Facile Preparation of CO 2 -Responsive Polymer Nano-Objects via Aqueous Photoinitiated Polymerization-Induced

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1 Communication Facile Preparation of CO 2 -Responsive Polymer Nano-Objects via Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA) Jianbo Tan,* Xuechao Zhang, Dongdong Liu, Yuhao Bai, Chundong Huang, Xueliang Li, Li Zhang* Carbon dioxide (CO 2 )-responsive polymer nano-objects are prepared by photoinitiated reversible addition-fragmentation chain transfer dispersion polymerization of 2-hydroxypropyl methacrylate and 2-(dimethylamino)ethyl methacrylate (DMAEMA) in water at room temperature using a poly(poly(ethylene glycol) methyl ether methacrylate) macromolecular chain transfer agent. Kinetic studies confirm that full monomer conversions are achieved in all cases within 10 min of visible-light irradiation (405 nm, 0.5 mw cm 2 ). The effect of DMAEMA on the polymerization is studied in detail, and pure higher order morphologies (worms and vesicles) are prepared by this particular formulation. Finally, CO 2 -responsive property of the obtained vesicles is investigated by dynamic light scattering, visual appearance, and transmission electron microscope. 1. Introduction Stimuli-responsive polymer nano-objects have attracted much attention due to their broad applications in a variety of areas such as nanoreactor, molecule separation, bioimaging, and nanomedicine. [1,2] To date, numerous external signals including light, [3] temperature, [4] and electric field, [5] as well as physiological factors, such as ph, [6] enzyme concentration, [7] and redox potential [8] have been explored for the synthesis of responsive polymer nanoobjects. In particular, carbon dioxide (CO 2 ) response is one Prof. J. Tan, X. Zhang, D. Liu, Y. Bai, C. Huang, X. Li, Prof. L. Zhang Department of Polymeric Materials and Engineering School of Materials and Energy Guangdong University of Technology Guangzhou , China tanjianbo@gdut.edu.cn; lizhang@gdut.edu.cn Prof. J. Tan, Prof. L. Zhang Guangdong Provincial Key Laboratory of Functional Soft Condensed Matter Guangzhou , China of the most exciting stimulus, which can apply to physiological environment. Moreover, CO 2 is a mild and green stimulus to the system due to the robust switchability of most CO 2 -responsive polymers, and thus no residue accumulation exists. [9,10] Solution self-assembly of block copolymer is one of the most commonly used strategies to prepare CO 2 - responsive polymer nano-objects with tertiary amine, guanidine, or amidine moieties containing in the block copolymers. [11 14] For example, Yan et al. [12] synthesized a poly(ethylene glycol)-b-poly((n-amidino)dodecyl acrylamide) (PEG-PAD) diblock copolymer, and carried out the self-assembly of the diblock polymer in aqueous media. They found that the obtained polymersomes reversibly responded to CO 2, which was able to realize hierarchical cargo release as well as selective separation of guest molecules. Wang et al. [15] reported the synthesis of polymer nano-objects by self-assembly of a 2-(diethylamino)- ethyl methacrylate (DEAEMA) based block copolymer. They found that bubbling CO 2 into the solution led to the transformation of giant worms into polymersomes, and (1 of 8) 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com DOI: /marc

2 Facile Preparation of CO 2 -Responsive Polymer Nano-Objects via Aqueous Photoinitiated... the polymersomes were converted into wormlike aggregates after nitrogen (N 2 ) bubbling. However, the solution self-assembly of block copolymers usually requires post polymerization processing via a solvent or ph switch. Moreover, the self-assembly is invariably conducted in a dilute aqueous solution (typically <1 wt%), which is difficult to implement on a large scale. [16 19] Over the past eight years or so, the development of reversible addition-fragmentation chain transfer (RAFT) mediated polymerization-induced self-assembly (PISA) has enabled the preparation of various diblock copolymer nano-objects at high solids contents (10% 50%). [20 23] This approach can be conducted via dispersion polymerization or aqueous emulsion polymerization. For the former case, the reaction medium can be water, [21] alcohol, [24 26] ionic liquid, [27] or alkane. [28] Aqueous RAFT dispersion polymerization is particularly attractive due to its mild reaction conditions, facile setup, as well as the preparation of various morphologies. [21] For aqueous RAFT dispersion polymerization, the monomer is required to be soluble in water, while the resulted polymer is water-insoluble. 2-Hydroxypropyl methacrylate (HPMA), [29] N-isopropylacrylamide (NIPAM), [30] 2-methoxyethyl acrylate (MEA), [31] and diacetone acrylamide (DAAM) [32] have been employed for the preparation of diblock copolymer nano-objects via aqueous RAFT dispersion polymerization. Despite the tremendous progress made in this area, however, CO 2 -responsive diblock copolymer nano-objects prepared by aqueous RAFT dispersion polymerization still remains unexplored. It should be noted that Dong et al. [33] reported a PISA formulation in isopropanol pressurized with CO 2. The presence of CO 2 had a significant effect on the PISA process, but the obtained polymer nano-objects did not exhibit typical CO 2 -responsive property. Photo-induced living polymerization is an attractive technique that can be carried out at low temperatures. In addition, the polymerization can be manipulated to a specific time or a specific area, which is particularly appealing for the preparation of functional materials. [34] Recently, photo-induced living polymerization has been applied to PISA taking advantage of the mild reaction conditions, as well as the high rate of polymerization. The Boyer group [35,36] reported two examples of visiblelight mediated alcoholic PISA of benzyl methacrylate (BzMA) at room temperature, and typical PISA morphologies (spheres, worms, and vesicles) could be obtained. However, the rate of polymerization was relatively low. Jiang et al. [37] reported a visible-light mediated dispersion polymerization of DAAM in water, and only spherical micelles were obtained. Our group [38,39] reported an aqueous photoinitiated polymerization-induced selfassembly (photo-pisa) formulation, and a diverse set of complex morphologies could be obtained in a short reaction time. The photo-pisa process can be carried out at room temperature or lower, a feature that is particularly beneficial for the preparation of thermo-responsive and bio-related polymer nano-objects. Herein, we demonstrate for the first time to prepare CO 2 -responsive polymer nano-objects via aqueous photo-pisa of HPMA. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) was employed as the comonomer to endow CO 2 -responsive moiety to the polymer nano-objects. The aqueous photo- PISA formulation reported here can be used for in situ encapsulation and subsequently CO 2 -triggered release of globular proteins and inorganic nanoparticles. It should be noted that the low temperature property of aqueous photo-pisa is critical to retain the activity of globular proteins during the encapsulation process, which is difficult for traditional PISA formulations. 2. Results and Discussion The macromolecular RAFT (macro-raft) agent was synthesized via solution polymerization of poly- (ethylene glycol) methyl ether methacrylate (PEGMA, 475 g mol 1 ) in 1, 4-dioxane at 70 C using 4-cyano- 4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (CDPA) as the RAFT agent and 2,2-azobisisobutyronitrile (AIBN) as the initiator. The polymerization was quenched at around 75% monomer conversion after 4.5 h to preserve the end-group fidelity. The obtained macro-raft agent is denoted as poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA 9 )-CDPA, and then chain-extended for aqueous photo-pisa of HPMA and DMAEMA, as shown in Scheme 1a. Sodium phenyl-2,4,6-trimethylbenzoylphosphinate (SPTP) was employed as the photoinitiator in this paper, which can decompose rapidly under 405 nm visible light irradiation. Kinetic studies of aqueous photo-pisa were conducted at a 10% w/w HPMA concentration with the target composition of PPEGMA 9 -P(HPMA 100 -co-dmaema n ) (n = 0, 5, 10, 20) (see Figure 1a), and monitored by 1 H NMR spectroscopy. Monomer conversions were calculated on the basis of the vinyl signals relative to the internal standard, as shown in the Supporting Information. In each case, quantitative monomer conversion could be achieved within 10 min of 405 nm visible-light irradiation. Increasing the amount of DMAEMA had no significant effect on the kinetic of aqueous photo-pisa. The first-order kinetic plots are shown in Figure 1b, and it indicates that nucleation occurred at around 4 min in each case. From 0 to 4 min, the polymerization occurs in the reaction medium, corresponding to the formation of molecularly dispersed diblock copolymer chains. From 4 to 10 min, a dramatic rate enhancement was observed, which corresponds to the onset of nucleation. During this stage, the unreacted HPMA monomer entered into the WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim (2 of 8)

3 J. Tan et al. Scheme 1. a) Synthesis of poly(poly(ethylene glycol) methacrylate)-b-poly(2-hydroxypropyl methacrylate-co-2-(dimethylamino)ethyl methacrylate) polymer nano-objects via photointiated RAFT dispersion polymerization in water; b) schematic illustration of CO 2 -responsive controlled disassembly process of the vesicles. cores to solvate the hydrophobic PHPMA chains, leading to a high local concentration of monomer, and thus the increase of rate of polymerization. For a target composition of PPEGMA 9 -P(HPMA 100 -co-dmaema 10 ), samples extracted during the kinetic study were also analyzed by tetrahydrofuran (THF) gel permeation chromatography (GPC), which confirmed the linear evolution of number-average molecular weight (M n ) with monomer conversion (Figure 1c). The values of M w /M n were less than 1.30 at low monomer conversions, and increased to 1.58 as the HPMA polymerization proceeded. The increase of M w /M n value can be ascribed to the occurrence of branching reaction during aqueous photo- PISA. Figure 1d shows GPC traces of PPEGMA 9 -CDPA and the PPEGMA 9 -P(HPMA 100 -co-dmaema n ) (n = 0, 5, 10, 20) polymers prepared by aqueous photo-pisa. When the amount of DMAEMA was low (n = 0 or 5), unimodal and symmetric GPC traces were formed with low M w /M n values ( 1.30). As the amount of DMAEMA increased (n = 10 or 20), GPC traces with a shoulder at high molecular weight region were formed (M w /M n 1.58). It is well-known that DMAEMA is a monomer that contains tertiary amine group, which may act as a co-initiator during photoinitiated polymerization process, and thus leading to broad molecular weight distributions. [40] We then attempted to investigate the effect of DMAEMA on the morphology of polymer nano-objects prepared via aqueous photo-pisa. It should be noted that all transmission electron microscope (TEM) samples were stained with a phosphotungstic acid solution (0.5 wt% in PBS, ph 7.4) to maintain the morphology of polymer nano-objects during the sample preparation process. For a target composition of PPEGMA 9 -PHPMA 120 (prepared at 20% w/w) (Figure 2a), worm-like micelles were obtained and the PPEGMA 9 -PHPMA 120 dispersion formed a free-standing soft worm gel due to worm-worm entanglements. For a target composition of PPEGMA 9 -P(HPMA co-dmaema 10 ), spheres and short worms were formed and the obtained dispersion became free-flowing (see Figure 2b). This is a result of enhanced hydrophilicity of the PHPMA chains upon the addition of DMAEMA into the reaction mixture: this lowers the packing parameter, leading to a morphology changes from long worms to spheres and short worms. Interestingly, the morphology changed to long worms and spheres when the target composition was PPEGMA 9 -P(HPMA 120 -co-dmaema 20 ) (Figure 2c); while the morphology changed to pure worms when the target composition was PPEGMA 9 - P(HPMA 120 -co-dmaema 40 ) (Figure 2d). For both cases, (3 of 8) 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4 Facile Preparation of CO 2 -Responsive Polymer Nano-Objects via Aqueous Photoinitiated... Figure 1. a) Kinetics of aqueous photo-pisa using PPEGMA 9 -CDPA as the macro-raft agent at a 10% w/w HPMA concentration with the target composition of PPEGMA 9 -P(HPMA 100 -co-dmaema n ) (n = 0, 5, 10, 20); b) plots of ln([m] 0 /[M]) versus irradiation time for aqueous photo-pisa at a 10% w/w HPMA concentration with the target composition of PPEGMA 9 -P(HPMA 100 -co-dmaema n ) (n = 0, 5, 10, 20); c) evolution of number-average molecular weight (M n ) and polydispersity (M w /M n ) with monomer conversion for aqueous photo-pisa with the target composition of PPEGMA 9 -P(HPMA 100 -co-dmaema 10 ); d) GPC traces of PPEGMA 9 -CDPA and PPEGMA 9 -P(HPMA 100 -co-dmaema n ) (n = 0, 5, 10, 20) copolymers prepared via aqueous photo-pisa at a 10% w/w HPMA concentration. free-standing physical gels were formed, indicating the entanglement of long worms. This is different from alcoholic PISA reported in the literature [41] that increasing the amount of solvophilic comonomer led to the formation of lower order morphologies. We reason this phenomenon is due to enhanced hydrogen bonding interaction between HPMA and DMAEMA as increasing the amount of DMAEMA, promoting the formation of higher order morphology. It should be noted that increasing the degree of polymerization (DP) of DMAEMA also increased the overall DP of the core-forming block, which may also affect the morphology. Similar phenomenon was also observed for the vesicles prepared via aqueous photo- PISA, as shown in Figure 2e h. For a target composition of PPEGMA 9 -PHPMA 200 (prepared at 20% w/w), pure vesicles were formed (Figure 2e). Adding DMEAMA to the formulation (target composition of PPEGMA 9 -P(HPMA co-dmaema 10 )) formed a mixed phase (spheres, worms, and vesicles) as shown in Figure 2f, which can be ascribed to the decrease of packing parameter as the incorporation of hydrophilic monomer (DMAEMA) to the hydrophobic chains. Further increasing the amount of DMAEMA (target composition of PPEGMA 9 -P(HPMA co-dmaema 40 ) and PPEGMA 9 -P(HPMA 200 -co-dmaema 80 )) led to the formation of pure vesicles again (Figure 2g,h). A free-standing gel was formed in the case of PPEGMA 9 - P(HPMA 200 -co-dmaema 80 ) vesicles, indicating strong hydrogen bonding interaction between vesicles. The preparation of pure vesicles containing DMAEMA moieties is very important, since this kind of vesicles can be used to release cargos for specific applications (e.g., drug delivery) via CO 2 trigger. Thermo-responsive behavior of the obtained physical soft worm gel was also investigated. A dispersion of PPEGMA 9 -PHPMA 120 worms was cooled to 4 C for 2 h and then warmed to 25 C for 36 h (see Figure 2e). The original dispersion was a free-standing physical gel at 25 C, and cooling from 25 to 4 C led to degelation. This has proved to be a common feature of diblock copolymer worms with PHPMA as the core-forming block. [42,43] The decrease of temperature results in a greater degree of hydration of the PHPMA chains, leading to the decrease of packing parameter and thus the observed degelation. After warming the cold dispersion at 25 C for 36 h, gel was reformed. In contrast, the dispersion of PPEGMA 9 -P(HPMA 120 -co- DMAEMA 40 ) worms did not exhibit any degelation on cooling at 4 C for 36 h. This may be ascribed to the strong hydrogen bonding between HPMA and DMAEMA that opposes a reduction in the packing parameter, which prevents the degelation WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim (4 of 8)

5 J. Tan et al. Figure 2. Polymer nano-objects prepared via aqueous photo-pisa at a 20% w/w HPMA concentration with the target composition of PPEGMA 9 -P(HPMA 120 -co-dmaema n ): a) n = 0, b) n = 10, c) n = 20, and d) n = 40. Polymer nano-objects prepared via aqueous photo-pisa at a 20% w/w HPMA concentration with the target composition of PPEGMA 9 -P(HPMA 200 -co-dmaema n ): e) n = 0, f) n = 10, g) n = 40, and h) n = 80. i) Digital photographs recorded for the thermo-responsive behavior of a 20% w/w aqueous dispersion of PPEGMA 9 -PHPMA 120 worms. j) Digital photographs recorded for the thermo-responsive behavior of a 20% w/w aqueous dispersion of PPEGMA 9 -P(HPMA 120 -co- DMAEMA 40 ) worms. In the next set of experiments, CO 2 -responsive property of the obtained vesicles was further investigated in detail by dynamic light scattering (DLS), visual appearance, and TEM. The mechanism of CO 2 -responsive disassembly of vesicles is shown in Scheme 1b. The tertiary amine group of DMAEMA becomes more hydrophilic after CO 2 treatment, which leads to the disassembly of vesicles. As a control experiment, the dispersion of PPEGMA 9 - PHPMA 200 vesicles was treated with CO 2 for 150 s and the hydrodynamic diameter did not show noticeable difference (Figure 3a). The dispersion remained milky white even after CO 2 treatment (Figure 3b), and TEM measurement confirmed the maintenance of vesicular morphology (Figure 3c). For the sample of PPEGMA 9 - P(HPMA 200 -co-dmaema 10 ), the hydrodynamic diameter drastically decreased from nm (0.055) to nm (0.228) after CO 2 treatment (Figure 3d), and the milky white dispersion became a turbid dispersion (Figure 3e), indicating the change of morphology. TEM measurement of the sample after CO 2 treatment shows that the original worms and vesicles (Figure 2h) disintegrated into irregular nanoparticles, as shown in Figure 3f. For the samples of PPEGMA 9 -P(HPMA 200 -co-dmaema 40 ) and PPEGMA 9 - P(HPMA 200 -co-dmaema 80 ) vesicles, the vesicles disassembled into soluble polymer chains after CO 2 treatment with no signal being detected by DLS (Figure 3g,j). The milky white dispersions became transparent (Figure 3h,k), and no nano-objects were observed by TEM measurement (Figure 3i,l). These results suggest that the presence of DMAEMA moiety is critical for the disassembly of vesicles via CO 2 treatment. The aqueous photo-pisa reported in this case can be carried out at room temperature, a feature that is particularly suitable for encapsulating bio-related species (5 of 8) 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

6 Facile Preparation of CO 2 -Responsive Polymer Nano-Objects via Aqueous Photoinitiated... Figure 3. DLS size distributions recorded for samples (prepared at 20% w/w HPMA concentration) with the target composition of PPEGMA 9 - P(HPMA 200 -co-dmaema n ) before and after CO 2 treatment: a) n = 0, d) n = 10, g) n = 40, and j) n = 80. Digital photographs of the PPEGMA9- P(HPMA200-co-DMAEMA n ) dispersions (prepared at 20% w/w HPMA concentration) before and after CO 2 treatment: b) n = 0, e) n = 10, h) n = 40, and k) n = 80. TEM images of the PPEGMA 9 -P(HPMA 200 -co-dmaema n ) polymer nano-objects (prepared at 20% w/w HPMA concentration) after CO 2 treatment: c) n = 0, f) n = 10, i) n = 40, and l) n = 80. (e.g., protein) into vesicles. Herein, we encapsulated a model protein (bovine serum albumin, BSA, 5% w/w) into PPEGMA 9 -P(HPMA 200 -co-dmaema 80 ) vesicles via aqueous photo-pisa and studied the CO 2 -triggered release behavior of the obtained vesicles. Mable et al. [44] reported that BSA absorbs at 278 nm and emits at 337 nm, and thus it is possible to calculate the loading efficiency by measuring the amount of BSA in the supernatant after centrifugation. Figure 4a shows fluorescence emission spectra (excited at 278 nm) obtained for a series of BSA solutions with the concentration ranging from % to 0.01% w/w. Increasing the concentration of BSA leads to stronger fluorescence intensity, and a calibration plot was constructed with fluorescence emission at 337 nm against Figure 4. a) Fluorescence emission spectra obtained for a series of BSA solutions with the concentration ranging from to 0.01% w/w (excited at 278 nm), b) plot of fluorescence emission at 337 nm versus the concentration of BSA, and c) fluorescence emission spectra obtained for supernatants of BSA-loaded PPEGMA 9 -P(HPMA 200 -co-dmaema 80 ) vesicles aqueous solution before and after CO 2 treatment (excited at 278 nm) WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim (6 of 8)

7 J. Tan et al. the concentration of BSA (see Figure 4b). The loading efficiency was around 24% by measuring the amount of BSA in the collected supernatant after three centrifugationredispersion cycles. The obtained BSA-loaded PPEGMA 9 - P(HPMA 200 -co-dmaema 80 ) vesicles were diluted with a certain amount of water and incubated at room temperature for 1 h, then split into two centrifuged tubes with one purged with CO 2 for 150 s. Both centrifuged tubes were then centrifuged at 2000 g for 40 min, and the supernatants were measured by spectrofluorometer (excited at 278 nm) as shown in Figure 4c. It is obvious that stronger fluorescence intensity was detected after CO 2 treatment, indicating the release of BSA from the vesicles. 4. Conclusions In conclusion, we report a facile method to prepare CO 2 - responsive polymer nano-objects by photoinitiated polymerization-induced self-assembly in water at room temperature. A PPEGMA macromolecular chain transfer agent was chain-extended with HPMA and DMAEMA. Kinetic studies confirmed that polymerizations proceeded rapidly in all cases, and quantitative monomer conversions were achieved within 10 min of 405 nm visible-light irradiation. GPC analysis indicated that the presence of DMAEMA led to branching of the final polymers. Adding a small amount of DMAEMA to aqueous photo-pisa limited the evolution of higher order morphologies. Further increasing the amount of DMAEMA promoted the formation of higher order morphologies due to the enhanced hydrogen bonding interaction. Finally, CO 2 -responsive property of the obtained vesicles was studied in detail. The results showed that DMAEMA was critical for the disassembly of vesicles prepared via aqueous photo-pisa. The aqueous photo-pisa formulation reported here provides a suitable platform for in situ encapsulation and subsequently CO 2 -triggered release globular proteins and inorganic nanoparticles. Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: The authors acknowledge support from the National Natural Science Foundation of China (Grant No ), Guangdong Natural Science Foundation (Grant No. 2016A ), and the Innovation Project of Education Department in Guangdong (Grant No. 2015KTSCX029). Received: August 16, 2016; Revised: September 18, 2016; Published online: October 25, 2016; DOI: /marc Keywords: CO 2 -responsive; diblock copolymers; photo-pisa [1] H. Che, J. C. M. van Hest, J. Mater. Chem. B 2016, 4, [2] A. Feng, J. Yuan, Macromol. Rapid Commun. 2014, 35, 767. [3] A. Bagheri, J. Yeow, H. Arandiyan, J. Xu, C. Boyer, M. Lim, Macromol. Rapid Commun. 2016, 37, 905. [4] J. Tan, M. Yu, X. Rao, J. Yang, Z. Zeng, Polym. Chem. 2015, 6, [5] X. Chang, Z. Cheng, B. Ren, R. Dong, J. Peng, S. Fu, Z. Tong, Soft Matter 2015, 11, [6] Z.-Y. Qiao, R. Ji, X.-N. Huang, F.-S. Du, R. Zhang, D.-H. Liang, Z.-C. 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