Preparation method of sprayable nano-oxidized cellulose hemostatic gel

Abstract

The invention relates to the technical field of haemostatic materials, in particular to a preparation method of a sprayable nano-oxidized cellulose haemostatic gel, comprising the following steps: 1, preparing nano-oxidized cellulose through a TEMPO-NaClO-NaBr oxidation system; step 2, carrying out water dispersion on the nano-oxidized cellulose prepared in step 1; step 3: preparing the nano-oxidized cellulose dispersed by water in step 2 into gel, and then adding ether and iodoform into the gel to obtain the nano-oxidized cellulose spray material. The spray nano-oxidized cellulose haemostatic gel of the invention quickly forms hydrogel when encountering blood after spraying nano-fibres, thus solving the problem that the traditional gauze of tourniquet cannot be used for packing and pressing haemostasis when bleeding at the joint boundary of the body. Moreover, iodoform and diethyl ether have anaesthetic, disinfection and anti-inflammatory effects.

Description

TECHNICAL FIELD The invention relates to the technical field of haemostatic materials, in particular to a preparation method of a sprayable nano-oxidized cellulose haemostatic gel. BACKGROUND At present, in the field ofwar trauma, the problem of timely and rapid haemostasis has not been effectively solved. Uncontrollable massive bleeding is the primary factor of battlefield casualties, which accounts for more than 90% of trauma deaths. Therefore, it is very important to stop bleeding quickly to reduce casualties in the battlefield. At present, traditional gauze such as tourniquet is commonly used to stop bleeding in war wounds, but traditional gauze such as tourniquet cannot be used to stop bleeding at the jointjunction, which has limited effect on stopping bleeding in severe bleeding and is sometimes prone to the risk of re-bleeding. Although the existing haemostatic materials have made remarkable progress in haemostasis effect and portability, their safety and effective haemostasis ability are still a major bottleneck in haemostasis. Oxidized regenerated cellulose has the characteristics of low immunogenicity, healing promotion and modifiable surface active sites for antibacterial and antiviral purposes. When the carboxyl content of oxidized regenerated cellulose is between 16% and 24%, it can show the best haemostasis and biodegradability. Oxidized regenerated cellulose is an effective haemostatic agent. The carboxyl group in oxidized regenerated cellulose structure can reduce the pH value of blood, provide an acidic environment, and attract Fe3+ ions in haemoglobin. At the same time, it can activate platelets in blood, make platelets aggregate and form thrombus, and then control massive bleeding. In addition, oxidized regenerated cellulose will swell after contact with blood, so that the ends of capillaries will be compressed and closed, which will accelerate haemostasis. The higher the carboxyl content in oxidized regenerated cellulose, the lower the polymerization degree and the better the haemostatic effect. Although oxidized regenerated cellulose has many excellent properties, it also has some disadvantages: oxidized surface can enhance haemostasis, but the active surface that can be provided without nano-dispersion is limited, and the pH is low, which is easy to damage some nervous systems and cannot be used for cerebral haemorrhage. In some published or authorized invention patents, natural or synthetic polymer materials, such as chitosan (CN102198288A), alginate (CN104013991A) and graphene oxide (CN104383578A), have been used to improve the haemostatic properties of oxidized regenerated cellulose. Although the above composite haemostatic materials are of great significance to the related research of oxidized regenerated cellulose, and at the same time, the haemostatic performance of the materials has been improved to a certain extent, but the improvement degree is small, that is, the improvement effect is poor. Moreover, the improvement methods in previous studies will also have a negative impact on the mechanical strength and bioabsorbability of oxidized regenerated cellulose haemostatic materials, and the modification effect is not worth the loss compared with the haemostatic performance that has been slightly improved. Moreover, the cross-linking strength between the existing nano-oxidized cellulose molecules based on hydrogen bonding is low, which makes its hydrogel weak and easy to be crushed under pressure. SUMMARY Aiming at the technical problems of slow haemostatic rate and small improvement of haemostatic performance of the existing modified nano-oxidized cellulose, the invention provides a preparation method of a sprayable nano-oxidized cellulose haemostatic gel. The nano-oxidized cellulose in the invention is a bioactive material which can quickly stop bleeding at a wound. When the nano-oxidized cellulose is sprayed with a large amount of gushing blood, its own characteristics make it quickly gel, so that it can effectively stop bleeding. In that invention, a low-molecular-weight organic substance or functional group is use to modify the surface of nano-oxidized cellulose, and chemical crosslinking among nano-celluloses based on the modified functional group can effectively improve the gel toughness, thereby well solving the problem. In the aspect of drug function, oxidized nanofibers can wrap small molecular substances such as drugs and proteins, which is beneficial to the rapid healing ofwounds after bleeding. At the same time, adding drugs such as ether and iodoform into nanofibers can alleviate the pain atwounds and ensure the antibacterial property of haemostatic materials. While improving the antibacterial property of the materials, the materials have good air permeability, water permeability and water retention characteristics. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a preparation method of a sprayable nano-oxidized cellulose haemostatic gel comprises the following steps: step 1: preparing nano-oxidized cellulose by a TEMPO-NaClO-NaBr oxidation system; step 2: carrying out water dispersion on the nano-oxidized cellulose prepared in step 1; step 3: preparing the nano-oxidized cellulose dispersed by water in step 2 into gel, and then adding ether and iodoform into the gel to obtain the nano-oxidized cellulose spray material. Preferably, the step 1 comprises: step 1.1: first, the pulp board is firstly treated in distilled water with a cooking machine to form a wet pulp, the pH of the pulp is adjusted to 2 by HCl to remove the minerals, then filtered and washed, the pH is adjusted to 7 by NaOH and finally dried at 120°C for 3h to determine the content of cellulose in it; step 1.2: mixing TEMPO, NaBr and deionized water according to the ratio of 0.016g TEMPO, 0.1g NaBr and 100 mL deionized water per gram of cellulose to prepare a solution; then adding a corresponding amount of the final product of step 1; then NaClO is added into the solution according to the ratio of 10 mmol / g; step 1.3: start the reaction, and add 0.5 M NaOH into the reaction solution to maintain the pH of the reaction at 10 until the reaction is finished; step 1.4: add sodium borohydride and ethanol to the solution after the reaction in step 1.3 according to 0.1 g sodium borohydride and 1 mL ethanol per gram of cellulose, and make it react for 3 h; step 1.5: wash and filter the solution after the reaction in step 1.4, and adjust the pH to neutral to obtain transparent gel-like nano-oxidized cellulose. Preferably, the step 2 comprises: step 2.1: dissolving the nano-oxidized cellulose prepared in step 1 in water, and processing by a homogenizer to uniformly disperse it; step 2.2: treating the solution dispersed in step 2.1 with an ultrasonic disperser for 50 min; step 2.3: centrifuge the solution after ultrasonic treatment in step 2.2 with a centrifuge, and take the supernatant after centrifugation to obtain the aqueous dispersion of nano-oxidized cellulose. Compared with the prior art, the invention has the beneficial effects that: 1. the sprayable nano-oxidized cellulose haemostatic gel of the present invention selectively oxidizes the prepared nano-cellulose through the TEMPO-NaClO-NaBr oxidation system, and introduces a sodium carboxylate structure on the C6 position of the nano-cellulose molecule, thereby increasing the specific surface area of oxidized regenerated cellulose, improving the haemostatic performance of the obtained sprayable nano-oxidized cellulose haemostatic gel, and overcoming the defect that the haemostatic performance of ordinary haemostatic materials is improved by a small margin; 2. the surface of the sprayable nano-oxidized cellulose haemostatic gel has carboxyl groups, which has a double haemostatic effect, so that the haemostatic time is greatly shortened, and the effect of rapid haemostasis is achieved; and drugs such as ether and iodoform are added, which can relieve the pain at the wound and ensure the antibacterial property of the haemostatic material; 3. the preparation process of the sprayable nano-oxidized cellulose haemostatic gel does not need special equipment, and the reaction conditions are mild, so that industrial production can be realized; 4. the raw materials of the preparation process of the sprayable nano-oxidized cellulose haemostatic gel are renewable resources, and the cost is low, so that the cost of the nano- cellulose / oxidized regenerated cellulose composite haemostatic material can be reduced; 5. using the sprayable nano-oxidized cellulose haemostatic gel prepared by the invention, the bleeding amount is reduced by 10-20% when the liver and artery bleed; 6. sprayable nano-oxidized cellulose haemostatic gel can quickly form hydrogel when it meets blood after spraying nano-fibres, which solves the problem that traditional gauze with tourniquet can not be used to stop bleeding when bleeding at the joint boundary of the body. BRIEF DESCRIPTION OF THE FIGURES The accompanying figures are provided to provide a further understanding of the invention and constitute a part of the specification. Together with the embodiments of the invention, they serve to explain the invention and do not constitute a limitation of the invention. In the attached figures: Fig. 1 is a flowchart of the method of the present invention. Fig. 2 (a) shows experimental results of haemostasis by nano-oxidized cellulose in rats, (b) shows experimental results of haemostasis of leg arteries of nano-oxidized cellulose SD rats. Fig. 3 shows the experimental results of animal liver haemostasis, (a) observation of haemostatic effect of nano-oxidized cellulose, (b) detection results of liver bleeding. Fig. 4 is a safety experiment of nano-oxidized cellulose prepared by the invention. Fig. 5 shows the results of ELSA detection of blood inflammatory factors after treatment with nano-oxidized cellulose prepared by the present invention. Fig. 6 shows test results ofwater contact angle of oxidized nano-cellulose material. Fig. 7 is observation results of macroscopic morphology of oxidized nano-cellulose material. Fig. 8 shows surface micro-morphology of oxidized nano-cellulose material before and after drying. Fig. 9 shows stress and strain results of oxidized nano-cellulose material and other haemostatic materials. Fig. 10shows comparative analysis of oxidized nano-cellulose material and other haemostatic materials. Fig. 11 shows viscosity observation chart of oxidized nano-cellulose material. DESCRIPTION OF THE INVENTION Preferred embodiments of the present invention are described below with reference to the accompanying figures, and it should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not used to limit the present invention. Embodiment As shown in Figs. 1-11, a preparation method of sprayable nano-oxidized cellulose haemostatic gel comprises: step 1: preparing nano-oxidized cellulose by a TEMPO-NaClO-NaBr oxidation system; step 1.1: first, the pulp board is firstly treated in distilled water with a cooking machine to form a wet pulp, the pH of the pulp is adjusted to 2 by HCI to remove the minerals, then filtered and washed, the pH is adjusted to 7 by NaOH and finally dried at 120°C for 3h to determine the content of cellulose in it; step 1.2: mixing TEMPO, NaBr and deionized water according to the ratio of 0.016g TEMPO, 0.1g NaBr and 100 mL deionized water per gram of cellulose to prepare a solution; then adding a corresponding amount of the final product of step 1; then NaClO is added into the solution according to the ratio of 10 mmol / g; step 1.3: start the reaction, and add 0.5 M NaOH into the reaction solution to maintain the pH of the reaction at 10 until the reaction is finished; step 1.4: add sodium borohydride and ethanol to the solution after the reaction in step 1.3 according to 0.1 g sodium borohydride and 1 mL ethanol per gram of cellulose, and make it react for 3 h; step 1.5: wash and filter the solution after the reaction in step 1.4, and adjust the pH to neutral to obtain transparent gel-like nano-oxidized cellulose. step 2: Carrying out water dispersion on the nano-oxidized cellulose prepared in step 1; step 2.1: dissolving the nano-oxidized cellulose prepared in step 1 in water, and processing by a homogenizer to uniformly disperse it; step 2.2: treating the solution dispersed in step 2.1 with an ultrasonic disperser for 50 min; step 2.3: centrifuge the solution after ultrasonic treatment in step 2.2 with a centrifuge, and take the supernatant after centrifugation to obtain the aqueous dispersion of nano-oxidized cellulose. step 3: preparing the nano-oxidized cellulose dispersed by water in step 2 into gel, and then adding ether and iodoform into the gel to obtain the nano-oxidized cellulose spray material. Experimental results 1. Solution viscosity measurement At the same concentration, it is measured with a rotary viscometer (NDJ-79), as shown in Table 1 below: Table 1 Viscosity ofcommon haemostatic materials It can be obtained from the above table: according to the data in the table, at the same concentration, the viscosity of oxidized nanocellulose is far higher than that of other materials. Fig. 11 shows the viscosity observation results of oxidized nano-cellulose, and it can be seen that it has good viscosity. 2. Measurement of surface tension of solution Measured by a contact angle measuring instrument (KRUSS, America), as shown in Fig. 6, the haemostatic mechanism of oxidized cellulose is mainly through the combination of its own - COOH structure with Fe3+ in blood to form a brown thrombus gel block, sealing blood vessels, and achieving the haemostatic effect. This process does not need to participate in the normal physiological coagulation mechanism. Secondly, oxidized cellulose with rough surface is easy to cause platelet aggregation and rupture, form platelet embolus, activate coagulation factors, activate coagulation system, and promote the transformation of fibrinogen into fibres. Furthermore, hydroxyl groups in oxidized cellulose can also cross-link with Ca2+ in plasma to form gel-like blood clots, blocking damaged blood vessels and stopping bleeding. According to the haemostatic mechanism of nano-oxidized cellulose, the control group and the experiment after combining with Fe3+ are designed. The water contact angle test is used to characterize the hydrophilicity of the material. As can be seen from Fig. 6, the hydrophilic effect of oxidized cellulose is the best, and the hydrophilicity after combining with Fe3+ is basically the same. 3. Macroscopic morphology observation The morphology ofRCMs is observed by optical microscope (BX41, OLYMPUS), and the shape of liquid at the tip of needle is photographed by digital camera (IXUS220HS, Canon). As shown in Fig. 7, observing the general state of nano-oxidized cellulose gel and the state after spraying proves that nano-oxidized cellulose can be made into spray state, which is beneficial to bleeding treatment. 4 Micro-morphology of surface The surface morphology is characterized by atomic force microscope. Atomic force microscopy (AFM) shows that the width of nano-oxidized cellulose prepared byTEMPO oxidation is 2-3 nm and the length is 500-1,000 nm. As shown in the right figure of Fig. 8, the surface of the oxidized cellulose membrane obtained by drying is covered with nano-oxidized cellulose. 5. Stress and strain of oxidized nano-cellulose material and other haemostatic materials The stress and strain of various materials are tested, and it can be seen from Fig. 9 that the maximum stress that oxidized cellulose can bear is higher than that of other materials. Young's modulus is obtained by calculating the stress-strain curve, as shown in Table 2 below: Table 2 Young's modulus of oxidized nano-cellulose material and other haemostatic materials membrane alginate membrane _ Maximum Young's The greater the Young's modulus, the less likely it is to deform. From the above table, we can see that the Young's modulus of oxidized nano-cellulose material is greater than that of other materials, which proves that oxidized nano-cellulose material has good performance and is not easy to deform. 6. The haemostatic experiment of the nano-oxidized cellulose gel material-rats. As shown in Fig. 2(a), the arrow points to the position of the blood vessels in the legs of rats. After the arterial vessels in the legs of rats are cut short, the nano-oxidized cellulose haemostatic gel material prepared by the invention is sprayed, observed for a period of time, and then sutured. As shown in Fig. 2(b), the left side is the control group, and the right side is the nano-oxidized cellulose gel group. It can be seen that there is obvious bleeding in the control group and it overflows, and the nano-oxidized cellulose gel group has a good haemostatic effect. 7. Animal visceral haemostasis experiment The internal organs of animals are taken out and divided into control group and nano- oxidized cellulose gel group, and the amount of bleeding is observed and detected. The observation result is shown in Fig. 3(a), and the blood loss detection result is shown in Fig. 3(b). From the results, it can be seen that the nano-oxidized cellulose of the invention has good haemostatic effect. 8. Safety experiment As shown in Fig. 4, the main organs, such as heart, liver, spleen, lung, kidney, and the experimental parts of nano-oxidized cellulose are stained by HE. The results show that nano- oxidized cellulose has good biological safety, and the last group is the staining results of bleeding blood vessel tissues. The results show that the material had no influence on tissues and is safe. 9. ELISA detection of inflammatory factors As shown in Fig. 5, the blood of experimental SD rats is used to detect inflammatory factors by ELISA, which show that nano-oxidized cellulose will not cause local and systemic inflammation. 10. Material structure analysis As shown in Fig. 10, it is the structure of oxidized cellulose, chitosan, sodium alginate and gelatine. From the point of structural similarity, sodium alginate is the closest to oxidized cellulose. The basic principle, main features and advantages ofthe present invention have been shown and described above. It should be understood by those skilled in the art that the present invention is not limited by the above-mentioned embodiments, and what is described in the above- mentioned embodiments and descriptions only illustrates the principles of the present invention. Vlthout departing from the spirit and scope of the present invention, there will be various changes and improvements in the present invention, which fall within the scope of the claimed invention. The scope of that present invention is define by the appended claim and their equivalents.

Claims

1. A method for preparing a sprayable nano-oxidized hemostatic cellulose gel, comprising: Step 1: Preparation of nano-oxidized cellulose using a TEMPO-NaClO NaBr oxidation system; Step 2: Converting the nano-oxidized cellulose prepared in Step 1 into an aqueous dispersion; Step 3: Converting the nano- dispersed with water in step 2 into a gel oxidized cellulose and then adding ether and iodoform to the gel to obtain the sprayable nano-oxidized cellulose material.

2. The method for preparing a sprayable nano-oxidized hemostatic cellulose gel according to claim 1, wherein step 1 comprises: Step 1.1: First, treat the boiled water in distilled water pulp sheet to form a wet pulp, bringing the pulp to a boil with the aid of HCI pH at 2 to remove minerals, then filter and wash of it, adjusting the pH to 7 using NaOH and finally drying for 3 hours at 120°C to determine the cellulose content in it; Step 1.2: Prepare a solution by mixing TEMPO, NaBr and deionized water in the ratio of 0.016 g TEMPO, 0.1 g NaBr and 100 ml deionized water per gram of cellulose; then add it to the solution adding a corresponding amount of the final product from step 1; then add NaClO in the ratio of 10 mmol / g; Step 1.3: Starting the reaction and adding 0.5 M to the reaction solution NaOH to maintain the pH of the reaction at 10 until the reaction is complete; step 1.4: adding sodium borohydride to the solution after the reaction in step 1.3 and ethanol in an amount of 0.1 g sodium borohydride and 1 ml ethanol per gram of cellulose, and let it react for 3 hours; Step 1.5: Washing and filtering the solution after the reaction in step 1.4, and bringing the pH to neutral value to transparent gel-like nano-oxidized cellulose available.

3. The method for preparing a sprayable nano-oxidized hemostatic cellulose gel according to claim 2, wherein step 2 comprises: Step 2.1: Dissolving the nano-oxidized cellulose prepared in step 1 in water and homogenize it to make it evenly dispersed; Step 2.2: Treating with an ultrasonic dispersing device for 50 min. the solution dispersed in step 2.1; step 2.3: centrifuging with a centrifuge after the ultrasonic treatment in step 2.2 the solution, and taking the supernatant liquid after centrifugation 5 to obtain the aqueous dispersion of nano-oxidized cellulose.