Synthesis of Poly(2-(methylsulfinyl)ethyl methacrylate) via Oxidation of Poly(2-(methylthio)ethyl methacrylate): Evaluation of the Sulfoxide Side Chain on Cryopreservation

Conventional cryopreservation solutions rely on the addition of organic solvents such as DMSO or glycerol, but these do not give full recovery for all cell types, and innovative cryoprotectants may address damage pathways which these solvents do not protect against. Macromolecular cryoprotectants are emerging, but there is a need to understand their structure–property relationships and mechanisms of action. Here we synthesized and investigated the cryoprotective behavior of sulfoxide (i.e., “DMSO-like”) side-chain polymers, which have been reported to be cryoprotective using poly(ethylene glycol)-based polymers. We also wanted to determine if the polarized sulfoxide bond (S+O– character) introduces cryoprotective effects, as this has been seen for mixed-charge cryoprotective polyampholytes, whose mechanism of action is not yet understood. Poly(2-(methylsulfinyl)ethyl methacrylate) was synthesized by RAFT polymerization of 2-(methylthio)ethyl methacrylate and subsequent oxidation to sulfoxide. A corresponding N-oxide polymer was also prepared and characterized: (poly(2-(dimethylamineoxide)ethyl methacrylate). Ice recrystallization inhibition assays and differential scanning calorimetry analysis show that the sulfoxide side chains do not modulate the frozen components during cryopreservation. In cytotoxicity assays, it was found that long-term (24 h) exposure of the polymers was not tolerated by cells, but shorter (30 min) incubation times, which are relevant for cryopreservation, were tolerated. It was also observed that overoxidation to the sulfone significantly increased the cytotoxicity, and hence, these materials require a precision oxidation step to be deployed. In suspension cell cryopreservation investigations, the polysulfoxides did not increase cell recovery 24 h post-thaw. These results show that unlike hydrophilic backboned polysulfides, which can aid cryopreservation, the installation of the sulfoxide group onto a polymer does not necessarily bring cryoprotective properties, highlighting the challenges of developing and discovering macromolecular cryoprotectants.


S3
were repeated 5 times with at least 10 measurements recorded for each run. Dh values were calculated using the Stokes-Einstein equation where particles are assumed to be spherical.

Size exclusion chromatography (SEC) in DMF. Size exclusion chromatography (SEC)
analysis was performed on an Agilent Infinity II MDS instrument equipped with differential refractive index (DRI), viscometry (VS), dual angle light scatter (LS) and variable wavelength UV detectors. The system was equipped with 2 x PLgel Mixed D columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The mobile phase used was DMF (HPLC grade) containing 5 mM NH4BF4 at 50 o C at flow rate of 1.0 mL.min -1 . Poly(methyl methacrylate) (PMMA) standards (Agilent EasyVials) were used for calibration between 955,000 -550 g.mol -1 . Analyte samples were filtered through a nylon membrane with 0.22 μm pore size before injection. Number average molecular weights (Mn), weight average molecular weights (Mw) and dispersities (ĐM = Mw/Mn) were determined by conventional calibration and universal calibration using Agilent GPC/SEC software.

Size exclusion chromatography (SEC) in THF. Size exclusion chromatography (SEC)
analysis was performed on an Agilent Infinity II MDS instrument equipped with differential refractive index (DRI), viscometry (VS), dual angle light scatter (LS) and multiple wavelength UV detectors. The system was equipped with 2 x PLgel Mixed C columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The eluent was THF containing 2 % triethylamine (TEA) and 0.01 % BHT (butylated hydroxytoluene) additives. Samples were run at 1 mL.min -1 at 30 °C.
Polystyrene (PS) standards (Agilent EasyVials) were used for calibration. Analyte samples were filtered through a GVHP membrane with 0.22 μm pore size before injection. Number average molecular weights (Mn), weight average molecular weights (Mw) and dispersities (ĐM = Mw/Mn) were determined by conventional calibration and universal calibration using Agilent GPC/SEC software.

Ice Recrystallisation Inhibition Assay
A 10 µL sample of polymer dissolved in PBS buffer (pH 7.4) is dropped 1.40 m onto a glass microscope coverslip, which is on top of an aluminium plate cooled to −78 °C using dry ice.
The droplet freezes instantly upon impact with the plate, spreading out and forming a thin wafer of ice. This wafer is then placed on a liquid nitrogen cooled cryostage held at −8 °C. The wafer is then left to anneal for 30 min at −8 °C. The number of crystals in the image is counted, again using ImageJ, and the area of the field of view divided by this number of crystals to give the average crystal size per wafer, and reported as a % of area compared to PBS control.

CF Leakage from Liposome Assay
DOPC in chloroform (1.0 mL, 25 mg/mL) was evaporated to produce a thin lipid film. The film was dried for an hour under a vacuum. The resulting lipid film was suspended in a 0.1 M CF/PBS solution (1.0 mL) to be sonicated for 30 minutes. The process of freezing and thawing the aqueous solution was repeated five times. The unilamellar vesicles were obtained using a mini-extruder set (Avanti Polar Lipids) and membranes with a 0.1 µm pore size. The excess of CF dye was removed by passing the liposome though a PD-10 column (GE Healthcare). The liposome size was measured by Dynamic light scattering (DLS) ( Figure S1). The resultant liposome solution (0.15 mL) and the polymer solution (0.15 mL) were mixed and separated to three Eppendorf tubes, 0.1 mL each. One of these tubes was frozen by liquid nitrogen for 10 min and then thawed at 37 ºC for 5 min. For complete dye leakage (100% leakage), Triton X-100 (3 mg) was added to other tube. The third tube was used for the measurement of the initial fluorescence. CF fluorescence was measured with BioTek Synergy HT Microplate Reader using an excitation wavelength of 485 nm and a detection wavelength of 528 nm. Fluorescence was quenched when the vesicles were protected after the freeze-thaw process and increase in S5 fluorescence intensity represented membrane damage due to the release of CF into the PBS solution.
Hydrogen peroxide (30% w/v aqueous solution, 2.8 g, 24.7 mmol, 100 eq) was added to the resulting polymer solution, and the flask was placed into an oil bath set to 40 ºC. After 18 hours, the polymer was precipitated into acetone, dissolved in water, and dialysed (1000 Da MWCO) for 48 hours (7 water changes), followed by lyophilisation. The
After 18 hours, the polymer was precipitated into acetone, dissolved in water, and dialysed (1000 MWCO) for 48 hours (7 water changes), followed by lyophilisation. The

Fluoresceinyl)Maleimide
Poly(2-(methylsulfinyl)ethyl methacrylate) (PMSEM100) (0.3 g, 0.017 mmol, 1 eq) and N-(5fluoresceinyl)maleimide (0.002 g, 0.034 mmol, 2 eq) were dissolved in a round bottom flask using 5 mL of anhydrous DCM. A stirrer bar was added, the flask sealed with a suba seal, and the solution bubbled with nitrogen overnight. Resulted reaction solution was precipitated in S7 acetone and then redissolved in water and dialysed for 3 days (MWCO = 500 Da) (7 water changes) followed by lyophilisation resulting to the formation of an orange polymer powder.
To confirm successful conjugation of fluorescent maleimide, UV-Vis analysis was performed on polymer solution before and after conjugation ( Figure S9).
To passage cells, these were dissociated from the flask by treating them with 0.25% Trypsin in EDTA (Gibco).

Cell suspension cryopreservation
After dissociation, A549 cells were centrifuged at 2000 rpm for 5 minutes and resuspended using freezing media, which was supplemented with 10% FBS only. An aliquot of cells was taken, and this aliquot was diluted 1:1 with 0.2% trypan blue. The number of cells with intact membranes (unstained cells) were counted using a haemocytometer (Sigma Aldrich) and the cell density was adjusted to 8 x 10 5 cells mL -1 . This cell counting method is referred to in text as the trypan blue exclusion test. The polymers were dissolved in freezing media supplemented with DMSO at twice the final concentration (for example, 40 mg mL -1 in 5% DMSO for a final concentration of 20 mg mL -1 in 2.5% DMSO) and sterilised using a 0.2 µm filter (Sartorius).
Cells were then diluted 1:1 in the freezing media supplemented with DMSO and the polymers, and a total of 4 x 10 5 cells were frozen per cryovial in 1 mL. Experimental conditions were S8 assessed in triplicates. Cryovials (Sigma-Aldrich) were then placed in a CoolCell LX vial freezing container (Corning) and into a -80 °C freezer to cool at a rate of -1 °C per minute.
After 24 hours, cryovials were thawed in a water bath at 37°C for 2-3 minutes, until no ice crystals were visible. The cell suspension from each cryovial was diluted in 9mL of warm cell culture media, centrifuged at 2000 rpm for 5 minutes, resuspended using 400 µL of cell culture media and transferred into a 24 well plate (Greiner). A549 cells were incubated at 37 °C and 5% CO2. After 24 hours, cell counts were performed using the trypan blue exclusion test as previously described and cell recovery (%) was calculated by dividing the number of unstained cells obtained post-thaw by the initial number of cells frozen. To assess cell viability 24 hours post-thaw, cell culture media from the supernatant was collected before cellular dissociation.
Non-attached cells were considered as dead as they failed to attach to the plate. Cell viability was therefore calculated by dividing the number of unstained cells post-thaw by the number of stained plus non-attached cells.

Polymer cytotoxicity assay
Polymer cytotoxicity was tested by measuring the metabolic reduction of resazurin to resorufin as an indicator of cell viability after either a 30-minute or 24-hour incubation period. A549 cells were seeded at 1 x 10 4 cells per well in 100 µL of cell culture media in 96-well plates.
After 24 hours, the cell culture media was replaced with 100 µL of media supplemented with decreasing polymer concentrations from 40 to 1.25 mg mL -1 . The cells were then incubated with the polymer solutions for either 30 minutes or 24 hours at 37 °C and 5% CO2. One tablet of resazurin sodium salt (Scientific Laboratory Supplies) was diluted 1 in 10 in cell culture media. After 24 hours, cell culture media was discarded and 100 μL of the resazurin sodium salt mixture was added to all wells. Cells were incubated for 1-4 hours at 37°C and 5% CO2 and the absorbance was measured hourly using the Synergy HTX Multi-Mode Reader (BioTek) at 570 and 600 nm. Measurements were performed until the control cells reached S9 approximately 80% resazurin reduction. To calculate cell viability, the treated cells were compared to the untreated control cells and the result was expressed as a percentage.

Cellular uptake studies using fluorescent microscopy
A549 cells were seeded in a 24 well plate at 5 x 10 4 cells per well in 500µL of cell culture media. After 24 hours, cell culture media was discarded and cells were incubated for 30 minutes at room temperature with 20 mg mL -1 of the fluorescently-labelled PMSEM100 in cell culture media containing 2.5% DMSO. Wells were then washed 3 times with Dulbecco's Phosphate Buffered Saline (DPBS) and cells were stained with 100 µg mL -1 Hoechst 33342 nuclear stain (Thermo Scientific) in DPBS for 5 minutes at room temperature. Wells were washed 3 times in DPBS before imaging. Negative control wells were incubated with 2.5% DMSO for 30 minutes and stained with Hoechst 33342 only. Positive control wells were incubated with 2.5% DMSO for 30 minutes, Hoechst 33342 and 1 µg mL -1 Rhodamine 123 (Vector Laboratories) for 20 minutes at room temperature to stain the mitochondria of live cells. This dye was chosen as it fluoresces at the same channel as the fluorescently labelled polymer (green/FITC channel).
Imaging was completed with a 10x objective lens, 380-405 nm and 451-488nm excitation filters and 416-452, 502-532 emission filters using an Olympus IX83 microscope. Images were analysed and overlayed using the Olympus ScanR analysis software.

Cellular uptake studies using flow cytometry
A549 cells were seeded in a 24 well plate at 5 x 10 4 cells per well in 500µL of cell culture media. After 24 hours, media was discarded and cells were incubated for 30 minutes at room temperature with different concentrations (20 mg mL -1 to 1.25 mg mL -1 ) of the fluorescently labelled PMSEM100 in cell culture media containing 2.5% DMSO. Then, cells were washed 3 times in DPBS. Negative controls were prepared by incubating cells in cell culture media containing 2.5% DMSO for 30 minutes. Cells were dissociated from the plate by treating them S10 with 0.25% trypsin in EDTA and subsequently, trypsin was inactivated by a 1:1 dilution with cell culture media. Flow cytometry of the cell suspension was then performed in a BD Accuri C6 flow cytometer. Fluorescence was measured using the 488 nm excitation laser and 530/30 nm emission filter and 20,000 events were acquired per sample. FlowJo was used to analyse and plot flow cytometry data.

Additional Data
Additional control experiments using liposomes were undertaken as simplistic cell models. Figure S1 shows DOPC liposomes with and without addition of PMSEM. This showed that the polymers did not disrupt nor aggregate the liposomes, which was essential to allow further freeze/thaw testing (see Figure S2).   fluorescently labelled polymer (FITC channel) using an Olympus IX83 microscope. S19 Figure S11. Relative cell-associated fluorescence measured by flow cytometry. Cells were incubated for 30 minutes at room temperature with varying concentrations (20 mg mL -1 to 1.25 mg mL -1 ) of fluorescently labelled PMSEM100 in cell culture media containing 2.5% DMSO.
Cell control was incubated for 30 minutes with media containing 2.5% DMSO. Fluorescence was detected using the 488 nm excitation laser and 530/30 nm emission filter using a BD Accuri C6 Plus flow cytometer.