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

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−versusPinkment-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.


General methods
General remarks. Chemicals were used as received unless otherwise indicated. All other reagents are of analytical purity and used without further treatment. Solvents used are of analytical grade. UV-absorbance spectra were carried out on a Varian Cary 500 spectrophotometer. Fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer. Isothermal titration calorimetric (ITC) measurement was performed with Affinity ITC. The fluorescence cell images were recorded by an Operetta high content imaging system (Perkinelmer, US) or a confocal laser scanning microscopy (Olympus, Japan; Leica TCS SP8, Germany).

Preparation of HSA/probe hybrids.
Stock solution of Pinkment-OAc and Pinkment-OH (1 mM) was prepared in DMSO. Stock solution of HSA (0.5 mM) was prepared in phosphate buffered saline (PBS, 0.01 M, pH 7.4). HSA/Pinkment-OAc and HSA/Pinkment-OH (5/5 μM) hybrid solution was prepared by simply mixing the probe with different concentrations of HSA, and the resulting mixture was incubated for 5 min to allow for sufficient host-guest binding.

Fluorescence microscopy.
The fluorescence measurements were carried out with a path length of 5 mm and an excitation wavelength of 545 nm by scanning the spectra between 545 nm and 800 nm. The bandwidth for both excitation and emission spectra was 5 nm. Unless otherwise mentioned, all the spectra were recorded at 25 °C.

Small Angle X-ray Scattering (SAXS) experiment
Small-angle X-ray scattering (SAXS) experiments were performed at beamline BL19U2 of the National Facility for Protein Science Shanghai (NFPS) at the Shanghai Synchrotron Radiation Facility (SSRF). The wavelength, , of the X-ray radiation was set at 1.033 Å. Scattered X-ray intensities were collected using a Pilatus 1M detector (DECTRIS Ltd). The sample-to-detector distance was set such that the detecting range of momentum transfer [q = 4 sin/, where 2 is the scattering angle] of the SAXS experiments was 0.01-0.40 Å -1 . To reduce the radiation damage, a flow cell made of a cylindrical quartz capillary with a diameter of 1.5 mm and a wall thickness of 10 µm was used. The protein HSA for the SAXS measurement was prepared at 10 μM in PBS (10 mM, pH 7.4) in the presence or absence of 10 molar equivalents of Pinkment-OAc. The scattering profiles for a matched buffer were subtracted from that of the protein. For each measurement, the SAXS data were collected as 20 x 1 sec exposures. The 2-D scattering images were converted to 1-D SAXS curves through azimuthally averaging after applying a solid angle correction and then normalizing with the intensity of the transmitted X-ray beam, using the software package BioXTAS RAW. 1 Background scattering was subtracted using the PRIMUS program in the ATSAS software package. 2 The conformational change of the hybrid site was observed using the SREFLEX program within the ATSAS software package. 3 The SUPCOMB program in the ATSAS software package was used to superimpose a three-dimensional SAXS structure onto a literature HSA atomic structure as detailed in the main text. 4

Protein and ligand preparation
The HSA-drug complex crystal structure 2BXI (2.5 Å) obtained from the RCSB PDB (Protein Data Bank) 5 was selected because the ligand azapropazone molecule (AZQ) in that structure is similar to Pinkment-OAc. Protein preparation was performed with the Protein Preparation Wizard panel in Maestro (Schrödinger, LLC, New York, NY, USA). 6 The HSA structure 2BXI was prepared by assigning charges and bond order, adding missing hydrogen atoms and disulfide bonds, and removing waters with fewer than 3 H-bonds, followed by structure minimization. Subsequently, Pinkment-OAc and Resorufin were prepared using the LigPrep module in Maestro package and the OPLS_2005 force field under a target pH of 7.0 ± 2.0.

Induced fit docking
The center of a docking box was defined according to the centroid of the workspace ligand AZQ, followed by grid generation using a 15 Å enclosing box. Molecular docking simulations were performed using the Induced Fit Docking (IFD) protocol in the Maestro package. 7 The IFD approach allows for dockings of a flexible ligand with flexible side chains of a protein (the backbone residues of the protein were kept rigid). As a result, twenty binding poses ranked according to the Glide score produced, from which the poses with docking score values of -8.786 and -9.704 were selected as the best binding arrangements of Pinkment-OAc and resorufin, respectively. PyMOL viewer (http://www.pymol.org/) was used to visualize binding poses and to generate molecular images.
Cell culture RAW264.7 and HeLa cells were maintained in a Dulbecco's Modified Eagle's Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Gland Island, NY, USA) in a humidified atmosphere of 5% CO 2 and 95% air at 37 o C and split when the cells reached 90% confluency.

High-content fluorescence imaging
Cells were seeded on a black 96-well microplate with an optically clear bottom (Greiner bioone, Germany) overnight. The cells were incubated with Pinkment-OAc (20 μM) or HSA/Pinkment-OAc (20/20 μM) for 30 min, followed by incubation with SIN-1 (500 μM) for 30 min. Then, cells were washed with PBS (phosphate buffered saline) three times. The fluorescence images were recorded using an Operetta high content imaging system (Perkin Elmer, USA) at an excitation wavelength of 520-550 nm and an emission wavelength of 580-650 nm. Data were quantified and plotted using the manufacturer's Columbus Analysis system (Perkin Elmer, USA).

Confocal laser scanning microscopy
Cells cultured in growth medium supplemented with 10% fetal bovine serum were plated onto 24-well microplates. Cells were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37°C overnight. Then, cells were incubated with Pinkment-OAc (20 μM) or HSA/Pinkment-OAc (20/20 μM) for 30 min, followed by incubation with SIN-1 (500 μM) for 30 min. The cells were then rinsed with warm PBS and fixed using 4% paraformaldehyde for 20 min. The cell nuclei were stained with Hoechst 33342 (5 μg mL -1 ) at room temperature for 5 min. After three rinses with PBS, the fluorescence was detected and photographed using a confocal laser scanning microscopy (Olympus, Japan; Leica TCS SP8, Germany).

Cell viability assay
Cells were plated on 96-well plates in growth medium. After 24 h, cells were treated with Pinkment-OAc and HSA/Pinkment-OAc at different concentrations for 24 h. Then, the cell viabilities were determined through a standard MTS cell proliferation assay.
In Vivo Imaging C57BL/6J mice were divided into two groups, the first group was given an intraperitoneal (i.p.) injection of saline (200 μL) as a control, the second group was injected i.p. with lipopolysaccharide (200 μL, 2 mg mL -1 in saline, Sigma-Aldrich, USA) for 4 h. The mice were anesthetized and the abdominal fur was removed using a razor. They were then injected i.p. with DMSO, Pinkment-OAc (100 μL, 200 μM in saline), or HSA/Pinkment-OAc (100 μL, 200/200 μM, in saline). Whole body images were acquired within 30 min using an IVIS Spectrum imaging system (Perkin Elmer, USA). The imaging parameters were set as follows: Ex. 550 nm, Em. 580-620 nm.