Light Responsive Vesicles Based on LinearâDendritic Block

Article pubs.acs.org/Macromolecules

Light Responsive Vesicles Based on Linear−Dendritic Block Copolymers Using Azobenzene−Aliphatic Codendrons Eva Blasco,† José Luis Serrano,‡ Milagros Piñol,*,† and Luis Oriol*,† †

Departamento de Química Orgánica, Facultad de Ciencias - Instituto de Ciencia de Materiales de Aragón (ICMA), Universidad de Zaragoza-CSIC, Pedro Cerbuna 12, 50009 Zaragoza, Spain ‡ Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain S Supporting Information *

ABSTRACT: We report on the synthesis and characterization of a series of amphiphilic linear−dendritic block copolymers (LDBCs) as well as their self-assembly in water. The LDBCs are composed of a 2000 g/ mol poly(ethylene glycol) (PEG) linear segment linked to the fourth generation of a 2,2-di(hydroxymethyl)propionic acid (bis-MPA)-based dendron containing 4-isobutyloxyazobenzene units (AZO) and hydrocarbon chains (C18) randomly connected to the periphery of the dendron. TEM and cryo-TEM images show that all the LDBCs form stable vesicles in water. The influence of AZO/C18 ratio in the photoresponse of the self-assemblies has been assessed as well as the encapsulation of both hydrophilic (Rhodamine B) and hydrophobic (Nile Red) fluorescent probes and the use of light as an external stimulus to trigger the release of the probes. The results show that by diluting the azobenzene content at the periphery of the dendron, the trans-to-cis photoisomerization rate can be substantially accelerated and the light-induced release activity can be tuned being the vesicles with a 50/50 AZO/C18 ratio the ones suffering the most significant changes upon irradiation.

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INTRODUCTION Polymeric vesicles, also known as polymersomes, represent a remarkable type of supramolecular structures formed by spontaneous self-assembly of amphiphilic block copolymers (BCs) in selective solvents whose growing interest is related to their potential for nanocontainers or nanoreactors applications among others. These structures are hollow spheres with a polymeric hydrophobic bilayer isolated from the internal cavity and outer space by hydrophilic coronas. Because of their internal aqueous compartment, they can encapsulate hydrophilic compounds within the interior and also integrate hydrophobic molecules within the membrane.1−7 The design of BCs has a direct influence on the morphology and behavior of the self-assemblies that can be tuned by adjusting variables such as block length, polymer architecture, or nature of the monomers. In particular, if stimulus-sensitive moieties are introduced in the chemical structure of the amphiphilic BCs, the resulting polymeric vesicles may have a controlled and programmed response to an external stimulus.7−9 Along with all possible stimuli, light is an especially attractive one because it can be remote, temporal, and spatially controlled.10−16 For the preparation of light-responsive systems, the most general and often used strategy is the incorporation of photochromic moieties as side groups in one of the BCs segment, usually the hydrophobic one. Several photochromic systems have been studied for this purpose including spiropyran,17−20 dithienylethene, or diazonaphthoquinone21,22 derivatives. Nevertheless, azobenzene has been © 2013 American Chemical Society

undoubtedly the most widely investigated group in the design of light-responsive systems based on amphiphilic BCs.23 The trans-to-cis photoinduced isomerization results in an increase of the dipole moment of the azobenzene moiety accompanied by a geometrical change from an elongated to a bent molecular shape, giving the possibility to provoke permeability changes in the membrane of the vesicle or even distortion of the selfassemblies.24,25 The described photoinduced effects constitute the basis for the promising application of light-responsive azobenzene BCs in the context of controlled delivery systems. Core−corona spherical micelles are the most common morphology reported for the self-assembling of azobenzenecontaining amphiphilic BCs in solution26−29 although several examples of azobenzene-containing polymeric vesicles have been reported. Su et al. described an amphiphilic BC composed of poly(acrylic acid) as the hydrophilic block and an azobenzene-containing poly(acrylate) as the hydrophobic block, which self-assembled into giant vesicles (micrometric scale) in a mixture of water and THF.30 The light-responsive behavior of the vesicular aggregates was studied by irradiation with 365 nm light and a photoinduced deformation of the vesicles was observed, changing from a spherical shape to an ear-like shape, due to trans-to-cis isomerization of the azobenzene side groups. The same authors reported an Received: May 10, 2013 Revised: July 4, 2013 Published: July 16, 2013 5951

dx.doi.org/10.1021/ma4009725 | Macromolecules 2013, 46, 5951−5960

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the self-assemblies under UV irradiation as well as encapsulation/release of fluorescence probes has been evaluated.

amphiphilic BC composed of the same azobenzene hydrophobic block and a thermoresponsive block of poly(Nisopropylacrylamide).31 The BC self-assembled into giant vesicles and fusion of the vesicles was observed upon irradiation with 365 nm UV light. Lin et al. described the formation of photoresponsive vesicles from an amphiphilic BC composed of PEG as the hydrophilic block and an azopyridine-containing poly(methacrylate).32 During the irradiation process using UV light, these vesicles suffered a photoinduced process involving fusion, disruption, disintegration and rearrangement. Most of the reported systems are linear−linear BC, but the use of novel macromolecular architectures can lead to interesting changes in the properties of the copolymers compared to linear analogues.33−35 Linear−dendritic block copolymers (LDBCs) formed by a linear polymeric segment linked to a dendron, which were introduced by Gitsov and Fréchet, are an example of these novel macromolecular architectures.36−38 In a previous work, we reported a series of amphiphilic LDBCs composed of PEG of different molecular weights and dendrons based on 2,2-di(hydroxymethyl)propionic acid (bis-MPA) functionalized on the periphery with 4-cyanoazobenzene moieties.24 Aqueous assemblies of different morphology (cylindrical micelles, sheet-like micelles, tubular micelles, and polymer vesicles) were formed depending on the dendron generation as well as the length of the hydrophilic PEG block. However, special attention was paid to the fourth generation of the azodendron having 16 peripheral units coupled to a PEG segment of 2000 g/mol average molar mass as this LDBC produced stable vesicles in water. The vesicles were constituted by a hydrophobic membrane with a bilayer organization of the azodendron solvated by a hydrophilic inner and outer corona formed by the PEG block. Our preliminary tests on the LDBC having 4-cyanoazobenzene units at the periphery demonstrated the photoresponsive behavior of the vesicles in which morphological changes in the membrane were observed under UV irradiation. Later, we showed that the incorporation of 16 4-isobutyloxyazobenzene units at the surface of the dendron facilitated the light-induced disruption of the membrane as it was found that lower intensity of light illumination was required to deform the vesicles.39 This might be related to the weaker aggregation tendency of 4alkoxybenzenes and to a larger photoinduced change in the dipolar moment of the azobenzene upon trans-to-cis isomerization when compared to 4-cyanoazobenzene. The vesicles were used to encapsulate both hydrophobic and hydrophilic molecules, and the light-stimulated delivery process of the encapsulated probes was verified. In this work, the chemical composition of the dendritic block is varied by incorporating 4-isobutyloxyazobenzene and hydrocarbon chains in different proportions randomly distributed on the periphery of the dendron. The purpose of this structural modification is to decrease the azobenzene content altering the interactions in the inner membrane and to investigate its possible influence in the uptake/release of fluorescent probes. Thereby, we report the synthesis, thermal properties and selfassembly in aqueous solutions of three LDBCs having a common linear PEG chain coupled to codendrons derived from the fourth-generation dendrons of bis-MPA. The LDBCs PEGb-d(isoAZO/C18)-75/25, PEG-b-d(isoAZO/C18)-50/50, and PEG-b-d(isoAZO/C18)-25/75 were prepared by varying the proportion of the peripheral groups, 4-isobutyloxyazobenzene units (isoAZO) and hydrocarbon chains (C18), in the dendritic block as is illustrated in Scheme 1. Light-responsive behavior of

Scheme 1. Synthesis of the Investigated LDBCs

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EXPERIMENTAL SECTION

Materials. Alkyne-functionalized PEG, the fourth-generation polyester dendron (d16OH), and 6-[4-(4′-isobutyloxyphenylazo)phenyloxy]hexanoic acid were prepared according to procedures previously reported.40,41 All other reagents were purchased from Sigma-Aldrich and used as received without further purification. Synthesis of the Azodendron d(isoAZO/C18)-75/25. d16OH(0.17 g, 0.09 mmol), 6-[4-(4′-isobutyloxyphenylazo)phenyloxy]hexanoic acid (0.50 g, 1.30 mmol), and stearic acid (0.12 g, 0.43 mmol) in 75:25 molar ratio and 4-(N,N-dimethylamino)pyridinium ptoluenesulfonate (0.42 g, 1.44 mmol) were dissolved in a mixture of dichloromethane (10 mL) and N,N-dimethylformamide (1 mL). The reaction flask was flushed with argon, and N,N′-dicyclohexylcarbodiimide (0.39 g, 1.91 mmol) was added. The mixture was stirred at room temperature (RT) for 48 h under an argon atmosphere. The white precipitate formed was filtered off, and the solvent was evaporated. The crude product was purified by column chromatography on silica gel and eluted with dichloromethane, gradually increasing the polarity to 1:10 ethyl acetate/dichloromethane. The codendron was obtained as an orange powder. Yield: 65%. IR (KBr), ν (cm−1): 2096, 1740, 1601, 1582, 1499, 1243, 1149, 844. 1H NMR (400 MHz, CDCl3) δ 5952


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(ppm): 7.8−7.80 (m, Ar), 6.96−6.90 (m, Ar), 4.3−4. (m, CH2− OCO) 3.93 (t, J = 6.3 Hz, CH2−OAr), 3.74 (d, J = 6.5 Hz, CH2− OAr), 3.25 (t, J = 6.8 Hz, CH2−N3), 2.33 (t, J = 7.5 Hz, CH2−CO), 2.27 (t, J = 7.4 Hz, CH2−CO), 2.15−2.04 (m, CH−(CH3)2) 1.84− 1.73 (m, CH2), 1.70−1.59 (m, CH2), 1.52−1.41 (m, CH2), 1.40−1.13 (m, CH2, CH3 (dendron)), 1.03 (d, J = 6.8 Hz, CH3(iso)), 0.87 (t, J = 6.6 Hz, CH3 (C18)). 13C NMR (101 MHz, CDCl3) δ (ppm): 173.3, 172.8, 161.2, 161.0, 146.9, 146.8, 124.3, 114.6, 74.6, 67.9, 46.4, 33.8, 31.9, 29.8, 29.7, 29.6, 29.4, 28.9, 28.3, 25.6, 24.9, 24.6, 22.7, 19.2, 17.8, 14.1. Anal. Calcd: C, 68.15%; H, 7.98%; N, 5.15%. Found: C, 68.12%; H, 8.24%; N, 8.42%. Synthesis of the Azodendron d(isoAZO/C18)-50/50. This codendron was synthesized following the procedure described for d(isoAZO/C18)-75/25 using 6-[4-(4′-isobutyloxyphenylazo)phenyloxy]hexanoic acid (0.30 g, 0.78 mmol) and stearic acid (0.22 g, 0.78 mmol) in 50:50 molar ratio. The product was obtained as an orange powder. Yield: 70%. IR (KBr), ν (cm−1): 2097, 1742, 1601, 1582, 1501, 1247, 1147, 840. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.85−7.82 (m, Ar), 6.98−6.91 (m, Ar), 4.34−4.06 (m, CH2−OCO) 3.96 (t, J = 6.3 Hz, CH2−OAr), 3.77 (d, J = 6.5 Hz, CH2−OAr), 3.25 (t, J = 6.8 Hz, CH2−N3), 2.34 (t, J = 7.5 Hz, CH2−CO), 2.27 (t, J = 7.4 Hz, CH2−CO), 2.16−2.06 (m, CH−(CH3)2) 1.84−1.73 (m, CH2), 1.72−1.62 (m, CH2), 1.60−1.42 (m, CH2), 1.40−1.13 (m, CH2, CH3 (dendron)), 1.03 (d, J = 6.8 Hz, CH3(iso)), 0.86 (t, J = 6.6 Hz, CH3 (C18)).13C NMR (101 MHz, CDCl3) δ (ppm): 173.1, 172.8, 161.3, 161.0, 146.9, 146.8, 124.3, 114.6, 114.6, 74.6, 67.9, 64.8, 46.6, 46.3, 34.0, 33.8, 31.9, 29.7, 29.7, 29.6, 29.6, 29.4, 29.2, 28.9, 28.3, 25.6, 24.9, 24.6, 22.7, 19.2, 17.8, 17.5, 14.1. Anal. Calcd: C, 69.32%; H, 8.89%; N, 3.83%. Found: C, 69.62%; H, 9.11%; N, 3.99%. Synthesis of the Azodendron d(isoAZO/C18)-25/75. This codendron was synthesized following the procedure described for d(isoAZO/C18)-75/25) using 6-[4-(4′-isobutyloxyphenylazo)phenyloxy]hexanoic acid (0.13 g, 0.35 mmol) and stearic acid (0.20 g, 0.69 mmol) in 25:75 molar ratio. The product was obtained as an orange powder. Yield: 70%. IR (KBr), ν (cm−1): 2096, 1742, 1601, 1582, 1500, 1247, 1148, 841. 1H NMR (400 MHz,CDCl3) δ (ppm): 7.83−7.80 (m, Ar), 6.96−6.90 (m, Ar), 4.36−4.06 (m, CH2−OCO) 3.93 (t, J = 6.3 Hz, CH2−OAr), 3.74 (d, J = 6.5 Hz, CH2−OAr), 3.25 (t, J = 6.8 Hz, CH2−N3), 2.33 (t, J = 7.5 Hz, CH2−CO), 2.27 (t, J = 7.4 Hz, CH2−CO), 2.15.2.03 (m, CH−(CH3)2) 1.83−1.72 (m, CH2), 1.72−1.57 (m, CH2), 1.55−1.40 (m, CH2), 1.38−1.10 (m, CH2, CH3 (dendron)), 1.03 (d, J = 6.8 Hz, CH3 (iso)), 0.87 (t, J = 6.6 Hz, CH3 (C18)). 13C NMR (101 MHz, CDCl3) δ (ppm): 173.0, 172.7, 161.3, 161.0, 146.9, 146.9, 124.2, 114.6, 74.6, 67.9, 64.9, 46.7, 46.5, 34.0, 33.8, 31.7, 29.7, 29.6, 29.5, 29.4, 29.2, 29.0, 28.3, 25.5, 24.9, 24.6, 22.8, 19.2, 17.8, 17.6, 14.2. Anal. Calcd: C, 70.62%; H, 9.93%; N, 2.35%. Found: C, 70.83%; H, 9.87%; N, 2.50%. Synthesis of the Block Copolymer PEG-b-d(isoAZO/C18)-75/ 25. Alkyne-functionalized PEG (53.0 mg, 26.6 μmol), d(isoAZO/ C18)-75/25) (150.4 mg, 20.4 μmol), and CuBr (5.8 mg, 40.8 μmol) were added to a Schlenk tube. N,N,N′,N″,N″-Pentamethyldiethylenetriamine (8.5 μL, 40.8 μmol) and deoxygenated N,N-dimethylformamide (2 mL) were added with an argon-purged syringe, and the flask was further degassed by three freeze−pump−thaw cycles and flushed with argon. The reaction mixture was stirred under an argon atmosphere at RT for 72 h. The mixture was diluted with THF and then passed through a short column of alumina. The solvent was partially evaporated, and the resulting polymer solution was carefully precipitated of cold ethanol. Yield: 85%. IR (KBr), ν (cm−1): 1737, 1601, 1581, 1500, 1246, 1148, 841. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.84−7.80 (m, Ar), 6.98−6.90 (m, Ar), 4.30−4.16 (m, CH2− OCO), 4.01−3.90 (m, CH2−OAr), 3.79−3.72 (m, CH2−OAr), 3.71− 3.55 (m, CH2−O(PEG)), 3.38 (s, CH3(PEG)), 3.02−2.95 (m, CH2− CO(PEG)), 2.76−2.70 (m, CH2−tet), 2.33 (t, J = 7.5 Hz, CH2−CO), 2.27 (m, CH2−CO), 2.15−2.04 (m, CH−(CH3)2) 1.83−1.71 (m, CH2), 1.70−1.55 (m, CH2), 1.5−1.39 (m, CH2), 1.36−1.13 (m, CH2, CH3(dendron)), 1.03 (d, J = 6.8 Hz, CH3(iso)), 0.86 (t, J = 6.6 Hz, CH3(C18)). Anal. Calcd: C, 65.32%; H, 8.16%; N, 4.02%. Found: C, 65.03%; H, 8.53%; N, 4.25%.

Synthesis of the Block Copolymer PEG-b-d(isoAZO/C18)-50/ 50. This LDBC was synthesized following the same procedure like PEG-b-d(isoAZO/C18)-75/25 using d(isoAZO/C18)-50/50 (150.0 mg, 21.6 μmol). Yield: 80%. IR (KBr), ν (cm−1): 1739, 1601, 1582, 1501, 1247, 1147, 841. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.83− 7.80 (m, Ar), 6.98−6.92 (m, Ar), 4.30−4.10 (m, CH2−OCO), 4.02− 3.92 (m, CH2−OAr), 3.80−3.73 (m, CH2−OAr), 3.71−3.54 (m, CH2−O(PEG)), 3.38 (s, CH3(PEG)), 3.01−2.97 (m, CH2−CO(PEG)), 2.75−2.69 (m, CH2−tet), 2.33 (t, J = 7.5 Hz, CH2−CO), 2.27 (t, J = 7.4 Hz, CH2−CO), 2.14−2.03 (m, CH−(CH3)2) 1.86− 1.73 (m, CH2), 1.72−1.61 (m, CH2), 1.60−1.42 (m, CH2), 1.40−1.10 (m, CH2, CH3 (dendron)), 1.03 (d, J = 6.8 Hz, CH3 (iso)), 0.85 (t, J = 6.6 Hz, CH3(C18)). Anal. Calcd: C, 66.05%; H, 8.92%; N, 2.96%. Found: C, 65.80%; H, 8.53%; N, 2.58%. Synthesis of the Block Copolymer PEG-b-d(isoAZO/C18)-25/ 75. This LDBC was synthesized following the same procedure described for PEG-b-d(isoAZO/C18)-75/25 using d(isoAZO/C18)25/75 (150.2 mg, 22.9 μmol). Yield: 80%. IR (KBr), ν (cm−1): 1739, 1600, 1581, 1500, 1246, 1147, 842. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.84−7.80 (m, Ar), 6.98−6.90 (m, Ar), 4.30−4.12 (m, CH2− OCO), 4.04−3.96 (m, CH2−OAr), 3.80−3.73 (m, CH2−OAr), 3.72− 3.55 (m, CH2−O(PEG)), 3.38 (s, CH3(PEG)), 3.02−2.97 (m, CH2− CO(PEG)), 2.74−2.70 (m, CH2−tet), 2.33 (t, J = 7.5 Hz, CH2−CO), 2.27 (t, J = 7.4 Hz, CH2−CO), 2.12−2.02 (m, CH−(CH3)2) 1.86− 1.72 (m, CH2), 1.70−1.61 (m, CH2), 1.60−1.42 (m, CH2), 1.40−1.13 (m, CH2, CH3(dendron)), 1.04 (d, J = 6,8 Hz, CH3 (iso)), 0.87 (t, J = 6.6 Hz, CH3(C18)). Anal. Calcd: C, 66.94%; H, 9.65%; N, 1.79%. Found: C, 66.50%; H, 9.59%; N, 2.08%. Characterization Techniques. IR spectra were obtained on a Nicolet Avatar 360-FT-IR spectrometer using KBr pellets. 1H NMR spectra were measured on a Bruker AV-400 spectrometer at 400 MHz. Elemental analysis was performed using a PerkinElmer 2400 microanalyzer. MALDI-TOF MS was performed on an Autoflex mass spectrometer (Bruker Daltonics) using dithranol as matrix. Number-average molecular weight and polydispersity of the LDBCs were calculated from the mass spectra using PolyTools 1.0 (Bruker Daltonics). Size exclusion chromatography (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. Measurements were performed in THF with a flow of 1 mL/min using polystyrene (PS) narrow molecular weight standards. Thermogravimetric analysis (TGA) was performed using a Q5000IR from TA Instruments under a nitrogen atmosphere and 2−5 mg of the sample. Thermal transitions were determined by differential scanning calorimetry (DSC) using a DSC Q-2000 from TA Instruments and powdered samples (2−5 mg) sealed in aluminum pans. Glass transition temperatures were determined at the midpoint of the baseline jump, and the isotropic temperatures were determined at the maximum of the corresponding peaks. Polarized optical microscopy (POM) was performed using an Olympus BH-2 polarizing microscope fitted with a Linkam THMS600 hot stage. UV−vis spectra were determined on an ATIUnicam UV4−200 spectrophotometer. Fluorescence measurements were recorded using a PerkinElmer LS 50B fluorescence spectrophotometer. General Procedure for the Preparation of the Vesicles. For the preparation of the self-assemblies, a solution of 5 mg/mL of the amphiphilic LDBC in THF was prepared, and Milli-Q water was gradually added while self-assembly was followed by measuring the turbidity. When a critical water content was reached, a sudden increase in turbidity occurred, indicating that the self-assembling process took place. Once turbidity reached an almost constant value, the mixture was dialyzed against water to remove the organic solvent using a Spectra/Por dialysis membrane (MWCO 1000) for 3 days. Water suspensions of the vesicles with a concentration around 2 mg/mL were obtained. Determination of the Critical Aggregation Concentration (CAC). Critical aggregation concentration was determined by fluorescence spectroscopy using Nile Red as the probe. For the 5953


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determination of CAC vesicles were loaded with Nile Red as follows: 119 μL of a solution of Nile Red in dichloromethane (5 × 10−6 M) was added into a series of flasks, and then the solvent evaporated. Afterward, a water suspension of the vesicles of a concentration ranging from 1.0 × 10−4 to 1.0 mg/mL prepared by diluting an initial 2 mg/mL vesicle suspension was added to each flask. In each flask a final concentration of 1.0 × 10−6 M of Nile Red was reached. These solutions were stirred overnight to reach equilibrium before fluorescence was measured. The emission spectra of Nile Red were registered from 560 to 700 nm while exciting at 550 nm. Loading of Rhodamine B into the Vesicle. In order to encapsulate the dye, vesicles formation was carried out following the same procedure described for the polymer vesicles but using a solution (volume of solution and concentration of dye accurately determined) of Rhodamine B in Milli-Q water. The copolymer was first dissolved in THF and a solution of Rhodamine B was gradually added to induce the self-assembly into vesicles. The charge ratio was 1:5 (moles of copolymer:moles of dye). Self-assembly was followed by measuring the turbidity, and the mixture was then dialyzed against water to remove THF and nonencapsulated Rhodamine B. The Rhodamine B dialyzed solution was analyzed by fluorescence measurements to determine the amount of nonencapsulated dye that was removed by dialysis and, as a consequence, the amount of dye encapsulated in the vesicles. Morphological Studies TEM and Cryo-TEM. The morphology of the block copolymer vesicles was studied by transmission electron microscopy in a JEOL-2000 FXIII electron microscope operating at 200 kV and by cryo-TEM in a JEM-2011 electron microscope. Preparation of samples for TEM inspection: 5 μL of a 0.5 mg/mL water dispersion of self-assemblies was applied to a TEM grid. Water of the sample was removed by capillarity using filter paper. Then, the sample was stained with uranyl acetate and the grid was dried overnight under vacuum. Preparation of samples for cryo-TEM inspection: 5 μL of a 2 mg/mL water dispersion of self-assemblies was applied to a suitable grid and then rapidly frozen in liquid ethane. Dynamic Light Scattering (DLS) Measurements. The 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 vesicles concentration was 0.05 mg/mL (concentration above CAC), and size measurements were performed at least three times on each sample to ensure consistency. Confocal Microscopy Studies. 5 μL of a 2 mg/mL water dispersion of self-assemblies with encapsulated Rhodamine B was applied to a glass slide, and a coverslip was placed on the top of the sample. The edges were sealed to avoid solvent evaporation during measurement. Fluorescence vesicles were observed with a 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. Irradiation Experiments. The water dispersions of self-assemblies were irradiated during with a compact low-pressure fluorescent lamp Philips PL-S 9W emitting between 350 and 400 nm. The samples were placed at a distance of 10 cm from the light source in quartz cuvettes at room temperature. After irradiation, the water suspensions were kept in dark.

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RESULTS AND DISCUSSION Synthesis and Characterization of the Amphiphilic Block Copolymers. The target amphiphilic LDBCs were prepared by coupling the PEG and the dendritic blocks via a copper(I)-catalyzed azide−alkyne [3 + 2] cycloaddition (CuAAC) (Scheme 1).39,40 The fourth-generation dendron derived from bis-MPA acid having 16 hydroxyl groups and an 6-azidohexyl group at the focal point (d16OH) was functionalized with isoAZO and C18 in different proportion by an esterification reaction using the corresponding acids in either 75:25, 50:50, or 25:75 molar ratios (molar ratio indicated as

Figure 1. MALDI-TOF spectra of the codendrons d(isoAZO/C18)75/25, d(isoAZO/C18)-50/50, and d(isoAZO/C18)-25/75 (from top to bottom). Assigned peaks correspond to the protonated species [M + H]+, although [M + Na]+ species are also detected.

AZO/C18 in Scheme 1). Different analytical techniques were used to gain full information about the composition of these 5954


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Table 1. Molecular Weight and Composition of the Synthesised Condendrons and LDBCs codendrons and LDBCs

Mn(theo)a

Mnb

Mnc

ĐM c

AZO/C18 ratiod

d(isoAZO/C18)-75/25 d(isoAZO/C18)-50/50 d(isoAZO/C18)-25/75 PEG-b-d(isoAZO/C18)-75/25 PEG-b-d(isoAZO/C18)-50/50 PEG-b-d(isoAZO/C18)-25/75

7349 6948 6548 9319 8918 8518

7350.2 6948.6 6548.7 9100 8800 8400

6300 6500 6000 8600 8400 8000

1.01 1.02 1.03 1.02 1.02 1.03

72/28 47/53 31/69

phobic/philic ratioe

79/21 78/22 76/24

a

Theoretical molecular weights of dendrons corresponding to the feed ratio AZO/C18. Theoretical weights of block copolymers calculated as the sum of the molecular weight of the PEG block (Mn = 1970.4) and the dendritic block. bMolecular weights calculated by MALDI-TOF. Data of the dendrons corresponding to the most intense peak of the protonated species [M + H]+ in dendron distributions (see MALDI-TOF spectra in Figure 1). cMn and ĐM were calculated by SEC using PS standars. dAZO/C18 ratio calculated by 1H NMR. eHydrophobic/hydrophophilic ratio was calculated considering the linear block (PEG) as the hydrophilic part and the dendritic block as the hydrophobic part.

Figure 2. 1H NMR spectra of d(isoAZO/C18)-75/25 showing the signals used to estimate the composition. See Figure S2 in the Supporting Information for a complete assignment of the signals.

codendrons. As expected, several peaks were registered in the MALDI-TOF mass spectra corresponding to a distribution of codendrons with different composition. The mass spectra indicated a statistical functionalization of the dendron periphery, being the most intense peaks corresponding to fully substituted codendrons. Nevertheless, peaks corresponding to codendrons with 15 peripheral substituents were also detected to a lesser extent (Figure 1). The maximum of this distribution (Table 1) corresponds to codendrons with a functionalization equal or similar to the acids’ proportion feed on the esterification reaction. Accordingly, for d(isoAZO/ C18)-75/25 the maximum of this distribution corresponds to fully functionalized condendron containing 12 AZO units and 4 alkyl chains (12AZO/4C18 composition) at the 16 peripheral groups, for d(isoAZO/C18)-50/50 there is a similar population of codendrons with 8AZO/8C18 and 9AZO/7C18 compositions, and for d(isoAZO/C18)-25/75 the majority corresponds to 4AZO/12C18 composition. 1H NMR spectroscopy was employed to study the average AZO/C18 composition by relative integration of azobenzene aromatic protons signals and the corresponding ones to the methylene protons (CH2−CO) of the functional units in the periphery (Figure 2). In all cases, the calculated AZO/C18 ratios are in agreement with the expected ones.

Figure 3. (a) SEC traces of d(isoAZO/C18)-50/50 (straight line) and PEG-b-d(isoAZO/C18)-50/50 (dashed line). (b) MALDI-TOF mass spectra of d(isoAZO/C18)-50/50 (top) and PEG-b-d(isoAZO/C18)50/50 (bottom).

Alkyne-functionalized PEG and azido-functionalized codendrons were finally coupled to give the corresponding LDBCs PEG-b-d(isoAZO/C18)-75/25, PEG-b-d(isoAZO/C18)-50/ 50, and PEG-b-d(isoAZO/C18)-25/75 . A slight excess of the PEG block was used in order to facilitate the completeness of the coupling reaction that was easily removed by precipitation of the LDBC in ethanol. The efficiency of the coupling was mainly corroborated by size exclusion chromatography (SEC) and MALDI-TOF mass spectrometry where residual traces of the noncoupled dendron were not detected (Figure 3). LDBCs average molecular weights calculated by MALDI-TOF fairly agree with theoretically estimated values; however, the values calculated by SEC using PS standards are underestimated (Table 1). This has been previously observed for other LDBCs and can be attributed to the smaller hydrodynamic volume of these copolymers in comparison with the linear standards.42 1H NMR and FTIR spectroscopies, and elemental analysis also 5955


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decomposition temperature associated with mass loss, calculated from the TGA curve, above 320 °C, being the stability of the LDBCs around 30 °C higher than the corresponding codendrons. The thermal transitions of the dendrons and BCs were studied by polarized optical microscopy (POM) and differential scanning calorimetry (DSC) (Table 2 and Figure S3). Upon cooling from the liquid isotropic state, crystallization of the codendrons was observed in all cases at approximately 30 °C, as calculated by DSC, being the crystallization temperature dependent on the C18 content. While crystallization of the dendron containing only 4-isobutyloxyazobenzene occurs at 51 °C,39 crystallization of codendrons decreases to 36 °C for PEGb-d(isoAZO/C18)-75/25 and to 29 °C for lower AZO/C18 contents. At the same time, crystallization enthalpy increases on decreasing the AZO/C18 proportion. This increase on the crystallinity content can be associated with the crystallization of the alkyl chain as it has been reported for bis-MPA-based hyperbranched polyesters containing terminal long alkyl chains.43 Similarly to the codendrons, the investigated LDBCs have tendency to crystallize. An exothermic crystallization process was recorded on the DSC curves at 31−38 °C associated with the codendron block being the reported crystallization temperature for LDBC with only azo moieties of 62 °C.39 Crystallization of the PEG block about 20 °C was not visible. Again, on decreasing the azobenzene content the crystallization temperature slightly decreases as the correlated crystallization enthalpy increases. Self-Assembly of the Linear−Dendritic Azobenzene BCs in Water. Vesicles were formed by dissolving the LDBCs in THF and adding water gradually while the process was followed by turbidimetry (Figure S4). The critical aggregation concentration (CAC) in water was determined using Nile Red as a polarity-sensitive fluorescent probe according to a procedure previously reported21,44 (Figure S5). Nile Red was equilibrated with LDBCs at several concentrations and the emission spectra registered from 560 to 700 nm. The relationship between the fluorescence and the concentration was nonlinear; at low concentrations of LDBC a weak emission was observed because Nile Red was dispersed in the aqueous medium, but at high concentrations of LDBCs the emission intensity increased, indicating that Nile Red was located in a more hydrophobic environment as a consequence of being encapsulated by stable polymeric self-assemblies. The values for PEG-b-d(isoAZO/C18)-75/25, PEG-b-d(isoAZO/C18)-50/ 50, and PEG-b-d(isoAZO/C18)-25/75 LDBCs were around 8−10 μg/mL, which are lower than those corresponding to the LDBC containing only azobenzene moieties (35 μg/mL) due to the incorporation of hydrophobic aliphatic chains. The morphology of the self-assemblies was studied by transmission electron microscopy (TEM). Self-assemblies were initially observed by TEM using dried samples stained with uranyl acetate. The presence of vesicular self-assemblies, in general with a deflated appearance due to dehydration during sample preparation, was confirmed (Figure 4a). Subsequently, vitrified samples of the self-assemblies without staining were analyzed by cryo-TEM (Figure 4b). Nondisturbed vesicles were observed with a clear membrane that showed a distribution of diameters ranging from 70 to 300 nm for all the LDBCs. The dark region of the membrane corresponds to the hydrophobic dendritic arrangement containing aromatic azobenzene moieties and hydrophobic aliphatic chains. The thickness of this inner part of the membrane was found to be around 8 nm

Table 2. Thermal Properties of the Codendrons and LDBCs TGAa

DSCb

codendrons and LDBCs

Td

Tc

ΔHc

d(isoAZO/C18)-75/25 d(isoAZO/C18)-50/50 d(isoAZO/C18)-25/75 PEG-b-d(isoAZO/C18)-75/25 PEG-b-d(isoAZO/C18)-50/50 PEG-b-d(isoAZO/C18)-25/75

331 335 327 359 365 360

36 29 29 38 32 31

128 228 258 139 173 232

Td (in °C): decomposition temperature associated with mass loss calculated by TGA, under a nitrogen atmosphere (10 °C/min) at the onset point in the weight loss curve. bTc (in °C) and ΔHc (in kJ per mol of polymer chain): crystallization temperature and associated enthalpy calculated in the first cooling scan (10 °C/min) from the isotropic molten state. Theoretical molecular weight of the codendrons and Mn of polymer (determined by MALDI-TOF) were used to calculate enthalpy values. a

Figure 4. (a) TEM images of nonirradiated dried vesicles. Cryo-TEM images of the vesicles: (b) before and (c) after irradiation. The length of the scale bar corresponds to 200 nm in (a) and 100 nm in (b) and (c).

Table 3. Mean Hydrodynamic Diameters (Dh) of the Vesicles before and after Irradiation Dh (nm)

a

vesicles

nonirradiated

irradiated

PEG-b-d(isoAZO/C18)-75/25 PEG-b-d(isoAZO/C18)-50/50 PEG-b-d(isoAZO/C18)-25/75

195 298 210

178 98/350a 190

Bimodal size distribution with two maxima (see Figure S6).

confirmed the expected structures (see the Experimental Section for further details and Figures S1 and S2). The thermal stability of the azodendrons and the BCs was studied by thermogravimetric analysis (TGA) using powdered samples. The results are presented in Table 2. All the samples exhibited a good thermal stability with the onset of 5956


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Figure 5. (a) Comparison of the UV−vis spectra of water suspension vesicles of PEG-b-d(isoAZO/C18)-75/25, PEG-b-d(isoAZO/C18)-50/50, and PEG-b-d(isoAZO/C18)-25/75 with the vesicles previously reported (100% AZO). Evolution of the UV−vis spectra of irradiated vesicles at different irradiation times: (b) PEG-b-d(isoAZO/C18)-75/25, (c) PEG-b-d(isoAZO/C18)-50/50, and (d) PEG-b-d(isoAZO/C18)-25/75.

fitting with a bilayer arrangement of the codendrons.24 Dynamic light scattering (DLS) measurements were also performed. The mean hydrodynamic diameters (Dh) were found to be around 200−300 nm (Table 3). Light Responsive Behavior of the Self-Assemblies. UV−vis spectra of vesicles suspensions (1 mg/mL in water) were recorded and compared with those previously recorded for LDBCs only containing 4-isobutyloxyazobenzene moieties (100% AZO) at the periphery39 (Figure 5a). The spectrum of PEG-b-d(isoAZO/C18)-75/25 is comparable to the spectrum of vesicles containing only AZO moieties, as expected due to the high content of azobenzene moieties. For these PEG-bd(isoAZO/C18)-75/25 vesicles the absorption maximum is located at 320 nm, which indicates the prevailing formation of azobenzene H-aggregates. Two shoulders present at 355 and 375 nm are attributed to nonaggregated trans-azobenzene and the presence of J-aggregates, respectively. A weak band at about 450 nm corresponding to n−π* transition was also detected. For PEG-b-d(isoAZO/C18)-50/50 and PEG-b-d(isoAZO/ C18)-25/75 vesicles, the spectra are very similar and showed a clear narrowing of the π−π* band accompanied by a bathochromic shift of the maximum, from 320 to 335 nm, indicating a lower tendency to aggregation of the azobenzene moieties. Vesicles suspensions (1 mg/mL) were exposed to UV irradiation (350−400 nm, 9W) while changes on the UV−vis spectra were registered at different irradiation times (Figure 5b−d). In all cases, a notable decrease of π−π* band as well as an increase of the absorbance at 450 nm were observed, indicating the presence of cis-azobenzene. PEG-b-d(isoAZO/ C18)-75/25, similarly to vesicles containing only AZO moieties, were irradiated up to 30 min, but only slight changes were detected in the UV−vis spectrum after 5 min of irradiation, indicating that a photostationary state has been reached. It was also observed that the lower is the AZO/C18

ratio less time is needed to reach the photostationary state, being approximately 2 min for PEG-b-d(isoAZO/C18)-50/50 vesicles and 1 min for PEG-b-d(isoAZO/C18)-25/75. Thus, a faster and more efficient photoinduced isomerization was achieved by decreasing azobenzene content in the codendrons. Once irradiated and maintained in darkness, the spectra of the vesicles were similar to the observed before irradiation due to the back cis-to-trans isomerization. Cryo-TEM microscopy (Figure 4c) and DLS measurements (Table 3 and Figure S6) were used to gain further information about morphological changes occurred by irradiation (samples were studied immediately after irradiation for 5 min). For PEGb-d(isoAZO/C18)-75/25 vesicles, a slight decrease in the Dh was detected. Similarly to the previously reported vesicles containing only AZO moieties, cryo-TEM images showed deformed vesicles with a distorted membrane. In the case of PEG-b-d(isoAZO/C18)-50/50, cryo-TEM images show drastic structural changes upon irradiation. An evident decrease in the number of vesicles accompanied by material without a clear morphology was observed. In the DLS plots, a marked change in the distribution curve was observed with the appearance of a new peak at smaller Dh (Figure S6). This new distribution could explain the disruption of some of the self-assemblies observed by cryo-TEM. By contrast, cryo-TEM images show that PEG-b-d(isoAZO/C18)-25/75 vesicles retained the morphology after irradiation, despite the modifications detected by UV−vis. Only minor modifications were observed by DLS measurements after irradiation. Encapsulation and Photoinduced Release of Molecular Probes. As mentioned above, vesicles are able to encapsulate hydrophobic molecules like Nile Red in the inner part of the membrane. In order to study the stimulated release of this type of molecules by the trans-to-cis photoisomerisation of the azobenzenes, vesicles with encapsulated Nile Red were irradiated using the same conditions that in the previous 5957


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Figure 7. Evolution of fluorescence intensity of the aqueous solution of vesicles with encapsulated Rhodamine B of PEG-b-d(isoAZO/ C18)-75/25 (top) and PEG-b-d(isoAZO/C18)-50/50 (bottom) at different irradiation times. The fluorescence value after 24 h of irradiation was considered as reference.

and, consequently, the fluorescent probe mainly remained encapsulated. In the case of PEG-b-d(isoAZO/C18)-75/25 and PEG-b-d(isoAZO/C18)-50/50, the initial Nile Red emission was not recovered, indicating the light triggered release of the trapped probe to the aqueous environment. It is necessary to mention that in the case of the vesicles containing only azobenzene previously reported the Nile Red emission was partially recovered on standing in the dark which might be related to a less efficient release of the fluorescence probe. Encapsulation and photoinduced release of Rhodamine B were also investigated that, as a hydrophilic probe, will be loaded at the internal cavity of the vesicle. Vesicles were prepared with loading contents of about 20 molecules of dye per LDBC molecule and irradiated for different time intervals. The evolution of the Rhodamine B release upon irradiation was investigated by confocal microscopy. Before irradiation, the green fluorescence due to the fluorescent dye is concentrated in some specific regions dispersed in a nonfluorescent background due to the encapsulation of the dye in the polymeric vesicles (see Figure S7). The appearance of a fluorescent background after irradiation is associated with the release of Rhodamine B.39 In an attempt to quantify the released dye versus the irradiation time, the intensity of the background fluorescence in the confocal images was measured after irradiating during different times. Values of fluorescence intensity were obtained averaging 200−250 randomly selected points of the background on the irradiated samples and comparing them with the corresponding value for nonirradiated samples. Figure 7 shows the evolution of fluorescence intensity of the aqueous solution at different irradiation times. Data recorded for PEG-b-d(isoAZO/C18)-25/75 revealed that almost no dye was released, which is in accordance with

Figure 6. Emission spectra of vesicles with encapsulated Nile Red of PEG-b-d(isoAZO/C18)-75/25, PEG-b-d(isoAZO/C18)-50/50, and PEG-b-d(isoAZO/C18)-25/75 (from top to bottom).

experiments. Before irradiation, an intense emission peak registered at 606 nm, under excitation at 550 nm, indicated that Nile Red was in a hydrophobic environment. Upon irradiation the emission of Nile Red abruptly decreased in all cases (Figure 6). This decrease of the emission of the dye can be due to both Nile Red migration from the membrane to the aqueous media and to the increase in the polarity of the inner membrane due to the change in net dipole moment associated with trans-to-cis isomerization. After standing the samples in the dark, and once thermal back cis to-trans isomerization took place according to the UV−vis spectra, Nile Red fluorescence was again evaluated. After 24 h, Nile Red emission was almost recovered to the initial value for PEG-b-d(isoAZO/C18)-25/75, indicating that the fluorescent probe was again in an hydrophobic environment 5958


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previous described results where no morphological change was detected by cryo-TEM in the irradiated vesicles of this LDBC. However, for PEG-b-d(isoAZO/C18)-75/25 and PEG-b-d(isoAZO/C18)-50/50, the intensity of the background fluorescence increases on increasing the irradiation time. While a gradual increase of the emission intensity over irradiation time was observed for PEG-b-d(isoAZO/C18)-75/ 25, a faster increase was found for PEG-b-d(isoAZO/C18)-50/ 50. Data collected for PEG-b-d(isoAZO/C18)-75/25 show that Rhodamine B is gradually liberated upon irradiation, and after 24 h in the dark, leaking of the uploaded dye still persist. However, a complete release was achieved by irradiation during 2 min in the case of vesicles derived from PEG-b-d(isoAZO/ C18)-50/50, which agrees with the vesicle collapsed observed by cryo-TEM.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M.P.)., [email protected] (L.O.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work 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 founding for her PhD. The authors acknowledge the Servicio de Microscopıá Electrónica and the Advanced Microscopy Laboratory of the Universidad de Zaragoza and the Servei de Microscòpia-Universitat Autònoma de Barcelona for the TEM and cryo-TEM observations. The authors additionally acknowledge the use of the Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza. The authors also thank Dra. Marı ́a Royo (Microscopy and Image Service) for technical assistance and help with the confocal microscope and the I+CS/IIS Aragon (Aragon Health Sciences Institute) for access to the microscope.

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CONCLUSIONS Light responsive vesicles have been prepared from LDBCs with a PEG of 2000 average molecular weight and new codendrons containing different percentages of 4-isobutyloxyazobenzene (AZO) and hydrocarbon chains (C18) randomly distributed at the periphery. It has been shown that diluting the azobenzene content using alky chains accelerates the trans-to-cis photoisomerization process at the inner membrane probably by frustrating the aggregation tendency of the azobenzenes and providing higher mobility. PEG-b-d(isoAZO/C18)-75/25 vesicles show similar photoresponse to previously reported vesicles containing only AZO. UV irradiation induces an evident deformation in the membrane and consequently and increase on its permeability. In this case, the release of the internal cargo molecule is constant and progressive. Nevertheless, the release is improved with respect to the LDBC with only azobenzene as demonstrated with hydrophobic cargo molecules retained at the membrane. For PEG-b-d(isoAZO/C18)-50/50 vesicles, trans-to-cis photoisomerisation causes important changes in the stability of the vesicles. Upon UV irradiation, large damages on the membrane of the vesicles are observed by cryo-TEM achieving a fast release of the encapsulated probes. When AZO content is diluted down to 25% in PEG-b-d(isoAZO/C18)-25/ 75, the vesicles do not suffer any modification upon irradiation. The absence of significant changes in the irradiated samples could be due to the fact that the morphological changes accompanied by the polarity change produced due to azobenzene isomerization were not enough to provoke a deformation in the polymeric membrane and the subsequent release of the fluorescence probes. Therefore, we have used the dendritic block of LDBCs as a platform to incorporate chemical changes and alter the properties of inner part of the vesicle membrane. By adjusting AZO/C18, the photoresponsive properties of the vesicles and consequently the release rate can be tailored.

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ASSOCIATED CONTENT

S Supporting Information *

FTIR and NMR spectra and DSC traces of the dendrons and the corresponding LDBC; turbidity plot, details of CAC calculation, DLS measurements, and confocal images. This material is available free of charge via the Internet at http:// pubs.acs.org. 5959


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Light Responsive Vesicles Based on LinearâDendritic Block

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