WO2021237543A1 - 基于海洋源明胶的可注射水凝胶止血剂及应用和应用方法 - Google Patents
基于海洋源明胶的可注射水凝胶止血剂及应用和应用方法 Download PDFInfo
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- WO2021237543A1 WO2021237543A1 PCT/CN2020/092737 CN2020092737W WO2021237543A1 WO 2021237543 A1 WO2021237543 A1 WO 2021237543A1 CN 2020092737 W CN2020092737 W CN 2020092737W WO 2021237543 A1 WO2021237543 A1 WO 2021237543A1
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- Prior art keywords
- marine
- hemostatic agent
- gelatin
- injectable hydrogel
- derived gelatin
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- 229940030225 antihemorrhagics Drugs 0.000 title claims abstract description 73
- 239000002874 hemostatic agent Substances 0.000 title claims abstract description 73
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
Definitions
- the invention belongs to the technical field of hemostatic agents, and in particular relates to an injectable hydrogel hemostatic agent based on marine-derived gelatin and its application and application method.
- the cross-linking reaction is uncontrollable.
- the working mechanism of this type of injectable hemostatic hydrogel is to use the cross-linking reaction of two precursor solutions to form a hydrogel to block the hemostatic point, but this cross-linking reaction is uncontrollable, and the gel will block if the operation time is too long. If the operation time of the syringe is too short, the precursor solution cannot be gelled in time, resulting in failure of hemostasis.
- the injectable fast light-curing hydrogel hemostatic agent based on natural polymer gelatin and hyaluronic acid will swell in the humid environment of the body, and the increase in volume will lead to the decrease of mechanical properties, which may also lead to the failure of wound closure. At the same time , The increase in volume will compress the surrounding tissues and cause additional damage.
- the present invention provides an injectable hydrogel hemostatic agent based on marine-source gelatin and an application and application method thereof, and aims to solve the mechanical performance drop, sealing failure, and sealing failure of the existing injectable fast light-curing hydrogel hemostatic agent due to swelling behavior. At least one problem such as additional damage to surrounding tissues.
- the present invention is realized as follows:
- the injectable hydrogel hemostatic agent based on marine-source gelatin, based on the weight percentage of each component being 100%, the injectable hydrogel hemostatic agent comprises the following components:
- the above-mentioned injectable hydrogel hemostatic agent based on marine-derived gelatin is used in the hemostasis of human or animal body tissues, organs accidental wounds or surgical wounds.
- the application method of the marine-source gelatin-based injectable hydrogel hemostatic agent as described above includes the following steps: injecting the marine-source gelatin-based injectable hydrogel hemostatic agent into the site to be hemostatic and using Ultraviolet light irradiation causes the injectable hydrogel hemostatic agent to solidify.
- the marine-derived gelatin-based injectable hydrogel hemostatic agent provided by the present invention includes chemically modified marine-derived gelatin, photocrosslinking gel factor, tissue adhesion factor, and photoinitiator.
- Injectable light curing can achieve curing effect within 20s, and exhibits strong tissue adhesion and mechanical properties, curing swelling and deformation are very small, so that the bleeding wound can be sealed quickly and lasting, and finally hemorrhage can be quickly stopped for acute wound bleeding. And there is no additional trauma effect on the surrounding tissues; in addition, because the material has degradable properties, it will gradually degrade as the wound is healed, and has good biosafety.
- the injectable hydrogel hemostatic agent based on marine-derived gelatin of the present invention has the effect of rapid hemostasis and no additional trauma to surrounding tissues, it can be applied to hemostasis of human or animal body tissues, organs accidental trauma or surgical wounds.
- the application method of the injectable hydrogel hemostatic agent based on marine source gelatin provided by the present invention has the characteristics of simple operation, short curing time, fast hemostasis and the like.
- Figure 1 is a NMR-400MHz nuclear magnetic resonance (NMR-400MHz) spectrum of methacrylic anhydride modified marine source fish skin gelatin provided in Example 1 of the present invention
- Figure 2 is a Pluronic F127-diacrylate nuclear magnetic resonance (NMR-400MHz) spectrum provided in Example 1 of the present invention
- Fig. 3 is a Pluronic F127-dibenzaldehyde formate nuclear magnetic resonance (NMR-400MHz) spectrum provided in Example 1 of the present invention
- Example 4 is a swelling picture of the injectable hydrogel hemostatic agent based on marine-source gelatin prepared in Example 1 of the present invention
- Example 5 is a comparison picture of the injectable hydrogel hemostatic agent based on marine-derived gelatin prepared in Example 1 of the present invention and the hydrogel provided in Comparative Example 1 before and after swelling;
- Example 6 is a picture showing changes before and after swelling of the injectable hydrogel hemostatic agent based on marine source gelatin prepared in Example 2 of the present invention
- Fig. 7 is a schematic diagram showing the percentage change in volume of the injectable hydrogel hemostatic agent based on marine-derived gelatin prepared in Example 1 of the present invention and the hydrogel provided in Comparative Example 1 after swelling;
- Example 8 is a schematic diagram of the mass change percentage of the injectable hydrogel hemostatic agent based on marine-derived gelatin prepared in Example 1 of the present invention and the hydrogel provided in Comparative Example 1 after swelling;
- Fig. 9 is a curve of diameter and mass change during the swelling process of the injectable hydrogel hemostatic agent based on marine-source gelatin prepared in Example 2 of the present invention.
- Fig. 10 is a comparative diagram of hemostatic experiment of liver in Example 1 of the present invention and blank control mice.
- the present invention provides an injectable hydrogel hemostatic agent based on marine-source gelatin, which is liquid before use, and only needs to be injected to the corresponding part to be hemostatic when used, and irradiated with ultraviolet light for 2s-20s.
- the hemostatic effect can be achieved.
- the marine-derived gelatin-based injectable hydrogel hemostatic agent of the present invention contains the following components:
- the marine source gelatin used in the chemically modified marine source gelatin is derived from marine source fish skin gelatin or marine source fish scale gelatin, and the modifier used is methacrylic anhydride.
- a methacrylic anhydride-modified marine-source gelatin that is, a methacrylic anhydride-modified marine-source fish skin gelatin or a methacrylic-anhydride-modified marine-source fish scale gelatin.
- the chemically modified marine-derived gelatin can be obtained in the following manner:
- the photo-crosslinking gel factor is a diacrylate modified poloxamer.
- the poloxamer mentioned in the present invention is a polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) triblock copolymer (English trade name is Pluronics), as long as it is a hydrophilic Pluronics Feasible, such as pluronics F127, pluronics F68, pluronics F87, pluronics F108, etc.
- the diacrylate modified poloxamer is Pluronics F127-diacrylate (abbreviation: PF127-DA).
- PF127-DA can be obtained as follows:
- the tissue adhesion factor involved in the present invention is selected from at least one compound that has the ability to react with amino groups and can be integrated into the hydrogel network through a photocuring reaction.
- the compounds that have the ability to react with amino groups and can be integrated into the hydrogel network through a photocuring reaction are dibenzaldehyde formate-modified poloxamer, acrylic acid-N-succinimide ester, 4-ethylene At least one of benzaldehyde, 3-methacrylamidopamine, and other compounds that have the ability to react with amino groups and can be integrated into the hydrogel network through a photocuring reaction.
- the dibenzaldehyde ester modified poloxamer is Pluronics F127-dibenzaldehyde formate (abbreviation: PF127-DF).
- a compound containing a catechol group such as 3-methacrylamide dopamine
- its weight content in the marine-source gelatin-based injectable hydrogel hemostatic agent does not exceed 0.5 %, otherwise it will affect the curing effect.
- PF127-DF can be obtained in the following manner:
- the total content of poloxamers does not exceed 20%.
- diacrylic acid The content of ester-modified poloxamer does not exceed 20%; when diacrylate-modified poloxamer and dibenzaldehyde formate-modified poloxamer are included, the diacrylate-modified poloxamer The total content of loxamer and benzaldehyde ester-modified poloxamer does not exceed 20%.
- the injectable hydrogel hemostatic agent based on marine-derived gelatin will lose liquid fluidity at room temperature and become a paste-like substance, which cannot be used in an injectable manner.
- the fluidity of the liquid can only be maintained at 10°C, which will cause cumbersome and negative effects on use.
- the photoinitiator used should be capable of curing the injectable hydrogel hemostatic agent based on marine-derived gelatin under (2-20) seconds of ultraviolet irradiation, if the photoinitiator used is effective If the curing time is too long, and the curing time exceeds 20s, it is difficult to exert the hemostatic effect.
- the photoinitiator is phenyl(2,4,6-trimethylbenzoyl) phosphate lithium salt (English abbreviation: LAP), which can make the marine-derived gelatin-based of the present invention
- LAP phenyl(2,4,6-trimethylbenzoyl) phosphate lithium salt
- the solvent used in the present invention as long as it can dissolve the chemically modified marine-derived gelatin, photocrosslinking gelatin factor, tissue adhesion factor and photoinitiator, and does not react before light exposure, and it is used
- the amount is not particularly limited, and can be formulated according to the actual concentration required for hemostasis.
- the solvent can be any one of deionized water, phosphate buffered saline (PBS buffer for short), and physiological saline.
- PBS buffer phosphate buffered saline
- the marine-derived gelatin-based injectable hydrogel hemostatic agent of the present invention can be prepared by the following method:
- the injectable hydrogel hemostatic agent based on marine-source gelatin of the present invention is chemically modified, the marine-source gelatin used has better biological activity and can better promote wound healing; and the marine-source gelatin-based hemostatic agent of the present invention
- the injectable hydrogel hemostatic agent has extremely low swelling deformation, can maintain sufficient mechanical properties and tissue adhesion for a long time, maintain the wound sealing effect for a long time, and will not compress the surrounding tissues. At the same time, due to its degradable properties, it will As the wound is healed and degraded gradually, the degradation product has no toxic effect on the organism and has better biological safety. Therefore, it can be widely used in the hemostasis of human or animal tissues, organs accidental wounds or surgical wounds.
- the injectable hydrogel hemostatic agent based on marine source gelatin of the present invention is applicable to any of liver, spleen, kidney, intestine, stomach, and lung.
- the injectable hydrogel hemostatic agent is injected into the site to be hemostatic, and ultraviolet light is used to irradiate the injectable hydrogel hemostatic agent to solidify.
- the wavelength of ultraviolet light does not exceed 405nm.
- the longer the wavelength the longer the curing time, which is not conducive to rapid hemostasis. If the wavelength of ultraviolet light is shorter, the surrounding tissues are more likely to be damaged.
- the preferred wavelength of ultraviolet light is 300 nm to 380 nm, for example, 365 nm ultraviolet light can be used for irradiation.
- a method for preparing an injectable hydrogel hemostatic agent based on marine source gelatin including the following steps:
- reaction solution was added to 1000 mL of absolute ethanol to precipitate, the precipitate was collected by centrifugation and dissolved in deionized water, and the solution was dialyzed in deionized water for 3 days and freeze-dried to obtain methacrylic anhydride modified marine source fish skin gelatin , Collect for later use.
- the crude product was dissolved in dichloromethane for a second time, precipitated in ice ether, centrifuged to collect the precipitate, vacuum dried, and repeated twice; and the product was dissolved in deionized water, freeze-dried, and collected for use.
- Figure 1 shows the NMR spectrum of chemically modified marine source fish skin gelatin.
- Figure 2 shows the NMR spectrum of PF127-DA.
- Figure 3 shows the NMR spectrum of PF127-DF.
- a method for preparing an injectable hydrogel hemostatic agent based on marine source gelatin including the following steps:
- a method for preparing an injectable hydrogel hemostatic agent based on marine source gelatin including the following steps:
- a method for preparing an injectable hydrogel hemostatic agent based on marine source gelatin including the following steps:
- a method for preparing an injectable hydrogel hemostatic agent based on marine source gelatin including the following steps:
- a method for preparing an injectable hydrogel hemostatic agent based on marine source gelatin including the following steps:
- a method for preparing an injectable hydrogel hemostatic agent based on marine source gelatin including the following steps:
- a hydrogel calculated as 100% by weight, and its chemical composition is:
- the obtained hydrogels are all colorless and highly transparent elastic solids.
- the colorless and highly transparent ensure that the real-time situation of bleeding points can be easily observed during the hemostasis process, which is beneficial to timely change the treatment plan.
- Example 1 Part of the products of Example 1 and Comparative Example 1 were cured by UV light under the same conditions, and immersed in PBS buffer at 37° C., and the volume and mass changes were observed. It can be seen that the volume of the phosphate buffer solution does not change after being soaked for 12 hours, and the swelling equilibrium is reached. The final changes in volume and mass are shown in Figures 7 and 8.
- the volume increase of the marine-derived gelatin-based injectable hydrogel hemostatic agent of Example 1 is 147% of the initial volume, and the mass increase is 161% of the initial.
- the volume growth was 300% of the initial volume, and the mass growth was 295% of the initial volume.
- the marine-derived gelatin-based injectable hydrogel hemostatic agent of the present invention has extremely low swelling deformation.
- hydrogel pressure burst testing equipment was used to perform tissue adhesion on the products of Examples 1-7 and Comparative Example 1.
- Example Pigskin wound number Barometer reading/mmHg Example 1 1# 261 Example 2 2# 262 Example 3 3# 273 Example 4 4# 326 Example 5 5# 291 Example 6 6# 276 Example 7 7# 277 Comparative example 1 8# 101
- the injectable hydrogel hemostatic agent based on marine-derived gelatin of Example 1 is dripped onto the bleeding site and irradiated with ultraviolet for 20 seconds, which can effectively stop bleeding and has the function of quickly stopping bleeding against acute bleeding.
- the marine-derived gelatin-based injectable hydrogel hemostatic agent of the present invention has strong tissue adhesion and mechanical properties, minimal curing and swelling deformation, and rapid hemostatic effects.
- the raw materials and degradation products have no irritation to the organism and are biologically safe.
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Abstract
一种基于海洋源明胶的可注射水凝胶止血剂及应用和应用方法，按照重量百分含量为100％计，所述基于海洋源明胶的可注射水凝胶止血剂包含以下组分；化学改性的海洋源明胶0.1％～10％；光交联凝胶因子10％～20％；组织粘附因子0.1％～15％；光引发剂0.1％～0.5％；以及溶解量的溶媒。该可注射水凝胶止血剂具有快速、良好的止血效果，且止血过程中对周围组织无额外创伤，具有可降解特性，可广泛应用于人体或动物体组织、脏器意外创伤或手术伤口止血中。
Description
本发明属于止血剂技术领域，尤其涉及基于海洋源明胶的可注射水凝胶止血剂及应用和应用方法。
意外创伤和外科手术导致的急性出血是世界普遍面临的重大医疗问题，它们会严重的威胁患者的生命健康。其中尤以人体脏器如肝脏和脾脏最甚，因为肝脏和脾脏中均有丰富的血管组织，在遭受创伤或者实施相关外科手术时，创面的急性出血不但十分常见而且非常棘手。如何找到快速而有效的止血处理方法已经是降低患者死亡率的最佳策略。
对于内脏破裂造成的急性出血以及不规则形状和较深窄的出血创面，目前常规的临床止血方法(如手术缝合或电刀止血等)或止血材料(如止血纱布或止血海绵等)，但是这种方法无法深入创面有效封堵出血点，导致止血效果不理想。此时，只有将性能优异的止血材料注射到出血创面并进行充分封堵，才能有效止血，从而降低患者死亡率。
随着生物材料科学的发展以及为了更好的应对医疗急性出血，研究人员开发出一系列可注射止血水凝胶，如，纤维蛋白胶和高分子水凝胶等。但是目前的可注射止血凝胶存在以下几方面的问题：
(1).交联反应不可控。这类可注射止血水凝胶的工作机理为利用两种前体溶液发生交联反应形成水凝胶来封堵止血点，但是这种交联反应不可控，如果操作时间过长凝胶会堵塞注射器，如果操作时间过短前体溶液无法及时凝胶化，造成止血失败。
(2).固化速度慢。注射后还未形成凝胶的前体溶液易被血液冲走，或产生渗漏和流失，无法实现快速止血。
(3).组织粘附性差，力学性能不足。这会导致无法在固化后有效封闭伤口，导致止血失败。
基于天然高分子明胶和透明质酸的可注射快速光固化水凝胶止血剂在体内潮湿环境中会发生溶胀行为，而体积变大会导致力学性能下降，同样可能会导致创伤封闭失败，与此同时，体积增大会压迫周围组织造成额外的伤害。
发明内容
针对上述问题，研究人员进一步研究开发了基于天然高分子明胶和透明质酸的可注射快速光固化水凝胶止血剂，这种可注射光固化水凝胶止血剂可以实现快速固化。
本发明提供基于海洋源明胶的可注射水凝胶止血剂及应用和应用方法，旨在解决现有可注射快速光固化水凝胶止血剂因具有溶胀行为而导致的力学性能下降、封闭失败、对周围组织有额外伤害等至少一种问题。
本发明是这样实现的：
基于海洋源明胶的可注射水凝胶止血剂，按照各组分重量百分含量为100％计，所述可注射水凝胶止血剂包含以下组分；
对应地，上述所述的基于海洋源明胶的可注射水凝胶止血剂在人体或动物 体组织、脏器意外创伤或手术伤口止血中的应用。
相应地，如上所述的基于海洋源明胶的可注射水凝胶止血剂的应用方法，包括以下步骤：将所述基于海洋源明胶的可注射水凝胶止血剂注射于待止血部位，并使用紫外光照射，使所述可注射水凝胶止血剂发生固化。
相对于现有技术，本发明提供的基于海洋源明胶的可注射水凝胶止血剂，由于包含化学改性的海洋源明胶、光交联凝胶因子、组织粘附因子以及光引发剂，实现可注射光固化，能在20s内达到固化效果，且表现出较强的组织粘附性和力学性能，固化溶胀形变极小，从而可以迅速并持久封闭出血创面，最终实现针对急性创面出血迅速止血且对周围组织无额外创伤的效果；此外，由于材料具有可降解特性，会随着伤口的愈合逐渐降解，具有很好的生物安全性。
正是由于本发明基于海洋源明胶的可注射水凝胶止血剂具有快速止血且对周围组织无额外创伤的效果，因而可以应用于人体或动物体组织、脏器意外创伤或手术伤口止血中。
本发明提供的基于海洋源明胶的可注射水凝胶止血剂的应用方法，具有操作简单、固化时间短、止血速度快等特点。
为了更清楚地说明本发明施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例1提供的甲基丙烯酸酐改性的海洋源鱼皮明胶核磁共振(NMR-400MHz)图谱；
图2是本发明实施例1提供的Pluronic F127-二丙烯酸酯核磁共振 (NMR-400MHz)图谱；
图3是本发明实施例1提供的Pluronic F127-二苯甲醛甲酸酯核磁共振(NMR-400MHz)图谱；
图4是本发明实施例1制备的基于海洋源明胶的可注射水凝胶止血剂溶胀图片；
图5是本发明实施例1制备的基于海洋源明胶的可注射水凝胶止血剂与对比例1提供的水凝胶溶胀前后比较图片；
图6是本发明实施例2制备的基于海洋源明胶的可注射水凝胶止血剂的溶胀前后变化情况图片；
图7是本发明实施例1制备的基于海洋源明胶的可注射水凝胶止血剂与对比例1提供的水凝胶溶胀后体积变化百分比示意图；
图8是本发明实施例1制备的基于海洋源明胶的可注射水凝胶止血剂与对比例1提供的水凝胶溶胀后质量变化百分比示意图；
图9是本发明实施例2制备的基于海洋源明胶的可注射水凝胶止血剂溶胀过程中的直径和质量变化情况曲线；
图10是本发明实施例1和空白对照小鼠肝脏止血实验对比图。
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明做进一步的详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提供的是一种基于海洋源明胶的可注射水凝胶止血剂，其在使用前为液态，使用时只需要将其注射至相应的待止血部位，并采用紫外光照射2s～20s，即可实现止血效果。
具体来说，本发明的基于海洋源明胶的可注射水凝胶止血剂，按照各组分重量百分含量为100％计，包含以下组分：
其中，化学改性的海洋源明胶中所使用的海洋源明胶来源于海洋源鱼皮明胶或者海洋源鱼鳞明胶，而使用的改性剂为甲基丙烯酸酐。经过甲基丙烯酸酐的改性，获得甲基丙烯酸酐改性的海洋源明胶(即甲基丙烯酸酐改性的海洋源鱼皮明胶或者甲基丙烯酸酐改性的海洋源鱼鳞明胶)。
在一些实施例中，化学改性的海洋源明胶可以通过如下方式获得：
将海洋源明胶溶于去离子水中，得到溶液，然后滴加一定量的甲基丙烯酸酐，用碱性溶液调节pH值，使得pH在8～9之间；
反应完成后，用乙醇沉淀，离心收集沉淀并进行透析，最后冷冻干燥，获得化学改性的海洋源明胶。
优选地，光交联凝胶因子为二丙烯酸酯改性的泊洛沙姆。本发明所说的泊洛沙姆为聚氧乙烯-聚氧丙烯-聚氧乙烯(PEO-PPO-PEO)三嵌段共聚物(英文商品名称为Pluronics)，只要是具有亲水性的Pluronics均可行，如pluronics F127、pluronics F68、pluronics F87、pluronics F108等。进一步优选地，所述二丙烯酸酯改性的泊洛沙姆为Pluronics F127-二丙烯酸酯(简称：PF127-DA)。
在一些实施例中，PF127-DA可以通过如下方式获得：
将Pluronics F127溶于无水二氯甲烷中，并加入一定量的三乙胺；
在冰水浴条件下滴加一定量的丙烯酰氯进行反应，反应完成后，用乙醚沉淀，离心收集沉淀并用水透析，最后冷冻干燥，即可获得PF127-DA。
优选地，本发明所涉及的组织粘附因子选自具备与氨基反应能力并且可以通过光固化反应整合到水凝胶网络的化合物中的至少一种。
具体地，具备与氨基反应能力并且可以通过光固化反应整合到水凝胶网络的化合物为二苯甲醛甲酸酯改性的泊洛沙姆、丙烯酸-N-琥珀酰亚胺酯、4-乙烯基苯甲醛、3-甲基丙烯酰胺基多巴胺、以及其他具备与氨基反应能力并且可以通过光固化反应整合到水凝胶网络的化合物中的至少一种。
进一步优选地，所述二苯甲醛甲酸酯改性的泊洛沙姆为Pluronics F127-二苯甲醛甲酸酯(简称：PF127-DF)。
当使用含有邻苯二酚基团的化合物(如3-甲基丙烯酰胺基多巴胺)作为组织粘附因子时，其在基于海洋源明胶的可注射水凝胶止血剂中的重量含量不超过0.5％，否则会影响固化效果。
在一些实施例中，PF127-DF可以通过如下方式获得：
将Pluronics F127溶于无水二氯甲烷，加入一定量的4-二甲氨基吡啶(英文简称：DMAP)和对醛基苯甲酸，并加入一定量的二环己基碳二亚胺(英文简称：DCC)；
待反应完成后，用乙醚沉淀，离心收集沉淀并用去离子水透析处理，最后冷冻干燥，获得PF127-DF。
进一步优选地，上述基于海洋源明胶的可注射水凝胶止血剂中，泊洛沙姆类化合物的总含量不超过20％，如只含二丙烯酸酯改性的泊洛沙姆时，二丙烯酸酯改性的泊洛沙姆的含量不超过20％；同时包含二丙烯酸酯改性的泊洛沙姆和二苯甲醛甲酸酯改性的泊洛沙姆时，二丙烯酸酯改性的泊洛沙姆和二苯甲醛 甲酸酯改性的泊洛沙姆的总含量不超过20％。如果泊洛沙姆类化合物的总含量超过20％，基于海洋源明胶的可注射水凝胶止血剂在室温下会丧失液体流动性，变成膏状物质，无法实现可注射的使用方式。泊洛沙姆类化合物的总含量超过20％后，只能在10℃条件下保持液体流动性，给使用造成繁琐的负面影响。
在本发明中，所使用的光引发剂应当能够使所述基于海洋源明胶的可注射水凝胶止血剂在(2～20)秒紫外照射下即发生固化，如果使用的光引发剂发挥作用的时间过长，并且固化时间超过20s，则难以发挥止血效果。
进一步优选地，所述光引发剂为苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐(英文简称为：LAP)，该引发剂可以使得本发明的基于海洋源明胶的可注射水凝胶止血剂在2s左右发生固化，从而可以快速有效的止血。
本发明所使用的溶媒，只要能够将化学改性的海洋源明胶、光交联凝胶因子、组织粘附因子以及光引发剂进行溶解，并且在光照前不发生反应，即可，并且其使用量没有特别的限定，可以根据实际止血需要的浓度进行配制。
优选地，所述溶媒可以是去离子水、磷酸盐缓冲液(英文简称PBS缓冲液)、生理盐水中的任一种。
本发明的基于海洋源明胶的可注射水凝胶止血剂可以采用如下的方法制备：
将化学改性的海洋源明胶、光交联凝胶因子、组织粘附因子按照目标比例溶于溶媒中，使得各组分完全溶解，形得到溶液，随后加入光引发剂，混匀，避紫外光照保存，待用。
由于本发明基于海洋源明胶的可注射水凝胶止血剂，使用的海洋源明胶经过化学改性，具有更好的生物活性，能够更好的促进伤口愈合；且本发明的基 于海洋源明胶的可注射水凝胶止血剂具备极低的溶胀形变，能够长时间保持足够的力学性能和组织粘附性，持久保持创伤封闭效果，并且不会压迫周围组织，同时由于其具有可降解特性，会随着伤口的愈合逐渐降解，降解物对生物体无毒害作用，具有更好的生物安全性。因此，可以广泛应用于人体或动物体组织、脏器意外创伤或手术伤口止血中。
本发明基于海洋源明胶的可注射水凝胶止血剂在止血过程中，适用的脏器为肝脏、脾脏、肾脏、肠、胃、肺中的任一种。
具体在使用是，可以按照如下方式进行操作：
将所述可注射水凝胶止血剂注射于待止血部位，并使用紫外光照射，使所述可注射水凝胶止血剂发生固化。
紫外光照射时，紫外光的波长不超过405nm，波长越长，固化的时间越长，越不利于快速止血，而如果紫外光波长越短，则越容易对周围组织产生伤害。比较优选的紫外光波长是300nm～380nm，如可以用365nm的紫外光照射。
为了更好的说明本发明的技术方案，下面结合若干具体实施例进行说明。
实施例1
一种基于海洋源明胶的可注射水凝胶止血剂的制备方法，包括以下步骤：
(1).化学改性海洋源明胶的合成：
将5.0g海洋鱼皮明胶溶于200mL去离子水中，60℃加热溶解；
降温至50℃，逐滴滴加1.0mL甲基丙烯酸酐，并用2mol/L的氢氧化钠溶液将pH值调节至约8.6；搅拌反应2h；
反应完成后将反应液加入1000mL无水乙醇中沉淀，离心收集沉淀并用去离子水溶解，并将溶液在去离子水中透析3天，冷冻干燥，得到甲基丙烯酸酐改性的海洋源鱼皮明胶，收集待用。
(2).PF127-DA的合成：
将12.6g Pluronics F127溶于100mL无水二氯甲烷中，加入1.2mL三乙胺，通氮气30min后，冰水浴降温至0℃，得到Pluronics F127溶液；
将650μL丙烯酰氯加入10mL无水二氯甲烷中，并逐滴滴加到Pluronics F127溶液中，氮气保护下至反应完成；
采用冰乙醚进行沉淀，离心收集沉淀，真空干燥得到粗产物；
最后，粗产物用去离子水溶解，4℃避光去离子水中透析3天，冷冻干燥，收集待用。
(3).PF127-DF的合成：
将12.6g Pluronics F127溶于100mL无水二氯甲烷中，加入900mg对醛基苯甲酸和500mg 4-二甲氨基吡啶，加入1.24g二环己基碳二亚胺，室温下搅拌反应至完全；
对反应产物进行滤，除去沉淀，将滤液滴加到500mL冰乙醚中沉淀，离心收集沉淀，真空干燥得到粗产物；
采用二氯甲烷对粗产物进行二次溶解，冰乙醚中沉淀，离心收集沉淀，真空干燥，重复2次；并采用去离子水溶解产物，冷冻干燥，收集待用。
(4).分别对步骤(1)～(3)得到的产物进行核磁共振(NMR-400MHz)图谱表征，结果如图1～3所示。
图1为化学改性的海洋源鱼皮明胶的核磁共振图谱。
从图1可以看到化学位移值在5.5附近的两个特征峰(星号标记)，对应图中化合物结构式中标识的星号，说明化学改性成功。
图2为PF127-DA的核磁共振图谱。
从图2可以看到化学位移值在1.0附近的特征峰(数字1标记)和6.0附近 的三个特征峰(数字2标记)，分别对应图2中化合物结构式中标识的数字1和数字2，说明PF127-DA材料制备成功。
图3为PF127-DF的核磁共振图谱。
从图3可以看到化学位移值在1.0附近的特征峰(数字1标记)，8.0附近的三个特征峰(数字2标记)，以及10.0附近的两个特征峰(数字3标记)，分别对应图3中化合物结构式中标识的数字1，数字2以及数字3，说明PF127-DF材料制备成功。
(5).基于海洋源明胶的可注射水凝胶止血剂的制备：
将10mg步骤(1)得到的甲基丙烯酸酐改性的海洋源鱼皮明胶，100mg步骤(2)得到的PF127-DA，50mg步骤(3)得到的产物PF127-DF和2.5mg苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入740μL PBS缓冲液(pH＝7.4)中，旋涡震荡至充分溶解，避紫外光保存。
实施例2
一种基于海洋源明胶的可注射水凝胶止血剂的制备方法，包括以下步骤：
(1).化学改性海洋源明胶、PF127-DA及PF127-DF的合成与实施例1的相同，所不同的是化学改性海洋源明胶中，海洋源明胶来源于海洋源鱼鳞明胶。
(2).基于海洋源明胶的可注射水凝胶止血剂的制备：
将100mg甲基丙烯酸酐改性的海洋源鱼磷明胶，100mg PF127-DA，50mg PF127-DF和2.5mg苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入750μL PBS缓冲液(pH＝7.4)中，旋涡震荡至充分溶解，避紫外光保存。
实施例3
一种基于海洋源明胶的可注射水凝胶止血剂的制备方法，包括以下步骤：
(1).化学改性海洋源明胶、PF127-DA及PF127-DF的合成与实施例1的相 同。
(2).基于海洋源明胶的可注射水凝胶止血剂的制备：
将100mg甲基丙烯酸酐改性的海洋源鱼皮明胶，100mg PF127-DA，100mg PF127-DF和5mg苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入695μL生理盐水中，旋涡震荡至充分溶解，避紫外光保存。
实施例4
一种基于海洋源明胶的可注射水凝胶止血剂的制备方法，包括以下步骤：
(1).化学改性海洋源明胶、PF127-DA的合成与实施例1的相同。
(2).基于海洋源明胶的可注射水凝胶止血剂的制备：
将10mg甲基丙烯酸酐改性的海洋源鱼皮明胶，150mg PF127-DA，15mg丙烯酸-N-琥珀酰亚胺酯和5mg苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入820μL去离子水中，旋涡震荡至充分溶解，避紫外光保存。
实施例5
一种基于海洋源明胶的可注射水凝胶止血剂的制备方法，包括以下步骤：
(1).化学改性海洋源明胶、PF127-DA与实施例1的相同。
(2).基于海洋源明胶的可注射水凝胶止血剂的制备：
将50mg甲基丙烯酸酐改性的海洋源鱼皮明胶，150mg PF127-DA，10mg丙烯酸-N-琥珀酰亚胺酯和5mg苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入785μL生理盐水中，旋涡震荡至充分溶解，避紫外光保存。
实施例6
一种基于海洋源明胶的可注射水凝胶止血剂的制备方法，包括以下步骤：
(1).化学改性海洋源明胶、PF127-DA的合成与实施例1的相同。
(2).基于海洋源明胶的可注射水凝胶止血剂的制备：
将50mg甲基丙烯酸酐改性的海洋源鱼皮明胶、150mg PF127-DA、1mg 4-乙烯基苯甲醛和5mg苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入794μL生理盐水中，旋涡震荡至充分溶解，避紫外光保存。
实施例7
一种基于海洋源明胶的可注射水凝胶止血剂的制备方法，包括以下步骤：
(1).化学改性海洋源明胶、PF127-DA的合成与实施例1的相同。
(2).基于海洋源明胶的可注射水凝胶止血剂的制备：
将50mg甲基丙烯酸酐改性的海洋源鱼皮明胶、150mg PF127-DA、5mg 3-甲基丙烯酰胺基多巴胺和5mg苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入790μL去离子水中，旋涡震荡至充分溶解，避紫外光保存。
对比例1
一种水凝胶，按照重量百分比为100％计，其化学组成为：
性能测试：
为验证实施例1～7得到的基于海洋源明胶的可注射水凝胶止血剂以及对比例1的水凝胶的性能，下面分别对其进行溶胀性能测试、组织粘附性能测试和动物止血实验。
1.溶胀性能测试
(1).分别取部分量的实施例1、2及对比例1产品进行365nm紫外光照射 20秒钟，固化得到水凝胶，结果如图4、5、6所示。
从图4～6可知，获得的水凝胶均呈无色高透明弹性固体状，无色高透明确保止血过程中可以方便观察出血点的实时情况，有利于及时变更治疗方案。
(2).将部分量的实施例1、对比例1的产品分别使用相同的条件的紫外光照固化，并浸泡在PBS缓冲液中，保持37℃，观察体积和测量质量变化情况。可以看到，磷酸盐缓冲液浸泡12小时后体积不再发生变化，达到溶胀平衡。体积和质量的最终变化情况如图7、8所示。
从图7、8可知，实施例1的基于海洋源明胶的可注射水凝胶止血剂的体积增长为初始的147％，质量增长为初始的161％。而对比例1的水凝胶，体积增长为初始的300％，质量增长为初始的295％。
将实施例2的部分产品浸泡在PBS缓冲液中，保持37℃。每12小时进行凝胶直径和湿重的测量，并与初始的水凝胶进行对比，结果如图9所示。从图9可知，实施例2的产品在12小时内就达到了溶胀平衡。
综上可见，本发明的基于海洋源明胶的可注射水凝胶止血剂具有极低的溶胀形变。
2.组织粘附性
利用常用的水凝胶压力爆破测试设备，对实施例1～7及对比例1的产品进行组织粘附性。
具体操作如下，在圆形猪皮表面制造8个直径均为2mm的孔状伤口，编号1#～8#，然后按照实施例1～7及对比例1的顺序在1#～8#伤口表面滴加对应的产品，用365nm紫外照射20秒固化。随后，将猪皮浸泡在磷酸盐缓冲液中12小时后取出。最后推动注射观察水凝胶破裂时气压计的读数，即水凝胶的粘附强度，结果见表1。
表1组织粘附性能测试
| 例别 | 猪皮伤口编号 | 气压计读数/mmHg |
| 实施例1 | 1# | 261 |
| 实施例2 | 2# | 262 |
| 实施例3 | 3# | 273 |
| 实施例4 | 4# | 326 |
| 实施例5 | 5# | 291 |
| 实施例6 | 6# | 276 |
| 实施例7 | 7# | 277 |
| 对比例1 | 8# | 101 |
由表1可知，实施例1～7的爆破强度可以达到270mm Hg及以上，远强于人体血压的120mm Hg，适用于紧急出血的快速止血。
3.止血实验
以两只C57小鼠为模型动物(雄性、8周龄、体重25g)，分别在小鼠肝脏部位制造一个长度1cm的缺损，模拟紧急出血，在出血伤口下方垫一张洁净干燥的滤纸便于观察。
出血5秒钟后，利用实施例1的基于海洋源明胶的可注射水凝胶止血剂进行止血，同时以不处理的伤口作为空白对照。对比观察伤口的出血情况，结果见附图10。
从图10可知，实施例1的基于海洋源明胶的可注射水凝胶止血剂滴加到出血部位并紫外照射20秒，就可以有效止血，具备针对急性出血快速止血的功能。
综上可见，本发明的基于海洋源明胶的可注射水凝胶止血剂具有较强的组织粘附性和力学性能、固化溶胀形变极小、快速止血的效果。同时，由于使用 的海洋源明胶、组织粘附因子本身可降解的特性，原料及降解产物对生物体无刺激，具有生物安全性。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (10)
- 基于海洋源明胶的可注射水凝胶止血剂，其特征在于，按照各组分重量百分含量为100％计，所述可注射水凝胶止血剂包含以下组分；
- 如权利要求1所述的基于海洋源明胶的可注射水凝胶止血剂，其特征在于，所述化学改性的海洋源明胶为甲基丙烯酸酐改性的海洋源明胶；所述光交联凝胶因子为二丙烯酸酯改性的泊洛沙姆；所述组织粘附因子选自二苯甲醛甲酸酯改性的泊洛沙姆、丙烯酸-N-琥珀酰亚胺酯、4-乙烯基苯甲醛、3-甲基丙烯酰胺基多巴胺、或者其他具备与氨基反应能力并且可以通过光固化反应整合到水凝胶网络的化合物中的至少一种。
- 如权利要求2所述的基于海洋源明胶的可注射水凝胶止血剂，其特征在于，所述甲基丙烯酸酐改性的海洋源明胶为甲基丙烯酸酐改性的海洋源鱼皮明胶或者鱼鳞明胶。
- 如权利要求1或2所述的基于海洋源明胶的可注射水凝胶止血剂，其特征在于，当所述光交联凝胶因子为二丙烯酸酯改性的泊洛沙姆且所述组织粘附因子为二苯甲醛甲酸酯改性的泊洛沙姆时，两者的总含量不超过20％。
- 如权利要求1所述的基于海洋源明胶的可注射水凝胶止血剂，其特征在于，所述光引发剂为能使所述可注射水凝胶止血剂在(2～20)秒紫外照射下即发生固化的光引发剂。
- 如权利要求1或5所述的基于海洋源明胶的可注射水凝胶止血剂，其特征在于，所述光引发剂为苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐。
- 如权利要求1所述的基于海洋源明胶的可注射水凝胶止血剂，其特征在于，所述溶媒为去离子水、磷酸盐缓冲液、生理盐水中的任一种。
- 如权利要求1～7任一项所述的基于海洋源明胶的可注射水凝胶止血剂在人体或动物体组织、脏器意外创伤或手术伤口止血中的应用。
- 如权利要求8所述的应用，其特征在于，所述脏器为肝脏、脾脏、肾脏、肠、胃、肺中的任一种。
- 如权利要求1～7任一项所述的基于海洋源明胶的可注射水凝胶止血剂的应用方法，其特征在于，包括以下步骤：将所述可注射水凝胶止血剂注射于待止血部位，并使用紫外光照射，使所述可注射水凝胶止血剂发生固化。
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