CN105477647A - 石墨烯量子点/壳聚糖干凝胶的制备并应用于荧光成像和药物缓释 - Google Patents
石墨烯量子点/壳聚糖干凝胶的制备并应用于荧光成像和药物缓释 Download PDFInfo
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Abstract
本发明涉及一步法合成石墨烯量子点/壳聚糖干凝胶并将其应用于裸鼠体内成像及药物载体，包括以下步骤：制备的石墨烯量子点/壳聚糖干凝胶、制备载药石墨烯量子点/壳聚糖干凝胶有益效果是：制备过程简单、反应温和，制成石墨烯量子点/壳聚糖干凝胶能够进行裸鼠体内成像，并且体外药物缓释性能与以往材料相比有大幅提升。
Description
技术领域
本发明涉及一种采用一步法合成石墨烯量子点/壳聚糖干凝胶并将其应用于裸鼠体内成像及药物载体，属于材料合成和生物医药领域。
技术背景
药物缓释是将小分子药物与高分子材料相结合，利用高分子的特性(如pH敏感性、温度敏感性等)使药物分子在高分子载体中缓慢释放出来。与传统给药体系相比较，药物缓释体系能够有效地提高药物的生物利用率，减少给药次数以及提高用药的稳定性。因此制备药物缓释材料引起研究人员的广泛兴趣。由于荧光成像能够跟踪药物分子在体内的分布情况，导致具有荧光效应的药物载体在生物医药中的发展更为重要。当今科研人员都在致力于研究具有靶向成像的药物缓释体系。
在以往的研究中，科研人员致力研究天然多糖，由于天然多糖的生物相容性较好，生物毒性低的特点，导致壳聚糖这种天然多糖在药物缓释体系中发挥着重要作用。本发明中将石墨烯量子点引入壳聚糖制备成石墨烯量子点/壳聚糖干凝胶，介于石墨烯量子点具有独特的荧光性质，并且在以往的报道中以证实石墨烯量子点在生物细胞中有着较好的生物相容性，且较低的生物毒性。在此我们发明了一种能够在裸鼠体内成像且具有pH敏感性的药物缓释载体石墨烯量子点/壳聚糖干凝胶。
发明内容
本发明的目的是在于提供一种新的药物载体石墨烯量子点/壳聚糖干凝胶，并将其应用于裸鼠体内成像及体外药物缓释。本发明还提供了一种新的石墨烯量子点/壳聚糖干凝胶的合成方法，将石墨烯量子点与醋酸配制的壳聚糖溶液混合，在一定的条件下一步法合成石墨烯量子点/壳聚糖干凝胶。
本发明所述一步法合成石墨烯量子点/壳聚糖干凝胶并将其应用于裸鼠体内成像及药物载体，包括以下步骤：
a、制备石墨烯量子点/壳聚糖干凝胶：磁力搅拌下将壳聚糖分散于醋酸溶液中，加入石墨烯量子点溶液。将混合溶液磁力搅拌并保持反应10min。反应结束后用胶头滴管缓慢滴加10％氢氧化钠到混合溶液中并调节pH至中性，静置过滤，超纯水洗涤，在-45℃下冷冻干燥24h，研磨备用，得到石墨烯量子点质量百分含量(wt％)不同的石墨烯量子点/壳聚糖干凝胶；
b、石墨烯量子点/壳聚糖干凝胶进体外药物释放实验：配制水杨酸钠溶液，加入石墨烯量子点/壳聚糖干凝胶进行载药，并将制得的载药石墨烯/壳聚糖干凝胶进行体外药物释放实验；
c、制备石墨烯量子点/壳聚糖干凝胶用于生物成像：将步骤a制得的石墨烯/壳聚糖干凝胶配制成分散液并注射到裸鼠皮下组织，进行荧光成像；
进一步，步骤a中石墨烯的含量为(0-43wt％)，反应时间为5-60min。
进一步，步骤a中壳聚糖浓度为1-10mgmL-1。
进一步，步骤b中石墨烯/壳聚糖干凝胶进行载药，药物为水杨酸钠浓度为1-2mgmL-1，载药时间为12-48h，载药后冷冻干燥的温度为-45℃。
进一步，步骤c中石墨烯/壳聚糖干凝胶浓度为1-10mgmL-1，注射体积为50-200μL。
本发明的有益效果是：石墨烯/壳聚糖干凝胶的制备方法简便易行，制备过程环保无污染，制备出的石墨烯/壳聚糖干凝胶能够进行裸鼠体内成像、较高的载药量、且缓释性能与以往材料相比也有了大幅的提升。
具体实施方式
实施例一：
制备石墨烯量子点/壳聚糖干凝胶包括以下几个步骤：
(1)将壳聚糖加入到0.1M醋酸溶液中并磁力搅拌分散，加入2mgmL-1石墨烯量子点溶液并磁力搅拌10min；在搅拌状态下用胶头滴管将10％氢氧化钠溶液缓慢滴加到石墨烯量子点、壳聚糖混合溶液中调节pH至中性。滴加完毕后，停止搅拌，静置过滤，并用超纯水洗涤，在-45℃下冷冻干燥24h，研磨备用，即得石墨烯量子点/壳聚糖干凝胶。制备出三个样品编号分别为C、D、E。
图1为石墨烯量子点/壳聚糖干凝胶的傅里叶红外谱图(FT-IR)，准确称取1mgA、B、C、D和E分别与200mg溴化钾碾磨压片。A为纯的壳聚糖FT-IR，有两个特征吸收带在1650和1604cm-1分别归因于-NHCO-中C＝O伸缩振动和-NH2中N-H振动。B为石墨烯量子点FT-IR，1766和1716cm-1归因于石墨烯量子点表面羧酸基团中C＝O伸缩振动。C～E为(wt％)不同的石墨烯量子点/壳聚糖干凝胶FT-IR，对比于壳聚糖和石墨烯量子点，1604cm-1处-NH2吸收振动和1766cm-1处C＝O振动在石墨烯量子点/壳聚糖干凝胶中消失。此外，石墨烯量子点中羧基的C＝O伸缩振动偏移至1704cm-1。通过FT-IR可证明成功制备出石墨烯量子点/壳聚糖干凝胶。图2为壳聚糖、石墨烯量子点及石墨烯量子点/壳聚糖干凝胶的场发射扫描电镜(FESEM)和透射电镜(TEM)，可以观察出随着石墨烯量子点含量的增加导致了石墨烯量子点/壳聚糖干凝胶的结构发生了很大的变化，通过FESEM可知A样品为片状结构、B样品为球状颗粒、C样品为团聚态结构、D样品为网络状结构、E样品出现了三维网络状的结构。通过TEM可知石墨烯量子点的含量在C-E中逐渐增加。
实施例二：
石墨烯量子点/壳聚糖干凝胶制备过程与实施例一相同。
将步骤(1)中的样品E在0.1MPBS(pH＝7.4)中配制成5mgmL-1分散液，将裸鼠右前腿两侧皮下分别注射100μL分散液，在裸鼠体内进行荧光成像实验。可以在激发波长为535nm发射波长为740nm处有荧光成像。说明制备的石墨烯量子点/壳聚糖干凝胶具有荧光成像性能。5小时后再次进行荧光成像实验，注射部位依旧能荧光成像，说明制备的石墨烯量子点/壳聚糖干凝胶能在裸鼠皮下靶向定点成像。
实施例三：
石墨烯量子点/壳聚糖干凝胶制备过程与实施例一相同。
分别将100mg步骤(1)中的样品C、D、E，分散到100mL的0.5mgmL-1水杨酸钠溶液中。避光磁力搅拌24h，停止搅拌，静置过滤，超纯水洗涤，在-45℃下冷冻干燥24h，研磨备用，即得到载药石墨烯量子点/壳聚糖干凝胶。样品分别编号为载药C、载药D和载药E。
分别将15mg载药石墨烯量子点/壳聚糖干凝胶进行体外药物缓释实验，在0.1MPBS(pH为4.5、7.4及8.5)进行药物缓释实验，缓释温度为37℃。图3为载药E体外缓释实验。可以观测出在酸性条件下(pH＝4.5)累计释药量为61％，在中性条件下(pH＝7.4)累计释药量为74％，在弱碱性条件下(pH＝8.5)累计释药量为85％。说明制备的石墨烯量子点/壳聚糖干凝胶具有pH敏感性。
分别将15mg载药C、载药D和载药E进行体外药物缓释实验，在0.1MPBS(pH＝7.4)进行体外药物缓释实验，缓释温度为37℃。图4中C、D和E分别为载药C、载药D和载药E体外缓释曲线。可以观测出载药E的累计释药量最高，载药D的累计释药量最低。说明石制备的E样品能够有效的提高载药含量。
对比例一：
制备壳聚糖干凝胶，制备过程如下：
(1)将壳聚糖粉末加入0.1M醋酸溶液中，磁力搅拌10min，在搅拌状态下滴加10％氢氧化钠溶液至溶液pH为中性，滴加完毕后，停止搅拌，静置过滤，并用超纯水洗涤，在-45℃下冷冻干燥24h，研磨备用，即得壳聚糖干凝胶，样品编号为载药A。
(2)将100mg步骤(1)分散到100mL的0.5mgmL-1水杨酸钠溶液中。避光磁力搅拌24h，静置过滤，超纯水洗涤，在-45℃下冷冻干燥24h，研磨备用，即得到载药壳聚糖干凝胶。
(3)将15mg载药A进行体外药物缓释实验，在0.1MPBS(pH＝7.4)进行药物缓释实验，缓释温度为37℃。图5中A的累计释药量分别低于图4中C、D和E累计释药量。
附图说明
下面结合附图对本发明进一步说明。
图1为A壳聚糖，B石墨烯量子点，C(25wt％)石墨烯量子点/壳聚糖干凝胶，D(30wt％)石墨烯量子点/壳聚糖干凝胶和E(43wt％)石墨烯量子点/壳聚糖干凝胶FT-IR。
图2为A壳聚糖，B石墨烯量子点，C(25wt％)石墨烯量子点/壳聚糖干凝胶，D(30wt％)石墨烯量子点/壳聚糖干凝胶和E(43wt％)石墨烯量子点/壳聚糖干凝胶的FESEM及TEM。
图3为E(43wt％)石墨烯量子点/壳聚糖干凝胶分别在0.1MPBS(pH＝4.5、7.4和8.5)体外药物缓释曲线图。
图4为C(25wt％)石墨烯量子点/壳聚糖干凝胶，D(30wt％)石墨烯量子点/壳聚糖干凝胶和E(43wt％)石墨烯量子点/壳聚糖干凝胶分别在0.1MPBS(pH＝7.4)体外缓释曲线图。
图5为A壳聚糖干凝胶在0.1MPBS(pH＝7.4)体外缓释曲线图。
Claims (5)
1.一步法合成石墨烯量子点/壳聚糖干凝胶及其应用于裸鼠体内成像及体外药物缓释，其特征在于：步骤如下：
a、制备石墨烯量子点/壳聚糖干凝胶：将壳聚糖分散于醋酸溶液中，加入石墨烯量子点溶液。将混合液磁力搅拌并保持反应5-60min。在搅拌状态下用胶头滴管将氢氧化钠溶液缓慢滴加到石墨烯量子点、壳聚糖混合溶液至pH为中性。滴加完毕后，静置过滤，超纯水洗涤后进行冷冻干燥，研磨备用，即得石墨烯量子点/壳聚糖干凝胶。在-45℃下冷冻干燥6-48h，研磨备用；
b、制备载药石墨烯量子点/壳聚糖干凝胶：配制水杨酸钠溶液，并加入步骤a制得的石墨烯量子点/壳聚糖干凝胶，避光并磁力搅拌24h。静置过滤，超纯水洗涤，在-45℃下进行冷冻干燥，研磨备用。即得到载药石墨烯量子点/壳聚糖干凝胶；
c、石墨烯量子点/壳聚糖干凝胶体内成像：配制石墨烯量子点/壳聚糖分散液，将石墨烯量子点/壳聚糖分散液注射到裸鼠皮下，进行裸鼠体内成像实验；
d、载药石墨烯量子点/壳聚糖干凝胶进行体外药物缓释实验。
2.根据权利要求1所述一步法合成石墨烯量子点/壳聚糖干凝胶并将其应用于裸鼠体内成像及体外药物缓释，其特征是：所述步骤a中石墨烯量子点/壳聚糖干凝胶中石墨烯量子点含量(0-43wt％)，壳聚糖浓度为1-10mgmL-1，反应时间为5-60min。
3.根据权利要求1所述一步法合成石墨烯量子点/壳聚糖干凝胶并将其应用于药物载体，其特征是：所述步骤b中载药分子为水杨酸钠，水杨酸钠的浓度为1-2mgmL-1。
4.根据权利要求1所述一步法合成石墨烯量子点/壳聚糖干凝胶并将其应用于裸鼠体内成像，其特征是：所述步骤c中配制浓度为5mgmL-1石墨烯量子点/壳聚糖干凝胶分散液并注射到裸鼠皮下进行成像实验。
5.根据权利要求1所述一步法合成石墨烯量子点/壳聚糖干凝胶并制备成载药石墨烯量子点/壳聚糖干凝胶将其应用于体外药物缓释，其特征是：所述步骤d中石墨烯量子点/壳聚糖干凝胶在37℃和pH分别为4.5、7.4及8.5下进行药物释放实验。
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