Visualizing semipermeability of the cell membrane using a pH-responsive ratiometric AIEgen

Abstract

In clinical chemotherapy, some basic drugs cannot enter the hydrophobic cell membrane because of ionization in the acidic tumor microenvironment, a phenomenon known as ion trapping. In this study, we developed a method to visualize this ion trapping phenomenon by utilizing a pH-responsive ratiometric AIEgen, dihydro berberine (dhBBR). By observing the intracellular fluorescence of dhBBR, we found that non-ionized dhBBR can enter cells more easily than ionized forms, which is in accordance with the concept of ion trapping. In addition, dhBBR shows superior anti-photobleaching ability to Curcumin thanks to its AIE properties. These results suggest that dhBBR can serve as a bioprobe for ion trapping.

Fig. 1. (A) Synthetic route to dhBBR. (B) Quantum yield of dhBBR in DMSO solution (10 μM) and single crystal states. Excitation wavelength: 365 nm. (C) Time-resolved emission decay curves of dhBBR in DMSO solution and single crystal states. Solution concentration: 10 μM; excitation wavelength: 365 nm.

Fig. 2. Single crystal packing of dhBBR. (A) Intramolecular torsional angle. (B) Intermolecular distance of adjacent molecules. (C) Intermolecular interactions.

Fig. 3. (A) Schematic illustration of dhBBR's fluorescent response to pH change. (B) Emission spectra of dhBBR in PBS buffer solutions with different pH values. [dhBBR] = 10 μM. (C) Plot of I₅₁₁/I₄₅₄versus pH. I₅₁₁ and I₄₅₄ denote the emission intensities of the solution at 511 and 454 nm, respectively. Excitation wavelength: 365 nm. (D) Fluorescence reversibility of dhBBR in PBS buffer between pH 2.0 and pH 12.0. [dhBBR] = 10 μM. (E) Ratiometric fluorescent responses of dhBBR (10 μM) to different potential interfering agents in pH 3.0 (red column) and 10.0 (black column) PBS buffer solutions: (0) control; (1) Na⁺; (2) K⁺; (3) Ca²⁺; (4) Mg²⁺; (5) Fe²⁺; (6) Cu²⁺; (7) Zn²⁺; (8) Mn²⁺; (9) Al³⁺; (10) Pb²⁺; (11) Ac^–; (12) SO₄^2–; (13) NO₃^–; (14) PO₄^2–; (15) S₂O₃^2–; (16) CO₃^2–; (17) Phe; (18) His; (19) Met; (20) Pro; (21) Arg; (22) Asp; (23) Cys; (24) Hcy; (25) GSH; (26) glucose; (27) H₂O₂; (28) NaClO. Conditions: λ_ex = 365 nm.

Fig. 4. CLSM images of A549 cells (A) and HEK 293T cells (C) stained with dhBBR (1 μM) in PBS buffer with different pH for 30 min. (B and D) Relative PL intensity of dhBBR treated A549 cells (B) and HEK 293T cells (D). Scale bar = 10 μm.

Fig. 5. (A) Photostability of dhBBR and Curcumin under continuous scanning at 405 nm. I₀ is the initial PL intensity, while I is that of the corresponding sample after a designated time of irradiation. (B and C) CLSM images of HeLa cells stained with (B) dhBBR (1 μM) and (C) Curcumin (1 μM) before and after 200 s of light irradiation. All the images share the same scale bar: 10 μm.