KR20230051799A - Multifunctional Nanoparticles Based on Polyphenol and Metallic Ions, and Use Thereof - Google Patents

Abstract

In the present disclosure, disclosed are methods for producing composites or composite particles, which, by inducing coordination and self-assembly with polyphenol-based compounds in a one-pot method while changing the composition of an aqueous solution containing metal cations and anions, manufactures polyphenol-based nanoparticles with a metal-phenol network, and by combining the polyphenol-based nanoparticles with hydrogel or introducing fluorescent substances or magnetic nanoparticles during a manufacturing process, can provide various functionalities.

Description

Multifunctional Nanoparticles Based on Polyphenol and Metallic Ions, and Use Thereof

The present disclosure relates to nanoparticles based on polyphenols and metal ions and uses thereof. More specifically, the present disclosure provides a metal-phenol network by inducing coordination and self-assembly with a polyphenol-based compound in a one-pot manner while changing the composition in an aqueous solution containing metal cations and anions. It relates to a method for preparing a composite or composite particle that can provide various functionalities by preparing polyphenol-based nanoparticles, combining them with a hydrogel, or introducing fluorescent materials or magnetic nanoparticles in the manufacturing process.

Vertebrate animals, including humans, have the ability to regenerate bone, but there are cases in which a part of bone tissue is lost and not restored due to disease, accident, aging, or the like. In general, in order to regenerate a defect in bone tissue, a bone graft material is filled in a specific area and then treated with a shielding film to increase the bone regeneration induction effect. In this guided bone regeneration, a bone graft material and a shielding film are needed to increase the natural regeneration process in case of bone defect and to control the proper environment for the adhesion, growth and differentiation of damaged bone cells, that is, the bone regeneration environment. do. As an example, synthetic bone graft materials made of materials similar to bone structures, such as hydroxyapatite and β-tricalcium phosphate (β-TCP), are used. Calcium and phosphoric acid for bone regeneration can be delivered at the same time as filling the space (eg, Adv. Healthcare Mater. 2019, 8, 1801106, RSC Adv. 7 13010-8, etc.). However, since most graft materials for bone regeneration are composed of calcium phosphate-based mineral components and are simply manufactured for the purpose of bone formation, various microenvironments that affect bone regeneration, such as inflammatory reaction, osteoclast reaction, infection, etc. at the site of bone defect There is a limit that cannot be controlled.

On the other hand, polyphenol compounds such as tannic acid are mainly found in fruits, vegetables, cacao, nuts, etc., and have attracted attention from biochemical or physiological aspects such as antioxidant properties. Tannic acid has 5 gallol groups and 5 catechol groups as shown in Formula 1 below, and forms strong non-covalent bonds with various molecules through hydrogen bonding and hydrophobic interaction.

[Formula 1]

In addition, a technique for layer-by-layer coating by alternately stacking polyphenols, metal ions, or peptides on a desired material using rapid bonding between materials such as polyphenols, metal ions, and peptides is also known. After coating with phenol and metal ions, the substrate is removed to form polyphenol nanoparticles (eg, Nano Today, 12, 2017, 136-148, etc.).

In this regard, polyphenol itself has a strong antioxidant effect, but an excessively high concentration of polyphenol may be toxic. In addition, in the case of water-soluble polyphenols, due to their high dissolution rate, they are easily diffused in the body, limiting their local application. Furthermore, in order to apply polyphenol as a functional biomaterial, a non-functional polymer may be required as a substrate depending on the method of forming the biomaterial in the form of coating or loading polyphenol. The method of forming a biomaterial by post-processing such a polymer is not only complicated in the process, but also has a possibility that the non-functional material induces an immune rejection reaction in vivo.

Therefore, there is a need for a polyphenol-based technology that can implement an optimized bone regeneration environment in a simple way and can furthermore provide various functions.

In one embodiment of the present disclosure, it is intended to provide a method for preparing an organic-inorganic composite capable of overcoming the above-mentioned limitations of the prior art and forming an optimized bone regeneration environment in a simple manner.

In another embodiment of the present disclosure, it is intended to provide a method for preparing multifunctional composite particles endowed with fluorescence or magnetism as well as effects derived from the phenol-metal network.

According to the first aspect of the present disclosure,

a) (i) a polyphenolic compound and (ii) combining an aqueous solution in which metal cations and anions are dissolved in an aqueous medium and adding a first base component to form polyphenolic nanoparticles having a metal-phenolic network; and

b) forming a nanoparticle-hydrogel composite by combining a biopolymer-based hydrogel solution and the nanoparticles;

A method for producing an organic-inorganic composite comprising a is provided.

According to an exemplary embodiment, the step of adding a second basic component to the aqueous solution containing the nanoparticles to stabilize the nanoparticles prepared in step a) may be further included prior to step b).

According to an exemplary embodiment, the aqueous solution may be a simulated body fluid or a concentrated simulated body fluid.

According to an exemplary embodiment, the polyphenolic compound is epigallocatechin gallate, catechin, catechin-3-gallate, gallocatechin, gallo Catechin-3-gallate, Catechin gallate, Epicatechin, Gallocatechin gallate, Epigallocatechin, Gallic acid , It may be at least one selected from the group consisting of tannic acid and quercetin.

According to an exemplary embodiment, the biopolymer may be at least one selected from the group consisting of gelatin, guar gum, xanthan gum, acacia gum, carrageenan gum, pectin, alginate, collagen, hyaluronic acid, chitosan, and agarose. .

According to an exemplary embodiment, the first basic component is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate and calcium bicarbonate, and

The second basic component may be at least one selected from the group consisting of sodium hydroxide, potassium hydroxide and calcium hydroxide.

According to an exemplary embodiment, the polyphenol content in the polyphenol-based nanoparticles may range from 5 to 90% by weight.

According to an exemplary embodiment, the size (DLS) of the polyphenol-based nanoparticles may range from 100 to 3000 nm.

According to a second aspect of the present disclosure,

preparing a composite aqueous solution by combining a phosphor and an aqueous solution in which metal cations and anions are dissolved in an aqueous medium; and

combining the complex aqueous solution and the polyphenol-based compound and adding a first basic component to form fluorescent material-containing polyphenol-based nanoparticles having a metal-phenol network;

A method for producing fluorescent composite particles comprising a is provided.

According to an exemplary embodiment, the fluorescent substance is umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, Tamra (TAMRA), Dichlorotriazinylamine fluorescein, dansyl chloride, quantum dots, phycoerythrin, FAM (fluorecein amidite), alexa fluor, Cy3, Cy5 , Cy7 and may be at least one selected from the group consisting of indocyanine green.

According to a third aspect of the present disclosure,

preparing a magnetic nanoparticle-containing aqueous solution by combining magnetic nanoparticles and an aqueous solution in which metal cations and anions are dissolved in an aqueous medium; and

combining the magnetic nanoparticle-containing aqueous solution with a polyphenol-based compound and adding a first basic component to form magnetic composite nanoparticles having a metal-phenol network;

A method for producing magnetic composite particles comprising a is provided.

According to an exemplary embodiment, the magnetic nanoparticles are at least one selected from the group consisting of magnetite and maghemite as iron oxide particles, and may have a size (diameter) range of 50 nm or less.

According to specific embodiments of the present disclosure, polyphenolic nanoparticles having a metal-phenolic network can be prepared by simply combining an aqueous solution containing metal cations with a polyphenolic compound, and combining it with a hydrogel The organic-inorganic complex can provide good in vivo bone regeneration or bone differentiation effects including antioxidant and anti-inflammatory effects. In particular, an environment optimized for bone regeneration can be provided by adjusting the concentration of specific ions in an aqueous solution containing metal cations and anions, such as a simulated body fluid, during the nanoparticle manufacturing process. Furthermore, when combined with fluorescent materials, magnetic nanoparticles, etc. in the process of manufacturing polyphenolic nanoparticles, utilization can be further increased in fields such as production of various functional biomaterials, drug delivery, and imaging.

Figure 1 is a self-assembly method according to a combination of a polyphenolic compound (tannic acid) and an aqueous solution containing metal cations and anions, according to an exemplary embodiment, schematically illustrating a process for preparing polyphenolic nanoparticles having a metal-phenolic network. It is a drawing shown as;
2a to 2c are diagrams showing optical pictures, SEM pictures, and DLS analysis results of calcium-containing nanoparticles prepared under conditions of various tannic acid concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL), respectively;
2d and 2e are graphs showing phenol content and calcium content in calcium-containing nanoparticles according to various tannic acid concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL), respectively;
Figure 2f is a graph showing the results of FT-IR analysis of calcium-containing nanoparticles prepared under conditions of various tannic acid concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL);
3a and 3b are diagrams showing SEM pictures and DLS analysis results of magnesium-containing nanoparticles prepared under conditions of various tannic acid concentrations (0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL), respectively;
3c and 3d are diagrams showing TEM images and EDS analysis results of magnesium-containing nanoparticles prepared under conditions of various tannic acid concentrations (0.1 mg/mL and 0.5 mg/mL), respectively;
Figure 3e is a graph showing the phenol content in magnesium-containing nanoparticles prepared under conditions of various tannic acid concentrations (0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL);
4a to 4c are graphs showing SEM pictures, DLS analysis results, and phenol content of copper-containing nanoparticles prepared under various tannic acid concentration conditions (1 mg/mL, 5 mg/mL, and 10 mg/mL), respectively. ;
5a and 5b show SEM images and DLS analysis results of calcium-containing nanoparticles prepared at various phosphate anion concentrations (10 mM, 20 mM, and 30 mM) in simulated body fluids containing calcium cations and phosphate anions, respectively. is a drawing,
5c and 5d are graphs showing the calcium content and phenol content in calcium-containing nanoparticles prepared at various phosphate anion concentrations (10 mM, 20 mM, and 30 mM) in simulated body fluids containing calcium cations and phosphate anions, respectively. ego;
Figures 6a and 6b respectively show fluorescence micrographs of FITC-containing polyphenolic nanoparticles prepared in Example and confocal microscopy after treating human adipose derived stem cells (hADSC) with polyphenolic nanoparticles incorporated with FITC. is a photograph;
7A to 7C are graphs showing optical photographs, DLS analysis results, and zeta-potential analysis results of calcium-containing particles into which magnetic nanoparticles are introduced in a dry state and a state dispersed in ethanol, respectively;
8a and 8b are Live/Dead staining photographs showing the results of cytotoxicity evaluation after culturing hADSCs to which polyphenolic nanoparticles (0 μg/ml, 10 μg/ml, and 50 μg/ml) of various concentrations were introduced, respectively. and a diagram showing the results of MTT analysis;
9a to 9c are diagrams showing Fe conversion test results, DCF-DA analysis results, and comparative test results using a rat abdominal cavity inflammation induction model for polyphenolic nanoparticles, respectively;
10 is a graph showing the results of an expression test of bone differentiation-related genes (RUNX2, OCN) through an experiment in which hADSCs were cultured in a bone differentiation medium for 14 days;
11a to 11c are graphs showing optical and SEM images of nanoparticle-hydrogel composites, and mechanical property measurement results, respectively;
12a to 12c are graphs showing the results of Fe conversion test of nanoparticle-hydrogel composites, Live/Dead staining pictures, and expression results of inflammation-related genes (TNF-α, IL-6, iNOS), respectively;
13a to 13d are graphs showing the results of fluorescence immunostaining using an antibody specific for OPN, an osteogenic differentiation-related gene, for nanoparticle-hydrogel composites, respectively, and gene expression rates for osteogenic differentiation-related genes (OPN, RUNX2, OSX) A graph showing RT-PCR results, Live/Dead staining pictures, and TRACP analysis results for confirming; and
14a and 14b are graphs showing the results of measuring the regenerated bone volume (BV/TV) and regenerated area and optical photographs analyzing the bone regeneration effect in the mouse skull defect model for the nanoparticle-hydrogel composite, respectively. am.

The present invention can all be achieved by the following description. The following description should be understood as describing preferred embodiments of the present invention, but the present invention is not necessarily limited thereto. In addition, the accompanying drawings are for understanding, and the present invention is not limited thereto, and details of individual components can be properly understood by the specific purpose of the related description to be described later.

"Hydrogel" in a broad sense is a macromolecular compound having a cross-linked network and exhibiting a high water absorption capacity, wherein the 3-particles formed by cross-linked covalent bonds in the form of hydrogen bonds or ionic bonds and weak cohesive forces. It can be understood as having a three-dimensional polymer network, and in a narrow sense, it can mean a biopolymer-based hydrogel.

The term "fluorescence" may refer to a type of light emission produced for a short period of time by electromagnetic excitation. That is, fluorescence refers to a phenomenon generated when a specific material absorbs light energy at a shorter wavelength and then emits light energy at a longer wavelength. Illustratively, the length of time between absorption and release is typically relatively short, for example on the order of about 10 ⁻⁹ to 10 ⁻⁸ seconds.

"Magnetism" is a concept including ferromagnetism and superparamagnetism, and ferromagnetism may mean a material that exhibits magnetism even in the absence of an external magnetic field, and superparamagnetism may mean a property that exhibits strong magnetism only in the presence of a magnetic field.

“Nanoscale” may mean having a morphology of, for example, about 3,000 nm or less, specifically about 2,000 nm or less, and more specifically about 1,000 nm or less.

When a component is referred to as "include", it means that it may further include other components unless otherwise stated.

The expressions "on" and "above" are used to refer to the concept of relative position, not only when other components or layers directly exist in the mentioned layer, but also other layers (intermediate layers) or components in between. Elements may or may not be present. Similarly, the expressions “under”, “under” and “below” and “between” may also be understood as relative concepts of position.

go. Organic-inorganic complex of polyphenolic nanoparticles and hydrogel

Preparation of polyphenolic nanoparticles having a metal-phenol network

According to one embodiment of the present disclosure, in order to form polyphenol-based nanoparticles having a metal-phenol network, first, an aqueous solution in which metal cations and anions are dissolved in an aqueous medium (specifically, a mineral-based aqueous solution) and a polyphenol-based nanoparticle Each compound is provided.

According to an exemplary embodiment, a schematic process for preparing polyphenol-based nanoparticles in which a metal-phenol network is formed by a self-assembly method according to a combination of a polyphenol-based compound (tannic acid) and a mineral-based aqueous solution is shown in FIG. same.

Referring to the figure, ions in the aqueous solution combined with the polyphenolic compound may be selected from the range of cations and anions that are typically contained in or can be contained in body fluids of the human body. As an example, the aqueous solution may contain at least one cation of a Group 1 metal and a Group 2 metal on the periodic table. In this case, an example of the Group 1 metal cation may be sodium ion, potassium ion, etc., and an example of the Group 2 metal cation is It may be a magnesium ion, a calcium ion, a strontium ion, or the like. In this regard, according to certain embodiments, the aqueous solution contains Group 2 metal cations, and may optionally contain Group 1 metal cations. Further, according to an exemplary embodiment, the aqueous solution may optionally contain copper (Cu), manganese (Mn), iron (Fe), cobalt (Co), zinc (Zn), tin (Sn), It may further contain a cation of at least one metal selected from gold (Au), silver (Ag), aluminum (Al), and the like, and more specifically, it may be a copper cation (Cu ²⁺ ). However, monovalent cations in an aqueous solution provide a buffer function due to relatively weak binding force, while divalent cations may be mainly involved in mineralization through metal-phenol coordination bonds.

On the other hand, the anion in the aqueous solution may affect the buffering effect (buffering) and / or mineral formation, for example, chloride anion (Cl ^- ), phosphate anion (H ₂ PO ₄ ^- ), carbonate anion (HCO ₃ ^- ), sulfate anion (SO ₄ ^2- ), and the like.

As an example. The monovalent cation in the aqueous solution is at least about 800 mM (specifically about 900 to 3000 mM, more specifically about 1000 to 2000 mM), and the divalent cation is at least about 10 mM (specifically about 20 to 200 mM, more specifically about 1000 to 2000 mM). 33 to 100 mM), and the phosphate anion may be adjusted within a range of at least about 5 mM (specifically about 8 to 100 mM, more specifically about 10 to 30 mM).

According to certain embodiments, the aqueous solution contains about 800 to 3000 mM (specifically 900 to 2000 mM, more specifically 1000 to 1500 mM) Na ⁺ , about 30 mM or less (specifically 2 to 10 mM, more specifically 2 to 10 mM, more Specifically, K ⁺ of about 4 to 7 mM), Mg ²⁺ of about 200 mM or less (specifically about 1 to 100 mM, more specifically about 5 to 50 mM), and about 200 mM or less (specifically about 10 to 100 mM). mM, more specifically about 30 to 50 mM) of Ca ²⁺ . In addition, the concentration of metal cations (eg, Cu ²⁺ ) that can be additionally contained is, for example, up to about 200 mM, specifically about 20 to 100 mM, more specifically about 30 to 50 mM. can

On the other hand, as an anion in an aqueous solution, about 3000 mM or less (specifically 900 to 2000 mM, more specifically 1000 to 1500 mM) Cl ^- , about 5 to 50 mM (specifically about 7 to 30 mM, more specifically about 10 mM) to 20 mM) PO ₄ ^{3 -} , about 3000 mM or less (specifically about 20 to 2000 mM, more specifically about 30 to 1000 mM) HCO ₃ ^- , and about 50 mM or less (specifically about 7 to 30 mM , more specifically 10 to 20 mM) of SO ₄ ^2- .

Further, in an exemplary embodiment, the pH of the ion-containing aqueous solution is, for example, about 4 to 5, specifically about 4.2 to 4.5, more specifically about 4.3 to 4.4, particularly specifically about 4.32 to 4.37. Possibly, this is to form a supersaturated state by dissolving all ions in the solution. As such, it may contain at least one selected from buffer components such as Tris, HEPES, TAPS, and Bicine for pH control.

According to an exemplary embodiment, the aqueous solution containing metal cations and anions may be a simulated body fluid (SBF). In this regard, the simulated body fluid may refer to an artificial plasma solution prepared similarly to the ionic state in human blood plasma and maintained at a mild pH and the same physiological temperature. The composition of an exemplary simulated body fluid is shown in Table 1 below (unit: mM).

ingredient Original
SBF c-SBF Tas-
SBF Bigi-
SBF r-SBF m-SBF I-SBF n-SBF Na ⁺ 142.0 142.0 142.0 141.5 142.0 142.0 142.0 142.0 K ⁺ 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Mg ²⁺ 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Ca ²⁺ 2.5 2.5 2.5 2.5 2.5 2.5 1.6 2.5 Cl ^- 103.0 148.8 147.8 125.0 124.5 103.0 103.0 103.0 HCO ₃ 27.0 4.2 4.2 27.0 27.0 10.0 27.0 4.2 H ₂ PO ₄ ^- 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 SO ₄ ^2- 0.5 0 0.5 0.5 0.5 0.5 0.5 0.5 buffer Tris Tris Tris HEPES HEPES HEPES HEPES Tris

As described in the table above, the simulated body fluid basically has an ion concentration similar to that of human blood plasma, and if necessary, at least one ion (specifically, one or two ions) is excluded and/or its concentration is changed. Even in one case, it can be understood that it is included in the scope of simulated bodily fluid.

In the case of simulated body fluids, since the concentrations of metal cations and anions contained therein may be relatively low, mineralization between the polyphenol-based compound and metal cations may not be easy when used as such. Therefore, it may be required to use a concentration of at least about 2 times, specifically about 5 to 20 times, and more specifically about 8 to 15 times so as to satisfy the concentration range of the above-mentioned aqueous solution (i.e., supersaturated ion solution).

Further, according to exemplary embodiments, the molar ratio of Ca/P in the simulated body fluid may be adjusted in the range of, for example, about 1 to 5, specifically about 2 to 4, and more specifically about 3 to 3.5.

On the other hand, polyphenol-based compounds combined with the above-mentioned aqueous solution are epigallocatechin gallate, catechin, catechin-3-gallate, gallocatechin, gallo Catechin-3-gallate, Catechin gallate, Epicatechin, Gallocatechin gallate, Epigallocatechin, Gallic acid , It may be at least one selected from the group consisting of tannic acid and quercetin. According to a specific embodiment, tannic acid can be used as the polyphenolic compound, since it can rapidly form a complex with metal cations and form more metal-phenolic networks than other polyphenolic compounds. Therefore, it may be advantageous to control the size of the formed particles.

According to this embodiment, an aqueous solution containing metal cations and anions as described above and an aqueous solution in which polyphenol-based compounds are respectively prepared are combined with polyphenol-based compounds.

At this time, the concentration of the polyphenolic compound in the combination may be adjusted in the range of, for example, about 0 to 40 mg/ml, specifically about 0.5 to 30 mg/ml, and more specifically about 1 to 20 mg/ml, In particular, the molar ratio of all cations/polyphenolic compounds may be adjusted in the range of, for example, about 0.5 to 300, specifically about 1 to 200, and more specifically about 5 to 100.

In addition, the combination temperature is not particularly limited, but may be, for example, about 20 to 50 ° C, specifically about 30 to 40 ° C, more specifically 32 to 38 ° C, and may be set to a temperature similar to body temperature. In addition, the combining process may be performed over a range of, for example, about 10 to 100 minutes, specifically about 15 to 80 minutes, and more specifically about 20 to 60 minutes, but this may be understood as an example.

Meanwhile, a first base component may be added for mineralization, that is, metal-phenol network formation, together with or after the combination of the ion-containing aqueous solution and the polyphenolic compound. At this time, the first basic component may be a kind that can increase the pH in the solution, for example, it may be at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate and calcium bicarbonate, specifically bicarbonate may be sodium. In addition, the amount of the first basic component added is within the range of, for example, about 0.005 to 0.1 M, specifically about 0.01 to 0.08 M, more specifically about 0.02 to 0.05 M, the concentration of the first basic component in the combination. can be regulated.

Through the above-described combination process, coordination bonds between the polyphenolic compound and metal cations in the aqueous solution occur, and a self-assembly reaction is induced to form a metal-phenol network, thereby forming nanoscale particles, that is, polyphenolic nanoparticles. is created As an example, the size (DLS) of the polyphenol-based nanoparticles may be, for example, about 100 to 3000 nm, specifically about 200 to 2000 nm, and more specifically about 250 to 1000 nm.

Meanwhile, as an optional step, a second base component may be added to stabilize the formed nanoparticles. To this end, a type capable of increasing the pH may be used as the second basic component, for example, at least one selected from the group consisting of sodium hydroxide, potassium hydroxide, and calcium hydroxide, and more specifically, sodium hydroxide may be used. can The amount of addition or treatment of the second base component is in the range of, for example, about 1 to 20 mM, specifically about 5 to 15 mM, more specifically about 7 to 12 mM, in the nanoparticle-containing medium (aqueous medium). can be adjusted so that In addition, stirring may be performed together with the stabilization step or before or after the stabilization step in order to suppress aggregation of the nanoparticles.

Meanwhile, according to an exemplary embodiment, a post-treatment process of separating and purifying nanoparticles from a medium in which polyphenolic nanoparticles are formed may be performed. As an example, after solid content is obtained by solid-liquid separation (centrifugation, filtering, magnetic separation, etc.), it may be washed with water (specifically, distilled water) at least once, and then subjected to a drying process. At this time, as the drying step, hot air drying, reduced pressure drying, freeze drying, etc. may be exemplified, and more specifically, a freeze drying method may be used. As the aforementioned separation and purification steps are well known in the art, detailed descriptions thereof are omitted.

According to exemplary embodiments, the content of polyphenol in the polyphenol-based nanoparticles is, for example, about 5 to 90% by weight, specifically about 10 to 80% by weight, more specifically about 20 to 70% by weight, particularly specifically It may range from about 30 to 50% by weight. In this regard, it may be advantageous to control the content of polyphenol in the nanoparticle within the aforementioned range as it affects the efficacy of the nanoparticle. In addition, the nanoparticles produced may exhibit the properties of spherical or irregular nanoparticles.

The advantages of the nanoparticle manufacturing method according to the present embodiment are largely (i) that metal-phenol network nanoparticles can be synthesized in a one-pot manner, and (ii) that they are combined with polyphenolic compounds. Through a simple manipulation of adjusting the concentration of metal cations and/or anions in the aqueous solution, it is possible to form an optimal regeneration environment for a composition optimized for subsequent use, specifically for bone regeneration use.

Preparation of polyphenolic nanoparticles and hydrogel composites

According to one embodiment of the present disclosure, polyphenol-based nanoparticles having a metal-phenol network can be combined with a hydrogel to prepare an organic-inorganic composite having bone regeneration ability, which is used to treat bone defects. It can be applied as a graft material for bone regeneration, a dental filling material, and a support for bone regeneration.

According to an exemplary embodiment, a solution in which a biopolymer-based hydrogel is dissolved in a solvent is prepared. In this regard, the biopolymer may be a water-soluble polymer, and specifically, at least one selected from the group consisting of gelatin, guar gum, xanthan gum, acacia gum, carrageenan gum, pectin, alginate, collagen, hyaluronic acid, chitosan, and agarose. It may be one, and more specifically, gelatin may be used. At this time, gelatin is particularly suitable because it is highly biodegradable, biocompatible, and has properties that facilitate cell attachment due to the presence of RGD peptides. In addition, the solvent may be at least one selected from water, PBS, DPBS, physiological saline, Tris buffer, and the like. Aqueous solvents with similar pH and density properties to bodily fluids may be advantageous. However, it is not limited to the types exemplified above. However, considering biocompatibility, water (specifically, distilled water) may be used.

According to exemplary embodiments, the concentration of the hydrogel solution may be adjusted in the range of, for example, about 5 to 30% by weight, specifically about 10 to 20% by weight, and more specifically about 12 to 17% by weight. If the content of the hydrogel is too high or low, it may be advantageous to control it within the above range because problems such as a decrease in the dispersion of nanoparticles, difficulty in shape control, and difficulty in controlling decomposition and physical properties may occur. However, the concentration range may be changed depending on the type of biopolymer used.

Then, the hydrogel solution and the previously prepared polyphenol-based nanoparticles are combined. In this regard, the concentration of the polyphenolic nanoparticles in the combined solution ranges, for example, from about 0.1 to 10 mg/ml, specifically from about 0.2 to 5 mg/ml, and more specifically from about 0.5 to 2 mg/ml. It can be adjusted in the bar, it can be determined in consideration of the content of the polyphenol-based nanoparticles in the final composite. However, this may be understood as an exemplary purpose.

After passing through the above steps, a process of preparing a crosslinked hydrogel may be performed. In this case, the crosslinking agent may use a kind having properties capable of crosslinking between amine groups. Illustratively, the crosslinking agent may be at least one selected from glyoxal, glutaraldehyde, formaldehyde, and genipin, and more specifically, glutaraldehyde may be used. In this case, the crosslinking agent may be used in an amount of, for example, about 0.01 to 1% by weight, specifically about 0.05 to 0.5% by weight, and more specifically about 0.1 to 0.3% by weight, based on the biopolymer in the hydrogel solution. According to an exemplary embodiment, the crosslinking agent may be introduced into the polyphenol-based nanoparticle-containing hydrogel solution in a form dissolved in a solvent rather than used alone. At this time, the concentration of the crosslinking agent solution may be, for example, about 0.2 to 1% by weight, specifically about 0.4 to 0.8% by weight, more specifically about 0.5 to 0.7% by weight, but this may be understood as an example. .

After performing the step of combining the nanoparticles and the hydrogel solution described above, a composite may be prepared by cooling. To this end, a rapid cooling method may be adopted, wherein the cooling temperature is, for example, about -10 ° C or less, specifically about -30 to -12 ° C, more specifically -20 to -15 ° C. In this case, it may be advantageous to have porosity inside the hydrogel by water crystallization at a cooling temperature.

According to exemplary embodiments, the content of the polyphenolic nanoparticles in the composite may be, for example, about 3 to 50% by weight, specifically about 10 to 40% by weight, and more specifically about 20 to 30% by weight. there is. At this time, if the content of the polyphenolic nanoparticles is too small or too high, phenomena such as incomplete crosslinking of biopolymers (eg, gelatin), precipitation of nanoparticles, and uneven distribution are induced, so control within the above range. It may be advantageous to do so, but this can be understood for illustrative purposes.

In addition, as in the present embodiment, as the nanoparticles are introduced into the hydrogel, superior mechanical properties can be exhibited compared to the case of using the hydrogel alone. As an example, the storage modulus measured by a rheometer may be at least about 10 kPa, specifically at least about 11 kPa, and more specifically in the range of about 11.4 to 15 kPa, compared to hydrogel alone, For example, the level may be increased by at least about 15%, specifically about 10 to 17%.

In addition, the nanoparticle-hydrogel composite can exhibit an antioxidant effect resulting from the metal-phenol network, and as a result of performing a Fe conversion test measured by a microplate reader, for example, about 40 to 48 hours. 80%, specifically about 50 to 70%, more specifically about 55 to 65% of the Fe conversion rate may be exhibited. In addition, the nanoparticles can exhibit good active oxygen scavenging ability, and when measured by the active oxygen measurement method (DCF-DA assay), the fluorescence signal is, for example, at least about 20%, specifically at least about 30%, more specifically A reduction of about 35 to 50% may be obtained.

Regarding the present embodiment, it is noteworthy that the nanoparticle-hydrogel composite can provide a superior bone regeneration effect compared to conventional materials. In particular, nanoparticles with characteristics optimized for bone regeneration can be prepared through the control of metal ions in an aqueous solution that is combined with a polyphenolic compound during nanoparticle production, and a composite in which the hydrogel is combined can produce high Combined with properties such as cell adhesion, biodegradability, biocompatibility, and porosity, the bone regeneration effect can be maximized.

me. Fluorescent material-containing polyphenolic nanoparticles

According to another embodiment of the present disclosure, a plurality of effects resulting from the metal-phenol network (eg, antioxidant, anti-inflammatory effect, bone differentiation effect, etc. ) and provides multifunctional nanoparticles that can be applied for imaging applications.

In the case of this specific embodiment, a method of adding a step of incorporating a fluorescent material during the manufacturing process of polyphenol-based nanoparticles having a metal-phenol network may be adopted. Specifically, a composite aqueous solution in which a fluorescent substance is added to an aqueous solution containing metal cations and anions can be prepared, and then combined with a polyphenolic compound as in the procedure described above.

According to an exemplary embodiment, the fluorescent material may be added in a form dissolved in a solvent, and the solvent may be, for example, at least one selected from ethanol, methanol, DMSO, DMF, acetone, chloroform, and the like. As an example, the fluorescent material may include, for example, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, TAMRA, Dichlorotriazinylamine fluorescein, dansyl chloride, quantum dots, phycoerythrin, FAM (fluorecein amidite), alexa fluor, Cy3, Cy5 , Cy7 and may be at least one selected from the group consisting of indocyanine green, and more specifically may be fluorescein isothiocyanate (FITC).

In an exemplary embodiment, the concentration of the fluorescent substance in the ion-containing aqueous solution is, for example, about 1 to 30 μg/ml, specifically about 3 to 25 μg/ml, and more specifically about 5 to 20 μg/ml. It can be adjusted within the range, but this can be understood as an example. However, if the concentration of the fluorescent substance in the ion-containing aqueous solution is too low, it may be difficult to observe the intended fluorescence, whereas if it is too high, particle formation may be hindered, it may be advantageous to properly adjust within the above range. .

all. magnetic composite particles

According to another embodiment of the present disclosure, a plurality of effects resulting from the metal-phenol network (eg, antioxidant, anti-inflammatory effect, It can be applied to fields such as biosensing and imaging by giving magnetism along with bone differentiation effect, etc.). In this way, when magnetism is applied, advantages such as easy separation of nanoparticles by external magnetic force can be achieved.

According to an exemplary embodiment, magnetic nanoparticles may typically be iron oxide particles. Iron oxide is largely known as eight types according to its oxidation state. For this embodiment, iron oxides exhibiting magnetism (ferromagnetic or superparamagnetic), typically hematite (α-Fe ₂ O ₃ ), maghemite (γ-Fe ₂ O ₃ ) and magnetite (magnetite, Fe ₃ O ₄ ) can be used. In the case of magnetite and maghemite crystal forms, they are iron ore crystal forms with strong magnetism and are advantageous for recovery using magnetism after the use of magnetic dendrimers.

According to an exemplary embodiment, the iron oxide particles may optionally further include at least one from manganese (Mn), cobalt (Co), nickel (Ni), zinc (Zn), and gadolinium (Gd). Double magnetite (Fe ₃ O ₄ ) is iron oxide that can be used in various fields such as pigment, MRI, and drug delivery. For the synthesis of iron oxide, specifically magnetite (Fe ₃ O ₄ ) particles, it is known in the art, for example, a co-precipitation method, a thermal decomposition method, a sol-gel method, a combustion method, a hydrothermal synthesis method, a synthesis using microwaves, a ball Milling, mechano-chemical processes, thermal decomposition of organometallic compounds, and the like can be used. According to exemplary embodiments, the shape of the iron oxide particles may be, for example, spherical, rod-shaped, cubic-shaped, hexagonal-shaped, triangular-shaped, hollow-shaped, flower-like structure-shaped, etc. , stability of surface energy, and surface area, it may be advantageous to have a spherical shape. However, this may be understood as an exemplary meaning.

According to an exemplary embodiment, the iron oxide, specifically magnetite (Fe ₃ O ₄ ) particles, are typically nanoscale particles, for example about 50 nm or less, specifically about 30 nm or less, more specifically about 5 to 50 nm. It can have a size (diameter) range in the 25 nm range, and can also exhibit superparamagnetism.

According to an exemplary embodiment, magnetic nanoparticles may be introduced during the manufacturing process of polyphenolic nanoparticles (ie, nanoparticles having a metal-phenol network). Specifically, an aqueous dispersion in which magnetic nanoparticles are added to an aqueous solution containing metal cations and anions may be prepared and then combined with a polyphenol-based compound. At this time, ultrasonic irradiation (or pulverization) treatment may be performed in parallel so that the magnetic nanoparticles can be uniformly dispersed in the aqueous medium.

In addition, the concentration of the magnetic nanoparticles in the aqueous dispersion may be adjusted in the range of, for example, about 0.05 to 1 mg/ml, specifically about 0.1 to 0.8 mg/ml, and more specifically about 0.2 to 0.5 mg/ml. However, this can be understood for illustrative purposes. However, if the concentration of the magnetic nanoparticles is less than a certain level, it may be difficult to expect a desired effect of imparting magnetism. However, when added in an excessive amount, it may be advantageous to adjust the concentration within the above-mentioned range as it may interfere with particle generation.

On the other hand, according to an exemplary embodiment, the size (diameter) of the finally prepared magnetic composite nanoparticles is, for example, about 100 to 3000 nm, specifically about 200 to 2000 nm, more specifically about 400 to 1000 nm range can be adjusted.

In an exemplary embodiment, the magnetic composite particle exhibits a negative (-) zeta-potential through the negatively charged characteristic of tannic acid, thereby suppressing inter-particle aggregation, for example, about -10 mV or less, Specifically, it may be about -15 mV or less, more specifically, about -30 to -20 mV, and it may be advantageous to adjust the content of tannic acid in the magnetic composite particles in consideration of this zeta-potential range.

The present invention can be more clearly understood by the following examples, which are only for illustrative purposes of the present invention and are not intended to limit the scope of the present invention.

Materials and equipment used in this example are as follows.

- matter

Magnesium chloride, sodium hydroxide, sodium chloride, and glutaraldehyde were obtained from Junsei.

Calcium chloride was obtained from DUKSAN, potassium chloride, potassium bromide, disodium phosphate, sodium bicarbonate, hydrogen peroxide (3%) and ferric chloride hexahydrate, pig skin-derived gelatin type A, tannic acid, tetrazolium bromide (MTT), fluoride Fluorescein isothiocyanate (FITC) and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich.

Adipose-derived stem cells (hADSC), Alexa Fluor ^TM 488 Phalloidin, were obtained from Invitrogen, and RAW 264.7 cells were obtained from Korea Cell Line Bank.

All reagents and chemicals used in this example were used as received without separate purification.

- Analytical equipment

Scanning electron microscope (FE SEM; JSM 7600F, JEOL, Tokyo, Japan)

Microplate reader (Varioskan LUX, Thermo Scientific, Waltham, MA, USA).

Supersonic crusher (BRANSON, St.Louis, USA)

Fourier transform infrared spectroscopy (FT-IR; Nicolet 6700, Thermo Fisher Scientific, Waltham, UK).

Rheometer (Discovery HR10, TA instruments, New Castle, DE, USA).

Confocal microscope (TCS SP5; Leica biosystems, Wetzlar, Germany)

Fluorescence microscopy (TE 2000; Nikon, Tokyo, Japan).

Energy dispersive X-ray spectrometer (EDS, JEM-ARM200F(HRP), JOEL, Tokyo, Japan)

Example 1

go. Preparation of polyphenolic nanoparticles using simulated body fluids with various ionic compositions

After dissolving tannic acid (TA) in concentrated simulated body fluid (10X SBF), self-assembly of nanoparticles was induced by treatment with 0.04 M NaHCO ₃ . At this time, as shown in Table 2 below, polyphenol-based nanoparticles were prepared by changing the composition of ions (Ca ²⁺ , Mg ²⁺ , Cu ²⁺ , and PO ₄ ^3- ) in the concentrated simulated body fluid.

Concentrated SBF Concentration (mM) Tannic Acid (TA) (mg/ml) Na ⁺ Ca ²⁺ Mg ²⁺ K ⁺ PO ₄ ^3- Cu ²⁺ One 1,000 33 5 5 10 0 1, 5, 10 2 1,000 0 38 5 10 0 0.1, 0.25, 0.5 3 1,000 0 5 5 10 33 1, 5, 10 4 1,000 33 5 5 10 0 One 5 1,000 33 5 5 20 0 One 6 1,000 33 5 5 30 0 One

Then, the mixture was stirred at room temperature for 10 minutes, and further stirred for 10 minutes after treatment with NaOH to stabilize the nanoparticles. Subsequently, centrifugation was performed at a speed of 4,000 rpm for 5 minutes using a high-speed centrifuge, washed several times using double distilled water, and then freeze-dried and stored in a vacuum state.

The phenol content of the obtained nanoparticles was measured through Folin-ciocalteu assay, and the structure of the nanoparticles was confirmed and the size was measured through SEM analysis and dynamic light scattering (DLS) analysis, respectively.

- Cytotoxicity evaluation of polyphenolic nanoparticles

In a culture dish to which 10,000 human adipose derived stem cells (hADSC) were added, polyphenol nanoparticles were diluted to 0 μg/ml, 10 μg/ml, and 50 μg/ml using Transwell, and then treated. , cultured and evaluated the cytotoxicity of the nanoparticles through Live/dead staining and MTT assay.

- Functional evaluation of polyphenolic nanoparticles

In order to confirm the antioxidant effect of the polyphenolic nanoparticles themselves, an Fe conversion test was performed. In addition, to confirm the antioxidant effect on cells, hADSCs were treated with 200 μM of H ₂ O ₂ and incubated with the nanoparticles for 24 hours. After that, the active oxygen removal effect of the polyphenolic nanoparticles in the cell medium was confirmed using the active oxygen measurement method (DCF-DA assay).

In order to confirm the anti-inflammatory effect of the polyphenolic nanoparticles, after inducing peritonitis using zymosan in the abdominal cavity of rats, the polyphenolic nanoparticles were diluted in distilled water at a concentration of 80 μg/ml and injected into the abdominal cavity. After 5 hours, intraperitoneal body fluid was collected and the content of inflammatory cytokines (TNF-α, IL-6) was confirmed by ELISA.

In order to confirm the osteogenic differentiation effect of polyphenolic nanoparticles, hADSCs were treated with calcium-containing polyphenolic nanoparticles (Ca-TP) at 2 μg per 10,000 cells for 14 days (osteogenic differentiation medium). medium, ODM). In the group for confirming bone differentiation under reactive oxygen conditions, treatment was performed with 200 μM of H ₂ O ₂ . The expression of bone differentiation-related genes (RUNX2, OCN) in each condition was confirmed through RT-PCR (Real time-polymerase chain reaction).

me. Preparation of polyphenolic nanoparticle-hydrogel composite

A nanoparticle-gelatin-based hydrogel composite was prepared using the previously prepared polyphenol-based nanoparticles. Specifically, after dissolving gelatin in secondary distilled water at a concentration of 15%, the concentrations of the polyphenolic nanoparticles prepared in Example 1 were 0 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1 mg/ml. A nanoparticle-gelatin solution was prepared to be ml. Subsequently, glutaraldehyde was diluted with double distilled water to a concentration of 0.5% to form a crosslinked hydrogel, which was mixed with the nanoparticle-gelatin solution 4:1 by volume and maintained at -18 °C for 24 hours. A polyphenol-based nanoparticle-hydrogel composite was prepared by freezing for a while.

The inside of the nanoparticle-hydrogel composite was observed through SEM, and the mechanical properties of the nanoparticle-hydrogel composite were measured using a rheometer.

- Evaluation of functionality of nanoparticle-hydrogel composites

In order to confirm the antioxidant effect of the nanoparticle-hydrogel composite itself, an Fe conversion test was performed. Specifically, each of the hydrogel and nanoparticle-hydrogel composite was aliquoted into hADSC, treated with 400 μM H ₂ O ₂ , cultured for 24 hours, and cell viability was confirmed through Live/Dead staining.

In order to confirm the anti-inflammatory effect of the nanoparticle-hydrogel composite, RAW 264.7 cells were seeded on the surface and treated with LPS (Lipopolysaccharide) to induce an inflammatory response, and genes related to the inflammatory response (TNF-α, IL-6, iNOS) was confirmed by RT-PCR.

- Effect of nanoparticle-hydrogel composite on bone regeneration environment

In order to confirm the osteogenic differentiation effect, after dispensing hADSC into nanoparticle-hydrogel complexes and culturing them in an osteogenic differentiation medium for 14 days, fluorescence staining (immunohistochemistry, IHC) was performed using an antibody specific for OPN, an osteogenic differentiation-related gene. performed. In addition, the expression level of bone differentiation related genes (OPN, RUNX2, OSX) was confirmed by RT-PCR.

In order to confirm the effect of inhibiting differentiation into osteoclasts, RAW 264.7 cells were seeded into nanoparticle-hydrogel complexes and treated with RANKL protein, which induces differentiation into osteoclasts, at a concentration of 100 ng/ml and cultured for 7 days. Then, TRACP assay was performed to confirm the degree of differentiation and activity into osteoclasts. At this time, the ratio of mature osteoclasts was visually confirmed through Live/Dead staining.

Example 2

Preparation of fluorescent material-containing polyphenolic nanoparticles

Fluorescein isothiocyanate (FITC) as a fluorescent substance was completely dissolved in DMSO, and then diluted to a concentration of 15 μg/ml in 10X SBF (concentrated SBF1 in Table 2). Then, it was treated with 10 mg/ml of tannic acid and 0.04 M of NaHCO ₃ , and reacted at room temperature for 10 minutes to prepare FITC-containing polyphenolic mineral nanoparticles.

Thereafter, it was treated with NaOH, stirred for 10 minutes, centrifuged using a high-speed centrifuge, and subsequently washed with double distilled water and then lyophilized and stored.

Fluorescence signals of the obtained nanoparticles were observed under a fluorescence microscope, and FITC signals in the cells were observed through confocal images by treating the cells with the nanoparticles.

Example 3

Preparation of magnetic composite particles

Magnetic nanoparticles (material: Fe ₃ O ₄ .; size: 20 nm) were dispersed in 10X SBF (concentrated SBF1 in Table 2) at a concentration of 0.3 mg/ml, where an ultrasonicator was used for good dispersion. . Then, after dissolving tannic acid at 10 mg/ml, the mixture was treated with 0.01 M NaHCO ₃ and stirred for 1 hour to form magnetic composite particles.

The size and surface charge of the magnetic composite particles were confirmed through DLS analysis and zeta-potential measurement of the obtained magnetic composite particles. In addition, in order to confirm the magnetism of the obtained magnetic composite particles, the interaction with the magnet was observed in each of the powder form and the form dispersed in ethanol.

Results and discussion

go. Analysis of properties of polyphenolic nanoparticles according to compositional changes of ions in simulated body fluids

- calcium ion

Optical images, SEM images, and DLS analysis results of the calcium-containing nanoparticles prepared under various tannic acid concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL) are shown in FIGS. 2A to 2C, respectively. Referring to the figure, the prepared nanoparticles decreased in proportion to the tannic acid concentration in the size (diameter) range of about 400 to 2000 nm.

Meanwhile, phenol content and calcium content in calcium-containing nanoparticles according to various tannic acid concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL) are shown in FIGS. 2D and 2E, respectively. According to FIG. 2d, through the folin-ciocalteu assay, it was confirmed that the phenol content in the nanoparticles increased as the tannic acid concentration increased, but the phenol content was constant when the tannic acid concentration was 5 mg/ml or more. In addition, referring to FIG. 2e, as a result of Ca assay, the calcium content in the nanoparticles decreased as the concentration of tannic acid increased, and in particular, the calcium content was constant when the concentration of tannic acid was 5 mg/ml or more.

The results of FT-IR analysis of calcium-containing nanoparticles prepared under conditions of various tannic acid concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL) are shown in FIG. 2f. According to the figure, characteristic peaks for PO ₄ ^3- bond, C=O bond, and -OH bond were observed in the sample, indicating that tannic acid and minerals are contained in the nanoparticles.

- Magnesium ion

SEM images and DLS analysis results of magnesium-containing nanoparticles prepared under conditions of various tannic acid concentrations (0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL) are shown in FIGS. 3a and 3b, respectively. Referring to the figure, magnesium-containing polyphenolic nanoparticles (Mg-TP) prepared under various tannic acid concentration conditions have a size (diameter) of about 200 to 4000 nm.

In addition, TEM images and EDS analysis results of magnesium-containing nanoparticles (Mg-TP) prepared under various tannic acid concentrations (0.1 mg/mL and 0.5 mg/mL) are shown in FIGS. 3c and 3d, respectively. According to the figure, it was formed as spherical particles, and it was confirmed that oxygen, magnesium, and phosphorus were contained in the Mg-TP particles.

The phenol content in magnesium-containing nanoparticles (Mg-TP) prepared under various tannic acid concentration conditions (0.1 mg/mL, 0.25 mg/mL and 0.5 mg/mL) is shown in FIG. 3e, as the tannic acid concentration increases Accordingly, it was confirmed that the phenol content in the Mg-TP particles also increased (folin-ciocalteu assay).

- copper ion

SEM images, DLS analysis results, and phenol content of the copper-containing nanoparticles prepared under various tannic acid concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL) are shown in FIGS. 4a to 4c, respectively.

Referring to the figure, the copper-containing nanoparticles (Cu-TP) prepared under various tannic acid concentration conditions increased in proportion to the tannic acid concentration in the size (diameter) range of about 300 to 2000 nm (FIGS. 4a and 4b) , it was confirmed that the phenol content in Cu-TP particles increased in proportion to the concentration of tannic acid when measured by the folin-ciocaltue assay (Fig. 4c).

- phosphate ion

SEM images and DLS analysis results of calcium-containing nanoparticles prepared under conditions of various phosphate anion concentrations (10 mM, 20 mM, and 30 mM) in simulated body fluids containing calcium cations and phosphate anions are shown in FIGS. 5A and 5B, respectively. , and the calcium content and phenol content in the calcium-containing nanoparticles are shown in FIGS. 5c and 5d, respectively.

Referring to the figure, as the concentration of phosphate anion in the simulated body fluid increased, it was confirmed that nanoparticles of a structurally non-uniform shape were formed, and their size increased (FIGS. 5a and 5b), and also Ca-TP particles While the calcium content within it increased, the phenol content showed a tendency to decrease (FIGS. 5c and 5d).

me. Characteristic analysis of fluorescent material-containing polyphenolic nanoparticles

Visual observation and fluorescence micrographs of the FITC-containing polyphenolic nanoparticles prepared in Example 2, and confocal micrographs after 24 hours of treatment of hADSCs with the FITC-containing polyphenolic nanoparticles are shown in FIGS. 6A and 6A. 6b, respectively. Referring to FIG. 6A, the FITC-containing nanoparticles were yellow when observed with the naked eye and exhibited green fluorescence under a fluorescence microscope. On the other hand, according to FIG. this has been manifested

all. Characteristic analysis of magnetic composite particles

Optical photographs, DLS analysis results, and zeta-potential analysis results of calcium-containing nanoparticles incorporated with magnetic nanoparticles (MNP) in a dry state and in a state dispersed in ethanol are shown in FIGS. 7A to 7C , respectively. In this regard, in the case of Ca-TP particles incorporated with magnetic nanoparticles, both the dry state and the ethanol dispersion reacted to a change in the external magnetic field, showing magnetic properties (FIG. 7a). In addition, as a result of DLS analysis of the Ca-TP particles incorporated with the magnetic nanoparticles, the size (diameter) was approximately 459 nm (FIG. 7b), and the zeta-potential of the surface at this time was about -23.33 mV (FIG. 7c).

la. Cytotoxicity evaluation of polyphenolic nanoparticles

Live/Dead staining photographs and MTT assay results showing the cytotoxicity evaluation results after culturing hADSCs introduced with various concentrations of polyphenolic nanoparticles (0 μg/ml, 10 μg/ml, and 50 μg/ml) are shown in FIG. 8A. and Fig. 8b, respectively. Referring to the figure, hADSC cultured with polyphenol-based nanoparticles at various concentrations showed little apoptosis signal after 24 hours, and there was no substantial difference in cell activity.

mind. Evaluation of multifunctionality of polyphenolic nanoparticles

- Antioxidant/anti-inflammatory effect

The Fe conversion test results for the polyphenolic nanoparticles, the DCF-DA analysis results, and the comparative test results using the rat abdominal cavity inflammation induction model are shown in FIGS. 9a to 9c, respectively. As a result of the analysis, the antioxidant effect of the polyphenolic nanoparticles increased in proportion to the concentration of the nanoparticles (FIG. 9a), and hADSCs exposed to H ₂ O ₂ for 24 hours showed active oxygen signals only when Ca-TP particles were present. did not appear (Fig. 9b). In addition, compared to the group treated only with zymogen (positive control; PC), the secretion of intraperitoneal inflammatory cytokines (TNF-α, IL-6) was significantly reduced in the group treated with TA and Ca-TP. (FIG. 9c).

- Bone differentiation effect

10 shows the results of the expression test of bone differentiation-related genes (RUNX2, OCN) through an experiment in which hADSCs were cultured in a bone differentiation medium for 14 days. Referring to the figure, the expression of bone differentiation-related genes (RUNX2, OCN) significantly increased in the group treated with Ca-TP particles, and the expression of bone differentiation markers even under active oxygen (H ₂ O ₂ ) treatment conditions. It was confirmed that the decrease was alleviated to some extent.

bar. Analysis of properties and functions of nanoparticle-hydrogel composites

- Confirmation of formation of nanoparticle-hydrogel composite and evaluation of mechanical properties

Optical and SEM images of the nanoparticle-hydrogel composite, and mechanical property measurement results are shown in FIGS. 11A to 11C , respectively.

Hydrogel composites were prepared with gelatin while varying the concentration of polyphenolic nanoparticles (0 mg/ml, 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml) (FIG. 11a), and nanoparticles in the composite were also prepared. It was confirmed that the particles were successfully introduced (FIG. 11b). In addition, according to FIG. 11c, as a result of measuring the mechanical properties of the composite using a rheometer, the storage modulus was 10.31 ± 0.2961 kPa when the nanoparticles were not contained, whereas the composite having a concentration of 1 mg / ml nanoparticles In the case of , the storage modulus was 11.46±0.7149 kPa.

- Evaluation of antioxidant and anti-inflammatory effects

As a result of the Fe conversion test of the nanoparticle-hydrogel composite, Live/Dead staining pictures and expression results of inflammation-related genes (TNF-α, IL-6, and iNOS) are shown in FIGS. 12A to 12C , respectively.

Referring to FIG. 12a, the antioxidant effect of the nanoparticle-hydrogel composite lasted up to 48 hours (Fe conversion rate of 56.583±3.887% based on 48 hours). In addition, according to FIG. 12b, after culturing cells on the surface of the nanoparticle-hydrogel composite, treatment with 400 μM H ₂ O ₂ and treatment for 24 hours, there was no significant difference in the Ca-TP nanoparticle-containing group. , which showed a high cell viability (99.31±0.5559%), whereas the control group showed a cell viability of 33.78±5.222%.

In addition, according to FIG. 12c, as a result of inducing an inflammatory response by dispensing RAW 264.7 cells on the surface of the nanoparticle-hydrogel composite and treating with Lipopolysaccharide (LPS) at a concentration of 1 μg/ml, Ca-TP particles containing In the group, the expression of inflammation-related genes (TNF-α, IL-6, iNOS) was decreased, and gene expression was found to be similar to that of the group that did not induce an inflammatory response.

- Evaluation of the effect of adjusting the bone regeneration environment

A graph showing the results of fluorescence immunostaining using an antibody specific for OPN, a gene related to bone differentiation, for nanoparticle-hydrogel composites, RT-PCR to confirm the gene expression rate for genes related to bone differentiation (OPN, RUNX2, OSX) Photos showing the results (cultivation for 14 days), Live/Dead staining photos, and TRACP analysis results are shown in FIGS. 13A to 13D , respectively.

According to FIG. 13a, hADSC were divided and cultured in an osteogenic differentiation medium for 14 days, and fluorescence immunostaining was performed using an antibody specific for OPN, a gene related to osteogenic differentiation. As a result, the OPN expression rate was 30.84±3.075% in the control group, whereas The nanoparticle-hydrogel composite showed an expression rate of 68.72±3.295%, indicating that the nanoparticle-hydrogel composite exhibited an effect of improving bone differentiation. In addition, FIG. 13B shows a significant increase in the expression of genes related to bone differentiation in the nanoparticle-hydrogel composite.

As a result of the test to confirm the effect of inhibiting differentiation into osteoclasts, the control group showed a multinucleated cell structure, which is a specific type of osteoclast, whereas in the nanoparticle-hydrogel complex group, cells at a similar level to cells grown in a general medium were obtained. structure was shown (Fig. 13c).

On the other hand, the TRACP assay was performed by dividing RAW 264.7 cells into the nanoparticle-hydrogel complex and culturing them in an osteoclast differentiation medium containing RANKL protein for 7 days. shown (Fig. 13d).

buy. Evaluation of the bone regeneration effect of nanoparticle-hydrogel composites

The nanoparticle-hydrogel composites are shown in Figs. 14a and 14b, respectively, for the optical photographs analyzing the bone regeneration efficacy in the rat skull defect model, and for measuring the regenerated bone volume (BV/TV) and regeneration area. .

Referring to FIG. 14a , the in-vivo bone regeneration effect of the biomaterial was confirmed by implanting the nanoparticle-hydrogel composite into the skull defect of mice. In particular, a group with only bone defects (Defect), a group implanted with a hydrogel containing no nanoparticles (Gel), and a group implanted with a calcium-containing nanoparticle-hydrogel complex (Ca-TP gel), respectively. After 8 weeks, the rat's skull was removed and the degree of regeneration was confirmed by microtomography. As a result, the Ca-TP gel group showed a remarkably high degree of regeneration.

In addition, according to FIG. 14b, when the regenerated bone volume (BV/TV) and regenerated area were quantified, the Ca-TP gel group showed a high bone regeneration effect, from which the calcium-containing nanoparticle-hydrogel composite It was confirmed that it exhibits high bone regeneration efficiency in vivo.

Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (18)

a) (i) a polyphenolic compound and (ii) combining an aqueous solution in which metal cations and anions are dissolved in an aqueous medium and adding a first base component to form polyphenolic nanoparticles having a metal-phenolic network; and
b) forming a nanoparticle-hydrogel composite by combining a biopolymer-based hydrogel solution and the nanoparticles;
Method for producing an organic-inorganic composite comprising a. The method of claim 1, further comprising the step of adding a second basic component to the aqueous solution containing the nanoparticles to stabilize the nanoparticles prepared in step a) prior to step b). - Manufacturing method of inorganic composite. The method of claim 1, wherein the aqueous solution contains at least 800 mM monovalent cation, at least 10 mM divalent cation, and at least 5 mM phosphate anion. The method of claim 3, wherein the aqueous solution is copper (Cu), manganese (Mn), iron (Fe), cobalt (Co), zinc (Zn), tin (Sn), gold (Au), silver (Ag), and A method for producing an organic-inorganic composite, characterized in that it further comprises up to 200 mM of cations of at least one metal selected from the group consisting of aluminum (Al). The method of claim 3 or 4, wherein the aqueous solution is a simulated body fluid or a concentrated simulated body fluid. The method of claim 1, wherein the polyphenolic compound is epigallocatechin gallate, catechin, catechin-3-gallate, gallocatechin, gallocatechin -3-gallate, catechin gallate, epicatechin, gallocatechin gallate, epigallocatechin, gallic acid, A method for producing an organic-inorganic complex, characterized in that at least one selected from the group consisting of tannic acid and quercetin. The method of claim 1, wherein the biopolymer is at least one selected from the group consisting of gelatin, guar gum, xanthan gum, acacia gum, carrageenan gum, pectin, alginate, collagen, hyaluronic acid, chitosan, and agarose. A method for producing an organic-inorganic composite. 3. The method of claim 2, wherein the first basic component is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate and calcium bicarbonate, and
The method for producing an organic-inorganic complex, characterized in that the second basic component is at least one selected from the group consisting of sodium hydroxide, potassium hydroxide and calcium hydroxide. The method of claim 1, wherein the polyphenol content in the polyphenol-based nanoparticles ranges from 5 to 90% by weight. The method of claim 1, wherein the size (DLS) of the polyphenol-based nanoparticles ranges from 100 to 3000 nm. preparing a composite aqueous solution by combining a phosphor and an aqueous solution in which metal cations and anions are dissolved in an aqueous medium; and
combining the complex aqueous solution and the polyphenol-based compound and adding a first basic component to form fluorescent material-containing polyphenol-based nanoparticles having a metal-phenol network;
Method for producing fluorescent composite particles comprising a. The method of claim 11, wherein the fluorescent substance is umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tamra (TAMRA), dichloro Triazinylamine fluorescein, dansyl chloride, quantum dots, phycoerythrin, FAM (fluorecein amidite), alexa fluor, Cy3, Cy5, A method for producing a fluorescent substance-containing composite particle, characterized in that at least one selected from the group consisting of Cy7 and indocyanine green. 12. The method of claim 11, wherein the concentration of the fluorescent substance in the composite aqueous solution is controlled in the range of 1 to 30 μg/ml. preparing a magnetic nanoparticle-containing aqueous solution by combining magnetic nanoparticles and an aqueous solution in which metal cations and anions are dissolved in an aqueous medium; and
combining the magnetic nanoparticle-containing aqueous solution with a polyphenol-based compound and adding a first basic component to form magnetic composite nanoparticles having a metal-phenol network;
Method for producing magnetic composite particles comprising a. 15. The method of claim 14, wherein the magnetic nanoparticles are at least one selected from the group consisting of magnetite and maghemite as iron oxide particles, and have a size of 50 nm or less. 16. The method of claim 15, wherein the magnetic composite nanoparticles have a size ranging from 100 to 3000 nm. 16. The method of claim 15, wherein the magnetic composite nanoparticles exhibit a zeta-potential of -10 mV or less. 15. The method of claim 14, wherein the concentration of the magnetic nanoparticles in the magnetic nanoparticle-containing aqueous solution is controlled in the range of 0.05 to 1 mg/ml.

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