A Novel Photoresponsive Azobenzene-Containing Miktoarm Star

Article pubs.acs.org/Macromolecules

A Novel Photoresponsive Azobenzene-Containing Miktoarm Star Polymer: Self-Assembly and Photoresponse Properties Eva Blasco,† Bernhard V. K. J. Schmidt,‡,§ Christopher Barner-Kowollik,*,‡,§ Milagros Piñol,† and Luis Oriol*,† †

Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Ciencia de Materiales de Aragón (ICMA), Universidad de Zaragoza-CSIC, 50009 Zaragoza, Spain ‡ Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76131 Karlsruhe, Germany § Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen, Germany S Supporting Information *

ABSTRACT: We report the synthesis and characterization of a novel azobenzene-containing miktoarm star polymer AB3 (Mn = 9700 g mol−1, ĐM = 1.10) as well as its self-assembly properties in water. The miktoarm copolymer is composed of a hydrophobic azopolymer and three hydrophilic PEG arms (Mn = 600 g mol−1). The hydrophobic/hydrophilic ratio of the amphiphilic miktoarm polymer is 78/22, leading to the formation of stable polymeric vesicles in water evidenced via TEM and cryo-TEM imaging. The photoresponse of these vesicles has been investigated by irradiation with UV light (λ = 350−400 nm) causing the disruption of the self-assemblies. Encapsulation of both hydrophilic and hydrophobic fluorescent probes, i.e., Nile Red and Rhodamine B, and the use of light as an external stimulus to trigger the release of the probes have also been demonstrated.

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INTRODUCTION Over the past years the development of powerful controlled/ living radical polymerization processes and highly efficient ligation techniques has enabled the preparation of functional and complex macromolecular architectures.1−7 In particular, the preparation of macromolecules with new architectures, topologies, or composition has been approached, leading to substantial changes in key properties such as solubility, intrinsic viscosity, or microphase separation in comparison with conventional copolymers based on linear architectures.8 Star polymers are the simplest branched polymers consisting of several linear chains emanating from a central core. Among them, miktoarm star polymersor merely miktoarm polymersare branched structures containing two or more arms with different chemical compositions and/or molecular weights.9−12 The most common type of miktoarm stars are A2B, A3B, AnBn, and ABC types, where A, B, and C are chemically different chains.13 The interest in miktoarm star polymers comes from the possibility of combining virtually any type and number of polymeric armsincluding functional moietiesinto a single macromolecule. In particular, amphiphilic miktoarm polymers containing both hydrophobic and hydrophilic arms are expected to generate nanostructures in water similar to amphiphilic block copolymers (BCs). In terms © 2014 American Chemical Society

of their synthesis, miktoarm polymers represent a challenge, but progress in reliable synthetic protocols has encouraged the study of their self-assembly in aqueous medium as well as their applications traditionally dominated by the linear−linear BC counterparts.14 Incorporation of photoresponsive moieties into amphiphilic copolymers makes these systems potentially useful as lightcontrolled delivery systems.14−17 Because of a rapid, reversible, and high quantum yield photoisomerization, azobenzene is one of the most widely investigated photoresponsive groups. To the best of our knowledge, only a few examples of azobenzene miktoarm polymers have been reported to date. Hizal, Tunca, and co-workers described the synthesis of (PS)2(PMMA)2 miktoarm copolymers from polystyrene (PS) and poly(methyl methacrylate) (PMMA) using an azobenzene moiety at the core.18 The synthesis of an azobenzene initiator with the required functionalities allowed the growth of the arms by two sequentially controlled radical polymerizations, i.e., atom transfer radical polymerization (ATRP) and nitroxide-mediated free radical polymerization (NMP), of methyl methacrylate and Received: February 3, 2014 Revised: April 3, 2014 Published: May 21, 2014 3693

dx.doi.org/10.1021/ma500254p | Macromolecules 2014, 47, 3693−3700

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Figure 1. Structure of (a) the LDBC previously reported26 and (b) the novel azobenzene containing miktarm star polymer described in the current study.

responsive carriers of hydrophobic cargoes, polymeric vesicles or polymersomes are more appealing nanocontainers as they can physically encapsulate hydrophobic as well as hydrophilic molecules due to their internal hydrophilic cavities and robust hydrophobic membranes. Thus, the incorporation of azobenzene chromophores into miktoarm polymer vesicles should provide systems of interest in materials and biological science as by using light as an external stimulus spatial resolution and temporal control of the photoinduced molecular changes on the chromophore would allow a release on demand.24 In our previous work on light-responsive amphiphilic linear− dendritic block copolymers (LDBCs), we have demonstrated that vesicles can be formed in water when an appropriate hydrophobic/hydrophilic balance is reached.25 Specifically, vesicles were prepared from LDBCs composed of the fourth generation of a polyester dendron having 16 peripheral 4isobutyloxyazobenzene units coupled to a PEG polymer of 2000 g mol−1 with a phobic/philic ratio of approximately 80/20 (Figure 1a).26 The vesicles were loaded with both hydrophobic and hydrophilic fluorescent probes to show that under lowintensity UV irradiation the distortion of the membrane increases its permeability to the entrapped molecules. On the basis of these precedents, the current work aims at the preparation of a novel macromolecular architecture having the same composition of the aforementioned LDBC and, consequently, an approximately 80/20 hydrophobic/hydrophilic balance ratio allowing for the formation of vesicular selfassemblies in water.25 To this end, an AB3 type miktoarm copolymer having one 4-isobutyloxyazobenzene side chain polymethacrylate (PAZO17) arm and three identical PEG arms (PEG12), i.e., (PAZO17)(PEG12)3 (Figure 1b), was targeted. The degree of polymerization of PAZO17 was on average 17, whereas in our previous study 16 azobenzene units were incorporated in the periphery of a dendron.26 To obtain the target 80/20 hydrophobic/hydrophilic ratio, three PEG12 arms of Mn = 600 g mol−1 (n = 12) were employed. A synthetic strategy was explored based on the connection of the PEG arms to the azopolymeric chain PAZO17. A novel three-arm star PEG ATRP macroinitiator was first synthesized by click chemistry and used for the subsequent polymerization of the azo

styrene, respectively. Wang and co-workers reported the synthesis and thermal behavior of a series of amphiphilic liquid crystalline miktoarm polymerscomposed of one poly(ethylene oxide) (PEO) and two azobenzene-containing polymethacrylate arms.19 The target miktoarm architecture was prepared by ATRP of the corresponding azobenzene methacrylate using a bifunctional PEO macroinitiator (PEOBr2). He and co-workers described a closely related series of miktoarm polymersfeaturing a diblock copolymer with poly(ethylene oxide) and polystyrene (PEO-b-PS) arm and two azobenzene-containing polymethacrylate arms. Again, a terminal difunctionalized PEO-b-PS arm was synthesized to initially construct the azobenzene-containing polymethacrylate arms.20,21 These polymers self-assembled into large vesicles, which showed a shape deformation with an elongation along the polarized direction upon irradiation with linear polarized light. More recently, the same authors reported a structurally related photoresponsive miktoarm polymer prepared by combining ATRP with the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction.22 These copolymers selfassembled into bowl-shaped self-assemblies that change to multibowl shapes on increasing the length of the hydrophobic arms. The photoisomerization of the azobenzene moieties within these self-assemblies was determined by the existence of H- and J-aggregates formed in the self-assembly process. In recent work, we reported a series of dual responsive azobenzene miktoarm polymers composed of an azobenzene poly(methacrylate) photoresponsive arm and three of poly(N,Ndiethylacrylamide) thermoresponsive arms.23 Stable micellar suspensions were obtained by carefully selecting the length of the poly(N,N-diethylacrylamide) arms, in particular for a 26/74 phobic/philic ratio. The target miktoarm copolymers were prepared by using a novel ATRP initiator to polymerize a triazido-functionalized azobenzene polymethacrylate and subsequently linking three alkyne-terminated poly(N,N-diethylacrylamide) arms by CuAAC. The thermo- and photoresponsive properties of these self-assemblies as well as the ability to act as controlled delivery systems were evidenced by encapsulation of a hydrophobic molecular probe. Even though these stable core−shell micellar self-assemblies can be used as stimuli3694


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Scheme 1. Synthetic Strategy for the Target Azobenzene Miktoarm Polymer (PAZO17)(PEG12)3

8.23 (s, 3H), 4.72 (s, 6H), 4.33 (s, 6H), 3.75−3.50 (m, 146H), 3.37 (s, 9H), 3.02 (s, 1H). Synthesis and Characterization of (PEG12)3-Br. A solution of the polymer (PEG12)3-OH (1.48 g, 0.76 mmol) in dry THF (20 mL) was prepared and cooled in an ice bath. The solution was stirred and triethylamine (0.5 mL, 3.80 mmol), and α-bromoisobutyryl bromide (0.4 mL, 3.80 mmol) was added dropwise under an argon atmosphere. The mixture was stirred at ambient temperature overnight, and methanol (1 mL) was added. The formed triethylammonium bromide was removed by filtration, and the solvent was removed under vacuum. Yield: 70%. IR (KBr), ν (cm−1): 1959, 1742, 1457, 1249, 1107. 1H NMR (250 MHz, CDCl3) δ (ppm): 8.18 (s, 3H), 4.70 (s, 6H), 4.46 (s, 8H), 3.75−3.50 (m, 144H), 3.37 (s, 9H), 2.03 (s, 6H). Synthesis of the Miktoarm Polymer (PAZO17)(PEG12)3. mAZO (200.0 mg, 456.3 μmol), (PEG12)3-Br (42.1 mg, 20.2 μmol), and CuBr (2.8 mg, 20.2 μmol) were added to a Schlenk tube. PMDETA (4.0 μL, 20.2 μmol) and deoxygenated anisole (1 mL) were added with an argon-purged syringe, and the flask was further degassed via three freeze−pump−thaw cycles and flushed with argon. The reaction mixture was stirred under an argon atmosphere at 80 °C for 24 h. The mixture was diluted with THF and passed through a short column of alumina. The solvent was partially evaporated, and the resulting polymer solution was carefully precipitated into cold methanol. Yield: 80% IR (KBr), v (cm−1): 1727, 1600, 1581, 1500, 1247, 1147, 841. 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.80−7.69 (m), 6.90−6.76 (m), 4.65 (s), 4.37 (s), 4.05−3.81 (m), 3.79−3.52 (m), 3.37 (s), 2.13−2.01 (m), 2.01−1.85 (m), 1.81−1.65 (m), 1.65−1.47 (m), 1.46−1.27 (m), 1.09−0.80 (m). Mn = 9700, ĐM =1.10 (PS standards). Anal. Calcd: C, 66.72%; H, 7.93%; N, 6.11%. Found: C, 67.21%; H, 8.04%; N, 6.26%. Characterization Techniques. IR spectra were recorded on a Bruker FT-IR spectrometer using KBr pellets. 1H NMR spectra were

monomer, providing a facile synthetic approach to the azobenzene block compared to the synthesis of azobenzenecontaining dendrimers. The self-assembly in water and photoresponsive behavior of the novel azobenzene-containing miktoarm star polymer were also evaluated.

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EXPERIMENTAL SECTION

Materials. 2,2,2-Tris(azidomethyl)ethanol solution (N3)3-OH and alkyne-functionalized PEG were prepared according to the procedure previously reported.27,28 Experimental details for the synthesis of the azobenzene-containing methacrylate (mAZO) are given in the Supporting Information. PEG monomethyl ether (Mn = 550 g mol−1) was purchased from Sigma-Aldrich and used without further purification. CuBr was used as received and handled in a drybox. All other reagents were purchased from Sigma-Aldrich and used as received without further purification. Synthesis and Characterization of (PEG12)3-OH. 2,2,2-Tris(azidomethyl)ethanol (N3)3-OH (3.42 g, 3.80 mmol, 24 wt % in toluene) and alkyne-functionalized PEG (9.00 g, 14.82 mmol) were placed into a Schlenk tube. PMDETA (160 μL, 0.76 mmol), CuBr (110.2 mg, 0.76 mmol), and deoxygenated toluene were added with an argon-purged syringe, and the flask was further degassed via three consecutive freeze−pump−thaw cycles and flushed with argon. The reaction mixture was stirred at ambient temperature overnight. Subsequently, the mixture was diluted with THF and passed through a short column of neutral alumina. The solvent was partially evaporated, and the resulting polymer solution was carefully precipitated into cold ethyl ether. Yield: 55%. IR (KBr), ν (cm−1): 3500, 1959, 1456, 1249, 1106. 1H NMR (250 MHz, CDCl3) δ (ppm): 3695


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previously reported methodology.27 Because of the high N/C ratio of this compound, it should be handled carefully. On the other hand, commercial PEG having a terminal hydroxyl group was etherified with propargyl bromide to obtain an alkyneterminated PEG28 of Mn = 600 g mol−1, as was corroborated by MALDI-TOF mass spectrometry (refer to Figure S2a in the Supporting Information). The PEG arms were coupled to the (N3)3-OH core via a CuAAC reaction using CuBr/PMDETA as catalytic system. A slight excess of the alkyne ended PEG was employed, which was subsequently removed by precipitation into cold diethyl ether. (PEG12)3-OH was subsequently modified by esterification of the hydroxyl group with αbromoisobutyryl bromide to install an ATRP initiation site into the remaining functionality of the core yielding the ATRP macroinitiator (PEG12)3-Br. The efficiency of the reaction and the average molecular weight (Table 1) were assessed by MALDI-TOF (refer to Figure S2b in the Supporting Information), SEC, and 1H NMR (refer to Figure S3 in the Supporting Information).

measured on a Bruker AV-400 spectrometer at 400 MHz and on a Bruker AM250 spectrometer at 250 MHz. Elemental analysis was performed using a PerkinElmer 2400 microanalyzer. MALDI-TOF MS was performed on an Autoflex mass spectrometer (Bruker Daltonics). Number-average molecular weight (Mn) and polydispersity were calculated from the mass spectra using PolyTools 1.0 (Bruker). SEC was carried out on a Waters e2695 Alliance liquid chromatography system equipped with a Waters 2424 eVaporation light scattering detector and a Waters 2998 PDA detector using two Ultrastyragel columns, HR4 and HR2 from Waters, of 500 and 104 Å pore size and on a Polymer Laboratories PL-GPC 50 Plus Integrated System, comprising an autosampler, a PLgel 5 mm bead-size guard column (50 × 7.5 mm) followed by three PLgel 5 mm MixedC columns (300 × 7.5 mm), and a differential refractive index detector. Measurements were performed in THF with a flow of 1 mL min−1 using narrow molecular weight PS standards. UV−vis spectra were recorded on an ATI-Unicam UV4-200 spectrophotometer. Fluorescence measurements were recorded using a PerkinElmer LS 50B fluorescence spectrophotometer. Thermogravimetric analyses (TGA) were performed using a Q5000IR from TA Instruments under nitrogen 50 atm using 2−5 mg of the sample. Thermal transitions were determined by differential scanning calorimetry (DSC) using a DSC Q-2000 from TA Instruments with powdered samples (2−5 mg) sealed in aluminum pans. Glass transition temperatures were determined at the midpoint of the baseline increase, and the melting temperatures were determined at the maximum of the corresponding peaks. The thermal behavior was additionally evaluated by polarizing optical microscopy (POM) using an Olympus BH-2 polarizing microscope fitted with a Linkam THMS600 hot stage. The morphologic study of the miktoarm polymer micelles was carried out by transmission electron microscopy (TEM) in a JEOL2000 FXIII electron microscope operating at 200 kV. Cryo-TEM observations were carried out in a JEM-2011 electronic microscope on samples rapidly frozen in liquid ethane. Dynamic light scattering (DLS) measurements were carried out in a Malvern Instrument Nano ZS using a He−Ne laser with a 633 nm wavelength and a detector angle of 173° at 25 °C. The self-assemblies concentration was close to 0.1 mg mL−1, and size measurements were performed at least three times on each sample to ensure consistency. The water dispersions of self-assemblies were irradiated with a compact low-pressure fluorescent lamp Philips PL-S (9 W) emitting between 350 and 400 nm. After the irradiation, the water suspensions were kept in dark. Fluorescent vesicles were observed with an Olympus FV10i confocal scanning microscope. Images were collected using a 60× oil immersion lens (lens specification, Plan S-APO 60xO, NA 1.35), a line average of 8, and a format of 1024 × 1024 pixels. The confocal pinhole was 1 airy unit.

Table 1. Molecular Weight of the Synthesized Polymers polymer

Mna

Mnb

Mnc

ĐM c

(PEG12)3-Br (PAZO17)(PEG12)3

2100

2200 9600

2900 9700

1.01 1.10

a

Mn calculated by MALDI. bMn calculated by 1H NMR (see text). cMn and polydispersity (ĐM) determined by SEC using PS standards.

In the final step, the azobenzene methacrylate mAZO was polymerized by ATRP using the (PEG12)3-Br macroinitiator in anisole and CuBr/PMDETA as catalytic system at 80 °C. As this macroinitiator was used for the first time, the polymerization conditions were optimized to obtain a target polymerization degree close to 16 (i.e., to have a number of photoresponsive moieties comparable to the previously described azobenzene functionalized dendrons).26 Monomer conversion was determined via 1H NMR by relative integration of the vinyl proton resonances, at 6.10 and 5.55 ppm, and the aromatic proton resonances associated with the azobenzene units, and subsequently ln([M]0/[M]) was plotted against polymerization time. Inspection of Figure 2a indicates that at the initial stages of polymerization the propagating species concentration was not constant. A constant radical population was only reached after approximately 2 h. The average molecular weights measured by SEC and 1H NMR increased linearly with monomer conversion, which is consistent with the polymerization proceeding in a controlled fashion (Figure 2b). Based on the kinetic analysis, the azomonomer polymerization was scaled up to produce a larger quantity of miktoarm polymer setting the reaction time on 24 h. 1H NMR spectroscopy was employed to estimate the average number of azobenzene units per macromolecular chain. The relative integration of the resonances corresponding to the end groups −CH3 of the PEG arms at 3.37 ppm (labeled as “a” in Figure 3) and the resonances corresponding to the aromatic protons of the repeating azobenzene unit gave 17 repeating units on average per polymer chain and consequently an average molecular weight of 9600 g mol−1. The molecular weight was additionally determined by SEC relative to PS standards and it was in good agreement with the 1H NMR data (Table 1). Figure 4 depicts the SEC curves corresponding to the macroinitiator and the miktoarm polymer (PAZO17)(PEG12)3. As can be observed, the polymerization gives rise to a shift of

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RESULTS AND DISCUSSION Synthesis and Characterization of the Amphiphilic Miktoarm Star Polymer. The target miktoarm star copolymer was prepared by a combination of CuAAC and ATRP following the three-step strategy depicted in Scheme 1. As starting compound, a tetrafunctional core having three azido and one hydroxyl group was employed. Three alkyne endfunctionalized PEG arms were first coupled by CuAAC to the core giving rise to a star PEG polymer. The remaining hydroxyl group was conveniently modified for achieving a new ATRP macroinitiator to grow the azopolymer arm, yielding the final miktoarm polymer. The tetrafunctional 2,2,2-tris(azidomethyl)ethanol core, (N3)3-OH, was prepared by substitution of the bromine groups of 2,2,2-tris(bromomethyl)ethanol by azide groups using a 3696


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Figure 2. (a) Relationship of ln([M]0/[M]) and monomer conversion with polymerization time and (b) evolution of Mn and ĐM with monomer conversion for the ATRP polymerization of mAZO in anisole at 80 °C. Note the good agreement between NMR and SEC determined numberaverage molecular weights. The initial increase in propagating center concentration in (a) leading to a rapid polymerization at early times is likely due to the active catalyst system employed.

the molecular weight distribution peak toward lower retention timesfrom 15.5 to 13.8 minindicating the successful polymerization of the mAZO and consequently the miktoarm star polymer formation. According to these data, the hydrophobic/philic ratio was 78/22 (considering PEG arms as the hydrophilic part and the rest of the macromolecule as the hydrophobic part) in accordance with the target proportion. TGA analysis of the polymer on powdered samples excluded the evolution of volatiles up to 300 °C (refer to Figure S4a in the Supporting Information). By POM, a melting process was observed, and no textures typical of mesomorphism were detected. The DSC analysis corroborated the crystalline character of the polymer (refer to Figure S4b in the Supporting Information). The miktoarm polymer (PAZO17)(PEG12)3 only presented a melting transition at around 110 °C in the heating process and the subsequent crystallization in the cooling scan at close to 100 °C. Self-Assembly in Water. Polymeric self-assemblies were prepared by the cosolvent method employing THF and water; the self-assembly process was followed by turbidimetry measurements. When water was gradually added to a solution of the miktoarm polymer in THF, an abrupt increase of scattered light was observed, indicating the formation of selfassemblies (refer to Figure S5a in the Supporting Information). Once the turbidity reached an almost constant value, the resulting dispersion was dialyzed against water to remove the organic solvent. After dialysis, a stable dispersion was obtained, although precipitation of the sample was observed after storing the suspension for a few weeks. The critical aggregation concentration (CAC) of (PAZO17)(PEG12)3 in water was determined by fluorescence spectroscopy using Nile Red as a probe, as its emission is sensitive to changes in polarity of the environment.29−32 In water, Nile Red exhibits a weak emission at 660 nm (with excitation at 550 nm) due to excimer formation, yet if the dye is located in a more hydrophobic environment its emission is blue-shifted and the intensity increases strongly. The relationship between the fluorescence and the concentration is nonlinear, and the CAC was determined at the onset of the fluorescence intensity increase (refer to Figure S5b in the Supporting Information). The calculated value of CAC was about 40 μg mL−1, which is similar to that calculated for the LDBC of similar composition (35 μg mL−1) previously reported.26

Figure 3. 1H NMR spectrum of (PAZO17)(PEG12)3 in CDCl3 (400 MHz) showing the resonances employed for Mn calculation.

Figure 4. SEC traces of the macroinitiator (PEG12)3-Br (dashed line) and the miktoarm polymer (PAZO17)(PEG12)3 (straight line).

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Figure 5. TEM image of (PAZO17)(PEG12)3 nonirradiated vesicles (a). Cryo-TEM images of (PAZO17)(PEG12)3 vesicles before (b) and after (c) irradiation for 5 min (350−400 nm, 9 W). The length of the scale bar corresponds to 1 μm in (a) and 200 nm in (b) and (c). (d) Schematic representation of a (PAZO17)(PEG12)3 vesicle.

Figure 6. (a) UV−vis spectra of (PAZO17)(PEG12)3 in a 5 × 10−6 mol L−1 solution in CHCl3 (straight line) and a water suspension of (PAZO17)(PEG12)3 vesicles (dashed line). (b) UV−vis spectra of (PAZO17)(PEG12)3 irradiated vesicles (concentration of 1 mg mL−1) for different times (350−400 nm, 9 W).

recorded (Figure 6a). The spectrum in solution was characterized by two absorption bands corresponding to the trans-isomer, a strong one centered at 360 nm attributed to the π−π* transition, and a weak one close to 450 nm corresponding to the n−π* transition. The spectrum of the vesicles showed a broader π−π* transition due to aggregation of the azobenzene moieties. A suspension of (PAZO17)(PEG12)3 vesicles of 1 mg mL−1 concentration was irradiated with a mercury low-pressure UV lamp (9 W), while recording the evolution of the UV−vis spectra (Figure 6b). During UV irradiation, a remarkable decrease in absorbance together with a hypsochromic shift of the π−π* transition took place accompanied by an increase of the absorbance at 450 nm corresponding to the n−π* transition. This change can be attributed to the photoisomerization of the trans-azobenzene to the cis-isomer. After 5 min of irradiation, no further changes in the UV−vis spectra were observed, indicating that a photostationary state was reached. After 24 h in the dark, the UV−vis spectra started to

The morphology of the (PAZO17)(PEG12)3 self-assemblies was investigated by TEM on dried samples stained with uranyl acetate. It was found that the miktoarm polymer self-assembled into vesicles, which appear deflated because of the drying process (Figure 5a). Cryo-TEM images showed spherical vesicles with diameters ranging from 300 to 700 nm having a membrane thickness around 9 nm (Figure 5b). A schematic representation of the vesicles is given in Figure 5d. The proposed model of these vesicles consists of a hydrophobic membrane with a bilayer organization of the PAZO17 arms solvated by a hydrophilic inner and outer corona formed by the PEG arms. The size of the polymeric vesicles was additionally evaluated by DLS measurements providing a mean hydrodynamic diameter (Dh) of 640 nm, larger than the values determined for LDBC vesicles, i.e. 365 nm, previously described.26 Photoresponsive Behavior of the Self-Assemblies. The UV−vis spectra of the miktoarm polymer (PAZO17)(PEG12)3 in solution and the vesicles suspension in water were 3698


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recover the initial shape due to thermal cis-to-trans backisomerization. Cryo-TEM observation of the polymeric micelles in combination with DLS measurements were performed to gain information about the morphological changes upon irradiation. The cryo-TEM image recorded after irradiation shows the presence of wrinkled vesicles (Figure 5c). Furthermore, a change of around 170 nm in the Dh was detected by DLS measurements (refer to Figure S6 in the Supporting Information) revealing a Dh of 470 nm after irradiation. This change also confirmed the deformation of the vesicles upon irradiation. The Dh was evaluated after 24 h of irradiation, and no evolution was found evidencing an irreversible morphological change. Encapsulation and Photoinduced Release of Molecular Probes. Once the photoresponsive properties of the vesicles were investigated, the possibility of encapsulation and controlled release was assessed using fluorescent probes. The fluorescent probes selected feature excitation/emission wavelengths (550 nm/600 nm) separated from the wavelengths required to induce the trans-to-cis photoisomerization of the azobenzene moieties (365 nm). Initially, Nile Red was encapsulated in the vesicles and the loaded vesicles were irradiated. Upon irradiation, a decrease of the Nile Red fluorescence intensity was observed as depicted in Figure 7.

number of molecules encapsulated in the vesicles. It was found that under these conditions vesicles were able to trap about 45 molecules of dye per miktoarm polymer chain. The evolution of the Rhodamine B release upon irradiation was investigated by confocal microscopy. Figure 8a displays the fluorescence

Figure 8. Fluorescence microscopy images of the water suspension of loaded (PAZO17)(PEG12)3 vesicles before (a) and after (b) irradiation for 5 min (350−400 nm, 9 W). The length of the scale bar corresponds to 5 μm.

before irradiation with the dye concentrated in specific regions due to encapsulation. Upon irradiation, fluorescent dots were still visible by fluorescence microscopy, yet also a fluorescent background was observed due to Rhodamine B release from the interior of the vesicles to the aqueous media (Figure 8b). These experiments evidenced that under UV illumination the vesicle membrane became permeable to the loaded fluorescent probe.

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CONCLUSIONS A combination of ATRP and CuAAC was employed for the preparation of a novel miktoarm polymer (PAZO17)(PEG12)3. Three alkyne-functionalized PEG arms were initially coupled by CuAAC to a tetrafunctional asymmetric core giving rise to a star PEG polymer that was subsequently used as macroinitiator for the polymerization of an azobenzene containing monomer, providing a significantly simpler synthetic approach compared to the former dendrimer-based design. By using PEG arms of a suitable length and adjusting the ATRP conditions, it was possible to reach a suitable phobic/philic ratio to obtain an azobenzene-containing miktoarm polymer that forms stable vesicles in water. These vesicles can be employed as nanocontainers to load both hydrophobic and hydrophilic cargo molecules. Upon UV irradiation, photoinduced morphological changes were observed by cryo-TEM due to azobenzene isomerization at the membrane that increases the permeability and allows the release of the entrapped probes. Therefore, formation of photoresponsive nanocarriers able to transport hydrophobic and hydrophilic cargo molecules has been demonstrated in miktoarm azopolymers with an adjustable structure and potential applications in controlled drug release.

Figure 7. Emission spectra of the Nile Red encapsulated micelles of (PAZO17)(PEG12)3 (concentration of 1 mg mL−1) recorded at different irradiation times.

The decrease on the emission of the dye can be due to both Nile Red migration from the membrane to the aqueous media and/or the increase in the polarity of the inner membrane due to the change in net dipole moment associated with trans-to-cis isomerization.26,33 After 24 h in the dark, the Nile Red emission recovered to its initial value, indicating that the fluorescent probe is again in a hydrophobic environment, and consequently the fluorescent probe mainly remained encapsulated. Subsequently, the encapsulation and photoinduced release of Rhodamine B were investigated as a hydrophilic probe to be loaded at the internal cavity of the vesicle. Initially, vesicles were formed in the presence of a Rhodamine B solution and dialyzed against water to remove the organic solvent and the nonencapsulated dye. The Rhodamine B dialyzed solution was analyzed by fluorescence measurements to determine the quantity of dye molecules in the solution and consequently the

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details for the synthesis of the azomonomer (mAZO), general procedures for the preparation of the selfassemblies, MALDI-TOF and NMR spectra, DSC traces of the miktoarm polymers, turbidity plot, and DLS of the selfassemblies. This material is available free of charge via the Internet at http://pubs.acs.org. 3699


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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected], fax +49 721 608 5740 (C.B.-K.). *E-mail [email protected] (L.O.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study was supported by the MINECO, Spain, under the project MAT2011-27978-C02-01, Fondo Europeo de Desarrollo Regional (FEDER), Gobierno de Aragon and Fondo Social Europeo. E. Blasco acknowledges the CSIC JAE-Pre contract funding for her PhD. The authors acknowledge the Servicio de Microscopia Electronica-Universidad de Zaragoza, the Advanced Microscopy Laboratory and the Servei de Microscopia-Universitat Autonoma de Barcelona for the TEM and cryo-TEM observations. The authors additionally acknowledge the use of the Servicio General de Apoyo a la Investigacion-SAI, Universidad de Zaragoza. The authors also thank the I+CS/IIS Aragon (Aragon Health Sciences Institute) for access to the confocal microscope. C.B.-K. acknowledges continued funding from the Karlsruhe Institute of Technology (KIT) in the context of the Helmholtz BioInterfaces and STN research programs, the German Research Council (DFG) as well as the ministry of science and arts of the state of BadenWürttemberg.

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