技术领域Technical field
本发明属于载药心肌补片技术领域，涉及一种免缝合的多层载药心肌补片及其制备方法。The invention belongs to the technical field of drug-loaded myocardial patches and relates to a suture-free multi-layer drug-loaded myocardial patch and a preparation method thereof.
背景技术Background technique
心肌梗死是一种我国常见的且高死亡率的心血管疾病，心梗发生后，大量的心肌细胞由于缺血缺氧而死亡，心肌组织将历经炎症期、增殖期和重塑期，坏死部位会通过形成纤维化瘢痕组织和进行左心室重塑来治愈梗死心肌，然而，梗死心肌所处的微环境仍然恶劣，如缺乏氧气和营养供应、炎症反应等，这对于梗死心肌及周围正常心肌的发育均是不利的，将会导致进一步的心肌纤维化和正常心肌细胞肥大性增长以代偿心功能，严重时将会导致心力衰竭。因此，促进坏死心肌处血管生成、减少炎症反应发生及抑制心肌纤维化是目前治疗心力衰竭的有效办法。Myocardial infarction is a common cardiovascular disease with high mortality in my country. After myocardial infarction, a large number of myocardial cells die due to ischemia and hypoxia. Myocardial tissue will go through the inflammation phase, proliferation phase and remodeling phase, and the necrotic area will The infarcted myocardium will be healed through the formation of fibrotic scar tissue and left ventricular remodeling. However, the microenvironment of the infarcted myocardium is still harsh, such as lack of oxygen and nutrient supply, inflammatory response, etc., which is detrimental to the development of the infarcted myocardium and surrounding normal myocardium. The development is unfavorable and will lead to further myocardial fibrosis and hypertrophic growth of normal myocardial cells to compensate for cardiac function. In severe cases, it will lead to heart failure. Therefore, promoting angiogenesis in necrotic myocardium, reducing inflammatory reactions and inhibiting myocardial fibrosis are currently effective methods for treating heart failure.
心肌补片修复策略是一种很有前途的替代生物治疗方法，其主要作用是可以为受损心肌部位提供力学支持，防止心室破裂，其次心肌补片能够作为运载药物、蛋白质和细胞的载体。The myocardial patch repair strategy is a promising alternative biological treatment method. Its main function is to provide mechanical support for the damaged myocardium and prevent ventricular rupture. Secondly, the myocardial patch can serve as a carrier for delivering drugs, proteins and cells.
姜黄素、环孢菌素A、葛根素、他汀类药物、重组人松弛素-2等药物，血管内皮生长因子、碱性成纤维生长因子、白细胞介素10等抗炎因子，均已被证明在心肌修复过程产生了有益的效果(Biomaterials loaded with growth factors/cytokines and stem cellsfor cardiac tissue regeneration.International Journal of Molecular Sciences,2020,21:5952.)。在心肌补片成型过程中加入一定比例的药物或因子可以实现装载的目的，如在静电纺丝液制备的过程中加入或在高分子前驱体溶液加入后进行均匀混合(CN201811636101.2一种心肌修复水凝胶材料及其制备方法)，再进行固化或者交联得到装载药物的成型心肌补片。然而，上述方法在药物装载及传递方面较为单一，应用于心外膜的心肌补片通常只递送一种药物或生长因子，难以满足心肌修复的复杂要求。水凝胶具有与天然细胞外基质相似的三维网络结构，并且可以通过溶胀、扩散等机制进行对药物的释放，通过对聚合物材料、浓度、交联反应和结构等参数的设计可以控制并持续地释放生长因子和药物。最近，有团队通过对水凝胶的材料及结构设计，制备了一种双层的水凝胶体系可以装载不同的药物，然后附着在心外膜上，延长了原位释放时间，有望减少纤维化和抑制瘢痕大小(A Bi-Layer Hydrogel Cardiac Patch Made of Recombinant FunctionalProteins.Advanced Materials,2022,34:2201411.)。心梗发生后，心肌梗死部位及所处环境十分复杂，心外膜承担着同时桥接内外环境的重要角色，上述心肌补片载药方法及应用方式只是在心外膜表面释放药物以修复受损心肌，忽略了心外膜内和受损心肌所处环境的特点，且药物的半衰期短以及在活体环境下的快速清除均为该领域的挑战。近年来，由微型针头组成的微针阵列贴片已经成为一种很有前景的工具，除了可以以微创的方式穿透组织，还可以高精度和高效率地递送药物。如有研究将血管内皮生长因子(VEGF)和白介素10(IL-10)包裹在微针阵列中，可以促进心脏受损区域的血管重建并抑制炎症反应(Inducedcardiomyocytes-integrated conductive microneedle patch for treatingmyocardial infarction.Chemical Engineering Journal,2021,414:128723.)。对水凝胶及微针进行设计及组合，并进行心外膜定位靶向治疗，有望实现在心肌修复过程中药物和生长因子的精准释放，以满足修复坏死心肌的多重要求。Curcumin, cyclosporine A, puerarin, statins, recombinant human relaxin-2 and other drugs, as well as anti-inflammatory factors such as vascular endothelial growth factor, basic fibroblast growth factor, interleukin 10, etc., have all been proven Produced beneficial effects in the myocardial repair process (Biomaterials loaded with growth factors/cytokines and stem cells for cardiac tissue regeneration. International Journal of Molecular Sciences, 2020, 21: 5952.). Adding a certain proportion of drugs or factors during the myocardial patch forming process can achieve the purpose of loading, such as adding during the preparation of the electrospinning solution or evenly mixing after adding the polymer precursor solution (CN201811636101.2 A myocardial patch Repair hydrogel material and preparation method thereof), and then solidify or cross-link to obtain a drug-loaded shaped myocardial patch. However, the above methods are relatively simple in terms of drug loading and delivery. Myocardial patches applied to the epicardium usually only deliver one kind of drug or growth factor, which is difficult to meet the complex requirements of myocardial repair. Hydrogel has a three-dimensional network structure similar to the natural extracellular matrix, and can release drugs through swelling, diffusion and other mechanisms. It can be controlled and sustained through the design of parameters such as polymer materials, concentration, cross-linking reaction and structure. to release growth factors and drugs. Recently, through designing the material and structure of hydrogel, a team has prepared a double-layered hydrogel system that can be loaded with different drugs and then attached to the epicardium, which prolongs the in-situ release time and is expected to reduce fibrosis. and inhibit scar size (A Bi-Layer Hydrogel Cardiac Patch Made of Recombinant FunctionalProteins. Advanced Materials, 2022,34:2201411.). After a myocardial infarction, the site of myocardial infarction and its environment are very complex. The epicardium plays an important role in bridging the internal and external environments at the same time. The above-mentioned drug-loading methods and application methods of myocardial patches only release drugs on the epicardial surface to repair damaged myocardium. , ignoring the characteristics of the environment within the epicardium and damaged myocardium, and the short half-life of the drug and its rapid clearance in the living environment are challenges in this field. In recent years, microneedle array patches composed of microneedles have emerged as a promising tool that, in addition to penetrating tissue in a minimally invasive manner, can also deliver drugs with high precision and efficiency. For example, studies have shown that vascular endothelial growth factor (VEGF) and interleukin 10 (IL-10) are wrapped in microneedle arrays, which can promote vascular reconstruction in damaged areas of the heart and inhibit inflammatory responses (Inducedcardiomyocytes-integrated conductive microneedle patch for treating myocardial infarction. Chemical Engineering Journal, 2021,414:128723.). The design and combination of hydrogels and microneedles, and epicardial targeted therapy, are expected to achieve precise release of drugs and growth factors during myocardial repair to meet the multiple requirements for repairing necrotic myocardium.
当将心肌补片固定到心外膜上发挥作用时，多数研究选择的都是运用缝合线将补片缝合到心外膜上。这种固定方式存在一些问题，第一，缝合针穿过心脏的时候势必会导致刺穿点的应力集中和该部位的组织损伤，进而形成疤痕组织。第二，心外膜经过缝合后心脏再次跳动后会产生一定的组织损伤和出血或感染，而这将均属于对心肌造成二次伤害。第三，将成型的补片缝合到动态的心脏上难度较大，花费时间长而且需要开胸缝合，不适用于微创手术。When fixing a myocardial patch to the epicardium is used, most studies choose to use sutures to sew the patch to the epicardium. There are some problems with this fixation method. First, when the suture needle passes through the heart, it will inevitably cause stress concentration at the puncture point and tissue damage at the site, thereby forming scar tissue. Second, after the epicardium is sutured and the heart beats again, there will be certain tissue damage, bleeding or infection, which will all cause secondary damage to the myocardium. Third, it is difficult to suture the formed patch to the dynamic heart, takes a long time and requires thoracotomy and suturing, which is not suitable for minimally invasive surgery.
针对于以上心外膜补片应用方式的弊端，有研究着眼于心肌补片的免缝合性能做了相应的材料和结构设计。如有研究设计了一系列兼具粘合剂和耗散矩阵两部分的粘合性水凝胶心肌补片，前者通过静电相互作用、共价键和物理相互渗透附着在心肌上，而后者通过滞后放大能量耗散，可以与动态心肌兼容(Tough adhesives for diverse wetsurfaces.Science,2017,357:378-381.)。进一步地，有学者报道了一种导电的粘合心肌补片，用于实现生物电子器件与各种湿动态组织之间快速、稳定、共形和导电的集成，并在猪的体内模型成功验证牢固的粘合性(Double-layered adhesive microneedle bandagebased on biofunctionalized mussel protein for cardiac tissueregeneration.Biomaterials,2021,278:121171.)。In response to the above drawbacks of the application of epicardial patches, some studies have focused on the suture-free performance of myocardial patches and made corresponding material and structural designs. For example, some research has designed a series of adhesive hydrogel myocardial patches that have both adhesive and dissipative matrix parts. The former is attached to the myocardium through electrostatic interactions, covalent bonds, and physical interpenetration, while the latter is attached to the myocardium through Hysteresis amplifies energy dissipation and is compatible with dynamic myocardium (Tough adhesives for diverse wetsurfaces. Science, 2017, 357:378-381.). Furthermore, some scholars reported a conductive adhesive myocardial patch that can be used to achieve fast, stable, conformal and conductive integration between bioelectronic devices and various wet dynamic tissues, and was successfully verified in a pig in vivo model. Strong adhesiveness (Double-layered adhesive microneedle bandagebased on biofunctionalized mussel protein for cardiac tissue regeneration. Biomaterials, 2021, 278:121171.).
针对于微针在心肌组织修复方面的研究多数集中在药物和细胞递送方面，而利用对微针的原料、结构设计实现心肌补片在心外膜上的锚定研究较少。主要是由于用于心肌修复的补片仍然存在着较多要求，需要将微针与心肌补片构建成有效的有机结合体以兼顾心肌补片的基本要求及免缝合性能。目前，有研究设计了一种交联的双相结构化微针贴片，包括可吸水并产生粘性的外部和基于丝素蛋白的刚性内部，当刺入心外膜时，微针的外层会吸水并产生膨胀，从而达到机械互锁的目的(记载于文献Double-layered adhesivemicroneedle bandage based on biofunctionalized mussel protein for cardiactissue regeneration)。专利CN202120534654.8提供了一种带微针的心肌补片，用于解决心肌补片固定到心脏表面时带来的技术问题，可以使心肌补片能够快速有效并且无微创伤地固定在目标组织处。然而，上述研究均采用了复杂的制备方法，如需提取多重原料并进行长时间的制备或需精确设计3D打印参数以获得微针表面的倒钩结构，工艺复杂且不易于规模化生产。Most research on microneedle repair of myocardial tissue focuses on drug and cell delivery, while there are fewer studies on the use of microneedle raw materials and structural design to anchor myocardial patches on the epicardium. Mainly because there are still many requirements for patches used for myocardial repair, it is necessary to construct an effective organic combination of microneedles and myocardial patches to take into account the basic requirements and suture-free performance of myocardial patches. Currently, research has designed a cross-linked biphasic structured microneedle patch, including an outer layer that absorbs water and creates adhesiveness and a rigid inner layer based on silk fibroin. When the epicardium is penetrated, the outer layer of the microneedle It will absorb water and swell to achieve mechanical interlocking (documented in the document Double-layered adhesive microneedle bandage based on biofunctionalized mussel protein for cardiac tissue regeneration). Patent CN202120534654.8 provides a myocardial patch with microneedle, which is used to solve the technical problems caused by fixing the myocardial patch to the heart surface, so that the myocardial patch can be fixed on the target quickly, effectively and without micro-trauma. Organization Department. However, the above-mentioned studies all used complex preparation methods. For example, multiple raw materials need to be extracted and prepared for a long time, or 3D printing parameters need to be precisely designed to obtain the barb structure on the surface of the microneedle. The process is complicated and not easy to be produced on a large scale.
因此，研发出简单可重复的生物制造工艺并制备出适用于心肌组织的免缝合心肌补片，并能够针对心肌梗死后微环境的特点按需精准定位释放药物以协助修复受损心肌，能够产生重要的意义。Therefore, it is possible to develop a simple and reproducible biomanufacturing process and prepare a suture-free myocardial patch suitable for myocardial tissue, and to accurately position and release drugs on demand according to the characteristics of the microenvironment after myocardial infarction to assist in repairing damaged myocardium. Significance.
发明内容Contents of the invention
本发明的目的是解决现有技术中心肌补片载药单一/无法精准定位释药以及需要复杂开胸缝合的问题，提供一种免缝合的多层载药心肌补片及其制备方法。The purpose of the present invention is to solve the problems in the prior art that myocardial patches carry only one drug, cannot accurately position the drug for release, and require complex thoracotomy and suturing, and provide a suture-free multi-layer drug-loaded myocardial patch and its preparation method.
为达到上述目的，本发明采用的技术方案如下：In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:
一种免缝合的多层载药心肌补片，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；A suture-free multi-layer drug-loaded myocardial patch, including a myocardial patch basal layer, an intermediate layer and a microneedle layer; the microneedle layer is a microneedle array, and the microneedle layer is evenly distributed on the same side of the intermediate layer and perpendicular to On the plane where the middle layer is located, the basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
心肌补片基底层装载具有抑制纤维化及保护心脏功能的他汀类药物，中间层装载具有抗炎和抑制左心室重构作用的姜黄素，微针层装载具有促进血管再生作用的血管内皮生长因子(VEGF)，上述三层分别作用于心梗部位所处环境、心外膜表面及心外膜内部；The basal layer of the myocardial patch is loaded with statins, which can inhibit fibrosis and protect cardiac function, the middle layer is loaded with curcumin, which has anti-inflammatory and inhibitory effects on left ventricular remodeling, and the microneedle layer is loaded with vascular endothelial growth factors, which can promote vascular regeneration. (VEGF), the above three layers respectively act on the environment of the myocardial infarction site, the epicardial surface and the inside of the epicardium;
心肌补片基底层和中间层材料为高分子凝胶，微针层材料为高分子凝胶或可降解高分子材料，在体内湿润环境的介导作用下，中间层和心肌补片基底层均溶胀释药，微针层根据材料特点缓慢释药(即当微针材料为可降解高分子材料时，是按照材料的缓慢降解释药的；当微针材料为高分子凝胶时，是按照溶胀机理释药的)；心肌补片基底层的溶胀速率＞中间层的溶胀速率，且当微针层的材料为高分子凝胶时，心肌补片基底层的溶胀速率＞中间层的溶胀速率＞微针层的溶胀速率；将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率＞中间层的溶胀率＞60％，以保证中间层和基底层在溶胀时对微针具有较大牵引力使其倾斜的同时短期内实现免缝合效果；The material of the basal layer and the middle layer of the myocardial patch is polymer gel, and the material of the microneedle layer is polymer gel or degradable polymer material. Under the mediation of the humid environment in the body, the middle layer and the basal layer of the myocardial patch are both Swelling releases the drug, and the microneedle layer slowly releases the drug according to the material characteristics (that is, when the microneedle material is a degradable polymer material, the drug is released according to the slow degradation of the material; when the microneedle material is a polymer gel, the drug is released according to the (swelling mechanism for drug release); the swelling rate of the basal layer of the myocardial patch > the swelling rate of the middle layer, and when the material of the microneedle layer is a polymer gel, the swelling rate of the basal layer of the myocardial patch > the swelling rate of the middle layer > Swelling rate of the microneedle layer; insert the multi-layered drug-loaded myocardial patch into the isolated pig heart and soak it in PBS buffer. After 20 minutes, the swelling rate of the basal layer of the myocardial patch > the swelling rate of the middle layer > 60% , to ensure that the middle layer and the basal layer have greater traction on the microneedles when they swell, causing them to tilt while achieving a suture-free effect in the short term;
心肌补片基底层与中间层的结合强度以及中间层与微针层的结合强度均大于20kPa，结合强度过小会导致微针变形失败。The bonding strength between the basal layer and the middle layer of the myocardial patch and the bonding strength between the middle layer and the microneedle layer are both greater than 20kPa. If the bonding strength is too small, the microneedle will fail to deform.
作为优选的技术方案：As the preferred technical solution:
如上所述的一种免缝合的多层载药心肌补片，将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟后测量微针轴向与中间层所在平面的夹角，微针轴向与中间层所在平面的夹角为30～70°；A suture-free multi-layered drug-loaded myocardial patch as described above. The multi-layered drug-loaded myocardial patch is inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the axial direction of the microneedle and the middle layer are measured. The angle between the plane where the microneedle is located and the plane where the middle layer is located is 30 to 70°;
将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为10～50cN，以最大拔脱力评价补片与组织的结合强度，从而表征免缝合效果，最大拔脱力的测试方法为：将心肌补片粘在电子万能材料试验机的上方传感器探头，猪心肌组织样本固定在下方夹具，多层心肌补片以0.05mm/s的速度向上驱动，直至微针完全脱离猪心肌组织。The myocardial patch was fixed on a porcine myocardial tissue sample with a thickness of 10 mm and then soaked in PBS buffer. After 20 minutes, the maximum pull-out force of the myocardial patch was measured to be 10-50 cN. The maximum pull-out force was used to evaluate the combination of the patch and the tissue. strength, thereby characterizing the suture-free effect. The test method for the maximum pull-out force is as follows: stick the myocardial patch to the upper sensor probe of the electronic universal material testing machine, fix the pig myocardial tissue sample to the lower clamp, and use the multi-layer myocardial patch with 0.05mm/ The microneedle is driven upward at a speed of s until the microneedle is completely separated from the pig myocardial tissue.
如上所述的一种免缝合的多层载药心肌补片，心肌补片基底层材料与中间层材料分别选自于甲基丙烯酸化透明质酸、甲基丙烯酸酰化明胶和聚乙二醇二丙烯酸酯中的一种，优选为甲基丙烯酸酰化明胶或聚乙二醇二丙烯酸酯；微针层材料为丝素蛋白、聚乙二醇二丙烯酸酯、甲基丙烯酸化透明质酸、聚乙烯醇或甲基丙烯酸酰化明胶(丝素蛋白为可降解高分子材料，其余材料为高分子凝胶)，优选为聚乙烯醇或聚乙二醇二丙烯酸酯。A suture-free multi-layered drug-loaded myocardial patch as described above, the base layer material and the middle layer material of the myocardial patch are respectively selected from methacrylated hyaluronic acid, methacrylated acylated gelatin and polyethylene glycol. One of the diacrylates, preferably methacrylated gelatin or polyethylene glycol diacrylate; the microneedle layer material is silk fibroin, polyethylene glycol diacrylate, methacrylated hyaluronic acid, Polyvinyl alcohol or methacrylic acid acylated gelatin (silk fibroin is a degradable polymer material, and the remaining materials are polymer gels), preferably polyvinyl alcohol or polyethylene glycol diacrylate.
如上所述的一种免缝合的多层载药心肌补片，微针形状为子弹头形、圆锥形或四棱锥形；微针分布密度为0.5～2针/mm；A suture-free multi-layered drug-loaded myocardial patch as described above, the microneedle shape is bullet-shaped, conical or quadrangular pyramid-shaped; the microneedle distribution density is 0.5 to 2 needles/mm;
微针形状为子弹头形时，底部半径为150～400微米，圆柱高度为600～1000微米，圆锥高度为300～500微米；When the microneedle shape is bullet-shaped, the bottom radius is 150-400 microns, the cylinder height is 600-1000 microns, and the cone height is 300-500 microns;
微针形状为圆锥形时，底部半径为150～400微米，微针长度为1000～1500微米；When the microneedle shape is conical, the bottom radius is 150 to 400 microns, and the microneedle length is 1000 to 1500 microns;
微针形状为四棱锥时，底部边长为150～400微米，微针长度为1000～1500微米。When the shape of the microneedle is a square pyramid, the length of the bottom side is 150 to 400 microns, and the length of the microneedle is 1000 to 1500 microns.
如上所述的一种免缝合的多层载药心肌补片，心肌补片基底层厚度为500～1200微米；中间层的厚度为400～800微米。A suture-free multi-layered drug-loaded myocardial patch as described above, the thickness of the basal layer of the myocardial patch is 500-1200 microns; the thickness of the middle layer is 400-800 microns.
如上所述的一种免缝合的多层载药心肌补片，多层载药心肌补片规格为0.8cm×0.8cm～1.5cm×1.5cm。A suture-free multi-layer drug-loaded myocardial patch as described above, the specifications of the multi-layer drug-loaded myocardial patch are 0.8cm×0.8cm～1.5cm×1.5cm.
本发明还提供如上所述的一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：The present invention also provides a method for preparing a suture-free multi-layer drug-loaded myocardial patch as described above, which includes the following steps:
(1)微针层的制备：将血管内皮生长因子(VEGF)均匀分散于微针层材料的溶液中得到铸模液A，将铸模液A浇筑到微针模具的微针部位(微针模具为具有微针孔和基底凹槽的PDMS模具)，干燥/紫外光固化得到微针层A，暂不脱模；(1) Preparation of the microneedle layer: Evenly disperse vascular endothelial growth factor (VEGF) in the solution of the microneedle layer material to obtain the casting liquid A, and pour the casting liquid A into the microneedle part of the microneedle mold (the microneedle mold is PDMS mold with micro-pinholes and base grooves), dry/UV-cured to obtain micro-needle layer A, do not demould yet;
(2)中间层的制备：将姜黄素均匀分散于中间层材料的溶液中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，紫外光固化得到中间层B，暂不脱模；(2) Preparation of the middle layer: Disperse curcumin evenly in the solution of the middle layer material to obtain the casting liquid B. Pour the casting liquid B into the base groove of the microneedle mold, and cure it with ultraviolet light to obtain the middle layer B. Not yet. demoulding;
(3)心肌补片基底层的制备：将他汀类药物均匀分散于心肌补片基底层材料的溶液中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，紫外光固化得到心肌补片基底层C；(3) Preparation of the myocardial patch basal layer: Evenly disperse statins in the solution of the myocardial patch basal layer material to obtain the casting liquid C, and pour the casting liquid C into the remaining parts of the base groove of the microneedle mold. The basal layer C of the myocardial patch is obtained by UV curing;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如上所述的方法，步骤(1)中微针层材料的溶液为丝素蛋白溶液、聚乙二醇二丙烯酸酯溶液、甲基丙烯酸化透明质酸溶液、甲基丙烯酸酰化明胶溶液或聚乙烯醇溶液；As mentioned above, the solution of the microneedle layer material in step (1) is silk fibroin solution, polyethylene glycol diacrylate solution, methacrylated hyaluronic acid solution, methacrylated acylated gelatin solution or polyethylene glycol diacrylate solution. vinyl alcohol solution;
丝素蛋白溶液的浓度为3％(w/v)～7％(w/v)，溶剂为水；The concentration of silk fibroin solution is 3% (w/v) to 7% (w/v), and the solvent is water;
聚乙二醇二丙烯酸酯溶液的浓度为60％(v/v)～80％(v/v)，溶剂为水；The concentration of the polyethylene glycol diacrylate solution is 60% (v/v) to 80% (v/v), and the solvent is water;
甲基丙烯酸化透明质酸溶液的浓度为5％(w/v)～10％(w/v)，溶剂为水；The concentration of the methacrylated hyaluronic acid solution is 5% (w/v) to 10% (w/v), and the solvent is water;
甲基丙烯酸酰化明胶溶液的浓度为30％(w/w)～50％(w/w)，溶剂为水；The concentration of the methacrylic acid acylated gelatin solution is 30% (w/w) to 50% (w/w), and the solvent is water;
聚乙烯醇溶液的浓度为15％(w/w)～30％(w/w)，溶剂为水；The concentration of the polyvinyl alcohol solution is 15% (w/w) to 30% (w/w), and the solvent is water;
步骤(2)中中间层材料的溶液为甲基丙烯酸化透明质酸溶液、聚乙二醇二丙烯酸酯溶液或甲基丙烯酸酰化明胶溶液；In step (2), the solution of the intermediate layer material is a methacrylated hyaluronic acid solution, a polyethylene glycol diacrylate solution or a methacrylated acylated gelatin solution;
甲基丙烯酸化透明质酸溶液的浓度为4％(w/v)～8％(w/v)，溶剂为水；The concentration of the methacrylated hyaluronic acid solution is 4% (w/v) to 8% (w/v), and the solvent is water;
聚乙二醇二丙烯酸酯溶液的浓度为40％(v/v)～60％(v/v)，溶剂为水；The concentration of the polyethylene glycol diacrylate solution is 40% (v/v) to 60% (v/v), and the solvent is water;
甲基丙烯酸酰化明胶溶液的浓度为20％(w/w)～40％(w/w)，溶剂为水；The concentration of the methacrylic acid acylated gelatin solution is 20% (w/w) to 40% (w/w), and the solvent is water;
步骤(3)中心肌补片基底层材料的溶液为甲基丙烯酸化透明质酸溶液、聚乙二醇二丙烯酸酯溶液或甲基丙烯酸酰化明胶溶液；In step (3), the solution of the base layer material of the central myocardial patch is a methacrylated hyaluronic acid solution, a polyethylene glycol diacrylate solution or a methacrylated acylated gelatin solution;
甲基丙烯酸化透明质酸溶液的浓度为3％(w/v)～7％(w/v)，溶剂为水；The concentration of the methacrylated hyaluronic acid solution is 3% (w/v) to 7% (w/v), and the solvent is water;
聚乙二醇二丙烯酸酯溶液的浓度为30％(v/v)～50％(v/v)，溶剂为水；The concentration of the polyethylene glycol diacrylate solution is 30% (v/v) to 50% (v/v), and the solvent is water;
甲基丙烯酸酰化明胶溶液的浓度为15％(w/w)～30％(w/w)，溶剂为水。The concentration of the methacrylic acid acylated gelatin solution is 15% (w/w) to 30% (w/w), and the solvent is water.
心肌补片基底层与中间层的材质和制备时材料的浓度至少有一处是不同的。The materials of the base layer and the middle layer of the myocardial patch and the concentration of the materials during preparation are different in at least one aspect.
如上所述的方法，步骤(1)铸模液A中血管内皮生长因子的浓度为0.5～3μg/mL；As described above, in step (1) the concentration of vascular endothelial growth factor in the casting liquid A is 0.5 to 3 μg/mL;
步骤(2)铸模液B中姜黄素的浓度为1～3mg/mL；In step (2), the concentration of curcumin in the casting liquid B is 1 to 3 mg/mL;
步骤(3)中他汀类药物为普伐他汀或阿托伐他汀，铸模液C中他汀类药物浓度为5～10mg/kg。In step (3), the statin drug is pravastatin or atorvastatin, and the concentration of the statin drug in the casting liquid C is 5 to 10 mg/kg.
如上所述的方法，干燥固化是指在温度为25～60℃的条件下干燥12～24h；As mentioned above, drying and curing refers to drying at a temperature of 25 to 60°C for 12 to 24 hours;
紫外光固化的时间为10s～24h，光引发剂为2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮，光引发剂用量为铸模液A、铸模液B或铸模液C的0.05～1％(v/v)。The UV curing time is 10s to 24h, the photoinitiator is 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, and the amount of photoinitiator is casting liquid A, casting liquid B or casting mold 0.05~1% (v/v) of liquid C.
铸模液中的高分子为丝素蛋白时，成型条件为干燥固化，干燥温度为25℃～60℃，干燥时间为12h～24h；When the polymer in the molding liquid is silk fibroin, the molding conditions are drying and solidification, the drying temperature is 25℃~60℃, and the drying time is 12h~24h;
铸模液中的高分子为聚乙烯醇时，成型条件为干燥固化，干燥温度为25℃～40℃，干燥时间为12h～24h；When the polymer in the molding liquid is polyvinyl alcohol, the molding conditions are drying and solidification, the drying temperature is 25℃~40℃, and the drying time is 12h~24h;
铸模液中的高分子为甲基丙烯酸化透明质酸或甲基丙烯酸酰化明胶时，成型条件为紫外光固化，光引发剂为2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮，光引发剂用量为铸模液的0.05％(v/v)～0.5％(v/v)，光固化时间为20min～6h；When the polymer in the casting liquid is methacrylated hyaluronic acid or methacrylated acylated gelatin, the molding conditions are UV curing and the photoinitiator is 2-hydroxy-4-(2-hydroxyethoxy)-2 - Methylpropiophenone, the photoinitiator dosage is 0.05% (v/v) ~ 0.5% (v/v) of the casting liquid, and the photocuring time is 20min ~ 6h;
铸模液中的高分子为聚乙二醇二丙烯酸酯时，成型条件为紫外光固化，光引发剂为2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮，光引发剂用量为铸模液的0.5％(v/v)～1％(v/v)，光固化时间为10s～40s。When the polymer in the molding liquid is polyethylene glycol diacrylate, the molding conditions are UV curing, the photoinitiator is 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, and the light The dosage of initiator is 0.5% (v/v) to 1% (v/v) of the casting liquid, and the photocuring time is 10s to 40s.
本发明的原理如下：The principle of the present invention is as follows:
针对于心肌补片的免缝合性能，现有技术通常是进行材料设计(如合成粘附性补片直接粘在心外膜上)，或者结构设计(如采用精细加工技术、3D打印、激光切割、铁磁流体控制等复杂的方法)将微针设计成倒钩状、蜜蜂刺状、箭头装、宝塔状等。而本发明提出了一种心肌补片在释药进行时便能达到意想不到的免缝合效果：即当将补片植入心外膜时，在体内湿润环境的介导作用下，中间层和心肌补片基底层均溶胀释药，微针层释药速率最慢，以达到长期的释放血管内皮生长因子以促进血管生成的目的；而中间层释药速率和溶胀速率次之，这种溶胀作用会导致中间层体积各向膨胀，又因为中间层与微针连接，微针又与心肌组织连接，在中间层的膨胀牵引作用力和心肌组织对微针的阻力作用下，会引发微针阵列由垂直结构转变为角度夹持结构，从而达到自固定的效果；同时，最外层(心肌补片基底层)释药速率和溶胀速率最快，体积各向膨胀较中间层快，因此会对微针层和中间层产生一定的压力作用，导致心肌补片在心外膜表面产生类似“自紧”的效果，紧紧贴附在心外膜表面，这种现象会进一步加强免缝合效果。Regarding the suture-free performance of myocardial patches, the existing technology usually involves material design (such as synthetic adhesive patches directly adhered to the epicardium), or structural design (such as the use of fine processing technology, 3D printing, laser cutting, Complex methods such as ferrofluid control) design microneedles into barb shapes, bee sting shapes, arrowhead shapes, pagoda shapes, etc. The present invention proposes a myocardial patch that can achieve an unexpected suture-free effect when drug release is in progress: that is, when the patch is implanted into the epicardium, under the mediation of the moist environment in the body, the middle layer and The basal layer of the myocardial patch all swells and releases the drug, and the microneedle layer releases the drug the slowest to achieve the purpose of long-term release of vascular endothelial growth factors to promote angiogenesis; while the middle layer has the second highest drug release rate and swelling rate. This kind of swelling The effect will cause the volume of the middle layer to expand in all directions, and because the middle layer is connected to the microneedles, and the microneedles are connected to the myocardial tissue, under the expansion traction force of the middle layer and the resistance of the myocardial tissue to the microneedles, the microneedles will be triggered. The array changes from a vertical structure to an angular clamping structure to achieve a self-fixing effect; at the same time, the outermost layer (basal layer of the myocardial patch) has the fastest drug release rate and swelling rate, and its volume expands faster than the middle layer, so it will A certain pressure is exerted on the microneedle layer and the middle layer, causing the myocardial patch to produce a "self-tightening" effect on the epicardial surface and tightly adhere to the epicardial surface. This phenomenon will further enhance the suture-free effect.
有益效果：Beneficial effects:
(1)本发明制备的多层载药心肌补片植入心肌后具有精准定位释药效果，即能够针对心外膜内、心外膜表面和心肌组织所处环境的特点分别进行释放对应的药物或生长因子；微针阵列层释放血管内皮生长因子(VEGF)以促进血管再生，中间层释放姜黄素以抗炎和抑制左心室重构，心肌补片基底层释放他汀类药物以抑制纤维化及保护心脏功能。本发明能够根据心梗后的不同治疗需求按需释放对应的药物，有利于促进坏死心肌处血管生成、减少炎症反应发生及抑制心肌纤维化，有利于梗死心肌修复。(1) The multi-layered drug-loaded myocardial patch prepared by the present invention has a precise positioning drug release effect after being implanted in the myocardium, that is, it can release corresponding drugs according to the characteristics of the environment in the epicardium, the epicardial surface and the myocardial tissue. Drugs or growth factors; the microneedle array layer releases vascular endothelial growth factor (VEGF) to promote vascular regeneration, the middle layer releases curcumin to anti-inflammation and inhibit left ventricular remodeling, and the basal layer of the myocardial patch releases statins to inhibit fibrosis. and protect heart function. The present invention can release corresponding drugs on demand according to different treatment needs after myocardial infarction, which is beneficial to promoting angiogenesis in necrotic myocardium, reducing the occurrence of inflammatory reactions and inhibiting myocardial fibrosis, and is beneficial to the repair of infarcted myocardium.
(2)本发明制备的多层载药心肌补片植入受损心肌组织时，微针阵列垂直于受损心肌组织，当刺入受损组织后，在体内湿润环境介导下，中间层和基底层即刻溶胀释药，宏观表现为发生体积膨胀，从而引发微针阵列由垂直结构转变为角度夹持结构，同时心肌补片基底层的膨胀力会导致对微针层和中间层产生一定的压力作用，导致心肌补片在心外膜表面产生类似“自紧”的效果，紧紧贴附在心外膜表面，实现免缝合效果。该操作可以通过胸腔镜手术进行，无需缝合固定，减小了手术难度和术后并发症，缩短了手术时间。(2) When the multi-layer drug-loaded myocardial patch prepared by the present invention is implanted into the damaged myocardial tissue, the microneedle array is perpendicular to the damaged myocardial tissue. After penetrating into the damaged tissue, under the guidance of the moist environment in the body, the middle layer The basal layer of the myocardial patch immediately swells and releases the drug. The macroscopic manifestation is volume expansion, which causes the microneedle array to change from a vertical structure to an angular clamping structure. At the same time, the expansion force of the basal layer of the myocardial patch will cause a certain impact on the microneedle layer and the middle layer. The pressure effect causes the myocardial patch to produce a "self-tightening" effect on the epicardial surface, tightly adhering to the epicardial surface, achieving a suture-free effect. This operation can be performed through thoracoscopic surgery without suture fixation, which reduces the difficulty of the operation and postoperative complications, and shortens the operation time.
(3)本发明的制备方法简单，可重复性高，适于规模性生产。(3) The preparation method of the present invention is simple, highly reproducible, and suitable for large-scale production.
附图说明Description of the drawings
图1为免缝合多层载药心肌补片的结构及应用示意图；Figure 1 is a schematic diagram of the structure and application of a suture-free multi-layer drug-loaded myocardial patch;
图2为免缝合多层载药心肌补片心肌补片的制备流程图。Figure 2 is a flow chart for the preparation of suture-free multi-layer drug-loaded myocardial patch myocardial patch.
具体实施方式Detailed ways
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.
本发明采用的微针模具为具有微针孔和基底凹槽的PDMS模具。The microneedle mold used in the present invention is a PDMS mold with microneedle holes and base grooves.
本发明采用如下测试方法：The present invention adopts the following test methods:
(1)结合强度：待测层长于另一层10～20mm，长出部分装于剥离试验机上夹具，其余部分固定在90度测试平台上，进行剥离强度实验，测试心肌补片基底层与中间层、中间层与微针层的界面结合强度；(1) Bonding strength: The layer to be tested is 10-20mm longer than the other layer. The extended part is installed on the clamp of the peel testing machine, and the remaining part is fixed on the 90-degree testing platform. The peeling strength experiment is performed to test the base layer and the middle layer of the myocardial patch. The interface bonding strength between the layer, the middle layer and the microneedle layer;
(2)最大拔脱力：将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后取出，并将心肌补片粘在电子万能材料试验机的上方传感器探头，猪心肌组织样本固定在下方夹具，多层心肌补片以0.05mm/s的速度向上驱动，直至微针完全脱离猪心肌组织，测得心肌补片的最大拔脱力。(2) Maximum pull-out force: Fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm, soak it in PBS buffer, take it out after 20 minutes, and stick the myocardial patch to the upper sensor probe of the electronic universal material testing machine , the pig myocardial tissue sample was fixed on the lower clamp, and the multi-layered myocardial patch was driven upward at a speed of 0.05mm/s until the microneedle completely separated from the pig myocardial tissue, and the maximum pull-out force of the myocardial patch was measured.
实施例1Example 1
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为3％(w/v)的丝素蛋白溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在60℃下干燥固化12h得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为0.5μg/mL；微针形状为子弹头形，底部半径为150微米，圆柱高度为800微米，圆锥高度为300微米，微针长度为1100微米；微针分布密度为2针/mm；The vascular endothelial growth factor is evenly dispersed in a silk fibroin solution with a concentration of 3% (w/v) (the solvent is water) to obtain the casting liquid A. The casting liquid A is poured into the microneedle part of the microneedle mold and heated at 60°C. The microneedle layer A was obtained by drying and solidifying for 12 hours without demoulding; the concentration of vascular endothelial growth factor in the casting liquid A was 0.5 μg/mL; the microneedle shape was bullet-shaped, the bottom radius was 150 microns, and the cylinder height was 800 microns, the cone height is 300 microns, the microneedle length is 1100 microns; the microneedle distribution density is 2 needles/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为40％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的0.05％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化6h得到厚度为500微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为2mg/mL；Curcumin is evenly dispersed in a methacrylic acid acylated gelatin solution (solvent is water) with a concentration of 40% (w/v) to obtain casting liquid B. Casting liquid B is poured into the base groove of the microneedle mold, and Under the conditions of adding 0.05% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid B, UV curing for 6 hours resulted in a thickness of The 500-micron middle layer B is not demoulded yet; among them, the concentration of curcumin in the molding liquid B is 2 mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(普伐他汀)均匀分散于浓度为7％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.5％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化4h得到厚度为500微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为10mg/kg；The statin drug (pravastatin) is evenly dispersed in a methacrylated hyaluronic acid solution (solvent is water) with a concentration of 7% (w/v) to obtain the casting liquid C, and the casting liquid C is poured into the microneedle mold The remaining parts in the base groove are added with a content of 0.5% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone of the casting liquid C. Under these conditions, ultraviolet light was cured for 4 hours to obtain a myocardial patch basal layer C with a thickness of 500 microns; among which, the statin concentration in the casting liquid C was 10 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为0.8cm×0.8cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the prepared suture-free multi-layer drug-loaded myocardial patch has a specification of 0.8cm × 0.8cm, including a basal layer, a middle layer and a microneedle layer of the myocardial patch; the microneedle layer is a microneedle array, and the microneedle layer is a microneedle array. The needle layer is evenly distributed on the same side of the middle layer and is perpendicular to the plane of the middle layer. The basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为350％和130％，微针轴向与中间层所在平面的夹角为60°；The multi-layer drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 350% and 130% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 60°;
心肌补片基底层与中间层的结合强度为30kPa，中间层与微针层的结合强度为25kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为30cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 30kPa, and the bonding strength between the middle layer and the microneedle layer is 25kPa; fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm and soak it in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 30 cN.
实施例2Example 2
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为7％(w/v)的丝素蛋白溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在25℃下干燥固化18h得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为3μg/mL；微针形状为子弹头形，底部半径为400微米，圆柱高度为600微米，圆锥高度为500微米，微针长度为1100微米；微针分布密度为0.5针/mm；The vascular endothelial growth factor is evenly dispersed in a silk fibroin solution with a concentration of 7% (w/v) (the solvent is water) to obtain the casting liquid A. The casting liquid A is poured into the microneedle part of the microneedle mold and heated at 25°C. The microneedle layer A was obtained by drying and solidifying for 18 hours without demoulding; the concentration of vascular endothelial growth factor in the casting liquid A was 3 μg/mL; the microneedle shape was bullet-shaped, with a bottom radius of 400 microns and a cylinder height of 600 Micron, the cone height is 500 micron, the microneedle length is 1100 micron; the microneedle distribution density is 0.5 needles/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为8％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的0.5％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化20min得到厚度为600微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为1mg/mL；Curcumin is uniformly dispersed in a methacrylated hyaluronic acid solution (solvent is water) with a concentration of 8% (w/v) to obtain casting liquid B, and casting liquid B is poured into the base groove of the microneedle mold. Under the condition of adding 0.5% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid B, the thickness was obtained by UV curing for 20 minutes. It is an intermediate layer B of 600 microns, which is not demolded yet; among them, the concentration of curcumin in the molding liquid B is 1 mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(普伐他汀)均匀分散于浓度为6％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.2％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化6h得到厚度为600微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为5mg/kg；The statin drug (pravastatin) is evenly dispersed in a methacrylated hyaluronic acid solution (solvent is water) with a concentration of 6% (w/v) to obtain the casting liquid C, and the casting liquid C is poured into the microneedle mold The remaining parts in the base groove are added with a content of 0.2% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid C. Under these conditions, ultraviolet light was cured for 6 hours to obtain a myocardial patch basal layer C with a thickness of 600 microns; among which, the statin concentration in the casting liquid C was 5 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为1.5cm×1.5cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the size of the prepared suture-free multi-layer drug-loaded myocardial patch is 1.5cm × 1.5cm, including the myocardial patch basal layer, middle layer and microneedle layer; the microneedle layer is a microneedle array, and the microneedle layer is a microneedle array. The needle layer is evenly distributed on the same side of the middle layer and is perpendicular to the plane of the middle layer. The basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为400％和300％，微针轴向与中间层所在平面的夹角为40°；The multi-layered drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 400% and 300% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 40°;
心肌补片基底层与中间层的结合强度为35kPa，中间层与微针层的结合强度为30kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为45cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 35kPa, and the bonding strength between the middle layer and the microneedle layer is 30kPa; the myocardial patch is fixed on a porcine myocardial tissue sample with a thickness of 10mm and then soaked in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 45 cN.
实施例3Example 3
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为5％(w/v)的丝素蛋白溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在40℃下干燥固化24h得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为1μg/mL；微针形状为子弹头形，底部半径为200微米，圆柱高度为1000微米，圆锥高度为400微米，微针长度为1400微米；微针分布密度为0.8针/mm；The vascular endothelial growth factor is evenly dispersed in a silk fibroin solution with a concentration of 5% (w/v) (the solvent is water) to obtain the casting liquid A. The casting liquid A is poured into the microneedle part of the microneedle mold and heated at 40°C. The microneedle layer A was obtained by drying and solidifying for 24 hours without demoulding; the concentration of vascular endothelial growth factor in the casting liquid A was 1 μg/mL; the microneedle shape was bullet-shaped, the bottom radius was 200 microns, and the cylinder height was 1000 Micron, the cone height is 400 micron, the microneedle length is 1400 micron; the microneedle distribution density is 0.8 needles/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为30％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的0.3％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化3h得到厚度为400微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为3mg/mL；Curcumin is evenly dispersed in a methacrylic acid acylated gelatin solution (solvent is water) with a concentration of 30% (w/v) to obtain casting liquid B. Casting liquid B is poured into the base groove of the microneedle mold. Under the conditions of adding 0.3% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone to the casting liquid B, UV curing was performed for 3 hours to obtain a thickness of The 400 micron middle layer B is not demoulded yet; among them, the concentration of curcumin in the molding liquid B is 3mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(普伐他汀)均匀分散于浓度为20％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.3％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化40min得到厚度为500微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为9mg/kg；The statin drug (pravastatin) is evenly dispersed in a methacrylic acid acylated gelatin solution (solvent is water) with a concentration of 20% (w/v) to obtain the casting liquid C. The casting liquid C is poured into the microneedle mold. The remaining parts in the base groove are under the condition of adding the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 0.3% (v/v) of the casting liquid C. Under UV curing for 40 minutes, a myocardial patch basal layer C with a thickness of 500 microns was obtained; among which, the statin concentration in the casting liquid C was 9 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为1.2cm×1.2cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the size of the prepared suture-free multi-layer drug-loaded myocardial patch is 1.2cm × 1.2cm, including the basal layer, middle layer and microneedle layer of the myocardial patch; the microneedle layer is a microneedle array, and the microneedle layer is a microneedle array. The needle layer is evenly distributed on the same side of the middle layer and is perpendicular to the plane of the middle layer. The basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为280％和180％，微针轴向与中间层所在平面的夹角为50°；The multi-layer drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 280% and 180% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 50°;
心肌补片基底层与中间层的结合强度为40kPa，中间层与微针层的结合强度为25kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为30cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 40kPa, and the bonding strength between the middle layer and the microneedle layer is 25kPa; the myocardial patch is fixed on a porcine myocardial tissue sample with a thickness of 10mm and then soaked in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 30 cN.
实施例4Example 4
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为70％(w/v)的聚乙二醇二丙烯酸酯溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在加入含量为铸模液A的1％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化10s得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为1.5μg/mL；微针形状为圆锥形，底部半径为150微米，微针长度为1000微米；微针分布密度为1.5针/mm；The vascular endothelial growth factor is evenly dispersed in a polyethylene glycol diacrylate solution with a concentration of 70% (w/v) (the solvent is water) to obtain the casting liquid A, and the casting liquid A is poured into the microneedle part of the microneedle mold. , obtained by adding the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 1% (v/v) of the casting liquid A, and curing it with UV light for 10 seconds. Microneedle layer A is not demolded yet; the concentration of vascular endothelial growth factor in the casting liquid A is 1.5 μg/mL; the shape of the microneedle is conical, the bottom radius is 150 microns, and the length of the microneedle is 1000 microns; the microneedle Distribution density is 1.5 pins/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为50％(w/v)的聚乙二醇二丙烯酸酯溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的0.5％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化40s得到厚度为500微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为1.5mg/mL；Curcumin is uniformly dispersed in a polyethylene glycol diacrylate solution (solvent is water) with a concentration of 50% (w/v) to obtain casting liquid B, and casting liquid B is poured into the base groove of the microneedle mold, Under the condition of adding 0.5% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid B, the thickness was obtained by UV curing for 40 seconds. It is an intermediate layer B of 500 microns, which is not demolded yet; among them, the concentration of curcumin in the molding liquid B is 1.5mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(阿托伐他汀)均匀分散于浓度为30％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.5％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化20min得到厚度为600微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为8mg/kg；The statin drug (atorvastatin) is evenly dispersed in a methacrylic acid acylated gelatin solution (solvent is water) with a concentration of 30% (w/v) to obtain the casting liquid C, and the casting liquid C is poured into the microneedle mold The remaining parts in the base groove are added with a content of 0.5% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone of the casting liquid C. Under the conditions, ultraviolet light was cured for 20 minutes to obtain a myocardial patch basal layer C with a thickness of 600 microns; among which, the statin concentration in the casting liquid C was 8 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为1cm×1cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the size of the prepared suture-free multi-layer drug-loaded myocardial patch is 1cm × 1cm, including the basal layer, the middle layer and the microneedle layer of the myocardial patch; the microneedle layer is a microneedle array, and the microneedle layer Evenly distributed on the same side of the middle layer and perpendicular to the plane of the middle layer, the myocardial patch basal layer is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为230％和100％，微针轴向与中间层所在平面的夹角为65°；The multi-layered drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 230% and 100%, respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 65°;
心肌补片基底层与中间层的结合强度为35kPa，中间层与微针层的结合强度为45kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为25cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 35kPa, and the bonding strength between the middle layer and the microneedle layer is 45kPa; fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm and soak it in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 25 cN.
实施例5Example 5
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为80％(w/v)的聚乙二醇二丙烯酸酯溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在加入含量为铸模液A的1％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化20s得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为1.2μg/mL；微针形状为圆锥形，底部半径为300微米，微针长度为1200微米；微针分布密度为1针/mm；The vascular endothelial growth factor is evenly dispersed in a polyethylene glycol diacrylate solution with a concentration of 80% (w/v) (the solvent is water) to obtain the casting liquid A, and the casting liquid A is poured into the microneedle part of the microneedle mold. , obtained by adding the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 1% (v/v) of the casting liquid A, and curing it with UV light for 20 seconds. Microneedle layer A is not demolded yet; the concentration of vascular endothelial growth factor in the casting liquid A is 1.2 μg/mL; the shape of the microneedle is conical, the bottom radius is 300 microns, and the length of the microneedle is 1200 microns; the microneedle The distribution density is 1 needle/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为40％(w/v)的聚乙二醇二丙烯酸酯溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的1％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化10s得到厚度为400微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为1.8mg/mL；Curcumin is evenly dispersed in a polyethylene glycol diacrylate solution (solvent is water) with a concentration of 40% (w/v) to obtain casting liquid B, and casting liquid B is poured into the base groove of the microneedle mold, Under the condition of adding the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 1% (v/v) of the casting liquid B, the thickness was obtained by UV curing for 10 seconds. It is an intermediate layer B of 400 microns, which is not demolded yet; among them, the concentration of curcumin in the molding liquid B is 1.8 mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(阿托伐他汀)均匀分散于浓度为25％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.1％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化3h得到厚度为500微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为7mg/kg；The statin drug (atorvastatin) is evenly dispersed in a methacrylic acid acylated gelatin solution (solvent is water) with a concentration of 25% (w/v) to obtain the casting liquid C, and the casting liquid C is poured into the microneedle mold The remaining parts in the base groove are added with a content of 0.1% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone of the casting liquid C. Under the conditions, ultraviolet light was cured for 3 hours to obtain a myocardial patch basal layer C with a thickness of 500 microns; among which, the statin concentration in the casting liquid C was 7 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为1.2cm×1.2cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the size of the prepared suture-free multi-layer drug-loaded myocardial patch is 1.2cm × 1.2cm, including the basal layer, middle layer and microneedle layer of the myocardial patch; the microneedle layer is a microneedle array, and the microneedle layer is a microneedle array. The needle layer is evenly distributed on the same side of the middle layer and is perpendicular to the plane of the middle layer. The basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为250％和120％，微针轴向与中间层所在平面的夹角为60°；The multi-layered drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 250% and 120% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 60°;
心肌补片基底层与中间层的结合强度为30kPa，中间层与微针层的结合强度为40kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为28cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 30kPa, and the bonding strength between the middle layer and the microneedle layer is 40kPa; fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm and soak it in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 28 cN.
实施例6Example 6
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为8％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在加入含量为铸模液A的0.05％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化20min得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为1.8μg/mL；微针形状为四棱锥形，底部边长为400微米，微针长度为1500微米；微针分布密度为1.2针/mm；The vascular endothelial growth factor is evenly dispersed in a methacrylated hyaluronic acid solution (the solvent is water) with a concentration of 8% (w/v) to obtain the casting liquid A, and the casting liquid A is poured into the microneedle part of the microneedle mold. , obtained by adding photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 0.05% (v/v) of the casting liquid A, and curing it with UV light for 20 minutes. The microneedle layer A is not demolded yet; the concentration of vascular endothelial growth factor in the casting liquid A is 1.8 μg/mL; the shape of the microneedles is a quadrangular pyramid, the bottom side is 400 microns, and the length of the microneedles is 1500 microns; The microneedle distribution density is 1.2 needles/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为6％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的0.4％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化1h得到厚度为500微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为2.2mg/mL；Curcumin is evenly dispersed in a methacrylated hyaluronic acid solution (solvent is water) with a concentration of 6% (w/v) to obtain casting liquid B, and casting liquid B is poured into the base groove of the microneedle mold. Under the condition of adding 0.4% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid B, the thickness was obtained by UV curing for 1 hour. It is an intermediate layer B of 500 microns, which is not demolded yet; among them, the concentration of curcumin in the molding liquid B is 2.2 mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(普伐他汀)均匀分散于浓度为5％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.05％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化5h得到厚度为500微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为6mg/kg；The statin drug (pravastatin) is evenly dispersed in a methacrylated hyaluronic acid solution (solvent is water) with a concentration of 5% (w/v) to obtain the casting liquid C, and the casting liquid C is poured into the microneedle mold The remaining parts in the base groove are added with a content of 0.05% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid C. Under the conditions, ultraviolet light was cured for 5 hours to obtain a myocardial patch basal layer C with a thickness of 500 microns; among which, the statin concentration in the casting liquid C was 6 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为0.8cm×0.8cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the prepared suture-free multi-layer drug-loaded myocardial patch has a specification of 0.8cm × 0.8cm, including a basal layer, a middle layer and a microneedle layer of the myocardial patch; the microneedle layer is a microneedle array, and the microneedle layer is a microneedle array. The needle layer is evenly distributed on the same side of the middle layer and is perpendicular to the plane of the middle layer. The basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为600％和450％，微针轴向与中间层所在平面的夹角为30°；The multi-layer drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 600% and 450% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 30°;
心肌补片基底层与中间层的结合强度为40kPa，中间层与微针层的结合强度为40kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为50cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 40kPa, and the bonding strength between the middle layer and the microneedle layer is 40kPa; fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm and soak it in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 50 cN.
实施例7Example 7
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为50％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在加入含量为铸模液A的0.5％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化6h得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为2μg/mL；微针形状为子弹头形，底部半径为300微米，圆柱高度为1000微米，圆锥高度为400微米，微针长度为1400微米；微针分布密度为1.8针/mm；The vascular endothelial growth factor is evenly dispersed in a methacrylic acid acylated gelatin solution with a concentration of 50% (w/v) (the solvent is water) to obtain the casting liquid A, and the casting liquid A is poured into the microneedle part of the microneedle mold. Under the condition of adding 0.5% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid A, UV curing for 6 hours resulted in micro Needle layer A is not demoulded yet; the concentration of vascular endothelial growth factor in the casting liquid A is 2 μg/mL; the microneedle shape is bullet-shaped, with a bottom radius of 300 microns, a cylinder height of 1000 microns, and a cone height of 400 Micron, the length of microneedles is 1400 microns; the distribution density of microneedles is 1.8 needles/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为40％(w/v)的聚乙二醇二丙烯酸酯溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的0.5％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化20s得到厚度为400微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为2.5mg/mL；Curcumin is evenly dispersed in a polyethylene glycol diacrylate solution (solvent is water) with a concentration of 40% (w/v) to obtain casting liquid B, and casting liquid B is poured into the base groove of the microneedle mold, Under the condition of adding 0.5% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid B, the thickness was obtained by UV curing for 20 seconds. It is an intermediate layer B of 400 microns and is not demolded yet; among them, the concentration of curcumin in the molding liquid B is 2.5mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(阿托伐他汀)均匀分散于浓度为20％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.5％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化1h得到厚度为800微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为9mg/kg；The statin drug (atorvastatin) is evenly dispersed in a methacrylic acid acylated gelatin solution (solvent is water) with a concentration of 20% (w/v) to obtain the casting liquid C, and the casting liquid C is poured into the microneedle mold The remaining parts in the base groove are added with a content of 0.5% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone of the casting liquid C. Under the conditions, ultraviolet light was cured for 1 hour to obtain a myocardial patch basal layer C with a thickness of 800 microns; among which, the statin concentration in the casting liquid C was 9 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为1.5cm×1.5cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the size of the prepared suture-free multi-layer drug-loaded myocardial patch is 1.5cm × 1.5cm, including the myocardial patch basal layer, middle layer and microneedle layer; the microneedle layer is a microneedle array, and the microneedle layer is a microneedle array. The needle layer is evenly distributed on the same side of the middle layer and is perpendicular to the plane of the middle layer. The basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为200％和100％，微针轴向与中间层所在平面的夹角为65°；The multi-layer drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 200% and 100% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 65°;
心肌补片基底层与中间层的结合强度为30kPa，中间层与微针层的结合强度为30kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为22cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 30kPa, and the bonding strength between the middle layer and the microneedle layer is 30kPa; fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm and soak it in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 22 cN.
实施例8Example 8
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为20％(w/v)的聚乙烯醇溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在25℃下干燥固化24h得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为2.5μg/mL；微针形状为圆锥形，底部半径为400微米，微针长度为1500微米；微针分布密度为1.5针/mm；The vascular endothelial growth factor is evenly dispersed in a polyvinyl alcohol solution with a concentration of 20% (w/v) (the solvent is water) to obtain the casting liquid A. The casting liquid A is poured into the microneedle part of the microneedle mold and heated at 25°C. The microneedle layer A was obtained by drying and solidifying for 24 hours without demoulding; the concentration of vascular endothelial growth factor in the casting liquid A was 2.5 μg/mL; the shape of the microneedle was conical, the bottom radius was 400 microns, and the length of the microneedle was 1500 microns; microneedle distribution density is 1.5 needles/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为60％(w/v)的聚乙二醇二丙烯酸酯溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的1％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化20s得到厚度为400微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为2.8mg/mL；Curcumin is evenly dispersed in a polyethylene glycol diacrylate solution (solvent is water) with a concentration of 60% (w/v) to obtain casting liquid B, and casting liquid B is poured into the base groove of the microneedle mold. Under the condition of adding the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 1% (v/v) of the casting liquid B, the thickness was obtained by UV curing for 20 seconds. It is an intermediate layer B of 400 microns and is not demolded yet; among them, the concentration of curcumin in the molding liquid B is 2.8 mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(普伐他汀)均匀分散于浓度为30％(w/v)的甲基丙烯酸酰化明胶溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.4％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化2h得到厚度为600微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为8mg/kg；The statin drug (pravastatin) is evenly dispersed in a methacrylic acid acylated gelatin solution (solvent is water) with a concentration of 30% (w/v) to obtain the casting liquid C. The casting liquid C is poured into the microneedle mold. The remaining parts in the base groove are under the condition of adding the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 0.4% (v/v) of the casting liquid C. Under UV curing for 2 hours, a myocardial patch basal layer C with a thickness of 600 microns was obtained; among which, the statin concentration in the casting liquid C was 8 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为1.2cm×1.2cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the size of the prepared suture-free multi-layer drug-loaded myocardial patch is 1.2cm × 1.2cm, including the basal layer, middle layer and microneedle layer of the myocardial patch; the microneedle layer is a microneedle array, and the microneedle layer is a microneedle array. The needle layer is evenly distributed on the same side of the middle layer and is perpendicular to the plane of the middle layer. The basal layer of the myocardial patch is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为260％和80％，微针轴向与中间层所在平面的夹角为70°；The multi-layer drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 260% and 80% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 70°;
心肌补片基底层与中间层的结合强度为35kPa，中间层与微针层的结合强度为25kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为12cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 35kPa, and the bonding strength between the middle layer and the microneedle layer is 25kPa; fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm and soak it in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 12 cN.
实施例9Example 9
如图2所示，一种免缝合的多层载药心肌补片的制备方法，包括如下步骤：As shown in Figure 2, a method for preparing a suture-free multi-layer drug-loaded myocardial patch includes the following steps:
(1)微针层的制备：(1) Preparation of microneedle layer:
将血管内皮生长因子均匀分散于浓度为80％(w/v)的聚乙二醇二丙烯酸酯溶液(溶剂为水)中得到铸模液A，将铸模液A浇筑到微针模具的微针部位，在加入含量为铸模液A的1％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化20s得到微针层A，暂不脱模；其中，铸模液A中血管内皮生长因子的浓度为2.2μg/mL；微针形状为四棱锥形，底部边长为150微米，微针长度为1000微米；微针分布密度为1针/mm；The vascular endothelial growth factor is evenly dispersed in a polyethylene glycol diacrylate solution with a concentration of 80% (w/v) (the solvent is water) to obtain the casting liquid A, and the casting liquid A is poured into the microneedle part of the microneedle mold. , obtained by adding the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 1% (v/v) of the casting liquid A, and curing it with UV light for 20 seconds. The microneedle layer A is not demolded yet; the concentration of vascular endothelial growth factor in the casting liquid A is 2.2 μg/mL; the shape of the microneedle is a quadrangular pyramid, the bottom side is 150 microns, and the length of the microneedle is 1000 microns; The microneedle distribution density is 1 needle/mm;
(2)中间层的制备：(2) Preparation of the middle layer:
将姜黄素均匀分散于浓度为7％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液B，将铸模液B浇筑到微针模具的基底凹槽内，在加入含量为铸模液B的0.2％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化4h得到厚度为500微米的中间层B，暂不脱模；其中，铸模液B中姜黄素的浓度为2mg/mL；Curcumin is evenly dispersed in a methacrylated hyaluronic acid solution (solvent is water) with a concentration of 7% (w/v) to obtain casting liquid B, and casting liquid B is poured into the base groove of the microneedle mold. Under the condition of adding 0.2% (v/v) of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the casting liquid B, the thickness was obtained by UV curing for 4 hours. It is an intermediate layer B of 500 microns, which is not demolded yet; among them, the concentration of curcumin in the molding liquid B is 2 mg/mL;
(3)心肌补片基底层的制备：(3) Preparation of the basal layer of myocardial patch:
将他汀类药物(阿托伐他汀)均匀分散于浓度为6％(w/v)的甲基丙烯酸化透明质酸溶液(溶剂为水)中得到铸模液C，将铸模液C浇筑到微针模具的基底凹槽内的剩余部位，在加入含量为铸模液C的0.45％(v/v)的光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮的条件下，紫外光固化4h得到厚度为800微米的心肌补片基底层C；其中，铸模液C中他汀类药物浓度为7mg/kg；The statin drug (atorvastatin) is evenly dispersed in a methacrylated hyaluronic acid solution (solvent is water) with a concentration of 6% (w/v) to obtain the casting liquid C, and the casting liquid C is poured into the microneedle To the remaining parts of the base groove of the mold, add photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a content of 0.45% (v/v) of the casting liquid C. Under the conditions of UV curing for 4 hours, a myocardial patch basal layer C with a thickness of 800 microns was obtained; among which, the statin concentration in the casting liquid C was 7 mg/kg;
(4)脱模，得到心肌补片A-B-C集合体，即为免缝合的多层载药心肌补片。(4) Demold and obtain the myocardial patch A-B-C aggregate, which is a suture-free multi-layered drug-loaded myocardial patch.
如图1所示，制得的免缝合的多层载药心肌补片规格为1cm×1cm，包括心肌补片基底层、中间层和微针层；微针层为微针阵列，微针层均匀分布在中间层的同一侧，且垂直于中间层所在平面，心肌补片基底层以原位界面粘结的方法位于中间层的另一侧；As shown in Figure 1, the size of the prepared suture-free multi-layer drug-loaded myocardial patch is 1cm × 1cm, including the basal layer, the middle layer and the microneedle layer of the myocardial patch; the microneedle layer is a microneedle array, and the microneedle layer Evenly distributed on the same side of the middle layer and perpendicular to the plane of the middle layer, the myocardial patch basal layer is located on the other side of the middle layer through in-situ interface bonding;
将多层载药心肌补片刺入离体猪心脏后浸泡在PBS缓冲液中，20分钟时心肌补片基底层的溶胀率和中间层的溶胀率分别为400％和350％，微针轴向与中间层所在平面的夹角为35°；The multi-layer drug-loaded myocardial patch was inserted into the isolated pig heart and soaked in PBS buffer. After 20 minutes, the swelling rate of the basal layer and the middle layer of the myocardial patch were 400% and 350% respectively. The microneedle shaft The angle between the direction and the plane of the middle layer is 35°;
心肌补片基底层与中间层的结合强度为35kPa，中间层与微针层的结合强度为25kPa；将心肌补片固定在厚度为10mm的猪心肌组织样本后浸泡在PBS缓冲液中，20分钟后测试得到心肌补片的最大拔脱力为48cN。The bonding strength between the basal layer and the middle layer of the myocardial patch is 35kPa, and the bonding strength between the middle layer and the microneedle layer is 25kPa; fix the myocardial patch on a porcine myocardial tissue sample with a thickness of 10mm and soak it in PBS buffer for 20 minutes. After testing, the maximum pull-out force of the myocardial patch was 48cN.
探究实施例1～9的免缝合多层载药心肌补片对心肌修复的影响，步骤如下：To explore the effect of the suture-free multi-layer drug-loaded myocardial patch of Examples 1 to 9 on myocardial repair, the steps are as follows:
(1)用中度乙醚麻醉大鼠，进行左胸切口以暴露心脏，然后在左心耳下方2mm处用8-0#缝合线结扎左冠状动脉，以建立大鼠心肌梗塞模型；(1) Anesthetize rats with moderate ether, make a left chest incision to expose the heart, and then ligate the left coronary artery with 8-0# suture 2mm below the left atrial appendage to establish a rat myocardial infarction model;
(2)将心肌梗死小鼠分为两组，其中一组植入多层载药心肌补片为实验组，未进行任何操作的为对照组；(2) Mice with myocardial infarction were divided into two groups. One group was implanted with multi-layer drug-loaded myocardial patches as the experimental group, and those without any operation were the control group;
(3)2周后，使用超声心动图观察大鼠心脏功能，处死大鼠，收集心脏并在4℃下用4％多聚甲醛固定，并在乙醇中脱水，对心肌组织切片进行Masson三色染色，以观察纤维化组织、梗死面积和左室壁厚度的变化；(3) After 2 weeks, use echocardiography to observe the heart function of the rats, sacrifice the rats, collect the hearts, fix them with 4% paraformaldehyde at 4°C, and dehydrate in ethanol, and perform Masson's tricolor on the myocardial tissue sections. Staining to observe changes in fibrotic tissue, infarct size, and left ventricular wall thickness;
实验结果表明，多层载药心肌补片的植入能够提高大鼠梗死心脏的射血分数和左室短轴缩短率，此外，收缩期左心室内部尺寸明显减小，并伴随着纤维化组织和梗死面积的减少。Experimental results show that the implantation of multi-layer drug-loaded myocardial patches can improve the ejection fraction and left ventricular short-axis shortening rate of infarcted hearts in rats. In addition, the internal size of the left ventricle during systole is significantly reduced, accompanied by fibrotic tissue. and reduction in infarct size.
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