Fabrication of a Thermoresponsive Biohybrid Double Hydrophilic Block Copolymer by a Cofactor Reconstitution Approach a

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1 Communication Fabrication of a Thermoresponsive Biohybrid Double Hydrophilic Block Copolymer by a Cofactor Reconstitution Approach a Xuejuan Wan, Shiyong Liu* The fabrication of a thermoresponsive biohybrid double hydrophilic block copolymer (DHBC) by a cofactor reconstitution approach is reported. Poly(-isopropylacrylamide) (PIPAM) bearing a porphyrin moiety at the chain terminal, PPIXZn-PIPAM, is synthesized by the combination of ATRP and a click reaction. The subsequent cofactor reconstitution process between apomyoglobin and PPIXZn-PIPAM affords well-defined myoglobin-b-pipam protein polymer bioconjugates. Behaving as typical responsive DHBCs, the obtained myoglobin-b- PIPAM biohybrid diblock copolymer exhibits thermoinduced aggregation behavior in aqueous solution as a Cofactor Reconstitution result of the presence of the thermoresponsive PIPAM Myoglobin Apomyoglobin block, as revealed by temperature-dependent transmittance, dynamic laser light scattering measurements, Heating transmission electron microscopy, and scanning electron Cooling microscopy. This work represents the first report of the Myoglobin-b-PIPAM Unimers at 25 preparation of responsive biohybrid DHBCs by the cofac- o C PIPAM-Core Micelles Stabilized tor reconstitution process. by Myoglobin at 45 o C Introduction X. Wan, S. Liu CAS Key Laboratory of Soft Matter Chemistry, Hefei ational Laboratory for Physical Sciences at the Micrometer-Scale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui , P. R. China Fax: (þ86) ; sliu@ustc.edu.cn a : Supporting information for this article is available at the bottom of the article s abstract page, which can be accessed from the journal s homepage at or from the author. Protein polymer bioconjugates have received ever increasing attention during the past decades because of their potential applications in biomedicine and biotechnology fields. [1 3] Generally speaking, the modification of native proteins with synthetic polymers can endow them with improved solubility and stability, increased plasma halflives and bioavailability, and reduced immunogenicity. Synthetic strategies typically employed for the preparation of protein polymer conjugates can be categorized into two main types. The conventional one relies on the covalent or non-covalent attachment of functionalized polymer precursors onto proteins. A variety of high-efficiency coupling protocols such as amidation reactions between amine residues and activated esters, [4] thiol-ene [5] and azide/ alkynyl click reactions, [6 8] and thiol/pyridyl disulfide exchange reactions [9] have been successfully employed to covalently conjugate well-defined synthetic polymer precursors to native proteins. As for non-covalent approaches, biotin/avidin, [10,11] saccharide/concanavalin A, [12] and other host guest [13] molecular recognition events or metal ligand coordination interactions [14] have been utilized for the preparation of polymer protein conjugates. The second strategy relies on well-developed controlled radical polymerization techniques [15 17] such as atom transfer radical polymerization (ATRP) and reversible 2070 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com DI: /marc

2 Fabrication of a Thermoresponsive Biohybrid Double Hydrophilic Block... addition fragmentation chain transfer (RAFT) polymerization, which allow for the direct synthesis of polymer protein conjugates from protein-based ATRP macroinitiators [18 20] and macro-raft mediating agents [5,9,21] in the presence of suitable monomers. Proteins modified with hydrophobic and hydrophilic polymer segments can be considered as biohybrid amphiphilic and double hydrophilic block copolymers (DHBCs), respectively. [22,23] The presence of bioactive natural macromolecular building blocks can endow these biohybrid block copolymers with novel aggregation and self-assembling properties. [24,25] Furthermore, if the synthetic polymer blocks are ph- or thermo-sensitive, the obtained responsive biohybrid block copolymers can exhibit extra advantages such as facile purification/recovery and external stimulitunable bioactivity. [26,27] In this aspect, a few research groups have reported the preparation of covalent conjugates of proteins such as bovine serum albumin (BSA), [6,28] lysozyme, [29] and streptavidin [10,11] with poly(-isopropylacrylamide) (PIPAM), which is well-known to possess a lower critical solution temperature (LCST) of 32 8C in aqueous solution. Highly efficient coupling reactions or controlled radical polymerization from initiator-derivatized proteins were typically employed. Sumerlin et al. [5] reported that BSA-PIPAM conjugates can be purified by thermal precipitation to remove free BSA by taking advantage of the thermoresponsiveness of PIPAM segments. The obtained polymer protein conjugates demonstrate > 90% retention of catalytic activity at 25 8C as compared to the native BSA. Moreover, the catalytic activity can be facilely tuned by temperature: upon heating to above the LCST of PIPAM segments, the activity decreases to 75%. Certain proteins contain cofactors that are bound together by specific non-covalent interactions to exhibit bioactivity, and the property of proteins can be modulated by cofactor modification. [30,31] This unique property has been utilized by olte and co-workers to synthesize polymer protein conjugates by the cofactor reconstitution approach, starting from cofactor-functionalized polymer precursors. [32 34] Under certain conditions, the obtained biohybrid block copolymers can still partially retain the activity and functionality of native proteins. They have prepared a variety of amphiphilic biohybrid block copolymers based on the modification of horseradish peroxidase (HRP) or myoglobin with synthetic segments such as polystyrene-block-poly(ethylene oxide) (PE-b-PS) copolymers or PS homopolymers, and investigated their selfassembling properties in aqueous solution. [32 34] To the best of our knowledge, the cofactor reconstitution approach has not been utilized to synthesize double hydrophilic thermoresponsive biohybrid block copolymers. Inspired by the work of olte and co-workers, [32 34] herein we report on the fabrication of thermoresponsive protein polymer bioconjugates by cofactor reconstitution and their thermo-induced aggregation behavior in aqueous solution (Scheme 1). PIPAM bearing a porphyrin moiety at the chain terminal, PPIXZn-PIPAM, was synthesized by the combination of ATRP and click reaction. The subsequent cofactor reconstitution process between apomyoglobin and PPIXZn-PIPAM led to the facile preparation of well-defined protein polymer bioconjugates. UV-Vis measurements and electrophoretic migration shift assays were employed to verify the successful reconstitution of apomyoglobin with PPIXZn-PIPAM. The obtained biohybrid diblock copolymer, myoglobin-b-pipam, behaves as a typical responsive DHBC, exhibiting thermo-induced aggregation in aqueous solution as a result of the presence of the thermoresponsive PIPAM segment. Experimental Part Materials -Isopropylacrylamide (IPAM, 95%, TCI) was recrystallized twice from benzene/hexane (2 : 1, v/v) prior to use. 2-(2-Aminoethoxy)- ethanol (98%) and di-tert-butyl dicarbonate (97%) were purchased from Acros and used without purification. Myoglobin from equine skeletal muscle ( 98%, SDS-PAGE) was purchased from Sigma. Protoporphyrin IX (PPIX, 98%) (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBP, 98%), diisopropylethylamine (DIPEA, 99%),,, 0, 00, 00 -pentamethyldiethylenetriamine (PMDETA, 98%), copper(i) bromide (CuBr, 99%), tris(2- aminoethyl)amine (TRE, 96%), propargyl alcohol (99%), and 2-chloropropionic acid (98%) were purchased from Aldrich and used as received. Tris(2-(dimethylamino)ethyl)amine (Me 6 TRE), [35] alkynyl-functionalized Wang resin, [36] and propargyl 2-chloropropionate [37] were synthesized according to literature procedures. All other chemicals were purchased from Shanghai Chemical Reagent Co. and used as received. Synthetic schemes employed for the preparation of PIPAM bearing a porphyrin moiety at the chain terminal, PPIXZn-PIPAM, and the protein polymer bioconjugates myoglobin-b-pipam are shown in Scheme 1. Detailed synthetic procedures for thepreparation of 2-(2-azidoethoxy)ethylamine (1), protoporphyrin IX mono t-butyl ester (2), azide-functionalized protoporphyrin IX (3), and azidefunctionalized PPIXZn (4) and their characterization data are described in the Supporting Information (Figures S1 and S2). Preparation of PPIXZn-PIPAM (5) PPIXZn-PIPAM was prepared by the click reaction of azidefunctionalized PPIXZn (4) with alkynyl-pipam. Alkynyl-PIPAM was prepared according to literature procedures by utilizing propargyl 2-chloropropionate as ATRP initiator. [37] Gel permeation chromatography (GPC) analysis revealed a number-average molecular weight, M n, of and an M w =M n of H MR analysis indicated a degree of polymerization (DP) of 90. Thus, the alkynyl-terminated PIPAM was denoted as alkynyl-pipam 90. Subsequently, azide-functionalized PPIXZn (44 mg, 0.06 mmol), alkynyl-pipam (0.55 g, 0.05 mmol), and PMDETA (17 mg, 0.1 mmol) were dissolved in 20 ml of,-dimethylformamide ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3 X. Wan, S. Liu a) H (i) HCl H 2 Cl (ii) SCl 2,CHCl 3 H 2 a 3,DMF 80 o C H H H H (Boc) 2, DMAP Pyridine H H H 3 H 2 1 (i) PyBP, CHCl 3 (ii) TFA, Formic acid H H H H 3 PPIX PPIX Mono t-butyl ester (+regioisomer) 2 Azide-functionalized PPIX (+regioisomer) 3 Zn(AC ) 2 Zn H H Cl n H Alkynyl-PIPAM CuBr / PMDETA Zn H H 3 PPIXZn-PIPAM (+regioisomer) 5 Azide-functionalized PPIXZn (+regioisome r) 4 b) Myoglobin Cofactor Reconstitution Apomyoglobin Heating Myoglobin-b-PIPAM Unimers at 25 o C Cooling PIPAM-Core Micelles Stabilized by Myoglobin at 45 o C Scheme 1. Schematic illustration of the preparation of a) poly(-isopropylacrylamide) (PIPAM) bearing a porphyrin moiety at the chain terminal, PPIXZn-PIPAM 90, and b) thermoresponsive DHBC copolymer, myoglobin-b-pipam 90, by the cofactor reconstitution approach. (DMF) in a Schlenk flask, and the mixture was subjected to three freeze/pump/thaw cycles. CuBr (14 mg, 0.1 mmol) was then added, and the Schlenk flask was sealed under vacuum. The reaction mixture was allowed to stir at room temperature overnight. Alkynyl-functionalized resin (0.2 g, 0.28 mmol theoretically) was then added and the reaction mixture was left to stir for another 4 h. Afterwards, the resin was filtered off and washed with CH 2 Cl 2 (3 20 ml). After removing all the solvents under reduced pressure, the crude product was dissolved in CH 2 Cl 2, and passed through a neutral alumina column to remove the catalyst. The product was dried at room temperature in a vacuum oven to constant weight (M n;gpc ¼ , M w =M n ¼ 1.10; Figure S3). Reconstitution of Apomyoglobin with PPIXZn- PIPAM Apomyoglobin was prepared from myoglobin according to literature procedures. [38,39] The reconstitution of apomyoglobin with PPIXZn-PIPAM 90 was conducted at 25 8C by mixing a 1 ml aqueous solution of PPIXZn-PIPAM (0.2 M PPIXZn moieties) into 10 ml of an aqueous phosphate-buffered solution ( M,pH 7.4) of apomyoglobin (0.01 M). After 24 h of gentle shaking, the excess PPIXZn-PIPAM was removed by dialysis against aqueous phosphate buffer ( M, ph 7.4) using 14 kda cut-off dialysis tubing ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DI: /marc

4 Fabrication of a Thermoresponsive Biohybrid Double Hydrophilic Block... Characterization All 1 H MR spectra were measured on a Bruker 300 MHz spectrometer using d 6 -DMS or CDCl 3 as solvents. Molecular weight distributions were determined by GPC using a series of three linear Styragel columns (HT2, HT4, and HT5) and a column temperature of 35 8C. A Waters 1515 pump and Waters 2414 differential refractive index detector (set at 30 8C) were used. The eluent was DMF þ 1gL 1 LiBr at a flow rate of 1.0 ml min 1.A series of six PS standards with molecular weights ranging from 800 to g mol 1 were used for calibration. All FT-IR spectra were measured on a Bruker Vector 22 Fourier transform infrared spectrometer using the KBr disk method. Absorbance measurements were performed on a Unico UV/vis 2802PCS spectrophotometer. The transmittance of the aqueous solutions was acquired at a wavelength of 700 nm. SDS-PAGE was conducted using 0.75 mm thick slab gels. Separating gels contained 10% acrylamide, 0.27% bis-acrylamide, and 1% SDS in 1.5 M Tris-HCl (ph 8.8). An aqueous solution containing 0.1% SDS, M Tris-HCl, and M glycine (ph 8.3) was used as the migration buffer. PAGE was conducted using 0.75 mm thick slab gels. Separating gels were composed of 10% acrylamide and 0.27% bis-acrylamide in 1.5 M Tris-HCl (ph 8.8). An aqueous solution containing M Tris-HCl and M glycine (ph 8.3) was used as the migration buffer. Gels were stained with Coomassie brilliant blue. Laser light scattering (LLS) measurements were conducted on a commercial spectrometer (ALV/DLS/SLS-5022F) equipped with a multi-taudigital time correlator (ALV5000) and a cylindrical 22 mw UIPHASE Hee laser (l 0 ¼ 632 nm) as the light source. Transmission electron microscopy (TEM) observations were conducted on a Hitachi H-800 electron microscope at an acceleration voltage of 200 kv. Scanning electron microscopy (SEM) images were taken on a JSM-6700F scanning electron microscope working at an acceleration voltage of 5 kev. Results and Discussion Synthetic schemes employed for the preparation of PIPAM bearing a porphyrin moiety at the chain terminal, PPIXZn-PIPAM, and the protein polymer bioconjugates myoglobin-b-pipam are shown in Scheme 1. Starting from PPIX, the reaction with di-tert-butyl dicarbonate led to PPIX mono t-butyl ester (2). This was followed by amidation of 2 with 2-(2-azidoethoxy)ethylamine. The subsequent coordination with Zn ions and click reaction with alkynyl- PIPAM leads to PPIXZn-PIPAM. The target responsive polymer protein conjugates were prepared by the reconstitution of apomyoglobin with PPIXZn-PIPAM under mild conditions, taking advantage of the highly specific interactions between apomyoglobin with the PPIXZn cofactors. Preparation of PPIXZn-PIPAM The coordination of compound 3 with zinc acetate afforded azide-functionalized PPIXZn (4, Scheme 1). From the UV-vis absorbance spectra of 3 and 4 as shown in Figure S2, we can clearly observe that the absorption maximum at l ¼ 400 nm for compound 3 is shifted to l ¼ 415 nm for compound 4. This is in agreement with previous literature reports. [32] The subsequent Cu I -mediated click reaction of azide-functionalized PPIXZn with an alkynyl-functionalized PIPAM homopolymer afforded PIPAM bearing a PPIXZn terminal functionality, PPIXZn-PIPAM. The coordination of azide-functionalized PPIX with zinc ions before the click step can avoid the binding of copper species into porphyrin rings. Azide-functionalized PPIXZn was then click conjugated with alkynyl-pipam to afford PPIXZn- PIPAM (5). UV-Vis measurements revealed that PPIXZn- PIPAM possesses the same absorption maximum (l ¼ 415 nm) as that of azide-functionalized PPIXZn (Figure S2), suggesting that the presence of the copper catalyst does not affect the preformed complexation between zinc ions and porphyrin moieties. Figure S4 shows the 1 H MR spectrum of PPIXZn- PIPAM 90, together with its peak assignments. Resonance signals at ppm are ascribed to the methenyl protons of the PIPAM repeating units (peak c). The integration area between peak f in the PPIXZn moieties and peak c was determined to be 45, which demonstrates the successful coupling of azide-functionalized PPIXZn and alkynyl- PIPAM. From the inset of Figure S4, the resonance signals ascribed to PPIX and the proton of the newly formed 1,2,3- triazole ring (peak h) can also be clearly discerned. GPC traces of alkynyl-pipam 90 and PPIXZn-PIPAM 90 are shown in Figure S3. After click conjugation, the GPC elution trace of PPIXZn-PIPAM 90 exhibits a shift towards higher molecular weight revealing an M n of and an M w =M n of Most importantly, the elution trace is mono-modal and quite symmetric, implying the successful coupling reaction. The efficiency of the click reaction between 4 and alkynyl-terminated PIPAM 90 was also checked by FT-IR analysis (Figure S5). Compared to the precursors, we can clearly observe the complete disappearance of the absorption band characteristic of the azide group at cm 1 and the alkynyl group at cm 1 in PPIXZn-PIPAM 90, which further confirms the successful preparation of PPIXZn-PIPAM 90. Cofactor-terminated PIPAM, PPIXZn- PIPAM 90, possesses a residual carboxylic acid functionality to effectively retain protein activity. [34,40] Moreover, a spacer group was incorporated between PPIXZn and the PIPAM chain, which is advantageous for the reconstitution of apomyoglobin with PPIXZn-PIPAM 90. [41] Preparation of Myoglobin-b-PIPAM Conjugates by Cofactor Reconstitution Direct chemical modification of the PPIX cofactor within the myoglobin molecule has been reported to be difficult because it is buried in the hydrophobic domain and the ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

5 X. Wan, S. Liu multistep reactions and purification process involved in chemical modifications might be detrimental to proteins. [39] The reconstitution of apomyoglobin with a prefunctionalized PPIX cofactor can be conducted under quite mild conditions. Taking advantage of this property, olte and co-workers successfully synthesized bioconjugates consisting of HRP or myoglobin and synthetic polymers such as PE-b-PS diblock copolymers or PS homopolymers. [32 34] It is worth noting that the PS segment is highly hydrophobic and the PE segment is hydrophilic, thus, the obtained bioconjugates are amphiphilic and can spontaneously self-assemble into nanostructured aggregates with a variety of morphologies. In this work, we aim to synthesize a thermoresponsive myoglobin-b-pipam biohybrid diblock copolymer, which can dissolve as unimers in aqueous media at room temperature and self-assemble into aggregates above the LCST of the PIPAM segments (Scheme 1b). The procedures employed for the preparation of apomyoglobin follows those reported by the Teale research group. [38,39] The UV-vis absorption spectrum obtained for apomyoglobin is shown in Figure 1. A comparison of the absorption spectra of myoglobin and apomyoglobin clearly reveals the disappearance of the absorption maximum at 400 nm, which suggests the cofactor has been successfully removed from myoglobin. The reconstitution of apomyoglobin with PPIXZn-PIPAM was conducted in aqueous media. The amount of PPIXZn-PIPAM added is approx. two times relative to that of apomyoglobin to ensure the complete reconstitution process. After incubation for 48 h, the reaction mixture was dialyzed against a phosphate buffer ( M, ph 7.4) to remove the excess PPIXZn- PIPAM. Compared to the UV-vis spectra of PPIXZn- PIPAM (Figure 1), we can observe the shift of absorption maximum from 415 to 428 nm for myoglobin-b-pipam conjugates, implying the successful preparation of a biohybrid diblock copolymer. In order to confirm the conjugation structure of myoglobin-b-pipam, electrophoresis migration-shift assays were carried out and the results are shown in Figure 2. Under SDS-PAGE conditions, proteins are denaturated and the cofactor apomyoglobin interaction is also disrupted. From Figure 2, we can judge that there exist almost no differences between myoglobin, apomyoglobin, and myoglobin-b-pipam conjugates, note that PPIXZn- PIPAM 90 alone does not exhibit any traces because of its inability to be stained by Coomassie brilliant blue. Under PAGE conditions, myoglobin, apomyoglobin, and myoglobin-b-pipam conjugates can be separated based on their structures and interactions with the gel matrix. We can clearly observe the differences in electrophoretic mobility between myoglobin and apomyoglobin. Most importantly, we can also tell that the electrophoretic mobility of myoglobin-b-pipam conjugates is quite different from those of myoglobin and apomyoglobin, although we currently do not understand why the biohybrid diblock copolymer exhibits a larger electrophoretic mobility compared to that of apomyoglobin. The protein cofactor interaction remains intact, and a difference is, therefore, expected between the native protein and the biohybrid macromolecule. It was worth noting that the results of the electrophoresis migration-shift measurement for myoglobin and apomyoglobin are quite different, because apomyoglobin has a remarkable difference to the native protein in its solution conformation and reactivity. Moreover, the electrophoresis strip of myoglobin-b-pi- PAM is quite stretched, as compared to that obtained under SDS-PAGE conditions. This should be ascribed to the polydisperse nature of the PPIXZn-PIPAM 90 chains. Based on the above electrophoresis migration-shift assay myoglobin apomyoglobin PPIXZn-PIPAM 90 myoglobin-b-pipam 90 Abs Wavelength / nm Figure 1. UV-Vis spectra recorded in phosphate buffer (ph 7.4) for a) myoglobin with l max at 409 nm, b) apomyoglobin with l max at 280 nm which is typical of protein, c) PPIXZn-PIPAM 90 with l max at 415 nm, and d) myoglobin-b-pipam 90 with l max at 428 nm. Figure 2. SDS-PAGE and PAGE of myoglobin-b-pipam and its precursors. Lane M: marker, lane 1: myoglobin, lane 2: apomyoglobin, lane 3: PPIXZn-PIPAM 90, and lane 4: myoglobin-b-pi- PAM ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DI: /marc

6 Fabrication of a Thermoresponsive Biohybrid Double Hydrophilic Block... results, we can conclude that the myoglobin-b-pipam conjugate was successfully obtained. Thermoresponsive Aggregation of Myoglobin-b-PIPAM Bioconjugates Taking advantage of the thermoresponsiveness of the PIPAM segments, the obtained protein polymer bioconjugates are expected to molecularly dissolve in aqueous solution at room temperature, and self-assemble into core shell aggregates at elevated temperatures. The temperature-dependent optical transmittance of a PPIXZn-PIPAM 90 aqueous solution was then determined (Figure 3a). The LCSTs were defined as the temperature corresponding to a 1% decrease in optical transmittance at 700 nm. For alkynyl-pipam 90, the transmittance exhibits abrupt changes at temperatures above 32 8C; whereas for PPIXZn-PIPAM, the LCST f (R h ) Transmittance % alkynyl-pipam 90 PPIXZn-PIPAM 90 myoglobin-b-pipam Temperature / o C 25 o C 45 o C R h /nm Figure 3. a) Temperature dependence of optical transmittance at 700 nm obtained for 2.0 g L 1 aqueous solutions (phosphate buffer, ph 7.4) of alkynyl-pipam 90, PPIXZn-PIPAM 90, and myoglobin-b-pipam 90. The LCST was defined as the temperature corresponding to a 1% decrease in optical transmittance. b) Hydrodynamic radius distributions, f(r h ), obtained for a 0.4 g L 1 aqueous solution of myoglobin-b-pipam 90 at 25 and 45 8C, respectively. Figure 4. a) TEM and b) SEM images of core shell aggregates formed from myoglobinb-pipam 90 at 45 8C. a) b) deceases to 30 8C, which suggests that the PPIXZn terminal moiety is slightly hydrophobic. Previous literature reports have established that the incorporation of hydrophilic and hydrophobic residues into PIPAM can accordingly increase or decrease its phase transition temperatures. [42] As compared to that of PPIXZn-PIPAM 90, the LCST of the myoglobin-b-pipam 90 biohybrid diblock copolymer considerably increases to 36 8C, which can be clearly ascribed to the attachment of highly hydrophilic myoglobin to the thermoresponsive PIPAM segment. Moreover, the biohybrid diblock copolymer can apparently form stable aggregates as the optical transmittance only decreases to 60% at elevated temperatures, which is in marked contrast to those of alkynyl-pipam 90 and PPIXZn-PI- PAM 90 (Scheme 1b). When the aqueous solution of PPIXZn- PIPAM 90 was subjected to repeated heating and cooling cycles, temperature-dependent optical transmittance exhibits full reversibility. This indicates that the thermoinduced assembly of PPIXZn-PIPAM 90 is completely reversible. We further employed dynamic LLS to characterize the aggregates formed from myoglobin-b-pipam 90 at elevated temperatures (Figure 3b). An aqueous solution of the biohybrid DHBC at 20 8C exhibits an intensity average hydrodynamic radius, <R h >,of5 6 nm, which confirms that the diblock copolymer molecularly dissolves in solution. Upon heating to 45 8C, which is above the LCST of the PIPAM segment, dynamic LLS reveals the formation of stable aggregates with a <R h > of 60 nm and a relatively narrow polydispersity of This clearly suggests the formation of PIPAM-core micelles stabilized by myoglobin coronas above the LCST of the PIPAM segments. TEM and SEM observations were further performed to examine the actual morphologies of the micelles formed from myoglobin-b-pipam at elevated temperatures (Figure 4). Both images clearly revealed the presence of spherical nanoparticles. Based on chemical intuition, these nanoparticles should consist of hydrophobic PIPAM cores stabilized by hydrophilic myoglobin coronas. ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

7 X. Wan, S. Liu Conclusion We fabricated thermoresponsive biohybrid DHBCs by the cofactor reconstitution approach and investigated their thermo-induced aggregation behavior in aqueous solution. A combination of ATRP and click reaction was utilized to synthesize PIPAM bearing a porphyrin moiety at the chain terminal, PPIXZn-PIPAM. The cofactor reconstitution of apomyoglobin with PPIXZn-PIPAM was then conducted to obtain well-defined protein polymer bioconjugates, myoglobin-b-pipam, which were characterized by UVvis spectroscopy and electrophoretic migration shift assays. The obtained myoglobin-b-pipam biohybrid diblock copolymer behaves as a typical responsive DHBC and self-assembles into PIPAM-core micelles stabilized with hydrophilic myoglobin coronas above the phase transition temperature of the PIPAM segment. This work represents the first report of the preparation of a responsive biohybrid double hydrophilic block copolymer by the cofactor reconstitution approach. Acknowledgements: The financial support of ational atural Science Foundation of China (SFC) Projects ( , , and ) and Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) is gratefully acknowledged. Received: June 8, 2010; Revised: August 4, 2010; Published online: ctober 13, 2010; DI: /marc Keywords: biohybrids; block copolymers; cofactor reconstitution; myoglobin; self-assembly [1] R. Duncan, at. Rev. Drug Discovery 2003, 2, 347. [2] Z. L. Ding, R. B. Fong, C. J. Long, P. S. Stayton, A. S. Hoffman, ature 2001, 411, 59. [3] L. A. Canalle, D. W. P. M. Lowik, J. C. M. van Hest, Chem. Soc. Rev. 2010, 39, 329. [4] M. J. Roberts, M. D. Bentley, J. M. Harris, Adv. Drug Delivery Rev. 2002, 54, 459. [5] P. De, M. Li, S. R. Gondi, B. S. Sumerlin, J. Am. Chem. Soc. 2008, 130, [6] M. Li, P. De, S. R. Gondi, B. S. Sumerlin, Macromol. Rapid Commun. 2008, 29, [7] M. van Dijk, D. T. S. Rijkers, R. M. J. Liskamp, C. F. van ostrum, W. E. 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