水溶性荧光聚合物纳米粒的制备及双光子成像

梁淑彩等
摘 要 报道了一种能用于活细胞双光子荧光(TPF)成像的水溶性聚合物纳米粒。首先以含双羧基的萘酰亚胺作为交联剂和荧光标记试剂,通过对聚乙烯亚胺发生交联反应制备纳米粒子,然后对其结构形态、单双光子荧光性能及细胞毒性进行测试。结果表明,获得的纳米粒为球形,粒径为5~10 nm;以443或800 nm为激发波长,荧光发射波长均为536 nm;在pH 4.0~9.0范围内,其荧光无明显变化;在pH 7.4 的溶液中和激发光为443 nm 的条件下,对其连续测定1.2万次后荧光强度变化不超过1%,说明其酸碱稳定性和光稳定性较好;浓度在 15 mg/L以下及与细胞作用时间在24 h以内细胞毒性较低。最后,用双光子共聚焦荧光显微镜观察了其在 Hela 细胞中的 TPF 成像性能。将Hela细胞与纳米粒共同孵育2 h后,在800 nm激光激发下,在细胞中可观察到其绿色荧光。此纳米粒可望用于靶向性双光子荧光成像探针的开发。关键词 萘酰亚胺; 聚乙烯亚胺;荧光聚合物纳米粒;双光子成像
1 引 言
双光子荧光(TPF)成像技术具有高的系统分辨率和信噪比,能对处在自然状态的样品进行无损观察与成像,在生物医学分析领域具有极大的应用空间。TPF成像应用的发展离不开性能优良的TPF材料。目前,有机小分子染料作为TPF材料的研究较多[1,2],但这些材料大多光稳定性和水溶性差,在体内清除较快,不利于生物体系的成像研究。采用纳米材料可以有效减少上述问题,最具有代表性的是无机发光量子点(QD)。QD的光稳定性和荧光性能俱佳,是荧光成像领域研究的热点[3,4],在细胞及组织中的TPF成像应用已有报道[5~7]。但其对人体健康与生态环境可能产生的不利影响限制了其大规模生产和应用[8,9]。
负载荧光染料的聚合物纳米材料具有好的生物相容性,并将大量染料包裹在一个纳米粒中,可大大提高检测灵敏度和材料的光稳定性,在单光子荧光成像方面已有广泛应用[10]。近年来,聚合物TPF材料的开发也开始引起注意,目前报道的多以疏水性聚合物包裹TPF染料形成聚合物纳米粒。这种纳米粒中TPF染料以非共价键方式存在,容易造成染料泄漏,同时得到的纳米材料亲水性差,使用时需分散至水溶液中或表面再进行亲水性修饰[11,12]。
聚乙烯亚胺(PEI)有良好的水溶性和生物相容性,是药物传递系统常用的载体材料[13,14]。萘酰亚胺衍生物具有好的荧光性能,如光稳定性,大的Stocks位移和高的荧光强度等,已用于开发测定FSymbolm@@ 、H2S及H2O2等的TPF探针[15~17]。本实验以含双羧基的萘酰亚胺染料对PEI进行交联制备了一种染料与聚合物共价键合的水溶性荧光聚合物纳米材料,对其结构、荧光性质及细胞毒性进行了测试,并考察了其在Hela细胞中的TPF成像。研究表明,此纳米粒为球形,粒径5~10 nm;具有好的pH和光稳定性,且浓度在15 mg/L以下有较低的毒性;能够进入细胞,在800 nm的激发光下,用共聚焦显微镜可以观察到细胞内纳米粒的绿色荧光,可望用于靶向性TPF成像探针的开发。
2 实验部分
2.1 仪器及试剂
4 结 论
本实验以含有两个羧基的萘酰亚胺衍生物TCN为交联剂与PEI中的氨基发生交联反应,得到了荧光聚合物纳米粒。该纳米粒具有如下特点:(1)具有水溶性,方便了其在生物体系应用; (2) 荧光团以共价的方式固定于纳米粒中,避免了其泄漏,提高了纳米粒的稳定性; (3)发射波长较长,能有效地避免生物体自发荧光的干扰; (4)具有好的pH和光稳定性; (5) 能够进入细胞,并成功实现细胞的TPF成像。这种在较长激发波长下进行的荧光成像,可以减少对细胞的损伤,提高成像质量; (6) 纳米粒中存在可进行功能化修饰的氨基,在靶向性TPF探针方面具有应用前景。
References
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余 慧, 梁淑彩, 钟海迪, 付 婷, 鄢国平. 分析化学, 2011, 39(3): 409-413
Preparation and TwoPhoton Imaging of a WaterSoluble
Fluorescent Nanoparticle
LIANG ShuCai1, CHEN XiaoHui2, LIU YanBin1, QIN Meng2, YAN GuoPing*2
1(College of Pharmacy, Wuhan University, Wuhan 430072, China)
2(School of Materials Science and Engineering, Wuhan Institute of Technology, Wuhan 430074, China)
Abstract A watersoluble polymeric fluorescent nanoparticle (PTCN) has been proposed for twophoton fluorescence (TPF) imaging of living cells. PTCN was firstly prepared by chemical crosslinking of polyethyleneimine using a dual carboxyl 1, 8naphthalimide derivative as the crosslinker. Subsequently, the investigations on its structure, morphology, one and twophoton fluorescence properties and cytotoxicity were carried out. It was found that PTCN was spherical with the diameters of 5-10 nm. It could emit fluorescence at 536 nm with the excitation wavelengths of 443 nm and 800 nm, respectively. PTCN possessed good pH stability and photostability. Its fluorescence did not change in the pH range of 4.0-9.0. In pH 7.4 buffer solution, when it was detected at λex/λem=443 nm/536 nm for 12000 times, the decrease in fluorescence intensity was less than 1%. Cytotoxic assays indicated it was low toxic upon interactions with Hela cells for 24 h at a concentration of 15.0 mg/L. Finally, the application of PTCN for imaging live cells was assessed by TPF microscopy technique. After Hela cells were incubated with PTCN for 2 h, the green fluorescence was observed in the cellsap at the excitation wavelength of 800 nm. The proposed nanoparticle is potential in the development of targeting TPF probes.
Keywords Naphthalimide; Polyethyleneimine; Fluorescent polymeric nanoparticles; Twophoton imaging
(Received 14 November 2013; accepted 12 January 2014)
This work was supported by the National Natural Science Foundation of China (Nos.20905059, 20835004)
贾鹏飞, 刘 恒, 张元红, 刘 鑫, 赵 宁, 于晓强. 高等学校化学学报 , 2010, 31(6): 1075-1077
7 Maestro L M, RamírezHernndez J E, Bogdan N, Capobianco J A, Vetrone F, García Sol J, Jaque D. Nanoscale, 2012, 4(1): 298-302
8 Hardman R. Environ. Health Persp., 2006, 114(2): 165-172
9 Mazumder S, Dey R, Mitra M K, Mukherjee S, Das G C. J. Nanomater., 2009, 815734
10 Vollrath A, Schubert S, Schubert U S. J. Mater. Chem. B, 2013, 1(15): 1994-2007
11 Maurin M, Vurth L, Vial J C, Baldeck P, Marder S R, Van der Sanden B, Stephan O. Nanotech., 2009, 20(23): 235102
12 Tian Y, Chen C Y, Cheng Y J, Young A C, Tucker N M, Jen A K Y. Adv. Funct. Mater., 2007, 17(10): 1691-1697
13 Godbey W T, Wu K K, Mikos A G. J. Control. Release, 1999, 60(23): 149-160
14 Zhang S, Wang G, Lin X. Biotechnol. Prog., 2008, 24(4): 945-956
15 Liu T, Zhang X, Qiao Q, Zou C, Feng L, Cui J, Xu Z. Dyes Pigments, 2013, 99(3): 537-542
16 Zhang J F, Lim C S, Bhuniya S, Cho B R, Kim J S. Org. Lett., 2011, 13(5): 1190-1193
17 Srikun D, Miller E W, Domaille D W, Chang C J. J. Am. Chem. Soc., 2008, 130(14): 4596-4597
18 YU Hui, LIANG ShuCai, ZHONG HaiDi, FU Ting, YAN GuoPing. Chinese J. Anal. Chem., 2011, 39(3): 409-413
余 慧, 梁淑彩, 钟海迪, 付 婷, 鄢国平. 分析化学, 2011, 39(3): 409-413
Preparation and TwoPhoton Imaging of a WaterSoluble
Fluorescent Nanoparticle
LIANG ShuCai1, CHEN XiaoHui2, LIU YanBin1, QIN Meng2, YAN GuoPing*2
1(College of Pharmacy, Wuhan University, Wuhan 430072, China)
2(School of Materials Science and Engineering, Wuhan Institute of Technology, Wuhan 430074, China)
Abstract A watersoluble polymeric fluorescent nanoparticle (PTCN) has been proposed for twophoton fluorescence (TPF) imaging of living cells. PTCN was firstly prepared by chemical crosslinking of polyethyleneimine using a dual carboxyl 1, 8naphthalimide derivative as the crosslinker. Subsequently, the investigations on its structure, morphology, one and twophoton fluorescence properties and cytotoxicity were carried out. It was found that PTCN was spherical with the diameters of 5-10 nm. It could emit fluorescence at 536 nm with the excitation wavelengths of 443 nm and 800 nm, respectively. PTCN possessed good pH stability and photostability. Its fluorescence did not change in the pH range of 4.0-9.0. In pH 7.4 buffer solution, when it was detected at λex/λem=443 nm/536 nm for 12000 times, the decrease in fluorescence intensity was less than 1%. Cytotoxic assays indicated it was low toxic upon interactions with Hela cells for 24 h at a concentration of 15.0 mg/L. Finally, the application of PTCN for imaging live cells was assessed by TPF microscopy technique. After Hela cells were incubated with PTCN for 2 h, the green fluorescence was observed in the cellsap at the excitation wavelength of 800 nm. The proposed nanoparticle is potential in the development of targeting TPF probes.
Keywords Naphthalimide; Polyethyleneimine; Fluorescent polymeric nanoparticles; Twophoton imaging
(Received 14 November 2013; accepted 12 January 2014)
This work was supported by the National Natural Science Foundation of China (Nos.20905059, 20835004)
贾鹏飞, 刘 恒, 张元红, 刘 鑫, 赵 宁, 于晓强. 高等学校化学学报 , 2010, 31(6): 1075-1077
7 Maestro L M, RamírezHernndez J E, Bogdan N, Capobianco J A, Vetrone F, García Sol J, Jaque D. Nanoscale, 2012, 4(1): 298-302
8 Hardman R. Environ. Health Persp., 2006, 114(2): 165-172
9 Mazumder S, Dey R, Mitra M K, Mukherjee S, Das G C. J. Nanomater., 2009, 815734
10 Vollrath A, Schubert S, Schubert U S. J. Mater. Chem. B, 2013, 1(15): 1994-2007
11 Maurin M, Vurth L, Vial J C, Baldeck P, Marder S R, Van der Sanden B, Stephan O. Nanotech., 2009, 20(23): 235102
12 Tian Y, Chen C Y, Cheng Y J, Young A C, Tucker N M, Jen A K Y. Adv. Funct. Mater., 2007, 17(10): 1691-1697
13 Godbey W T, Wu K K, Mikos A G. J. Control. Release, 1999, 60(23): 149-160
14 Zhang S, Wang G, Lin X. Biotechnol. Prog., 2008, 24(4): 945-956
15 Liu T, Zhang X, Qiao Q, Zou C, Feng L, Cui J, Xu Z. Dyes Pigments, 2013, 99(3): 537-542
16 Zhang J F, Lim C S, Bhuniya S, Cho B R, Kim J S. Org. Lett., 2011, 13(5): 1190-1193
17 Srikun D, Miller E W, Domaille D W, Chang C J. J. Am. Chem. Soc., 2008, 130(14): 4596-4597
18 YU Hui, LIANG ShuCai, ZHONG HaiDi, FU Ting, YAN GuoPing. Chinese J. Anal. Chem., 2011, 39(3): 409-413
余 慧, 梁淑彩, 钟海迪, 付 婷, 鄢国平. 分析化学, 2011, 39(3): 409-413
Preparation and TwoPhoton Imaging of a WaterSoluble
Fluorescent Nanoparticle
LIANG ShuCai1, CHEN XiaoHui2, LIU YanBin1, QIN Meng2, YAN GuoPing*2
1(College of Pharmacy, Wuhan University, Wuhan 430072, China)
2(School of Materials Science and Engineering, Wuhan Institute of Technology, Wuhan 430074, China)
Abstract A watersoluble polymeric fluorescent nanoparticle (PTCN) has been proposed for twophoton fluorescence (TPF) imaging of living cells. PTCN was firstly prepared by chemical crosslinking of polyethyleneimine using a dual carboxyl 1, 8naphthalimide derivative as the crosslinker. Subsequently, the investigations on its structure, morphology, one and twophoton fluorescence properties and cytotoxicity were carried out. It was found that PTCN was spherical with the diameters of 5-10 nm. It could emit fluorescence at 536 nm with the excitation wavelengths of 443 nm and 800 nm, respectively. PTCN possessed good pH stability and photostability. Its fluorescence did not change in the pH range of 4.0-9.0. In pH 7.4 buffer solution, when it was detected at λex/λem=443 nm/536 nm for 12000 times, the decrease in fluorescence intensity was less than 1%. Cytotoxic assays indicated it was low toxic upon interactions with Hela cells for 24 h at a concentration of 15.0 mg/L. Finally, the application of PTCN for imaging live cells was assessed by TPF microscopy technique. After Hela cells were incubated with PTCN for 2 h, the green fluorescence was observed in the cellsap at the excitation wavelength of 800 nm. The proposed nanoparticle is potential in the development of targeting TPF probes.
Keywords Naphthalimide; Polyethyleneimine; Fluorescent polymeric nanoparticles; Twophoton imaging
(Received 14 November 2013; accepted 12 January 2014)
This work was supported by the National Natural Science Foundation of China (Nos.20905059, 20835004)
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