CN108452315B - 一种透皮性酪氨酸酶抑制剂的制备方法 - Google Patents
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Abstract
本发明公开了一种透皮性酪氨酸酶抑制剂的制备方法,特点是包括以下步骤:(1)利用凝胶‑溶胶法在碱性的乙醇溶液中合成氧化锌量子点,再进行氨基化得到氨基化氧化锌量子点;(2)将BQ‑788与氨基化氧化锌量子点连接形成BQ‑788/NH2@ZnO;(3)将鞣花酸和N,N'‑羰基二咪唑溶解于N,N‑二甲基甲酰胺中,室温搅拌2小时后加入到含有BQ‑788/NH2@ZnO的N,N‑二甲基甲酰胺溶液中,在氮气保护下室温反应7小时,分别用无水乙醇和去离子水清洗3遍,即得到透皮性酪氨酸酶抑制剂BQ‑788/EA@ZnO,优点是透皮率高、特异性靶向黑素细胞。
Description
技术领域
本发明涉及一种酪氨酸酶抑制剂,尤其是涉及一种透皮性酪氨酸酶抑制剂的制备方法。
背景技术
黑色素是由黑素细胞产生的天然色素,能起到吸收紫外线,防止光损伤的作用。但是,黑色素的过度沉积会影响人体皮肤美观,更严重地会诱发黑色素瘤。黑色素的生成过程分为两步,第一步:L-酪氨酸经酪氨酸酶催化生成L-多巴,多巴再经酪氨酸酶催化生成多巴醌;第二步:多巴醌经两个非酶促反应途径生成黑色素。可见酪氨酸酶是黑色素形成的关键限速酶。当酪氨酸酶过量表达时会导致黑色素的大量沉积,引发色素紊乱相关疾病。因此,使用高效低毒的酪氨酸酶抑制剂可有效防止黑色素的过度沉积。至今已开发出如,黄酮及其衍生物,有机酸类,苷类,萜类,酯类等酪氨酸酶抑制剂,均对酪氨酸酶有一定的抑制作用。然后,透皮率低、缺乏靶向性这两个缺点严重限制了上述酪氨酸酶抑制剂在化妆品和临床上的使用效果。
发明内容
本发明所要解决的技术问题是提供一种透皮率高、特异性靶向黑素细胞的透皮性酪氨酸酶抑制剂的制备方法。
本发明解决上述技术问题所采用的技术方案为:一种透皮性酪氨酸酶抑制剂的制备方法,包括以下步骤:
(1)氨基化氧化锌量子点(NH2@ZnO)的合成
A.利用凝胶-溶胶法在碱性的乙醇溶液中合成氧化锌量子点:将0.41g的醋酸锌溶解在10ml的无水乙醇中,在68℃下搅拌反应1.5小时,然后冰浴冷却得到醋酸锌溶液;将0.16g的氢氧化钠溶解到5 mL无水乙醇中后,逐滴加入到醋酸锌溶液中,将混合溶液继续搅拌至在波长为365nm紫外灯下观察溶液呈绿色荧光,然后加入10mL正庚烷,静置后离心取沉淀,即得到氧化锌量子点;
B.将氧化锌量子点加入到15mL的乙醇溶液中,滴入0.08 mL APTES,50 ℃恒温搅拌反应7小时,取沉淀分别用无水乙醇和去离子水清洗3遍,即得到氨基化氧化锌量子点,然后真空干燥待用;
(2)BQ-788/NH2@ZnO的合成
将5.31mg的BQ-788溶解在0.1 mL的N,N-二甲基甲酰胺(DMF)中,然后加入2.87mg的O-(7氮苯并三氮唑)- N,N,N',N'-四甲基脲六氟磷酸酯,室温搅拌反应1小时得到含有BQ-788混合液;同时将12.72mg的氨基化氧化锌量子点完全分散在1mLN,N-二甲基甲酰胺(DMF)中,逐滴加入0.02mLN,N-二异丙基乙胺充分混合后,加入到含有BQ-788混合液中,继续搅拌1小时,反应结束后,将产物分别用无水乙醇和去离子水清洗3遍,即得到BQ-788/NH2@ZnO,然后在真空中干燥待用;
(3)鞣花酸的负载
将0.09g鞣花酸和0.05g N,N'-羰基二咪唑溶解于2 mL N,N-二甲基甲酰胺中,室温搅拌2小时后加入到含有0.41g BQ-788/NH2@ZnO的10 mL N,N-二甲基甲酰胺溶液中,在氮气保护下室温反应7小时,分别用无水乙醇和去离子水清洗3遍,即得到透皮性酪氨酸酶抑制剂BQ-788/EA@ZnO。
与现有技术相比,本发明的优点在于:本发明首次公开了一种透皮性酪氨酸酶抑制剂的制备方法,其采用的氧化锌是生物安全性良好的金属氧化物,并且当环境pH值小于5.5时可以溶解为锌离子。因此,当氧化锌作为药物载体材料时,非常适合于进行递释药物进入细胞内,借由内涵体的酸性环境,控制释放负载在其上的药物。同时,研究表明,氧化锌量子点具有很好的皮肤渗透性,通过表面改性连接上靶向黑素细胞的基团,此载药系统具有将酪氨酸酶抑制剂透皮运输到黑素细胞的优势。
附图说明
图1为ZnO的X射线光谱图
图2为ZnO的能量色散X射线光谱图
图3为ZnO量子点透射电镜照片;
图4为BQ-788 / EA @ ZnO透射电镜照片;
图5为ZnO量子点、NH2@ZnO、BQ-788/NH2@ZnO和BQ-788/EA@ZnO的红外光谱图;
图6为BQ-788/EA@ZnO热重分析结果示意图;
图7为EA、ZnO量子点和BQ-788/EA@ZnO的透皮实验结果;
图8 为BQ-788/EA@ZnO与黑素细胞形成细胞培养,10min后PBS缓冲液冲洗细胞在荧光显微镜下观察结果;
图9为BQ-788/EA@ZnO与角质形成细胞培养,10min后PBS缓冲液冲洗细胞在荧光显微镜下观察结果;
图10为不同pH值对BQ-788/EA@ZnO中EA累积释放率的影响‘
图11为BQ-788/EA@ZnO浓度与酪氨酸酶活性抑制率的关系图;
图12为BQ-788/EA@ZnO浓度与抗黑素生成率的关系图。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
一、具体实施例
1、氨基化氧化锌量子点(NH2@ZnO)的合成
A.利用凝胶-溶胶法在碱性的乙醇溶液中合成氧化锌量子点:将0.41g的醋酸锌溶解在10ml的无水乙醇中,在68℃下搅拌反应1.5小时,然后冰浴冷却得到醋酸锌溶液;将0.16g的氢氧化钠溶解到5 mL无水乙醇中后,逐滴加入到醋酸锌溶液中,将混合溶液继续搅拌至在波长为365nm紫外灯下观察溶液呈绿色荧光,然后加入10mL正庚烷,静置后离心取沉淀,即得到氧化锌量子点;
利用X射线衍射分析合成的产物,其图1X射线光谱与氧化锌标准PDF卡片(JCPDS36-1451)吻合,证明合成产物即为ZnO。同时,图2能量色散X射线光谱的分析结果也验证了这个结果;
B.将氧化锌量子点加入到15mL的乙醇溶液中,滴入0.08 mL APTES,50 ℃恒温搅拌反应7小时,取沉淀分别用无水乙醇和去离子水清洗3遍,即得到氨基化氧化锌量子点,然后真空干燥待用;
2、BQ-788/NH2@ZnO的合成
将5.31mg的BQ-788溶解在0.1 mL的N,N-二甲基甲酰胺(DMF)中,然后加入2.87mg的O-(7氮苯并三氮唑)- N,N,N',N'-四甲基脲六氟磷酸酯,室温搅拌反应1小时得到含有BQ-788混合液;同时将12.72mg的氨基化氧化锌量子点完全分散在1mLN,N-二甲基甲酰胺(DMF)中,逐滴加入0.02mLN,N-二异丙基乙胺充分混合后,加入到含有BQ-788混合液中,继续搅拌1小时,反应结束后,将产物分别用无水乙醇和去离子水清洗3遍,即得到BQ-788/NH2@ZnO,然后在真空中干燥待用;
3、鞣花酸的负载
将0.09g鞣花酸和0.05g N,N'-羰基二咪唑溶解于2 mL N,N-二甲基甲酰胺中,室温搅拌2小时后加入到含有0.41g BQ-788/NH2@ZnO的10 mL N,N-二甲基甲酰胺溶液中,在氮气保护下室温反应7小时,分别用无水乙醇和去离子水清洗3遍,即得到BQ-788/EA@ZnO,然后在真空中干燥待用。
当ZnO量子点接上靶向基团BQ-788和酪氨酸酶抑制药物鞣花酸后,构建新型的酪氨酸酶抑制剂(BQ-788 / EA @ ZnO)。图3为ZnO量子点透射电镜照片显示ZnO量子点呈单分散状态,粒径分布窄,均在4 nm左右。图4为BQ-788/EA@ZnO透射电镜照片显示,BQ-788/EA@ZnO由于表面修饰,粒径稍稍有所增大,同时也发生了一定程度的聚集。
如图5所示,利用红外光谱仪表征了ZnO量子点、NH2@ZnO、BQ-788/NH2@ZnO和BQ-788/EA@ZnO的红外光谱,由图可知,相对于ZnO量子点,NH2@ZnO的红外光谱在3400cm-1和1619cm-1处出现两个吸收峰,是氨基的特征峰,证明ZnO量子点表面已成功接枝上氨基。BQ-788/NH2@ZnO红外光谱中在2926cm-1左右出现的吸收峰,以及1600~1400cm-1之间出现的数个吸收峰分别是甲基和苯环的震动吸收峰,证明BQ-788已连接在BQ-788/NH2@ZnO上。BQ-788/EA@ZnO红外光谱中1200cm-1左右的吸收峰是EA分子中羟基的吸收峰。以上的所有结果都证明BQ-788/EA@ZnO已被成功合成。
如图6所示,用热重分析仪测量BQ-788/EA@ZnO中各成分所占的质量比,结果显示ZnO占35.53%,BQ-788占7.25%,EA占33.05%。
二、应用实施例
1、实验方法
1)透皮实验
利用透皮测试仪测量BQ-788/EA@ZnO的透皮性能,选择豚鼠皮肤为透皮模型。在35℃条件下,样品室中放置一定浓度的BQ-788/EA@ZnO,一定时间点(1 h、2 h、4 h、8 h、 16h和24 h)于样品室收集透过的BQ-788/EA@ZnO,并测定浓度以计算累积渗透量。设置纯EA 和纯 ZnO量子点对照组。
2)BQ-788/EA@ZnO的靶向作用
将黑素细胞和角质形成细胞分别以每孔2×10 4个细胞的密度接种到预铺设盖玻片的24孔板中。12小时后,抽出细胞培养基并用含有浓度为1mg/mL的BQ-788/EA@ZnO的新鲜培养基代替。细胞再孵育5分钟或10分钟。然后抽出培养基。细胞用PBS缓冲液剧烈洗涤3次并用4wt%多聚甲醛固定15分钟。用PBS缓冲液洗涤3次后,用Hochest染色细胞5分钟,取出盖玻片在荧光显微镜下观察。
3)EA的体外释放行为
在37℃下在两种不同介质中研究BQ-788/EA@ZnO的释放曲线:(a)酸性缓冲液(pH5.0);(b)中性缓冲液(pH 7.4)。将5.20mgBQ-788/EA@ZnO分散在2mL不同的PBS缓冲液中并密封在透析袋(MW = 3000)中。将透析袋分别浸入3mL的不同pH值的PBS缓冲液中并在37℃下搅拌12小时。在预定时间点用紫外分光光度计测量透析液中EA的浓度,以计算其累积释放率。
4)酪氨酸酶活性抑制
将黑素细胞以每孔8000个的密度接种到96孔板中。12小时后,加入BQ-788/EA@ZnO使得最终ZnO的当量浓度为3.75,6.25和12.5 μg/mL。继续培养72小时,用PBS缓冲液洗涤黑素细胞两次,并加入50μL1wt%TritonX-100。将黑素细胞置于-80℃冰箱中30分钟,然后在室温下取出直至破裂。裂解的细胞升温至37℃,加入50μL0.2wt%左旋多巴。37℃保温2 h,测定475 nm处的吸光度,计算对酪氨酸酶活性的抑制率。同时设置相应浓度的EA和BQ-788/NH2@ZnO的对照组。使用未经任何处理的黑素细胞作为空白对照以计算黑色素的含量,如下:Ea(%)= At/Ab×100% ,其中Ea是TYR的酶活性,At是处理组的吸光度,Ab是空白对照组的吸光度。所有实验组重复六次。
5)黑色素含量测定
将黑素细胞以每孔5000个细胞的密度接种到96孔板中,并用不同浓度的BQ-788/EA@ZnO(ZnO的当量浓度为3.75、6.25和12.5 μg/mL)处理72小时。处理后,加入1mL 1mol/LNaOH在80℃下保温30 min,裂解细胞,测定475 nm处的吸光度,对黑色素进行定量。与此同时,设置相应浓度的EA和BQ-788/NH2@ZnO处理的实验对照组。使用未经任何处理的黑素细胞作为空白对照以计算黑色素的含量,如下:Cm(%)=Am/Ac×100%,其中Cm为黑色素的含量,Am为处理组的吸光度,Ac为空白对照组的吸光度。所有实验组重复六次。
2、实验结果分析
图7为 EA、ZnO量子点和BQ-788/EA@ZnO的透皮实验结果,由图7可知,利用具有良好透皮性能的ZnO量子点来透皮递送EA,虽然BQ-788/EA@ZnO的透皮效率相对于ZnO量子点有所下降,但显著提高了EA的透皮率。目前所开发出来包括EA在内的酪氨酸酶抑制剂的皮肤透过率都非常低,这也是限制其在化妆品和临床上应用的主要原因。所以,提高酪氨酸酶抑制剂的透皮率,将会极大地提升其抑制效率。
图8和图9分别为利用BQ-788/EA@ZnO分别与黑素细胞和角质形成细胞培养,10min后PBS缓冲液冲洗细胞,在荧光显微镜下观察结果,只有黑素细胞旁有ZnO发射的荧光(如图8所示),而角质细胞旁没有(如图9所示),结果证明BQ-788/EA@ZnO透过皮肤后,能特异性靶向黑素细胞,可以将EA靶向递送到黑素细胞。
图10为不同pH值对BQ-788/EA@ZnO中EA累积释放率的影响。由图可知,BQ-788/EA@ZnO在pH为7.4的缓冲液中释放缓慢,12 h的累积释放率只有5.65±1.47%,而在pH为5.0的缓冲液中EA快速释放,2 h时累积释放率已达63.28±1.62%。8h时累积释放率已达94.45±1.21%,之后释放趋势趋于平缓。EA通过共价键与ZnO QDs相连,能稳定吸附在ZnO QDs表面。只有当BQ-788/EA@ZnO置于酸性缓冲液中,ZnO溶解成Zn2+,与EA的结合被破坏,EA迅速释放到缓冲液中。
图11为不同BQ-788/EA@ZnO浓度对黑素细胞中酪氨酸酶活性的抑制率,图12为不同BQ-788/EA@ZnO浓度对黑素细胞中抗黑素素生成效果,整体结果是呈浓度依赖型,BQ-788/EA@ZnO浓度越大,抑制效果越好。当BQ-788/EA@ZnO的浓度达到12.5μg/mL,酪氨酸酶活性抑制率达到44.23%,抗黑色素生成率达37.50%。这一数据要显著高于BQ-788/NH2@ZnO实验组。虽然没有显著高于单纯EA实验组,但考虑到EA的低透皮渗透率,BQ-788/EA@ZnO对于皮肤外用更加有效。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,作出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (1)
1.一种透皮性酪氨酸酶抑制剂的制备方法,其特征在于包括以下步骤:
(1)氨基化氧化锌量子点的合成
A.利用凝胶-溶胶法在碱性的乙醇溶液中合成氧化锌量子点:将0.41g的醋酸锌溶解在10ml的无水乙醇中,在68℃下搅拌反应1.5小时,然后冰浴冷却得到醋酸锌溶液;将0.16g的氢氧化钠溶解到5 mL无水乙醇中后,逐滴加入到醋酸锌溶液中,将混合溶液继续搅拌至在波长为365nm紫外灯下观察溶液呈绿色荧光,然后加入10mL正庚烷,静置后离心取沉淀,即得到氧化锌量子点;
B.将氧化锌量子点加入到15mL的乙醇溶液中,滴入0.08 mL APTES,50 ℃恒温搅拌反应7小时,取沉淀分别用无水乙醇和去离子水清洗3遍,即得到氨基化氧化锌量子点,然后真空干燥待用;
(2)BQ-788/NH2@ZnO的合成
将5.31mg的BQ-788溶解在0.1 mL的N,N-二甲基甲酰胺中,然后加入2.87mg 的O-(7氮苯并三氮唑)- N,N,N',N'-四甲基脲六氟磷酸酯,室温搅拌反应1小时得到含有BQ-788混合液;同时将12.72mg的氨基化氧化锌量子点完全分散在1mLN,N-二甲基甲酰胺中,逐滴加入0.02mLN,N-二异丙基乙胺充分混合后,加入到含有BQ-788混合液中,继续搅拌1小时,反应结束后,将产物分别用无水乙醇和去离子水清洗3遍,即得到BQ-788/NH2@ZnO,然后在真空中干燥待用;
(3)鞣花酸的负载
将0.09g鞣花酸和0.05g N,N'-羰基二咪唑溶解于2 mL N,N-二甲基甲酰胺中,室温搅拌2小时后加入到含有0.41g BQ-788/NH2@ZnO的10 mL N,N-二甲基甲酰胺溶液中,在氮气保护下室温反应7小时,分别用无水乙醇和去离子水清洗3遍,即得到透皮性酪氨酸酶抑制剂BQ-788/EA@ZnO。
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