Synthesis and Characterization of a Micelle-Based pH Nanosensor

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Synthesis and Characterization of a Micelle-Based pH Nanosensor with an Unprecedented Broad Measurement Range Pramod Kumar E.K.,†,‡ Lise N. Feldborg,†,‡ Kristoffer Almdal,† and Thomas L. Andresen*,†,‡ †

DTU Nanotech, Department of Micro-and Nanotechnology, Technical University of Denmark, Building 423, 2800 Lyngby, Denmark ‡ Center for Nanomedicine and Theranostics, Technical University of Denmark, Building 423, 2800 Lyngby, Denmark ABSTRACT: A new cross-linked micelle pH nanosensor design was investigated. The nanosensor synthesis was based on self-assembly of an amphiphilic triblock copolymer, poly(ethylene glycol)-b-poly(2-amino ethyl methacrylate)-bpoly(coumarin methacrylate) (PEG-b-PAEMA-b-PCMA), which was synthesized by isolated macroinitiator atom transfer radical polymerization. Micelles were formed by PEG-bPAEMA-b-PCMA self-assembly in water, giving micelles with an average diameter of 45 nm. The PCMA core was employed to utilize coumarin-based photoinduced cross-linking in the core of the micelles, which was performed by UV irradiation (320 nm < λ < 500 nm), and the progress of the cross-linking was monitored by UV spectroscopy. The formed cross-linked core−shell−corona micelle was converted into ratiometric pH nanosensors by binding the pH-sensitive fluorophores oregon green 488 and 2′,7′-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein and a reference fluorophore Alexa 633 to the PAEMA shell region of the micelles. Fluorescence measurements show that these pH nanosensors are sensitive in a surprisingly broad pH range of 3.4−8.0, which is hypothesized to be due to small differences in the individual fluorophores’ local environement. It was found that the utilization of selforganization principles to form the nanoparticles, followed by cross-linking to ensure sensor integrity, provides a fast and highly flexible method that can be utilized in a broad range of nanosensor designs. KEYWORDS: nanosensor, cross-linked micelle, coumarin, core−shell−corona micelle, ratiometric sensor, pH sensor

I. INTRODUCTION Nanosensors can be used in a range of applications and have recently gained considerable interest for measuring intracellular metabolites and pH.1 A number of polymeric nanoparticle matrices have been investigated for use in ratiometric fluorescence based pH nanosensors for biological studies at the single-cell level.1,2 These include polystyrene,2 polyacrylamide,3−6 and polysaccharide,7 and recently, we published a nanosensor system based on micelles.8 Nanosensors for pH measurements ex vivo in cells and/or in vivo in tissues should not only be capable of measuring the fluorescent intensity changes with pH but should also have the capability of targeting the compartment of interest. Today, we do not possess a sufficient understanding of nanoparticle internalization and trafficking in cells to reach this objective.1 However, nanosensors can provide new knowledge, and one of the long-term objectives of this research should be to allow intracellular compartment and tissue-specific measurements of metabolites in living cells and organisms. To research this goal, new sensor designs are needed,9 as well as an increased knowledge of nanoparticle cellular trafficking. Poly(ethylene glycol) (PEG) is often used to surface coat nanoparticles in the drug delivery area as it reduces interactions with blood proteins, resulting in long circulating properties after intravenous administration.10 Polymeric micelles that are formed by spontaneous selfassembly of amphiphilic block copolymers provide a scaffold © 2013 American Chemical Society

for nanosensor designs where a PEG corona can be implemented and allow for sensor designs that, in principle, can be used both in vitro and in vivo. A sensor with a PEG corona could, as an example, be used to measure the pH in tumor tissue by utilizing the EPR effect to secure high accumulation or in other tissues by use of antibody or peptide targeting ligands.11 In the present article, we have been focusing on providing a new sensor architecture based on micelles formed by triblock copolymers that, in the future, could allow for metabolite measurements ex vivo and in vivo. Radical polymerization techniques, such as ATRP (atom transfer radical polymerization),12 allow synthesis of welldefined functional block copolymers having narrow molecular weight distributions and a controlled architecture. Various selfassembled morphologies can result from amphiphilic triblock copolymers depending on the mass ratio of hydrophilic and hydrophobic blocks; for example, functional amphiphilic triblock copolymers with comparable hydrophilic-to-hydrophobic block ratios can form well-defined core−shell−corona functional micelles in a spherical or cylindrical morphology.13 Furthermore, regioselective cross-linking can enhance micelle applications by preventing its dissociation below the critical Received: September 11, 2012 Revised: January 1, 2013 Published: January 18, 2013 1496

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micelle concentration (CMC).14−16 A number of reversible micelle cross-linking strategies have been reported,17−20 based on photochemistry, which avoids the use of cross-linking reagents and provides very short reaction times. Furthermore, radiation-induced reversible photochemical dimerizations of coumarin21 have been used for the dimensional synthetic modifications of block copolymer micelles.22−24 The cyclobutane ring resulting from the 2πS + 2πS cycloaddition between the coumarin-containing unimers of the self-assembly are reversible and hence provide possibilities of photo-crosslinking and de-cross-linking of the micelles by illuminating the samples at two different wavelengths. In this paper, we utilize photochemical cross-linking of a micelle core to form a nanosensor system with an unprecedented broad pH measurement range. The use of micelle self-assembly, followed by photochemical cross-linking, provides a highly versatile method for nanosensor design and allows for good control of nanosensor surface chemistry, which is important to control biological interactions. The synthesized sensor uses fluorophores with excitation at 488 and 633 nm, which is appropriate for ex vivo cellular measurements.

Table 1. Molecular Weight of PEG120-b-PAEMA(Boc)9-bPCMA80 (1) Mna

Mwa

Mw/Mna

Mnb

nc

mc

pc

22 100

26 800

1.21

30 390

120

9

80

a

Determined by GPC. bDetermined by NMR. cNumber of repeating units of PEG block (n), PAEMA(Boc) block (m), and PCMA block (p).

removed from PEG-b-PAEMA(Boc)-b-PCMA by treatment with trifluoroacetic acid to obtain the PEG-b-PAEMA-b-PCMA (2). Complete deprotection of the copolymer was confirmed by the disappearance of Boc-methyl groups at 1.39 ppm by 1H NMR (Figure 2). The sizes of the three blocks were optimized

II. RESULTS AND DISCUSSION Figure 1 shows the photochemical synthesis of core crosslinked pH nanosensors. The amphiphilic triblock copolymer

Figure 2. 1H NMR spectra of (a) PEG120-b-PAEMA(Boc)9-b-PCMA80 (1) and (b) PEG120-b-PAEMA9-b-PCMA80 (2) in CDCl3. Figure 1. Schematic illustration of photo-cross-linked pH nanosensor synthesis, Fp = pH sensitive and reference fluorophore conjugation.

to ensure micelle formation and size (ratios between the blocks and Mw), ensure high micelle stability (size of the hydrophobic block), and to ensure enough sensor fluorophores in the constructs for reliable measurements (size of the middle block used for conjugation of fluorophores). The amphiphilic triblock PEG-b-PAEMA-b-PCMA core− shell−corona functional micelle (3), having a radially compartmentalized corona, was prepared by dissolving PEGb-PAEMA-b-PCMA (2) in DMF, followed by slow displacement of the common solvent (DMF) by a selective solvent for the hydrophilic block (water). Micelles were formed, and the mixture was subjected to dialysis against Milli-Q water. Dynamic light-scattering measurements (DLS) showed a number-average hydrodynamic diameter (Dh) = 45 ± 2 nm, and zeta potential (ξ) measurements gave ξ = 24 ± 1 mV (Table 2).

poly (PEG-b-PAEMA(Boc)-b-PCMA) (1) was synthesized by isolated macroinitiator ATRP (Scheme 1). The number-average molecular weight of the triblock measured by GPC was found to be lower than that calculated by 1H NMR (CDCl3) peak integration. The polydispersity index (Mw/Mn = 1.21) showed a narrow molecular weight distribution. The results are summarized in Table 1. The Boc-protection group was Scheme 1. Synthesis of Amphiphilic Triblock Copolymer PEG-b-PAEMA-b-PCMA by Isolated Macroinitiator ATRP

Table 2. Characterization Data for Micelles 3 and 4 and pH Nanosensors 5 and 6 micelle 3 4 5 6

DLS (Dh)a (nm) 45 40 41 38

± ± ± ±

2 1 2 3

zeta (ξ)b (mV) 24 22 17 16

± ± ± ±

1 2 2 1

a

Number-average hydrodynamic diameter of aqueous micelle solution measured by DLS (average of three independent measurements). b Average zeta potential from 10 measurements. 1497

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Cross-linking of the PCMA core of the PEG-b-PAEMA-bPCMA micelle (3) was achieved by UV-radiation-induced photochemical cross-linking (Scheme 2). The micelle solution Scheme 2. Synthesis of Core−Shell−Corona Micelle Core Cross-Linked Ratiometric pH Nanosensors

Figure 4. (a) IR spectra of non-cross-linked PEG-b-PAEMA-b-PCMA micelle (3) and (b) core cross-linked micelle (4).

formation. Additionally, the cross-linked micelles show an increase in absorption around 1500 cm−1 and a broad absorption peak at 1150 and 1240 cm−1. These changes are also supporting the cyclobutane ring formation at the PCMA micelle core during photoirradiation of the aqueous PEG-bPAEMA-b-PCMA micelle solution. The synthesis of core cross-linked micelle nanosensor (5) is shown in Scheme 2. The PEG-b-PAEMA-b-PCMA micelle (3) (1.28 mg/mL) was irradiated with λ > 320 nm using the same UV photocuring setup. After 55% of photodimerization (determined from Figure 3), DLS and zeta potential measurements show only a slight decrease in hydrodynamic diameter and no change in surface charge density of the micelles (Table 2, micelle 4). This indicates that intramicelle cross-linking occurred rather than intermicelle cross-linking, which would cause larger aggregates. That intramicelle cross-linking seems to occur exclusively may be attributed to the steric stabilization offered by the PEG corona.25 The positive surface charge (Table 2) further confirms the availability of free amino groups at the inner corona (shell) of the core cross-linked micelles. The photo-cross-linked micelle was then converted into a ratiometric pH nanosensor by covalently conjugating pHsensitive BCECF and oregon green 488 isothiocyanate (OGITC), together with a reference fluorophore Alexa Fluor 633 carboxylic acid succinimidyl ester (Alexa 633 SUC) at the inner corona of the micelle. The resulting nanosensor was purified by dialysis; DLS and zeta potential measurements show that the sensor (5) maintained the same size as that of the cross-linked micelle (4), however, with a slightly decreased surface charge density due to the fluorophore conjugation (Table 2). The pH sensitivity range of the pH nanosensor was determined by fluorescence measurements. The nanosensor (0.126 mg/mL) was excited (λex = 490 nm for OG and BCECF; λex = 625 nm for Alexa 633) in buffers of different pHs. The pH-dependent fluorescence emission spectra were collected at 508−565 nm and pH-independent reference spectra at 635−720 nm (Figure 5a). The fluorescence intensity (I) ratio ((I(OG) + I(BCECF))/I(Alexa 633)) was plotted against the corresponding pH to obtain the pH calibration curves (Figure 5b). OG and BCECF were chosen as the pH-sensitive fluorophores due to their complementary pKa values of 4.8 and 7.0, respectively,2 which allows for formation of nanosensors with an extended measurement range when used in the right ratios.4,6 However, the pH calibration curve shows that the nanosensor is sensitive in a range of pH 3.4−8.0, which is a significantly broader sensitivity range than expected from having two fluorophores with two pKa values. The broad measurement range is attributed to that the local fluorophore environment must be creating a broadness in the individual fluorophore pKa values, which is a very interesting effect for

(0.03 mg/mL) was exposed to UV radiation (2 W/cm2) (320 nm < λ < 500 nm) from a UV−vis photocuring system, and the decrease in coumarin absorption (320 nm) as a function of irradiation time was monitored by UV−vis spectroscopy. The degree of coumarin photodimerization (PD) at the PCMA core was also calculated from this UV−vis spectrum. PD % = (A0 − At)/A0 × 100, where A0 is the UV absorption of the coumarin core of the micelles before UV irradiation (t = 0) and after the time t, At (Figure 3).

Figure 3. (a) Decrease in UV absorption of PEG-b-PAEMA-b-PCMA micelles during photoirradiation (λ > 320 nm) measured by UV−vis at different irradiation times. (b) The increase in photodimerization degree during UV irradiation.

The micelle cross-linking by coumarin photochemical dimerization was further confirmed by FT-IR spectroscopy (Figure 4). Compared to the non-cross-linked micelles, the FTIR spectra of the core cross-linked micelles show a shift in the carbonyl stretching absorption at ∼1729 cm−1 to higher energy with an increase in peak broadness. This indicates that, as the photochemical cross-linking increases, the number of conjugated carbonyls in the pyrone subunits of the coumarins decrease due to the double bond dimerization at the micelle core. The alkenes CC stretching at 1390 cm−1 decreased considerably after UV irradiation, which confirms the consumption of double bonds due to cyclobutane ring 1498

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6b). The photo-de-dimerization studies show that complete decross-linking was not possible, indicating the presence of a dynamic dimerization-de-dimerization equilibrium in the PCMA core of the micelle at λ < 255 nm. DLS and zeta potential measurements of the de-cross-linked micelle sensor (6) showed no significant diffrence in the number-average hydrodynamic diameter (Dh) nor in the surface charge density of the micelles (Table 2). AFM images was recorded of the non-cross-linked (3) and cross-linked micelles (4) (Figure 7). The images show a dramatic difference in size and monodispersity before and after cross-linking. The non-cross-linked micelles seemed to aggregate more on the surface, and an analysis of the height of the individual particles in several images was made and represented in a histogram (Figure 7). The cross-linked micelles showed a very narrow height distribution relative to non-cross-linked micelles and an apparent low degree of aggregation, indicating that cross-linking at the coumarin core results in a more robust and uniform spherical micelle on the hydrophilic silicon surface. Many self-organized nanoparticles aggregate or dissociate when dried on the solid supports used for AFM (as the non-cross-linked micelles), and the integrity of the cross-linked micelles on the surface shows that the covalent bonds that were formed “lock” the micelle into a stable particle. It should further be noted that the observed height in AFM is smaller than what is observed in solution as the drying on the surface and the AFM tip during measurement “flattens” the soft micelle particles, which also gives them a larger diameter in the x,y plane. Lastly, we tested the colloidal storage stability of the micelles under a biologically relevant salt concentration at different pHs. The cross-linked micelles were stable for weeks at biological relevant pH in isotonic salt, and storage for more than 4 months at pH 7.4 did not lead to any change in sensor properties evaluated by DLS, UV absorption, and fluorescence emission. At pH 9.1, some aggregation was observed after 3 months, which seemed to be due to hydrolysis of the micelle core.

Figure 5. Representative fluorescence emission spectra for the pH nanosensor (a) and the pH calibration curve (b). Fluorescence intensity ratio = ((I(OG) + I(BCECF))/I(Alexa 633)) of three independent batch preparations of the pH nanosensor to show the reproducibility of sensor synthesis (sensors 1−3).

future sensor designs. We have earlier shown that the fluorophore microenvironment can be used to tune fluorophore pKa values,4 but it is the first time we have been able to expand the measurement range. To test the robustness of the approach, we synthesized three independent batches of the nanosensors, and all showed the same broad measurement range (Figure 5b). Since intracellular pH is generally between 6.8 and 7.4 in the cytosol of cells, up to 8.0 in the mitochondria matrix, and 3.8−6.5 in the cell’s acidic organelles (endosome/lysosome system),1 the newly developed nanosensor will potentially be capable of monitoring the intracellular pH of all cell organelles if they can be directed to individual cellular compartments in a controlled way. To further characterize the nanosensor material properties, we investigated the photo-de-cross-linking of the cross-linked micelle to evaluate reversibility. Three milliliters of the pH nanosensor dispersion (0.03 mg/mL) in a quartz cuvette was exposed to UV radiation (λ < 255 nm) from a 6 W 254 nm UVGL-58 UV lamp, and the increase in UV absorption (ca. 320 nm) as a result of cyclobutane ring scission was monitored by UV−vis spectroscopy (Figure 6a). The degree of photo-dedimerization was also calculated from the UV spectra (Figure

III. CONCLUSION Polymeric core−shell−corona micelles were synthesized by photo-cross-linking of a coumarin-containing core. The micelles were utilized as a scaffold for ratiometric pH nanosensors. The chemical design allowed conjugation of pH-sensitive and reference fluorophores at the shell region of the core cross-linked micelle with a protective shell of a PEG corona surrounding it. A unique sensitivity range was achieved between pH 3.4 and 8.0, which, to our knowledge, is the broadest measurement range achieved in a nanoparticle sensor. We attribute this broad range to be due to small differences in the microenvironment in the micelle shell of the pH-sensitive fluorophores, which gives a pKa distribution of the individual fluorophores and thereby a wider pH sensitivity range of the sensor. The sensor design may, therefore, be interesting in a number of applications, including monitoring of pH in cells as the pH of all cellular compartments is within the sensitivity range of the sensor. The use of photoinduced cross-linking should furthermore be able to prevent the disintegration of micelle nanosensors in biological environments under infinitely diluted conditions, which could make this design interesting in future in vivo applications.

Figure 6. (a) Increase in UV absorption of core cross-linked micelle nanosensor (0.03 mg/mL) during exposure to UV radiation (λ < 255 nm) and (b) the corresponding photo-de-dimerization degree of the nanosensor. 1499

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Figure 7. AFM images of micelles. 2D and 3D images are presented, including histograms of height distributions of (a) non-cross-linked micelle, 3, and (b) cross-linked micelle, 4. XY scan size is 1 μm × 1 μm. degassed with three freeze−pump−thaw cycles to remove oxygen, and the polymerization was carried out at 40 °C for 15 h under an argon atmosphere. The reaction was quenched by exposing the flask to air, and the reaction mixture was then passed through a silica gel column to remove the copper catalyst using methanol as eluent. On exposure to air, the dark brown reaction mixture turned blue, indicating oxidation of the Cu(I) catalyst. After removing most of the methanol by rotary evaporation, the polymer was precipitated into excess cold diethyl ether. The product was isolated by filtration and dried under vacuum: PEG-b-PAEMA(Boc)Cl (1.1 g, 64%). 1H NMR (300 MHz, CDCl 3 ) δ (ppm): 5.50 (broad s, −NH), 4.0 (broad s, −OCH 2 CH 2 NH), 3.63 (s, −CH 2 CH 2 O), 3.37 (broad s, −OCH2CH2NH, −OCH3), 1.82 (broad s, −CH2 backbone), 1.43 (s, −C(CH3)3), 1.11−0.81 (m, −C(CH3)2, −CH3 backbone). FTIR (cm−1): 3387, 2892, 1716, 1520, 1466, 1391, 1361, 1342, 1279, 1241, 1150, 1112, 1060, 996, 965, 843. PEG-b-PAEMA(Boc)-b-PCMA (1). PEG-b-PAEMA(Boc)-Cl (1 g, 0.11 mmol), 7-(2-methacryloyloxyethoxy)-4-methylcoumarin (CMA) (2.6 g, 9.02 mmol), CuCl2 (12 mg, 0.088 mmol), PMDETA (0.068 mL, 0.33 mmol), and 10 mL of DMF were added to a 25 mL Schlenk flask equipped with a magnetic stir bar. The flask was frozen in liquid nitrogen, and CuCl catalyst (11 mg, 0.11 mmol) was added. The reaction mixture was degassed with three freeze−pump−thaw cycles to remove oxygen, and the polymerization was carried out at 80 °C for 24 h under an argon atmosphere. The reaction mixture was concentrated under vacuum, and the polymer was precipitated into an excess of cold methanol, filtered, and dried: PEG-b-PAEMA(Boc)b-PCMA (2 g, 56%). 1 H NMR (300 MHz, CDCl3) δ (ppm): 7.40−5.90 (Coumarin H), 4.38−4.04 (−OCH2CH2O-Coumarin), 4.02−3.86 (−OCH2CH2NH), 3.58 (s, −CH2CH2O), 3.39−3.25 (−OCH2CH2NH), 2.22 (s, −CH3 of coumarin), 2.04−1.55 (−CH2 backbones of PAEMA and PCMA blocks), 1.39 (s, −C(CH3)3), 1.15−0.77 (−CH3 backbones of PAEMA and PCMA blocks). PEG-b-PAEMA-b-PCMA (2). PEG-b-PAEMA(Boc)-b-PCMA (500 mg, 0.016 mmol) was treated with a 1:1 TFA/DCM mixture (8 mL) for 15 h at room temperature. After removing most of the solvent under rotary evaporation, the polymer was precipitated into excess cold diethyl ether, filtered, and dried. The complete deprotection of tert-butyloxycarbonyl (Boc) was confirmed by the disappearance of the Boc signal at 1.39 ppm in 1H NMR (300 MHz, CDCl3). Self-Assembly of PEG-b-PAEMA-b-PCMA Micelle (3). The amphiphilic triblock copolymer (PEG-b-PAEMA-b-PCMA) (100 mg,

IV. EXPERIMENTAL SECTION Materials and Measurements. 2′,7′-Bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein free acid (BCECF) was purchased from Biotium. Oregon green 488 isothiocyanate (OGITC) and Alexa Fluor 633 carboxylic acid succinimidyl ester (Alexa 633 SUC) were obtained from Invitrogen. 2,2′-Bipyridyl (bpy) (99%), PMDETA (99%), CuCl2 (99.995%), trifluoroacetic acid (TFA) (99%), N-(3-dimethyl amino propyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) (98%), Nhydroxysuccinimide (NHS) (98%), dialysis tubing (MWCO = 12 kDa), and all other solvents and chemicals were purchased from Sigma Aldrich and used as received. CuCl (99.995%) was washed with glacial acetic acid, followed by absolute ethanol and diethyl ether, dried, and stored under argon. Solvents used for ATRP were purified by distillation over drying agents, as indicated in parentheses, stored under molecular sieves, and were transferred under argon: MeOH Mg(OMe)2, DMF (CaH2). The argon atmosphere (99.9999%) used in the reactions was provided by AGA Denmark. NMR spectra were recorded by using a 300 MHz Varian Mercury 300 BB spectrometer; IR spectra were recorded using a Perkin-Elmer Spectrum 100 FT-IR spectrometer. GPC measurements were carried out with an RID10ASHIMADZU refractive index detector and Mixed-D GPC column from Polymer Laboratories with a flow rate of 0.5 mL/min at 25 °C using DMF with 50 mM LiCl as eluent. UV−vis spectra were recorded on a Unicam Helios Uni 4923 spectrophotometer. Fluorescence measurements were carried out on an Olis upgraded SLM based spectrofluorometer. Atomic force microscopy (AFM) images were obtained by a PSIA XE-150 scanning force microscope using noncontact tapping mode close to the resonance frequency of the silicon cantilever (NanoWorld, Pointprobe type; force constant = 42 N/m and tip radius < 12 nm) of around 320 kHz. Images were recorded on a silica surface and under atmospheric conditions. Dynamic light-scattering (DLS) and zeta potential measurements were carried out using a Brookhaven Zeta PALS instrument. Synthesis of PEG-b-PAEMA-b-PCMA (2). The macroinitiator PEG-Br26 and the monomers 2-[N-(tert-butoxycarbonyl)amino]ethyl methacrylate (AEMA(Boc)) and 7-(2-methacryloyloxyethoxy)-4methylcoumarin (CMA) were synthesized by previously reported procedures.27,28 PEG-b-PAEMA(Boc)Cl. PEG-Br (1 g, 0.19 mmol), AEMABoc (522 mg, 2.28 mmol), 2,2′-bipyridyl (62 mg, 0.40 mmol), and 5 mL of methanol were added to a 25 mL Schlenk flask equipped with a magnetic stir bar. The flask was frozen in liquid nitrogen, and CuCl catalyst (21 mg, 0.21 mmol) was added. The reaction mixture was 1500

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(9) Lee, Y.-E. K.; Smith, R.; Kopelman, R. Annu. Rev. Anal. Chem. 2009, 2, 57. (10) Andresen, T. L.; Thompson, D. H.; Kaasgaard, T. Mol. Membr. Biol. 2010, 27, 353. (11) Kaasgaard, T.; Andresen, T. L. Expert Opin. Drug Delivery 2010, 7, 225. (12) Matyjaszewski, K.; Tsarevsky, N. V. Nat. Chem. 2009, 1, 276. (13) Lei, L.; Willet, N.; Zhang, J.; Varshney, S.; Je, R. Macromolecules 2004, 37, 1089. (14) Read, E. S.; Armes, S. P. Chem. Commun. 2007, 3021. (15) O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L. Chem. Soc. Rev. 2006, 35, 1068. (16) van Nostrum, C. F. Soft Matter 2011, 7, 3246. (17) Li, Y.; Lokitz, B. S.; Armes, S. P.; Mccormick, C. L.; Hill, B.; Sheffield, S. Macromolecules 2006, 39, 2726. (18) Xu, X.; Flores, J. D.; Mccormick, C. L. Macromolecules 2011, 44, 1327. (19) Lee, H.-I.; Wu, W.; Oh, J. K.; Mueller, L.; Sherwood, G.; Peteanu, L.; Kowalewski, T.; Matyjaszewski, K. Angew. Chem., Int. Ed. 2007, 46, 2453. (20) Jiang, J.; Qi, B.; Lepage, M.; Zhao, Y. Macromolecules 2007, 40, 790. (21) Trenor, S. R.; Shultz, A. R.; Love, B. J.; Long, T. E. Chem. Rev. 2004, 104, 3059. (22) Lepage, M.; Jiang, J.; Babin, J.; Qi, B.; Tremblay, L.; Zhao, Y. Phys. Med. Biol. 2007, 52, N249. (23) Jiang, J.; Shu, Q.; Chen, X.; Yang, Y.; Yi, C.; Song, X.; Liu, X.; Chen, M. Langmuir 2010, 26, 14247. (24) Zhao, Y.; Bertrand, J.; Tong, X.; Zhao, Y. Langmuir 2009, 25, 13151. (25) Napper, D. J. Colloid Interface Sci. 1977, 58, 390. (26) Liu, S.; Weaver, J. V. M; Tang, Y.; Billingham, N. C.; Armes, S. P.; Tribe, K. Macromolecules 2002, 35, 6121. (27) Dufresne, M.-H.; Leroux, J.-C. Pharm. Res. 2004, 21, 160. (28) Obi, M.; Morino, S.; Ichimura, K. Chem. Mater. 1999, 11, 656.

0.0033 mmol) was dissolved in 20 mL of DMF under stirring. To the solution was added 2 mL of Milli-Q water over a time interval of 30 min. The stirring was continued, and an additional 40 mL of Milli-Q water was added dropwise. The cloudy micelle solution was transferred to a dialysis tubing of MWCO 12 kDa and dialyzed against Milli-Q water for 3 days. The final micelle concentration was 1.28 mg/mL. DLS showed a hydrodynamic diameter of (Dh) = 45 ± 2 nm, and zeta potential (ξ) measurements showed ξ = 24 ± 1 mV. Photochemical Cross-Linking of the Micelle Core (4). Two millliliters of PEG-b-PAEMA-b-PCMA aqueous micelle solution (0.03 mg/mL) in a quartz cuvette was irradiated from a Omnicure UV photocuring system (S 2000) with a UV intensity of 2 W/cm2 (measured by a radiophotometer) using a standard filter (320−500 nm). The sample was irradiated at different time intervals, and the decrease in absorption of the coumarin moiety with time was monitored at 320 nm. pH Nanosensor Synthesis (5). A 3.9 mL portion of PEG-bPAEMA-b-PCMA micelle solution (1.28 mg/mL) under stirring was exposed to UV radiation (2 W/cm2) from the photocuring system. Progress of the photodimerization was monitored by UV spectroscopy (at a concentration of 0.03 mg/mL). After 55% of photodimerization (Dh = 40 ± 1 nm and ξ = 22 ± 2 mV), the sample was transferred to a dialysis tube of MWCO = 12 kDa and dialyzed against a carbonate buffer of pH 9.0. To this alkaline colloidal dispersion was added activated BCECF (4 μL). BCECF (0.25 mg/mL) was activated with EDC·HCl and NHS in water. OG-ITC (1 μL) (1 mg/mL in dry DMSO) and Alexa 633 SUC (2 μL) (1 mg/mL in dry DMSO) were also added and stirred at RT for 14 h. The reaction mixture was transferred to a dialysis tube of MWCO = 12 kDa and dialyzed against a PBS buffer of pH 7.4 for 3 days and then against Milli-Q water for another 3 days. The final nanosensor concentration was 1.26 mg/mL; Dh = 41 ± 2 nm and ξ = 17 ± 2 mV. Photochemical De-Cross-Linking of the Micelle Nanosensor (6). The core cross-linked micelle nanosensor (3 mL) (0.03 mg/mL) in a quartz cuvette was exposed to UV radiation (λ < 255 nm). The cuvette was placed vertically 16 cm away from the 6 W 254 nm UVGL-58 UV lamp. The progress of de-cross-linking was monitored by UV−vis spectroscopy. The de-cross-linked micelle showed a Dh = 38 ± 3 nm and ξ = 16 ± 1 mV.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 45887762. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Kræftens Bekæmpelse and the Danish Research Council (FTP grant 274-07-0172) and the NanoMorph consortium for financial support.

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REFERENCES

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dx.doi.org/10.1021/cm302922d | Chem. Mater. 2013, 25, 1496−1501