Rapid Formation of Cell Aggregates and Spheroids Induced by a ...
Supporting Information
Rapid Formation of Cell Aggregates and Spheroids Induced by a “Smart” Boronic Acid Copolymer
Adérito J. R. Amaral, and George Pasparakis*
UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK
*
E-mail: [email protected]
S-1
Instrumentation 1
H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 MHz instrument. Chromatographic parameters of the polymer were determined using high performance size exclusion chromatography (SEC) equipped with a differential viscometer, right-angle laser-light scattering, low-angle laser-light scattering and refractive index detectors (Viscotek). The system was equipped with an on-line degasser and the chromatograms were recorded at 30 ºC. The eluent (tetrahydrofuran) was used at a flow rate of 1 mL/min. The number-average molecular weight (Mn,SEC) of the polymers was determined by triple detection calibration using OmniSEC software. Cloud point temperature was measured by using a SpectraMax M2e spectrophotometer (Molecular Devices, UK). An EVOS® FL Imaging System (Life Technologies) was used for optical and fluorescence microscopy studies.
Polymer synthesis Synthesis of PNIPAAm homopolymer. Briefly, NIPAAm (1.13 g, 10 mmol) and AIBN (16 mg, 0.1 mmol) were dissolved in a DMSO/EtOH mixture (5 mL) in a 25 mL roundbottom flask. The flask was sealed with a rubber septum and purged with argon before placing it in an oil bath at 75 ºC to initiate the polymerization. The reaction was left under magnetic stirring for 15 hours. The polymer was isolated by cooling the reaction down to room temperature followed by precipitation into excess of diethyl ether. The polymer was dried in vacuum with the use of a rotary evaporator to afford the final polymer product as a white powder (yield 74%). The same procedure was followed for the synthesis of fluorescent PNIPAAm-APBA (P1) with additional co-monomer feed of fluorescein O-methacrylate (fluorescein-MA) with the ratio PNIPAAm:APBA:fluorescein-MA 98.9:1:0.1.
Cloud point measurements The lower critical solution temperature (LCST) turbidity assay was performed by measuring the absorbance of polymer solutions in respect to temperature. P1 solutions (5 mg/mL) were prepared in phosphate buffered saline (PBS, pH 7.4, 0.01 M) and Dulbecco’s modified eagle’s medium (DMEM). The LCST was considered to be the initial onset of a sharp increase in absorbance at 500 nm.
S-2
Glucose-polymer competition assay The competition assay was carried out by preparing a series of P1–cells suspensions containing increasing amounts of glucose. The samples were examined for potential reduction of aggregate size due to APBA-glucose binding competition.
Supplementary Figures
Figure S1. 1H NMR spectrum of P1 (DMSO-d6, 400 MHz). Relative integration of the isopropyl peak (c, d) of PNIPAAm to the phenyl protons (e) of APBA gives a NIPAAm:APBA 98.3:1.7 final ratio.
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Figure S2. Fluorescence microscopy image of H9c2 CAs incubated with the fluorescent P1 after 1 h incubation period (scale bar = 400 µm).
Figure S3. Glucose competition assay (scale bars = 1 mm). The minimum glucose concentration required to totally inhibit the formation of cell aggregates is 0.1 M, which is higher than the glucose concentration that already exists in the culture medium (0.02 M). Note that free glucose at 0.05 M partially inhibits cell aggregation as shown by the small cell clusters formed (incubation time is 2 h).
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Figure S4. HepG2 cell aggregation experiments. (a-e) The microscopy images show the increase of cell density over time at 15, 30, 45, 60 min and 24 h, respectively; (f) control image is from a polymer-free cell suspension (scale bars = 1 mm).
Figure S5. Live/dead assay images of spheroids after 5 days incubation with (a) 50 µg/mL and (b) 100 µg/mL of P1, and (c) polymer-free cells (scale bars = 200 µm). (d) The percentage of dead cells found in the spheroids for different concentrations of P1 (1x104 cells per well). Data
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represented as mean ± SD from triplicates. (e) Spheroid pathophysiological gradient model showing the depletion of vital nutrients at the center of the spheroids, resulting in increased cell death.
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Rapid Formation of Cell Aggregates and Spheroids Induced by a “Smart” Boronic Acid Copolymer
Adérito J. R. Amaral, and George Pasparakis*
UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK
*
E-mail: [email protected]
S-1
Instrumentation 1
H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 MHz instrument. Chromatographic parameters of the polymer were determined using high performance size exclusion chromatography (SEC) equipped with a differential viscometer, right-angle laser-light scattering, low-angle laser-light scattering and refractive index detectors (Viscotek). The system was equipped with an on-line degasser and the chromatograms were recorded at 30 ºC. The eluent (tetrahydrofuran) was used at a flow rate of 1 mL/min. The number-average molecular weight (Mn,SEC) of the polymers was determined by triple detection calibration using OmniSEC software. Cloud point temperature was measured by using a SpectraMax M2e spectrophotometer (Molecular Devices, UK). An EVOS® FL Imaging System (Life Technologies) was used for optical and fluorescence microscopy studies.
Polymer synthesis Synthesis of PNIPAAm homopolymer. Briefly, NIPAAm (1.13 g, 10 mmol) and AIBN (16 mg, 0.1 mmol) were dissolved in a DMSO/EtOH mixture (5 mL) in a 25 mL roundbottom flask. The flask was sealed with a rubber septum and purged with argon before placing it in an oil bath at 75 ºC to initiate the polymerization. The reaction was left under magnetic stirring for 15 hours. The polymer was isolated by cooling the reaction down to room temperature followed by precipitation into excess of diethyl ether. The polymer was dried in vacuum with the use of a rotary evaporator to afford the final polymer product as a white powder (yield 74%). The same procedure was followed for the synthesis of fluorescent PNIPAAm-APBA (P1) with additional co-monomer feed of fluorescein O-methacrylate (fluorescein-MA) with the ratio PNIPAAm:APBA:fluorescein-MA 98.9:1:0.1.
Cloud point measurements The lower critical solution temperature (LCST) turbidity assay was performed by measuring the absorbance of polymer solutions in respect to temperature. P1 solutions (5 mg/mL) were prepared in phosphate buffered saline (PBS, pH 7.4, 0.01 M) and Dulbecco’s modified eagle’s medium (DMEM). The LCST was considered to be the initial onset of a sharp increase in absorbance at 500 nm.
S-2
Glucose-polymer competition assay The competition assay was carried out by preparing a series of P1–cells suspensions containing increasing amounts of glucose. The samples were examined for potential reduction of aggregate size due to APBA-glucose binding competition.
Supplementary Figures
Figure S1. 1H NMR spectrum of P1 (DMSO-d6, 400 MHz). Relative integration of the isopropyl peak (c, d) of PNIPAAm to the phenyl protons (e) of APBA gives a NIPAAm:APBA 98.3:1.7 final ratio.
S-3
Figure S2. Fluorescence microscopy image of H9c2 CAs incubated with the fluorescent P1 after 1 h incubation period (scale bar = 400 µm).
Figure S3. Glucose competition assay (scale bars = 1 mm). The minimum glucose concentration required to totally inhibit the formation of cell aggregates is 0.1 M, which is higher than the glucose concentration that already exists in the culture medium (0.02 M). Note that free glucose at 0.05 M partially inhibits cell aggregation as shown by the small cell clusters formed (incubation time is 2 h).
S-4
Figure S4. HepG2 cell aggregation experiments. (a-e) The microscopy images show the increase of cell density over time at 15, 30, 45, 60 min and 24 h, respectively; (f) control image is from a polymer-free cell suspension (scale bars = 1 mm).
Figure S5. Live/dead assay images of spheroids after 5 days incubation with (a) 50 µg/mL and (b) 100 µg/mL of P1, and (c) polymer-free cells (scale bars = 200 µm). (d) The percentage of dead cells found in the spheroids for different concentrations of P1 (1x104 cells per well). Data
S-5
represented as mean ± SD from triplicates. (e) Spheroid pathophysiological gradient model showing the depletion of vital nutrients at the center of the spheroids, resulting in increased cell death.
S-6
