Supporting Information

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1 Supporting Information 2-(Methylthio)ethyl Methacrylate: A Versatile Monomer for Stimuli Responsiveness and Polymerization-Induced Self-Assembly In The Presence Of Air Sihao Xu, a Gervase Ng, a Jiangtao Xu, a Rhiannon P. Kuchel, b Jonathan Yeow, a and Cyrille Boyer a [a] Centre for Advanced Macromolecular Design and Australian Centre for NanoMedicine, School of Chemical Engineering, The University of New South Wales, Sydney NSW 2052 (Australia) [b] Electron Microscope Unit, Mark Wainwright Analytical Centre, The University of New South Wales, Sydney NSW 2052 (Australia) EXPERIMENTAL SECTION Materials 2-(methylthio)ethyl methacrylate (MTEMA, Sigma-Aldrich) was used as received. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA) (M n = 300 g mol -1 ) (Sigma-Aldrich), 5,10,15,20- tetraphenyl -21H,23H-porphine (ZnTPP, Sigma-Aldrich), 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CDTPA, Boron Molecular) and all the other reagents were used as received unless otherwise specified. 2,2 -azobis(isobutyronitrile) (AIBN, Fluka, 98%) was purified by recrystallization from methanol. Methyl methacrylate (MMA, Sigma-Aldrich) was deinhibited by passing through a column of basic alumina. Instrumentation All 1 H-NMR spectra were recorded using a Bruker 300 or 400 MHz spectrometer. All chemical shifts are S1

2 reported in ppm (δ) relative to tetramethylsilane, referenced to the chemical shifts of residual solvent resonances. The molecular weight and dispersity of the prepared polymers were measured by GPC. The eluent was DMAc (containing 0.03% w/v LiBr and 0.05% w/v 2,6-dibutyl-4-methylphenol (BHT)) at 50 o C (flow rate of 1 ml/min) with a Shimadzu modular system comprising an SIL-10AD auto-injector, a Polymer Laboratories 5.0 µm bead-size guard column ( mm 2 ) followed by four linear PL (Styragel) columns (105, 104, 103, and 500 Å) and an RID-10A differential refractive-index (RI) detector and UV-Vis detector. The calibration of the system was based on narrow molecular weight distribution of polystyrene standards with molecular weights of g mol 1. DLS measurements were performed using a Malvern Zetasizer Nano Series running DTS software and using a 4 mw He Ne laser operating at a wavelength of 633 nm and an avalanche photodiode (APD) detector. The scattered light was detected at an angle of 173. TEM studies of self-assembled block copolymers were conducted using a Transmission Electron Microscope at an accelerating voltage of either 100 kv (JEOL-1400) or 200 kv (FEI Tecnai G2 20). The polymerization dispersions were diluted with methanol (or water for aqueous dispersions) to give wt% dispersions and deposited onto carbon-coated copper grids. Phosphotungstic acid staining was applied to all samples. Online Fourier Transform near-infrared (FTNIR) spectroscopy was used to determine monomer conversions by following the decrease of the vinylic C H stretching overtone of the monomer at 6100 cm -1. A Bruker Vertex 70 Fourier transform spectrometer equipped with a CaF 2 beam splitter, and room temperature DLaTGS detector was used. Spectra were analyzed with OPUS software (Version 7.5). Photopolymerization reactions were carried out under RS Component PACK LAMP RGB LED lights (5 W, λ max = 635 nm (red)) as shown below. The distance of the samples to the light source was 10 cm. The light intensity was measured using a light meter Newport Power Meter Model 843-R. The RGB multi-colored LED light bulb with remote control was purchased from RS Components Australia. S2

3 Typical photopolymerization setup used in this study. The visible light initiated oxidation of POEGMA-b-PMTEMA nanoparticles was performed using a VeraSol LED solar simulator consisting of a LSS-7120 Oriel VeraSol LED controller and LSH-7520 LED head. UV-Vis absorption spectra were recorded using a CARY 300 spectrophotometer (Varian) equipped with a temperature controller. Kinetic studies of PET-RAFT polymerization of MTEMA For FTNIR kinetic studies of MTEMA, a typical polymerization mixture ([MTEMA]:[CDTPA]:[ZnTPP] = 200:1:0.01) was set up as follows: ZnTPP (93.62 µg, mol, µl of a 1 mg/ml tetrahydrofuran (THF) stock solution) was added to a 21 ml glass vial. After evaporating the residual THF under a stream of N 2, CDTPA (5.57 mg, mol), MTEMA ( mg, mol) and DMF (0.474 ml) ([M] = 50 wt%) were further added to the vial. 900 µl of the reaction mixture was transferred to an 0.9 ml FT-NIR quartz cuvette (1 cm 2mm) which was sealed with a rubber septum and parafilm. For deoxygenated experiments only, N 2 was bubbled through the reaction mixture for 15 min at 0 o C before sealing with vacuum grease. The sealed cuvette was then irradiated with red LED light (λ max = 635 nm, 3.0 mw/cm 2 ) at room temperature. At predetermined time points, the cuvette was transferred from the reactor to the sample holder for FT-NIR measurement. The monomer conversions were calculated using the ratio of the integral of the wavelength region cm -1 at different time points relative to the integral of the initial polymerization mixture (0 % monomer conversion). At regular intervals, a degassed syringe was used to extract ~50 µl aliquots for GPC and NMR analysis. S3

4 Kinetic studies of PET-RAFT polymerization of MMA For FTNIR kinetic studies of MMA, a typical polymerization mixture ([MMA]:[CDTPA]:[ZnTPP] = 200:1:0.01) was set up as follows. ZnTPP (110.3 µg, mol, µl of a 1 mg/ml tetrahydrofuran (THF) stock solution) was added to a 21 ml glass vial. After evaporating the residual THF under a stream of N 2, CDTPA (6.57 mg, mol), MMA ( mg, mol) and ml DMF ([M] = 50 w/w%) were further added to the vial. 900 µl of the reaction mixture was transferred to an 0.9 ml FTNIR quartz cuvette (1 cm 2 mm) which was sealed with a rubber septum and parafilm. For deoxygenated experiments only, N 2 was bubbled through the reaction mixture for 15 min at 0 o C before sealing with vacuum grease. The sealed cuvette was then irradiated with red LED light (λ max = 635 nm, 3.0 mw/cm 2 ) at room temperature. At predetermined time points, the cuvette was transferred from the reactor to the sample holder for FTNIR measurement. The monomer conversions were calculated using the ratio of the integral of the wavenumber region cm -1 at different time points relative to the integral of the initial polymerization mixture (0 % monomer conversion). At regular intervals, a degassed syringe was used to extract ~50 µl aliquots for GPC analysis. Synthesis of POEGMA macro-cta via RAFT polymerization A typical synthesis of a POEGMA macro-cta by RAFT polymerization was set up as follows: OEGMA (9.0 g, 0.03 mol), CDTPA ( g, mol), AIBN (20.53 mg, mol) and 37.5 ml toluene were added to a 200 ml round bottom flask which was sealed with a rubber septum and purged with nitrogen for 30 min at 0 o C. The polymerization was carried out for 5 h at 70 o C before quenching in an ice bath and exposing to air. The resulting polymer was purified by precipitation in a diethyl ether and petroleum spirit (boiling range of o C) mixture (30:70, v/v). GPC analysis using DMAc as a mobile solvent and polystyrene standards indicated M n,gpc = g mol -1 and Đ = H NMR indicated a monomer conversion of 57 % which was calculated using the following equation α = 100 [p / (p+m)], where m = I ppm and p = [( I ppm / 2) - ( I ppm )]. The theoretical molecular weight was determined to be M n,theo = g/mol using the following equation: M n,theo = MW CDTPA + [α [M] 0 /[CDTPA] 0 MW OEGMA ] where MW CDTPA is the molecular weight of the RAFT agent, α is the monomer conversion, [M] 0 is the initial monomer concentration, [CDTPA] 0 is the initial concentration of the RAFT agent and MW OEGMA is the molecular weight of OEGMA (300 g/mol). S4

5 Kinetic studies of PET-RAFT dispersion polymerization of MTEMA For FTNIR kinetic studies of the PET-RAFT dispersion polymerization of MTEMA, a typical polymerization mixture ([MTEMA]:[POEGMA]:[ZnTPP] = 120:1:0.01 was set up as follows. ZnTPP (35.21 µg, mol, µl of a 1mg/mL tetrahydrofuran (THF) stock solution) was added to a 21 ml glass vial. After evaporating the residual THF under a stream of nitrogen, POEGMA (25.96 mg, mol), MTEMA (99.83 mg, mol) and ml MeOH (total solids content = 15 wt%) were further added to the vial. 900 µl of the reaction mixture was transferred to an 0.9 ml FTNIR quartz cuvette (1 cm 2 mm) which was sealed with a rubber septum and parafilm. For deoxygenated experiments only, N 2 was bubbled through the reaction mixture for 15 min at 0 o C before sealing with vacuum grease. The sealed cuvette was then irradiated with red LED light (λ max = 635 nm, 1.7 mw/cm 2 ) at room temperature. At predetermined time points, the cuvette was transferred from the reactor to the sample holder for FTNIR measurement. The monomer conversions were calculated using the ratio of the integral of the wavenumber region cm -1 at different time points relative to the integral of the initial polymerization mixture (0% monomer conversion). At regular intervals, a degassed syringe was used to extract ~50 µl aliquots for GPC analysis. Note: 1 H NMR analysis of the final samples was used to confirm the conversion of MTEMA obtained by NIR due to possible effects of light scattering on the NIR signal intensity. PET-RAFT dispersion polymerization of MTEMA under red light A typical experiment ([MTEMA]:[POEGMA]:[ZnTPP] = 120:1:0.01 and total solids content of 15 wt%) was set up as follows: POEGMA macro-cta (M n, theo = g mol -1 and Đ = 1.17) (11.54 mg, mol) was added to a 1.6 ml glass vial followed by ZnTPP (15.65 µg, mol, µl of a 1 mg/ml tetrahydrofuran (THF) stock solution). After evaporating the residual THF under a stream of nitrogen, MTEMA (44.37 mg, mol and ml MeOH were further added to the vial which was subsequently sealed with a rubber septum and parafilm. For deoxygenated experiments only, N 2 was bubbled through the reaction mixture for 15 min at 0 o C before sealing with vacuum grease. The sealed vial was then irradiated with red LED light (λ max = 635 nm, 1.7 mw/cm 2 ) at room temperature for 24 h before it was quenched by exposure to air (and storage in the dark). 1 H NMR (CDCl 3 ) of the crude polymerization mixture was used to measure the monomer conversion by comparing the relative intensities of the vinyl protons of MTEMA at ppm with the methylene protons adjacent to the sulfur centre in (P)MTEMA at ppm. GPC chromatograms were also used to determine the molecular weight (M n,gpc ) and polymer dispersity. S5

6 Finally, TEM analysis was used to determine the size and the morphology of the self-assembled block copolymers. Oxidation of POEGMA-b-PMTEMA nanoparticles under visible light A typical procedure for the oxidation of the POEGMA-b-PMTEMA nanoparticles (PISA-7) was set up as follows: an aqueous solution of the purified POEGMA-b-PMTEMA nanoparticles (1 ml, 0.5 wt%) was added to a 4 ml glass vial followed by irradiation with yellow light (λ max = 560 nm, 9.7 mw/cm 2 ) in the presence of oxygen and with constant stirring. At predetermined time points, a 100 µl aliquot was withdrawn and the residual solvent evaporated under nitrogen. The degree of oxidation was assessed by 1 H NMR of the irradiated sample (in DMSO-d 6 ) by comparing the integral of the peak at 2.15 ppm at different time points relative to the integral of the initial nanoparticle mixture (0 % thioether conversion). Transmittance studies of the oxidation of POEGMA-b-PMTEMA nanoparticles In order to monitor the transmittance of the nanoparticle solutions under visible light, the same irradiation procedure was performed but in a quartz cuvette (path length = 1 cm) and with a nanoparticle concentration of 0.05 wt%. At predetermined time points, the transmittance through the nanoparticle solution at a wavelength of 500 nm was quantified by UV-Vis spectroscopy. For the oxidation of the POEGMA-b-PMTEMA nanoparticles by H 2 O 2, 300 µl of aqueous H 2 O 2 (30 w/w%) was added to the purified nanoparticles (1 ml, 0.05 wt%) which were incubated in a quartz cuvette (path length = 1 cm) with constant stirring in the dark. At predetermined time points, the transmittance through the nanoparticle solution at a wavelength of 500 nm was quantified by UV-Vis spectroscopy. Quantification of ZnTPP loading and encapsulation efficiency Theoretical ZnTPP loading of POEGMA-b-PMTEMA nanoparticles The theoretical ZnTPP loading relative to polymer ({ZnTPP} theoretical ) was determined based on the initial reaction mixture stoichiometry using: {ZnTPP} theoretical = (mass(zntpp) 0 ) / (α mass(mtema) 0 + mass(poegma) 0 + mass(zntpp) 0 )), where α is the monomer conversion, and mass(zntpp) 0, mss(mtema) 0 and mass[poegma] 0 are the initial masses of ZnTPP, MTEMA and POEGMA in the initial reaction mixture respectively. For PISA-7, {ZnTPP} theoretical was calculated to be 0.17 wt% relative to polymer S6

7 Experimental ZnTPP concentration relative to polymer The amount of encapsulated ZnTPP within the purified aqueous nanoparticles was calculated by comparison of the UV-Vis absorbance at 428 nm against a suitable calibration curve acquired in DMSO. The experimental extinction coefficient (ε 428 ) of ZnTPP at 428 nm in DMSO with a 1 cm pathlength was determined to be ε 428 = (mg/ml) -1. To determine the experimental ZnTPP loading ({ZnTPP} experimental }), the ZnTPP loaded nanoparticles were firstly dried to remove water and the nanoparticles with encapsulated ZnTPP dissolved in DMSO. {ZnTPP} experimental was calculated as follows: {ZnTPP} experimental = (A 428 / ε 428 ) / ({polymer + ZnTPP}), where A 428 is the experimental absorbance of the ZnTPP/polymer sample at 428 nm, ε 428 is the extinction coefficient of ZnTPP in (mg/ml) -1 and {polymer+ ZnTPP} is the combined polymer + ZnTPP concentration in mg/ml (determined gravimetrically). For PISA-4, {ZnTPP} experimental was calculated to be 0.15 wt% relative to polymer Encapsulation efficiency of ZnTPP into nanoparticles The encapsulation efficiency (EE) was calculated using EE = 100 ({ZnTPP} experimental / {ZnTPP} theoretical ) For PISA-7, the EE was calculated to be approximately 85 % S7

8 Table S1. Experimental and characterization data for polymerization kinetics of MTEMA performed with and without prior deoxygenation. PET-RAFT homopolymerizations were performed in DMF under red light (λ max = 635 nm, 3.0 mw/cm 2 ) using a [MTEMA]:[CDTPA]:[ZnTPP] = 200:1:0.01. S8

9 Figure S1. Polymerization kinetics of MMA conducted with and without prior deoxygenation. Photopolymerizations were performed at room temperature in DMF under red light (λ max = 635 nm, 3.0 mw/cm 2 ) using a [MMA]:[CDTPA]:[ZnTPP] = 200:1:0.01. Variation of (A) ln([m] 0 /[M] t ) with irradiation time and (B) GPC derived molecular weight and dispersity with conversion. Corresponding molecular weight distributions at different irradiation times for polymerizations conducted (C) with and (D) without deoxygenation. *Note: Polymerization was performed under deoxygenated conditions at 60 o C. Comment: we observed a similar discrepancy between the theoretical and experimental molecular weights for conventional PET-RAFT polymerization of methyl methacrylate (MMA) in the non- and de-oxygenated polymerization (SI, Figure S1B). To elucidate if this is effect could be attributed to the temperature, we performed MMA polymerization at 60 o C under similar condition in deoxygenated system. Interestingly, we observed a better correlation between experimental and theoretical molecular weight values suggesting better RAFT control is achieved at elevated temperatures. S9

10 Figure S2. (A) 1 H NMR spectrum (acquired in CDCl 3 of vinyl protons of MTEMA after PET-RAFT polymerization in the presence of oxygen. Polymerization was performed in DMF under red light (λ max = 635 nm, 3.0 mw cm -2 ) using a [MTEMA]:[CDTPA]:[ZnTPP] = 200:1:0.01. Additional 1 H NMR spectra (acquired in CDCl 3 ) of (B) unreacted MTEMA monomer, (C) MTEMA after the addition of an excess of H 2 O 2 (performed in MeOD) and (D) MTEMA after irradiation under red light (λ max = 635 nm, 3.0 mw cm -2 ) in the presence of ZnTPP and oxygen. S10

11 Figure S3. FTIR spectrum of MTEMA after irradiation with visible light (λ max = 560 nm, 9.7 mw/cm 2 ) in the presence of ZnTPP and air. S11

12 Figure S4 Typical 1 H NMR spectrum (recorded in CDCl 3 ) of purified PMTEMA homopolymer after PET-RAFT polymerization in DMF under red light (λ max = 635 nm, 3.0 mw/cm 2 ) and in the presence of oxygen. S12

13 Figure S5. Copolymerization kinetics of MMA and MTEMA in the presence of oxygen. Polymerizations were performed under red light (λ max = 635 nm, 3.0 mw/cm 2 ) using either [MMA]:[MTEMA]:[CDTPA]:[ZnTPP] = 190:10:1:0.01 or [MMA]:[MTEMA]:[CDTPA]:[ZnTPP] = 180:20:1:0.01: variation of (A) ln([m] 0 /[M] t ) with irradiation time and (B) GPC derived molecular weight and dispersity with total monomer conversion (the dashed lines represent the evolution of the theoretical molecular weight for the respective polymerizations). Corresponding molecular weight distributions at different irradiation times for polymerizations conducted using (C) [MMA]:[MTEMA] = 190:10 or (D) [MMA]:[MTEMA] = 180:20. S13

14 Figure S6. TEM image for POEGMA-b-PMTEMA block copolymer (PISA-7) synthesized using PET-RAFT dispersion polymerization in MeOH. Polymerizations were performed for 24 h under red light (λ max = 635 nm, 1.0 mw/cm 2 ) and at a total solids content of 15 wt%. S14

15 Figure S7. UV-Vis absorption spectra of ZnTPP loaded nanoparticles obtained (A) directly in water or (B) in DMSO after evaporation of water. Spectra were acquired at a polymer concentration of 0.05 wt%. The loading of ZnTPP relative to polymer ({ZnTPP} experimental ) and encapsulation efficiency were calculated using equations given in the experimental section. S15

16 Figure S8. 1 H NMR spectra (recorded in d 6 -DMSO) of POEGMA-b-PMTEMA vesicles (PISA-7) after irradiation with visible light (λ max = 560 nm, 9.7 mw/cm 2 ) in the presence of oxygen. Note: the region from ppm is shown in more detail in the Main text, Figure 4. S16

17 Figure S9. 1 H NMR spectrum of (A) purified POEGMA-b-PMTEMA nanoparticles in water, (B) after irradiation of nanoparticles (0.5 wt%) with visible light (560 nm, 9.7 mw/cm 2 ) in the presence of oxygen and (C) after treatment of nanoparticles (0.5 wt%) with H 2 O 2. In both cases, peaks corresponding to the sulfoxide polymer (PMSEMA) can be seen at 2.65 ppm (methyl protons adjacent to the sulfoxide), ppm (methylene protons adjacent to the sulfoxide) and 4.30 ppm (methylene protons adjacent to the methacrylate ester). S17

18 Scheme S1. Reaction scheme demonstrating the formation of thioether-functionalized nanoparticles (using a PISA approach) and their subsequent disassembly under visible light. S18

19 Figure S10. Transmittance measurements of POEGMA-b-PMTEMA vesicles (PISA-7) measured at 500 nm at a nanoparticle concentration of 0.05 wt% in water. (A) Change in turbidity of vesicles under visible light irradiation (560 nm, 9.7 mw/cm 2 ) in the presence of oxygen and (B) digital photographs showing the corresponding reaction mixture (left) before and (right) after irradiation. (C) Change in turbidity of vesicles in the presence of H 2 O 2 and (D) digital photographs showing the corresponding reaction mixture (left) before and (right) after incubation with H 2 O 2. S19