CN116459383B - 一种用于促进糖尿病伤口愈合的铈基mof水凝胶及其制备方法和应用 - Google Patents
一种用于促进糖尿病伤口愈合的铈基mof水凝胶及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种常温下铈基MOF水凝胶的生成工艺，并以此制备了一种用于促进糖尿病伤口愈合的3D打印铈基MOF水凝胶，并提供了其制备方法和应用。本发明使用铈基MOF的原位生成工艺实现了常温下铈基MOF水凝胶的3D打印，极大的简化了铈基MOF的的制备流程，具有尺寸稳定、规模化、操作简便及经济高效等优点。本发明通过3DMax软件设计任意尺寸的水凝胶模型，并导出STL格式用于生物打印机(STL)进行打印，打印出的水凝胶网络先进行紫外光的初步交联形成初级网络，之后进行离子溶液的浸泡同时完成最终的水凝胶网络交联及铈基MOF的原位合成。本发明的制备方法大大降低了铈基MOF的制备时间及成本，同时所打印的铈基MOF水凝胶也具有良好的生物相容性及抗菌性并且能有效的促进糖尿病伤口的愈合。
Description
技术领域
本发明属于生物材料技术领域，具体涉及一种用于促进糖尿病伤口愈合的铈基MOF水凝胶及其制备方法和应用。
背景技术
随着生活水平的日渐提高，全球糖尿病的患病人数不断增加。而糖尿病患者引发皮肤损伤后常常伴随着伤口的反复感染导致伤口愈合缓慢，严重者可能会造成截肢，脓毒血症等最终威胁到患者的生命安全。糖尿病患者由于其胰岛素的相对或绝对缺乏造成糖脂代谢异常，从而使其伤口创面长期处于高糖环境造成创面愈合过程中的氧化应激水平增高，过量的活性氧会导致创面促炎细胞增加，抗炎细胞减少，降低血液的抗感染能力，新生血管生成减少，造成循环障碍，血流量减少，肉芽组织生成减少，组织长期处于低氧状态，厌氧菌数量增多等，同时高糖环境也为细菌的定植、增长提供了有利环境。目前针对糖尿病伤口的治疗措施主要是在控制全身血糖的基础上进行，主要有清创法、负压伤口治疗(NPWT)、高压氧疗(HBOT)、自体富含血小板凝胶(APG)、光动力疗法(PDT)等。但由于受到设备、治疗费用、远期疗效等因素影响，现阶段最主要的治疗方式依旧为清创术。
伤口敷料作为清创术中重要的辅助材料，在保护创面和促进伤口愈合的过程中起到了重要的作用，传统敷料由于其抗感染性不强，无法提供湿性愈合环境等特点无法满足糖尿病伤口在清创术后的环境要求。水凝胶材料因具有高含水量的聚合物网络，与天然细胞外基质相似等特性，被人们视作良好的生物材料而广泛应用于组织工程，在伤口敷料方面有着广泛的应用前景。并且基于水凝胶材料合成的一系列敷料被认为是促进糖尿病伤口愈合的有效方法。然而现阶段的水凝胶材料多通过天然或人工材料附载药物或生长因子实现促进糖尿病伤口愈合的效果，但因其生产成本较高，生产工艺复杂，难以实现大规模普及。同时由于人体对于药物的敏感程度不同，异源性生长因子可能产生的免疫排斥反应等因素也限制了该类敷料的应用范围。
金属-有机骨架(Metal-Organic Framework，MOF)，是一种有机配体与金属离子自组装形成的一种具有周期性网络结构的骨架材料，因其具有高结晶度，高吸附性、高孔隙率等特性已经在吸附、催化等方面表现出广泛的应用前景。铈(Cerium，Ce)作为地表含量最多的稀土元素，在各个领域内有着较为广泛的应用前景，William T.Self等研究发现铈离子不同价态间的相互转化可以产生类SOD和类过氧化物酶的特性，Wu等发现Ce4+4f轨道上的低能量状态也更有利于其催化特性的表达，Zhang等发现氧化铈纳米颗粒具有优良的抗菌特性，例如，发明专利CN108969757B公开了负载microRNA的氧化铈纳米复合水凝胶，复合水凝胶包括交联后的胶原蛋白，分散在胶原蛋白内的阳离子聚合物修饰的氧化铈纳米晶；所述阳离子聚合物修饰的氧化铈纳米晶通过静电吸附作用负载有microRNA。该负载microRNA的氧化铈纳米复合水凝胶具备良好的清除活性氧自由基作用，不仅可以减少创伤部位的氧化应激损伤，同时抑制氧化微环境对负载的microRNA的破坏作用，提高microRNA激活血管新生的活性。但现阶段铈基MOF的主要是通过DMF作为去质子剂在高温条件下制得，时间长，成本高，操作复杂，无法一步合成在水凝胶中的分散程度较差。
如何在常温下制备一种铈基MOF水凝胶，且原位合成均匀分散，步骤简单，成本低廉，制备得到的水凝胶具有良好的生物相容性，优秀的抗菌性能，能够有效的促进糖尿病伤口的愈合，降低糖尿病的致残性和致死率，是本领域技术人员亟待解决的技术问题。
发明内容
针对上述技术问题，本发明提供了一种用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，包括如下步骤：
(1)将海藻酸钠和丙烯酰胺溶解于去离子水中，得到初级水凝胶溶液；
(2)在步骤(1)所得溶液中加入光引发剂和N,N'-亚甲基双丙烯酰胺，避光搅拌；
(3)将1’3’5-苯三甲酸和三乙胺加入步骤(2)所得溶液中，避光搅拌过夜；
(4)将步骤(3)所得混合物装入3D打印料筒中，3D打印机进行打印，打印过程中进行初级交联；
(5)打印完成后将步骤(4)所得样品再次进行交联，得到水凝胶；
(6)将步骤(5)制得水凝胶浸泡于铈离子溶液中，过夜保存，即得。
优选的，步骤(1)所述的加入海藻酸钠和丙烯酰胺的物质的量比例为1～3：2～6；海藻酸钠终浓度为1.5％～3％。
优选的，步骤(2)所述的光引发剂为I2959，I1173，I907和二苯甲酮中的任一种，终浓度为0.4～0.8wt％；所述的N,N'-亚甲基双丙烯酰胺的浓度为步骤(1)所述丙烯酰胺6～8‰。
优选的，步骤(2)所述的光引发剂为2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮(I2959)。
优选的，步骤(3)所述的1’3’5-苯三甲酸和三乙胺物质的量为0.1～1:0.3～3；1’3’5-苯三甲酸的终浓度为0.1～1％(w：v)。
优选的，步骤(4)所述的初级交联和步骤(5)所述的交联均采用物理交联。
优选的，所述的物理交联为采用UV灯光光照交联，波长范围为296-325nm。
优选的，步骤(6)所述的铈离子溶液浓度为10～50mg/mL。
本发明的第二目的是提供一种用于促进糖尿病伤口愈合的铈基MOF水凝胶，通过所述的制备方法制备得到。
本发明的第三目的是提供所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法制备得到的产品和/或所述的铈基MOF水凝胶在制备促进糖尿病伤口愈合药物中的应用。
本发明的有益效果是：(1)本发明改善传统辅料制作繁琐，模式固定且难以更改的缺陷等问题，创造性的发明了一种基于3D打印技术快速、批量化、规模化、个性化制作铈基MOF水凝胶的方法。
(2)本发明提供一种常温下铈基MOF原位合成水凝胶的3D打印技术，实现了铈基MOF水凝胶的快速高效、批量化、规模化、个性化的制作，该方法具有经济高效、操作简单、模式灵活、高度可重复性的优点。
(3)改善铈基MOF的合成步骤过于繁琐，成本较高，水凝胶中的分散性较差等问题，提供了一种常温下步骤简单，成本低廉，原位合成均匀分散的铈基MOF水凝胶及其制备方法，该水凝胶具有良好的生物相容性，优秀的抗菌性能，能够有效的促进糖你尿病伤口的的愈合.
(4)所制备的铈基MOF水凝胶采用海藻酸钠-丙烯酰胺双网络结构，丙烯酰胺提供水凝胶初级网络的支撑作用，海藻酸钠起到提供MOF附着位点和终网支撑的双重作用，原位合成的铈基MOF则负责提供抗菌性能及促进糖尿病伤口愈合的双重作用，同时MOF的存在还可以辅助增强水凝胶的物理性能。
(5)本发明促进糖尿病伤口愈合的常温下3D打印铈基MOF水凝胶的制备为铈基MOF水凝胶的批量化、规模化生产提供了可能并探索了一套最简便、省时的常温下铈基MOF水凝胶的生产工艺流程。
附图说明
下面结合附图对本发明作进一步详细的描述。
图1制备得到的铈基MOF水凝胶实物图
注：A：MVC-MOF水凝胶(黄色)；B：Ce-BTC水凝胶(白色)
图2铈基MOF水凝胶抗菌效果图注：A为水凝胶对大肠杆菌及金黄色葡萄球菌的抗菌率；B为水凝胶对大肠杆菌及金黄色葡萄球菌共培养前后菌落实物图。
图3铈基MOF水凝胶治疗糖尿病小鼠伤口效果图
图4铈基MOF水凝胶的H&E染色图
图5铈基MOF水凝胶的PAS染色图
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明提供的铈基MOF水凝胶，以质量为基准，3D打印墨水里水：海藻酸钠：丙烯酰胺：1’3’5-苯三甲酸的质量比为(100～1000):(1～30):(2～60):(0.1～10)。
实施例1：CBH水凝胶的制备
(1)将海藻酸钠(3g)和丙烯酰胺(6g)溶解于去离子水(100mL)中，得到初级水凝胶溶液；
(2)在步骤(1)所得溶液中加入光引发剂(0.4g)和N,N'-亚甲基双丙烯酰胺(0.036g)，避光搅拌；
(3)将1’3’5-苯三甲酸(1g)和三乙胺(4mL)加入步骤(2)所得溶液中，避光搅拌过夜；
(4)将步骤(3)所得混合物装入3D打印料筒中，3D打印机进行打印，打印过程中使用波长范围在296-325nm的UV灯光照10～30min进行初级交联；
(5)打印完成后将步骤(4)所得样品再次使用波长范围在296-325nm的UV灯光照10～30min进行交联，得到水凝胶；
(6)将步骤(5)制得水凝胶浸泡于铈离子(Ce3+)溶液(50mg/mL)中，过夜保存，即得。
实施例2：C3H水凝胶的制备
该实施例与实施例1相同，不同之处在于：无步骤S3。
实施例3：MMH水凝胶的合成
该实施例与实施例1相同，不同之处在于：取步骤S7所得样品置于H2O2浸泡，铈离子溶液中保存。
实施例4：C4H水凝胶的合成
该实施例的制备方法与实施例3相同，不同之处在于：无步骤S3，步骤(6)所述的铈离子溶液为Ce4+溶液。
实施例5水凝胶的实物图
实施例1和实施例3制备得到的铈基MOF水凝胶实物图如图1所示，从图中可以看出，实施例1制备得到的铈基MOF水凝胶颜色为透明状；实施例3制备得到的浸泡H2O2溶液的铈基MOF水凝胶颜色变为橙色。
实施例6水凝胶的抗菌性能
将实施例1-4中的CBH、MMH、C3H和C4H作为实施对象，用于以下实验。
实验方法如下：
本实验采用平板计数法检测CBH、MMH、C3H和C4H的抗菌性能。在Luria Bertani液体培养基上培养大肠杆菌和金黄色葡萄球菌。首先用无菌接种环挑取适量大肠杆菌和金黄色葡萄球菌菌落在装有适量液体培养基的离心管中摇晃接种，制作菌悬液，用作实验用菌液。在旋转振荡器上以50r/min于37℃条件下培养24h。通过磷酸盐缓冲溶液(PBS)分别将两个菌株稀释103倍，实验环境为无菌。分别用Ce-BTC和MVC-MOF水凝胶与菌液混合，未加入任何材料的菌液作为对照组，37℃下进行共培养。24h后每组取100μL菌液，稀释至106倍。取200μL菌种稀释液，将其涂布在LB琼脂平板上，每组涂3个平板。将涂布好的平板置于37℃恒温培养箱中培养24h，观察并计数每个平板上的菌落数(即菌落形成单位：CFU)。由此计算出大肠杆菌和金黄色葡萄球菌的失活率，并以此来表示样品的抗菌性能。其计算公式如下：
抗菌率＝((N-N’)/N)×100％
其中，N为对照组(未加入样品的大肠杆菌和金黄色葡萄球菌悬液)的菌落数；N’为实验组(加入样品的大肠杆菌和金黄色葡萄球菌悬液)的菌落数。
实验结果：如图2所示，CBH、MMH和C3H均为完全抗菌，C4H能杀死92％的大肠杆菌和99％的金黄色葡萄球菌。
实施例6铈基MOF水凝胶促进糖尿病伤口愈合的效果
受试对象：雄性Waster大鼠，6至8周龄，体重180-220g，购于兰州大学生物医学实验中心，应用STZ法建立大鼠糖尿病动物模型，建模成功后在大鼠背部制备大小为1*1cm2的糖尿病伤口模型。
实验分组：将建立的糖尿病伤口大鼠按治疗方式分为空白对照组(3只)、CBH组(3只)、C3H组(3只)、MMH组(3只)和C4H组(3只)。
治疗方案：空白对照组不予任何处理，治疗组分别使用本发明实施例1、实施例2、实施例3与实施例4的3D打印水凝胶处理，每周1次，治疗周期为21天。
疗效评价：分别于用药后7天、14天、21天观察背部伤口愈合情况。21天后处死大鼠，切背部伤口，放置于4％多聚甲醛内固定,酒精脱水后二甲苯透明石蜡包埋、切片，行H&E及PAS染色，观察各组大鼠皮肤组织病理变化。
实验结果：如图3所示，大体观显示7d时CBH组的伤口愈合程度明显快于另外四组，从14d开始MMH组的伤口愈合程度渐渐接近于CBH组，且明显高于另外三组，至第21d，CBH组基本完全愈合，MMH组愈合率达92％，其余组愈合率为80％左右。
如图4所示，H&E染色显示结果与大体观结果基本相同，7d只有CBH组形成了较为连续的上皮组织，并且炎症细胞明显少于另外四组，14d时CBH组形成了初级腺泡结构且纤维结缔组织较致密，MMH组的炎症细胞明显减少，纤维结缔组织致密，21d时CBH组基本形成了正常的皮肤组织，上皮薄，有成熟腺泡组织，也存在毛细血管，MMH组的上皮组织变薄，皮下结缔组织致密，表面渗出物减少，反观对照组、C3H组、C4H组仅仅形成了连续的上皮组织和相对致密的纤维结缔组织并且依旧存在部分炎症细胞。
如图5所示，PAS染色显示7d所有组的伤口均表现为高糖环境，14d时MMH组的伤口含糖量较其他组明显减少，21d除了对照组和C3H组以外另外三组的伤口环境的含糖量均有所降低。
本发明公开了一种基于3D打印技术常温下制备的铈基MOF水凝胶，并在此基础上制备了用于促进糖尿病伤口愈合的伤口敷料，是海藻酸钠、丙烯酰胺、1’3’5-苯三甲酸、三乙胺混合溶液先通过3D打印和UV预交联，然后再浸泡铈离子溶液交联并原位合成铈基MOF形成的复合材料，并提供了其制备方法和应用。本发明的有益效果为：本发明采用3D打印技术制作的铈基MOF水凝胶，大大降低了传统辅料在制备过程中模式单一，人工成本高等问题，具有经济高效、操作简单、模式灵活、个性化等优点。同时本发明基于3D打印常温下制备的铈基MOF水凝胶，也解决了传统铈基MOF制备过程中步骤繁琐，水凝胶中的分散性差等问题，除此之外，原位合成的铈基MOF可以提供增强抗菌、促进糖尿病伤口愈合等作用。故本发明常温下3D打印铈基MOF水凝胶对于铈基MOF水凝胶批量化、规模化生产提供了可能并探索了一套最简便、省时的工艺流程。
最后应说明的是：以上仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，包括如下步骤：
(1)将海藻酸钠和丙烯酰胺溶解于去离子水中，得到初级水凝胶溶液；
(2)在步骤(1)所得溶液中加入光引发剂和N,N'-亚甲基双丙烯酰胺，避光搅拌；
(3)将1’3’5-苯三甲酸和三乙胺加入步骤(2)所得溶液中，避光搅拌过夜；
(4)将步骤(3)所得混合物装入3D打印料筒中，3D打印机进行打印，打印过程中进行初级交联；
(5)打印完成后将步骤(4)所得样品再次进行交联，得到水凝胶；
(6)将步骤(5)制得水凝胶浸泡于铈离子溶液中，过夜保存，即得。
2.如权利要求1所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，步骤(1)所述的加入海藻酸钠和丙烯酰胺的物质的量比例为1～3：2～6；海藻酸钠终浓度为1.5％～3％。
3.如权利要求1所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，步骤(2)所述的光引发剂为2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮(I2959)，I1173，I907和二苯甲酮的任一种，终浓度为0.4～0.8wt％；所述的N,N'-亚甲基双丙烯酰胺的浓度为步骤(1)所述丙烯酰胺6～8‰。
4.如权利要求1所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，步骤(2)所述的光引发剂为2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮(I2959)。
5.如权利要求1所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，步骤(3)所述的1’3’5-苯三甲酸和三乙胺物质的量为0.1～1:0.3～3；1’3’5-苯三甲酸的终浓度为0.1～1％(w：v)。
6.如权利要求1所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，步骤(4)所述的初级交联和步骤(5)所述的交联均采用物理交联。
7.如权利要求6所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，所述的物理交联为采用UV灯光光照交联，波长范围为296-325nm。
8.如权利要求6所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法，其特征在于，步骤(6)所述的铈离子溶液浓度为20～50mg/mL。
9.一种用于促进糖尿病伤口愈合的铈基MOF水凝胶，其特征在于，通过权利要求1-8任一项所述的制备方法制备得到。
10.如权利要求1-8任一所述的用于促进糖尿病伤口愈合的铈基MOF水凝胶的制备方法制备得到的产品和/或如权利要求9所述的铈基MOF水凝胶在制备促进糖尿病伤口愈合药物中的应用。
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