WO2018070734A1 - Marking material having contrasting effect and tissue adhesion characteristics and comprising core/shell structure and method for manufacturing same - Google Patents

Abstract

The present invention relates to a marking material having a contrasting effect and tissue adhesion characteristics and comprising core/shell nanoparticles, which are structured to comprise a core having a contrasting effect and a silica shell formed on the surface of the core, and a method for manufacturing the same. More particularly, the present invention relates to core/shell nanoparticles configured such that the core portion has a contrasting effect, and the silica shell portion has tissue adhesion characteristics, thereby giving the core/shell nanoparticles both the tissue adhesion characteristics and the contrasting effect. Therefore, according to the present invention, the nanoparticles can be utilized as a tissue adhesive and as a contrast medium, and can be used as a marking material for a real-time multi-mode image-based technology.

Description

Labeling substance having contrast effect and tissue adhesion property including coreshell structure and preparation method thereof

The present invention relates to a labeling substance having a contrast effect and a tissue adhesion property including a core shell and a method for producing the same. More particularly, the present invention relates to an application in a real-time multimodal imaging based technology of a labeling substance having a contrasting effect and a tissue adhesive property including a core and a silica shell having a contrasting effect.

The use of tissue adhesives used in surgery and procedures allows tissue to be attached with a simpler process and faster sealing than threaded sutures, lower infection rates, and reduce pain felt by the patient.

For this reason, many tissue adhesives are used in the medical field for attaching tissues. Tissue adhesives are based on proteins or synthetic polymers, including fibrin, gelatin, polyurethane and polyethylene glycol, and tissue adhesives are being developed for various surgical procedures.

In particular, tissue adhesives can effectively seal wounds in limited areas that are difficult to access by conventional wound closure methods, and can reduce postoperative complications, thus making them convenient for incision surgery or image-based surgery.

Nanoparticles have a large surface area and high absorption energy between the tissue and the surface of the nanoparticles and thus have excellent tissue adhesion, and the nanoparticles have various applications such as hemostasis or wound closure. For example, silica in nanoparticles has high adhesive strength to tissues or hydrogels and is used as tissue adhesives.

Various nanoparticles have also been used as contrast agents for fluorescence imaging, magnetic resonance imaging (MRI), computed tomography (CT), optoacoustic tomography and surface enhanced Raman scattering.

Surgery to a minimal extent is performed by imaging the treatment in real time using X-ray transmission, computed tomography, ultrasonography or fluorescence imaging. Therefore, in order to perform the treatment accurately and safely, it is necessary to have a characteristic that the treatment position can be visualized in the imaging treatment.

Each imaging modality has its own advantages and disadvantages. For example, CT has advantages in terms of high resolution and three-dimensional visual reconstruction of tissues, but the contrast effect on soft tissues is poor due to inherent low sensitivity.

T ₂ -weighted MRIs using magnetic nanoparticles as contrast agents show high sensitivity and excellent soft tissue contrast effects, but the dark signal of the contrast agent is often confused with other low intensity sites such as air, bleeding, calcification or metal deposition.

To overcome these drawbacks, multimode imaging can be used to combine the advantages of each imaging modality. In multimodal imaging, nanoparticles can be modified to suit the application and can be easily labeled with biomolecules, fluorescent dyes or radioisotopes, and thus can be developed as multifunctional nanoprobes for multimodal imaging.

Tantalum oxides in nanoparticles include high refractive index, thermal and chemical stability, catalytic activity, radiopacity and biocompatibility and are therefore used as antireflective coatings, water separation catalysts, fixed metal oxide catalysts or X-ray contrast agents.

The labeling substance is a substance for marking a position to be treated during an imaging medical surgery or a procedure, and the substance should simultaneously have tissue adhesiveness and contrast effect properties.

Nanoparticles can be used as a contrast agent using a contrast effect or a tissue adhesive having a tissue adhesiveness capable of bonding biological tissue, respectively, but nanoparticles themselves have limitations to simultaneously include a contrast effect and a tissue adhesiveness.

Therefore, there is a demand for technology development that can solve the above-mentioned disadvantages of the prior art and can be applied as a labeling material.

[Preceding technical literature]

[Patent Documents]

(Patent Document 1) Republic of Korea Patent Publication No. 2013-0037664

An object of the present invention is to provide a labeling material having a contrast effect and a tissue adhesion property including a core shell structure and a method for producing the labeling material.

The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned above may be clearly understood by those skilled in the art from the following description. There will be.

In order to achieve the above technical problem, one embodiment of the present invention has a contrast effect and tissue adhesion characteristics comprising a core shell nanoparticles having a structure comprising a core having a contrast effect and a silica shell formed on the surface of the core Provide a labeling substance.

In an embodiment of the present invention, the core may be a label having a contrast effect and a tissue adhesion characteristic, characterized in that it has a contrast effect of radiopaque properties in X-ray transmission or computed tomography.

In an embodiment of the present invention, the core may be a labeling material having a contrast effect and tissue adhesion properties, characterized in that it comprises tantalum oxide, manganese oxide, iron oxide, gold, cadmium selenide or zinc sulfide.

In an embodiment of the present invention, the silica shell may be a labeling material having a contrast effect and a tissue adhesion characteristic, characterized in that it has a high adhesive properties to the hydrogel or tissue.

In an embodiment of the present invention, the silica shell may be a labeling material having a contrast effect and a tissue adhesion characteristic, characterized in that the bonding by one or more selected from the electrostatic interaction and hydrogen bonding with the tissue.

In an embodiment of the present invention, it may be a labeling material having a contrast effect and tissue adhesion characteristics further comprising a fluorescent material located on the surface of the core-shell nanoparticles.

In an embodiment of the present invention, the fluorescent material includes Rhodamine B, tetramethyltamine, indocyanine green (ICG) or cyanine 5.5 (Cyanine 5.5), the contrast effect and tissue adhesion, characterized in that It may be a label having a characteristic.

In order to achieve the above technical problem, another embodiment of the present invention is to prepare a microemulsion by adding a surfactant, ethanol and aqueous sodium hydroxide solution to the solvent, to form a core by adding tantalum ethoxide to the microemulsion Forming a shell by adding ammonia water and a silicon compound to the solution obtained in the core forming step, forming a shell, and reacting the core and the shell formed in the shell forming step to form a core-shell nanoparticle structure. It provides a labeling material manufacturing method having a contrasting effect and tissue adhesion properties comprising.

In an embodiment of the present invention, the solvent may be a labeling material manufacturing method having a contrast effect and tissue adhesion characteristics, characterized in that it comprises hexane, cyclohexane or toluene.

In an embodiment of the present invention, the silicon compound may be a method for producing a labeling substance having a contrast effect and tissue adhesion properties, characterized in that it comprises tetraethoxysilane, triethoxysilane or trimethoxysilane.

In an embodiment of the present invention, it may be a method for producing a labeling material having a contrast effect and tissue adhesion characteristics, characterized in that the thickness of the shell is adjusted by adjusting the amount of the silicone compound.

In an embodiment of the present invention, the core and the shell are reacted for 12 hours to 24 hours by a sol-gel method, and the contrast effect and the tissue, characterized in that carried out by drying at 50 ℃ to 70 ℃ It may be a method for preparing a labeling substance having adhesive properties.

In an embodiment of the present invention, after forming the core-shell nanoparticle structure, further comprising the step of removing the surfactant adsorbed on the coreshell nanoparticles, the contrast effect and tissue adhesion characteristics Eggplant may be a method for preparing a labeling substance.

In an embodiment of the present invention, the step of removing the surfactant adsorbed to the core shell nanoparticles, the coreshell nanoparticles are separated from the dispersion step and dispersed in a solvent and from the coreshell nanoparticle solution obtained in the dispersion step It may be a method for preparing a labeling substance having a contrast effect and tissue adhesion properties, including removing and washing the adsorbed surfactant.

In an embodiment of the present invention, in the step of separating and dispersing the coreshell nanoparticles in a solvent, the coreshell nanoparticles are separated by centrifugation, and the contrast effect and tissue adhesion characteristics, characterized in that dispersed in ethanol solvent It may be a labeling material manufacturing method having a.

In an embodiment of the present invention, in the step of removing and washing the surfactant adsorbed from the coreshell nanoparticle solution, the coreshell nanoparticle solution is characterized in that the surfactant adsorbed with hydrochloric acid is removed, washed with ethanol It may be a labeling material manufacturing method having a contrast effect and tissue adhesion properties.

In order to achieve the above technical problem, another embodiment of the present invention comprises the steps of preparing a core shell nanoparticles, dissolving a fluorescent dye and fluorescent dye adhesive in a solvent to prepare a dye solution, core shell nano in the dye solution Adding particles, and then dispersing them in a buffer to prepare a fluorescent conjugated coreshell nanoparticle solution and separating unreacted dye molecules from the fluorescent conjugated coreshell nanoparticle solution, wherein the coreshell nanoparticles The present invention provides a fluorescent labeling material manufacturing method having a contrasting effect and a tissue adhesion characteristic, which has a structure including a core having a contrasting effect and a silica shell formed on the surface of the core.

In an embodiment of the present invention, the core may be a fluorescent labeling method having a contrast effect and a tissue adhesion characteristic, characterized in that it comprises tantalum oxide, manganese oxide, iron oxide, gold, cadmium selenide or zinc sulfide. .

In an embodiment of the present invention, the fluorescent dye is an imaging effect and tissue adhesion, characterized in that it comprises Rhodamine B, tetramethyltamine, indocyanine green (ICG) or cyanine 5.5 (Cyanine 5.5) It may be a method of manufacturing a fluorescent label having a characteristic.

In an embodiment of the present invention, the fluorescent dye adhesive may be a method for producing a fluorescent labeling material having a contrast effect and tissue adhesion characteristics comprising tetraethoxysilane, triethoxysilane or trimethoxysilane. .

In an embodiment of the present invention, the solvent may be a method for producing a fluorescent label having a contrast effect and tissue adhesion properties, characterized in that it comprises dimethylformamide or dimethyl sulfoxide.

In an embodiment of the present invention, the buffer may be a fluorescent labeling method having a contrast effect and tissue adhesion characteristics, characterized in that the phosphate buffer.

According to an embodiment of the present invention, there is provided a labeling material having a contrast effect and a tissue adhesion property comprising a nanoparticle having a core shell structure comprising a core having a contrast effect and a silica shell formed on the surface of the core.

Therefore, the silica shell portion of the nanoparticles can be used as a tissue adhesive with excellent tissue adhesion and provides a feature that can bleed blood leaking from the wound portion of the tissue.

In addition, the core portion of the nanoparticles have a contrast effect, which provides a feature that can be used in image-based technology as a contrast agent.

In addition, the nanoparticles provide a feature that is used as a marker to display the location of real-time multi-mode imaging-based surgery and procedures in vivo using tissue adhesion and contrast effects.

The effects of the present invention are not limited to the above-described effects, but should be understood to include all the effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a schematic diagram of a labeling substance having a contrast effect and tissue adhesion characteristics of the present invention.

Figure 2 is a flow chart schematically showing a method for producing a labeling material having a contrast effect and tissue adhesion characteristics according to an embodiment of the present invention.

Figure 3 is a flow chart schematically showing a method for producing a fluorescent label having a contrast effect and tissue adhesion properties according to an embodiment of the present invention.

4 is a TEM image of a TSN manufactured according to an embodiment of the present invention (FIG. 4 (a)), an EELS image (FIG. 4 (b)) of tantalum of TSN, and an EELS image of silicon of TSN (FIG. 4). (c)) and EELS merged images of tantalum and silicon of TSN (FIG. 4 (d)).

FIG. 5 shows TEM images of TSNs synthesized according to a change in a molar ratio of a silica precursor to a tantalum ethoxide precursor according to an embodiment of the present invention.

6 is a graph of shell thickness according to a molar ratio of a silica precursor to a tantalum ethoxide precursor according to an embodiment of the present invention.

7 is an ultraviolet and visible light spectral graph of TSN-dye according to an embodiment of the present invention.

8 is a graph (FIG. 8 (b)) of a lab joint share test procedure picture (FIG. 8 (a)) and TSN analysis by the test according to an embodiment of the present invention.

9 is a graph showing the average value of the load when the adhesive portion of the sample is separated in the lab joint share inspection process according to an embodiment of the present invention.

10 is a SEM image of the calf liver tissue with TSN attached according to an embodiment of the present invention.

11 is a graph showing the average value of the hemorrhage amount of the experimental rat liver according to the hemostatic material during the hemostatic effect test picture (TS 11 (a)) and TSN hemostatic effect test process of the TSN according to an embodiment of the present invention (Fig. ))to be.

12 is an image (FIG. 12 (a)) comparing the degree of contrast of water, TSN, SiO ₂ NP or CA-Lp in X-ray transmission method according to an embodiment of the present invention and the degree of contrast calculated by SNR It is a graph (FIG. 12 (b)).

FIG. 13 is a graph comparing the degree of contrast of TSN, SiO ₂ NP or iopamidol in a computed tomography according to an embodiment of the present invention (FIG. 13 (a)) and a graph of X-ray absorption coefficient according to concentration (FIG. 13). (b)).

FIG. 14 is an image (FIG. 14 (a)) and an ultrasound intensity graph for each concentration (FIG. 14 (b)) comparing the contrast level of TSN or SiO ₂ NP in the ultrasonic test method according to the exemplary embodiment of the present invention.

FIG. 15 is an imaging image of TSN or CA-Lp injected into the liver of experimental rats using X-ray transmission according to an embodiment of the present invention.

16 is a contrast image of TSN or CA-Lp injected into the liver of a laboratory mouse using computed tomography according to an embodiment of the present invention.

FIG. 17 is an imaging image of SiO ₂ NP, TSN, or CA-Lp injected between calves using an ultrasound test according to an exemplary embodiment of the present invention.

FIG. 18 is a diagram illustrating an image of culturing TSN or CA-Lp with a cell suspension according to an embodiment of the present invention (FIG. 18 (a)) and a degree of viability of cells according to TSN, CA-Lp, or SiO ₂ NP concentrations. It is a graph (FIG. 18 (b)).

FIG. 19 shows images after 3 days (FIG. 19 (a)) and 14 days (FIG. 19 (b)) of TSN injected into the liver of a laboratory rat according to an embodiment of the present invention, and 3 days of CA-Lp (FIG. And images after 19 (c) and 14 days (Fig. 19 (d)).

20 is an image showing TSN-dye as a label in lung tumors of rats for experiments using X-ray transmission according to an embodiment of the present invention.

Figure 21 is an image showing the TSN-dye as a label in the lung tumor of the rat rat using a computed tomography according to an embodiment of the present invention.

FIG. 22 is an image showing TSN-dye as a label in a lung tumor of a laboratory mouse using fluorescence imaging according to an embodiment of the present invention.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and like reference numerals designate like parts throughout the specification.

Throughout the specification, when a part is said to be "connected (connected, contacted, coupled)" with another part, it is not only "directly connected" but also "indirectly connected" with another member in between. "Includes the case. In addition, when a part is said to "include" a certain component, this means that it may further include other components, without excluding the other components unless otherwise stated.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms “comprise” or “have” are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described in the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Hereinafter, the tissue adhesive having a contrast effect will be described.

1 is a schematic diagram of a labeling substance having a contrast effect and tissue adhesion characteristics of the present invention.

Referring to FIG. 1, the labeling material having the contrast effect and the tissue adhesion property of the present invention may be composed of coreshell nanoparticles having a structure including a core having a contrast effect and a silica shell formed on the surface of the core.

The core shell is a particle composed of a core that is a functional nanomaterial and a shell that is an outer structure of the core.

Coreshells are generally nanoparticles that are prepared when complex functional nanoparticles having two or more properties are needed, and the properties of the coreshell may include both the properties of the core and the properties of the shell.

In an embodiment of the present invention, the core may be a label having a contrast effect and a tissue adhesion characteristic, characterized in that it has a contrast effect of radiopaque properties in X-ray transmission or computed tomography.

In an embodiment of the present invention, the core may include, but is not limited to, tantalum oxide, manganese oxide, iron oxide, gold, cadmium selenide or zinc sulfide.

Tantalum oxide nanoparticles in the core material include high refractive index, thermal and chemical stability, catalytic activity, radiopacity and biocompatibility, and thus are used as antireflective coatings, water separation catalysts, fixed metal oxide catalysts or X-ray contrast agents. have.

In addition, tantalum oxide nanoparticles are about 200 times cheaper than gold, but the X-ray attenuation coefficient is similar to gold, and no special cytotoxicity has been reported.

In an embodiment of the present invention, the silica shell may include a feature that has a high adhesion to the hydrogel or tissue.

Silica is an important component of skeletal material in bone or cartilage and is used as a medical material because of its excellent biocompatibility.

In an embodiment of the present invention, the silica shell may include being bonded to any one or more selected from electrostatic interaction with the tissue and hydrogen bonding.

When a solution containing nanoparticles containing a silica shell is applied to an interface of a tissue, molecules constituting the tissue are adsorbed and fixed by electrostatic interaction on the surface of the nanoparticle. At the same time, as the molecules constituting the tissue around the nanoparticles are rearranged, the silica shell and the tissue may be bonded while the nanoparticles serve as a bridge.

In an embodiment of the present invention, it may be a labeling material having a contrast effect and tissue adhesion characteristics further comprising a fluorescent material located on the surface of the core-shell nanoparticles.

In an embodiment of the present invention, the fluorescent material may include, but is not limited to, rhodamine B, tetramethyltamine, indocyanine green (ICG) or cyanine 5.5 (Cyanine 5.5). Specify it.

Unlike other fluorescent dyes, rhodamine-based compounds are excellent in light stability and have high quantum yield and long absorption and emission wavelengths. Indocyanine Green is a near-infrared fluorescent dye that has high stability in and around the body when used as a nanomaterial. Cyanine is easy to synthesize compounds of various absorption and excitation wavelengths and has high molar extinction coefficient characteristics.

With these properties, the fluorescent materials can be widely used in laser dyes, biosensors, fluorescent chemical sensors, medical fields and the like.

Hereinafter, a method of preparing a labeling substance having the above-mentioned contrast effect and tissue adhesion characteristics will be described.

Figure 2 is a flow chart schematically showing a method for producing a labeling material having a contrast effect and tissue adhesion characteristics according to an embodiment of the present invention.

Referring to FIG. 2, the method for preparing a labeling substance having a contrast effect and a tissue adhesion property according to an embodiment of the present invention includes preparing a microemulsion by adding a surfactant, ethanol and an aqueous sodium hydroxide solution to a solvent (S101), Core formation step (S102) of forming a core by adding tantalum ethoxide to the microemulsion, shell formation step (S103) of forming a shell by adding ammonia water and silicon compound to the solution obtained in the core formation step and the shell formation The method may include a method for preparing a tissue adhesive having a contrasting effect including reacting the core and the shell formed in the step to form a core-shell nanoparticle structure (S104).

First, the step (S101) of preparing a microemulsion by adding a surfactant, ethanol, and aqueous sodium hydroxide solution to a solvent may include, but is not limited to, hexane, cyclohexane, or toluene as a solvent.

The ethanol and sodium hydroxide aqueous solutions have a hydrophilic structure, and the hexane, cyclohexane, and toluene solvents have a lipophilic structure, so that the ethanol, aqueous sodium hydroxide solution and the solvent cannot be mixed together.

Surfactants have both lipophilic and hydrophilic groups in a molecule.

In the step (S101), the surfactant may serve to disperse ethanol and sodium hydroxide in a stable state in hexane, cyclohexane or toluene as small particles, thereby preparing a microemulsion.

Next, tantalum ethoxide is added to the microemulsion to form a core (S102).

Next, the silicon compound is tetraethoxysilane, triethoxysilane or trimethoxysilane in step S103 of forming a shell by adding ammonia water and a silicon compound to the solution obtained in the core forming step (S102). It may include, but is not limited to.

In an embodiment of the present invention, by adjusting the amount of the silicone compound may include adjusting the thickness of the shell.

Since the shell of the core-shell nanoparticles of the present invention is composed of a silica component, as the amount of the silicon compound increases, the thickness of the shell may become thick, whereas as the amount of the silicon compound decreases, the thickness of the shell Can be thinned.

Next, the core and the shell in the step of forming the core shell nanoparticle structure by reacting the core and the shell formed in the shell forming step (S103) (S104) for 12 hours to the sol-gel (sol-gel) method The reaction may be performed for 24 hours, and may be performed by drying at 50 ° C. to 70 ° C. If the reaction time is too short, the core shell may be inhomogeneously formed because sufficient time is not given for the core and the shell to be combined. On the other hand, if the reaction time is too long, the shell precursor enters the core and may be formed of a single component instead of the core shell structure. It may be made of nanoparticles and may not be desirable. In addition, when the drying time is too short or long, it is out of the proper drying time of the solvent, it may be undesirable because the uniform formation of the core shell may be limited.

For example, the core shell nanoparticles may have a diameter of 9.6 nm to 12.0 nm, and the silica shell may have a thickness of 1.8 nm to 2.6 nm, but is not limited thereto.

Labeling method according to an embodiment of the present invention has a contrast effect and a tissue adhesive properties after the step of forming the core shell nanoparticle structure (S104), to remove the surfactant adsorbed to the coreshell nanoparticles Step S105 may be further included.

Removing the surfactant adsorbed to the core shell nanoparticles according to an embodiment of the present invention (S105), separating the coreshell nanoparticles and dispersing in a solvent and the coreshell nanoparticle solution obtained in the step It may be a method for preparing a tissue adhesive having a contrast effect comprising the step of removing and washing the adsorbed surfactant from the.

First, in the step of removing the surfactant adsorbed on the core shell nanoparticles (S105), the coreshell nanoparticles may be separated by centrifugation and dispersed in an ethanol solvent to form a coreshell nanoparticle solution.

Centrifugal separation is a method of rotating a mixture about an axis and applying a centrifugal force to the mixture to separate the mixture according to density.

The centrifugation method can separate the liquid contained in the core-shell nanoparticles of the gel (gel) to separate the pure coreshell nanoparticles.

Next, in an embodiment of the present invention, the core shell nanoparticle solution may be removed with a surfactant adsorbed with hydrochloric acid, and washed with ethanol.

Surfactant-adsorbed coreshell nanoparticles may act as impurities when the interaction between the coreshell nanoparticles and the tissue proceeds, thereby reducing tissue adhesion of the coreshell nanoparticles. It may be desirable to remove.

Hereinafter will be described a method for producing a fluorescent label having the contrast effect and tissue adhesion properties.

Figure 3 is a flow chart schematically showing a method for producing a fluorescent label having a contrast effect and tissue adhesion properties according to an embodiment of the present invention.

Referring to Figure 3, the method for producing a fluorescent labeling material having a contrast effect and tissue adhesion characteristics according to an embodiment of the present invention preparing a core-shell nanoparticles (S201), fluorescent dyes and fluorescent dye adhesives in a solvent Dissolving to prepare a dye solution (S202), adding core shell nanoparticles to the dye solution and dispersing it in a buffer to prepare a fluorescent conjugated coreshell nanoparticle solution (S203) and the fluorescent conjugated coreshell nanoparticle solution Isolating the dye molecules not reacted at (S204), wherein the core shell nanoparticles have a contrast effect, characterized in that having a structure comprising a core having a contrast effect and a silica shell formed on the surface of the core Eggplant can provide a method for producing a fluorescent tissue adhesive.

First, in preparing the core shell nanoparticles (S201), the core shell nanoparticles may have a structure including a core having a contrast effect and a silica shell formed on the surface of the core.

Next, in the step of preparing a dye solution by dissolving a fluorescent dye and a fluorescent dye adhesive in a solvent (S202) the fluorescent dye is rhodamine B, tetramethyl rhodamine, indyan green or cyanine 5.5 (Cyanine 5.5) It may include, but is not limited to.

In an embodiment of the present invention, the fluorescent dye adhesive may include that characterized in that tetraethoxysilane, trimethoxysilane or trimethoxysilane.

Tetraethoxysilane, trimethoxysilane, and trimethoxysilane are silane-based coupling agents having both organic and inorganic reactivity, and may improve adhesion of inorganic materials such as metals or organics, adhesives, paints or It is applied to a sealant or the like to improve adhesion with the resin.

The tetraethoxysilane, trimethoxysilane and trimethoxysilane may be used as an adhesive for attaching the fluorescent dye and coreshell nanoparticles.

In an embodiment of the present invention, the solvent may be a fluorescent tissue adhesive manufacturing method having a contrast effect, characterized in that dimethylformamide.

Next, in the step of preparing a fluorescent conjugated coreshell nanoparticle solution by adding coreshell nanoparticles to the dye solution and dispersing it in a buffer (S203), the buffer may include a phosphate buffer. .

A buffer is generally a solution in which the hydrogen ion concentration (pH) of the solution does not change significantly due to the addition of an acid or a base.

Phosphate buffer is a solution that suppresses the pH change by adding phosphate to the phosphate solution.

When the dye solution and the core shell nanoparticles are added to the phosphate buffer solution to react, the phosphate buffer solution can control the pH change when the reaction is performed.

Next, in the embodiment of the present invention, the dye molecules which do not react in the fluorescent conjugated core shell nanoparticle solution may be characterized in that the separation by centrifugation.

Hereinafter, Examples and Experimental Examples of the present invention will be described. However, these Examples and Experimental Examples are intended to explain the configuration and effects of the present invention in more detail, and the scope of the present invention is not limited thereto.

[Production Example 1]

Preparation of labeling material (Tantalum oxide / Silica core / shell Nanoparticles, TSN) with contrast effect and tissue adhesion characteristics

46 g of Igepal CO-520, 5 ml of ethanol and 5 ml of sodium hydroxide aqueous solution were added to 800 ml of cyclohexane and stirred to prepare a microemulsion. Next, 1 ml of tantalum ethoxide was added to the prepared microemulsion and stirred at room temperature for 30 minutes, and then 5 ml of ammonia water and 1 ml of tetraethoxysilane, which were silica precursors, were sequentially added and stirred at room temperature for 24 hours. Reacted. The reaction solution was evaporated at 60 ℃. The synthesized coreshell nanoparticles were separated by centrifugation, dispersed in ethanol, 5 ml of hydrochloric acid was added, separated by centrifugation, and washed twice with ethanol. The core shell nanoparticles washed with ethanol were washed twice with phosphate buffer and 20 ml of distilled water, respectively, to prepare a labeling material having a contrast effect and a tissue adhesion property to form a tantalum oxide / silica core / shell nanoparticle structure. A labeling substance having the contrast effect and tissue adhesion property was prepared at 40 wt%.

[Production Example 2]

Preparation of fluorescent marker ( rhodamine- attached TSN, TSN-dye) with contrast effect and tissue adhesion

0.44 mg of tetramethyltamine and 0.18 mg of trimethoxysilane were dissolved in 0.5 ml of dimethylformamide and shaken for 30 minutes to prepare a tetramethyltamine isothiocyanate dye solution. The tantalum oxide / silica core / shell nanoparticles prepared in Preparation Example 1 was added to the prepared dye solution by dispersing in a phosphate buffer solution and reacted by shaking for at least 8 hours. Unreacted dye molecules are separated by centrifugation several times, and prepared by dispersing the fluorescent label having the completed contrast effect and tissue adhesion properties in water.

Comparative Example 1

Preparation cyanoacrylate iodide and oil mixture radiopacity (cyanoacrylate and radiopaque iodized oil (Lipiodol ), CA-Lp)

Histoarcyl and Lipiodol Ultra-Fluid were mixed in 360 mgI / ml of iodine in a volume ratio of 1: 3.

Comparative Example 2

Preparation of Silica Nanoparticles (SiO ₂ Nanoparticles, SiO ₂ NP)

Silica nanoparticles were prepared with commercially available Ludox ™ -50.

Experimental Example 1

Shape and component analysis of TSN

In order to confirm the shape and constituent elements of the prepared TSN, the analysis was performed using a transmission electron microscope (TEM) and an electron energy loss spectroscopy (EELS). TEM images of TSN are shown in FIG. 4 (a) and EELS images are shown in FIGS. 4 (b) to 4 (d).

Through TEM analysis, the shape of the TSN prepared according to Preparation Example 1 of the present invention was confirmed to be spherical, the diameter of the TSN is 9.6 nm to 12.0 nm, it can be seen that the shell thickness of TSN is 1.8 nm to 2.6 nm. have. In addition, it can be determined that the TSN is uniformly synthesized by EELS analysis, as the mapping of tantalum and silicon elements in the core-shell structure overlaps.

Experimental Example 2

TSN thickness analysis according to silica precursor content

In order to confirm the change of shell thickness of TSN according to the content of silica, which is a constituent of TSN, the molar ratio of the silica precursor to the tantalum ethoxide precursor in the preparation method of Preparation Example 1 was 0, 0.5, 1, 2, 3, 4, The shell thickness change of TSN was analyzed using TEM while changing to 6, 8 or 12, and the results are shown in order in FIGS. 5 (a) to 5 (i).

5 (a) to 5 (i), it can be seen from the contrast difference between tantalum oxide and silica in the TEM image that the shell thickness of TSN becomes thicker as the molar ratio of the silica precursor to the tantalum ethoxide precursor increases. It was analyzed that the silica precursor plays a role in forming the shell of TSN when preparing TSN. 6 illustrates the shell thickness according to the molar ratio of the silica precursor to the tantalum ethoxide precursors of the TSNs prepared according to the exemplary embodiment. (The scale bars in Figures 5 (a) to 5 (i) are all 20 nm.)

Experimental Example 3

Ultraviolet / Visible Spectroscopic Analysis of TSN-dye

Ultraviolet and visible light spectroscopic characteristics of TSN-dye prepared in Preparation Example 2 were analyzed using an ultraviolet / visible light spectrophotometer, and the results thereof are shown in FIG. 7.

Referring to FIG. 7, it was confirmed that the absorption and emission spectra of the tetramethyltamine isothiocyanate dye solution and TSN-dye in which the dye solution was bound to TSN are identical, and the wavelength of the spectrum was analyzed at 576 nm. . It can be determined that the dye solution is bound without changing the structure in the process of combining TSN-dye and the dye solution.

Experimental Example 4

Tissue Adhesion Analysis of TSN

A lap joint share test was performed to analyze the tissue adhesion of TSN.

To make a sample, we first make several pieces of fresh calf between 45 mm long, 10 mm long and 4 mm high, two of which are 15 μl using TSN, SiO ₂ NP or CA-Lp. Attach. The length of the two pieces of adhesive part attached was 10 mm, and the two pieces of adhesive parts were fixed by applying pressure for 10 seconds by hand to prepare a sample.

The sample was analyzed using a universal testing machine to measure the adhesive strength, and after fixing both sides of the sample up and down, the sample was separated by applying a 50 N load upwards at a rate of 30 mm / min.

8 (a) to 8 (b) are lap joint share inspection process photographs and TSN analysis graphs based on the inspection.

According to Figures 8 (a) to 8 (b), the adhesive strength of the sample is confirmed to be measured through the process of lifting a piece, a load is applied, the maximum point and separation of the load and the sliding process It was.

9 is a graph showing the average value of the load when the adhesive part of the sample is separated.

According to FIG. 9, it was analyzed that the load appeared larger when the adhesive part was separated from the sample using the adhesive than the sample without the adhesive (control, Ctrl.), And similar loads were found in TSN, CA-Lp, or SiO ₂ NP. Confirmed. This can be judged that the adhesive strength of the adhesive influenced the increase in tissue adhesion.

Experimental Example 5

Tissue Adhesion Shape Analysis of TSN

Samples are first prepared to confirm the tissue adhesion shape of TSN.

Samples for TSN tissue adhesion shape were fixed with 0.1 M sodium cacodylate hydrochloride buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde, and the cacodyl hydrochloride buffer Wash with In order to more securely fix the tissue washed with buffer, fixate with Cacodyl hydrochloride buffer containing 1% osmium tetraoxide solution and wash three times with distilled water to remove residual fixation reagent. Tissues washed with distilled water were colored overnight with 2% uranyl acetate solution and soaked in ethanol at 30%, 50%, 70%, 80% and 90% concentrations for 10 minutes to dry the moisture of the colored tissues, respectively. . Finally, immerse with 100% ethanol three times to prepare the water removed.

Tissue samples to which TSN was attached were subjected to shape analysis using a scanning electron microscope (SEM). SEM images of the tissues with TSN attached are shown in FIG. 10.

Through SEM analysis, the shape of the TSN-attached tissue prepared according to Experimental Example 5 of the present invention can be identified as a shape in which the TSN and the tissue are bound and adsorbed. This can be judged to enable adhesion of liver tissues with which spontaneous network formation due to adsorption of TSN in tissues is cut off.

Experimental Example 6

Analysis of the hemostatic effect of TSN

Prepare a punctured wound with an 18-gauge needle sterilized in the liver of a laboratory rat, and then apply TSN or CA-Lp to the wound as a hemostatic substance, respectively, or compress the wound directly. In order to compare the amount of bleeding according to the hemostatic substances, the bleeding caused by hemostasis of the liver of the rats by direct compression or hemostasis using the hemostatic substance was absorbed into the filter paper, and the amount of blood loss was measured by the weight difference before and after the absorption of the filter paper. Figure 11 (a) of the hemostatic effect experiment of TSN is shown in Figure 11 (b), a graph showing the average value of the hemorrhage amount of the rat liver according to the hemostatic material in the TSN hemostatic effect experiment.

11 (a) to 11 (b), it was confirmed that bleeding stopped when TSN acted as a hemostatic substance. In addition, hemorrhage of the liver with TSN or CA-Lp hemostatic material was analyzed to reduce the amount of bleeding similarly. It can be determined that the degree of hemostatic effect of TSN or CA-Lp is similar.

Experimental Example 7

Analysis of TSN Contrast Effect in X-ray Transmission

Samples were prepared by adding water (Ref.), 40 wt% TSN, 50 wt% SiO ₂ NPs or 75 wt% CA-Lp into a 2-ml tube. X-ray imaging effects were compared using the Allura Xper FD20 instrument from Philips. To quantify the contrast, the mean attenuation of each material (S) was calculated by dividing by the standard deviation of the background attenuation (N) (signal-to-noise ratio, SNR).

12 (a) is an image comparing the contrast of water, TSN, SiO ₂ NP or CA-Lp in the X-ray transmission method according to an embodiment of the present invention.

Referring to FIG. 12 (a), when X-rays were transmitted through water, TSN, SiO ₂ NP, and CA-Lp, X-rays transmitted through TSN were weakened in intensity. It was analyzed that the role of contrast agent of TSN is effective.

A graph calculated by SNR to quantify the contrast level of the contrast agent is shown in Figure 12 (b).

Referring to FIG. 12 (b), it was confirmed that TSN showed 15 times higher SNR value than SiO ₂ NP, which was determined that the tantalum oxide core portion of TSN weakened X-rays to show contrast enhancement effect.

Experimental Example 8

Analysis of TSN Contrast Effects on Computed Tomography (CT)

Commercially available contrast agents, Iopamidol, TSN or SiO ₂ NP are placed in 2-ml barrels, 1 wt% of agarose is dispersed and diluted with water, 0, 1.51, 3.12, 6.25, 12.5, 25 and 50 mg ml Prepare samples by submerging ^-1 concentration of material into 200 μl microtubes each. CT contrast effects were analyzed by contrast images using a Philips medical CT scanner. To determine the CT detection limit, the X-ray absorption coefficient (CT numbers, (Hounsfield unit, HU)) was measured according to the concentration of each substance.

Figure 13 (a) is an image comparing the degree of contrast of TSN, SiO ₂ NP or iopamidol in computed tomography according to an embodiment of the present invention.

Referring to FIG. 13 (a), TSN and iopamidol were observed to be opaque when X-rays were transmitted through SiO ₂ NP, confirming that the contrast effect was excellent.

In the graph measuring the X-ray absorption coefficient according to the concentration of TSN, SiO ₂ NP or iopamidol shown in FIG. 13 (b), the X-ray absorption coefficient is proportional as the concentration of TSN or iopamidol increases. It was confirmed that the increase. In addition, when comparing the X-ray absorption coefficient based on the concentration of 1 mg ml ^-1 , it was confirmed that TSN is 1.5 times higher than Iopamidol and 150 times higher than SiO ₂ NP. It can be judged that the X-ray absorption coefficient of TSN shows high contrast enhancement effect as a contrast agent in CT.

Experimental Example 9

Analysis of TSN Contrast Effect in Ultrasonography

To compare the ultrasound intensity, dilute to 1 wt% agarose in TSN or SiO ₂ NP to prepare 200 μl of samples at concentrations of 0, 4, 8, 16, 32 and 64 mg ml ⁻¹ , and inject between fresh calves B-mode ultrasound image was analyzed using Accuvix V10, and ultrasonic intensity was measured. Ultrasonic images according to TSN or SiO ₂ NP concentrations are shown in FIG. 14 (a), and ultrasound intensities according to the concentrations are shown in FIG. 14 (b).

According to FIG. 14 (a), the ultrasonic signal of TSN starts to be distinguished from the background from 8 mg ml ⁻¹ , and the ultrasonic intensity at this time is 3 times higher than that of SiO ₂ NP as shown in FIG. 14 (b). Confirmed.

In ultrasonography, the degree of contrast can vary depending on the size, compressibility and density of the material. TSN has a hydrodynamic size of 20.4 nm, which is larger than 16.4 nm SiO ₂ NP and the density of tantalum oxide (~ 8.18 g). Since cm ^-3 ) is high, the ultrasonic detection limit is lower than that of SiO ₂ NP, which may increase the contrast effect.

Experimental Example 10

In Vivo Contrast Effect Analysis of TSN

Insert the TSN or CA-Lp into the liver of the rat, and analyze the X-ray contrast effect by X-ray transmission and computed tomography, and insert the TSN, SiO ₂ NP or CA-Lp into the calf Ultrasound contrast effect was analyzed.

FIG. 15 is an imaging image of TSN or CA-Lp injected into the liver of experimental rats using X-ray transmission according to an embodiment of the present invention.

According to FIG. 15, since the contrast effect of TSN or CA-Lp was similar, it was confirmed that the degree of contrast appeared similarly in the X-ray transmission image.

16 is a contrast image of TSN or CA-Lp injected into the liver of a laboratory mouse using computed tomography according to an embodiment of the present invention.

According to Figure 16, the X-ray absorption coefficient of TSN was 1.8 times higher than the vertebral bone of the rat, it was confirmed that the level similar to CA-Lp.

FIG. 17 is an imaging image of SiO ₂ NP, TSN, or CA-Lp injected between calves using an ultrasound test according to an exemplary embodiment of the present invention.

According to FIG. 17, it was confirmed that the ultrasonic signal of TSN or SiO ₂ was sufficiently distinguished from the background.

As a result, the TSN may be determined to include the contrast effect that the contrast agent should have in X-ray and ultrasound image-based technologies.

Experimental Example 11

Biocompatibility Analysis of TSN

20 μl of TSN or CA-Lp was dropped in the center of the bottom of the culture dish, followed by incubation for 24 hours by adding a HeLa cell suspension thereon to prepare a sample and analyzed by reverse confocal microscopy.

Figure 18 (a) is an image of the cell suspension cultured TSN or CA-Lp according to an embodiment of the present invention, Figure 18 (b) is the viability of the cells according to the TSN, CA-Lp or SiO ₂ NP concentration It is a graph showing the degree.

According to FIG. 18 (a), most of the cells cultured with TSN are attached to the substrate, but the cells cultured with CA-Lp have a circular shape and fall from the substrate. It can be determined that TSN has better biocompatibility than CA-Lp.

According to Figure 18 (b), the viability of the cells cultured CA-Lp rapidly decreased as the concentration is higher, the cells cultured SiO ₂ NP was reduced at a high concentration of 800 μg ml ^-1 . However, it was confirmed that the cells cultured with TSN showed excellent cell viability even at high concentration. It can be judged that TSN has excellent biocompatibility without showing cytotoxicity even when cultured with cells. (The scale bars in FIGS. 18A-18B are all 200 μm.)

Experimental Example 12

In vivo biocompatibility analysis of TSN

Laboratory mice were used in vivo to analyze the in vivo biocompatibility of TSN. The rats were abdominally incised, and the liver tissue images were analyzed 3 or 14 days after the injection of TSN or CA-Lp into a 1.27 mm diameter and 1.5 cm length wound in the left lobe of the rat.

19 (a) to 19 (d) are images and CA after 3 days (FIG. 19 (a)) or 14 days (FIG. 19 (b)) of TSN injected into livers of experimental rats according to one embodiment of the present invention. Image after 3 days (Fig. 19 (c)) or 14 days (Fig. 19 (d)) of -Lp.

According to Figs. 19 (a) to 19 (d), in the liver of CA-Lp-injected mice, immune cells observed in purple until 3 to 14 days are continuously produced, and in the liver of TSN-injected mice, slightly It was confirmed that only immune cell response occurs. TSN has a better biocompatibility in vivo than CA-Lp, so it can be determined that the immune cell response appears only temporarily. (The scale bars in Figures 19 (a) to 19 (d) are all 100 μm.)

Experimental Example 13

Application of TSN as a Labeling Material in Real Time Multi-mode Image-Based Technology

To create a homogeneous lung cancer model, lung tumor chips measuring 0.5 cm wide, 0.5 cm long and 0.5 cm high are injected into the tail vein of a rat. After 10 days, TSN-dye was injected into the occlusion site with a 25-gauge needle to confirm tumor chip growth and occlusion of the lung. As a marker of TSN-dye using micro-CT, real-time transmission and fluorescence imaging, The application was analyzed.

FIG. 20 is an image showing TSN-dye as a label in a lung tumor of a laboratory rat using X-ray transmission according to an embodiment of the present invention, and FIG. 21 is a lung tumor of a laboratory rat using computed tomography. Is an image showing TSN-dye as a labeling substance.

According to Figures 20 to 21, it was confirmed that TSN-dye is distinguished from the ribs and vertebrae by the difference in the degree of contrast while being fixed as a labeling material. In addition, TSN-dye was injected to confirm that the experimental rats did not show any adverse effects on breathing and behavior.

FIG. 22 is an image showing TSN-dye as a label in a lung tumor of a laboratory mouse using fluorescence imaging according to an embodiment of the present invention.

According to FIG. 22, TSN-dye may be determined to be suitable as a labeling material due to characteristics of radiopaque and high fluorescence signals in fluorescence imaging.

Therefore, referring to Preparation Examples and Experimental Examples of the present invention, the core-shell nanoparticles having a structure including a core having a contrast effect and a silica shell formed on the surface of the core have excellent tissue adhesiveness and contrast effect, thereby providing a tissue adhesive and a contrast agent. It can be used as. Due to these characteristics, the core-shell nanoparticles may be used as markers when performing surgery and procedures using real-time multimodal imaging-based techniques such as X-ray transmission, computed tomography and fluorescence imaging.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is represented by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.

Claims (22)

Core with contrast effect; And Labeling material having a contrast effect and tissue adhesion properties comprising a core shell nanoparticles having a structure comprising; a silica shell formed on the surface of the core. The method of claim 1, The core has a contrast effect and a tissue adhesion characteristic, characterized in that it has a contrast effect of radiopaque properties in X-ray transmission or computed tomography. The method of claim 1, The core has a contrast effect and tissue adhesion characteristics, characterized in that it comprises tantalum oxide, manganese oxide, iron oxide, gold, cadmium selenide or zinc sulfide. The method of claim 1, The silica shell is a labeling material having a contrast effect and tissue adhesion characteristics, characterized in that it has a high adhesive property to the hydrogel or tissue. The method of claim 4, wherein The silica shell is a labeling substance having a contrast effect and tissue adhesion characteristics, characterized in that the bonding by any one or more selected from the electrostatic interaction and hydrogen bonding with the tissue. The method of claim 1, Labeling material having a contrast effect and tissue adhesion characteristics further comprising a fluorescent material located on the surface of the core-shell nanoparticles. The method of claim 6, The fluorescent material is a label having a contrast effect and tissue adhesion properties, characterized in that it comprises Rhodamine B, tetramethyl rhodamine, indocyanine green (ICG) or cyanine 5.5 (Cyanine 5.5). Preparing a microemulsion by adding a surfactant, ethanol and aqueous sodium hydroxide solution to the solvent; Forming a core by adding tantalum ethoxide to the microemulsion; A shell forming step of forming a shell by adding ammonia water and a silicon compound to the solution obtained in the core forming step; And Forming a core-shell nanoparticle structure by reacting the core and the shell formed in the shell forming step; Labeling method having a contrast effect and tissue adhesion properties comprising a. The method of claim 8, The solvent is hexane, cyclohexane or toluene comprising a labeling material having a contrast effect and a tissue adhesion characteristic characterized in that the manufacturing method. The method of claim 8, Said silicon compound is tetraethoxysilane, triethoxysilane or trimethoxysilane characterized in that it comprises a contrasting material and a tissue adhesive property characterized in that the manufacturing method. The method of claim 8, Method for producing a labeling material having a contrast effect and tissue adhesion characteristics, characterized in that for controlling the thickness of the shell by adjusting the amount of the silicone compound. The method of claim 8, The core and the shell are reacted for 12 to 24 hours by a sol-gel method, and the method for producing a labeling substance having a contrast effect and tissue adhesion characteristics, characterized in that it is carried out by drying at 50 ℃ to 70 ℃ . The method of claim 8, After the step of forming the core shell nanoparticle structure, the method of producing a labeling material having a contrast effect and tissue adhesion characteristics further comprising the step of removing the surfactant adsorbed on the coreshell nanoparticles. The method of claim 13, Removing the surfactant adsorbed on the core shell nanoparticles, A dispersion step of separating and dispersing the coreshell nanoparticles in a solvent; And Removing and washing the adsorbed surfactant from the core shell nanoparticle solution obtained in the dispersing step; Labeling method having a contrast effect and tissue adhesion properties comprising a. The method of claim 14, In the separating and dispersing the coreshell nanoparticles in a solvent, the coreshell nanoparticles are separated by centrifugation and dispersed in an ethanol solvent. The method of claim 14, In the step of removing and washing the adsorbed surfactant from the coreshell nanoparticle solution, the coreshell nanoparticle solution is characterized in that the surfactant adsorbed with hydrochloric acid is removed, washed with ethanol contrast effect and tissue adhesion characteristics Label production method having a .. Preparing core-shell nanoparticles; Dissolving a fluorescent dye and a fluorescent dye adhesive in a solvent to prepare a dye solution; Adding coreshell nanoparticles to the dye solution, and then dispersing them in a buffer to prepare a fluorescent conjugated coreshell nanoparticle solution; And And separating the unreacted dye molecules from the fluorescent conjugated coreshell nanoparticle solution. The core shell nanoparticles have a structure including a core having a contrast effect and a silica shell formed on the surface of the core, characterized in that the fluorescent labeling material having a contrast effect and tissue adhesion properties. The method of claim 17, The core is a tantalum oxide, manganese oxide, iron oxide, gold, cadmium selenide or zinc sulfide, characterized in that it comprises a fluorescent labeling material having a method and tissue adhesive properties. The method of claim 17, The fluorescent dye comprises a rhodamine B, tetramethyl rhodamine, indocyanine green (ICG) or cyanine 5.5 (Cyanine 5.5), characterized in that it comprises a fluorescent labeling material having a contrast effect and tissue adhesion characteristics . The method of claim 17, The fluorescent dye adhesive is tetraethoxysilane, triethoxysilane or trimethoxysilane, characterized in that it comprises a fluorescent labeling material having a contrast effect and tissue adhesion characteristics characterized in that it comprises. The method of claim 17, The solvent is a method for producing a fluorescent label having a contrast effect and tissue adhesion characteristics, characterized in that it comprises dimethylformamide or dimethyl sulfoxide. The method of claim 17, The buffer solution is characterized in that the phosphate buffer solution, characterized in that the fluorescent labeling material having a contrast effect and tissue adhesion properties.

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