CN114191568B - 一种基于多酚的凝聚层颗粒制备方法 - Google Patents
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Abstract
本发明属于生物技术领域,公开了一种基于多酚的凝聚层颗粒制备方法。利用多酚类化合物与一种或多种含氢键受体的聚合物,通过氢键相互作用组装成颗粒,通过调控多酚类化合物与聚合物的摩尔比例,调控其尺寸与形貌。将颗粒与细胞共孵育,利用多酚类化合物的抗氧化性质,清除细胞内自由基,提高细胞应激条件下的抗氧化能力。此外利用多酚具有大量相互作用位点的特性,实现颗粒对多种物质的包封,可用作纳米载体。本发明的凝聚层颗粒形貌尺寸可控,兼具动态与稳定性,无有机溶剂与表面活性剂的加入,生物相容性好,且不需要特殊设备,制备方法简单,成本低,因此在生物医药及化妆品领域具有广泛的应用前景。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种基于多酚的凝聚层颗粒制备方法,所制备的颗粒尺寸形貌动态性质均可调,既可用于提高细胞抗氧化能力,又可用作纳米载体,实现多种物质的包封。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
在生物医药领域,特定大小或者形貌的组装体可以实现不同的生物功能。例如:与药物共组装用于药物递送,将组装体吸附在两相界面实现乳液稳定,或作为生物微反应器进行酶促反应。当赋予组装体一定的动态响应性时(例如:酸响应性),可实现药物可控释放。因此,制备生物相容性好、形貌可控、兼具动态稳定性的组装体在生物医药领域具有重要意义。二元双水相体系,由于无表面活性剂与有机溶剂的加入,因此,具有良好的生物相容性。二元双水相体系主要分为两种,第一种是选取相互作用力较弱的两种聚合物,即:形成传统的水包水乳液,可通过引入微流控设备来调节粒径,而微流控设备对操作技术要求较高,导致其在工程化制备方面存在一定挑战。第二种是使二元混合物之间存在较强的相互作用力,这种通过共混合的方式,制备方式更简单。现有研究中利用聚电解质的静电吸附,阳离子-π相互作用,以及特定的官能团在使用或制备时存在一定的限制。如:静电吸附的颗粒对盐的浓度敏感,阳离子-π相互作用需要较高的盐浓度屏蔽阳离子之间的静电排斥。而含氢键受体的聚合物和多酚存在大量氢键足够实现颗粒的自组装,现有的研究多利用二者相互作用在模板上沉积,制备胶囊,但在尺寸的调控方面依靠模板,因此存在局限性。有研究利用多种相互作用,如:氢键与金属配位作用协同,或疏水相互作用协同,但在制备纳米级颗粒的过程中,用到了有机溶剂的预先溶解(专利文献CN103948934A,专利文献CN107441513A),或预先调节溶液pH后逐步滴加酸的方式缓慢形成颗粒。在制备方式上,有机溶剂的引入降低了材料生物相容性,并且制备步骤更繁琐。
发明内容
本发明的目的解决现有技术存在的上述问题,提供了一种基于多酚的凝聚层颗粒制备方法,本发明提供的基于多酚的凝聚层颗粒可用于清除细胞内自由基,提高细胞的抗氧化能力,也可利用多酚存在的多种作用位点实现生物活性大分子、小分子或无机材料的包封。
为实现上述技术目的,本发明采用如下技术方案:
本发明的第一个方面,提供了一种基于多酚的凝聚层颗粒制备方法,包括:将多酚化合物与至少一种含有氢键受体的聚合物在溶剂中混合均匀,得到凝聚层颗粒。本发明开发了一种普适性方法来制备生物相容性良好、尺寸形貌可控、动态性质可调、具有生物功能的颗粒,具有重要意义。
本发明的基本原理是基于多酚与聚合物之间存在的大量氢键,通过调控形成氢键的密度来调节凝聚层颗粒的尺寸和形貌,通过调节多酚与聚合物的摩尔比例控制凝聚层颗粒的动态性质。
本发明的第二个方面,提供了一种功能化颗粒的制备方法,包括:将荧光染料、小分子药物、无机颗粒或蛋白质,与含有氢键受体的聚合物溶液先预混,得到聚合物复合溶液;将所述聚合物复合溶液与多酚溶液共混,即得功能化颗粒,用作纳米载体,实现多种物质的包封。
本发明的第三个方面,提供了上述的方法制备的功能化颗粒在细胞自由基清除中的特性。多酚类化合物(例如:茶多酚)广泛存在于药用植物中,是一种良好的抗氧化剂,已有部分多酚类化合物被美国视频药品监管管理局(FDA)批准使用,因此具有良好的生物相容性。将其与一种或几种聚合物共混制备成颗粒,与细胞共孵育使其被细胞内吞,利用多酚的抗氧化性质,降低应激条件下(例如:双氧水刺激)细胞内的自由基水平,实现细胞抗氧化。
本发明的有益效果在于:
(1)本发明的操作方法简单、成本低、具有普适性,易于规模化生产。
(2)与现有技术相比,本发明最突出的优点是不需要微流控等特殊设备的使用,不加入有机溶剂,不需要预先调节溶液的pH,原料易得,价格低廉,具有良好的生物相容性。制备的凝聚层颗粒生物相容性好,具有尺寸、形貌、动态性质可调节性,且实现了从纳米级到微米级再到宏观块状材料的跨尺度尺寸调节。
(3)本发明以功能化化合物多酚作为组成成份,其组装体能够利用其抗氧化性,降低由于双氧水刺激而升高的细胞内自由基水平,提高细胞抗氧化能力;同时,功能化颗粒能够作为纳米载体,实现多种物质的包封,因此,在生物医药及化妆品领域具有广泛的应用前景。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中得到的基于多酚的凝聚层颗粒透射电子显微镜图像。
图2为实施例2中得到的基于多酚的凝聚层颗粒光学显微镜照片。
图3为实施例3中得到的基于多酚具有胶囊形貌的凝聚层颗粒光学显微镜图片。
图4为实施例4中得到的基于多酚具有胶囊形貌的凝聚层颗粒激光共聚焦图片。
图5为实施例8中牛血清白蛋白负载于凝聚层颗粒的激光共聚焦图片。
图6为实施例10中细胞体内代表自由基水平的荧光数据。空白组为在细胞培养基中孵育的细胞,对照组为经双氧水刺激的细胞,实验组为经双氧水刺激后与功能化颗粒共孵育的细胞。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
基于多酚的凝聚层颗粒制备方法的步骤如下:
(1)将一种或几种聚合物溶解在超纯水中,通过涡旋仪使聚合物混合均匀,呈澄清透明的溶液,制得浓度为总浓度0.01-300.0mg/mL的聚合物溶液,在室温下将一定体积的聚合物水溶液置于离心管中。
所述聚合物包括聚乙烯吡咯烷酮(PVP),聚乙烯醇(PVA),聚乙烯亚胺(PEI),聚乙烯醇(PVA),不同分子量的聚乙二醇(PEG,1~1000kDa),含聚乙二醇的嵌段共聚物,带有不同末端官能团的聚乙二醇(巯基,氨基,羟基,羧基,甲氧基,琥珀酰亚胺碳酸酯,马来酰亚胺,甲基丙烯酸酯,活性酯NHS,Cy3-NHS,Cy5-HNS,Cy7-NHS),不同臂数的多臂聚乙二醇(四臂,六臂,八臂)等。
(2)将多酚溶解于超纯水中,配置成浓度为0.01-500.0mg/mL的多酚水溶液。取与步骤(1)相同体积的多酚水溶液置于步骤(1)的离心管中,通过倒置正置离心管5次,使多酚溶液与聚合物溶液混合均匀,得到不同尺寸、形貌、性质的凝聚层颗粒。
所述的多酚包括,邻苯二酚,邻苯三酚,没食子酸,表没食子儿茶素没食子酸酯(EGCG),鞣花酸,儿茶素水合物,白藜芦醇,没食子酸乙酯,单宁酸(TA)。
在一些实施例中,通过调节多酚与聚合物的质量分数与摩尔及混合液pH能够制备100纳米至10微米的球型实心液滴,冻干后为颗粒。也能够制备1-10微米的中空胶囊。聚合物与多酚的摩尔比为1:(0.01~1000),溶液pH为2~8.5。
在一些实施例中,所述多酚的加入速度为0.1~50mL/s。
下面结合具体的实施例,对本发明做进一步的详细说明,应该指出,所述具体实施例是对本发明的解释而不是限定。
实施例1
配置20mL聚乙烯吡咯烷酮(PVP)的水溶液,其质量浓度为0.1mg/mL,在涡旋及超声的条件下溶解。配置20mL没食子酸乙酯的水溶液,其质量浓度为0.1mg/mL,通过涡旋及超声使没食子酸乙酯快速溶解。将20mL没食子酸乙酯水溶液加入到20mL的PVP溶液中,正置倒置离心管5次将二者混合液混匀,即可得到稳定的凝聚层纳米颗粒。所得纳米颗粒的透射电子显微镜图片如图1,具有良好的分散性及稳定性,衬度均匀表示颗粒质地均匀且为实心颗粒,利用纳米粒度仪跟踪颗粒的尺寸,其尺寸具有单分散性,且能稳定一周以上。
实施例2
配置20mL聚乙二醇(2kDa)与聚乙烯亚胺(PEI)的混合水溶液,其总质量浓度为50mg/mL,二者质量比为1:1,在涡旋及超声的条件下溶解。配置20mL单宁酸的水溶液,其质量浓度为50mg/mL,通过涡旋及超声使单宁酸快速溶解。将20mL单宁酸水溶液加入到20mL聚合物混合液中,正置倒置离心管5次将二者混合液混匀,即可得到分散性良好的凝聚层纳米颗粒,如图2。
实施例3
配置20mL末端官能团为羧基的聚乙二醇(10kDa)的水溶液,其质量浓度为300mg/mL,在涡旋及超声的条件下溶解。配置20mL单宁酸的水溶液,其质量浓度为500mg/mL,通过涡旋及超声使单宁酸快速溶解。将20mL单宁酸水溶液加入到20mL末端官能团为羧基的聚乙二醇溶液中,正置倒置离心管5次将二者混合液混匀,即可得到胶囊形状的凝聚层纳米颗粒,如图3,可控制两胶囊的动态融合速率。
实施例4
配置20mL末端官能团为Cy5-NHS的聚乙二醇(10kDa)的水溶液,其质量浓度为300mg/mL,在涡旋及超声的条件下溶解。配置20mL单宁酸的水溶液,其质量浓度为500mg/mL,通过涡旋及超声使单宁酸快速溶解。将20mL单宁酸水溶液加入到20mL末端官能团为羧基的聚乙二醇溶液中,正置倒置离心管5次将二者混合液混匀,即可得到胶囊形状的凝聚层纳米颗粒,如图4。
实施例5
配置20mL的聚乙烯吡咯烷酮(PVP)水溶液,其质量浓度为0.1mg/mL,在涡旋及超声的条件下溶解。配置20mL邻苯三酚的水溶液,其质量浓度为10mg/mL。将20mL邻苯三酚水溶液加入到PVP溶液中,正置倒置离心管5次将二者混合液混匀,即可得到分散性良好的凝聚层纳米颗粒。
实施例6
配置20mL直链聚乙二醇(2kDa)的水溶液,其质量浓度为5mg/mL,在涡旋及超声的条件下溶解。配置20mL茶多酚(EGCG)的水溶液,其质量浓度为10mg/mL。将20mL的EGCG水溶液加入到20mL直链聚乙二醇溶液中,正置倒置离心管5次将二者混合液混匀,即可得到分散性良好的凝聚层纳米颗粒。
实施例7
配置5mL聚乙烯醇(10kDa)的乙腈溶液,其质量浓度为20mg/mL,在涡旋及超声的条件下溶解。配置5mL单宁酸的乙腈分散液,其质量浓度为20mg/mL。将5mL单宁酸乙腈分散液加入到5mL聚乙二醇乙腈分散液中,正置倒置离心管5次将二者混合液混匀,即可得到微米级的凝聚层颗粒。
实施例8
配置5mL聚乙二醇-甲氧基(40kDa)的水溶液,其质量浓度为50mg/mL,在涡旋及超声的条件下溶解。向其中加入FITC标记的牛血清白蛋白(BSA),制成含0.1mg/mL BSA的聚合物水溶液。配置5mL单宁酸的水溶液,其质量浓度为100mg/mL。将5mL单宁酸水溶液加入到5mL含有BSA的聚合物溶液中,正置倒置离心管5次将二者混合液混匀,即可得到封装了BSA的凝聚层颗粒,具有良好的分散性。通过激光共聚焦证明了BSA的成功封装,将溶液13400g离心后检测上清液的FITC浓度,得出BSA的封装率达90%。
实施例9
配置1mL聚乙二醇-羟基(10kDa)和聚乙二醇-巯基(10kDa)的混合水溶液,总质量浓度为1mg/mL,二者质量比为1:1,在涡旋及超声的条件下溶解。向其中加入金纳米棒(50nm*10nm),制成含5mg/mL金纳米棒的聚合物混合溶液。配置1mL EGCG的水溶液,其质量浓度为1mg/mL,通过涡旋及超声使EGCG快速溶解。将1mL的EGCG水溶液加入到1mL含有金纳米棒的聚合物溶液中,正置倒置离心管5次将二者混合液混匀,即可得到封装了金纳米棒的凝聚层颗粒,具有良好的分散性。通过透射电子显微镜证明了金纳米棒的成功封装。
实施例10
配置20mL聚乙二醇(1kDa)的水溶液,其质量浓度为1mg/mL,在涡旋及超声的条件下溶解。配置20mL单宁酸的水溶液,其质量浓度为1mg/mL,通过涡旋及超声使单宁酸快速溶解。将20mL单宁酸水溶液加入到20mL聚乙二醇溶液中,正置倒置离心管5次将二者混合液混匀,即可得到分散性良好的凝聚层纳米颗粒。将巨噬细胞孵育12小时,使细胞贴壁生长。细胞分为三组,空白组继续细胞与细胞培养液孵育12小时,实验组细胞预先用含有纳米颗粒的细胞培养液共培养1小时,而后用过氧化氢溶液刺激1小时,对照组细胞与细胞培养液共培养1小时后,用等量过氧化氢溶液刺激1小时。用灭菌的磷酸盐缓冲溶液(pH=7.4)冲洗孔板,每组加入DCFH-DA荧光探针,检测细胞内自由基产生情况。用流式细胞仪对荧光强度进行定量分析,证明颗粒的加入能够降低由过氧化氢刺激的细胞内自由基水平,使其降至普通细胞水平,实现细胞抗氧化。
最后应该说明的是,以上所述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,其依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分进行等同替换。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 (3)
1.一种基于多酚的凝聚层颗粒制备方法,其特征在于,包括:
将多酚化合物水溶液与至少一种含有氢键受体的聚合物水溶液混合均匀,即得;
所述多酚化合物为邻苯三酚、茶多酚、没食子酸乙酯或单宁酸;
所述聚合物为聚乙烯吡咯烷酮、聚乙烯醇、聚乙烯亚胺、聚乙二醇、带有不同末端官能团的聚乙二醇;所述带有不同末端官能团的聚乙二醇中,末端官能团为巯基,羟基,羧基,甲氧基或Cy5-NHS;
所述混合的具体方法如下:
(1)将一种或几种聚合物溶解在超纯水中,通过涡旋仪使聚合物混合均匀,呈澄清透明的溶液,制得浓度为总浓度0.01-300.0 mg/mL的聚合物溶液,在室温下将一定体积的聚合物水溶液置于离心管中;
(2)将多酚化合物溶解于超纯水中,配置成浓度为0.01-500.0 mg/mL的多酚水溶液,取与步骤(1)相同体积的多酚水溶液置于步骤(1)的离心管中,多酚水溶液的加入速度为0.1~50 mL/s;通过倒置正置离心管5次,使多酚溶液与聚合物溶液混合均匀;
通过调节多酚与聚合物的质量分数与摩尔比及混合液pH能够制备100纳米至10微米的球型实心液滴及1-10微米的中空胶囊;所述实心液滴冻干后为颗粒;聚合物与多酚化合物的摩尔比为1:(0.01~1000);溶液pH为2~8.5。
2.权利要求1的方法制备的凝聚层颗粒,其特征在于,所述凝聚层颗粒为球型实心颗粒或中空胶囊。
3.权利要求1所述的方法制备的凝聚层颗粒在制备提高细胞氧化应激能力药物运载体中的应用,其特征在于,制备凝聚层颗粒的聚合物为聚乙二醇,多酚为单宁酸。
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