WO2025085041A1

WO2025085041A1 - A method for the production of exosome-containing acellular skin patch

Info

Publication number: WO2025085041A1
Application number: PCT/TR2024/051209
Authority: WO
Inventors: Burak DERKUS
Original assignee: TC Ankara Universitesi
Current assignee: TC Ankara Universitesi
Priority date: 2023-10-20
Filing date: 2024-10-18
Publication date: 2025-04-24
Anticipated expiration: 2026-04-20

Abstract

The invention relates to a method for the production of a skin patch for the rapid healing of erosions or wounds or burns, obtained from decellularized (decellularized, acellular) skin tissue doped with exosomes.

Description

A METHOD FOR THE PRODUCTION OF EXOSOME-CONTAINING

ACELLULAR SKIN PATCH

Technical Field

The invention relates to a method for the production of a skin patch for the rapid healing of erosions or wounds or burns, obtained from skin tissue doped and decellularized (de-cellularized, acellular) with exosomes.

Prior Art

Wound healing is a multi-stage process including hemostasis phase, inflammation phase, proliferation phase, maturation and remodeling phase and heals at different times depending on the shape of the wound and the layer it affects. Acute wounds can heal relatively quickly, but if inflammation or infection occurs, this process can be prolonged and can become a chronic wound. With the emergence of the discipline of tissue engineering, skin patches and fillers have been produced from a variety of synthetic and natural polymers/biopolymers with different properties, but the existing techniques and products do not have all the necessary properties for effective and natural wound healing by being natural, allowing vascularization, immunoregulatory function, increasing collagen production and accelerating cell proliferation.

Acellular or decellularized or decellularized tissues and organs have been used for some time in tissue engineering and regenerative medicine to regenerate damaged tissues and organs. The extracellular matrix (ECM), which is the remaining part of the decellularized tissue, is used alone or in composite with other materials in forms such as films, sponges, fibers, particles, hydrogels, etc. to accelerate tissue regeneration. For example, many tissues such as blood vessels, spinal cord, cartilage and heart have already been decellularized and used as tissue engineering tools. Skin tissue has also been decellularized and used as a wound dressing material. Exosomes are nanoparticles released into the extracellular space by all types of cells, ranging in size from 30 to 150 nm, spherical, doughnut-like, with a unique morphology, containing a wide variety and dense biomolecules such as mRNA, microRNA, snRNA, DNA fragments, protein, lipids, metabolites, growth factors and cytokines. Exosomes are responsible for cell-cell communication and are primarily involved in many cellular functions such as cell differentiation, migration, proliferation, immune response, apoptosis and metastasis. In particular, there are studies reporting that exosomes obtained from mesenchymal stem cells accelerate cell proliferation, increase collagen production, immunoregulate, increase vascularization and enhance wound healing.

Acellular extracellular matrix (aECM) has emerged as promising biomaterials for tissue regeneration. The advantage of decellularized tissues over scaffolds derived from synthetic materials is that the native structure of the tissue ECM is largely preserved. Their natural structure, which is difficult to mimic by synthetic materials, is increasingly attracting attention due to their relatively high bioactivity, low immunogenicity and good biodegradability. Exosome-doped materials are superior to the known technique due to the aforementioned potentials and advantages.

US Patent US2020397945A1, which is in the known state of the art, mentions biological formulations and methods developed for the treatment of cardiac tissues and disorders.

US Patent No. US2019314551 Al, which is in the known state of the art, describes tissue prostheses having a basic structure and physiological sensor system. The tissue prostheses are adapted and configured to induce remodeling of damaged tissue and regeneration of new tissue, and to simultaneously detect and monitor physiological properties when implanted in the subject.

US Patent US2022047370A1, which is in the known state of the art, mentions a method and graft material for preventing or reducing the formation and/or development of a hernia in a subject at risk of developing a hernia. US Patent No. US10864233B2, which is in the known state of the art, discloses methods and structures for the treatment of cardiovascular disorders, comprising advanced bio-remodeling of damaged cardiovascular tissue and methods and structures for administering an ECM-based composition to damaged cardiovascular tissue in combination with a therapeutic drug such as ventricular assist and transmyocardial revascularization (TMR) that induces regeneration of new cardiovascular tissue.

In the Turkish Patent document numbered 2021/017956, which is in the known state of the art, a product containing tomato-derived exosomes that can be used as an aid in wound healing treatment is mentioned. With the invention, it is aimed to obtain a product that does not cause toxic effects on the human body, accelerates cell division and cell migration in skin injuries, ensures the continuity of cell viability, does not cause damage to healthy cells, does not create a risk of infection since it does not require any procedure such as surgery, and has a low cost.

When the existing studies in the art were examined, there was a need to realize a skin patch production method that carries the histological / biochemical structure of the skin tissue and accelerates cell proliferation and helps the wound to heal faster in an effective and natural way.

Objectives of the Invention

The aim of the present invention is to provide a method of producing a skin patch that carries the histological/biochemical structure of the skin tissue and accelerates cell proliferation and helps the wound to heal faster in an effective and natural way.

Another aim of this invention is to realize a skin patch production method that, in addition to its rapid wound healing properties, provides immunoregulation, intercellular communication, increases vascularization and increases collagen production in fibroblasts. Detailed Description of the Invention

The results of a method of producing a leather patch for the purposes of the present invention are shown in the accompanying figures.

These figures are

Figure 1: Graphical representation of DNA quantification in native and decellularized skin samples.

Figure 2: Graphical representation of GAG quantification in native and decellularized skin samples.

Figure 3: Graphical representation of HP quantification in native and decellularized skin samples.

Figure 4: View of the size distribution graph of human umbilical cord Wharton's jelly mesenchymal stem cell exosomes.

Figure 5: View of the size distribution scatter plot of human umbilical cord Wharton's jelly mesenchymal stem cell exosomes.

Figure 6: Transmission electron microscopy (TEM) image of human umbilical cord Wharton's jelly mesenchymal stem cell exosomes.

Figure 7: Scanning electron microscopy (SEM) image of acellular dermal patch containing exosomes.

Figure 8: Graphical view of exosome release kinetics of exosome-containing acellular dermal patch.

Figure 9: View of Col 1 Al gene expression analysis in human dermal fibroblasts cultured on exosome-containing acellular dermal patch.

The invention relates to a method of producing acellular skin patches containing exosomes, comprising the steps - isolation of exosomes from human umbilical cord mesenchymal stem cells,

- cleaning the skin obtained from bovine or human cadaver in a buffer solution from hair, fatty tissue, epidermis and hypodermis layers,

- cleaned skin samples were processed in nuclease-containing buffer solution at 37 °C for 90 minutes to remove nuclear material,

- the samples were then processed in 0.25 wt% trypsin solution in distilled water for 6 hours on a mixer,

- following washing, the samples were sterilized in 70% ethanol by volume on a mixer for 10-15 hours,

- followed by treatment of the skin samples obtained in 3% H2O2 for 15 minutes,

- following washing, skin samples were processed in a solution containing 1 wt% Triton-x-100, 0.26 wt% (ethylenediamine tetraacetic acid) EDTA and 0.69 wt% Tris for 6 hours,

- following washing, decontaminated skin samples (aECM) were processed in 0.1% peracetic acid and 4% ethanol by volume for 2 hours,

- following the washing steps, the skin aECM is ground until no small pieces remain, frozen at -80 °C and lyophilized,

- the resulting acellular skin extracellular matrix (aECM) is combined with carbohydrate and protein biopolymers to improve its mechanical properties.

In the inventive method;

Autologous or allogeneic human umbilical cord mesenchymal stem cells (hUMSC), autologous or allogeneic Wharton's jelly mesenchymal stem cells, autologous or allogeneic human adipose tissue mesenchymal stem cells, autologous or allogeneic human bone marrow mesenchymal stem cells, all kinds of autologous or allogeneic human multipotent stem cells, Isolation of exosomes from all kinds of autologous or allogeneous pluripotent stem cells, autologous or allogeneous fibroblasts and other connective tissue cells, all kinds of plants and plant sources or microalgae is carried out by gradual centrifugation/ultra-centrifugation technique, biopolymerbased commercial isolation reagents or other isolation methods and tools. Scanning electron microscopy (TEM) image, particle size distribution and scatter plot of exosomes isolated from human umbilical cord mesenchymal stem cells are shown in Figure 6, Figure 4 and Figure 5, respectively.

Acellular skin extracellular matrix (aECM) is then produced. The skin is obtained from bovine or human cadavers. It is preferably of bovine origin. In a general application, skin samples are first placed in buffer solution. pH can be between 5.5- 9.0, preferably 7.4. The buffer solution can be phosphate buffer, acetate buffer, carbonate buffer or sulfonate buffers. The skin is then cleaned of hair and adipose tissue, as well as the epidermis and hypodermis layers, and only the dermis is further processed. Samples are cleared of nuclear material by processing for 90 minutes at 37 °C in buffer solution containing nucleases, preferably DNAase and RNAase (Figure 1).

Following the washing steps, the samples were processed in 0.25 wt% trypsin solution in distilled water for 6 hours on a mixer. Following the washing steps, the samples were processed in 70% ethanol by volume on a mixer for 10-15 hours. The skin samples obtained are then processed in 3% H2O2 by volume for 15 minutes. After washing, the skin samples were processed in a solution containing 1 wt% Triton-x-100, 0.26% EDTA and 0.69% Tris for 6 hours. The skin samples obtained are processed again in the same solution, this time for 16 hours. Following the washing steps, decellularized skin samples (aECM) are decontaminated by processing in 0.1% peracetic acid and 4% ethanol by volume for 2 hours. Following the washing steps, the skin aECM is ground until no small pieces remain, frozen at -80°C and lyophilized. Acellular skin ECM is expected to retain collagen and glycosaminoglycan (GAG) content. The results of hydroxyproline and GAG test to measure the amount of collagen in aECM obtained in an application are presented in Figure 2 and Figure 3. It is seen that aECM preserves the collagen and GAG content to a great extent. The obtained aECM can be combined with some carbohydrate and protein biopolymers to improve the mechanical properties of one of the end products, the skin patch. These biopolymers can be cellulose, oxidized cellulose, carboxymethyl cellulose, alginate, hyaluronic acid, chitin, chitosan, their derivatives and nanoparticles and other ECMs in protein structure, gelatin, collagen types, silk fibroin, fibrin, keratin, elastin, soy proteins, milk proteins, wheat proteins or vegetable proteins and their derivatives and modifications.

In an exemplary embodiment of the inventive method, dermal fillers containing exosomes were prepared.

After preparation of aECM in powder form as described in [2], it is digested according to the pepsin digestion protocol. According to this protocol, aECM is dissolved in 0.01 M HC1 solution containing pepsin (600-1200 U/mg) at 0.01%- 5%, preferably 2%, by weight and stirred in a magnetic stirrer. Digestion can take place for 12-72 hours, preferably 21 hours. Exosomes as described in [1], preferably umbilical cord mesenchymal stem cell exosomes, 0.0001-1 mg/ml, preferably 100 pg/ml in the preferred embodiment, are added to the aECM in the resulting gel and mixing is performed for 30 min. For neutralization and hydrogelization, 10% by volume of 0.1 N NaOH and 1/9 of lOx PBS were added, followed by gentle pipetting 20-30 times and finally thermal treatment at 37 °C for 1 hour. The resulting exosome-containing dermal filler is washed several times and filled into tubes, syringes, vials, etc. for use.

In another exemplary embodiment of the method of the invention, a skin patch containing exosomes is prepared.

The aECM+exosome gel obtained before the neutralization step described in Example 1 is transferred to suitable containers with a thickness of 1-10 mm, preferably 2 or 3 mm. In preferred practice, 10 cm diameter petri dishes, 6-mesh plates (surface area 9.6 cm²), 10x10 cm square dishes made of borosilicate glass, polycarbonate or polystyrene are used. This is followed by the addition of 10% by volume of 0.1 N NaOH and 1/9 of 1 Ox PBS for neutralization and hydrogelization, followed by gentle stirring and finally thermal treatment at 37 °C for 1 hour. The solid-phase, i.e. hydrogelized, aECM+exosome mixture was frozen at -80 °C followed by lyophilization, i.e. freeze-drying. Dermal patches containing exosomes are obtained.

In another exemplary embodiment of the method of the invention, a skin patch containing crosslinked exosomes was prepared.

The crosslinkers to be used for crosslinking the end product obtained in Example 2 may be glutaraldehyde, formaldehyde, carbodiimide compounds and polyepoxy compounds. In a general embodiment the crosslinker concentration can be between 0.01-1000 mM, in preferred embodiments between 1 and 10 mM. After the crosslinking process, washing is performed several times. The scanning electron microscopy (SEM) image of the cross-linked dermal patch containing exosomes obtained in one application is presented in Figure 7. In addition, the amount of exosomes released into the environment was measured by the BCA test, which is a protein quantification test, and it was observed that the exosome release reached approximately 80% at 168 hours (day 7). (Figure 8) When human dermal fibroblast cells were seeded on the obtained exosome-containing dermal patches, it was observed that the cells increased the expression of Alpha- 1 type I collagen (COL 1A1) on the exosome-containing patches, that is, they started to produce more collagen. (Figure 9)

In another exemplary embodiment of the method of the invention, a skin patch containing exosomes combined with cellulosic biopolymers is prepared.

The aECM+exosome solution obtained before the neutralization step described in Example 1 is mixed with carboxymethyl cellulose (CMC) solution. In general practice, the concentration of aECM in the final formulation can be between 0.001% and 5% by mass, the concentration of CMC between 0.001% and 10% by mass, and the concentration of exosomes between 0.0001-1 mg/ml. In preferred practice, these concentrations are 2% aECM, 0.5% CMC and 100 pg/ml exosome. In the remainder of the application, CMC is added as a powder or solution into the aECM+exosome solution and mixed until homogenization is achieved. Transfer to suitable containers with a thickness of 1-10 mm, preferably 2 or 3 mm. In preferred practice, 10 cm diameter petri dishes, 6-mesh plates (surface area 9.6 cm²), 10x10 cm square dishes made of borosilicate glass, polycarbonate or polystyrene are used. This is followed by the addition of 10% by volume of 0.1 N NaOH and 1/9 of lOx PBS for neutralization and hydrogelization, followed by gentle stirring and finally thermal treatment at 37 °C for 1 hour. The solid-phase, i.e. hydrogelized, aECM+exosome mixture was frozen at -80 °C followed by lyophilization, i.e. freeze-drying. Functional and mechanically durable but biodegradable dermal patches based on CMC/aECM and containing exosomes are obtained.

Claims

1. The invention relates to a method for the production of acellular skin patches containing exosomes, comprises the steps

- isolation of exosomes from human umbilical cord mesenchymal stem cells,

- cleaning the skin obtained from bovine or human cadavers from hair, adipose tissue, epidermis and hypodermis layers by placing the skin in a buffer solution,

- cleaned skin samples were processed in buffer solution containing nuclease to remove nuclear material,

- then the samples were processed in distilled water and trypsin solution on a mixer,

- washed and sterilized in ethanol on a mixer,

- then the skin samples were processed in H2O2,

- following washing, skin samples were mixed and processed in a solution containing Triton-x-100, (ethylenediamine tetraacetic acid) EDTA and Tris,

- washing steps followed by decontamination of decellularized skin samples (aECM) by treatment in peracetic acid and ethanol,

- washing steps followed by grinding, freezing and lyophilization of acellular extracellular skin matrix (aECM) until no small pieces remain,

- combining acellular skin extracellular matrix (aECM) with carbohydrate and protein biopolymers to improve its mechanical properties.

2. The invention relates to a method of producing an acellular skin patch comprising an exosome according to claim 1, wherein the exosomes used are autologous or allogeneic Wharton's jelly mesenchymal stem cells, autologous or allogeneic human adipose tissue mesenchymal stem cells, autologous or allogeneic human bone marrow mesenchymal stem cells, characterized by being derived from any autologous or allogeneic human multipotent stem cells, any autologous or allogeneic pluripotent stem cells, autologous or allogeneic fibroblasts and other connective tissue cells, or from any plant or vegetative source.

3. The invention relates to a method for producing an acellular skin patch comprising an exosome according to claim 1, characterized in that the cleaned skin samples are treated in a buffer solution containing nuclease at 37 °C for 90 minutes.

4. The invention relates to a method of producing an acellular skin patch comprising an exosome according to claim 1, characterized in that it is mixed in a 0.25 wt% trypsin solution.

5. The invention relates to a method for producing an acellular leather patch comprising an exosome according to claim 1, characterized in that the leather samples are processed in 70% ethanol by volume for 10-15 hours.

6. The invention relates to a method of producing an acellular skin patch comprising an exosome according to claim 1, characterized by the biopolymers used for improving the mechanical properties being cellulose, oxidized cellulose, carboxymethyl cellulose, alginate, hyaluronic acid, chitin, chitosan, their derivatives and nanoparticles and other ECMs in protein structure, gelatin, collagen types, silk fibroin, fibrin, keratin, elastin, soy proteins, milk proteins, wheat proteins or vegetable proteins and their derivatives and modifications.