开发用于肿瘤微环境成像的pH敏感荧光探针

doi:10.3969/j.issn.1674-8115.2022.07.005

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (7): 875-884.doi: 10.3969/j.issn.1674-8115.2022.07.005

• 论著 · 基础研究 • 上一篇

开发用于肿瘤微环境成像的pH敏感荧光探针

王雨心(), 孙瑞琪, 刘坚华(), 何伟娜()

上海交通大学基础医学院药物化学与生物信息学中心，上海 200025

收稿日期:2022-03-19 接受日期:2022-06-25 出版日期:2022-07-28 发布日期:2022-09-04
通讯作者: 刘坚华,何伟娜 E-mail:yuxinwang@sjtu.edu.cn;jhliu7912@sjtu.edu.cn;hewn0319@sjtu.edu.cn
作者简介:王雨心（1995—），女，硕士生；电子信箱yuxinwang@sjtu.edu.cn。
基金资助:
上海交通大学基础医学院前沿研究中心原创探索类青年支持项目(YCTSQN2021001);上海交通大学“交大之星”计划医工交叉研究基金(YG2016MS22)

Development of pH-responsive fluorescent probe for tumor microenvironment imaging

WANG Yuxin(), SUN Ruiqi, LIU Jianhua(), HE Weina()

Medical Chemistry and Bioinformatics Center, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Received:2022-03-19 Accepted:2022-06-25 Online:2022-07-28 Published:2022-09-04
Contact: LIU Jianhua,HE Weina E-mail:yuxinwang@sjtu.edu.cn;jhliu7912@sjtu.edu.cn;hewn0319@sjtu.edu.cn
Supported by:
Original Exploration Youth Support Project of Frontier Research Center, Shanghai Jiao Tong University College of Basic Medical Sciences(YCTSQN2021001);“Star of SJTU” Program of Shanghai Jiao Tong University, Medical-Engineering Cross Research Fund(YG2016MS22)

摘要/Abstract

摘要：

目的·合成水溶性pH响应性花菁类近红外荧光探针，评估其光学性能，并将其应用于模拟肿瘤微环境的在体成像。方法·应用两步经典的化学反应，合成水溶性pH响应性近红外荧光探针R2S，同步合成脂溶性探针R2Z作为对照。应用磁共振氢谱、质谱及高效液相色谱验证探针的化学结构及纯度。利用紫外-可见、光致发光光谱学测试评价探针的pH响应性、响应可逆性、光稳定性及结构稳定性。利用细胞荧光共定位成像评价探针的细胞膜通透性，使用HCT-116细胞、HeLa细胞进行细胞毒性实验，使用BALB/c健康雌性小鼠进行小动物在体成像实验，评估探针的安全性。通过在小鼠背部左侧和右侧分别皮下注射pH 6.50和pH 7.40的PBS溶液模拟肿瘤微酸性环境和正常的细胞环境，随后注射探针R2S进行在体成像实验，比较R2S在pH 6.50侧和pH 7.40侧的荧光强度。结果·成功合成了水溶性pH响应性近红外荧光探针R2S及其脂溶性类似物R2Z。随着溶液酸性逐渐增强（从pH 11.10到pH 3.47），R2S的最大吸收波长由642 nm红移至774 nm，最大发射波长亦由794 nm红移至808 nm。探针R2S的酸解离常数（pK_a）值约为6.88。与脂溶性探针R2Z相比，R2S在保留了较好细胞膜通透性的前提下，展现出更大的斯托克斯位移、更好的响应可逆性、更高的稳定性。R2Z在12.5 μmol/L浓度下，细胞相对存活率低于80%；而R2S在100 μmol/L浓度下，仍未表现出明显的细胞生长抑制现象，表明其具有更高的生物安全性。R2S在模拟肿瘤微酸性环境的低pH组织和正常pH组织中的成像具有明显的区分度。结论·水溶性pH响应性花菁类近红外荧光探针R2S对pH变化响应灵敏、稳定，最佳响应范围与肿瘤所处微环境的pH相符，在肿瘤成像领域展现出了较强的在体成像应用潜力。

关键词: 肿瘤成像, 近红外荧光探针, 肿瘤微环境, 花菁类染料, 水溶性, pH敏感

Abstract:

Objective·To synthesize a water-soluble pH-responsive near-infrared cyanine-based fluorescent probe, evaluate its optical properties, and conduct in vivo imaging analysis of the quasi-tumor microenvironment.

Methods·The water-soluble pH-responsive near-infrared fluorescent probe R2S was synthesized via two classic chemical transformation steps and the liposoluble probe R2Z was synthesized in the similar way. ¹H nuclear magnetic resonance spectroscopy, mass spectroscopy and high performance liquid chromatography were used to verify the structure and the purity of the probes synthesized. Ultraviolet-visible and photoluminescence spectroscopy were used to evaluate its pH responsiveness, response reversibility and stability. Then, cell permeability of the probe was measured by cell imaging. The cytotoxicity of the probe was evaluated with HCT-116 cells and HeLa cells, and in vivo imaging experiments were performed by using healthy BALB/c female mice. Finally, imaging experiments of quasi-tumor-acidic-microenvironment were carried out by separately injecting PBS solution with pH 6.50 and pH 7.40 into the back of the mice, and the fluorescence intensity of R2S at pH 6.50 and pH 7.40 was compared.

Results·The water-soluble pH-responsive near-infrared fluorescent probe R2S and its liposoluble analog R2Z were synthesized successfully. As the acidity of the probe solution increased (from pH 11.10 to pH 3.47), the maximum absorption wavelength of R2S shifted from 642 nm to 774 nm, while the maximum emission wavelength shifted from 794 nm to 808 nm, with a pK_a value of 6.88. Compared to the liposoluble probe R2Z, R2S showed a larger Stokes shift, higher stability without affecting the cell membrane permeability. At the high administration concentration of 100 μmol/L, R2S did not exhibit the inhibition of proliferation, while for R2Z at the concentration of 12.5 μmol/L, the relative cell viability already descended to below 80%, indicating R2S had higher biosecurity than R2Z. Imaging of R2S in low pH tissue (simulating tumor tissue) and normal pH tissue exhibited a high degree of discrimination.

Conclusion·The water-soluble pH-responsive near-infrared cyanine fluorescent probe R2S exhibits sensitive and stable responses to pH changes, and the optimal response range is consistent with the pH of the tumor acidic microenvironment, illustrating a strong potential for its in vivo tumor imaging applications.

Key words: tumor imaging, near-infrared fluorescence probe, tumor microenvironment (TME), cyanine dye, water solubility, pH responsivity

中图分类号:

R313

王雨心, 孙瑞琪, 刘坚华, 何伟娜. 开发用于肿瘤微环境成像的pH敏感荧光探针[J]. 上海交通大学学报（医学版）, 2022, 42(7): 875-884.

WANG Yuxin, SUN Ruiqi, LIU Jianhua, HE Weina. Development of pH-responsive fluorescent probe for tumor microenvironment imaging[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 875-884.

图/表 8

图1 探针R2S与R2Z的合成路线Note： NaOAc—sodium acetate; HOAc—acetic acid; DMF—N, N-dimethylformamide; Ar—argon.

Fig 1 Synthetic routes of the target probes R2S and R2Z

图2 R2S与R2Z在不同pH值下的吸收光谱及荧光光谱Note： A/B. Absorption spectra of 20 μmol/L R2S (A) and 20 μmol/L R2Z (B). C/D. Fluorescence spectra of 1 μmol/L R2S (C) and 20 μmol/L R2Z (D). All samples were measured in PBS at pH 3.47, 4.48, 5.20, 5.71, 6.10, 6.50, 6.80, 7.03, 7.44, 8.20, 9.47, 10.40 and 11.10 (A/B) or pH 3.54, 4.53, 5.35, 6.02, 6.48, 6.82, 7.36, 7.64, 8.57, 9.58, 10.11 and 11.13 (C) or pH 3.69, 4.55, 5.50, 6.20, 6.66, 6.98, 7.49, 7.80, 8.63, 9.04, 10.08 and 11.11 (D).

Fig 2 Absorption spectra and fluorescence spectra of the probes R2S and R2Z at different pH values

表1 R2S与R2Z的光学参数

Tab 1 Spectroscopic parameters of probes R2S and R2Z

Parameter	R2S/nm	R2Z/nm
λ_Abs_max （protonated）	744	760
λ_Abs_max （deprotonated）	642	662
λ_Em_max （protonated）	808	804
λ_Em_max （deprotonated）	794	790
Stokes shift （protonated）	64	44

图3 R2S与R2Z在不同pH值下的相对荧光强度Note： A. Changes in fluorescence ratio I/Imax of R2S at 750 nm excitation, 808 nm emission in PBS at the pH values of Fig. 2C. B. Changes in fluorescence ratio I/Imax of R2Z at 750 nm excitation, 804 nm emission in PBS at the pH values of Fig. 2D.

Fig 3 Relative fluorescence intensity of probes R2S and R2Z upon different pH values

图4 R2S与R2Z的pH响应可逆性和稳定性Note： A/B. Changes of absorption spectra of probes R2S (A) and R2Z (B) toward cyclically adjusted pH between 3.68 and 10.31. C. Time-dependent changes of fluorescence intensity at 805 nm of probes R2S and R2Z under UV light irradiation with a wavelength of 254 nm. D. Time-dependent changes of absorbance at 750 nm of probes R2S and R2Z under 37 ℃. E—emission intensity of probes at different time; A—absorbance of probes at different time.

Fig 4 Reversible changes toward cyclically adjusted pH and stabilities of probes R2S and R2Z

图5 R2S与R2Z对HCT-116细胞和HeLa细胞的毒性Note： A/B. Cytotoxicity of HCT-116 cells after being treated with R2S (A) and R2Z (B) at different concentrations for 24 h. C/D. Cytotoxicity of HeLa cells after being treated with R2S (C) and R2Z (D) at different concentrations for 24 h. ①P=0.007, ②P=0.009, compared with the control group.

Fig 5 Cytotoxicity evaluation in HCT-116 and HeLa cells of probes R2S and R2Z

图6 R2S与R2Z的HeLa细胞成像Note： Confocal fluorescence imaging for 10 μg/mL Hoechst (blue channel, 430?470 nm) and 10 μmol/L R2S or R2Z (red channel, 700?800 nm) in HeLa cells.

Fig 6 Imaging of probes R2S and R2Z in HeLa cells

图7 R2S与R2Z的体外与体内NIR成像Note： A. NIR images of 20 μmol/L R2S and R2Z in PBS at pH 6.00, 6.50, 7.00, 7.50 and 8.00. B/C. Radiant efficiency of R2S (B) and R2Z (C) at above pH values. D. In vivo NIR images of mice after intravenous injection of R2S and R2Z (10 μmol/L, 200 μL). E. In vivo NIR images of a mouse labeled with R2S. PBS solution (50 μL, pH 6.50) and PBS solution (50 μL, pH 7.40) were injected into two adjacent spots on the back of the mouse. Then, R2S (5 μL, 50 μmol/L) was subcutaneously injected into these two different locations. F. Radiant efficiency of the spot at pH 6.50 and spot at pH 7.40.

Fig 7 In vitro and in vivo NIR imaging of probes R2S and R2Z

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开发用于肿瘤微环境成像的pH敏感荧光探针

Development of pH-responsive fluorescent probe for tumor microenvironment imaging

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