Nitric Oxide (NO) Cleavable Biomimetic Thermoresponsive Double

Article pubs.acs.org/Macromolecules

Nitric Oxide (NO) Cleavable Biomimetic Thermoresponsive Double Hydrophilic Diblock Copolymer with Tunable LCST Jinming Hu,† Michael R. Whittaker,† Sul Hwa Yu,† John F. Quinn,*,† and Thomas P. Davis*,†,‡ †

ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia ‡ Department of Chemistry, University of Warwick, Coventry, ULCV4 7AL, U.K.

Downloaded by UNIV OF PENNSYLVANIA on August 23, 2015 | http://pubs.acs.org Publication Date (Web): June 11, 2015 | doi: 10.1021/acs.macromol.5b00996

S Supporting Information *

ABSTRACT: The fabrication of responsive biomimetic polymers that can respond to externally applied stimuli has received considerable attention over the past few years due to the variety of potential applications. Herein, we report a convenient method to fabricate a thermoresponsive diblock copolymer that is sensitive to a biological messenger molecule, nitric oxide (NO). A well-defined thermoresponsive double hydrophilic block copolymer (DHBC) of poly(ethylene glycol)-b-poly(N-isopropylacrylamide) (PEG-b-PNIPAM) was initially synthesized via atom radical transfer polymerization (ATRP) using a PEG-Cl macroinitiator including an o-nitroaniline motif. The o-nitroaniline derivative was subjected to reduction in the presence of a reducing agent (zinc powder), yielding DHBC with a single amide-functionalized o-phenylenediamine moiety (an efficient NO-reactive group) at the chain junction point. Upon addition of a near equivalent amount of NO (relative to the o-phenylenediamine residues), the o-phenylenediamine groups were transformed to benzotriazoles, resulting in spontaneous hydrolysis (as confirmed by UV−vis and GPC measurements). This resulted in scission of the original diblock copolymers and therefore a substantial decrease in the lower critical solution temperature (LCST) (due to the loss of the hydrophilic PEG chains). NO-triggered cleavage of the hydrophilic block of a DHBC may have potential application in NOmediated drug release.

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INTRODUCTION Stimuli-responsive polymers have been the subject of considerable research over the past few decades due to their diverse applications in a number of fields.1−8 In addition to the well-documented materials that are sensitive to temperature,9−11 pH,12−14 ionic strength,15−17 irradiation with light,18−21 redox environment,22−24 and external fields,25−27 new stimuli-responsive polymers are constantly being explored. Recently, polymers for which solubility or reactivity can be manipulated by exposure to biorelated gases have emerged as a new class of stimuli-responsive polymers. In particular, much attention has been paid to the development of carbon dioxide (CO2) responsive materials which exploit the mildly acidic conditions generated by dissolving CO2 in aqueous media. Specifically, polymers with pH-responsive moieties including (tertiary) amines, amidine groups, and carboxylate motifs etc. can undergo protonation or deprotonation when exposed to CO2 in solution, thereby switching their aqueous solubility.28−35 On the other hand, oxygen (O2) responsive polymers which use the noncovalent interaction between O2 and fluorinated groups have also been explored.36−38 Our group recently devised several kinds of nitric oxide (NO) responsive polymers by exploiting the high reactivity between o-phenylenediamine derivatives and NO.39−41 In most of these cases the applied gas concentrations were relatively high, and saturated © 2015 American Chemical Society

gas solutions (formed by sparging the gases through the polymer solutions) were necessary to stimulate a response. This represents an obvious limitation should these materials be applied in a physiological setting, given the relatively low physiological gas concentrations. As such, it is important to fabricate novel gas-responsive polymers that can sensitively and selectively respond to substantially lower gas concentrations. An important challenge in achieving this outcome is to markedly decrease the concentration of gas-responsive moieties within the polymer chain while simultaneously preserving the gas-sensitive behavior. In previous reports regarding CO2 and O2-responsive polymers, the reactive groups have typically been included as pendent chains,28,33,39,41 although there is at least one example where the responsive residue has been located at the chain end.36 The latter design significantly decreased the concentration of gas-responsive moieties without compromising the gas-responsive characteristics of the polymer. We envisioned that highly gas-sensitive polymers could be achieved by inserting gas-responsive moieties into specific locations within the polymeric chain (e.g., chain ends, junction points and so on). Considering the highly reactive nature of oReceived: May 11, 2015 Revised: June 1, 2015 Published: June 11, 2015 3817

DOI: 10.1021/acs.macromol.5b00996 Macromolecules 2015, 48, 3817−3824

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affecting other functional groups in the polymer. The ophenylenediamine moiety was readily transformed to an amidederived benzotriazole group in the presence of an equivalent amount of NO (relative to the o-phenylenediamine) by taking advantage of the high reactivity between o-phenylenediamine and NO. Significantly, the amide-functionalized benzotriazole was labile in the aqueous milieu and was spontaneously hydrolyzed, cleaving the original PEG-b-PNIPAM diblock copolymer and generating benzotriazole-terminated PEG and carboxyl-terminated PNIPAM. This resulted in a pronounced decrease in the lower critical solution temperature (LCST) compared to the initial diblock copolymers (Scheme 1).


phenylenediamine moieties toward NO and the labile nature of the amide-functionalized benzotriazole derivative,41 we surmised that if a single NO-responsive o-phenylenediamine unit could be installed into the chain junction point of a block copolymer (rather than in the pendent chains), exposure to NO could facilitate chain scission. Specifically, the assynthesized diblock copolymer could react with NO to form an amide-derived benzotriazole motif, which would undergo spontaneous hydrolysis resulting in cleavage of the original diblock copolymer. In this case, a more sensitive NOresponsive polymer can be achieved due to the use of a single NO-responsive unit per polymer chain. As a proof-of-concept, herein we report the fabrication of a thermoresponsive double hydrophilic diblock copolymer (DHBC) incorporating a single NO-reactive unit at the chain junction point (Scheme 1). The key design element was to

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MATERIALS AND METHODS Materials. N-Isopropylacrylamide (NIPAM) was purchased from Sigma-Aldrich and recrystallized from toluene/n-hexane (v/v = 1:3) mixture twice prior to use. 4-Amino-3-nitrophenol, propargyl bromide solution (80 wt% in toluene), azideterminated poly(ethylene glycol) methyl ether (PEG45-N3), copper(I) bromide (CuBr), copper(I) chloride (CuCl), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%), α-bromoisobutyryl bromide (98%), 2-chloropropionyl chloride (97%), and potassium carbonate (K2CO3) were purchased from Sigma-Aldrich and used as received without further purification. All solvents (EMSURE) were of analytical grade and were used as received unless otherwise noted. Tris[2(dimethylamino)ethyl]amine (Me6tren) was synthesized according to a previously published protocol.42,43 NO gas and saturated NO solution (∼1.8 mM as determined by a Griess assay) were prepared according to previous protocols.44 Synthesis. The preparation of small molecule precursor (2), PEG-NH2 precursor, PEG-based macroinitiators (PEG-Br and PEG-Cl), and PEG-b-PNIPAM DHBCs are shown in Scheme 2. Synthesis of 2-Nitro-4-(prop-2-yn-1-yloxy)aniline (Scheme 2). 4-Amino-3-nitrophenol (4.62 g, 30 mmol, 1), K2CO3 (8.29 g, 60 mmol), propargyl bromide solution (5.0 mL, 45 mmol), and acetone (200 mL) were charged to a 500 mL round bottle flask. The solution was heated to reflux and maintained under reflux overnight. The solution was then cooled to room temperature and filtered. The filtrate was concentrated under vacuum using a rotary evaporator and washed with an excess amount of petroleum ether to remove unreacted propargyl bromide. The resulting solid was collected and purified by column chromatography using petroleum ether/ethyl acetate (v/v = 3:1) as the eluent. After removing the solvent, the product was isolated as an orange solid (4.29 g, yield: 74%). 1H NMR (CDCl3, δ, ppm, Figure S1a): 7.61 (d, 1H), 7.06 (t, 1H), 7.04 (d, 1H), 6.72 (d, 1H), 6.70 (s, 1H), 5.87 (s, 2H), 4.60 (d, 2H), 2.48 (t, 1H). 13C NMR (DMSO-d6, δ, ppm, Figure S1b): 147.3, 142.8, 129.4, 128.0, 121.1, 107.7, 79.4, 78.9, 56.6. Synthesis of 2-Nitro-4-(prop-2-yn-1-yloxy)aniline-Functionalized Poly(ethylene glycol) (PEG-NH2, Scheme 2). 2Nitro-4-(prop-2-yn-1-yloxy)aniline-functionalized PEG (PEGNH2) precursor was synthesized via the copper-catalyzed cycloaddition reaction between 2 and azide-terminated PEG (PEG45-N3). In a typical experiment, PEG45-N3 (2.0 g, ∼1 mmol), 2 (288 mg, 1.5 mmol), PMDETA (173 mg, 1 mmol), and DMF (4.0 mL) were charged into a 20 mL polymerization vial capped with a rubber septum. The mixture was carefully deoxygenated by sparging with nitrogen for 20 min. CuBr (143 mg, 1 mmol) was added under a blanket of nitrogen and the vial was then capped. The reaction was conducted at 50 °C for

Scheme 1. Incorporation of an NO Responsive Group into PEG-b-PNIPAM Diblock Copolymer, Followed by Reduction with Zinc Powder and Treatment with NOa

a

The nitrobenzene linker group was reduced in the presence of reducing agent (zinc powder), providing a diblock copolymer which was reactive to NO. Exposing the diblock copolymer to NO led to conversion of the linker to a benzotriazole motif, which was spontaneously hydrolyzed in an aqueous milieu, thereby facilitating cleavage of the diblock copolymer.

synthesize a multifunctional linker that can form a reactive bridge between two block chains and which will react with NO in a highly specific fashion. Starting from commercially available 4-amino-3-nitrophenol, the phenol group was first modified with propargyl bromide without recourse to protecting group chemistry. The incorporated alkynyl group was then functionalized with azide-terminated hydrophilic poly(ethylene glycol) (PEG-N3) via a copper-catalyzed cycloaddition reaction, and the residual aniline group was used to incorporate an atom transfer radical polymerization (ATRP) initiator via an amidation reaction with α-bromoisobutyryl bromide or 2chloropropionyl chloride (affording PEG-Br or PEG-Cl macroinitiator, respectively). Using the PEG-Cl macroinitiator, a thermoresponsive hydrophilic diblock copolymer, PEG-b-poly(N-isopropylacrylamide) (PEG-b-PNIPAM) equipped with an o-nitroaniline moiety at the chain junction point was readily achieved using ATRP. The nitro group could be reduced under mildly reductive conditions to generate an o-phenylenediamine derivativewhich has well-established NO-reactivitywithout 3818


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Scheme 2. Synthetic Routes Employed for the Preparation of PEG-Based Macroinitiators (PEG-Cl and PEG-Br) and Subsequent Preparation of PEG-b-PNIPAM DHBCs via ATRP

mL) were charged into a 20 mL polymerization vial equipped with a stirring bar and rubber septum. The mixture was carefully deoxygenated by sparging with nitrogen for 20 min. After that, CuCl (10 mg, 0.1 mmol) was added under a blanket of nitrogen and the polymerization vial was capped. The polymerization was carried out at 35 °C for 3.5 h. After that, the polymerization vial was opened, and the contents diluted with 10 mL of THF and stirred at room temperature for 30 min. The copper catalyst was removed by passing the organic solution through a silica gel column. The organic solution was then concentrated under vacuum using a rotary evaporator and precipitated into an excess amount of diethyl ether three times. PEG-b-PNIPAM diblock copolymers were achieved as yellowish powder. According to a similar procedure, PEG-b-PNIPAM DHBCs were also synthesized using PEG-Br macroinitiator instead of PEG-Cl macroinitiator. Reduction of PEG-Br Macroinitiator with Zinc Powder (Scheme S1). Typically, PEG-Br (120 mg, [NO2] = ∼50 μmol) was dissolved in a mixture of methanol and HCl (1 N) (20 mL, v:v = 1/1) at 0 °C. Zinc powder (650 mg, 10 mmol) was then added progressively over 5 min. The yellowish solution turned colorless within minutes, suggesting the successful reduction of NO2 groups. After 1 h stirring at 0 °C, the mixture was passed through a neutral aluminum oxide column and concentrated under vacuum using a rotary evaporator. The aqueous solution was neutralized with NaHCO3 and extracted with CH2Cl2 three times. The organic phase was combined and dried over anhydrous Na2SO4. After precipitation into diethyl ether, the reduced product was obtained as a white solid (∼100 mg). The reduction of PEG-b-PNIPAM DHBCs was also conducted by a similar procedure. Treatment of the Reduced PEG Macroinitiator with NO (Scheme S1). The reduced PEG-Br macroinitiator (56 mg, ∼ 25 μM) was dissolved in 5 mL of deionized water and charged with NO gas for 5 min, followed by stirring at room

24 h. After that, the mixture was precipitated into an excess amount of diethyl ether to remove DMF and unreacted 2. The solid was collected and redissolved in 20 mL THF, and the resulting solution was passed through a silica gel column to remove the copper catalyst. The THF solution was concentrated under vacuum using a rotary evaporator and precipitated into an excess amount of diethyl ether until the supernatant was colorless. After drying in a vacuum oven at room temperature, the final product was afforded as a yellowish solid (1.75 g, yield: ∼80%, Figure S3). Synthesis of PEG-Based Macroinitiators (Scheme 2). The PEG-based macroinitiators, PEG-Br and PEG-Cl, were synthesized via amidation of the PEG-NH2 precursor with either α-bromoisobutyryl bromide or 2-chloropropionyl chloride in the presence of K2CO3. Using the preparation of PEG-Br as an example, PEG-NH2 (1.1 g, 0.5 mmol) and K2CO3 (695 mg, 5 mmol) were dissolved in 100 mL of dry CH2Cl2 (dried by molecular sieves). To the resulting solution α-bromoisobutyryl bromide (0.81 mmol, 100 μL) in 5 mL of dry CH2Cl2 was added dropwise. After addition, the reaction was stirred gently at room temperature for a further 1 h. After removal of undissolved K2CO3 by vacuum filtration, the CH2Cl2 solution was then washed successively with saturated NaHCO3 and saline solutions, dried over sodium sulfate, concentrated under vacuum using a rotary evaporator, and precipitated into diethyl ether three times. PEG-Br macroinitiator was obtained as yellowish powder (0.83 g, yield: ∼73%, Figure S4a). PEG-Cl macroinitiator was synthesized via a similar protocol using 2-chloropropionyl chloride instead of α-bromoisobutyryl bromide. The resulting NMR spectrum of PEG-Cl is shown in Figure S4b. Synthesis of PEG-b-PNIPAM Diblock Copolymers (Scheme 2). PEG-b-PNIPAM diblock copolymers were synthesized via ATRP using either PEG-Br or PEG-Cl macroinitiator. In a typical experiment, PEG-Cl (228 mg, 0.1 mmol), NIPAM (1.13 g, 10 mmol), Me6tren (23 mg, 0.1 mmol), and isopropanol (4.0 3819


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backbone and characteristic peaks of aromatic rings was determined to be ∼180:1 (Figure S3). Subsequently, PEGbased macroinitiators, PEG-Br and PEG-Cl, were synthesized via amidation of the aniline residue of the PEG-NH2 precursor with α-bromoisobutyryl bromide or 2-chloropropionyl chloride. The chemical structures of the resulting macroinitiators were confirmed by NMR spectroscopy (Figure S4). It is well-known that o-phenylenediamine derivatives can react with NO in an aerobic environment with the generation of benzotriazole motifs, and this has been extensively employed for the development of fluorescent NO probes.46,47 Moreover, our group recently employed this chemistry to construct NOresponsive polymers.39−41 While an o-phenylenediamine containing molecule is a more tempting starting material to prepare NO-responsive polymers, we selected to start with 4amino-3-nitrophenol (rather than a diaminophenol derivative) for the following reasons: (1) 4-amino-3-nitrophenol is more stable than diaminophenol derivatives during synthetic procedures; (2) ensuring that only one amino group is functionalized in a diaminophenol derivative would be difficult, and the isolation of monofunctionalized product would be time-consuming; and (3) the residual free amino group could be problematic during the subsequent ATRP step. Given that the nitro groups can be converted to amino with use of a mild reducing agent, we hypothesized that the reduced product could further react with NO to form amide-derived benzotriazole moieties, which in turn may be susceptible to water and undergo spontaneous hydrolysis in an aqueous milieu.41 To verify our hypothesis, we first examined the chemical transitions of PEG-Br macroinitiator in the presence of reducing agent, followed by NO exposure. Specifically, the UV−visible absorbance of PEG-Br in aqueous solution was monitored before and after reduction with zinc powder, and after exposure to NO. Initially, there was an intense absorption peak centered at ∼348 nm, which was tentatively ascribed to the absorbance of nitrobenzene moieties. Upon addition of zinc powder into the acidic aqueous/alcoholic solution of PEG-Br, the initial yellowish solution faded within several minutes and a colorless solution was achieved after 1 h incubation, suggesting the consumption of nitro motif on the PEG-Br macroinitiator. This visible transition was reflected by changes in the UV− visible absorbance, with the complete loss of absorbance at ∼348 nm (Figure S5). Meanwhile, a new absorbance peak centered at ∼288 nm became evident, in accordance with the absorbance of the amide-functionalized o-phenylenediamine derivative (Figure S5).41 The successful reduction reaction was further confirmed by NMR analysis (Figure S6), revealing a shift to high field for the protons of the aromatic ring due to the removal of electron-withdrawing NO2 group. It is also important to note that the α-bromide containing initiator was labile under a reductive environment and the removal of the αbromine was observed (Figure S6b). Subsequently, the reactivity of the reduced PEG-Br toward NO was examined. Upon sparging NO gas to the aqueous solution of reduced PEG-Br macroinitiator, a remarkable absorbance change was observed (Figure S5), indicating that the reduced PEG macroinitiator with amide-functionalized ophenylenediamine motif was sensitive to NO. Importantly, we found that the NO-adduct product derived from reduced PEGBr macroinitiator was not stable in aqueous media, and that the hydrolysis process could be readily monitored by UV−vis spectrophotometry. As shown in Figure S7, the absorbance

temperature for 24 h. The aqueous solution was freeze-dried and the isolated polymer precipitated into an excess amount of diethyl ether before further characterization. Characterization. Nuclear Magnetic Resonance (NMR) Spectra. 1H and 13C NMR spectra were recorded on a Bruker AC400F (400 MHz) spectrometer. Deuterium oxide (D2O), chloroform (CDCl3), and DMSO-d6 were used as the solvents, depending on the particular substance being analyzed. Gel Permeation Chromatography (GPC). GPC analyses of polymer samples were performed in N,N-dimethylacetamide (DMAc with 0.03% w/v LiBr and 0.05% 2,6-dibutyl-4methylphenol (BHT) using a Shimadzu modular system comprising a DGU-12A degasser, an SIL-10AD automatic injector, and a 5.0 μm bead-size guard column (50 × 7.8 mm) followed by four 300 × 7.8 mm linear Phenogel columns (bead size: a 5.0 μm; pore sizes: 105, 104, 103, and 500 Å) and an RID-10A differential refractive-index detector. The temperature of columns was maintained at 50 °C using a CTO-10A oven, and the flow rate was kept at 1 mL/min using a LC-10AT pump. A molecular weight calibration curve was produced using commercial narrow molecular weight distribution polystyrene standards with molecular weights ranging from 500 to 106 g/mol. Polymer solutions at 2−3 mg/mL were prepared in the eluent and filtered through 0.45 μm filters prior to injection. UV−Visible Spectrophotometry. All UV−vis spectra were acquired on a Shimadzu UV-3600 UV−vis-NIR spectrophotometer in quartz cuvettes of 10 mm path length. The UV−vis spectra were recorded immediately after NO solution addition unless otherwise noted. For the turbidity measurements, the optical transmittance of the aqueous PEG-b-PNIPAM solutions (2.0 g/L in all cases) before and after reduction and further treatment with NO were acquired on the same spectrophotometer equipped with a Shimadzu temperature controller and Tm Analysis software, using quartz cuvettes (path length = 10 mm) at a wavelength of 500 nm with a heating rate of 0.5 °C/ min. The LCST was defined as the temperature corresponding to 50% transmittance.45 Fourier Transform Infrared (FT-IR) Spectra. Fourier transform infrared (FT-IR) spectra were recorded on a Shimadzu IRTracer-100 FT-IR spectrometer under attenuated total reflectance (ATR). The spectra were collected over 64 scans with a spectral resolution of 4 cm−1.

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RESULTS AND DISCUSSION A thermoresponsive DHBC, PEG-b-PNIPAM, incorporating a single NO-responsive unit at the junction of the two blocks was synthesized via ATRP using PEG-based macroinitiators (Scheme 2). To prepare the PEG-based macroinitiator, commercially available 4-amino-3-nitrophenol was first functionalized at the hydroxyl group with propargyl bromide to form 2-nitro-4-(prop-2-yn-1-yloxy)aniline (2). The chemical structure of 2 was confirmed by 1H and 13C NMR analysis (Figure S1). In the next step, a slight excess amount of 2 (1.5 equiv) was reacted with azide-terminated PEG (PEG-N3) via the copper-catalyzed click reaction. After removing the unreacted 2 by precipitating into diethyl ether, the PEG-NH2 precursor was isolated as yellowish powder (Scheme 2). Full conversion of PEG-N3 was verified by FT-IR spectra, which revealed the disappearance of the peak at ∼2100 cm−1 corresponding to N3 moieties (Figure S2). Additionally, the 1 H NMR spectrum further supported the complete functionalization of the PEG-N3, as the ratio of integrals for the PEG 3820


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Macromolecules Table 1. Structural Parameters of the DHBCs Used in This Study entry

sample

Mna/kDa

Mnb/kDa

Mw/Mnb

LCSTc/oC

P1 BP1-R BP1-NO BP2 BP2-R BP2-NO

PEG45-b-(NO2)PNIPAM61 PEG45-b-(NH2)PNIPAM61 PEG45-BT/PNIPAM61-COOH PEG45-b-(NO2)PNIPAM83 PEG45-b-(NH2)PNIPAM83 PEG45-BT/PNIPAM83-COOH

9.2 9.2 − 11.7 11.7 −

16.7 17.1 − 19.8 19.7 −

1.08 1.08 − 1.10 1.09 −

48.6 49.8 38 43.2 45.1 36.5


a Determined by NMR analysis. bDetermined by GPC analysis using DMAc as the eluent. cDetermined by temperature-dependent transmittance changes of the aqueous solutions (2.0 g/L) at the wavelength of 500 nm. Lower critical solution temperature (LCST) was defined as the temperature corresponding to a 50% decrease in transmittance.45

macroinitiator residue after polymerization, suggesting that PEG-Cl was a feasible initiator for the ATRP of NIPAM. Subsequently, a similar protocol to that used for the PEG-Br macroinitiator was followed to appraise the NO-responsive behavior of the DHBCs and their corresponding thermoresponsive transitions upon NO treatment. Using BP1 as an example, reduction in the presence of zinc powder was carried out in an ice−water bath, and this led to the expected disappearance of yellowish color, and accompanying changes in the UV−vis spectrum (Figure S10). The 1H NMR result further confirmed the successful reduction of BP1, with the characteristic resonance signals of the aromatic rings shifting to upfield, likely due to the conversion of the NO2 group to NH2 (Figure S11). These observations were consistent with the PEG-Br macroinitiator after reduction (Figures S5 and S6). Importantly, there was no noticeable shift in GPC elution times for BP1 before and after reduction (Figure 1), which suggests

peaks at 245 and 325 nm underwent a steady decrease, whereas the absorbance originating from free benzotriazole gradually increased. The hydrolysis reaction reached a plateau within ∼5 h incubation in aqueous solution (Figure S7b), which was slower than for an NO-responsive monomer consisting of an amide-functionalized benzotriazole entity.41 After recovery of the final hydrolyzed product by lyophilization, the 1H NMR spectra demonstrated the formation of benzotriazole-containing PEG derivative (Figure S6c). Taken together, these results indicate that the NO2 group-containing PEG-based macroinitiator (i.e., PEG-Br) can be successfully reduced with the generation of amide-decorated o-phenylenediamine derivatives. Further, the reduced product is intrinsically sensitive to NO exposure (resulting in formation of amide-functionalized benzotriazole moieties at the chain end). Finally, benzotriazole-terminated PEG was isolated as the final product due to the spontaneous hydrolysis of the amide-functionalized benzotriazole derivative (Scheme S1). On the basis of the above results, we attempted to extend the unique NO-responsive behavior to block copolymer systems having an NO sensitive junction (i.e., using the NO-responsive PEG-Br as the macroinitiator). However, it was not possible to prepare well-defined PEG-b-PNIPAM diblock copolymers when using PEG-Br macroinitiator, as evidenced by GPC analysis which revealed two distinct peaks and a relatively broad polydispersity (>1.2, Figure S8). This was somewhat surprising given that amide-based initiators have been successfully employed in the polymerization of (meth)acrylates and styrene monomers.48−52 The failure of polymerization of NIPAM using PEG-Br macroinitiator can be attributed to a low equilibrium ATRP constant, unfavorable dynamics, and possible side reactions of C-Br bonds as suggested by Matyjaszewski and co-workers.53 Considering the successful ATRP of (meth)acrylamide monomers54,55 and the results reported by Xia et al.,56 indicating that chloropropionamide-based small molecule initiator could be successfully used to polymerize NIPAM monomer in 2-propanol with excellent control, we proposed substituting PEG-Cl for PEG-Br macroinitiator in order to achieve well-defined PEG-b-PNIPAM diblock copolymers. The PEG-Cl macroinitiator can be readily synthesized via a similar protocol to PEG-Br, using 2-chloropropionyl chloride instead of α-bromoisobutyryl bromide (Scheme 2). The chemical structure of PEG-Cl was identified by 1H NMR spectroscopy (Figure S4b). Interestingly, the polymerizations of NIPAM using PEG-Cl macroinitiator exhibited very narrow polydispersities (≤1.10, Table 1, Figure S8) and the detailed polymerization kinetic study (shown in Figure S9) is in agreement with a previous report where a small molecule-based ATRP initiator was applied.56 Moreover, there was no unreacted PEG-Cl

Figure 1. GPC traces recorded for PEG-b-PNIPAM (BP1) (a) before reduction, (b) after reduction, and (c) after reduction, with NO treatment for 5 min and standing at room temperature for 24 h. (d) PEG-Br macroinitiator after reduction and NO treatment for 5 min and standing at room temperature for 24 h.

that the reduction process was exclusively toward NO2 groups and did not adversely affect the stabilities of other functional groups in the polymer (such as amide bonds and the 1,2,3triazole ring originating from the click reaction). The successful reduction to an o-phenylenediamine functionalized amide allows for further manipulation of the thermoresponsive transitions of the DHBCs by exposure to NO. Next, the NO-responsive performance of reduced BP1 was examined. Upon addition of varying amounts of NO (0−1.0 equiv. relative to the o-phenylenediamine derivative) into the aqueous solution of reduced BP1, the absorbance peaks at 266 and 325 nm steadily increased, whereas the absorbance peak at 289 nm underwent a slight decrease (Figures 2a and 2b). The 3821


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Figure 2. (a) Absorbance spectra and (b) corresponding absorbance intensity changes for PEG-b-PNIPAM (BP1, 1.0 g/L, ∼100 μM) after reduction with zinc powder and adding varying amounts of NO (0−1.0 equiv. relative to the o-phenylenediamine derivative). (c) Time-dependent absorbance spectra and (d) absorbance intensity changes for PEG-b-PNIPAM (BP1, 1.0 g/L, ∼100 μM) after reduction with zinc powder and subsequent treatment with 100 μM NO.

Scheme 3. Proposed Mechanism for Reaction of the PEG-b-PNIPAM Diblock Copolymer upon Sequential Exposure to (i) Reducing Agent and (ii) NOa

a The nitro group was first reduced to an amino group in the presence of zinc powder, thus generating o-phenylenediamine motifs. The resulting ophenylenediamine motif was capable of reacting with NO to form amide-functionalized benzotriazole groups, which spontaneously hydrolyzed and thus resulted in the cleavage of PEG-b-PNIPAM diblock copolymer.

absorption spectra leveled off after addition of ∼1.0 equiv of NO (relative to the o-phenylenediamine motif), suggesting a highly specific reaction between NO and the polymer. By contrast, the BP1 precursor which had not been reduced

exhibited negligible absorbance changes upon NO addition (Figure S12a), confirming that the o-nitroaniline precursor was intrinsically insensitive to NO. Further, after reaction with NO, the BP1 diblock copolymer (like the PEG-Br macroinitiator), 3822


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Macromolecules was labile in aqueous solution. Spontaneous hydrolysis of the benzotriazole after NO treatment was evidenced by the decreased absorbance at 325 nm and the increased absorbance at 284 nm, respectively (Figure 2c,d). Hydrolysis of the BP1 diblock copolymer after NO treatment was complete within ∼10 h, this being slightly slower than that of PEG-Br macroinitiator (Figure S7). We tentatively attributed the slower hydrolysis to the steric hindrance of the diblock chains. Significantly, compared with previously reported gas-responsive polymers and our own work regarding NO-responsive polymers,39−41 this PEG-b-PNIPAM DHBC incorporating a single NO-responsive o-phenylenediamine motif at the block junction displayed better responsiveness to a much lower gas concentration. This may be beneficial for biomedical applications where the concentration of endogenous gases can be very low.57 After 24 h standing in aqueous solution, the BP1 diblock copolymers subjected to reduction and treatment with NO were recovered from aqueous solution via lyophilization, and analyzed via GPC. The GPC elution profile, shown in Figure 1, exhibits two peaks rather than the single symmetrical peak evident prior to NO addition. Moreover, the elution time of the second peak corresponded well with that of benzotriazoleterminated PEG originating from PEG-Br macroinitiator after reduction and NO treatment (Figure 1). Importantly, there was no appreciable shift in the elution time if the reduction process was skipped, even upon exposure to a much higher NO concentration (Figure S12b). This demonstrates that the selective generation of the o-phenylenediamine motif under zinc reduction played an essential role in achieving NOresponsive behavior. It is worth noting that the final absorbance of BP1 diblock copolymer, after reduction with zinc powder and following NO addition, was quite comparable to that of the PEG-Br macroinitiator treated with the same procedure (Figure S13). Moreover, the 1H NMR spectrum of BP1 after reduction and treatment with NO is strongly suggestive of the formation of free benzotriazole-terminated PEG (BT-PEG) and carboxylterminated PNIPAM (Figure S14). As such, we propose that the BP1 diblock copolymers undergo a similar transition to PEG-Br macroinitiator upon reduction and NO treatment. More specifically, the nitro group is first reduced in the presence of zinc powder, generating an o-phenylenediamine derivative at the junction of the blocks in the PEG-b-PNIPAM diblock copolymer. The o-phenylenediamine moiety can then be transformed to an amide-functionalized benzotriazole in the presence of NO, which then spontaneously hydrolyzes leading to scission of the diblock copolymer chain (Scheme 3). Since PNIPAM exhibits a lower critical solution temperature (LCST) around 32 °C in aqueous solution,58 we surmised that the LCST of the current NO-responsive diblock copolymers would be significantly altered by the NO-induced cleavage of hydrophilic PEG. Using turbidimetry, the LCSTs of BP1 and BP2 (with varying PNIPAM lengths) before reduction were determined to be 48.6 and 43.2 °C, respectively (Table 1 and Figure 3). These were much higher than that of PNIPAM homopolymer,55,59 as might be expected due to the hydrophilic PEG chain which serves to elevate the phase transition temperature. The reduction of NO2 groups in the presence of zinc powder led to a slight increase in the LCSTs, presumably due to the formation of more hydrophilic ophenylenediamine moieties. Notably, the increased LCSTs confirmed that the diblock copolymer remained intact after reduction, in accordance with the GPC result (Figure 1).

Figure 3. Temperature-dependent transmittance changes recorded at a wavelength of 500 nm for aqueous solutions (2.0 g/L) of (a) BP1 before reduction, (b) BP1 after reduction, (c) BP1 after reduction and treatment with NO and standing for 24 h, (d) BP2 before reduction, (e) BP2 after reduction, and (f) BP2 after reduction and NO treatment and standing for 24 h.

However, significant drops in LCST were observed after the reduced DHBCs were treated with NO. Specifically, the LCSTs of BP1 and BP2, after reduction and NO addition, were reduced to 38 and 36.5 °C, respectively (Table 1 and Figure 3). These results are consistent with previous results regarding the LCSTs of PNIPAM homopolymers.59 The dramatic reduction of the LCSTs can be attributed to the loss of hydrophilic PEG after the DHBCs were subjected to NO addition (Scheme 3).

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CONCLUSION In conclusion, a well-defined thermoresponsive DHBC, PEG-bPNIPAM, was successfully synthesized via conventional ATRP using a PEG-Cl macroinitiator incorporating an o-nitroaniline precursor. The PEG-b-PNIPAM DHBC underwent reduction in the presence of reducing agent (in this case zinc powder) resulting in generation of an o-phenylenediamine moiety at the junction of the two blocks. The resulting polymers were highly sensitive to exposure to nitric oxide. Specifically, upon exposure to NO, the o-phenylenediamine derivative in the chain junction point was readily transformed to an amide-derived benzotriazole motif, which spontaneous hydrolyzed leading to scission of the diblock copolymer. The cleavage of the DHBCs, with the generation of benzotriazole-terminated PEG and carboxyl-functionalized PNIPAM, led to substantially decreased LCSTs as compared to the block copolymer prior to NO treatment. This novel NO-responsive chemistry could be utilized to develop novel drug nanocarriers which exploit endogenous NO as the stimulus to trigger release of embedded cargo through scission of supramolecular assembly constituents.

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ASSOCIATED CONTENT

S Supporting Information *

Conversion of the PEG-Br macroinitiator, detailed NMR spectroscopy, ATR FT-IR spectra, UV−visible spectroscopy, and GPC elution profiles. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b00996.

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AUTHOR INFORMATION

Corresponding Authors

*(T.P.D.) E-mail:[email protected]. *(J.F.Q.) E-mail: [email protected]. 3823


Article

Macromolecules Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was conducted within the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036). T.P.D. gratefully acknowledges the award of an Australian Laureate Fellowship.


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Nitric Oxide (NO) Cleavable Biomimetic Thermoresponsive Double

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