CN113663118B

CN113663118B - Application of esterified modified starch hemostatic material

Info

Publication number: CN113663118B
Application number: CN202111230333.XA
Authority: CN
Inventors: 孙云波; 赵新红; 窦桂芳; 孟志云; 甘慧; 顾若兰; 吴卓娜; 朱晓霞; 孙文种
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2022-02-18
Anticipated expiration: 2041-10-22
Also published as: CN113663118A

Abstract

The invention discloses application of esterified modified starch in preparation of a hemostatic material. The esterified modified starch is prepared by preparing holes through enzymolysis of starch, performing acyl chloride reaction on tranexamic acid protected by BOC, and performing esterification reaction on the enzymolysis starch and BOC-carbamoyl chloride, and comprises the following raw materials: starch, BOC-tranexamic acid, thionyl chloride and amylase. The esterified modified starch hemostatic material provided by the invention has the advantages of abundant main raw material sources, good safety and no residue, can be widely applied to hemostasis of irregular bleeding such as parenchymal viscera in vivo and diffuse bleeding, does not need debridement after hemostasis, and can be degraded and absorbed by a human body after the hemostasis.

Description

Application of esterified modified starch hemostatic material

Technical Field

The invention belongs to the field of medical materials, and particularly relates to application of esterified modified starch in preparation of a hemostatic material.

Background

The hemostatic materials are mainly divided into two categories of hemostatic instruments and hemostatic medicaments, wherein the hemostatic instruments mainly comprise a tourniquet, a hemostatic forceps and a hemostatic dressing, and the hemostatic medicaments are divided into a chemical hemostatic, a biological hemostatic and a traditional Chinese medicine. The hemostatic materials have the advantages and disadvantages of hemostatic performance, and the tourniquet and the hemostatic forceps can be used for the hemostasis of the aorta, but are not suitable for the hemostasis of special parts, and tissue necrosis can be caused by overtightening; the hemostatic dressing can be used for hemostasis of irregular wounds, but debridement may cause secondary bleeding of the wound. The chemical hemostat has quick effect but unstable property and obvious side effect on the whole body; the biological hemostatic has good hemostatic effect, but is easy to generate immunoreaction and high in cost; the traditional Chinese medicine hemostatic has rich sources and low cost, but takes effect slowly. Although various hemostatic materials are available at present and have good clinical effects, the development of novel hemostatic materials with high efficiency, biosafety, low cost, easy preparation, good biodegradability and biocompatibility has become a urgent task. The domestic and foreign trauma curing and treating experts are dedicated to research a quick-acting hemostatic material with low cost, quick response and no debridement.

Disclosure of Invention

The invention aims to provide application of esterified modified starch.

The application of the esterified modified starch provided by the invention is the application of the esterified modified starch in preparing hemostatic materials.

The esterified modified starch has the advantages of rich main raw material sources, good safety and no residue, can be widely applied to hemostasis of irregular bleeding such as parenchymal viscera in vivo and diffuse bleeding, does not need debridement after hemostasis, can continue the hemostasis effect to the operation, and can be degraded and absorbed by a human body after the operation.

The esterified modified starch, namely the tranexamic acid-loaded cross-linked porous starch, can be prepared by the following steps:

1) dissolving starch in disodium hydrogen phosphate citric acid buffer solution, heating in water bath, and mechanically stirring; then adding alpha amylase and saccharifying enzyme, and continuing stirring; adding an alkali solution to enable the pH value of the system to be 10-14, stirring to inactivate alpha amylase and saccharifying enzyme, centrifuging, washing, and freeze-drying to obtain enzymolysis starch;

2) reacting BOC-tranexamic acid with N, N-dimethylformamide and thionyl chloride in dichloromethane to obtain BOC-carbamoyl chloride;

3) reacting the enzymolysis starch obtained in the step 1) with the BOC-carbamoyl chloride obtained in the step 2), centrifuging, washing and freeze-drying after the reaction is finished to obtain the tranexamic acid-carrying cross-linked porous starch.

In the step 1) of the method, the ratio of the starch to the disodium hydrogen phosphate citric acid buffer solution is (2-3) g: (50-100) ml; the pH value of the disodium hydrogen phosphate citric acid buffer solution is 4-5. The rotating speed of the mechanical stirring is 300-600 rpm.

In the step 1) of the method, the temperature of the water bath is 45-55 ℃;

in the step 1) of the method, the alpha amylase and the saccharifying enzyme are added, and the stirring is continued for 5-10 hours.

In the step 1), the dosage of the alpha amylase is 25-75 KNU/g starch (specifically 50KNU/g starch); the dosage of the saccharifying enzyme is 5000-15000U/g starch (specifically 10000U/g starch).

In step 1) of the above method, the alkali solution may be specifically a sodium hydroxide solution, such as a 0.5M sodium hydroxide solution.

In the step 1), after the alkali solution is added, the stirring time can be 20-40 min.

In step 1) of the above method, the centrifugation conditions may be 5500rpm for 5 min.

In step 1) of the above method, the washing is washing with deionized water, and specifically, the washing may be carried out three times.

In the step 1) of the method, the freeze drying condition is-60 ℃ and 24 hours.

The step 1) of the method can be specifically carried out according to the following steps: weighing 2-3 g of starch, dissolving in 50-100 ml of disodium hydrogen phosphate citric acid buffer solution, performing mechanical stirring at 50 ℃ in a water bath at 500rpm, adding an appropriate amount of alpha amylase and glucoamylase, stirring for 5-10 h, adding 15-20 ml of 0.5M sodium hydroxide solution to adjust the pH value to 10-14, stirring for 30min, centrifuging at 5500rpm for 5min, washing with deionized water for three times, and freeze-drying to obtain the enzymatic starch.

In the step 2), the ratio of the BOC-tranexamic acid to the N, N-dimethylformamide to the thionyl chloride to the dichloromethane is (5-10) g: (200-400) μ L: (5-11) ml: (50-100) ml.

In the step 2), the reaction temperature is 25-30 ℃; the reaction time is 2-4 h. The reaction is carried out in a stirring state, and the stirring speed is 300-600 rpm.

The step 2) of the method can be specifically carried out according to the following steps: dissolving 5-10 g of BOC-tranexamic acid in 50-100 ml of dichloromethane, refluxing at 30 ℃ and 500rpm, magnetically stirring for 10min, adding 200-400 mu L N of N-dimethylformamide and 5-11 ml of thionyl chloride, and stirring for 2-4 h. And (5) performing rotary evaporation to obtain BOC-carbamyl chloride powder.

In the step 3) of the method, the mass ratio of the enzymolysis starch to the BOC-carbamoyl chloride is 1-2 g: 5-10 g.

In the step 3), the reaction temperature is 40-80 ℃ (specifically 40 ℃, 60 ℃ or 80 ℃) and the reaction time is 4-12 h (specifically 4h, 8h or 12 h). The reaction is carried out in a stirring state, and the stirring speed is 300-600 rpm.

In step 3) of the above method, the centrifugation conditions may be 5500rpm for 5 min.

In step 3), the washing is performed by using deionized water, and specifically, the washing can be performed three times.

In the step 3), the freeze drying is carried out at-60 ℃ for 24 h.

The step 3) of the method can be specifically carried out according to the following steps: 1-2 g of enzymolysis starch, magnetically stirring at 40-80 ℃ and 500rpm, adding 1-10 g of BOC-carbamoyl chloride, stirring for 4-12 h, centrifuging at 5500rpm for 10min, washing for 3 times with deionized water, centrifuging, and freeze-drying for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Drawings

FIG. 1 is a graph showing the results of IR spectroscopy on tranexamic acid-supported crosslinked porous starch prepared in example 1;

FIG. 2 is a scanning electron microscope result chart of the tranexamic acid-loaded crosslinked porous starch prepared in example 1: a1, A2 and A3 potato starch; b1, B2 and B3: performing enzymolysis on starch; c1, C2 and C3: tranexamic acid-loaded cross-linked porous starch;

FIG. 3 is a graph showing the results of the liquid absorption rate of tranexamic acid-loaded crosslinked porous starch prepared in example 1;

FIG. 4 is a graph showing the results of an in vitro cytotoxicity test of tranexamic acid-loaded cross-linked porous starch prepared in example 1;

FIG. 5 is a graph showing the results of acute toxicity test of tranexamic acid-loaded crosslinked porous starch prepared in example 1;

FIG. 6 is a graph showing the in vitro clotting time results of tranexamic acid-loaded cross-linked porous starch prepared in example (P is less than 0.05, and the results are compared with positive control euphoric particles);

fig. 7 is a photograph of hemostasis of each group of hemostatic materials after hemostasis in the mouse tail-broken bleeding model: a carries tranexamic acid cross-linked porous starch, B Xin Suanzu hemostatic granule group, C blank gauze group;

FIG. 8 is the statistics of blood loss during hemostasis in the mouse tail-severed hemorrhage model (P < 0.05, P < 0.01, results compared to positive control euphoric particles).

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

The sources of enzymes used in the following examples are as follows:

alpha-amylase was purchased from Shanghai Aladdin Biotechnology, Inc., lot number: l2008186, the enzyme activity is more than or equal to 500 KNU/g.

Saccharifying enzymes were purchased from Shanghai Aladdin Biotechnology Ltd, lot number: d2102059, the enzyme activity is 100000U/ml.

The Xin Su Zhi Xue Keli used in the following examples was purchased from Hangzhou cooperative medical supplies, Inc. with a specification of 1.0 g/count.

The starches used in the examples below are all potato starches.

Example 1: preparation of tranexamic acid-loaded cross-linked porous starch

(1) Weighing 2g of starch, dissolving in 100ml of disodium hydrogen phosphate citric acid buffer solution with pH =4.5, carrying out water bath at 50 ℃, mechanically stirring at 500rpm, adding 50KNU/g alpha amylase and 10000U/g glucoamylase, stirring for 6h, adding 17ml of 0.5M sodium hydroxide solution to adjust the pH value to 12, stirring for 30min, centrifuging at 5500rpm for 5min, washing with deionized water for three times, and carrying out freeze drying to obtain the enzymatic starch.

(2) 10 g BOC-tranexamic acid, 100ml dichloromethane dissolved, 30 ℃, 500rpm reflux magnetic stirring for 10min, adding 200 u L N, N-dimethylformamide, 5.646 ml thionyl chloride and stirring for 3 h. And (5) performing rotary evaporation to obtain BOC-carbamyl chloride powder.

(3) 1g of enzymolysis starch, magnetically stirring at 40 ℃ and 500rpm, adding 3g of BOC-carbamoyl chloride, stirring for 8h, centrifuging at 5500rpm for 10min, washing for 3 times with deionized water, centrifuging, and freeze-drying at-60 ℃ for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Example 2: preparation of tranexamic acid-loaded cross-linked porous starch

(3) 1g of enzymolysis starch, magnetically stirring at 60 ℃ and 500rpm, adding 3g of BOC-carbamoyl chloride, stirring for 8h, centrifuging at 5500rpm for 10min, washing for 3 times with deionized water, centrifuging, and freeze-drying at-60 ℃ for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Example 3: preparation of tranexamic acid-loaded cross-linked porous starch

(3) 1g of enzymolysis starch, stirring magnetically at 80 ℃ and 500rpm, adding 3g of BOC-carbamoyl chloride, stirring for 8h, centrifuging at 5500rpm for 10min, washing for 3 times with deionized water, centrifuging, and freeze-drying at-60 ℃ for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Example 4: preparation of tranexamic acid-loaded cross-linked porous starch

(3) 1g of enzymolysis starch, magnetic stirring at 80 ℃ and 500rpm, adding 3g of BOC-carbamyl chloride, stirring for 4h, centrifuging at 5500rpm for 10min, washing for 3 times with deionized water, centrifuging, and freeze-drying at-60 ℃ for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Example 5: preparation of tranexamic acid-loaded cross-linked porous starch

(3) 1g of zymolytic starch, magnetically stirring at 80 ℃ and 500rpm, adding 3g of BOC-carbamoyl chloride, stirring for 12h, centrifuging at 5500rpm for 10min, washing for 3 times by deionized water, centrifuging, and freeze-drying at-60 ℃ for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Example 6 preparation of Tranexamic acid-loaded crosslinked porous starch

(3) 1g of zymolytic starch, stirring magnetically at 80 ℃ and 500rpm, adding 5g of BOC-carbamyl chloride, stirring for 8h, centrifuging at 5500rpm for 10min, washing for 3 times by deionized water, centrifuging, and freeze-drying at-60 ℃ for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Example 7: preparation of tranexamic acid-loaded cross-linked porous starch

(3) 1g of zymolytic starch, magnetically stirring at 80 ℃ and 500rpm, adding 1g of BOC-carbamyl chloride, stirring for 8h, centrifuging at 5500rpm for 10min, washing for 3 times by deionized water, centrifuging, and freeze-drying at-60 ℃ for 24h to obtain the tranexamic acid-carrying cross-linked porous starch.

Comparative examples 1,

Mixing potato starch and tranexamic acid according to the mass ratio of 1:5 to obtain a physical mixture.

Material characterization of tranexamic acid-loaded crosslinked porous starch prepared in example 8 and example 6

1 infrared spectrum:

infrared spectrum analysis adopts a tabletting-free method, a sample of about 1mg is ground and uniformly spread on a sample carrying table to scan the spectrum, and the wave number range is measured to be 4000-600 cm^-1. The results of the infrared spectroscopy are shown in FIG. 1.

The infrared spectrum of the tranexamic acid-loaded cross-linked porous starch synthesized by a series of chemical reactions is greatly different from that of raw material potato starch. The potato starch is 3326cm^-1O-H stretching vibration peak associated with hydrogen bond at 2910cm^-1Is the asymmetric stretching vibration peak of C-H at 1640cm^-1C = C peak of stretching vibration at 1411cm^-1Is a-OH in-plane bending vibration peak at 1146cm^-1、1076cm^-1And the peak appearing at 995cm-1 was an asymmetric C-O-C stretching vibration peakAnd C-O stretching vibration peak. Tranexamic acid at 2921cm^-1Is represented by-CH₂Symmetric expansion peak at 1635cm^-1The peak of the stretching vibration is C = O and is 1566cm^-1Is represented by-NH₂Has a bending vibration peak of 1491cm^-1Is represented by-CH₂Bending vibration of 1379cm^-1Is represented by-CH₂Peak of flexural vibration of 1328cm^-1Bending vibration peak, CC stretching vibration peak and-CH of-CH₂Has a bending vibration peak of 1278cm^-1Bending vibration peak and CO stretching vibration peak at-OH, and the vibration peak is 1228cm^-1Is treated as CNH₂Bending vibration peak, CH₂Peak of flexural vibration at 1194cm^-1Bending vibration peak, CH, of CO₂Bending vibration peak of (2) at 1089cm^-1The peak of stretching vibration, the peak of COH bending vibration and NH are positioned at CC₂Peak of flexural vibration at 1070cm^-1Is located at CC bending vibration peak and CH₂The bending vibration peak is CH bending vibration peak at 970cm-1 and at 921cm^-1is-NH₂Bending vibration peak, -CH₂Has a bending vibration peak of 842, 768cm^-1Is the peak of CC flexural vibration, CH₂A bending vibration peak. The tranexamic acid-loaded cross-linked porous starch is 3288cm^-1The absorption peak is sharp and is shifted due to the shift of O-H stretching vibration peak in starch, and is within 1710cm^-1The peak is the stretching vibration peak of C = O in ester bond and is 1633cm^-1Is represented by-NH₂In a shear mode of vibration of 1553cm^-1Is represented by-CH₂In a shear mode of vibration of 1398cm^-1An in-plane bending vibration peak and a CO stretching vibration peak at-OH of 1203cm^-1Tensile vibration peak at CC, COH in-plane bending vibration peak, NH₂Symmetric bending vibration peak at 1025cm^-1The stretching vibration peak of C-O of dehydrated glucose ring C-O-C is at 935cm^-1Is represented by-NH₂Rocking peak, -CH₂The rocking vibration peak of (1). To sum up, the tranexamic acid-carrying cross-linked porous starch is 1710cm^-1The typical characteristic absorption peak of the ester bond is shown, the esterification reaction is fully illustrated, and the substitution degree reaches the detection range of the instrument.

2 field emission scanning electron microscope:

the testing steps are as follows: potato starch, enzymatic starch and amino-loaded cyclic acid crosslinked porous starch samples are dispersed on a sample loading table, and after gold spraying and vacuum pumping, scanning and photo collection are carried out by using a Hitachi S-4800 type electron microscope.

The scanning electron microscope results are shown in FIG. 2, wherein A1, A2 and A3 are potato starch; b1, B2 and B3: performing enzymolysis on starch; c1, C2 and C3: tranexamic acid-loaded cross-linked porous starch.

The scanning electron microscope result picture shows that: the potato starch granules are oval and have smooth surfaces. The surface of the enzymolyzed starch becomes rough, some particles begin to separate, and the surface of the particles is decomposed into a plurality of shallower holes. The surface of the tranexamic acid-loaded cross-linked starch is rough, after the starch esterification process, the crystal structure and the granular structure of the starch are damaged, and the breakage of the hydrogen bonds of the starch can also cause the rough surface of the starch, so that the acidic environment can also damage the ordered structure of the starch during the esterification reaction, and the esterified starch granules with rough and porous surfaces are obtained.

3 degree of substitution:

weighing 1-2 g of tranexamic acid-loaded cross-linked porous starch, placing the starch in a 50ml conical flask, adding 15-30 ml of deionized water, 5-10 ml of absolute ethyl alcohol and 20-40 ml of 0.5M NaOH solution, stirring for 3h in a water bath at 50 ℃ at 600rpm, adding 100-. The enzyme-hydrolyzed starch was used as a blank and the measurement was carried out 3 times in parallel.

Ester group content A = [ (V-V)_{Blank space}）×10-3×C_HCl×M_{Tranexamic acid residue}×100%]/W_{Weighing of}=[（19.2-13.8）×10-3×0.5×140.21×100%]/1=37.612%

Degree of substitution =162A/[ M_{Tranexamic acid residue}×100-（M_{Tranexamic acid}-1）×A]

=162 × 0.37612/[14021- (140.21-1) × 0.37612] =0.436 (medium substitution)

The measurement of the substitution degree of the tranexamic acid-carrying cross-linked porous starch is measured by an acid-base titration method, the substitution degree is less than 0.2, the low substitution is determined, the medium substitution is determined between 0.2 and 2, the high substitution is determined when the substitution degree is greater than 0.2, and the substitution degree of the tranexamic acid-carrying cross-linked porous starch is 0.436, the medium substitution is determined.

4 liquid absorption rate:

(1) 0.1g of the potato starch, 0.1g of the enzymolysis starch and 0.1g of the tranexamic acid-carrying cross-linked porous starch are respectively weighed for later use.

(2) Principle of communicating vessels: the sieve plate of the sand core funnel is flush with the pipette, and the funnel and the rubber tube are all filled with PBS (see FIG. 3 (left) in the experimental device).

(3) Samples were evenly sprinkled onto the sand core funnel sieve plate, and the volume was recorded for an initial 20s, followed by recording every 10 s.

The results are shown in FIG. 3 (right). According to the liquid absorption rate experiment result, the following results are obtained: compared with the enzymolysis process, the influence of the esterification process on the starch imbibition rate is large, and the scanning electron microscope result picture shows that the particle surface is rough after the starch is esterified, and more micropores are formed on the particle surface, so that the specific surface area of the material is greatly increased, and the imbibition volume is increased.

Example 9 safety testing of Tranexamic acid-loaded crosslinked porous starch

1 in vitro cytotoxicity assay:

(I) leaching the sample

Reference "national standards for medical device biological evaluation-twelfth section: preparing a sample and a reference sample (GB/T16886.12-2017), adding 0.4g of tranexamic acid-loaded cross-linked porous starch into 4.4mL of serum-free DMEM medium, leaching for 24h at 37 ℃, adding 10% fetal calf serum into the supernatant of the obtained leaching liquor according to the volume ratio, uniformly mixing to obtain 100% leaching liquor, and diluting and uniformly mixing the 100% leaching liquor by the DMEM medium containing 10% fetal calf serum in an equal ratio to obtain 75% leaching liquor, 50% leaching liquor and 25% leaching liquor.

Adding 1g potato starch into 5mL serum-free DMEM medium, leaching at 37 ℃ for 24h, and carrying tranexamic acid to crosslink the porous starch in a leaching solution treatment mode to respectively obtain 100% leaching solution, 75% leaching solution, 50% leaching solution and 25% leaching solution.

(II) experiment steps:

(1) taking out growthVigorous L929 cells, conventional 0.25% pancreatin-EDTA solution digestion, blood cell plate count, complete medium dilution to 1X 10⁵Cell suspension/mL, 100. mu.L per well was seeded in 96-well culture plates. Placing the mixture in a 5% (v/v) carbon dioxide mixed air and 37 ℃ constant-temperature cell culture box for pre-culture for 24 hours.

(2) Culturing for 24h, discarding old culture medium, adding 100 μ L100% leaching solution, 75% leaching solution, 50% leaching solution and 25% leaching solution into experimental group, adding 100 μ L10% peptide bovine serum containing culture medium into negative control group, adding 100 μ L0.2% phenol solution into positive control group, and continuously culturing for 24 h.

(3) And taking out the culture plate after 24h of culture, discarding the liquid in each hole, adding 50 mu L of freshly prepared MTT solution into each hole, and continuously putting the holes into an incubator for culture for 3-5 h.

(4) The plate was removed, the liquid in each well was discarded, 100. mu.L of isopropanol was added to each well, shaken well and dissolved well. The absorbance (o.d. value) was measured by a microplate reader at a wavelength of 570 nm. The reference wavelength is 650 nm.

The Relative cell proliferation Rate (RGR) was calculated as follows.

RGR (%) = (test sample 100% leach liquor optical density average/blank optical density average) × 100%

Note: the test samples are potentially more cytotoxic at lower survival rates. If the survival rate drops to < 70% of the blank, it is potentially cytotoxic.

The results are shown in FIG. 4. As can be seen from fig. 4, the cell proliferation rate of the negative control group is 100% as a reference, and the relative cell proliferation rate of the positive control group is only 2.0% of that of the negative control group, which indicates that the modeling is successful and the result data is reliable. The mean relative cell proliferation rates of 100% leaching solution, 75% leaching solution, 50% leaching solution and 25% leaching solution of the tranexamic acid-loaded cross-linked porous starch and the potato starch are all larger than 70% relative to the negative control group, which indicates that the sample of the tranexamic acid-loaded cross-linked porous starch has no cytotoxicity to L929 fibroblasts in vitro.

2 acute toxicity test:

(I) leaching the sample

Reference GB/T16886.12-2017 medical device biology evaluation twelfth part sample preparation and reference sample. 0.88 g of tranexamic acid-loaded crosslinked porous starch was accurately weighed and extracted with 10.4ml of physiological saline at 37 ℃ for 24 hours. 2g of potato starch was weighed out accurately and extracted with 10ml of physiological saline at 37 ℃ for 24 hours.

(II) Experimental procedure

(1) Healthy Kunming mice, 18-22 g and males are randomly divided into 3 groups, and are respectively randomly set as a normal saline solution group, a potato starch experimental group and a tranexamic acid-carrying cross-linked porous starch experimental group.

(2) Dose and route of administration: the experimental group is injected into the abdominal cavity according to the dosage of 50 mL/kg and is provided with the normal saline leaching liquor of the corresponding group; the animals in the control group were administered with normal saline by intraperitoneal injection at a dose of 50 mL/kg.

(3) And (4) observation: after administration, general conditions (mental state, appearance, stool and urine, eating condition) and toxicity of the animals in each experimental group and control group were observed, and the number of dead animals was recorded. Animal body weights were recorded one week after dosing.

The results are shown in FIG. 5. The general state of the animals in the two experimental groups and the control group is good, the general conditions of the diet, behavior and the like of the mice are not abnormal, and the animals in each group are not dead. As shown in figure 5, after the leaching liquor of the experimental group of potato starch and tranexamic acid-loaded cross-linked porous starch is injected into the abdominal cavity of a mouse, the weight of the mouse shows a stable rising trend after 1 day, and the reaction is consistent with that of a control normal saline group mouse, which shows that the potato starch and the tranexamic acid-loaded cross-linked porous starch have no systemic acute toxic reaction.

EXAMPLE 10 effectiveness of Tranexamic acid-loaded crosslinked porous starch

1 in vitro clotting time assay:

tranexamic acid-loaded cross-linked starch was coagulated in vitro and time determined:

(1) taking a plurality of 5ml centrifuge tubes with the specification for standby. 30mg of the obtained tranexamic acid-loaded crosslinked porous starch sample is accurately weighed and placed in a centrifuge tube, a blank centrifuge tube is used as a negative control, euphoric speed hemostatic powder is used as a positive control (the dosage is 30 mg), and the dosage of the physical mixture in the comparative example 1 is 30 mg.

(2) New Zealand rabbits (male, 2.0-2.5 kg) were weighed, injected at 1ml/kg dose into the ear margin, the rabbits were fixed supine, the femoral artery was bled quickly, 1ml of fresh blood was added immediately into each centrifuge tube, gently shaken to bring the material into full contact with the blood, the tubes were tilted once every 15s in a 37 ℃ water bath condition until the blood did not flow, and the clotting time was recorded.

The results are shown in FIG. 6. As can be seen from fig. 6, the blood coagulation time of the blank group of new zealand rabbits was 830s, and the coagulation time was significantly shortened in the other experimental groups compared to the blank group. Compared with the positive control group of quick-acting hemostatic particles, the blood coagulation time of the tranexamic acid-loaded cross-linked porous starch in the example 6 is obviously reduced (p < 0.05). And the tranexamic acid-carrying cross-linked porous starch can be fully contacted with blood to play a role in hemostasis, and a film cannot be formed, so that the contact of the blood and a hemostatic material is prevented, and the hemostatic effect is played on the surface of the material.

Stopping bleeding by breaking the tail of the mouse:

the blank gauze, the Xinkuanzu hemostatic particles and the tranexamic acid-loaded cross-linked porous starch are accurately weighed to be 100mg respectively and placed in a 2ml centrifuge tube to be used as a sample. About 33-39g of male Kunming mice, 30 mice are randomly divided into 3 groups according to body weight, 10 mice in each group are anesthetized by intraperitoneal injection of 3% pentobarbital sodium aqueous solution according to the dose of 50mg/kg, the mice are fixed on a mouse board, the tail is drooped freely, the length of the tail is measured, the tail is cut off from the tail tip, the length of the tail is 1/2 by using surgical scissors, free bleeding is 10s (recording the free bleeding amount), then the mice are inserted into a centrifuge tube and are pressed lightly. Pressing for 2min, taking out, inserting into another sample if bleeding continues, stopping bleeding for 2min, taking out, and observing. The rat tail naturally droops, and within ten minutes, no active bleeding is marked as successful hemostasis; if the bleeding is still not stopped after the hemostasis is pressed twice, the failure of the hemostasis is recorded, and the hemostasis process of each mouse is pressed twice at most; if the hemostasis is successful after the hemostasis is pressed for the first time, the hemostasis is not pressed for the second time. The hemostatic photographs of the groups of hemostatic materials after hemostasis in the mouse tailed-bleeding model are shown in fig. 7.

Table 1 comparison of the blood stopping effect of hemostatic materials in mouse tail-biting model (n = 10)

As can be seen from FIG. 8 and Table 1, there was no significant difference (P > 0.05) between the free bleeding volume groups within 10s of the tail-broken of each group of mice, indicating that the model was successfully created, the reproducibility was good, and the experimental groups were comparable. The tranexamic acid-loaded cross-linked porous starch has the advantages of rapid hemostasis, stable effect and 100 percent of hemostasis success rate, and the hemostasis pressing frequency is less than that of the other two groups of hemostasis materials, and the bleeding amount is obviously less than that of euphoria-accelerated hemostasis granules (P is less than 0.05). In conclusion, in a mouse tail-breaking model hemostasis experiment, compared with domestic similar hemostasis materials, the tranexamic acid-loaded cross-linked porous starch can effectively deal with the bleeding condition of small arteriovenous vessels.

Claims

1. The application of the esterified modified starch in preparing the hemostatic material;

the esterified modified starch, namely the tranexamic acid-carrying cross-linked porous starch, is prepared by the method comprising the following steps:

1) dissolving starch in disodium hydrogen phosphate citric acid buffer solution, heating in water bath, and mechanically stirring; then adding alpha amylase and saccharifying enzyme, and continuing stirring; adding an alkali solution, adjusting the pH value of the system to 10-14, inactivating alpha amylase and saccharifying enzyme, centrifuging, washing, and freeze-drying to obtain enzymolysis starch;

3) reacting the enzymolysis starch obtained in the step 1) with the BOC-carbamoyl chloride obtained in the step 2), centrifuging, washing and freeze-drying after the reaction is finished to obtain the tranexamic acid-carrying cross-linked porous starch;

in the step 1), the ratio of the starch to the disodium hydrogen phosphate citric acid buffer solution is (2-3) g: (50-100) mL;

the pH value of the disodium hydrogen phosphate citric acid buffer solution is 4-5;

the temperature of the water bath is 45-55 ℃;

in the step 1), the dosage of the alpha amylase is 25-75 KNU/g starch; the dosage of the saccharifying enzyme is 5000-15000U/g starch;

adding alpha amylase and saccharifying enzyme, and continuously stirring for 5-10 h;

in the step 2), the mixture ratio of BOC-tranexamic acid, N-dimethylformamide, thionyl chloride and dichloromethane is (5-10) g: (200-400) μ L: (5-11) mL: (50-100) mL;

in the step 2), the reaction temperature of the reaction is 25-30 ℃; the reaction time is 2-4 h;

the reaction is carried out in a stirring state, and the stirring speed is 300-600 rpm;

in the step 3), the mass ratio of the enzymolysis starch to the BOC-carbamoyl chloride is 1-2 g: 5-10 g;

the reaction temperature is 40-80 ℃, and the reaction time is 4-12 h; the reaction is carried out in a stirring state, and the stirring speed is 300-600 rpm.

2. Use according to claim 1, characterized in that: in the step 1), the alkali solution is a sodium hydroxide solution; and after the alkali solution is added, stirring for 20-40 min.

3. Use according to claim 1, characterized in that: in the step 1) described above, the step of,

the centrifugation conditions were 5500rpm for 5 min;

the washing is carried out by using deionized water, and the washing times are three times;

the freeze drying condition is freezing at-60 ℃ for 24 h.

4. Use according to claim 1, characterized in that: in the step 3), the step of the method comprises the following steps,

the centrifugation conditions were 5500rpm for 5 min;

the freeze drying condition is freezing at-60 ℃ for 24 h.