pH-reversible vesicles based on the “supramolecular amphiphilies” formed by cyclodextrin and anthraquinone derivate

Colloids and Surfaces A: Physicochem. Eng. Aspects 375 (2011) 87–96

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pH-reversible vesicles based on the “supramolecular amphiphilies” formed by cyclodextrin and anthraquinone derivate Tao Sun, Yueming Li, Huacheng Zhang, Jianye Li, Feifei Xin, Li Kong, Aiyou Hao ∗ School of Chemistry and Chemical Engineering and Key Laboratory of Colloid and Interface Chemistry of Ministry of Education, Shandong University, Jinan 250100, PR China

a r t i c l e

i n f o

Article history: Received 24 July 2010 Received in revised form 21 November 2010 Accepted 27 November 2010 Available online 10 December 2010 Keywords: Vesicles pH-responsive Cyclodextrin Anthraquinone Inclusion complex Cell staining

a b s t r a c t Aggregates assembled by “supramolecular amphiphilies” are more promising in developing responsive materials. First pH-reversible vesicles based on “supramolecular amphiphilies” were prepared from the supramolecular inclusion of cyclodextrins (CDs) and anthraquinone derivate (1-((3(dimethylamino)propyl)amino)anthracene-9,10-dione, 1). 1, as the guest molecule, was synthesized by the direct reaction of 1-nitroanthraquinone with N1 ,N1 -dimethylpropane-1,3-diamine. The vesicles were characterized in detail by transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), and epi fluorescence microscope (EFM). 1 H NMR, 2D NMR ROESY, UV–vis spectrum, and FT-IR were further employed to study the formation mechanism of the vesicles. The vesicles’ responsive property, especially the pH-responsive property was tested. We also tried to use the vesicle system as a new kind of fluorescence staining material for living cells and mouse prostate carcinoma cells (RM-1) were found to be stained effectively by the vesicles. Our research may provide new references in exploiting novel intelligence materials and biomaterials. © 2010 Elsevier B.V. All rights reserved.

1. Introduction In recent five years, “supramolecular amphiphilies”, different from the formal amphiphilies in the structure (Fig. 1), has been studied and applied in building multi aggregates, including nanotubes [1–5], gels [6,7], nanoparticles [8–10], as well as vesicles [11–26]. In the “supramolecular amphiphilies”, the hydrophilic head and the hydrophobic tail are not connected by covalent bonds, but through the supramolecular interaction (generally the inclusion phenomenon). A primary criterion for the inclusion phenomenon of a guest molecule within the host’s cavity is obviously its size and then other factors such as strict fit, van der Waals’ interactions, hydrogen bond, hydrophobic interaction and so on. It can be inferred that the inclusion phenomenon plays a key role in combining the hydrophobic tail and the hydrophilic head together. Cyclodextrins, a series of ␣-1,4-linked cylic oligosaccharides composed of 6, 7, or 8 d-(+)-glucose repeat units (corresponding to ␣-, ␤-, and ␥-CDs, respectively), are usually chosen as the hydrophilic guest molecule because it is cheap, water-soluble and biocompatible [12,13].

∗ Corresponding author. Tel.: +86 531 88363306; fax: +86 531 88564464. E-mail address: [email protected] (A. Hao). 0927-7757/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2010.11.067

Vesicles, enclosing a volume with membranes consisting of a bilayer or multilayer of specific molecules, have drawn increasing attention for the hope of promising applications in drug and gene delivery [27], nanoreactors [28], and artificial cell membranes [29]. If the vesicles can be achieved by the assembling of the “supramolecular amphiphilies”, it is not only simple and effective but also more promising in developing responsive drugcarrier systems with target-release ability. It is known that the supramolecular inclusion is easily affected by some stimuli [30–32], which also means the vesicles based on the supramolecular amphiphilies are responsive and sensitive to the exoteric stimuli. Our group prepared a series of vesicles that have the redoxresponsive property based on CDs and ferrocene [11–16]. Recently, some scientists reported vesicles formed by the inclusion of CDs and azo-compound with satisfactory photo-switched properties [17–23]. In this paper, we describe in detail the preparation, characterization and application of a pH-reversible vesicular system based on the complex of 1 and ␤-CD (or (2-O-hydroxypropyl-␤-cyclodextrin, HP-␤-CD)) for the first time. The vesicles were indicated by TEM, SEM, DLS, and the mechanism was suggested based on the experimental results of UV, 1 H NMR, 2D NMR ROESY and FT-IR. The vesicles’ pH-reversible property was also studied by TEM, SEM and UV. We also managed to use the vesicles as new kind of fluorescence staining materials for living cancer cells. The study might

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Fig. 1. Comparison between general amphiphilies and supramolecular amphiphilies.

1-Nitroanthraquinone was a gift from Shandong Aokete Chemical Reagent Co. Ltd., China. ␤-CD purchased from Guangdong Yunan Chemical Reagent Co. Ltd., China was recrystallized twice from distilled water and dried in vacuum for 12 h. HP-␤-CD, with a n = 4.8 substituting degree, was purchased from Zibo Xinda Chemical Reagent Co. Ltd., China. N,N-dimethylformamide (DMF) was firstly dried over MgSO4 for one day and then was distilled in 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 ethyl acetate and petroleum ether (1:5, by volume). Mouse prostate carcinoma cells (RM-1) were cultured in DMEM medium, under 36.5 ± 0.5 ◦ C, 5% CO2 , pH = 7.0–7.4, permeate pressure = 0.26–0.32 mol/L. Cell suspensions which meet counting requirements were filled in corresponding flasks with proper culture mediums, placed in the educated box for three hours, then cultured continuity after replacing the medium. The PBS buffer solution was prepared by adding 8.5 g NaCl, 2.85 g Na2 HPO4 ·12H2 O, 0.2 g KCl and 0.27 g KH2 PO4 into 1000 mL distilled water and then sonicated for 20 min at 300 K.

were filtered through a 0.45 ␮m filter before detection. UV/visible spectra were recorded at room temperature with a TU-1800pc UV/visible spectrophotometer. Fluorescence spectrum was taken using a Hitachi F-4500 spectrophotometer (Japan) operated at 488 nm of excitation wavelength and 10 nm of slit width. QDs at a concentration of 1.0 × 10−9 M was measured both in phosphate buffered saline (PBS, pH = 7.4) solution and borate buffer solution (pH = 8.3). All measurements were performed at room temperature. Fluorescence spectra were measured using a LS-55 instrument in a 0.2 cm × 1 cm quartz cell. The excitation wavelength of the fluorescence spectra was 430 nm. EFM imaging was performed with an Olympus IX81 fluorescence microscope (Tokyo, Japan) equipped with high-numerical-aperture 60× (1.45 NA) and 100× (1.40 NA) oil-immersion objective lens, a mercury lamp source, a mirror unit consisting of a 330–385 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). Fluorescence microscope experiments were performed with an Olympus IX71 fluorescence microscope (Tokyo, Japan) equipped with high-numerical-aperture 100× (1.40 NA) oil-immersion objective lens, a mercury lamp source, ultraviolet light emission and fluorescence detection mode. The pH of the solution was measured on a PHS-3TC pH-meter, which had been calibrated at 25 ◦ C with pH standard solutions of pH 6.28 ± 0.01 and 9.18 ± 0.01. Surface tension was measured by KRUSS K-100 (Germany) surface tension meter. The sonication was performed with a KQ116 ultrasonic cleaner, Kunshan ultrasonic apparatus Co. Ltd., China.

2.2. Analytical measurements and methods

2.3. Synthesis and characterization of 1

1 H NMR and 13 C NMR spectra were carried out on an API Bruker Avance 400 M NMR at room temperature (room temperature) with CD3 COCD3 as the solution and tetramethylsilane (TMS) as the reference. The experiment of chemical shifts of H between 1/␤-CD and ␤-CD was carried out in a solution of 10−4 mol/L in D2 O. FT-IR spectra were obtained on an Avatar 370 FT-IR Spectrometer. 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 room temperature by using a 12-angle replaced detector in a scintillation vial and a 50 mW solid-state laser ( = 658.0 nm). All solutions for DLS

1, as a new chemical entity, was prepared by an improved method. N1 ,N1 -dimethylpropane-1,3-diamine directly reacted with 1-nitroanthraquinone in DMF without catalyst, which is more convenient than the previous method for the analogous compound [33]. Here, DMF was chosen as the solution for its excellent solubility. 1-Nitroanthraquinone (2.01 g, 8 mmol) was allowed to react with N1 ,N1 -dimethylpropane-1,3-diamine (16 mmol) in DMF (8 mL) at 125 ◦ C, refluxing for 10 h. The reaction was monitored by TLC. The reaction mixture was then cooled down and poured into water, filtered under reduced pressure. The crude product was washed with water, and further purified by silica gel column chromatography with a mixture eluent of ethyl acetate with petroleum ether (1:5, by volume). Data of 1: red powder; m.p. = 60.5–60.8 ◦ C; Yield, 91%; Rf = 0.55; 1 H NMR (TMS, ı

extend the application of vesicles in biosimulation, drug-delivery and smart materials. 2. Experimental 2.1. Materials

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Scheme 1.

ppm): 8.296–8.267 (q, 1H, H-6AQ), 8.204–8.174 (q, 1H, H-7AQ), 7.910–7.795 (m, 2H, H-5AQ, H-8AQ), 7.661–7.608 (q, 1H, H3AQ), 7.518–7.494 (d, 1H, H-4AQ), 7.294–7.266 (d, 1H, H-2AQ), 3.518–3.454 (m, 2H, NHCH2 CH2 CH2 N(CH3 )2 ), 2.450–2.405 (t, 2H, NHCH2 CH2 CH2 N(CH3 )2 ), 2.227 (s, 6H, NHCH2 CH2 CH2 N(CH3 )2 ), 1.943–1.852 (m, 2H, NHCH2 CH2 CH2 N(CH3 )2 ); 13 C NMR (400 MHz, CD3 COCD3 , r.t., TMS, ı ppm): 184.38(C O), 182.91(C O), 151.76, 135.32, 134.96, 134.56, 134.06, 133.08, 132.95, 126.48, 126.24, 118.09, 114.85, 112.57 (C of benzene rings), 42.56 (NHCH2 CH2 CH2 N(CH3 )2 ), 40.41 (NHCH2 CH2 CH2 N(CH3 )2 ), 29.70, 29.45 (NHCH2 CH2 CH2 N(CH3 )2 ), 28.93 (NHCH2 CH2 CH2 N(CH3 )2 ); FT-IR (KBr plate, v cm−1 ): 3263.7 (m, N–H), 1626.9 (m, ıN–H), 1660.3 (vs, br, C O). ESIMS calcd for C19 H20 N2 O2 H+ m/z 308.15, found m/z 308.55. Anal. calcd for C19 H20 N2 O2 : C, 74.00; H, 6.54; N, 9.08. Found: C, 74.11; H, 6.57; N, 9.01 (Scheme 1). 2.4. Preparation of vesicles Two equimolar solutions (2 × 10−4 mol/L), one of 1 and the other of CD, were prepared with triply distilled water. All sample solutions for the investigation were freshly prepared by diluting the stock solution. The sample solutions were mixed by the same volume of 1 and of CD solutions, and then sonicated for 20 min at 300 K before detection. The effect of an external stimulus was investigated by adding external substances (acetic acid) to the sample solutions. 2.5. Preparation of the solid inclusion complex and physical mixture of 1 and ˇ-CD ␤-CD (1 mmol) was dissolved in water of 60 ◦ C (15 mL) and a clear solution was formed. 1 (1 mmol, 0.5 mL) was added slowly and stirred for half an hour then cooled down. The aqueous solution of 1/␤-CD was distilled under reduced pressure at room temperature to remove the water and obtain the solid inclusion complex. In order to make the FT-IR comparison, a physical mixture of 1 and ␤-CD was needed. To make the physical mixture, 1 (1 mmol) and ␤CD (1 mmol) were ground with KBr separately, then well mixed and pelleted in order to avoid the inclusion of guest and host molecule during the grinding procedure [12]. 2.6. Detection of the stoichiometries of 1/ˇ-CD complex Two equimolar stock solutions (10−3 mol/L), 1 (g) and the other of CD (h), were prepared. A set of working solutions was then obtained by mixing Vg mL of the stock 1 solution with (Vt − Vh ) mL of the stock CD’s solution, where Vt is a fixed total volume and Vg is a variable value (from 0 to 10 mL, 0 ≤ Vg ≤ Vt ). 2.7. Stain of living cells In a separate keeper, 10 mL mouse prostate carcinoma cells with a density of 105 /mL were cultured in a wall-adhesive mode. 20 ␮L of vesicle solution of 1/␤-CD (10−4 mol/L) were added into the liquid medium in culture flask and well mixed. The cells were then successively cultured in the cell incubator for three hours. Basic

culture medium was then removed and PBS buffer solution was used to wash the cells (5 mL × 3). 3. Results and discussion 3.1. Morphologies and sizes of the vesicles The microscopic morphologies of the mixture of 1/␤-CD and 1/HP-␤-CD (molar ratio = 1:1) in water (10−4 mol/L) were observed by negatively stained TEM (Fig. 2a and b), which is a reliable and versatile method in studying micro-aggregates [34,35]. The spheres with core–shell structures were observed. These observations are similar to the vesicular morphologies obtained by Zhang et al. [13,23], Perro et al. [36] and Zou et al. [21]. According to the literatures of Darcy and co-workers [37], these micro-aggregates have a typical vesicular structure. The average diameters of these micro-aggregates assembled by 1/␤-CD and 1/HP-␤-CD are 350 nm and 200 nm, respectively. In order to further confirm the vesicular structures, scanning electron microscopy (SEM, Fig. 2c and d) was employed to characterize the vesicle samples. The technology of sputter-coating with gold to the micro-aggregates surface can be applied to prepare organic samples for SEM observation [34–36,21,37–40]. The differences between the two images were due to the different preparation method. Samples for Fig. 2c were prepared by transferring a drop of 1/␤-CD aqueous solution onto a base plate followed by air drying and sputter-coating with gold. The color contrast between the gray and white surfaces reveals the spherical structure of the vesicles. This method seems beneficial to preserve the morphology and the onion-type multilayer of the vesicles could not be recognized clearly for few spherical structures were damaged. Meanwhile, the sample for Fig. 2d was obtained after directly sputter-coating the 1/HP-␤-CD TEM samples with gold. As observed in Fig. 2d, clear hollow spheres were found to form. The solid vesicular structure may collapse to form holes as observed in SEM for the high energy impacts. The vesicles can be sustainable for about 2 weeks at 20 ◦ C. In addition, no vesicles were observed in individual 1, ␤-CD or HP-␤-CD. The sizes and size distribution of these vesicles were further confirmed by dynamic light scattering (DLS, Fig. 3), which gave the average hydrodynamicradius (Rh) 270.0 nm and 170.9 nm. The half of diameters measured by TEM is much smaller than hydrodynamic radius obtained from DLS. Since TEM and DLS were applied to measure solid and swollen vesicles, respectively, the diameter of the vesicles measured by TEM is smaller than twice of hydrodynamic radius obtained by DLS [14,21]. The resultant assemblies were also confirmed by epi fluorescence microscope (EFM). In Fig. 4, the particles in 1/␤-CD aqueous solution emitted fluorescence when excited at 430 nm by mercury lamp. The fluorescence should be attributed to the particles containing anthraquinone derivative. Anthraquinone (AQ) and its derivatives, widely used as dyes, can be applied as probes in biochemistry [41–43], analytical chemistry [44], and physical chemistry [45,46] attributed to their strong fluorescence. Several drips of 1/␤-CD solution on the glass slide was observed under the fluorescence microscope, and microspheres with strong fluorescence were clearly observed and found to be in a typical

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Fig. 2. TEM images of the micro-morphology of the samples of 1 and CD (molar ratio = 1:1, (a) 1:␤-CD; (b) 1:HP-␤-CD, scale bars = 200 nm) in water (10−4 mol/L) with phosphotungstic acid as the negative staining agent; SEM Images of the micro-morphology of the samples of 1 and CD (molar ratio = 1:1, (c) 1:␤-CD; (d) 1:HP-␤-CD, scale bars = 500 nm) in water (10−4 mol/L) at room temperature.

Brownian movement (Fig. 4). The study on the Brownian movement of nanoparticles may serve useful in the analysis of vesicle-gel transformation process [47]. Owing to the limited resolving ability of a light microscope (100×), the bilayers could not be recognized clearly [48–51]. However, it is clear that sphere structures formed and dispersed homogeneously in the solution. The property of fluorescence was successfully implanted into the vesicles. The molar ratio of the host and the guest molecules also plays an important role in the formation of supramolecular associates.

The concentration of the guest molecule was set as a constant 10−4 mol/L and ␤-CD was as the host molecule. When G:H was 1:0.5, both vesicles and irregular solid were found (Fig. 5a). It is understandable that 1 molecules which were not included by ␤-CD might assemble together to form the irregular solid, since 1 is not so soluble in water. When G:H was 1:1, clear vesicle phase was found. When G:H was 1:2, no obvious changes were detected from the TEM images (Fig. 5b). If we keep increasing the amount of host molecule to G:H = 1:5, no regular assembly was

Fig. 3. DLS size distributions of 1 and CDs (molar ratio = 1:1) in water (10−4 mol/L) at room temperature (a) 1:␤-CD; (b) 1:HP-␤-CD.

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Table 1 The apparent inclusion constants between 1/␤-CD and 1/HP-␤-CD at the maximum absorption. Composition

Complex stoichiometry

1/␤-CD 1/HP-␤-CD

1:1 1:1

Apparent inclusion constant (K) 419 L mol−1 527 L mol−1

Table 2 The comparison of ␤-CD

1

R 0.9763 0.9835

H NMR in the absence and the presence of 1.

.

Fig. 4. Images of particles by LCSM of 1:␤-CD sample in aqueous solution (10−4 mol/L) at room temperature excited at 430 nm with mercury lamp. Scale bar = 5 ␮m.

found, which may be due to the formation of pseudo rotaxane like assembly. 3.2. The possible formation mechanism of the vesicles The complex stoichiometry was confirmed by UV spectroscopy using Job’s plot method. The Y axis, which represents A (absorbency) and in Fig. 6a (1/␤-CD) and Fig. 6b (1/HP-␤-CD), were obtained by choosing different wavelengths (see supporting information, 266 nm and 516 nm were chosen, respectively.) to measure the stoichiometry in order to make sure the universal results. Therefore, the Y axis scale of Fig. 5a is much larger than the one of Fig. 5b, because of the different As in the wavelengths of 266 nm and 516 nm. The experimental curves (Fig. 6), describing the interactions between 1 and CDs in the aqueous solution, go through a maximum at a molar fraction of 0.5, which indicates that the complex stoichiometries of 1/␤-CD and 1/HP-␤-CD in aqueous solution are 1:1 [52,53]. The intermolecular interactions between 1 (guest) and CDs (host) in water (10−4 mol/L) can be represented as follows: G + H = G·H. The apparent inclusion constants of 1/␤-CD and 1/HP-␤-CD were determined by UV double-reciprocal method based on the Eq. (1) [11–13], as listed in Table 1. 1 1 1 = + n ˛ A ˛K[CMˇ-CD]0

(1)

ı␤-CD ımixture ıa a

H1

H3

H5

H6

H2

H4

4.958 5.352 0.394

3.844 3.911 0.067

3.763 3.878 0.115

3.727 3.823 0.096

3.520 3.595 0.075

3.457 3.518 0.061

ı = ımixture −ı␤-CD .

where A is the change of absorbance of 1 in the present of ␤-CD, ˛ is a constant, [␤-CD]0 is the initial concentration of ␤-CD is the apparent constant for the formation of the 1:n inclusion complex, n which could be calculated from a plot of 1/A versus 1/[ˇ-CD]0 . The surface tension value (60.74 mN m−1 , 10−4 mol/L, 25 ◦ C) is lower than pure water (71.19 mN m−1 , 25 ◦ C), which suggests that 1/␤-CD, as a ‘supramolecular amphiphile’, is surface active. The most direct and dependable evidence for the supramolecular inclusion of 1 and ␤-CD is from 1 H NMR, which is one of the most powerful tools for realizing supramolecular assemblies in solution [54–56]. Clear chemical shifts of ␤-CD in the absence and in the presence of 1 were observed, which will demonstrate the inclusion phenomenon (Table 2). Almost all the hydrogen resonances of ␤CD in 1/␤-CD (molar ratio = 1:1) sample showed clear upfield shifts in comparison with those individual ␤-CD. These values demonstrate that 1 is included by ␤-CD [57]. The anthraquinone moity is relatively large compared with other regular guest molecules, such as benzene, ferrocene and adamantane, thus when included into the cavity of ␤-CD, clear shifts of H-1 to H-6 were all observed. The spatial conformations of the inclusion complex can be further confirmed by 2D NMR ROESY, which has a maximal observation limit at a spatial proximity of 5 A˚ [58–61]. The selected region of the 2D NMR ROESY (600 MHz) spectrum of 1 with a concentration of 10−4 mol/L in the presence of ␤-CD in D2 O is shown in Fig. 7. The peaks at 8.01, 7.80, 7.55 and 7.23 ppm belong to H-7 , H-8 , H-3 and H-2 on the substituted anthraquinone rings, respectively. Correla-

Fig. 5. TEM images of the micro-morphology of the samples (a) 1:␤-CD, molar ratio = 1:0.5; (b) 1:␤-CD, molar ratio = 1:2, in water ([1] was set as 10−4 mol/L) with phosphotungstic acid as the negative staining agent (scale bars = 200 nm).

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Fig. 6. Job’s curve of the inclusion complex of (a) 1/␤-CD in the aqueous solution, the A in the wavelength of 266 nm was picked to obtain the Job’s curve; (b) 1/HP-␤-CD in the aqueous solution by UV, the A in the wavelength of 516 nm was picked to obtain the Job’s curve.

tions between the hydrogen of anthraquinone and cyclodextrin can be clearly observed, which demonstrates the interactions between anthraquinone moity and ␤-CD. The correlations demonstrate that the anthraquinone moiety on 1 are recognized by the ␤-CD cavity to form the inclusion complex [62–64]. The spatial conformations of the inclusion complex can be further confirmed by FT-IR spectra, which is a useful method in studying the supramolecular inclusion complex. A comparison of the FT-IR spectra of the solid inclusion of 1/␤-CD and the physical mixture of 1/␤-CD was also undertaken (see supporting information). In the physical mixture, stretching vibration of the hydroxyl group appears in the region of 3386.4 cm−1 , while in the solid inclusion complex the peak shifts to 3406.6 cm−1 , which verified the formation of hydrogen bonding between 1 and ␤-CD. The characteristic absorption band of C O in the physical mixture which appeared in the region of 1659.8 cm−1 changed much both in the peak form and intensity. The differences between the physical mixture and the inclusion complex indicate that 1 was included into the cavity of ␤-CD to form the inclusion complex [62,63].

No vesicles were detected by TEM in aqueous solutions of ␤-CD or 1. Thus, the combination of 1 and ␤-CD is crucial for the formation of vesicles. In fact, the NMR, UV and FT-IR data indicate that ␤-CD can bind 1 in a 1:1 manner, which is similar to the analogous inclusion complexes in literature [11–16]. CDs, with hydrophobic cavities, have the capability to bind substrates selectively to form inclusion complexes [30]. The compound of anthraquinone is a famous kind of dye because of its delocalized conjugated ␲ system. The research on the inclusion of anthraquinone and cyclodextrin serves useful in analytical chemistry [65], physical chemistry [65–67], photochemistry, etc. A possible inclusion of 1 into ␤-CD, with a cavity opening size of 6.5 A˚ would be an axial and partial inclusion, due to a suitable size of the guest molecules in this direction [65–67]. In this case, the unsubstituted benzene ring was inside the host’s cavity while the central ring bearing two C O groups as well as the alkyl-substituted aminobenzene ring remained outside the cavity, and participated in H-bonding with the secondary hydroxyl groups of ␤-CD. The proposed inclusion complex is shown in Scheme 2. Anthraquinone moity of 1 would enter the ␤-CD’s cavity to construct the hydrophilic “head”, and the alkyl chain outside the cavity would perform the role of hydrophobic “tail” to form a peculiar type of “amphiphilic surfactant”. While being ultrasonicated and well dispersed in the solution, the supramolecular complex could self-assemble into vesicles. 3.3. pH-reversible property of the vesicles

Fig. 7. Selected regions of 2D NMR ROESY (600 MHz) spectrum of 1/␤-CD (1.0 × 10−4 mol/L) in D2 O at ambient temperature.

The self-aggregates with responsive, especially reversible property, have great potential in building up molecular machines and novel drug-release system. Covalently attached functional groups on surfactants that are responsive to external stimuli—for example, pH [68], temperature [69], redox state [70] and even light [71]—can control the assembly and disassembly of these amphiphilic surfactants. However, since now there were still no reports on the pH-reversible vesicles based on supramolecular amphiphilies. The vesicles disappeared upon the addition of equimolar acetic acid (pH = 4.4). Solid irregular particles ranging from 10 nm to 50 nm existed in the system observed by TEM (Fig. 7a). When pH was adjusted to 7.0 by NaOH, vesicle-like particles with regular rims and homogeneous sizes were observed again (Fig. 8b and c). The TEM and SEM images show that the vesicles are slightly smaller than primary ones, which may be attributed to the effect of generated salt [72]. According to the transition mechanism (Scheme 3), addition of acid can cause tertiaryamine being protonated into ammonium. Ionic interactions could damage the aggregation of

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Scheme 2.

Fig. 8. (a) TEM images of the mixture of 1/␤-CD and acetic acid in aqueous solution (both 10−4 mol/L, pH = 4.4). (b) TEM images of the 1/␤-CD solution after the pH adjust (pH = 7.0). (c) SEM images of the 1/␤-CD solution after the pH adjust (pH = 7.0). Scale bars = 200 nm.

hydrophobic parts into bilayers [22] (Scheme 1). The observed solid particles may aggregate only by ionic interaction. When excess H+ was neutralized, which made the guest structure from ammonium back to tertiaryamine and vesicles would form again. The whole process resembles the pH-reversible property of vesicles formed by CD and methyl orange [73]. This pH-reversible phenomenon can be repeated at least 5 times. Meanwhile, the variation of peaks in the UV spectra corresponding to 1 in the presence and absence of acetic acid indicates that the secondary amine may change into a quaternary ammonium. It is anticipated that this research will provide a model system that introduces a vesicle with pH-reversible property into host–guest chemistry (Fig. 9).

3.4. Application of the vesicles in cell-staining Vesicles have drawn increasing attention for the hope of applications in the target-release fields [27]. However, vesicles, to a certain extent, seem to have more potential than practical application. Cell staining and location is becoming a focus because of their applications in cellular localization and cancer cure [74,75]. From the above experimental results, it is clear that the vesicles are multiresponsive, or in other words, the vesicles are sensitive. Hence, we can deduce that when introduced into the cell suspension with a complicated composition vesicles may collapse, and the guest molecules may be released to stain the cells.

Scheme 3.

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Fig. 9. UV spectra of 1 (blue line), 1/␤-CD (red line), the mixture of 1/␤-CD and acetic acid (black line). [18-AQ] = [18-AQ/␤-CD] = [acetic acid] = 10−4 mol/L. T = 300 K. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Mouse prostate carcinoma cells (RM-1) were used to test the fluorescence staining effect. In the view of fluorescence microscope, it is found that the cells can be stained with strong luminous intensity and good dyeing evenness (Fig. 10a and b). Images of cells by fluorescence microscope are in good accordance with the images under natural lighting. Since it is known that cells which

are not fluorescently stained cannot be observed by microscope under a fluorescence condition, we could deduce that the vesicles were collapsed and 1 may be adsorbed by the cell membrane. Control experiments were also carried out and it was found that sole 1 (10−4 mol/L in aqueous solution) could only stain the cells with very weak fluorescence (Fig. 10c and d), which can further demonstrate the important role of the formation of vesicles plays. Our methods may provide a new approach to detect the cellular localization. This may also lead a new way to achieve targeted release of some anti-cancer drugs carried by the vesicles, especially when CDs, which have molecule recognition property, are as the key composition of the building block of the vesicles. The samples have been observed by the fluorescence microscope for more than 3 h and the fluorescent intensity did not decrease. Our method offers a longer fluorescence lifetime and better homogeneity compared with other cell-stainers, such as Rhodamine B. The progress of staining can be mediated by the noncovalent bond between CDs and glycoprotein or other molecules in the cell membrane. Then, vesicles are collapsed and 1 is released from CD cavity and adsorbed by the cell membrane, for their low solubility in aqueous solution. The sensitive property of the supramolecular vesicles formed by “amphiphilic surfactant” was successfully applied here. The study on the affect of fluorescent vesicles to the inner cell organelles is still in process.

Fig. 10. Microscope images of living cells after treating by the vesicular solution: RM-1 cells stained by vesicles of 1/␤-CD (10−4 mol/L in aqueous solution) (a) under natural lighting; (b) in fluorescence microscope; RM-1 cells stained by vesicles of 1 (10−4 mol/L in aqueous solution) (c) under nature light; (d) in fluorescence microscope.

T. Sun et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 375 (2011) 87–96

4. Conclusion In summary, we report the preparation and application of the first pH-reversable fluorescent vesicle system based on the “supramolecular amphiphilies”. As the inclusion of 1 and ␤-CD, the supramolecular complex can self-assembly into vesicular structures in aqueous solution, which were revealed by TEM, SEM, DLS and EFM. The possible mechanism of vesicle-formation was suggested based on the results of 1 H NMR, 2D NMR ROESY, UV, and FT-IR. The vesicles collapse when acetic acid was added. When pH was adjusted to 7.0, vesicles appeared again. The vesicles were successfully applied in the staining of living cells and the possible staining approach was also suggested. We believe the supramolecular vesicles will serve useful in the fields of biomaterials, cell-mimic, design of intelligent materials and molecule machines. Acknowledgements This work was supported by the NSFC (grant no. 20625307), National Basic Research Program of China (973 Program, 2009CB930103) and Graduate Independent Innovation Foundation of Shandong University (GIIFSDU, yzc09057). We thank Mr. Guanghui Cheng from Department of Medical Genetics and Key Laboratory for Experimental Teratology of the Ministry of Education for helping the cell-staining process and Dr. Z. Yang from Université Pierre et Marie Curie: Paris 6 for editing the manuscript for English corrections. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfa.2010.11.067. References [1] C. Park, M.S. Im, S. Lee, J. Lim, C. Kim, Tunable fluorescent dendron–cyclodextrin nanotubes for hybridization with metal nanoparticles and their biosensory function, Angew. Chem. 120 (2008) 10070–10074. [2] L. Jiang, Y. Peng, Y. Yan, M. Deng, Y. Wang, J. Huang, Annular ring microtubes formed by SDS 2␤-CD complexes in aqueous solution, Soft Matter 6 (2010) 1731–1736. [3] C. Park, I. Lee, S. Lee, Y. Song, M. Rhue, C. Kim, Cyclodextrin-covered organic nanotubes derived from self-assembly of dendrons and their supramolecular transformation, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 1199–1203. [4] Y. Tang, L. Zhou, J. Li, Q. Luo, X. Huang, P. Wu, Y. Wang, J. Xu, J. Shen, J. Liu, Giant nanotubes loaded with artificial peroxidase centers: self-assembly of supramolecular amphiphiles as a tool to functionalize nanotubes, Angew. Chem. 122 (2010) 4012–4016. [5] Z. Wang, Z. Li, Z. Liu, Photostimulated reversible attachment of gold nanoparticles on multiwalled carbon nanotubes, J. Phys. Chem. C 113 (2009) 3899–3902. [6] A. Maciollek, M. Munteanu, H. Ritter, New generation of polymeric drugs: copolymer from NIPAAM and cyclodextrin methacrylate containing supramolecular-attached antitumor derivative, Macromol. Chem. Phys. 211 (2010) 245–249. [7] Y. Takashima, T. Nakayama, M. Miyauchi, Y. Kawaguchi, H. Yamaguchi, A. Harada, Complex formation and gelation between copolymers containing pendant azobenzene groups and cyclodextrin polymers, Chem. Lett. 33 (2004) 890–891. [8] X. Li, Z. Qi, K. Liang, X. Bai, J. Xu, J. Liu, J. Shen, An artificial supramolecular nanozyme based on ␤-cyclodextrin-modified gold nanoparticles, Catal. Lett. 124 (2008) 413–417. [9] C. Luo, F. Zuo, Z. Zheng, X. Cheng, X. Ding, Y. Peng, Tunable smart surface of gold nanoparticles achieved by light-controlled molecular recognition effection, Macromol. Rapid Commun. 29 (2008) 149–154. [10] J. Liu, R. Sondjaja, K.C. Tam, Alpha-cyclodextrin induced self-assembly of a double-hydrophilic block copolymer in aqueous solution, Langmuir 23 (2007) 5106–5109. [11] H. Zhang, J. Shen, Z. Liu, Y. Bai, W. An, A. Hao, Controllable vesicles based on unconventional cyclodextrin inclusion complexes, Carbohydr. Res. 344 (2009) 2028–2035. [12] H. Zhang, W. An, Z. Liu, A. Hao, J. Hao, J. Shen, X. Zhao, H. Sun, L. Sun, Redoxresponsive vesicles prepared from supramolecular cyclodextrin amphiphiles, Carbohydr. Res. 345 (2009) 87–96.

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