Protein encapsulation: a new approach for improving the capability of small-molecule fluorogenic probes

Abstract

Herein, we report a protein-based hybridization strategy that exploits the host-guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO^- fluorescent probe Pinkment-OAc. Formation of a HSA/Pinkment-OAc supramolecular hybrid was confirmed by SAXS and solution-state analyses. This HSA/Pinkment-OAc hybrid provided an enhanced fluorescence response towards ONOO^- versus Pinkment-OAc alone, as determined by in vitro experiments. The HSA/Pinkment-OAc hybrid was also evaluated in RAW 264.7 macrophages and HeLa cancer cell lines, which displayed an enhanced cell permeability enabling the detection of SIN-1 and LPS generated ONOO^- and the in vivo imaging of acute inflammation in LPS-treated mice. A remarkable 5.6 fold (RAW 264.7), 8.7-fold (HeLa) and 2.7-fold increased response was seen relative to Pinkment-OAc alone at the cellular level and in vivo, respectively. We anticipate that HSA/fluorescent probe hybrids will soon become ubiquitous and routinely applied to overcome solubility issues associated with hydrophobic fluorescent imaging agents designed to detect disease related biomarkers.

Scheme 1. (A) Reaction mechanism of Pinkment-OAc (R = Ac) and Pinkment-OH (R = H) in the presence of ONOO⁻. (B) Human serum albumin (HSA)-encapsulation of Pinkment enabling the detection of ONOO⁻in vitro and in vivo.

Fig. 1. Fluorescence spectra of (A) Pinkment-OAc (5 μM) and (B) HSA/Pinkment-OAc (5/5 μM) with the addition of ONOO⁻ (0–10 μM); phosphate buffered saline – PBS (pH 7.4), λ_ex = 545 nm. (C) Comparison of the fluorescence intensities of Pinkment-OAc (5 μM) and HSA/Pinkment-OAc (5/5 μM) with the addition of ONOO⁻ (0–10 μM). Fluorescence spectra of (D) Pinkment-OH (5 μM) and (E) HSA/Pinkment-OH (5/5 μM) with the addition of ONOO⁻ (0–10 μM); phosphate buffered saline – PBS (pH 7.4), λ_ex = 545 nm. (F) Comparison of the fluorescence intensities of Pinkment-OH (5 μM) and HSA/Pinkment-OH (5/5 μM) with the addition of ONOO⁻ (0–10 μM). Error bars represent S. D. (n = 3).

Fig. 2. X-Ray scattering patterns and molecular docking of Pinkment-OAc to HSA. (A) X-ray scattering pattern of HSA. (B) Fitting of atomic models of the hybrid (model refined with scattering data, top), HSA (model refined with scattering data, middle), and the crystal structure of HSA (PDB id 1n5u; bottom). (C) HSA crystal structure superimposed on an atomic model of HSA/Pinkment-OAc (w/w = 1 : 10) (NMA simulation; crystal structure used to fit the data: PDB id 1n5u). (D) Chemical structure of Pinkment-OAc and its reaction with ONOO⁻. (E) X-ray scattering pattern of HSA/Pinkment-OAc (w/w = 1 : 10). (F) Interatomic distance distribution function, P(r), of the X-ray scattering patterns of HSA and HSA/Pinkment-OAc (w/w = 1 : 10). (G) HSA crystal structure superimposed with an atomic model of HSA (NMA simulation; crystal structure used to fit the data: PDB id 1n5u). (H) Proposed binding of Pinkment-OAc and resorufin to HSA showing interaction mode between Pinkment-OAc, resorufin, and selected amino acid residues of HSA.

Fig. 3. Fluorescence imaging experiments. (A) Confocal images and fluorescence quantification of (B) RAW 264.7 and (C) HeLa cells treated with Pinkment-OAc (20 μM, 1% DMSO in PBS) or HSA/Pinkment-OAc (20/20 μM, 1% DMSO in PBS) with or without added SIN-1 (500 μM). The excitation and emission wavelengths for Pinkment-OAc are 559 nm and 580–650 nm, respectively. The cell nuclei were stained with Hoechst 33342. ****P < 0.0001, ***P < 0.001. Error bars represent S. D. (n = 3).

Fig. 4. Fluorescence imaging experiments. (A) Confocal images and (B) fluorescence quantification of RAW 264.7 cells treated with Pinkment-OAc (20 μM, 1% DMSO in PBS) or HSA/Pinkment-OAc (20/20 μM, 1% DMSO in PBS) with or without added LPS (lipopolysaccharide, 1 μg mL⁻¹). The excitation and emission wavelengths for Pinkment-OAc are 559 nm and 580–650 nm, respectively. The cell nuclei were stained with Hoechst 33342. ***P < 0.001. Error bars represent S. D. (n = 3).

Fig. 5. Demonstrating the effectiveness of the HSA/Pinkment-OAc hybrid for the in vivo imaging of ONOO⁻. (A) Fluorescence images and (B) quantification of C57BL/6J mice treated with Pinkment-OAc (100 μL, Pinkment-OAc = 200 μM in saline) or HSA/Pinkment-OAc (100 μL, HSA/Pinkment-OAc = 200/200 μM in saline) in the absence and presence of LPS (200 μL, 2 mg mL⁻¹ in saline). **P < 0.01, ***P < 0.001. Error bars represent S. D. (n = 3).