CN113876950B - 一种适用于no协同光动力治疗的微针贴片及其制备方法 - Google Patents

一种适用于no协同光动力治疗的微针贴片及其制备方法 Download PDF

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CN113876950B
CN113876950B CN202111312255.8A CN202111312255A CN113876950B CN 113876950 B CN113876950 B CN 113876950B CN 202111312255 A CN202111312255 A CN 202111312255A CN 113876950 B CN113876950 B CN 113876950B
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计剑
王幽香
余伟江
贾凡
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Zhejiang University ZJU
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Abstract

本发明公开了一种适用于NO协同光动力治疗的微针贴片及其制备方法。微针贴片采用高分子可溶解聚合物作为基质，微针贴片包括基底层和位于基底层上的针体，针体中装载了复合纳米粒子，复合纳米粒子包含通过静电作用组装的NO载体和光敏剂，NO载体包括载体粒子和接枝在载体粒子上且可释放NO的NO供体。制备方法包括：将NO供体接枝到载体粒子上，接着将接枝了NO供体的NO载体和光敏剂通过静电组装形成复合纳米粒子；将复合纳米粒子和高分子可溶解聚合物混合，所得混合溶液浇筑在微针模板里，经过刮除残余部分后干燥形成针尖；将高分子可溶解聚合物浇筑在微针模板里，干燥后形成基底，剥离得到微针贴片。

Description

一种适用于NO协同光动力治疗的微针贴片及其制备方法
技术领域
本发明涉及微针技术领域，具体涉及一种适用于NO协同光动力治疗的微针贴片及其制备方法。
背景技术
光动力疗法是利用光敏剂和激光活化治疗肿瘤疾病的一种新方法，因其侵袭性小、无耐药性等优点在皮肤癌的治疗中具有独到的优势。
在标准的光动力治疗过程中，光敏剂经静脉进行注射，在肿瘤部位累积到一定浓度后，通过光进行激活以产生破坏肿瘤的活性氧自由基。
然而，光敏剂的全身给药导致富集在肿瘤中的药物有限，同时不可避免在健康的器官或组织中产生大量的积累，从而带来全身性的光毒性。
此外，肿瘤细胞内具有较高水平的谷胱甘肽。作为一种天然存在的还原剂，谷胱甘肽能够清除各种氧化剂，从而保护癌细胞免于活性氧自由基的消融，削弱光动力疗法的效果。
因此，开发具有更高递送精度和谷胱甘肽清除能力的新材料策略以增强光动力疗法功效的需求长期存在。
近年来，聚合物可溶解微针因其药物递送效率高，对患者侵入性小，在透皮给药领域受到了大量的关注，如公开号为CN 111544758 A、CN 111544756 A的专利说明书等。它们通过直接物理刺破的方式在皮肤角质层中形成大量微米级别的孔洞，借助聚合物溶解的方式将药物快速地递送至皮肤内，因此适用于各类药物的递送。更重要的是，微针能够通过自助的方式作用于皮肤病变部位，将药物直接递送至病灶内，因此成为光动力疗法透皮药物递送的绝佳选择。
另一方面，S-亚硝基硫醇(RSNO)是一大类化合物，是人体中天然存在的一类NO供体，被认为具有良好的生物安全性。S-亚硝基硫醇可以通过消耗细胞内的谷胱甘肽来释放NO，生成的NO可以通过各种生物代谢进一步降低细胞内的谷胱甘肽水平。同时NO能够与活性氧自由基反应产生过氧亚硝酸根阴离子或其他活性氮物质，它们比ROS或NO更致命，能够极大地提升肿瘤细胞的杀灭效率。
发明内容
本发明的目的之一是提供一种适用于NO协同光动力治疗的微针贴片。
一种适用于NO协同光动力治疗的微针贴片，所述微针贴片采用高分子可溶解聚合物作为基质，所述微针贴片包括基底层和位于所述基底层上的针体，所述针体中装载了复合纳米粒子，所述复合纳米粒子包含通过静电作用组装的NO载体和光敏剂，所述NO载体包括载体粒子和接枝在所述载体粒子上且可释放NO的NO供体。
在一优选例中，所述针体的高度为0.1～1mm，所述基底层上的针体密度为1～1000针每平方厘米。
在一优选例中，所述高分子可溶解聚合物选自透明质酸、聚乙烯基吡咯烷酮、聚乙烯醇、聚乙二醇中的一种或多种。
在一优选例中，所述复合纳米粒子的粒径为1～500nm。
所述NO供体包括但不限于L-精氨酸、硝酸酯类、呋咱氮氧化物类、S-亚硝基硫醇类，优选为S-亚硝基硫醇类化合物。
所述S-亚硝基硫醇类包括但不限于S-亚硝基-N-乙酰半胱氨酸、S-亚硝基谷胱甘肽、S-亚硝基-N-乙酰青霉胺。
所述NO供体的接枝率优选为2％～90％，进一步优选为30％～90％，优选方案可使NO有足够的负载量的同时有足够的表面正电荷量。
在一优选例中，所述载体粒子选自聚酰胺-胺(聚酰胺胺型树枝状聚合物)、二氧化硅(可以是纳米粒子、微球等)、壳聚糖中的一种或多种。
在一优选例中，所述光敏剂表面带负电，可与接枝有NO供体的NO载体通过静电作用进行组装，优选自亚甲基蓝、吲哚菁绿、二氢卟吩e6、血卟啉中的一种。
在一优选例中，所述复合纳米粒子中光敏剂的质量分数为1％～40％。
本发明另一目的是提供所述的微针贴片的一种优选制备方法，包括步骤：
(1)将NO供体接枝到载体粒子(可以是载体分子、纳米粒子等形式)上，接着将接枝了NO供体的NO载体和光敏剂通过静电组装形成复合纳米粒子；
(2)将复合纳米粒子和高分子可溶解聚合物混合，所得混合溶液浇筑在微针模板里，经过刮除残余部分后干燥形成针尖；
(3)将高分子可溶解聚合物浇筑在微针模板里，干燥后形成基底，剥离得到微针贴片。
本发明与现有技术相比，主要优点包括：
本发明提供的一种适用于NO气体和光动力协同治疗的微针贴片解决了光动力治疗中药物递送效率低的问题以及治疗产生的活性氧自由基受细胞内高水平谷胱甘肽还原的影响。当所制备的微针贴片作用于皮肤病变部位后，聚合物针尖快速溶解将复合纳米粒子高效地递送至病灶内。该复合纳米粒子由NO载体和光敏剂组装而成，能够释放NO并大量消耗细胞内的谷胱甘肽。而生成的NO可以与光动力疗法产生的活性氧自由基反应形成活性氮物质，进一步提升治疗效果。
附图说明
图1为实施例1制备的复合纳米粒子的紫外可见光吸收光谱图；
图2为实施例1制备的复合纳米粒子的水动力学直径图；
图3为实施例1的制备的复合纳米粒子与细胞培养后胞内谷胱甘肽的含量图；
图4为实施例1制备微针贴片的流程示意图；
图5为实施例1制备的微针贴片照片；
图6为实施例1制备的微针贴片应用于荷瘤裸鼠的肿瘤治疗效果图。
具体实施方式
下面结合附图及具体实施例，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。下列实施例中未注明具体条件的操作方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1
(1)先制备N-乙酰-D-青霉胺硫内酯(NAP-TL)：将1.5g N-乙酰基-D-青霉胺在0℃下溶解在5mL吡啶中，再将10mL乙酸酐吡啶(体积比1:1)的混合溶液逐滴加入；过夜搅拌后，通过减压蒸发除去溶剂，并将粗产物溶于氯仿中，用1M盐酸洗涤3次，收集有机相层并用硫酸镁干燥；将固体滤去后将所得溶液蒸发以除去溶剂。剩余的固体分散在己烷中通过真空过滤以获得结晶产物NAP-TL。
(2)制备接枝S-亚硝基-N-乙酰青霉胺的聚酰胺-胺型树枝状聚合物(PAMAM-SNAP)：将0.3g末端基团为氨基的第四代聚酰胺-胺型树枝状大分子和0.31g NAP-TL加入5mL二甲基甲酰胺(DMF)中，并在室温下搅拌24h；将过量的乙醚滴入所得溶液，离心后将产物溶解在蒸馏水中透析过夜，冷冻干燥获得接枝了N-乙酰青霉胺的聚酰胺-胺型树枝状大分子(PAMAM-NAP)；在0℃下，将0.3g PAMAM-NAP和1mL亚硝酸叔丁酯(TBN)添加到5mL的DMF中，并在黑暗中搅拌过夜；所得溶液在乙醚中沉淀，所得固体溶解于蒸馏水中透析24h，冷冻干燥得到PAMAM-SNAP，SNAP的接枝率为86％。
(3)取100mg步骤(2)得到的PAMAM-SNAP和10mg光敏剂二氢卟吩e6(Ce6)加入到5mL二甲基亚砜(DMSO)和水混合溶液(体积比1:1)中搅拌过夜，透析24后冷冻干燥得到复合纳米粒子PAMAM-SNAP/Ce6，PAMAM-SNAP/Ce6的水动力学直径为21nm，Ce6的质量分数为3％。
(4)取50mg步骤(3)得到的PAMAM-SNAP/Ce6加入到0.5mL 50％(w/v)的聚乙烯基吡咯烷酮(PVP)溶液中，搅拌均匀后浇筑到PDMS微针模板内，刮除模板针孔外残余浇筑液后干燥得到载药针体。
(5)取0.1mL 100％(w/v)的PVP溶液浇筑到微针模板的中作为基底部分，干燥后将微针贴片剥离。
上述步骤(1)～(3)制备的复合纳米粒子的紫外可见光吸收光谱如图1所示，水动力学直径如图2所示，其对胞内谷胱甘肽的消耗如图3所示，图3里的对照组是纯培养基培养的A375黑色素瘤细胞胞内谷胱甘肽浓度。
上述步骤(4)和步骤(5)的微针制备过程如图4所示。
上述步骤(5)制得的微针贴片的显微形貌如图5所示，针高度为1000μm，密度为25针每平方厘米。
上述步骤(5)制得的微针贴片对荷瘤裸鼠的抑瘤效果如图6所示，微针作用5min，在光照(660nm，24J/cm2)处理后，18天的抑瘤率为88％，明显优于空白对照组和其它类型的微针(或缺少SNAP，或缺少Ce6)，体现了本发明微针贴片NO和光动力协同治疗作用。
实施例2
(1)先制备接枝S-亚硝基谷胱甘肽的壳聚糖(CS-GSNO)：将100mg壳聚糖加入10mL1％的乙酸溶液中搅拌1h充分溶解；将70mg谷胱甘肽(GSH)溶解在5mL蒸馏水中，加入50mg(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和40mg N-羟基硫代琥珀酰亚胺，活化1h后，加入10mL壳聚糖溶液避光室温反应24h，避光透析2天后冷冻干燥获得接枝了谷胱甘肽的壳聚糖(CS-GSH)；取100mg CS-GSH加入到10mL的0.1M盐酸溶液中，再加20mg亚硝酸钠，在0℃下搅拌反应40min后加丙酮20mL，继续搅拌10min；沉淀减压过滤后，分别用丙酮和乙醚各洗涤3次，得到产物CS-GSNO。GSNO的接枝率为38％。
(2)取80mg步骤(1)得到的CS-GSNO和10mg光敏剂吲哚菁绿(ICG)加入到5mL和0.1M的盐酸溶液中搅拌过夜，透析24h后冷冻干燥得到复合纳米粒子CS-GSNO/ICG。CS-GSNO/ICG的水动力学直径为942nm，ICG的质量分数为4％。
(3)取100mg步骤(2)得到的CS-GSNO/ICG加入到0.5mL 40％(w/v)的聚乙烯醇(PVA)溶液中，搅拌均匀后浇筑到微针模板内，刮除模板针孔外残余浇筑液后干燥得到载药针体。
(4)取0.1mL 50％(w/v)的PVA溶液浇筑到微针模板的中作为基底部分，干燥后将微针贴片剥离。所制备的针高度为750μm，密度为200针每平方厘米。
实施例3
(1)先制备N-乙酰-D-青霉胺硫内酯(NAP-TL)：将1.5g N-乙酰基-D-青霉胺在0℃下溶解在5mL吡啶中，再将10mL乙酸酐吡啶(体积比1:1)的混合溶液逐滴加入；过夜搅拌后，通过减压蒸发除去溶剂，并将粗产物溶于氯仿中，用1M盐酸洗涤3次，收集有机相层并用硫酸镁干燥；将固体滤去后将所得溶液蒸发以除去溶剂。剩余的固体分散在己烷中通过真空过滤以获得结晶产物NAP-TL。
(2)制备接枝S-亚硝基-N-乙酰青霉胺的二氧化硅(SiO2-SNAP)：将0.4g直径为40nm的氨基化二氧化硅微球和0.25g NAP-TL加入5mL二甲基甲酰胺(DMF)中，并在室温下搅拌24h；所得溶液通过离心获得沉淀，再用蒸馏水洗涤三次后冷冻干燥获得接枝了N-乙酰青霉胺的二氧化硅微球(SiO2-NAP)；在0℃下，将0.15g SiO2-NAP和2.2mL亚硝酸叔丁酯(TBN)添加到5mL的DMF中,并在黑暗中搅拌过夜；所得溶液通过离心获得沉淀，再用蒸馏水洗涤三次，冷冻干燥后获得SiO2-SNAP，SNAP的接枝率为62％。
(3)取50mg步骤(2)得到的SiO2-SNAP和10mg光敏剂亚甲基蓝(MB)加入到5mL蒸馏水中，透析24后冷冻干燥得到复合纳米粒子SiO2-SNAP/MB。SiO2-SNAP/MB水动力学直径为45nm，MB的质量分数为7％。
(4)取100mg步骤(3)得到的SiO2-SNAP/MB加入到0.5mL 30％(w/v)的透明质酸(HA)溶液中，搅拌均匀后浇筑到微针模板内，刮除模板针孔外残余浇筑液后干燥得到载药针体。
(5)取0.1mL 60％(w/v)的HA溶液浇筑到微针模板的中作为基底部分，干燥后将微针贴片剥离。所制备的针高度为800μm，密度为100针每平方厘米。
实施例4
(1)先制备接枝S-亚硝基-N-乙酰半胱氨酸的聚酰胺胺型树枝状大分子(PAMAM-SNAC)：将70mg乙酰半胱氨酸溶解在5mL蒸馏水中，加入50mg(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和40mg N-羟基硫代琥珀酰亚胺，活化1h后，加90mg末端基团为氨基的第三代聚酰胺胺型树枝状大分子避光室温反应24h，避光透析2天后冷冻干燥获得接枝了N-乙酰半胱氨酸的聚酰胺-胺型树枝状大分子(PAMAM-NAC)；取90mg PAMAM-NAC加入到10mL的0.1M盐酸溶液中，再加60mg亚硝酸钠，在0℃下搅拌反应20min后冷冻干燥；产物用乙醚和丙酮各洗涤3次，得到产物PAMAM-SNAC。PAMAM-SNAC的水动力学直径为50nm，SNAC的接枝率为43％。
(2)取60mg步骤(1)得到的PAMAM-SNAC和6mg光敏剂血卟啉(HP)加入到5mL和乙醇和水混合溶液(体积比1:1)中搅拌过夜，透析24h后冷冻干燥得到复合纳米粒子PAMAM-SNAC/HP。PAMAM-SNAC/HP水动力学直径为30nm，HP的质量分数为8％。
(3)取75mg步骤(2)得到的PAMAM-SNAC/HP加入到0.4mL 30％(w/v)的透明质酸(HA)溶液中，搅拌均匀后浇筑到微针模板内，刮除模板针孔外残余浇筑液后干燥得到载药针体。
(4)取0.1mL 50％(w/v)的HA溶液浇筑到微针模板的中作为基底部分，干燥后将微针贴片剥离。所制备的针高度为400μm，密度为500针每平方厘米。
实施例5
(1)先制备接枝S-亚硝基-N-乙酰半胱氨酸的二氧化硅微球(SiO2-SNAC)：将55mg乙酰半胱氨酸溶解在6mL蒸馏水中，加入40mg(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和25mg N-羟基硫代琥珀酰亚胺，活化2h后，加0.6g直径为50nm的氨基化二氧化硅微球避光室温反应36h，避光透析1天后冷冻干燥获得接枝了N-乙酰半胱氨酸的二氧化硅微球(SiO2-NAC)；取85mg SiO2-NAC加入到10mL的0.1M盐酸溶液中，再加45mg亚硝酸钠，在0℃下搅拌反应20min后冷冻干燥；产物用乙醚和丙酮各洗涤3次，得到产物SiO2-SNAC。SNAC的接枝率为55％。
(3)取65mg步骤(2)得到的SiO2-SNAC和15mg光敏剂二氢卟吩e6(Ce6)加入到10mL二甲基亚砜(DMSO)和水混合溶液(体积比1:1)中搅拌过夜中，透析24h后冷冻干燥得到复合纳米粒子SiO2-SNAC/Ce6。SiO2-SNAC/Ce6水动力学直径为55nm，Ce6的质量分数为5％。
(4)取65mg步骤(3)得到的SiO2-SNAC/Ce6加入到0.5mL 70％(w/v)的聚乙烯基吡咯烷酮(PVP)溶液中，搅拌均匀后浇筑到微针模板内，刮除模板针孔外残余浇筑液后干燥得到载药针体。
(5)取0.1mL 80％(w/v)的PVP溶液浇筑到微针模板的中作为基底部分，干燥后将微针贴片剥离。所制备的针高度为600μm，密度为121针每平方厘米。
实施例6
(1)先制备接枝S-亚硝基谷胱甘肽的聚酰胺胺型树枝状大分子(PAMAM-GSNO)：将80mg谷胱甘肽(GSH)溶解在5mL蒸馏水中，加入50mg(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和40mg N-羟基硫代琥珀酰亚胺，活化1h后，加入0.3g末端基团为氨基的第四代聚酰胺-胺型树枝状大分子避光室温反应24h；避光透析2天后冷冻干燥获得接枝了谷胱甘肽的聚酰胺-胺型树枝状大分子(PAMAM-GSH)；取60mg PAMAM-GSH加入到10mL的0.1M盐酸溶液中，再加20mg亚硝酸钠，在0℃下搅拌反应30min后加丙酮15mL，继续搅拌15min；沉淀减压过滤后，分别用丙酮和乙醚各洗涤3次，得到产物PAMAM-GSNO。GSNO的接枝率为85％。
(2)取55mg步骤(1)得到的PAMAM-GSNO和10mg光敏剂血卟啉(HP)加入到10mL和0.1M的盐酸溶液中搅拌过夜，透析24h后冷冻干燥得到复合纳米粒子PAMAM-GSNO/HP。PAMAM-GSNO/HP水动力学直径为30nm，HP的质量分数为6％。
(3)取100mg步骤(2)得到的PAMAM-GSNO/HP加入到0.5mL 20％(w/v)的聚乙烯醇(PVA)溶液中，搅拌均匀后浇筑到微针模板内，刮除模板针孔外残余浇筑液后干燥得到载药针体。
(4)取0.1mL 30％(w/v)的PVA溶液浇筑到微针模板的中作为基底部分，干燥后将微针贴片剥离。所制备的针高度为850μm，密度为36针每平方厘米。
此外应理解，在阅读了本发明的上述描述内容之后，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。

Claims (4)

1.一种适用于NO协同光动力治疗的微针贴片，其特征在于，所述微针贴片采用高分子可溶解聚合物作为基质，所述微针贴片包括基底层和位于所述基底层上的针体，所述针体中装载了复合纳米粒子，所述复合纳米粒子包含通过静电作用组装的NO载体和光敏剂，所述NO载体包括载体粒子和接枝在所述载体粒子上且可释放NO的NO供体；
所述复合纳米粒子为PAMAM-SNAP/Ce6，水动力学直径为21nm，Ce6的质量分数为3％，制备方法为：
(1)先制备N-乙酰-D-青霉胺硫内酯NAP-TL：将1.5g N-乙酰基-D-青霉胺在0℃下溶解在5mL吡啶中，再将10mL体积比1:1的乙酸酐吡啶的混合溶液逐滴加入；过夜搅拌后，通过减压蒸发除去溶剂，并将粗产物溶于氯仿中，用1M盐酸洗涤3次，收集有机相层并用硫酸镁干燥；将固体滤去后将所得溶液蒸发以除去溶剂，剩余的固体分散在己烷中通过真空过滤以获得结晶产物NAP-TL；
(2)制备接枝S-亚硝基-N-乙酰青霉胺的聚酰胺-胺型树枝状聚合物PAMAM-SNAP：将0.3g末端基团为氨基的第四代聚酰胺-胺型树枝状大分子和0.31g NAP-TL加入5mL二甲基甲酰胺DMF中，并在室温下搅拌24h；将过量的乙醚滴入所得溶液，离心后将产物溶解在蒸馏水中透析过夜，冷冻干燥获得接枝了N-乙酰青霉胺的聚酰胺-胺型树枝状大分子PAMAM-NAP；在0℃下，将0.3g PAMAM-NAP和1mL亚硝酸叔丁酯TBN添加到5mL的DMF中，并在黑暗中搅拌过夜；所得溶液在乙醚中沉淀，所得固体溶解于蒸馏水中透析24h，冷冻干燥得到PAMAM-SNAP，SNAP的接枝率为86％；
(3)取100mg步骤(2)得到的PAMAM-SNAP和10mg光敏剂二氢卟吩e6 Ce6加入到5mL体积比1:1的二甲基亚砜DMSO和水混合溶液中搅拌过夜，透析24后冷冻干燥得到复合纳米粒子PAMAM-SNAP/Ce6。
2.根据权利要求1所述的微针贴片，其特征在于，所述针体的高度为0.1～1mm，所述基底层上的针体密度为1～1000针每平方厘米。
3.根据权利要求1所述的微针贴片，其特征在于，所述高分子可溶解聚合物选自透明质酸、聚乙烯基吡咯烷酮、聚乙烯醇、聚乙二醇中的一种或多种。
4.根据权利要求1～3任一权利要求所述的微针贴片的制备方法，其特征在于，包括步骤：
(1)将NO供体接枝到载体粒子上，接着将接枝了NO供体的NO载体和光敏剂通过静电组装形成复合纳米粒子；
(2)将复合纳米粒子和高分子可溶解聚合物混合，所得混合溶液浇筑在微针模板里，经过刮除残余部分后干燥形成针尖；
(3)将高分子可溶解聚合物浇筑在微针模板里，干燥后形成基底，剥离得到微针贴片。
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