水溶性荧光聚合物纳米粒的制备及双光子成像

Preparation and Two-Photon Imaging of a Water-Soluble Fluorescent Nanoparticle

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。

A water-soluble polymeric fluorescent nanoparticle （ PTCN ） has been proposed for two-photon fluorescence （ TPF ） imaging of living cells. PTCN was firstly prepared by chemical crosslinking of polyethyleneimine using a dual carboxyl 1, 8-naphthalimide derivative as the crosslinker. Subsequently, the investigations on its structure, morphology, one-and two-photon 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.