Exosome-containing, rapidly cross-linking, modified hyaluronic acid-based dermal filler material and production method

Abstract

The invention is based on hyaluronic acid-tyramine (hyaluronic acid-tyramine, HA-Tyr), a modified derivative of hyaluronic acid (HA), which is a fast cross-linking, biologically safe, anti-wrinkle / reducing agent in aesthetic applications, cheek, lip, under-eye, forehead and other facial area applications, It is related to a dermal filler material and production method that contains stem cell exosomes and thus creates a natural and longer-lasting filling environment by increasing collagen production at the injection site and also reduces inflammation thanks to the immunoregulatory properties of exosomes.

Description

[0001] EXOSOME-CONTAINING, RAPIDLY CROSS-LINKING, MODIFIED HYALURONIC ACID-BASED DERMAL FILLER MATERIAL AND

[0002] PRODUCTION METHOD

[0003] Technical Field

[0004] The invention is based on hyaluronic acid-tyramine (hyaluronic acid-tyramine, HA- Tyr), a modified derivative of hyaluronic acid (HA), which is a fast cross-linking, biologically safe, anti-wrinkle / reducing agent in aesthetic applications, cheek, lip, under-eye, forehead and other facial area applications, It is related to a dermal filler material and production method that contains stem cell exosomes and thus creates a natural and longer-lasting filling environment by increasing collagen production at the injection site and also reduces inflammation thanks to the immunoregulatory properties of exosomes.

[0005] Prior Art

[0006] Early examples of dermal fillers were made from synthetic polymers such as poly- L-lactic acid (PLA), calcium hydroxyapatite and polymethylmethacrylate (PMMA). Currently, dermal fillers are usually derived from the basic structural protein or glycosaminoglycan molecules of natural tissues such as collagen and hyaluronic acid (HA) and macromolecular structures are obtained by different cross-linking methods to obtain viscous, injectable gels with a certain mechanical strength. Thanks to the mechanical strength provided by cross-linking, the structure obtained takes the volume of the area when injected under the skin and can remain in the area for a certain period of time (up to 12 months with current technology) without disintegrating, after which time it degrades, disintegrates and metabolizes. Thanks to its natural components, it does not show any adverse effects both during the time it functions as a filler and after it is degraded. The main features that distinguish existing dermal fillers are HA source, HA concentration, cross-linking molecule, cross-linking strategy, mono- or poly-phasic and the presence of different additives such as anesthetics or lubricants in the syringe. Among the existing fillers, the sources of HA are usually cocklebug and bacterial (synthetic fermentation of Staphylococcus equine).

[0007] Microbial HA has recently attracted more attention due to the potential allergenic effect of rooster ibis-derived HA. Structurally, fillers prepared with high HA concentrations (>100 kDa) are able to replace more tissue volume and function longer. HA concentrations of 20 mg / g and above are ideal concentrations, but very high concentrations make syringe injection difficult. Higher HA weight allows the production of more cross-linked and therefore firmer HA gels, which can extend the life of the filling up to 12 months. However, very high molecular weight HA can cause immune reactions.

[0008] Existing technologies cross-link HA with the use of various chemicals. For example, Juvederm products usually use a mixture of long-chain high molecular weight HA molecules (10%) and low molecular weight and short-chain HA molecules (90%). The products are manufactured with Hylacross or VyCross crosslinking technologies. These approaches use the cross-linker 1,4-butanediol diglycidyl ether (BDDE). Restylane family products generally use a gel base containing HA microparticles of the same particle size. The products are manufactured with XpresHAn crosslinking technology. The XpresHAn approach is based on particle size and crosslinker calibration. Optimum softness and flexibility are targeted with particle size and crosslinker concentration within a certain range. NAS-HA technology is another technology used. In this approach, BDDE crosslinker is used. The degree of cross-linking is around 1-2%. The microparticles obtained are 250 pm in diameter. Revanesse Versa is produced with Thiofix technology. In this technology, HA is ground into microparticles by wetmilling process. HA concentration is 22-28 mg / ml. Contains 3 mg / ml Lidocaine. The cross-linking agent is BDDE, pH=7. In Prevelle Silk dermal filler, the amount of HA is 5.5 mg / mL, lidocaine is used as anesthetic (0.3%), and the cross-linking agent is divinyl sulfone (DVS, 12%). Prevella Puragen dermal filler uses 2,7,8- diepoxy octane (DEO) as cross-linking technology. Exosomes are nano- saccharides released into the extracellular space by almost 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, ONA fragments, protein, lipids, metabolites, growth factors and cytokines. Exosomes are responsible for cell-cell communication and are primarily involved in cell differentiation, migration, proliferation, immune response, apoptosis, metastasis and many other cellular functions. In particular, there are studies reporting that exosomes obtained from mesenchymal stem cells accelerate cell proliferation, increase collagen production, immunoregulate, increase vascularization and accelerate wound healing.

[0009] The presence of the epoxy group, which enables effective cross-linking of HA in the production process by BDDE cross-linking, above the safety level of 2 ppm may cause skin irritation and allergic reactions. Since dermal fillers are classified as biomedical material 111 (MDR3), they should be tested biologically in detail. Accordingly, they should be tested for genotoxicity, carcinogenicity, toxicity and long-term tissue defects according to ISO 10993 standards. Accordingly, BDDE is expected to be below 2 ppm. DVS, another commonly used cross-linker, has been shown to be toxic in human colorectal carcinoma cells with an IC50 of 34 pM, above which they cause protein alkylation and protein cross-linking. However, the IC50 value of this crosslinker (30 mg / kg) is lower than that of bisepoxides. It has also been reported that diethyl sulfone groups formed after crosslinking do not cause toxicity up to 10 mM.

[0010] The cross-linking protocol of existing dermal fillers can take up to 24 hours, which is another disadvantage of the prior art. This is a disadvantage both in terms of the length of the manufacturing process and the fact that hyaluronic acid is subjected to chemical reactions for long periods of time, which can cause structural changes. Another important technical problem is that the existing dermal fillers are not bioactive and do not create a natural and long-lasting effect by increasing collagen production as well as being a filling material. They also do not play important roles in the post-implantation process such as tissue regeneration and immunoregulation. In the US Patent document numbered US2003157113A1, which is in the known state of the art, immunotherapeutic compositions and methods developed for the treatment of tumors and cancer in general are mentioned.

[0011] In the US Patent document numbered US2012177604A1, which is in the known state of the art, hydrogel formation in the presence of peroxidase and at low hydrogen peroxide concentration is mentioned.

[0012] In the Korean Patent No. KR20200024801 A, which is in the known state of the art, a nano hydrogel containing exosomes obtained from stem cell culture and having improved biocompatibility is mentioned for the filler material.

[0013] When the studies available in the art are examined, it is a hyaluronic acid-tyramine (hyaluronic acid-tyramine, HA-Tyr) based, rapidly cross-linkable, biologically safe, hyaluronic acid-tyramine (HA-Tyr), a modified derivative of hyaluronic acid (HA), with wrinkle-reducing / reducing properties in aesthetic applications, cheek, lip, under-eye, forehead and other facial area applications, There was a need to develop a dermal filler material and production method that contains stem cell exosomes and thus creates a natural and longer-lasting filling environment by increasing collagen production at the injection site and also reduces inflammation thanks to the immunoregulatory properties of exosomes.

[0014] Objectives of the Invention

[0015] The aim of the present invention is to provide a fast cross-linkable, biologically safe, hyaluronic acid-tyramine (HA-Tyr) based hyaluronic acid-tyramine (HA- Tyr), a modified derivative of hyaluronic acid (HA), which has anti- wrinkle / reducing properties in aesthetic applications, cheek, lip, under eye, forehead and other facial area applications, The aim is to realize a dermal filler material and production method that contains stem cell exosomes in its structure and thus creates a natural and longer-lasting filling environment by increasing collagen production at the injection site and also reduces inflammation thanks to the immunoregulatory properties of exosomes.

[0016] Detailed Description of the Invention

[0017] The results of the dermal filler material and the production method for achieving the objects of the present invention are shown in the accompanying figures.

[0018] These figures are as follows;

[0019] Figure 1: Transmission electron microscopy (TEM) image of umbilical cord Wharton's jelly mesenchymal stem cell exosomes.

[0020] Figure 2: Size distribution graph of human umbilical cord Wharton's jelly mesenchymal stem cell exosomes.

[0021] Figure 3: Scanning electron microscopy (SEM) image of dermal filler material containing exosomes.

[0022] Figure 4: Graphical representation of COL 1A1 expression of hDF cells in dermal fillers containing different doses of exosomes.

[0023] Figure 5: Graphical representation of collagen production capacity of hDF cells in dermal fillers containing different doses of exosomes evaluated by hydroxyproline assay.

[0024] The invention relates to an exosome-containing, rapidly cross-linking, modified hyaluronic acid-based dermal filler, which comprises;

[0025] - hyaluronic acid-tyramine as a biopolymeric base,

[0026] - horseradish peroxidase for the polymerization process,

[0027] - hydrogen peroxide (H2O2) as a polymerization initiator,

[0028] - exosome as a bioactive component, - lidocaine hydrochloride as a pain reliever,

[0029] - mannitol to reduce inflation and stabilize HA.

[0030] The invention relates to a method of manufacturing an exosome-containing, fast cross-linking, modified hyaluronic acid-based dermal filler, which comprises the steps;

[0031] - preparing a hyaluronic acid-tyramine (HA-Tyr) solution in deionized distilled water or phosphate buffer saline,

[0032] - applying steam sterilization to the prepared HA-Tyr solution,

[0033] - adding horseradish peroxidase (HRP) enzyme to the prepared solution,

[0034] - isolation of exosomes from autologous or allogeneic human umbilical cord mesenchymal stem cells (hUMSCs) by graded centrifugation / ultracentrifugation using biopolymer-based commercial isolation reagents or other isolation methods and tools,

[0035] - add to the prepared solution the exosomes described in the previous step, preferably exosomes isolated from human umbilical cord ligament / Wharton's jelly mesenchymal stem cells,

[0036] - add hydrogen peroxide (H2O2) to the prepared solution and wait for cross-linking to complete,

[0037] - washing the obtained cross-linked material 3 times with deionized ultrapure water or PBS,

[0038] - adding lidocaine hydrochloride (3 mg / ml) to the prepared solution for pain relief,

[0039] - adding steam sterilized mannitol (9 mg / ml) to the prepared solution to reduce inflammation and stabilize HA, - preparation of hyaluronic acid (HA) solution as lubricant in deionized distilled water or phosphate buffer saline and steam sterilization,

[0040] - mixing the prepared hyaluronic acid (HA) solution into the final formulation.

[0041] In the inventive method, a hyaluronic acid-tyramine (HA-Tyr) solution obtained from microbially produced hyaluronic acid (HA), preferably 290 kD, with a molecular mass between 10 kDa-lMDa, preferably 290 kD, was prepared as 10-40 mg / ml, preferably 23 mg / ml, by weight in deionized distilled water or phosphate buffer saline. Steam sterilization was applied to the prepared HA-Tyr solution. Horseradish peroxidase (HRP) enzyme was added to the prepared solution at 1-10 U / mL, preferably 2 U / mL, under sterile conditions.

[0042] 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, any 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 by gradual centrifugation / ultracentrifugation technique and biopolymer-based commercial isolation reagents or other isolation methods and tools.

[0043] Exosomes described in the previous step, preferably exosomes isolated from human umbilical cord ligament / Wharton's jelly mesenchymal stem cells, are added to the prepared solution at 0.1-200 pg / ml, preferably 10 pg / ml, or 105-1012particles / ml, preferably 109particles / ml.

[0044] Hydrogen peroxide (H2O2) between 0.1-10 mM, preferably 1 mM, is added to the prepared solution and the cross-linking is allowed to complete for at least 2 minutes. The resulting crosslinked material is washed 3 times with deionized ultrapure water or PBS. Lidocaine hydrochloride (3 mg / ml) is added to the prepared solution as a painkiller. Steam sterilized mannitol (9 mg / ml) is added to the prepared solution to reduce inflammation and stabilize HA.

[0045] As a lubricant, tyramine unmodified hyaluronic acid (HA) solution is prepared as 15-30 mg / ml, preferably 23 mg / ml, by weight in deionized pure water or phosphate buffered saline (PBS) and steam sterilized.

[0046] The prepared HA solution is mixed into the final formulation at 15% by weight. The pH of the final mixture is adjusted to 7 and filled into 1-5 ml syringes under vacuum. Finally, the final product is obtained.

[0047] Furthermore, in an alternative embodiment of the method of the invention, an exosome-free dermal filler can be prepared by skipping the exosome addition step.

[0048] In one application, exosomes were isolated from human umbilical cord ligament mesenchymal stem cells, their morphology and size were examined by transmission electron microscopy (TEM) (Figure 1), and size distribution was determined by nanoparticle tracking analysis (NTA). (Figure 2)

[0049] Exosome-containing dermal filler material containing 10 pg / ml exosomes was prepared and the microscopic structure of the material, which was brought to solid phase by freeze-drying technique, was examined by scanning electron microscopy (SEM). (Figure 3)

[0050] In order to demonstrate the function of the obtained dermal filler material, filler materials containing exosomes at 3 different doses of 10, 50 and 100 pg / ml were prepared and human dermal fibroblast (hDF) cells were cultured in 3-dimensional dermal filler material for up to 7 days. Alpha-1 type I collagen (COL 1 Al) gene expression was examined on the 2nd and 7th day of cultivation to evaluate the collagen production efficiency of hDF cells. It was observed that the cells expressed COL 1 Al gene at a higher rate in filler materials containing exosomes than in filler materials without exosomes (Figure 4). While there was an increase in COL 1 Al gene expression with increasing exosome dose, this increase was not significant between cells cultured in fillers containing 50 and 100 pg / ml. Cells cultured in fillers containing 10 pg / ml exosomes showed a statistically significant difference compared to cells cultured in fillers without exosomes. To confirm this result at the protein level, hydroxyproline assay, which indirectly measures the amount of collagen protein, was applied. The results were similar to the gene expression profile, collagen production was found to be statistically significantly higher in all groups containing exosomes compared to the control group without exosomes and this increase was greater in the groups containing 50 and 100 pg / ml exosomes and there was no significant difference between these two groups. (Figure 5)

[0051] In existing techniques, HA-based dermal fillers are produced with long processing times (24 hours on average) and with the use of cross-linkers such as BDDE or DVS, the presence of which above certain levels can cause toxic effects. In the present invention, the crosslinking time is reduced to a few minutes due to the fact that the HA-Tyr molecule functionalized with tyramine, which is biologically safe as it is an amino acid derivative, allows rapid enzymatic orthogonal covalent crosslinking and non-toxic hydrogen peroxide is used as the crosslinking trigger. Hydrogen peroxide is converted to water by the enzyme horseradish peroxidase (HRP) during the radical polymerization reaction and subsequent washing makes the final product safer than those described in the prior art.

[0052] Another advantage of the present invention over the existing technique is that it contains biological material in its structure and thanks to this biological material, it increases collagen production and shows immunoregulatory effect after implantation. hUMSC exosomes have been previously reported to increase collagen production and immunoregulate.

[0053] Hyaluronic acid-tyramine (HA-Tyr) hydrogels synthesized by functionalizing hyaluronic acid with tyramine, which is an amino acid derivative and biologically safe, do not show toxicity, increase cellular viability, gel in less than 2 minutes following cross-linker application and become ready for medical application; these aspects indicate that they will be suitable for use as dermal filling materials. Considering that the dermal filler material to be used per application is approximately 70 mg and the degree of tyraminization of HA-Tyr is approximately 3%, it can be estimated that the toxicity will be very low. In this way, the potential toxicity of existing products is reduced.

[0054] Existing techniques require reaction times of up to 24 hours for effective covalent crosslinking with crosslinkers such as BDDE or DVS. In the present invention, when the HA-Tyr molecule is treated with non-toxic and physiological levels of hydrogen peroxide (H2O2) in the presence of the enzyme horseradish peroxidase, the oxidative crosslinking process takes place via radical polymerization and a rapid gelation is achieved. In this way and by functionalization of amino acid derivative molecules, the cross-linking process, which requires long times in the existing technique, has been reduced to 2 minutes.

[0055] Another technical problem of the existing technique and products is that they are not bioactive. The present invention describes the integration of exosomes, a subtype of extracellular vesicles released by stem cells into the extracellular environment, into the dermal filler material to obtain a longer-lasting and natural structure by increasing collagen production and providing regenerative and immunoregulatory functions to the dermal filler material.

Claims

CLAIMS1. The invention relates to an exosome-containing, rapidly cross-linking, modified hyaluronic acid-based dermal filler, characterized by comprising- hyaluronic acid-tyramine as a biopolymeric base,- horseradish peroxidase for the polymerization process,- hydrogen peroxide (H2O2), as a polymerization initiator,- exosome as a bioactive component,- lidocaine hydrochloride as a pain reliever, - mannitol to reduce inflation and stabilize HA.

2. The invention relates to a method of manufacturing an exosome-containing, fast cross-linking, modified hyaluronic acid-based dermal filler, characterized by comprising the steps- preparation of hyaluronic acid-tyramine (HA-Tyr) solution in deionized distilled water or phosphate buffered saline,- application of steam sterilization to the prepared hyaluronic acid-tyramine (HA-Tyr) solution,- addition of horseradish peroxidase (HRP) enzyme into the prepared solution,- isolation of exosomes from autologous or allogeneic human umbilical cord mesenchymal stem cells (hUMSCs) by gradedcentrifugation / ultracentrifugation using biopolymer-based commercial isolation reagents or other isolation methods and tools,- addition of the exosomes described in the previous step, preferably exosomes isolated from human umbilical cord ligament / Wharton's jelly mesenchymal stem cells, into the prepared solution,- adding hydrogen peroxide (H2O2) to the prepared solution and waiting for cross-linking to complete,- washing the resulting cross-linked material with deionized ultrapure water or phosphate buffered saline (PBS),- adding lidocaine hydrochloride for pain relief to the prepared solution,- adding steam sterilized mannitol to the prepared solution to reduce inflammation and stabilize hyaluronic acid (HA),- preparation of hyaluronic acid (HA) solution as lubricant in deionized distilled water or phosphate buffer saline and steam sterilization,- mixing of the prepared hyaluronic acid (HA) solution into the final formulation.

3. The invention according to claim 2, which relates to a method of producing a dermal filler, characterized in that the hyaluronic acid-tyramine (HA-Tyr) solution is prepared at 10-40 mg / ml by weight in deionized distilled water or phosphate buffered saline.

4. The invention according to claim 2, which relates to a method of producing a dermal filler, characterized in that 1-10 U / mL horseradish peroxidase (HRP) enzyme is added to the prepared solution.he invention according to claim 2, which relates to a method of producing a dermal filler, characterized in that the exosomes are added to the solution at 0.1-200 pg / ml or 105-1012particles / ml.

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