KR102401238B1 - Bentonite-hydrogel bio-nano-composites and preparing method thereof - Google Patents

Abstract

The present invention relates to a bentonite-hydrogel composite and a preparation method thereof, wherein the bentonite-hydrogel composite is bonded to 7-15% (w/v) of hydrogel and 0.05-1% (w/v) of bentonite, and the preparation method comprises the steps of: i) mixing bentonite nanoparticles with a gelatin prepolymer solution; ii) sonicating the mixed solution; iii) adding a photoinitiator to the mixture; and iv) performing photocuring by ultraviolet rays or visible rays. Accordingly, it is possible to provide a nanocomposite material optimized for tissue engineering which is free of any irritation, has excellent biocompatibility, and has improved properties such as elastic strength and pore size, and the like.

Description

Bentonite-hydrogel composite and its manufacturing method {Bentonite-hydrogel bio-nano-composites and preparing method thereof}

The present invention relates to a bentonite-hydrogel composite and a method for manufacturing the same, and more specifically, to prepare a nanocomposite optimized for tissue engineering by controlling the content of photocurable gelatin and bentonite, elastic strength, pore size, etc. It relates to a nanocomposite material with improved properties and a method for manufacturing the same.

As a natural clay mineral, bentonite plays an important role in tissue engineering due to its excellent properties such as high cation exchange and high surface area. Its microstructure is composed of a laminated plate structure. Each plate consists of three sandwiched layers, a central octahedral alumina (Al ₂ O ₃ ) layer, and two tetrahedral silica (SiO ₂ ) layers, (Al ₂ -mMgm)Si ₄ O ₁₀ (OH) ₂ ·nH ₂ O is formed. In addition, bentonite has a high swelling ratio, high adsorption capacity and drug transport capacity. The properties of bentonite include large interlayer space, cation exchange capacity, antibacterial activity, drug-transport and release, swelling and hygroscopicity, gel formation, and can serve as cell center adsorption sites to enhance cell adhesion and cell proliferation. have the ability

In addition, as compared to carbon-based materials and gold nanoparticles, bentonite is a natural clay and can be decomposed into chemical elements such as magnesium and calcium, so it is harmless to the human body and has no reported toxicity, so it is suitable for tissue regeneration studies.

On the other hand, gelatin methacroyloyl (GelMA) is widely used in the field of tissue engineering due to its properties. GelMA aids in cell proliferation and adhesion, and the mechanical stiffness of GelMA can be controlled through the concentration of macromers and crosslinking density. GelMA's fabrication techniques are versatile and can be easily prepared, making it suitable for a variety of applications. Various nanomaterials have been used to improve their physical and biological properties, and there are studies focusing on carbon-based materials such as graphene oxide to improve mechanical stiffness. Gold nanoparticles are also used to enhance cell proliferation and adhesion. Since silicate clays have been widely used in antacid, cosmetic and pharmaceutical applications, there has been a recent focus on investigating their role. Thus, bentonite is one of these clays that can be used in drug delivery, bone regeneration, regenerative medicine and imaging technology, consistent with this recent focus.

Hydrogel (gelatin) is fundamentally required to have the ability to absorb a large amount of moisture, proper physical strength, and form stability to maintain a constant shape. In addition, it must be non-toxic, and can prevent contamination from the outside during use. , it is characterized by easy adhesion to wounds or skin, as well as sterilization, excellent storage, and easy handling.

However, polyacrylic acid and polyvinyl alcohol, which are currently widely used as hydrogel materials, have a high water absorption rate and excellent shape stability, but when attached to the skin for a long time, they stimulate the skin and cause itching. has a problem.

In order to solve these problems and provide a material that is renewable, environmentally friendly, harmless to the human body and has a high water absorption rate, the present invention is a bentonite having no irritation and excellent mechanical strength and biocompatibility by mixing a hydrogel and bentonite. -Provides a hydrogel nanocomposite material and a method for manufacturing the same.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to an embodiment of the present invention, a bentonite formed by combining a hydrogel and bentonite-provides a hydrogel composite.

According to another embodiment of the present invention, in the method for preparing a bentonite-hydrogel composite,

i) mixing the bentonite nanoparticles with a gelatin prepolymer solution;

ii) performing sonication on the mixed solution;

iii) adding a photoinitiator to the mixture; and

iv) performing photocuring by ultraviolet or visible light;

It provides a method for producing a bentonite-hydrogel composite, characterized in that it comprises a.

The present invention relates to a bentonite-hydrogel composite and a method for manufacturing the same, and it is possible to provide a nanocomposite material optimized for tissue engineering, which has no irritation and excellent biocompatibility according to the content of photocurable gelatin and bentonite, and has elasticity The effect of providing a nanocomposite material with improved properties such as strength and pore size can be expected. In addition, the nanocomposite material of the present invention has the effect that it can be used for 3D printing.

1 is a view showing a manufacturing process of bentonite-GelMA having a NIH 3 T3 cell.
2 is a diagram showing the contact mechanical test of bentonite-GelMA, (A) is a photograph of the experimental stage, (B) is a force-displacement (force) of various concentrations of Na-Bent-GelMA and control samples ( displacement) curve, and quantified elastic modulus values of 7% GelMA with 0.05%, 0.1%, 0.2% Na and Ca-Bent (n=5).
3 is a view showing a bentonite treatment process.
4 is a view showing a synthetic process of chemically treated nano-bentonite particles.
5 is a view showing the compression test results of chemically treated bulk-bentonite with MgCl ₂ and an alkali activator.
6 is a view showing the characterization results of bentonite-GelMA nanocomposite hydrogel, (A) shows the elastic modulus of Ca-Bent (treated and untreated) with GelMA, (B) shows the FTIR spectral analysis results of GelMA, Na-Bent and Ca-Bent, (C) shows the elemental analysis of bentonite-GelMA at the white arrow point at the Si and Ca peaks, (D) is 7% SEM images of different concentrations of Ca-Bent with GelMA are shown.
7 is a view showing a nanocomposite material manufacturing process according to an embodiment of the present invention.
8 is a view showing the analysis of the physical property value (elastic modulus) of the nanocomposite material according to an embodiment of the present invention.
9 is a view showing the analysis of physical properties (pore size) of the nanocomposite material according to an embodiment of the present invention.
10 and 11 are views showing the biocompatibility analysis of the nanocomposite material.

According to one aspect of the present invention, a bentonite formed by combining a hydrogel and bentonite provides a hydrogel composite.

At this time, the content of the hydrogel forming the complex may be 7 to 15% (w / v), the content of bentonite may be 0.05 to 1% (w / v).

When the content of the hydrogel is less than 7% (w/v), the elastic strength is too low, the difference in elastic strength with the hydrogel not containing bentonite may be insignificant, and the content is 15% (w/v) If it exceeds, the difference in pores with the hydrogel that does not contain bentonite may be insignificant, and the degree of expansion of the composite may appear too large, which is not preferable in terms of physical properties of the nanocomposite material.

In addition, even if the content of the bentonite is less than 0.05% (w/v) or exceeds 1% (w/v), it may not be preferable in terms of elastic strength.

On the other hand, the bentonite-type of bentonite forming the hydrogel composite may be Na, Ca, or Mg bentonite, and in terms of elastic strength, Na bentonite may be more preferable.

According to another aspect of the present invention, in the method for preparing a bentonite-hydrogel composite,

i) mixing the bentonite nanoparticles with a gelatin prepolymer solution;

ii) performing sonication on the mixed solution;

iii) adding a photoinitiator to the mixture; and

iv) performing photocuring by ultraviolet or visible light;

It provides a method for producing a bentonite-hydrogel composite, characterized in that it comprises a.

At this time, provided according to an aspect of the present invention, the gelatin solution,

i) adding gelatin to phosphate buffered saline (PBS);

ii) stirring until the gelatin is completely dissolved;

can be manufactured through

In addition, in the bentonite-hydrogel composite prepared according to the manufacturing method, as described above, the content of the hydrogel in the bentonite-hydrogel composite is 7 to 15% (w/v), and the content of the bentonite is 0.05 to It may be 1% (w/v), and the effect thereof is the same as described above.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all modifications, equivalents and substitutes for the embodiments are included in the scope of the rights.

The terms used in the examples are used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

In addition, in the description with reference to the accompanying drawings, the same components are given the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

In addition, in describing the components of the embodiment, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only for distinguishing the elements from other elements, and the essence, order, or order of the elements are not limited by the terms.

Components included in one embodiment and components having a common function will be described using the same names in other embodiments. Unless otherwise stated, a description described in one embodiment may be applied to another embodiment, and a detailed description in the overlapping range will be omitted.

Next, a nanocomposite material and a manufacturing process thereof according to an embodiment of the present invention will be described with reference to the drawings.

- Examples

1) GelMA synthesis

In the manufacturing process, 10 g of gelatin obtained from pig skin (Sigma Aldrich, St. Louis, MO, USA) was added to sterile phosphate buffered saline (PBS, VWR, Monroeville, PA, USA) in a flask. It is a 7-day process that starts by performing magnetic stirring at 50 ° C until the gelatin is completely dissolved. 8 mL of methacrylic anhydride (100 mL) (Sigma Aldrich, St. Louis, MO, USA) was added to the gelatin solution, and the mixture was magnetically stirred (240) at 50 °C for 2 hours for synthesis. rpm). Another 100 mL of sterile PBS was added to the synthesized GelMA to make the final solution volume 200 mL. As a next step, GelMA was transferred to the dialysis membrane, and the sealed dialysis membrane was stirred with a magnetic stirrer (~500 rpm) at 40 °C for at least 5 days. Water was changed twice a day. On day 5, 200 mL of ultrapure water was added to the empty conical flask prior to the addition of GelMA, and the solution was heated for 15 min and sealed at 40 °C. As a final step, the solution was filtered using a sterile vacuum (0.22 μm) Millipore filtration cup (Millipore Sigma, Burlington, MA, USA), the GelMA was transferred to a falcon tube, and the tube was frozen until it was frozen. Stored at 80° C. for at least two days.

2) Bentonite-GelMA hydrogel preparation

Ca-Bent and Na-Bent nanoparticles were obtained from Korea Institute of Geoscience and Mineral Resources. The bentonite used in this experiment is classified as low swelling bentonite and its components are 1.67% sodium oxide (Na ₂ O), 5.57% magnesium oxide (MgO), 17.09% aluminum oxide (Al ₂ O ₃ ), 57.12% silicon dioxide ( SiO ₂ ), 0.09% phosphorus pentoxide (P ₂ O ₅ ), 0.14% potassium oxide (K ₂ O), 1.66% calcium oxide (CaO), 0.23% titanium oxide (TiO ₂ ), 0.06% manganese(II) oxide ( MnO), 3.83% iron oxide (Fe ₂ O ₃ ) and 12.84% loss on ignition (LOI). Various concentrations of Na-Bent and Ca-Bent nanoparticles were mixed with GelMA prepolymer solution. First, 0.05%, 0.1%, and 0.2% bentonite nanoparticles were mixed in 2% GelMA prepolymer solution, respectively. In order to disperse the nanoparticles well in the mixture and to avoid aggregation and aggregation of nanoparticles, a probe sonicator (Q700, QSonica, CT, USA) was used for 30 minutes. Then, 0.5% photoinitiator (PI) (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, Sigma Aldrich, St. Louis, MO, USA) and 5% GelMA were added to the mixture, each A final solution (5 mL) of 7% GelMA and 0.5% PI, with 0.05%, 0.1% and 0.2% bentonite was made.

1 shows a schematic diagram comprising bentonite nanoparticles in GelMA using a photoinitiator that produces free radicals to polymerize and form irreversibly crosslinked 3D cell capsule hydrogels.

3) Mechanical property characterization

A contact mechanical testing method was used to improve the reliability of the mechanical analysis and to minimize sample variability. This method uses a specimen with a cylindrical probe and analyzes the data with a Hertz contact mechanical model. Measurement of the liquid environment has the following advantages: i) the wet state of the material is closer to the actual cell culture conditions in the medium, ii) the hydration rate of the material is always constant, and the hydrogel properties of the material because of the hydration rate no change As an advantage of the aforementioned contact mechanical testing method, a custom-made aluminum cylindrical probe end with a flat surface that can be attached to a mechanical testing machine (Biomomentum Mach-1, QC, Montreal, Canada) is shown in Figure 2 It was developed as shown in (A). Measurements were performed with samples in PBS. The force-displacement curve Fig. 2(B) was measured at more than 10 points of the specimen. The slope of linear regression was used to calculate the Young's modulus of the material. The following well-known Hertz contact mechanical model of a flat cylindrical probe was used to calculate the Young's modulus of the material:

Here, F is the force, E is the Young's modulus, a is the radius of the probe, d is the displacement, and v is the Poisson's ratio, and the Poisson's ratio corresponds to 0.5 in the hydrogel. For each measurement, n ≒ 5 points were considered for calculation. The maximum load of data (kN) was used to calculate the maximum compressive force of the sample. The height and radius of each sample were measured before the compaction test.

As a result, as shown in FIG. 2(C), the 0.05% Na-Bent and 7% GelMA nanocomposite hydrogels exhibited a Young's modulus value of 20.6 kPa, which was about twice higher than that of 7% GelMA, 10.2 kPa. It was confirmed that the numerical values were shown, and 0.1% and 0.2% Na-Bent showed 12.5 and 10.1 kPa, respectively, and it was confirmed that as the Na-Bent content increased, the mechanical properties of GelMA were negatively affected. In addition, it was found that Ca-Bent did not significantly affect the mechanical properties of GelMA.

4) Chemical treatment of bulk bentonite

Chemical treatment of bentonite with MgCl ₂ with or without alkali activator was investigated. The alkali activator is a mixture of Na ₂ SiO ₃ and NaOH (Fisher Scientific International, Haverhill, MA, USA) in a ratio of 3:1. This ratio was obtained from a previous study (Muhammad et al., 2018). The mixed solution for the alkali activator was kept at room temperature overnight. To prepare bentonite samples with MgCl ₂ without alkali activator, as shown in FIG. 3 , 194 g of Ca-Bent was mixed with 6 g of MgCl ₂ before manual mixing with 58 mL of water. A sample treated with MgCl ₂ and alkali activator was prepared by mixing 194 g of bentonite, 4.2 mL of alkali activator solution, 6 g of MgCl ₂ , and 53.8 mL of RO water. The mixture was thoroughly mixed for 5 minutes and stored in a plastic bag overnight for chemical reaction. The samples were then compressed using unconfined compression strength test equipment (UCS) (GDS instruments, United Kingdom) to prepare cylindrical specimens with diameters of 50 mm and 20 mm. Cylindrical samples were packaged to maintain the moisture content of the samples and cured for 3, 7, 10, and 14 days.

Meanwhile, another bentonite treatment method of increasing the viscosity and gelling properties of bentonite by treating Ca-Bent with MgO and Na ₂ CO ₃ (Fisher Scientific International, Haverhill, MA, USA) was investigated. MgO powder was mixed with Na ₂ CO ₃ powder and dissolved in 20 mL of RO water, then the mixture was added to 193 g of Ca-Bent and 35 mL of RO water. Cylindrical samples were prepared using the same method as mentioned above and the samples were packaged and stored at room temperature for curing.

5) Preparation of bentonite nanoparticles

Since bentonite is agglomerated due to chemical treatment, an additional process is required to prepare bentonite nanoparticles through chemical treatment. Through this process, nanoparticles in the 500 nm range were prepared by processing Ca-Bent treated with 1% MgO and Na ₂ CO ₃ (Sarkar et al., 2017). As shown in FIG. 4 , the nanoparticle synthesis process starts with wet pulverizing of bentonite using zirconium balls. In this step, in order to prevent swelling of the bentonite, 7 g of bentonite was ground with isopropanol alcohol (IPA) with 2 mm zirconium balls for 30 minutes. The pulverized bentonite was sieved with a 1.8 μm mesh to remove zirconium balls. The second step was carried out by using 0.1 mm zirconium balls for 1 hour to convert the bentonite into smaller particles. Then, 10 g of crushed bentonite was dissolved in 950 mL and mixed for 10 minutes using a vortex mixer. The solution was then sonicated using a probe ultrasound with a probe diameter of 15 mm. The sonicated bentonite sample was centrifuged at 4000 rpm for 30 minutes to obtain a supernatant of bentonite particles (Sarkar et al., 2017). The collected supernatant was further filtered using a 0.2 μm vacuum filter to obtain bentonite nanoparticles, and then dried on a hot plate at 100° C. until the excess water was completely evaporated. Then, the bentonite sample is placed in an oven to dry completely overnight at 105° C. and all residual moisture is removed for a dry grinding step. The final step is to further decompose the agglomerated bentonite nanoparticles by dry grinding the bentonite for 30 minutes using a 2 mm zirconium ball. Finally, in order to confirm the size of the particles, the bentonite nanoparticles were measured using a Zetasizer Nano ZS (ZEN 3600 from Malvern). Zetasizer was used to measure the size of bentonite nanoparticles pulverized by chemical treatment to prevent agglomeration of bentonite nanoparticles and increase bentonite dispersion in GelMA chains in 2% GelMA solution. Sonication of bentonite nanoparticles in 2% GelMA prepolymer solution for 30 min was the optimal condition used to maintain a uniform distribution of nanoparticles.

6) Structural properties of nanocomposite hydrogels

Microstructure analysis was obtained using a field emission scanning electron microscope (FESEM), and a 10 nm platinum-palladium (Pt-Pd) alloy was sputtered onto the cross-linked hydrogel. The instrument used was (TESCAN, BRNO, Czechia) MIRA3 XMU FESEM, Aztec X-max EDS system (Oxford Instruments, Abingdon, UK). Elemental analysis of the composition was obtained using energy-dispersive X-ray spectroscopy. (Shimadzu, Kyoto, Japan) Fourier transform infrared spectroscopy of the IR Prestige-21 device was used to evaluate the functional groups of bentonite-GelMA samples and to study the oscillations of hydrogen bonds and silicon-oxygen bonds in nanocomposites. Spectra were recorded in the range of 500-4000 cm ^-1 . The results are shown in FIG. 6 .

7) In vitro cytocompatibility study of bentonite-GelMA hydrogel

The biocompatibility of bentonite-GelMA was tested by encapsulating NIH 3 T3 fibroblasts, a cell line isolated from Swiss albino mouse embryonic tissue, in bentonite-GelMA hydrogels. Cells were trypsinized, resuspended in bentonite-GelMA prepolymer and cross-linked using UV light for 60 s. Various concentrations of Ca-Bent (0.05 %, 0.1 %, 0.2 % w/v) dispersed in 7% GelMA prepolymer solution were mixed in 2.5×10 ⁶ cells/mL fibroblasts. A droplet of 120 μl cell-bentonite-GelMA prepolymer was cross-linked in a petri dish, and Dulbecco's Modified Eagle's Medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin ( The cell-containing hydrogel cultured in Dulbecco's modified eagle medium (DMEM)) was incubated at 37 °C and 5% CO ₂ for 5 days.

Characterization of cell-containing hydrogels was performed using a standard live/dead cell viability assay (Biotium, Fermont, CA, USA) with calcein-AM/ethidium homodimer. To prepare the assay, 5 mL of sterile PBS was mixed with 0.04% calcein AM and 2% ethidium homodimer. Samples containing cells were washed twice with PBS and stained with an assay solution at 37° C. for 20 minutes under dark conditions. Live and dead cells were imaged using an inverted fluorescence microscope (Axio Observer 7, Carl Zeiss Canada Ltd., North York, ON, Canada), and live cells were imaged using ImageJ software (NIH, Bethesda, MD, USA). number was calculated.

As shown in FIGS. 10 and 11 , it was confirmed that the cell viability exceeded 90% at each different concentration of bentonite, and from this, it can be seen that the bentonite-GelMA nanocomposite provides an environment suitable for cell growth. have.

As described above, although the embodiments have been described with reference to the limited drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the described techniques are performed in an order different from the described method, and/or the described components of the system, structure, apparatus, circuit, etc. are combined or combined in a different form than the described method, or other components Or substituted or substituted by equivalents may achieve an appropriate result.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (7)

It is formed by combining hydrogel and bentonite,
The content of the hydrogel is 7 to 15% (w / v),
The content of the bentonite is 0.05 to 1% (w / v),
The bentonite is Na bentonite or Ca bentonite bentonite-hydrogel composite.
delete delete delete In the method for preparing a bentonite-hydrogel composite,
i) mixing the bentonite nanoparticles with a gelatin prepolymer solution;
ii) performing sonication on the mixed solution;
iii) adding a photoinitiator to the mixture; and
iv) performing photocuring by ultraviolet or visible light;
includes,
The content of the hydrogel is 7 to 15% (w / v),
The content of the bentonite is 0.05 to 1% (w / v),
The bentonite is Na bentonite or Ca bentonite, characterized in that, bentonite-a method of producing a hydrogel composite.
6. The method of claim 5,
The gelatin prepolymer solution,
i) adding gelatin to phosphate buffered saline (PBS);
ii) stirring until the gelatin is completely dissolved;
Which will be prepared through, bentonite-method of producing a hydrogel composite.
delete

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