Electronic Supporting Information

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1 Electronic Supporting Information A Polymerization-Induced Self-Assembly Approach To Nanoparticles Loaded With Singlet Oxygen Generators Jonathan Yeow, [a] Sivaprakash Shanmugam, [a] Nathaniel Corrigan, [a] Rhiannon P. Kuchel, [b] Jiangtao Xu [a] and Cyrille Boyer* [a] a Centre for Advanced Macromolecular Design (CAMD) and Australian Centre for NanoMedicine (ACN), 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 * cboyer@unsw.edu.au

2 EXPERIMENTAL SECTION Materials Benzyl methacrylate (BzMA, Aldrich) was deinhibited by passing through a column of basic alumina. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA) (M n = 300 g mol -1 ) (Sigma-Aldrich) was used as received. 2,2 -azobis(isobutyronitrile) (AIBN, Fluka, 98%) was purified by recrystallization from methanol. 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CDTPA, Boron Molecular), 5,10,15,20-Tetraphenyl-21H,23H-porphine (ZnTPP, Sigma-Aldrich) and anthracene-9,10-dipropionic acid disodium salt (ADPA, Sapphire Bioscience) were used as received. All other reagents were used as received unless otherwise specified. All volumes were measured using micropipettes. Instrumentation All 1 H-NMR spectra were recorded using a Bruker 300 or 400 MHz spectrometer. All chemical shifts are 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 (10 5, 10 4, 10 3, 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.

3 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 ethanol (or water for aqueous dispersions) to give wt% dispersions and deposited onto carbon-coated copper grids. Uranyl acetate 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. A Bruker IFS 66/S Fourier transform spectrometer equipped with a tungsten halogen lamp, a CaF 2 beam splitter, and liquid nitrogen cooled InSb detector was used. Spectra were analyzed with OPUS software. Singlet oxygen quenching experiments were 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. Synthesis of POEGMA macro-cta via RAFT polymerızatıon A typical synthesis of a POEGMA macro-cta by RAFT polymerization was set up as follows: OEGMA (6.0 g, 0.02 mol), CDTPA ( g, mol), AIBN (8.21 mg, mol) and 25 ml toluene were added to a 50 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 4 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, Đ = H NMR indicated a monomer conversion of 46 % 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

4 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). Catalyst study of ZnTPP mediated PET-RAFT dispersion polymerization under red light A typical experiment (Exp. iv [BzMA]:[POEGMA]:[ZnTPP] = 200:1:0.1 and total solids content of 10 wt%) was set up as follows: POEGMA macro-cta (M n, theo = g mol 1 and Đ = 1.16) (30 mg, mol) was added to a 4 ml glass vial followed by ZnTPP (221.1 µg, mol, ul of a 1 mg/ml tetrahydrofuran (THF) stock solution). After evaporating the residual THF under a stream of nitrogen, BzMA (114.9 mg, mol and ml EtOH (90 wt%) were further added to the vial which was subsequently sealed with a rubber septum and purged with nitrogen for 20 min in an ice bath at 0 o C. The polymerization mixture was irradiated with red LED light (λ max = 635 nm, 0.7 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 (P)BzMA methylene protons adjacent to the ester bond in the monomer (5.17 ppm) and polymer ( ppm) respectively. The number average molecular weight (M n, NMR ) of POEGMA- PBzMA block copolymers was found by comparing the same PBzMA peak ( ppm) with the POEGMA methylene protons at 4.1 ppm. GPC chromatograms were also used to determine the molecular weight (M n,gpc ) and polymer dispersity. Finally, DLS and TEM analysis were used to determine the size and the morphology of the self-assembled block copolymers. Note: For all experiments, photocatalyst stock solutions were firstly prepared in THF (due to the limited solubility of ZnTPP in ethanol) and the residual THF removed before addition of monomer and solvent.

5 Kinetic studies of ZnTPP mediated PET-RAFT dispersion polymerization of BzMA by on-line Fourier transform near-infrared (FTNIR) spectroscopy For FTNIR kinetic studies, a polymerization mixture of [BzMA]:[POEGMA 29.5 ]:[ZnTPP] = 200:1:0.02 and total solids content of 10 wt% in either EtOH or 90:10 v/v EtOH:MeCN was employed. Briefly, a stock solution of POEGMA 29.5 macro-cta (M n, NMR = g mol 1 and Đ = 1.16) (30 mg, mol), BzMA (114.9 mg, mol), ZnTPP (44.2 µg, mol) and ml solvent (either EtOH or 90:10 v/v EtOH:MeCN) was prepared and a 800 µl aliquot added to a 900 µl FTNIR quartz cuvette (1 cm 2 mm). The cuvette was sealed with a rubber septum and purged with nitrogen for 20 min. The polymerization mixture was irradiated with red LED light (λ max = 635 nm, 0.7 mw/cm 2 ) at room temperature. At predetermined time points (approximately every 30 min) the cuvette was transferred from the light reactor to the sample holder manually for FTNIR measurement. The monomer conversions were calculated by 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). Synthesis of POEGMA-b-PBzMA nanoparticles at high conversion using ZnTPP mediated PET- RAFT dispersion polymerization under red or yellow light A typical procedure for the synthesis of nanoparticles at high conversion (Exp. ix [BzMA]:[POEGMA 23 ]:[ZnTPP] = 150:1:0.01 and total solids content of 10 wt%) was set up as follows: POEGMA macro-cta (M n, theo = g mol 1 and Đ = 1.16) (15 mg, mol) was added to a 4 ml glass vial followed by ZnTPP (14.1 µg, mol, 14.1 ul of a 1 mg/ml THF stock solution). After evaporating the residual THF under a stream of nitrogen, BzMA (73.4 mg, mol and ml EtOH:MeCN (90:10 v/v%) were further added to the vial which was subsequently sealed with a rubber septum and purged with nitrogen for 20 min in an ice bath at 0 o C. The polymerization mixture was irradiated with red (λ max = 635 nm, 2.1 mw/cm 2 ) or yellow (λ max = 560 nm, 3.1 mw/cm 2 ) LED light at room temperature for 48 h before it was quenched by exposure to air (and storage in the dark).

6 Monitoring of singlet oxygen production by reaction with ADPA The ZnTPP loading within aqueous vesicles of Exp. xiii was determined by comparison of its visible absorption at 560 nm with a suitable calibration curve of free ZnTPP in DMSO. Absorptions were measured in DMSO (after removal of residual water) to eliminate the effects of nanoparticle induced scatter on the observed absorption by ZnTPP. The ZnTPP loading was determined to be 0.2 wt% relative to the polymer mass. Monitoring of singlet oxygen production was determined by measuring the change in absorbance of the singlet oxygen quencher, anthracene dipropionic acid disodium salt (ADPA) at 378 nm using a modified literature procedure. [1] Briefly, 400 ul of 2.5 mm ADPA stock solution in 0.06 M NaOH was added to 9.2 ml of an aqueous dispersion of ZnTPP loaded vesicles (adjusted to 1 mg/ml in deionized water). 1 ml aliquots were then irradiated with yellow light (λ max = 560 nm, 9.7 mw/cm 2 ) for different times (0, 5, 10, 15, 20, 30 and 40 min) before centrifugation twice at rpm for 20 min. The supernatant was collected and analysed by visible absorption spectroscopy. Synthesis of POEGMA-b-PBzMA nanoparticles using ZnTPP mediated PET-RAFT dispersion polymerization without prior deoxygenation A typical procedure for the synthesis of nanoparticles without deoxygenation (Exp. xiv [BzMA]:[POEGMA 26.5 ]:[ZnTPP] = 100:1:0.005 and total solids content of 10 wt%) was set up as follows: POEGMA macro-cta (M n, theo = g mol 1 and Đ = 1.16) (45 mg, mol) was added to a 4 ml glass vial followed by ZnTPP (18.2 µg, mol, 18.2 ul of a 1 mg/ml THF stock solution). After evaporating the residual THF under a stream of nitrogen, BzMA (94.4 mg, mol) and ml EtOH were further added to the vial. 1.5 ml of the solution was transferred to a 1.5 ml glass vial containing ascorbic acid (2.5 mg, mol). The mixture was sealed with a rubber septum and immediately irradiated with red LED light (λ max = 635 nm, 2.1 mw/cm 2 ) at room temperature for 48 h before it was quenched by exposure to air (and storage in the dark).

7 Figure S1. Polymerization kinetics of POEGMA homopolymers synthesized using CDTPA as RAFT agent showing the evolution of: (A) GPC-derived molecular weight and dispersity versus monomer conversion and (B) Ln([M] 0 /[M] t ) with irradiation time. (C) Summary of POEGMA homopolymers used in this study.

8 Figure S2. (A) Characterization, (B) molecular weight distributions and (C) DLS intensity-average diameter distributions of POEGMA b-pbzma diblock copolymers synthesized using [BzMA]:[macro-CTA] = 200:1 and [macro-cta]:[zntpp] ratios of (Exp. i), (Exp. ii), (Exp. iii) or (Exp. iv). (D) TEM micrographs of self-assembled nanoparticles synthesized at different catalyst concentrations. Polymerizations were irradiated under red light (λ max = 635 nm, 0.7 mw/cm 2 ) for 24 h at a total solids content of 10 wt% in ethanol.

9 Figure S3. (A, C) GPC molecular weight distributions and (B, D) DLS intensity-average diameter distributions of POEGMA 23 -b-pbzma diblock copolymers formed at 10 wt% under visible red light (λ max = 635 nm, 2.1 mw/cm 2 ) in EtOH:MeCN (90:10 v/v). Polymerization (Exp. xii) performed under low intensity red light (λ max = 635 nm, 0.7 mw/cm 2 ). *Polymerization (Exp. xiii) performed under yellow light (λ max = 560 nm, 3.1 mw/cm 2 ).

10 Figure S4. TEM micrograph of WLM (Exp. xiii) synthesized under yellow light (λ max = 560 nm, 3.1 mw/cm 2 ).

11 Figure S5. (A) UV-Vis absorption spectra of aqueous dispersions of ZnTPP-loaded nanoparticles purified by dialysed against ethanol followed by water (red) or dialyzed directly against water (blue). (B) UV-Vis absorption spectra of the same dispersions after complete solubilisation of the nanoparticles in DMSO. (C) TEM micrographs of aqueous ZnTPP-loaded nanoparticles with spherical (Exp. v), worm-like (Exp. vii) and vesicular (Exp. ix) morphologies.

12 Figure S6. (A) Characterization and (B) GPC-derived molecualar weight distribution of a POEGMA-b- PBzMA copolymer synthesized using [BzMA]:[macro-CTA]:[ZnTPP] = 150:1:0.5 at 10 wt% with low red light intensity (λ max = 635 nm, 0.45 mw/cm 2 ). (C) Corresponding TEM micrographs of Exp. xiv in (left) EtOH and (right) after direct dialysis against water.

13 Table S1: Comparison between PET-RAFT PISA experiments performed with and without ascorbic acid as a sacrificial singlet oxygen quencher. Polymerizations were performed using a [ZnTPP]:[macro-CTA] = and without conventional deoxygenation prior to red light (λ max = 635 nm, 2.1 mw/cm 2 ) irradiation for 48 h. The induction period was estimated using online FTNIR monitoring (SI, Figure S7).

14 Figure S7. Online FTNIR polymerization kinetics of a ZnTPP mediated dispersion polymerization performed without degassing and in the presence of ascorbic acid. Polymerizations were performed using a [ZnTPP]:[macro-CTA] = and without conventional deoxygenation prior to red light (λ max = 635 nm, 2.1 mw/cm 2 ) irradiation for 48 h (Exp. xv).

15 Additional references [1] a) C. Conte, A. Scala, G. Siracusano, N. Leone, S. Patane, F. Ungaro, A. Miro, M. T. Sciortino, F. Quaglia, A. Mazzaglia, RSC Advances 2014, 4, ; b) C. Conte, F. Ungaro, G. Maglio, P. Tirino, G. Siracusano, M. T. Sciortino, N. Leone, G. Palma, A. Barbieri, C. Arra, A. Mazzaglia, F. Quaglia, Journal of Controlled Release 2013, 167,