Thermo- and Light-Regulated Formation and Disintegration of Double

Feb 20, 2013 - negatively charged carboxylate residues, leading to elevated CMT (∼46 °C). Thus, thermo-induced mixed micelles in the intermediate ...
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Thermo- and Light-Regulated Formation and Disintegration of Double Hydrophilic Block Copolymer Assemblies with Tunable Fluorescence Emissions Yonghao Wu, Huamin Hu, Jinming Hu, Tao Liu, Guoying Zhang, and Shiyong Liu* CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *

ABSTRACT: We report on thermo- and light-regulated formation and disintegration of double hydrophilic block copolymer (DHBC) micelles associated with tunable fluorescence emissions by employing two types of DHBCs covalently labeled with fluorescence resonance energy transfer (FRET) donor and acceptor moieties, respectively, within the light and temperature dually responsive block. Both DHBCs are molecularly soluble at room temperature in their aqueous mixture, whereas, upon heating to above the critical micellization temperature (CMT, ∼31 °C), they coassemble into mixed micelles possessing hydrophilic coronas and mixed cores containing FRET donors and acceptors. Accordingly, the closer spatial proximity between the FRET pair (NBDAE and RhBEA moieties) within micellar cores leads to substantially enhanced FRET efficiency, compared to that in the non-aggregated unimer state. Moreover, upon UV irradiation, the light-reactive moieties undergo light-cleavage reaction and transform into negatively charged carboxylate residues, leading to elevated CMT (∼46 °C). Thus, thermo-induced mixed micelles in the intermediate temperature range (31 °C < T < 46 °C) undergo light-triggered disintegration into unimers, accompanied with the decrease of FRET efficiency. Overall, the coassembly and disassembly occurring in the mixed DHBC solution can be dually regulated by temperature and UV irradiation, and most importantly, these processes can be facilely monitored via changes in FRET efficiency and distinct emission colors.

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INTRODUCTION In the past two decades, ever-increasing attention has been paid to the dynamic self- assembly of stimuli-responsive block copolymers,1−5 which can orderly aggregate into micelles, rods, or vesicles under a specified solution condition and undergo further structural disintegration or reorganization upon exerting an alternate external stimulus such as temperature,6−9 pH,10−12 light irradiation,13−15 ultrasound,16,17 enzyme,18−20 or redox potential.21,22 By virtue of the stimuli-tunable assembly and disassembly behavior, responsive block copolymer assemblies have been broadly utilized and exploited in a variety of fields such as drug/gene delivery and controlled release,23−26 sensing matrix,27 smart coatings,28,29 nanoreactors30 and templates,31,32 and switchable catalysis.33,34 Among various types of stimuli which can be utilized to modulate block copolymer assemblies, light irradiation is an attractive one due to its easy operation, low cost, fast responsiveness, facile on/off switch, site-specificity, tunable wavelengths and irradiation intensity, and no need of introducing additional chemical agents.13 Therefore, polymeric assemblies based on light-responsive amphiphilic or double hydrophilic block copolymers (DHBCs) have received extensive investigations. The general strategy employed is to incorporate photolabile,35−37 photoisomerizable,38−40 or photoionizable moieties41,42 into the hydrophobic block of © 2013 American Chemical Society

amphiphilic diblock copolymers. Upon exposure to UV light or pulsed infrared laser irradiation, the hydrophobic block can be rendered water-soluble via specific phototriggered reactions. Consequently, the initially formed self-assembled aggregates will dissociate into molecularly dissolved unimers in aqueous solution. If the photoreactions involved are reversible, the processes of light-regulated assembly/disassembly of self-assembled aggregates can be repeated, as reported in those cases that involve light-ionizable or light-isomerizable spiropyran,41,42 malachite green,43 and azobenzene moieties.38,39 However, most types of light-responsive moieties, which can be covalently incorporated into block copolymers and endow them with light-triggered micelle-to-unimer characteristics, undergo irreversible photolabile reactions. In this aspect, Zhao research group have presented a series of pioneering works by employing amphiphilic block copolymers with the hydrophobic block functionalized with light-cleavable moieties such as ester derivatives of pyrenylmethyl, 35 o-nitrobenzyl,37 and 7(diethylamino)coumarin.36 In a typical example, they synthesized amphiphilic light-responsive diblock copolymers, poly(ethylene oxide)-b-poly(2-nitrobenzyl methacrylate) (PEO-bReceived: May 22, 2012 Published: February 20, 2013 3711

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Scheme 1. Thermo- and Light-Regulation of FRET Efficiency in the Aqueous Mixture of PEO-b-P(NIPAM-co-DMNA-coNBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) DHBCs via Reversible Thermo-Induced Comicellization (above CMT1) and UV Irradiation-Induced Disintegration of Mixed Micelles at Temperatures between CMT1 (Non-Irradiated Aqueous Mixture) and CMT2 (UV Irradiated Aqueous Mixture)a

a

NBDAE and RhBEA serve as FRET donor and acceptor moieties, respectively.

PNBM),37 which can self-assemble into core−corona type micelles. Upon irradiation with either one-photon UV (∼365 nm) or two-photon near-infrared light (∼700 nm), micelles disintegrated due to light cleavage of o-nitrobenzyl moieties and the generation of carboxylate residues within the initially hydrophobic block.44 This led to transformation of an amphiphilic diblock copolymer into a DHBC, as demonstrated by the light-triggered release of Nile Red physically encapsulated within hydrophobic cores. In their recent work, light-labile 7-(diethylamino)coumarin ester derivatives were employed for more effective two-photon triggered micelle disintegration.36 In addition, the combination of NaYF4:TmYb upconverting nanoparticles (UCNPs) with micelles containing photolabile 4,5-dimethoxy-2-nitrobenzyl methacrylate residues also allowed triggered micellar disassembly under continuouswave near-infrared laser irradiation.45 Other photoresponsive moieties such as 2-diazo-1,2- naphthoquinone (DNQ) were also employed to achieve phototriggered micellar disintegration, as reported by Fréchet et al.46 in 2005. They prepared DNQ-terminated PEG lipids, which self-assemble into micelles above the critical micellization concentration (CMC) of ∼0.15 g/L. Under 365 nm UV or 795 nm pulse laser irradiation, the DNQ terminal functionality undergoes Wolff rearrangement and transforms into 3-indenecarboxylic acid (pKa ∼4.5), which is highly hydrophilic, and this renders light-triggered micellar dissociation. It is worth noting that the above examples exclusively utilized light irradiation as the sole stimulus to trigger micellar disintegration. Recently, diblock copolymer assemblies responsive to two or more external stimuli9,18,19 have emerged to be another hot topic.8,9,19−22 Specifically, certain DHBCs can possess both thermo- and light-responsiveness. Zhao et al.9 reported in 2008 the synthesis of poly(ethylene oxide)-bpoly(ethoxytri(ethylene glycol) acrylate-co-o-nitrobenzyl acrylate) diblock copolymers, PEO-b-P(TEGEA-co-NBA), which molecularly dissolve in water at room temperature but selfassemble into micelles above the critical micellization temperature (CMT). Upon UV irradiation, the newly generated

carboxylate residues resulting from the photocleavage of onitrobenzyl moieties lead to elevated CMT, and this process is accompanied by micellar dissociation. For copolymers of Nisopropylacrylamide (NIPAM) and NBA, UV irradiation also leads to elevated lower critical solution temperature (LCST), which can be further exploited to develop PNIPAM-based thermoresponsive photoresists. In the above examples, the thermo- and light-triggered micellar formation/disintegration processes were typically monitored by changes in optical transmittance, light scattering intensities, and aggregate sizes.9,47,48 Though the changes of fluorescence emission intensity of physically embedded dyes37,43 (e.g., Nile Red, pyrene) have also been utilized to monitor and verify micellar assembly/disassembly events, it is only an indirect and qualitative technique due to the nature of noncovalent encapsulation. If these responsive diblock copolymer micelles are to be used as in vivo drug delivery and controlled-release nanocarriers, the above approaches might restrict the direct monitoring of micelle formation/ disintegration processes. We have recently been interested in the development of responsive polymer-based sensing and detection systems, which involves the construction of ratiometric fluorescent probes of pH, temperature, and other analytes by utilizing the fluorescence resonance energy transfer (FRET) principle.49 The FRET efficiency typically depends on many factors,50,51 such as the extent of spectral overlap between the emission band of the FRET donor and the excitation band of the FRET acceptor, the orientation of the transition dipole moments of the two fluorophores, and, most importantly, the spatial distance (∼1−10 nm) between them. We expect that, if thermo- and light-responsive diblock copolymers were respectively labeled with FRET donors and acceptors, thermo-induced nanosized polymeric assemblies can offer a distinct nanoplatform for the FRET process; additionally, lighttriggered disassembly of polymeric micelles into unimers will lead to decreased FRET efficiency and emission color changes, which can be facilely utilized for monitoring these processes. 3712

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In our previous work, 49 thermo-responsive nanogels covalently labeled with FRET donors and acceptors and DMNA (tertiary amine-containing NBA derivative) were fabricated and the thermal phase transition of nanogels can be monitored via FRET efficiency changes. However, due to the fact that FRET donors and acceptors are initially covalently colabeled into microgels, the change in FRET efficiency can only be incurred by volumetric phase-transition-induced modulation of the relative distance between FRET donors and acceptors; thus, the emission intensity ratios (F588/F527) only changed ∼1.8-fold (from ∼2.2 to 1.2) upon UVirradiation-incurred nanogel swelling. In this work, we report on the thermo- and light-regulated formation and disintegration of DHBC micelles associated with tunable fluorescence emission characteristics by using two types of DHBCs covalently labeled with FRET donor and acceptor moieties, respectively, within the thermo- and light-responsive block (Scheme 1). PEO-b-P(NIPAM-co-DMNA-co-NBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) DHBCs were synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization at first (Scheme 2), where DMNA

Mw/Mn = 1.06) was purchased from Aldrich and used as received. NIsopropylacrylamide (NIPAM, 97%, Tokyo Kasei Kagyo Co.) was purified by recrystallization from a mixture of benzene and n-hexane (1/3, vol/vol). 5-(2′-(Dimethylamino)ethoxy)-2-nitrobenzyl acrylate (DMNA), 49 3-(benzylthiocarbonothioyl- thio)propanoicacid (BTPA),52 4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBDAE), 53,54 and rhodamine-B-based fluorescent monomer (RhBEA)49 were prepared according to previously reported literature procedures. 2,2′-Azoisobutyronitrile (AIBN) was recrystallized from 95% ethanol. Dichloromethane (CH2Cl2) was dried over CaH2 and distilled just prior to use. N,N′-Dicyclohexylcarbodiimide (DCC), 4dimethylaminopyridine (DMAP), and all other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Water was deionized with a Milli-Q SP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ cm. Sample Preparation. Synthetic routes employed for the preparation of dually thermo- and light-responsive DHBCs labeled with NBDAE (FRET donor) and RhBEA (FRET acceptor), respectively, are shown in Scheme 2. Synthesis of PEO-Based macroRAFT Agent. PEO-based macroRAFT agent was prepared according to literature reports. In a typical procedure, PEO113−OH (10.0 g, 2.0 mmol) was dissolved in anhydrous toluene (50 mL) and azeotropic distillation was carried out at 50 °C under reduced pressure to remove most of the solvent. Then, BTPA (1.09 g, 4.0 mmol) and dry CH2Cl2 (100 mL) were added. After cooling to 0 °C in an ice−water bath, a mixture of DCC (0.83 g, 4.0 mmol) and DMAP (49 mg, 0.4 mmol) in dry CH2Cl2 (20 mL) was added dropwise over ∼1 h. The reaction mixture was then stirred at room temperature for 48 h. After that, the insoluble salts formed in the reaction mixture were removed by filtration, and the filtrate was concentrated on a rotary evaporator and precipitated into an excess of cold diethyl ether. The collected precipitates were dissolved in CH2Cl2 and then precipitated again into anhydrous diethyl ether. The above dissolution−precipitation cycle was repeated three times, and the final precipitates were collected and dried in a vacuum oven overnight at room temperature to afford PEO113 macroRAFT agent as a slightly yellowish powder (8.4 g, yield 79.7%; Mn,GPC = 5.2 kDa, Mw/Mn = 1.07) (Figure 1a). The degree of

Scheme 2. Schematic Illustration for the RAFT Synthesis of Well-Defined Thermo- and Light-Responsive DHBCs, PEOb-P(NIPAM-co-DMNA-co-NBDAE), and PEO-b-P(NIPAMco-DMNA-co-RhBEA)

is a light-cleavable monomer, 5-(2-(dimethylamino)ethoxy)-2nitrobenzyl acrylate, and NBDAE and RhBEA are 4-(2acryloyloxyethylamino)-7-nitro-2,1,3- benzoxadiazole and rhodamine-B-based polymerizable monomers acting as FRET donor and acceptor, respectively. Due to the thermo-responsiveness of the NIPAM-containing block, both DHBCs are molecularly soluble at room temperature in their aqueous mixture (1:1, wt/ wt), whereas, upon heating to above the CMT, the two DHBCs coassemble into mixed micelles possessing PEO coronas and mixed cores of P(NIPAM-co-DMNA-co-NBDAE) and P(NIPAM-co-DMNA-co-RhBEA). Accordingly, the closer spatial proximity between the FRET pair (NBDAE and RhBEA moieties) within micellar cores led to substantially enhanced FRET efficiency compared to the unimer state below the CMT. Moreover, upon UV irradiation, DMNA moieties undergo light-cleavage reactions, affording negatively charged carboxylate residues and leading to prominently elevated CMT. Thus, thermo-induced mixed micelles formed in the intermediate temperature range can undergo light-triggered micelle-tounimer transformation, accompanied with the decrease of FRET efficiency and emission color changes. Overall, the introduction of controllable FRET processes into responsive DHBCs allows for facile in situ monitoring of triggered micellar formation and disintegration processes.

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Figure 1. DMF GPC traces recorded for (a) PEO113-based macroRAFT agent, (b) PEO113-b-P(NIPAM0.91-co-DMNA0.09-coRhBEA) 68 , and (c) PEO 113 -b-P(NIPAM 0.91 -co-DMNA 0.09 -coNBDAE)90 block copolymers. end-group functionalization was calculated to be nearly quantitative, as evidenced by 1H NMR analysis (Figure S1, Supporting Information; CDCl3, δ, ppm, TMS: 7.33 (5H, ArH), 4.60 (2H, ArCH2), 4.27 (2H, CH2OCOCH2), 3.83−3.58 (452H, CH2CH2O), 3.54 (3H, CH3O), 3.38 (2H, CH2OCOCH2CH2SC(S)), 2.82 (2H, CH2OCOCH2CH2SC(S)). Synthesis of PEO-b-P(NIPAM-co-DMNA-co-NBDAE) and PEO-bP(NIPAM-co-DMNA-co-RhBEA). Thermo- and light-responsive DHBCs covalently labeled with NBDAE or RhBEA moieties were synthesized via RAFT polymerization. For the synthesis of PEO-bP(NIPAM-co-DMNA-co-NBDAE) diblock copolymer, a typical procedure was as follows. Into a reaction tube equipped with a magnetic stirring bar, NIPAM (0.99 g, 8.75 mmol), DMNA (0.23 g,

EXPERIMENTAL SECTION

Materials. Poly(ethylene oxide) monomethyl ether with a mean degree of polymerization (DP) of 113 (PEO113−OH, Mn = 5.0 kDa, 3713

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0.78 mmol), NBDAE (13 mg, 0.05 mmol), PEO-based macroRAFT agent (0.50 g, 0.095 mmol), AIBN (2.4 mg, 0.015 mmol), and 1,4dioxane (4.0 g) were charged. The reaction tube was carefully degassed by three freeze−pump−thaw cycles and then sealed under a vacuum. After being immersed into an oil bath thermostatted at 70 °C and stirred for 12 h, the reaction tube was quenched into liquid nitrogen, and 1,4-dioxane was added. The mixture was precipitated into an excess of diethyl ether. The above dissolution−precipitation cycle was repeated three times, and PEO-b-P(NIPAM-co-DMNA-co-NBDAE) diblock copolymer was obtained as a slightly yellowish powder after drying in an vacuum oven overnight at room temperature. The DP and NIPAM molar content of P(NIPAM-co-DMNA-co-NDBAE) block were determined to be 91 mol % from 1H NMR analysis (Figure S1, Supporting Information). Thus, the diblock copolymer was denoted as PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-NBDAE)90. The NBDAE molar content in the thermoresponsive block was determined to be 0.43 mol % via fluorescence measurement using NBDAE monomer as the calibration standard. GPC analysis revealed an Mn of 16.0 kDa and an Mw/Mn of 1.14 for PEO-b-P(NIPAM-co-DMNA-co-NBDAE) diblock copolymer (Figure 1b). PEO 113 -b-P(NIPAM 0.91 -coDMNA0.09-co-RhBEA)68 was also synthesized (Figure S2, Supporting Information; Mn = 15.1 kDa, Mw/Mn of 1.18, Figure 1c; RhBEA molar content in the thermoresponsive block: 0.40 mol %). On the basis of similar protocols, two control diblock copolymers without DMNA moieties, PEO113-b-P(NIPAM-co- NBDAE)87 (Mn = 15.6 kDa, Mw/Mn = 1.13) and PEO113-b-P(NIPAM-co-RhBEA)84 (Mn = 15.2 kDa, Mw/ Mn = 1.14), were also synthesized. Characterization. All 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV300 NMR spectrometer operating at a resonance frequency of 300 MHz in the Fourier transform mode. CDCl3 was used as the solvent. The molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC) equipped with a Waters 1515 pump and Waters 2414 differential refractive index detector (set at 30 °C). A series of three linear Styragel columns (HR2, HR3, and HR4) were used at an oven temperature of 45 °C. DMF was used as the eluent at a flow rate of 1.0 mL/min. A series of low polydispersity polystyrene standards were employed for calibration. All UV−vis spectra were acquired on a Unico UV/vis 2802PCS spectrophotometer. The transmittance of the aqueous solutions as a function of temperature was acquired at a wavelength of 700 nm. A thermostatically controlled couvette was employed, and the heating rate was 0.2 °C min−1. The critical micellization temperatures (CMTs) of diblock copolymers were defined as the temperature at which ∼1% decrease in the optical transmittance can be discerned. Dynamic laser light scattering (LLS) measurements were conducted on a commercial spectrometer (ALV/DLS/SLS-5022F) equipped with a multitau digital time correlator (ALV5000) and a cylindrical 22 mW UNIPHASE He−Ne laser (λ0 = 632 nm) as the light source. Scattered light was collected at a fixed angle of 90° for a duration of ∼3 min. Distribution averages and particle size distributions were computed using cumulants analysis and CONTIN routines. All data were averaged over three measurements. Fluorescence spectra were recorded using a RF-5301/PC (Shimadzu) spectrofluorometer. The temperature of the water-jacketed cell holder was controlled by a programmable circulation bath. Both excitation and emission slit widths were set equal to 5 nm. Atomic force microscopy (AFM) measurements were performed on a Digital Instrument Multimode Nanoscope IIID operating in the tapping mode under ambient conditions. A silicon cantilever (RFESP) with a resonance frequency of ∼80 kHz and a spring constant of ∼3 N/m was used. The set-point amplitude ratio was maintained at 0.7 to minimize sample deformation induced by the tip. The samples were prepared by dip coating 0.2 g/L aqueous micellar solution before and after UV irradiation (365 nm, 1.0 mW/cm2) onto the surface of freshly cleaved mica.

DMNA-co-NBDAE) and PEO-b-P(NIPAM-co-DMNA-coRhBEA) diblock copolymers with the dually responsive block covalently labeled with FRET donor (NBDAE) and acceptor (RhBEA), respectively, were synthesized via RAFT copolymerization of NIPAM, DMNA, and NBDAE or RhBEA by employing PEO-based macroRAFT agent (Scheme 2). The RAFT polymerization of NIPAM and its copolymerization with other acrylate monomers has been well-documented in literature reports. GPC analysis revealed an Mn of 16.0 kDa and Mw/Mn of 1.14 for PEO113-b-P(NIPAM0.91-co-DMNA0.09co-NBDAE)90 and an Mn of 15.1 kDa and Mw/Mn of 1.18 for PEO113-b-P(NIPAM0.91-co-DMNA0.09- co-RhBEA)68 diblock copolymer (Figure 1). In both cases, a clear shift to the higher molecular weight side for the elution profiles of diblock copolymers compared to that of PEO macroRAFT agent can be discerned, suggesting the successful RAFT synthesis of dually responsive diblock copolymers. The chemical structures of two types of DHBCs were characterized by 1H NMR analysis (Figures S1 and S2, Supporting Information). According to peak area integral ratios of resonance signals characteristic of PEO (δ ∼ 3.6 ppm), NIPAM (−CH(CH3)2, peak g at δ ∼ 4.0 ppm), and DMNA moieties (benzyl CH2 protons, peak c at δ ∼ 5.45 ppm), respectively, the DPs of P(NIPAM-co-DMNA-coNDBAE) and P(NIPAM-co-DMNA-co-RhBEA) diblock copolymers were determined to be ∼90 and ∼68, respectively. In addition, NIPAM molar contents in the responsive dually responsive block were determined to be ∼91 mol % for both diblock copolymers. Thus, the two diblock copolymers were denoted as PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-NBDAE)90 and PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-RhBEA)68, respectively. Since the molar contents of NBDAE and RhBEA moieties in the hydrophobic block were too low to be accurately quantified by 1H NMR analysis, fluorescence measurements using NBDAE and RhBEA monomers in ethanol as the calibration standards were conducted, revealing ∼0.43 and ∼0.40 mol % dye labeling contents in the responsive blocks, respectively. Thermo- and Light-Regulated Formation and Disintegration of DHBC Micelles. It is well-known that the critical micellization temperature (CMT) of a thermoresponsive diblock copolymer can be facilely modulated to a desired value by incorporating hydrophilic or hydrophobic repeating units into the thermoresponsive block such as PNIPAM; besides, the CMT can also be tuned by other appropriate factors, such as pH, light, ionic strength, and terminal group hydrophobicity or hydrophilicity.55,56 In Zhao’s previous work concerning temperature and pH dually sensitive water-soluble diblock copolymers, poly(ethylene oxide)-b-poly(methoxydi(ethylene glycol) methacrylate-co-methacrylic acid),57 the CMTs can be tuned from 24 to 60 °C by changing the solution pH from 4.0 to 6.7. Ionov et al.28 copolymerized hydrophobic o-nitrobenzyl ester monomeric repeating units into PNIPAM, and the obtained P(NIPAM-co-NBA) copolymers exhibit considerably elevated LCST upon light-cleavage reaction which transforms hydrophobic NBA moieties into ionized carboxylate residues. In the current work, we synthesized temperature and light dually responsive DHBCs, PEO-b-P(NIPAM-co-DMNA-coNBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA). Thermo- and light-regulated self-assembling and disassembly behavior was then investigated. Temperature-dependent optical transmittance was employed at first to determine the CMTs (Figure 2a). At pH 8.5, PEO-b-P(NIPAM-co-DMNA-co-

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RESULTS AND DISCUSSION Synthesis of Dye-Labeled Dually Responsive Double Hydrophilic Block Copolymers. PEO-b-P(NIPAM-co3714

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in the range 330−400 nm and its intensity increased with irradiation duration. These results confirmed the light cleavage of o-nitrobenzyl moieties under UV 365 nm irradiation. As described above, the CMTs of PEO-b-P(NIPAM-coDMNA-co-NBDAE) and PEO-b-P(NIPAM-co-DMNA-coRhBEA) diblock copolymer solutions were ∼30 and ∼32 °C before UV irradiation and ∼46 and ∼51 °C after UV irradiation, respectively. Thus, at an intermediate temperature, such as at 37 or 40 °C, UV irradiation will lead to micelle-tounimer transformation due to the fact that the CMTs of UVirradiated diblock solutions get to be considerably higher (Scheme 1). Since both DHBCs possess covalently labeled dyes and NBDAE and RhBEA dyes were located within a more hydrophobic microenvironment in the micellar state and in a hydrophilic microenvironment in the unimer state, fluorescence measurements should be capable of monitoring the lighttriggered micellar disintegration process. As shown in Figures 3a and 4, upon UV irradiation, the fluorescence emission

Figure 2. (a) Temperature-dependent optical transmittance recorded at a wavelength of 700 nm for 1.0 g/L aqueous solutions (pH 8.5) of PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-NBDAE)90 (●) before and (○) after UV 365 nm irradiation for 30 min and PEO113-bP(NIPAM0.91-co-DMNA0.09-co-RhBEA)68 (■) before and (□) after UV irradiation for 30 min. (b) Temperature-dependent optical transmittance at a wavelength of 700 nm recorded for 1.0 g/L aqueous mixtures (pH 8.5) of PEO113-b-P(NIPAM0.91-co-DMNA0.09co-NBDAE)90 and PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-RhBEA)68 (1:1, wt/wt) (▲) before and (△) after UV irradiation for 30 min.

NBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) solutions appeared clear and transparent at room temperature, suggesting a molecularly dissolved state. Upon heating, a slightly bluish tinge characteristic of colloidal dispersion appeared for the aqueous solution. The CMTs of PEO-bP(NIPAM-co-DMNA-co-NBDAE) and PEO-b-P(NIPAM-coDMNA-co-RhBEA) diblock copolymer solution at pH 8.5 were determined to be ∼30 and ∼32 °C, respectively. Considering the light-cleavable feature of DMNA moieties within the NIPAM-containing block, aqueous solutions of the two DHBCs at pH 8.5 were subjected to UV light (365 nm) irradiation. As we can see from Figure 2a, after UV irradiation for ∼30 min, the optical transmittance of aqueous solutions of PEO-b-P(NIPAM-co-DMNA-co-NBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) did not change significantly until the temperature was increased to above ∼46 and ∼51 °C, respectively. Obviously, the observed elevated CMTs for both DHBCs after UV irradiation can be ascribed to the light cleavage of DNMA moieties, which resulted in the formation of carboxylate residues (pKa ∼ 4.5) in the NIPAM-containing block. At pH 8.5, these newly generated carboxylate residues are in the ionized state, which are highly hydrophilic. Further evidence supporting that the photolysis of o-nitrobenzyl moieties occurred in DHBC micellar solution was obtained via UV−vis spectroscopy, as shown Figure S3 (Supporting Information), together with the irradiation-duration-dependent UV−vis spectra of DMNA monomer serving as a reference. As we can tell, after UV irradiation for varying time periods, the absorption band at ∼307 nm ascribed to o-nitrobenzyl moieties decreased, whereas the absorption band corresponding to onitrosobenzaldehyde, i.e., the photolysis product, was detected

Figure 3. Time-dependent fluorescence emission spectra (λex = 470 nm, slit widths: Ex = 5 nm, Em = 5 nm) recorded at 40 °C for 0.1 g/L aqueous solutions of (a) PEO113-b-P(NIPAM0.91-co-DMNA0.09-coNBDAE) 90 and (b) PEO 113 -b-P(NIPAM 0.91 -co-DMNA 0.09 -coRhBEA)68 upon UV irradiation (365 nm) for varying time durations (0−30 min).

intensity at 532 nm recorded for PEO-b-P(NIPAM-co-DMNAco-NBDAE) micellar solution at 40 °C decreased gradually and reached a plateau after ∼30 min of irradiation (Figure 4), exhibiting a total intensity decrease of ∼62%. It has been wellestablished that the microenvironment polarity can significantly affect the quantum yields of fluorescent dyes (Figure S5, Supporting Information). Our previous reports also confirmed that emission intensities of certain dyes, including NBD and rhodamine B, are higher when they are located within hydrophobic domains (e.g., micellar cores) compared to those when they are molecularly dissolved in the aqueous media.58 It is worth noting that, for PEO-b-P(NIPAM-coDMNA-co-RhBEA) diblock copolymer micelles, UV irradiation can similarly lead to ∼58% decrease in the RhBEA emission intensity at ∼588 nm (Figures 3b and 4). 3715

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an abrupt increase of scattered intensity associated with the appearance of a bluish tinge can be observed. As shown in Figure 5, the aqueous mixture at 40 °C exhibited an intensity-

Figure 4. Time-dependent fluorescence emission intensities (λex = 470 nm, slit widths: Ex = 5 nm, Em = 5 nm) recorded at 40 °C for 0.1 g/L aqueous solutions of (■) PEO113-b-P(NIPAM0.91-co-DMNA0.09-coNBDAE)90 (λem = 532 nm) and (●) PEO113-b-P(NIPAM0.91-coDMNA0.09-co-RhBEA)68 (λem = 588 nm) upon UV irradiation (365 nm) for varying time durations (0−30 min).

Figure 5. Typical hydrodynamic radius distribution, f(Rh), recorded at 40 °C for the aqueous mixture (pH 8.5) of PEO113-b-P(NIPAM0.91-coDMNA0.09-co-NBDAE)90 and PEO113-b- P(NIPAM0.91-co-DMNA0.09co-RhBEA)68 (1:1, wt/wt).

Another possible implication for UV-irradiation-induced decrease of emission intensities (Figures 3 and 4) might come from photobleaching. To decouple the effect of photobleaching from UV-incurred micellar disintegration, the emission intensity of aqueous solutions of PEO113-b-P(NIPAMco-NBDAE)87 and PEO113-b-P(NIPAM-co-RhBEA)84 at 25 °C was monitored upon UV irradiation (365 nm, 1.0 mW/cm2) (Figure S4, Supporting Information). For 0−30 min of UV irradiation, ∼3 and 2% decreases in emission intensities were observed, respectively. This should be ascribed to photobleaching of labeled dyes. However, considering that ∼62 and 58% decreases of emission intensities were observed for micellar solutions of PEO-b- P(NIPAM-co-DMNA-coNBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) at 40 °C upon UV irradiation, the dominating mechanism can be safely ascribed to light-triggered micelle-to-unimer transition (Scheme 1). Thermo- and Light-Regulated Assembly and Disassembly of Mixed DHBC Micelles Associated with Emission Color Changes. The emission band of NBDAE overlaps well with the absorbance band of RHBAE moieties; thus, they can form an ideal FRET pair (Figure S6, Supporting Information). Since PEO-b-P(NIPAM-co-DMNA-co-NBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) diblock copolymers possess covalently labeled NBDAE and RhBEA moieties in the thermo- and light-responsive block, the above-described thermo- and light-regulated micellar formation and disintegration might be monitored via changes in FRET efficiency if the two diblock copolymers coassemble into mixed micelles. The closer proximity between the FRET pair in mixed micelles can lead to enhanced FRET efficiency. In addition, the subsequent light-triggered micellar disintegration results in the decrease of FRET efficiency due to the fact that FRET donor and acceptor dyes fall apart from each other in the unimer state (Scheme 1). Figure 2b shows temperature-dependent optical transmittance recorded at a wavelength of 700 nm for a 1.0 g/L aqueous mixture (pH 8.5) of PEO-b-P(NIPAM-co-DMNA-coNBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) (1:1, wt/wt). The CMT of the aqueous mixture before UV irradiation was determined to be ∼31.5 °C and shifted to ∼46 °C after exposure to UV light for 30 min. Thus, heating the aqueous mixture to above the CMT led to the formation of mixed micelles (Scheme 1). Accordingly, dynamic LLS characterization of the aqueous mixture revealed quite low scattering intensity at 25 °C, whereas, upon heating to 40 °C,

average hydrodynamic radius, ⟨Rh⟩, of ∼70 nm and a polydispersity index, μ2/Γ2, of 0.074. A typical AFM height image of thermo-induced micelles is shown in Figure 6a, revealing the presence of robust spherical micelles with diameters in the range of ∼80−100 nm. We then investigated the thermo- and light-responsive coassembly and disassembly of DHBC micelles in the aqueous mixture via changes in FRET efficiencies and emission colors. Figure 7a shows temperature-dependent fluorescence emission spectra recorded for the aqueous mixture. The two emission peaks at ∼532 and ∼585 nm can be clearly observed at 25 °C, which correspond to the emission bands of NBDAE donor and RhBEA acceptor moieties, respectively. For the aqueous mixture before UV irradiation, both types of DHBC are molecularly dissolved at temperatures below the CMT, resulting in a large spatial separation between the FRET pair as they are covalently attached to different chains; thus, the FRET efficiency is low initially. Upon heating to above the CMT, the two DHBCs coassemble into mixed micelles and a much closer proximity between the FRET pair can be achieved accompanied with enhanced FRET efficiency. Apparently, we can observe from Figure 7a that the emission intensities of NBDAE and RhBEA moieties exhibit an abrupt decrease and increase, respectively. This is in stark contrast to the temperature-dependent emission intensities observed for aqueous solutions of PEO-b-P(NIPAM-co-DMNA-coNBDAE) or PEO-b-P(NIPAM-co-DMNA-co-RhBEA) diblock copolymer; in both cases, emission intensities considerably increase above the CMT (Figure S5, Supporting Information). This clearly confirms the occurrence of effective FRET processes within mixed micelles formed above the CMT. From Figure 8, we can tell that the emission intensity ratio, F585/F520, exhibited an abrupt increase above ∼31 °C, which corresponds to the CMT value as determined by temperaturedependent optical transmittance measurements (Figure 2b). It is worth noting that the most prominent increase of F585/F520 during the heating of the aqueous mixture occurred in the temperature range of ∼32−40 °C, which reasonably agrees with the thermo-induced micellization temperature range for typical PEO-b-PNIPAM diblock copolymers as determined by the temperature-dependent LLS technique.8,48 This also suggests that the thermo-induced micellization process can also be wellmonitored by the fluorescence technique involving FRET 3716

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Figure 6. AFM height images recorded by drying the aqueous mixture (pH 8.5, 40 °C) of PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-NBDAE)90 and PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-RhBEA)68 (1:1, wt/wt) (a) before and (b) after UV irradiation for 30 min.

Figure 8. The variation of emission intensity ratios, F585/F520, recorded in the temperature range 25−65 °C for a 0.1 g/L aqueous mixture (pH 8.5) of PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-NBDAE)90 and PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-RhBEA)68 (1:1, wt/wt): (●) before UV irradiation and (○) after UV irradiation (365 nm) for 30 min.

transmittance measurements (Figure 2b). A comparison of the temperature-dependent changes of emission intensity ratios for the aqueous mixture before and after UV irradiation for 30 min revealed that, in the latter case, F585/F520 values exhibit much less changes over the thermo-induced micellization temperature range. Specifically, for the aqueous mixture before UV irradiation, F585/F520 values increase ∼6.25-fold (from 0.8 at 25 °C to 5.0 at 45 °C), whereas, after UV irradiation, an increase of ∼3.79-fold (from 0.95 at 45 °C to 3.60 at 60 °C) was observed. This phenomenon might be interpreted according to the following two aspects. First, light-generated carboxylate residues in the thermo- and light-responsive block exist in the ionized form; thus, in the unimer and micellar states at low and elevated temperatures, respectively, FRET donor and acceptor dyes were located within different microenvironments compared to the aqueous mixture before UV irradiation, which will affect the relative quantum yields. Second, the presence of negatively charged carboxylate moieties within thermo-induced micelles formed in the aqueous mixture after UV irradiation will lead to a less dense chain packing compared to those without UV irradiation. This will lead to a different spatial distance between FRET donors and acceptors in the two micellar states. As discussed above, the CMT value of the aqueous mixture (1:1 wt/wt, pH 8.5) was determined to be 31.5 and 46 °C before and after UV irradiation, respectively. At intermediate temperatures between these two critical values, we expect that

Figure 7. Fluorescence emission spectra (λex = 470 nm, slit widths: Ex = 5 nm, Em = 5 nm) recorded in the temperature range 25−65 °C for 0.1 g/L aqueous mixtures (pH 8.5) of PEO113-b-P(NIPAM0.91-coDMNA0.09-co-NBDAE)90 and PEO113-b-P(NIPAM0.91-co-DMNA0.09co-RhBEA)68 (1:1, wt/wt) (a) before UV irradiation and (b) after UV irradiation for 30 min.

processes. Another feature we can observe from Figure 7a is that, during heating of the aqueous mixture, the emission bands characteristic of NBDAE and RhBEA exhibit blue and red shifts, respectively, relative to that at low temperatures. This might partially reflect the microenvironmental polarity changes, i.e., enhanced hydrophobicity within micellar cores, associated with thermo-induced micelle formation and different extent of spectral overlap between emission and absorption bands in the micellar and unimer states. For the aqueous DHBC mixture after being subjected to UV irradiation for 30 min, the temperature-dependent fluorescence emission spectra were also recorded (Figures 7b and 8). As compared to that before irradiation, we can clearly tell that the transition temperature, i.e., the temperature above which the intensity ratio F585/F520 exhibits an abrupt increase, apparently shifts to a higher temperature (∼46 °C). This correlates quite well with that determined by temperature-dependent optical 3717

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UV irradiation will directly result in the micelle-to-unimer transition, accompanied with the decrease of FRET efficiency (Scheme 1). This has been obviously confirmed by the AFM image recorded by drying the aqueous mixture after 30 min of UV irradiation (Figure 6b), revealing the almost complete disappearance of spherical nanoparticles as compared to that before UV irradiation (Figure 6a). Time-dependent fluorescence emission spectra recorded at 40 °C for the mixed micellar solution upon UV irradiation are shown in Figure 9, together Figure 10. Optical images recorded under inverted fluorescence microscopy equipped with a temperature-regulated incubator (450− 480 nm exciter filter and long pass 515 nm barrier filter) for a 0.5 g/L aqueous mixture (pH 8.5) of PEO113-b-P(NIPAM0.91-co-DMNA0.09-coNBDAE)90 and PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-RhBEA)68 (1:1, wt/wt) at varying conditions: (a) 25 °C and (b) 40 °C before UV irradiation, (c) 40 °C after UV irradiation for 30 min.

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CONCLUSIONS In summary, we reported the fabrication of dynamic selfassembling systems from dye-labeled dually responsive DHBCs in which micellar formation and disintegration can be wellmodulated by temperature and light irradiation. Moreover, mixed micelle formation from PEO-b-P(NIPAM-co-DMNA-coNBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) DHBCs allows the construction of fluorescent systems involving FRET processes, which can be facilely utilized to monitor the thermo-induced micelle formation and lighttriggered micelle dissociation processes by taking advantage of the different proximity between FRET donor and acceptorlabeled DHBC chains in micellar and unimer states. Compared to other conventional techniques capable of monitoring micellar formation and disintegration such as optical transmittance and LLS, the introduction of the FRET mechanism renders to be a more facile and noninvasive technique with low background interference, which is suitable for in situ and in vivo monitoring when temperature and light dually responsive polymeric assemblies, as described in this work, are to be used as drug delivery and controlled release nanovehicles. We are currently also utilizing a hybrid mixture of PEO-b-P(NIPAMco-DMNA-co-NBDAE) and PEO-b-P(NIPAM-co-DMNA-coRhBEA) as gene delivery carriers by taking advantage of partially protonated tertiary amine-containing DMNA moieties; thus, UV-triggered decomplexation and release of DNA can be achieved, in addition to the advantage of monitoring complexation and decomplexation processes via changes in FRET efficiency and emission colors.

Figure 9. (a) Time dependence of fluorescence emission spectra (λex = 470 nm, slit widths: Ex = 5 nm, Em = 5 nm) and (b) fluorescence intensity ratio changes, F585/F520, recorded at 40 °C for a 0.1 g/L aqueous mixture (pH 8.5) of PEO113-b-P(NIPAM0.91-co-DMNA0.09-coNBDAE)90 and PEO113-b-P(NIPAM0.91-co-DMNA0.09-co-RhBEA)68 (1:1, wt/wt) upon UV irradiation for varying time durations.

with the emission intensity ratio (F585/F520) changes as a function of irradiation time. A gradual decrease of F585/F520 with extended irradiation duration was clearly evident, suggesting that the average donor−acceptor distance progressively increases due to light-triggered micelle-to-unimer transition. After ∼20−30 min of UV irradiation, the emission intensity ratio, F580/F520, reached a plateau, suggesting the completion of the micellar disassembly process. Thus, the FRET technique can be facilely employed to in situ monitor the processes of thermo-induced comicellization and light-induced micellar disassembly. Finally, thermo-induced coassembly and light-triggered disassembly occurring in the aqueous mixture of PEO-b-P(NIPAM-co-DMNA-co-NBDAE) and PEO-b-P(NIPAM-co-DMNA-co-RhBEA) diblock copolymers can also be visually checked by the naked eye. As shown in Figure 10, upon increasing the temperature from 25 to 40 °C, an apparent orange-to-red fluorescence emission transition can be discerned under inverted fluorescence microscopy, which can be ascribed to enhanced FRET efficiency resulting from thermo-induced comicellization (Figure 8). On the other hand, upon UV irradiation of the micellar solution formed at 40 °C for 30 min, a red-to-orange emission color change can be clearly discerned, which originates from light-triggered micelle-to-unimer transition and decreased FRET efficiency (Scheme 1, Figure 9).

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra, UV−vis spectra, irradiation durationdependent normalized emission intensities, temperaturedependent relative fluorescence emission intensities, and normalized fluorescence emission and normalized absorption spectra. 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. 3718

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ACKNOWLEDGMENTS The financial support from National Natural Scientific Foundation of China (NNSFC) Project (21274137, 91027026, and 51033005), Fundamental Research Funds for the Central Universities, and Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, 20123402130010) is gratefully acknowledged.

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