CN105602276B - 可聚合近红外荧光染料单体及其制备方法、用途 - Google Patents
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Abstract
本发明公开了一种可聚合关于近红外（NIR）荧光染料单体及其制备方法、用途。所述的近红外功能荧光染料单体为 (NFM)是以氟硼荧光染料先与甲基丙烯酰氯反应，再经过提纯合成得到的。本发明所合成的新的NIR荧光单体，为近红外荧光聚合物的可控合成及应用提供了新方法，在生物体内近红外荧光成像方面具有广阔的应用前景。
Description
技术领域
本发明属于本发明属于有机化合物和高分子材料的技术领域，具体涉及一类新型单体，即NFM及其合成的近红外荧光功能染料单体。
背景技术
近红外荧光成像是目前光化学分子成像领域研究的热点，以合适的荧光探针标记细胞、组织和器官等，用特定波长的 红光激发荧光染料，发出近红外荧光，应用近红外荧光成像设备进行检测，即可直接检测生物体内发生的生理过程和病理状态。近红外（650 -900 nm）染料在化学生物学中的应用前景广泛，因为其对生物的光损伤最小，可以探查深层组织，并可将背景自发荧光的影响最小化。
传统的有机NIR染料，由于发展的较早，所以易获得，成本低，物理/化学性质研究的比较透彻，所以是目前这一领域应用的最为广泛的一类NIR荧光材料。但该类探针也存在许多明显的缺点，比如弱亲水性，静电复印性，低量子产率，生物系统不稳定性和低探测敏感性等等，这些都直接影响着它们在生物研究中的应用。最近进行的设计策略已经克服了一些传统近红外有机试剂的限制，比如通过将这些近红外染料封装成纳米颗粒或者和特定的配体结合，尽管这些纳米粒子是一个好的平台，可以进行特定的成像和癌症治疗的多功能探测，但它仍然只是处于早起的临床应用。大范围生产和无毒的良好的生物相容性的困难，网状内皮组织系统的滞后的影响和单核吞噬细胞系统，由于在这些方面的困难，胶囊染料纳米粒子在纳米药物方面的运用仅限于细胞和实验动物层面。本发明合成的单体可以通过聚合或者与其他结构通过化学键结合，再形成多功能结构发明有新的应用。例如在体内荧光成像，用于肿瘤的治疗等。
发明内容
要解决的技术问题：本发明的目的在于提供可聚合近红外荧光染料单体及其制备方法、用途，即可直接检测生物体内发生的生理过程和病理状态，在体内荧光成像，应用于肿瘤治疗等。
技术方案：针对上述问题，本发明公开了可聚合近红外荧光染料单体，所述的单体的化学结构式如下：
。
一种可聚合近红外荧光染料单体的制备方法，制备方法包括下述步骤：
以氟硼荧光染料、甲基丙烯酰氯和三乙胺按摩尔比1：2：3投料，以无水四氢呋喃为溶剂，在温度为0-30℃的条件下将三乙胺与氟硼荧光染料搅拌，再将四氢呋喃稀释过的甲基丙烯酰氯滴加到混合溶剂中，滴加完毕后，常温继续搅拌反应4-10 h；通过200-300目的硅胶柱，再用体积比为1:8的乙酸乙酯和石油醚混合液进行洗脱，收集洗脱液后减压蒸馏，得到近红外荧光功能染料单体NFM。
所述的可聚合近红外荧光染料单体，所述的单体在溶剂为四氢呋喃，浓度为5 μgmL-1的情况下，紫外吸收峰为649 nm，其荧光光谱的最强激发峰的波长为649.5 nm，最强的发射波长在697 nm。
优选的，所述的一种可聚合近红外荧光染料单体的制备方法，所述的温度为0-5℃。
优选的，所述的一种可聚合近红外荧光染料单体的制备方法，所述的温度为滴加完毕后，常温继续搅拌反应5h。
一种可聚合近红外荧光染料单体的用途，是以染料单体为反应物，与大分子链转移剂聚甲基丙烯酸聚乙二醇单甲醚酯和疏水性单体2 -（4 -（十二烷氧基）苯基）1，3二恶烷-甲基丙烯酸（DBAM）进行可逆加成断裂转移自由基聚合，制备近红外荧光功能染料的聚合物进行荧光成像。
有益效果：
该合成方法利用甲基丙烯酰氯的亲核反应，合成步骤简单，通过后处理柱层析，就可得到产率较高较纯净的近红外荧光单体，借助较成熟的聚合技术，使该近红外荧光单体能在生物体内荧光成像方面具有较大的应用，对于肿瘤的治疗成像也有很大的应用前景。
附图说明
图 1 是本发明的近红外荧光染料单体NIM的氢核磁谱图；
图 2是本发明的近红外荧光染料单体NIM的紫外荧光图；
图3为DDP的合成路线；
图4为PPEGMA与DDP的GPC流出曲线；
图5为编号a和b是DDP在Hela细胞中的成像，编号c是DOX的成像；
图6为编号a和b是DDP在V79细胞中的成像，编号c和d是在KB细胞中的成像；
图7为DDP在小鼠体内不同时间点的成像情况。
具体实施方式
下面结合附图及实施案例对本发明作进一步描述。下文中的描述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围。
化学试剂：
甲基丙烯酸聚乙二醇单甲醚酯(PEGMA, Mn = 500 g mol-1) 购于Aldrich公司；偶氮二异庚腈（AIBN），AR；四氢呋喃，AR；二氯甲烷，AR；三乙胺，AR；1，4-二氧六环，AR都购于中国医药（集团）上海化学试剂公司；阿霉素盐酸盐(DOX•HCl)购于BBI生命科学公司。透析袋(截留分子量Mw=3500)购于美国Biosharp公司。甲基丙烯酰氯(95%)购于Aladdin公司。2 -（4 -（十二烷氧基）苯基）1，3二恶烷-甲基丙烯酸（DBAM）, 二硫代-1-萘甲酸异丁氰酯
(CPDN)和氟硼荧光染料都依据文献合成。
测试仪器及条件：
凝胶渗透色谱仪：日本东曹公司（TOSOH）HLC-8320型GPC；测试条件：Tskgel SuperMultiporeHZ-N（4.6*150）两柱联用，示差检测器，流动相为四氢呋喃（0.35 mL min-1），柱温40℃，
核磁共振：Bruker 300MHz核磁仪，以(CD3)2SO.为溶剂测定；
紫外可见分光光度计：岛津UV-2600紫外可见分光光度计。
荧光分光光度计：HITACHI F-2500荧光分光光度计，激发和发射的狭缝宽为10.0和10.0 nm。
实施例1
将化合物氟硼荧染料105 mg，三乙胺84 μL加到20 mL无水四氢呋喃中搅拌混合，在冰水浴的条件下慢慢滴加1 mL 无水四氢呋喃稀释的39 μL的甲基丙烯酰氯。1 h之后撤去冰水浴，在室温的条件下继续搅拌5 h。然后真空旋转蒸馏除去溶剂，二氯甲烷溶解固体后用饱和食盐水和蒸馏水洗涤，再用无水硫酸钠干燥，减压蒸馏除尽溶剂，通过200-300目的硅胶柱，再用体积比为1:8的乙酸乙酯和石油醚混合液进行洗脱，收集洗脱液后减压蒸馏，得到近红外荧光功能染料单体NFM。由图3所示，单体在溶剂为四氢呋喃，浓度为5 μgmL-1的情况下，紫外吸收峰（图3中的实线）为649 nm，其荧光光谱的最强激发峰的波长为649.5 nm，最强的发射波长在697 nm（图3中的虚横线）。
实施例2
近红外荧光单体的应用
大分子链转移剂PPEGMA由聚（乙二醇）甲基丙烯酰氯（PEGMA）进行RAFT聚合得到的。PEGMA (3087 μL, 6.5 mmol), CPDN (36 mg, 0.13 mmol)和AIBN (10.7 mg, 6.5 ×10-2 mmol)加到安瓿瓶中，加入2 mL1，4-二氧六环作为溶剂，除氧后60 ℃搅拌反应5 h。再用大量冰乙醚沉淀，离心（10,000 rpm，13 min）得到PPEGMA（Mn = 20900 g/mol。再以PPEGMA (209 mg, 0.010 mol)为大分子链转移剂，加入所合成的单体NFM (6.5 mg, 0.011mmol)和疏水性单体DBAM (69.1 mg, 0.159 mmol),再加入AIBN (0.82 mg, 0.005 mmol)和 1 mL的1，4-二氧六环℃搅拌反应11.75 h，用大量正己烷沉淀之后，再离心（10,000rpm，13 min）得到聚合物DDP，分子量Mn = 27700 g/mol。
将聚合物DDP(20 mg) 和DOX•HCl (4 mg)溶于2 ml DMSO并以1.3 mL h-1滴加到8mL的二次水中，搅拌10 h后装入透析袋中透析48 h,之后冷冻干燥除去水，得到的胶束冷冻保存。培养了三种细胞进行成像，分别是人宫颈癌细胞（Hela）、中国仓鼠肺细胞（V79）和人口腔上皮癌细胞（KB）。胶束中加入二次水，配成70 μg mL-1，并分别加入到培养的细胞中，用共聚焦显微镜进行荧光成像观察图5和图6。其中细胞核用Hoechst 33324染色，呈蓝色，在图中第一列，激发光谱405 nm，发射光谱在435–480 nm。DOX在绿色通道，在图中第二列，激发光谱488 nm，发射光谱在520–600 nm。DDP在红色通道，在图中第三列，激发光谱640 nm，发射光谱在700–800 nm。随着时间的增长，细胞的成像越清晰，DOX在细胞的红色通道中不能成像，意味着红色通道的成像正是聚合物的成像。在癌细胞较正常细胞成像清晰，这是因为癌细胞的酸性环境使得胶束解离。
将培养4-6周的小鼠的右后腿上种上Hela癌细胞，等长到2 至5 mm时，将8 mg mL-1的胶束从尾部静脉注射200 μL，然后用活体荧光体内成像系统观测小鼠，结果如图7。以630nm为激发，发射光谱为710 nm，聚合物先聚集在肝脏部位，然后进入肺等呼吸系统，在注射8h后，可清晰的观察到肿瘤部位的荧光成像。
Claims (5)
1.可聚合近红外荧光染料单体，其特征在于，所述的单体的化学结构式如下：
。
2.根据权利要求1所述的一种可聚合近红外荧光染料单体的制备方法，其特征在于制备方法包括下述步骤：
以氟硼荧光染料、甲基丙烯酰氯和三乙胺按摩尔比1：2：3投料，以无水四氢呋喃为溶剂，在温度为0-30℃的条件下将三乙胺与氟硼荧光染料搅拌，再将四氢呋喃稀释过的甲基丙烯酰氯滴加到混合溶剂中，滴加完毕后，常温继续搅拌反应4-10 h；通过200-300目的硅胶柱，再用体积比为1:8的乙酸乙酯和石油醚混合液进行洗脱，收集洗脱液后减压蒸馏，得到近红外荧光功能染料单体NFM。
3.权利要求1的所述的可聚合近红外荧光染料单体，其特征在于所述的单体在溶剂为四氢呋喃，浓度为5 μg mL-1的情况下，紫外吸收峰为649 nm，其荧光光谱的最强激发峰的波长为649.5 nm，最强的发射波长在697 nm。
4.根据权利要求2所述的一种可聚合近红外荧光染料单体的制备方法，其特征在于所述的温度为0-5℃。
5.根据权利要求2所述的一种可聚合近红外荧光染料单体的制备方法，其特征在于滴加完毕后，常温继续搅拌反应5h。
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