CN113637472B - 具有良好生物相容性的脂质体葡萄糖荧光探针及其制备方法 - Google Patents
具有良好生物相容性的脂质体葡萄糖荧光探针及其制备方法 Download PDFInfo
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- CN113637472B CN113637472B CN202110889477.XA CN202110889477A CN113637472B CN 113637472 B CN113637472 B CN 113637472B CN 202110889477 A CN202110889477 A CN 202110889477A CN 113637472 B CN113637472 B CN 113637472B
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Abstract
本发明公开了一种具有良好生物相容性的脂质体葡萄糖荧光探针及其制备方法。该纳脂质体探针是通过使用两亲性分子(DSPE‑PEG2000)将有机硼酸葡萄糖分子探针包封在一起,形成的具有脂质双分子层膜的纳米囊泡。有机硼酸分子中的二硼酸识别位点可以与葡萄糖分子中的顺式‑邻二醇结构特异性的结合,从而使纳米探针具备了对葡萄糖的高亲和力和选择性。脂质体纳米探针的构建使分子探针兼具有脂质体所具的有良好特性。相比有机硼酸分子,该脂质体纳米探针具有更好的生物相容性、更快的响应时间和良好的荧光稳定性,从而能够对体外和体内葡萄糖进行快速、准确的识别和检测。同时,该脂质体纳米探针具有制备方法简便,稳定且高效等优点。
Description
技术领域
本发明属于荧光分析检测领域,具体涉及一种具有良好生物相容性的脂质体葡萄糖荧光探针及其制备方法。
背景技术
葡萄糖是一种重要的食药类化工原料,同时,是维持生命的重要能源物质。葡萄糖作为一种重要的生理信号物质,其代谢状态与多种疾病的发展息息相关,比如糖尿病、肿瘤、肥胖症、帕金森和阿尔兹海默症等。因此,葡萄糖的识别和检测在临床医学研究中具有重要的应用价值。
葡萄糖的检测方法主要包括酶类传感器及有机硼酸类传感器等。酶类葡萄糖传感器基于酶的特异性反应,具有特异性强的特点,但识别反应具有不可逆性,因此,酶类探针无法对葡萄糖实现实时性监测。有机硼酸化合物能够与邻二羟基化合物在水溶液中迅速且可逆性地共价结合,形成五元或六元环状酯,因此,有机硼酸类探针成为葡萄糖传感器领域研究的热点。但目前,有机硼酸类葡萄糖探针普遍存在着水溶性低、时间响应性长、选择性和灵敏度低的问题,设计具有良好生物相容性的硼酸类葡萄糖传感器依然是一个巨大的挑战。
脂质体(Liposome)是将目标分子包封于类脂质双分子层内形成的微型囊泡状的结构,呈球形,直径25~1000nm不等。脂质体通常由各种天然磷脂、合成磷脂或胆固醇等组成,这些成分具有非常好的生物相容性,研究发现脂质体纳米粒子具有良好的生物相容性。脂质体已经广泛的应用于新药输送制剂的开发,脂质体作药物运送的载体能够改善药物的溶解度、减少包载药物的毒性、优化药代动力学性质、提高药理学作用,防止局部的刺激。
近年来,将荧光探针与脂质体结合制备的脂质体荧光探针在体外和体内的荧光检测和成像方面取得了显著的进展。Deissler等(Deissler V et al.SMALL,2010,4(8):1240-1246)以胆固醇和卵磷脂为原料,采用薄膜水化和挤出法将近红外荧光染料DY-676-C18酯包载入脂质双分子层中,制备出粒径均一的脂质体荧光探针。结果表明,该脂质体荧光探针具有良好的稳定性、生物相容性以及药代动力学参数,最后探针成功应用于巨噬细胞以及体内炎症的成像。Jin等(Peng J et al.BIOSENS BIOELECTRON,2017,94:278-285)设计合成了基于蒽酰亚胺的硼酸酯类的H2O2分子探针,采用溶剂注入法使用DSPE-PEG2000包载该探针合成了具有磷脂双分子层的脂质体纳米粒子(NPs-A)。结果表明,NPs-A具有较小的细胞毒性和良好的生物相容性。然后,系统的考察了该纳米探针的大小、形态以及细胞毒性,最后成功应用于体外和体内H2O2的检测和荧光成像。研究表明脂质体荧光探针具有良好的信噪比,有利于生物体内的高分辨成像;具有显著优化探针分子溶解度、改善药代动力学参数、减低毒性等特点,能够实现实时、原位、高灵敏的荧光成像,对于体外和体内的荧光检测与成像具有广阔的应用前景。
发明内容
本发明的目的在于针对现有有机硼酸分子探针水溶性低、响应时间长、灵敏度和选择性低等不足,提供一种具有良好生物相容性、响应时间快的脂质体葡萄糖荧光探针及其制备方法。
上述具有良好生物相容性的脂质体葡萄糖荧光探针包括有机硼酸分子探针和脂质体两个部分。其中,有机硼酸分子作为葡萄糖特异性识别探针。脂质体是通过两亲性分子通过自组装形成的纳米囊泡状结构。本发明通过对有机硼酸分子的脂质体纳米化修饰,从而避免了有机硼酸分子水溶性差、响应速度慢等的不足。
上述具有良好生物相容性的脂质体葡萄糖荧光探针中的有机硼酸分子为具有结构式(Ⅰ)的化合物:
其中:
R1、R2、R3各自独立选自氢、C1-C18烷基、C2-C18烯基、C2-C18炔基、C3-C18异烃基、吸电子基团和供电子基团;
作为优选,所述吸电子基团选自C(O)R4、COOR4、C(O)NH2、NHC(O)R4、C(O)NR4R5、CF3、CN、SO3H、SO2CF3、SO2R4、SO2NR4R5、铵、乙酰基、羧基、卤素、烷基铵和NO2,其中R4和R5各自独立选自H或C1-C6烷基。
作为优选,所述供电子基团选自NR6R7、OR6、NHC(O)R6、OC(O)R6、巯基、羟基、苯基和乙烯基,其中R6和R7各自独立选自H或C1-C6烷基。
上述具有良好生物相容性的脂质体葡萄糖荧光探针,所述的两亲性分子具有亲水的头部和疏水的尾部,其头部可由胆碱、乙醇胺等形成,尾部可由两条脂肪酸链形成。在水溶液中它们能自动形成双分子层结构,使疏水的尾部埋藏在里面,即膜的中央,亲水的头部露在外面,优选为磷脂和功能化磷脂分子。
上述具有良好生物相容性的脂质体葡萄糖荧光探针,所述的磷脂包含磷脂酰胆碱、脂酰乙醇胺、磷脂酰甘油、磷脂酰丝氨酸、磷脂酸、磷脂酰肌醇的一种或多种,优选为二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰磷脂酰胆碱(DSPC)、二肉豆蔻酰磷脂酰胆碱(DMPC)、1-棕榈酰-2-亚油酰-sn-甘油-3-磷脂酰胆碱(PLPC)、二油酰磷脂胆碱(DOPC)、蛋黄磷脂酰胆碱(EPC)、二芥酰磷脂胆碱(DEPC)、二月桂酰磷脂酰胆碱(DLPC)、氢化的大豆磷脂酰胆碱(HSPC)、二硬脂酰磷脂酰乙醇胺(DSPE)、二肉豆蔻酰磷脂酰乙醇胺(DMPE)、二棕榈酰磷脂酰乙醇胺(DPPE)中的一种或多种。
上述具有良好生物相容性的脂质体葡萄糖荧光探针,所述的功能化磷脂包含不同磷脂的聚乙二醇衍生物、不同磷脂的聚乙二醇偶联靶向多肽衍生物,优选为二硬脂酰磷脂酰乙醇胺-聚乙二醇共聚(mPEG-DSPE)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺共聚(DSPE-PEG-NHS)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺共聚(DSPE-PEG-Maleimide)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-生物素共聚(DSPE-PEG-Biotin),二硬脂酰磷脂酰乙醇胺-聚乙二醇-氨基共聚(DSPE-PEG-NH2)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧酸共聚(DSPE-PEG-COOH)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-羟基共聚(DSPE-PEG-OH)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-巯基共聚(DSPE-PEG-SH)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-叠氮共聚(DSPE-PEG-N3)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-荧光素共聚(DSPE-PEG-FITC)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-硅烷共聚(DSPE-PEG-Silane)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-NBD标记共聚(DSPE-PEG-NBD)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-丙酰胺双巯基吡啶共聚(DSPE-PEG-PDP)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-叶酸共聚(DSPE-PEG-FA)中的一种或多种。
上述具有良好生物相容性的脂质体葡萄糖荧光探针,所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇共聚优选为二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)。
上述具有良好生物相容性的脂质体葡萄糖荧光探针的制备包括有机硼酸分子探针的合成和两亲性分子纳米化修饰两个部分。下面以脂质体探针NPs-Ac-CDBA为例,说明该类脂质体探针的部分合成路线及制备方法。
1)脂质体探针NPs-Ac-CDBA中化合物AC-CDBA的合成:
反应步骤为:
步骤i:9,10-二甲基蒽与三氯化氯作为起始原料发生傅克酰基化反应生成乙酰化产物C7;步骤ii:化合物C7与N-溴代丁二酰亚胺发生溴代反应生成化合物C8;步骤iii:化合物C8与与甲胺发生亲核取代反应生成二胺产物C9;步骤iv:二胺产物C9通过与两分子的化合物C3发生亲核取代反应生成目标化合物Ac-CDBA。
2)脂质体探针NPs-Ac-CDBA的制备:
采用超声法制备脂质体探针NPs-Ac-CDBA,首先,将Ac-CDBA和DSPE-PEG2000按5:1的质量比溶于适量的甲醇中。在超声条件下,将上述甲醇混合溶液注入一定量的去离子水中,将混合溶液继续超声一定时间。然后,取出该混合溶液进行透析,除去残留的有机溶剂。使用超滤管将透析后的溶液浓缩至一定体积,最终获得了脂质体荧光探针NPs-Ac-CDBA。
上述具有良好生物相容性的脂质体葡萄糖荧光探针可应用于体外或体内生物样品中葡萄糖的行定性和定量检测,应用于对葡萄糖的特异性识别、标记、传感、富集、分离、分析、检测和成像等,可应用于开展临床医学诊断以及生命医学等研究应用。
与现有技术相比,本发明的有益效果为:
上述具有良好生物相容性的脂质体葡萄糖荧光探针能够特异性的识别葡萄糖,有机硼酸分子中二硼酸识别位点的设计使其可以特异性的结合葡萄糖分子中的顺式-邻二醇结构,从而保证了探针对葡萄糖的高亲和力和选择性。
上述具有良好生物相容性的脂质体葡萄糖荧光探针是通过两亲性分子自组装为脂质体纳米探针,从而使葡萄糖探针兼具有脂质体的优良特性,包括低毒性、良好的溶解度以及良好的药代动力学参数等。
上述具有良好生物相容性的脂质体葡萄糖荧光探针经研究后发现,脂质体纳米探针比有机分子探针具有更好的水溶性、更快的响应时间和良好的荧光稳定性,从而能够实现对葡萄糖快速、准确的识别和检测。同时,本发明的制备方法简便、定量范围宽、稳定且高效。
附图说明
图1(a)为纳米探针NPs-CN-DBA的粒径分布图(动态光散射);图1(b)为纳米探针NPs-CN-DBA的透射电镜照片;
图2(a)为纳米探针NPs-Ac-CDBA的粒径分布图(动态光散射);图2(b)为纳米探针NPs-Ac-CDBA的透射电镜照片;
图3(a)为分子探针CN-DBA在不同葡萄糖浓度(0–0.4M)条件下的荧光光谱图;图3(b)为分子探针CN-DBA与不同糖类物质的荧光响应图,糖类物质包括葡萄糖、果糖、核糖、麦芽糖、甘露糖、半乳糖、乳糖、葡萄糖胺和蔗糖,数据使用平均值±标准差表示(n=3),10μMCN-DBA在0.5%MeOH/PBS(pH 7.4)中测试,25℃,λex=375nm,λem=427nm;
图4(a)为分子探针Ac-CDBA在不同葡萄糖浓度(0–0.4M)条件下的荧光光谱图;图4(b)为分子探针Ac-CDBA与不同糖类物质的荧光响应图,糖类物质包括葡萄糖、果糖、核糖、麦芽糖、甘露糖、半乳糖、乳糖、葡萄糖胺和蔗糖,数据使用平均值±标准差表示(n=3),10μM Ac-CDBA在0.5%MeOH/PBS(pH 7.4)中测试,25℃,λex=405nm,λem=482nm;
图5(a)为脂质体探针NPs-CN-DBA在PBS缓冲液中与不同浓度葡萄糖(0–0.1M)的荧光光谱图;图5(b)为脂质体探针NPs-CN-DBA与不同浓度葡萄糖(0.1μM–12.5mM)的Benesi–Hildebrand线性回归曲线图;10μM NPs-CN-DBA在0.1M PBS缓冲液(pH 7.4)中测试,25℃,λex=375nm,λem=427nm;
图6(a)为脂质体探针NPs-Ac-CDBA在PBS缓冲液中与不同浓度葡萄糖(0–0.1M)的荧光光谱图;图7(b)为脂质体探针NPs-Ac-CDBA与不同浓度葡萄糖(0.1μM–12.5mM)的Benesi–Hildebrand线性回归曲线图;10μM NPs-CN-DBA在0.1M PBS缓冲液(pH 7.4)中测试,25℃,λex=405nm,λem=482nm;
图7(a)为分子探针CN-DBA在有或无葡萄糖(0.1M)情况下的荧光强度随时间变化的关系图(0.5%MeOH/PBS缓冲液,pH 7.4);(b)为脂质体探针NPs-CN-DBA在有或无葡萄糖(0.1M)情况下的荧光强度随时间变化的关系图(0.1M PBS缓冲液,pH 7.4);
图8(a)为分子探针Ac-CDBA在有或无葡萄糖(0.1M)情况下的荧光强度随时间变化的关系图(0.5%MeOH/PBS缓冲液,pH 7.4);(b)为脂质体探针NPs-Ac-CDBA在有或无葡萄糖(0.1M)情况下的荧光强度随时间变化的关系图(0.1M PBS缓冲液,pH 7.4);
图9为HeLa细胞中加入200μM纳米探针NPs-Ac-CDBA后于0s、375s、750s、1125s、1500s时拍摄的共聚焦荧光图,λex/em=405/410–600nm,标尺为50μm;
图10为HeLa细胞中加入200μM纳米探针NPs-Ac-CDBA后1500s内的荧光变化图,λex/em=405/410–600nm,数据使用平均值±标准差表示(n=3);
图11为本发明所述的脂质体葡萄糖探针的结构示意图。
具体实施方式
下面将结合实施例,对本发明所述的具有良好生物相容性和荧光稳定性的脂质体葡萄糖荧光探针及制备方法作进一步详细说明。
本发明的目的是针对现有有机硼酸类葡萄糖探针存在的水溶性和稳定性差的不足,提供了一种基于有机硼酸的脂质体葡萄糖荧光探针及其制备方法,利用该纳米探针实现了在水溶性基质中对葡萄糖高灵敏性的识别和检测。
各实施案例的有机硼酸分子探针分别命名为CN-DBA和Ac-CDBA,其纳米化修饰后的脂质体探针分别命名为NPs-CN-DBA和NPs-Ac-CDBA。
实施例1:
分子探针CN-DBA的合成,其合成路线如下:
1)化合物C2的合成:
取500mL圆底烧瓶,加入4-氰基-2-甲基苯硼酸(5.00g,31.06mmol),新戊二醇(3.88g,37.25mmol)和200mL甲苯,加装Dean-Stark分水器,然后回流反应20h,TLC检测反应,反应结束后减压干燥除去反应溶剂。将粗产物溶于二氯甲烷中,以二氯甲烷为洗脱剂进行快速柱层析纯化,得6.76g油状化合物C2,收率为95.0%。1H NMR(400MHz,Chloroform-d)δ7.79(d,J=7.5Hz,1H),7.40(d,J=8.0Hz,2H),3.78(s,4H),2.52(s,3H),1.04(s,6H).
2)化合物C3的合成:
取500mL圆底烧瓶,加入化合物C2(6.76g,29.51mmol),N-溴代丁二酰亚胺(5.52g,30.99mmol),AIBN(0.13g,0.79mmol)和四氯化碳(150mL)中,搅拌回流16h,TLC检测反应,反应结束后冷却至室温,过滤。反应溶剂真空干燥得8.96g黄色油状化合物C3,收率为98.6%。1HNMR(400MHz,Chloroform-d)δ7.85(d,J=7.7Hz,1H),7.58(d,J=1.1Hz,1H),7.50–7.47(m,1H),4.83(s,2H),3.78(s,4H),1.03(s,6H).
3)化合物C5的合成:
取250mL三颈圆底烧瓶,加入9,10-二甲基蒽(1.13g,5.48mmol),N-溴代丁二酰亚胺(2.16g,12.14mmol),过氧化苯甲酰(20mg,82.57μmol),20mL氯仿和40mL四氯化碳,搅拌回流1.8h,TLC检测反应,反应结束后减压干燥,加入50mL甲醇搅拌,过滤并用5mL甲醇洗涤,所得产物真空干燥,得1.79g黄色固体化合物C5,收率为89.8%。1H NMR(400MHz,DMSO-d6)δ8.50(dd,J=6.9,3.3Hz,4H),7.73(dd,J=6.9,3.2Hz,4H),5.81(s,4H).
4)化合物C6的合成:
取化合物C5(1.65g,4.53mmol)溶于80mL氯仿中,加入甲胺甲醇(30%,w/v,16.58mL,135.90mmol),室温搅拌4h。TLC检测反应,反应结束后减压干燥除去反应溶剂,粗产物以甲醇/氯仿作洗脱剂进行快速柱层析纯化,得0.69g化合物C-6,收率为57.6%。1HNMR(400MHz,Chloroform-d)δ8.39(dd,J=6.9,3.3Hz,4H),7.54(dd,J=6.9,3.2Hz,4H),4.69(s,4H),2.68(s,6H).
5)化合物CN-DBA的合成:
取100mL三颈圆底烧瓶,加入化合物C-6(600mg,2.27mmol),化合物C-3e(1.92g,6.23mmol),碳酸钾(0.94g,6.81mmol),碘化钾(31.54mg,0.19mmol)和15.0mL DMF,氮气保护,室温搅拌16h后,TLC检测反应,反应结束后用40mL氯仿稀释反应液,饱和氯化钠溶液洗涤,合并有机相,减压干燥。将固体溶解于甲醇中,振摇过程中逐滴加入水,体系逐渐析出淡黄色沉淀,过滤,产物经真空干燥得410.6mg黄色固体CN-DBA,收率为31.1%。1H NMR(400MHz,Methanol-d4)δ8.39(d,J=7.9Hz,4H),7.76(d,J=7.3Hz,2H),7.70–7.60(m,8H),5.18(s,4H),4.45(s,4H),2.47(s,6H).13C NMR(101MHz,Methanol-d4)δ134.42,132.86,131.24,130.67,126.82,124.68,118.67,109.85,61.73,49.63,39.38.HRMS(ESI+):calcdfor C34H34B2N4O4[M+2H]+584.2761,found 584.2765.
实施例2:
分子探针Ac-CDBA的合成,其合成路线如下:
合成步骤:
1)化合物C7的合成:
取250mL圆底烧瓶,加入氯化铝(2.61g,19.57mmol),9,10-二甲基蒽(2.79g,13.52mmol),无水乙酰氯(1.5mL,21.1mmol)和150mL二硫化碳,室温搅拌12h。然后将反应体系加热致45℃反应2h。TLC检测反应,反应结束后,加入45mL含2.4mL盐酸的冰水,将反应体系冷却致室温,用二氯甲烷提取有机相,无水硫酸钠干燥,过滤,减压除去反应溶剂,粗产物用二氯甲烷作洗脱剂进行快速柱层析纯化,得1.59g化合物C-8,收率为47.3%。1H NMR(400MHz,Chloroform-d)δ9.00(d,J=1.5Hz,1H),8.37–8.31(m,3H),8.00(dd,J=9.2,1.8Hz,1H),7.61–7.54(m,2H),3.17(s,3H),3.08(s,3H),2.79(s,3H).
2)化合物C8的合成:
取100mL三颈圆底烧瓶,加入化合物C7(1.36g,5.48mmol),N-溴代丁二酰亚胺(2.10g,11.80mmol),过氧化苯甲酰(20mg,82.57μmol),20mL三氯甲烷和40mL四氯化碳,搅拌回流反应1.8h,TLC检测反应,反应结束后冷却至室温,加入50mL甲醇,过滤,用5mL甲醇洗涤。产物经真空干燥得到1.63g棕色固体化合物C8,收率为73.2%。1H NMR(400MHz,chlorform-d)δ9.00(s,1H),8.38(d,J=7.4Hz,3H),8.17(d,J=9.2Hz,1H),7.73(p,J=8.0,7.5Hz,2H),5.53(s,2H),5.47(s,2H),2.82(s,3H).
3)化合物C9的合成:
取250mL圆底烧瓶,加入化合物C8(0.60g,1.48mmol),氯仿(80mL),甲胺甲醇(30%,w/v.15mL,114.72mmol),室温搅拌反应4h。TLC检测反应,反应结束后,反应溶液减压干燥除去反应溶剂,粗产物使用甲醇/氯仿作洗脱剂进行快速柱层析纯化,得0.24g棕色固体化合物C9,收率为52.9%。1H NMR(400MHz,DMSO-d6)δ9.12(s,1H),8.49(t,J=12.3Hz,3H),7.95(d,J=9.2Hz,1H),7.62(p,J=6.0Hz,2H),4.61(d,J=37.8Hz,4H),2.78(s,3H),2.51(s,6H).
4)化合物Ac-CDBA的合成:
取100mL三颈圆底烧瓶,加入化合物C9(716.4mg,2.34mmol),化合物C3(2.16g,7.02mmol),碳酸钾(485.1mg,3.51mmol),碘化钾(77mg,0.46mmol)和15.0mL DMF,氮气保护,室温搅拌16h后,TLC检测反应,反应结束后用40mL氯仿稀释反应液,饱和氯化钠溶液洗涤,合并有机相,真空干燥。将产物溶解于甲醇中,振摇过程中逐滴加入水,体系逐渐析出淡黄色沉淀,过滤,产物经真空干燥得697.2mg黄色固体化合物Ac-CDBA,收率为47.7%。1HNMR(400MHz,Methanol-d4)δ9.11(s,1H),8.44(dt,J=17.5,9.1Hz,3H),8.12(d,J=9.3Hz,1H),7.79–7.50(m,8H),5.20(d,J=31.1Hz,4H),4.32(d,J=51.7Hz,4H),2.84(s,3H),2.46(d,J=10.3Hz,6H).13C NMR(101MHz,MeOD)δ198.79,134.38,134.23,132.63,132.34,131.89,131.82,130.58,130.34,130.30,127.84,127.79,127.03,125.50,125.29,124.85,123.80,118.64,118.58,110.31,109.98,61.71,61.58,49.97,40.53,39.70,25.69.HRMS(ESI+):calcd for C36H35B2N4O5[M+H]+625.2788,found 625.2790.
实施例3:
本实施例中所述的脂质体葡萄糖荧光探针NPs-CN-DBA和NPs-Ac-CDBA的制备方法如下:
1)采用超声法制备脂质体荧光探针NPs-CN-DBA。首先,将实施例1制备的CN-DBA(2.91mg)和DSPE-PEG2000(14.55mg,DSPE-PEG2000和CN-DBA的质量比为5:1)溶于1mL甲醇中。在细胞超声破碎仪(XL2000,Misonix Incorporated,NY)的超声条件下,使用注射器(1mL)将上述甲醇混合溶液注入去离子水(15mL)中,将混合溶液继续超声15s。然后,取出该混合溶液转移到截留分子量为3500的再生纤维素透析袋(MW Cut-off:3500)中透析24小时。使用超滤管将透析后的溶液浓缩至一定体积,最终获得了脂质体荧光探针NPs-CN-DBA。
2)采用超声法制备脂质体荧光探针NPs-Ac-CDBA。首先,将实施例2制备的Ac-CDBA(3.12mg)和DSPE-PEG2000(15.6mg,DSPE-PEG2000和Ac-CDBA的质量比为5:1)溶于1mL甲醇中。在细胞超声破碎仪(XL2000,Misonix Incorporated,NY)的超声条件下,使用注射器(1mL)将上述甲醇混合溶液注入去离子水(15mL)中,将混合溶液继续超声15s。然后,取出该混合溶液转移到截留分子量为3500的再生纤维素透析袋(MW Cut-off:3500)中透析24小时。使用超滤管将透析后的溶液浓缩至一定体积,最终获得了脂质体荧光探针NPs-Ac-CDBA。
实施例4:
本实施例中通过马尔文粒径仪和冷冻透射电镜对脂质体探针NPs-CN-DBA和NPs-Ac-CDBA的粒径和形态进行表征,如图1和图2所示。
将实施例3制备的脂质体探针NPs-CN-DBA或NPs-Ac-CDBA加入PS比色皿中,在25℃的条件下,将比色皿置于Zetasizer Nano-ZS90型纳米分析仪(英国马尔文公司)内稳定5分钟后测试,每个NPs-CN-DBA或NPs-Ac-CDBA样品分别取样三次,每个样品测试三次,获得了样品中NPs-CN-DBA(图1a)和NPs-Ac-CDBA(图2a)的强度、数量以及粒径的分布图。发现脂质体探针NPs-CN-DBA和NPs-Ac-CDBA均有良好的均一性,其水合粒径约在100–150nm。
将实施例3制备的脂质体探针NPs-CN-DBA或NPs-Ac-CDBA滴到超薄碳膜的铜网上,使用滤纸将多余的溶液吸干,然后,经一定时间减压干燥后于Talos L120C G2透射电子显微镜(捷克)下拍摄,获得了NPs-CN-DBA(图1b)和NPs-Ac-CDBA(图2b)的基本形貌特征。脂质体探针NPs-CN-DBA的粒径约为55nm,NPs-Ac-CDBA的粒径约为91nm。
实施例5:
本实施例中考察了分子探针CN-DBA和Ac-CDBA在0.5%MeOH/PBS缓冲液中对葡萄糖的荧光响应,如图3和图4所示。
将实施例1和2制备的分子探针CN-DBA和Ac-CDBA溶于甲醇中,配置浓度为2mM的探针母液。取1mL探针母液转移至100mL容量瓶中,使用0.1M PBS(pH 7.4)缓冲液定容至100mL,得到探针浓度为20μM的溶液。然后探针溶液与不同浓度的葡萄糖溶液按1:1比例混合,充分摇匀,得不同浓度葡萄糖的待测溶液(探针10μM,0.5%MeOH)。按相同的方法配置分子探针CN-DBA和Ac-CDBA与半乳糖、甘露糖、果糖、核糖、麦芽糖、葡萄糖胺、乳糖和蔗糖的混合溶液。取1mL待测溶液加入容量为3.5mL的石英荧光比色皿(四通光)中,设置分子探针CN-DBA(λex=375nm)和Ac-CDBA(λex=405nm)的激发波长,测试相应区间内的荧光发射光谱。
在0.5%MeOH/PBS缓冲液中测试了探针CN-DBA(λex=375nm)和Ac-CDBA(λex=405nm)在不同葡萄糖浓度条件下的荧光响应。发现随着葡萄糖的加入,分子探针CN-DBA在427nm处的荧光发射逐渐增强(图3a)。探针Ac-CDBA的荧光发射显著红移,随着葡萄糖的增加,Ac-CDBA在482nm处的荧光信号显著增强。结果表明CN-DBA和Ac-CDBA对葡萄糖具有高灵敏的荧光响应(图4a)。同时,在0.5%MeOH/PBS缓冲液中考察了CN-DBA和Ac-CDBA针对不同糖类物质的选择性,如葡萄糖、果糖、核糖、麦芽糖、甘露糖、半乳糖、乳糖、葡萄糖胺和蔗糖。发现CN-DBA(图3b)和Ac-CDBA(图4b)对葡萄糖的选择性明显优于其它糖类物质。在该选择性的荧光测试中,除葡萄糖外,其探针对D-果糖也具有一定的荧光响应,但血液中的葡萄糖浓度(3.6–5.8mM)远高于果糖以及其它糖类物质(其它糖类物质在血液中的含量均<0.1mM)。因此,在实际检测应用中其它糖类对CN-DBA和Ac-CDBA开展葡萄糖检测的影响可基本忽略。
实施例6:
本实施例中考察了脂质体探针NPs-CN-DBA和NPs-Ac-CDBA在PBS缓冲液(0.1M,pH 7.4)中对葡萄糖的荧光响应,如图5和图6所示。
将实施例3制备的脂质体探针NPs-CN-DBA和NPs-Ac-CDBA溶于PBS缓冲液中,配置浓度为20μM的探针溶液,然后,然后脂质体探针NPs-CN-DBA和NPs-Ac-CDBA溶液与不同浓度的葡萄糖溶液按1:1比例混合。取1mL待测溶液加入容量为3.5mL的石英荧光比色皿(四通光)中,设置分子探针的激发波长,测试相应区间内的荧光发射光谱。发现脂质体NPs-CN-DBA(图5a)和NPs-Ac-CDBA(图6a)对葡萄糖的荧光响应与分子探针CN-DBA和Ac-CDBA在0.5%MeOH/PBS缓冲液中相一致,均可以灵敏的检测葡萄糖水平的变化。表明脂质体纳米化修饰有效解决了分子探针CN-DBA和Ac-CDBA水溶性的问题,同时,脂质体探针NPs-CN-DBA和NPs-Ac-CDBA保持了对葡萄糖高灵敏的识别能力。
在0.1M PBS缓冲液(pH 7.4)中,脂质体探针NPs-CN-DBA(图5b)和NPs-Ac-CDBA(图6b)荧光变化值的倒数(1/(F–F0))与葡萄糖浓度的倒数(1/C[Glucose])在0.1μM–12.5mM范围内存在良好的线性关系。发现NPs-CN-DBA(图5b)和NPs-Ac-CDBA的葡萄糖定量范围(0.1μM–12.5mM)远远宽于人体正常的血糖范围(3–7mM)。表明其脂质体探针NPs-CN-DBA和NPs-Ac-CDBA在临床医学检验上具有良好的应用潜力。
实施例7:
本实施例中考察了分子探针CN-DBA和Ac-CDBA,脂质体探针NPs-CN-DBA和NPs-Ac-CDBA与葡萄糖反应的时间响应性,如图7和图8所示。
将分子探针CN-DBA(20μM,1%MeOH)或Ac-CDBA(20μM,1%MeOH)加入都5mL的EP管中,然后,按1:1比例加入0.2M葡萄糖溶液,迅速摇匀,取1mL CN-DBA或Ac-CDBA混合体系溶液于比色皿中,在25℃条件下立刻测试CN-DBA(λex/em=375/380–600nm)或Ac-CDBA(λex/em=405/410–600nm)与葡萄糖混合体系的荧光光谱,每个样品每分钟连续测试3次,测试该样品20min内的荧光发射光谱,并单独测试分子探针CN-DBA(10μM,0.5%MeOH/PBS)和Ac-CDBA(10μM,0.5%MeOH/PBS)的荧光变化。发现在0.5%MeOH/PBS缓冲液体系中,分子探针CN-DBA(图7a)或Ac-CDBA(图8a)本身荧光背景较低,当加入0.1M葡萄糖后产生荧光增强响应,在5分钟左右达到饱和荧光信号,说明分子探针CN-DBA和Ac-CDBA对葡萄糖的反应速度较快。
将脂质体探针NPs-CN-DBA(20μM)或NPs-Ac-CDBA(20μM)与0.2M葡萄按1:1比例混合均匀后,立刻测试探针NPs-CN-DBA(λex/em=375/380–600nm)或NPs-Ac-CDBA(λex/em=405/410–600nm)与葡萄糖混合体系的荧光光谱,每个样品每分钟连续测试3次,测试20min内的荧光发射光谱,并单独测试脂质体探针NPs-CN-DBA(10μM)和NPs-Ac-CDBA(10μM)的荧光变化。发现当加入0.1M葡萄糖后,脂质体探针NPs-CN-DBA(λem=427)和NPs-Ac-CDBA(λem=482)的荧光信号显著增强,且它们首次测试的荧光结果在前20min内保持稳定,即脂质体探针NPs-CN-DBA和NPs-Ac-CDBA与葡萄糖的时间响应性更快(<1min),且在20min内的荧光信号基本保持不变,体现出脂质体探针比分子探针对葡萄糖具有更快的时间响应性,且荧光稳定性更优。
实施例7:
本实施例中初步考察了脂质体探针NPs-Ac-CDBA在细胞内的荧光成像能力,如图9和图10所示。
将Hela细胞接种于共聚焦培养皿(35mm)中,加入1mL培养基,于培养箱中培养过夜。次日,将培养基弃去,用PBS清洗细胞三次,然后加入脂质体探针NPs-Ac-CDBA浓度为200μM的无糖DMEM培养基孵育,分别测试了Hela细胞在未加入探针孵育时以及孵育了0s、375s、750s、1125s和1500s后的荧光共聚焦图像。成像条件:使用405nm的激发光,收集410–600nm波段的荧光信号。如图9所示,在对照组细胞内中并未观测到荧光信号,因此排除了细胞自身背景信号的影响。随着脂质体探针NPs-Ac-CDBA(200μM)孵育时间的延长,Hela细胞内的荧光强度在1–1000s的时间范围内显著增加,体现出强烈的荧光信号,1125s后达到饱和(图10)。表明脂质体纳米NPs-Ac-CDBA(图11)具有良好的生物相容性和细胞膜透过性,具有应用于细胞及体内葡萄糖荧光检测和成像的良好潜力。
Claims (2)
1.一种具有良好生物相容性的脂质体葡萄糖荧光探针,其特征在于,所述的脂质体葡萄糖荧光探针包括有机硼酸分子和脂质体两个部分,所述的有机硼酸分子作为葡萄糖特异性识别探针,所述的脂质体是利用两亲性分子通过自组装形成的纳米囊泡结构,所述的有机硼酸分子为具有结构式(Ⅰ)的化合物,其中,R3选自C(O)R4、C(O)NR4R5、NHC(O)R4,其中R4和R5各自独立选自H或C1-C6烷基,所述的两亲性分子选自二硬脂酰磷脂酰乙醇胺-聚乙二醇共聚(mPEG-DSPE)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺共聚(DSPE-PEG-NHS)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺共聚(DSPE-PEG-Maleimide)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-生物素共聚(DSPE-PEG-Biotin),二硬脂酰磷脂酰乙醇胺-聚乙二醇-氨基共聚(DSPE-PEG-NH2)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧酸共聚(DSPE-PEG-COOH)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-羟基共聚(DSPE-PEG-OH)、二硬脂酰磷脂酰乙醇胺-聚乙二醇-巯基共聚(DSPE-PEG-SH)中的一种或多种
2.如权利要求1所述的脂质体葡萄糖荧光探针,其特征在于,所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇共聚为二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)。
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