Boronate ester cross-linked PVA hydrogels for the capture and H2O2-mediated release of active fluorophores

A new set of H2O2-responsive PVA hydrogels were formed using the boronate fluorescent probe PF1 and the novel boronate fluorescent probe PT1 as the covalent crosslinkers.


General Information
All solvents and reagents were purchased from commercial suppliers and used without further purification unless otherwise noted. Analytical thin-layer chromatography (TLC) was performed using commercial precoated silica gel plates with an aluminum backing. Column chromatography was carried out using silica gel (0.040-0.063 mm). Proton and carbon NMR spectra were recorded at 298K using a Bruker Advance 500 spectrometer. Chemical shifts are reported in ppm using TMS or solvent residual signals as the internal reference standards. Highresolution mass spectrometry (HRMS) analyses were performed on an Agilent 6545 LC/Q-TOF instrument. UV-Vis spectra were recorded from 220 nm to 800 nm using a BMG Labtech CLARIOstar plate reader at room temperature using Costar U bottom 96 well microplates. Fluorescence spectra were recorded on a BMG Labtech CLARIOstar at room temperature using Greiner Bio-One microplates (Black-walled, flat bottom (chimney well), polystyrene 96-well plates).

Gel Synthesis (Pment and Gment)
PVA (mw 13,000 -23,000 kDa; purchased from Sigma Aldrich) was dissolved in DMSO to form a 10% w/v solution. Solutions of PF1 and PT1 were prepared at 100 mM in DMSO. Aliquots of the PVA solution (0.5 mL) were combined in a vial with either PF1 or PT1 solutions (0.5 mL). These solutions were stirred and heated with a heat gun, inducing gelation within 30 s. The gels were then heated at 60 °C overnight in an oven. The gels were subsequently washed with petroleum ether (5 mL) twice, and PBS (5 mL, pH 7.4) twice and then stored in PBS until used. These materials, based on PF1 or PT1, are referred to as Gment and Pment, respectively, as noted in the main text.

Stability studies of Gment and Pment
Hydrogels with mass 200 mg (± 10 mg) were placed in 1 mL of aqueous PBS (pH 7.4) contained in a 25 mL scintillation vial. A 100 µL aliquot of this solution was removed every day for 7 days and its UV-Vis spectrum was recorded to ensure there was no change. The 100 µL aliquot was replaced with fresh PBS to maintain a constant volume within the vial. Results indicated stability in this aqueous medium for over 7 days.

S3
Cell proliferation studies A549 cells (ATCC ® CCL-185 ™ ) were harvested and seeded into 96-well culture plates  in 100 µL of culture media. They were allowed to incubate overnight at 37 o C in the presence of 5% CO 2 . A549 cells were seeded at a density of 1500 cells/well. The next day, appropriate serial dilutions of drug stocks in culture media were made. To each well of a 96 well plate was added 100 µL of the appropriate solution. After a total of three days, a 50 mL aliquot of 3 mg/mL tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Alfa Aesar L11939) was added to each well, followed by a 4 h incubation period at 37 o C. After removal of the medium, the resulting formazan was dissolved in 50 mL DMSO and the respective absorbances were measured at 560-650 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). Absorbance values were corrected for background and then normalized to wells containing untreated cells to allow for plate-to-plate comparisons. Resulting dose response curves were subjected to linear regression analysis (Origin by OriginLab, Inc.) for determination of IC 50 values. The data are shown as mean inhibition of proliferation or growth as a percentage of control cells and are from 2-3 replicate experiments

Limit of detection
The limit of detection was calculated using the formula shown below: Limit of detection (LOD) = 3σ/slope σ = Standard deviation of the lowest concentration Using the graph shown below, the limit of detection for Gment for the detection of H 2 O 2 at 5 minutes was calculated: