Thermoresponsive Dynamic Covalent Polymers with Tunable

Aug 26, 2013 - (3, 4) The reversible nature of dynamic covalent linkages enables ... with the adaptive behavior of constitutional dynamic systems. ...
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Thermoresponsive Dynamic Covalent Polymers with Tunable Properties Jingyi Li, Shixia Yang, Lin Wang, Xiaobei Wang, and Li Liu* Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, P. R. China S Supporting Information *

ABSTRACT: A bisaldehyde-containing trithiocarbonate chain transfer agent was prepared and mediated the synthesis of polymers with bisaldehyde-functionalized α-termini by reversible addition−fragmentation chain transfer (RAFT) radical polymerization. The α-termini of RAFT-derived thermoresponsive poly(N-isopropylacrylamide) was conjugated with hydrazides via reversible acylhydrazone bonds. Introduction of hydrophilic end-groups generated a dynamic covalent polymer with “isothermal” lower critical solution temperature (LCST) response to medium pH and dynamic chain exchange character. A biofunctional dynamic covalent polymer was prepared by conjugation with biotin hydrazide. Under appropriate reaction conditions, a dynamic covalent block copolymer with an aldehyde group at the junction point was constructed through a reversible arylhydrazone linkage by coupling with acylhydrazide-terminated poly(ethylene glycol). The dynamic covalent block copolymer exhibited pH-dependent LCST. The remaining aldehyde group was used to react with amino-containing molecules. Bioconjugated dynamic covalent block copolymers containing reversible imine and arylhydrazone linkages were constructed by conjugation of the block copolymer to glucosamine and protein.

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acid catalyst.10,14 Functional materials with tunable mechanical, optical, and thermoresponsive properties were developed. Moreover, they incorporated biologically relevant moieties into dynamic polymers to generate biodynamers, which combine the functional properties (recognition, catalysis) of naturally occurring polymers with the adaptive behavior of constitutional dynamic systems.15 Zhu and co-workers prepared dynamic covalent block copolymers via acylhydrazone or oxime linkages and applied these polymers in the pH-triggered drug delivery.16 Fulton and co-workers utilized reversible addition− fragmentation chain transfer (RAFT) polymerization to prepare a series of polymer building blocks possessing either aldehyde or alkoxyamine end groups.17 Dynamic covalent diblock copolymers were generated through a single reversible oxime bond, and the dynamic nature was highlighted. On the basis of acylhydrazone or imine bonds, they also constructed dynamic polymer scaffolds,18 responsive nanoparticles,19 and singlechain polymer nanoparticles.20 In this research, we utilized RAFT polymerization technique to prepare polymers possessing bisaldehyde functions as α-end groups. By reactions with hydrazides, dynamic covalent polymers were constructed through the reversible acylhydrazone linkage. RAFT polymerization is a versatile approach to the synthesis of well-defined polymers with predetermined end groups that are particularly useful for preparation of block

INTRODUCTION The construction of new advanced supramolecular materials possessing responsive and adaptable properties has attracted increasing interest in recent years.1,2 To achieve this aim, dynamic covalent polymers, or dynamers, have been constructed via reversible covalent interactions between building blocks by applying concepts from dynamic covalent chemistry.3,4 The reversible nature of dynamic covalent linkages enables polymers to reconfigure and optimize their structures under appropriate conditions even after polymerization. Therefore, polymers containing dynamic covalent bonds can tune their properties by exchanging and reshuffling their building blocks. A number of dynamic covalent bonds, such as thermally activated alkoxyamine bonds,5 Diels−Alder adducts,6 chemosensitive imines,7 boronic esters,8 and disulfide bonds9 are utilized in the synthesis of dynamers with thermoresponsive, chemoresponsive, photoresponsive, or electroresponsive properties and self-/rehealing gels.1 Among the known reversible covalent bonds, acylhydrazone bonds are particularly attractive because the acylhydrazone functionality provides both dynamic character through the reversibility of the imino bond and hydrogen-bonding sites through the amide group.10 The formation of acylhydrazone by condensation of hydrazides with carbonyl groups has been implemented in the generation of biologically active substances,11 self-healing gels,12 and pH-responsive micelles as drug carriers.13 Lehn and co-workers have pioneered in the synthesis of polyacylhydrazones by the condensations of the corresponding dihydrazide and dialdehyde in the presence of © 2013 American Chemical Society

Received: May 6, 2013 Revised: August 4, 2013 Published: August 26, 2013 6832

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copolymers, bioconjugates, and fluorescently labeled polymers.21 Polymerization from functional RAFT chain transfer agents (CTAs) can lead to near-quantitative incorporation of a desired functionality at the α-end of each chain. This approach is straightforward and highly efficient for the synthesis of polymers with α-end group functionalization. Functional CTAs have been extensively used to prepare polymers that have carboxylic acid, alcohol, alkyne, azide, and pyridyl disulfidefunctionalized α-termini.22 A functional CTA containing bisaldehyde groups on the reinitating “R” group was synthesized in this research, and RAFT polymerization of N-isopropylacrylamide (NIPAM) in the presence of the CTA resulted in well-defined poly(Nisopropylacrylamide) (PNIPAM) chains with bisaldehyde groups at the α-termini. The high retention of the aldehyde group facilitated the conjugation of small hydrazides to the polymer termini via acylhydrazone bonds and production of ploymers with pH-dependent thermoresponsive properties. A dynamic covalent block copolymer was also constructed through arylhydrazone linkage by a coupling reaction with acylhydrazide-functionalized poly(ethylene glycol). By controlling the reaction conditions, PNIPAM-dyn-PEG block copolymer with an aldehyde group at the junction point was prepared. Conjugation of the block copolymer to amino-containing small molecules and protein generated polymer conjugates with reversible arylhydrazone and imine bonds.

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Synthesis of Bisaldehyde Functionalized Polymers. Typically, bisaldehyde-functionalized PNIPAM (PNIPAM-bisCHO) homopolymer was synthesized using the following procedure: NIPAM (0.5 g, 4.4 mmol), CTA-bisCHO (48 mg, 0.07 mmol), and AIBN (1.3 mg, 8 × 10−3 mmol) were dissolved in 1.5 mL of dioxane. The solution was degassed by three freeze−thaw cycles. The polymerization was carried out at 60 °C for 24 h. The reaction was stopped by cooling in ice water and exposing the solution to air. The solution was concentrated under vacuum, and PNIPAM was precipitated into cold ethyl ether. The polymer was centrifuged, washed, and dried under vacuum to a constant weight. The polymer was obtained as pale yellow solid, with a yield of 84%. In the case of styrene, the polymerization was carried out in bulk. General Procedure for the Conjugation of Bisaldehyde-Functionalized PNIPAM (PNIPAM-bisCHO) with Hydrazides. PNIPAMbisCHO (120 mg, 8.7 × 10−3 mmol) and Girard’s reagent T (GT, 29.3 mg, 0.175 mmol) were dissolved in 5 mL of ethanol. The reaction was stirred for 24 h at 25 °C after the addition of a catalytic amount of acetic acid. The excess GT was removed by dialysis against distilled water for 2 days at 37 °C (MWCO 1000). The purified polymer PNIPAM-dyn-GT was recovered by lyophilization. The bioconjugation of PNIPAM-bisCHO and biotin hydrazide was carried out in DMSO for 24 h at 25 °C using trifluoroacetic acid (TFA) as the catalyst. After dialysis against DMSO/H2O (50 vol %) for 2 days and subsequently against H2O for 3 days, the polymer was recovered by lyophilization. Chain Exchange Reaction of PNIPAM-dyn-GT with Biotin Hydrazide. PNIPAM-dyn-GT (20 mg, 1.4 × 10−3 mmol) was dissolved in 2 mL of deionized water. The solution of biotin hydrazide (0.371 mg, 1.4 × 10−3 mmol) in 0.5 mL of DMSO was added. After the pH was adjusted to 3−4, the solution was stirred at 25 °C for 24 h. The solution was dialyzed against DMSO/H2O (50 vol %)) for 1 day and subsequently against distilled water for 2 days. The polymer was recovered by lyophilization. Synthesis of Dynamic Covalent Diblock of PNIPAM-dyn-PEG. The PNIPAM-bisCHO (200 mg, 0.016 mmol) and acylhydrazide-functionalized PEG (62 mg, 0.029 mmol) were dissolved in DMF. After the addition of a catalytic amount of TFA, the reaction proceeded for 24 h at 25 °C, then the solution was concentrated under reduced pressure, and dried under vacuum to yield a solid product. To remove the possible free PEG, the product was purified by thermal precipitation. Briefly, the sample was dissolved in sodium sulfate aqueous solution (0.5 M) at a concentration of 60 mg/mL. The solution was maintained at 35 °C for 1 h and then centrifuged at 35 °C. The precipitate was dissolved in deionized water then dialyzed against water for 2 days (MWCO 1000) and recovered by lyophilization. Conjugation of PNIPAM-dyn-PEG with Amine-Containing Molecules. Fluorescently labled PNIPAM-dyn-PEG was prepared by reaction with fluoresceinamine. PNIPAM-dyn-PEG (15 mg) and fluoresceinamine (0.4 mg) were dissolved in PBS (pH 8.0). The reaction was carried out in the dark at 25 °C for 24 h. The solution was dialyzed against PBS (pH 8.0) in the dark for 2 days to remove free fluoresceinamine. After dialysis, the solution was characterized directly by fluorescence spectroscopy. Glucose-conjugated PNIPAM-dyn-PEG was prepared by the reaction with glucosamine. PNIPAM-dyn-PEG (6.5 mg) and glucosamine HCl (0.1 mg) were dissolved in PBS (pH 8.0). The reaction was carried out at 25 °C for 24 h. The solution was dialyzed against PBS (pH 8.0) for 2 days to remove free glucosamine. After dialysis, the conjugation of glucosamine was verified by alizarin red S (AR) assay.25 Bioconjugation of PNIPAM-dyn-PEG to Lysozyme. PNIPAM-dynPEG (8 mg) and lysozyme (0.98 mg) were dissolved in PBS (pH 8.0 or pH 6.6). The solution was stirred for 24 h at 25 °C. The free lysozyme was removed by ultrafiltration (Mcutoff = 50 kDa). Characterizations. 1H NMR measurements were performed on a Varian UNITYplus 400 M nuclear magnetic resonance spectrometer using CDCl3 or DMSO-d6 as the solvents. The number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity (Mw/Mn) of the polymers were determined by gel permeation chromatography (GPC) at 35 °C with a Waters 1525

EXPERIMENTAL PART

Materials. N-Isopropylacrylamide (NIPAM, Aldrich, 97%) was recrystallized from the mixture of benzene and hexane before use. Styrene was washed with 5% NaOH, dried over anhydrous MgSO4, and then distilled under reduced pressure. N,N-Dimethylacrylamide (DMA, Aldrich, 99%) was purified through a basic aluminum oxide column. HABA/Avidin reagent (Sigma), poly(ethylene glycol) monomethyl ether (CH3O−PEG−OH, Mn = 2000, Alfa Aesar), Girard’s reagent T (Aldrich, 99%), 4-hydroxybenzaldehyde (Acros, 99%), biotin hydrazide (Aldrich, 97%), fluoresceinamine (Sigma), and lysozyme (Sigma) were used directly. Azobis(isobutyronitrile) (AIBN) was recrystallized twice from methanol. All other reagents were commercial chemicals and used directly, except specially claimed. 2Dodecysulfanylthiocarbonylsulfanl-2-methyl propionic acid (DMP) was synthesized according to the literature.23 Acylhydrazide-functionalized PEG was prepared by the modification of poly(ethylene glycol) monomethyl ether (CH3O−PEG−OH) according to the literature.12a All solvents were redistilled before use. Synthesis of 2-[2-(2-Hydroxyethoxy)-ethoxy]ethyl-4-formylbenzoate.24 Briefly, 4-hydroxybenzaldehyde (16.4 g, 134.3 mmol) was added to a solution of sodium hydroxide (5.37 g, 134.3 mmol) in water (246 mL). The solution was warmed to 60 °C, and epichlorohydrin (4.3 mL, 66.8 mmol) was added slowly over 2 h. The reaction was then allowed to stir an additional 3 h at 60 °C. The precipitate was recrystallized from methanol/water (1:1, v/v), with a yield of 87%. Synthesis of Bisaldehyde-Functionalized Chain Transfer Agent (CTA-bisCHO). A solution of DMP (0.63 g, 1.73 mmol) and 2-[2-(2hydroxyethoxy)-ethoxy]ethyl-4-formylbenzoate (0.49 g, 1.73 mmol) in methylene chloride (20 mL) was cooled to 0 °C. A solution of 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC· HCl) (0.37 g, 1.91 mmol) and 4-dimethylaminopyridine (DMAP) (0.23 g, 1.91 mmol) in methylene chloride (10 mL) was added dropwise. After being stirred overnight under N2, the solution was washed with saturated NaHCO3 solution and water and then dried over MgSO4. After evaporation of the solvent, the crude product was purified by column chromatography (silica gel, petroleum ether/ethyl acetate (3:1, v/v)) to obtain CTA-bisCHO as a yellow solid, with a yield of 41%. 6833

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Scheme 1. Outline of Synthesis of Bisaldehyde-Containing PNIPAM (PNIPAM-bisCHO) and Hydrazone Conjugation

Table 1. Characterization of a Series of Bisaldehyde-End-Functionalized Polymers polymer P4 P6 P7 P10 P11 P13

CTA-bisCHO 1 1 1 1 1 1

(1 (1 (1 (1 (1 (1

equiv) equiv) equiv) equiv) equiv) equiv)

monomer styrene (100 equiv) NIPAM (40 equiv) NIPAM (100 equiv) NIPAM (60 equiv) DMA (50 equiv) DMA (50 equiv)

initiator 0.1 0.1 0.07 0.1 0.05 0.07

chromatograph equipped with a Waters 2414 refractive index detector. Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 1 mL min−1. Dynamic light scattering (DLS) measurements were conducted on a Zetasizer Nano ZS from Malvern Instruments equipped with a 10 mW HeNe laser at a wavelength of 633 nm. All samples were filtered through a Millipore 0.45 μm filter prior to measurements. Fluorescence spectroscopy was performed on a Shimadzu RF-5301 PC spectrofluorophotometer. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS−PAGE). SDS−PAGE was performed with 14% polyacrylamide gels. Electrophoresis was carried out at 80 V voltage, 16 mA current for 2 h. Staining was accomplished using Coomassie Brilliant Blue R-250 solution. Lower Critical Solution Temperature (LCST) Measurement. The transmittance of polymer aqueous solution was determined at λ = 600 nm by a Shimadzu UV-2450 UV−visible spectrophotometer equipped with a temperature control unit. The LCST was defined as the intercept of the tangent of a transmittance−temperature curve at the onset of turbidity. HABA/Avidin Assay. The amount of available biotin on polymer was determined by HABA/avidin binding assay. The HABA/avidin reagent (Sigma) was reconstituted with 10 mL of deionized water. In a 1 mL cuvette, 900 μL HABA/avidin reagent was pipetted, and the ) by UV−vis absorbance was measured at λ = 500 nm (AHABA/avidin 500 spectrophotometer. To this solution, 100 μL of sample was added, the solution was mixed by inversion, and the absorbance at λ = 500 ) was read. The amount of the available biotin is (AHABA/avidin+sample 500 calculated by the following formula:26,27 μmole biotin/mL = (ΔA500/ 34) × 10, which corresponds to the micromoles of biotin per milliliter blank ) + Asample of the sample solution, where ΔA500 = 0.9 × (AHABA/avidin 500 500 HABA/avidin+sample − A500 .

(equiv) (equiv) (equiv) (equiv) (equiv) (equiv)

t (h)

Mn,NMR

MnGPC

PDI

24 3 24 6 24 24

7500 3900 13800 8600 5200 7400

6400 3400 15800 8600 4900 8300

1.09 1.02 1.10 1.10 1.13 1.06

construct the dynamic covalent polymers because of its reversibility and adaptive character.10 RAFT polymerization was used to prepare polymers with end-functional bisaldehyde groups. RAFT polymerization provides a convenient approach to the synthesis of a wide range of well-defined polymers possessing high levels of end-group functionality. By incorporation of a bisaldehyde group into the R group of the chain transfer agent, polymers with an α-end bisaldehyde functional group can be generated via RAFT polymerization. The bisaldehyde-containing chain transfer agent CTA-bisCHO was prepared by a carbodiimide-mediated coupling reaction between 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (DMP) and 2-[2-(2-hydroxyethoxy)-ethoxy]ethyl4-formylbenzoate (Scheme 1). 1H NMR characterization confirmed the successful synthesis of CTA-bisCHO (Supporting Information, Figure S1). The RAFT polymerization of styrene, NIPAM and N,Ndimethylacrylamide (DMA) mediated by CTA-bisCHO afforded a series of bisaldehyde-terminated polymers. The polymers were characterized by GPC, and the results indicated that the polymers possessed unimolecular weight distribution and low polydispersities. The Mn calculated from NMR data matched well with those obtained by GPC (Table 1). The presence of the bisaldehyde end group was confirmed by the singlet signal at δ 9.9 ppm, corresponding to the formyl proton in the 1H NMR spectrum (Figure 1a). Modification of Polymers with Hydrazides via Acylhydrazone Conjugation. The conjugations of PNIPAM-bisCHO with Girard’s reagent T (GT), and biotin hydrazide were investigated. Acetic acid was added to a 1:20 (mol/mol) mixture of PNIPAM122-bisCHO (P7 in Table 1) and Girard’s reagent T in ethanol. The solution was allowed to

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RESULTS AND DISCUSSION Synthesis of Bisaldehyde-End-Functionalized Polymers. In our study, the acylhydrazone bond was chosen to 6834

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Figure 1. 1H NMR spectra of (a) PNIPAM-bisCHO, (b) PNIPAM-dyn-GT, (c) biotin-conjugated PNIPAM, and (d) PNIPAM-dyn-GT after exchange with biotin hydrazide.

which represented an increase of ∼14.6 °C compared with the precursor polymer PNIPAM122-bisCHO (LCST 25.1 °C), illustrating that simple end-group modification can remarkably influence the solution properties of PNIPAM122-bisCHO without the chemical modification of the polymer backbone. At 45 °C, the ξ-potential of PNIPAM122-bisCHO was −10.96 mV; however, after conjugation with GT, the ξ-potential increased to 16.07 mV because of the positive charge of GT, confirming the successful coupling of PNIPAM-bisCHO with GT. To monitor the pH-responsive character of PNIPAM122-dynGT and reversibility of acylhydrazone bonds in the polymer, an “isothermal” turbidimetry experiment was conducted. At 30 °C, the pH value of the aqueous solution of PNIPAM122-dyn-GT (2

react at room temperature for 24 h. After dialysis against H2O for 2 days, the polymer was recovered by lyophilization. 1H NMR spectrum showed significant reduction in the intensity of the signal corresponding to the formyl proton and the appearance of the trans-acylhydrazone proton at 8.4 ppm (Figure 1b). These changes were accompanied by a reduction in intensity of the aromatic signals at 7.84 ppm and the appearance of signals corresponding to the aromatic moiety of acylhydrazone at 7.65 ppm. The signals of methylene and methyl protons of GT moiety were observed at 4.73 and 3.52 ppm, respectively. On the basis of the 1H NMR result, the conjugation yield was calculated to be about 98%. The transmittance measurement of PNIPAM122-dyn-GT in aqueous solution indicated a LCST of 39.7 °C (Figure 2), 6835

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linkage was reformed, and the polymer became more hydrophilic. The “isothermal” turbidimetry experiment demonstrates that the change of medium pH can modulate the hydrophilic/hydrophobic balance of the polymer, which allows the LCST behavior to be triggered without a temperature gradient. The change of LCST with the pH of the aqueous solution was also investigated. First, PNIPAM122-dyn-GT was dissolved in deionized water (pH = 6.4) at a concentration of 2 mg/mL. Subsequently, the pH of the aqueous solution was adjusted to 3.5, and finally, the pH was adjusted back to 5.0. At each pH, the solution was allowed to stir for 24 h at room temperature, and the LCST was measured by UV−vis spectrophotometry (Figure 2). As the pH changed from 6.4 to 3.5, the LCST decreased from 39.7 to 26.2 °C, almost the same as that of original PNIPAM. Next, as the pH increased from 3.5 to 5.0, the LCST shifted to 35 °C. These results indicated that with the decreasing of pH value, the acylhydrazone bond between the PNIPAM and GT moiety cleaved, which resulted in the lowering of the LCST. As the medium became less acidic, the GT was conjugated to PNIPAM homopolymer again via an acylhydrazone bond, and the LCST of the polymer shifted to a higher temperature. These phenomena demonstrate that the LCST of PNIPAM122-dyn-GT solution can be tuned by the pH of the solution. The pH-mediated cleavage/coupling of the end-group triggers a change in the hydrophilic/hydrophobic balance of the PNIPAM. The coupling of the hydrophilic GT to the end of the PNIPAM increases the hydrophilicity and raises the LCST, whereas under acidic conditions, the cleavage of the acylhydrazone linkage results in a more hydrophobic PNIPAM and lower LCST. As illustrated in Scheme 2, the

Figure 2. Temperature dependence of the transmittance of polymer aqueous solutions. PNIAM122-bisCHO in H2O (a, pH 6.4) and PNIPAM122-dyn-GT in H2O (b, pH 6.4) at (c) pH 3.5 and at (d) pH 5.0 (adjusted from pH 3.5 back to pH 5.0).

mg/mL) was changed from 6.4 to 5.0 and 3.5, respectively. Under each pH, the solution was incubated at 30 °C, and the transmittance was measured at specified time intervals (Figure 3). It was found that at pH 5.0, the solution transmittance

Scheme 2. Illustration of the Reversibility of Acylhydrazone Linakge in PNIPAM-dyn-GT with pH and Chain Exchange with Biotin Hydrazide

alternation of the end-groups with the pH can dramatically change the hydrophilic/hydrophobic balance of the PNIPAM without any effect on the polymer backbone. As a result, the PNIPAM122-dyn-GT shows a pH-dependent thermoresponsive property. By introducing a hydrophilic end-group via an acylhydrazone linker to aldehyde-end-functionalized thermoresponsive polymers, it can generate pH-sensitive “isothermal” responsive polymers. The acylhydrazone-terminated PNIPAMs may find use as double-responsive materials in controlled release systems. The bioconjugation of bisaldehyde-end-functionalized PNIPAM122 and biotin hydrazide (aldehyde/hydrazide = 1:1 mol/ mol) was carried out in DMSO for 24 h at 25 °C using a trace amount of TFA as the catalyst. After purification by dialysis against DMSO/H2O (50 vol %) and subsequently against H2O, the polymer was recovered by lyophilization. The 1H NMR spectrum showed that the intensity of the formyl proton reduced to 17% relative to the precursor PNIPAM122-bisCHO (Figure 1c). The signal of the acylhydrazone proton appeared

Figure 3. Isothermal response of PNIPAM122-dyn-GT to medium pH at 30 °C.

slowly changed from 100% to 90% in 5 h and kept falling to 70% in the next 3 h then leveled off (curve a). However, at pH 3.5, the solution gradually became turbid, and the transmittance decreased remarkably with time and reached 39% in 5 h (curve b). After incubation at pH 3.5 for 5 h, an aqueous solution of NaOH was added to the polymer solution, and the pH value was controlled at 5.0. The transmittance was found to increase slowly with time and reached 71% after 4 h (curve c). These phenomena indicate that the acylhydrazone linkage is gradually hydrolyzed with the decreasing of medium pH, which results in the cleavage of hydrophilic GT moiety from PNIPAM chain and the increasing hydrophobicity of polymer. The hydrolysis rate at pH 5.0 is much slower than that at pH 3.5. As the pH was adjusted from 3.5 back to pH 5.0, the acylhydrazone 6836

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Figure 4. (A) UV−vis spectra of (a) HABA/avidin complex blank solution and (b) after the addition of biotinylated PNIPAM solution. (B) DLS curves of aggregates formed by biotinylated PNIPAM in the aqueous solution at 25 °C before (curve a) and after (curve b) incubation with streptavidin. TEM images of aggregates formed by biotinylated PNIPAM in the aqueous solution at 25 °C (C) before and (D) after incubation with streptavidin.

Figure 5. (A) UV−vis spectra of (a) HABA/avidin complex blank solution and (b) after the addition of PNIPAM122-dyn-GT solution. (B) UV−vis spectra of (a) HABA/avidin complex blank solution and (c) after the addition of the obtained polymer via chain exchange with biotin hydrazide.

the HABA/avidin complex at 500 nm before and after mixing with the solution of biotinylated polymers. The absorption decreased proportionately to the biotin present in the system because biotin displaced HABA as a result of its high affinity for avidin.26 The change in absorbance can be used to calculate the amount of biotin available to avidin. Figure 4A shows UV−vis

at 8.1 ppm. The characteristic signals of biotin were observed at 6.5 and 4.5 ppm. The biconjugation yield was calculated to be 83% on the basis of the 1H NMR analysis. The content of biotin was ∼1.8 × 10−2 mg/mg of polymer. The bioavailability of biotin was evaluated by a HABA/avidin competitive binding assay. The assay was conducted by measuring the absorbance of 6837

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Scheme 3. Illustration of the Preparation of PNIPAM-dyn-PEG Block Copolymer through Arylhydrazone Linkage

covalent polymers by direct bioconjugation to various biomolecules or dynamic chain exchange with various biomolecules. Preparation of Dynamic Covalent Block Copolymers of PNIPAM and PEG. As illustrated in Scheme 3, the dynamic covalent block copolymers of PNIPAM and PEG were prepared by mixing of bisaldehyde-end-functionalized PNIPAM122 polymer and acylhydrazide-terminated PEG45 in DMF with TFA or p-toluenesulfonic acid (p-TsOH) as the catalyst.10,29 After the reaction, the block copolymer was analyzed by 1H NMR in DMSO-d6 (Figure 6). In the spectrum,

spectra of the HABA/avidin complex before and after the addition of biotinylated PNIPAM polymer solution. Upon addition of the biotinylated PNIPAM solution, the absorbance of the HABA/avidin complex at 500 nm decreased, indicating that the biotin moieties of the polymer competitively bound to avidin by replacing HABA molecules. The amount of biotin available to avidin was calculated to be 8 × 10−3 mg/mg of polymer. The recognition of biotinylated PNIPAM for protein was further demonstrated by the interaction of biotinylated PNIPAM and streptavidin (SAv). As shown in Figure 4B, DLS data indicated that biotinylated PNIPAM formed particles with an average diameter of 134 nm in the aqueous solution at 25 °C. However, after incubation with SAv solution for 1 h, large particles appeared at 600−2000 nm on DLS curve, which was formed by SAv-induced cross-linking of interparticles.28 In the TEM image, it was observed that the particle size of biotinylated PNIPAM was ∼120 nm (Figure 4C), which was smaller than that obtained by DLS because of the dehydrated state of the TEM specimen. After the addition of SAv, large aggregates were clearly observed in the TEM image (Figure 4D). DLS and TEM results demonstrate that biotinylated PNIPAM can effectively bind to SAv. The bioconjugation of biotin hydrazide to PNIPAM-bisCHO via acylhydrazone linkage can endow the dynamic covalent polymer with biological function. To demonstrate the dynamic nature of PNIPAM122-dyn-GT, 1 equiv of biotin hydrazide was added to the aqueous solution of PNIPAM122-dyn-GT at pH 4, and the solution was stirred at room temperature for 24 h. After dialysis, the sample was lyophilized and analyzed by 1H NMR. The 1H NMR spectrum (Figure 1d) revealed the presence of the signal at δ 8.03 ppm associated with hydrazone linkage between PNIPAM-bisCHO and biotin hydrazide. The bioavailability of biotin was evaluated by a HABA/avidin competitive binding assay. After the addition of PNIPAM-dyn-GT to HABA/Avidin solution, there was no decrease in absorbance at 500 nm (Figure 5A), indicating that PNIPAM-dyn-GT cannot bind to avidin. However, a decrease from 0.73 to 0.55 was clearly observed for the obtained polymer via exchange with biotin hydrazide (Figure 5B), indicating that the biotin moiety in the polymer displaced HABA from the avidin/HABA complex. This result shows that by reshuffling the end group of PNIPAM via chain exchange (Scheme 2), the polymer can interact with avidin. The amount of available biotin to avidin was calculated to be 2.64 × 10−3 mg/mg of polymer. The formed dynamic covalent polymer PNIPAM-dyn-GT can be endowed with biological function via chain exchange with a biomolecule. Therefore, it is possible to modulate and tune the biological properties of dynamic

Figure 6. 1H NMR spectrum of dynamic covalent block copolymer PNIPAM109-dyn-PEG45 (copolymer D2 in Table 2) in DMSO-d6.

it was observed that two new signals appeared at δ 11.6 ppm corresponding to the NH of arylhydrazone10 and 8.39 ppm corresponding to the ArCHN of arylhydrazone along with a reduction in the intensity of the signal at 9.9 ppm corresponding to the formyl proton. The characteristic signal of PEG presented at δ 3.5 ppm, corresponding to CH2CH2O. All these results indicate the formation of dynamic covalent block copolymer PNIPAM122-dyn-PEG45 via arylhydrazone linkage. On the basis of 1H NMR analysis, the reacted percentage of aldehyde groups using different catalysts was calculated; the data are listed in Table 2. By changing the feed ratio of the PNIPAM-bisCHO to acylhydrazide-terminated PEG45, the reaction degree of aldehyde group can be controlled. With p-TsOH (4 wt %) as catalyst, aldehyde end groups can be consumed completely under the condition of 1 equiv excess of acylhydrazideterminated PEG45 (Supporting Information Figure S4). To further modify the PNIPAM122-dyn-PEG45 copolymer using the 6838

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Table 2. Reaction of Bisaldehyde-End-Functionalized PNIPAM122 and Acylhydrazide-Terminated PEG45 under Different Conditions copolymer

catalyst

D1 D2

TFA pTsOH pTsOH

D3

−CHO: −NHNH2 (mol/mol)

reacted percentage of aldehyde groupa (%)

2:3 1:1

73 50b

1:2

100

a

Calculated by 1H NMR data. bDetermined by 1H NMR after purified by thermal precipitation.

remaining aldehyde group, copolymer D2 was purified by thermal precipitation to remove free PEG homopolymer. After lyophilization, 1H NMR analysis indicated that one aldehyde group remained at the junction point of two blocks (Figure 6). GPC analysis showed that the monomodal peak shifted to a higher molecular weight relative to those of precursors PNIPAM122-bisCHO and acylhydrazide-terminated PEG45 homopolymers (Figure 7). GPC and 1H NMR results demonstrate the formation of PNIPAM-dyn-PEG block copolymer with an aldehyde group at the junction point.

Figure 8. Temperature dependence of the transmittance of block polymer aqueous solutions under various pH values. (a) PNIAMbisCHO at pH 6.4; PNIPAM-dyn-PEG at pH (b) 6.4, (c) 5.5, (d) 3.5, and (e) 4.6 (adjusted from 3.5 to 4.6).

bisCHO. After 24 h incubation at pH 3.5, the polymer was lyophilized and characterized by GPC analysis. Figure 7d showed that the main peak of GPC curve shifted to a lower molecular weight and a new shoulder peak corresponding to precursor PEG appeared. These results indicate the cleavage of arylhydrazone linkage of PNIPAM122-dyn-PEG45 at pH 3.5. After the pH was adjusted from 3.5 to 4.6, the polymer solution was stirred at pH 4.6 for 24 h. It was found that the LCST shifted back to 30 °C (Figure 8e), indicating the reformation of arylhydrazone linkage between PNIPAM and PEG. These results confirm that the change of pH value can modulate the hydrophilic/hydrophobic balance of dynamic covalent block copolymer PNIPAM122-dyn-PEG45 and exert an effect on the thermoresponsive property of the polymer solution. DLS was employed to characterize the thermoresponsive micellization of PNIPAM122-dyn-PEG45. Figure 9 shows the temperature dependence of hydrodynamic diameter for 0.5 mg/mL aqueous solution of PNIPAM122-dyn-PEG45. Below 30 °C, PNIPAM122-dyn-PEG45 copolymer had an average hydrodynamic diameter of ∼10 nm, implying that the copolymer existed as unimer. A sharp increase of hydrodynamic diameter

Figure 7. Gel permeation chromatographic traces of (a) acylhydrazideterminated PEG45, (b) PNIPAM122-bisCHO, (c) PNIPAM122-dynPEG45 (copolymer D2), and (d) copolymer after 24 h incubation at pH 3.5.

The temperature-responsive behavior of the block copolymer was studied by UV−vis spectroscopy. Because of the effect of hydrophobic end groups,30 PNIPAM122-bisCHO homopolymer with a long alkyl chain, C12H25, and two aromatic groups had a lower LCST (25.1 °C). However, after coupling with hydrophilic PEG, the LCST of PNIPAM122-dyn-PEG45 was expected to shift to a higher temperature. It was found that the LCST of PNIPAM122-dyn-PEG45 in H2O (pH 6.4) was 33 °C (Figure 8), higher than PNIPAM122-bisCHO, demonstrating the successful coupling of PNIPAM-bisCHO and PEG. To demonstrate the reversibility of arylhydrazone linkage, hydrochloric acid was added to the aqueous solution of PNIPAM122dyn-PEG45. The pH of the solution was controlled at 5.5 and 3.5. At each pH, the solution was stirred for 24 h. It was found that the LCST decreased to 30.5 °C at pH 5.5 and further shifted to 27.5 °C at pH 3.5, close to the original PNIPAM122-

Figure 9. Dependence of the hydrodynamic diameter of PNIPAM122dyn-PEG45 block copolymer on temperature. 6839

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was observed above 30 °C, which meant the occurrence of micellization. Beyond 45 °C, the micellar size remained almost constant at ∼45 nm. This clearly indicates the thermo-induced formation of micelles with PNIPAM block as the hydrophobic cores and PEG block as the hydrophilic shells. The DLS autocorrelation function curves are shown in Supporting Information Figure S5. TEM image showed that the micelles at 45 °C were spherical with an average diameter of ∼30 nm (Supporting Information Figure S6). Modification of PNIPAM-dyn-PEG Block Copolymer with Amine-Containing Molecules. The remaining aldehyde group at the junction point of PNIPAM122-dyn-PEG45 block copolymer can be utilized for further conjugation with other functional molecules, as illustrated in Scheme 4. It is wellScheme 4. Conjugation of PNIPAM-dyn-PEG Block Copolymer to Amine-Containing Molecules via an Imine Bond Figure 10. Fluorescence emission spectra of (a) PNIPAM122-dynPEG45 and (b) fluoresceinamine-labeled PNIPAM122-dyn-PEG45 at 25 °C. All spectra were measured in pH 8.0 PBS at λex = 490 nm.

Figure 11 shows the fluorescence emission spectra of AR/PBA and after the addition of glucose-conjugated block copolymer

known that the aldehyde group can react with the amine group to form a reversible imine bond. To prove the reactivity of the aldehyde group for amine-containing molecules, the solution of fluoresceinamine was added to the solution of PNIPAM122-dynPEG45 block copolymer in PBS (pH 8.0). After it reacted for 24 h at room temperature, the solution was dialyzed against PBS buffer in the dark for 2 days to remove free fluoresceinamine. The fluorescence emission spectra were measured at an excitation wavelength of 490 nm (Figure 10). There was no absorption in the fluorescence spectrum of PNIPAM122-dynPEG45; however, the characteristic emission peak of fluoresceinamine at λ = 518 nm was clearly present after the modification, which confirmed the attachment of fluoresceinamine to the junction point of block copolymer. On the basis of the preparation of fluorescently labeled block copolymer, glucose-modified block copolymer was prepared by the reaction of PNIPAM122-dyn-PEG45 block copolymer and glucosamine in PBS (pH 8.0). The conjugation of glucose to copolymer was verified by alizarin red S (AR) assay,25 a threecomponent system consisting of AR, phenylboronic acid (PBA), and sugar-modified polymer. AR is inherently nonfluorescent but fluoresces strongly when bound to PBA under alkaline conditions. The reversible covalent binding of PBA with the catechol diol groups induces emission at 578 nm.

Figure 11. Fluorescence emission spectra of (a) AR/PBA solution, (b) mixed solution of AR/PBA and glucose-conjugated PNIPAM122-dynPEG45, and (c) mixed solution of AR/PBA and PNIPAM122-dynPEG45 (λex = 460 nm).

(λex = 460 nm). The reduction of the fluorescence intensity at 578 nm was clearly observed, which was caused by the competition for diol-binding sites on PBA between AR and polymer-bound glucose because of the high affinity of glucose to PBA. The modifications of PNIPAM-dyn-PEG diblock copolymer with fluoresceinamine and glucosamine proved the accessibility of the remaining aldehyde group at the junction point to amines. Bioconjugation of PNIPAM-dyn-PEG Diblock Copolymer with Lysozyme. Bioconjugation of lysozyme to PNIPAM122-dyn-PEG45 block copolymer was achieved via the reaction of aldehyde group at the junction point and the amine groups present on the protein. The reaction was carried out at a ratio of CHO/NH2 = 3:1 (mol/mol) under pH 6.6. The 6840

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unreacted lysozyme was removed by ultrafiltration (Mcutoff = 50 kDa). Lysozyme bioconjugation was confirmed by SDS−PAGE and DLS. Lanes E and F in Figure 12 show a broad band

Figure 12. SDS−PAGE of lysozyme-bioconjugated PNIPAM122-dynPEG45. Lanes: (A) protein marker; (B) lysozyme; (C) blank; (D) PNIPAM122-dyn-PEG45 solution; (E, F) lysozyme-bioconjugated PNIPAM122-dyn-PEG45 (E, low concentration; F, high concentration).

corresponding to a molecular weight of 30−90 kDa. The average molecular weight of PNIPAM122-dyn-PEG45 is 15 kDa, and lysozyme has a molecular weight of 14.3 kDa. Since lysozyme contains six ε-amino groups in the side chains of the lysine residues and one α-amino at its N terminus, the SDS− PAGE result indicates that more than one block copolymer chain is bioconjugated to lysozyme. At 25 °C, DLS data demonstrated that the hydrodynamic diameter of lysozymebioconjugated block copolymer became larger compared with that of PNIPAM122-dyn-PEG45 (Figure 13A). At 45 °C, because of the collapse of PNIPAM block, lysozyme-bioconjugated block copolymer formed particles (as illustrated in Scheme 4) with an average diameter, determined by DLS, of 48 nm. TEM image (Figure 13B) revealed that lysozyme-bioconjugated PNIPAM122-dyn-PEG45 copolymer formed spherical particles with a size range from 21 to 30 nm at 45 °C, which was smaller than that determined by DLS. Because in the DLS measurement, the particles were in a hydrated state, the size determined by DLS was bigger than that measured by TEM.

Figure 13. (A) Hydrodynamic size distributions of PNIPAM122-dynPEG45 (a, 25 °C; a′, 45 °C), lysozyme-bioconjugated PNIPAM122-dynPEG45 (b, 25 °C; b′, 45 °C), and lysozyme (c, 25 °C; c′, 45 °C). (B) TEM image of aggregates formed by lysozyme-bioconjugated PNIPAM122-dyn-PEG45 copolymer at 45 °C.

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linkage was revealed by the tunable thermoresponsive behavior of the polymer under various pH conditions. The remaining aldehyde group was utilized to conjugate other aminecontaining molecules through the imine bond. Fluoresceinamine-labeled, glucose-conjugated, and lysozyme-conjugated PNIPAM-dyn-PEG block copolymers were prepared, confirming the accessibility of the aldehyde group at the joint point to amines. Such bioconjugated dynamers combine the functional properties of biomolecules with the reversibile and stimuliresponsive characters of dynamic covalent polymers, which may have numerous applications in biotechnology and biomedical areas. This work demonstrates that the hydrazone covalent coupling to bisaldehyde−functionalized PNIPAM copolymer can be a versatile methodology to prepare conjugates with pHdependent thermoresponsive behavior and dynamic character.

CONCLUSIONS Thermoresponsive PNIPAM homopolymers possessing bisaldehyde functions as end groups were synthesized by modified CTA-mediated RAFT polymerization. We have demonstrated effective and practical hydrazone conjugation of bisaldehydeend-functionalized PNIPAM with Girard’s reagent T and biotin hydrazide. Dynamic covalent polymer with an “isothermal” LCST response to medium pH and dynamic chain exchange character was generated via reversible acylhydrazone linkage. The alternation of the end groups with medium pH can dramatically change the solution behavior of the PNIPAM homopolymer without any effect on the polymer backbone. By reshuffling the end group of PNIPAM via chain exchange, the functional property was tuned. Thermoresponsive dynamic covalent block copolymer was constructed by the reaction of acylhydrazide-modified PEG and bisaldehyde-modified PNIPAM. Under appropriate conditions, PNIPAM-dyn-PEG block copolymer with one aldehyde group at the junction point of the blocks was prepared. The reversibility of the arylhydrazone

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of chain transfer agent, acylhydrazideterminated PEG and copolymer samples in Table 2, DLS 6841

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autocorrelation function curves, and TEM image of PNIPAM122-dyn-PEG45 block copolymer micelles at 45 °C. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was financially supported by the National Natural Science Foundation of China under Contract No. 21174066 and PCSIRT (IRT1257).

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