Biological material impregnated with lipid vesicles

Abstract

Biological material impregnated with lipid vesicles The present invention relates to a biological material impregnated with at least one active principle characterized in that said active principle is a solution comprising lipid vesicles.

Description

Title of the invention: Biological material impregnated with lipid vesicles

[0001] The present invention relates to the field of biological materials impregnated with active principles and their uses in therapeutic treatments. PREVIOUS ART

[0002] Exosomes are a form of extracellular vesicles. Exosomes are vesicles with a diameter between 30 and 100 nm. They are surrounded by a lipid bilayer and float at a density of 1.13-1.19 g / ml in sucrose gradients. Vesicles with the characteristics of exosomes have been isolated from multiple different body fluids, including semen, blood, saliva, breast milk, amniotic fluid, ascitic fluid, cerebrospinal fluid, and bile. Over the past decade, much research has focused on exosomes as a mode of intercellular communication since exosomes are secreted by various cell types, including stem cells. Some studies indicate that mesenchymal stem cells secrete exosomes in vitro and that these exosomes possess therapeutic properties.

[0003] Exosomes contain many proteins or nucleic acids of interest such as growth factors, cytokines, heat shock proteins (HSPs), amino acids, nucleic acids (DNAs, RNAs), metabolites, enzymes etc. They also include membrane proteins that can act as receptors / ligands in different cell signaling pathways.

[0004] The publication Bakhtyar et al. Stem Cell Research & Therapy (2018) 9:193 discloses that exosomes derived from mesenchymal cells present in Wharton's jelly have beneficial properties on healing.

[0005] The chorioamniotic membrane, which separates the fetus from the mother's endometrium in mammals, includes the amniotic membrane, or amnion, and the chorionic membrane, or chorion. These two membranes are connected by a spongy tissue membrane, also called the spongy layer, consisting of, among other things, collagen and proteoglycans, the spongy tissue membrane comprising protein bridges, attached on either side to the amnion on the one hand, and to the chorion on the other hand.

[0006] The amniotic membrane is the innermost layer of the chorioamniotic membrane. Its role is to protect the fetus and maintain the amniotic fluid around it. This amniotic membrane is a very thin, transparent tissue which has several layers, namely an epithelial layer, a basement membrane, a layer compact and a fibroblastic layer.

[0007] Non-vascularized and non-innervated, the amniotic membrane is rich in collagen and various growth factors, and has properties that contribute to the healing process.

[0008] Amniotic membrane, obtained from the placenta after childbirth, is a tissue that has been used for over a hundred years in the treatment of burns and wounds. Indeed, as early as 1910, Davies was using fetal membranes on both burns and ulcerated tissue. Trelford and Trelford-Sauder report that in 1935 authors published clinical applications of amniotic membrane in vaginoplasty, conjunctival reconstruction, treatment of burns or wounds, and treatments related to intra-abdominal adhesion. Trelford et al. also report that in 1952 Douglas used amnion to treat extensive wounds. For the first time, it was indicated that the stromal layer of the amniotic membrane, including the compact layer and the fibroblastic layer, allowed for greater adhesion of the graft, and therefore its effectiveness. In 1972, the work of Trelford et al. confirmed this fact. Gindraux et al.report that since 1972, and especially since its rediscovery in 1995, other authors have confirmed all the clinical applications previously presented, and have also reported new indications such as the genitourinary tract, stomach, larynx, oral cavity, head and neck, whether in clinical trials or case reports.

[0009] Amniotic membranes are particularly rich in growth factors such as EGF (Epidermal Growth Factor), TGF (Transforming Growth Factor) and KGF (Keratinocyte Growth Factor), as well as hyaluronic acid. These growth factors and hyaluronic acid are particularly suitable for promoting biological healing processes.

[0010] Wharton's jelly is a gelatinous connective tissue surrounding the vein and the two arteries of the umbilical cord of mammals. Wharton's jelly is a substance particularly rich in constituent elements of the extracellular matrix of connective tissues, in particular glycosaminoglycans and proteoglycans; as well as collagen fibers (types I, III, IV and V). Wharton's jelly also includes numerous growth factors such as, in particular, fibroblast growth factors (FGFs), insulin-like growth factors I (IGF-I), transforming growth factors (TGFs), platelet-derived growth factors (PDGFs) and epidermal growth factors (EGFs).

[0011] These structural properties of Wharton's jelly can be used for their properties of helping to heal lesions, in particular skin lesions or lesions of the ocular surface. Indeed, these constituent elements of Wharton's jelly participate in the improvement of the biological processes of healing and reduction inflammation in a patient.

[0012] Tissues from umbilical cord or placenta can serve as ideal matrices for the impregnation of active ingredients which will then be released when these tissues are used for various therapeutic treatments.

[0013] WO2020245324 discloses a biological lens filled with wharton jelly and impregnated with an active ingredient, gentamicin. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a biological material impregnated with at least one active principle, characterized in that said active principle is a solution comprising lipid vesicles.

[0015] By “biological material” we mean any material derived from human, animal or plant tissues.

[0016] By "impregnation" we mean that the active ingredient penetrates the material and spreads there, diffuses there.

[0017] Lipid vesicles contain or include in their structure molecules / proteins of interest depending on the pathology to be treated.

[0018] In one embodiment, the biological material according to the invention is characterized in that said lipid vesicles are of biological origin.

[0019] By “lipid vesicles of biological origin” we mean vesicles which originate from cells and which are not artificial assemblies.

[0020] In one embodiment, the biological material according to the invention is characterized in that said lipid vesicles are exosomes.

[0021] The content of exosomes is dependent on the type of cells from which they were isolated.

[0022] Exosomes can be isolated from different cell types of interest depending on the pathology to be treated. Isolation can be carried out by any technique known to those skilled in the art, for example centrifugation, ultrafiltration or immunoprecipitation.

[0023] The active ingredient may be a solution comprising exosomes from a single cell type or a mixture of exosomes from different cell types.

[0024] In one embodiment, the biological material according to the invention is characterized in that said exosomes are derived from mesenchymal stem cells.

[0025] In one embodiment, the biological material according to the invention is characterized in that said mesenchymal stem cells are derived from umbilical cord.

[0026] In one embodiment, the biological material according to the invention is characterized in that said biological material is derived from one or more tissues of human origin.

[0027] In one embodiment, the biological material according to the invention is characterized in that said biological material is derived from placenta or umbilical cord.

[0028] In one embodiment, the biological material according to the invention is characterized in that said biological material is Wharton's jelly.

[0029] By "Wharton's jelly" is meant the gelatinous biological tissue present in the umbilical cord of mammals from which the vein and the two arteries naturally included within said gelatinous biological tissue have been removed. In the present invention, the term "Wharton's jelly" may be understood as including or not including the amniotic membrane surrounding the Wharton's jelly of the umbilical cord. In the present invention, the term "Wharton's jelly" is understood as not including thick collagen fibers and / or vascular lacunae and walls (villi and intervillous chambers).

[0030] In one embodiment, said biological material in the form of Wharton jelly is characterized in that it is obtained according to the method described in WO2019 / 038411.

[0031] In one embodiment, the biological material according to the invention is characterized in that said biological material is an amniotic membrane.

[0032] The term "amniotic membrane" refers to the tissue envelope that develops around the embryo, then the fetus, in mammals during pregnancy. Its role is to protect the developing organism by maintaining the amniotic fluid around it. It is attached to the second membrane, the chorion. The amniotic membrane includes the following physiological sub-layers: the epithelial cell layer, the basement membrane, the compact layer, the fibroblastic layer, and the spongy layer.

[0033] In one embodiment, said biological material in the form of amniotic membrane is characterized in that it is obtained according to the method described in WO2017 / 140914.

[0034] In one embodiment, the biological material according to the invention is characterized in that said biological material is sterile.

[0035] Since amniotic membranes and Wharton's jelly are biological tissues, there is a significant risk of transmission of infection from a donor to a recipient, or of contamination during collection of the tissue at delivery, during preparatory treatments of the tissue, or during transport of this tissue. In fact, an effective process for both disinfection and preservation of these tissues is vital for patient safety.

[0036] In one embodiment, the biological material according to the invention is characterized in that said biological material is virally inactivated.

[0037] By "viro-inactivation" we mean a technique which makes it possible to strongly reduce or completely and definitively, the capacity of action of viruses. These, defined as inactivated, lose their pathogenic and replication capacities by a decrease in their population of 4 log during residual titrations which follow one or two independent chemical steps, whether on enveloped or non-enveloped viruses, DNA or RNA.

[0038] Such a method is for example described in documents WO2017 / 140914 or WO2019 / 038411 in the name of TBF GENIE TISSULAIRE.

[0039] In one embodiment, the device according to the invention is characterized in that said biological material is viro-inactivated according to the two chemical viro-inactivation steps of the method described in WO2017 / 140914 or WO2019 / 038411.

[0040] In one embodiment, the biological material according to the invention is characterized in that said biological material is dehydrated.

[0041] In one embodiment, the biological material according to the invention is characterized in that said biological material is lyophilized.

[0042] By "freeze-drying" is meant a technique aimed at drying a previously frozen product by sublimation. More precisely, the liquid to be removed from the product is first transformed into ice by freezing; then by primary drying, under vacuum, the ice is sublimated; finally by secondary drying, the water molecules on the surface of the product are extracted by desorption.

[0043] Viro-inactivation treatments as well as lyophilization destroy the membranes of exosomes which would be naturally present in the tissues serving as matrices, the environment of these matrices after these treatments is therefore suitable for being impregnated by solutions comprising exosomes, these exosomes being able to come from many different cell types.

[0044] In one embodiment, the biological material according to the invention is characterized in that said biological material is impregnated with at least one second active principle.

[0045] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of antibiotics, antiseptics, antivirals, monoclonal antibodies, semi-synthetic inhibitors of metalloproteases, immunosuppressive agents, anti-inflammatories, antifungals, anti-allergics, anesthetics, or immunoadhesive proteins, agents for preventing dry eyes, alone or in combinations.

[0046] The at least second active ingredient may be an active ingredient which is naturally present or not in said biological material.

[0047] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of antibiotics. Some examples of antibiotics that can be used are given below: tetracyclines (daunomycin, tetracycline, chloretetracycline, oxytetracycline, etc.), glycopeptides (vancomycin, etc.), aminoglycosides (gentamycin, etc.), aminoglycosides (tobramycin, neomycin, etc.), fluoroquinolones (ciprofloxacin, moxifloxacin, etc.), quinolones (gatifloxacin, etc.), polypeptides (bacitracin, polymyxin, etc.), phenicols (chloramphenicol, etc.), macrolides (erythromycin, etc.), sulfonamides (sulfacetamide, sulfamethoxazole, sulfisoxazole, etc.), cephalosporins (cefradoxil, ce-foxitin, etc.), and any other antibiotics.

[0048] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of steroidal anti-inflammatory drugs (ISAs) and / or non-steroidal anti-inflammatory drugs (NSAIDs). Some examples of ISAs are given below: triamcinolone, dexamethasone, prednisolone, hydrocortisone, corticosterone, fluocinolone, prednisolone, methylprednisolone, fluo-rometholone, betamethasone, tetrahydrocortisol, rimexolone, etc. Some examples of NSAIDs are given below: indomethacin, nepafenac, diclofenac, bromfenac, ketorolac, suprofen, etc.

[0049] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of immunosuppressants, a non-limiting list is given below: dexamethasone, betamethasone, etc.

[0050] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of antivirals, a non-limiting list is given below: ganciclovir, trifluorothymidine, aciclovir, DDI, AZT, foscamet, vidarabine, trifluorouridine, idoxuridine, ri-bavirin, protease inhibitors, anti-cytomegalovirus agent, etc.

[0051] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of antifungals, a non-limiting list is given below: fluconazole, nitro-furazone, amphotericin B, ketoconazole, etc.

[0052] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of antiallergics, a non-limiting list is given below: methapyriline, chlorpheniramine, pyrilamine, prophenpyridamine, etc.

[0053] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of anesthetics, a non-limiting list is given below: lidocaine, mepivacaine, etc.

[0054] In one embodiment, the biological material according to the invention is characterized in that said at least one second active ingredient is chosen from the group consisting of agents to prevent dry eyes, a non-exhaustive list is given below: azithromycin, cyclosporine, lubricants etc. EXAMPLES

[0055] Example 1: Provision of a biological material being a virus-inactivated amniotic membrane:

[0056] A properly informed and consenting donor in accordance with the requirements of the Declaration of Helsinki offers placental tissue from childbirth as a donation. This childbirth may have taken place by caesarean section or vaginally. Due to health requirements relating to donations of tissues and cells of human origin, upstream qualification of the donor is systematic. This qualification involves a search for HIV, hepatitis B, C, HTLV and the pale treponema bacterium responsible for syphilis.

[0057] Placental tissue is collected as soon as possible in the delivery room following delivery, whether vaginal or by caesarean section. It can be advantageously placed in a sterile box and then frozen at a temperature of -20°C.

[0058] This isolated placental tissue can be stored dry at a temperature of -80°C for up to two years or be directly processed.

[0059] In the laboratory, in a sterile room, the following procedure is applied:

[0060] The amniotic membrane is isolated from the placenta and cleaned.

[0061] The amniotic membrane undergoes a succession of baths ensuring treatment chemical removal of it. This treatment aims to disinfect the amniotic membrane, and in particular its viral inactivation. Gentle agitation at approximately 30 rpm of the liquid medium is applied during each bath to ensure homogeneous penetration of the solvents into the tissues.

[0062] Firstly, the amniotic membrane is placed in a bath of purified water at room temperature for approximately 3 hours. This step ensures both the thawing of the physiological tissue and a first step of cell lysis by osmotic pressure.

[0063] Then, the amniotic membrane is transferred into a decontaminating bath composed of 70% v / v ethanol at room temperature for approximately 1 hour.

[0064] A wash is carried out in purified water for approximately 15 minutes at room temperature to remove the ethanol.

[0065] In order to ensure the second stage of decontaminating treatment, the amniotic membrane is transferred into a bath composed of 30% w / v hydrogen peroxide at room temperature for approximately 15 minutes.

[0066] Then the amniotic membrane is transferred into a decontaminating bath composed of 3% w / v hydrogen peroxide at room temperature for approximately 1 hour.

[0067] The chemical action applied to the amniotic membrane can then be neutralized in at least one bath comprising, for example, dilute sodium hydroxide around a pH of 8.5. The at least one neutralization bath is carried out at room temperature for approximately 15 minutes. Interesting results have been found at this neutralization stage using two baths of dilute sodium hydroxide at pH 8.5.

[0068] In order to ensure a rebalancing of the pH and to best eliminate the organic residues detaching from the tissue of interest, the amniotic membrane is transferred into at least one bath of physiological buffer, chosen appropriately, in order to ensure its physiological rebalancing. The at least one bath is carried out at room temperature for approximately 15 minutes. Interesting results have been found at this stage using two successive baths in PBS solution.

[0069] Finally, the amniotic membrane can be transferred to a final bath of purified water, at room temperature, for at least 15 minutes and up to approximately 1 hour.

[0070] At this stage, it can be considered that the amniotic membrane obtained according to this chemical treatment can be defined as an amniotic membrane that is largely disinfected, in particular virally inactivated, and decellularized.

[0071] Example 2: Supply of a biological material being a ground Wharton jelly:

[0072] A properly informed and consenting donor in accordance with the requirements of the Declaration of Helsinki offers the umbilical cord from childbirth as a donation. Due to health requirements relating to donations of tissues and cells of human origin, upstream qualification of the donor is systematic. This qualification involves a search for HIV, hepatitis B, C, HTLV viruses and the pale treponema bacterium responsible for syphilis.

[0073] The umbilical cord is recovered as soon as possible in the delivery room. It is advantageously placed in a sterile box, containing a NaCl solution at +4°C.

[0074] In the laboratory, in a sterile room, the following procedure is applied:

[0075] A segment of 20 cm to 50 cm in length is isolated by cutting the umbilical cord.

[0076] The umbilical cord is rinsed and hydrated in successive purified water baths, with gentle stirring, for 4 hours.

[0077] The blood vessels of the umbilical cord segment are identified and separated from the rest of the segment in order to retain only the Wharton's jelly and the surrounding amniotic membrane. The Wharton's jelly and the amniotic membrane are used in the remainder of the process under the general name of Wharton's jelly, because the amount by weight of membrane is negligible compared to the amount by weight of Wharton's jelly.

[0078] Wharton's jelly is frozen dry at a temperature of -80°C.

[0079] During the chemical treatment, Wharton's jelly is thawed in the open air, at room temperature, for a period of 5 minutes. This freezing step, followed by thawing, ensures significant decellularization of the biological tissue.

[0080] Wharton's jelly undergoes a series of baths ensuring chemical treatment. The objective of this treatment is to disinfect the Wharton's jelly, and in particular to inactivate it virologically. Gentle linear agitation of the liquid medium is applied during each bath to ensure homogeneous penetration of the solvents into the tissues.

[0081] Firstly, the Wharton jelly is placed in a bath of purified water at room temperature for approximately 3 hours. This step ensures both the end of thawing of the physiological tissue and a step of cell lysis by osmotic pressure.

[0082] Then, the Wharton jelly is transferred into a decontaminating bath composed of 70% v / v ethanol at room temperature for approximately 1 hour.

[0083] A wash is carried out in purified water for approximately 15 minutes at room temperature to remove the ethanol.

[0084] In order to ensure the second decontamination treatment step, the Wharton jelly is transferred into a bath composed of 30% w / v hydrogen peroxide at room temperature for approximately 15 minutes.

[0085] Then, the Wharton jelly is transferred to a decontaminating bath composed of 3% w / v hydrogen peroxide at room temperature for about 1 hour. The Wharton jelly obtained is virus-inactivated. The chemical action applied to the virus-inactivated Wharton jelly is then neutralized, in a bath containing diluted sodium hydroxide around a pH of 8.5. The treatment of the neutralization bath is carried out at room temperature for about 15 minutes.

[0086] In order to ensure a rebalancing of the pH and to best eliminate the organic residues detaching from the tissue of interest, the virally inactivated Wharton's jelly is transferred into a physiological buffer bath (PBS), in order to ensure its physiological rebalancing. The bath is carried out at room temperature for approximately 15 minutes.

[0087] Finally, the Wharton jelly is transferred to two successive baths of purified water, at room temperature, for at least 15 minutes and up to approximately 1 hour.

[0088] At this stage, it can be considered that the Wharton jelly obtained according to this chemical treatment is a Wharton jelly that is largely disinfected, in particular virally inactivated, and decellularized.

[0089] Following this part relating to chemical treatments, the virus-inactivated Wharton jelly undergoes a grinding step.

[0090] Wharton's jelly is fed into a Retsch MM400 vibrating ball mill, equipped with a 35 mL zirconium oxide bowl. Wharton's jelly occupies approximately 1 / 3 of the bowl. A 20 mm diameter zirconium oxide ball is added to the bowl along with the Wharton's jelly. Grinding is performed for 1 minute at a frequency of 3 Hz. The 20 mm diameter ball is recovered, and 9 zirconium oxide balls with a diameter of 10 mm are added to the bowl. A second 3-minute grinding is performed at a frequency of 30 Hz. A third grinding is performed with 60 5 mm balls for 3 minutes at a frequency of 30 Hz.

[0091] The substance obtained is a homogeneous gelled liquid substance.

[0092] Example 3: Method for obtaining an exosome solution:

[0093] Mesenchymal stem cells are multiplied with bovine serum.

[0094] The media are collected, Amicon 0.5 ultra filtration tubes (Merk) are used to avoid the need for ultracentrifugation.

[0095] The exosomes after filtration are centrifuged and collected.

[0096] A diagnosis of exosome recovery is performed by the ExoAb Antobody Kit system.

[0097] Example 4: Method for obtaining a biological material impregnated with a solution comprising exosomes:

[0098] Three supports are used and at least 3 fabrics are tested (matrices): • Crushed Wharton's Jelly • The umbilical cord wall • The spongy layer of the amniotic membrane

[0099] The exosomes are mixed with PBS or NaCl and the quantity / volume fixed is related to the recovery (example 3).

[0100] A positive control is kept for dosing over several days to verify the absence of degradation of exosomes over 48 hours in NaCL or PBS. PBS is the classic medium.

[0101] After impregnation and total absorption, the matrices will be immersed in NaCL for times of 5 minutes, 30 minutes, 3 hours, 5 hours, 24 hours and 48 hours. The excess NaCl is completely recovered and centrifuged.

[0102] The presence of exosomes in the saline solution is tested by Elisa with the ExoAb Antobody Kit test.

[0103] It is necessary to carry out a negative control over 5 minutes, 5 hours, 24H and 48h on non-impregnated tissues because it is possible that traces of exosomes are present in each matrix.

Claims

Claims

1. Biological material impregnated with at least one active ingredient characterized in that said active ingredient is a solution comprising lipid vesicles, said biological material being lyophilized and derived from placenta of human origin and / or derived from umbilical cord of human origin.

2. Biological material according to claim 1 characterized in that said lipid vesicles are of biological origin.

3. Biological material according to claim 2 characterized in that said lipid vesicles are exosomes.

4. Biological material according to claim 3 characterized in that said exosomes are derived from mesenchymal stem cells.

5. Biological material according to any one of the preceding claims characterized in that said biological material is Wharton's jelly.

6. Biological material according to any one of claims 1 to 4 characterized in that said biological material is an amniotic membrane.

7. Biological material according to any one of the preceding claims, characterized in that said biological material is virus-inactivated.