技术领域technical field
本发明属于生物医用材料技术领域，涉及一种止血材料及其制备方法，具体而言，本发明涉及一种可直接施用于外科手术(含微创手术)以及外伤等原因造成的人及其它哺乳动物的组织器官创面，用于止血、封闭伤口、减少组织渗出、促进组织修复以及保护创面组织等目的的明胶基止血粉剂和止血凝胶材料的制备方法。The invention belongs to the technical field of biomedical materials, and relates to a hemostatic material and a preparation method thereof. Specifically, the invention relates to a hemostatic material that can be directly applied to human and other breastfeeding patients caused by surgical operations (including minimally invasive operations) and trauma. The preparation method of gelatin-based hemostatic powder and hemostatic gel material for the purposes of hemostasis, wound sealing, tissue exudation reduction, tissue repair promotion and wound tissue protection for tissue and organ wounds of animals.
背景技术Background technique
外科手术过程中需要对不同原因造成的伤口进行止血和封闭，因此止血材料是非常重要的一类生物医用材料，具有重要的研发和应用意义。目前临床常用的止血材料从作用机制上可分为三类：第一类止血材料是通过材料的物理或化学作用使伤口部位加速凝血(如止血纱布、高分子多糖类、无机类沸石等)；第二类是直接或间接提供外来的凝血成分、利用激发自身凝血机制加速实现凝血(如纤维蛋白类止血材料、含有凝血酶和凝血因子类止血材料)；第三类是利用材料对组织很强的粘着力直接封闭创面，从而实现止血(如α氰基丙烯酸酯类材料)。然而不同类型的止血材料都存在诸多瓶颈。During surgical operations, it is necessary to stop bleeding and seal wounds caused by different reasons. Therefore, hemostatic materials are a very important class of biomedical materials, which have important research and application significance. At present, the hemostatic materials commonly used in clinical practice can be divided into three categories from the mechanism of action: the first type of hemostatic material accelerates blood coagulation at the wound site through the physical or chemical action of the material (such as hemostatic gauze, polymer polysaccharides, inorganic zeolites, etc.) The second category is to provide external coagulation components directly or indirectly, and to accelerate coagulation by stimulating its own coagulation mechanism (such as fibrin hemostatic materials, hemostatic materials containing thrombin and coagulation factors); the third category is to use materials that are very effective on tissues. Strong adhesive force directly seals the wound surface, thereby achieving hemostasis (such as α-cyanoacrylate materials). However, there are many bottlenecks in different types of hemostatic materials.
对于利用止血机制来实现血凝的之血制品而言，其缺点是它们仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。另外，载有凝血酶类凝血剂的止血制品在使用过程中，凝血酶类蛋白分子易外溢到正常血管中，因此存在诱发正常血流发生凝血产生血栓的风险，因此不依赖机体凝血机制并能迅速封闭创面的止血制品在外科手术中更受青睐。For blood products that use the hemostatic mechanism to achieve blood coagulation, the disadvantage is that they are only effective for bleeding with a small flow rate, so they must be used with a hemostatic clamp, which makes the above-mentioned hemostatic materials often require surgeons to use them. Predict the amount of bleeding in advance or use the hemostat repeatedly. In addition, during the use of hemostatic products loaded with thrombin-like coagulants, thrombin-like protein molecules are easy to spill into normal blood vessels, so there is a risk of inducing normal blood flow to coagulate and produce thrombus. Therefore, it does not rely on the body's coagulation mechanism and can Hemostatic products that quickly seal wounds are more popular in surgical operations.
而通过物理作用实现止血的止血制品，如止血纱布、海绵等，在止血过程需要通过对出血创面进行物理压迫，同时材料通过吸收血液中的水，促使凝血，止血速度慢，效果较差。并且，这类材料仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。同时，传统止血材料(如棉纱、绷带)对于不规则形状、深、窄、动脉破裂等现场常见创伤的止血效果很不理想。另外，还存在其他的缺点：1)影响术后缝合，增加新的创面出血；2)不能自然降解，在体内形成残留；3)止血过程需要长时间的外力按压实现物理止血，对于脆弱的神经或脑组织不适用。另一类无机物止血材料，包括沸石、高岭土等，这类材料利用了天然硅铝酸盐材料多孔结构、高比表面积等特性，通过吸收血液中的水分，浓缩创面局部的凝血成分，从而实现快速止血。而人工制备的介孔硅材料同样具有多孔结构、高比表面积等特性，而且相比天然硅铝酸盐材料具有成分、粒径、孔径可控等优势，同样可实现有效止血。然而，这类材料对于血流大的出血无能为力，对于大面积创面效果也有限；并且这类材料虽然介局部组织反应是良性的，但是进入脉管和组织器官的介孔硅颗粒会引起严重的全身毒性。因此，开发针对现场和院前急救用的，快速、安全、有效的新型止血材料成为医学和生物材料科学领域中的重要课题。However, hemostatic products that achieve hemostasis through physical action, such as hemostatic gauze, sponge, etc., need to physically compress the bleeding wound during the hemostatic process. Moreover, this type of material is only effective for bleeding with a small flow rate, so it must be used with a hemostatic clamp, which makes the above-mentioned hemostatic material often require the surgeon to predict the amount of bleeding in advance or repeatedly use the hemostatic clamp. At the same time, traditional hemostatic materials (such as cotton gauze and bandages) are not ideal for the hemostatic effect of common wounds such as irregular shapes, deep, narrow, and ruptured arteries. In addition, there are other disadvantages: 1) It affects postoperative suture and increases new wound bleeding; 2) It cannot be degraded naturally, and residues are formed in the body; 3) The process of hemostasis requires long-term external pressure to achieve physical hemostasis. Or brain tissue does not apply. Another type of inorganic hemostatic materials, including zeolite, kaolin, etc., these materials take advantage of the porous structure and high specific surface area of natural aluminosilicate materials, and absorb the water in the blood to concentrate the blood coagulation components in the wound, so as to achieve Quickly stop bleeding. The artificially prepared mesoporous silicon material also has the characteristics of porous structure and high specific surface area, and has the advantages of controllable composition, particle size, and pore size compared with natural aluminosilicate materials, and can also achieve effective hemostasis. However, this type of material is powerless for bleeding with large blood flow, and has limited effect on large-area wounds; and although this type of material has a benign local tissue reaction, the mesoporous silicon particles entering the blood vessels and tissues and organs will cause serious damage. Systemic toxicity. Therefore, the development of fast, safe and effective new hemostatic materials for on-site and pre-hospital first aid has become an important topic in the field of medicine and biomaterial science.
可注射的凝胶类止血制品因其临床易操作性、适于微创手术近年来渐渐进入临床止血应用。目前国内临床上使用的止血凝胶类产品主要依靠进口，其中美国强生公司的占据了主要的市场份额。该材料由明胶组成，水溶后快速形成泡沫状凝胶物，产品具有很好的可注射性。但在止血过程中存在诸多问题。首先，凝胶力学强度差，流变仪测得弹性模量<1kPa，对于出血量大、血管压力高的出血组织无法实现止血；其次，该材料与组织间黏性差，对于临床出血量稍大的创面，难以实现快速止血。因此，开发具有良好可注射性、可塑性等临床操作性能，可用于血流量大、血压高的组织器官创面止血的新型凝胶材料具有重要的实用意义。Injectable gel-type hemostatic products have gradually entered clinical hemostasis applications in recent years because of their clinical ease of operation and suitability for minimally invasive surgery. At present, the hemostatic gel products used clinically in China mainly rely on imports, among which Johnson & Johnson’s Occupy a major market share. The material is composed of gelatin, which quickly forms a foamy gel after being dissolved in water, and the product has good injectability. But there are many problems in the process of hemostasis. First, the mechanical strength of the gel is poor, and the elastic modulus measured by the rheometer is less than 1kPa, so hemostasis cannot be achieved for bleeding tissues with large bleeding volume and high vascular pressure; It is difficult to achieve rapid hemostasis on the wound surface. Therefore, it is of great practical significance to develop new gel materials with good injectability, plasticity and other clinical performance, which can be used for hemostasis on tissue and organ wounds with large blood flow and high blood pressure.
发明内容Contents of the invention
鉴于上述所述的止血材料在现有技术中存在的问题，本发明提供一种可注射型自愈合止血材料及其制备方法。本发明的方法制备得到的止血材料具有良好的可注射性、自愈合性能和较好的力学强度，对于出血量大、血管压力高的出血组织也可以实现快速止血。In view of the problems of the above-mentioned hemostatic material in the prior art, the present invention provides an injectable self-healing hemostatic material and a preparation method thereof. The hemostatic material prepared by the method of the present invention has good injectability, self-healing performance and good mechanical strength, and can also realize rapid hemostasis for bleeding tissues with large bleeding volume and high blood vessel pressure.
本发明的技术方案如下：Technical scheme of the present invention is as follows:
一种可注射型自愈合止血材料，由如下方法制备得到：An injectable self-healing hemostatic material is prepared by the following method:
(1)以明胶为原料，使其在去离子水中加热溶解，配置浓度为1～10w/v％的明胶水溶液，调pH值为1-6或8-14，向溶液中加入>2倍溶液体积的极性有机溶剂，生成明胶微凝胶颗粒分散液，加入交联剂交联反应1～12h，离心、清洗得到明胶微凝胶颗粒；(1) Use gelatin as a raw material, heat and dissolve it in deionized water, configure a gelatin aqueous solution with a concentration of 1-10w/v%, adjust the pH value to 1-6 or 8-14, and add >2 times the solution to the solution volume of polar organic solvent to generate a dispersion of gelatin microgel particles, add a crosslinking agent for crosslinking reaction for 1 to 12 hours, centrifuge and wash to obtain gelatin microgel particles;
其中，所述明胶微凝胶颗粒的zeta电势为-30～+30mV，所述明胶微凝胶颗粒的直径为20nm～2μm；Wherein, the zeta potential of the gelatin microgel particles is -30～+30mV, and the diameter of the gelatin microgel particles is 20nm～2μm;
(2)将步骤(1)制备得到的表面zeta电势>+10mV的明胶微凝胶颗粒，分散在pH<5的酸性水溶液或pH>9的碱性水溶液中，得分散有带正电荷的明胶微凝胶颗粒的分散液，再与带负电荷的高分子颗粒分散液按照颗粒数比1:10～10:1共混，或者与等电点<6的带负电荷的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，用pH调节剂调pH至7.0，冷冻干燥，得明胶微凝胶颗粒冻干粉末I；(2) The gelatin microgel particles with the surface zeta potential>+10mV prepared in step (1) are dispersed in the acidic aqueous solution of pH<5 or the alkaline aqueous solution of pH>9, and positively charged gelatin is dispersed. The dispersion of microgel particles is then blended with the dispersion of negatively charged polymer particles according to the particle number ratio of 1:10 to 10:1, or with negatively charged hydrophilic polymers with an isoelectric point <6 The aqueous solution is blended according to the mass ratio of the gelatin microgel particles to the hydrophilic polymer in the range of 1:10 to 10:1, the pH is adjusted to 7.0 with a pH regulator, and freeze-dried to obtain the gelatin microgel particle freeze-dried powder I;
(3)将步骤(1)制备得到的表面zeta电势<-10mV的明胶微凝胶颗粒，分散在pH<5的酸性水溶液或pH>9的碱性水溶液中，得明胶微凝胶颗粒的分散液，再与带正电荷的高分子颗粒分散液按照颗粒数比1:10～10:1混合，或者与等电点>8的带正电荷的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，用pH调节剂调pH至7.0，冷冻干燥，得明胶微凝胶颗粒冻干粉末II；(3) The gelatin microgel particles with surface zeta potential<-10mV prepared in step (1) are dispersed in the acidic aqueous solution of pH<5 or the alkaline aqueous solution of pH>9 to obtain the dispersion of gelatin microgel particles liquid, and then mixed with the positively charged polymer particle dispersion according to the particle number ratio of 1:10 to 10:1, or mixed with the positively charged hydrophilic polymer aqueous solution with an isoelectric point>8 according to the gelatin microgel particles Blend with the hydrophilic polymer at a mass ratio of 1:10 to 10:1, adjust the pH to 7.0 with a pH regulator, and freeze-dry to obtain gelatin microgel particle freeze-dried powder II;
(4)将步骤(1)制备得到的表面zeta电势为-10～+10mV的明胶微凝胶颗粒分散在中性水溶液中，再与另一种表面zeta电势在-10～+10mV的高分子颗粒分散液按照颗粒数比1:10～10:1共混，或者与等电点6～8的亲水性高分子水溶液按照明胶微凝胶颗粒与亲水性高分子的质量比1:10～10:1共混，冷冻干燥，得明胶微凝胶颗粒冻干粉末Ⅲ；(4) Disperse the gelatin microgel particles with a surface zeta potential of -10 to +10mV prepared in step (1) in a neutral aqueous solution, and then mix with another polymer with a surface zeta potential of -10 to +10mV The particle dispersion is blended according to the particle number ratio of 1:10-10:1, or the hydrophilic polymer aqueous solution with an isoelectric point of 6-8 is used according to the mass ratio of gelatin microgel particles to hydrophilic polymer 1:10 ~ 10:1 blending, freeze-drying to obtain gelatin microgel particle freeze-dried powder III;
(5)将明胶微凝胶颗粒冻干粉末I、明胶微凝胶颗粒冻干粉末II或明胶微凝胶颗粒冻干粉末Ⅲ分别与水性溶液共混，得可注射型自愈合止血材料；(5) respectively blending the freeze-dried powder of gelatin microgel particles I, the freeze-dried powder of gelatin microgel particles II or the freeze-dried powder of gelatin microgel particles III with an aqueous solution to obtain an injectable self-healing hemostatic material;
其中，所述的带正电荷的高分子颗粒的表面电荷为+5～+60mV，带负电荷的高分子颗粒的表面电荷为-5～-60mV，所述高分子颗粒的直径为100nm～20μm，在步骤(5)中得到的可注射型自愈合止血材料中胶体颗粒占总体积的百分比为50vol％～150vol％；在步骤(3)、(4)和(5)中所述的亲水性高分子的分子量为1k～500kDa。Wherein, the surface charge of the positively charged polymer particles is +5～+60mV, the surface charge of the negatively charged polymer particles is -5～-60mV, and the diameter of the polymer particles is 100nm～20μm , in the injectable self-healing hemostatic material obtained in step (5), the percentage of colloid particles accounting for the total volume is 50vol%～150vol%; The molecular weight of the water-based polymer is 1k-500kDa.
本发明上述可注射型自愈合止血材料的制备方法中，将步骤(1)制备得到的明胶微凝胶颗粒，冷冻干燥得到的明胶微凝胶颗粒冻干粉末与水性溶液共混，也可以得到本发明所述的可注射型自愈合止血材料，但其性能差于本发明中其他方法制备得到的可注射型自愈合胶体凝胶。In the preparation method of the above-mentioned injectable self-healing hemostatic material of the present invention, the gelatin microgel particles prepared in step (1), the freeze-dried gelatin microgel particle lyophilized powder obtained by freeze-drying, and the aqueous solution can also be blended. The injectable self-healing hemostatic material described in the present invention is obtained, but its performance is inferior to the injectable self-healing colloid gel prepared by other methods in the present invention.
本发明上述可注射型自愈合止血材料的制备方法中，在步骤(1)中，根据调整明胶水溶液的浓度、极性有机溶剂的加入量、交联反应时间等，可以制备得到带有不同zeta电势和直径大小的明胶微凝胶颗粒。本发明上述技术方案中，明胶水溶液浓度优选为1～10w/v％，更优选为2.5～5w/v％；极性有机溶剂的加入量优选为明胶水溶液的>2倍，更优选为3～6倍，明胶微凝胶颗粒直径优选为20nm～5μm，更优选为100nm～2000nm。In the preparation method of the above-mentioned injectable self-healing hemostatic material of the present invention, in step (1), according to adjusting the concentration of the gelatin aqueous solution, the addition amount of the polar organic solvent, the cross-linking reaction time, etc., it can be prepared with different Zeta potential and diameter size of gelatin microgel particles. In the above technical scheme of the present invention, the concentration of the aqueous gelatin solution is preferably 1 to 10w/v%, more preferably 2.5 to 5w/v%; the amount of the polar organic solvent is preferably >2 times that of the aqueous gelatin solution, more preferably 3 to 6 times, the gelatin microgel particle diameter is preferably 20nm-5μm, more preferably 100nm-2000nm.
本发明上述可注射型自愈合止血材料的制备方法中，步骤(2)、(3)和(4)中，明胶微凝胶颗粒的分散液和各有机高分子颗粒分散液中的颗粒数的比例影响所制备胶体凝胶的弹性模量和自修复效率，本发明中两种分散液中颗粒数的比例优选为1:10～10:1，更优选为1:5～5:1，共混的两种颗粒直径比为1:5～5:1时，可得到具有更高弹性模量和自修复效率的可注射型自愈合止血材料，若直径的差异过大，则所得到的胶体凝胶弹性模量下降，自修复效率降低。In the preparation method of the above-mentioned injectable self-healing hemostatic material of the present invention, in steps (2), (3) and (4), the number of particles in the dispersion of gelatin microgel particles and the dispersion of each organic polymer particle The ratio affects the elastic modulus and self-repairing efficiency of the colloidal gel prepared, the ratio of the number of particles in the two dispersions in the present invention is preferably 1:10～10:1, more preferably 1:5～5:1, When the diameter ratio of the two blended particles is 1:5 to 5:1, an injectable self-healing hemostatic material with higher elastic modulus and self-repairing efficiency can be obtained. If the difference in diameter is too large, the obtained The elastic modulus of the colloidal gel decreases and the self-healing efficiency decreases.
在本发明的上述技术方案中，将制备得到的明胶微凝胶颗粒冻干粉末I、明胶微凝胶颗粒冻干粉末II或明胶微凝胶颗粒冻干粉末Ⅲ直接作为止血材料来应用，也可以与适量水性溶液共混，制备得到胶体凝胶状的可注射型自愈合止血材料，其弹性模量为1Pa～100kPa，优选20kPa～100kPa。In the above technical scheme of the present invention, the prepared gelatin microgel particle freeze-dried powder I, gelatin microgel particle freeze-dried powder II or gelatin microgel particle freeze-dried powder III are directly applied as a hemostatic material. It can be blended with an appropriate amount of aqueous solution to prepare a colloidal gel-like injectable self-healing hemostatic material, and its elastic modulus is 1Pa-100kPa, preferably 20kPa-100kPa.
进一步地，在上述技术方案中，所述的带正电荷的高分子颗粒分散液以壳聚糖、A型明胶、聚丙烯酰胺、聚(N-异丙基丙烯酰胺)、聚乙烯亚胺中的一种或几种作为原料制备得到，所述的带负电荷的高分子颗粒分散液以透明质酸、海藻酸、A型明胶、B型明胶或聚丙烯酸中的一种或几种作为原料制备得到，所述的表面zeta电势在-10～+10mV的高分子颗粒分散液以胶原蛋白、白蛋白、明胶中的一种或几种作为原料制备得到。所述带正电荷的高分子颗粒分散液或带负电荷的高分子颗粒分散液或表面zeta电势在-10～+10mV的高分子颗粒分散液，本领域技术人员可以根据高分子颗粒的常规制备技术来制备得到，在本申请中不再详细陈述。Further, in the above-mentioned technical scheme, the described positively charged macromolecular particle dispersion liquid is mixed with chitosan, type A gelatin, polyacrylamide, poly(N-isopropylacrylamide), polyethyleneimine One or more of them are prepared as raw materials, and the negatively charged polymer particle dispersion is prepared from one or more of hyaluronic acid, alginic acid, A-type gelatin, B-type gelatin or polyacrylic acid The polymer particle dispersion liquid whose surface zeta potential is -10-+10mV is prepared by using one or more of collagen, albumin and gelatin as raw materials. The positively charged polymer particle dispersion or the negatively charged polymer particle dispersion or the polymer particle dispersion with a surface zeta potential of -10 to +10 mV can be prepared by those skilled in the art according to the conventional preparation of polymer particles Technology to prepare, no longer detailed statement in this application.
所述的等电点>8的带正电荷的亲水性高分子为壳聚糖、A型明胶、聚丙烯酰胺、聚(N-异丙基丙烯酰胺)、聚乙烯亚胺中的一种或几种，所述等电点<6的带负电荷的亲水性高分子为透明质酸、海藻酸、A型明胶、B型明胶或聚丙烯酸中的一种或几种，所述的等电点6～8的高分子为胶原蛋白、白蛋白、明胶、聚乙烯醇、聚乙二醇中的一种或几种。The positively charged hydrophilic polymer with an isoelectric point>8 is one of chitosan, type A gelatin, polyacrylamide, poly(N-isopropylacrylamide), polyethyleneimine or several, the negatively charged hydrophilic macromolecule with an isoelectric point<6 is one or more of hyaluronic acid, alginic acid, type A gelatin, type B gelatin or polyacrylic acid, the described The polymer with an isoelectric point of 6-8 is one or more of collagen, albumin, gelatin, polyvinyl alcohol, and polyethylene glycol.
进一步地，在上述技术方案中，在步骤(1)中所述的极性有机溶剂为甲醇、乙醇、异丙醇、丁醇、丙酮、乙腈、四氢呋喃中的一种或几种的组合；所述的交联剂为戊二醛、甘油醛、甲醛、碳二亚胺、二卤代烷、异氰酸酯、二异氰酸酯、谷氨酰胺转胺酶、京尼平中的一种或几种。Further, in the above technical scheme, the polar organic solvent described in step (1) is one or more combinations of methanol, ethanol, isopropanol, butanol, acetone, acetonitrile, tetrahydrofuran; The crosslinking agent is one or more of glutaraldehyde, glyceraldehyde, formaldehyde, carbodiimide, dihaloalkane, isocyanate, diisocyanate, transglutaminase, and genipin.
进一步地，在上述技术方案中，在步骤(1)中所述的交联反应的反应体系中交联剂与明胶中氨基基团的摩尔比为0.1～10。交联剂与明胶中氨基基团的摩尔比影响所形成的明胶微凝胶颗粒的交联度，交联度过高明胶微流强度更高、表面电荷更倾向于带负电，交联度过低明胶微球强度低、表面电荷取决于明胶原料的等电点，本发明优选的技术方案中交联度较低时较为好，优选的，本发明中所述交联剂与明胶中氨基基团的摩尔比要控制在0.5～5。Further, in the above technical solution, the molar ratio of the cross-linking agent to the amino groups in the gelatin in the reaction system of the cross-linking reaction described in step (1) is 0.1-10. The molar ratio of cross-linking agent to amino groups in gelatin affects the degree of cross-linking of the formed gelatin microgel particles. If the cross-linking is too high, the microfluidic strength of gelatin is higher, and the surface charge tends to be negatively charged. The strength of low gelatin microspheres is low, and the surface charge depends on the isoelectric point of the gelatin raw material. It is better when the degree of crosslinking is lower in the preferred technical solution of the present invention. Preferably, the crosslinking agent described in the present invention and the amino group in the gelatin The molar ratio of the group should be controlled at 0.5-5.
进一步地，在上述技术方案中，在步骤(2)和步骤(3)中所述的酸性水溶液和碱性水溶液中所含有的离子浓度均小于200mM。所述的酸性水溶液和碱性水溶液中所含有的离子的种类不特别限定，可以采用本领域常规的用于调节酸性或碱性的试剂，如盐酸、硫酸、醋酸、氢氧化钙、氢氧化钾、氨水、碳酸钠等。Further, in the above technical solution, the ion concentrations contained in the acidic aqueous solution and the alkaline aqueous solution described in step (2) and step (3) are both less than 200 mM. The types of ions contained in the acidic aqueous solution and alkaline aqueous solution are not particularly limited, and conventional reagents in the art for adjusting acidity or alkalinity can be used, such as hydrochloric acid, sulfuric acid, acetic acid, calcium hydroxide, potassium hydroxide , ammonia, sodium carbonate, etc.
进一步地，在上述技术方案中，在步骤(2)和步骤(3)中所述的pH调节剂包括酸性物质和碱性物质，所述酸性物质为葡萄糖酸内酯、HCl、HNO3、H2SO4中的一种或几种，所述碱性物质为尿素和脲酶的组合、或者氢氧化钠、氢氧化钙、氢氧化钾、氨水中的一种或几种。Further, in the above technical scheme, the pH regulators described in step (2) and step (3) include acidic substances and alkaline substances, and the acidic substances are gluconolactone, HCl, HNO3 , H2 SO4 one or more, the alkaline substance is a combination of urea and urease, or one or more of sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonia water.
进一步地，在上述技术方案中，在步骤(5)中所述的水性溶液是离子浓度120～200mM、pH值为6～8的任意水溶液、亲水性高分子的水溶液、不水溶的纳米颗粒分散液中的一种或几种的组合。Further, in the above technical solution, the aqueous solution described in step (5) is any aqueous solution with an ion concentration of 120-200mM and a pH value of 6-8, an aqueous solution of a hydrophilic polymer, or a water-insoluble nanoparticle One or a combination of several dispersions.
进一步地，在上述技术方案中，所述水性溶液中含有生物活性物质和/或赋形剂。其中所述赋形剂为溶剂、分散介质、包被剂、表面活性剂、抗氧化剂、防腐剂、等渗剂、粘合剂、润滑剂、颜料，以及它们的组合或类似物；所述生物活性物质为凝血剂、抗感染药物、消炎药、生物活性蛋白药物中的至少一种。所述凝血剂选自胶原蛋白、明胶、氧化纤维素、羧甲基纤维素、壳聚糖、透明质酸、海藻酸钠、高岭土、凝血酶、纤维蛋白、钙剂、鱼精蛋白、多肽、缩氨酸、氨基酸中的一种或其组合；所述抗感染药物选自抗生素、抗菌制剂、抗病毒制剂、抗真菌药物、抗溃疡药物、中药制剂的一种或其组合；所述消炎药物选自非类固醇类、固醇类消炎药、抗溃疡药物、中药制剂的一种或其组合。Further, in the above technical solution, the aqueous solution contains bioactive substances and/or excipients. Wherein the excipient is solvent, dispersion medium, coating agent, surfactant, antioxidant, preservative, isotonic agent, binder, lubricant, pigment, and their combination or analog; The active substance is at least one of blood coagulants, anti-infection drugs, anti-inflammatory drugs, and biologically active protein drugs. The coagulant is selected from collagen, gelatin, oxidized cellulose, carboxymethyl cellulose, chitosan, hyaluronic acid, sodium alginate, kaolin, thrombin, fibrin, calcium agent, protamine, polypeptide, One or a combination of peptides and amino acids; the anti-infective drug is selected from one or a combination of antibiotics, antibacterial agents, antiviral agents, antifungal drugs, anti-ulcer drugs, and traditional Chinese medicine preparations; the anti-inflammatory drug One selected from non-steroids, steroid anti-inflammatory drugs, anti-ulcer drugs, traditional Chinese medicine preparations or a combination thereof.
本发明还提供上述所述的可注射型自愈合止血材料在制备用于体表组织、体腔内组织器官的有血创面的止血、防黏连、防感染、促进组织愈合和/或封闭伤口的止血制品中的应用。其中所述的止血制品可采用止血粉剂、止血颗粒、止血球、止血气雾剂、止血海绵、止血凝胶、止血膜、止血栓或止血贴等方式。The present invention also provides that the above-mentioned injectable self-healing hemostatic material is used for hemostasis, anti-adhesion, anti-infection, promotion of tissue healing and/or sealing of wounds on bloody wounds of body surface tissues and tissues and organs in body cavities. application in hemostatic products. The hemostatic products mentioned therein can be in the form of hemostatic powder, hemostatic granule, hemostatic ball, hemostatic aerosol, hemostatic sponge, hemostatic gel, hemostatic membrane, hemostatic plug or hemostatic patch.
本发明的有益效果：Beneficial effects of the present invention:
1.和传统止血材料的区别和优势；1. Differences and advantages from traditional hemostatic materials;
外科手术过程中需要对不同原因造成的伤口进行止血和封闭，因此止血材料是非常重要的一类生物医用材料，具有重要的研发和应用意义。目前临床常用的止血材料从作用机制上可分为三类：第一类止血材料是通过材料的物理或化学作用使伤口部位加速凝血(如止血纱布、高分子多糖类、无机类沸石等)；第二类是直接或间接提供外来的凝血成分、利用激发自身凝血机制加速实现凝血(如纤维蛋白类止血材料、含有凝血酶和凝血因子类止血材料)；第三类是利用材料对组织很强的粘着力直接封闭创面，从而实现止血(如α氰基丙烯酸酯类材料)。然而不同类型的止血材料都存在诸多瓶颈。During surgical operations, it is necessary to stop bleeding and seal wounds caused by different reasons. Therefore, hemostatic materials are a very important class of biomedical materials, which have important research and application significance. At present, the hemostatic materials commonly used in clinical practice can be divided into three categories from the mechanism of action: the first type of hemostatic material accelerates blood coagulation at the wound site through the physical or chemical action of the material (such as hemostatic gauze, polymer polysaccharides, inorganic zeolites, etc.) The second category is to provide external coagulation components directly or indirectly, and to accelerate coagulation by stimulating its own coagulation mechanism (such as fibrin hemostatic materials, hemostatic materials containing thrombin and coagulation factors); the third category is to use materials that are very effective on tissues. Strong adhesive force directly seals the wound surface, thereby achieving hemostasis (such as α-cyanoacrylate materials). However, there are many bottlenecks in different types of hemostatic materials.
对于利用止血机制来实现血凝的止血制品而言，其缺点是它们仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。另外，载有凝血酶类凝血剂的止血制品在使用过程中，凝血酶类蛋白分子易外溢到正常血管中，因此存在诱发正常血流发生凝血产生血栓的风险，因此不依赖机体凝血机制并能迅速封闭创面的止血制品在外科手术中更受青睐。For the hemostatic products that use the hemostatic mechanism to achieve blood coagulation, the disadvantage is that they are only effective for bleeding with a small flow rate, so they must be used with a hemostatic clamp, which makes the above-mentioned hemostatic materials often require the surgeon to pre-deploy Predict the amount of bleeding or use the hemostat repeatedly. In addition, during the use of hemostatic products loaded with thrombin-like coagulants, thrombin-like protein molecules are easy to spill into normal blood vessels, so there is a risk of inducing normal blood flow to coagulate and produce thrombus. Therefore, it does not rely on the body's coagulation mechanism and can Hemostatic products that quickly seal wounds are more popular in surgical operations.
而通过物理作用实现止血的止血制品，如止血纱布、海绵等，在止血过程需要通过对出血创面进行物理压迫，同时材料通过吸收血液中的水，促使凝血，止血速度慢，效果较差。并且，这类材料仅对流速较小的出血有效，因此使用时必须配合止血钳夹，这就使得上述止血材料在使用时常常需要外科医生提前预测出血量或者反复多次使用止血钳。同时，传统止血材料(如棉纱、绷带)对于不规则形状、深、窄、动脉破裂等现场常见创伤的止血效果很不理想。另外，还存在其他的缺点：1)影响术后缝合，增加新的创面出血；2)不能自然降解，在体内形成残留；3)止血过程需要长时间的外力按压实现物理止血，对于脆弱的神经或脑组织不适用。另一类无机物止血材料，包括沸石、高岭土等，这类材料利用了天然硅铝酸盐材料多孔结构、高比表面积等特性，通过吸收血液中的水分，浓缩创面局部的凝血成分，从而实现快速止血。而人工制备的介孔硅材料同样具有多孔结构、高比表面积等特性，而且相比天然硅铝酸盐材料具有成分、粒径、孔径可控等优势，同样可实现有效止血。然而，这类材料对于血流大的出血无能为力，对于大面积创面效果也有限；并且这类材料虽然介局部组织反应是良性的，但是进入脉管和组织器官的介孔硅颗粒会引起严重的全身毒性。However, hemostatic products that achieve hemostasis through physical action, such as hemostatic gauze, sponge, etc., need to physically compress the bleeding wound during the hemostatic process. Moreover, this type of material is only effective for bleeding with a small flow rate, so it must be used with a hemostatic clamp, which makes the above-mentioned hemostatic material often require the surgeon to predict the amount of bleeding in advance or repeatedly use the hemostatic clamp. At the same time, traditional hemostatic materials (such as cotton gauze and bandages) are not ideal for the hemostatic effect of common wounds such as irregular shapes, deep, narrow, and ruptured arteries. In addition, there are other disadvantages: 1) It affects postoperative suture and increases new wound bleeding; 2) It cannot be degraded naturally, and residues are formed in the body; 3) The process of hemostasis requires long-term external pressure to achieve physical hemostasis. Or brain tissue does not apply. Another type of inorganic hemostatic materials, including zeolite, kaolin, etc., these materials take advantage of the porous structure and high specific surface area of natural aluminosilicate materials, and absorb the water in the blood to concentrate the blood coagulation components in the wound, so as to achieve Quickly stop bleeding. The artificially prepared mesoporous silicon material also has the characteristics of porous structure and high specific surface area, and has the advantages of controllable composition, particle size, and pore size compared with natural aluminosilicate materials, and can also achieve effective hemostasis. However, this type of material is powerless for bleeding with large blood flow, and has limited effect on large-area wounds; and although this type of material has a benign local tissue reaction, the mesoporous silicon particles entering the blood vessels and tissues and organs will cause serious damage. Systemic toxicity.
而本发明提供的可注射型自愈合止血材料为使用带表面电荷的高分子微凝胶颗粒，通过颗粒间的静电相互作用自组装得到的胶体凝胶材料。由于微凝胶之间的静电作用是物理交联且具有可逆性，因此当这种胶体凝胶受到破坏性的剪切力破坏时，微凝胶颗粒间的静电相互作用被外力破坏，胶体凝胶从具有刚性的固体材料向具有流动性的流体材料转变，这一过程被称为剪切变稀行为。当外力取消时，胶体间的相互作用快速恢复，胶体颗粒通过重新形成物理交联组装成胶体凝胶。因此本发明所述的止血材料具有可注射性和自愈合性能，自愈合性能测试显示，剪切破坏后的自修复率超过85％以上。更重要的是，本发明的止血材料在注射和固化过程无需引入化学交联反应，不同于化学高分子，也没有引入小分子交联剂，生物相容性好，可降解吸收，无毒副作用，安全性好，使其在生物医学领域的广泛应用成为可能。The injectable self-healing hemostatic material provided by the present invention is a colloidal gel material self-assembled by using surface-charged polymer microgel particles through electrostatic interaction between particles. Since the electrostatic interaction between microgels is physically cross-linked and reversible, when the colloidal gel is destroyed by destructive shear force, the electrostatic interaction between microgel particles is destroyed by external force, and the colloidal gel The transformation of the glue from a rigid solid material to a fluid fluid material is known as shear thinning behavior. When the external force is removed, the interaction between the colloids is quickly restored, and the colloidal particles assemble into a colloidal gel by re-forming physical crosslinks. Therefore, the hemostatic material described in the present invention has injectability and self-healing performance, and the self-healing performance test shows that the self-repairing rate after shear damage exceeds 85%. More importantly, the hemostatic material of the present invention does not need to introduce chemical cross-linking reactions during the injection and curing process. Unlike chemical polymers, it does not introduce small molecule cross-linking agents. It has good biocompatibility, can be degraded and absorbed, and has no toxic and side effects , good security, making it possible for it to be widely used in the biomedical field.
另外，本发明提供的止血材料有较好的力学强度，凝胶状止血材料的弹性模量大于10kPa，对于出血量大、血管压力高的出血组织也可以实现快速止血。还有本发明止血材料的胶体颗粒的高比表面积、胶体凝胶的稳定强度有利于创口的封闭。In addition, the hemostatic material provided by the present invention has better mechanical strength, and the elastic modulus of the gel-like hemostatic material is greater than 10 kPa, which can also achieve rapid hemostasis for bleeding tissues with large bleeding volume and high blood vessel pressure. In addition, the high specific surface area of the colloid particles of the hemostatic material of the present invention and the stable strength of the colloid gel are beneficial to the sealing of the wound.
2.在药物缓释方面的优势2. Advantages in drug sustained release
止血制品是外科手术中必不可少的生物医用材料之一，为促进止血、控制手术创面感染等风险、加速催化创口愈合，将止血材料与功能性药物联合使用是提高临床止血、促进创面愈合的有效手段之一。这些凝血药物、抗菌消炎药物、或促进组织修复再生的生物活性药物包括小分子药物和蛋白类大分子药物，这些药物在与止血制品复合使用的过程中都存在药物释放不可控，分子类药物易失活的问题。因此，开发可加载药物并可控释放药物的止血材料具有重要的医学应用价值。传统的止血材料通常利用预加工好的止血制品(如止血膜、止血粉、止血海绵等)，将材料直接混入药物溶液，通过药物在支架的表面物理吸附实现药物的加载。这种方式通常导致止血材料在使用时，所载药物的暴释，对于促进创伤修复的蛋白类药物不能实现长期持续的释放。Hemostatic products are one of the essential biomedical materials in surgical operations. In order to promote hemostasis, control the risk of surgical wound infection and accelerate wound healing, the combination of hemostatic materials and functional drugs is an important way to improve clinical hemostasis and promote wound healing. one of the effective means. These coagulation drugs, antibacterial and anti-inflammatory drugs, or bioactive drugs that promote tissue repair and regeneration include small molecule drugs and protein macromolecular drugs. These drugs have uncontrollable drug release in the process of compound use with hemostatic products, and molecular drugs are prone to inactivation problem. Therefore, the development of hemostatic materials that can be loaded with drugs and release them in a controlled manner has important medical application value. Traditional hemostatic materials usually use pre-processed hemostatic products (such as hemostatic film, hemostatic powder, hemostatic sponge, etc.), and the material is directly mixed into the drug solution, and the drug is loaded by physical adsorption of the drug on the surface of the stent. This method usually leads to the burst release of the drug contained in the hemostatic material when it is used, and it cannot achieve long-term sustained release of the protein drug that promotes wound repair.
本发明的止血材料对比与传统载体材料具有更多优势。1)具有微纳米尺寸的微凝胶颗粒为基本单元，相比传统多孔支架材料具有更高比表面积，因此可表面吸附的蛋白量更高；2)生长因子的加载是将微凝胶颗粒的冻干粉直接与生长因子水溶液共混，在颗粒溶胀过程中蛋白分子在渗透压作用下进入微凝胶颗粒网络内部，因此蛋白的释放主要是由微凝胶的降解速率控制的；3)生长因子的释放速率主要由明胶微凝胶的降解速率调控，因此通过控制微凝胶的交联度可以实现对加载的生长因子的释放速率进行调控，进一步将不同的生长因子加载入不同交联度的胶体颗粒，可以实现多种生长因子的有序可控释放。Compared with traditional carrier materials, the hemostatic material of the present invention has more advantages. 1) The microgel particles with micro-nano size are the basic units, which have a higher specific surface area than traditional porous scaffold materials, so the amount of protein that can be adsorbed on the surface is higher; 2) The loading of growth factors is to make the microgel particles The freeze-dried powder is directly blended with the growth factor aqueous solution, and the protein molecules enter the microgel particle network under the action of osmotic pressure during the particle swelling process, so the release of the protein is mainly controlled by the degradation rate of the microgel; 3) growth The release rate of factors is mainly regulated by the degradation rate of gelatin microgels, so the release rate of loaded growth factors can be regulated by controlling the degree of cross-linking of microgels, and further loading different growth factors into different cross-linking degrees The colloidal particles can realize the orderly and controllable release of various growth factors.
附图说明Description of drawings
图1为实施例1方法制备的A型明胶微凝胶颗粒的扫描电镜照片。Fig. 1 is the scanning electron micrograph of the type A gelatin microgel particles prepared by the method in Example 1.
图2为实施例1方法制备的由带相反电荷的A型和B型明胶胶体颗粒组成的复合胶体凝胶的自修复行为的流变学测试结果。Fig. 2 is the rheological test result of the self-healing behavior of the composite colloidal gel prepared by the method of Example 1 composed of oppositely charged type A and type B gelatin colloidal particles.
图3为实施例2所述的方法制备的带相反电荷的海藻酸钙和A型明胶胶体颗粒组成的复合胶体凝胶的自修复行为的流变学测试结果。3 is the rheological test result of the self-healing behavior of the composite colloidal gel composed of oppositely charged calcium alginate and type A gelatin colloidal particles prepared by the method described in Example 2.
图4表示实施例6中所述方法制备的止血材料实现生物活性蛋白药物的有序释放。Figure 4 shows that the hemostatic material prepared by the method described in Example 6 realizes the ordered release of bioactive protein drugs.
图5表示实施例7所述的通过动物实验证实本发明止血材料的止血功效。Figure 5 shows the demonstration of the hemostatic efficacy of the hemostatic material of the present invention through animal experiments as described in Example 7.
具体实施方式detailed description
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学公司购买。The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical companies.
下面实施例中将胶体颗粒冷冻干燥后得各胶体颗粒的冻干粉末，其中所述冷冻干燥条件为：将胶体颗粒在-60℃、<300Pa下冷冻干燥2-3天。In the following examples, the colloidal particles were freeze-dried to obtain freeze-dried powders of the colloidal particles, wherein the freeze-drying conditions were: the colloidal particles were freeze-dried at -60° C. and <300 Pa for 2-3 days.
实施例1Example 1
以A型明胶为原料，使其在去离子水中加热40℃溶解，配置浓度为5w/v％的明胶水溶液，调节明胶水溶液pH值为3，随后向溶液中加入3倍溶液体积的乙醇，生成明胶微凝胶颗粒的分散液；分别向分散液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量分别为每克明胶对应25wt％戊二醛66μL，交联反应时间12hr，离心清洗得到表面zeta电势位正的A型明胶微凝胶颗粒的分散液。同样方法，以B型明胶为原料制备得到表面zeta电势为负的B型明胶微凝胶颗粒的分散液。利用激光粒度仪测得制备微凝胶颗粒尺寸和zeta电势数据如表1所示。经冷冻干燥分别得到A型明胶颗粒的冻干粉末(标记为GelA)和B型明胶颗粒的冻干粉末(标记为GelB)。通过扫描电子显微镜对明胶颗粒的形貌进行观测，结果如图1所示。制备得到的A型明胶颗粒的粒径分布窄，尺寸在150-300nm范围内。Using type A gelatin as raw material, heat it in deionized water at 40°C to dissolve it, prepare a gelatin aqueous solution with a concentration of 5w/v%, adjust the pH value of the gelatin aqueous solution to 3, and then add ethanol three times the volume of the solution to the solution to generate Dispersion of gelatin microgel particles; add 25wt% glutaraldehyde aqueous solution to the dispersion respectively to crosslink the gelatin microgel particles, the amount of glutaraldehyde added is respectively 66 μL of 25wt% glutaraldehyde per gram of gelatin, The cross-linking reaction time is 12 hours, centrifuged and washed to obtain a dispersion of type A gelatin microgel particles with positive surface zeta potential. In the same way, a dispersion of B-type gelatin microgel particles with a negative surface zeta potential was prepared using B-type gelatin as a raw material. The particle size and zeta potential data of the prepared microgel measured by laser particle size analyzer are shown in Table 1. The freeze-dried powder of type A gelatin particles (marked as GelA) and the freeze-dried powder of type B gelatin particles (marked as GelB) were respectively obtained by freeze-drying. The morphology of the gelatin particles was observed by a scanning electron microscope, and the results are shown in Figure 1. The prepared type A gelatin particles have a narrow particle size distribution, and the size is in the range of 150-300nm.
表1.不同类型明胶颗粒的性能参数Table 1. Performance parameters of different types of gelatin particles
将A型明胶和B型明胶微凝胶颗粒分别分散在20mM的NaOH碱性水溶液中，分别得到分散有带正电荷的A型明胶微凝胶颗粒和带负电荷的B型明胶微凝胶颗粒的分散液，将两者充分混合、搅拌，得分散有两种不同微凝胶颗粒的分散液，其中A型明胶和B型明胶混合的颗粒数量比为1:1；向分散液中加入100mM的盐酸调节pH值至7.0，搅拌混合，冷冻干燥，得到含有两种不同明胶胶体颗粒的冻干粉末，标记为GelA+B，可作为止血粉剂。将上述混合物冻干粉末分别与适当量的1mM的NaCl溶液共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，得到含不同微凝胶胶体颗粒体积分数的胶体凝胶，得到可注射型止血凝胶。制备所得不同组分的胶体凝胶，通过流变仪对所得止血凝胶材料的粘弹性能进行评价。结果如表2所示，胶体体积分数增加，凝胶的弹性模量增加；在体积分数相同时，带相反电荷胶体颗粒组成的凝胶弹性模量显著强于单一组分的胶体凝胶。在微凝胶胶体颗粒体积分数为120vol％时，GelA+B组分的胶体凝胶弹性模量约为38kPa。Disperse Type A gelatin and Type B gelatin microgel particles in 20mM NaOH alkaline aqueous solution respectively to obtain positively charged Type A gelatin microgel particles and negatively charged Type B gelatin microgel particles The dispersion liquid of the two is fully mixed and stirred to obtain a dispersion liquid with two different microgel particles, wherein the number ratio of the mixed particles of type A gelatin and type B gelatin is 1:1; add 100mM gelatin to the dispersion liquid hydrochloric acid to adjust the pH value to 7.0, stir and mix, and freeze-dry to obtain a freeze-dried powder containing two different gelatin colloid particles, labeled as GelA+B, which can be used as a hemostatic powder. The lyophilized powder of the above mixture was blended with an appropriate amount of 1mM NaCl solution, and mixed rapidly to obtain an injectable self-healing colloidal gel, and colloidal gels containing different volume fractions of microgel colloidal particles were obtained. Injectable hemostatic gel. The obtained colloidal gels with different components were prepared, and the viscoelastic properties of the obtained hemostatic gel materials were evaluated by a rheometer. The results are shown in Table 2. As the colloidal volume fraction increases, the elastic modulus of the gel increases; when the volume fraction is the same, the elastic modulus of the gel composed of oppositely charged colloidal particles is significantly stronger than that of a single component colloidal gel. When the volume fraction of microgel colloidal particles is 120vol%, the colloidal gel elastic modulus of GelA+B component is about 38kPa.
胶体凝胶的自修复行为是通过流变仪进行表征，具体测试方法如下。对胶体凝胶进行连续的流变测试：首先进行震荡时间扫描，对样品施加频率为1Hz和应变为0.5％的外力，测试样品的储能模量(或弹性模量，G’)和损耗模量(或粘性模量，G”)，此时凝胶在低剪切力情况下表现出固体的刚性行为，因此储能模量G’大于损耗模量G”且保持稳定。这一阶段的G’值即为样品的初始弹性模量。随后逐渐增加施加的应变从0.1％至1000％，此过程中通过施加外力将样品破坏，弹性模量G’逐渐降低，最终低于G”，即胶体体系从刚性固体向粘性流体发生转变，结构被破坏。随后立即取消外力作用，考察样品弹性模量的恢复情况。将外力释放后，样品恢复的储能(弹性)模量与其初始储能弹性模的百分比(％)定量考察凝胶的自修复效率。凝胶的自修复效率如表3所示，由带相反电荷胶体颗粒组成的凝胶弹性模量显著强于单一组分的胶体凝胶。GelA+B胶体凝胶的自修复过程如图2所示，凝胶在剪切破坏后其弹性模量瞬间恢复，5分钟内自修复弹性模量恢复到初始模量超过85％。并且这样自修复行为可以反复发生：在对样品施加多个循环的剪切破坏过程中，每次取消外力后，凝胶的弹性模量都会快速恢复，并恢复到初始弹性模量的80％以上。The self-healing behavior of the colloidal gel is characterized by a rheometer, and the specific test method is as follows. Continuous rheological test of the colloidal gel: firstly, an oscillation time sweep is performed, and an external force with a frequency of 1 Hz and a strain of 0.5% is applied to the sample, and the storage modulus (or elastic modulus, G') and loss modulus of the sample are tested Quantity (or viscous modulus, G"), at this time, the gel exhibits the rigid behavior of a solid under low shear force, so the storage modulus G' is greater than the loss modulus G" and remains stable. The G' value at this stage is the initial elastic modulus of the sample. Then gradually increase the applied strain from 0.1% to 1000%. During this process, the sample is destroyed by applying an external force, and the elastic modulus G' gradually decreases, and finally is lower than G", that is, the colloidal system changes from a rigid solid to a viscous fluid. Destroyed. Immediately cancel the external force, investigate the recovery of the sample elastic modulus. After the external force is released, the percentage (%) of the storage (elastic) modulus of the sample recovery and its initial storage elastic modulus is quantitatively investigated. Repair efficiency. The self-repair efficiency of the gel is shown in Table 3, and the elastic modulus of the gel composed of oppositely charged colloidal particles is significantly stronger than that of a single component. The self-repair process of GelA+B colloidal gel is as follows As shown in Figure 2, the elastic modulus of the gel recovers instantaneously after shear failure, and the self-healing elastic modulus returns to more than 85% of the initial modulus within 5 minutes. And such self-healing behavior can occur repeatedly: after applying multiple During the shear failure process of the first cycle, the elastic modulus of the gel will recover rapidly and recover to more than 80% of the initial elastic modulus each time the external force is removed.
表2.实施例1中制备的不同胶体凝胶含有不同体积分数的微凝胶颗粒所得到凝胶材料的弹性模量G'The elastic modulus G' of the gel material obtained by the different colloidal gels prepared in Table 2. containing different volume fractions of microgel particles
表3.实施例1中制备的不同胶体凝胶不同体积分数的微凝胶颗粒所得的凝胶材料的自修复效率Table 3. The self-healing efficiency of the gel material obtained by the microgel particles of different colloidal gels with different volume fractions prepared in Example 1
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。*Note: The self-healing efficiency is the percentage (%) of elastic modulus recovery within 5 minutes after the stress is released after you shear the gel material for 60 seconds with 1000% strain.
实施例2Example 2
以A型明胶为原料，在加热40℃下溶解，配置得到浓度为5w/v％的A型明胶水溶液，调节pH值为11，随后向溶液中加入3.5倍体积的乙醇，生成A型明胶微凝胶颗粒悬浮液；向悬浮液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量为每g明胶对应25wt％戊二醛66μL，交联反应时间12hr，随后加入甘氨酸中和未反应的醛基，离心清洗得到A型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表4所示。Use type A gelatin as the raw material, dissolve it under heating at 40°C, prepare a type A gelatin aqueous solution with a concentration of 5w/v%, adjust the pH value to 11, and then add 3.5 times the volume of ethanol to the solution to generate type A gelatin microparticles. Gel particle suspension; add 25wt% glutaraldehyde aqueous solution to the suspension to crosslink the gelatin microgel particles, the amount of glutaraldehyde added is 66 μL of 25wt% glutaraldehyde per g of gelatin, and the crosslinking reaction time is 12hr , followed by adding glycine to neutralize unreacted aldehyde groups, and centrifuging to obtain type A gelatin microgel particles. The particle size and surface zeta point parameters are shown in Table 4.
通过乳液法制备海藻酸微凝胶颗粒，具体制备方法如下：将1wt％的海藻酸钠的水溶液逐滴加入50mM氯化钙水溶液中并持续高速搅拌(搅拌速度>5000rpm)，即得到海藻酸钙微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表4所示。将A型明胶和海藻酸钙微凝胶颗粒分别分散在10mM的醋酸酸性水溶液中，分别得到带正电荷的A型明胶微凝胶颗粒的分散液Ⅰ、带负电荷的海藻酸微凝胶颗粒的分散液Ⅱ，将分散液Ⅰ和分散液Ⅱ充分混合、搅拌，得分散液III，其中A型明胶和海藻酸钙微凝胶颗粒混合的颗粒数量比为2:1；向分散液III中加入100mM的氢氧化钠调节pH值至7.0，搅拌混合，冷冻干燥，得到混有两相不同胶体颗粒的冻干粉末。将上述微凝胶颗粒冻干粉末与一定体积的1mM的NaCl盐溶液共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，即本发明所述的止血材料，其中微凝胶胶体颗粒体积分数占胶体凝胶体积的50vol％或100vol％。使用流变仪对胶体凝胶的力学参数进行评价，结果如图3和表5所示，带相反电荷的海藻酸钙和A型明胶胶体颗粒共混得到的胶体凝胶的弹性模量随着胶体颗粒体积分数的增加而增加，体积分数100vol％时，储存(弹性)模量G’超过12kPa。剪切破坏后5分钟内自愈合效率同样随着胶体颗粒体积分数的增加而增加，体积分数100vol％时，弹性模量G’自愈合效率超过80％。Prepare alginic acid microgel particles by emulsion method, the specific preparation method is as follows: add 1wt% sodium alginate aqueous solution dropwise into 50mM calcium chloride aqueous solution and continue high-speed stirring (stirring speed> 5000rpm) to obtain calcium alginate The microgel particles, particle size and surface zeta point parameters are shown in Table 4. Disperse type A gelatin and calcium alginate microgel particles in 10mM acetic acid aqueous solution to obtain dispersion I of positively charged type A gelatin microgel particles and negatively charged alginate microgel particles Dispersion II, fully mixed and stirred Dispersion I and Dispersion II to obtain Dispersion III, wherein the particle number ratio of type A gelatin and calcium alginate microgel particles mixed is 2:1; Add 100 mM sodium hydroxide to adjust the pH value to 7.0, stir and mix, and freeze-dry to obtain a freeze-dried powder mixed with two different colloidal particles. Blend the freeze-dried powder of the above-mentioned microgel particles with a certain volume of 1mM NaCl salt solution, and stir and mix quickly to obtain an injectable self-healing colloidal gel, which is the hemostatic material described in the present invention, wherein the microgel The volume fraction of colloidal particles accounts for 50vol% or 100vol% of the volume of the colloidal gel. The mechanical parameters of the colloidal gel were evaluated using a rheometer, and the results were shown in Figure 3 and Table 5. The elastic modulus of the colloidal gel obtained by blending oppositely charged calcium alginate and type A gelatin colloidal particles increased with The volume fraction of colloidal particles increases, and when the volume fraction is 100vol%, the storage (elasticity) modulus G' exceeds 12kPa. The self-healing efficiency within 5 minutes after shear failure also increases with the increase of the volume fraction of colloidal particles. When the volume fraction is 100vol%, the elastic modulus G' self-healing efficiency exceeds 80%.
表4.实施例2中制备的A型明胶和海藻酸微凝胶的性能参数Table 4. Performance parameters of type A gelatin and alginic acid microgel prepared in Example 2
表5.实施例2中制备的自愈合胶体凝胶在不同胶体体积分数情况下的力学强度(流变测试弹性模量G')和自愈合效率。Table 5. Mechanical strength (rheological test elastic modulus G') and self-healing efficiency of the self-healing colloidal gel prepared in Example 2 at different colloidal volume fractions.
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。*Note: The self-healing efficiency is the percentage (%) of elastic modulus recovery within 5 minutes after the stress is released after you shear the gel material for 60 seconds with 1000% strain.
实施例3Example 3
以B型明胶为原料，在加热40℃下溶解，配置得到浓度为5w/v％的B型明胶水溶液，调节pH值为3，随后向溶液中加入3倍体积的乙醇，生成B型明胶微凝胶颗粒悬浮液；向悬浮液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量为每g明胶对应25wt％戊二醛80μL，交联反应时间12hr，反复离心并在去离子水中重悬，得到B型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表6所示。Use B-type gelatin as raw material, dissolve it under heating at 40°C, prepare a B-type gelatin aqueous solution with a concentration of 5w/v%, adjust the pH value to 3, and then add 3 times the volume of ethanol to the solution to generate B-type gelatin microparticles. Gel particle suspension; add 25wt% glutaraldehyde aqueous solution to the suspension to crosslink the gelatin microgel particles, the amount of glutaraldehyde added is 80 μL of 25wt% glutaraldehyde per g of gelatin, and the crosslinking reaction time is 12hr , repeatedly centrifuged and resuspended in deionized water to obtain type B gelatin microgel particles, the particle size and surface zeta point parameters are shown in Table 6.
将壳聚糖季铵盐溶于去离子水配置浓度为2.5w/v％壳聚糖季铵盐水溶液。将明胶B微凝胶颗粒分散在20mM的醋酸酸性水溶液中，进一步将壳聚糖季铵盐水溶液与明胶B微凝胶颗粒分散液充分共混，搅拌，得分散液III，其中B型明胶微凝胶颗粒与高分子壳聚糖季铵盐的质量比为10:1，向分散液III中加入100mM的氢氧化钠调节pH值至7.0，然后冷冻干燥，得到B型明胶胶体颗粒和壳聚糖季铵盐的复合冻干粉末。将上述冻干粉末与一定体积的10mM的NaCl盐溶液共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，即本发明所述的止血材料，其中明胶微凝胶颗粒体积分数占自愈合凝胶体积分数为100vol％。使用流变仪对胶体凝胶的力学参数进行评价，结果如表7所示，带相反电荷的B型明胶颗粒和壳聚糖季铵盐共混得到的胶体凝胶在胶体颗粒体积分数为100vol％时，储存(弹性)模量G’约48kPa。剪切破坏后5分钟内自愈合效率在胶体颗粒体积分数为100vol％时，弹性模量G’自愈合效率超过89％。The chitosan quaternary ammonium salt is dissolved in deionized water to prepare a 2.5w/v% chitosan quaternary ammonium salt aqueous solution. Disperse the gelatin B microgel particles in 20mM acetic acid aqueous solution, further fully blend the chitosan quaternary ammonium salt solution with the gelatin B microgel particle dispersion, and stir to obtain dispersion III, wherein the B-type gelatin microgel particles The mass ratio of the gel particles to the polymer chitosan quaternary ammonium salt is 10:1, adding 100mM sodium hydroxide to the dispersion III to adjust the pH value to 7.0, then freeze-drying to obtain B-type gelatin colloid particles and chitosan Composite lyophilized powder of sugar quaternary ammonium salt. Blend the above freeze-dried powder with a certain volume of 10mM NaCl salt solution, and stir and mix quickly to obtain an injectable self-healing colloidal gel, which is the hemostatic material of the present invention, wherein the gelatin microgel particle volume fraction The volume fraction of the self-healing gel is 100vol%. Rheometer is used to evaluate the mechanical parameters of colloidal gel, and the results are shown in Table 7. The colloidal gel obtained by blending B-type gelatin particles with opposite charges and chitosan quaternary ammonium salt has a particle volume fraction of 100vol %, the storage (elastic) modulus G' is about 48kPa. When the self-healing efficiency within 5 minutes after shear failure is 100vol% of the colloidal particles, the elastic modulus G' self-healing efficiency exceeds 89%.
表6.实施例3中制备的B型明胶微凝胶颗粒的性能参数The performance parameter of the B-type gelatin microgel particle prepared in the embodiment 3 of table 6.
表7.实施例3中制备的B型明胶微凝胶颗粒和壳聚糖季铵盐共混制备的自愈合凝胶的力学强度(流变测试弹性模量G')和自愈合效率。Table 7. The mechanical strength (rheological test elastic modulus G') and self-healing efficiency of the self-healing gel prepared by blending the B-type gelatin microgel particles prepared in Example 3 and the chitosan quaternary ammonium salt .
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。*Note: The self-healing efficiency is the percentage (%) of elastic modulus recovery within 5 minutes after the stress is released after you shear the gel material for 60 seconds with 1000% strain.
实施例4Example 4
以A型明胶为原料，在加热40℃下溶解，配置得到浓度为10w/v％的A型明胶水溶液，调节pH值为11，随后向溶液中各加入2倍体积的乙醇，生成A型明胶微凝胶颗粒悬浮液；向悬浮液中加入25wt％戊二醛水溶液，使明胶微凝胶颗粒交联，加入戊二醛的量为每g明胶对应25wt％戊二醛264μL，交联反应时间12hr，反复离心并在去离子水中重悬，得到A型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表8所示。Use type A gelatin as the raw material, dissolve it under heating at 40°C, prepare a type A gelatin aqueous solution with a concentration of 10w/v%, adjust the pH value to 11, and then add 2 times the volume of ethanol to the solution to generate type A gelatin Microgel particle suspension; add 25wt% glutaraldehyde aqueous solution to the suspension to crosslink the gelatin microgel particles. The amount of glutaraldehyde added is 264 μL of 25wt% glutaraldehyde per gram of gelatin, and the crosslinking reaction time After 12 hours, repeated centrifugation and resuspension in deionized water to obtain type A gelatin microgel particles, the particle size and surface zeta point parameters are shown in Table 8.
将聚乙二醇(PEG，分子量2kDa)溶于去离子水配置浓度为5w/v％PEG水溶液。将上述方法制备的A型明胶微凝胶颗粒分散得到的PEG水溶液中，进一步将PEG水溶液与A型明胶微凝胶分散液充分共混，搅拌，得分散液III，其中A型明胶微凝胶颗粒与PEG的质量比为1:2。随后将分散液III的pH值调节至7.0，然后冷冻干燥，得到A型明胶胶体颗粒和PEG的复合冻干粉末。将上述冻干粉末与一定体积的10mM的NaCl盐溶液共混，并快速搅拌混合均匀得到自愈合胶体凝胶，即本发明所述的止血材料，其中明胶微凝胶颗粒体积分数占自愈合凝胶体积分数为100vol％。使用流变仪对胶体凝胶的力学参数进行评价，结果如表9所示，当胶体凝胶中A型明胶胶体颗粒体积分数为100vol％时，储存(弹性)模量G’约19kPa。剪切破坏后5分钟内自愈合效率在胶体颗粒体积分数为100vol％时，弹性模量G’自愈合效率约为83％。Polyethylene glycol (PEG, molecular weight 2kDa) was dissolved in deionized water to prepare a 5w/v% PEG aqueous solution. In the PEG aqueous solution obtained by dispersing the type A gelatin microgel particles prepared by the above method, further fully blend the PEG aqueous solution with the type A gelatin microgel dispersion, and stir to obtain dispersion III, wherein the type A gelatin microgel The mass ratio of particles to PEG is 1:2. Subsequently, the pH value of the dispersion III was adjusted to 7.0, and then freeze-dried to obtain a composite freeze-dried powder of type A gelatin colloid particles and PEG. The above freeze-dried powder is blended with a certain volume of 10mM NaCl salt solution, and quickly stirred and mixed to obtain a self-healing colloidal gel, which is the hemostatic material of the present invention, wherein the gelatin microgel particle volume fraction accounts for the self-healing colloidal gel. The combined gel volume fraction is 100vol%. Use a rheometer to evaluate the mechanical parameters of the colloidal gel, and the results are as shown in Table 9. When the volume fraction of type A gelatin colloidal particles in the colloidal gel is 100vol%, the storage (elasticity) modulus G' is about 19kPa. The self-healing efficiency within 5 minutes after shear failure is about 83% when the volume fraction of colloidal particles is 100vol%.
表8.实施例5中制备的A型明胶微凝胶颗粒的性能参数Table 8. Performance parameters of type A gelatin microgel particles prepared in Example 5
表9.实施例4中制备的A型明胶微凝胶颗粒和PEG共混制备的自愈合凝胶的力学强度(流变测试弹性模量G')和自愈合效率。Table 9. Mechanical strength (rheological test elastic modulus G') and self-healing efficiency of the self-healing gel prepared by blending the type A gelatin microgel particles prepared in Example 4 and PEG.
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。*Note: The self-healing efficiency is the percentage (%) of elastic modulus recovery within 5 minutes after the stress is released after you shear the gel material for 60 seconds with 1000% strain.
实施例5Example 5
以A型明胶为原料，在加热40℃下溶解，配置得到浓度为5w/v％的A型明胶水溶液，调节pH值为11，随后向溶液中各加入3.5倍体积的乙醇，生成A型明胶微凝胶颗粒悬浮液；随后加入25wt％戊二醛水溶液交联明胶微凝胶颗粒，加入戊二醛的量为每g明胶对应25wt％戊二醛66μL，交联反应时间12hr，离心清洗得到A型明胶微凝胶颗粒，颗粒尺寸和表面zeta点位参数如表10所示。Use type A gelatin as the raw material, dissolve it under heating at 40°C, prepare a type A gelatin aqueous solution with a concentration of 5w/v%, adjust the pH value to 11, and then add 3.5 times the volume of ethanol to the solution to generate type A gelatin Microgel particle suspension; then add 25wt% glutaraldehyde aqueous solution to cross-link gelatin microgel particles, the amount of glutaraldehyde added is 66 μL of 25wt% glutaraldehyde per g gelatin, the crosslinking reaction time is 12hr, and centrifuged to obtain Type A gelatin microgel particles, particle size and surface zeta point parameters are shown in Table 10.
通过乳液法制备海藻酸微凝胶颗粒，具体制备方法如下：将1wt％的海藻酸钠的水溶液加入氯化钙水溶液中并持续高速搅拌(搅拌速度>5000rpm)，即得到海藻酸钙颗粒，颗粒尺寸和表面zeta点位参数如表10所示。Prepare alginate microgel particles by emulsion method, the specific preparation method is as follows: add 1wt% sodium alginate aqueous solution into calcium chloride aqueous solution and continue high-speed stirring (stirring speed>5000rpm), that is, calcium alginate particles, particles The size and surface zeta point parameters are shown in Table 10.
将A型明胶和海藻酸钙微凝胶颗粒分别分散在10mM的醋酸酸性水溶液中，分别得到带正电荷的A型明胶微凝胶颗粒的分散液Ⅰ、带负电荷的海藻酸微凝胶颗粒的分散液Ⅱ，将分散液Ⅰ和分散液Ⅱ充分混合、搅拌，得分散液III，其中A型明胶和海藻酸微凝胶颗粒混合的颗粒数量比为2:1；向分散液III中加入100mM的氢氧化钠调节pH值至7.0，搅拌混合，冷冻干燥，得到混有两相不同胶体颗粒的复合冻干粉末。将聚乙二醇(PEG，分子量2kDa)溶于去离子水配置浓度为5w/v％PEG水溶液，与上述复合冻干粉末共混，并快速搅拌得到自愈合凝胶，即本发明所述的止血材料，其中两种胶体颗粒体积分数占胶体凝胶体积的100vol％。使用流变仪考察胶体凝胶的力学参数，结果如表11所示，当胶体颗粒体积分数100vol％时，储存(弹性)模量G’超过87kPa。剪切破坏后5分钟内自愈合效率同样随着胶体颗粒体积分数的增加而增加，体积分数100vol％时，弹性模量G’自愈合效率超过91％。Disperse type A gelatin and calcium alginate microgel particles in 10mM acetic acid aqueous solution to obtain dispersion I of positively charged type A gelatin microgel particles and negatively charged alginate microgel particles Dispersion II, fully mixed and stirred Dispersion I and Dispersion II to obtain Dispersion III, in which the particle number ratio of type A gelatin and alginic acid microgel particles mixed is 2:1; add to Dispersion III 100 mM sodium hydroxide was used to adjust the pH value to 7.0, stirred and mixed, and freeze-dried to obtain a composite freeze-dried powder mixed with two phases of different colloidal particles. Dissolve polyethylene glycol (PEG, molecular weight 2kDa) in deionized water to prepare a 5w/v% PEG aqueous solution, blend with the above-mentioned composite freeze-dried powder, and stir rapidly to obtain a self-healing gel, which is described in the present invention A hemostatic material, wherein the volume fraction of two colloidal particles accounts for 100vol% of the volume of the colloidal gel. Use a rheometer to investigate the mechanical parameters of the colloidal gel. The results are shown in Table 11. When the volume fraction of the colloidal particles is 100vol%, the storage (elasticity) modulus G' exceeds 87kPa. The self-healing efficiency within 5 minutes after shear failure also increases with the increase of the volume fraction of colloidal particles. When the volume fraction is 100vol%, the elastic modulus G' self-healing efficiency exceeds 91%.
表10.实施例5中制备的A型明胶和海藻酸钙微凝胶的性能参数Table 10. Performance parameters of type A gelatin and calcium alginate microgels prepared in Example 5
表11.实施例5中制备的自愈合胶体凝胶在不同胶体体积分数情况下的力学强度(流变测试弹性模量G')和自愈合效率。Table 11. Mechanical strength (rheological test elastic modulus G') and self-healing efficiency of the self-healing colloidal gel prepared in Example 5 at different colloidal volume fractions.
*注：自修复效率是采用1000％的应变持续你剪切凝胶材料60s后，检测应力释放后5min内的弹性模量恢复的百分比(％)。*Note: The self-healing efficiency is the percentage (%) of elastic modulus recovery within 5 minutes after the stress is released after you shear the gel material for 60 seconds with 1000% strain.
实施例6Example 6
以A型明胶为原料，通过实施例1所述反溶剂法制备A型明胶微凝胶颗粒，戊二醛交联浓度为每g明胶使用25wt％戊二醛的量为66μL，制备得到带正电荷的A型明胶微凝胶颗粒；以B型明胶为原料，通过实施例1所述的反溶剂法制备B型明胶微凝胶颗粒，其中戊二醛交联浓度为每g明胶使用25wt％戊二醛的量为264μL，制备得到带负电荷的B型明胶微凝胶颗粒，制备参数和所得微凝胶颗粒尺寸和zeta点位数据如表12。Using type A gelatin as raw material, prepare type A gelatin microgel particles by the anti-solvent method described in Example 1, the crosslinking concentration of glutaraldehyde is 66 μL of 25wt% glutaraldehyde per g of gelatin, and the obtained Charged type A gelatin microgel particles; using type B gelatin as raw material, prepare type B gelatin microgel particles by the anti-solvent method described in Example 1, wherein the glutaraldehyde crosslinking concentration is 25wt% per g of gelatin The amount of glutaraldehyde was 264 μL, and negatively charged B-type gelatin microgel particles were prepared. The preparation parameters and the obtained microgel particle size and zeta point data are shown in Table 12.
表12.实施例6中制备的不同类型明胶微凝胶颗粒的性能参数Table 12. Performance parameters of different types of gelatin microgel particles prepared in Example 6
将A型明胶颗粒分散在含100ng/ml浓度的碱性成纤维生长因子(bFGF)的水溶液中，将B型明胶颗粒分散在含100ng/ml浓度的骨形态发生蛋白-2(BMP-2)的水溶液中，分别得到载有bFGF的A型明胶颗粒和载有BMP-2的B型明胶微凝胶颗粒的分散液。将上述两种明胶颗粒分散液按照颗粒数比1:1充分混合，并冷冻干燥，得到载有不同生长因子的两种明胶颗粒冻干粉末。将上述微凝胶颗粒冻干粉末与一定体积的1mM的NaCl盐溶液共混，并快速搅拌混合均匀，得到载有不同生长因子的、可注射型自愈合胶体凝胶，其中微凝胶胶体颗粒体积分数占胶体凝胶体积的100vol％。两种不同的生长因子从胶体凝胶载体材料中体外释放动力学的释放曲线如图4所示，载有bFGF的A型明胶由于交联度低降解速率较快，因此bFGF释放速率较快，载有BMP-2的B型明胶由于交联度高降解速率慢，因此BMP-2释放速率较为缓慢；结果表明本发明所述的胶体凝胶可实现不同生长因子药物的有序释放。Disperse type A gelatin particles in an aqueous solution containing 100ng/ml concentration of basic fibroblast growth factor (bFGF), and disperse type B gelatin particles in an aqueous solution containing 100ng/ml concentration of bone morphogenetic protein-2 (BMP-2) In the aqueous solution, the dispersions of type A gelatin particles loaded with bFGF and type B gelatin microgel particles loaded with BMP-2 were obtained respectively. The above two gelatin particle dispersions were fully mixed according to the particle number ratio of 1:1, and freeze-dried to obtain two kinds of gelatin particle freeze-dried powders loaded with different growth factors. Blend the freeze-dried powder of the above-mentioned microgel particles with a certain volume of 1mM NaCl salt solution, and stir and mix evenly to obtain injectable self-healing colloidal gels loaded with different growth factors, wherein the microgel colloids The particle volume fraction accounts for 100vol% of the colloidal gel volume. The release curves of the in vitro release kinetics of two different growth factors from the colloidal gel carrier material are shown in Figure 4. The type A gelatin loaded with bFGF degrades faster due to the low degree of cross-linking, so the release rate of bFGF is faster. B-type gelatin loaded with BMP-2 has a slow degradation rate due to a high degree of cross-linking, so the release rate of BMP-2 is relatively slow; the results show that the colloidal gel of the present invention can realize the orderly release of different growth factor drugs.
实施例7Example 7
通过实施1中的制备方法得到A型明胶和B型明胶微凝胶颗粒分散液，将两种明胶颗粒分别分散在20mM的盐酸水溶液中，分别得到分散有带正电荷的A型明胶微凝胶颗粒和带负电荷的B型明胶微凝胶颗粒的分散液，将两者充分混合、搅拌，得分散有两种不同微凝胶颗粒的分散液，其中A型明胶和B型明胶混合的颗粒数量比为1:1；向分散液中加入100mM的氢氧化钠调节pH值至7.0，搅拌混合，冷冻干燥，得到含有两种不同明胶胶体颗粒的冻干粉末。将上述混合物冻干粉末与磷酸缓冲溶液PBS共混，并快速搅拌混合均匀得到可注射型自愈合胶体凝胶，即本发明所述的止血材料，其中两种胶体颗粒体积分数占胶体凝胶体积的100vol％。以新西兰兔背部脊柱骨缺损出血创面为动物实验模型，制造2.5-4mm的出血创口，将明胶基止血凝胶通过普通医用注射器直接注射在出血创面，轻轻压迫1min后出血点被封闭，止血时间为1min。动物实验的过程如图5所示。Obtain type A gelatin and type B gelatin microgel particle dispersion liquid by implementing the preparation method in 1, two kinds of gelatin particles are dispersed in the hydrochloric acid aqueous solution of 20mM respectively, obtain the positively charged A type gelatin microgel dispersed respectively Particles and negatively charged B-type gelatin microgel particle dispersion, the two are fully mixed and stirred to obtain a dispersion of two different microgel particles, in which A-type gelatin and B-type gelatin mixed particles The quantity ratio is 1:1; 100 mM sodium hydroxide is added to the dispersion to adjust the pH value to 7.0, stirred and mixed, and freeze-dried to obtain a freeze-dried powder containing two different gelatin colloidal particles. Blend the freeze-dried powder of the above mixture with phosphate buffer solution PBS, and stir and mix quickly to obtain an injectable self-healing colloidal gel, which is the hemostatic material of the present invention, wherein the volume fraction of the two colloidal particles accounts for the colloidal gel 100vol% of the volume. Taking the bleeding wound of spinal bone defect on the back of New Zealand rabbits as an animal experimental model, a bleeding wound of 2.5-4mm was made, and the gelatin-based hemostatic gel was directly injected into the bleeding wound through an ordinary medical syringe, and the bleeding point was sealed after being lightly pressed for 1 minute. 1min. The process of the animal experiment is shown in Figure 5.
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| CN201710335184.0ACN106983905B (en) | 2017-05-12 | 2017-05-12 | A kind of injectable self-healing hemostatic material and its preparation method and application |
| PCT/CN2018/086502WO2018205996A1 (en) | 2017-05-12 | 2018-05-11 | Injectable type self-healing hemostatic material, preparation method therefor and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108310448A (en)* | 2018-02-07 | 2018-07-24 | 广州迈普再生医学科技有限公司 | A kind of preparation method of fluid gelatin hemostatic material |
| WO2018205996A1 (en)* | 2017-05-12 | 2018-11-15 | 深训华诺生物科技有限公司 | Injectable type self-healing hemostatic material, preparation method therefor and use thereof |
| CN109438727A (en)* | 2018-10-17 | 2019-03-08 | 中国人民解放军总医院 | A kind of self-healing property hydrogel of fluorescence response and preparation method thereof |
| CN110527115A (en)* | 2019-08-01 | 2019-12-03 | 华南理工大学 | Multi-functional selfreparing hydrogel and preparation method thereof and the application in determination of biogenic amines |
| CN110743038A (en)* | 2019-11-06 | 2020-02-04 | 大连理工大学 | Double-network structure composite hydrogel and preparation method and application thereof |
| CN110760076A (en)* | 2019-11-06 | 2020-02-07 | 大连理工大学 | An injectable, high-strength composite hydrogel based on colloidal particle-iPRF double network structure and its preparation method and application |
| CN111068111A (en)* | 2018-10-19 | 2020-04-28 | 胡尚秀 | Injectable self-assembled microsphere gel, its use and preparation method |
| CN111386132A (en)* | 2017-10-02 | 2020-07-07 | 阿莱奥生物医学工程有限公司 | water-based tissue adhesive |
| CN113134113A (en)* | 2021-04-07 | 2021-07-20 | 赛克赛斯生物科技股份有限公司 | Preparation method of absorbable hemostatic fluid gelatin and absorbable hemostatic fluid gelatin |
| CN114028606A (en)* | 2021-10-26 | 2022-02-11 | 浙江理工大学 | Chitosan and protamine antibacterial hemostatic microsphere and preparation method thereof |
| CN114848668A (en)* | 2021-01-20 | 2022-08-05 | 香港中文大学 | Composition with wound healing promoting and quick hemostasis functions |
| CN115845121A (en)* | 2022-12-07 | 2023-03-28 | 华南理工大学 | Hemostatic, antibacterial and healing promoting microgel assembly powder and preparation method thereof |
| CN117018268A (en)* | 2023-07-26 | 2023-11-10 | 深圳大学 | Preparation method and application of 3D (three-dimensional) biological printing ink based on gelatin particles |
| WO2024217056A1 (en)* | 2023-04-17 | 2024-10-24 | 上海交通大学 | High-efficiency hemostatic material, and preparation method therefor and use thereof |
| WO2025176196A1 (en)* | 2024-02-23 | 2025-08-28 | 欧承健股份有限公司 | Gel, manufacturing method therefor, and use thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115819803B (en)* | 2022-12-26 | 2025-09-02 | 杭州钧兴生物科技有限公司 | A self-healing collagen hydrogel material and preparation method thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007120818A2 (en)* | 2006-04-12 | 2007-10-25 | Massachusetts Institute Of Technology | Compositions and methods for inhibiting adhesions |
| CN105796478A (en)* | 2016-03-22 | 2016-07-27 | 王华楠 | High-strength, self-healing, injectable composite colloidal gel material assembled from nano-colloidal particles and its preparation method and application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688525B (en)* | 2012-05-07 | 2013-11-27 | 东南大学 | A kind of biomacromolecule hydrogel and preparation method thereof |
| US8680240B1 (en)* | 2012-11-01 | 2014-03-25 | George David Falus | Tissue sealant for use in non-compressible hemorrhage |
| US20160121017A1 (en)* | 2014-11-04 | 2016-05-05 | Biomedica Management Corp | SINGLE SOLUTION of Gel-LIKE FIBRIN HEMOSTAT |
| CN106983905B (en)* | 2017-05-12 | 2019-10-11 | 深圳华诺生物科技有限公司 | A kind of injectable self-healing hemostatic material and its preparation method and application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007120818A2 (en)* | 2006-04-12 | 2007-10-25 | Massachusetts Institute Of Technology | Compositions and methods for inhibiting adhesions |
| CN105796478A (en)* | 2016-03-22 | 2016-07-27 | 王华楠 | High-strength, self-healing, injectable composite colloidal gel material assembled from nano-colloidal particles and its preparation method and application |
| Title |
|---|
| HUANA WANG, ET.AL.: "Oppositely Charged Gelatin Nanospheres as Building Blocks for Injectable and Biodegradable Gels", 《ADV. MATER.》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018205996A1 (en)* | 2017-05-12 | 2018-11-15 | 深训华诺生物科技有限公司 | Injectable type self-healing hemostatic material, preparation method therefor and use thereof |
| CN111386132A (en)* | 2017-10-02 | 2020-07-07 | 阿莱奥生物医学工程有限公司 | water-based tissue adhesive |
| CN108310448A (en)* | 2018-02-07 | 2018-07-24 | 广州迈普再生医学科技有限公司 | A kind of preparation method of fluid gelatin hemostatic material |
| CN108310448B (en)* | 2018-02-07 | 2021-03-02 | 广州迈普再生医学科技股份有限公司 | Preparation method of fluid gelatin hemostatic material |
| CN109438727B (en)* | 2018-10-17 | 2020-08-07 | 中国人民解放军总医院 | Fluorescence-responsive self-healing hydrogel and preparation method thereof |
| CN109438727A (en)* | 2018-10-17 | 2019-03-08 | 中国人民解放军总医院 | A kind of self-healing property hydrogel of fluorescence response and preparation method thereof |
| CN111068111A (en)* | 2018-10-19 | 2020-04-28 | 胡尚秀 | Injectable self-assembled microsphere gel, its use and preparation method |
| CN110527115A (en)* | 2019-08-01 | 2019-12-03 | 华南理工大学 | Multi-functional selfreparing hydrogel and preparation method thereof and the application in determination of biogenic amines |
| CN110743038A (en)* | 2019-11-06 | 2020-02-04 | 大连理工大学 | Double-network structure composite hydrogel and preparation method and application thereof |
| CN110760076A (en)* | 2019-11-06 | 2020-02-07 | 大连理工大学 | An injectable, high-strength composite hydrogel based on colloidal particle-iPRF double network structure and its preparation method and application |
| CN114848668A (en)* | 2021-01-20 | 2022-08-05 | 香港中文大学 | Composition with wound healing promoting and quick hemostasis functions |
| CN114848668B (en)* | 2021-01-20 | 2024-03-26 | 香港中文大学 | Composition with functions of promoting wound healing and rapid hemostasis |
| CN113134113A (en)* | 2021-04-07 | 2021-07-20 | 赛克赛斯生物科技股份有限公司 | Preparation method of absorbable hemostatic fluid gelatin and absorbable hemostatic fluid gelatin |
| CN113134113B (en)* | 2021-04-07 | 2022-07-01 | 赛克赛斯生物科技股份有限公司 | Preparation method of absorbable hemostatic fluid gelatin and absorbable hemostatic fluid gelatin |
| CN114028606A (en)* | 2021-10-26 | 2022-02-11 | 浙江理工大学 | Chitosan and protamine antibacterial hemostatic microsphere and preparation method thereof |
| CN115845121A (en)* | 2022-12-07 | 2023-03-28 | 华南理工大学 | Hemostatic, antibacterial and healing promoting microgel assembly powder and preparation method thereof |
| WO2024217056A1 (en)* | 2023-04-17 | 2024-10-24 | 上海交通大学 | High-efficiency hemostatic material, and preparation method therefor and use thereof |
| CN117018268A (en)* | 2023-07-26 | 2023-11-10 | 深圳大学 | Preparation method and application of 3D (three-dimensional) biological printing ink based on gelatin particles |
| WO2025176196A1 (en)* | 2024-02-23 | 2025-08-28 | 欧承健股份有限公司 | Gel, manufacturing method therefor, and use thereof |
| Publication number | Publication date |
|---|---|
| CN106983905B (en) | 2019-10-11 |
| WO2018205996A1 (en) | 2018-11-15 |
| Publication | Publication Date | Title |
|---|---|---|
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| TA01 | Transfer of patent application right | Effective date of registration:20181010 Address after:518104 Houting Community, Shajing Street, Baoan District, Shenzhen City, Guangdong Province Applicant after:Shenzhen Sino Biological Technology Co., Ltd. Address before:116000 College of life sciences, Dalian University of Technology, 2 linggong Road, hi tech park, Dalian, Liaoning Applicant before:Wang Huanan | |
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