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Enzyme-Assisted Photoinitiated Polymerization-Induced SelfAssembly: An Oxygen-Tolerant Method for Preparing Block Copolymer Nano-Objects in Open Vessels and Multiwell Plates Jianbo Tan,*,†,‡ Dongdong Liu,† Yuhao Bai,† Chundong Huang,† Xueliang Li,† Jun He,† Qin Xu,† and Li Zhang*,†,‡ †

Department of Polymeric Materials and Engineering, School of Materials and Energy, and ‡Guangdong Provincial Key Laboratory of Functional Soft Condensed Matter, Guangdong University of Technology, Guangzhou 510006, China S Supporting Information *

ABSTRACT: High-throughput synthesis of well-defined polymer nano-objects has long been an attractive yet challenging topic in the area of polymer chemistry and material science. Herein, we report an enzyme-assisted photoinitiated polymerizationinduced self-assembly (photo-PISA) approach to prepare well-defined AB diblock or ABC triblock copolymer nano-objects at room temperature in open vessels and multiwell plates. Kinetic studies indicated that the presence of glucose oxidase (GOx) endowed the polymerizations with excellent oxygen tolerance. Good control was maintained during the enzyme-assisted photoPISA process. This method facilitates high-throughput PISA, allowing for the construction of a detailed phase diagram in a rather short time. We also demonstrate the potential bio-related application of this method by the successful encapsulation of horseradish peroxidase (HRP) and bovine serum albumin (BSA) into self-assembled polymer vesicles without compromising protein activities. This robust oxygen-tolerant PISA approach leads to unprecedented access to well-defined polymer nanoobjects for nonexperts.

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INTRODUCTION Polymer scientists have been continuously interested in making well-defined block copolymer nano-objects due to their many potential applications including catalysis, nanoreactor, bioimaging, drug/gene delivery, etc.1−4 Cosolvent self-assembly of block copolymers is the most commonly used strategy to prepare block copolymer nano-objects with different morphologies such as spheres, worms, lamellae, sunflowers, and vesicles.5 Nevertheless, one major drawback of this method is that the self-assembly is typically conducted in very dilute solutions ( 1.80) were found when targeting PGMA40-PHPMA300-PGlyMAn (n = 100, 200, 300). Because of the poor aqueous solubility of GlyMA, the chain extension of GlyMA was conducted under seeded emulsion polymerization conditions. Although SPTP is a water-soluble photoinitiator, both a hydrophobic radical and a hydrophilic radical are generated upon visible light irradiation. The hydrophobic radical would lead to uncontrolled polymerization in monomer droplets, resulting in broad molar mass distributions. To justify this hypothesis (vide supra), we then

conducted a control experiment that focused on photoinitiated emulsion polymerization of GlyMA using PGMA45-CTA as the macro-CTA and SPTP as the photoinitiator. GPC analysis revealed a bimodal trace with a broad molar mass distribution (Figure S7). These results verified that the broad molar mass distributions of PGMA40-PHPMA300-PGlyMAn (n = 100, 200, 300) were attributed to the inherent nature of photoinitiated emulsion polymerization using SPTP as the photoinitiator. In our group, we are currently working on the design and synthesis of new photoinitiators that are favorable for photoinitiated emulsion polymerization. Enzyme-Assisted Photo-PISA in Multiwell Plates. The above results proved that the enzyme-assisted photo-PISA is an oxygen-tolerant approach that can be carried out in open vessels. Given that the degassing mechanism of GOx is independent of volume,36,38,39 we expected that the enzymeassisted photo-PISA can be potentially performed in multiwell plates, giving rise to high-throughput synthesis of polymer nano-objects. Scheme 2 shows the enzyme-assisted photo-PISA is conducted in 96-well plates simply by pipet addition. The volume of each polymerization was scaled down to 250 μL, and the reaction can be finished within 30 min. In preliminary study, presynthesized PPEGMA8-CTA was chain-extended with HPMA (10% w/w) via the enzyme-assisted photo-PISA targeting different DPs of PHPMA. As shown in Figure 5a, DMF GPC indicated high blocking efficiencies with symmetric GPC curves. A linear increase in Mn as well as low Mw/Mn values were observed as targeted DPs of PHPMA increased (Figure 5b). To further expand the scope of this technique, a E

DOI: 10.1021/acs.macromol.7b01219 Macromolecules XXXX, XXX, XXX−XXX

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Figure 6. (a−d) Representative TEM images recorded for PPEGMA8-PHPMAn obtained via the one-pot enzyme-assisted photo-PISA of HPMA in a 96-well plate at different HPMA concentrations. (e) GPC profiles of PPEGMA8-CTA and PPEGMA8-PHPMAn obtained via the one-pot enzymeassisted photo-PISA of HPMA in a 96-well plate at different HPMA concentrations. In the TEM images, P stands for PPEGMA and H stands for PHPMA.

Figure 7. (a) Digital photograph of PPEGMA8-PHPMAn diblock copolymers prepared via enzyme-assisted photo-PISA of HPMA by varying the monomer concentration and DP of PHPMA. (b) Detailed phase diagram constructed for the preparation of PPEGMA8-PHPMAn diblock copolymer nano-objects via enzyme-assisted photo-PISA of HPMA by varying the monomer concentration and DP of PHPMA. Phase regions consist of spheres (S), worms (W), vesicles (V), and mixed morphologies.

copolymer nano-objects with predetermined molar mass and well-defined morphology. One particularly useful feature of this new technique is to generate phase diagram of self-assembled polymer nano-objects in a timely fashion. Phase diagram provides important insight for preparing well-defined polymer nano-objects with certain morphologies. However, the building-up of phase diagram is typically tedious and time-consuming.45,48 In comparison with traditional PISA setup, our new technique greatly facilitates the construction of highly detailed phase diagram (up to tens of recipes) in a quite short time period. As a proof-of-concept experiment, we constructed a phase diagram of PPEGMA8PHPMAn polymer nano-objects via enzyme-assisted photoPISA in a 96-well plate in which the monomer concentration and target DP of PHPMA were varied (containing 27 recipes). According to Figure 7a, the digital photograph of reaction mixtures after polymerization showed an increase in turbidity with the increment in DP of PHPMA or monomer

one-pot photopolymerization was conducted in a 96-well plate. First, PPEGMA8-CTA was prepared via photopolymerization in a 96-well plate with near-quantitative monomer conversion (>99%) being achieved under visible light irradiation. Diblock copolymer nano-objects were then prepared by simply adding HPMA to the same well following by the enzyme-assisted photo-PISA. Typical PISA morphologies including spheres, worms, intermediate jellyfish, and vesicles were obtained by this one-pot approach in a 96-well plate as shown in Figure 6a−d. Figure 6e shows GPC traces of PPEGMA8-CTA and the corresponding PPEGMA8-PHPMAn diblock copolymers at different HPMA concentrations. Narrow molar mass distributions (Mw/Mn < 1.25) were observed in all cases, while small amounts of PPEGMA8-CTA residue were still present. The values of Mn, Mw, and Mw/Mn are summarized in Table S1. These results suggest that the enzyme-assisted photo-PISA is well-suited for the high-throughput synthesis of diblock F

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Figure 8. (a) Evolution of Mn with DP of PHPMA according to the GPC measurement of samples prepared via enzyme-assisted photo-PISA of HPMA by varying the monomer concentration and DP of PHPMA. (b) GPC profiles of PPEGMA8-PHPMA160 diblock copolymers prepared via four runs of enzyme-assisted photo-PISA at 10% w/w HPMA.

Figure 9. (a) Amounts of HRP and GOx in the supernatant, after each centrifugation−redispersion cycle, as determined by a calibration curve. (b) Kinetic study of HRP (released from the vesicles) by varying ABTS concentration. The red line in (b) is fit from the Michaelis−Menten model.

proteins) to be retained. To demonstrate potential biomedical applications of the present technique, we loaded proteins into polymer vesicles in situ via the enzyme-assisted photo-PISA. As a proof-of-concept experiment, horseradish peroxidase (HRP) was encapsulated in situ into PGMA40-PHPMA250 vesicles (at 20% w/w HPMA concentration) via the enzyme-assisted photo-PISA in a 96-well plate. As shown in Figure S8, vesicular morphology was observed, indicating the presence of HRP did not affect the photo-PISA process. Free HRP was removed via multiple centrifugation−redispersion cycles. It is reasonable to assume that the loading efficiency of GOx is equal to that of HRP, since the loading efficiency should be dependent on the ratio of the total vesicle lumen volume to the solution volume. The amount of HRP and GOx in the supernatant was monitored by a Bradford assay (Figure S9), which revealed that free HRP and GOx can be completely removed after two centrifugation−redispersion cycles (Figure 9a). A loading efficiency of 50.2% was calculated based on this data of Bradford assay. We also encapsulated a commonly used protein (bovine serum albumin (BSA)) into PGMA40-PHPMA250 vesicles (at 20% w/w HPMA concentration) via the enzymeassisted photo-PISA in a 96-well plate. Free BSA was removed via centrifugation−redispersion cycles. As BSA absorbs at 278 nm and then emits at 337 nm;49 thus, it is possible to calculate the amount of BSA in solution according to a calibration plot (Figure S10). The loading efficiency of BSA was around 52.3% as determined by measuring the amount of BSA in supernatants after centrifugation, which is similar to that of HRP. We then studied the catalytic behavior of the encapsulated HRP using H2O2 and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrates. First, HRP was released from the vesicles by incubating the HRP-loaded vesicles at 4 °C for 30

concentration. The obtained samples were then characterized by TEM, and the constructed phase diagram was exhibited in Figure 7b. This phase diagram is rather similar to that reported by our group for a PPEGMA14-CTA stabilizer block via photoPISA of HPMA degassed with nitrogen.32 In each case, the final block copolymer morphology is strongly concentrationdependent, with spheres preferentially formed at low monomer concentrations, since the probability of particle−particle fusion is reduced. In contrast, pure worms and vesicles were only obtained at high monomer concentrations. Moreover, a large region of mixed phases was observed in this phase diagram. The obtained samples were then analyzed by DMF GPC, producing a linear increase in Mn as higher PHPMA DPs were targeted (Figure 8a). Notably, the plots of Mn values were almost identical at different monomer concentrations. To demonstrate the reproducibility of enzyme-assisted photo-PISA, one recipe (i.e., PPEGMA8-PHPMA160, 10% w/w HPMA concentration) was repeated for four times in a 96-well plate and thoroughly characterized by 1H NMR and GPC. 1H NMR measurement indicated that full monomer conversions were achieved in all cases. More importantly, the GPC traces of the final diblock copolymer products are almost identical as shown in Figure 8b. This technique not only extends the PISA technique by expanding the scope of possible monomers that can be used in PISA for nonexperts but also greatly improves the reproducibility of PISA results due to its oxygen-tolerant feature. Loading Proteins into Vesicles. Since the enzymeassisted photo-PISA reported in this paper is conducted under mild conditions (visible light, aqueous medium, short reaction time, and room temperature) without inert atmosphere or freeze−pump−thaw process, we reasoned that this approach would allow the activity of biomolecules (e.g., G

DOI: 10.1021/acs.macromol.7b01219 Macromolecules XXXX, XXX, XXX−XXX

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min.49 Then initial rates of HRP-catalyzed oxidation of ABTS were measured at different ABTS concentrations, and the data were fitted using the Michaelis−Menten kinetic model (Figure 9b). Based on the data in Figure 9b, a Km value of 0.48 mM was obtained, which is similar to Km values of HRP reported in literature (0.36−0.68 mM).50 Because of the presence of GOx, it may lead to the generation of H2O2 during the catalytic measurement, although the amount of H2O2 should be very small. To demonstrate the presence of GOx has no effect on the measurement, the catalytic behavior of native HRP (in the absence of GOx) was measured. A Km value of 0.48 mM was found for the native HRP (Figure S11), which is the same as the released HRP from vesicles. These results confirmed that the enzyme-assisted photo-PISA process did not compromise the binding affinity of HRP to the substrate.

CONCLUSIONS In summary, we have presented a robust oxygen-tolerant method to prepare polymer nano-objects by combining enzyme catalytic reaction and photo-PISA. A diverse set of well-defined polymer nano-objects (sphere, worms, jellyfish, vesicles, and framboidal vesicles) were accessible by this method in open vessels and 96-well plates without any degassing. The effect of stirring rates was investigated, indicating that a moderate level of stirring rates is crucial to ensure efficient polymerizations. More importantly, this approach enables high-throughput PISA synthesis and the building-up of detailed phase diagrams within a rather short time, which would potentially lighten the PISA research. In addition, HRP and BSA were successfully loaded into the self-assembled polymer vesicles by the enzyme-assisted photo-PISA. Encapsulation into vesicles did not affect the activity of HRP and BSA, which exemplified the potential application of this method for preparing bio-related polymer nano-objects. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b01219. Full experimental section and additional results (PDF)

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REFERENCES

(1) Wang, Z.; van Oers, M. C. M.; Rutjes, F. P. J. T.; van Hest, J. C. M. Polymersome Colloidosomes for Enzyme Catalysis in a Biphasic System. Angew. Chem., Int. Ed. 2012, 51 (43), 10746−10750. (2) Louzao, I.; van Hest, J. C. M. Permeability Effects on the Efficiency of Antioxidant Nanoreactors. Biomacromolecules 2013, 14 (7), 2364−2372. (3) Liu, T.; Hu, J.; Jin, Z.; Jin, F.; Liu, S. Two-Photon Ratiometric Fluorescent Mapping of Intracellular Transport Pathways of pHResponsive Block Copolymer Micellar Nanocarriers. Adv. Healthcare Mater. 2013, 2 (12), 1576−1581. (4) Lee, J. S.; Feijen, J. Polymersomes for Drug Delivery: Design, Formation and Characterization. J. Controlled Release 2012, 161 (2), 473−483. (5) Mai, Y.; Eisenberg, A. Self-Assembly of Block Copolymers. Chem. Soc. Rev. 2012, 41 (18), 5969−5985. (6) Blanazs, A.; Madsen, J.; Battaglia, G.; Ryan, A. J.; Armes, S. P. Mechanistic Insights for Block Copolymer Morphologies: How Do Worms Form Vesicles? J. Am. Chem. Soc. 2011, 133 (41), 16581− 16587. (7) Warren, N. J.; Armes, S. P. Polymerization-Induced SelfAssembly of Block Copolymer Nano-Objects via RAFT Aqueous Dispersion Polymerization. J. Am. Chem. Soc. 2014, 136 (29), 10174− 10185. (8) Liu, G.; Qiu, Q.; Shen, W.; An, Z. Aqueous Dispersion Polymerization of 2-Methoxyethyl Acrylate for the Synthesis of Biocompatible Nanoparticles Using a Hydrophilic RAFT Polymer and a Redox Initiator. Macromolecules 2011, 44 (13), 5237−5245. (9) Zhou, W.; Qu, Q.; Xu, Y.; An, Z. Aqueous PolymerizationInduced Self-Assembly for the Synthesis of Ketone-Functionalized Nano-Objects with Low Polydispersity. ACS Macro Lett. 2015, 4, 495−499. (10) Charleux, B.; Delaittre, G.; Rieger, J.; D’Agosto, F. Polymerization-Induced Self-Assembly: From Soluble Macromolecules to Block Copolymer Nano-Objects in One Step. Macromolecules 2012, 45 (17), 6753−6765. (11) Tan, J.; Huang, C.; Liu, D.; Li, X.; He, J.; Xu, Q.; Zhang, L. Polymerization-Induced Self-Assembly of Homopolymer and Diblock Copolymer: A Facile Approach for Preparing Polymer Nano-Objects with Higher-Order Morphologies. ACS Macro Lett. 2017, 6 (3), 298− 303. (12) He, W.-D.; Sun, X.-L.; Wan, W.-M.; Pan, C.-Y. Multiple Morphologies of PAA-B-PSt Assemblies throughout RAFT Dispersion Polymerization of Styrene with PAA Macro-CTA. Macromolecules 2011, 44 (9), 3358−3365. (13) Wan, W.-M.; Hong, C.-Y.; Pan, C.-Y. One-Pot Synthesis of Nanomaterials via RAFT Polymerization Induced Self-Assembly and Morphology Transition. Chem. Commun. 2009, No. 39, 5883−5885. (14) Lowe, A. B. RAFT Alcoholic Dispersion Polymerization with Polymerization-Induced Self-Assembly. Polymer 2016, 106, 161−181. (15) Semsarilar, M.; Jones, E. R.; Blanazs, A.; Armes, S. P. Efficient Synthesis of Sterically-Stabilized Nano-Objects via RAFT Dispersion Polymerization of Benzyl Methacrylate in Alcoholic Media. Adv. Mater. 2012, 24 (25), 3378−3382. (16) Gao, C.; Li, S.; Li, Q.; Shi, P.; Shah, S. A.; Zhang, W. Dispersion RAFT Polymerization: Comparison between the Monofunctional and Bifunctional Macromolecular RAFT Agents. Polym. Chem. 2014, 5 (24), 6957−6966. (17) Ding, Z.; Gao, C.; Wang, S.; Liu, H.; Zhang, W. Macro-RAFT Agent Mediated Dispersion Polymerization: The Monomer Concentration Effect on the Morphology of the in Situ Synthesized Block Copolymer Nano-Objects. Polym. Chem. 2015, 6 (46), 8003−8011. (18) Li, S.; He, X.; Li, Q.; Shi, P.; Zhang, W. Synthesis of Multicompartment Nanoparticles of Block Copolymer through Two Macro-RAFT Agents Co-Mediated Dispersion Polymerization. ACS Macro Lett. 2014, 3 (9), 916−921. (19) Zhang, Q.; Zhu, S. Ionic Liquids: Versatile Media for Preparation of Vesicles from Polymerization-Induced Self-Assembly. ACS Macro Lett. 2015, 4 (7), 755−758.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (J.T.). *E-mail [email protected] (L.Z.). ORCID

Jianbo Tan: 0000-0002-5635-7178 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from the National Natural Science Foundation of China (Grant 21504017), Guangdong Natural Science Foundation (Grant 2016A030310339), Science and Technology Planning Project of Guangdong Province (Grant 2017A010103045), and Science and Technology Program of Guangzhou (Grant 201707010420). H

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Macromolecules (20) Fielding, L. A.; Lane, J. A.; Derry, M. J.; Mykhaylyk, O. O.; Armes, S. P. Thermo-Responsive Diblock Copolymer Worm Gels in Non-Polar Solvents. J. Am. Chem. Soc. 2014, 136 (15), 5790−5798. (21) Derry, M. J.; Fielding, L. A.; Armes, S. P. PolymerizationInduced Self-Assembly of Block Copolymer Nanoparticles via RAFT Non-Aqueous Dispersion Polymerization. Prog. Polym. Sci. 2016, 52, 1−18. (22) Pei, Y.; Thurairajah, L.; Sugita, O. R.; Lowe, A. B. RAFT Dispersion Polymerization in Nonpolar Media: Polymerization of 3Phenylpropyl Methacrylate in N-Tetradecane with Poly(stearyl Methacrylate) Homopolymers as Macro Chain Transfer Agents. Macromolecules 2015, 48 (1), 236−244. (23) Tan, J.; Sun, H.; Yu, M.; Sumerlin, B. S.; Zhang, L. Photo-PISA: Shedding Light on Polymerization-Induced Self-Assembly. ACS Macro Lett. 2015, 4 (11), 1249−1253. (24) Tan, J.; Huang, C.; Liu, D.; Zhang, X.; Bai, Y.; Zhang, L. Alcoholic Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA): A Fast Route toward Poly(isobornyl Acrylate)-Based Diblock Copolymer Nano-Objects. ACS Macro Lett. 2016, 5, 894− 899. (25) Tan, J.; Bai, Y.; Zhang, X.; Huang, C.; Liu, D.; Zhang, L. LowTemperature Synthesis of Thermoresponsive Diblock Copolymer Nano-Objects via Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA) Using Thermoresponsive Macro-RAFT Agents. Macromol. Rapid Commun. 2016, 37 (17), 1434−1440. (26) Tan, J.; Zhang, X.; Liu, D.; Bai, Y.; Huang, C.; Li, X.; Zhang, L. Facile Preparation of CO2-Responsive Polymer Nano-Objects via Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA). Macromol. Rapid Commun. 2017, 38 (13), 1600508. (27) Tan, J.; Liu, D.; Zhang, X.; Huang, C.; He, J.; Xu, Q.; Li, X.; Zhang, L. Facile Preparation of Hybrid Vesicles Loaded with Silica Nanoparticles via Aqueous Photoinitiated Polymerization-Induced Self-Assembly. RSC Adv. 2017, 7 (37), 23114−23121. (28) Tan, J.; Liu, D.; Huang, C.; Li, X.; He, J.; Xu, Q.; Zhang, L. Photoinitiated Polymerization-Induced Self-Assembly of Glycidyl Methacrylate for the Synthesis of Epoxy-Functionalized Block Copolymer Nano-Objects. Macromol. Rapid Commun. 2017, 1700195. (29) Yeow, J.; Xu, J.; Boyer, C. Polymerization-Induced SelfAssembly Using Visible Light Mediated Photoinduced Electron Transfer−Reversible Addition−Fragmentation Chain Transfer Polymerization. ACS Macro Lett. 2015, 4 (9), 984−990. (30) Yeow, J.; Sugita, O. R.; Boyer, C. Visible Light-Mediated Polymerization-Induced Self-Assembly in the Absence of External Catalyst or Initiator. ACS Macro Lett. 2016, 5 (5), 558−564. (31) Yu, Q.; Ding, Y.; Cao, H.; Lu, X.; Cai, Y. Use of Polyion Complexation for Polymerization-Induced Self-Assembly in Water under Visible Light Irradiation at 25 °C. ACS Macro Lett. 2015, 4 (11), 1293−1296. (32) Tan, J.; Bai, Y.; Zhang, X.; Zhang, L. Room Temperature Synthesis of Poly(poly(ethylene Glycol) Methyl Ether Methacrylate)Based Diblock Copolymer Nano-Objects via Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA). Polym. Chem. 2016, 7 (13), 2372−2380. (33) Matyjaszewski, K.; Coca, S.; Gaynor, S. G.; Wei, M.; Woodworth, B. E. Controlled Radical Polymerization in the Presence of Oxygen. Macromolecules 1998, 31 (17), 5967−5969. (34) Xu, J.; Jung, K.; Atme, A.; Shanmugam, S.; Boyer, C. A Robust and Versatile Photoinduced Living Polymerization of Conjugated and Unconjugated Monomers and Its Oxygen Tolerance. J. Am. Chem. Soc. 2014, 136 (14), 5508−5519. (35) Li, J.; Ding, C.; Zhang, Z.; Pan, X.; Li, N.; Zhu, J.; Zhu, X. Visible Light-Induced Living Radical Polymerization of Butyl Acrylate: Photocatalyst-Free, Ultrafast, and Oxygen Tolerance. Macromol. Rapid Commun. 2017, 38, 1600482. (36) Chapman, R.; Gormley, A. J.; Herpoldt, K.-L.; Stevens, M. M. Highly Controlled Open Vessel RAFT Polymerizations by Enzyme Degassing. Macromolecules 2014, 47 (24), 8541−8547.

(37) Lv, Y.; Liu, Z.; Zhu, A.; An, Z. An, Z. Glucose Oxidase Deoxygenation−redox Initiation for RAFT Polymerization in Air. J. Polym. Sci., Part A: Polym. Chem. 2017, 55 (1), 164−174. (38) Chapman, R.; Gormley, A. J.; Stenzel, M. H.; Stevens, M. M. Combinatorial Low-Volume Synthesis of Well-Defined Polymers by Enzyme Degassing. Angew. Chem. 2016, 128 (14), 4576−4579. (39) Oytun, F.; Kahveci, M. U.; Yagci, Y. Sugar Overcomes Oxygen Inhibition in Photoinitiated Free Radical Polymerization. J. Polym. Sci., Part A: Polym. Chem. 2013, 51 (8), 1685−1689. (40) Iwata, H.; Hata, Y.; Matsuda, T.; Ikada, Y. Initiation of Radical Polymerization by Glucose Oxidase Utilizing Dissolved Oxygen. J. Polym. Sci., Part A: Polym. Chem. 1991, 29 (8), 1217−1218. (41) Yeow, J.; Shanmugam, S.; Corrigan, N.; Kuchel, R. P.; Xu, J.; Boyer, C. A Polymerization-Induced Self-Assembly Approach to Nanoparticles Loaded with Singlet Oxygen Generators. Macromolecules 2016, 49 (19), 7277−7285. (42) Ng, G.; Yeow, J.; Xu, J.; Boyer, C. Application of Oxygen Tolerant PET-RAFT to Polymerization-Induced Self-Assembly. Polym. Chem. 2017, 8 (18), 2841−2851. (43) Yeow, J.; Chapman, R.; Xu, J.; Boyer, C. Oxygen Tolerant Photopolymerization for Ultralow Volumes. Polym. Chem. 2017, DOI: 10.1039/C7PY00007C. (44) Canning, S. L.; Smith, G. N.; Armes, S. P. A Critical Appraisal of RAFT-Mediated Polymerization-Induced Self-Assembly. Macromolecules 2016, 49 (6), 1985−2001. (45) Blanazs, A.; Ryan, A. J.; Armes, S. P. Predictive Phase Diagrams for RAFT Aqueous Dispersion Polymerization: Effect of Block Copolymer Composition, Molecular Weight, and Copolymer Concentration. Macromolecules 2012, 45 (12), 5099−5107. (46) Chambon, P.; Blanazs, A.; Battaglia, G.; Armes, S. P. Facile Synthesis of Methacrylic ABC Triblock Copolymer Vesicles by RAFT Aqueous Dispersion Polymerization. Macromolecules 2012, 45 (12), 5081−5090. (47) Ratcliffe, L. P. D.; Ryan, A. J.; Armes, S. P. From a WaterImmiscible Monomer to Block Copolymer Nano-Objects via a OnePot RAFT Aqueous Dispersion Polymerization Formulation. Macromolecules 2013, 46 (3), 769−777. (48) Tan, J.; Liu, D.; Bai, Y.; Huang, C.; Li, X.; He, J.; Xu, Q.; Zhang, X.; Zhang, L. An Insight into Aqueous Photoinitiated PolymerizationInduced Self-Assembly (Photo-PISA) for the Preparation of Diblock Copolymer Nano-Objects. Polym. Chem. 2017, 8, 1315. (49) Mable, C. J.; Gibson, R. R.; Prevost, S.; McKenzie, B. E.; Mykhaylyk, O. O.; Armes, S. P. Loading of Silica Nanoparticles in Block Copolymer Vesicles during Polymerization-Induced SelfAssembly: Encapsulation Efficiency and Thermally Triggered Release. J. Am. Chem. Soc. 2015, 137 (51), 16098−16108. (50) Morawski, B.; Quan, S.; Arnold, F. H. Functional Expression and Stabilization of Horseradish Peroxidase by Directed Evolution in Saccharomyces Cerevisiae. Biotechnol. Bioeng. 2001, 76 (2), 99−107.

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DOI: 10.1021/acs.macromol.7b01219 Macromolecules XXXX, XXX, XXX−XXX