KR20190066936A - 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, which has remarkably improved cell viability, cell proliferation rate, and collagen formation ability as well as increased stability of levan hydrogel by adding HAp to the levan hydrogel; and a method for producing the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a hybrid filler and a method for producing the hybrid filler,

The present invention relates to a Levan / HAp organic hybrid filler and a method for producing the same. More specifically, the present invention relates to a Levan / HAp-free hybrid filler having a remarkably improved cell survival rate, cell proliferation rate and collagen forming ability as well as an increase in the stability of the Levan hydrated gel by adding HAp to the Levan hydrogel, and a process for producing the same will be.

The first filler used for the purpose of soft tissue enlargement for filling soft tissue defects was considered to be autologous fat and in 1893 Neuber was the first to implant autologous fat from the patient's arm into the face defect It is an example. Collagen is the most commonly found protein in the human body and is the most abundant protein in mammals, accounting for about 25-35% of the total protein. In particular, it is a major component of bone, tendon, and ligament, and plays a role in maintaining the structure of organs. It was easily extracted from the skin of cattle and pigs. Collagen filler extracted from cattle entered the market with the approval of US FDA for the first time in 1981. Conventional collagen fillers are pig skin or bovine collagen products, and since an immune response occurs in 3 to 5% of patients, there is a problem that they must be transplanted after an allergic test is performed.

Cosmoderm® and Cosmoplast® were developed to solve the hassle of skin test when using collagen from cattle or pigs. It uses human collagen obtained through cultivation of fibroblasts. Since it is human-derived, there is no fear of allergic reaction, and since it is convenient to have skin reaction test in advance, it became a representative product of collagen filler after obtaining FDA approval in 2003. However, these autologous cell culture collagen can be applied to patients with fear of animal-derived collagen, but since it is necessary to collect a large area of skin, only those patients who have extra skin or those who have to undergo surgery for other reasons It is a procedure. In addition, the collagen filler prepared by purifying the collagen secreted from the culture solution through human fibroblast culture can solve the immune reaction, but there still remains a problem in the stability part.

In order to avoid the problem of collagen, a method of using hyaluronic acid as a filler is also widely studied. In other words, unlike collagen, hyaluronic acid does not act as an antigen because there is no difference in chemical structure between bacteria and mammals. Therefore, hyaluronic acid has been 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 is a problem of the collagen filler is small. However, since hyaluronic acid also contains a small amount of animal protein in the manufacturing process, the problem of foreign matter reaction is not completely solved. Hyaluronic acid is degraded by two pathways in the body. First, it is degraded by hyaluronidase. Second, it attaches to the cell receptor and is fused into the cell and degraded by enzymes in the lysosome . The biodegradation of hyaluronic acid is very rapid and is known to be completely degraded within 0.5 to several days. In order to overcome this short biodegradation, cross-linking of hyaluronic acid has been introduced. When the molecular weight is increased by cross-linking, the phagocytosis by leukocytes, especially monocytes, can be suppressed firstly, and the action rate of hyaluronidase is also reduced. However, medical fillers prepared by treating a hyaluronic acid with a cross-linking agent have limitations in that side effects (such as cell or tissue necrosis, and immune reaction) may occur due to the use of a cross-linking agent.

Therefore, there is a need to develop new formulations that have superior properties of biocompatibility and biodegradability and are capable of correcting the texture and shape naturally due to viscosity and elasticity, and in particular, hyaluronic acid and its efficacy It is required to develop a material having a similar and low cost.

Accordingly, the present inventors have made extensive studies to improve the stability of Levan / carboxymethylcellulose / pluronic organic complexes for use in medical biopharmaceutical materials having excellent biocompatibility and biodegradability disclosed in Patent Application No. 10-2017-0132657 As a result of efforts, it has been confirmed that the stability of Levan / carboxymethylcellulose / pluronic organic complex HAp (Hydroxyapatite) can be improved, thereby further improving the stability of Levan hydrated gel.

It is an object of the present invention to provide a Levan hydrated gel having improved stability, which comprises HAp (Hydroxyapatite).

It is another object of the present invention to provide a process for producing a Levan hydrated gel having improved stability, which comprises HAp (Hydroxyapatite).

As used herein, the term " levan " is a water-soluble fructose polymer composed mainly of beta-2,6 linkages and is produced from sugar by the folate transfer reaction of levansucrase. Levan according to the present invention may have excellent biodegradability as well as biocompatibility.

As used herein, the term " biocompatibility " is a property required for a biomedical material, and refers to a property of a material that can coexist with a living body while performing an 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, and can be decomposed by, for example, body fluids or microorganisms in vivo in mammals including humans .

According to a first embodiment,

The present invention

To provide a Levan hydration gel having improved stability including Levan, thermosensitive phase transfer polymer, carboxymethyl cellulose and hydroxyapatite.

In the Levan hydrated gel having improved stability according to the present invention, the molecular weight of the levan is 150 to 200 kDa.

In the Levan hydrogel having improved stability according to the present invention, the thermosensitive phase transition polymer is selected from the group consisting of polyethylene oxide (PEO) -Polypropylene Oxide (PPO) copolymer, polyethylene oxide (PEO) -Polylactic acid (PLA) (PEO) -polyglycolic lactic acid (PLGA) copolymer or a polyethylene oxide (PEO) -polycaprolactone (PCL) copolymer. For example, the polyethylene oxide (PEO) -Polypropylene Oxide (PPO) copolymer can be used in a variety of applications including Pluronic series (BASF, USA), specifically Pluronic F-38, Pluronic F- May include, for example, Lonicin F-77, Pluronic F-87, Pluronic F-88, Pluronic F-98, Pluronic F-108, Pluronic F-127, . The polymer solution having the phase transition phenomenon of the present invention has a sol phase at a sol-gel phase transition temperature and a gel phase at a sol-gel phase transition temperature or higher. In one embodiment, Pluronic F-127 was used as the thermosensitive phase transfer polymer. The levan and pluronic F-127 may be mixed at a weight ratio of 1: 2 to 1:20 by weight, preferably 1: 3 to 1: 4.

In the Levan hydrogel having improved stability according to the present invention, Levan, Pluronic F-127 and carboxymethylcellulose are mixed in a weight ratio of 1: 2 to 20: 1. Preferably, Levan, Pluronic F-127 and Carboxymethylcellulose are mixed in a weight ratio of 1: 3-4: 1.

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

In the Levan hydrated gel having improved stability according to the present invention, the Levan hydrated gel is used as a biopharmaceutical material for medical use.

According to a second embodiment,

The present invention

A temperature-responsive phase transition polymer and a Levans hydrogel containing ozone by carboxymethylcellulose in an amount of 0.1 to 5 wt% based on the weight of the Levan hydrated gel and mixing the hydroxyapatite in an amount of 0.1 to 5 wt% To provide a method for producing an improved Levan hydration gel.

In the method for preparing a Levan hydrogel according to the present invention, the molecular weight of levan is 150 to 200 kDa.

In the method for preparing the Levans hydrogel according to the present invention, the thermosensitive phase transition polymer may be selected from the group consisting of polyethylene oxide (PEO) -Polypropylene Oxide (PPO) copolymer, polyethylene oxide (PEO) -Polylactic acid (PLA) , Polyethylene oxide (PEO) -polyglycolic lactic acid (PLGA) copolymer or polyethylene oxide (PEO) -polycaprolactone (PCL) copolymer. For example, the polyethylene oxide (PEO) -Polypropylene Oxide (PPO) copolymer can be used in a variety of applications including Pluronic series (BASF, USA), specifically Pluronic F-38, Pluronic F- May include, for example, Lonicin F-77, Pluronic F-87, Pluronic F-88, Pluronic F-98, Pluronic F-108, Pluronic F-127, .have.

In the method for preparing a Levan hydrogel according to the present invention, Levan, Pluronic F-127 and carboxymethyl cellulose are mixed in a weight ratio of 1: 2 to 20: 1. Preferably, Levan, Pluronic F-127 and Carboxymethylcellulose are mixed in a weight ratio of 1: 3-4: 1.

In the method for manufacturing a Levan hydrated gel having improved stability according to the present invention, the Levan hydrated gel is used as a biopharmaceutical material for medical use.

Fig. 1 shows the stability evaluation results of Levan hydrated gel according to the amount of HAp added in Example 1. Fig.
Fig. 2 shows the results of the rheological evaluation of the Levan / HAp organic hybrid filler of Example 2. Fig.
Fig. 3 shows the cytotoxicity evaluation results of the Levan / HAp organic hygroscopic hydrogel of Experimental Example 1. Fig. (A) PF127 / Lev5 / CMC0.5, B) PF127 / Lev5 / CMC0.5 / HAp0.1, C) PF127 / Lev5 / CMC0.5 / HAp0.5 )
4 shows the cell survival rate of the Levan / HAp organic hybrid hydrogel of Experimental Example 2. FIG.
Fig. 5 shows the cell growth rate evaluation results of the Levan / HAp organic hybrid filler of Experimental Example 3.
Fig. 6 shows the results of evaluating collagen-forming ability of the Levan / HAp organic hybrid filler of Experimental Example 4. Fig.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be described in more detail with reference to the following examples, which are illustrated in the accompanying drawings. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

< Example >

Example 1.HAp's  Depending on the content Levan Hydrated gel  Stability evaluation

(Lev5 / CMC0.5 / PF127) containing 5 wt% of Levan, 0.5 wt% of a pluronic polymer (PF127, 17 wt%) and carboxymethylcellulose (CMC) And physically mixed, and then 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 FIG. 1, when HAp was added to Lev5 / CMC0.5 / PF127, Levan hydrated gel was found to be more stable. In particular, when the content of HAp is 0.1 to 1 wt%, it is confirmed that the stability of Levan hydrated gel is increased and the optimum Levan hydrated gel is formed.

Example 2. Levan / HAp  Abs hybrid Filler Rheological  Evaluation of rheological property

In order to evaluate the rheological properties or mechanical properties of Levan hydrogel, HAp was added to the semi-hydrated gel (Lev5 / CMC0.5 / PF127) at 4 ℃ at different concentrations of 0.1wt% to 5wt% and physically mixed. The sol state was injected into the plate on the rheometer instrument and the rheological properties of the Levan hydrated gel 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 FIG. 2, the Levan hydration gel (Lev5 / CMC0.5 / PF127) is present as a sol at low temperature and can be transformed into a gel state at 37 ° C, Interestingly, when HAp was added to the Levan hydrated gel, the strength of the Levan hydrated gel decreased as the HAp content increased. And the physical binding force of HAp decreased with the addition of HAp.

< Experimental Example >

Experimental Example 1. Levan / HAp  Abs hybrid Hydrated gel  Cytotoxicity Assessment

Low molecular weight chitosan (1 wt% in 1% acetic acid) was applied on a round coverglass (12 mm diameter), dried for 12 hours, and the remaining acetic acid was removed using DIW. Then, hADF cells (5x10 ⁴ cells / well) were coated and cultured for about 12 hours to form a cell layer (cell culture medium: DMEM (FBS 10%, including 1% antibiotics)). (Live cell staining, green fluorescence staining) and PI (dead cell staining, red fluorescence) to determine whether the cell layer is in contact with the cell layer on a Levan / HAp organic hybrids gel after 24 hours, 띔) dyeing reagent). The results are shown in Fig.

As can be seen from Fig. 3, it was confirmed that there was no cytotoxicity in all the groups. In other words, the fluorescence of red (dead cells) was observed to be less than 5%, so that the Levan / HAp organic hygroscopic gel did not cause intracellular toxicity, and based on these results, .

Experimental Example 2. Levan / HAp  Abs hybrid Filler  cell Survival rate  evaluation

The cell viability of the Levan / HAp organic hybrid gel was measured by WST-8 analysis and the results are shown in FIG.

As can be seen from FIG. 4, the Levan / HAp organic hygroscopic gel showed no biocompatibility without cytotoxicity in all groups. More specifically, the cell viability of 95% or more in a 24-hour cell culture proved to be excellent in the biocompatibility of the levan / HAp organic hygroscopic gel according to the present invention.

Experimental Example 3. Levan / HAp  Abs hybrid Filler  Evaluation of cell proliferation rate

The cells were treated with low molecular weight chitosan (1 wt% in 1% acetic acid) on a round coverglass (12 mm diameter), dried for 12 hours, and the remaining acetic acid was removed using DIW. Human Adult Dermal Fibroblasts (5x104 cells / well) and cultured for about 12 hours to form a cell layer (cell culture medium: DMEM (FBS 10%, including 1% antibiotics)). After the cell layer was contacted with the Levan / HAp organic hygroscopic gel, the cell proliferation rate over time (1-4 days) was analyzed using the WST-8 evaluation method, and the results are shown in FIG.

As can be seen from FIG. 5, cell proliferation rate was significantly increased from day 4 when HAp was charged to Levan hydrogel (Lev5 / CMC0.5 / PF127). Therefore, the addition of HAp to Levan hydrated gels has been shown to affect the proliferation rate of dermal fibroblasts.

Experimental Example 4. Levan / HAp  Abs hybrid Filler  Collagen Formability  evaluation

HADF (human Adult Dermal Fibroblasts) were brought into contact with the Levan / HAp organic hygroscopic hydrogel, and then the intracellular RNA was extracted and analyzed for collagen type 1 formation ability using qPCR (control gene: beta-actin) . The results are shown in Fig.

As can be seen from FIG. 6, when HAp was added to levan hydrogel (Lev5 / CMC0.5 / PF127), intracellular collagen-forming ability was increased. Particularly, when the amount of HAp was 0.5 wt% and 1 wt%, it was confirmed that the collagen-forming ability was 4 times higher than that of the control group. Therefore, Levan hydration gel containing HAp has been proved to be a material capable of promoting collagen formation in vivo and exhibiting wrinkle reducing effect.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

Levan, a temperature-responsive phase transfer polymer, carboxymethylcellulose, and hydroxyapatite.
The method according to claim 1,
Wherein the levan has a molecular weight of 150 to 200 kDa.
The method according to claim 1,
The thermosensitive phase transfer polymer is selected from the group consisting of polyethylene oxide (PEO) -polypropylene oxide (PPO) copolymer, polyethylene oxide (PEO) -poly lactic acid (PLA) copolymer, polyethylene oxide (PEO) -polyglycolic lactic acid (PLGA) And a polyethylene oxide (PEO) -copolaporrolactone (PCL) copolymer. The Levan hydrogel gel having improved stability is characterized in that it is a mixture of at least one member selected from the group consisting of polyethylene oxide (PEO) and polycaprolactone (PCL).
The method according to claim 1,
Wherein the hydroxyapatite is mixed in an amount of 0.1 to 5% by weight based on the weight of the Levan hydrated gel.
The method according to claim 1,
Wherein the Levan hydrated gel is used as a medical biofiller material.
A temperature-responsive phase transition polymer and a Levans hydrogel containing ozone by carboxymethylcellulose in an amount of 0.1 to 5 wt% based on the weight of the Levan hydrated gel and mixing the hydroxyapatite in an amount of 0.1 to 5 wt% (EN) METHOD FOR MANUFACTURING GEL.
The method according to claim 6,
And the molecular weight of the levan is 150 to 200 kDa.
The method according to claim 6,
The thermosensitive phase transfer polymer is selected from the group consisting of polyethylene oxide (PEO) -polypropylene oxide (PPO) copolymer, polyethylene oxide (PEO) -poly lactic acid (PLA) copolymer, polyethylene oxide (PEO) -polyglycolic lactic acid (PLGA) And a mixture of at least one selected from the group consisting of polyethylene oxide (PEO) -copolaporrolactone (PCL) copolymer, and the like.
The method according to claim 6,
Wherein the hydroxyapatite is mixed in an amount of 0.1 to 5% by weight based on the weight of the Levan hydrated gel.
The method according to claim 6,
Wherein the Levan hydrated gel is used as a biopharmaceutical material for medical use.

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