Self-Assembled Vesicles Prepared from Amphiphilic Cyclodextrins as

Publication Date (Web): May 18, 2012 ... of well-defined aggregation with functional host sites with great potential applications in drug-carrier syst...
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Self-Assembled Vesicles Prepared from Amphiphilic Cyclodextrins as Drug Carriers Tao Sun,† Qie Guo,§ Cai Zhang,§ Jingcheng Hao,† Pengyao Xing,† Jie Su,† Shangyang Li,† Aiyou Hao,*,† and Guangcun Liu*,‡ †

School of Chemistry & Chemical Engineering and Key Laboratory of Colloid and Interface Chemistry of Ministry of Education, Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan 250100, PR China ‡ Qianfoshan Hospital Affiliated to Shandong University, Jinan 250018, PR China §

S Supporting Information *

ABSTRACT: Controlled self-assembly of amphiphilic cyclodextrin is always a challenging topic in the field of supramolecular chemistry, since it provides the spontaneous generation of well-defined aggregation with functional host sites with great potential applications in drug-carrier systems. β-Cyclodextrin modified with an anthraquinone moiety (1) was successfully synthesized. In the aqueous solution, 1 was found able to self-assemble into vesicles, which was characterized in detail by TEM, SEM, EFM, and DLS. The formation mechanism of the vesicles was suggested based on the 2D ROESY and UV−vis results, and further verified by the MD simulation. Subsequently, the stimuli response property of the vesicles, including to Cu2+ and H+, was also studied. The vesicles can efficiently load Paclitaxel inside the membrane with functional macrocyclic cavities available, which can further carry small molecules, such as ferrocene. The vesicles loading with Paclitaxel have remarkable anticancer effects. This work will provide new strategy in drug-carrier systems and tumor treatment methods.

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INTRODUCTION Cyclodextrins, a series of α-1,4-linked cyclic oligosaccharides composed of 6, 7, or 8 D -(+)-glucose repeat units (corresponding to α-, β-, and γ- cyclodextrins, respectively), are widely used as the functional macrocyclic host molecules in supramolecular chemistry for their low cost, water solubility, and biocompatible properties.1 Due to the special cyclic molecular structure, the glucose units in cyclodextrin are oriented in a way that causes the outer surface of cyclodextrin to be hydrophilic while its inner cavity hydrophobic. As potential candidates for drug delivery in vivo, chemically modified cyclodextrins can form inclusion complexes with specific drugs to alter their physical, chemical, and biological properties.2 Our group recently reported water-soluble 6-oligo (lactic acid) cyclomaltoheptaose (6-OLA-β-cyclodextrin) as kinetically controlled release system for amoxicillin.3 However, these traditional drug delivery and release systems that mainly employed the β-cyclodextrin derivatives were rarely concerned with the condition of their self-assembled nanostructures in solutions. In fact, the microstructure types and sizes would greatly affect the drug loading efficiency and the drug-release process.4 For example, it has been well-reported that micelles with a size smaller than 200 nm may be preferentially delivered into tumors due to their enhanced permeability and retention (EPR) effect.5,6 © 2012 American Chemical Society

As one important self-assembled nanostructure, vesicles, enclosing a volume with membranes consisting of a bilayer or multilayer of specific molecules, have attracted increasing attention for the hope of promising applications in drug and gene delivery,7 nanoreactors,8 and artificial cell membranes.9 Vesicles based on cyclodextrin would combine the properties of bilayer structures and macrocyclic host molecules, especially if the cyclodextrin cavities can function as independent host sites for molecule recognition when they are confined to vesicles.10 Herein, a modified β-cyclodextrin containing an anthraquinone moiety, mono[6-deoxy-N-ethylamino-(N′-1-anthraquinone)]-β-cyclodextrin (1), as a potential building block for nanostructures, was synthesized (Scheme 1). From TEM (transmission electron microscopy), SEM (scanning electron microscopy), and DLS (dynamic light scattering) observations, 1 was able to form vesicles in aqueous medium. The mechanism of vesicle formation was suggested based on the 2D NMR ROESY, UV−vis, and MD simulation. Furthermore, the vesicles were responsive to external stimuli, including H+ and Cu2+. The vesicles were found able to carry Paclitaxel (PTX) via the hydrophobic center of the membrane. With the macrocyclic cavities confined in the bilayer available, the Received: April 13, 2012 Revised: May 18, 2012 Published: May 18, 2012 8625

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Scheme 1

mixing time of 0.200 s, a relaxation delay time of 1.000 s, and an acquisition time of 0.228 s were used. All pulse sequences were set according to the company standards. Briefly, the solutions containing different IR spectra were obtained on an Avatar 370 FT-IR Spectrometer. Elemental analysis was carried out on a Vario EL III elemental analyzer from Elementar Company, standard deviation: absolute deviation ≤0.1%, decomposition temperature 1000 °C, 2.5 L He (99.995%), and 40 mL O2 (99.995%) gas consuming per 10 min. Mass spectroscopy was run on a 6510 Q-TOF MS instrument from Agilent Technologies Company. Temperature for ESI is 350 °C, drying gas is 10 L/min, nebuilizer is 40 psig, and Dual ESI voltage is 4000 V. The muffle furnace is a SX-4−10 model (800 °C, 220 V, 4 kW) from TAISITE instrument Co. Ltd., Tianjin, China. All samples for TEM were prepared by the phosphotungstic acid staining technique. The JEM-100CX electron microscope was employed. SEM images were obtained with a Hitachi S-4800 scanning electron microscope by coating the vesicular solution to the base plate and then dried and sputter-coated with gold. DLS measurements were carried out with a Wyatt QELS Technology DAWN HELEOS instrument poised at constant room temperature (25 °C) by using a 12-angle replaced detector in a scintillation vial and a 50 mW solid-state laser (λ = 658.0 nm). All solutions for DLS were filtered through a 0.45 μm filter before detection. UV−vis spectra were recorded at room temperature with a TU-1800pc UV−vis spectrophotometer. Epifluorescence microscopy (EFM) imaging was performed with an Olympus IX81 fluorescence microscope (Tokyo, Japan) equipped with highnumerical-aperture 60× (1.45 NA) oil-immersion objective lens, a mercury lamp source, a mirror unit consisting of a 330 to 385 nm excitation filter (BP330−385), a 455 nm dichromatic mirror (DM 455), emission filter (IF510−550), and a 16 bit thermoelectrically cooled EMCCD (Cascade 512B, Tucson, AZ, USA). Imaging acquisition and data analysis were performed using MetaMorph software (Universal Imaging, Downingtown, PA, USA). The experiments of XRD were performed on a German Bruker/D8 ADVANCE diffractometer with Cu Ka radiation (λ = 0.15406 nm, 40 kV, 40 mA). The surface tension measurements were carried out on a Processor Tensiometer K100 (Krüss) using the Wilhelmy plate method at a temperature of 25.0 ± 0.1 °C. Before each measurement, the plate was cleaned with deionized water and flamed, while the surface tension of deionized water (the resistivity is 18.25 MΩ·cm) was measured to calibrate the tensiometer and check the cleanliness of the sample pool. The sonication was performed with a KQ 318 ultrasonic cleaner (40

vesicles can further carry other molecules, such as ferrocene. The mechanism of the dual-loading process was also studied. Furthermore, it was found that the vesicles loading with PTX have remarkable anticancer effects compared with natural PTX dissolved in DMSO. We regard the vesicle system with independent host sites on the bilayer as promising materials to meet the requirements for functional, regular, well-defined, and stimuli-responsive nanoarchitectures, which could be used in the fields of molecular devices, smart materials, and biomaterials. Especially, our research may pave the way for delivery vehicles with dualloading and releasing ability.

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EXPERIMENTAL DETAILS

Materials. 1-Nitroanthraquinone was purchased from Shandong Aokete Chemical Reagent Co. Ltd., China. β-Cyclodextrin was purchased from Binzhou Zhiyuan Biotechnology Co. Ltd., China, recrystallized twice from distilled water and dried under vacuum for 12 h. PTX was purchased from Beijing Noezer Pharmaceutical Co. Ltd., China. N,N-Dimethylformamide (DMF) was first dried over MgSO4 for one day and then distilled under vacuum. Other reagents were all commercially available from Country Medicine Reagent Co. Ltd., Shanghai, China. All other organic reagents were of analytical purity and used as received without further purification. Thin layer chromatography (TLC) analysis was performed on glass plates precoated with silica gel F254 obtained from Qingdao Haiyang Chem., China. The developer was a mixture of isopropanol, water, and aqueous ammonia (30%) (5:2:1, by volume). The silica gel for the purification, 2CX II gross porosity model with a 200−300 mesh number, is from Qingdao Haiyang Chem., China. Human hepatocellular carcinoma cell lines HepG2 and BEL-7402 were obtained from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, P.R. China), They were both maintained in RPMI-1640 medium (Gibco BRL, Gaithersburg, MD), containing 10% fetal bovine serum (FBS) and antibiotics (100 mg/mL penicillin and 100 mg/mL streptomycin) with 5% CO2 at 37 °C. Analytical Measurements and Methods. 1H NMR and 13C NMR spectra were carried out on an API Bruker Avance 400 M NMR at room temperature with D2O as the solution and the solvent peak as the reference. 2D 1H−1H ROESY experiments were recorded using an INOVA-600 (600 MHz) spectrometer at ambient temperature. A 8626

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Annexin V/PI Assay. Alternatively, we further verified apoptosis by using annexin V/PI double staining of unfixed cells, distinguishing between early apoptotic cells and late apoptotic/necrotic cells. Both floated and attached cells were suspended in 300 μL binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA). Annexin VFITC (5 μL) and PI (5 μL) were then added into each sample. After 15 min incubation in the dark, the flow cytometric analysis was carried out.

kHz, 200 W), Kunshan ultrasonic apparatus Co. Ltd., China. The ChemBio3D software is version 11.0, Ultra level by CambridgeSoft. Fluorescence microscope were performed with an Olympus IX71 fluorescence microscope (Tokyo, Japan) equipped with highnumerical-aperture 100× (1.40 NA) oil-immersion objective lens, a mercury lamp source, ultraviolet light emission, and fluorescence detection mode. Synthesis and Characterization of 1. Mono[6-(2-aminoethylamino)-6-deoxy]-β-cyclodextrin (2) was prepared according to the literature.11,12 To a solution of dry DMF (10 mL) containing 2.35 g (2 mmol) of mono [6-(2-aminoethylamino)-6-deoxy]-β-cyclodextrin, 0.61 g (2.4 mmol) 1-nitroanthraquinone was added. The reaction mixture was heated to 110 °C and stirred for 5 h and monitored by TLC, then cooled and poured into 25 mL acetone. The mixture was filtered and the cake was washed with acetone for three times. The crude product was further purified by silica gel column chromatography with an mixed eluent of isopropanol, water, and 30% aqueous ammonia (10:2:1, by volume) to give the product 1 (489 mg), a yield of 18%. 1, red powder, Rf = 0.6 (with an mixed developer of isopropanol, water, and 30% aqueous ammonia (5:2:1, by volume)), 1 H NMR (300 MHz, D2O, 300 K, δ ppm): 8.201−8.073 (m, 2H, H6′H-7′), 8.000−7.946 (m, 2H, H-5′H-8′), 7.760 (t, 1H, H-3′), 7.253− 7.223 (m, 2H, H-2′H-4′), 4.967−4.924 (m, 7H, H-1), 3.935−3.479 (m, 43H, H-2 to H-6), 1.074−1.054 (d, 4H, (CH2)2). FT-IR (KBr plate, υ cm−1): 3408.56 (vs, br, υOH), 2930.18 (s, υCH2), 1675.32(m, δNH), 1056.14 (s, δCH). ESI-MS calcd. for C58H82N2O36H+ m/z 1383.47, found m/z 1383.26. Anal. Calcd. for C58H82N2O36·C3H8O (isopropyl alcohol): C, 50.76; H, 6.28; N, 1.94. Found: C, 50.79; H, 5.98; N, 1.87. Preparation of the Vesicles. Molar quantified mother solution of 1, 1 × 10−3 mol/L, was prepared by adding 0.138 g of 1 into 100 mL triple-distilled water at 300 K. All sample solutions for the vesicle investigation were freshly prepared by diluting the stock solution and then sonicating for 20 min at 300 K before detection. The effect of an external stimulus was investigated by adding external substances (acetic acid and Cu2+) to the sample solutions. Atomic-Based MD Simulation. In order to construct the intersimulation model, a bilayer containing nine pairs of 1 units was placed in the middle of a tetragonal box of 48 × 48 × 60 Å3, surrounded by a mixture consisting of H2O molecules (Figure 5). The α, β, and γ angles are 90°, 60°, and 90°, respectively. The system yields 10−4 molar solutions according to the experiment data. Application of periodic boundary conditions with such a unit cell produces a vesicle bilayer perpendicular to the z-axis. The system was first minimized (10 000 steps of steepest descent minimization) to avoid bad contacts. Then, the MD simulation was carried out in the NVT ensemble (constant particle number, volume, and temperature) at a temperature of 298 K with a time step of 1 fs. The nonbonded interactions were cut off at 12 Å, and long-range electrostatic interactions were accounted using Ewald method. The COMPASS force field was employed in the simulation. The molecular modeling software Materials Studio 4.3 from Accelrys, Inc., was used in this study to perform the calculations. Loading of PTX to the Vesicles. PTX (0.0025 mmol) was dissolved into 0.5 mL methanol and slowly added dropwise into the prepared vesicular solution (25 mL, 10−4 mol/L) under vigorous stirring, and then the mixture was sonicated for 20 min at 300 K. The resulting turbid liquid was carefully filtered to remove the solid PTX to obtain the vesicular solution loaded with PTX. The NMR samples were prepared in the same way (with D2O as the solvent). The solid samples of 1/PTX inclusion were obtained by evaporating the new vesicular solution carefully under reduced pressure. Assay for Cell Viability. To evaluate cytotoxicity targeting tumor cells mediated by PTX, the HCC cell lines mentioned above were trypsinized, and 4 × 103 cells were plated to each well of the 96-well plate. They were treated with increased concentrations of PTX or PTX-coated vesicles 1/PTX (from 12.5 to 100 nM) for 12, 24, and 48 h, respectively. MTT assay was employed to determine the cell viability via measuring the activity of enzymes that reduce MTT to formazan dyes which have an absorbance at OD450.

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RESULTS AND DISCUSSION Synthesis and Characterization. Mono [6-(2-aminoethylamino)-6-deoxy]-β-cyclodextrin (2) was prepared according to the literature,11,12 while 1 was prepared via the direct reaction of 2 and 1-nitroanthraquinone in DMF (Scheme 1) without any catalyst. In order to rule out the possibility of the competitive host− guest inclusion phenomenon, which is very common in the preparation of macrocyclic compound derivates,13 the comparison of FTIR, X-ray diffraction (XRD), TLC, and MS among 1, 2, 3 (the mixture of 2 and 1-aminoanthraquinone), and 1aminoanthraquinone was carried out, in which typical distinguishing patterns were found (see Supporting Information). Favorable solubility in water is the basis of the application in biological systems. The water solubility of 1 was measured to be as much as 17 mg/mL (25 °C). It should be emphasized that 1 has similar water solubility to that of β-cyclodextrin. As this molecule possesses well-identified hydrophobic and hydrophilic parts, the behavior of this cyclodextrin−anthraquinone coupling molecule was also investigated by surface tension measurements. The surface tension value (60.67 mN·m−1, 10−4 mol/L, 25 °C) is lower than that of pure water (72 mN·m−1, 25 °C), which shows that 1, as a conjugate of a hydrophilic βcyclodextrin moiety with a hydrophobic anthraquinone moiety, is surface active. Morphologies and Sizes of the Vesicles. Negatively stained TEM (Figure 1a), which is a reliable and versatile method for studying microaggregates, was employed to

Figure 1. (a) TEM images of the micromorphology of the samples of 1 in water (scale bars = 200 nm, 10−4 mol/L) with phosphotungstic acid as the negative staining agent. (b) Images of particles by EFM of 1 sample in aqueous solution (10−4 mol/L) at room temperature excited at 430 nm with mercury lamp (scale bar = 10 μm); SEM images of the micromorphology of the samples of 1 in water (10−4 mol/L) at room temperature; (c) scale bar = 2 μm; (d) scale bar = 300 nm. 8627

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that the thickness of the vesicular outer layer is about 10 nm considering the sputter-coated gold (15 nm minus 2 × 2 nm). Interestingly, the vesicles deformed greatly when the electronscanning time lasted for a relatively long time during the SEM observation process. We speculated that the vesicle deformation was induced by the sliding of the π−π stackers, which are easily affected by the electron beams.15 It is notable that, in both TEM and SEM observations, some cross-linked vesicles were found. Microspheres with strong fluorescence were observed by EFM. Several drops of a solution of 1 on a glass slide were observed under the fluorescence microscope, and microspheres with strong fluorescence were clearly observed. The fluorescence should be attributed to the anthraquinone moiety. Apparently, the spherical structures were dispersed homogeneously in the solution. Meanwhile, it is easy to understand that the bilayer could not be clearly distinguished owing to the limited amplification of EFM (60×). We are also excited to find no obvious fluorescence quenching, which is common when fluorophores are confined in the aggregates.16−18 We infer that, in the aggregates, the intramolecular rotation is restricted, and a nonradiative transfer of energy can be avoided to some extent.19−21 Also, the free motion of the molecules with fluoresent chromophores is limited in the aggregates. The collisions with each other, which may quench the fluorecence, are restricted.22−24 It is also interesting that the spheres were found to undergo a kind of Brownian movement. Since then, TEM, cryo-TEM, FF-TEM, and SEM are widely used to character static vesicles or other nanoparticles. Few reports are concerned about the moving trajectory of these particles in solution.25 The study on the Brownian movement of nanoparticles will be of great help in the analysis of the accurate location of vesicles, in knowing exactly how, when, and where the vesicles are moving, in studying the dynamics of vesicles, and in tracking one special or chosen vesicle. The sizes and size distribution of these vesicles were further confirmed by DLS (Figure 2). The DLS data gave an average hydrodynamic radius (Rh) of 135 nm in the aqueous solution, meaning a diameter of about 270 nm. It is reasonable that the diameter measured by TEM (200 nm) is smaller than that

investigate the nanostructures formed by 1 in the aqueous solution (10−4 mol/L). Figure 1a shows the spherical structures of the microaggregates of about 200 nm in diameter. The particles show a strong contrast between the center and the periphery, which is a characteristic typical of vesicular structures.14 The core−shell morphologies by TEM indicate that these vesicular spheres with hollow cores collapsed during the drying procedure of the TEM sample preparation.15 In order to further confirm the vesicular structures, SEM was also applied to study the samples. The technology of sputter-coating with gold to the microaggregate surfaces can be applied to prepare organic samples for SEM observation. The sample for Figure 2c,d was obtained after directly sputter-coating the 1

Figure 2. DLS of the vesicles assembled from 1 in aqueous solution (1.0 × 10−4 mol/L) at room temperature.

TEM sample with gold. The central and edge portions of these particles showed a clear color contrast (Figure 1c,d), indicating a concave central area in these aggregates. The diameters of the vesicular spheres were in good agreement with those observed by TEM. The detailed SEM observations (Figure 1d) indicate

Figure 3. Relationship between the concentration of 1 in aqueous solution and the UV−vis absorbance: (a) the UV−vis absorbance of 1 with different concentrations; (b) the linear relationships at the peak of wavelength 312 nm; (c) the linear relationships at the peak of wavelength 502 nm. 8628

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obtained from DLS, because TEM and DLS were applied to measure solid and swollen vesicles, respectively.26 It should be further pointed out that the colloidal stability of the vesicles is excellent, for no morphology and size changes were observed for two months after the vesicle solution was prepared. Possible Formation Mechanism of the Vesicles. Cyclodextrin derivates are easy to form the intermolecular inclusion one by one (head to tail) in the aqueous solution, especially at a high concentration.27−31 In this condition, the hydrophobic tail is included into another cyclodextrin cavity. As a result, the natural properties of the cyclodextrin amphiphile were greatly affected and vesicles will not be able to form. Since it was reported that anthraquinone can form inclusion complex with β-cyclodextrin,32−36 the possible intermolecule inclusion model cannot be ignored. A series of UV−vis spectra of 1 in aqueous solution with increasing concentration (from 10−6 to 10−3 mol/L) was undertaken (Figure 3a). The peak values of 502 and 312 nm wavelength were picked to check the linearity of UV absorbance and the concentrations. Apparently, two different linear relationships were observed when 312 nm peak values were picked and treated: from 10−6 to 10−4 mol/L, slope = 4146, adj. R2 = 0.996; from 2 × 10−4 to 10−3 mol/L, slope = 2681, adj. R2 = 0.993 (Figure 3b). The similar result was obtained when 502 nm peak values were picked and treated (Figure 3c, from 10−6 to 10−4 mol/L, slope = 3226, adj. R2 = 0.998; from 2 × 10−4 to 10−3 mol/L, slope = 2187, adj. R2 = 0.991). We deduced that there was a sudden change of aggregating models between 10−4 and 2 × 10−4 mol/L, which lead to the different linear relationships of UV absorbance and the concentrations.37 Considering that in the ROSEY results (the concentration of 1 is 10−4 mol/L in D2O, see Supporting Information), no correlations between anthraquinone and cyclodextrin moiety were found, we can speculate that 1 molecules tend to exist in a π−π stacking state when concentration is 10−4 mol/L. Anthraquinone derivates are well-known components of dyestuffs because of their delocalized conjugated π system. Anthraquinone compounds can easily form the self-assembled aggregation by π−π stacking, which is similar to other reported π systems with solubilizing substituents.38,39 The UV−vis spectra of 1 in different solutions further demonstrate the π−π stacking phenomenon. In aqueous solution, 1 has an obviously weak absorbance compared with in other organic solvents (Figure 3). The enhancement of the absorbance in organic solvents may be due to the good solubility of the chromophore.40,41 On the other hand, the aggregates may enhance the scattering of light, resulting in a decrease of the absorbance in aqueous solution.42,43 Considering that the anthraquinone moieties are as the chromophores, we deduce that the anthraquinone moieties in aqueous solution tend to exist in a π−π stacking status. On the basis of the UV−vis and 2D NMR ROESY results, we can indicate that anthraquinone moiety of 1 would aggregate via π−π stacking behavior in aqueous solution with a 10−4 mol/ L concentration. The organized π−π stackers can be regarded as “flagpoles” fastened with the cyclodextrin moieties as the “waving flags”. While being ultrasonicated and well-dispersed in the aqueous solution, 1 could self-assemble into bilayer and further into regular vesicles. This result was further confirmed by the MD simulation. A ChemBio3D minimization44 gives a simple estimate of about 2 nm for the size of 1 molecule. The thickness of the vesicles with the value of 10 nm observed by

Figure 4. UV−vis spectra of 1 (1.0 × 10−4 mol/L) in different solvents at 25 °C, the corresponding solvents were as the base lines respectively.

SEM is approximately 2- or 3-fold the length of the bilayer (approximately 4 nm), suggesting that the objects observed are lamellar vesicles consisting of several bilayers. Simulation of the Vesicle Model. In order to indicate the possibility of the mechanism model above, a molecular dynamic simulation was performed. Atomic-based MD simulations can play a powerful role in indicating the properties at a microscopic level and are considered as complements to experiments. In this paper, a classical molecular dynamics simulation of the vesicular structure was performed to investigate the possibility of the mechanism model mentioned above. Since it could be very time-consuming to obtain the spontaneous bilayer structure from a random initial configuration, we started the simulations from the preassembled state, as has been done in most other simulation systems.45,46 As shown in Figure 5, after a relatively long time to achieve the

Figure 5. Results of the molecule dynamic simulation of 1 in the aqueous solution (10−4 mol/L).

balance the bilayer is stable. As in the space-extension principle, the part of the bilayer shown in Figure 5 can be generalized to the whole vesicle. The obtained results are similar to the suggested mechanism shown in Scheme 2, which further verifies our presumption. It should be noted that obvious π−π 8629

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stacking phenomenon was found from the result as shown in the green cycle in Figure 5. Scheme 2. Illustration of the Formation Mechanism of the Vesicles

Properties of the Vesicles. Response to External Stimuli. The stimuli-responsive property of materials has attracted much attention both in theory and in application. The research on the stimuli-responsive materials may greatly extend their ability to mimic some biological process and the application of smart molecular devices.47 TEM tests were done after equimolar CuCl2 or acetic acid was added into the vesicular solution, and we learned that the vesicles would disappear upon the addition of equimolar CuCl2 or acetic acid. It is known that Cu2+ easily coordinates with N atoms.48 The changes of the UV−vis spectra of 1 in the presence of Cu 2+ with different concentrations indicate that 1 can complex with this ion (Figure 6a). The vesicles are also responsive to H+. The UV spectrum, which is similar to the results of the samples upon the addition of CuCl2, also have obvious variation upon the addition of different amount of acetic acid, perhaps because of the change from a secondary amine on the aromatic ring to a quaternary ammonium species. Both Cu2+ and H+ could impede the formation of selfassembled aggregates, which may be due to the increase of the electrostatic repulsive force between the “building blocks” (Scheme 3). The results described here may provide new opportunities in mimicking some biological process. Since we know that the cancer cells can cause an acidic environment for their excessive proliferation,49,50 H+-responsive vesicles have potential applications in building new pH-triggered anticancer drug-release systems and pH-sensitive materials. Loading of PTX. In order to address this application issue, PTX was employed to investigate the drug-loading property of the vesicles. PTX (Scheme 4), isolated from the bark of Taxus brevifolia, is a well-known anticancer agent, which has provided effective treatment for a wide range of carcinomas by interfering with microtubule breakdown during cell division.51 However, the strong hydrophobicity of PTX has drastically limited its use in natural form. To date, several attempts have been made to carry PTX.52,53 However, complicated preparation procedures are still significant concerns for these sophisticated formulations. Therefore, an easy, effective, and body-friendly system is highly desirable on the way to translating insoluble drugs from the bench to the clinic. The quantitative PTX in ethanol solution (10−2 mol/L) was added to the vesicular solution by droplet under stirring. As expected, after long-term equilibrium, vesicles are still stable

Figure 6. (a) UV spectra of 1 (green line), the mixture of 1 (10−4 mol/L), and CuCl2 with increasing concentrations (10−4, 2 × 10−4, 5 × 10−4, 10 × 10−4, 20 × 10−4 mol/L; the CuCl2 solution with corresponding concentration was baseline), T = 300 K; (b) UV spectra of 1 (red line), the mixture of 1 (10−4 mol/L), and acetic acid with increasing concentrations (10−4, 2 × 10−4, 5 × 10−4, 10 × 10−4, 20 × 10−4 mol/L; the acetic acid solution with corresponding concentration was baseline), T = 300 K.

Scheme 3. Illustration of the Disappearance of the Vesicles upon Addition of Cu2+ Ions and Acetic Acid

based on the TEM, SEM, and DLS observation. From the TEM and SEM observation, the vesicles loaded with PTX (named 1/ PTX) are slightly different from the native vesicles. 1/PTX 8630

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Scheme 4. Structure of PTX and Illustration of the Mechanism of the Vesicles Loaded with PTX

Figure 8. DLS result of the new vesicles treated by PTX in aqueous solution at T = 300 K.

vesicles are a little larger, and the edges are not so regular. The particle size distribution obtained by TEM ranged from 100 nm to 300 nm, which is more asymmetric than the formal one. DLS experiment further confirmed the size distribution. The DLS data showed an average hydrodynamic radius (Rh) of 171 nm in the aqueous solution, which means a diameter of about 342 nm (the formal average vesicular diameter is 270 nm by DLS). It is understandable that, after incorporating with PTX, the hydrophilicity of the amphiphile might decrease to result in a loose structure of 1/PTX vesicle. The obtained 1/PTX vesicle can be sustainable for at least one month at 20 °C. It is interesting because it means that loading of the drug does not greatly affect the formation of the spheres. This would agree with a mechanism in which anthraquinone is not included, either intra- or intermolecularly to produce the spheres, since PTX inclusion would have competed and hampered sphere formation.

solubilization. Furthermore, the FT-IR and XRD results also confirmed the complex of 1 and PTX (see Supporting Information). At first, we guessed that PTX might be included by the cyclodextrin cavity confined in the membrane of vesicles; however, the 2D NMR ROSEY results showed that there were only slight correlations between PTX and H-4/H-2 of cyclodextrin moiety (Figure 9). It is known that H-4/H-2 are

Figure 9. Selected region of the 2D NMR ROESY (600 MHz) spectrum of 1 (1.0 × 10−4 mol/L) treated by PTX in D2O at T = 300 K.

Figure 7. (a) TEM images of the micromorphology of the samples of the PTX-treated vesicular solution (scale bar = 200 nm) with phosphotungstic acid as the negative staining agent; (b) SEM images of the micromorphology of the samples of the PTX-treated vesicular solution at room temperature (scale bar = 5 μm).

outer cavity protons, which probably shows that PTX was not located in the cyclodextrin cavity but loaded inside the hydrophobic-rich region of the vesicles containing the anthraquinones and that the remaining aromatics are located in between the cyclodextrins. In order to further investigate the assuming PTX position, the comparison of the UV−vis spectra of 1 and 1/PTX with a 10−4 concentration in aqueous solution was undertaken (Figure 10), from which we can see obvious differences between the spectra of 1 and 1/PTX. The UV−vis spectra of PTX in ethanol/H2O (1:1, 10−4 mol/L) solution was also undertaken, and there is no UV absorbance among the wavelengths from 350 to 700 nm. Obviously, the anthraquinone moiety linked to cyclodextrin is the only chromophore in both cases. The

Possible Mechanism of the Load of PTX. 1H NMR is one of the most powerful and direct tools for analyzing supramolecular assemblies in solution. Owing to its fairly poor water solubility, PTX is nearly transparent to 1H NMR under most conditions when D2O is used as solvent.37 In our experiment, new peaks, different from the formal ones of anthraquinone, were found to appear, which are in accordance with the reported PTX NMR peaks in D2O.38 Therefore, assessment of the PTX complexes by 1H NMR demonstrates the presence of the structural protons of the PTX molecule consistent with significant 8631

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important in the secondary molecular loading and theory research. Ferrocene (FC) was chosen as the testing species, for its biological activity to be applied to pharmaceutics55−57 and properties to be included by cyclodextrins.58,59 Excitingly, 1/ PTX vesicles treated by ferrocene (Figure 9, named 1/PTX/ FC) still exist based on the TEM and SEM results. The 1/ PTX/FC vesicles have slightly smaller diameters than the 1/ PTX vesicles.

Figure 10. UV−vis absorbance of 1 and 1/PTX (red line: 1; black line: 1/PTX) T = 300 K in aqueous solution.

Figure 11. TEM (a) and SEM (b) characterizations for the 1/PTX/ FC vesicles, T = 300 K.

stronger intensity may be attributed to the insertion of PTX into the π−π stackers, which is similar to the UV−vis results of 1 in different solvents. Considering that π−π stacking is relatively weak interaction (0−50 kJ/mol), we deduce that the π−π stackers were wrecked to an extent due to the insertion of PTX, which may be because the hydrophobic interaction was stronger than the π−π interaction here. The more independent anthraquinone moieties will generate stronger UV absorbance. This also can explain why we obtain a more loose vesicular structure when loaded with PTX. Meanwhile, the comparison of 1H NMR of 1 and 1/PTX (D2O, 10−4 mol/L) was undertaken: Δδ of the cyclodextrin part: H-1, 0.001; H-2, 0.001; H-3, 0.001; H-4, 0.009; H-5, −0.002; H-6, 0.003 (Table 1). There are no obvious shifts

2D NMR ROSEY of 1/PTX/FC was also undertaken to study the mechanism of secondary loading. Clear correlations between the FC protons and H-3 to H-6 belonging to cyclodextrin moiety were observed, which suggests that FC molecules enter the cyclodextrin cavities. From the 1H NMR, we can clearly find the single peak of ferrocene at δ = 4.24 ppm. It is known that ferrocene is not soluble in water, and it is not possible to observe the ferrocene peak with D2O as the solution employing NMR without any solubilizers. We can deduce that it is the existence of 1 that resulted the solubilization of ferrocene. The comparison of 1H NMR among 1, 1/FC, 1/PTX, and 1/PTX/FC Δδ of the cyclodextrin part was undertaken to further justify the secondary loading mechanism. The comparison between 1 and 1/FC yields Δδ: H-1, 0.014; H-2, 0.034; H-3, 0.055; H-4, 0.031; H-5, 0.018; H-6, −0.007. There is an obvious chemical shift of H-3, which strongly suggested that ferrocene entered the cyclodextrin cavity of 1, possibly from the wide rim.60 The comparison between 1/PTX and 1/ PTX/FC yields Δδ: H-1, 0.012; H-2, 0.034; H-3, 0.053; H-4, 0.038; H-5, 0.018; H-6, −0.011. We obtained a similar result for the comparison between 1 and 1/FC, which further suggests that there is no influence on the inclusion of 1 and ferrocene with the existence of PTX. In other words, PTX and ferrocene complex with 1 in different positions. The comparison between 1/FC and 1/PTX/FC yeilds Δδ: H-1, −0.001; H-2, −0.001; H3, −0.002; H-4, −0.002; H-5, −0.002; H-6, −0.001; There are almost no chemical shifts, which means that, when the cyclodextrin cavity included with ferrocene, the addition of PTX will not influence the inclusion complex. Apparently, the inclusion constant of 1/FC is much larger than that of 1/PTX; therefore, PTX cannot replace the included ferrocene in the cyclodextrin cavity and tends to insert in the anthraquinone moieties. Furthermore, after comparing the area integration of H-1 (single peak, easy to integrate) and ferrocene, we obtain the complex stoichiometry Host/Guest = 2:1 (the 1H NMR spectra, the integration and the calculation steps can be seen in the Supporting Information). Application of the Vesicle System in Cell Staining and Anticancer Effects. To figure out whether 1/PTX can be

Table 1. 1H NMR Comparison entrya

1

1/PTX

1/FC

1/PTX/FC

H-1 H-2 H-3 H-4 H-5 H-6

5.094 3.662 3.942 3.605 3.891 3.882

5.095 3.661 3.941 3.596 3.889 3.885

5.108 3.696 3.997 3.636 3.909 3.875

5.107 3.695 3.995 3.634 3.907 3.874

a

All the samples were measured in a solution of D2O, T = 300 K. The peak from DHO (4.790 ppm) are indicated as the interior label.

except for the H-4. While H-4 is outside the cavity, we also judge that the aromatic parts of PTX are located in between the cyclodextrins but not inside the cavities. This is also in agreement with the result of 2D NMR ROESY results. According to the data, we could judge that 1 and PTX combined together to form the supramolecular ″exclusion complex″ (Scheme 4). In addition, 1H NMR spectra provides not only direct evidence for the formation of 1/PTX inclusion complexes, but also information about their stoichiometry. The integration of the areas associated with the phenyl protons of PTX and the H-1 protons of 1 suggests a 2:1 stoichiometry for 1/PTX complex, which means the corresponding loading efficiency is about 50%.54 Secondary Molecular Loading. In the exclusion complex vesicle model, the cyclodextrin cavities are still available to small and proper molecules. It is not only interesting, but also 8632

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Figure 12. Selected region of the 2D NMR ROESY (600 MHz) spectrum of 1 (1.0 × 10−4 mol/L) treated by PTX and then FC in D2O, T = 300 K.

Figure 13. Comparison of the inhibition rate of HepG2 cells induced by PTX and 1/PTX. HepG2 cells were treated with increased concentrations of 1/PTX or PTX (from 0 to 100 nM) for 12 (a), 24 (b), and 48 h (c), respectively. Cell vitality was measured via MTTAssay. (d) Inhibition rate of cell vitality (12 h) = (ODctrl − ODPTX/(1/PTX))/ODctrl × 100%.

apoptotic cells. The Annexin V−PI double positive cells which can be depicted as the death cells were also increased after PTX and 1/PTX stimulation. Moreover, we noticed that 1/PTX administration could induce more tumor cells to be apoptotic via calculating the total proportion of tumor cells located in the early or late stage of apoptosis. Therefore, we could draw a conclusion that 1/PTX was a good attempt to counteract the tumor growth. The control experiment was carried out using PTX dissolved in DMSO. Since some PTX solid appears during the procedure of 1/PTX vesicle preparation, it is obvious that the

regarded as a strategy to increase the specific antitumor efficiency of chemotherapeutic drug PTX, we confirmed the cytotoxicity of 1/PTX and PTX targeting different tumor cells in vitro. Our results from cell viability assay suggested that 1/ PTX showed more significant cytotoxicity than PTX, followed by inhibition of the proliferation in HepG2 cells (Figure 13). Alternatively, we further verified apoptosis via Annexin VFITC staining. Data shown in Figure 14 indicated that both PTX and 1/PTX could promote the apoptosis of HepG2 cells as evidenced by the increase of a proportion of Annexin VFITC-positive cells which represented the early stage of 8633

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Figure 14. 1/PTX enhances cytotoxicity to tumor cells compared with PTX in vitro. HepG2 cells were treated with increased concentrations of 1/ PTX or PTX (from 0 to 100 nM) for 12 h respectively, and then cells were stained with Annexin V/PI dye for the identification of apoptotic cells via flow cytometric analysis.

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concentration of single PTX in 1/PTX vesicular solution is less than 10−4 mol/L (about 5 × 10−5 mol/L). Even so, we obtain an exciting result that, with a smaller amount, 1/PTX still has better anticancer effects than natural PTX completely dissolved into DMSO (10−4 mol/L), which demonstrates that the vesicles with macrocyclic host sites confined in the membranes can effectively load PTX and greatly increase its anticancer effects, probably by supplying a medium size proper for the uptake of the cancer cells and the molecule-recognition ability of the unoccupied cyclodextrin cavity to the cell membrane. Meanwhile, it cannot be neglected that cyclodextrin can provide a sustained-release effect to normal drugs. In order to prove that 1/PTX can provide effective treatment for a wide range of carcinomas, the anticancer effects of 1/PTX to BEL7402 were also evaluated, and the results can be seen in the Supporting Information.

*A. Hao: Tel. +86-531-88363306; Fax +86-531-88564464; Email: [email protected]. G. Liu.: Tel. +86-531-82968900; Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the NSFC (Grant No. 20625307), National Basic Research Program of China (973 Program, 2009CB930103). We greatly thank Mr. Zhijie Yang from Université Pierre et Marie Curie: Paris 6 for editing the manuscript on English corrections and Ms. Zhaozhen Cao for the careful EFM characterization of vesicles.

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CONCLUSION In conclusion, a facile approach to design vesicles with functional macrocyclic host sites was developed: 1, as a modified cyclomaltoheptaose (β-cyclodextrin) containing an anthraquinone moiety, was synthesized via 3 steps. In aqueous solution, 1 can self-assemble into vesicles, which was characterized in detail by TEM, SEM, EFM, and DLS. The possible vesicular model with functional host sites on the bilayers was suggested based on the results of 2D NMR ROESY and UV−vis spectrum and simulated by molecular dynamic software. The vesicles are responsive to the external stimuli, such as H+ and Cu2+. PTX and ferrocene were successfully carried in order via different locations, and both loading mechanisms were suggested. The vesicles loaded with PTX have remarkable anticancer effects, even better than natural PTX. We believe this work will cast a new light on the delivery materials confined within macrocyclic host sites.

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AUTHOR INFORMATION

Corresponding Author

REFERENCES

(1) Szejtli, J. Introduction and General Overview of Cyclodextrin Chemistry. Chem. Rev. 1998, 98, 1743−1754. (2) Khan, A. R.; Forgo, P.; Stine, K. J.; D’Souza, V. T. Methods for Selective Modifications of Cyclodextrins. Chem. Rev. 1998, 98, 1977− 1996. (3) Shen, J.; Hao, A.; Du, G.; Zhang, H.; Sun, H. A Convenient Preparation of 6-Oligo (lactic acid) Cyclomaltoheptaose as Kinetically Degradable Derivative for Controlled Release of Amoxicillin. Carbohydr. Res. 2008, 343, 2517−2522. (4) Zhang, H.; Liu, Z.; Xin, F.; An, W.; Hao, A.; Li, J.; Li, Y.; Sun, L.; Sun, T.; Zhao, W.; Li, Y.; Kong, L. Successively-Responsive DrugCarrier Vesicles Assembled by ’Supramolecular Amphiphiles’. Carbohydr. Res. 2011, 346, 294−304. (5) Matsumura, Y.; Maeda, H. A New Concept for Macromolecular Therapies in Cancer Chemotherapy: Mechanisms of Tumortropic Accumulation of Proteins and the Antitumor Agents Smancs. Cancer Res. 1986, 46 (12), 6387−6392. (6) Tu, C.; Zhu, L.; Li, P.; Chen, Y.; Su, Y.; Yan, D.; Zhu, X.; Zhou, G. Supramolecular Polymeric Micelles by the Host-Guest Interaction of Star-Like Calix[4]arene and Chlorin e6 for Photodynamic Therapy. Chem. Commun. 2011, 47, 6063−6065. (7) Allen, T. M.; Cullis, P. R. Drug Delivery Systems: Entering the Mainstream. Science 2004, 303, 1818−1822. (8) Christensen, S. M.; Stamou, D. Surface-Based Lipid Vesicle Reactor Systems: Fabrication and Applications. Soft Matter 2007, 3, 828−836.

ASSOCIATED CONTENT

S Supporting Information *

Purification and characterization of 1, more NMR information, and the anticancer effect to BEL-7402 cells. This material is available free of charge via the Internet at http://pubs.acs.org. 8634

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(9) Lehn, J. M. Toward Self-Organization and Complex Matter. Science 2002, 295, 2400−2403. (10) Sun, T.; Zhang, H.; Kong, L.; Qiao, H.; Li, Y.; Xin, F.; Hao, A. Controlled Transformation from Nanorods to Vesicles Induced by Cyclomaltaoheptaoses (β-cyclodextrins). Carbohydr. Res. 2011, 285− 293. (11) Brady, B.; Lynam, N.; O’Sullivan, T.; Ahern, C.; Darcy, R. 6A-Op-Toluensulfonyl-β-Cyclodextrin. Org. Synth. 2004, 10, 686. (12) Samal, S.; Geckeler, K. E. Unexpected Solute Aggregation in Water on Dilution. Chem. Commun. 2001, 21, 2224−2225. (13) Samal, S.; Geckeler, K. E. Cyclodextrin−Fullerenes: A New Class Of Water-Soluble Fullerenes. Chem. Commun. 2000, 13, 1101− 1102. (14) Wang, L.; Liu, H.; Hao, J. Stable Porphyrin Vesicles Formed in Non-Aqueous Media and Dried to Produce Hollow Shells. Chem. Commun. 2009, 11, 1353−1355. (15) Mazzaglia, A.; Ravoo, B. J.; Darcy, R.; Gambadauro, P.; Mallamace, F. Aggregation in Water of Nonionic Amphiphilic Cyclodextrins with Short Hydrophobic Substituents. Langmuir 2002, 18, 1945−1948. (16) Jenekhe, S. A.; Osaheni, J. A. Excimers and Exciplexes of Conjugated Polymers. Science 1994, 265, 765−768. (17) Cornil, J.; dos Santos, D. A.; Crispin, X.; Silbey, R.; Bredas, J. L. Influence of Interchain Interactions on the Absorption and Luminescence of Conjugated Oligomers and Polymers: A QuantumChemical Characterization. J. Am. Chem. Soc. 1998, 120, 1289−1299. (18) Jayanty, S.; Radhakrishnan, T. P. Enhanced Fluorescence of Remote Functionalized Diaminodicyanoquinodimethanes in the Solid State and Fluorescence Switching in a Doped Polymer by Solvent Vapors. Chem.Eur. J. 2004, 10, 791−797. (19) Ikeda, M.; Takeuchi, M.; Shinkai, S. Unusual Emission Properties of A Triphenylene-Based Organogel System. Chem. Commun. 2003, 12, 1354−1355. (20) Ryu, S. Y.; Kim, S.; Seo, J.; Kim, Y. W.; Kwon, O. H.; Jang, D. J.; Park, S. Y. Strong Fluorescence Emission Induced by Supramolecular Assembly and Gelation: Luminescent Organogel from Nonemissive Oxadiazole-Based Benzene-1,3,5-Tricarboxamide Gelator. Chem. Commun. 2004, 1, 70−71. (21) An, B. K.; Kwon, S. K.; Jung, S. D.; Park, S. Y. Enhanced Emission and Its Switching in Fluorescent Organic Nanoparticles. J. Am. Chem. Soc. 2002, 124 (48), 14410−14415. (22) Yam, V. W. W.; Wong, K. M. C.; Zhu, N. Solvent-Induced Aggregation through Metal···Metal/π···π Interactions: Large Solvatochromism of Luminescent Organoplatinum(II) Terpyridyl Complexes. J. Am. Chem. Soc. 2002, 124 (23), 6506−6507. (23) Seo, S. H.; Chang, J. Y. Organogels from 1H-Imidazole Amphiphiles: Entrapment of a Hydrophilic Drug into Strands of the Self-Assembled Amphiphiles. Chem. Mater. 2005, 17, 3249−3254. (24) An, B. K.; Lee, D. S.; Lee, J. S.; Park, Y. S.; Song, H. S.; Park, S. Y. Strongly Fluorescent Organogel System Comprising Fibrillar SelfAssembly of A Trifluoromethyl-Based Cyanostilbene Derivative. J. Am. Chem. Soc. 2004, 126, 10232−10233. (25) Jesorka, A.; Markstrom, M.; Karlsson, M.; Orwar, O. Controlled Hydrogel Formation in the Internal Compartment of Giant Unilamellar Vesicles. J. Phys. Chem. B 2005, 109 (31), 14759−14763. (26) Maurice, R.; Eftink, M. L.; Bystrom, K.; Perlmutter, H. D.; Kristol, D. S. Cyclodextrin Inclusion Complexes: Studies of the Variation in the Size of Alicyclic Guests. J. Am. Chem. Soc. 1989, 111, 6765−6772. (27) Terao, J.; Ikai, K.; Kambe, N.; Seki, S.; Saeki, A.; Ohkoshi, K.; Fujihara, T.; Tsuji, Y. Synthesis of A Head-to-Tail-Type CyclodextrinBased Insulated Molecular Wire. Chem. Commun. 2011, 47, 6816− 6818. (28) Tran, D. N.; Legrand, F. X.; Menuel, S.; Bricout, H.; Tilloy, S.; Monflier, E. Cyclodextrin−Phosphane Possessing A Guest-Tunable Conformation for Aqueous Rhodium-Catalyzed Hydroformylation. Chem. Commun. 2012, 48, 753−755. (29) Terao, J. Permethylated Cyclodextrin-Based Insulated Molecular Wires. Polym. Chem. 2011, 2, 2444−2452.

(30) Zhang, Z.; Luo, Y.; Chen, J.; Dong, S.; Yu, Y.; Ma, Z.; Huang, F. Formation of Linear Supramolecular Polymers That Is Driven by CHπ Interactions in Solution and in the Solid State. Angew. Chem., Int. Ed. 2011, 50, 1397−1401. (31) Machut-Binkowski, C.; Legrand, F. X.; Azaroual, N.; Tilloy, S.; Monflier, E. New Phosphane Based on a β-Cyclodextrin, Exhibiting a Solvent-Tunable Conformation, and its Catalytic Properties. Chem. Eur. J. 2010, 16, 10195−10201. (32) Dermody, D. L.; Peez, R. F.; Bergbreiter, D. E.; Crooks, R. M. Chemically Grafted Polymeric Filters for Chemical Sensors: Hyperbranched Poly(acrylic acid) Films Incorporating β-Cyclodextrin Receptors and Amine-Functionalized Filter Layers. Langmuir 1999, 15, 885−890. (33) D’Anna, F.; Riela, S.; Gruttadauria, M.; Meo, P. L.; Noto, R. A Spectrofluorimetric Study of Binary Fluorophore−Cyclodextrin Complexes Used as Chiral Selectors. Tetrahedron 2005, 61, 4577− 4583. (34) Petrova, S. S.; Kruppa, A. I.; Leshina, T. V. Photochemical Intracomplex Reaction between β-Cyclodextrin and Anthraquinone2,6-Disulfonic acid Disodium Salt in Water Solution. Chem. Phys. Lett. 2005, 407, 260−265. (35) Dang, X. J.; Nie, M. Y.; Tong, J.; Li, H. L. Inclusion of the Parent Molecules of Some Drugs with β-Cyclodextrin Studied by Electrochemical and Spectrometric Methods. J. Electroanal. Chem. 1998, 448, 61−67. (36) Jiang, H.; Sun, H.; Zhang, S.; Hua, R.; Xu, Y.; Jin, S.; Gong, H.; Li, L. NMR Investigations of Inclusion Complexes between βCyclodextrin and Naphthalene/Anthraquinone Derivatives. J. Inclusion Phenom. Macrocyclic Chem. 2007, 58, 133−138. (37) Thomas, S.; Milanesi, L. Hydrophobically Self-Assembled Nanoparticles as Molecular Receptors in Water. J. Am. Chem. Soc. 2009, 131, 6618−6623. (38) Zang, L.; Che, Y.; Moore, J. S. One-Dimensional Self-Assembly of Planar π-Conjugated Molecules: Adaptable Building Blocks for Organic Nanodevices. Acc. Chem. Res. 2008, 41, 1596−1608. (39) Liu, Y.; Wang, K.; Guo, D.; Jiang, B. Supramolecular Assembly of Perylene Bisimide with β-Cyclodextrin Grafts as a Solid-State Fluorescence Sensor for Vapor Detection. Adv. Funct. Mater. 2009, 19, 2230−2235. (40) Würthner, F. Perylene Bisimide Dyes as Versatile Building Blocks for Functional Supramolecular Architectures. Chem. Commun. 2004, 14, 1564−1579. (41) Hulvat, J. F.; Sofos, M.; Tajima, K.; Stupp, S. I. Self-Assembly and Luminescence of Oligo(p-phenylene vinylene) Amphiphiles. J. Am. Chem. Soc. 2005, 127 (1), 366−372. (42) Messmore, B. W.; Hulvat, J. F.; Sone, E. D.; Stupp, S. I. Synthesis, Self-assembly, and Characterization of Supramolecular Polymers from Electroactive Dendron Rodcoil Molecules. J. Am. Chem. Soc. 2006, 126 (44), 14452−14458. (43) Schenning, A. P. H. J.; Jonkheijm, P.; Peeters, E.; Meijer, E. W. Hierarchical Order in Supramolecular Assemblies of HydrogenBonded Oligo(p-phenylene vinylene)s. J. Am. Chem. Soc. 2001, 123, 409−416. (44) Zhong, D. Y.; Franke, J.; Blomker, T.; Erker, G.; Chi, L. F.; Fuchs, H. Manipulating Surface Diffusion Ability of Single Molecules by Scanning Tunneling Microscopy. Nano Lett. 2009, 9, 132−136. (45) Wang, Z.; Larson, R. G. Molecular Dynamics Simulations of Threadlike Cetyltrimethylammonium Chloride Micelles: Effects of Sodium Chloride and Sodium Salicylate Salts. J. Phys. Chem. B 2009, 113, 13697−13710. (46) Yakovlev, D. S.; Boek, E. S. Molecular Dynamics Simulations of Mixed Cationic/Anionic Wormlike Micelles. Langmuir 2007, 23, 6588−6597. (47) Wang, Y.; Xu, H.; Zhang, X. Tuning the Amphiphilicity of Building Blocks: Controlled Self-Assembly and Disassembly for Functional Supramolecular Materials. Adv. Mater. 2009, 21, 2849− 2864. 8635

dx.doi.org/10.1021/la301497t | Langmuir 2012, 28, 8625−8636

Langmuir

Article

(48) Masakatsu, S.; Motomu, K. Asymmetric Synthesis of Tertiary Alcohols and Tertiary Amines via Cu-Catalyzed C-C Bond Formation to Ketones and Ketimines. Chem. Rev. 2008, 108, 2853−2873. (49) Bhujwalla, Z. M.; Mccoy, C. L.; Glickson, J. D.; Gillies, R. J.; Stubbs, M. Estimations of Intra- and Extracellular Volume and PH by 31P Magnetic Resonance Spectroscopy: Effect of Therapy on RIF-1 Tumours. J. Brit. Cancer 1998, 78, 606−611. (50) Yamagata, M.; Tannock, I. F. The Chronic Administration of Drugs that Inhibit the Regulation of Intracellular PH: in Vitro and Anti-Tumour Effects. J. Brit. Cancer 1996, 73, 1328−1334. (51) Xiao, K.; Luo, J.; Fowler, W. L.; Li, Y.; Lee, J. S.; Xing, L.; Cheng, R. H.; Wang, L.; Lam, K. S. A Novel Self-Assembling Nanoparticle for Paclitaxel Delivery in Ovarian Cancer Treatment. Biomaterials 2009, 30, 6006−6016. (52) Danhier, F.; Lecouturier, N.; Vroman, B.; Jérome, C.; Marchand-Brynaert, J.; Feron, O.; Préat, V. Paclitaxel-Loaded PEGylated PLGA-Based Nanoparticles: In Vitro and in Vivo Evaluation. J. Controlled Release 2009, 133, 11−17. (53) Yu, D. H.; Lu, Q.; Xie, J.; Fang, C.; Chen, H. Z. PeptideConjugated Biodegradable Nanoparticles as a Carrier to Target Paclitaxel to Tumor Neovasculature. Biomaterials 2010, 31, 2278− 2292. (54) Liu, Y.; Chen, G.; Chen, Y.; Cao, D.; Ge, Z.; Yuan, Y. Inclusion Complexes of Paclitaxel and Oligo(ethylenediamino) Bridged bis (βCyclodextrin)s: Solubilization and Antitumor Activity. Bioorg. Med. Chem. 2004, 12, 5767−5775. (55) Baramee, A.; Coppin, A.; Mortuaire, M.; Pelinski, L.; Tomavo, S.; Brocard, J. Synthesis and in Vitro Activities of Ferrocenic Aminohydroxynaphthoquinones against Toxoplasma Gondii and Plasmodium Falciparum. Bioorgan. Med. Chem. 2006, 14, 1294−1302. (56) Gormen, M.; Plazuk, D.; Pigeon, P.; Hillard, E. A.; Plamont, M. A.; Top, S.; Vessieres, A.; Jaouen, G. Comparative Toxicity of [3]Ferrocenophane and Ferrocene Moieties on Breast Cancer Cells. Tetrahedron Lett. 2010, 51, 118−120. (57) Neuse, E. W.; Meirin, M. G.; Blom, N. F. MetalloceneContaining Platinum Complexes as Potential Antitumor Agents. 1. Dichloro(1,6-differrocenyl-2,5-diazahexane) Platinum(II) and cisDichlorobis(1-ferrocenylethylamine) Platinum(II). Organometallics 1988, 7, 2562−2565. (58) Zhang, H.; An, W.; Liu, Z.; Hao, A.; Hao, J.; Shen, J.; Zhao, X.; Sun, H.; Sun, L. Redox-Responsive Vesicles Prepared from Supramolecular Cyclodextrin Amphiphiles. Carbohydr. Res. 2010, 345, 87− 96. (59) Zhang, H.; Shen, J.; Liu, Z.; Bai, Y.; An, W.; Hao, A. Controllable Vesicles Based on Unconventional Cyclodextrin Inclusion Complexes. Carbohydr. Res. 2009, 344, 2028−2035. (60) Smith, V. K.; Ndou, T. T.; Warner, I. M. Spectroscopic Study of the Interaction of Catechin with alpha.-, .beta.-, and gamma.Cyclodextrins. J. Phys. Chem. 1994, 98, 8627−8631.

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