta and / or TGF-beta. In a particular embodiment PDGF-beta is bound by the membranes of the modified membrane vesicles. In another embodiment TGF-beta is bound by the membranes of the modified membrane vesicles.

[0115] The effect of PDGF-beta and TGF-beta on proliferation and fibrotic pathways Growth factors, such as platelet-derived growth factor BB (PDGF-BB) and transforming growth factor p (TGF ), are key regulators of cellular functions, including proliferation, migration, and differentiation. Growth factor signalling is modulated by context-dependent cross-talk between different signalling pathways. Regulation of pro- fibrotic cytokines PDGF-BB and TGF is interrelated and their connected and similar role in cancer has also been observed. For example, TGF induces phosphorylation of Smad2, a downstream mediator of the canonical TGF pathway, in primary dermal fibroblasts.

[0116] Autocrine activation of PDGF signalling pathways is involved in certain gliomas, sarcomas, and leukaemias. Paracrine PDGF signalling is commonly observed in epithelial cancers, where it triggers stromal recruitment and may be involved in epithelial-mesenchymal transition, thereby affecting tumor growth, angiogenesis, invasion, and metastasis. PDGF s drive pathological mesenchymal responses in vascular disorders, as well as in fibrotic diseases, including pulmonary fibrosis, liver cirrhosis, scleroderma, glomerulosclerosis, and cardiac fibrosis. PDGF signalling has been implicated in several fibrotic conditions and is assumed to play a role in driving proliferation of cells with a fibroblast phenotype. Tissue fibrosis also involves excessive deposition of extracellular matrix, which may in part reflect increased PDGF activity, but likely also increased TGFp function. Together, excessive mesenchymal cell proliferation and matrix deposition lead to tissue scarring and progressive loss of organ function. PDGF-beta has been found in particular as an important pro-fibrotic factor. Different PDGF isoforms, including PDGF-BB and also other factors such as TGFp play a significant role in the activation of fibroblasts, such as their proliferation, migration, and the production of the ECM.

[0117] The transforming growth factor-p (TGF-P) family of proteins comprises secreted, homodimeric and heterodimeric proteins, and controls the differentiation. Enhanced TGF-P signalling contributes to cancer and fibrosis. The activation of the TGF-P signalling pathway initiates collagen accumulation that can lead to progressive fibrosis, which has earned TGF-P the title of “the master regulator of fibrosis”. In fact, transforming growth factor beta 1 (TGF- pi), was found to be the most potent inducer of pathological fibrosis. TGF-pi level correlates with collagen deposition.

[0118] Ishmaeel A et al. (Endothelial cell-derived pro-fibrotic factors increase TGF-pi expression by smooth muscle cells in response to cycles of hypoxia-hyperoxia. Biochim Biophys Acta Mol Basis Dis. 2022 Jan 1;1868(1):166278) have found in a normoxia-hypoxia-hyperoxia model that in an in vitro model of ischemia / reperfusion increased secretion of pro-fibrotic paracrine factors PDGF-BB (and CTGF) by endothelial cells was predominantly driving TGF-P 1 -mediated expression by smooth muscle cells. They confirmed that cycles of ischemia / reperfusion (I / R) would induce fibrosis by oxidative damage to endothelial cells, and the released pro-fibrotic growth factors act to increase dedifferentiation of SMCs to a synthetic phenotype that releases TGF-pi. The authors suggest that targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of I / R. TGF-pi is a pleiotropic cytokine that activates hepatic stellate cell (HSC) proliferation but inhibits parenchymal cell proliferation. Shah R. et al. (TGF-pi up-regulates the expression of PDGF-P receptor mRNA and induces a delayed PI3K-, AKT-, and p70(S6K) -dependent proliferative response in activated hepatic stellate cells. Alcohol Clin Exp Res. 2013 Nov;37(ll): 1838-48.) have found that the action of TGF-P 1 involves the activation of PDGF - P receptor via the PI3K / AKT / p70(S6K) signalling pathway and suggest that TGF-pi-mediated oxidative stress causes the transdifferentiation of HSC and primes them for extracellular matrix (ECM) deposition and scar contraction. The authors conclude that liver injury up-regulates TGF-pi that inhibits parenchymal cell proliferation but stimulates HSC proliferation leading to the production of ECM and type I collagen resulting in fibrosis. These growth factors and the fibroblasts also play a role in the development of different tumours and metastasis of them. The pathophysiological roles of cancer-associated fibroblasts (CAFs) in the heterogeneous tumour microenvironment have attracted increasing interest. CAFs play crucial roles in tumour progression and the response to chemotherapy.

[0119] The mechanism of fibrosis is the same or very similar in any tissue or organ. Thus, taken together, the modified membrane vesicles are expectably useful in the treatment of fibrosis or of fibroproliferative disease of any tissue or organ of a subject.

[0120] The modified membrane vesicles used herein were found to inhibit cell proliferation in MTT assays. The membrane vesicles significantly reduced fibroblast proliferation and thus provided an antifibrotic effect.

[0121] In a particular embodiment the modified membrane vesicles are modified to have an altered, impaired innate cargo (e.g. proteome) in comparison with a native EV. In the modified membrane vesicles polypeptide, in particular protein cargo is affected and changed, in particular impaired. Such impairment may be due to a kind of denaturing effect like heating or mechanical forces upon grinding, freeze-thaw or extrusion. Enzymatic effect like treatment with a protease, in particular trypsin is also a method to impair the proteome of the starting membrane vesicle upon modifying. Moreover, in certain modifying methods, the protein content is greatly reduced, like in case of lipid extract preparations. The term cargo refers to compounds inside the MV, such as an added active agent (e.g. a small molecule antiproliferative agent), may refer to the polypeptide content of the membrane of the MV or may refer to the protein corona of the MV. Preferably the term cargo refers to compounds inside the MV.

[0122] In a preferred embodiment the modified membrane vesicles originate from prokaryotic organisms. In a preferred embodiment the modified membrane vesicles originate from fungi, in particular yeast. In a preferred embodiment the modified membrane vesicles originate from fungi, in particular yeast, preferably Yarrowia sp., preferably Y. lipolytica, preferably Saccharomyces sp., preferably S. pastorianus or S. pombe, preferably Kluyveromyces sp., preferably K. lactis. In a preferred embodiment the modified membrane vesicles originate from bacterial sources, e.g. from E. coli, preferably Lactobacillus or Lactococcus sp., preferably Lactobacillus bulgaricus or Lactococcus lactis. In a particular embodiment processed bacterial vesicles are applied. In an embodiment, as biological membranes, EVs are applied the protein content of which is impaired e.g. by physical or chemical or biochemical treatment, in particular by heat treatment. In a preferred embodiment the membrane vesicles originate from plants. In a preferred embodiment the membrane vesicles originate from plants, in particular multicellular plants, in particular monocotyledons. Alternatively, the membrane vesicles originate from dicotyledons. In a preferred embodiment the membrane vesicles originate from plants, in particular unicellular plants, in particular algae. In a preferred embodiment the membrane vesicles originate from organisms listed in the numbered paragraphs below. Preferably a “modified MV” may be i) a synthetic MV, that is an MV synthesized de novo from molecular components or ii) artificial, cell derived MV or artificial, cell organelle derived MV, that is an MV produced by a process comprising a non-naturally occurring step of disrupting a cell or cell organelle or iii) lipid MV (or sometimes referred to as “lipid extract” herein), that is an MV produced by a process comprising a non-naturally occurring step of lipid isolation preferably from a cell, that is preferably followed by vesicle formation or iv) an impaired MV, that is an MV obtained from a cell or an nEV by a process comprising at least one non- naturally occuring step that results in - a decrease in the diversity of components or a decreased amount of a component of the modified MV compared to the nEV obtained by the same process lacking the at least one non-naturally occuring step, or

[0123] - a decrease in the diversity of biologically active components or a decreased amount of a biologically active component of the modified MV compared to the nEV obtained by the same process lacking the at least one non- naturally occuring step, or

[0124] - the loss of a biological function of a component of the modified MV compared to the nEV obtained by the same process lacking the at least one non-naturally occuring step, or v) an induced MV, that is an MV obtained from a cell by a process comprising at least one non-naturally occuring step that results in

[0125] - a decrease in the diversity of components or a decreased amount of a component of the modified MV compared to the cell, or

[0126] - a decrease in the diversity of biologically active components or a decreased amount of a biologically active component of the modified MV compared to the cell, or

[0127] - the loss of a biological function of a component of the modified MV compared to the cell, or vi) a supplemented MV, that is an MV obtained from a cell or an nEV by a process comprising at least one non- naturally occuring step that results in

[0128] - an increase in the diversity of components or an increased amount of a component of the modified MV compared to the nEV obtained by the same process lacking the at least one non-naturally occuring step, or

[0129] - an increase in the diversity of biologically active components or an increased amount of a biologically active component of the modified MV compared to the nEV obtained by the same process lacking the at least one non- naturally occuring step, or

[0130] - gain of a biological function of a component of the modified MV compared to the nEV obtained by the same process lacking the at least one non-naturally occuring step, or vii) a hybrid or combination of nEVs derived from different species or different individuals of the same species or different tissues from the same individual or viii) a combination of (more than one) mMVs or a combination of (more than one) nEV(s) and mMV(s). Preferably the mMV is i). Preferably the mMV is ii). Preferably the mMV is iii). Preferably the mMV is iv). Preferably the mMV is v). Preferably the mMV is vi). Preferably the mMV is vii). Preferably the mMV is viii). Preferably the membrane of mMVs, preferably mMVs have a decreased complexity of components compared to naturally occuring MVs. Preferably the membrane of mMVs, preferably mMVs contain a low amount of functional polypeptides or peptides or proteins or essentially lack functional polypeptides or peptides or proteins. Preferably mMVs contain a low amount of functional nucleic acids or essentially lack functional nucleic acids. Preferably the membrane of mMVs, preferably mMVs have a molecular composition that is different from EVs generated naturally from the same type of cell or cell organelle. Preferably mMVs essentially lack or lack or express in a smaller extent the molecular markers typical for EVs generated naturally from the same type of cell or cell organelle (such marker may be any one of the following markers: CD9, D63, CD81, CD82, Alix, TSG101, HSP70, HSP90, Flo- tillin-1, Annexins, integrinbl, integrina6, P selectin, ARF6, VAMP, preferably CD9, CD81 or ALIX). Preferably mMVs are depleted in (i.e. contain less) ubiquitinated proteins compared to EVs generated naturally from the same type of cell or cell organelle. Preferably, the molecular composition of a plurality of mMVs is less heterogeneous compared to a plurality of EVs generated naturally from the same type of cell. Preferably the size distribution of a plurality of mMVs is less heterogeneous compared to a plurality of EVs generated naturally from the same type of cell.

[0131] Preferably i) Synthetic MVs have a decreased complexity of components compared to naturally occuring MVs. Synthetic MVs contain a low amount of functional polypeptides or peptide s / proteins or essentially lack functional polypeptides or peptides. Synthetic MVs contain a low amount of functional nucleic acids or essentially lack functional nucleic acids. Synthetic MVs contain a low amount of polysaccharide or sugar components or essentially lack polysaccharide or sugar components.

[0132] Preferably ii) Artificial cell derived MVs may be e.g. MVs obtained by the extrusion of cells. Artificial, cell derived MVs or artificial cell organelle derived MVs have a different molecular composition from EVs generated naturally from the same type of cell or cell organelle. Artificial cell derived MVs or artificial cell organelle derived MVs lack or express in a small extent the molecular markers typical for EVs generated naturally from the same type of cell or cell organelle (such marker may be any one of the following markers: CD9, D63, CD81, CD82, Alix, TSG101, HSP70, HSP90, Flotillin-1, Annexins, integrinbl, integrin a6, P selectin, ARF6, VAMP). The molecular composition of a plurality of artificial cell derived MVs or artificial cell organelle derived MVs is less heterogeneous compared to a plurality of EVs generated naturally from the same type of cell or cell organelle. The cells may be treated with a peptidase to eliminate or essentially eliminate functional polypeptides / proteins, with nuclease, a RNAase, DNAse or nucleotidase to eliminate or essentially eliminate functional nucleic acids, may be treated to lower the polysaccharide content thereof prior to extrusion. Artificial cell derived MVs may be prepared by the disruption of cells from organisms of different taxonomical units (e.g. species, e.g. a human cell and an algal or yeast cell) and combining the membrane structures of the cells from the organisms of different taxonomical units. Accordingly, artificial cell derived MVs may comprise components from different organisms.

[0133] Artificial, cell or tissue derived MVs may be prepared by the disruption of cells or tissues belonging to different organism and also by the disruption of different cells or tissues from the same organism and combining the membrane structures of the cells belonging to different cells or tissues. Accordingly, artificial cell derived MVs may comprise components from different tissues.

[0134] Preferably iii) the membrane of lipid MVs consists essentially of lipids. Lipid MVs are depleted in polypeptides and proteins. Preferably the membrane of lipid MVs, preferably lipid MVs contain a low amount of functional polypeptides or peptides / proteins or essentially lack functional polypeptides orpeptides / proteins. Preferably the membrane of lipid MVs, preferably lipid MVs contain a low amount of ftmctional nucleic acids or essentially lack functional nucleic acids. Preferably the membrane of lipid MVs, preferably lipid MVs contain a low amount of polysaccharide or sugar components or essentially lack polysaccharide or sugar components. Preferably lipid MVs are supplemented with a component (preferably protein component) of human origin, in particular when the lipid MV is of nonhuman origin. Preferably lipid mMVs essentially lack or lack or express in a smaller extent the molecular markers typical for nEVs generated naturally from the same type of cell (such marker may be any one of the following markers: CD9, D63, CD81, CD82, Alix, TSG101, HSP70, HSP90, Flotillin-1, Annexins, integrinbl, integrin a6, P selectin, ARF6, VAMP), preferably CD9, CD81 or ALIX.

[0135] Preferably iv) Impaired MVs are MVs artificially depleted in functional polypeptides, nucleic acids or polysaccharides. Impaired MVs may be generated from nEVs by treating the nEVs with a peptidase to eliminate or essentially eliminate functional polypeptides, by treating the nEVs with nuclease, a RNAase, DNAse or nucleotidase to eliminate or essentially eliminate functional nucleic acids. Impaired MVs may be generated from nEVs by treatment with acids (eg.: HC1) to eliminate or essentially eliminate fimctional polypeptides / proteins. Impaired MVs may be generated from nEVs by heat treatment to eliminate or essentially eliminate functional polypeptides / proteins Preferably v) An induced MV may be e.g. an MV that is obtained by plasma membrane vesiculation induced by chemical or physical means (giant plasma membrane vesicle, gMV). An induced MV or a gMV is an MV that has a size in the micrometer range (e.g. >0.2 pM or>0.5 pM or preferably >1 pM and <1 mm or preferably < 100 pM or preferably < 50 pM), preferably > 1 pM and < 50 pM. The median size of a plurality of an induced MV may be >0.5 or > 1 pM and <1 mm or < 100 pM.

[0136] Preferably vi) A supplemented MV may be an MV, mMV or an nEV of any kind to which an additional component is added. The additional component may be a polypeptide, e.g. a polypeptide / protein that is heterologous to the MV, an active agent, a polysaccharide (sugar moiety). Preferably the additional component is derived from a different or the same organism than the MV. Examples of supplemented MVs may be: algal nEV to which human serum or human serum albumin is added, lipid MV to which human serum or human serum albumin is added. Preferably the additional component is not a receptor of a growth factor. Preferably the supplemented MV is a lipid MV to which a polypeptide / protein is added. Preferably the supplemented MV is a lipid MV of non-human origin to which a component of human origin (e.g. a polypeptide / protein of human origin) or a component that is a component of the human body is added. Preferably the supplemented MV is of human origin to which a component derived from another human subject is added.

[0137] Preferably vii) A hybrid is an entitity generated by the fusion of the EVs or a combination of the EVs.

[0138] Preferably viii) A hybrid is an entitity generated by the fiision of different type of MVs or a combination different type of MVs. A hybrid is an entitity generated by the fusion of different type of mMVs or a combination different type of mMVs. A hybrid is an entitity generated by the fiision of different type of mMVs or a combination different type of nEVs. A hybrid is an entity generated by the combination or fusion of MVs from different tissues, from different individuals of the same species or from different species. A hybrid is an entity generated by the combination of an MV and a macromolecule from a cell, tissue or organism that is not the same as the cell, tissue or organism from which the MV is obtained. Therefore a hybrid MV preferably comprises components from different cells, tissues or organisms. The terms hybrid and combination may be used interchangeably herein.

[0139] Some preferred examples of i) synthetic MVs: A synthetic MV may be e.g. a liposome. A synthetic MV may be an MV obtained by the extraction of lipids from a cell or tissue and mixing (some of) the lipids under conditions that allow the formation of a vesicle. ii) artificial cell derived MVs: Artificial cell derived MVs may be e.g. MVs obtained by the extrusion of cells herein sometimes referred to as “extrudate”. Extrusion may be performed as described herein, or by any method known to the skilled person. Extrudates may be generated from algal, human, yeast or bacterial cells. It is also possible to make an extrudate (i.e. an artificial cell derived MV) from a mixture of cells of different types, e.g. a mixture of human and algal cells). Artificial cell derived MVs may further be treated or processes. For example to an algal extrudate may be added human serum albumin or human serum. Such artificial cell derived MVs contain molecular components from different species (taxonomical units), typically contain human polypeptides / proteins besides algal lipids (or lipids of other origin). iii) Lipid MVs: a lipid MV may be e.g. an MV composed of (some) of the lipids isolated from a cell or from different types of cells. The vesicles described herein as “lipid extracts” or “lipid” are examples of the lipid MV (1MV) subtype of the mMVs. The cell, the isolated lipids or the lipid MV may be further treated. The vesicles described herein as trypsinised lipid are examples of lipid MVs. Lipid MVs may be generated from a mixture of lipids from different types of cells, e.g. from a mixture of lipids that are from human and algal cells. Lipid MVs may be generated from a mixture of lipids from different organisms, different individuals or different cells or tissues of the same individual. iv) impaired MVs: an impaired MV may be generated by a process comprising: enzyme treatment (e.g. peptidase, e.g. trypsin) of a cell (e.g. algal cell) and generating MVs by any method (e.g. extrusion, lipid isolation). The vesicles described herein as trypsinised lipid are examples of impaired MVs. vii and viii) combinations and hybrids: Combinations and hybrids may be generated by fiising or mixing the components of the hybrid or the combination (e.g. a human nEV and an algal nEV or an extrudate (artificial cell derived MV) derived from a plant and a gMV from a human cell).

[0140] The invention relates to modified membrane vesicles (mMVs) for use in therapy, preferably in therapy of a disorder.

[0141] Preferably, the invention relates to mMVs for use in therapy of a disorder in a subject. The subject is an animal, in particular a human subject.

[0142] In a preferred embodiment the mMVs are provided in the form of a medical device, pharmaceutical, cosmetic or nutraceutical composition comprising one or more pharmaceutically or nutraceutically acceptable excipients.

[0143] In a preferred embodiment the mMVs are provided in the form of a pharmaceutical or nutraceutical composition comprising one or more pharmaceutically or nutraceutically acceptable excipients.

[0144] In a preferred embodiment mMV types from multiple sources are combined.

[0145] In a preferred embodiment mMVs used as active agents are combined with medicaments or other active agents against the same disease.

[0146] Preferably the mMVs are for use in the therapy of a disorder associated with cell proliferation, in particular excessive cell proliferation, and / or cell migration, in particular pathological cell migration and / or ECM-formation impairment, in particular impairment of regulation of ECM-formation, more particularly non-physiological (abnormal, non-healthy) ECM production and / or physiological ECM production, or deposition, in particular to regulate ECM production and / or deposition. Preferably the disorder is manifested due to cell proliferation, and / or migration and / or ECM formation impairment, e.g. ECM production and / or deposition, in particular excessive cell proliferation, and / or pathological cell migration and / or non-physiological ECM production / deposition.

[0147] In an embodiment mMVs are for use in the therapy of a fibrotic, in particular a fibroploriferative disorder. In an embodiment the fibrotic, in particular a fibroploriferative disorder is related to cell migration and / or cell proliferation and / or ECM-formation impairment. In an embodiment rnMVs are for use in the therapy of a neoplastic, preferably tumorogenic disorder. In particular the neoplastic disorder is related to cell migration and / or cell proliferation and / or ECM-formation impairment. Preferably the disorder is a proliferation / migration disorder.

[0148] A proliferation / migration disorder is a disorder wherein both cell proliferation and migration play an important part in the condition (in particular a non-healthy condition) to develop (e.g. in disease etiology and / or manifestation) together with non-physiological ECM production or deposition in certain stage(s) of the condition. Typically such proliferation / migration disorders are progressive fibrosis or tissue scarring, neoplastic conditions or cancers, in particular solid tumours and conditions and stages leading or potentially leading to their manifestation.

[0149] In a particular embodiment the proliferation / migration disorders are progressive fibrosis or tissue scarring, neoplastic conditions or cancers.

[0150] In a particular embodiment the rn Vs for use according to the invention have antiproliferative effect.

[0151] Preferably the disorder e.g. the proliferation / migration disorder is fibrosis comprising progressive fibrosis or is a fibroproliferative disease, in particular a fibroproliferative disease involving progressive fibrosis.

[0152] Preferably the fibrosis involvs fibrosis of the skin, or fibrosis in the gastrointestinal organs, in particular the intestine, or of the peritoneum or of abdominal organs, or of the kidney or of the lung or of the heart.

[0153] In a particular embodiment fibrosis is the fibrosis of the lung. In a particular embodiment fibrosis is the fibrosis of the peritoneum.

[0154] The fibrosis (e.g. progressive) as defined herein may occur at any part of the subject’s body where fibrosis, in particular progressive fibrosis may occur.

[0155] In an embodiment the rnMVs inhibit ECM production.

[0156] In a further embodiment fibrosis is considered herein as a dynamic process of ECM production or degradation. An abnormal process (like injury on the one hand and progressive fibrosis on the other) shifts the equilibrium of this dynamic process to the extreme or removes from equilibrium. In a variant the ECM production / degradation becomes irregulated. In a preferred embodiment the rnMVs or the compositions described herein improve the regulation of ECM production, preferably to maintain the dynamic nature thereof or maintain the normal equilibrium. Preferably the disorder e.g. the proliferation / migration disorder is a neoplastic disease, preferably cancer, preferably tumourgenesis, in particular a solid tumour.

[0157] Preferably the disorder is a disorder of the skin, (preferred), gastrointestinal organs, in particular the intestine, (preferred) (colon), abdominal organs (liver), cardiovascular, in particular the heart, peritoneum, urinary system in particular the kidney, respiratory system in particular the lung, brain.

[0158] In a particular embodiment the rnMVs penetrate into the cells of the animal, in particular vertebrate (e.g. as defined in paragraph 1) subject, preferably cells of any of the organs as defined herein. wherein preferably proliferation and migration of fibroblasts contribute to the manifestation of the disease. rnMVs can pass (or get through) barriers of the body, like blood-brain barrier, the blood-eye-barrier, epithelial layer, endothelial layers etc. Thus fibrosis or cancer may be present in the brain or in the eye etc.

[0159] Preferably the rnMVs are for use in inhibiting the activity of a signalling pathway selected from the group consisting of

[0160] - PDE - PDGF receptor alpha and / or PDGF receptor beta signaling pathways, preferably PDGF-induced signaling, preferably PDGF-BB induced signalling pathway, wherein if PDGF-BB induced signalling pathway is mentioned herein it can be understood this broader way,

[0161] - TGFp, preferably TGFbeta-1 induced signalling pathways, wherein if TGFbeta-1 or TGFbeta induced signalling pathway is mentioned herein it can be understood this broader way,

[0162] - EGF induced signalling pathway.

[0163] In a particular embodiment

[0164] - the EVs are for use in inhibiting cell migration, preferably EGF induced cell migration,

[0165] - the EVs are for use in inhibiting cell proliferation, preferably PDGF-BB induced cell proliferation, and / or

[0166] - the EVs are for use in inhibiting ECM production, preferably TGFp induced ECM production / deposition.

[0167] - the EVs are for use in inhibiting a condition selected from cell migration, cell proliferation and / or ECM production, preferably a growth factor mix induced or in particular PDE induced condition.

[0168] In particular embodiments the mMVs are for use in inhibiting PDE, EGF, FGF PDGF-BB and / or TGFp induced cell migration. In particular embodiments the mMVs are for use in inhibiting PDE, EGF, FGF, PDGF-BB and / or TGFp induced cell proliferation. In particular embodiments the mMVs are for use in inhibiting PDE, EGF, FGF- 1 PDGF-BB and / or TGFp induced ECM production / deposition. In particular embodiments the mMVs are for use in inhibiting cell migration, cell proliferation, and / or ECM production induced by an agent comprising multiple growth factors or a mixture of growth factors, such as a mixture of EGF, FGF-1, PDGF-BB and / or TGFp, like PDE. In particular embodiments: The mMVs are for use in therapy of disorders caused by cell migration wherein cell migration is induced by PDE EGF signalling pathway activation. Preferably, administration of mMVs reduce cell migration. The mMVs are for use in therapy of disorders caused by cell proliferation wherein cell proliferation is induced by PDE PDGF-BB signalling pathway activation is activated to induce cell proliferation and wherein administration of mMVs reduce cell proliferation. The mMVs for use in therapy of disorders caused by ECM- production wherein ECM-production is induced by PDE TGFp signalling pathway activation. Preferably, administration of mMVs reduce / modify ECM production. Preferably, administration of algal EVs regulate ECM production. Preferably, administration of mMVs increase physiological ECM production and reduce non-physiolog- ical ECM production.

[0169] Signalling pathway activation involves activation of a part of said pathway (partial activation) provided that said activation results in activation of receptors of PDGF-BB and / or TGF-P and / or EGF (i.e. as used herein PDGF-BB and / or TGF-P and / or EGF receptors).

[0170] Preferably the mMVs are for use in inhibiting the activity of a signalling pathway selected from the group consisting of

[0171] - a signalling pathway induced by multiple growth factors or a mixture of growth factors like PDE

[0172] - PDGF-BB induced signalling pathway,

[0173] - TGFp induced signalling pathways,

[0174] - EGF induced signalling pathway,

[0175] - FGF induced signalling pathway, wherein in said disorder one or more of the PDGF-BB and / or TGF-P and / or FGF and / or EGF receptors are activated. Preferably the disorder is selected from the groups consisting of

[0176] - a fibroproliferative disorder, - a neoplastic disorder.

[0177] Preferably the rnMVs are for use in the treatment of processes or symptom of the disease mediated by PDGF (e.g. PDGF-BB, PDGF receptor), TGF-P, TGF- receptor, EGF, PDE, FGF, FGF-1 or a receptor thereof, preferably PDGF (preferably PDGF-BB, PDGF receptor). Preferably the rnMVs are for use in the therapy of a tumour by the inhibition of ECM production and / or deposition. Preferably the rnMVs are for use in inhibiting the activity of a PDGF-BB induced signaling pathway, a TGF induced signaling pathway, EGF induced signaling pathway, PDE induced signaling pathway, FGF (FGF-1) induced signaling pathway. Preferably the rnMVs are for use in inhibiting cell migration, preferably EGF or PDE induced cell migration, and / or the rnMVs are for use in inhibiting cell proliferation, preferably PDGF-BB or PDE induced cell proliferation. Preferably the rnMVs are for use in regulating ECM production / deposition, preferably inhibiting ECM production / deposition, preferably TGFp or PDE induced ECM production.

[0178] Pharmaceutical compositions -formulation and administration

[0179] Formulation of pharmaceutical compositions from membrane vesicles are well known in the art and are described, among others, in the following publications: Sun C, Qin Y, Zhuang H, Zhang Y, Wu Z, Chen Y. Membrane Vesicles as Drug Delivery Systems: Source, Preparation, Modification, Drug Loading, In Vivo Administration and Biodistribution, and Application in Various Diseases. Pharmaceutics. 2023; 15(7):1903.

[0180] Aytar elik P, Erdogan-Gover K, Barut D, Enuh BM, Amasya G, Sengel-Turk CT, Derkus B, Qabuk A. Bacterial Membrane Vesicles as Smart Drug Delivery and Carrier Systems: A New Nanosystems Tool for Current Anticancer and Antimicrobial Therapy. Pharmaceutics. 2023; 15(4):1052.; Qiong Long, Peng Zheng, Xiao Zheng, Weiran Li, Liangqun Hua, Zhongqian Yang, Weiwei Huang, Yanbing Ma, Engineered bacterial membrane vesicles are promising carriers for vaccine design and tumor immunotherapy, Advanced Drug Delivery Reviews, Volume 186, 2022, 114321.

[0181] Nevertheless, pharmaceutical compositions may not comprise any other active agent except the modified membrane vesicles themselves. In an embodiment the pharmaceutical compositions consist essentially of the modified membrane vesicles and pharmaceutically acceptable excipients.

[0182] The following paragraphs describe different aspects of the invention.

[0183] 1. A modified membrane vesicle for use in a method of treatment of a cell proliferative disease.

[0184] A pharmaceutical preparation (composition) for use in a method of treatment of a cell proliferative disease, preferably a neoplasm and / or fibrosis, said preparation comprising an modified membrane vesicle capable of binding a growth factor, for use in a method of treatment of a cell proliferative disease; and a pharmaceutically acceptable excipient.

[0185] A method for the treatment of a cell proliferative disease, preferably a neoplasm and / or fibrosis, said method comprising the administration of a plurality of modified membrane vesicles to a subject in need thereof.

[0186] A modified membrane vesicle capable of binding thereby neutralizing a protein, in particular a growth factor, for use in a method of treatment of a cell proliferative disease. Neutralization of the growth factors result in an inhibitory or inhibition-equivalent effect on the respective receptors.

[0187] A modified membrane vesicle capable of binding a growth factor selected from the group consisting of PDGF- beta and TGFbeta, preferably PDGF-beta, for use in a method of treatment of a cell proliferative disease. A modified membrane vesicle capable of binding PDGF-beta for use in a method of treatment of a cell proliferative disease. In a preferred embodiment the cell proliferative disease is a neoplastic disease, preferably cancer or tumorous disease. In a fiirther preferred embodiment the cell proliferative disease is a fibroproliferative disease. In a further preferred embodiment the modified membrane vesicle is for use in a method of treatment of fibrosis or a fibrotic condition.

[0188] The modified membrane vesicle, preferably engineered membrane vesicle is preferably free of cargo, preferably free of added cargo (as an additional active agent). Preferably the level of innate cargo level is reduced or impaired.

[0189] 2. Preferably the modified membrane vesicle is of biomembrane origin. The modified membrane vesicle for use according to numbered paragraph 1 wherein said modified membrane vesicle is an engineered membrane vesicle, preferably of biomembrane origin.

[0190] Preferably the non-lipid membrane composition (or content) of the engineered membrane vesicle is impaired in comparison with the original biomembrane from which it is engineered. Preferably, the protein content of said modified membrane vesicle is impaired.

[0191] 3. Preferably the protein content of said engineered membrane vesicle is impaired. Preferably vesicle is of biomembrane origin and which is depleted in functionally active polypeptides. Preferably vesicle is of biomembrane origin and which is depleted in native polypeptides but may be supplemented with a homo (auto)- and heterogenous protein.

[0192] Preferably the lunction of the proteins in the membrane vesicle is impaired or reduced. Preferably the proteins are non-ftmctional. In an embodiment the proteins are inactivated. Inactivation is preferably carried out by heat treatment. Inactivation may be carried out by high-shear force, e.g. by grinding, extrusion or homogenization. Inactivation may be carried out by heat-thaw cycles. Inactivation may be carried out by chemical means. Inactivation may be carried out by enzymatic means e.g. by peptidase, e.g. protease digestion or treatment. In a preferred embodiment trypsin treatment is applied. Additionally or alternatively (depending on the cargo to be inactivated) RNase and / or glucosidase treatment may be applied.

[0193] Preferably the protein content is reduced (lowered) significantly, if measured by any of the following methods: gel electrophoresis by Coomassie Brilliant Blue staining or by silver staining, by UV spectrophotometry or by a protein assay, e.g. as disclosed herein. In an embodiment the protein content is not detectable by any of these methods. Preferably the RNA content of said engineered membrane vesicle is impaired. Preferably the modified membrane vesicle is of biomembrane origin and which is depleted in functionally active innate RNA in particular native RNA. The membrane vesicle for use according to numbered paragraph 1 or 2 wherein said vesicle is of biomembrane origin and which is depleted in native RNA molecules.

[0194] In other aspect the modified membrane vesicles are depleted in innate cargo compound, at least in non-membrane bound bioactive molecules, and preferably both in membrane-bound and non-membrane bound molecules, preferably at least in proteins; optionally additionally in RNA and sugars (saccharides) which would possibly considered as providing activity to the vesicles. However, in the present invention activity of modified membrane vesicles is attributed to the membrane of the modified membrane vesicles, preferably of the engineered membrane vesicles.

[0195] 4. Preferably the cell proliferative disease is a disease mediated by a growth factor selected from the group consisting of PDGF-beta and TGFbeta, preferably PDGF-beta. Preferably the cell proliferative disease is a disease mediated by a growth factor selected from the group consisting of PDGF-beta and TGFbeta, preferably PDGF- beta. Preferably the cell proliferative disease is a PDGFR-alpha or a PDGFR-beta mediated disease, i.e. preferably a disease which is affected by PDGFR-alpha or a PDGFR-beta ligand. Preferably the cell proliferative disease is a TGFbeta receptor mediated disease, i.e. preferably a disease which is affected by a TGF-beta receptor.

[0196] The membrane vesicle for use according to numbered paragraph 1, 2 or 3 or an engineered membrane vesicle as defined herein, wherein said membrane vesicles are for use in the therapy of a disorder associated with cell proliferation, in particular excessive cell proliferation, and / or ECM-formation impairment, in particular impairment of regulation of ECM-formation, more particularly non-physiological (abnormal, non-healthy) ECM production and / or physiological ECM production, or deposition, in particular to regulate ECM production and / or deposition. The membrane vesicle for use according to numbered paragraph 1, 2 or 3 or an engineered membrane vesicle as defined herein, wherein said membrane vesicles are for use in the therapy of a neoplastic disorder and / or fibrotic, in particular a fibroploriferative disorder. In a particular embodiment wherein said membrane vesicles are for use in the therapy of a fibroproliferative disorder associated with fibroblast activation. In a particular embodiment the membrane vesicles are for use in the therapy of a fibrotic, in particular a fibroploriferative disorder preferably as defined herein. In particular the fibrotic, in particular a fibroploriferative disorder is related to cell proliferation and / or ECM-formation impairment. In a particular embodiment the membrane vesicles are for use in the therapy of a neoplastic, preferably tumorogenic disorder preferably as defined herein. In particular the neoplastic disorder is related to cell proliferation and / or ECM-formation impairment.

[0197] In a particular embodiment the vesicle is not for use as an adjuvant. In a particular embodiment the vesicle is not for use as an antiviral agent.

[0198] 5. Preferably the modified, preferably engineered membrane vesicle, preferably said engineered membrane vesicle of biomembrane origin is selected from the group consisting of, giant plasma membrane vesicles, extruded membrane vesicles, lipid extract and peptidase-treated variants thereof (peptidase treated artificial membrane vesicle of biomembrane origin), e.g. peptidase-treated lipid extract. In an embodiment the engineered membrane vesicle, preferably said engineered membrane vesicle of biomembrane origin is or is prepared from extracellular vesicles with denatured polypeptide content.

[0199] In a particular embodiment hybride vesicles as modified membrane vesicles are applied. Preferably modified membrane vesicles comprise membranes of biological membrane origin as disclosed herein whereas further membrane composing agents like lipids are added thereto. Moreover, hybrid vesicle composed of membranes from different sources may be prepared. As an example, alga, yeast or bacterial; and mammalian, e.g. human hybrid vesicles may be prepared and used wherein the human membrane component may serve as humanization (making acceptable to human immune system) or targeting whereas the other, e.g. algal, yeast or bacterial part can be effectively produced.

[0200] 6. Preferably the modified preferably engineered membrane vesicle, preferably said modified preferably engineered membrane vesicle of biomembrane origin wherein said the polypeptides in said membrane vesicles are impaired in comparison with the starting biomembrane, preferably impaired by heat treatment or peptidase or protease treatment or by the manufacturing process. In a particular embodiment the membrane vesicle is selected from extruded membrane vesicles, lipid extract and peptidase-treated variants thereof, peptidase treated artificial membrane vesicle of biomembrane origin, e.g. peptidase-treated lipid extract. In an embodiment the engineered membrane vesicle of biomembrane origin are obtained by chemical treatment from cells or membranes of cells. In an embodiment the engineered membrane vesicle of biomembrane origin are obtained by physical methods from cells or membranes of cells. 7. Preferably the vesicles originate from, i.e. prepared from prokaryotic organisms.

[0201] 8. Preferably the vesicles originate from fungi, in particular yeast. Preferably the vesicles originate from bacteria, in particular lactobacilli. In another embodiment the bacteria are different from lactobacilli. Preferably the vesicles originate from algae. In a preferred embodiment the algae are Chlorophyta.

[0202] 9. Preferably the vesicles originate from plants, in particular multicellular plants, in particular monocotyledon cells. In a particular embodiment the plants are dicotyledon cells. In a particular embodiment the vesicles are from cells of unicellular plants.

[0203] In a particular embodiment the vesicles are from cells of multicellular plants. In a particular embodiment the vesicles are from cells of angiospermal plants. In a particular embodiment the vesicles are from cells of gymnospermous plants.

[0204] 10. Preferably the vesicles originate from animal cell mebranes. In an particular embodiment the vesicles are from mammalian cells. In a particular embodiment the vesicles are from non-human mammalian cells. In a particular embodiment the vesicles are from cells of body fluids of mammals, preferably milk. In a particular embodiment the vesicles are from human cells. In a particular embodiment the vesicles are from human cells, preferably tissue stem cells, preferably MSCs or IPS cells.

[0205] In a particular embodiment the vesicles are from human cells different from stem cells. In a particular embodiment the vesicles are from cells different from stem cells. In a particular embodiment the vesicles are from vertebrate cells. In a particular embodiment the vesicles are from invertebrate cells. In a particular embodiment the vesicles are from mollusc cells.

[0206] 11. In a preferred embodiment the pharmaceutical preparations consist essentially of the membrane vesicles and one or more pharmaceutically acceptable excipients. In the present invention in an embodiment the pharmaceutical preparations may not comprise any other active agent except the membrane vesicles themselves. In a further embodiment the invention relates to a combination of a pharmaceutical preparation for use in a method of treatment according to the invention and an active agent for a cell proliferative disease.

[0207] 12. Preferably, the pharmaceutical preparation comprises a pharmaceutically acceptable carrier suitable for membrane vesicles.

[0208] 13. The pharmaceutical preparation for use according to any of numbered paragraph 11 to 15 which comprises a pharmaceutically acceptable excipient. In a preferred embodiment the excipient is selected from the following substances: stabilizers, light-protective agents, fillers, nebulizers, buffers and salts, aggregation and / or oxidation inhibitors etc.

[0209] 18. The pharmaceutical preparation for use according to any of numbered paragraph 11 to 17, said preparation being for use in therapy of a disorder preferably in the vertebrate subject according to any of numbered paragraph 1 to 6 wherein the disorder is a disorder of the skin, gastrointestinal organs, in particular the intestine, (preferably colon), abdominal organs (preferably liver), cardiovascular, in particular the heart, peritoneum, urinary system, in particular the kidney, respiratory system in particular the lung, preferably, gastrointestinal organs, in particular the intestine, (preferably colon), abdominal organs (preferably liver), cardiovascular, in particular the heart, peritoneum, urinary system in particular the kidney, respiratory system in particular the lung, preferably cardiovascular system, in particular the heart, peritoneum, urinary system in particular the kidney, respiratory system in particular the lung or the brain. 14. In a particular embodiment the number of membrane vesicles in a dose unit of the composition is at least 103preferably at least 105, or at least 106or at least 107or at least 108or at least 109or at least IO10. In an embodiment the number of membrane vesicles in a dose unit of the composition is at most IO20preferably at most 1015, or at most 1012or at most 1011. In particular the number of membrane vesicles in a dose unit of the composition is at least or at least 3 x 106or at least 3 xlO7or at least 3 x 108or at least 3 x 109. Preferably, the number of algal EVs in the composition is at least 3 xlO7or at least 3 x 108or at least 3 x 1 x 109or at least 3 x IO10.

[0210] In an embodiment the above data relate to concentration and 1 ml of dose unit.

[0211] In a particular example a dose for the treatment of an animal is at least 3 x 107membrane vesicles or 3 x 108(or possibly 3 x 109) membrane vesicles per treatment, e.g. in a mice as a model animal. In mice the proposed dose corresponds to about at least 1.5 x 109membrane vesicles or at least 1.5 x IO10membrane vesicles per kilogram body weight (kgbw). In larger animals like in humans the dose may be smaller per kgbw, e.g. 1.5 x 108membrane vesicles or at least 1.5 x 109membrane vesicles per kgbw.

[0212] In a particular embodiment the dose range in a dose unit of the composition in case of mammals e.g. in humans is at least 105membrane vesicles per dose or at least 106and at most 109membrane vesicles per dose or at least 107and at most IO10membrane vesicles per dose or at least 108and at most 1011or at most 1012or at most 1013or at most 1014membrane vesicles per dose, preferably daily dose. In an embodiment this is a daily dose.

[0213] 15. Preferably the MVs are administered

[0214] - orally, preferably in the form of a composition for oral administration,

[0215] - topically, preferably in the form of a composition for topical administration,

[0216] - intraperitoneally, preferably in the form of a composition for intraperitoneal administration,

[0217] - intranasally, preferably in the form of a composition for intranasal administration,

[0218] - subcutan / intracutan preferably in the form of a composition for subcutan administration,

[0219] - intramuscular, preferably in the form of a composition for intramuscular administration, or

[0220] - intravenous, preferably in the form of a composition for intravenous administration.

[0221] - In a particular embodiment administration is intratumoral. In a particular embodiment administration is intraocular. In a particular embodiment administration is oral administration. In a particular embodiment administration is intraperitoneal administration. In a particular embodiment administration is intravenous administration. In a particular embodiment administration is intracutan administration.

[0222] 16. Preferably the biological membrane is from a yeast; in a particular embodiment the yeast species is selected from the group consisting of Scheffersomyces stipitis, Yarrowia lipolytica, Dekkera bruxellensis, Saccha- romyces pastorianus, Saccharomyces eubayanus, Zygosaccharomyces bailii, Saccharomyces paradoxus, Saccha- romyces cerevisiae (var. diastaticus), Saccharomyces kudravzevii, Saccharomyces mikitae, Schizosaccharomyces pombe, Torulaspora delbrueckii, Saccharomyces sensu stricto, Saccharomyces mikatae, Candida krusei, Candida humilis, Candida auris, Hansenula anomala, Kluyveromyces marxianus, Yarrowia lipolytica, Saccharomyces uvarum, Aspergillus fumigatus.

[0223] Preferably the vesicle is obtained from cells of algae. In a particular embodiment the alga is Chlorella.

[0224] Preferably the vesicle is obtained from cells of bacteria. In a particular embodiment the bacterium is E. coli, Lactobacillus, preferably L. bulgaricus (L. delbrueckii subsp. bulgaricus) or Lactococcus, preferably L. lactis.

[0225] 17. A combination of a pharmaceutical preparation for use in a method of treatment of a cell proliferative disease according to the invention and a further agent for use in the treatment of said proliferative disease. Preferably the disease is selected from a neoplasm and / or fibrosis, said preparation comprising an artificial membrane vesicle capable of binding a growth factor, for use in a method of treatment of a cell proliferative disease; and a pharmaceutically acceptable excipient.

[0226] An artificial membrane vesicle capable of binding a growth factor is as defined in claim 11, or in any of numbered paragraphs 1 to 20. The disease is as defined in any of numbered paragraphs 1 to 20. In a particular case the active agent is selected from an active agent used in the Examples herein.

[0227] EXAMPLES

[0228] Methods

[0229] Algae cultivation

[0230] Chlorella sp. MACC-360 was grown in Tris acetate phosphate (TAP; UTEX, USA) and MACC-1, MACC-3 in Bristol (UTEX, USA) and the Spirulina sp. MACC-908 in spirulina (UTEX, USA) media, in 250 ml Erlenmeyer flasks on an orbital shaker set at 150 rpm at room temperature. Autotrophic cultivation was provided by optimized conditions, ensuring 12 hours-day / 12 hours-night photoperiods using white light bulb until cell count reached approximately 7xl07cell / ml. Algal strain was kindly provided by Zoltan Molnar (Mosonmagyarovar Algal Culture Collection, Mosonmagyarovar, Hungary).

[0231] Chlorella vulgaris (Jongerius Ecoduna GmbH, Austria), and Spirulina platensis powder (Jongerius Ecoduna GmbH, Austria, Hangzhou Natur Foods Co., China) was purchased as edible powder and rehydrated in phosphate buffered saline (PBS) at 20 mg / ml concentration at room temperature under gently agitation for 1 hour.

[0232] Bacteria and yeast cultivation

[0233] To start a new culture from a colony with a sterile loop a small piece of inoculums were put into 10-20 ml sterile liquid medium (yeast minimal media, Yeast minimal nitrogen base (Y0626, Merck Kft, Hungary) 6.7 g / 970 ml, Yeast minimal nitrogen base supplement (Y2001, Merck, Hungary) 1.4 g / 970 ml, 2% D Glucose (G8270, Merck, Hungary) 20 g / 970 ml)) and incubated at 30°C. When the culture’s OD (600 nm) reached at least 1.5A, lOOx dilutions were made by adding 300 mL medium to 3 mL inoculums and cultured overnight. BY4741 strain was kindly provided by Ildiko Unk (HUN-REN Biological Research Centre, Szeged), Escherichia coli, and Pseudomonas aeruginosa strains were kindly provided by Eva Kenesei (Semmelweis University, Budapest, Hungary), S. pombe, S. pastorianus, Y. lipolytica, K. lactis, L. lactis and L. bulgaricus were purchased from Hungarian University of Agriculture and Life Sciences, National Collection of Agricultural and Industrial Microorganisms.

[0234] Human samples

[0235] Peritoneal dialysis (PD) effluents (PDE) were taken from patient receiving PD at the Pediatric Center, Semmelweis University, Budapest, Hungary. Peritoneal samples were collected at the time of the first insertion of Tenckhoff peritoneal catheter and at the time of PD catheter removal at the Pediatric Center, Semmelweis University, Budapest, Hungary (loss of ultrafiltration capacity of the peritoneal membrane) (31224-5 / 2017 / EKU). Colon samples were taken at the Pediatric Center, Semmelweis University Budapest, Hungary during routine endoscopy and the residues of these samples were used for our work (19048-4 / 2018 / EKU).

[0236] Mammalian cells

[0237] Primary human peritoneal (pFB), colon (cFB), skin (sFB) and primary mouse lung (1FB) fibroblasts were prepared by enzymatic digestion of the related tissue using 1 mg / ml collagenase type II (Life Technologies Kft, Hungary). The isolated cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium / Nutrient Mixture F12 (DMEM- F12; Life Technologies Kft, Hungary) medium supplemented with 10% heat-inactivated foetal calf serum (FCS; Life Technologies Kft, Hungary), 100 pg / ml streptomycin and 100 U / ml penicillin (Life Technologies Kft, Hungary) at 37°C in a humidified atmosphere of 5% CO2 in air. Monolayers were identified as FBs by their morphology, a-SMA positivity investigated by immunofluorescent staining.

[0238] HK-2 and A549 were cultured in DMEM (Life Technologies Kft, Hungary), HCT-116 were cultured in RPMI 1640 (Life Technologies Kft, Hungary), and HT-29 were cultured in McCoy’s 5A medium (Life Technologies Kft, Hungary) at 37°C supplemented with 10% heat-inactivated foetal calf serum (FCS; Life Technologies Kft, Hungary), 100 pg / ml streptomycin and 100 U / ml penicillin (Life Technologies Kft, Hungary) at 37°C in a humidified atmosphere of 5% CO2 in air. Monolayers were identified as FBs by their morphology, a-SMA positivity investigated by immunofluorescent staining.

[0239] Mesenchymal stem cells (MSCs) from PDE were pelleted by centrifugation (1200 rpm for 20 minutes) [Yangchun Du et al., (2021). Comparison of mesenchymal stromal cells from peritoneal dialysis effluent with those from umbilical cords: characteristics and therapeutic effects on chronic peritoneal dialysis in uremic rats. Stem Cell Research & Therapy, 12, Article number: 398] and were cultured at 37°C in DMEM-F12 medium supplemented with 10% FCS, 100 pg / ml streptomycin and 100 U / ml penicillin (Life Technologies Kft, Hungary) at 37°C in a humidified atmosphere of 5% CO2 in air. iPS(IMR90)-4 (IPS) were cultured in Essential 8 TM media (Life Technologies Kft, Hungary) medium supplemented with 10% heat-inactivated foetal calf serum (FCS; Life Technologies Kft, Hungary), lOpM Rock inhibitor (ROCKi, Y-27632 Dihydrochloride, Merck Kft, Hungary), 100 pg / ml streptomycin and 100 U / ml penicillin (Life Technologies Kft, Hungary) at 37°C in a humidified atmosphere of 5% CO2 in air on matrigel-coated (Coming® Matrigel® Basement Membrane, Life Technologies Kft, Hungary) surface.

[0240] Membrane vesicle isolation (nEVs of different origin)

[0241] To remove cell debris, human MSCs of peritoneal origin, IPSs, MACC-360, BY4741, E. coli, and Pseudomonas aeruginosa cells or rehydrated Spirulina platensis powder were centrifuged for 20 minutes at 2000 rpm (algae, MSCs, IPSs) or 4600 rpm 30 minutes (rehydrated Spirulina platensis powder, bacteria, and yeast), then the supernatants were filtrated with 0.22 pm pore size filter (Millipore 35 Express® PLUS, Merck, Germany). Subsequently samples were ultrafiltered and concentrated by tangential flow filtration, using TFF-easy filters (Hansa BioMed Life Sciences, BIOCENTER Laboratory Supplier Ltd, Hungary). MV samples were further purified by size exclusion chromatography (SEC) on IZON qEVoriginal / 70 nm pore size columns (IZON, USA). Fractions (1-2) were collected and analysed by NTA to measure the particle size and number of the isolated MVs.

[0242] EV from IPS was treated with 0,25% trypsin-EDTA (TFF:trypsin-EDTA = 2:1 ratio, (Gibco, Life Technologies Kft, Hungary) for 1 hour at room temperature, then SEC (70 nm pore size columns, IZON, USA) was carried out to remove the “residual” trypsin-EDTA.

[0243] Extradates (exMVs)

[0244] Human red blood cells (RBCs), MSCs, HK-2, A549, MACC-360, MACC-908, Y. lipolytica, K. lactis or L. bul- garicus cell pellet was extruded after multiple steps of sonication (Kerry Steel Probe Sonicator Ultrasonic, immersed in liquid) and tissue homogenization in 2 ml Potter-Elvehjem Tissue Grinder and freeze-thaw cycle (-80- 60°C) by LIPEX ® Extruder (Evonic Industries, Germany) with polycarbonate membrane filters (Whatman, UK) at 60°C. Samples were further purified by size exclusion chromatography (SEC) on IZON qEVoriginal / 70 nm pore size columns (IZON, USA). Fractions (1-2) were collected and analysed by NTA to measure the particle size and number of the isolated MVs. In case of wheat grass, after grinding it by a slow fruit juicer (Star-Light SJB-150R) at 55 rpm, the homogenizated sample was centrifuged at 4600 rpm for 30 minutes then fdtrated with 0.22 pm pore size filter (Millipore 35 Express® PLUS, Merck, Germany). Subsequently samples were ultrafiltered and concentrated by tangential flow filtration, using TFF-easy filters (Hansa BioMed Life Sciences, BIOCENTER Laboratory Supplier Ltd, Hungary). Samples were further purified by size exclusion chromatography (SEC) on IZON qEVoriginal / 70 nm pore size columns (IZON, USA).

[0245] Giant plasma membrane vesicles (gMVs)

[0246] To induce giant plasma membrane vesicles (gMVs), MSCs or pFBs were rinsed with PBS then with GPMV buffer, containing 10 mM HEPES, 150 mM NaCl, 2 mM CaC12, pH 7.4 (Sigma-Aldrich, Hungary). Thereafter cells were incubated in 25 mM PF A, 2 mM DTT diluted in GPMV puffer at 37°C for 1 hour. GPMVs were harvested by centrifugation the supernatant of the cells (3000 rpm for 10 minutes) and resuspended in 1 ml of PBS.

[0247] Lipid isolation and lipid vesicle formation (IMVs)

[0248] To extract the lipids from MSCs, cFB, pFBs, RBCs, HTC-116, HK-2, A549, MACC-1, MACC-3, MACC-360, MACC-908, Chlorella vulgaris poweder, Spirulina platensis powder, Saccharomyces pombe, Saccharomyces pas- torianus, Kluyveromyces lactis, Lactococcus lactis, Yaeeowia lipolytica or Lactobacillus bulgaricus the cells or the algal powder were resuspended in 2 ml of isopropanol-methanol (1: 1) mixture (Sigma- Aldrich, Hungary). Samples were lysed in a Sonorex RK 52 H ultrasonic bath (Bandelin, Germany) for 15 minutes, then centrifuged (15000 rpm for 10 minutes) and the supernatant was dried at room temperature overnight. Lipid extracts were resuspended in 500 pl of PBS and sonicated for 5 minutes to form vesicels (LVs), thereafter purified by SEC (see above). Fractions (1-2) were collected and analysed by NTA to measure the particle size and number of the isolated EVs.

[0249] Trypsin-digested LVs (t lMV) originated from MACC-360, pFBs, L. lactis, or L. bulgaricus were also produced by incubating 1MV in 0.25% Trypsin-EDTA (Gibco, Life Technologies Kft, Hungary) at 37 °C for 30 minutes, centrifuged (3000 rpm for 10 minutes).

[0250] Hybrid MVs

[0251] Hybrid MVs were created by the mixing of the lipids of different origin (Table L). Briefly, lipides of pFBs, MSCs, S. pastorianus, Spirulina platensis powder, Y. lipolitica, MACC-360 or L. bulgaricus were extracted as previously described. Thereafter, hybrid MVs were generated from a 1: 1 mixture of the lipids isolated from different origin. Briefly the lipid mixtures of a human and a different organism were resuspended in 500 pl of PBS, sonicated for 5 minutes, thereafter purified by SEC (see above). Fractions (1-2) were collected and analysed by NTA to measure the particle size and number of the isolated MVs.

[0252] Table 1. Hybride IMVs (Co lMV) were prepared by the 1:1 mixture of IMVs of different origin.

[0253] TABLE 1. List of different hybride IMVs.

[0254] MV Source MV Type

[0255] MSC lipid + MACC-360 lipid CoJMV

[0256] MSC lipid + Spirulina platensis powder lipid Co lMV

[0257] MSC lipid + Y. lipolytica lipid Co lMV

[0258] MSC lipid + L. bulgaricus lipid Co lMV pFB lipid + S. pastorianus lipid Co lMV Hybride MVs were also generated by the mixing of the lipids of MSCs, MACC-360, K. lactis or L. lactis with cholesterol in 1: 1 ratio.

[0259] Hybrid MVs were generated from lipid based MVs by the creation of human albumin (HSA) crown, as well. Briefly, lipids of MAC-360, MACC-908, Chlorella vulgaris powder, S. pastorianus or S. pombe were extracted, then MVs were created as described above. Then the lipid based MVs were incubated with HSA (50g / L) for 1 h at room temperature. Finally the unbound human albumin were removed from the MVs by SEC.

[0260] Hybride MVs were also generated by the co-extrudatation of MSCs and MACC-360 Briefly, the MSC and MACC- 360 cells were mixed, pelleted, and then extruded after multiple steps of sonication (Kerry Steel Probe Sonicator Ultrasonic, immersed in liquid) and tissue homogenization in 2 ml Potter-Elvehjem Tissue Grinder and freezethaw cycle (-80-60°C) by LIPEX ® Extruder (Evonic Industries, Germany) with polycarbonate membrane filters (Whatman, UK) at 60°C. Finally the resulted extrudates were further purified by size exclusion chromatography (SEC) on IZON qEVoriginal / 70 nm pore size columns (IZON, USA).

[0261] Hybride MVs were also generated by the co-extrudatation of cell extrudates. Briefly, human MSCs, MACC-360, MACC-908 and K. lactis were separately extruded, as above described, then the extrudates of MSC and MACC- 360 or extrudates of MSC and MACC-908 or extrudates of MSC and K. lactis were mixed and extruded again as above described.

[0262] Nanoparticle Tracking Analysis (NTA)

[0263] In NTA, the sample is illuminated by a laser beam and the light scattered by individual particles moving under Brownian motion is recorded by a microscope camera. Individual particles are tracked, and their hydrodynamic size is obtained based on a modified Stokes-Einstein equation. For NTA of particles, the measurable size range is between about 10 to 1000 nm in diameter, preferably 100 nm to about 600 nm. As particle concentrations can be calculated, albeit with moderate accuracy, NTA may be used to observe relative changes in particle concentration. The measurement was performed with a ZetaView PMX-120 (Particle Metrix GmbH, Meerbusch, Germany).

[0264] Microscopy

[0265] The average size of gMVs was determined by microscopy. Briefly following isolation of gMVs, 5 ul-droplets of the suspensions were places on a glass slide. Multiple DIC images were taken by Olympus 1X81 microscope system using x 20 magnification. The size distribution of the gMVs was determined by analyzing the diameter of 250 individual particles by Image! graphical analysis software.

[0266] FACS

[0267] Native EVs and modified MV samples were incubated with the anti-CD9 (sc-13118, Bio-Kasztel Kft, Hungary) or anti-CD81 primary antibodies (SAB3500454, Merck Life Science Kft.) or after permeabilization of the samples with BD cytofix / cytoperm (554714, Bio-Kasztel Kft, Hungary) with anti- ALIX (SAB4200477, Bio-Kasztel Kft, Hungary) primary antibody for Ih / RT.

[0268] After washing step with PBS the samples were incubated with the proper secunder antibodies for Ih / RT (A-21200, and A21206, Life technologies Kft, Hungary). FACS analysis was performed by CytoFLEX Flow cytometer (Beckman Coulter Life Sciences) after systematic use of Triton lysis as a control for microvesicle labeling. Protein analysis

[0269] The protein-content of native nanoparticle-containing samples was investigated by UV spectrophotometry (DeNo- vix DS-11 spectrophotometer, DeNovix Inc., Wilminutesgton, DE, USA) measuring their optical density at 280 nm, and by Qubit Protein Assay Kit (Invitrogen, Hungary) according to the instructions of the manufacturer. For silver staining, nanoparticle-containing samples (the same working solutions used in in vitro experiments) were denatured in Leammli buffer, then equal volumes were loaded and separated on 4-20% gradient SDS polyacrylamide gel (BioRad, Hercules, USA). Briefly, gels were fixed in 20% ethanol - 10% acetic acid solution, sensitized in 0.8 mM sodium thiosulfate, impregnated in 12 mM silver nitrate, thereafter developed in solution containing potassium carbonate, formalin and sodium thiosulfate (Silver staining of proteins in polyacrylamide gels 2006, DOI: 10.1038 / nprot.2006.288). All reagents were purchased from Merck.

[0270] MVs uptake experiments in vitro

[0271] For testing the in vitro internalization of MVs of MACC-360, Sirulina platensi powder, BY4741 EV, E.coli, MSC and lipid extract of pFB origin, samples were labelled by using the liphophilic fluorescence dye, Dil (DiIC18(3), l,r-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Molecular Probes, UK) following the manufacturer’s instructions. Briefly, 200 pl MVs were incubated with 2 pl of 1 mg / ml Dil dye for 60 minutes at 37 °C. Unbound dye was eliminated by SEC after washing the labelled EVs with 400 pl PBS. As negative control the same volume of the dye was incubated in PBS (in the absence of EVs) and subjected to same incubation periods, SEC was carried out as previously described.

[0272] Finally pFB and cFB cells were seeded in 4-well cell culture slide (Coming Costar, Sigma Aldrich, Hungary) at 80% confluence. Cells were then incubated with cell culture medium containing 3xl07-5.9xl010particles of the Dil-labelled EVs for 24 hours at 37°C in a humidified atmosphere of 5% CO2. Subsequently, nuclei were stained with Hoechst 33342 (1 : 10000, Merck Kft, Hungary) for 10 minutes at room temperature and the slides were coverslipped with ProLongTM Gold antifade mountant (Invitrogen, Hungary). Internalized MVs were visualised using Olympus IX-81 fluorescent microscope system (Olympus Corporation, Japan).

[0273] Confocal microscopy to confirm FRET results PDGF-B binding

[0274] 200 pl MVs were incubated with 2 pl of 1 mg / ml DiD dye for 60 minutes at 37°C. Unbound dye was eliminated by SEC after washing the labelled MVs with 400 pl PBS. In parallel same volume of the dye was incubated in PBS (in the absence of MVs) and subjected to same incubation periods, then after wash step SEC was carried out as previously described. This latter sample was used as negative control. To test PDGF-B binding 10 ng / ml rhPDGF-B was added for the samples and incubated for 60 minutes at 37 °C. Unbound rhPDGF-B was eliminated by SEC after washing the labelled MVs with 400 pl PBS.

[0275] 3x104 pFBcells were seeded in 8-well cell culture slide (IBIDI-8 well high glass bottom chamber, Biocenter Kft, Hungary) at 80% confluence. Cells were immediately incubated with cell culture medium containing CellLight™ Lysosomes-GFP, BacMam 2.0 (Life Technologies Kft, Hungary) according to the manufacturers ’s instruction than after 24h with 3xl07-5.9x1010 particles of the DiD-labelled-rhPDGF bound MVs for 24 hours at 37°C in a humidified atmosphere of 5% CO2. After fixation step by BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit (Bio-Techne R&D Systems Ltd, Hungary), chambers were incubated firstly with primary antibodies against PDGF-B (1:250, Abeam, Bio-Kasztel Kft, Hungary) for 24 hours at 4°C. After multiple washing with PBS slides were incubated with the corresponding abberior STAR 580 -conjugated secondary antibodies (1:250, abberior GmbH, Germany) for 1 hour at room temperature. Nuclei were stained with Hoechst 33342 (1 : 10000, Merck Kft, Hungary) for 10 minutes at room temperature. Internalized MVs were visualised Nikon Eclipse TI2 (France), Abberior Expert Line (abberior GmbH, Germany).

[0276] Fluorescence Resonance Energy Transfer (FRET) FRET was carried out to test the PDGF-B binding of nEVs of MSC, MACC-360, Spirulina platensis, BY4741, E. coli, LVs of MACC-360 and pFB, t_LV of pFB, and g_MV of pFB origin. TGF-B binding of nEVs of Spirulina platensis powder, BY4741, and E. coli was in cell free environment, as well (Table 2.). Briefly, 200 pl of the above mentioned vesicles were incubated with 2 pl of 1 mg / ml Dil dye for 60 minutes at 37 °C. Unbound dye was eliminated by SEC after washing the labelled EVs with 400 pl PBS. To test PDGF-B or TGF-P binding 10 ng / ml of human recombinant PDGF-B (rhPDGF-B) or 10 ng / ml of human recombinant TGF-P (rhTGF-P) was added to the samples and incubated for 60 minutes at 37°C. Subsequently samples were incubated firstly with primary antibodies against PDGF-B (1: 1000, abl81341 Abeam, Bio-Kasztel Kft, Hungary) or primary antibodies against TGF-P (1:1000, sc-130348 Santa Cruz, Bio-Kasztel Kft, Hungary) for 1 hour at room temperature, then were incubated with the corresponding Alexa Fluor®-488 conjugated secondary antibodies (1 : 1000, Life Technologies Kft, Hungary) for 1 hour at room temperature. Dil labelled samples without the „linker” rhPDGF-B or TGF-P but incubated with the anti-PDGF-B or the anti-TGF-p primary antibody and the corresponding secondary antibodies served as control. The fluorescence lifetime of the donor dye (Dil) was measured with a Chronos BH Time Correlated Single Photon Counting lifetime spectrometer (ISS Inc., Illinois) and was determined by an iterative reconvolution procedure employing least-squares fitting.

[0277] Table 2. List of the membrane vesicles (MV) of different origin investigated by Fluorescence Resonance Energy Transfer (FRET). nEV: native EV; MV: membrane vesicle; 1MV: isolated lipid based MV; t lMV: trypsine treated 1MV; gMV: giant plasma MV. Yes = the investigated vesicle binds PDGF-BB or TGF-B.

[0278] TABLE 2. The PDGF-BB, or TGF- binding of nEVs, 1MV, t lMVs, and g_MVs was investigated by FRET.

[0279] MV Source MV Type Assay Treatment Binding

[0280] MSC nEV FRET PDGF-BB yes pFB 1MV FRET PDGF-BB yes pFB t lMV FRET PDGF-BB yes pFB gMV FRET PDGF-BB yes

[0281] MACC-360 nEV FRET PDGF-BB yes

[0282] MACC-360 1MV FRET PDGF-BB yes

[0283] Spirulina platensis powder nEV FRET PDGF-BB yes

[0284] BY4741 nEV FRET PDGF-BB yes

[0285] E. coli nEV FRET PDGF-BB yes

[0286] Spirulina platensis powder nEV FRET TGF-B yes

[0287] BY4741 nEV FRET TGF-B yes

[0288] E. coli nEV FRET TGF-B yes

[0289] Optical Waveguide Lightmode Spectroscopy (OWLS)

[0290] OWLS 210 instrument (Microvacuum Kft., Budapest, Hungary) was used to investigate the EGF, FGF-1, PDGF- BB, and HSA binding of nEVs of MSC and MACC-360 cells, and that of the LVs isolated from MSC, MACC- 360 cells and Spirulina platensis and Chlorella vulgaris powder (Table 3.). Briefly, the method relies on the coupling of plane polarised He-Ne laser light into the waveguide layer by a diffraction grating. The measurements were done in 42 mM Tris pH 7.4 buffer at 25 °C. The instrument is a flow injection analysis (FIA) system, first the waveguide surfaces were coated with poly-L-lysine (PLL) (P3543, MERCK, Hungary) and the investigated MVs were injected onto this surface after the stabilization of the baseline signal. Finally, the proteins to be examined were injected. As a control measurement, the proteins were injected directly onto the PLL-treated waveguide surface leaving out the MVs. During the measurements the shift of the coupling angle was investigated after each injection. From the angle shifts refractive index, the thickness of the layer formed can be calculated and the mass per area of the bound material can be determined.

[0291] Table 3. List of the membrane vesicles (MV) of different origin investigated by Optical Waveguide Lightmode Spectroscopy (OWLS). nEV: native EV; 1MV: isolated lipid based MV; HSA 1MV: HSA treated 1MV. Yes = the investigated vesicle binds EGF, FGF-1, HAS, Human serum, PDGF-BB or TGF-B.

[0292] TABLE 3. The EGF, FGF-1 human serum albumin (HSA), human serum, PDGF-BB, or TGF- binding of nEVs, 1MV, and HSA 1MV of MSC, MACC-360, Chlorella vulgaris powder or spirulina platensis powder was investigated by OWLS.

[0293] MV Source MV Type Assay Treatment Binding

[0294] MSC nEV OWLS EGF yes

[0295] MSC nEV OWLS FGF-1 yes

[0296] MSC nEV OWLS HSA yes

[0297] MSC nEV OWLS PDGF-BB yes

[0298] MSC nEV OWLS Human serum yes

[0299] MSC nEV OWLS TGF-B yes

[0300] MACC-360 nEV OWLS EGF yes

[0301] MACC-360 nEV OWLS FGF-1 yes

[0302] MACC-360 nEV OWLS HSA yes

[0303] MACC-360 nEV OWLS PDGF-BB yes

[0304] MACC-360 nEV OWLS Human serum yes

[0305] MACC-360 nEV OWLS TGF-B yes

[0306] MACC-360 1MV OWLS EGF yes

[0307] MACC-360 1MV OWLS FGF-1 yes

[0308] MACC-360 1MV OWLS HSA yes

[0309] MACC-360 1MV OWLS PDGF-BB yes

[0310] MACC-360 1MV OWLS Human serum yes

[0311] MACC-360 1MV OWLS TGF-B yes

[0312] MSC 1MV OWLS EGF yes

[0313] MSC 1MV OWLS FGF-1 yes

[0314] MSC 1MV OWLS HSA yes

[0315] MSC 1MV OWLS PDGF-BB yes

[0316] Chlorella vulgaris powder 1MV OWLS EGF yes

[0317] Chlorella vulgaris powder 1MV OWLS FGF-1 yes

[0318] Chlorella vulgaris powder 1MV OWLS HSA yes

[0319] Chlorella vulgaris powder 1MV OWLS PDGF-BB yes

[0320] Chlorella vulgaris powder 1MV OWLS TGF-B yes Chlorella vulgaris powder HSA 1MV OWLS TGF-B yes

[0321] Spirulina platensis powder 1MV OWLS EGF yes

[0322] Spirulina platensis powder 1MV OWLS FGF-1 yes

[0323] Spirulina platensis powder 1MV OWLS HSA yes

[0324] Spirulina platensis powder 1MV OWLS PDGF-BB yes

[0325] MTT (cell proliferation and viability) assay

[0326] MTT assay was performed on platelet derived growth factor B (PDGF-B, 10 ng / ml, R&D Systems, USA) treated primary pFBs, cFBs, sFBs in the presence or absence of MVs from different sources (Table 4., 5., 6. ). In some cases MV treatment was supplemented with nintedanib (0.1 pM), axitinib (0.01 pM, Merck Kft, Hungary), or ponatinib (0.01 pM, Merck Kft, Hungary) treatment (Table 6.). Vehicle treated cells served as controls. 24 hours after treatments, cell proliferation / viability was determined by a colorimetric method, based on the intracellular mitochondrial dehydrogenase activity of the attached cells. Briefly, 10 pl of MTT reagent, containing 5 mg / ml thiazolyl blue 30 tetrazolium bromide (diluted in sterile H2O) was added into each well including cells and 100 pl of supernatant as well, then incubated at 37 °C for 4 hours. Thereafter, the supernatants were removed from cells using a pipette, and the intracellular MTT crystals were dissolved by adding 100 pl 1:1 mixture of DMSO and ethanol (all reagents were purchased from Merck, Germany). Absorbance was recorded at 570 nm and at 690 nm as background in a SPECTROstar Nano microplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, 35 Germany). Results were normalized and determined as percentage ratio of control group values. Table 4. Antiproliferative effect of nativ extracellular vesicles (nEV) and modified membrane vesicels (mMVs), including nEVs, exMVs, gMVs, IMVs, tJMVs, t_nEVs of MSC, IPS, pFB, cFB, RBC, A549, HCT-116, HK-2, MACC-1, MACC-3, MACC-360, MACC-908, Chlorella vulgaris powder, Spirulina platensis powder, BY4741, K. lactis, S. pastorianus, S. pombe, Y. lipolytica, L. bulgaricus, L. lactis, E. coli, Pseudomonas aeruginosa or wheat grass origin was investigated on human recombinant PDGF-BB or human peritoneal dialysis effluent (PDE) treated cFBs, IFBs, pFBs, sFBs, A549, or HT-29 cells by MTT cell proliferation assay (n=5). To investigate the difference between control vs. control + EV or treatment vs. treatment + EV groups t-test was performed. The statistical difference was considered to be significant if p<0.05. Yes=the investigated MVs inhibited the PDE or PDGF-BB induced cell proliferation. No= the investigated MVs had no effect or induced the PDE or PDGF-BB induced cell proliferation.

[0327] TABLE 4. Effect of MVs of different type and origin on the proliferation of different fibroblasts

[0328]

[0329] Table 5. The antiproliferative effect of hybride modified membrane vesicels (mMVs), including Co exMVs, Co lMVs, Ch lMVs, and HSA-lMVs of MSC, pFB, MACC-360, MACC-908, Chlorella vulgaris powder, Spi- rulina platensis powder, Y. lipolytica, S. pastorianus, S. pombe, K. lactis, L. lactis, or L. bulgaricus origin was investigated on recombinant PDGF-BB induced cFBs or sFBs by MTT cell proliferation assay (n=5 / 6). The effect of Co lMVs of MSCs and pFBs on the collagen production of TGF-B treated cFBs was investigated by SiriusRed assay (n=5). To investigate the difference between control vs. control + EV or treatment vs. treatment + EV groups t-test was performed. The statistical difference was considered to be significant if p<0.05. Yes=the investigated hybride MVs inhibited the PDGF-BB induced cell proliferation or TGF-B induced collagen production of the fibroblasts. No= the investigated MVs had no effect or induced the PDGF-BB induced cell proliferation or on the TGF-B induced collagen production of the fibroblasts. TABLE 5. Effect of different type of hybride / combined MVs of different origin on the proliferation or collagen production of different fibroblasts. inhibition inhibition

[0330] MV Source 1 MV Source 2 MV Type Treatment Cell Assay on untre- on treated ated

[0331] MSC MACC-360 Co exMV PDGF-BB cFB MTT yes yes

[0332] MSC MACC-908 Co exMV PDGF-BB sFB MTT yes yes

[0333] MSC K. lactis Co exMV PDGF-BB sFB MTT yes yes

[0334] MSC MACC-360 Co 1MV PDGF-BB cFB MTT yes yes

[0335] MSC L. bulgaricus Co 1MV PDGF-BB cFB MTT yes yes

[0336] Spirulina platen-

[0337] MSC sis powder Co 1MV PDGF-BB cFB MTT yes yes

[0338] MSC Y. lipolytica Co 1MV PDGF-BB cFB MTT yes yes pFB S. pastorianus Co 1MV PDGF-BB cFB MTT yes yes

[0339] MSC cholesterol Ch 1MV PDGF-BB cFB MTT no yes

[0340] K. lactis cholesterol Ch 1MV PDGF-BB cFB MTT yes yes

[0341] L. lactis cholesterol Ch 1MV PDGF-BB cFB MTT yes yes

[0342] MACC-360 HSA HSA 1MV PDGF-BB sFB MTT yes yes

[0343] MACC-908 HSA HSA 1MV PDGF-BB sFB MTT yes yes

[0344] Chlorella vulgaris powder HSA HSA 1MV PDGF-BB sFB MTT yes yes

[0345] S. pastorianus HSA HSA 1MV PDGF-BB sFB MTT yes yes

[0346] S. pombe HSA HSA 1MV PDGF-BB sFB MTT yes yes

[0347] MSC cholesterol Co 1MV TGF-B cFB SR no yes pFB S. pastorianus Co 1MV TGF-B cFB SR no yes

[0348] Table 6. The antiproliferative effect of nativ extracellular vesicles (nEV) or modified membrane vesicels (mMVs), including IMVs and t lMVs of pFB or MACC-360 origin combined with nintedanib, ponatinib or with axitinib was investigated on recombinant PDGF-BB induced pFBs by MTT cell proliferation assay (n=5 / 6).

[0349] To investigate the difference between control vs. control + EV or treatment vs. treatment + EV groups t-test was performed. The statistical difference was considered to be significant if p<0.05. Yes = combined treatment with the investigated MVs and drug was significantly more effective to inhibit the PDGF-BB induced fibroblast proli- feratino than treatment with the investigated MV or drug alone.

[0350] TABLE 6. Effect of different type of MVs of different origin and small molecular weight drugs on the proliferation of fibroblasts.

[0351] MV Source MV Type Treatment Cotreatment Cell Assay inhibition on treated pFB 1MV PDGF-BB nintedanib pFB MTT yes pFB t 1MV PDGF-BB nintedanib pFB MTT yes pFB 1MV PDGF-BB axitinib pFB MTT yes pFB 1MV PDGF-BB ponatinib pFB MTT yes

[0352] MACC-360 nEV PDGF-BB nintedanib pFB MTT yes

[0353] MACC-360 1MV PDGF-BB nintedanib pFB MTT yes

[0354] MACC-360 t 1MV PDGF-BB nintedanib pFB MTT yes

[0355] Sirius Red (collagen detection) assay

[0356] Sirius Red assay was performed on transforming growth factor beta (TGF-P, 1 nM, R&D Systems, USA) treated cFBs in the presence or absence of MVs from different sources (Table 7.). Vehicle treated cells served as controls. 48 hours after treatments, collagen deposition was determined based on a basic histological dye SiriusRed, incorporating into the triple helical collagen molecules. After removing supernatants, cells were incubated in a fixative solution containing ethanol, 3.7% formaldehyde, 2% glacial acetic acid for 15 minutes at room temperature. Samples were stained for 1 hour at room temperature with 0.1% solution of SiriusRed (DirectRed80) dissolved in 1% acetic acid, then washed three times with 200 pl of 0.1 M HC1, and finally the bounded dye was dissolved by adding 100 pl of 0.1 M NaOH (all reagents were purchased from Merck, Germany). Absorbance was recorded at 544 nm and at 690 nm as background in a SPECTROstar Nano microplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, Germany). Results were normalized and determined as percentage ratio of control group values.

[0357] Table 7. The effect of nEVs, t_nEVs, exMVs, gMVs, and IMVs of MSC, IPS, pFB, cFB, RBC, A549, HCT-116, HK-2, MACC-360, MACC-908, Chlorella vulgaris powder, Spirulina platensis powder, BY4741, K. lactis, S. pastorianus, S. pombe, Y. lipolytica, E. coli on the collagen production of TGF-B treated cFBs or sFBs was investigated by SiriusRed assay (n=5). To investigate the difference between control vs. control + EV or treatment vs. treatment + EV groups t-test was performed. The statistical difference was considered to be significant if p<0.05. Yes=the investigated MVs inhibited the PDE or TGF-B induced collagen production of the fibroblasts. No= the investigated MVs had no effect or induced on the TGF-B induced collagen production of the fibroblasts.

[0358] TABLE 7. Effect of different type of MVs of different origin on the collagen production of different fibroblasts.

[0359] MV Source MV type _ Treat .men *t / C-.e ilil Assay inhibition . o .n unt- inhibition on reated t .reat .ed .

[0360] MSC nEV TGF-B cFB SR no yes

[0361] MSC 1MV TGF-B cFB SR no yes

[0362] IPS nEV TGF-B sFB SR no yes

[0363] IPS t nEV TGF-B sFB SR no yes

[0364] IPS nEV TGF-B cFB SR no yes pFB 1MV TGF-B cFB SR no yes pFB 1MV PDE cFB SR no yes pFB gMV PDE cFB SR yes yes cFB 1MV TGF-B cFB SR no yes

[0365] RBC 1MV TGF-B cFB SR no yes

[0366] A549 1MV TGF-B cFB SR no yes

[0367] A549 exMV TGF-B cFB SR no yes

[0368] HCT-116 1MV TGF-B cFB SR no yes

[0369] HK-2 1MV TGF-B cFB SR no yes

[0370] MACC-360 1MV TGF-B cFB SR no yes

[0371] MACC-360 exMV TGF-B cFB SR yes yes

[0372] MACC-360 nEV TGF-B cFB SR no yes

[0373] MACC-908 exMV TGF-B cFB SR no yes

[0374] Chlorella vulgaris powder 1MV PDE cFB SR yes no

[0375] Chlorella vulgaris powder 1MV TGF-B cFB SR no yes

[0376] Spirulina platensis powder 1MV PDE cFB SR yes yes

[0377] Spirulina platensis powder 1MV TGF-B cFB SR no yes

[0378] Spirulina platensis powder nEV TGF-B cFB SR no yes

[0379] BY4741 nEV TGF-B cFB SR yes yes

[0380] K. lactis 1MV TGF-B cFB SR no yes

[0381] S. pastorianus 1MV TGF-B cFB SR no yes S. pastorianus 1MV PDE cFB SR no yes

[0382] S. pombe 1MV TGF-B cFB SR no yes

[0383] Y. lipolytica 1MV TGF-B cFB SR no yes

[0384] Y. lipolytica exMV TGF-B cFB SR no yes

[0385] E. coli nEV TGF-B cFB SR yes yes

[0386] TAS (cell migration) assay

[0387] TAS assay was performed as we previously described (PMID: 35216230) on epidermal growth factor (EGF, 10 ng / ml, R&D Systems, USA) or PDE treated human cFBs in the presence or absence of 109particles / ml MVs. Vehicle treated (PBS in case of EGF and EV, 30% PDF in case of PDE) cells served as controls (Table 8.). Briefly, cells were seeded at near-full density into 96 well-plates, containing non-toxic gel barriers to create cell-free zones. After 24 hours of incubation, barriers were removed and wells were washed with PBS, thereafter cells were treated. Bright-field images of each well were taken using Olympus IX-81 microscope system (Olympus, Japan) at 0, 24, 48 and 72 hours after treatment. Cell-free gap area was measured using ImageJ 1.48v software and determined as a ratio of initial gap area at 0 hour.

[0388] Table 8. The effect of IMVs of MACC-360, Spirulina platensis powder, K. lactis, Y. lipolytica, L. lactis, and L. bulgaricus on the EGF or PDE induced migration of cFBs was investigated by TAS assay (n=5). To investigate the difference between control vs. control + EV or treatment vs. treatment + EV groups t-test was performed. The statistical difference was considered to be significant if p<0.05. Yes=the investigated MVs inhibited the PDE or EGF induced migration of the fibroblasts. No= the investigated MVs had no effect or induced on the EGF induced migration of the fibroblasts.

[0389] TABLE 8. Effect of IMVs of different origin on the migration of fibroblasts.

[0390] „ , „7. _ . * / -. n * inhibition on inhibition on

[0391] MV Source MV type Treatment Cell Assay . . . . . . untreated treated

[0392] MACC-360 1MV EGF cFB TAS no yes

[0393] MACC-360 1MV PDE cFB TAS no yes

[0394] Spirulina platensis powder 1MV EGF cFB TAS yes no

[0395] K. lactis 1MV PDE cFB TAS no yes

[0396] K. lactis 1MV EGF cFB TAS no yes

[0397] Y. lipolytica 1MV PDE cFB TAS yes yes

[0398] Y. lipolytica 1MV EGF cFB TAS no yes

[0399] L. lactis 1MV EGF cFB TAS no yes

[0400] L. lactis 1MV PDE cFB TAS no yes

[0401] L. bulgaricus 1MV EGF cFB TAS no yes

[0402] L. bulgaricus 1MV PDE cFB TAS no yes

[0403] Statistical Analysis

[0404] Data were analyzed using GraphPad Prism 8.0. software (GraphPad Software Inc., USA). After testing normality by Kolmogorov-Smirnov test, analysis of significance was performed by unpaired t-test or Mann- Whitney test. In case of the EV-EV and EV- combination testing two-way ANOVA was used to analyse the differences. Results were illustrated as mean+SD of the corresponding groups. The applied tests, significances, and number of elements (n) are indicated in the Brief description of figures.

[0405] RESULTS

[0406] Isolation of native extracellular vesicles (nEVs) The present inventors have initially isolated native extracellular vesicles (nEVs) from different sources (see section methods). The size distribution, median size, and particle concentration of the nEVS were determined using nanoparticle tracking analysis (NTA). According to NTA measurements the average size of the nEVs was 144.87 ± 9.88 nm.

[0407] Preparation of modified membrane vesicles

[0408] Modified (artificial) membrane vesicles, including exMVs, gMVs, IMVs, t nEVs, t lMVs, Ch lMVs, and HSA lMVs were isolated from different sources (see section methods). The size distribution, median size, and particle concentration of the MVs were determined using nanoparticle tracking analysis (NTA) or microscopy. The average size of the MVs was 146.93 ± 12.10 nm except that of gMVs average size of which was 9.0 ± 2.0 pm.

[0409] Characterisation

[0410] Figure 1 shows protein analysis of nanoparticle-samples by (a) silver staining, (b) UV-spectrophotometry, and (c) protein assay. As can be seen therein, the lipid extract of primary human peritoneal fibroblast from patient A (phPFB / A); the trypsin treated lipid extract of pFB; the lipid extract of MACC-360 and the trypsin treated lipid extract of MACC-360 contained no protein. The giant plasma membrane vesicles (gMVs) of pFB, EV of MSCs from PDE origin, EVs isolated from the peritoneal effluent of a patient with peritoneal dyalisis, native EV of Chlorella sp. MACC-360 origin, and MACC-360 extrudate contained some protein.

[0411] FACS

[0412] While nEVs and gMVs of MSC origin were immunopositive for known EV markers including CD9, CD81, and Alix exMVs or IMVs of MSC origin were immunonegative for the investigated nEV markers (Table 9.). These data, in accordance with protein analyisis and supplemented with NTA / microscopic analysis of MVs demonstrate that modified MVs can be clearly discriminated from nEVs based on their size and protein content.

[0413] Table 9. The molecular markers of the different type of MVs.

[0414] > nEV of MSC origin exMV of MSC origin 1MV of MSC origin

[0415] CD9 _ + _ - _ - _

[0416] CD81 + - > - >

[0417] ALIX - > - >

[0418] Penetration:

[0419] Penetration of nEVs, IMVs and gMVs were investigated into cFB, pFB cell. Each investigated MVs can penetrate into the fibroblasts, exept gMVs (Table 10). In vivo intraperitoneally or intravenously administrated nEVs of MACC-360 origin were present in the brain, spleen, live, lung, kidney and intestine of the C57BF6 mice.

[0420] Table 10. The in vitro penetration of different type of MVs into different fibroblasts.

[0421] MV Source MV Type Target cell Penetration

[0422] MSC nEV pFB Yes

[0423] MACC-360 nEV cFB Yes

[0424] MACC-360 1MV cFB Yes

[0425] Spirulina platensis powder nEV cFB Yes

[0426] BY4741 nEV sFB Yes BY4741 nEV cFB Yes

[0427] E. coli nEV pFB Yes pFB gMV pFB No

[0428] FRET:

[0429] It was demonstrated by fluorescence resonance energy transfer (FRET) that native EVs from different sources, namely from MSCs, MACC-360 EV, Spirulina platensis powder, BY4741 EV, E.coli, and MVs from pFBs (1MV, t lMV, gMV) and MACC-360 (1MV) bound PDGF-B, and native EVs from Spirulina platensis powder, BY4741 EV, E.coli, bond TGF-B (Table 2).

[0430] OWLS:

[0431] It was revealed by optical waveguide lightmode spectroscopy (OWLS) that nEVs originated from MSCs, MACC- 360 and IMVs originated from MACC-360 bund EGF, FGF-1, HSA, PDGF-BB, Human serum, and TGF-B. It was also demonstrated that 1_MV of MSC, Chlorella vulgaris and Spirulina platesnis powder bind EGF, FGF-1, HSA, andPDGF-BB andthati MV andHSA lMV originated from Chlorella vulgaris powder bind TGF-B (Table 3).

[0432] M i l :

[0433] It was demonstrated that different MVs, including exMV, gMV, 1MV, nEV, t lMV, t nEV, Co exMV, Co lMV, Ch lMV, HAS 1MV of MSC, IPS, pFB, cFB, RBC, A549, HCT-116, HK-2, MACC-1, MACC-3, MACC-360, MACC-908, Chlorella vulgaris powder, Spirulina platensis powder, BY4741, K. lactis, S. pastorianus, S. pombe, Y. lipolytica, L. bulgaricus, L. lactis, E. coli, Pseudomonas aeruginosa, and wheat grass origin altered the PDGF- BB induced proliferation of pFB, cFBs, sFBs and IFBs (Table 4, 5, and 6).

[0434] SR:

[0435] It was demonstrated that different MVs, including exMV, gMV, 1MV, nEV, t nEV, Co lMV of MSC, IPS, pFB, cFB, RBC, A549, HCT-116, HK-2, MACC-360, MACC-908, Chlorella vulgaris powder, Spirulina platensis powder, BY4741, K. lactis, S. pastorianus, S. pastorianus, S. pombe, Y. lipolytica, E. coli origin altered the TGF-P induced collagen production of cFBs and sFBs (Table 5 and 7)

[0436] TAS:

[0437] It was demonstrated that IMVs of MACC-360, Spirulina platensis powder, K. lactis, Y. lipolytica, L. lactis, L. bulgaricus origin altered the proliferation the EGF or PDE induced migration of cFBs (Table 8).

Claims

CLAIMS1. Modified membrane vesicles (rn Vs) for use in inhibiting the activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathways, wherein the rnMVs are capable of binding the growth factor when the rnMVs are administered to a subject, and wherein the rnMVs are produced by a process comprising at least one non-naturally occurring step.

2. The rnMVs for use according to claim 1, wherein the rnMVs are selected from the group consisting of i) a synthetic MV, wherein the synthetic MV is an MV synthesized de novo from molecular components, ii) an artificial, cell derived MV wherein the artificial, cell derived MV is an MV produced by a process comprising a non-naturally occurring step of disrupting a cell, iii) a lipid MV, wherein the lipid MV is an MV produced by a process comprising a non-naturally occurring step of lipid isolation, iv) an impaired MV, wherein the impaired MV is an MV produced from a cell or a native extracellular vesicle (nEV) by a process comprising at least one non-naturally occurring step that results in- a decrease in the diversity of component(s) or decreased amount of component(s) of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, or- a decrease in the diversity of biologically active component(s) or a decreased amount of biologically active component(s) of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, or- the loss of a biological function of component(s) of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, v) an induced MV, wherein the induced MV is an MV obtained from a cell by a process comprising at least one non-naturally occurring step that results in- a decrease in the diversity of components or a decreased amount of a component of the mMV compared to the cell, or- a decrease in the diversity of biologically active components or a decreased amount of a biologically active component of the mMV compared to the cell, or- the loss of a biological function of a component of the mMV compared to the cell, or vi) a supplemented MV, wherein the supplemented MV is an MV obtained from an nEV or a cell by a process comprising at least one non-naturally occurring step that results in- an increase in the diversity of components or an increased amount of a component(s) of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, or- an increase in the diversity of biologically active components or an increased amount of a biologically active component(s) of the mMV compared to the nEV obtained by the same process lacking the at least one non-naturally occurring step, or- gain of a biological function(s) of a component of the mMV compared to an nEV obtained by the same process lacking the at least one non-naturally occurring step, vii) a hybrid of nEVs derived from different species, viii) a combination of rnMVs or a combination of nEV(s) and nEV(s), or a combination of nEV(s) and mMV(s).

3. The mMVs for use according to claim 1 or 2, wherein the membrane of the mMVs essentially lacks functional protein components.

4. The mMVs for use according to claim 2 or 3, wherein the rnMVs are selected from ii) an artificial, cell derived MV, wherein the artificial, cell derived MV is an MV produced by the extrusion of the cell, iii) a lipid MV, wherein the lipid MV is produced by a process comprising a non-naturally occurring step of lipid isolation from a cell, followed by vesicle formation, iv) an impaired MV, wherein the impaired MV is depleted in functional proteins, v) an induced MV, wherein the induced MV is a giant plasma membrane vesicle, vii) a hybrid or combination of any one of ii)-v).

5. The mMVs for use according to any one of claims 2-4, wherein the mMVs are selected from a lipid MV, wherein the membrane of the lipid MV essentially lacks functional protein components, an impaired MV, wherein the membrane of the impaired MV essentially lacks functional protein components, a supplemented MV, which is a lipid MV with a membrane that essentially lacks functional protein components and which is supplemented with a human protein, lipid or polysaccharide, wherein the human protein is not a receptor of the growth factor, a hybrid or combination thereof.

6. The mMVs for use according to any one of the preceding claims, for use in the treatment of a cell proliferative disorder.

7. The mMVs for use according to claim 6, wherein the cell proliferative disorder is selected from a neoplasm or a fibroproliferative disease.

8. The mMVs for use according to claim 7, wherein the cell proliferative disorder is a fibroproliferative disease.

9. The mMVs for use according to any one of the preceding claims, wherein the growth factor is selected from PDGF, TGF, EGF, FGF.

10. The mMVs for use according to claim 9, wherein the growth factor is selected from PDGF-B and TGF-beta.

11. The mMVs for use according to any one of the preceding claims, wherein the mMVs are free of an added active agent for use in inhibiting the activity of a growth factor, a receptor of a growth factor or in modulating, preferably inhibiting the activity of a growth factor mediated signaling pathway.

12. The mMVs for use according to any one of the preceding claims, wherein the mMVs are free of an added active agent for use in the treatment of a cell proliferative disorder.

13. The mMVs for use according to claim 5, for use in the treatment of a cell proliferative disorder, wherein the mMVs are free of an added active agent for use in the treatment of a cell proliferative disorder.

14. The mMVs for use according to claim 13, wherein the growth factor is PDGF-BB.

15. The mMVs for use according to claim 1, for use in the treatment of a cell proliferative disorder, wherein the mMVs are free of an added active agent for use in the treatment of a cell proliferative disorder, the mMVs are artificial, cell derived MVs produced by extrusion of a cell and the growth factor is PDGF-BB.

16. The mMVs for use according to claim 1, for use in the treatment of a cell proliferative disorder, wherein the mMVs are free of an added active agent for use in the treatment of a cell proliferative disorder, the mMVs are giant plasma mebrane vesicles and the growth factor is PDGF-BB.

17. The mMVs for use according to any one of the preceding claims, for use in inhibiting ECM production, preferably collagen production.

18. The mMVs for use according to claim 17, wherein the growth factor is TGF-beta.

19. The mMV s for use according to claim 5 , for use in inhibiting ECM production, preferably collagen production.

20. The mMVs for use according to any one of the preceding claims, for use in inhibiting cell migration.

21. The mMVs for use according to claim 20, wherein the growth factor is EGF.

22. The mMVs for use according to claim 5, for use in inhibiting cell migration.

23. Modified membrane vesicles obtainable by mixing isolated lipids of a human cell and isolated lipids of an algal cell, a bacterial cell or a yeast cell under conditions allowing the formation of vesicles.

24. Modified membrane vesicles obtainable by combining membrane components of a human cell and membrane components of an algal cell, a bacterial cell or a yeast cell.

25. Extracellular vesicles (EVs) for use in the treatment of a cell proliferative disease, wherein the EVs are from algal cells, yeast cells or bacterial cells.

26. The EVs for use according to claim 25, wherein the EVs are from yeast cells, preferably from Saccharomyces sp. cells or from Kluyveromyces sp. cells, preferably from Saccharomyces cerevisiae cells, preferably BY4741 cells or from Saccharomyces pombe cells or from Saccharomyces pastorianus cells or from Kluyveromyces lactis cells.

27. The EVs for use according to claim 25 or 26, wherein the EVs are supplemented with a component of human origin, preferably with a human protein.

28. The EVs for use according to any one of claims 25-27, for use in inhibiting PDGF-BB induced activation.

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