CN111870693B - 一种黑磷促进精氨酸快速释放no的方法 - Google Patents
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Abstract
本发明公开一种黑磷促进精氨酸快速释放NO的方法,取4‑二甲氨基吡啶、含胍基化合物和黑磷反应;分离得到保护的黑磷BP‑含胍基化合物;取1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺(EDC)、N‑羟基琥珀酰亚胺(NHS)和葡萄糖氧化酶(GOx)在PB中反应;加入BP‑含胍基化合物,离心得到GOx和BP‑含胍基化合物偶联物BP‑含胍基化合物‑GOx;将BP‑含胍基化合物‑GOx溶解于葡萄糖溶液中,用近红外光照射黑磷,产生过氧化氢(H2O2),H2O2氧化含胍基化合物快速产生NO。用黑磷光热效应,促进葡萄糖氧化酶产生过氧化氢、精氨酸氧化快速产生NO。产品稳定性好,酶活高,NO释放快速,产量高。
Description
技术领域
本发明涉及二维材料制备方法,特别涉及一种黑磷促进精氨酸快速释放NO的方法。
背景技术
一氧化氮(nitric oxide,NO)是生物体内一种重要的生物信号分子和效应分子,是由NO合酶(NOS)催化精氨酸产生,生物体内各组织中起到各种生理作用如扩张血管,抗肿瘤等,但受限于内源性NO的产生量,往往难以满足机体应对病变的需要,所以外源性NO供体已被开发作为其主要来源。NO供体类型主要分为有机硝酸酯、S-亚硝基硫醇、硝普盐、呋咱N-氧化物、偶氮鎓二醇盐等。这些小分子NO供体在疾病治疗中已经有广泛的应用,但也存在半衰期短,特异性差,代谢产物毒副作用大等缺点。这些都在不同程度上阻碍了其在生物医药领域的应用,因此,为了弥补外源性NO供体带来的不足,对于内源性NO的开发极为重要。
精氨酸在体内是一种重要的氨基酸,精氨酸不仅可以在NOS的作用下产生NO,在H2O2作用下,精氨酸也能被氧化产生NO。A.R.Ray等(Journal of Molecular Catalysis A:Chemical,2007:207-214.)设计一种简单易行的负载铁(III)卟啉催化剂,用铁(III)卟啉模拟一氧化氮合酶的作用来催化L-Arg与H2O2的仿生氧化产生NO。Fan等(Angew.Chem.Int.Ed.2016(55):1-6.)设计一种新型的葡萄糖反应体系(L-Arg-HMON-GOx),利用中空介孔有机二氧化硅纳米粒子(HMON)作为载体,在葡萄糖氧化酶(GOx)的作用下将其转化为葡萄糖酸和H2O2,H2O2进一步氧化L-精氨酸产生NO。
精氨酸作为NO供体已被用于各种抗菌及抗肿瘤治疗中,但该过程存在释放缓慢且释放量少等缺陷,因此亟需一种有效策略来弥补现存不足。黑磷(BP)由于其紫外到近红外光的宽吸收,近红外消光系数大和光热转换效率高等特点,可以作为良好的光热试剂。
发明内容
发明目的:本发明目的是提供加速H2O2与Arg反应快速产生大量NO的方法。
技术方案:本发明提供一种黑磷促进精氨酸快速释放NO的方法,包括如下步骤:
(1)取4-二甲氨基吡啶(DMAP)、含胍基化合物和黑磷,常温下在有机溶剂和水的混合液中反应;
(2)上述反应完成后,去除未反应的含胍基化合物;
(3)将反应后的分散液离心分离得到保护的黑磷(BP-含胍基化合物);
(4)取1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS)和葡萄糖氧化酶(GOx),在PB中常温反应;
(5)将BP-含胍基化合物加入上步反应中,反应后离心得到GOx和BP-含胍基化合物偶联物(BP-含胍基化合物-GOx);
(6)将BP-含胍基化合物-GOx溶解于葡萄糖溶液中,用近红外光照射黑磷,促进GOx产生过氧化氢(H2O2),H2O2氧化含胍基化合物快速产生NO。
BP-含胍基化合物-GOx的分子结构式如下:
进一步地,所述含胍基化合物为精氨酸(Arg)、刀豆氨酸。
进一步地,所述步骤(1)中的有机溶剂为N,N’-二甲基甲酰胺(DMF)或二甲亚砜。
进一步地,所述步骤(2)中用透析袋去除未反应的可发生亲核取代的化合物。
进一步地,所述近红外光波长为808nm。
进一步地,所述近红外光照射后黑磷的温度范围为37℃-60℃。
有益效果:本发明基于黑磷纳米片(BPNSs)的GOx和含胍基化合物(如精氨酸)共负载体系(BP-含胍基化合物-GOx),利用BPNSs优异的光热性能,在近红外光照射下,提升GOx酶活性,有效地将光能转化为热能,促进GOx催化葡萄糖生成H2O2,加快氧化含胍基化合物产生NO,该体系稳定性好,NO释放量高,光热效果好。共价偶联物生物相容性高,黑磷稳定性提高,且不影响其光热性能。
附图说明
图1为二维黑磷TEM图谱;
图2为BP-Arg-GOx的TEM图谱;
图3为二维黑磷的粒径图谱;
图4为BP-Arg-GOx粒径图谱;
图5为BP-Arg-GOx粒径稳定性图谱;
图6为BP-Arg-GOx的NO释放图谱。
具体实施方式
本发明缩写含义列于下表1
表1化合物定义
DMAP | 4-二甲氨基吡啶 |
EDC | 1-(3-二甲氨基丙基)-3-乙基碳二亚胺 |
NHS | N-羟基琥珀酰亚胺 |
GOx | 葡萄糖氧化酶 |
Arg | 精氨酸 |
DMF | N,N’-二甲基甲酰胺 |
实施例1
BP-Arg-GOx的合成与表征
(1)BP-Arg合成:量取1mg/mL黑磷的DMF(或为二甲亚砜)溶液2mL,加入6mg精氨酸(还可以使用刀豆氨酸等能氧化产生NO的含胍基化合物)和3mgDMAP,再加入200μL去离子水,该混合液过夜反应8h。反应完成后将混合液透析6h除去未反应的精氨酸,透析完成后,将所得产物17000g离心4min,并用去离子水清洗三遍,最后将所得产物重悬于去离子水中保存。
(2)BP-Arg-GOx合成:称取2mg GOx,加入5mg EDC和10mg NHS,在PB中活化GOx上的羧基,30min后向活化好的GOx里加入5mg BP-Arg,过夜反应后,离心取沉淀,重悬于PB中,以备后续使用。
将BP-Arg-GOx溶解于葡萄糖溶液中,用近红外光(波长为808nm)照射黑磷,促进GOx产生过氧化氢(H2O2),H2O2氧化Arg快速产生NO。
实施例2
粒径图谱测定:各取适量的黑磷、BP-Arg-GOx,分别测定其在PB 7.4中的水动力直径。另外,在第1、5、10天时测定BP-Arg的水动力直径,监测其稳定性。
实施例3
NO释放图谱测定:取一定量BP-Arg-GOx,测定其NO释放量。
如附图1-4所示,修饰后的产物BP-Arg-GOx与原料BP经TEM和DLS分析可知,修饰后的BP表面表现出与原料BP明显的不同,DLS结果也证明,BP-Arg-GOx与BP相比,粒径有一定的增大。
如附图5所示,修饰后的产物BP-Arg-GOx经DLS分析可知,BP-Arg-GOx在PBS溶液中5天后依然保持了良好的稳定性。
如附图6所示,修饰后的产物BP-Arg-GOx经近红外光照后显示了与游离GOx相似的酶活力,表明BP-Arg-GOx修饰的成功性以及对酶活力的最大保留。
Claims (6)
1.一种黑磷促进精氨酸快速释放NO的方法,其特征在于:包括如下步骤:
(1)取4-二甲氨基吡啶(DMAP)、含胍基化合物和黑磷,常温下在有机溶剂和水的混合液中反应;
(2)上述反应完成后,去除未反应的含胍基化合物;
(3)将反应后的分散液离心分离得到保护的黑磷BP-含胍基化合物;
(4)取1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS)和葡萄糖氧化酶(GOx),在PB中常温反应;
(5)将BP-含胍基化合物加入上步反应中,反应后离心得到GOx和BP-含胍基化合物偶联物BP-含胍基化合物-GOx;
(6)将BP-含胍基化合物-GOx溶解于葡萄糖溶液中,用近红外光照射黑磷,促进GOx产生过氧化氢(H2O2),H2O2氧化含胍基化合物快速产生NO。
2.根据权利要求1所述的黑磷促进精氨酸快速释放NO的方法,其特征在于:所述含胍基化合物为精氨酸、刀豆氨酸。
3.根据权利要求1所述的黑磷促进精氨酸快速释放NO的方法,其特征在于:所述步骤(1)中的有机溶剂为N, N’-二甲基甲酰胺(DMF)或二甲亚砜。
4.根据权利要求1所述黑磷促进精氨酸快速释放NO的方法,其特征在于:所述步骤(2)中用透析袋去除未反应的可发生亲核取代的化合物。
5.根据权利要求1所述的黑磷促进精氨酸快速释放NO的方法,其特征在于:所述近红外光波长为808 nm。
6.根据权利要求1所述的黑磷促进精氨酸快速释放NO的方法,其特征在于:所述近红外光照射后黑磷的温度范围为37 ℃-60 ℃。
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