WO2023044689A1 - 一种免疫代谢心梗贴片及其制备方法与应用 - Google Patents
一种免疫代谢心梗贴片及其制备方法与应用 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
Definitions
- the invention belongs to biological material technology, and relates to an immunometabolic myocardial infarction patch and its preparation method and application, in particular to the application of 2-deoxy-D-glucose patch in the preparation of biological materials for treating myocardial infarction with synergistic stem cells.
- MI Myocardial infarction
- Ischemic myocardial infarction leads to necrosis and scarring of cardiomyocytes, which in turn affects cardiac function.
- Most of the current drug or device treatments can only relieve the symptoms, but they cannot reverse the damage to the heart tissue.
- heart transplantation can completely improve the state of the heart, it is difficult to be widely used clinically due to factors such as scarcity of donor sources, immune rejection, and expensive treatment costs.
- Mesenchymal stem cells are present in almost all tissues of the human body with specific stem cell niches.
- Bone marrow-derived mesenchymal stem cells are the first mesenchymal stem cells to be discovered, and they are also the most commonly used stem cells for the treatment of myocardial infarction.
- Chinese patent application CN2021107004588 discloses the application of 2-deoxy-D-glucose in the preparation of drugs for the treatment of myocardial infarction with synergistic stem cells. Animal experiments have confirmed that after injection of 2-deoxy-D-glucose, the survival of injected mesenchymal stem cells is enhanced rate and enhance the therapeutic effect of mesenchymal stem cells. However, the method of injection makes it difficult to determine the site of administration, and there are obvious side effects.
- the present invention discloses an immunometabolic myocardial infarction patch and its preparation method and application.
- the 2-deoxy-D-glucose (2-DG) efficacy is provided.
- an immunometabolic myocardial infarction patch which includes a patch matrix and 2-deoxy-D-glucose; the 2-deoxy-D-glucose is located in the patch matrix.
- the invention discloses a preparation method of the above-mentioned immunometabolic myocardial infarction patch.
- the immune metabolic myocardial infarction patch is obtained by mixing 2-deoxy-D-glucose, chitosan and gelatin and then drying. Specifically, after the chitosan solution and the gelatin solution are mixed, 2-deoxy-D-glucose is added, and then dried to obtain an immunometabolic myocardial infarction patch. Drying is performed at 40° C. to 60° C. for 15 to 30 hours.
- the patch matrix is prepared by mixing chitosan and gelatin, and the weight of 2-deoxy-D-glucose is 35%-40% of the patch matrix weight.
- the mass ratio of chitosan, gelatin and 2-deoxy-D-glucose is 4: (3.5-4.5): (2.5-4).
- the invention discloses the application of the above immunometabolic myocardial infarction patch in the preparation of biological materials for treating myocardial infarction.
- the invention discloses the application of the above immunometabolic myocardial infarction patch in the preparation of biological materials for treating myocardial infarction with synergistic stem cells.
- the invention discloses the application of the above immunometabolic myocardial infarction patch and stem cells in the preparation of drugs for synergistic treatment of myocardial infarction.
- the invention discloses a new application of using 2-deoxy-D-glucose patch to improve the therapeutic effect of stem cells after myocardial infarction.
- the biggest problem of MSCs treatment of myocardial infarction is the low retention rate and poor survival of transplanted cells in the myocardial infarction site.
- the invention discloses that the therapeutic effect of 2-deoxy-D-glucose patch on mesenchymal stem cells after myocardial infarction is obviously improved.
- the 2-DG patch involved in the present invention in the preparation of biological materials for the prevention or treatment of myocardial infarction
- the 2-DG patch can be used for the preparation of myocardial infarction protection materials, to realize the 2-deoxy-D-glucose patch especially Synergistic stem cells, especially mesenchymal stem cells, can improve cardiac function after myocardial infarction, slow down the expansion of ventricles after myocardial infarction, and reduce the technical effect of myocardial infarction size.
- the specific treatment method of the prepared biological material disclosed in the present invention can be attached to the ischemic area of the heart.
- MI local intramyocardial transplantation may be superior to intravenous injection or intracoronary transplantation, but regardless of the transplantation route, the ultimate goal is still to optimize the strategy to enhance the survival rate of injected MSCs.
- the effect of the existing mesenchymal stem cell injection in the treatment of myocardial infarction is not good.
- the operation of this method is complicated.
- Myocardial infarction is a cardiovascular disease that seriously endangers human health.
- the present invention uses 2-deoxy-D-glucose patch for the first time to improve cardiac function after myocardial infarction, especially combined with mesenchymal stem cells to reduce the area of myocardial infarction, thereby preventing or treating myocardial infarction.
- 2-DG the immunometabolic myocardial infarction patch disclosed by the present invention has a better therapeutic effect, especially significantly lower side effects.
- Figure 1 shows that 2-DG patch reduces the proportion of inflammatory macrophages in myocardial infarction.
- FIG. 2 shows that 2-DG patches significantly increased the survival of transplanted MSCs.
- FIG. 3 shows that the 2-DG patch improves the cardiac function of mice after myocardial infarction treated with MSCs.
- FIG. 4 shows that 2-DG patch improves the ventricular remodeling of mice after myocardial infarction treated with MSCs.
- Figure 5 is a comparison of related side effects of 2-DG patch and injection of 2-DG.
- 2-deoxy-D-glucose patch (2-DG patch) is made of 4% chitosan and 2% gelatin in a weight ratio of 1:1 as the patch matrix, and the patch is loaded with 1% 2-Deoxy-D-glucose (2-DG), the latter is a glucose analogue, which has the effects of interfering with the synthesis of virus-specific glycoproteins, inhibiting herpes monoviruses, RAN and DNA enveloped viruses, and cancer cell proliferation; But so far there is no research report on 2-deoxy-D-glucose in myocardial infarction, especially no research on the relationship between 2-DG and stem cells.
- mice C57BL/6J male mice of about 25 g were selected as the experimental subjects, and the myocardial infarction model was established by ligation of the left anterior descending coronary artery. After intraperitoneal injection of anesthesia, orotracheal intubation, connected to an air ventilator, respiratory rate 110 times/min, tidal volume 2.5ml, breath-to-breath ratio 1:1.3.
- the outer skin was incised through the left thoracic longitudinal incision, the pectoralis major muscle was peeled off, the chest was opened through the third and fourth intercostal transverse incisions, the heart was exposed, and the pericardium was torn off with tweezers.
- the left coronary artery can be roughly coursed with the aid of an operating microscope.
- the anterior descending coronary artery (LAD) and a small amount of myocardial tissue were ligated, the depth of the needle was about 1 mm, and the width was controlled within 3 mm; the chest was closed layer by layer.
- the sham operation group only passed under the LAD without knotting, and the rest were the same as the model group.
- the ligation area to the apex of the heart became white with the naked eye.
- the left ventricle tissue was taken for cardiac tissue staining, and obvious fibrosis could be seen, which proved that the myocardial infarction model was successfully established.
- the extracted MSCs were injected at three points at the edge of the myocardial infarction immediately after ligation of the LAD, and the total number of injected cells was 5x10 5 /20ml PBS; 2-DG (500 mg/kg/day) was injected intraperitoneally on the 1st day and 2nd day after surgery, and the rest were the same as in the MSCs group; -The DG patch was attached to the myocardial infarction area, and the rest were the same as the MSCs group.
- mice in the control group normal saline group
- mice in the control group normal saline group
- mice in the control group normal saline group
- the feeding methods of the three groups were the same, and then the cardiac function and myocardial infarction area were detected after myocardial infarction.
- Example 1 Reduce the proportion of inflammatory macrophages in the myocardial infarction area: take the heart on the 3rd day after operation to prepare frozen sections, and make immunofluorescent tissue sections of macrophages. Green marks iNOS, red marks F4/80, and blue represents DAPI marks nucleus. The experimental results are shown in Figure 1, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- Example 2 Increase the survival of mesenchymal stem cells: conventional method to prepare mesenchymal stem cells (Fluc-MSCs) transfected with luciferase gene lentivirus, and use luciferase to react with the substrate to produce bioluminescent characteristics to observe the transplanted mesenchymal stem cells Stem cell viability. Specifically: on the 1st, 3rd, and 7th day after the operation, live imaging detection was performed on the mice to observe the bioluminescent signal (BLI) in the heart of the mice. , continuously detect the BLI signal for 10 minutes, and measure it every 1 minute until the signal reaches the peak. The experimental results are shown in Figure 2, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- Example 3 Effective improvement of cardiac function after myocardial infarction detected by echocardiography. Mice were anesthetized (the same method as before), placed in the left lateral position after depilation, placed the probe of the cardiac ultrasound diagnostic instrument on the anterior wall of the heart, and took a two-dimensional short-axis view of the left ventricle at the level of the papillary muscle, and recorded M-scan at the same time, for 3 consecutive Cardiac cycle measurements of left ventricular ejection fraction (EF) and fractional shortening (FS). The experimental results are shown in Figure 3, * P ⁇ 0.05.
- Example 4 Effective improvement of ventricular remodeling heart weight and Masson staining: the mice were sacrificed 28 days after the operation, and the body weight (BW) and heart weight (HW) of the mice were measured; HW/BW was calculated.
- Masson staining the mice were sacrificed 28 days after the operation, and the left ventricle tissue was taken for heart tissue staining to observe the therapeutic effect.
- the image analysis software Image J was used to analyze the area of each part, and the myocardial infarction area/heart area was calculated. The experimental results are shown in Figure 4, *P ⁇ 0.05, **P ⁇ 0.01.
- Example 5 2-DG patch has no side effects: Liver and kidney were collected on the 3rd day after operation, ALT, AST and BUN were measured, frozen sections of liver and kidney were stained with HE; body weight and random blood glucose of mice were continuously measured after operation. The experimental results are shown in Figure 5, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ns means no statistical difference.
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Abstract
一种免疫代谢心梗贴片及其制备方法与应用，免疫代谢心梗贴片可作为增效干细胞治疗心梗的生物材料，动物实验证实，应用2-脱氧-D-葡萄糖贴片后，增强注射的间充质干细胞的存活率，增强间充质干细胞的治疗效果，且降低了注射2-脱氧-D-葡萄糖的副作用。
Description
本发明属于生物材料技术，为一种免疫代谢心梗贴片及其制备方法与应用，具体涉及2-脱氧-D-葡萄糖贴片在制备增效干细胞治疗心梗的生物材料中的应用。
心肌梗死（MI）是一种严重危害人类健康的心血管疾病，随着我国人民生活水平的不断提高，缺血性心肌梗死的发病率也在不断上升。缺血性心肌梗死会导致心肌细胞坏死和瘢痕形成，进而影响心脏功能。目前药物或器械治疗大多只能缓解症状，但却不能逆转心脏组织损伤。心脏移植虽能彻底改善心脏状态，但因供体来源稀缺、免疫排斥以及昂贵的治疗费用等因素，在临床上很难广泛应用。间充质干细胞几乎存在于人体所有具有特定干细胞龛位的组织中。骨髓来源的间充质干细胞(BM-MSCs)是第一个被发现的间充质干细胞，也是目前最多的用于心梗治疗的干细胞。中国专利申请CN2021107004588公开了2-脱氧-D-葡萄糖在制备增效干细胞治疗心梗的药物中的应用，动物实验证实，注射2-脱氧-D-葡萄糖后，增强注射的间充质干细胞的存活率，增强间充质干细胞的治疗效果。但是注射的方法使得用药部位难以确定，而且存在明显副作用。
为了提高2-脱氧-D-葡萄糖治疗方法的操作便利性，本发明公开了一种免疫代谢心梗贴片及其制备方法与应用，采用常规粘贴的方法就可发挥2-脱氧-D-葡萄糖（2-DG）的功效。
本发明采用如下技术方案：一种免疫代谢心梗贴片，包括贴片基质与2-脱氧-D-葡萄糖；2-脱氧-D-葡萄糖位于贴片基质内。
本发明公开了上述免疫代谢心梗贴片的制备方法，将2-脱氧-D-葡萄糖、壳聚糖、明胶混合后干燥，得到免疫代谢心梗贴片。具体的，壳聚糖溶液与明胶溶液混合后，加入2-脱氧-D-葡萄糖，然后干燥，得到免疫代谢心梗贴片。干燥为40℃～60℃干燥15～30小时。
本发明中，贴片基质由壳聚糖、明胶混合制备，2-脱氧-D-葡萄糖的重量为贴片基质重量的35%～40%。壳聚糖、明胶和2-脱氧-D-葡萄糖的质量比为4∶（3.5～4.5）：（2.5～4）。
本发明公开了上述免疫代谢心梗贴片在制备治疗心梗的生物材料中的应用。
本发明公开了上述免疫代谢心梗贴片在制备增效干细胞治疗心梗的生物材料中的应用。
本发明公开了上述免疫代谢心梗贴片和干细胞在制备增效治疗心梗的药物中的应用。
本发明公开了一种利用2-脱氧-D-葡萄糖贴片改善心梗后干细胞治疗效果的新用途。MSCs治疗心梗的最大问题是移植细胞在心梗部位的滞留率低、存活性差。本发明公开了2-脱氧-D-葡萄糖贴片对心肌梗死后间充质干细胞的治疗效果有明显提升。
本发明涉及的2-DG贴片在制备预防或治疗心梗生物材料中的应用，具体的是2-DG贴片可用于制备心梗保护材料，实现2-脱氧-D-葡萄糖贴片尤其是增效干细胞，特别是间充质干细胞改善心梗后心功能、减缓心梗后心室扩张、减小心梗面积的技术效果。
本发明公开的制备的生物材料，具体的治疗方式可采取贴附于心脏缺血区。考虑到MSCs的移植成功率，MI局部心肌内移植可能比静脉注射或冠状动脉内移植更为优越，但无论采用何种移植途径，最终目的仍然是优化策略，以增强注射的MSCs的存活率。现有技术下，由于MI导致心脏部位环境的复杂，使得现有间充质干细胞注射治疗心梗的效果欠佳，研究者采用各种方法以提高其对心梗的治疗效果，比如体外采用细胞因子培养间充质干细胞后回输等，此方法操作复杂。心肌梗死是一种严重危害人类健康的心血管疾病，随着我国人民生活水平的不断提高，缺血性心肌梗死的发病率也在不断上升。本发明首次采用2-脱氧-D-葡萄糖贴片改善心梗后心功能，尤其是与间充质干细胞结合，减小心梗面积，从而可以预防或者治疗心梗。与注射2-DG相比，本发明公开的免疫代谢心梗贴片治疗效果好，尤其是副作用明显降低。
图1为2-DG贴片减少心梗炎性巨噬细胞比例。
图2为2-DG贴片明显增加移植MSCs存活。
图3为2-DG贴片改善MSCs治疗心梗后小鼠的心功能。
图4为2-DG贴片改善MSCs治疗心梗后小鼠的心室重构。
图5为2-DG贴片、注射2-DG的相关副作用比较。
作为具体的例子，2-脱氧-D-葡萄糖贴片（2-DG贴片）由4%壳聚糖和2%明胶按照重量比1:1混合作为贴片基质，贴片负载有1%的2-脱氧-D-葡萄糖（2-DG），后者是一种葡萄糖类似物，具有干扰病毒特异性糖蛋白的合成、抑制疱疹单病毒、RAN和DNA包膜病毒、癌症细胞增殖等功效；但迄今为止尚未见2-脱氧-D-葡萄糖在心肌梗死方面的研究报道，尤其未见2-DG与干细胞作用关系的研究。
以下所列实施例，仅为帮助本领域技术人员更全面理解本发明，但不以任何方式限制本发明。本发明涉及的原料试剂以及建模、测试都是本领域常规技术。
所使用的主要材料和来源分别如下：C57BL/6J小鼠（昭衍（苏州）新药研究中心提供，此实验由苏州大学伦理委员会批准）；小动物呼吸机（上海奥尔科特生物科技有限公司，上海）；手术器械（六六视觉公司，苏州）；缝合针线（上海浦东金环医疗用品有限公司，上海）；小动物超声影像系统（Visual Sonics Vevo 2100）；间充质干细胞，采用现有技术从8周雄性C57BL/6J小鼠股骨骨髓中提取，常规培养第六代以后用于实验；2-脱氧-D-葡萄糖（2-DG，苏州天可贸易有限公司，苏州）；壳聚糖（国药集团化学试剂有限公司，上海）；明胶（国药集团化学试剂有限公司，上海）。
小鼠心肌梗死模型的建立：选用25g左右的C57BL/6J雄性小鼠为实验对象，采用左冠状动脉前降支结扎法制作心梗模型。腹腔注射麻醉后，经口气管插管，接空气呼吸机，呼吸频率110次/min，潮气量2.5ml，吸呼比1:1.3。右侧卧位，左胸纵切口切开外层皮肤，剥离胸大肌，第三、四肋间横切口开胸，暴露心脏，用镊子撕开心包膜。借助手术显微镜可见左冠状动脉大致走行。在左心耳下缘约1.5mm处，将冠状动脉前降支（LAD） 连同少量心肌组织一起结扎，进针深度约1 mm，宽度控制在3 mm以内；逐层关胸。
假手术组仅穿过LAD下方不打结，其余同模型组。
结扎后肉眼可见结扎处至心尖变白，1 周后取左心室组织进行心脏组织染色，可看到明显的纤维化，证明心梗模型建立成功。
MSCs组在结扎LAD后当即在心梗边缘区分三点注射提取的MSCs，注射细胞总数量为5x10
5/20ml PBS；2-DG注射组（2-DG
inj）在心梗建立前6h、心梗术后第1天、术后第2天腹腔注射2-DG（500 mg/kg/天），其余同MSCs组；2-DG贴片组（2-DG
pat）在心梗建立后立即将2-DG贴片贴附于心梗区，其余同MSCs组。对照组（生理盐水组）小鼠在结扎LAD后注射等量无菌生理盐水作为对照；三组喂养方式一致，然后进行心梗后心功能、心梗面积检测。
制备例 免疫代谢心梗贴片（2-DG贴片）的制备方法：1mL乙酸用水稀释至100mL，得到1%乙酸水溶液，作为溶剂；将4g壳聚糖加入100 mL的1%乙酸水溶液中，得到壳聚糖溶液；将2g明胶加入100 mL的1%乙酸水溶液中，得到明胶溶液。取1mL壳聚糖溶液与2mL明胶溶液混合，再加入30mg的2-DG，然后3500rpm离心15分钟，再取1.5mL加入3.5cm培养皿，置于烘箱中，50℃过夜（24小时）烘干，再应用 2ml 2%NaOH-80%乙醇溶液浸泡30分钟进行脱酸，之后应用2ml 80%乙醇溶液清洗三次，每次10分钟，用去离子水洗至中性；取直径为3.5mm的贴片作为贴片基质，用于心梗治疗。
实施例1 减少心梗区炎性巨噬细胞比例：术后第3天取心脏制备冰冻切片，做巨噬细胞的组织切片免疫荧光，绿色标记iNOS，红色标记F4/80，蓝色表示DAPI标记细胞核。实验结果见图1，*P < 0.05，**P < 0.01，***P < 0.001。
实施例2 增加间充质干细胞存活：常规方法制备带有荧光素酶基因慢病毒转染的间充质干细胞（Fluc-MSCs），利用荧光素酶与底物反应产生生物发光特性观察移植间充质干细胞的存活率。具体为：术后第1、3、7天对小鼠进行活体成像检测，观察小鼠心脏内生物发光信号（BLI），具体为：小鼠吸入麻醉，腹腔注射D-荧光素150mg/kg体重，连续检测BLI信号10分钟，每隔1分钟测一次，直到信号达到顶点。实验结果见图2，*P<0.05，**P<0.01，***P<0.001。
实施例3 有效改善心梗后心功能：心脏超声检测心梗后心功能。小鼠麻醉（方法同前），脱毛后左侧卧位，将心脏超声诊断仪探头置于心前壁，于乳头肌水平取左室二维短轴观，同时记录M型扫描，连续3个心动周期测量左室射血分数（EF）和缩短分数（FS）。实验结果见图3，*
P < 0.05。
实施例4 有效改善心室重构：心脏重量和Masson染色：术后28天处死小鼠，测量小鼠体重（body weight，BW）、心脏重量（heart weight，HW）；计算HW/BW。Masson染色：术后28天处死小鼠，取左心室组织进行心脏组织染色，观察治疗效果。按常规Masson染色步骤进行，在普通光学显微镜下观察并拍照。采用图像分析软件Image J分析各部分面积，计算心梗面积/心脏面积。实验结果见图4，*P < 0.05，**P < 0.01。
实施例5 2-DG贴片没有副作用：术后第3天取肝脏肾脏，测ALT、AST和BUN，肝脏肾脏冰冻切片HE染色；术后连续检测小鼠体重和随机血糖。实验结果见图5，*P < 0.05，**P < 0.01，***P < 0.001，ns表示没有统计学差异。
【结论】上述实验的结果综合证明：2-DG贴片能有效提高MSCs改善心梗的功能。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
- 一种免疫代谢心梗贴片，其特征在于，包括贴片基质与2-脱氧-D-葡萄糖；2-脱氧-D-葡萄糖位于贴片基质内。
- 根据权利要求1所述免疫代谢心梗贴片，其特征在于，2-脱氧-D-葡萄糖的重量为贴片基质重量的35%～40%。
- 根据权利要求1所述免疫代谢心梗贴片，其特征在于，贴片基质由壳聚糖、明胶混合制备。
- 权利要求1所述免疫代谢心梗贴片的制备方法，其特征在于，将2-脱氧-D-葡萄糖、壳聚糖、明胶混合后干燥，得到免疫代谢心梗贴片。
- 根据权利要求4所述免疫代谢心梗贴片的制备方法，其特征在于，干燥为40℃～60℃干燥15～30小时。
- 根据权利要求5所述免疫代谢心梗贴片的制备方法，其特征在于，壳聚糖溶液与明胶溶液混合后，加入2-脱氧-D-葡萄糖，然后干燥，得到免疫代谢心梗贴片。
- 根据权利要求6所述免疫代谢心梗贴片的制备方法，其特征在于，壳聚糖、明胶和2-脱氧-D-葡萄糖的质量比为4∶（3.5～4.5）：（2.5～4）。
- 权利要求1所述免疫代谢心梗贴片在制备增效干细胞治疗心梗的生物材料中的应用或者在制备治疗心梗的生物材料中的应用。
- 权利要求1所述免疫代谢心梗贴片和干细胞在制备增效治疗心梗的药物中的应用。
- 权利要求1所述免疫代谢心梗贴片在制备低副作用增效干细胞治疗心梗的生物材料中的应用。
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