Thioanisole ester based logic gate cascade to control ROS-triggered micellar degradation

In certain tumor and diseased tissues, reactive oxygen species (ROS), such as H2O2, are produced in higher concentrations than in healthy cells. Drug delivery and release systems that respond selectively to the presence of ROS, while maintaining their stability in “healthy” biological conditions, have great potential as on-site therapeutics. This study presents polymer micelles with 4-(methylthio)phenyl ester functionalities as a ROS-responsive reactivity switch. Oxidation of the thioether moieties triggers ester hydrolysis, exposing a hydrophylic carboxylate and leading to micellar disassembly. At 37 °C, the micelles fall apart on a timescale of days in the presence of 2 mM H2O2 and within hours at higher concentrations of H2O2 (60–600 mM). In the same time frame, the nanocarriers show no hydrolysis in oxidant-free physiological or mildly acidic conditions. This logic gate cascade behavior represents a step forward to realize drug delivery materials capable of selective response to a biomarker input.


S1. Materials
All reagents were obtained from commercial suppliers (Sigma Aldrich, TCI Chemicals or Acros Organics) and used without further purification unless otherwise specified. Reference compounds 4-(methylthio)phenol and 4-(methylsulfonyl)phenol were purchased respectively from Sigma Aldrich and TCI. SDS of these compounds reports that chemical, physical, and toxicological properties have not been thoroughly investigated. 4-(Methylmercapto)phenol: this substance/mixture contains no components considered to be either persistent, bioaccumulative and toxic (PBT), or very persistent and very bioaccumulative (vPvB) at levels of 0.1% or higher. Air and moisture sensitive reagents were transferred via syringe. All air and/or moisture sensitive reactions were carried out in oven-dried glassware under a positive pressure of argon gas with commercially available anhydrous solvents. Petroleum ether refers to the fraction boiling in the range 40 -60 °C. Reactions were monitored by analytical thin-layer chromatography (TLC) on silica gel plates (Merck 60F254) and either visualized by UV light (254 nm) or by staining with a solution of KMnO4/K2CO3/AcOH in water followed by heating. Flash chromatography was performed on 230-400 mesh silica gel (Sigma Aldrich). 1 H NMR and 13 C NMR spectra were recorded on an Agilent-400 MR DD2 (400 MHz and 101 MHz for 1 H and 13 C, respectively) spectrometer at 298 K. Chemical shifts are reported in ppm relative to the residual solvent peak, the multiplicity is reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and J-couplings (J) are reported in Hertz (Hz). To suppress the water peak, PRESAT configuration (suppress one highest peak) was used. NMR spectra were processed by MNova NMR software (Mestrelab Research). Infrared spectra were recorded on a FT-IR Thermo Fisher Nicolet 6700 spectrophotometer and are reported in wavenumbers. GC-MS samples were analyzed using an Agilent 5977 GC/MSD equipped with a Stabilwax MS column (oven temperature: 250 °C, flow: 2.5 mL/min). ESI-MS was performed using LTQ XL spectrometer equipped with Shimadzu HPLC setup operating at 0.2 mL/min flow rate with water/MeCN mobile phase containing 0.1 vol% formic acid and Discovery C18 column. Gel permeation chromatography (GPC) was performed on a Shimadzu system equipped with a LC-20AD liquid chromatograph and a RID-10A refractive index detector. Fluorescence release was measured in 96 well plates using a micro plate reader (Biotek Synergy H1). Fluorescence spectra of Nile Red loading were recorded with a fluorescence spectrometer Spex Fluorolog-3 equipped with a standard 90° setup. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano-ZS equipped with a 4 mW laser operating at 633 nm. TEM and Cryo-EM measurements were performed on a Jeol JEM 1400 Transmission Electron Microscope with an operating voltage of 120 keV. No unexpected or unusually high safety hazards were encountered.

S2.1 Synthesis of 4-(methylsulfinyl)phenol 1
To 4-(methylthio)phenol (1.0 mmol) was added solution of 30% H2O2 (1.2 equiv., 0.04 g) and boric acid (10 mol%, 0.1 mmol, 0.006 g), and the mixture was stirred at room temperature for 30 min. The mixture was extracted with CH2Cl2 (5 × 10 mL) and the organic layers washed with brine (15 mL). The brine was extracted additional 5 times with CH2Cl2. The combined organics was dried over Na2SO4 and the solvent was removed through rotatory evaporation. The crude product was purified by flash chromatography over silica gel (methanol/ethyl acetate 2:98) and crystalized in ethyl acetate to obtain 4-

S2.2 Synthesis of 4-(methylthio)phenylacrylate
Triethylamine (Et3N) (6.27 mL, 1.50 equiv.) was added dropwise to a solution of 4-(methylthio)phenol (4.20 g, 30.0 mmol, 1.00 equiv.) and acryloyl chloride (3.64 mL, 1.50 equiv.) in dry CH2Cl2 at 0 °C and stirred overnight for 16 hours, slowly increasing the temperature to 20 °C. The reaction mixture was S4 diluted with CH2Cl2 (250 mL) and washed with water (500 mL) and brine (500 mL). The combined organic layers were dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography over silica gel (ethyl acetate/petroleum ether 1:9 to 1:4) to afford 4-(methylthio)phenylacrylate (MTPA) (4.30 g, 22.1 mmol, 74% yield) as a light yellow oil.  Table S1. N,N-dimethylacrylamide (DMA), which was filtered over basic alumina prior to use, and 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) were dissolved in DMF and degassed for 15 minutes by bubbling argon gas. The resulting solution was stirred in a light reactor (444 nm), samples were taken regularly to follow the conversion with 1 H NMR. The reaction was stopped after 10 hours when the desired conversion was obtained. The reaction mixture was diluted with DMF (10.0 mL) and precipitated three times in diethyl ether (500 mL). The precipitated product was dried in a vacuum oven at 50 °C for three hours to afford p(DMA102) macroDDMAT (2.46 g) and p(DMA130) macroDDMAT (3.37 g) as yellow solids.

S2.4 Synthesis of p(DMAn-b-MTPAm)
Specific conditions and data are shown in Table S2. Macro chain transfer agent p(DMAn) macro-DDMAT and 4-(methylthio)phenyl acrylate were dissolved in DMF (1.0 mL) and degassed for 15 minutes by bubbling argon gas. The resulting solution was stirred in a light reactor (444 nm) for the given time. The reaction mixture was diluted with DMF (5.00 mL) and precipitated three times in diethyl ether (300 mL). The precipitated product was dried in a vacuum oven at 50 °C for three hours to afford p(DMA102-b-MTPA32) (0.54 g) and p(DMA130-b-MTPA16) (1.13 g) as white solids.

S3. Polymer Characterization
For all the polymers, 1 H NMR spectra were recorded in CDCl3, and the molecular weight was measured through gel permeation chromatography (GPC) in DMF. p(DMAn-b-MTPAm) structures were derived by 1 H NMR as reported below. Polymerization conversion (ρ) was calculated by monitoring reduction in the 1 H NMR integrals of the monomer unsaturated protons (∫M: 5.60 -6.80 ppm for DMA, 6.12 -6.55 ppm for MTPA) and aromatic protons in case of MTPA (7.32 ppm) relative to the proton (7.95 ppm) of the reaction solvent DMF. The 1 H NMR spectra to follow the polymerization conversion were taken in DMSO-d6. In the case of a copolymerization with both DMA and MTPA the conversion of both monomers was calculated according Equation S1.

Equation S1
For a polymerization containing z monomers, Mn,conv was calculated according to Equation S2

S3.2 GPC data of the block copolymers
The average molecular weight and dispersity Ð (Mw/Mn) of the synthesized polymers was measured using a Shimadzu GPC with DMF LiBr (25 mM) as eluent. The system was equipped with a Shimadzu CTO-20AC Column oven, a Shimadzu RID-10A refractive index detector, a Shimadzu SPD-20A UV-Vis detector, PL gel guard column (MIXED, 5 µm), 50 mm x 7.5 mm, and 1× Agilent PLGel (MIXED, 5 µm), 300 mm × 7.5 mm, providing an effective molar mass range of 200 to 2 x 10 6 g/mol. DMF LiBr (25 mM) was used as an eluent with a flow rate of 1.0 mL/min at 50 °C. The GPC columns were calibrated with low dispersity PMMA standards (Sigma Aldrich) ranging from 800 to 2.2 x 10 6 g/mol, and molar masses are reported as PMMA equivalents. A 3rd-order polynomial was used to fit the log Mp vs. time calibration curve for both systems, which was near linear across the molar mass ranges.

S4.2 DLS measurements of the polymeric micelles before and after H2O2 treatment
To 1.0 mL of a 1.0 mg/mL micellar dispersion of p(DMAm-b-MTPAn) prepared as previously described were added 66 µL of stock solutions of hydrogen peroxide in PB with variable concentration to yield a final H2O2 concentration of 0.0, 0.007, 0.2, 2.0 wt%. The size distribution and the scattering intensity at 37°C were followed by DLS as a function of time. The curves are drawn for the different data sets as a guide for the eye.

S4.4 Cryo-EM images of the polymeric micelles before and after H2O2 treatment
To two vials was added 2.0 mL each of a 1.0 mg/mL micellar dispersion of p(DMAm-b-MTPAn) prepared as previously described. To one of these vials, 132 μL of a stock solution of H2O2 in PB (100 mM, pH = 7.4) was added to reach final H2O2 concentration of 0.2 wt% to take Cryo-EM pictures after 24 hours. To the second vial, 132 μL of phosphate buffer (100 mM, pH = 7.4) was added to obtain the sample without H2O2. The samples were centrifuged (4000 rpm for 15 minutes) using 10 kDa filters and concentrated to 20 mg/mL afterwards. The concentrated in 100 μL volume was washed with additional 100 μL H2O, obtaining a final concentration of 10 mg/mL. Cryo-TEM images were obtained by adding 4 μL of the 10 mg/mL micellar solution onto a Quantifoil 1.2/1.3 200 mesh Cu grid. The drop was blotted for four seconds with filter paper to obtain a thin layer on the grid, and vitrified by rapid immersion in liquid ethane (Leica EM GP version 16222032). The grid was finally inserted into a cryo-holder (Gatan model 626) and then transferred to the Jeol JEM 1400 TEM. For the statistical analysis of the micelles diameters, about 20 images were made of each of the samples. Cryo-EM images were analyzed manually using ImageJ.

Figure S6
Cryo-EM images and particle analysis of PM16 micelles A) Normalized frequency distribution based on Cryo-EM images analysis of PM16 micelles. B) Cryo-EM image (Scale bar = 100 nm) of PM16 micelles.

Figure S7
Cryo-EM images and particle analysis of PM32 micelles before and 24 h after the addition of 0.2 wt% H2O2. A) Normalized frequency distribution based on Cryo-EM images analysis of PM32 micelles. B) Normalized frequency distribution based on Cryo-EM images analysis of PM32 micelles 24 h after the addition of 0.2 wt% H2O2. C) Cryo-EM image (Scale bar = 100 nm) of PM32 micelles. D) Cryo-EM image (Scale bar = 100 nm) of PM32 micelles 24 h after the addition of 0.2 wt% H2O2.

S5.1 1 H NMR study of p(DMAn-b-MTPAm) micelles before and after H2O2 treatment
To 0.5 ml of p(DMAm-b-MTPAn) micelles (8.0 mg/mL) in a NMR tube was added 55 μL D2O and 33 μL H2O2 (30 wt%). NMR tubes were kept at 37 °C during all the experiments. The first 1 H NMR spectrum was taken right after the addition of H2O2 (t=0) and, subsequently, a measurement is taken every hour until no change in conversion was detected. The conversion (%) of 1 and 2 was measured calculating the percentage of the integral of the respective aromatic peaks at 7.64 and 7.77 ppm for each time point against the total integral value obtained at the end of the acquisitions. The peak between 2.87 and 3.24 ppm corresponding to the protons of p(DMAn) was used as reference.

S5.2 1 H NMR study of p(DMAn-b-MTPAm) micelles at different pH
p(DMAm-b-MTPAn) micelles (8.0 mg/mL) were prepared separately in PB (100 mM) with different pH: 5.0, 6.0, 7.4. From each micellar dispersion, 0.5 mL were added in a NMR tube together with 55 μL D2O and 33 μL of the appropriate phosphate buffer at 37 °C. 1 H NMR measurements were taken every 24 h for 6 days. The conversion (%) of 1 was measured calculating the percentage of the difference between the integral of the peak in the aromatic region at 7.64 ppm for each time point and the integral of the same region at t=0, divided by the expected integral for the complete removal of 1. The peak between 2.87 and 3.24 ppm corresponding to the protons of p(DMAn) was used as reference.

S6.1 Determination of micelle loading with Nile Red
A Nile Red solution in THF (20 µL, 1.0 mg/mL) was added to the previously prepared micellar dispersions of p(DMAm-b-MTPAn) (1.0 mL, 1.0 mg/mL) and incubated in the dark in an open vial to evaporate the organic solvent. The nonencapsulated payload was removed through centrifugation (5000 rpm, 10 minutes) and 900 µL DMF were added to 100 µL of the Nile Red-loaded micellar dispersions. The fluorescence of the solution was measured at an excitation wavelength of 540 ± 20 nm and emission wavelength 620 ± 30 nm and compared to the calibration curve of known concentrations Nile Red in PB (100 mM, pH = 7.4)/ DMF 1:9 ( Figure S9), to determine the Nile Red loading per mg of polymer. Drug loading (DL) and encapsulation efficiency (EE) were calculated as follows (Equations S3 and S4, respectively).

S6.2 H2O2-triggered release of Nile Red from p(DMAn-b-MTPAm) micelles
A Nile Red solution in THF (20 µL, 1.0 mg/mL) was added to the previously prepared micellar dispersions (1.0 mL, 1.0 mg/mL) and incubated in the dark in an open vial to evaporate the organic solvent. The micellar dispersions were divided over three vials for each polymer and hydrogen peroxide solution was added to a final concentration of 0, 0.2 and 2.0 wt% for each series. The fluorescence of the solution was measured on a Synergy H1 (Biotek) microplate reader at 37 °C, using an excitation wavelength of 540 ± 20 nm and emission wavelength 620 ± 30 nm. The release percentage for each sample at specific time point was determined by subtracting the fluorescent value (Ft) from that of the sample before the addition of H2O2 (Ft0), and the percent fluorescence remaining was determined by normalization to the same value (Ft0).

S7. Cell viability assay on p(DMAn-b-MTPAm) micelles
HeLa cells in DMEM culture medium supplemented with 10% fetal bovine serum (Gibco, life technologies TM ) and 1% Penicillin/Streptomycin (100x, Biowest) under humidified normoxic (95% air, 5% CO2) were plated at 2000 cells/well (suspended in 200 μL cell culture medium) in a 96-well plate and incubated at 37 °C. After 3 days, 20 μL of both PM16 and PM32 micelles (0.0-11 mg/mL) in PBS (phosphate buffer saline, pH = 7.4) was added to each well, to reach final micelles concentrations in the range (0.0-1000 μg /mL). After 24 hours, the micellar solutions were removed, the cells were washed with PBS for three times, and 200 μL of fresh culture medium was added. The cells have been allowed to grow for an additional 3 days, then their cytotoxicity was evaluated using the WST-8 assay Dojindo Laboratories,. For this test, 10 μL of CCK-8 reagent was added to each well and incubated for 3 hours, then the absorbance at 450 nm was measured using a microplate scanning spectrophotometer (PowerWave XSTM, Bio-Tek). The surviving fraction (SF) of the Hela Cells was calculated according equation S5.