技术领域technical field
本发明涉及一种多孔的多糖类细胞吸附材料及其制备方法与应用。The invention relates to a porous polysaccharide cell adsorption material and its preparation method and application.
背景技术Background technique
目前市面上的止血材料主要分为纤维蛋白类、多糖类以及微孔沸石类。止血材料的止血原理多为在材料的物理或化学作用下增加伤口附近血细胞、血小板、凝血因子的浓度，形成凝血，从而达到止血的目的。然而纵观目前上市的止血材料，存在诸如制作过程复杂、生产成本昂贵、保存条件严格(干燥低湿)等问题，而且有的止血材料在达到止血目的的同时还会伴随其他副作用，比如微孔沸石类止血剂，在凝血的过程中会伴随着因吸附作用导致的放热烧伤等有害作用。At present, the hemostatic materials on the market are mainly divided into fibrin, polysaccharide and microporous zeolite. The hemostatic principle of hemostatic materials is mostly to increase the concentration of blood cells, platelets, and coagulation factors near the wound under the physical or chemical action of the material to form blood coagulation, so as to achieve the purpose of hemostasis. However, looking at the hemostatic materials currently on the market, there are problems such as complicated production process, high production cost, strict storage conditions (dry and low humidity), and some hemostatic materials will be accompanied by other side effects while achieving the purpose of hemostasis, such as microporous zeolite Hemostatic agents, in the process of blood coagulation, will be accompanied by harmful effects such as exothermic burns caused by adsorption.
发明内容Contents of the invention
为了克服现有的止血材料制作过程复杂、生产成本昂贵以及毒副作用多等缺点，本发明提供了一种新型的多孔有机无机杂化多糖类细胞吸附材料及其制备方法。In order to overcome the disadvantages of the existing hemostatic materials such as complex production process, high production cost and many side effects, the present invention provides a novel porous organic-inorganic hybrid polysaccharide cell adsorption material and a preparation method thereof.
本发明所提供的多孔有机无机杂化多糖类细胞吸附材料是按照下述步骤的方法制备得到的：将二氧化硅溶胶、壳聚糖溶液与交联剂水溶液混合均匀，得到水凝胶，然后再将所述水凝胶依次进行陈化、冷冻干燥直至恒重，得到所述多孔有机无机杂化多糖类细胞吸附材料；其中，所述交联剂水溶液中的交联剂为对醛基苯甲酸封端的聚乙二醇。The porous organic-inorganic hybrid polysaccharide cell adsorption material provided by the present invention is prepared according to the following steps: uniformly mix silica sol, chitosan solution and cross-linking agent aqueous solution to obtain a hydrogel, Then, the hydrogel is aged and freeze-dried until constant weight to obtain the porous organic-inorganic hybrid polysaccharide cell adsorption material; wherein, the cross-linking agent in the cross-linking agent aqueous solution is p-aldehyde Benzoic acid-terminated polyethylene glycol.
其中，所述水凝胶中二氧化硅、壳聚糖与交联剂的质量比可为(30-80)∶(10-70)∶(20-70)，优选质量比为60∶15∶25。Wherein, the mass ratio of silicon dioxide, chitosan and crosslinking agent in the hydrogel can be (30-80): (10-70): (20-70), preferably the mass ratio is 60:15: 25.
本发明中，所述二氧化硅溶胶按照下述方法制备得到：将正硅酸酯在pH值为2-6(以3.5-4.5为佳)的酸性条件下进行水解，搅拌直至分层现象消失，得到均相水溶液即为二氧化硅溶胶。所述二氧化硅溶胶中二氧化硅的质量含量可为5-21％。In the present invention, the silica sol is prepared according to the following method: hydrolyze orthosilicate under acidic conditions with a pH value of 2-6 (preferably 3.5-4.5), and stir until the stratification phenomenon disappears , to obtain a homogeneous aqueous solution that is silica sol. The mass content of silicon dioxide in the silicon dioxide sol may be 5-21%.
所述壳聚糖溶液是将壳聚糖溶于浓度为0.005-0.1g/ml(以0.02g/ml为佳)的醋酸溶液中得到的。所述壳聚糖溶液中壳聚糖的质量浓度为0.5％-5％。The chitosan solution is obtained by dissolving chitosan in an acetic acid solution with a concentration of 0.005-0.1 g/ml (preferably 0.02 g/ml). The chitosan mass concentration in the chitosan solution is 0.5%-5%.
本发明所用壳聚糖的分子量可为1000Da-500000Da，脱乙酰度可为75％-100％。The molecular weight of the chitosan used in the invention can be 1000Da-500000Da, and the degree of deacetylation can be 75%-100%.
所述交联剂对醛基苯甲酸封端的聚乙二醇(聚乙二醇对醛基苯甲酸二酯)是聚乙二醇与对醛基苯甲酸经酯化反应得到，具体合成方法参见文献Biomacromolecules，2011，12(8)，pp 2894-2901。所用聚乙二醇的分子量为200-10000Da。The polyethylene glycol (polyethylene glycol p-aldehyde benzoic acid diester) terminated by p-aldehyde benzoic acid of the cross-linking agent is obtained through esterification reaction between polyethylene glycol and p-aldehyde benzoic acid, and the specific synthesis method can be found in Literature Biomacromolecules, 2011, 12(8), pp 2894-2901. The polyethylene glycol used has a molecular weight of 200-10000 Da.
所述交联剂水溶液中交联剂的质量浓度为5％-65％，以20％-30％为佳。The mass concentration of the crosslinking agent in the crosslinking agent aqueous solution is 5%-65%, preferably 20%-30%.
所述陈化的条件为室温放置2天以上或40-60℃(以50℃为佳)恒温放置24小时The aging condition is to place it at room temperature for more than 2 days or at a constant temperature of 40-60°C (preferably 50°C) for 24 hours
上述方法还包括将制备的多孔有机无机杂化多糖类细胞吸附材料进行研磨粉碎的步骤。粉碎后的材料粒径在20-300目为宜。The above method also includes the step of grinding and pulverizing the prepared porous organic-inorganic hybrid polysaccharide cell adsorption material. The particle size of the pulverized material is preferably 20-300 mesh.
本发明提供了一种新型的多孔有机无机杂化细胞吸附材料，其主要组成为壳聚糖、二氧化硅和含醛基聚乙二醇交联剂。制备时将交联剂水溶液与二氧化硅溶胶(即正硅酸酯的水解溶液)混合均匀，然后将此混合溶液与壳聚糖溶液混合，通过搅拌或者涡旋振动等操作使其混合均匀。壳聚糖上丰富的胺基与聚乙二醇交联剂两端的醛基形成亚胺键(席夫碱)，链段间相互穿插交联体系能够在1分钟内很快形成水凝胶。二氧化硅溶胶水凝胶经过缩聚，原位形成二氧化硅网络骨架，得到有机无机杂化的壳聚糖多孔材料。该材料具备良好红细胞吸附能力，是一种潜在的极佳的止血材料。所得止血材料在体外血细胞吸附实验中五分钟吸附率为80％以上。The invention provides a novel porous organic-inorganic hybrid cell adsorption material, which is mainly composed of chitosan, silicon dioxide and aldehyde group-containing polyethylene glycol cross-linking agent. During preparation, the crosslinking agent aqueous solution and the silica sol (that is, the hydrolysis solution of orthosilicate) are uniformly mixed, and then the mixed solution is mixed with the chitosan solution, and the mixture is uniformly mixed by stirring or vortex vibration. The abundant amine groups on chitosan form imine bonds (Schiff bases) with the aldehyde groups at both ends of the polyethylene glycol cross-linking agent, and the interpenetrating cross-linking system between chain segments can quickly form a hydrogel within 1 minute. The silica sol hydrogel undergoes polycondensation to form a silica network skeleton in situ to obtain an organic-inorganic hybrid chitosan porous material. The material has good red blood cell adsorption capacity and is a potential excellent hemostatic material. The obtained hemostatic material has an adsorption rate of more than 80% in five minutes in an in vitro blood cell adsorption experiment.
本发明制备吸附材料所需原料来源广泛、价格低廉、制备方法简单。所提供的吸附材料绿色环保，不仅能够满足止血的要求，还能克服现售止血材料的多种不足，易于实现大规模的工业生产。The raw materials required for preparing the adsorption material in the invention have wide sources, low price and simple preparation method. The provided adsorption material is green and environment-friendly, can not only meet the requirements of hemostasis, but also overcome various deficiencies of currently available hemostatic materials, and is easy to realize large-scale industrial production.
本发明所提供的细胞吸附材料具有如下突出特点：The cell adsorption material provided by the present invention has the following prominent features:
1、制备本发明细胞吸附材料所需的原料来源丰富，价格低廉。1. The raw materials required for the preparation of the cell adsorption material of the present invention are rich in sources and low in price.
2、本发明细胞吸附材料的制备工艺简单，工艺参数较易控制。2. The preparation process of the cell adsorption material of the present invention is simple, and the process parameters are easier to control.
3、本发明细胞吸附材料的绿色环保，且止血能力较强，止血速度较快，副作用少，易于实现大规模的工业生产。3. The cell adsorption material of the present invention is green and environmentally friendly, has strong hemostatic ability, fast hemostatic speed, few side effects, and is easy to realize large-scale industrial production.
具体实施方式Detailed ways
下面通过具体实施例对本发明进行说明，但本发明并不局限于此。The present invention will be described below through specific examples, but the present invention is not limited thereto.
下述实施例中所述实验方法，如无特殊说明，均为常规方法。The experimental methods described in the following examples are conventional methods unless otherwise specified.
下述实施例中所用材料除对醛基苯甲酸封端的聚乙二醇交联剂外均可通过商业途径获得。交联剂的制备方法为末端为羟基的聚乙二醇与对醛基苯甲酸通过酯化反应合成，具体参见文献Biomacromolecules，2011，12(8)，pp 2894-2901。The materials used in the following examples are all commercially available except for the p-aldehydebenzoic acid-terminated polyethylene glycol cross-linking agent. The preparation method of cross-linking agent is that polyethylene glycol with terminal hydroxyl group and p-aldehyde benzoic acid are synthesized by esterification, specifically refer to literature Biomacromolecules, 2011, 12 (8), pp 2894-2901.
实施例、多孔有机无机杂化的多糖类细胞吸附材料的制备Example, Preparation of Porous Organic-Inorganic Hybrid Polysaccharide Cell Adsorption Material
1.将3g壳聚糖溶解在97g 0.02g/ml的醋酸溶液中，得到质量浓度为3％的壳聚糖溶液；1. 3g chitosan is dissolved in the acetic acid solution of 97g 0.02g/ml, obtains the chitosan solution that mass concentration is 3%;
2.正硅酸乙酯在酸性(pH值为3.5)条件下水解，搅拌直至分层现象消失，得到3ml均相水溶液(即二氧化硅溶胶，其中，二氧化硅的质量含量为21％；2. Ethyl orthosilicate was hydrolyzed under acidic (pH value 3.5) conditions, stirred until the stratification disappeared, and 3ml of homogeneous aqueous solution (i.e. silica sol, wherein the mass content of silica was 21%;
3.将1ml对醛基苯甲酸封端的聚乙二醇的水溶液(质量浓度20％)与步骤2中的二氧化硅溶胶混合均匀，然后将此混合溶液与壳聚糖溶液混合，通过搅拌使其混合均匀。3. the aqueous solution (mass concentration 20%) of the polyethylene glycol of 1ml p-aldehyde benzoic acid end-blocking is mixed with the silica sol in step 2, then this mixed solution is mixed with chitosan solution, makes by stirring It mixes well.
4.对分散在水凝胶中的溶胶进行陈化(陈化条件50℃恒温放置24小时)，在陈化过程中二氧化硅溶胶原位进行缩聚形成二氧化硅网络支架。4. Aging the sol dispersed in the hydrogel (the aging condition is kept at a constant temperature of 50° C. for 24 hours). During the aging process, the silica sol undergoes polycondensation in situ to form a silica network scaffold.
5.将得到的二氧化硅网络支架真空冻干直至恒重，再研磨成粉末状得到粒径为60-100目的具有多孔结构的有机-无机杂化止血材料。5. Vacuum freeze-dry the obtained silica network scaffold until constant weight, and then grind it into powder to obtain an organic-inorganic hybrid hemostatic material with a particle size of 60-100 mesh and a porous structure.
体外血细胞吸附实验：In vitro hemadsorption experiment:
取30毫克止血材料粉末，加入至1毫升磷酸盐缓冲溶液之中(pH为7.2，质量浓度为0.9％)，然后加入50微升的新鲜小鼠全血(保存于抗凝管中)，轻轻摇晃，等固体稍沉降，每隔一定时间取上层液体进行细胞计数。在5分钟内，上层清液的细胞数目减少到初始的20％，本发明在5分钟内吸附了血液中的80％以上的血细胞及血小板等。Take 30 mg of hemostatic material powder, add it to 1 ml of phosphate buffer solution (pH 7.2, mass concentration 0.9%), then add 50 microliters of fresh mouse whole blood (stored in an anticoagulant tube), gently Shake gently, wait for the solid to settle slightly, and take the upper layer liquid at regular intervals for cell counting. Within 5 minutes, the number of cells in the supernatant is reduced to 20% of the initial value, and the present invention absorbs more than 80% of blood cells and platelets in the blood within 5 minutes.
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