Nanoflower-Shaped Biocatalyst with Peroxidase Activity Enhances the

Jan 9, 2018 - ABSTRACT: Organic−inorganic hybrid nanoflowers, facilely made from bovine serum albumin and copper phosphate (BSA−. Cu3(PO4)2·3H2O)...
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Nanoflower-Shaped Biocatalyst with Peroxidase Activity Enhances the Reversible Addition−Fragmentation Chain Transfer Polymerization of Methacrylate Monomers Xing-Huo Wang, Ming-Xue Wu, Wei Jiang, Bo-Lei Yuan, Jun Tang,* and Ying-Wei Yang* International Joint Research Laboratory of Nano-Micro Architecture Chemistry (NMAC), College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, P. R. China S Supporting Information *

ABSTRACT: Organic−inorganic hybrid nanoflowers, facilely made from bovine serum albumin and copper phosphate (BSA− Cu3(PO4)2·3H2O), have attracted considerable attention for the application of biocatalysts in recent years. The improved stability and activity of above-mentioned nanoflowers enhanced the efficiency of reversible addition−fragmentation chain transfer (RAFT) polymerization of functional methacrylate monomers with the assistance of acetylacetone (ACAC) and hydrogen peroxide (H2O2) in a mixed solvent of DMF and H2O. Such RAFT strategy can be employed for the polymerization of N,Ndimethylaminoethyl methacrylate (DMAEMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA500), in which both poly(DMAEMA) and poly(PEGMA500) could be synthesized in a controllable manner with typical RAFT features, e.g., precise control of molecular weight, specific molecular structure, and narrow polydispersity index (Mw/Mn). Significantly, the low-cost nanoflowers could be easily separated from reaction mixture after polymerization and will not adhere to resulting polymers as same as enzymes. Moreover, 1H NMR characterization of the retaining end groups of the resultant polymers and the chain extension experiments confirmed the mechanism of RAFT polymerization. The present biocatalytic system can serve as optimal alternatives of free enzymes in RAFT polymerization, which will hopefully enrich the methodology toward the construction of vinyl-based polymers with controlled radical polymerization (CRP).

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INTRODUCTION Controlled/living radical polymerization (CRP) as a robust and versatile polymerization strategy for the synthesis of various kinds of functional polymer materials has gained significant attention in the past few decades, which can be classified as atom transfer radical polymerization (ATRP), reversible addition−fragmentation chain transfer (RAFT) polymerization, and so on.1−4 RAFT polymerization possesses many advantages, such as its simple way to trigger polymerization,5 decent tolerance to a variety of functional vinyl monomers,6,7 high percentage of end-group functionalization, and good compatibility with a broad range of reaction conditions.8−11 Recently, many approaches have been developed, such as zerovalent metal/RAFT agent mediated CRP12,13 and RAFT photopolymerization,14−18 to overcome some of the limitations of RAFT polymerization, e.g., terminating of growing chains and unavoidable use of thermal initiator. In general, the thermal decomposition initiator, e.g., azodiisobutyronitrile (AIBN) used in polymerization system, demands relatively high temperature (≥60 °C).19 It is preferable to conduct polymerization at room temperature to effectively avoid thermal polymerization as well as irreversible chain transfer.20,21 Enzymatic biocatalysis has been proven to be an efficient and selective approach, as it was responsible for constructing polymers with well-defined structures at ambient temperature.22−26 However, enzymatic © XXXX American Chemical Society

biocatalysis has certain limitations, that is, high cost, harsh application conditions, and difficulty in separation, etc.27−29 Thus, exploration of more optimized catalysts is in urgent need. As the most potential alternative, hybrid nanoflower-shaped nanocrystals of bovine serum albumin−copper phosphate, i.e., BSA−Cu3(PO4)2·3H2O, comprised of organic entity and inorganic component with hierarchical flowerlike spherical architecture, will be a promising biomimetic catalyst candidate to provide a novel approach to this goal.30−32 So far, a large variety of enzymes have been immobilized on Cu3(PO4)2·3H2O to fabricate nanoflower-shaped biocatalysts, such as horseradish peroxidase,33,34 glucose oxidase,35 lipase,36 amino acid,37 and even mimic enzyme.38 Compared with free enzymes, such nanoflower-shaped biocatalysts exhibited enhanced enzyme activity and stability due to their high surface area and good confinement of organic entities. Additionally, previous studies have demonstrated that Cu3(PO4)2·3H2O frameworks intrinsically show peroxidase-like activity,31,39 the same function as laccase40 and HRP,41 because copper/iron compounds in those nanoflowers can function as Fenton-like reagents to catalyze substrates with the assistance of hydrogen peroxide (H2O2). Received: December 14, 2017 Revised: January 9, 2018

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DOI: 10.1021/acs.macromol.7b02650 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 1. Mechanism of RAFT Polymerization Initiated by BSA−Cu3(PO4)2·3H2O Hybrid Nanoflowers

Table 1. RAFT Polymerization Initiated by BSA-Cu3(PO4)2·3H2O Hybrid Nanoflowers under Different Conditions entry 1 2 3 4 5 6 7 8c a

[DMAEMA]:[CPDB]:[H2O2]:[ACAC] 400:1:0.0028:5.57 400:1:0.0028:4.18 400:1:0.0028:2.79 200:1:0.0028:5.57 200:1:0.0014:5.57 200:1:0.00056:5.57 200:1:0.00028:5.57 62:1:0.0028:5.57

nanoflower (mg) 4 4 4 4 4 4 4 4

kpapp a (min−1) 6.04 5.40 3.89 4.91 1.29 n.d. n.d. 7.97

× × × × ×

−3

10 10−3 10−3 10−3 10−3

× 10−3

time (h)

convb (%)

Mn,GPC (kg/mol)

Mw/Mn

3 3 3 3 3 24 24 2.5

63.57 58.42 42.31 56.25 28.25 7.30 0 90.71

48.05 40.50 29.39 23.73 17.12 10.40 n.d. 53.22

1.13 1.13 1.17 1.18 1.17 1.12 n.d. 1.07

calculated by the slope of the linearly increasing section of the semilogarithmic plot. bDMAEMA and PEGMA500 conversion determined by 1H NMR. cPEGMA500 used as monomer. kpapp

Hence, we envision that BSA−Cu3(PO4)2·3H2O organic− inorganic hybrid nanoflowers could catalytically generate radicals mediated by H2O2 and acetylacetone (ACAC), which could be used as initiation step in RAFT polymerization, and the biomimetic catalyst could have great potential to induce RAFT polymerization and play a role as same as HRP initiated RAFT polymerization.22 On the basis of the above considerations, herein we first present a promising strategy to construct well-defined macromolecules by combining the advantages of RAFT polymerization and the initiation step that catalytically generate radicals by BSA−Cu3(PO4)2·3H2O hybrid nanoflowers, ACAC, and H2O2 ternary system. Significantly, unlike free enzymes usually

adhere to products,23 employing Cu3(PO4)2·3H2O hybrid nanoflowers in a heterogeneous system could avoid complicated purification processes after polymerization. We take advantage of Cu3(PO4)2·3H2O hybrid nanoflowers to produce primary radicals in the chain initiation step (Scheme 1). More specifically, Cu3(PO4)2·3H2O hybrid nanoflowers could catalyze ACAC to generate ACAC radicals (ACAC•) under the presence of H2O2, which could be further used to initiate RAFT polymerization in a mixed solvent of DMF and H2O. Such nanobiocatalysts enjoy the advantages of mild reaction temperature, good control over polymer synthesis, low cytotoxicity, easy separation, and high efficiency activity. We B

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Figure 1. BSA−Cu3(PO4)2·3H2O hybrid nanoflowers initiated RAFT polymerization for poly(DMAEMA) polymers. (a) First-order kinetic plots for semilogarithmic kinetic plot of ln([M]0/[M]) (■) as a function of reaction time. (b) Monomer conversion (●) as a function of reaction time. (c) Mn (■) and Mw/Mn (●) as a function of monomer conversion. (d) Corresponding GPC traces of poly(DMAEMA) synthesized through RAFT polymerization. [DMAEMA]:[CPDB]:[H2O2]:[ACAC] = 400:1:0.0028:5.57 at 30 °C.

applied the prepared Cu3(PO4)2·3H2O hybrid nanoflowers with their intrinsic peroxidase-like activity of copper phosphate frameworks to generate ACAC radicals that could be further employed to initiate RAFT polymerization (Scheme 1). Poly(DMAEMA) Prepared by RAFT Polymerization. First of all, the initiation system was employed to polymerize N,N-dimethylaminoethyl methacrylate (DMAEMA), a typical type of functional methacrylate monomer that has been widely explored in gene delivery.42−44 To evaluate the potential of Cu3(PO4)2·3H2O hybrid nanoflowers in the catalysis of ACAC to generate ACAC•, primary polymerization of DMAEMA was conducted by monitoring the kinetics (entry 1 in Table 1). The feed molar ratio of [DMAEMA]:[CPDB]:[H2O2]:[ACAC] was maintained at 400:1:0.0028:5.57, and the polymerization was triggered by injecting 10 μL of H2O2 at 30 °C. The semilogarithmic plot of ln([M]0/[M]) vs reaction time exhibited excellent linearly increasing tendency (Figure 1a), meeting the feature of typical polymerization and demonstrating that a constant concentration of chain propagating radicals presents in the system during polymerization. The observed inhibition period was about 23 min, and the apparent propagating rate constant (kpapp) was determined to be 6.04 × 10−3 min−1. With the elongation of reaction time, monomer conversion was increasing, and 64% monomer conversion was achieved in 180 min. High monomer conversion (>83%) could be attained with further proceeding polymerization to 17 h (Figure 1b). Simultaneously, monomer conversion up to nearquantitative conversion and molecular weight (Mn) increased linearly as expected for a pseudoliving polymerization. On the other hand, the molecular weight distribution (Mw/Mn) decreased from 1.24 to 1.13, demonstrating that such RAFT polymerization initiated by Cu3(PO4)2·3H2O hybrid nanoflowers performed in a well-controlled manner (Figure 1c). The

believe that this new strategy will pave a new way in the green synthesis of functional polymeric architectures.

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RESULTS AND DISCUSSION The Cu3(PO4)2·3H2O hybrid nanoflowers materials created by a simple and facile method through self-assembly were first Table 2. Different Target DPs of Poly(DMAEMA) Synthesized by RAFT Polymerization Initiated by BSA− Cu3(PO4)2·3H2O Hybrid Nanoflowersc entry DPtarget 1 2 3 4 5 6

50 100 200 400 700 1000

DPtrue

conva (%)

time (h)

Mn,thb (kg/mol)

Mn,GPC (kg/mol)

Mw/Mn

24 55 142 331 457 816

48.72 54.55 70.81 82.67 65.22 81.58

24 24 24 24 24 24

4.05 8.79 22.49 52.18 71.96 128.41

7.10 14.60 25.93 55.48 71.68 119.68

1.06 1.18 1.18 1.19 1.16 1.23

a

DMAEMA conversion determined by 1H NMR spectroscopy. bMn,th = [DMAEMA]/[CTA] × monomer conversion ×155 + 221. c [DMAEMA] = 35% w/v, 30 °C.

reported by Zare and co-workers.27 Briefly, an aqueous solution of copper sulfate (CuSO4) was added to phosphate buffered saline (PBS) containing bovine serum albumin (BSA), followed by incubating at 25 °C for 3 days. The scanning electron microscopy (SEM) image of the Cu3(PO4)2·3H2O hybrid nanoflowers showed hierarchical architectures with high surface-to-volume ratios (Figure S1). Meanwhile, the inorganic component, copper phosphate, possessed high peroxidase-like activity because copper compounds in nanoflowers could act as Fenton-like reagents under the presence of H2O2. Thus, we C

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Figure 2. RAFT polymerization of DMAEMA initiated by BSA−Cu3(PO4)2·3H2O hybrid nanoflowers under different cycles. (a) Monomer conversion of DMAEMA during different cycles. (b) Mn and Mw/Mn during different cycles. Conditions: [DMAEMA]:[CPDB]:[H2O2]:[ACAC] = 200:1:0.0028:5.57 at 30 °C.

Figure 3. Poly(DMAEMA) synthesized by BSA−Cu3(PO4)2·3H2O hybrid nanoflowers initiated RAFT polymerization with different feed volumes of ACAC. (a) First-order kinetic plots for semilogarithmic kinetic plot of ln([M]0/[M]) as a function of reaction time. (b) Mn and Mw/Mn as a function of reaction time. Conditions: [DMAEMA]:[CPDB]:[H2O2] = 400:1:0.0028 at 30 °C.

Figure 4. First-order kinetic plots for semilogarithmic kinetic plot of ln([M]0/[M]) as a function of reaction time for the synthesis of poly(DMAEMA) by BSA−Cu3(PO4)2·3H2O hybrid nanoflowers initiated RAFT polymerization with different feed volumes of ACAC. Conditions: [DMAEMA]:[CPDB]:[ACAC] = 200:1:5.57 at 30 °C.

Figure 5. 1H NMR spectrum (400 MHz, D2O) of poly(DMAEMA) polymer synthesized by RAFT polymerization initiated by BSA− Cu3(PO4)2·3H2O hybrid nanoflowers.

Mw/Mn value was determined to be 1.24 when polymerization was conducted for 45 min, larger than that of polymerization for 180 min (Mw/Mn = 1.13). The diverse chain lengths may be caused by continuous generating radicals through the Cu3(PO4)2·3H2O hybrid nanoflowers in the initial catalytic stage. With the prolongation of the polymerization time, the obtained products possessed narrow Mw/Mn. The Mw/Mn of

the final product after polymerization for 17 h was determined to be 1.19, a little bit higher than that of the product from 3 h reaction (Mw/Mn = 1.13), which may be attributed to the higher viscosity of reaction mixture that caused the termination during last stage of polymerization reaction. Figure 1d confirmed that all polymer chains with narrow Mw/Mn shifted D

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target DPs ranged from 50 to 1 000. The inconformity between Mn,th and Mn,GPC of poly(DMAEMA) with target DPs of 50 and 100 (entries 1 and 2 in Table 2) might ascribe to the radicals incompletely transferred to chain transfer agent (CTA). However, this phenomenon could be weakened when the target DP was increased to 200 (entry 3 in Table 2), and the consistency of values between Mn,th and Mn,GPC was able to be maintained until target DP up to 1 000. Although the mixture became so viscous that the magnetic stir bar could not rotate during the last stage of polymerization, the value of Mn,GPC of target DP = 1000 (entry 6 in Table 2) determined by GPC was still close to Mn,th with the value of Mw/Mn (1.23), indicating the living characteristic of RAFT polymerization initiated by Cu3(PO4)2·3H2O hybrid nanoflowers in preparation of poly(DMAEMA) with ultrahigh Mn, more than 100 kg/mol. The recyclability of Cu3(PO4)2·3H2O hybrid nanoflowers was evaluated using DMAEMA as a model monomer of RAFT polymerization. All three cycles of polymerization exhibited low values of Mw/Mn, along with a slightly drop in monomer conversion because of the irreversible destruction to Cu3(PO4)2·3H2O hybrid nanoflowers in the mixed solvent of DMF and H2O (Figure 2). However, it is worth noting that monomer conversion was still kept in a relatively high level, more than 65%, and it was also the first time that Cu3(PO4)2· 3H2O hybrid nanoflowers were employed in the process of polymerization. Overall, compared with free enzyme separated from reaction mixture by complicated process, it is convenient for Cu3(PO4)2·3H2O hybrid nanoflowers to be separated from reaction solution by centrifugation with desirable reutilization that plays an important role in the preparation of biomedical polymers at an industrial kilogram or pilot plant scale.

Figure 6. GPC traces of chain extension of poly(DMAEMA) synthesized by RAFT polymerization with macro-CTA. Curve a: Mn = 25.93 kg/mol, Mw/Mn = 1.15. Curve b: Mn = 34.19 kg/mol, Mw/Mn = 1.15. Conditions: [DMAEMA]:[macro-CTA]:[H2O2]:[ACAC] = 200:1:0.0028:5.57 at 30 °C.

continuously to high molecular weight as measured by GPC in DMF. To evaluate the wide applicability of such a typical RAFT method, the present strategy was further employed for synthesizing various degrees of polymerization (DPs) of poly(DMAEMA) (Table 2). Almost all of the products exhibited actual molecular weights (Mn,GPC) in accord with theoretical molecular weights (Mn,th) with low Mw/Mn, less than 1.23; meanwhile, the values of Mn of the synthesized poly(DMAEMA) ranged from 7.10 to 119.68 kg/mol, and the

Figure 7. BSA−Cu3(PO4)2·3H2O hybrid nanoflowers initiated RAFT polymerization for Poly(PEGMA500) polymers. (a) First-order kinetic plots for semilogarithmic kinetic plot of ln([M]0/[M]) (■) as a function of reaction time. (b) Monomer conversion (●) as a function of reaction time. (c) Mn (■) and Mw/Mn (●) as a function of monomer conversion. (d) Corresponding GPC traces of poly(PEGMA500) synthesized through RAFT polymerization. Conditions: [PEGMA500]:[CPDB]:[H2O2]:[ACAC] = 62:1:0.0028:5.57 at 60 °C. E

DOI: 10.1021/acs.macromol.7b02650 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Effects of Substrate Concentrations on Polymerization. To gain further insight into the polymerization mechanism, the kinetics of the polymerization initiated by Cu3(PO4)2·3H2O hybrid nanoflowers with different concentrations of ACAC and H2O2 were investigated. The polymerization rate was determined by the concentration of initial radicals generated by ACAC under mild condition in the system. So, it is important to manipulate polymerization rate by adjusting the feed volume of ACAC (Figure 3a and entries 1−3 in Table 1). The semilogarithmic kinetic plot of ln([M]0/[M]) was consistent with living-like characteristics because there is a linear increase as a function of reaction time, and kpapp of feed volume of 10, 15, and 20 μL were determined to be 3.89 × 10−3, 5.40 × 10−3, and 6.51 × 10−3 min−1, respectively. The GPC traces of poly(DMAEMA) synthesized through RAFT polymerization with different feed volumes of ACAC are shown in Figures S2 and S3. The induction time of RAFT polymerization initiated by Cu3(PO4)2·3H2O hybrid nanoflowers decreased as the ACAC feed volume increased because the concentration of ACAC• might influence the induction time of RAFT process. The lower concentration of radicals needs longer equilibrium time to activate all CTAs, as a result, leading to much longer induction time. To further understand the effect of concentration of radicals generated by ACAC, Mn values as a function of reaction time were investigated to monitor the lengths of macromolecule chains in the system (Figure 3b). When the polymerization was conducted for a certain period of time, the obtained products exhibited higher values of Mn with the increase of ACAC feed volume from 10 to 20 μL. In addition, the Mw/Mn values in all three systems were low to 1.13 after polymerizing for 3 h, demonstrating that the polymers regulated by feed volume of ACAC were all propagating in a living fashion. However, it was interesting that when the feed volume of ACAC was 10, 15, and 20 μL, Mn of the final obtained products was measured to be 34.23, 48.41, and 55.48 kg/mol, respectively, with an increasing monomer conversion of 48.32%, 74.72%, and 82.67%, respectively. The concentration of another cosubstrate H2O2 could also act as a limiting factor during such RAFT polymerization,20 and changing the feed volume of H2O2 will also make a difference in polymerization kinetics behavior (entries 4−7 in Table 1). The polymerization rate was characterized by the semilogarithmic kinetic plot of ln([M]0/[M]) as a function of reaction time, and kpapp values of 5 and 10 μL of H2O2 were determined to be 1.29 × 10−3 and 4.91 × 10−3 min−1, respectively (Figure 4). The higher slope proves the faster polymerization rate, demonstrating that the feed volume of H2O2 has an impact on kinetics behavior of the RAFT process; more specifically, by adjusting the feed volume of H2O2 from 10 to 5 μL, monomer conversion was decreased from 56.25% to 28.25% after 180 min of polymerization. These results suggested that no matter how sufficient the ACAC added, the feed volume of H2O2 was capable of acting as a limiting factor to confine polymerization rate. Further experiments were conducted to validate this point: (a) if the feed volume of H2O2 was decreased to 2 μL (entry 6 in Table 1), monomer conversion could only reach 7.30% even after polymerization for 24 h, and (b) no polymer was detected while the feed volume of H2O2 was only 1 μL (entry 7 in Table 1). Taking the above experimental results into consideration and comparing with other conventional RAFT polymerization, the polymerization rate of the present system could be regulated by

controlling the feed volume of ACAC and H2O2; that is to say, both substrate concentrations could act as limiting factors to restrict the polymerization rate, which provides a new insight for kinetics studies of RAFT polymerization. Proof of “Living” Feature of Polymerization. The representative structure of poly(DMAEMA) was characterized by 1H NMR spectroscopy after dialysis and lyophilization (Figure 5). The signals at δ = 0.94 ppm (a) and δ = 1.93 ppm (b) were assigned to methyl and methylene in the backbone chain, suggesting that successful RAFT polymerization was initiated by Cu3(PO4)2·3H2O hybrid nanoflowers with the assistance of ACAC and H2O2. Moreover, the chemical shifts observed at δ = 7.66−8.05 ppm were derived from Z group (phenyl group) of CTA, indicating good retention of the end group of CTA at the end of macromolecular chains. However, the proton resonances of terminal group of CTA at the end of macromolecule chain were overlapped with the proton resonances of polymer, so that the methyl signals were hardly distinguished. In order to further prove the existence of terminal groups at the end of polymer chain after RAFT polymerization initiated by Cu3(PO4)2·3H2O hybrid nanoflowers, chain extension experiment was performed with immediate separation step after macro-CTA (poly(DMAEMA)) synthesized to a target Mn. First, RAFT polymerization of DMAEMA initiated by Cu3(PO4)2·3H2O hybrid nanoflowers was performed with a feed molar ratio of [DMAEMA]:[CPDB]:[H2O2]:[ACAC] = 200:1:0.0028:5.57, and Cu3(PO4)2·3H2O hybrid nanoflowers in the mixture were separated immediately by centrifugation after polymerization, followed by dialysis against distilled water prior to lyophilization to obtain pure polymer. The values of Mn and Mw/Mn of the resulting macro-CTAs were determined to be 25.93 kg/mol and 1.18, respectively, with a monomer conversion of 70.81%. Then, the chain extension experiment was conducted with feed molar ratio of [DMAEMA]:[macroCTA]:[H2O2]:[ACAC] = 200:1:0.0028:5.57, Mn and Mw/Mn of the products were evaluated by GPC. Compared with macroCTA, the Mn of chain-extented poly(DMAEMA) increased to 34.91 kg/mol, while the Mw/Mn decreased to 1.15. These results clearly indicated that a successful chain extension occurred in polymerization and confirmed high percentage of end-group functionalization of polymers (Figure 6). Poly(PEGMA500) Prepared by RAFT Polymerization. Poly(PEGMA500) was prepared through typical RAFT process initiated by Cu3(PO4)2·3H2O hybrid nanoflowers with higher polymerization reaction temperature. Poly(PEGMA) bears different lengths of oligo(ethylene oxide) as side chains and represents one of the most promising biomaterials due to its desirable biocompatibility and nontoxic.45 Thus, it is very appealing to construct such polymer through our newly developed green catalytic method.46 To obtain poly(PEGMA500), we need to increase temperature to 60 °C. It is amazing that Cu3(PO4)2·3H2O hybrid nanoflowers could bear such a high temperature to catalyze ACAC to generate radicals, which is responsible for activating CTA to promote RAFT polymerization. The feed molar ratio of [PEGMA500]:[CPDB]:[H2O2]:[ACAC] was maintained at 62:1:0.0028:5.57, and the polymerization was triggered by injecting 10 μL of H2O2 (entry 8 in Table 1). When the monomer conversion ranged from 5.66% to 90.97% in RAFT polymerization, the obtained semilogarithmic kinetic plot of ln([M]0/[M]) vs reaction time exhibited a linear change during the reaction, which was in accordance with the F

DOI: 10.1021/acs.macromol.7b02650 Macromolecules XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (51473061), Major Science and T ec h nol o g y R es e ar ch Pro je c t o f J il in P r o vi n c e (20140203012G X), Jilin Province-University Cooperative Construction Project − Special Funds for New Materials (SXGJSF2017-3), Jilin University Talents Cultivation Program, and the Fundamental Research Funds for the Central Universities.

RAFT feature (Figures 7a and 7b). The induction period was about 8 min, a much shorter period than RAFT polymerization of DMAEMA, with kpapp = 7.97 × 10−3 min−1. Compared with the polymerization process of DMAEMA (Figure 1), the polymerization rate of PEGMA500 was faster. In addition, the monomer conversion was increased abruptly after polymerization for 120 min, yet, the value of Mw/Mn was determined to be 1.07, indicating that the controllability could maintain during the whole process of polymerization and almost no free radicals termination occurred between two radicals. This phenomenon could be attributed to the high viscosity of the resulting poly(PEGMA500) when monomer conversion reached a high level during the end stage of polymerization. The values of Mn and Mw/Mn of the first sample obtained from the reaction mixture were 15.71 kg/mol and 1.11, respectively. With the elongation of reaction time, the Mn increased linearly with the rise of monomer conversion in the entire polymerization reaction, which reached 53.22 kg/mol at 150 min with 1.07 Mw/Mn (Figure 7c). GPC traces are monomodal and shifted continuously toward high molecular weight (Figure 7d). The Mn of poly(PEGMA500) measured by GPC using DMF as the eluent was higher than its theoretical value, which was caused by large hydrodynamic radius of poly(PEGMA500) in DMF.

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CONCLUSIONS In conclusion, RAFT polymerization was carried out by a new initiation approach, in which radicals were generated by ACAC under the catalyzation of Cu3(PO4)2·3H2O hybrid nanoflowers. BSA−Cu3(PO4)2·3H2O hybrid nanoflowers endowed RAFT polymerization of DMAEMA and PEGMA500 with narrow Mw/ Mn, well-controlled Mn, and definite structures. Moreover, the kinetics of polymerization could be manipulated by changing the feed volumes of ACAC and H2O2. 1H NMR spectroscopy and chain experiment further proved the existence of Z group (phenyl group) of CTA. Well-defined poly(DMAEMA) with DPs ranged from 50 to 1000 were synthesized with low values of Mw/Mn. Most important of all, the Cu3(PO4)2·3H2O hybrid nanoflowers could be separated by simple purification processes and recycled for at least three times. We expect that Cu3(PO4)2· 3H2O hybrid nanoflower-based biocatalytic system will perfectly improve RAFT techniques and hopefully provide a promising and intriguing direction for polymer synthesis. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b02650. Experimental details including materials, characterization, and synthesis; SEM and GPC data (Figures S1−S3) (PDF)

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Corresponding Authors

*E-mail: [email protected] (J.T.). *E-mail: [email protected] (Y.-W.Y.). ORCID

Ying-Wei Yang: 0000-0001-8839-8161 Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acs.macromol.7b02650 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.7b02650 Macromolecules XXXX, XXX, XXX−XXX