KR102009172B1 - Medicinal bio-fillers with levan hydrogels - Google Patents

Abstract

The present invention relates to a medical biofiller material using Levan. More specifically, the present invention relates to a hydrated gel for a medical bio-filler material comprising the levane as a medical bio-filler material having excellent biocompatibility and biodegradability, and the levane and the temperature sensitive phase transition polymer.

Description

Medical Bio-Filler Material using Levan Hydration Gel {MEDICINAL BIO-FILLERS WITH LEVAN HYDROGELS}

The present invention relates to a medical biofiller material using Levan. More specifically, the present invention relates to a hydrated gel for a medical bio-filler material comprising the levane as a medical bio-filler material having excellent biocompatibility and biodegradability, and the levane and the temperature sensitive phase transition polymer.

The first filler used to expand soft tissue to fill soft tissue defects is reported as autologous fat.In 1893, Neuber first implanted autologous fat from a patient's arm into a defect in the face. This is an example. Collagen is the most common protein found in the body and is the largest protein in mammals, accounting for about 25-35% of the total protein. In particular, it is a major component of the bones, tendons, ligaments and mainly serves to maintain the structure of the organs. Easily extracted from cow or pig skin, the collagen filler from cows entered the market in 1981 with the approval of the US FDA. Conventional collagen filler is a pig skin or bovine-derived collagen product, because the immune response occurs in 3 to 5% of patients, there is a problem that must be transplanted after performing an allergy test.

Cosmoderm® and Cosmoplast® have been developed to address the hassle of testing skin reactions when using bovine or pig collagen. Human collagen obtained from culturing fibroblasts is used. Since it is derived from humans, there is no concern about allergic reactions, and there is no need to perform skin reaction tests beforehand, and it has become a representative product of collagen filler after obtaining approval from the FDA in 2003. However, these autologous cell culture collagens can be applied to patients who are afraid of animal-derived collagen.However, since a large area of skin must be collected, only those patients who have a lot of extra skin or who have to undergo surgery for other reasons can use it. It is a procedure. In addition, the collagen filler prepared by purifying collagen secreted into the culture medium through human fibroblast culture can solve the immune response, but there is still a problem in stability.

In order to avoid the problem of collagen, a method of using hyaluronic acid as a filler is also widely studied. That is, unlike collagen, hyaluronic acid does not act as an antigen because there is no difference in chemical structure between bacteria and mammals, so it is developed and used as a filler material to replace collagen. Since hyaluronic acid has the same structure in all species, there is an advantage that the immune response, which was a problem of the collagen filler, is small. However, hyaluronic acid also contains a small amount of animal protein in the manufacturing process, so the problem of foreign body reaction is not completely solved. Hyaluronic acid is broken down into two pathways in the body: first, by hyaluronidase, and second, by attaching to cell receptors, phagocytosing into cells, and by enzymes in lysosomes. . Biodegradation of hyaluronic acid is known to be so fast that it decomposes within 0.5 to several days. In order to overcome such short biodegradation, the crosslinking of hyaluronic acid was introduced. When the molecular weight is increased by the crosslinking, the phagocytosis of leukocytes, especially monocytes, can be suppressed first, and the action rate of hyaluronidase is also reduced. However, medical fillers prepared by treating a hyaluronic acid with a crosslinking agent may have side effects (eg, cell or tissue necrosis, inducing an immune response) due to the use of the crosslinking agent, and thus are limited.

Therefore, there is a need for the development of a new formulation that has excellent biocompatibility and biodegradability as a method for improving the sustainability, and has a good viscosity and elasticity so that it can naturally correct touch and shape, and particularly hyaluronic acid and efficacy The development of similar and inexpensive materials is required.

Prior literature

1. International Patent Publication WO2012-144678

The present inventors have made diligent efforts to develop new materials capable of naturally correcting touch and shape as well as biocompatibility and biodegradability as well as good viscosity and elasticity. As a result, it is confirmed that Levan is a suitable material to replace the conventional hyaluronic acid. The invention has been completed.

An object of the present invention is to provide a levan as a medical biofiller material excellent in biocompatibility and biodegradability.

It is also an object of the present invention to provide a hydrogel for a medical biofiller material comprising a levane and a temperature sensitive phase change polymer.

As used herein, the term "biocompatibility" is a property required for biomedical materials, and refers to a property of a material that can coexist with a living body while exhibiting its original function without adversely affecting the living body.

As used herein, the term “biodegradable” means a property that can be degraded when exposed to a physiological solution, for example, can be degraded by body fluids or microorganisms in the body of a mammal, including humans. I mean the property that is.

As used herein, the term “levan” is a water-soluble fructose polymer, which is mainly composed of beta-2,6 bonds and is produced from sugar by the fructose transfer reaction of levansucase.

According to the first embodiment,

The present invention is to provide a Levan as a medical bio-filler material excellent in biocompatibility and biodegradability.

In the levan as a medical bio-filler material according to the present invention, the molecular weight of the levan may be 150 to 200 kDa.

According to the second embodiment,

The present invention is to provide a hydrogel for medical bio-filler material containing the levane and temperature sensitive phase-transfer polymer.

In the hydrogel for medical bio-filler material according to the present invention, the molecular weight of the levan may be 150 to 200 kDa.

In the hydrogel for medical biofiller material according to the present invention, the temperature sensitive phase transition polymer is polyethylene oxide (PEO) -polypropylene oxide (PPO) copolymer, polyethylene oxide (PEO) -polylactic acid (PLA) copolymer, It may be, but is not limited to, at least one mixture selected from the group consisting of polyethylene oxide (PEO) -polyglycolic acid (PLGA) copolymer and polyethylene oxide (PEO) -polycaprolactone (PCL) copolymer. For example, the polyethylene oxide (PEO) -polypropylene oxide (PPO) copolymer is Pluronic series (Pluronic series, BASF, USA), specifically Pluronic F-38, Pluronic F-68, Pluronic Tronic F-77, Pluronic F-87, Pluronic F-88, Pluronic F-98, Pluronic F-108, Pluronic F-127, or mixtures thereof. . The polymer solution having a phase transition phenomenon of the present invention has a sol phase below the sol-gel phase transition temperature and a gel phase above the sol-gel phase transition temperature. In one embodiment, Pluronic F-127 was used as the temperature sensitive phase change polymer. The levan and Pluronic F-127 may be mixed in a weight ratio of 1: 2 to 1:20, preferably 1: 3 to 1: 4.

In the hydrogel for medical bio-filler material according to the present invention, the hydrogel is an alginic acid, carboxymethyl cellulose in the aqueous solution, in order to improve the viscosity and stability of the aqueous solution of the temperature sensitive phase transition polymer, It may further comprise at least one compound selected from the group consisting of dextran, collagen, gelatin, elastin. In one embodiment, carboxymethylcellulose was used to improve the viscosity and stability of Pluronic F-127. The Levan, Pluronic F-127 and carboxymethylcellulose may be mixed in a weight ratio of 1: 2-20: 1, preferably 1: 3-4: 1.

It was confirmed that the levan for the biofiller material according to the present invention may exhibit a cell proliferation rate and collagen formation ability comparable or superior to hyaluronic acid as a candidate material that can replace hyaluronic acid. In addition, the medical bio-filler material including the levane and the temperature sensitive phase transition polymer according to the present invention can stably form a hydration gel, and has a high viscosity compared to hyaluronic acid to impart viscoelasticity to the skin and improve the role of shock and lubricant. It has been shown to promote the production of major substances such as collagen and to help maintain skin tissue. Accordingly, the levan for biofiller material according to the present invention is expected to be very valuable as a semi-permanent filler with biocompatibility and biodegradability.

1 shows the cytotoxicity evaluation results of Levan and hyaluronic acid according to Example 1 of the present invention.
Figure 2 shows the cell proliferation comparison evaluation results of Levan and hyaluronic acid according to Example 2 of the present invention.
Figure 3 shows the comparative evaluation results of collagen formation ability of levane and hyaluronic acid according to Example 3 of the present invention.
Figure 4 shows the hydrogel stability evaluation results according to the Leban concentration according to Experimental Example 1-1 of the present invention.
5 shows the results of evaluation of the stability of the Levan hydrogel according to the carboxymethyl cellulose concentration according to Experimental Example 1-2 of the present invention.
6 shows the results of evaluating the viscoelastic properties of Levan hydration gel according to Experimental Example 2-1 of the present invention.
7 shows the results of evaluating the stability of the Levan hydration gel according to Experimental Example 2-2 of the present invention.
8 shows the cytotoxicity evaluation results of Levan hydration gel according to Experimental Example 3 of the present invention.

With reference to the accompanying drawings the present invention will be described in more detail by the following examples. However, these Examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

< Example >

Example  One. Levan  Cytotoxicity Assay

hADF (human Adult Dermal Fibroblasts, passage # 10, 1x10 ⁴ cells / well) was applied to 96 well DMEM (containing 10% FBS, 1% antibiotics) and stabilized for about 12 hours. Hyaluronic acid was prepared at various concentrations (1-5000 μg / mL) as a levan and comparative example according to the present invention, added to the cells, and then incubated in DMEM (containing 1% antibiotics) for 24 and 48 hours, respectively. The effects of hyaluronic acid on cellular metabolism as an example of levan and fertilizer according to the present invention were analyzed using the WST-8 assay, and the results are shown in FIG. 1.

As can be seen from Figure 1, it was confirmed that both the leban and hyaluronic acid according to the present invention is excellent in biocompatibility without causing cytotoxicity up to 5000μg / mL. Meanwhile, in order to confirm that the experimental method was properly established, cytotoxicity was observed after applying PEI (polyethylenimine, 250 kDa, 200 μg / mL) having high cytotoxicity to the cells using the same method. By showing cell viability of less than 5% even in culture, the biocompatibility of Levan proved excellent.

Example  2. Levan  Cell Proliferation Rate Analysis

hADF (human Adult Dermal Fibroblasts, passage # 10, 1x10 ⁴ cells / well) was applied to 96 well DMEM (containing 10% FBS, 1% antibiotics) and stabilized for about 12 hours. Hyaluronic acid was prepared in 0.2% and 0.5%, respectively, as a levan and a comparative example according to the present invention, and then added to the cells and analyzed for hourly (1-4 days) cell proliferation rate using the WST-8 assay. The results are shown in FIG.

As can be seen from Figure 2, when treated with hyaluronic acid for more than three days as a levan and a comparative example according to the present invention was confirmed to exhibit a high cell proliferation rate. Even when Leban was used at a concentration of 0.2%, sufficient cell proliferation was shown. In particular, by showing no statistically significant difference in the cell growth rate of hyaluronic acid as a comparative example with the levane according to the present invention, it was proved that the levane according to the present invention is a candidate substance that can replace hyaluronic acid.

Example  3. Levan  Collagen Formation ability  analysis

hADF (human Adult Dermal Fibroblasts, passage # 10, 1x10 ⁴ cells / well) was applied to 96 well DMEM (containing 10% FBS, 1% antibiotics) and stabilized for about 12 hours. As a levan and a comparative example according to the present invention, hyaluronic acid was added to the cells at 0.2% and 0.5%, respectively, and after 24 hours, the intracellular RNA was extracted and analyzed for collagen (collagen type 1) formation ability using qPCR. (Control gene: beta-actin). The results are shown in FIG.

As can be seen from Figure 3, it was confirmed that the intracellular collagen formation ability increases with increasing the concentration of hyaluronic acid as a levan and comparative example according to the present invention. In particular, by showing no statistically significant difference in collagen formation ability of hyaluronic acid as a comparative example with the levane according to the present invention, it was proved that the levane according to the present invention is a candidate substance that can replace hyaluronic acid.

< Experimental Example ＞

Experimental Example １．Levan Hydrogel  Manufacturing and Stability Assessment

1-1. Hydrogel with Levan and Pluronic Polymer

1wt%, 5wt% and 10wt% of levane and pluronic polymer (PF127, 17wt%) were physically mixed to prepare a hydrogel containing the levane according to the present invention, and then similar conditions to the in vivo environment (PBS (pH 7.4), 37 ° C.), the stability was evaluated. The results are shown in FIG.

As can be seen from Figure 4, Pluronic (PF127, 17wt%) itself, as is well known, the hydrogel was formed in a 37 ℃ environment. On the other hand, low molecular weight Levan (Lev, 10wt%) was slightly viscous, but no gel was formed. Interestingly, it was confirmed that the stability was increased as the amount of Levan mixed with Pluronic increased. On the other hand, Pluronic polymer and high concentration (10wt%) of Levan formulation is difficult to control and may cause problems in the mass production process in the future, it was determined that Pluronic Polymer and Levan formulation of 5wt% would be most suitable.

1-2. Levan, Pluronic Polymer and Carboxymethylcellulose Hydrogel

After varying the concentration of carboxymethyl cellulose (CMC) to 0.2 wt% and 0.5 wt%, respectively, to the Pluronic polymer and the Levan formulation of 5 wt%, the stability was evaluated using the same method as in Experimental Example 1-1. It was. The results are shown in FIG.

As can be seen from Figure 5, it was confirmed that the addition of carboxymethyl cellulose flu to the Pluronic polymer and 5 wt% Levan formulation can be further improved stability. In particular, the stability of the hydrogel was found to be the best at 5wt% carboxymethylcellulose conditions.

Experimental Example 2. Levan , Pluronic  Polymer and Carboxymethyl Cellulose  contain Hydrogel Rheological  Rheological Property Evaluation

2-1. Viscoelasticity Evaluation of Levan Hydrogel

In order to evaluate the rheological or mechanical properties of the Levan hydrogel, the Levan, Pluronic polymer and carboxymethylcellulose were physically mixed according to the mixing ratio at 4 ° C. After injecting the sol state into the plate on the rheometer device, the rheological properties of the levane hydrogel were measured under the following conditions. The results are shown in FIG.

Rheometer measurement conditions:

(1) 20 mm parallel plate geometry & 2 mm gap size,

(2) 0.1 rad / s & 0.1% strain,

(3) 4 ° C. for 4 min & 37 ° C. for 30 min, and

(4) frequency sweep test (from 0.1 to 100 rad / s at 0.1% strain)

As can be seen from Figure 6, it was confirmed that the levane hydration gel is present in a sol (sol) state at a low temperature can be transformed into a gel (gel) state at 37 ℃ in vivo environment. Increasing the amount of levane up to 10wt% impeded the pulsonic micelle assembly and lowered the gel's strength (elastic modulus). These results indicate that the hydrophobic action force between the Levan and the Pluronic polymer is not large. On the other hand, it was confirmed that the strength of the gel was increased by increasing the amount of carboxymethyl cellulose. This phenomenon is believed to be due to the occurrence of strong hydrogen bonds between the carboxy group of carboxymethylcellulose and the hydroxy group of Levan.

2-2. Stability Evaluation of Levan Hydrogel

In order to evaluate the stability of the levane hydrogel, which is a physical binding gel rather than a chemical binding gel, frequecy of 0.1 to 100 rad / s was added, and the results are shown in FIG. 7.

As can be seen from FIG. 7, it was confirmed that the levane hydrogel was stably maintained.

Experimental Example 3. Levan , Pluronic  Polymer and Carboxymethyl Cellulose  contain Hydrogel  Toxicity Assessment

Low molecular weight chitosan (1 wt% in 1% acetic acid) was applied on the round coverglass (12 mm diameter), dried for 12 hours, and the remaining acetic acid was removed using DIW. Then, hADF cells (5 × 10 ⁴ cells / well) were applied and incubated for about 12 hours to form a cell layer (cell medium: DMEM (containing 10% of FBS, 1% of antibiotics)). After 24 hours of contact with the cell layer on the Levan hydrogel, Live / Dead assay (live cell staining, green fluorescence) and PI (dead cell staining, red fluorescence) staining reagent ). The results are shown in FIG.

As can be seen from Figure 8, it was confirmed that there is no cytotoxicity in all groups. In other words, red fluorescence (dead cells) is observed at less than 5%, Levan hydration gel does not cause intracellular toxicity, and based on these results, it is judged that it will not cause toxicity even when implanted in vivo as a biofiller. On the other hand, in the PEI group, which is known to be highly cytotoxic, no green fluorescence (living cells) was observed, confirming that it was highly cytotoxic.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (9)

A) Levan for medical bio-fillers with excellent biocompatibility and biodegradability,
B) a temperature sensitive phase transition polymer, and
C) carboxymethylcellulose
Hydrogel for medical bio-filler comprising a.
The method of claim 1,
The molecular weight of the levan is 150 to 200 kDa, the medical bio-filler hydrogel.
delete The method of claim 1,
The temperature sensitive phase transition polymer is polyethylene oxide (PEO) -polypropylene oxide (PPO) copolymer, polyethylene oxide (PEO) -polylactic acid (PLA) copolymer, polyethylene oxide (PEO) -polyglycolic acid (PLGA) copolymer And polyethylene oxide (PEO) -polycaprolactone (PCL) copolymer, at least one mixture selected from the group consisting of hydrogels for medical bio-fillers.
The method of claim 1,
The temperature sensitive phase transition polymer is Pluronic F-127, the medical bio-filler hydrogel.
The method of claim 5,
The levan and the Pluronic F-127 is 1: 2 to 1: 20 that will be mixed in a weight ratio, medical gel bio filler hydrogel.
The method of claim 1,
The medical bio-filler hydrogel is 1 selected from the group consisting of alginic acid, carboxymethyl cellulose, dextran, collagen, gelatin, elastin. It further comprises at least a compound, hydrogel for medical bio-filler.
The method of claim 1,
The hydrogel for medical bio-filler is a levane, a hydrosensitive gel for medical bio-filler that includes Pluronic F-127 and carboxymethyl cellulose as a temperature-sensitive phase-transfer polymer.
The method of claim 8,
The hydrogel for medical bio-filler is a levane, a temperature sensitive phase-transfer polymer containing Pluronic F-127 and carboxymethyl cellulose in a weight ratio of 1: 2-20: 1, gel for medical bio-filler.

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