CN113288881B - 一种pH响应型活性因子载运体系的制备方法及其在肠靶向中的应用 - Google Patents
一种pH响应型活性因子载运体系的制备方法及其在肠靶向中的应用 Download PDFInfo
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Abstract
本发明提供了pH响应型的活性因子载运体系制备方法及应用，该方法利用微流控芯片内流体精确控制的特性，使活性因子的良溶剂和反溶剂在流体汇合区域交汇，活性因子颗粒以纳米尺度分散在水中，冻干后形成紫胶‑活性因子纳米颗粒。本发明制备的紫胶‑活性因子纳米颗粒平均粒径小于200nm，可在结肠液中溶解缓释，以达到结肠靶向释放活性因子的功能，与现有技术相比，本发明具有稳定性好、生物相容性好等优点；且制备原料均为食品级材料，安全无毒副作用，制备过程中不涉及高压高热，可有效保护活性因子的生物活性。
Description
技术领域
本发明涉及微流控技术领域，更具体地说，涉及一种基于微流控芯片的pH响应型活性因子载运体系的制备方法及其在靶向中的应用。
背景技术
岩藻黄质(Fucoxanthin)也被称为褐藻黄素、岩藻黄素，是类胡萝卜素中叶黄素类的一种天然色素，颜色呈淡黄至褐色，广泛存在于各种藻类、海洋浮游植物中，是褐藻、硅藻及金藻种主要所含有的色素。岩藻黄质具有强抗氧化能力，并具有多种生物功效，如抗肿瘤、抗肥胖、调节血糖等功效。然而岩藻黄质在实际应用中有许多问题等待解决：1)稳定性差，在加工、运输和储藏过程中易受光、热、氧气等环境因素影响；2)通过口腔进入人体后对消化液的低pH值和酶的作用敏感，难以保证其能到达相应的靶向位点发挥作用，生物利用率低。目前，通常构建岩藻黄质载运体系来提升其稳定性。
目前常用的载运体系构建方法包括高压均质法、超声破碎法、喷雾干燥法等，然而这些方法存在一定局限。例如，由于传统方法在载运体系制备过程的控制力不足，用传统方法制备的聚合物颗粒通常表现为各批次之间的物理化学性质变化较大，例如平均粒径、粒度分布、表面电荷等等。要精确控制载运体系有效的释放，制备具有可控尺寸和粒度分布的聚合物颗粒十分关键，因为这些指标对于载荷的释放速率有很大影响。微流控芯片技术是通过微通道、微反应室和其他某些功能部件，可将生物和化学等领域中所涉及的样品制备、反应、分离、检测等基本操作单元集成分析。其特有的微纳米及尺寸结构、灵活的芯片设计和功能集成特征，是进行功能化可控载运体系合成和制备研究的一种理想平台。
紫胶是一种经过FDA和CFDA认证的天然的生物相容性材料，由紫胶虫寄生于植物分泌的天然树脂提纯而得。紫胶通常为紫色或黄色，易溶于乙醇和碱性溶液，但对酸和水的渗透率很低，不溶于酸性或中性的水溶液。
发明内容
本发明提供了一种基于微流控芯片的pH响应肠靶向活性因子载运体系制备方法，以解决现有技术的岩藻黄质对消化液的低pH值和酶的作用敏感导致稳定性差的问题，并使其具备结肠靶向递送岩藻黄质的功能。
为达到上述目的，本发明提供一种pH响应型的活性因子载运体系，以紫胶作为壁材基质形成包裹活性因子的纳米颗粒。
优选的，所述活性因子包括岩藻黄质、叶黄素、姜黄素、大蒜素、虾青素、原花青素、活性肽。
一种上述载运体系的制备方法，包括以下步骤：
S1、制备紫胶-活性因子的乙醇溶液：将紫胶与活性因子溶于乙醇当中，搅拌混匀，得到活性因子终浓度0.5～5mg/mL、紫胶终浓度5～50mg/mL的紫胶-活性因子溶液；
S2、制备样品：利用微流控装置将紫胶-活性因子溶液以0.5～5mL/h流速、反溶剂相以10～100mL/h的流速汇集，反应结束后，得到并收集活性因子纳米颗粒。
S3、冻干：将步骤S2所述活性因子纳米颗粒置于-20～-80℃条件下预冷2～8h，在-40～-60℃、真空度40～60Pa、避光条件下真空冷冻干燥48～72h，得到紫胶-活性因子纳米颗粒。
优选的，步骤S2中所述微流控装置使用方法包括：将所述紫胶-活性因子溶液通过溶剂导入口(3)导入内径为0.5～10mm的溶剂导入通道(4)、将所述反溶剂相通过反溶剂导入通道(2)导入内径为0.5～10mm的反溶剂液体导入通道(2)，所述反溶剂导入通道(2)和所述溶剂导入通道(4)通过汇集口(5)与内径为0.5～10mm的液体导出通道(6)连通，反应结束后，通过所述液体导出通道(6)导出所述紫胶-活性因子纳米颗粒。
优选的，所述反溶剂相包括纯净水。
优选的，所述紫胶-活性因子纳米颗粒于4～25℃条件下真空密封保藏。
一种载运体系的应用，用于靶向释放。
优选的，用于结肠中的靶向释放。
优选的，用于制备结肠中的抗炎、抗氧化药品。
本发明的有益效果是：
本发明利用紫胶在不同pH条件下溶解性不同的特点，构建紫胶-活性因子纳米颗粒，使其具备结肠靶向递送岩藻黄质的功能。
以岩藻黄质作为模式活性因子，FDA和CFDA认证的天然的生物相容性材料紫胶为壁材，利用紫胶在低pH的胃液中，表面的大多数羧基被质子化，电离被抑制，紫胶的疏水性导致纳米颗粒聚集并沉降，在碱性的结肠液中，紫胶表面大部分的羧基的解离，使紫胶可溶，并释放岩藻黄质以达到结肠靶向释放的功能。
本发明进一步采用微流控技术利用反溶剂沉淀法即活性因子和壁材溶解在良溶剂中，然后将良溶剂相加入不良溶剂中，其中良溶剂和不良溶剂是互溶的，随着良溶剂的扩散，活性物质和聚合物在良溶剂和不良溶剂混合液中的溶解度降低，并且通过微流控技术在控制组分混合、调控纳米颗粒性能共同沉淀析出，同时活性因子被嵌入壁材基质中形成包裹活性物质的纳米颗粒；与传统方法相比，微流控装置中的颗粒制备过程微型化，从而降低了试剂的消耗。此外，微流控方法能够连续在线合成聚合物颗粒，这可以减少所获得的颗粒的物理化学性质在批次之间的变化。
本发明操作简单，只需要控制各相的流速即可，成本低廉，包埋条件温和，易于规模化生产；本发明制备过程中不涉及高压高热，可有效保护岩藻黄质的生物活性。
附图说明
图1是本发明制备纳米颗粒的使用的微流控芯片示意图；
图2是本发明实施例1制备的纳米颗粒的扫描电镜图；
图3是本发明实施例1制备的纳米颗粒的透射电镜图；
图4是本发明实施例1制备的纳米颗粒的粒径分布图；
图5是本发明实施例1制备的纳米颗粒的红外光谱图；
图6是本发明实施例1制备的纳米颗粒在不同消化液中的溶解性；
图7是本发明实施例1制备的纳米颗粒在不同消化液中的释放率；
图8是本发明实施例1制备的纳米颗粒的细胞毒性测定；
其中1、反溶剂导入口，2、反溶剂导入通道，3、溶剂导入口，4、溶剂导入通道，5、汇集口，6、液体导出通道，7、收集口。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
一种pH响应型的活性因子载运体系，以紫胶作为壁材基质形成包裹活性因子的纳米颗粒；所述活性因子包括岩藻黄质、叶黄素、姜黄素、大蒜素、虾青素、原花青素、活性肽。
所述载运体系的制备方法，包括以下步骤：
S1、制备紫胶-活性因子溶液：将紫胶与活性因子溶于乙醇等极性溶剂或非极性溶剂当中，搅拌混匀，得到活性因子终浓度0.5～5mg/mL、紫胶终浓度5～50mg/mL的紫胶-活性因子溶液；
S2、制备样品：利用微流控装置将紫胶-活性因子溶液以0.5～5mL/h流速、反溶剂相以10～100mL/h的流速汇集，反应结束后，得到并收集活性因子纳米颗粒。
S3、冻干：将步骤S2所述活性因子纳米颗粒置于-20～-80℃条件下预冷2～8h，在-40～-60℃、真空度40～60Pa、避光条件下真空冷冻干燥48～72h，得到紫胶-活性因子纳米颗粒。所述紫胶-活性因子纳米颗粒于4～25℃条件下真空密封保藏。
所述微流控装置使用方法包括：将所述紫胶-活性因子溶液通过溶剂导入口(3)导入内径为0.5～10mm的溶剂导入通道(4)、将所述反溶剂相通过反溶剂导入通道(2)导入内径为0.5～10mm的反溶剂液体导入通道(2)，所述反溶剂导入通道(2)和所述溶剂导入通道(4)通过汇集口(5)与内径为0.5～10mm的液体导出通道(6)连通，反应结束后，通过所述液体导出通道(6)导出所述紫胶-活性因子纳米颗粒。
实施例1
一种基于微流控技术制备具有pH响应功能的紫胶-岩藻黄质肠道靶向纳米载运体系的方法，包括以下步骤：
S1、制备紫胶-岩藻黄质的乙醇溶液：将紫胶与活性因子岩藻黄质溶于乙醇当中，搅拌混匀，得到含有紫胶-岩藻黄质的混合溶液A。所述溶液A中的岩藻黄质终浓度是3mg/mL，紫胶终浓度是10mg/mL；
S2、采用微流控技术制备样品：用注射泵将步骤S1的所述溶液A与纯净水分别通过溶剂导入口3和反溶剂导入口1注射到微流控芯片内，将收集软管接入所述芯片的收集口7处，以导出收集液；所述注射泵流速为：溶液A的流速为1.5mL/h，纯净水的流速为20mL/h，反应结束后，收集含有岩藻黄质纳米颗粒的收集液B。所述芯片由由PDMS或PMMA材料制作，包括内径为0.5mm的溶剂导入通道4；内径为1mm的反溶剂液体导入通道2；内径为1mm的液体导出通道6。
S3、冻干：步骤S2所述收集液B置于-80℃预冷8h，在-50℃、真空度50Pa条件下真空冷冻干燥72h，干燥过程避光，得到紫胶-岩藻黄质复合纳米颗粒，真空密封，置于25℃保藏。
图1为本发明中制备纳米颗粒的使用的微流控芯片示意图，紫胶-岩藻黄质的乙醇溶液作为良溶剂相，纯净水作为反溶剂相，在微流控芯片中通过良溶剂与不良溶剂的快速混合，使岩藻黄质和紫胶共同沉淀析出形成包裹岩藻黄质的紫胶纳米颗粒。
紫胶-岩藻黄质纳米颗粒的形貌观察、尺寸大小、光谱表征及模拟消化实验
S1、紫胶-岩藻黄质纳米颗粒的扫描电镜图
使用扫描电镜观察本发明实施例1制备的纳米颗粒，图2是紫胶-岩藻黄质纳米颗粒的扫描电镜图，结果显示，经过冷冻干燥后的纳米颗粒形态似球型，分布均匀，粒子表面也相对比较光滑，颗粒的粒径大小在100-200nm范围之间。
S2、紫胶-岩藻黄质纳米颗粒的透射电镜图
使用透射电镜观察本发明实施例1制备的纳米颗粒，图3是紫胶-岩藻黄质纳米颗粒的透射电镜图，结果显示，纳米颗粒具有核壳结构，岩藻黄质被包裹在紫胶之中。
S3、紫胶-岩藻黄质纳米颗粒的粒径分布
使用激光粒度仪测定本发明实施例1制备的纳米颗粒的粒径分布，结果如图4所示，纳米颗粒的粒径分布在100～300nm之间，粒径集中在130～180nm，与扫描电镜观察结果相对应。
S4、紫胶-岩藻黄质纳米颗粒的红外光谱表征
使用傅里叶红外光谱仪测试本发明实施例1制备的纳米颗粒，图5是纳米颗粒的傅里叶红外光谱表征，曲线(a)为岩藻黄质，曲线(b)为紫胶，曲线(c)为紫胶-岩藻黄质纳米颗粒。针对岩藻黄质的曲线：在3432cm-1处的峰为O-H拉伸，3000～2800cm-1(C-H拉伸)和1339～1452cm-1(C-H剪切和弯曲)，1726cm-1处是羰基C＝O伸缩振动。1928cm-1处的峰为丙烯键，被认为是岩藻黄质的独特官能团。紫胶曲线在3430cm-1附近的宽带是由酸性和羟基侧链的-OH振动引起的。在2930cm-1和2855cm-1附近的两个峰分别归因于-CH3和-CH2基团的C-H伸缩振动，1729和1636cm-1处的强带分别为酯结构和羧酸结构的羰基特征。与紫胶相比，紫胶-岩藻黄质纳米颗粒的大多数特征峰保持不变。然而，在1580cm-1处出现了代表R-COO-的新峰。这是由于在紫胶-岩藻黄质纳米颗粒形成过程中氢键的产生导致紫胶羧酸羰基中的C＝O伸缩振动向低频转移，从而证实岩藻黄质凭借氢键成功装载到紫胶当中。
S5、紫胶-岩藻黄质纳米颗粒的模拟消化评价
在模拟消化液中进行紫胶-岩藻黄质的释放实验。将5mg本发明实施例1制备的纳米颗粒分别浸泡在5mL人工模拟胃液(pH＝1.34)、人工模拟小肠液(pH＝6.68)和人工模拟结肠液(pH＝7.92)中37℃水浴孵育2h后，离心取上清。采用紫外-可见分光光度法，在446nm波长处测定纳米颗粒在模拟消化液中的释放量。
图6是纳米颗粒在不同消化液中的溶解性，结果可见，纳米颗粒在酸性的胃液和近中性小肠液中呈聚集沉淀的状态，岩藻黄质包裹在纳米颗粒当中。在碱性的结肠液中，纳米颗粒溶解，岩藻黄质被释放到溶液当中。图7是纳米颗粒在不同消化液中释放率，结果表明岩藻黄质在胃液中的释放率为2.53％，在小肠液中的释放率为12.21％，而在结肠液中的释放率达到83.34％，由此表明，紫胶-岩藻黄质纳米颗粒可以在胃和小肠中保护岩藻黄质，而在结肠中靶向释放。
紫胶-岩藻黄质纳米颗粒对于细胞毒性作用
测定实施例1制备的紫胶-岩藻黄质纳米颗粒对细胞的毒性作用；选用Caco-2细胞和含有10％(V胎牛血清/VDMEM培养基＝1/9)的胎牛血清的高糖DMEM培养基。将细胞以3.5105个细胞/孔的密度接种于96孔板，在体积分数为5％的CO2培养箱中孵育12h。使用细胞培养级二甲基亚砜溶解紫胶-岩藻黄质纳米颗粒，添加于培养基中使得紫胶-岩藻黄质纳米颗粒在培养级中的终浓度为0、4、8、12、16、20μg/mL。将含有紫胶-岩藻黄质纳米颗粒的培养基加入细胞中，培养12h。之后每孔加入MTT(0.5mg/mL)溶液20μL，继续培养4h。之后去除培养基，在每孔加入150μL二甲基亚砜，并在摇床上低速振荡10min，使结晶物充分溶解，在酶联免疫检测仪OD 490nm处测量各孔的吸光值。同时设置调零孔(培养基、MTT、二甲基亚砜)，对照孔(细胞、相同浓度的药物溶解介质、培养液、MTT、二甲基亚砜)。通过以下公式计算细胞活力。
细胞活力＝[(对照-空白)-(给药-空白)]/(对照-空白)×100％
图8为不同浓度纳米颗粒对Caco-2细胞的毒性作用，结果表明，在最高浓度为20μg/mL的紫胶-岩藻黄质纳米颗粒的作用下，Caco-2细胞的活力没有降低，证明本文所述紫胶-岩藻黄质纳米颗粒不存在细胞毒性，具有良好的生物相容性，这是保证其在制备结肠中的抗炎、抗氧化药品领域应用的必要条件。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (2)
1.一种基于微流控芯片的pH响应型的活性因子载运体系的制备方法，其特征在于，以紫胶作为壁材基质形成包裹岩藻黄质的纳米颗粒；包括以下步骤：
S1、制备紫胶-岩藻黄质溶液：将紫胶与岩藻黄质溶于乙醇中，搅拌混匀，得到岩藻黄质终浓度0.5～5mg/mL、紫胶终浓度5～50mg/mL的紫胶-岩藻黄质溶液；
S2、采用微流控技术制备样品：用注射泵将步骤S1所述紫胶-岩藻黄质溶液与纯净水分别通过溶剂导入口(3)和反溶剂导入口(1)注射到微流控芯片内，将收集软管接入所述芯片的收集口(7)处，以导出收集液；所述注射泵流速为：紫胶-岩藻黄质溶液的流速为1.5mL/h，纯净水的流速为20mL/h，反应结束后，收集含有岩藻黄质纳米颗粒的收集液B；所述芯片由PDMS或PMMA材料制作，包括内径为0.5mm的溶剂导入通道(4)；内径为1mm的反溶剂液体导入通道(2)；内径为1mm的液体导出通道(6)；
S3、冻干：将步骤S2所述收集液B置于-20～-80℃条件下预冷2～8h，在-40～-60℃、真空度40～60Pa、避光条件下真空冷冻干燥48～72h，得到紫胶-岩藻黄质纳米颗粒。
2.一种权利要求1所述制备方法制备的载运体系在制备具有抗氧化功能的药品中的应用。
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