Development of pH-responsive fluorescent probe for tumor microenvironment imaging

doi:10.3969/j.issn.1674-8115.2022.07.005

Abstract

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

CLC Number:

R313

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.

Figures/Tables 8

Fig 1 Synthetic routes of the target probes R2S and R2Z

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

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

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

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

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

Fig 6 Imaging of probes R2S and R2Z in HeLa cells

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

TrendMD

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