KR102029388B1 - Levan/hydroxyapatite based organic-inorganic hybrid filler and method for preparing the same - Google Patents

Abstract

The present invention relates to a Levan / HAp organic-inorganic hybrid filler and a method for producing the same. More specifically, the present invention relates to a Levan / HAp organic-inorganic hybrid filler and a method for preparing the same, which not only increases the stability of the Levan hydrogel, but also significantly improves cell viability, cell proliferation rate, and collagen formation ability by adding HAp to the Levan hydrogel. will be.

Description

LEVAN / HYDROXYAPATITE BASED ORGANIC-INORGANIC HYBRID FILLER AND METHOD FOR PREPARING THE SAME}

The present invention relates to a Levan / HAp organic-inorganic hybrid filler and a method for producing the same. More specifically, the present invention relates to a Levan / HAp organic-inorganic hybrid filler and a method for preparing the same, which not only increases the stability of the Levan hydrogel, but also significantly improves cell viability, cell proliferation rate, and collagen formation ability by adding HAp to the Levan hydrogel. will be.

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.

Accordingly, the present inventors have made intensive efforts to improve the stability of the Levan / carboxymethylcellulose / Pluronic organic complex for use in the biocompatible and biodegradable medical biofiller material disclosed in Patent Application No. 10-2017-0132657. As a result, the present invention was completed by confirming that the stability of the levane hydrogel can be further improved when the levane / carboxymethylcellulose / pluronic organic complex HAp (Hydroxyapatite) is combined.

An object of the present invention is to provide an improved stability of the levane hydrogel, characterized in that it comprises HAp (Hydroxyapatite).

It is another object of the present invention to provide a method for producing a stable levane hydrogel, characterized in that it comprises HAp (Hydroxyapatite).

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. Levan according to the present invention may be excellent in biocompatibility as well as biocompatibility.

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 the 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.

According to the first embodiment,

The present invention

It is an object of the present invention to provide an improved stability of Levan hydrogel including levane, a temperature sensitive phase transition polymer, carboxymethylcellulose and hydroxyapatite.

In the levan hydrogel having improved stability according to the present invention, the molecular weight of the levan is characterized in that 150 to 200 kDa.

In the Levan hydration gel with improved stability 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, polyethylene oxide (PEO) -polyglycolic acid (PLGA) copolymer or 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 Levan hydrogel with improved stability according to the present invention, the Levan, Pluronic F-127 and carboxymethyl cellulose is characterized in that the 1: 2-20: 1 by weight. Preferably the Levan, Pluronic F-127 and carboxymethylcellulose may be mixed in a weight ratio of 1: 3-4: 1.

In the levan hydrogel having improved stability according to the present invention, the hydroxyapatite is mixed in an amount of 0.1 to 5 wt% based on the weight of the levan hydrogel. Preferably the hydroxyapatite may be mixed in an amount of 0.1 to 1% by weight based on the weight of the levane hydrogel.

In the levan hydrogel having improved stability according to the present invention, the levan hydrogel is characterized in that it is used as a medical bio-filler material.

According to the second embodiment,

The present invention

Stability including the step of mixing hydroxyapatite in the amount of 0.1 to 5wt% based on the weight of the Levan hydration gel to the Levan hydration gel containing the levane, the temperature-sensitive phase transition polymer and carboxymethyl cellulose An object of the present invention is to provide an improved method of preparing a levane hydrogel.

In the method of preparing the stability of the levane hydrogel according to the present invention, the molecular weight of the levane is characterized in that 150 to 200 kDa.

In the method for preparing stability of the levane hydrogel 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 , Polyethylene oxide (PEO) -polyglycolic acid (PLGA) copolymer or 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. .have.

In the method of preparing stability levane hydrogel according to the present invention, the levane, Pluronic F-127 and carboxymethyl cellulose is characterized in that 1: 2-20: 1 by weight. Preferably the Levan, Pluronic F-127 and carboxymethylcellulose may be mixed in a weight ratio of 1: 3-4: 1.

In the manufacturing method of the improved stability of the levan hydrogel according to the present invention, the levan hydrogel is characterized in that it is used as a medical bio-filler material.

1 shows the results of stability evaluation of the levane hydrogel according to the amount of HAp added in Example 1. FIG.
2 shows the results of rheological evaluation of the Levan / HAp organic-inorganic hybrid filler of Example 2.
Figure 3 shows the cytotoxicity evaluation results of the Levan / HAp organic-inorganic hybrid hydrogel of Experimental Example 1. (A) PF127 / Lev5 / CMC0.5, B) PF127 / Lev5 / CMC0.5 / HAp0.1, C) PF127 / Lev5 / CMC0.5 / HAp0.5, D) PF127 / Lev5 / CMC0.5 / HAp1 )
Figure 4 shows the cell viability of the Levan / HAp organic-inorganic hybrid hydrogel of Experimental Example 2.
5 shows the results of cell proliferation evaluation of the Levan / HAp organic-inorganic hybrid filler of Experimental Example 3.
6 shows collagen formation ability evaluation results of the Levan / HAp organic-inorganic hybrid filler of Experimental Example 4. FIG.

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 １．of HAp  According to the content Levan Hydrogel  Stability evaluation

The amount of HAp in the Levan hydrogel (Lev5 / CMC0.5 / PF127) containing 5 wt% of Levan, Pluronic polymer (PF127, 17wt%) and 0.5wt% of carboxymethylcellulose (CMC) was 0.1wt% to 5wt% Differently added and physically mixed, the stability was evaluated under conditions similar to the in vivo environment (PBS (pH 7.4), 37 ° C.). The results are shown in FIG.

As can be seen from Figure 1, when the addition of HAp to Lev5 / CMC0.5 / PF127 it was shown that the levane hydrogel is formed more stably. Particularly, when the content of HAp is 0.1 to 1wt%, the stability of the levane hydrogel is increased and it is confirmed that the optimal levane hydrogel is formed.

Example 2. Levan Of HAp  Organic weapons hybrid Of filler Rheological  Rheological Property Evaluation

In order to evaluate the rheological or mechanical properties of the levane hydrogel, HAp was added to the semi-hydrogel (Lev5 / CMC0.5 / PF127) at 4 ° C. in a range of 0.1 wt% to 5 wt%, and physically mixed. 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 2, the levane hydrogel (Lev5 / CMC0.5 / PF127) is a sol (sol) at a low temperature can be transformed to a gel (gel) state at 37 ℃ in vivo environment Interestingly, when HAp is added to the levane hydrogel, the strength of the levane hydrogel decreases as the HAp content increases, which is an inorganic substance in the levane hydrogel in which the hydrogel is formed through physical bonding. As the HAp is distributed, the physical binding force is lowered.

< Experimental Example >

Experimental Example 1. Levan Of HAp  Organic weapons hybrid Hydrogel  Cytotoxicity 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 / HAp organic-inorganic hybrid hydrogel, Live / Dead assay (AO (live cell staining, green fluorescence) and PI (dead cell staining, red fluorescence) Iii) using a staining reagent). The results are shown in FIG.

As can be seen from Figure 3, it was confirmed that there is no cytotoxicity in all groups. That is, red fluorescence (dead cells) is observed at less than 5%, so the Levan / HAp organic-inorganic hybrid hydrogel does not cause intracellular toxicity, and based on these results, it will not cause toxicity even when implanted in vivo as a biofiller. I think that.

Experimental Example 2. Levan Of HAp  Organic weapons hybrid Of filler  cell Survival rate  evaluation

Cell viability of the Levan / HAp organic-inorganic hybrid hydrogel was measured by the WST-8 assay, and the results are shown in FIG. 4.

As can be seen from Figure 4, Levan / HAp organic-inorganic hybrid hydrogel was found to be excellent in biocompatibility without causing cytotoxicity in all groups. More specifically, it showed that the biocompatibility of the Levan / HAp organic-inorganic hybrid hydrogel according to the present invention is excellent by showing a cell viability of at least 95% in 24 hours cell culture.

Experimental Example 3. Levan Of HAp  Organic weapons hybrid Of filler  Cell proliferation evaluation

After applying low molecular weight chitosan (1 wt% in 1% acetic acid) on a round coverglass (12 mm diameter), it was dried for 12 hours and the remaining acetic acid was removed using DIW, followed by human adult dermal fibroblasts cells (5x104 cells / well) was applied and cultured for about 12 hours to form a cell layer (cell medium: DMEM (10% FBS, 1% antibiotics)). After contacting the cell layer on the Levan / HAp organic-inorganic hybrid hydrogel, hourly (1-4 days) cell growth rate was analyzed using the WST-8 assay, and the results are shown in FIG. 5.

As can be seen from Figure 5, when the HAp charged to Levan hydration gel (Lev5 / CMC0.5 / PF127) was shown to significantly increase the cell proliferation rate from day 4. Therefore, it has been demonstrated that the addition of HAp to the Levan hydrogel can affect the proliferation rate of dermal fibroblasts.

Experimental Example 4. Levan Of HAp  Organic weapons hybrid Of filler  Collagen Formation ability  evaluation

Human Adult Dermal Fibroblasts (hADF) were contacted on a Levan / HAp organic-inorganic hybrid hydrogel, the RNA was extracted, and collagen (collagen type 1) formation was analyzed using qPCR (control gene: beta-actin). . The results are shown in FIG.

As can be seen from Figure 6, the addition of HAp to the levane hydration gel (Lev5 / CMC0.5 / PF127) was found to increase the intracellular collagen formation ability. In particular, when the amount of HAp is 0.5wt% and 1wt% it was confirmed that it shows more than four times higher collagen formation ability than the control. Therefore, it has been proved that the levane hydrogel containing HAp is a material capable of promoting collagen formation in vivo and exhibiting wrinkle improvement efficacy.

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 (10)

Levan hydrogel with improved stability for use as a medical biofiller,
The levane hydrogel contains levane, a temperature sensitive phase transition polymer, carboxymethylcellulose and hydroxyapatite,
The hydroxyapatite is characterized in that it is mixed in an amount of 0.1 to 1% by weight based on the weight of the levane hydration gel, the stability of the levane hydration gel.
The method of claim 1,
The molecular weight of the Levan is characterized in that 150 to 200 kDa, the stability of the improved Levan hydrogel.
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 a mixture of one or more selected from the group consisting of polyethylene oxide (PEO) -polycaprolactone (PCL) copolymers.
delete delete According to claim 1 to claim 3, wherein the improved stability of the levan hydration gel for use as a medical bio-filler, the method is:
Stability including the steps of adding hydroxyapatite in an amount of 0.1 to 1 wt% based on the weight of the Levan hydration gel to the levane hydration gel containing the levane, the temperature-sensitive phase transition polymer and carboxymethylcellulose Method for preparing this improved Levan hydrogel.
The method of claim 6,
The molecular weight of the Levan is characterized in that 150 to 200 kDa, the method of producing a stable stability of the Levan hydrogel.
The method of claim 6,
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, wherein the mixture is one or more selected from the group consisting of.
delete delete

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