Hybrid nanoclay, method of making and use thereof

Abstract

A kind of hybrid nanoclay and its preparation method and application, including hybrid nanoclay particles with adhesion, the hybrid nanoclay particles include nanoclay with metal polyphenol network on surface, the metal polyphenol network is formed by Zn 2+ And tannic acid self-assembly;The mass percentage of metal polyphenol network in hybrid nanoclay particles is not less than 33%. The hybrid nanoclay particles provided by the present application have antioxidant, antibacterial and hemostatic capacity;Can adhere to stomach tissue, can efficiently remove free radicals in vivo;Can stimulate the secretion of gastric mucosa protective factor, maintain the complete form of gastric mucosa, significantly improve the level of stomach health for all-round protection and repair of damaged gastric mucosa.

Description

Technical Field

[0001] This invention patent relates to the field of nanoclay technology, and in particular to a hybrid nanoclay, its preparation method, and its application. Background Technology

[0002] Gastric ulcer (GU) is one of the most common digestive system diseases, characterized by gastric mucosal damage, primarily caused by an imbalance between mucosal attack factors (gastric acid, reactive oxygen species, etc.) and mucosal defense factors (gastric mucus and bicarbonate secretion, prostaglandins, nitric oxide, etc.). Ethanol-induced gastric mucosal damage is mainly caused by oxidative stress, manifesting as gastric mucosal erosion and bleeding, accompanied by the generation of large amounts of reactive oxygen species (ROS). Furthermore, ROS participate in the inflammatory response of ulcers, leading to more severe hemorrhagic ulcers and tissue necrosis, thereby exacerbating the degree of gastric mucosal damage.

[0003] Oral formulations have become an ideal drug dosage form choice due to their significant advantages such as high patient compliance and low cost. Focusing on the development of such oral medications with specific efficacy is of great significance for improving the treatment level of gastric ulcers and improving patient prognosis. Therefore, there is an urgent need to develop a safe and effective gastric mucosa protectant that combines antioxidant effects (scavenging free radicals) with hemostatic effects. Summary of the Invention

[0004] This invention provides a hybrid nanoclay with antioxidant, antibacterial, and hemostatic properties. It adheres to gastric tissue, efficiently scavenging free radicals in the body and stimulating the secretion of gastric mucosal protective factors, maintaining the integrity of the gastric mucosa, and providing comprehensive protection and repair for damaged gastric mucosa, significantly improving gastric health. It can be applied to the preparation of drugs for the prevention and treatment of ethanol-induced acute gastric mucosal injury.

[0005] This invention provides a hybrid nanoclay comprising hybrid nanoclay particles (Clay@ZT) with adhesive properties. X Hybridized nanoclay particles include nanoclay with a surface modified with a metal polyphenol network, wherein the metal polyphenol network is composed of Zn 2+ It is formed by self-assembly with tannic acid (TA); the mass percentage of the metal polyphenol network in the hybrid nanoclay particles is not less than 33%.

[0006] Preferably, the metal polyphenol network is modified at least once to form a metal polyphenol network shell on the surface of nanoclay.

[0007] Preferably, the nanoclay is layered nanoclay or rod-shaped nanoclay, wherein the layered nanoclay is kaolinite (KAL) and the rod-shaped nanoclay is attapulgite (PGS).

[0008] Furthermore, the nanoclay can also be hollow tubular halloysite nanotubes (HNTs).

[0009] A method for preparing hybrid nanoclay, the method comprising the following steps:

[0010] Step (1): Add the acidic solution to the nano clay, stir evenly, wash with pure water until the supernatant is neutral, discard the supernatant, dry and grind the precipitate to obtain the acidified nano clay.

[0011] Step (2): Resuspend the acidified nano-clay, add tannic acid solution and Zn 2+ The solution was adjusted to pH 8, and a self-assembly reaction was carried out under stirring for 1-2 hours. After washing with pure water, a precipitate was obtained, completing a single modification operation. The above modification operation was repeated 1-3 times on the precipitate to obtain the hybrid clay nanoparticles Clay@ZT. X X represents the number of modifications.

[0012] Preferably, in step (2), X≥2, and the mass percentage of the metal polyphenol network in the hybrid nanoclay particles is not less than 43%.

[0013] Preferably, in step (2), the tannic acid solution and Zn 2+ The molar concentration ratio of the solution is 1:1.

[0014] Preferably, the stirring conditions in step (2) are magnetic stirring at 400 rpm.

[0015] Application of hybrid nanoclays in the preparation of drugs for treating gastric mucosal damage.

[0016] Specifically, the gastric mucosal injury is stress-related gastric mucosal injury, including ethanol-induced acute gastric mucosal injury. The hybrid nanoclay provided by this invention alleviates oxidative stress by increasing GSH in gastric tissue, enhancing SOD and CAT activity, and reducing MDA content.

[0017] The application of hybrid nanoclays in the preparation of drugs for preventing acute gastric mucosal injury, wherein acute gastric mucosal injury includes acute gastric mucosal injury caused by stress or non-stress factors, and the non-stress factors include acute gastric mucosal injury caused by drugs, alcohol, irritating foods, etc.

[0018] The beneficial effects of this invention are:

[0019] This invention selects three natural raw materials: nano-clay, zinc, and polyphenols. A new hybrid nano-clay particle is prepared by modifying the surface of nano-clay with a metal polyphenol network through a one-step mixing method and at least one self-assembly reaction. Under the same dosage and reaction time, the shell produced by the step-by-step modification is more uniform and significantly better than that produced by the single modification. At the same time, the mass ratio of the metal polyphenol network in the step-by-step modification product is significantly improved.

[0020] The hybrid nano-clay particles provided by this invention have antioxidant, antibacterial and hemostatic capabilities; they can adhere to gastric tissue and efficiently remove free radicals in the body; they can stimulate the secretion of gastric mucosal protective factors, maintain the integrity of the gastric mucosa, and provide comprehensive protection and repair for damaged gastric mucosa, significantly improving gastric health.

[0021] The hybrid nanoclay particles provided by this invention, due to their tiny particle size, can embed themselves in the fine folds and depressions of the gastric mucosa. Furthermore, because their surface is rich in hydroxyl groups, they can adhere tightly to gastric tissue through hydrogen bonds, thus achieving stable retention. In therapeutic research, in addition to the above reasons, the negatively charged surface of the hybrid particles allows them to specifically bind to positively charged proteins in the ulcer region, based on the principle of opposite charges attracting. During this process, the hybrid particles continuously release ZT, synergistically exerting antioxidant, hemostatic, and other biological properties, and can be applied to the preparation of drugs for the prevention and treatment of gastric mucosal damage. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the preparation route of the hybrid nanoclay of the present invention.

[0023] Figure 2 Characterization of the products of Comparative Example 1 and Comparative Example 2. Among them, Figure 2 A in the middle is Clay@ZT 1A and Clay@ZT 2B TEM image, scale bar 500nm; Figure 2 B is Clay@ZT 1A and Clay@ZT 2B The statistical results of ZT content.

[0024] Figure 3 Clay@ZT in Example 2 X Image taken under a transmission electron microscope (TEM), scale bar 500 nm.

[0025] Figure 4 For Clay@ZT X Fourier transform infrared spectral image.

[0026] Figure 5 Clay@ZT in Example 2 X The results of the component proportion analysis.

[0027] Figure 6 Clay@ZT in Example 2 X Zeta potential diagram.

[0028] Figure 7 Clay@ZT as in Example 3 X Release curve of TA at 37℃ and pH = 7.4.

[0029] Figure 8 Clay@ZT in Example 3 2 Release curves of TA at 37℃ and pH = 7.4 / 4.0 / 2.2.

[0030] Figure 9 Clay@ZT in Example 3 2 Chinese Zn 2+ Release curves at 37℃ and pH = 7.4 / 4.0 / 2.2.

[0031] Figure 10 Images of red blood cells after centrifugation in Example 4 and images of red blood cells observed under an optical microscope (scale bar of centrifuge tube image is 5 mm; scale bar of red blood cell image is 10 μm).

[0032] Figure 11 Clay@ZT in Example 4 X Figure showing the results of the live / dead cell staining experiment.

[0033] Figure 12 Example 5 Clay@ZT X The diagram shows the experimental results of antibacterial properties, with a scale bar of 5 mm.

[0034] Figure 13 Example 6 Clay@ZT 2 Statistical results of bleeding volume in liver hemostasis experiments.

[0035] Figure 14 Clay@ZT in Example 7 2 The results of the adhesion force experiment are shown in the figure. Among them, Figure 14 In the middle, A represents the force-displacement curve of the lap shear. Figure 14 B in the figure represents a quantitative statistical graph of adhesion strength.

[0036] Figure 15 This is a diagram showing the experimental results of preventing acute gastric mucosal injury in Example 8. Among them, Figure 15 Image A in the middle is a representative image of mouse stomach tissue. Figure 15 Figure B is a quantitative statistical graph of the area of ​​gastric mucosal hemorrhage ulcers in mice.

[0037] Figure 16 This is a line graph showing the weight changes of mice during the modeling process in Example 8.

[0038] Figure 17 This is a histological evaluation image of the mouse gastric tissue from Example 8. Figure 17 Image A is a representative image of H&E staining of mouse gastric mucosa tissue. Figure 17 Image B is a representative image of PAS staining of mouse gastric mucosa tissue, with a scale bar of 100 μm.

[0039] Figure 18 This is a graph showing the detection results of oxidative stress-related indicators in the gastric tissue of mice in Example 8.

[0040] Figure 19 This is a diagram showing the experimental results of acute gastric mucosal injury in Example 9. Among them, Figure 19 Image A in the middle is a representative image of mouse stomach tissue. Figure 19 Figure B is a quantitative statistical graph of the area of ​​gastric mucosal hemorrhage ulcers in mice.

[0041] Figure 20 This is a line graph showing the weight changes of mice during the modeling process in Example 9.

[0042] Figure 21 This is a histological evaluation image of the mouse gastric tissue from Example 9. Among them, Figure 21 Image A is a representative image of H&E staining of mouse gastric mucosa tissue. Figure 21 Image B is a representative image of PAS staining of mouse gastric mucosa tissue, with a scale bar of 100 μm.

[0043] Figure 22 This is a graph showing the detection results of oxidative stress-related indicators in the gastric tissue of mice in Example 9. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0045] All raw materials used in the embodiments of this invention were commercially available. Kaolin (KAL) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., attapulgite (PGS) was purchased from Xuyi County Junda Attapulgite Materials Co., Ltd., and halloysite (HNT) was purchased from Guangzhou Runwo Materials Technology Co., Ltd.

[0046] The mice used in this invention were SPF-grade male KM mice provided by the Beijing Vital River Laboratory Animal Center. All animal procedures were carried out in accordance with the Guidelines for Laboratory Animal Care and Use of Wenzhou Medical University and were approved by the Animal Ethics Committee of Wenzhou Medical University.

[0047] Preparation of ZT (metal polyphenol network) experimental material: Tannic acid was weighed and dissolved in pure water to prepare a 24 mM tannic acid solution; zinc oxide was weighed and dissolved in pure water, and zinc hydroxide was dissolved in 1 M dilute hydrochloric acid to prepare a 24 mM ZnO2 solution. 2+ Solution. Mix 24 mM tannic acid solution and 24 mM Zn... 2+ The solutions were mixed at a volume ratio of 1:1, and the pH of the solution was adjusted to 8 using tris-HCl with pH = 8.8. The mixture was then magnetically stirred at 400 rpm for 2 h, followed by washing twice with pure water. The precipitate was dried at 65 ℃, ground, and stored at 4 ℃.

[0048] Example 1: Hybridized nanoclay particles Clay@ZT X Preparation

[0049] Step (1): Weigh 5.0 g of nano clay (HNT, PGS, KAL) respectively, add 100 ml of 1M dilute hydrochloric acid solution, stir magnetically at 500 rpm for 3.5 h, wash with pure water until the supernatant is neutral, discard the supernatant, dry the precipitate at 65 ℃, grind and store at room temperature to obtain acidified nano clay.

[0050] Step (2): Weigh 50 mg of acidified nano-clay, add 5 mL of pure water, sonicate for 1 h, and then add 250 μL of 24 mM tannic acid solution and 250 μL of 24 mM Zn solution successively. 2+ The solution was adjusted to pH 8 using tris-HCl (pH=8.8), and the self-assembly reaction was carried out for 1 h under magnetic stirring at 400 rpm. The solution was then washed twice with pure water to obtain the precipitate Clay@ZT. 1 (HNT@ZT) 1 PGS@ZT 1 KAL@ZT 1 ).

[0051] The precipitate was resuspended in 5 mL of pure water, followed by the addition of 250 μL of 24 mM tannic acid solution and 250 μL of 24 mM Zn. 2+ The solution was adjusted to pH 8 using tris-HCl (pH=8.8), and the self-assembly reaction was carried out for 1 h under magnetic stirring at 400 rpm. The solution was then washed twice with pure water to obtain the precipitate Clay@ZT. 2 (HNT@ZT) 2 PGS@ZT 2 KAL@ZT 2 ).

[0052] The precipitate was resuspended in 5 mL of pure water, followed by the addition of 250 μL of 24 mM tannic acid solution and 250 μL of 24 mM Zn. 2+ The solution was adjusted to pH 8 using tris-HCl (pH=8.8), and the self-assembly reaction was carried out for 1 h under magnetic stirring at 400 rpm. The solution was then washed twice with pure water to obtain the precipitate Clay@ZT. 3 (HNT@ZT) 3 PGS@ZT 3 KAL@ZT 3 ).

[0053] The precipitate was resuspended in 5 mL of pure water, followed by the addition of 250 μL of 24 mM tannic acid solution and 250 μL of 24 mM Zn. 2+ The solution was adjusted to pH 8 using tris-HCl (pH=8.8), and the self-assembly reaction was carried out for 1 h under magnetic stirring at 400 rpm. The solution was then washed twice with pure water to obtain the precipitate Clay@ZT. 4 (HNT@ZT) 4 PGS@ZT 4 KAL@ZT 4 ).

[0054] The precipitate was dried at 65 ℃, ground, and stored at 4 ℃.

[0055] Comparative Example 1 Clay@ZT 1A Preparation

[0056] With Clay@ZT 1 The difference lies in the fact that step (2) includes weighing 100 mg of acidified nano-clay, adding 10 mL of pure water, sonicating for 1 h, and then adding 1 mL of 24 mM tannic acid solution and 1 mL of 24 mM Zn solution respectively. 2+ The solution was adjusted to pH 8 using tris-HCl (pH=8.8), and the self-assembly reaction was carried out at 400 rpm with magnetic stirring for 2 h. The solution was then washed twice with pure water to obtain the precipitate Clay@ZT. 1A (HNT@ZT) 1A PGS@ZT 1A KAL@ZT 1A ).

[0057] The precipitate was dried at 65 ℃, ground, and stored at 4 ℃.

[0058] Comparative Example 2 Clay@ZT 2B Preparation

[0059] With Clay@ZT 2The difference lies in the fact that step (2) includes weighing 100 mg of acidified nano-clay, adding 10 mL of pure water, sonicating for 1 h, and then adding 0.5 mL of 24 mM tannic acid solution and 0.5 mL of 24 mM Zn solution respectively. 2+ The solution was adjusted to pH 8 using tris-HCl (pH=8.8), and the self-assembly reaction was carried out for 1 h under magnetic stirring at 400 rpm. The solution was then washed twice with pure water to obtain the precipitate Clay@ZT. 1B ;

[0060] The precipitate was resuspended in 10 mL of pure water, and then 0.5 mL of 24 mM tannic acid solution and 0.5 mL of 24 mM Zn were added successively. 2+ The solution was adjusted to pH 8 using tris-HCl (pH=8.8), and the self-assembly reaction was carried out for 1 h under magnetic stirring at 400 rpm. Afterwards, the solution was washed twice with pure water, and the precipitate was Clay@ZT. 2B (HNT@ZT) 2B PGS@ZT 2B KAL@ZT 2B ).

[0061] The precipitate was dried at 65 ℃, ground, and stored at 4 ℃.

[0062] Example 2 Clay@ZT X The representation

[0063] 2.1 Morphological observation

[0064] A small amount of hybrid nanoclay particles from Example 1, Comparative Example 1, and Comparative Example 2 were dispersed in anhydrous ethanol and dropped onto a copper grid. After air drying, the microstructure of the hybrid nanoclay particles was observed using a transmission electron microscope.

[0065] The morphology of hybrid nanoclay particles was observed using TEM. For example... Figure 2 As shown, compare Clay@ZT 2B and Clay@ZT 1A Under the same dosage and self-assembly reaction time, the effect of staged modification is significantly better than that of single modification, and the distribution of the metal polyphenol network shell is more uniform.

[0066] like Figure 3As shown, one ZT modification is not enough to completely modify the surface of the nano-clay; after two ZT modifications, the surfaces of HNT and KAL are almost completely coated with ZT. Among them, PGS may be due to its non-planar structure and complex morphology, which leads to greater steric hindrance. ZT needs to overcome greater steric barriers to reach the active site during the modification process; after three or four ZT modifications, the surface of the nano-clay is almost completely coated with ZT.

[0067] 2.2 Fourier Transform Infrared Spectroscopy Analysis

[0068] The change in characteristic peaks was observed using Fourier transform infrared spectroscopy to determine whether MPN was successfully modified into Clay. Small amounts of ZT, Clay, and Clay@ZT from Example 1 were taken respectively. X Placed on the sample stage, the 4000-400 cm⁻¹ values ​​were measured using Fourier transform infrared spectroscopy. -1 The infrared spectrum between them.

[0069] like Figure 4 As shown, Clay@ZT X In the infrared spectrum at 1700 cm⁻¹ -1 Absorption peaks of C=O appear on both sides, at 1340 cm⁻¹. -1 The bending vibration peaks of CO appear on both sides, and at 1200 cm⁻¹ -1 The stretching vibrations of CO (phenol) are observed at the position, and these peaks coincide with TA, with the peak intensity increasing with the number of ZT modifications. (Clay@ZT) X The infrared spectra of both ZT and Clay (HNT / PGS / KAL) showed absorption peaks, indicating that ZT was successfully modified into Clay.

[0070] 2.3 Component Proportion Analysis

[0071] Clay@ZT was calculated using the acid dissolution method. X The proportions of Clay and ZT in the formula. Weigh out Clay@ZT. X Place the contents into a 1.5 mL centrifuge tube and record the mass (m0) of the centrifuge tube and the amount of Clay@ZT added. X The mass (m1) was recorded. 1 mL of 1 M dilute hydrochloric acid solution was added to dissolve the metal polyphenol network. The mixture was centrifuged at 6000 rpm for 5 minutes, and the supernatant was discarded. 1 mL of dilute hydrochloric acid solution (1 M) was added again to dissolve ZT. This process was repeated three times. The mixture was then washed three times with pure water, centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded. The mixture was then dried and weighed. The mass (m2) was recorded.

[0072] The formula for calculating the proportion of Clay is as follows: Nano clay content (%) = [(m2−m0) / (m1−m0)] × 100%.

[0073] like Figure 5 As shown, with the increase of the number of metal polyphenol network modifications, the mass percentage of Clay decreases, while the mass percentage of ZT increases. TEM results show that PGS@ZT 2 The ZT in the middle does not completely wrap the PGS, but compared with other Clays, the quality ratio results of Clay and ZT are no different, possibly because some ZT modifiers are applied to the ZT. Clay@ZT 1 The ZT content is no less than 33%, Clay@ZT 2 The ZT content is not less than 45%.

[0074] like Figure 2 As shown, under the same dosage and reaction time, the mass percentage of ZT in the two modified hybrid particles was significantly increased compared to Clay@ZT. 1A Clay@ZT 2B The ZT content in each group consistently exceeded 43%, and this balanced proportion is conducive to the full synergistic effect of the two. Therefore, Clay@ZT was selected as the primary ZT. 2 As a typical example, subsequent experiments were conducted.

[0075] 2.4 Zeta potential

[0076] For nanomaterials, testing the zeta potential can characterize their stability. The larger the absolute value of the zeta potential, the stronger the electrostatic repulsion between particles, and the more stable the dispersion system.

[0077] Weigh out ZT, Clay, and Clay@ZT respectively. X Prepare a stock solution of 1 mg / mL, disperse it by ultrasonication, take 400 μL of the stock solution and add 3600 μL of pure water, dilute and continue ultrasonic dispersion, and measure its Zeta potential using a laser particle size analyzer.

[0078] like Figure 6 As shown, Clay@ZT X Both indicate negative charge, which facilitates tight bonding with wound ulcer areas. Among them, HNT, after ZT modification, becomes HNT@ZT. 1 HNT@ZT 2 and HNT@ZT 4 The Zeta potential of HNT was almost statistically different from that of HNT, while HNT@ZT 3 The zeta potential of PGS@ZT increased compared to HNT, from -42.10 ± 1.71 mV to -36.57 ± 1.16 mV. This is likely due to the unique hollow tubular structure of HNT, with some ZT modification occurring in the hollow regions, leading to different zeta potential changes. After ZT modification, the zeta potential of PGS@ZT was significantly higher than that of HNT. X and KAL@ZTX The zeta potential of ZT was lower than that of PGS and KAL, indicating a more stable dispersion system. This may be because PGS has a rod-like structure and KAL has a layered structure, but there is no cation exchange between the layers. As a result, ZT is modified on the surface of both PGS and KAL, leading to a decreasing trend in the zeta potential.

[0079] Example 3: Hybrid nano-clay particles Clay@ZT X TA and Zn 2+ Release

[0080] Release rate (%) = (release amount / total amount) × 100%.

[0081] Clay@ZT under neutral conditions X TA release in the middle. Weigh 3 mg of Clay@ZT respectively. X Place the sample in a 1.5 mL centrifuge tube, add 1 mL of PBS solution (pH=7.4), and incubate at 37 ℃ to release TA. Centrifuge at 4000 rpm for 4 min at 1, 2, 4, 8, 12, 24, 36, and 48 h. Take 500 μL of the supernatant, add 500 μL of PBS solution (pH=7.4), resuspend, and continue to incubate at 37 ℃ to release TA. Store the supernatant at 4 ℃. After all samples have been collected, measure the amount of TA released by UV. Weigh 3 mg of Clay@ZT. X Dissolve in 1 mL of dilute hydrochloric acid solution (1 M), shake on a shaker for 3 h, then centrifuge (10000 rpm, 10 min) and collect the supernatant. Measure the total TA content in the particles using ultraviolet light. Figure 7 As shown, the release rates of the HNT, PGS, and KAL groups decreased after multiple shell modifications, indicating that the multiple shell modification method is beneficial for Clay@ZT. X The stability and slow release of TA.

[0082] Clay@ZT under acidic conditions 2 TA release from the molecule. Buffer solutions with pH values ​​of 2.2 and 4.0 were prepared using 0.1 M citric acid solution and 0.2 M disodium hydrogen phosphate solution, respectively; 3 mg of Clay@ZT was weighed out for each solution. 2Place the sample in a 1.5 mL centrifuge tube, add 1 mL of buffer solution (pH = 2.2 / 4.0), and incubate at 37 ℃ to release TA. Centrifuge at 4000 rpm for 4 min at 1, 2, 4, 8, 12, 24, 36, and 48 h, and collect 500 μL of the supernatant. Add another 500 μL of buffer solution (pH = 2.2 / 4.0), resuspend, and continue to incubate at 37 ℃ to release TA. Store the supernatant at 4 ℃. After all samples have been collected, measure the TA release amount using UV light. After 48 h of sampling, discard the supernatant, add 1 mL of dilute hydrochloric acid solution (1 M), shake on a shaker for 3 h, then centrifuge at 10000 rpm for 10 min and collect the supernatant. Measure the remaining TA content in the particles using UV light. The total TA is the total released TA plus the remaining TA content. Figure 8 The image shows the release under acidic conditions. At pH 7.4, Clay@ZT... 2 The release of TA from the sample was slow, with a release rate of approximately 20% after 48 hours; however, at pH 4.0 and 2.2, the release of TA from Clay@ZT was significantly higher. 2 The TA is released rapidly, with the release rate reaching almost 100% after 4 hours. (Clay@ZT) 2 The rapid release of TA under acidic conditions is beneficial for its application in the stomach.

[0083] Clay@ZT under different pH conditions 2 Chinese Zn 2+ Release: Buffer solutions with pH values ​​of 2.2 and 4.0 were prepared using 0.1 M citric acid solution and 0.2 M disodium hydrogen phosphate solution, respectively; 12 mg of Clay@ZT was weighed out for each solution. 2 Place the solution in a 15 mL centrifuge tube, add 4 mL of buffer (pH = 2.2 / 4.0) / PBS solution (pH = 7.4), and incubate at 37 °C to release Zn. 2+ At 1, 2, 4, 8, 12, 24, 36, and 48 h, the supernatant was centrifuged (4000 rpm, 4 min), and 3 mL of the supernatant was collected. 3 mL of buffer (pH = 2.2 / 4.0) / PBS solution (pH = 7.4) was added, and the mixture was resuspended and incubated at 37 ℃ to release Zn. 2+ The supernatant was stored at 4 °C, and Zn was determined by atomic absorption spectrometry after all samples were collected. 2+ Release amount. After 48 h of sampling, the supernatant was discarded, 1 mL of citric acid solution (0.1 M) was added, and the mixture was shaken on a shaker for 3 h. Then, it was centrifuged (10000 rpm, 10 min), and the supernatant was collected. The remaining Zn in the particles was determined by atomic absorption spectrometry. 2+ Content, Zn2+ The total amount is the total amount released plus the remaining Zn. 2+ Content. For example... Figure 9 As shown, when the pH is 7.4, Zn 2+ Slow release, at 48 h, Zn 2+ The release rate is approximately 20%; however, at pH 4.0 and 2.2, the release rate of Zn is... 2+ Almost 100% released at 2 hours, Clay@ZT 2 Chinese Zn 2+ Rapid release under acidic conditions is advantageous for use in the stomach.

[0084] The stability of the particles under neutral conditions was verified, and that of TA and Zn was also verified. 2+ The release rate is relatively low; it is unstable under acidic conditions, and TA and Zn... 2+ It is fully released in about 4 hours.

[0085] Example 4 Clay@ZT X biocompatibility

[0086] For ZT, Clay, and Clay@ZT X Hemolytic performance test was performed. 1 mg each of ZT, Clay, and Clay@ZT were weighed out. X Add 500 μL of physiological saline and 500 μL of red blood cell solution to a 1.5 mL centrifuge tube. A positive control was set up with 500 μL of physiological saline and 500 μL of red blood cell solution; a negative control was set up with 500 μL of purified water and 500 μL of red blood cell solution. Incubate the red blood cells at 37 ℃ for 1 h, then centrifuge at 3000 rpm for 5 min. Calculate the hemolysis rate based on the absorbance of the supernatant. Figure 10 As shown, ZT, Clay, Clay@ZT X The supernatant from the pure water group was similar in clarity to that from the saline group, indicating no significant difference. This suggests that the red blood cells did not rupture or hemolysis. However, the supernatant from the pure water group showed a distinct red color, indicating that the red blood cells ruptured after co-incubation with pure water, resulting in significant hemolysis. Microscopic observation of the red blood cells revealed that the red blood cells in the pure water group ruptured, while those in the saline group, ZT, Clay, and Clay@ZT groups showed rupture. X The red blood cells in the group were intact. Hemolysis rate calculations showed that ZT, Clay, and Clay@ZT... X The hemolysis rate was less than 5% in all groups, indicating that the blood compatibility of each group was good.

[0087] GES-1 cells and Clay@ZT were detected by MTT assay. X Cell viability after co-incubation was assessed using ZT, Clay, and Clay@ZT. XCytotoxicity. ZT, Clay, and Clay@ZT were tested at concentrations of 100 μg / mL. X After incubation with GES-1 cells for 24 h, the absorbance at 490 nm was measured using a microplate reader. Data analysis showed that the cell viability in each group exceeded 80%, with no statistically significant difference compared to the control group, indicating good biocompatibility. This demonstrates that ZT, Clay, and Clay@ZT are effective inhibitors of the novel coronavirus. X It has good cell compatibility.

[0088] GES-1 cells were used for cell viability / deadness staining assays. GES-1 cells were seeded at a density of 20,000 cells / well in 24-well plates, with 1 mL per well. The plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 h, and cell growth was continuously observed. ZT, Clay, and Clay@ZT cells, sterilized for 3 h under UV light, were weighed separately. X 2 mg of each sample was added to 5 mL centrifuge tubes, along with 2.5 mL of DMEM complete medium to prepare a stock solution of 800 μg / mL. This stock solution was then diluted to 100 μg / mL using DMEM complete medium. After cell adhesion, the old medium was aspirated, and the cells were washed 1-2 times with PBS. 1 mL of the diluted sample solution was added to each well, and the cells were incubated at 37 °C in a 5% CO2 incubator for 24 h. Cells were then stained with calcein and PI dye and incubated at 37 °C in a 5% CO2 incubator in the dark for 30 min. The old medium was aspirated, and the cells were washed 1-2 times with PBS. 200 μL of basal medium was added to each well, and the cells were imaged using an inverted fluorescence microscope. Figure 11 As shown, ZT, Clay, and Clay@ZT X Incubation of GES-1 cells at this drug concentration for 24 h did not affect normal cell growth and showed no cytotoxicity.

[0089] Example 5 Clay@ZT X Antibacterial properties

[0090] Methicillin-resistant Staphylococcus aureus (MRSA) was selected to test the antimicrobial properties of the hybrid particles. MRSA was scraped into LB broth using a sterile inoculation loop in a laminar flow hood and incubated overnight at 37 °C with shaking incubator (150 rpm). The LB broth was then centrifuged (8000 rpm, 5 min), the supernatant was discarded, and the broth was diluted with sterile physiological saline to obtain a 10-1 concentration. 6 Prepare a bacterial suspension at CFU / mL. Use the bacterial suspension, physiological saline, and Clay@ZT... X Three different concentrations (50, 100, 150 μg / mL) of Clay@ZT were prepared. XThe suspension was incubated at 37 ℃ with shaking for 12 h (150 rpm). After the incubation, 5 μL was dropped onto an agar plate and incubated in a 37 ℃ constant temperature incubator for 8 h before observation and photography.

[0091] Antibacterial performance results as follows Figure 12 As shown, when Clay@ZT X At a concentration of 50-150 μg / mL, Clay@ZT X It has good antibacterial properties, and the more times it is modified, the better the antibacterial properties.

[0092] Example 6 Clay@ZT X hemostatic properties

[0093] Select Clay@ZT 2 Liver hemostasis experiment was performed. First, the weight of the filter paper was weighed and recorded as m1. After anesthetizing the mice, they were placed ventrally, and the tissue fluid near the liver was wiped clean. Filter paper was placed under the liver, and a wound was created in the liver using the needle of a 10 mL syringe. Bleeding was observed, and after 10 seconds, 5 mg of Clay@ZT was injected. 2 The filter paper was placed on the wound, while the control group's wound was left untreated. The time was 1 minute. A photo was taken and the weight of the blood-absorbing filter paper was recorded as m2. The difference between m2 and m1 was the amount of bleeding.

[0094] In the control group, no bleeding stopped within 1 minute, while the area of ​​bloodstains in the treatment group was significantly smaller than that in the control group. Figure 13 As shown, the bleeding volume in the treatment group was significantly less than that in the control group, indicating that Clay@ZT X It has a hemostatic effect.

[0095] Example 7 Clay@ZT X Adhesion force

[0096] Apparent adhesion assays used porcine skin as the substrate. Clay@ZT at a concentration of 100 mg / mL was prepared using PBS. 2 Granular solution: Two 1 × 2.5 cm pieces of pigskin were treated with 20 μL of Clay@ZT solution. 2 The particles were bonded together with either the solution or PBS (media control group). After pressing with a 50 g weight for 30 seconds, one piece of pigskin was lifted off the plate, and the results were observed in each group. 2 The two pieces of pigskin were successfully glued together, while the PBS group failed to glue the pigskin together.

[0097] Overlap shear tests were conducted using a universal testing machine equipped with a 1000 N loading unit. Similar to the apparent adhesion test, overlap shear test samples were prepared and placed between the clamps of the universal testing machine. The top clamp moved upwards at a speed of 10 mm / min, and the force changes were recorded during the process to quantitatively analyze the adhesion force of the hybrid particles. Figure 14 As shown, HNT@ZT 2 KAL@ZT has the strongest adhesion strength. 2 The adhesion strength is the weakest. Hybrid particles possess adhesive force, which is beneficial for adhering to the gastric mucosa. This not only prolongs their residence time in the stomach but also ensures stable drug release, thereby achieving a more efficient therapeutic effect.

[0098] Example 8 Clay@ZT 2 Prevention of gastric mucosal damage by nano-clay

[0099] 8.1 Establishment of an ethanol-induced acute gastric mucosal injury model in mice

[0100] Thirty-four male KM mice weighing 35-40g were randomly divided into six groups. Five experimental groups each contained six mice, and a separate normal control group consisted of four mice. After one week of acclimatization, experiments were conducted. On day 0, the normal control and model groups were administered 0.25 mL of pure water by gavage, while the drug-treated groups were administered 0.25 mL of Clay@ZT by gavage. 2 The solution (0.50 mg / mL) was administered and weighed. After 6 consecutive days of gavage and weighing, the mice were fasted but allowed free access to water for 24 hours. One hour after gavage administration of pure water and granular solution, the model group and the treatment group were given anhydrous ethanol (0.1 mL / 10 g) to induce acute gastric mucosal injury, based on body weight. Two hours later, the mice were anesthetized with 1% sodium pentobarbital solution, and samples were taken (ocular blood, gastric tissue, heart, liver, spleen, lungs, and kidneys) using autoclaved surgical instruments.

[0101] After cleaning the stomach tissue with physiological saline, it was placed on filter paper and photographed for observation. The stomach contents were removed and placed in a 1.5 mL centrifuge tube and stored at -80 ℃ for later use. Then, the stomach tissue was dissected along the greater curvature of the stomach, and the residue on the surface of the gastric mucosa was cleaned with physiological saline. It was then placed on filter paper and photographed for observation of the gastric mucosa and gastric serosa. The heart, liver, spleen, lungs and kidneys were cleaned with physiological saline and dried. They were then weighed, recorded and photographed for observation.

[0102] The removed stomach, heart, liver, spleen, lung, and kidney tissues were fixed, embedded, and sectioned for subsequent staining experiments.

[0103] Figure 15Image A shows a representative image of mouse gastric tissue. The model group exhibited numerous hemorrhagic streaks, and the mucosa was also red, indicating significant inflammation and ulceration. The drug-treated group showed significantly fewer hemorrhagic streaks than the model group, and the mucosa appeared the same flesh color as the normal group, without significant inflammation or ulceration. The ulcer area was statistically analyzed. Figure 15 As shown in Figure B, the ulcer area ratio in the model group was 53.91 ± 6.18%, HNT@ZT 2 The ulcer area ratio in the group was 15.40 ± 4.71%, PGS@ZT 2 The ulcer area ratio in the group was 9.30 ± 5.48%, KAL@ZT 2 The ulcer area ratio in the group was 15.94 ± 5.06%, and both representative images and quantitative statistical results indicated that Clay@ZT 2 It has a certain preventive effect on gastric mucosal damage.

[0104] Figure 16 This is a line graph showing the change in mouse body weight during the modeling process. The body weight of mice in different groups gradually increased. On day 5, the mice were fasted, so on day 6, their body weight decreased by about 5-6 g. Compared with the normal group, there was no statistically significant difference between the model group and the drug-treated group.

[0105] 8.2 Histological evaluation of gastric tissue

[0106] Gastric tissue sections were stained with H&E and PAS.

[0107] Hematoxylin and eosin (H&E) staining is used to observe the integrity and orderly arrangement of cells in the gastric mucosa to assess the protective capacity of the gastric mucosa. Figure 17 In the normal group (Clay@ZT), the glands of the mice were neatly arranged, the gastric tissue structure was intact, and there was no edema, congestion, or cell shedding in the gastric mucosa. In the model group, gastric epithelial cell shedding and gastric mucosal damage could be observed. In the drug-treated group, the gastric tissue was relatively intact, with no epithelial cell shedding or mucosal damage observed; the glands were intact and orderly arranged, indicating that Clay@ZT 2 It can prevent damage to the gastric mucosa.

[0108] PAS staining is used to assess gastric mucus secretion levels. Gastric mucus, as a mucosal defense factor, plays a crucial role in preventing gastric mucosal damage. In PAS staining, the purple portion represents PAS-positive areas. Figure 17 As shown in Figure B, no PAS-positive areas were observed in the normal group mice, a small number of PAS-positive areas were observed in the model group, and a large number of positive areas were observed in the drug-treated group, indicating that Clay@ZT 2 It can promote the secretion of gastric mucus and play a role in protecting the gastric mucosa.

[0109] 8.3 Detection of oxidative stress indicators in gastric tissue

[0110] Ethanol can directly damage the gastric mucosa through disruption, dehydration, and mucosal cytotoxicity. Then, ethanol can indirectly damage the gastric mucosa by inducing inflammation, oxidative stress, and apoptosis through leukocyte recruitment.

[0111] Take an appropriate amount of gastric tissue stored at -80 ℃, thaw it, rinse it with 4 ℃ physiological saline, blot it dry with filter paper, weigh it, and prepare a 10% (w / w) gastric tissue homogenate with PBS. Centrifuge the homogenate at 4 ℃ (10000 rpm, 10 min), collect the supernatant, aliquot it, and store it at -80 ℃. Detect SOD activity, CAT activity, GSH level, and MDA content in the gastric tissue supernatant according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd.) to assess the relevant indicators of oxidative stress in each group of gastric tissue.

[0112] like Figure 18 As shown, the gastric mucosal MDA content in the model group was significantly increased, while SOD and CAT activities and GSH levels decreased, indicating that lipid peroxidation is involved in the pathophysiological process of ethanol-induced gastric mucosal damage in mice. The drug-treated group reduced oxidative stress by increasing GSH production, increasing SOD and CAT activity, and decreasing MDA content. These results demonstrate that Clay@ZT... 2 By enhancing antioxidant capacity, it exerted a good preventive effect on gastric mucosal damage in mice.

[0113] 8.4 Biosafety Analysis

[0114] Visceral indices for the heart, liver, spleen, lungs, and kidneys were calculated for each group. Statistical results showed that, compared with the normal group, the model group and Clay@ZT 2 There were no statistically significant differences in visceral indices among the groups, demonstrating good biocompatibility.

[0115] Further biosafety assessment involved H&E staining of tissue sections from the heart, liver, spleen, lungs, and kidneys of each group. No damage or excessive inflammation was found, confirming the biosafety of Clay@ZT. 2 It exhibits low biotoxicity and good biosafety.

[0116] 8.5 Summary

[0117] The modeling results showed that the bleeding in the treatment group was significantly less than that in the model group, and the ulcer area was also significantly reduced. H&E staining showed that the gastric mucosa in the treatment group was more intact. PAS staining results showed that the treatment group had a large number of PAS positive areas, indicating that it can promote gastric mucus secretion and prevent gastric mucosal damage. Oxidative stress index detection showed that the treatment group reduced oxidative stress and prevented gastric mucosal damage by increasing GSH, enhancing SOD and CAT activity, and reducing MDA content. Biosafety assessment showed that there was no statistically significant difference in visceral indices between the treatment group and the normal group, and H&E staining of the heart, liver, spleen, lungs and kidneys showed no damage or excessive inflammation, proving that it has low biotoxicity and good safety.

[0118] Example 9 Clay@ZT 2 Treatment of gastric mucosal damage with nano-clay

[0119] 9.1 Establishment of an ethanol-induced acute gastric mucosal injury model in mice

[0120] Forty male KM mice weighing 35-40g were randomly divided into seven groups. Six experimental groups each contained six mice, and a separate control group of four mice was established. After one week of acclimatization, the mice were weighed on day -2 and then fasted for 24 hours with free access to water. On day -1, the mice were weighed again. The experimental groups were administered anhydrous ethanol (0.1 mL / 10g) by gavage, while the control group was administered pure water (0.1 mL / 10g) by gavage. Modeling was completed two hours later.

[0121] On day 0, the normal group and the model group were administered 0.25 mL of pure water by gavage, the positive control group was administered 0.25 mL of omeprazole (3.6 mg / mL) by gavage, and the treatment group was administered 0.25 mL of Clay@ZT by gavage. 2 The solution (0.50 mg / mL) was administered by gavage for 4 consecutive days. On the 4th day, the mice were anesthetized with 1% sodium pentobarbital solution, and surgical instruments sterilized by autoclaving were used to collect samples (eyeballs, blood, stomach tissue, heart, liver, spleen, lungs, and kidneys).

[0122] After cleaning the stomach tissue with physiological saline, it was placed on filter paper and photographed for observation. The stomach contents were removed and placed in a 1.5 mL centrifuge tube and stored at -80 ℃ for later use. Then, the stomach tissue was dissected along the greater curvature of the stomach, and the residue on the surface of the gastric mucosa was cleaned with physiological saline. It was then placed on filter paper and photographed for observation of the gastric mucosa and gastric serosa. The heart, liver, spleen, lungs and kidneys were cleaned with physiological saline and dried. They were then weighed, recorded and photographed for observation.

[0123] The removed stomach, heart, liver, spleen, lung, and kidney tissues were fixed, embedded, and sectioned for subsequent staining experiments.

[0124] Figure 19Image A shows a representative image of mouse gastric tissue; large areas of red hemorrhage were observed in the gastric mucosa of the model group; the omeprazole group and Clay@ZT... 2 The group only showed localized redness, with most of the mucosa appearing the same flesh color as the normal group; the ulcer area ratio was statistically analyzed, such as... Figure 19 As shown in Figure B, the ulcer area ratio in the model group was 16.77 ± 3.24%, while the ulcer area ratio in the omeprazole group was 1.44 ± 1.56%. (HNT@ZT) 2 The ulcer area ratio in the group was 0.71 ± 1.07%, PGS@ZT 2 The ulcer area ratio in the group was 0.25 ± 0.38%, KAL@ZT 2 The ulcer area ratio of the treatment group was 0.17 ± 0.28%. Representative images and quantitative statistical results showed that the treatment group only had local redness and the ulcer area was significantly reduced.

[0125] Figure 20 This is a line graph showing the change in mouse body weight during the modeling process. During the experiment, there was no statistically significant difference between the omeprazole group and the treatment group compared with the normal group. However, by day 3, the body weight of the model group was smaller than that of the normal group, indicating that gastric mucosal damage affected the mice's eating ability, which indirectly demonstrates the therapeutic effect of the omeprazole group and the treatment group on gastric mucosal damage.

[0126] 9.2 Histological evaluation of gastric tissue

[0127] Gastric tissue sections were stained with H&E and PAS.

[0128] Hematoxylin and eosin (H&E) staining is used to observe the integrity and orderly arrangement of cells in the gastric mucosa to assess the protective capacity of the gastric mucosa. Figure 21 As shown in Figure A, the glands of the normal group mice were neatly arranged, the gastric tissue structure was intact, and there were no edema, congestion, or cell shedding in the gastric mucosa epithelium. In the model group, gastric epithelial cell shedding and gastric mucosal damage could be observed. In the omeprazole group and the drug-treated group, the gastric tissue was relatively intact. Among them, compared with the drug-treated group, milder epithelial cell shedding and mucosal damage could be observed in the omeprazole group.

[0129] PAS staining was used to assess gastric mucus secretion levels. Gastric mucus, as a mucosal defense factor, plays an important role in preventing gastric mucosal damage. Figure 21 As shown in Figure B, no PAS-positive areas were observed in the normal group mice, a small number of PAS-positive areas were observed in the model group, and a large number of positive areas were observed in the omeprazole group and the drug-treated group. The drug-treated group had more positive areas, which means that it can better promote gastric mucus secretion and gastric mucosal repair.

[0130] 9.3 Detection of oxidative stress indicators in gastric tissue

[0131] Take an appropriate amount of gastric tissue stored at -80 ℃, thaw it, rinse it with 4 ℃ physiological saline, blot it dry with filter paper, weigh it, and prepare a 10% (w / w) gastric tissue homogenate with PBS. Centrifuge the homogenate at 4 ℃ (10000 rpm, 10 min), collect the supernatant, aliquot it, and store it at -80 ℃. Detect SOD activity, CAT activity, GSH level, and MDA content in the gastric tissue supernatant according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd.) to assess the relevant indicators of oxidative stress in each group of gastric tissue.

[0132] like Figure 22 As shown, the MDA content in the gastric mucosa of the model group was significantly increased, while the activities of SOD and CAT and the level of GSH decreased, indicating that lipid peroxidation is involved in the pathophysiological process of ethanol-induced gastric mucosal damage in mice. (Clay@ZT) 2 It exhibits therapeutic effects against oxidative stress by increasing GSH production, increasing SOD and CAT activity, and reducing MDA content.

[0133] The above results indicate that Clay@ZT 2 By reducing oxidative stress in the gastric mucosa, omeprazole exerted a good therapeutic effect on gastric mucosal damage in mice. Omeprazole mainly treats gastric mucosal damage by inhibiting gastric acid secretion; therefore, the oxidative damage in the omeprazole group was better than that in the model group, but not as good as that in Clay@ZT. 2 Group.

[0134] 9.4 Biosafety Analysis

[0135] Visceral indices for the heart, liver, spleen, lungs, and kidneys were calculated for each group. Statistical results showed that, compared with the normal group, the model group, omeprazole group, and Clay@ZT group had significantly lower visceral indices. 2 There were no statistically significant differences in visceral indices among the groups, demonstrating good biocompatibility.

[0136] Further biosafety assessment involved H&E staining of heart, liver, spleen, lung, and kidney tissue sections from each group. No damage or excessive inflammation was observed, confirming the biosafety of Clay@ZT. 2 It exhibits low biotoxicity and good biosafety.

[0137] 9.5 Summary

[0138] The modeling results showed that the drug-treated group only had localized redness, and the ulcer area was significantly reduced. H&E staining showed that the drug-treated group had less epithelial cell shedding and mucosal damage. PAS staining results showed that both the omeprazole group and the drug-treated group had a large number of PAS positive areas, with the drug-treated group having more positive areas, indicating that it could better promote gastric mucus secretion and gastric mucosal repair. Oxidative stress index detection showed that the drug-treated group reduced oxidative stress by increasing GSH, enhancing SOD and CAT activity, and reducing MDA content, which was more effective than the omeprazole group. Biosafety assessment showed that there was no statistically significant difference in visceral indices between the drug-treated group and the normal group, and H&E staining of the heart, liver, spleen, lungs, and kidneys showed no damage or excessive inflammation, proving that it has low biotoxicity and good safety.

[0139] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Use of a hybrid nanoclay for the preparation of a medicament for the treatment of gastric ulcer, characterized in that, The hybrid nanoclay comprises hybrid nanoclay particles with adhesion, the hybrid nanoclay particles comprising a nanoclay surface-modified with a metal polyphenol network, the metal polyphenol network being formed from Zn 2+ and tannic acid self-assembly; the mass percentage of the metal polyphenol network in the hybrid nanoclay particles is not less than 33%.

2. Use of a hybrid nanoclay for the preparation of a medicament for the prevention of gastric ulcer, characterized in that, The hybrid nanoclay comprises hybrid nanoclay particles with adhesion, the hybrid nanoclay particles comprising a nanoclay surface-modified with a metal polyphenol network, the metal polyphenol network being formed from Zn 2+ and tannic acid self-assembly; the mass percentage of the metal polyphenol network in the hybrid nanoclay particles is not less than 33%.

3. Use of the hybrid nanoclay according to claim 1 or 2, characterized in that, The metal polyphenol network forms a metal polyphenol network shell on the surface of the nanoclay through not less than one modification.

4. Use of the hybrid nanoclay according to claim 3, characterized in that, It is characterized in that, The nanoclay is a layered nanoclay or a rod-like nanoclay.

5. Use of the hybrid nanoclay according to claim 1 or 2, characterized in that, The preparation method of the hybrid nanoclay comprises the following steps: Step (1): adding an acid solution into the nanoclay to obtain an acidized nanoclay; Step (2): Resuspend the acidized nanoclay, add tannic acid solution and Zn 2+ solution, adjust the solution pH to 8, and conduct a self-assembly reaction under stirring conditions for 1-2 h, obtain a precipitate after washing with pure water, and complete a single modification operation; repeat the above modification operation 1-3 times on the precipitate to obtain the hybrid nanoclay particles Clay@ZT X , and X represents the number of modifications.

6. Use of the hybrid nanoclay according to claim 5, characterized in that, In the step (2), X≥2.

7. Use of the hybrid nanoclay according to claim 5, characterized in that, The molar concentration ratio of the tannic acid solution and Zn 2+ solution was 1:

1.

8. Use of the hybrid nanoclay according to claim 5, characterized in that, The stirring condition in the step (2) is 400 rpm magnetic stirring.

Images