CN115382013B - 一种压电纳米颗粒及其制备方法 - Google Patents
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Abstract
本发明属于纳米材料技术领域,具体涉及一种压电纳米颗粒及其制备方法。本发明一种压电纳米颗粒BCG‑NPs,为核壳型压电纳米颗粒,主要由BaTiO3,负载在BaTiO3表面作为正电部分的壳聚糖,以及负载在壳聚糖表面作为负电部分的氧化石墨烯组成。
Description
技术领域
本发明属于纳米材料技术领域,具体涉及一种压电纳米颗粒及其制备方法。
背景技术
仿生骨植入材料是骨再生领域的热门领域,包括模拟结构、成分和力学(L.L.Hench,I.Thompson,Journal of The Royal Society Interface 2010,7,379.)。之前的研究已经证实,生物电可以作为一种细胞调节器,对骨内稳态发挥有益的作用,尤其是承重骨(C.Bassett,R.J.Pawluk,R.O.Becker,Nature 1964,204,652;b)C.Bassett,R.O.Becker,Science 1962,137,1063.)。因此,模拟骨电特性的生物材料是再生医学领域极具吸引力的试剂(A.Dk,B.Bb,A.Akd,Biomaterials 258.)。目前,这方面的文献有限,主要集中在利用种植体表面电荷和外部微电流进行骨再生(W.Chrzanowski,J.H.Lee,A.Kondyurin,M.S.Lord,J.H.Jang,H.W.Kim,M.M.M.Bilek,Advanced FunctionalMaterials 2015,25,193.)。因此,需要研究如何在生物材料和组织界面生成最佳且稳定的生理电微环境以诱导新骨(Y.Liu,X.Zhang,C.Cao,Y.Zhang,J.Wei,Y.J.Li,W.Liang,Z.Hu,J.Zhang,Y.Wei,Advanced Functional Materials 2017,27,1703771.1.)。
近年来,压电材料因其在生物医学领域的潜在应用而受到了广泛关注(B.Tandon,J.J.Blaker,S.H.Cartmell,Acta Biomaterialia2018,S1742706118302290.)。压电材料可以被压力极化,这样压电材料两端的表面就会出现符号相反的束缚电荷。纳米钛酸钡(BaTiO3)是一种常用的压电材料,其制备工艺成熟,粒径可控,形状可控,压电性能好,价格低廉(KenjiUchino,Advanced piezoelectric materials:science and technology,Advanced piezoelectric materials:science and technology,2010.)。然而,作为一种生物材料,钛酸钡的生物相容性和分散性较差,从而限制了其在骨再生策略中的应用(U.Yasutomo,U.Takahiro,H.Hwishim,H.Yoshiaki,T.Kazuyoshi,N.Eiji,Journal of theSociety of Materials Science Japan 2008,57,899.)。
静电逐层自组装(LBL)是一种基于正负聚电解质电荷相互作用的超分子技术。这是一种低成本、简单的方法,可以将各种有机、无机和生物材料组装到固体基底上,无需复杂的仪器和苛刻的条件(Z.Xi,H.Chen,H.Zhang,Chemical Communications 2007,p.1395)。LBL技术在生物医学领域有着广泛的应用,如薄膜包衣、微模式、纳米生物反应器、人工细胞、包衣粒子和药物递送系统(H.Ai,S.A.Jones,Y.M.Lvov,Cell Biochemistry&Biophysics 2003,39,23.)。
壳聚糖(CS)具有良好的生物相容性、亲水性和表面正电荷,可作为阳离子共聚物组分使用(a)I.Y.Kim,S.J.Seo,H.S.Moon,M.K.Yoo,I.Y.Park,B.C.Kim,C.S.Cho,Biotechnology Advances 2008,26,1;b)Y.Wang,Q.Hong,Y.Chen,X.Lian,Y.Xiong,Colloids and surfaces B:Biointerfaces2012,100,77.)。目前已有学者将导电材料引入压电材料领域,研究结果证实,加入导电相的压电材料在极化后呈现更佳的压电性能(Fangwei Qi,Zichao Zeng,Jia Yao,Materials Science&Engineering C 2021)。原子薄层氧化石墨烯(GO)片是导电性能极佳的材料,同时可用作生物相容性平台,有可能介导组织再生应用的干细胞谱系规范;其高亲水性和高血清蛋白吸收能力增强细胞粘附、增殖和成骨分化((a)W.C.Lee,C.Lim,H.Shi,L.Tang,Y.Wang,C.T.Lim,K.P.Loh,Acs Nano 2011,5,7334;b)C.Chung,Y.K.Kim,D.Shin,S.R.Ryoo,D.H.Min,Acc Chem Res 2013,46,2211.)。GO片材中的高水平亲水性基团提供了良好的分散性和稳定性,此外,GO不仅抑制细菌(例如金黄色葡萄球菌、大肠杆菌等)附着,而且还包含固有的抗菌活性(X.Zou,Z.Li,Z.Wang,Y.Luo,Journal of the American Chemical Society 2016,138,2064.)。GO在硬组织再生和骨组织工程中可能是一个很有前途的候选者(H.Y.Mao,S.Laurent,W.Chen,O.Akhavan,M.Imani,A.A.Ashkarran,M.Mahmoudi,Chemical Reviews 2013,113,3407.)。因此,选择GO作为具有负表面电荷的壳层,赋予共聚物骨诱导电位。GO的加入有望与压电效应协同工作,进而促进骨再生。
发明内容
针对现有问题的不足,本发明的目的是提供一种压电纳米颗粒BCG-NPs及其制备方法。
本发明解决其技术问题采用的技术方案是:
第一方面,本发明保护一种压电纳米颗粒BCG-NPs,为核壳型压电纳米颗粒,主要由BaTiO3,负载在BaTiO3表面作为正电部分的壳聚糖,以及负载在壳聚糖表面作为负电部分的氧化石墨烯组成。
第二方面,本发明保护前述压电纳米颗粒BCG-NPs的制备方法,主要包括如下步骤:
步骤1,钛酸钡-壳聚糖NP(BC-NPs)的合成:将BaTiO3粉末加入到水中,超声分散;然后,将2-4%(w/v)壳聚糖乙酸溶液缓慢滴入到BaTiO3分散液中;搅拌4-6小时后,将混合物离心以去除未反应的试剂,沉淀物为BC-NP;洗涤,干燥;
步骤2,钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)合成:将步骤1所得BC-NP加入到水中,超声分散,并缓慢滴入到2%(w/v)氧化石墨烯水溶液中;搅拌8~15小时后,生成悬浮液;离心,洗涤,干燥。
作为本申请的优选技术方案,所述步骤1中,BaTiO3与水的质量体积比为0.5~3g:50mL。
优选的,BaTiO3与水的质量体积比为1g:50mL。
作为本申请的优选技术方案,所述步骤1中,壳聚糖乙酸溶液的浓度为2%、3%、4%中的任一种,搅拌时间为4,5,6小时中的任一种。
优先的,所述步骤1中,壳聚糖乙酸溶液的浓度为2%,搅拌时间4小时。
作为本申请的优选技术方案,所述步骤2中,搅拌时间为12小时。
作为本申请的优选技术方案,超声分散的时间为30~60min。
作为本申请的优选技术方案,所述超声分散的时间为30min。
本发明还保护前文所述的制备方法制备得到的压电纳米颗粒BCG-NPs。
第三方面,本发明还保护前文所述的压电纳米颗粒BCG-NPs在制备仿生骨材料中的应用。
第四方面,本发明保护一种对植入仿生骨材料的患者进行超声处理的装置,其包括:
(1)用于接触患者患病部位刺激仿生骨材料产生微电流的超声探头;
(2)用于对超声条件进行控制的控制单元;
优选的,所述仿生骨材料为前文所述的压电纳米颗粒BCG-NPs;
优选的,超声功率为0.6~2.0W/cm2,优选的为1.3W/cm2。
有益效果
(1)改性后纳米颗粒分散性能显著提升;纳米颗粒在医学方面的应用范围很广,例如:改性各种植入材料、作为纳米胶囊搭载药物、通过各种机制抗菌、成骨、抑制肿瘤等等。这些功能都要求纳米颗粒具有良好的分散性能以维持纳米尺寸,这是纳米颗粒具有较大的比表面积及发挥小尺寸效应的基础。
(2)改善细胞相容性;自组装改性后得到的钛酸钡-壳聚糖-氧化石墨烯纳米颗粒(BCG-NPs)相较未改性的钛酸钡纳米颗粒促细胞增殖效果更好,具有更佳的细胞相容性。这在生物材料领域是非常重要的。
(3)BCG-NPs在超声刺激下能够产生微电流;
(4)BCG-NPs对比空白对照组在超声刺激下促成骨作用更佳。
附图说明
图1为BCG-NPs制备工艺示意图;
图2为不同制备条件获得的BCG-NPs的透射电镜图。2%(w/v)的CS溶液,组装4h条件下得到的BCG-NPs分散性最佳;
图3为实施例1的BCG-NPs与改性前样品对比的形态和大小分布。(a)BaTiO3-NPs,(b)BC-NPs,(c)BCG-NPs的透射电子显微镜显微照片。(d–f)分别测量BaTiO3-NPs、BC-NPs、BCG-NPs的粒径分布;扫描电子显微镜显微照片显示(g)BaTiO3-NPs,(h)BC-NPs,(i)BCG-NPs。纳米颗粒的大小报告为平均值±标准偏差(SD);
图4:(a)BC-NPs,BCG-NPs,GO和壳聚糖(CS)、BaTiO3标准PDF卡片:PDF#39-1894(国际衍射数据中心(ICDD),PDF2-2004,CS),PDF#75-0583(国际衍射数据中心(ICDD),PDF2-2004,BaTiO3四方相;(b)显示了BaTiO3-壳聚糖-氧化石墨烯纳米颗粒和BaTiO3-壳聚糖纳米颗粒在2θ=10°时的局部放大图。
图5为BCG-TI的电滞回线,显示出典型的铁电特性;
图6为自组装前后纳米颗粒对细胞增殖的效果,钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)对细胞增殖的促进作用更强,具有更佳的细胞相容性。
图7:(a)超声装置及微电流测量装置示意图。(b)上图为空白培养基在超声下产生的微电流大小;下图为相同功率下200μg ml-1BCG-NPs产生的微电流大小。(c)不同浓度BCG-NPs在不同功率下对细胞增殖的作用;
图8:1.3W/cm2功率下200μg ml-1BCG-NPs对早期成骨指标碱性磷酸酶ALP活性的影响。
图9:1.3W/cm2功率下200μg ml-1BCG-NPs对晚期成骨指标细胞外基质矿化结节(茜素红法)的影响。
具体实施方式
以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的,均视为可以通过市场购买的常规产品。
钛酸钡(BaTiO3)粉体(中国南京宏德纳米材料有限公司)。它的平均粒径为≤100纳米,作为核壳纳米结构的核。
氧化石墨烯(GO)片(≤500纳米,厚度=0.335–1纳米)从碳烯技术(中国深圳)有限公司购买。
壳聚糖(CS)(脱乙酰度>95%,粘度=100–200mPa)购自上海麦克林生化有限公司(中国上海)。
实施例1.钛酸钡-壳聚糖-氧化石墨烯纳米颗粒(BCG-NPs)的合成
(1)钛酸钡-壳聚糖NP(BC-NPs)的合成。
将1g BaTiO3粉末分散到50ml去离子水中,并在Bransonic超声波仪中对溶液进行30分钟的超声波处理。然后,将2%(w/v)CS(1g)乙酸溶液(50mL)缓慢滴入到BaTiO3分散液中。搅拌4小时,将混合物离心以去除未反应的试剂,沉淀物为BC-NPs。将合成的BC-NPs重新分散在去离子水中,超速离心,并丢弃上清液。该冲洗步骤进行三次。
(2)钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)合成。
将BC-NPs分散在去离子水中,Bransonic超声波仪超声30min,并缓慢滴入到100mL2%(w/v)GO水溶液中。搅拌12小时后,生成悬浮液。由此,对BCG-NPs按以上相同方法进行离心、洗涤和干燥。
实施例2钛酸钡-壳聚糖-氧化石墨烯纳米颗粒(BCG-NPs)的合成
(1)钛酸钡-壳聚糖NPs(BC-NPs)的合成。
将1g BaTiO3粉末分散到50ml去离子水中,并在Bransonic超声波仪中对溶液进行30分钟的超声波处理。然后,将2%(w/v)CS(1g)乙酸溶液(50mL)缓慢滴入到BaTiO3分散液中。搅拌6小时,将混合物离心以去除未反应的试剂,沉淀物为BC-NP。将合成的BC-NP重新分散在去离子水中,超速离心,并丢弃上清液。该冲洗步骤进行三次。
(2)钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)合成。
将BC-NPs分散在去离子水中,Bransonic超声波仪超声30min,并缓慢滴入到100mL2%(w/v)GO水溶液中。搅拌12小时后,生成悬浮液。由此,对BCG-NPs按以上相同方法进行离心、洗涤和干燥。钛酸钡-壳聚糖NPs(BC-NPs)的合成。
实施例3钛酸钡-壳聚糖-氧化石墨烯纳米颗粒(BCG-NPs)的合成
(1)钛酸钡-壳聚糖NPs(BC-NPs)的合成
将1g BaTiO3粉末分散到50ml去离子水中,并在Bransonic超声波仪中对溶液进行30分钟的超声波处理。然后,将4%(w/v)CS(1g)乙酸溶液(50mL)缓慢滴入到BaTiO3分散液中。搅拌4小时,将混合物离心以去除未反应的试剂,沉淀物为BC-NPs。将合成的BC-NPs重新分散在去离子水中,超速离心,并丢弃上清液。该冲洗步骤进行三次。
(2)钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)合成。
钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)合成。将BC-NPs分散在去离子水中,Bransonic超声波仪超声30min,并缓慢滴入到100mL 2%(w/v)GO水溶液中。搅拌12小时后,生成悬浮液。由此,对BCG-NPs按以上相同方法进行离心、洗涤和干燥。
实施例4钛酸钡-壳聚糖-氧化石墨烯纳米颗粒(BCG-NPs)的合成
(1)钛酸钡-壳聚糖NPs(BC-NPs)的合成
将1g BaTiO3粉末分散到50ml去离子水中,并在Bransonic超声波仪中对溶液进行30分钟的超声波处理。然后,将4%(w/v)CS(1g)乙酸溶液(50mL)缓慢滴入到BaTiO3分散液中。搅拌6小时,将混合物离心以去除未反应的试剂,沉淀物为BC-NPs。将合成的BC-NPs重新分散在去离子水中,超速离心,并丢弃上清液。该冲洗步骤进行三次。
(2)钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)合成。
钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)合成。将BC-NPs分散在去离子水中,Bransonic超声波仪超声30min,并缓慢滴入到100mL 2%(w/v)GO水溶液中。搅拌12小时后,生成悬浮液。由此,对BCG-NPs按以上相同方法进行离心、洗涤和干燥。
钛酸钡-壳聚糖-氧化石墨烯纳米颗粒(BCG-NPs)表征
在所有实验中,样品在Bransonic超声波仪20W的条件下超声处理30分钟。
(1)形貌及分散性表征:在200目多孔碳膜铜网上加入5μl NPs溶液制备TEM样品。将10μl NPs溶液滴在导电胶带上。干燥和喷金后,制备扫描电镜观察样品。使用扫描电子显微镜(SEM,TESCAN MAIA3,捷克)和透射电子显微镜(TEM,JEM-2100F,日本)生成样品显微照片。
(2)粒径分布:使用TEM像分析软件(Nano Measurer,1.2.5,复旦大学,上海,中国)计算NP尺寸。
(3)物相分析:使用X射线衍射仪(XRD;日本Rigaku Smartlab 9)鉴定NPs晶体结构和晶相纯度。在40kV和150mA,衍射角5°和90°之间的条件下,使用铜阳极源的Cu Kα辐射
结果如图2-图4所示,BCG-NPs为单分散球体,是所有样品中分散最好的颗粒;层层自组装改性很大程度上提高了纳米颗粒的分散性,而分散性又很大程度影响着纳米颗粒的未来应用;且2%(w/v)的CS溶液,组装4h条件下得到的BCG-NPs分散性最佳。
(4)压电性能测量
1)电滞回线
在极化和24小时老化后,对放置在钛基底上的BCG-NPs进行测试。使用铁电分析仪(TF analyzer 2000E,德国Aix ACCT Systems GmbH)在最大磁场振幅为10kV/mm、频率为1Hz的条件下测量极化电场回路。
2)超声作用下微电流测试
纳米颗粒分散在培养基中,正负电极通入悬浮液,在超声作用下通过示波器测试实时微电流大小。
3.细胞相容性及成骨性能测试
(1)实验方案由南京医科大学伦理委员会批准,经供体书面知情同意,人类脐带间充质干细胞的提取步骤(hMSCs)如下。脐带在含有1%青霉素和链霉素的无菌磷酸盐缓冲液(Biosharp,Beijing Labgic Technology Co.,Ltd.,中国北京)中清洗(上海碧云天生物科技有限公司,中国上海)。去除脐静脉和动脉后,间充质切成2-3mm块,放入25mL培养瓶中(Corning,美国纽约康宁公司),并在DMEM/F12培养基中培养(Gibco,赛默飞世尔科技(中国)有限公司,上海,中国)含有10%胎牛血清(Gibco,赛默飞世尔科技(中国)有限公司,中国上海)和100U/mL青霉素和链霉素(上海碧云天生物科技有限公司,中国上海)。3天后去除未贴壁的组织块,按常规方法收集、培养、扩增hMSCs。
(2)细胞增殖
使用细胞计数试剂盒-8(CCK-8,中国上海东仁化工科技有限公司)比色法测定细胞活力。HWJ-MSCs以1×105细胞mL-1接种于96孔培养板中。细胞贴壁后,分别将1000μg mL-1的BTO-NPs、BC-NPs、BCG-NPs加入培养孔中,培养24h后按照制造商的说明进行CCK-8分析。在微孔板阅读器(Spectramax 190,Molecular Devices,USA)上,在450nm处评估Formazan吸光度。进行三次独立实验的重复。
结果如图6所示,相较于改性前的钛酸钡纳米颗粒(BaTiO3-NPs)及钛酸钡-壳聚糖纳米颗粒(BC-NPs),钛酸钡-壳聚糖-氧化石墨烯(BCG-NPs)对细胞增殖的促进作用更强,具有更佳的细胞相容性。自组装改性有利于提高纳米颗粒的细胞相容性。
为选出促进成骨的最优浓度及最佳超声功率。分别添加含有BCG-NPs的培养基(0μgmL-1、50μg mL-1、100μg mL-1、200μg mL-1、500μg mL-1和1000μg mL-1),对所有浓度的孔板分别进行0.6、1.3、2.0W/cm2不同的超声功率进行处理。细胞分别培养1天、3天后,按照制造商的说明进行CCK-8分析。在微孔板阅读器(Spectramax 190,Molecular Devices,USA)上,在450nm处评估Formazan吸光度。进行三次独立实验的重复。
结果如图7所示,培养1天、3天后,1.3W/cm2功率下200μg ml-1BCG-NPs对细胞增殖的促进作用最强。采取最优浓度及最佳超声功对BCG-NPs产生的微电流大小进行测量,相较空白培养基组具有显著差异,进一步证实BCG-NPs的压电特性、构建生理微电环境的潜力。
(3)成骨性能
1)碱性磷酸酶(alkaline phosphatase,ALP)活性
为评价1.3W/cm2功率下,200μg mL-1BCG-NPs对hWJ-MSCs ALP活性的影响,将hWJ-MSCs以3×104/孔接种在24孔板的样本上。培养1d后,加入纳米颗粒,并将培养基更换为成骨培养基,该培养基为基础培养基中加入由10mMβ-甘油磷酸盐、50μg/mL维生素C和0.1μM地塞米松组成的成骨诱导液配制而成。每天进行5分钟超声培养,培养7d及14d后,用5-溴-4-氯-3-吲哚磷酸/硝基四氮唑蓝(BCIP/NBT)ALP比色试剂盒对hWJ-MSCs进行染色并拍照。
结果如图8所示,培养7天、14天后,BCG-NPs具有显著的促成骨分化能力。
2)细胞外基质(extracellular matrix,ECM)矿化
通过茜素红(Alizarin red S,ARS)染色检测1.3W/cm2功率下,200μg mL-1BCG-NPs对hWJ-MSCs矿化结节形成的影响。将hWJ-MSCs以2×104/孔接种在24孔板中的样本上。培养1d后,加入纳米颗粒,并将培养基更换为成骨培养基。每天进行5分钟超声培养,培养21d。用PBS洗涤2遍,无水乙醇固定15min,室温风干后加入1%(w/v)茜素红(pH=4.2)染色5min,双蒸水洗涤2遍,在显微镜下观察并拍照。
结果如图9所示,培养21天后,BCG-NPs具有显著的促成骨分化能力。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求为保护范围。
Claims (8)
1.一种压电纳米颗粒BCG-NPs,其特征在于,所述为压电纳米颗粒为核壳型压电纳米颗粒,主要由BaTiO3,负载在BaTiO3表面作为正电部分的壳聚糖,以及负载在壳聚糖表面作为负电部分的氧化石墨烯组成;
所述压电纳米颗粒BCG-NPs按照如下步骤制备:
步骤1,钛酸钡-壳聚糖BC-NPs的合成:将BaTiO3粉末加入到水中,超声分散;然后,将2-4%壳聚糖乙酸溶液缓慢滴入到BaTiO3分散液中;搅拌4-6小时后,将混合物离心以去除未反应的试剂,沉淀物为BC-NPs;洗涤,干燥;
步骤2,钛酸钡-壳聚糖-氧化石墨烯BCG-NPs的合成:将步骤1所得BC-NPs加入到水中,超声分散,并缓慢滴入到2%w/v氧化石墨烯水溶液中;搅拌8~15小时后,生成悬浮液;离心,洗涤,干燥;
其中,BaTiO3的平均粒径≤100纳米。
2. 根据权利要求1所述的压电纳米颗粒BCG-NPs,其特征在于,所述步骤1中,BaTiO3与水的质量体积比为0.5~3g:50 mL。
3. 根据权利要求2所述的压电纳米颗粒BCG-NPs,其特征在于,所述步骤1中,BaTiO3与水的质量体积比为1g:50 mL。
4.根据权利要求1所述的压电纳米颗粒BCG-NPs,其特征在于,所述步骤1中,所述壳聚糖乙酸溶液的浓度为2%、3%、4%中的任一种,搅拌时间为4,5,6小时中的任一种。
5.根据权利要求1所述的压电纳米颗粒BCG-NPs,其特征在于,所述步骤2中,搅拌时间为12小时。
6. 根据权利要求1所述的压电纳米颗粒BCG-NPs,其特征在于,超声分散的时间为30~60 min。
7. 根据权利要求6所述的压电纳米颗粒BCG-NPs,其特征在于,超声分散的时间为30min。
8.权利要求1-7任一所述的压电纳米颗粒BCG-NPs在制备仿生骨材料中的应用。
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