WO2022127662A1 - 一种冰冻微针阵列及其制备方法和应用 - Google Patents

一种冰冻微针阵列及其制备方法和应用 Download PDF

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WO2022127662A1
WO2022127662A1 PCT/CN2021/136345 CN2021136345W WO2022127662A1 WO 2022127662 A1 WO2022127662 A1 WO 2022127662A1 CN 2021136345 W CN2021136345 W CN 2021136345W WO 2022127662 A1 WO2022127662 A1 WO 2022127662A1
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microneedle
frozen
holes
raw material
material solution
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PCT/CN2021/136345
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English (en)
French (fr)
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赵远锦
张筱萱
王月桐
张大淦
商珞然
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南京鼓楼医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0023—Drug applicators using microneedles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0046—Solid microneedles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0053—Methods for producing microneedles

Definitions

  • the invention relates to the technical field of biomedical materials, in particular to a frozen microneedle array and a preparation method and application thereof.
  • the microneedle array is mainly composed of a base and micron-sized needle tips, which can penetrate the epidermis without touching the capillaries and nerve endings, so it can significantly enhance the skin permeability and drug absorption rate, providing a micro Invasive, painless transdermal drug delivery.
  • the field of microneedling has developed rapidly, however, it still faces some challenges.
  • the material constituting the microneedle array needs to have a certain mechanical strength to pierce the epidermis, which greatly limits the choice of materials, such as some materials with high water content cannot be used.
  • microneedles can be used to carry and deliver some biologically active substances, but these biologically active substances are often easily inactivated, and have different requirements on the environment and preparation process. Therefore, a universal microneedle that can carry a variety of biologically active substances needs to be developed urgently.
  • Water can form hard ice after freezing, and the freezing process in nature is a typical example of softening into rigidity.
  • the hardness of the water-containing material constituting the microneedle array is significantly increased, enough to penetrate the epidermis, breaking the material limitation of the microneedle.
  • many biologically active substances are stored in a freezing environment and resume activity after recovery at room temperature. Therefore, frozen microneedles will not cause damage to these biologically active substances, and are expected to become a universal delivery platform for different biologically active substances.
  • the technical problem to be solved by the present invention is to provide a frozen microneedle array and a preparation method and application thereof in view of the above-mentioned deficiencies of the prior art.
  • the material is limited, and the versatility is good.
  • the technical solution adopted in the present invention is: a preparation method of a frozen microneedle array, comprising the following steps:
  • the holes of the hole array are inverted cones or inverted regular quadrangular pyramids.
  • the hole spacing is 200-800 ⁇ m
  • the hole depth is 300-850 ⁇ m
  • the hole diameter is 150-600 ⁇ m
  • the depth of the hole is 300-850 ⁇ m
  • the length of the bottom of the hole is 150-600 ⁇ m.
  • the microneedle raw material solution is selected from one or more materials selected from methacrylate gelatin, methylated hyaluronic acid, methylated alginic acid, and methylated silk fibroin,
  • the microneedle raw material solution is cured by mixing a photoinitiator with a volume fraction of 1% and irradiating it with ultraviolet light for 20s to 1min, and the photoinitiator is 2-hydroxy-2-methylpropiophenone.
  • the microneedle raw material solution is selected from sodium alginate material, and the microneedle raw material solution is solidified by mixing the calcium chloride solution with an excess volume fraction of 10%, and sucking off the upper layer calcium chloride solution after fully reacting. .
  • step S1 the microneedle raw material liquid is selected from Matrigel, and the microneedle raw material liquid is cured by standing at 37° C. for a period of time.
  • step S1 the method for fully filling the pores with the microneedle raw material solution is centrifugation at 1000 rpm for 3 min, or vacuum treatment for 5 min.
  • step S3 the conditions of the freezing treatment are freezing below -20°C for more than 12 hours, and the storage temperature of the frozen microneedle array is below 0°C.
  • the present invention also provides a frozen microneedle array, which is prepared by the above-mentioned preparation method of the frozen microneedle array.
  • the present invention also provides the application of the above frozen microneedle array in the preparation of drug-loaded frozen microneedles, wherein the drug-loaded frozen microneedles are biologically active substances carried inside the microneedles of the frozen microneedle array, and the biologically active substances include small Molecules, proteins, probiotics.
  • the present invention provides a frozen microneedle array, which can be made of a variety of water-containing materials such as hydrogel, while maintaining a certain mechanical strength, breaking the limitation of materials on traditional microneedles.
  • the frozen microneedle array proposed by the present invention can carry a variety of biologically active substances, including small molecules, proteins, and probiotics, and ensures the activity of the biologically active substances, and can be used in many biomedical fields.
  • the present invention provides a method for preparing a frozen microneedle array by adding a freezing process during the template replication process.
  • the method is simple, easy to operate, has low technical requirements, and freezes
  • the process has almost no harm to the vitality of biologically active substances, and has universality, which is conducive to popularization and industrialized preparation.
  • Fig. 1 is the preparation flow chart of the frozen microneedle array of the present invention
  • Fig. 2 is the structure diagram of the frozen methacrylate gelatin microneedle carrying Rhodamine B according to the embodiment of the present invention 1;
  • Fig. 3 is the actual picture of the frozen calcium alginate microneedle carrying green fluorescent labeled insulin in Example 2 of the present invention
  • Fig. 4 is the fluorescence image after the frozen calcium alginate microneedle carrying green fluorescent labeled insulin in Example 2 of the present invention punctured the mouse skin;
  • A is the frozen methacrylate gelatin microneedle carrying rhodamine B in Example 1; B is the frozen microneedle carrying green fluorescent labeled insulin in Example 2 Calcium alginate microneedles, C is the frozen Matrigel microneedles carrying Bacillus subtilis of Example 3.
  • microneedle raw material solution 1, polydimethylsiloxane template 2, gauze 3.
  • the present invention provides a frozen microneedle array, see FIG. 1 , and its preparation includes the following steps:
  • microneedle raw material solution 1 Dropwise to the surface of the polydimethylsiloxane template 2 with the hole array, and make it fully fill the holes of the polydimethylsiloxane template 2, and remove the holes other than the holes. Excess microneedle stock solution 1, and then solidify the microneedle stock solution 1 in the hole array;
  • the holes of the hole array are inverted conical or inverted regular quadrangular pyramid; when the holes are inverted conical holes, the hole spacing is 200-800 ⁇ m, the hole depth is 300-850 ⁇ m, and the hole diameter is 150-600 ⁇ m; For regular quadrangular pyramid-shaped holes, the hole spacing is 200-800 ⁇ m, the hole depth is 300-850 ⁇ m, and the bottom side length of the hole is 150-600 ⁇ m.
  • the method for fully filling the microneedle raw material liquid 1 in the holes is centrifugation at 1000 rpm for 3 minutes, or vacuum treatment for 5 minutes;
  • the microneedle raw material solution 1 is selected from one or both of methacrylate gelatin, methylated hyaluronic acid, methylated alginic acid, methylated silk fibroin, sodium alginate, and matrigel. the above materials;
  • the microneedle raw material solution 1 is selected from one or more materials selected from methacrylate gelatin, methylated hyaluronic acid, methylated alginic acid, and methylated silk fibroin
  • the microneedle raw material solution 1 is mixed by mixing The photoinitiator with a volume fraction of 1% is cured by ultraviolet irradiation for 20s to 1min, and the photoinitiator is 2-hydroxy-2-methylpropiophenone
  • the microneedle raw material solution 1 is made of sodium alginate, the microneedle The raw material solution 1 is solidified by mixing the calcium chloride solution with an excess volume fraction of 10%, and sucking off the upper layer of calcium chloride solution after sufficient reaction
  • the microneedle raw material solution 1 is selected from Matrigel, the microneedle raw material solution 1 passes It was cured by standing at 37°C for a period of time.
  • the present invention also provides the application of the above frozen microneedle array in the preparation of drug-loaded frozen microneedles.
  • the biologically active substances include small molecules, proteins, and probiotics.
  • a frozen methacrylate gelatin microneedle carrying Rhodamine B the preparation of which comprises the following steps:
  • a frozen calcium alginate microneedle carrying green fluorescent labeled insulin the preparation of which comprises the following steps:
  • a frozen matrigel microneedle carrying Bacillus subtilis the preparation of which comprises the following steps:
  • Example 4 Mechanical strength verification of the frozen microneedle array prepared by the present invention
  • the needle tip of the frozen microneedle array has a certain mechanical strength, which can pierce the mouse skin and release the drug in it, which gets rid of the limitation of the material selection of the microneedle.
  • Example 5 Drug-loading verification of the frozen microneedle array prepared by the present invention
  • Example 1 In order to verify that the frozen microneedle array prepared by the present invention can effectively carry biologically active substances, we used the frozen methacrylate gelatin microneedles carrying rhodamine B in Example 1 and the frozen seaweed carrying green fluorescent labeled insulin in Example 2. The calcium carbonate microneedles were observed under a fluorescence microscope, and the results are shown in A and B in Figure 5.

Abstract

一种冰冻微针阵列及其制备方法和应用，基于冰冻可以使含水物质由软变硬的原理，冰冻微针阵列通过在模板复制过程中引入冰冻工艺的方法制备得到，操作简单、便于推广、能够大规模生产，制备的冰冻微针阵列可以由各种溶液或水凝胶制备而成，摆脱了微针在材料选择上的限制，此外，冰冻工艺对生物活性物质的活力几乎没有伤害，因此，这种冰冻微针有能力搭载多种生物活性物质，更具有通用性，有望用于多种生物及医学领域。

Description

一种冰冻微针阵列及其制备方法和应用 技术领域
本发明涉及生物医学材料技术领域，尤其涉及一种冰冻微针阵列及其制备方法和应用。
背景技术
微针阵列主要由一个基底和微米级大小的针尖构成，其可以在不接触毛细血管和神经末梢的同时穿透表皮层，因此可以显著增强皮肤的渗透性和药物吸收率，提供了一种微创、无痛的经皮给药方式。近年来，微针领域得到迅速的发展，然而仍面临着一些挑战。首先，构成微针阵列的材料需要有一定的机械强度以刺破表皮，这大大局限了材料的选择范围，如一些含水量较高的材料就无法使用。另外，微针可以用来搭载并递送一些生物活性物质，但这些生物活性物质往往易失活，对所处环境及制备过程要求高且不尽相同。因此，一种可以搭载多种生物活性物质的通用微针亟需被开发出来。
水在冰冻后可以形成坚硬的冰，自然界中的结冰过程就是化柔为刚的一个典型的例子。受此启发，将微针阵列进行冰冻处理后，组成微针阵列的含水材料的硬度显著增加，足够穿破表皮，打破了材料方面对微针的限制。另外，许多生物活性物质在冰冻环境下保存，常温复苏后恢复活性，因此，冰冻微针不会对这些生物活性物质造成损害，有望成为不同生物活性物质的通用递送平台。
发明内容
本发明所要解决的技术问题是针对上述现有技术的不足，提供一种冰冻微针阵列及其制备方法和应用，冰冻微针阵列在通过模板复制中引入冰冻工艺制得，制备的微针摆脱了材料的限制，通用性好。
为实现上述技术目的，本发明采取的技术方案为：一种冰冻微针阵列的制备方法，包括以下步骤：
S1、将微针原料液滴加到具有孔洞阵列的聚二甲基硅氧烷模板表面，并使其充分填充在聚二甲基硅氧烷模板的孔洞中，去除孔洞之外的多余微针原料液，然后固化孔洞阵列中微针原料液；
S2、将一层湿润的纱布进一步覆盖在聚二甲基硅氧烷模板的孔洞阵列上面，作为微针基底；
S3、将聚二甲基硅氧烷模板、微针原料液和微针基底整体置于低温下进行冰冻处理，然后将冰冻后的材料从聚二甲基硅氧烷模板中剥离，得到冰冻微针阵列。
进一步地，所述孔洞阵列的孔洞为倒圆锥形或倒正四棱锥形。
进一步地，当孔洞为倒圆锥形孔洞时，孔洞间距为200-800μm，孔洞深度为300-850μm，孔洞直径为150～600μm；当孔洞为倒正四棱锥形孔洞时，孔洞间距为200～800μm，孔洞深度为300～850μm，孔洞底边长为150～600μm。
进一步地，步骤S1中，所述微针原料液选自甲基丙烯酸酯明胶、甲基化透明质酸、甲基化海藻酸、甲基化丝素蛋白中的一种或两种以上材料，微针原料液通过混合体积分数为1％的光引发剂并紫外照射20s～1min而固化，所述光引发剂为2-羟基-2-甲基苯丙酮。
进一步地，步骤S1中，所述微针原料液选用海藻酸钠材料，微针原料液通过混合过量体积分数为10％的氯化钙溶液，待充分反应后吸除上层氯化钙溶液而固化。
进一步地，步骤S1中，所述微针原料液选自基质胶，微针原料液通过在37℃下静置一段时间而固化。
进一步地，步骤S1中，微针原料液充分填充在孔洞中的方法为1000rpm离心3min，或真空处理5min。
进一步地，步骤S3中，冰冻处理的条件为-20℃以下冰冻12h以上，冰冻微针阵列的保存温度为0℃以下。
本发明还提供了一种冰冻微针阵列，采用上述冰冻微针阵列的制备方法制得。
本发明还提供了上述冰冻微针阵列在制备载药冰冻微针上的应用，所述载药冰冻微针为生物活性物质搭载在冰冻微针阵列的微针内部，所述生物活性物质包括小分子、蛋白、益生菌。
与现有技术相比，本发明的有益效果在于：
一、本发明提供了一种冰冻微针阵列，其可以由水凝胶等多种含水材料制成，同时维持一定的机械强度，打破了材料对传统微针的限制。
二、本发明提出的冰冻微针阵列可搭载多种生物活性物质，包括小分子、蛋白、益生菌，且保证生物活性物质的活性，可用于诸多生物医学领域。
三、本发明基于冰冻可以使含水物质由软变硬的原理，提供了一种通过在模板复制过程中添加冰冻工艺制备冰冻微针阵列的方法，方法简便、容易操作、技术要求低，而且冰冻工艺对生物活性物质的活力几乎没有伤害，具有通用性，利于推广和工业化制备。
附图说明
图1为本发明的冰冻微针阵列的制备流程图；
图2为本发明实施例1的搭载罗丹明B的冰冻甲基丙烯酸酯明胶微针结构图；
图3为本发明实施例2的搭载绿色荧光标记胰岛素的冰冻海藻酸钙微针的实物图；
图4为本发明实施例2的搭载绿色荧光标记胰岛素的冰冻海藻酸钙微针穿刺鼠皮后的荧光图；
图5为本发明的冰冻微针阵列的俯视荧光图：其中，A为实施例1的搭载罗丹明B的冰冻甲基丙烯酸酯明胶微针；B为实施例2的搭载绿色荧光标记胰岛素的冰冻海藻酸钙微针，C为实施例3的搭载枯草芽孢杆菌的冰冻基质胶微针。
其中的附图标记为：微针原料液1、聚二甲基硅氧烷模板2、纱布3。
具体实施方式
为了使本领域技术领域人员更好地理解本发明的技术方案，下面结合附图对本发明的实施例作进一步详细描述。
下述实施例中所使用的实验方法，如无特殊说明，均为常规方法，所用的试剂、方法和设备，如无特殊说明，均为本技术领域常规试剂、方法和设备。
本发明提供了一种冰冻微针阵列，参阅图1，其制备包括以下步骤：
S1、将微针原料液1滴加到具有孔洞阵列的聚二甲基硅氧烷模板2表面，并使其充分填充在聚二甲基硅氧烷模板2的孔洞中，去除孔洞之外的多余微针原料液1，然后固化孔洞阵列中微针原料液1；
其中，孔洞阵列的孔洞为倒圆锥形或倒正四棱锥形；当孔洞为倒圆锥形孔洞时，孔洞间距为200-800μm，孔洞深度为300-850μm，孔洞直径为150～600μm；当孔洞为倒正四棱锥形孔洞时，孔洞间距为200～800μm，孔洞深度为300～850μm，孔洞底边长为150～600μm。
进一步，微针原料液1充分填充在孔洞中的方法为1000rpm离心3min，或真空处理5min；
S2、将一层湿润的纱布3进一步覆盖在聚二甲基硅氧烷模板2的孔洞阵列上面，作为微针基底；
S3、将聚二甲基硅氧烷模板2、微针原料液1和微针基底整体置于低温下进行冰冻处理，冰冻处理条件为-20℃以下冰冻12h以上，然后将冰冻后的材料从聚二甲基硅氧烷模板2中剥离，得到冰冻微针阵列，冰冻微针阵列在0℃以下保存。
步骤S1中，微针原料液1选自甲基丙烯酸酯明胶、甲基化透明质酸、甲基化海藻酸、甲基化丝素蛋白、海藻酸钠、基质胶中的一种或两种以上材料；
当微针原料液1选自甲基丙烯酸酯明胶、甲基化透明质酸、甲基化海藻酸、甲基化丝素蛋白中的一种或两种以上材料，微针原料液1通过混合体积分数为1％的光引发剂并紫外照射20s～1min而固化，所述光引发剂为2-羟基-2-甲基苯丙酮；当微针原料液1选用海藻酸钠材料时，微针原料液1通过混合过量体积分数为10％的氯化钙溶液，待充分反应后吸除上层氯化钙 溶液而固化；当微针原料液1选自基质胶时，微针原料液1通过在37℃下静置一段时间而固化。
本发明还提供了上述冰冻微针阵列在制备载药冰冻微针上的应用，载药冰冻微针为生物活性物质搭载在冰冻微针阵列的微针内部，具体为生物活性物质混合在微针原料液中，所述生物活性物质包括小分子、蛋白、益生菌。
实施例1
一种搭载罗丹明B的冰冻甲基丙烯酸酯明胶微针，其制备包括如下步骤：
S1、配置载药微针原料液A：配置含有质量分数为15％的甲基丙烯酸酯明胶和体积分数为1％的2-羟基-2-甲基苯丙酮的水溶液，并向其中混合1mg/mL的罗丹明B；
S2、制备载药冰冻微针阵列
S21、用移液枪将100μl载药微针原料液A滴加在具有倒圆锥形孔洞阵列(孔洞间距为200μm，孔洞深度为300μm，孔洞直径为150μm)的聚二甲基硅氧烷模板表面，真空处理5min，使其充分填充在倒圆锥形孔洞中，用移液枪吸走倒圆锥形孔洞至外的多余载药微针原料液A，紫外照射20s，使倒圆锥形孔洞内的载药微针原料液A固化，得到载药尖端A；
S22、将一层湿润的纱布覆盖在上述聚二甲基硅氧烷模板的孔洞阵列上面，作为微针基底；
S23、将聚二甲基硅氧烷模板、载药尖端A和微针基底整体置于-80℃冰冻12h，然后将冰冻后的材料从聚二甲基硅氧烷模板中剥离，即得到如图2所示的搭载罗丹明B的冰冻甲基丙烯酸酯明胶微针，并在0℃以下保存待用。
实施例2
一种搭载绿色荧光标记胰岛素的冰冻海藻酸钙微针，其制备包括如下步骤：
S1、配置载药微针原料液B：配置含有质量分数为4％的极低黏度海藻酸钠溶液，并向其中混合绿色荧光标记胰岛素(最终含量0.5mg/mL)；
S2、制备载药冰冻微针阵列
S21、用移液枪将400μl载药微针原料液B滴加在具有倒圆锥形孔洞阵列(孔洞间距为700μm，孔洞深度为850μm，孔洞直径为600μm)的聚二甲基硅氧烷模板表面，通过1000rpm离心3min，使其充分填充在倒圆锥形孔洞中，用移液枪吸走倒圆锥形孔洞至外的多余载药微针原料液B，向孔洞表面滴加400μl质量分数为10％的氯化钙溶液，待充分反应15min后，吸走氯化钙溶液，载药微针原料液B固化成型，得到载药尖端B；
S22、将一层湿润的纱布覆盖在上述聚二甲基硅氧烷模板的孔洞阵列上面，作为微针基底；
S23、将聚二甲基硅氧烷模板、载药尖端B和微针基底整体置于-20℃冰冻48h，然后将冰冻后的材料从聚二甲基硅氧烷模板中剥离，得到如图3所示的搭载绿色荧光标记胰岛素的冰冻海藻酸钙微针，并在0℃以下保存待用。
实施例3
一种搭载枯草芽孢杆菌的冰冻基质胶微针，其制备包括如下步骤：
S1、配置载药(活体微生物)微针原料液C：将枯草芽孢杆菌(购自北纳生物有限公司)混合在基质胶(美国康宁(corning)公司)中，其中枯草芽孢杆菌的最终含量为10 8/mL；
S2、制备载药冰冻微针阵列
S21、用移液枪将300μl载药微针原料液C滴加在具有倒正四棱锥形孔洞阵列(孔洞间距为400μm，孔洞深度为800μm，孔洞直径为600μm)的聚二甲基硅氧烷模板表面，通过1000rpm离心3min，使其充分填充在倒正四棱锥形孔洞中，用移液枪吸走倒正四棱锥形形孔洞至外的多余载药微针原料液C，将其在37℃下放置20min，载药微针原料液C固化成胶，得到载药尖端C；
S22、将一层湿润的纱布覆盖在上述聚二甲基硅氧烷模板的孔洞阵列上面，作为微针基底；
S23、将聚二甲基硅氧烷模板、载药尖端C和微针基底整体置于-80℃冰冻24h，然后将冰冻后的材料从聚二甲基硅氧烷模板中剥离，即得到载枯草芽孢杆菌的冰冻基质胶微针，并在0℃以下保存待用。
实施例4：本发明制备的冰冻微针阵列的机械强度验证
为了验证本发明制备的冰冻微针阵列有足够的机械强度并能刺穿皮肤，我们将小白鼠背部备皮，并将实施例2制备的搭载绿色荧光标记胰岛素的冰冻海藻酸钙微针倒扣在酒精擦拭过的小白鼠背部皮肤上；随后立刻用10N的力按压2min，待冰融化后移去微针基底。
如图4所示的鼠皮荧光照片可见，冰冻微针阵列的针尖具有一定的机械强度，可以刺穿鼠皮并释放其中的药物，摆脱了微针在材料选择上的限制。
实施例5：本发明制备的冰冻微针阵列的载药性验证
为了验证本发明制备的冰冻微针阵列可以有效地搭载生物活性物质，我们将实施例1的搭载罗丹明B的冰冻甲基丙烯酸酯明胶微针、实施例2的搭载绿色荧光标记胰岛素的冰冻海藻酸钙微针置于荧光显微镜下观察，结果如图5中A和B所示。
由图5中A和B可见，两种冰冻微针均显示出较强的荧光，说明本申请的冰冻微针阵列可成功负载药物并保存其荧光性质。
进一步地，我们将实施例3的搭载枯草芽孢杆菌的冰冻基质胶微针重新倒扣在聚二甲基硅氧烷模板上，恢复至室温后浸泡在含有活细菌染色剂SYTO(体积分数0.1％)的磷酸盐缓冲液中30min，并随后置于荧光共聚焦显微镜下观察，结果如图5中C所示。
由图5中C可见，枯草芽孢杆菌大量被染色，证明其在冰冻微针中的成功上载以及活性保持。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。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前提下的若干改进和润饰，应视为本发明的保护范围。

Claims (10)

  1. 一种冰冻微针阵列的制备方法，其特征在于，包括以下步骤：
    S1、将微针原料液滴加到具有孔洞阵列的聚二甲基硅氧烷模板表面，并使其充分填充在聚二甲基硅氧烷模板的孔洞中，去除孔洞之外的多余微针原料液，然后固化孔洞阵列中微针原料液；
    S2、将一层湿润的纱布进一步覆盖在聚二甲基硅氧烷模板的孔洞阵列上面，作为微针基底；
    S3、将聚二甲基硅氧烷模板、微针原料液和微针基底整体置于低温下进行冰冻处理，然后将冰冻后的材料从聚二甲基硅氧烷模板中剥离，得到冰冻微针阵列。
  2. 根据权利要求1所述的冰冻微针阵列的制备方法，其特征在于：所述孔洞阵列的孔洞为倒圆锥形或倒正四棱锥形。
  3. 根据权利要求2所述的冰冻微针阵列的制备方法，其特征在于：当孔洞为倒圆锥形孔洞时，孔洞间距为200-800μm，孔洞深度为300-850μm，孔洞直径为150～600μm；当孔洞为倒正四棱锥形孔洞时，孔洞间距为200～800μm，孔洞深度为300～850μm，孔洞底边长为150～600μm。
  4. 根据权利要求1所述的冰冻微针阵列的制备方法，其特征在于：步骤S1中，所述微针原料液选自甲基丙烯酸酯明胶、甲基化透明质酸、甲基化海藻酸、甲基化丝素蛋白中的一种或两种以上材料，微针原料液通过混合体积分数为1％的光引发剂并紫外照射20s～1min而固化，所述光引发剂为2-羟基-2-甲基苯丙酮。
  5. 根据权利要求1所述的冰冻微针阵列的制备方法，其特征在于：步骤S1中，所述微针原料液选用海藻酸钠材料，微针原料液通过混合过量体积分数为10％的氯化钙溶液，待充分反应后吸除上层氯化钙溶液而固化。
  6. 根据权利要求1所述的冰冻微针阵列的制备方法，其特征在于：步骤S1中，所述微针原料液选自基质胶，微针原料液通过在37℃下静置一段时间而固化。
  7. 根据权利要求1所述的冰冻微针阵列的制备方法，其特征在于：步骤S1中，微针原料液充分填充在孔洞中的方法为1000rpm离心3min，或真空处理5min。
  8. 根据权利要求1所述的冰冻微针阵列的制备方法，其特征在于：步骤S3中，冰冻处理的条件为-20℃以下冰冻12h以上，冰冻微针阵列的保存温度为0℃以下。
  9. 一种冰冻微针阵列，其特征在于，采用权利要求1-8任一项所述冰冻微针阵列的制备方法制得。
  10. 权利要求9所述冰冻微针阵列在制备载药冰冻微针上的应用，其特征在于，所述载药冰冻微针为生物活性物质搭载在冰冻微针阵列的微针内部，所述生物活性物质包括小分子、蛋白、 益生菌。
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