CN113788482B

CN113788482B - Modified montmorillonite, preparation method thereof and application thereof in hemostasis

Info

Publication number: CN113788482B
Application number: CN202110978705.0A
Authority: CN
Inventors: 马飞; 孟志云; 孙云波; 窦桂芳; 甘慧; 顾若兰; 朱晓霞; 吴卓娜; 孙文种; 韩鹏
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2023-04-07
Anticipated expiration: 2041-08-25
Also published as: CN113788482A

Abstract

The invention providesA modified montmorillonite, its preparation method and application in hemostasis are provided. The modified montmorillonite is prepared from montmorillonite and tranexamic acid. According to the structural characteristics of montmorillonite and tranexamic acid, the invention utilizes the intercalation composite mechanism, selects montmorillonite as an intercalation host material and tranexamic acid as an intercalation guest material to prepare the modified montmorillonite. The invention adopts tranexamic acid to modify the montmorillonite and replaces Na in the montmorillonite ⁺ 、Ca ²⁺ The crystal structure and the crystallinity of the cationic polymer are optimized, the properties of the cationic polymer are changed, the cationic polymer shows more excellent in-vitro hemostatic capability in an in-vitro blood coagulation time experiment, and the hemostatic effect is better compared with that of montmorillonite; can also reduce the damage of montmorillonite to erythrocytes, has no cytotoxicity, and is safer to use than montmorillonite.

Description

Modified montmorillonite, preparation method thereof and application thereof in hemostasis

Technical Field

The invention relates to the technical field of hemostatic materials, in particular to modified montmorillonite, a preparation method thereof and application thereof in hemostasis.

Technical Field

With the increasing aging of the population in China and the influence of factors such as environment and the like, the incidence of malignant tumors is increasing day by day, radiotherapy is currently used as one of effective methods for treating tumors, plays a vital role in the comprehensive treatment of complicated abdominal and pelvic cavity malignant tumors, and plays an important role in the aspects of improving the local control rate of the tumors, improving the life quality of patients and the like. Radioactive Enteritis (RE) is mostly intestinal radioactive injury caused by abdominal or pelvic malignant tumors after receiving radiotherapy, is a characteristic toxic reaction of the radiotherapy and is also a common complication of the radiotherapy, and the incidence rate of the Radiation Enteritis (RE) is increased year by year along with the wide application of radiotherapy technology. Studies have shown that small intestinal epithelial cells are one of the most vulnerable sites to radiation, and that people exposed to doses above 10Gy suffer from a rather severe gastrointestinal syndrome (GIS), manifested by diarrhea, electrolyte disturbances, hematochezia and even subacute death. At present, no clear standard treatment scheme exists for radiation enteritis, the combination is mainly used for combined treatment and comprehensive treatment, and the intestinal hemostasis is vital in the prevention and treatment process.

Montmorillonite is a natural aluminosilicate clay ore with a two-dimensional plane layer structure, which is composed of silicon-oxygen tetrahedron and aluminum-oxygen octahedron, tetrahedral sheet (T) octahedron sheets (O) are arranged alternately to form a most main structural unit layer, the unit layer is 2:1 layer, namely two tetrahedral sheets sandwich a T-O-T layer composed of octahedron sheets, each structure is about 1nm thick and 100nm long, simultaneously, the top oxygen of tetrahedron points to the center of the structural layer and shares with the octahedron, and finally the three layers are connected together. Water molecules or other polar molecules tend to intrude between the montmorillonite layers, causing swelling. The montmorillonite can rapidly absorb water in wound blood, enrich platelets and blood coagulation factors to activate human blood coagulation cascade reaction, and form a clay layer to seal a wound to play a role in stopping bleeding. The activated blood coagulation factors VII, VIII and XI activate human blood coagulation cascade reaction to promote blood coagulation system, and has the advantages of no heat release, no animal or human originated protein, low cost, etc.

However, the existing montmorillonite has a certain damage effect on erythrocytes in the hemostasis process, and the hemostasis performance of the montmorillonite is still to be further improved. If the montmorillonite can be modified, the hemostatic and safety performance of the montmorillonite can be further improved, and the montmorillonite has better application value.

Disclosure of Invention

Constraints to the above-mentioned existenceThe invention provides modified montmorillonite, a preparation method thereof and application thereof in hemostasis. According to the structural characteristics of montmorillonite and tranexamic acid, the invention utilizes the intercalation composite mechanism, selects montmorillonite as an intercalation host material and tranexamic acid as an intercalation guest material to prepare the modified montmorillonite. The invention adopts tranexamic acid to modify the montmorillonite and replaces Na in the montmorillonite ⁺ 、Ca ²⁺ The crystal structure and the crystallinity of the montmorillonite are optimized, the properties of the montmorillonite are changed, the montmorillonite-montmorillonite composite material shows more excellent in-vitro hemostatic ability in an in-vitro coagulation time experiment, and the montmorillonite composite material has a better hemostatic effect; can also reduce the damage of the montmorillonite to erythrocytes, has no cytotoxicity effect and is safer to use compared with the montmorillonite.

In order to achieve the purpose, the invention adopts the following technical scheme:

one of the purposes of the invention is to provide modified montmorillonite prepared from raw materials including montmorillonite and tranexamic acid.

Preferably, the first and second liquid crystal materials are,

in the modified montmorillonite, tranexamic acid is inserted into the interlayer of the montmorillonite to replace cations among the montmorillonite layers; the cation comprises Ca ²⁺ 、Mg ²⁺ 、Na ⁺ 。

Preferably, the first and second liquid crystal materials are,

in the modified montmorillonite, the mass ratio of tranexamic acid to montmorillonite is (0.075-0.15): 1.

The second purpose of the invention is to provide a preparation method of the modified montmorillonite which is the first purpose of the invention, wherein the modified montmorillonite is prepared by carrying out intercalation reaction on raw materials including montmorillonite and tranexamic acid.

Preferably, the first and second liquid crystal materials are,

(1) Uniformly dispersing the montmorillonite in water, and performing swelling treatment to obtain montmorillonite dispersion liquid;

(2) And adding tranexamic acid into the montmorillonite dispersion liquid, then adding a pH regulator to regulate the pH value to be acidic, heating for reaction, and carrying out post-treatment to obtain the modified montmorillonite.

Preferably, the first and second liquid crystal materials are,

in the step (1), the step (c),

the montmorillonite is medicinal montmorillonite;

the mass volume percentage concentration of the montmorillonite dispersion liquid is 1-5w/v%; preferably 1.5-3.5w/v%;

the conditions of the swelling treatment are as follows: heating and swelling under stirring.

In the present invention, the conditions for the swelling treatment are not limited so much as to enable the swelling treatment.

Preferably, the first and second liquid crystal materials are,

in the step (1), the step (c),

the heating temperature of the swelling treatment is 50-80 ℃, and the treatment time is 4-8h.

Preferably, the first and second liquid crystal materials are,

in the step (2),

adding 0.005-0.03mol of tranexamic acid per gram of montmorillonite based on montmorillonite contained in the montmorillonite dispersion liquid; preferably 0.008 to 0.02mol;

the pH regulator is inorganic acid.

Adjusting the pH to 3-5;

the post-treatment comprises centrifugation, washing, freeze drying and grinding.

Preferably, the first and second liquid crystal materials are,

in the step (2),

the pH adjusting agent comprises nitric acid; other pH-adjusting substances commonly used in the art may also be used, such as hydrochloric acid, sulfuric acid, and the like.

In the present invention, the conditions of the intercalation reaction are not particularly limited so as to enable the intercalation reaction.

Preferably, the first and second liquid crystal materials are,

the stirring speed of the heating reaction is 250-500r/min;

the heating temperature of the heating reaction is 50-70 ℃, and the reaction time is 40-55h.

The third purpose of the invention is to provide the application of the modified montmorillonite which is one of the purposes of the invention in hemostasis.

Preferably, the first and second liquid crystal materials are,

the hemostasis includes intestinal hemostasis.

The reaction principle is as follows:

in a water system, montmorillonite has fine and irregular surfaces and negative charges because montmorillonite unit cell particles are connected with water in a hydrogen bond mode through hydroxyl groups between layers, and finally, continuous network structures are formed by the connection among the particles to form uniform liquid. When the stirring is carried out by external force, the liquid can present good fluidity, and once the external force is removed, the liquid can automatically recover into gel. Montmorillonite is dispersed in a solution mainly by a colloidal state, has a small mineral particle size, can be dissociated into particles or small unit cells in water, and each unit cell has the same negative charge and is mutually repulsive, so that the unit cells cannot be aggregated into large particles in a dilute solution, and thus the montmorillonite can show excellent dispersibility and suspensibility in water. The isoelectric point of tranexamic acid is 6.2-6.8, tranexamic acid has positive charge under acidic condition (pH less than pI), and free H under acidic condition ⁺ OH bound to montmorillonite ^- The reaction causes more positive ions to be exposed, and the tranexamic acid can generate intercross ions Ca with the montmorillonite under the action of external force ²⁺ 、Mg ²⁺ And the like, thereby preparing the novel intercalated modified montmorillonite.

Compared with the prior art, the invention has the following advantages:

the invention adopts tranexamic acid to modify the montmorillonite and replaces Na in the montmorillonite ⁺ 、Ca ²⁺ When cations are used, the crystal structure and the crystallinity of the montmorillonite are optimized, the properties of the montmorillonite are changed, the montmorillonite composite material shows more excellent in-vitro hemostatic capability in an in-vitro blood coagulation time experiment, and the montmorillonite composite material has a better hemostatic effect.

In the safety research, the modified montmorillonite can reduce the hemolytic effect of the montmorillonite, reduce the damage of the montmorillonite to erythrocytes, has no cytotoxicity effect and is safer to use compared with the montmorillonite.

The modified montmorillonite can reduce the content of endotoxin in the serum of a radiated mouse in a mouse experiment for intervening the radioactive enteritis, and meanwhile, the histopathology of small intestine discovers that the modified montmorillonite has a certain effect on the protection and repair of the epithelial cells of the small intestine of the mouse with the radioactive enteritis and the maintenance of the integrity of the intestinal mucosa.

The modified montmorillonite of the invention is a safer and more effective hemostatic which can be used for treating intestinal bleeding.

Drawings

FIG. 1 is an infrared spectrum of three of MMT (montmorillonite), TXA (tranexamic acid) and TXA-MMT (modified montmorillonite);

FIG. 2 is an XRD data pattern of three MMT (montmorillonite), TXA (tranexamic acid) and TXA-MMT (modified montmorillonite);

FIG. 3 is a plot of the thermal weight loss of MMT (montmorillonite);

FIG. 4 is a thermogravimetric plot of TXA (tranexamic acid);

FIG. 5 is a thermogravimetric plot of TXA-MMT (modified montmorillonite);

FIG. 6 is a Zeta potential test plot of MMT (montmorillonite), TXA (tranexamic acid) and TXA-MMT (modified montmorillonite);

FIG. 7 is an SEM electron microscope test chart of the MMT (montmorillonite), TXA (tranexamic acid) and TXA-MMT (modified montmorillonite);

FIG. 8 is the results of an in vitro cytotoxicity test of TXA-MMT (modified montmorillonite);

FIG. 9 is the results of in vitro hemolytic assays for both MMT (montmorillonite) and TXA-MMT (modified montmorillonite);

FIG. 10 shows the in vitro clotting time results for MMT (montmorillonite), TXA (tranexamic acid) and TXA-MMT (modified montmorillonite);

FIG. 11 is a graph of histopathological changes in the mouse ileum (HE stain X200);

wherein, in FIG. 7, (a-b) is an SEM electron microscope test chart of TXA-MMT (modified montmorillonite); (c-d) SEM electron microscope test picture of MMT (montmorillonite); (e-f) is an SEM (scanning electron microscope) test picture of TXA (tranexamic acid);

wherein, in FIG. 10, (a) is a graph of in vitro coagulation time result data; (b) is the picture of the in vitro coagulation time experiment;

wherein, in FIG. 11, (a) is a pathological change map of normal mouse ileum tissue; (b) Is a pathological change map of the mouse ileum tissue three days after irradiation; (c) Is a pathological change graph of mouse ileum tissue after montmorillonite intervention; (d) Is a pathological change graph of mouse ileum tissue after modified montmorillonite intervention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It is to be understood that the description herein is only illustrative of the present invention and is not intended to limit the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention. The reagents and instruments used in the present invention are commercially available, and the characterization means involved can be referred to the description in the prior art, which is not repeated herein.

For a further understanding of the present invention, reference will now be made in detail to the preferred embodiments of the present invention.

The raw material sources are as follows:

experimental reagent and animal

Montmorillonite (pharmaceutical grade), shanghai alading biotechnology limited;

tranexamic acid (99%), bailingwei technologies, beijing;

nitric acid, isopropanol and phenol are analytically pure, national medicine group chemical reagent limited;

PBS, DMEM medium, sigma, usa;

fetal bovine serum, 0.25% pancreatin-EDTA, siemer fei ltd, usa;

MTT, beijing Sorley technologies, inc.

Experimental animals: healthy male New Zealand rabbits (2.0-2.5 kg), healthy female C57BL/6J mice (18-22 g), purchased from Beijing Keyu animal breeding center, license number: SCXK (Jing) 2018-0010.

Example 1

Preparation of modified montmorillonite

Accurately weighing medicinal montmorillonite, dispersing in deionized water to obtain 2% (w/v) suspension, placing in a flask, and swelling at 60 deg.C under stirring at 350r/min for 6 hr to obtain montmorillonite dispersion. Adding 0.01mol of tranexamic acid into each gram of montmorillonite according to the montmorillonite contained in the montmorillonite dispersion liquid, adjusting the pH to be about 4 by using nitric acid, continuously reacting for 48 hours under the stirring condition of 60 ℃ and 350r/min, centrifuging a product after the reaction is finished, washing the product with deionized water for three times, freezing the product in a refrigerator at the temperature of-80 ℃, and then freeze-drying and grinding the product to obtain a white powdery sample, namely the modified montmorillonite.

In the modified montmorillonite prepared by the method, the mass ratio of tranexamic acid to montmorillonite is 0.1.

Example 2

Preparation of modified montmorillonite

Accurately weighing medicinal montmorillonite, dispersing in deionized water to obtain 1.5% (w/v) suspension, placing in a flask, and swelling at 50 deg.C under stirring at 300r/min for 4 hr to obtain montmorillonite dispersion. Adding 0.008mol of tranexamic acid into each gram of montmorillonite according to the montmorillonite contained in the montmorillonite dispersion liquid, adjusting the pH to about 3 by using nitric acid, continuously reacting for 50 hours under the stirring conditions of 70 ℃ and 400r/min, centrifuging a product after the reaction is finished, washing the product with deionized water for three times, freezing the product in a refrigerator at the temperature of-80 ℃, and then freeze-drying and grinding the product to obtain a white powdery sample, namely the modified montmorillonite.

In the modified montmorillonite prepared by the method, the mass ratio of tranexamic acid to montmorillonite is 0.075.

Example 3

Preparation of modified montmorillonite

Accurately weighing medicinal montmorillonite, dispersing in deionized water to obtain 3% (w/v) suspension, placing in a flask, and swelling at 78 deg.C under stirring at 400r/min for 7 hr to obtain montmorillonite dispersion. Adding 0.02mol of tranexamic acid into each gram of montmorillonite according to the weight of the montmorillonite contained in the montmorillonite dispersion liquid, adjusting the pH to be about 4 by using nitric acid, continuously reacting for 55 hours under the stirring conditions of 55 ℃ and 300r/min, centrifuging a product after the reaction is finished, washing the product with deionized water for three times, freezing the product in a refrigerator at the temperature of-80 ℃, and then freeze-drying and grinding the product to obtain a white powdery sample, namely the modified montmorillonite.

In the modified montmorillonite prepared by the method, the mass ratio of tranexamic acid to montmorillonite is 0.12.

Example 4

Preparation of modified montmorillonite

Accurately weighing medicinal montmorillonite, dispersing in deionized water to obtain 3.5% (w/v) suspension, placing in a flask, and swelling at 65 deg.C under 500r/min stirring for 7h to obtain montmorillonite dispersion. Adding 0.025mol of tranexamic acid into each gram of montmorillonite according to the montmorillonite contained in the montmorillonite dispersion liquid, adjusting the pH to be about 5 by using nitric acid, continuously reacting for 40h under the stirring conditions of 50 ℃ and 300r/min, centrifuging a product after the reaction is finished, washing the product with deionized water for three times, freezing the product in a refrigerator at the temperature of-80 ℃, and then freeze-drying and grinding the product to obtain a white powdery sample, namely the modified montmorillonite.

In the modified montmorillonite prepared by the method, the mass ratio of tranexamic acid to montmorillonite is 0.15.

The characterization method comprises the following steps:

1. fourier infrared spectroscopy (FTIR)

The structural characteristics of the samples were measured using a NicoleetiS 5 type Infrared Spectroscopy from ThermoFisher, USA. And (3) performing a potassium bromide tabletting test, grinding the KBr and the sample according to the mass ratio of 100 to 1, pressing the ground KBr and the sample into a uniform and transparent sheet, scanning for 32 times, and testing the wavelength range of 4000-500 cm ^-1 The ThermoScientificOMNIC software performed the analysis.

5363 ray diffraction (XRD) 2.X

The method adopts a German BRUKERD8-Focus type X-ray diffractometer, the tube voltage is 35kV, the tube current is 30mA, a Cu target is used as a radioactive source, the radiation wavelength is lambda =0.154nm, and the continuous recording spectrum scanning is carried out. The test angle is 5-90 degrees, the scanning step is 0.02 degree, and the scanning speed is 4.5 degrees/min ^-1 。

3. Thermogravimetric analysis (TG-DTG)

Grinding the sample, weighing about 10mg, placing into a crucible for testing, and adopting a Japanese Hitachi TG-DTA7200 instrument at a temperature ranging from room temperature to 800 ℃ and a heating rate of 10 ℃ min ^-1 。

Zeta potential

Adopting a British Marvens Zetasizer NanoS90 nanometer particle size tester, wherein the particle size measurement range of the tester is 0.6-6000 nm; the Zeta potential measurement range is 0 mV-150 mV; the temperature measuring area is 2-90 ℃. Dispersing a sample in a deionized water medium (pH = 7.0), performing ultrasonic oscillation, stabilizing for a period of time, directly testing if the solution is in a suspension state and does not generate precipitates, and continuously diluting until no precipitates are generated if the precipitates are generated; 3ml of sample was injected into the cuvette and the cuvette was inserted into the instrument and the test was performed after the temperature had stabilized (data was recorded).

5. Scanning Electron Microscope (SEM)

The instrument model is an American FEI Quanta250FEG scanning electron microscope, and the sample is tested after gold spraying treatment because the material does not have conductivity.

6.X fluorescence Spectroscopy (XRF)

Qualitative and semi-quantitative tests were performed using a pyroelectric ARLAdvant' XINtellipower model 3600X-ray fluorescence spectrum with a tube voltage of 50kV, a tube current of 50mA, and a full elemental scan.

7. In vitro cytotoxicity

In vitro cytotoxicity test L929 cells were used and the MTT method was used for the test. After the sample is irradiated and sterilized by Co-6025kGy, a serum-free DMEM medium is taken as an extraction medium, and extraction proportion of 0.2g/mL is adopted to extract for 24 hours at the temperature of 37 ℃. Adding 10% fetal calf serum into the obtained supernatant of the leaching liquor according to the volume ratio, and uniformly mixing to obtain 100% leaching liquor; diluting the 100% leaching solution to 75%, 50%, and 25%. Taking vigorous L929 cells, digesting with conventional 0.25% pancreatin-EDTA solution, counting blood cell plates, diluting with complete DMEM medium containing 10% fetal calf serum to 1 × 10 ⁵ Cell suspension/mL, 100. Mu.L per well was seeded in 96-well culture plates. Placing in 5% (v/v) carbon dioxide mixed air at 37 deg.CCulturing in a warm cell culture box for 24h. (to reduce the edge effect, the wells of the outer circle of the 96-well plate were not loaded, but only blank DMEM medium was added). And after 24h of culture, discarding the old culture medium, and respectively arranging a material experimental group, a negative control group and a positive control group on each 96-well plate. Adding material leaching liquor with different concentrations into the material groups respectively; adding DMEM medium containing 10% fetal calf serum into the negative control group; the positive control group was 0.2% (w/v) phenol solution prepared in DMEM containing 10% fetal bovine serum, 100. Mu.L per well, and cultured in an incubator for 24 hours and 48 hours. After which the liquid in each well was discarded and 50. Mu.L of freshly prepared 1mg/mL MTT solution was added to each well and placed in the incubator for further 2h. After 2h, the liquid in each well is discarded, 100. Mu.L of isopropanol solution is added into each well, and vortex shaking is carried out for 10min until the solution is fully dissolved. The absorbance (OD value) was measured at a wavelength of 570nm or 650nm (calibration wavelength) using a microplate reader.

Relative cell proliferation (Relative growth rate RGR) was calculated by the following formula, RGR (%) = (mean absorbance value of experimental group/mean absorbance value of blank group) × 100% = OD sample [ OD570-OD650]/OD blank [ OD570-OD650] × 100%, and the final result is expressed as mean. + -. Standard deviation.

8. In vitro hemolysis

Rabbit blood was used to evaluate the effect of modified montmorillonite on in vitro hemolytic properties. 5mL of fresh anticoagulated New Zealand rabbit blood is taken and added with 5mL of normal saline for dilution for standby. The sample components are MMT (montmorillonite) and TXA-MMT (modified montmorillonite), 5mL of physiological saline, 200 mu L of diluted anticoagulated rabbit blood and 50mg of material are mixed in each branch of the sample group, and 3 branches are arranged in parallel in each group. And meanwhile, deionized water and normal saline are respectively used as a positive control and a negative control. All the tubes were incubated at 37 ℃ for 1h, centrifuged at 3000r/min for 5min, and the supernatant was aspirated. The absorbance was measured at 545nm using an ultraviolet-visible spectrophotometer. The hemolysis rate was calculated according to the following equation: hemolysis (%) = (experimental mean absorbance value-negative control mean absorbance value)/(positive control mean absorbance value-negative control mean absorbance value) × 100%.

9. Time of coagulation in vitro

Taking a plurality of 5mL centrifuge tubes with the same specification, sterilizing for 30min by high-pressure steam at 121 ℃, and drying for later use. Accurately weighing 20mg of the test sample and placing the test sample in a centrifuge tube, taking a blank centrifuge tube as a negative control, and accurately weighing 20mg of tranexamic acid and placing the test sample in the centrifuge tube as a positive control. The rabbit was anesthetized by intravenous injection at the ear margin of New Zealand rabbit with 3% (w/v) sodium pentobarbital at a dose of 1mL/kg, the rabbit was fixed supine, the femoral artery was bled quickly, 1mL of fresh whole rabbit blood was immediately added to each tube, and the tube was gently shaken to bring the material into full contact with the blood. The tube was placed in a 37 ℃ water bath and observed obliquely every 15 seconds until the blood was completely coagulated, and the coagulation time was recorded. The above experiment was repeated three times and the final clotting time results are expressed as mean ± standard deviation.

10. Ionizing radiation and material intervention

Healthy female C57 mice weighing 18-22 g were selected for the experiment and then randomized into 4 groups of 6 mice each. 12h before irradiation, the following treatments were received: 1) Blank group (300 μ L of deionized water for intragastric administration); 2) Model group (300 μ L of deionized water for intragastric administration); 3) Montmorillonite group (8 mg medicinal montmorillonite added with 300 μ L deionized water for intragastric administration); 4) Modified montmorillonite group (8 mg modified montmorillonite added with 300 μ L deionized water for intragastric administration). Before irradiation, the mice are anesthetized by intraperitoneal injection of 1% (w/v) sodium pentobarbital (50 mg/kg), and then fixed on an irradiation plate, and the rest parts of the abdomen irradiation of the mice are shielded by lead to avoid hematopoietic damage caused by radiation. Except for the normal group, the model group and the administration group receive radiation of a Co-60 radioactive source, the absorbed dose is 14Gy, the dose rate is 66.92cGy/min, and the distance between an animal and the radioactive source is 3.0m; after irradiation, each group was intervened 1 time per day following pre-irradiation treatment. Animals were sacrificed 3 days after irradiation, the anterior orbital blood was collected, placed in a sterile tube, serum was isolated (sterile procedure as possible), and endotoxin in serum was detected using a limulus kit, while TNF- α, IL-1 β, IL-6 inflammatory factors in serum were detected. After the animals were sacrificed by taking blood from the orbit, the intestinal tissues were taken back and washed with physiological saline until no fecal residue remained, fixed with 4% formaldehyde, dehydrated conventionally, embedded in paraffin, cut into 5 μm thick sections transversely, stained with Hematoxylin and Eosin (HE), and the pathological changes were observed under the optical lens.

11. Statistical analysis

All statistical data were analyzed using SPSS19.0 software. Results are mean ± standard deviation. Statistical comparisons were made using the variance of the t-test, with P <0.05 being considered significant differences and P <0.01 being considered very significant differences.

Results and discussion:

material characterization:

the following smectites were used as commercially available medicinal grade smectites hereinbefore; the modified montmorillonite was the modified montmorillonite prepared in inventive example 1.

1.FTIR

By observing the infrared spectrum shown in FIG. 1, it was found that the concentration of montmorillonite was 1639cm ^-1 The absorption peak is caused by vibration of Al-OH and hydroxyl in the adsorbed water, 1034cm ^-1 The absorption peak at (A) is caused by the vibration of the Si-O plane. Tranexamic acid 1637cm ^-1 The absorption peak is caused by N-H bending vibration, 2925cm ^-1 And 2862cm ^-1 The absorption peak at (B) is caused by stretching vibration of-OH on-COOH. The infrared spectrum of the modified montmorillonite is observed to be 1632cm ^-1 The absorption peak is 1639cm relative to montmorillonite ^-1 The absorption peak of (A) was shifted and probably related to N-H bending vibration after intercalation of tranexamic acid into montmorillonite, and was found to be 1719cm ^-1 、2945cm ^-1 Two new absorption peaks are generated, and the analysis shows that the peak length is 1719cm ^-1 The vibration is caused by stretching and contracting of carbonyl group, and the length of the vibration is 2945cm ^-1 The C-H stretching vibration on saturated carbon causes that carbonyl and C-H are groups on tranexamic acid, which indicates that the tranexamic acid reacts with montmorillonite to generate a new product.

2.XRD

The layers of montmorillonite are bound by van der waals force, there are many metal cations and hydroxyl hydrophilic groups in the unit cell of montmorillonite, so it will show strong hydrophilicity, and water molecules can enter into the layers to cause the interlayer spacing (d 001) to increase, resulting in swelling of montmorillonite. In order to investigate whether TXA-MMT (modified montmorillonite) is an intercalated structure, XRD experiments were performed and XRD data pattern results of montmorillonite and its composites are shown in fig. 2. According to the bragg equation: 2dsin theta = n lambda (d: interplanar spacing, theta: angle between incident X-ray and interplanar plane, lambda: X-ray wavelength, n: diffraction)A step map) may calculate the layer spacing. At the diffraction angle 2 theta of about 6 degrees, the diffraction peaks of the montmorillonite and the modified montmorillonite are not shifted, which indicates that the interlayer spacing is not changed, probably because the interplanar spacing d of the medical montmorillonite is larger and the spacing is not changed after the intercalation reaction occurs. However, the diffraction peak of the modified montmorillonite becomes sharp because the crystal form arrangement is changed from disorder to order, which shows that the crystal structure and the crystallinity are better than the montmorillonite. Meanwhile, TXA-MMT (modified montmorillonite) has diffraction peaks changed at the positions of 34.9 degrees, 55 degrees, 61.8 degrees, 73.4 degrees and 76 degrees of 2 theta, and it is speculated that tranexamic acid and hydroxyl on the surface of montmorillonite form hydrogen bonds or that tranexamic acid and interlayer ions Ca of montmorillonite are formed ²⁺ 、Mg ²⁺ And the exchange is carried out to form a new product.

TG-DTG thermogravimetric analysis

Thermogravimetric analysis is carried out on the sample, as shown in fig. 3, which is an analysis graph of MMT (montmorillonite), the thermogravimetric loss curve mainly has two obvious weightlessness stages, the weight loss before 100 ℃ is mainly caused by free hydrothermal decomposition on the surface of the montmorillonite, the second weightlessness occurs at 500-700 ℃, the second weightlessness is mainly caused by the loss of bound water of the montmorillonite, and the phase transformation occurs, and the DTG graph also shows that the weightlessness peaks occur at about 100 ℃ and at 500-700 ℃.

As shown in fig. 4, which is an analysis diagram of TXA (tranexamic acid), the boiling point of tranexamic acid is about 300 ℃, and at this temperature, the distance between tranexamic acid molecules increases, which results in that gas volatilizes and a large amount of weight loss is caused; TXA (tranexamic acid) has poor thermal stability, and six-membered rings, carboxyl groups and the like of structural functional groups of the TXA are decomposed after the temperature is continuously raised, so that the quality of the TXA is further lost; DTG picture shows that tranexamic acid generates weight loss peak at about 300 deg.C and 400-500 deg.C correspondingly.

As shown in FIG. 5, which is an analytical graph of TXA-MMT (modified montmorillonite), weight loss before 100 ℃ is mainly due to the surface free hydrothermal decomposition of TXA-MMT (modified montmorillonite), significant mass loss in the range of 300 to 500 ℃ is due to the change in the state of tranexamic acid in the composite, and a small mass loss at 500 to 700 ℃ is caused by the phase transition due to the structural denaturation of montmorillonite.

Zeta potential

The Zeta potential is an indicator of the amount of charge on the surface of the particles, is related to the stability of the particle system, and is generally used for evaluating or predicting the physical stability of the particle dispersion system, and the higher the absolute value is, the larger the electrostatic repulsion force between the particles is, and the better the physical stability is. As shown in FIG. 6, the Zeta potentials of TXA-MMT (modified montmorillonite), MMT (montmorillonite) and TXA (tranexamic acid) show a negative potential, in which MMT (montmorillonite) is due to the silicon oxy tetrahedron Si ⁴⁺ Is covered with Al ³⁺ Substitutional, octahedral Al of Al aluminium ³⁺ Is coated with Mg ²⁺ 、Fe ²⁺ The crystal layer is replaced to generate an excessive negative charge to become a permanent negative charge. The structure of the tranexamic acid contains an amino group and a carboxyl group, the isoelectric point of the tranexamic acid is between 6.2 and 6.8, the tranexamic acid is negatively charged in deionized water (pH = 7), and the Zeta potential of the tranexamic acid is negative. When the TXA-MMT (modified montmorillonite) charge is-28.47 mv after the complex reaction of montmorillonite and tranexamic acid, and the MMT (montmorillonite) and TXA (tranexamic acid) charge are-14.87 mv and-21.33 mv respectively, the success of the tranexamic acid montmorillonite reaction is demonstrated.

5.SEM

The purpose of SEM was to observe the microscopic morphological changes of the material before and after modification. SEM images of TXA-MMT (modified montmorillonite), as shown in FIG. 7, (a-b) is SEM electron microscopy test image of TXA-MMT (modified montmorillonite); (c-d) SEM electron microscope test picture of MMT (montmorillonite); and (e-f) is an SEM electron microscope test picture of (tranexamic acid). From fig. 7, it can be seen that the MMT (montmorillonite) has curled sheet edges, large particles and easy aggregation, and is a blocky structure with stacked sheets, and the particle size is less than 1 μm; TXA-MMT (modified montmorillonite) is still a blocky structure with stacked sheets, the particle size of the blocky structure is not greatly changed with montmorillonite, but many tiny particles appear on the surface of the modified montmorillonite, and a small amount of tranexamic acid in a reaction system is attached to the surface of the MMT (montmorillonite). Comparing the electron microscope images of TXA-MMT (modified montmorillonite) with MMT (montmorillonite), TXA-MMT (modified montmorillonite) was found to be more compact than MMT (montmorillonite), probably due to the replacement of Na by TXA (tranexamic acid) ⁺ 、Ca ²⁺ Isocationals, so that the crystal structure and the crystallinity are more optimized.

6.XRF

The results of the element content analysis of MMT (montmorillonite) and TXA-MMT (modified montmorillonite) are shown in Table 1, in units of: (wt%)

Table 1 results of content analysis

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XRF analysis results As shown in Table 1, the montmorillonite is microcrystalline kaolinite, is a natural aluminosilicate clay ore, is composed of elements and compounds such as silicon, aluminum, calcium, magnesium, iron, sodium and the like, and has Ca which can be exchanged among MMT (montmorillonite) layers ²⁺ ，Mg ²⁺ ，Na ⁺ And the like. After TXA (tranexamic acid) and MMT (montmorillonite) are subjected to intercalation reaction under certain conditions, the content percentage of Ca in the MMT (montmorillonite) is reduced from 2.120 to 0.850; the content percentage of Mg is reduced from 3.200 to 2.980; the percentage of Na content decreased from 0.145 to 0.063, indicating that TXA (tranexamic acid) replaced Ca between MMT (montmorillonite) layers ²⁺ 、Mg ²⁺ And Na +, etc., and TXA (tranexamic acid) and MMT (montmorillonite) are subjected to intercalation reaction to generate a new composite product.

7. In vitro cytotoxicity

The in vitro cytotoxicity test is an in vitro test for simulating the growth environment of organisms in an in vitro state, and detecting cytolysis, cell growth inhibition and other toxic effects of medical instruments and biological materials after contacting organism tissues. We measured the in vitro cytotoxicity [ mean ± SD (n = 6) ] of TXA-MMT (modified montmorillonite) using the MTT method. As shown in fig. 8, the cellular activities of TXA-MMT (modified montmorillonite) at concentrations of 25%, 50%, 75% and 100% were 93%, 84%, 78% and 78% at 24 h; the positive control phenol group had less than 10% cell activity at 24h. When the cellular activity >75% meets the biological evaluation criteria, we consider the biological material to be safe. Therefore, TXA-MMT (modified montmorillonite) has no obvious cytotoxicity and is a safe biomedical material.

8. In vitro hemolysis

The hemocompatibility of MMT (montmorillonite) and TXA-MMT (modified montmorillonite) was measured by in vitro hemolysis method, and the result is shown in fig. 9, the hemolysis rate of montmorillonite was most obvious 56.2%, and the hemolysis rate of modified montmorillonite was 2.9%, indicating that the formation of modified montmorillonite reduced the damage of erythrocytes. The reason for the reduction of the hemolysis rate of the modified montmorillonite is probably that the MMT (montmorillonite) property is changed due to the TXA (tranexamic acid) intercalation between MMT (montmorillonite), and on the other hand, a small amount of TXA (tranexamic acid) is attached to the surface of MMT (montmorillonite) to play a certain protection role; this result also further substantiates the non-cytotoxic properties of TXA-MMT (modified montmorillonite).

9. Time of coagulation in vitro

The in vitro blood coagulation time is mainly used for evaluating the capability of the hemostatic material for promoting blood coagulation after contacting with blood, and the shorter the blood coagulation time is, the better the hemostatic effect of the material is. Blood rapidly coagulates after exiting a blood vessel, and the coagulation process is a chain reaction chemistry that occurs in plasma and is involved by a variety of coagulation factors, with the result that the blood is converted from a liquid state to a gelatinous state. In vitro coagulation was measured by a test tube method, and the in vitro coagulation time of the material is shown in fig. 10, mean ± SD (n = 3). P <0.05 and. P <0.01, the normal coagulation time of the blank rabbit blood is about 500s, and compared with the blank negative, the in vitro coagulation time of other groups is significantly reduced. TXA-MMT (modified montmorillonite) has significantly reduced in vitro coagulation compared to MMT (montmorillonite) and TXA (tranexamic acid), all with significant differences. This is because MMT (montmorillonite) and TXA-MMT (modified montmorillonite) have a certain water-absorbing ability, and TXA-MMT (modified montmorillonite) and TXA (tranexamic acid) ensure effective clot formation by competitively inhibiting the hydrolysis of fibrin by plasmin, promoting blood coagulation. The results show that the TXA-MMT (modified montmorillonite) modified montmorillonite has good in vitro coagulation ability.

10. Small intestine histopathology detection

Observed under an optical microscope, ileum tissues of mice in a normal group have abundant intestinal villi, the intestinal villi are clearly visible, the arrangement of glands in an inherent layer is compact, pan cells can be seen at the bottom, and no obvious gland injury is seen; no significant inflammatory cell infiltration and bleeding was seen, as shown in fig. 11 (a). Three days after irradiation, the mouse ileum tissue lesions were severe, as shown in fig. 11 (b), local glandular cell atrophy was seen in the mucosal lamina propria, pangolin cell depletion, and accompanied by bleeding symptoms (arrow 1), slight loose edema of submucosa (arrow 3), and thinning of the muscularis of intestinal wall (arrow 2). Given the post-treatment with montmorillonite and modified montmorillonite, the ileal histological changes were alleviated, as shown in fig. 11 (c) for the ileal histological changes in MMT (montmorillonite) group mice, with a small amount of intestinal villous mucosal epithelium exfoliation (arrow 4), congestion of capillaries (arrow 6), tight arrangement of lamina propria glands, no apparent gland damage, and slight edema of submucosa (arrow 5); the intestinal villi of the ileum tissue of mice in the TXA-MMT (modified montmorillonite) group are abundant, the intestinal villi are clearly visible, individual glands of the lamina propria are atrophied, the morphology disappears to form connective tissue (arrow 8), and no obvious hemorrhage is seen; the submucosa was slightly edematous (arrow 7) as shown in fig. 11 (d).

Under normal physiological state, the apoptosis and the shedding of the intestinal epithelial cells and the proliferation and the differentiation of the stem cells are in dynamic balance, and the integrity of the intestinal barrier function and the repair after the damage are maintained. The proliferated intestinal stem cells are located at the bottom of the crypt of the small intestine epithelium, migrate upwards along the axis of crypt villus to the top of the villus in the process of maturation and complete differentiation. In the area with radiation enteritis, after being irradiated by rays, undifferentiated cells at the bottom of the crypt excessively proliferate, are most easily damaged, denature and necrose, and interrupt the cell supply on the surface of intestinal villi, thereby causing the deletion of the epithelial layer of the intestinal tract and the increase of the intestinal permeability. In the study, the C57 mouse has serious pathological changes of small intestine tissues after being irradiated by 14Gy, and the intestinal tract has bleeding symptoms; after the montmorillonite and the modified montmorillonite are subjected to dry prognosis, the pathological change degree of small intestine tissues is relieved, and the protective and repair effects on small intestine epithelial cells of mice are achieved; the modified montmorillonite has better effect on intestinal mucosa bleeding and capillary vessel hemostasis.

From the above results, the characterization by FTIR, XRD, TG-DTG and XRF shows that the invention successfully prepares TXA-MMT (modified montmorillonite) by using intercalation technology; meanwhile, the particle sizes of the modified montmorillonite are all smaller than 1 mu m under SEM observation, and Zeta potential shows that the negative charge of the modified montmorillonite is enhanced after TXA (tranexamic acid) reacts with MMT (montmorillonite).

The invention adopts tranexamic acid to modify montmorillonite and replace Na in the montmorillonite ⁺ 、Ca ²⁺ And the like, so that the crystal structure and the crystallinity of the montmorillonite are optimized, the properties of the montmorillonite are changed, the montmorillonite-montmorillonite composite material shows more excellent in-vitro hemostasis capability in an in-vitro coagulation time experiment, and the montmorillonite composite material has a better hemostasis effect compared with montmorillonite.

In a mouse experiment for intervening in the radiation enteritis, the modified montmorillonite can reduce the endotoxin content in the serum of a mouse after radiation, and can reduce the expression of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the serum of the mouse, and meanwhile, the histopathology of the small intestine discovers that the modified montmorillonite has a certain effect on the protection and repair of the epithelial cells of the small intestine of the mouse with the radiation enteritis and the maintenance of the integrity of the intestinal mucosa.

The result shows that the modified montmorillonite is a safer and more effective hemostatic which can be used for treating intestinal bleeding.

It should be noted that, in order to research the substitute of tranexamic acid, the inventor of the present invention conducted research by changing the ring structure of tranexamic acid, and found that, when other structures are matched with montmorillonite, no synergistic effect or insignificant synergistic effect is produced, and only tranexamic acid used in the present application needs a small amount of tranexamic acid to surprisingly increase and improve various properties of montmorillonite, and the effect is significant.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A modified montmorillonite is characterized in that the modified montmorillonite is prepared from raw materials including montmorillonite and tranexamic acid; the modified montmorillonite is prepared from montmorillonite and tranexamic acid through intercalation reaction;

in the modified montmorillonite, tranexamic acid is inserted into the interlayer of the montmorillonite to replace cations among the montmorillonite layers; under acidic condition, pH is less than pI, tranexamic acid is positively charged, and free H under acidic condition ⁺ OH bound to montmorillonite ^- The reaction causes more positive ions to be exposed, and the tranexamic acid can exchange with interlayer ions of the montmorillonite under the action of external force; the cation comprises Ca ²⁺ 、Mg ²⁺ 、Na ⁺ 。

2. The modified montmorillonite according to claim 1,

3. The process for producing modified montmorillonite according to any one of claims 1 to 2, characterized in that,

the modified montmorillonite is prepared from montmorillonite and tranexamic acid through intercalation reaction.

4. The method for producing a modified montmorillonite according to claim 3, comprising the steps of:

5. The method for producing modified montmorillonite according to claim 4,

in the step (1), the step (c),

the montmorillonite is medicinal montmorillonite;

the mass volume percentage concentration of the montmorillonite dispersion liquid is 1-5w/v%;

the conditions of the swelling treatment are as follows: heating and swelling under stirring; the heating temperature of the swelling treatment is 50-80 ℃, and the treatment time is 4-8h.

6. The method for producing modified montmorillonite according to claim 5, wherein the mass volume percent concentration of the montmorillonite dispersion is 1.5-3.5w/v%.

7. The process for producing modified montmorillonite according to claim 4,

in the step (2), the step (3),

adding 0.005-0.03mol of tranexamic acid per gram of montmorillonite based on montmorillonite contained in the montmorillonite dispersion liquid;

the pH regulator is inorganic acid;

adjusting the pH value to 3-5;

8. The method for producing modified montmorillonite according to claim 7, wherein 0.008 to 0.02mol of tranexamic acid per gram of montmorillonite is added.

9. The process for producing modified montmorillonite according to claim 7,

in the step (2), the step (3),

the pH adjustor comprises nitric acid;

the heating reaction is carried out under the stirring condition, and the stirring speed is 250-500r/min;

10. Use of a modified montmorillonite according to any of claims 1-2 in hemostasis.

11. Use according to claim 10, wherein the haemostasis comprises intestinal haemostasis.