Self-Assembly of Morphology-Tunable Architectures from

The WCU Center for Synthetic Polymer Bioconjugate Hybrid Materials, ... National Research and Development Center for Hepatobiliary Cancer, Pusan Natio...
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Self-Assembly of Morphology-Tunable Architectures from Tetraarylmethane Derivatives for Targeted Drug Delivery Xinhua Huang,† Young-Il Jeong,‡ Byeong Kyu Moon,† Lidong Zhang,† Dae Hwan Kang,‡ and Il Kim*,† †

The WCU Center for Synthetic Polymer Bioconjugate Hybrid Materials, Department of Polymer Science and Engineering, Pusan National University, Pusan 609-735, Korea ‡ National Research and Development Center for Hepatobiliary Cancer, Pusan National University, Yangsan Hospital, Yangsan 626-870, Korea S Supporting Information *

ABSTRACT: Tetraarylmethane compounds consisting of two pyrogallol and two aniline units, namely, Ar2CAr′2 {Ar = 3,4,5C6H2(OH)3 and Ar′ = 3,5-R2-4-C6H2NH2 [R = Me (1), iPr (2)]} exhibit excellent self-assembly behavior. Compound 1 yields size-tunable hollow nanospheres (HNSs) with a narrow size distribution, and 2 yields various morphologies ranging from microtubules to microrods via self-assembly induced by hydrogen bonding and π−π stacking interactions. On the basis of the experimental results, a plausible mechanism for morphology tunability was proposed. As a means of utilizing the self-assembled HNSs for targeting controlled drug delivery, folic acid (FA) and rhodamine 6G (Rh6G) were grafted onto compound 1 to yield the FA−Rh6G−1 complex. The HNSs fabricated with FA−Rh6G−1 showed low cytotoxicity against human embryonic kidney 293T cells and CT26 colon carcinoma cells and good doxorubicin (DOX) loading capacity (9.6 wt %). The FA receptor-mediated endocytosis of FA−Rh6G−1 HNSs examined by using a confocal laser scanning microscope and a flow cytometer revealed that the uptake of FA−Rh6G−1 HNSs into CT26 cells was induced by FA receptor-mediated endocytosis. In vitro drug delivery tests showed that the DOX molecules were released from the resulting HNSs in a sustainable and pH-dependent manner, demonstrating a potential application for HNSs in targeted drug delivery for cancer therapy.

1. INTRODUCTION The ability to control the morphology of organic nanomaterials by means of molecular design and synthesis is increasingly gaining attention.1 Since nanomaterials show atypical properties such as surface effect, quantum size effect, macroscopic quantum tunneling effect, and size dependent optoelectronic properties, attempts have been made to use them as highly efficient catalysts,2 in biolabeling3 and functionalization,4,5 as novel luminescent materials,6 nonlinear optics,7 chemical sensors,8 and in superhigh-density information storage.9 Organic nanomaterials can be fabricated from organic macromolecules and from low-molecular-weight organic compounds (LMWOCs). Specifically, 0D and 1D nanomaterials composed of organic macromolecules such as polymers, biomolecules, graphenes, and dendrimers have also been investigated extensively during the past decade. Since Nakanishi and co-workers10 introduced a facile reprecipitation method to fabricate organic nanomaterials, a number of efficient protocols for constructing organic nanostructures with different morphologies, crystallinities, and optoelectronic properties have been developed, recognizing that many properties of the organic molecular aggregates exhibit a size dependence.11,12 In addition, the optical and © 2013 American Chemical Society

electronic properties of organic nanomaterials are fundamentally different from those of inorganic materials owing to the weak supramolecular interactions such as hydrogen bonding, π−π stacking, and van der Waals forces. Aggregates produced by these weak interactions are readily disturbed by environmental factors (e.g., surfactants, solvents, concentration, and temperature). In other words, the morphologies of organic micro/nanocrystals can be easily controlled using the selfassembly conditions. Furthermore, some researchers reported that the cohesive energy of organic polyhedral crystal surfaces is sensitive to the molecular architectures;13,14 however, the self-assembly of nonplanar molecules with multiple aromatic planes has rarely been reported.15 It is believed that nonplanar molecules with multiple aromatic planes could offer additional factors to control the formation of various polyhedral micro/nanocrystals by manipulating the different π−π stacking interactions, tuning the cohesive energies of the crystal facets, and controlling the kinetic growth process. Received: December 23, 2012 Revised: February 15, 2013 Published: February 20, 2013 3223

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hydrogen bonding, π−π stacking, and van der Waals forces during the self-assembly. This work may provide a facile strategy to design and prepare organic micro/nanocrystals for drug carrier applications.

In the case of a nonplanar molecule with multiple aromatic planes, the anisotropic cohesive energy will be recognized as the directional π−π stacking from each aromatic plane. Recently, Hasell and co-workers16 synthesized a porous cagelike organic nanocrystal in a controlled way from a rigid organic cage module with nonplanar interactions. This work encouraged us to employ a nonplanar molecule with multiple aromatic planes to control the anisotropic cohesive energy and the self-assembly of the nanoparticles. Nanospheres with hollow or porous structures have enhanced applications in targeted delivery, energy storage, catalysis, and artificial cells.17−20 The simple and efficient protocol for the immobilization of active ingredients such as dyes, inks, cells, drugs, and proteins into the interior cavities is a key factor for the achievement of these promising applications.21−23 Generally, for structures such as porous spheres with good dispersity, proper size, and ideal surface permeability, easy access into the interior of the materials for guest materials can be achieved with higher feasibility. In this article, we present the fabrication of 0D and 1D nanomaterials based on tetraarylmethane derivatives (two pyrogallol and two alkyl-substituted aniline fragments) bearing six hydroxyl groups, two amine groups, and two methyl or isopropyl groups. These nonplanar multiaryl molecules are different from those of most molecules previously used for making organic micro/nanostructures in that they have one main aromatic plane for the π−π stacking.24,25 In addition, the multiple hydroxyl and amine groups, combined with the π−π stacking interactions, also play a role in tuning the cohesive energies under self-assembly. After conjugating folic acid (FA) and rhodamine 6G (Rh6G) and encapsulating doxorubicin (DOX), we attempted to apply the self-assembled hollow nanospheres in controlled drug delivery for cancer therapy. To the best of our knowledge, there have been no reports on the fabrication of hollow nanospheres based on cruciform-shaped tetrahedral aromatic hydrocarbons, which have four conjugated aromatic planes connected via a common sp3-hybridized carbon (Figure 1A). These LMWOCs were designed to induce

2. MATERIALS AND METHODS 2.1. Materials. POCl3 (98%), N,N-dimethylformamide (DMF) (99%), gallic acid (99%), pyrogallic acid (98%), 2,6-dimethylaniline (99%), 2,6-diisopropylaniline (99%), folic acid (FA) (98%), trifluoromethanesulfonate (CF3SO3H; 98%), dicyclohexylcarbodiimide (DCC) (99%), and N-hydroxysuccinimide (NHS) (99%) were obtained from Sigma-Aldrich and used without further purifications. Tetrahydrofuran (THF), acetonitrile (CH3CN), 1,2-dichloroethane (DCE), dichloromethane (DCM), ethanol (EtOH), and dimethylformamide (DMF) were distilled before use. ZnCl2 was dried under a vacuum at 120 °C to remove moisture before use. The immobilization solution, Immu-Mount, was purchased from Thermo Electron Corporation (Pittsburgh, PA). 2.2. Characterization. 1H NMR and 13C NMR spectra were recorded on a 400 MHz 1H (100 MHz 13C) Varian Unity Plus spectrometer. Elemental analysis was performed on an Elementar Analysensysteme GmbH (Germany) elemental analyzer. The fastatom bombardment mass spectra (FAB-MS) were recorded on a JEOL JMS DX300 apparatus (JEOL, Tokyo, Japan). Dynamic and static light scattering (DLS) experiments were performed on a DLS-7000 instrument (Otsuka Electronics) using an argon ion laser operating with vertically polarized light at λ = 488 nm. The surfaces of the selfassembled samples were observed using a scanning electron microscope (SEM) on an S-3000H apparatus (Hitachi, Japan) or a JSM-6700F (JEOL, Tokyo, Japan). Transmission electron microscopy (TEM) was performed using a 1200 EX (JEOL, Tokyo, Japan) at 120 keV. Samples for TEM were deposited onto carbon-coated copper electron microscope grids and dried in air. Fourier transform infrared (FTIR) spectra were obtained at a resolution of 1 cm−1 with a Shimadzu IR Prestige-21 (Kyoto, Japan) spectrophotometer between 4000 and 400 cm−1. The IR measurements of the powder samples were performed in the form of KBr pellets. UV−vis absorption spectra of the samples were recorded at room temperature on a Shimadzu UV1650PC spectrometer (Kyoto, Japan). The fluorescence spectra were obtained using a spectrofluorometer model F-4500 (Hitachi, Japan). X-ray diffraction (XRD) patterns of the neat powder were measured on a Bruker D8 Focus diffractometer using Cu Kα radiation. X-rays were generated with a Cu anode, and the Cu Kα beam (λ = 1.5406 Å) was passed through a graphite monochromator. The long spacing d was obtained from the typical peak. 2.3. Synthesis of Compounds (Figure S1, Supporting Information). Exifone was synthesized using gallic and pyrogallic acid according to the method reported in the literature.26 The tetraarylmethane compounds were also synthesized by employing previously reported methods.27 A typical procedure for compound 1 is as follows: The mixture of 2,6-dimethylaniline (1.74 g, 14.4 mmol) and exifone (3.0 g, 7.19 mmol) dissolved in DMF (10 mL) was introduced into a 100 mL round flask under a nitrogen atmosphere. The reaction mixture was slowly heated in an oil bath to 120 °C under vigorous stirring. CF3SO3H (10 mL) was then added dropwise over 1 h. After 24 h of reaction at 110 °C, the mixture was slowly cooled to room temperature, and then, 20 mL of saturated aqueous NaHCO3 was added. The organic phase was extracted with CHCl3 three times, and the combined organic layers were washed with brine. The solution was then dried over MgSO4 and concentrated by evaporation. The residue was purified by recrystallization in ethanol to give a white solid product (1) in a 27.8% yield. Melting point (mp) 170−171 °C. 1H NMR (CDCl3, 400 MHz, δ ppm): 8.37 (1H, s, ArH), 8.09−8.07 (1H, m, ArH), 7.29−7.20 (m, 1H, ArH), 6.98−6.85 (m, 4H, ArH), 6.79−6.67 (m, 1H, ArH), 2.2 (m, 12H, CH3). 13C NMR (CDCl3, 100 MHz, δ ppm) aromatic-C: 164.8, 157.2, 135.2, 132.3, 127.7; center-C: 24.7; methyl-C: 18.6. Fab-MS m/z (%): 448.56 (100) [M-4CH2]+. Anal. Calcd for C29H30N2O6: C, 69.31; H, 6.02; N, 5.57;

Figure 1. Molecular structures and their energy-minimized density functional theory (DFT) computed structures of Ar2CAr′2 {Ar = 3,4,5C6H2(OH)3 and Ar′ = 3,5-R2-4-C6H2NH2 [R = Me (1), iPr (2)]} using Accelrys Materials Studio version 4.2 and DMol3 at the DFT GGA-PW91/DNP (fine) level of theory (DNP-Double numeric quality basis including polarization functions, equivalent to 6-31G** basis sets). The electrostatic potential (ESP) derived charges of the respective atoms are given on the optimized structures. 3224

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was then replaced with 200 μL of DMSO, the absorbance (560 nmtest/630 nm-reference) was determined using an automated computer-linked microplate reader (Molecular Device Co.), and the relative cell viability was calculated. 2.9. FA-Receptor Mediated Endocytosis of HNS against CT26 Cells. For fluorescence observation, CT26 cells (1 × 106 cells/ well) were seeded onto a cover-glass and incubated overnight at 5% CO2 and 37 °C. Media were exchanged with RPMI1640 media with FA (2 mM) (FA+) or without FA (FA−) and then further incubated for 3 h. The FA−Rh6G−1 HNSs were added in RPMI1640 media bearing the pretreated CT26 cells and incubated for 1 h at 5% CO2 and 37 °C. The resulting cells were washed with PBS (pH 7.4, 0.1 M), treated with 4% paraformaldehyde, and fixed with an immobilization solution. These cells were observed with a confocal laser scanning microscope (CLSM, TCS-SP2; Leica, Wetzlar, Germany). CT26 colon carcinoma cells (1 × 106 cells/well) were seeded in 6well plates and incubated overnight at 5% CO2 and 37 °C. Media were exchanged with serum-free RPMI1640 media containing 2 mM FA (FA+) or no FA (FA−) and then further incubated for 3 h. FA− Rh6G−1 HNSs were added to these cell culture media and incubated for 1 h at 5% CO2 and 37 °C. The cells were harvested and immediately analyzed using a flow cytometer (FACScan, Becton Dickinson Biosciences, San Jose, CA). 2.10. In Vitro Drug Loading and Release. The DOX@FA− Rh6G−1 HNS solution (1 mL) was transferred to a dialysis membrane (MWCO 12 000) and dialyzed against 20 mL of PBS solutions with different pH values of 5.0, 6.5, and 7.4 at 37 °C. Drug release was monitored by UV−vis spectroscopy. 2.11. Molecular Dynamics Simulations and Polymorph Prediction. Molecular models of compounds 1 and 2 were taken from DFT isolated-molecule geometry optimizations using the DMol3 code, as implemented in the Accelrys package Materials Studio (version 4.3),32 and the PW91 functional33 with the double numerical polarized (DNP) basis set.34 Lattice energies were evaluated using an empirically derived atom−atom potential (W99)35 for the description of repulsion−dispersion contributions and atomic point charges or multipoles for the electrostatics. In the simplest electrostatic model, atom-centered charges were derived to reproduce the molecular electrostatic potential (ESP charges),36 using the fitting procedure implemented in DMol3. Atomic multipoles were derived from a distributed multipole analysis37 of a B3LYP/6-31G** electron density. The center of interaction for all hydrogen atoms was shifted by 0.1 Å along the X−H bond (X = C, N, O) toward the heavy atom; the X−H distances were shortened by 0.1 Å after DFT optimization. All charge− charge, charge−dipole, and dipole−dipole contributions to the lattice energy were evaluated using Ewald summation, while higher order terms (up to R−5) and the repulsion−dispersion interactions were summed to a 15 Å cutoff. These optimized gas-phase conformations were used as the starting points for the crystal structure prediction using the Materials Studio Polymorph Predictor (PP).32 The optimized structure was used for PP calculations. Crystal structures were generated in the 10 most common space groups38 found in organic crystals registered in the CSD (P21/c, P1, P212121, P21, C2/c, Pbca, Pna21, Pbcn, Cc, and C2). The crystal structures were generated using the simulated annealing algorithm as implemented in the PP module. The computer-generated crystal structures were lattice energy minimized (using the atomic point charge model potential described above) and clustered to remove duplicates. Independent searches in each space group were performed until no new structures were generated. The COMPACK algorithm was used for the final clustering of the structures.39 Finally, the computer-generated crystal structures were energy minimized using the atomic multipole model potential. The PP module was set to its default fine setting with the force field Dreiding 2.21 with ESP charges. This setting sets the simulated annealing algorithm to a temperature range of 300−100000.0 K with a heating factor of 0.025, requiring 12 consecutive steps to be accepted before cooling and a maximum of 7000 steps.

O, 19.10. Found: C, 69.29; H, 6.05; N, 5.59; O, 19.07. FT-IR (KBr) (cm−1): 3237, 3170, 2881, 1629, 1229, 1029, 783, 697. Compound 2 was synthesized by a similar procedure using 2,6diisopropylaniline to generate a gray solid in 12.8% yield. mp 156−157 °C. 1H NMR (DMSO, 400 MHz, δ ppm): 8.28 (s, NH), 7.27−7.17 (m, 8H, ArH), 3.11−2.94 (m, 4H, CH2), 1.12−1.07 (m, 24H, CH3). 13 C NMR (DMSO, 100 MHz, δ ppm) aromatic-C: 161.0, 146.0, 131.7, 128.0, 123.3; center-C: 28.5; methylene-C: 28.2; methyl-C: 23.8. FabMS m/z (%): 616.7 (100) [M+H]+. Anal. Calcd for C37H46N2O6: C, 72.29; H, 7.54; N, 4.56; O, 15.61. Found: C, 72.26; H, 7.57; N, 4.59; O, 15.58. FT-IR (KBr) (cm−1): 3445, 3223, 2982, 2867, 1652, 1531, 1407, 1393, 739. 2.4. Determination of the Critical Micelle Concentration (CMC). The CMC of compounds 1 and 2 was determined by UV−vis spectroscopy using Rh6G as the fluorescent probe. Samples for UV− vis measurements were prepared according to procedures described in the literature.28 The concentration of the solution of 1 (or 2) in water was between 2.0 and 2 × 10−4 mg mL−1. A 2 μL Rh6G solution (0.1 mM in EtOH) was added to a 2.0 mL solution. The resulting solution was incubated in a dark place for 5 h. The UV−vis absorption of the incubated solution was measured in the range 250−600 nm, and the absorbance at 527 nm was selected to determine the CMC. 2.5. Fabrication of Tunable Morphology by Self-Assembly. For the fabrication of hollow nanospheres (HNSs), high-purity water (200 μL) was slowly injected into the solution of 1 in THF (10 mM, 1.8 mL) with vigorous stirring. After stirring for 5 h at 40 °C, the homogeneous solution was immediately cooled to room temperature, and then left undisturbed for approximately 4 h to stabilize the nanostructure. The self-assembly behavior of 2 was also investigated using the same procedure. 2.6. FA and Rh6G Conjugation onto DOX-Loaded HNS (DOX@FA−Rh6G−1). DOX-loaded HNSs were prepared by the dialysis method.29 Compound 1 (10 mg) was dissolved in THF (18.0 mL) and then added to 2 mg of DOX (neutralized by Et3N) in water (2.0 mL) while stirring for 12 h. Next, 0.5 mL of an activated FA solution and 0.5 mL of hydrolyzed Rh6G30 were added into the DOXloaded HNS solution and stirred for 24 h at room temperature. The resulting mixture was dialyzed by membrane (MWCO 3500) against distilled water to remove the organic solvent and unreacted organic compounds. 2.7. Drug Encapsulation Efficiency. To determine drug loading,31 10 μL of a DOX-loaded HNS water solution was diluted with 2 mL of water. Then, the drug concentration was determined using a UV−vis spectrometer at 483 nm (Thermo Spectronic). The DOX concentration was calculated on the basis of UV−vis absorbance using a standard curve generated in water for drug concentrations ranging from 0 to 10 mg mL−1. The drug encapsulation efficiency and the drug loading capacity were then determined using the following equations:

drug encapsulation efficiency (%) amount of drug loaded = × 100 amount of drug added drug loading capacity (%) amount of drug loaded = × 100 amount of self‐assembled compound 2.8. In Vitro Cytotoxicity of Compound 1 and FA−Rh6G−1 Micelles to 293T Cells. In vitro cytotoxicity of compound 1 and FA− Rh6G−1 HNS was tested using a thiazolyl blue tetrazolium bromide (MTT) assay. The 293T cells (human embryonic kidney cells) were seeded in a 96-well plate at a density of 1000 cells/well and cultured in 5% CO2 at 37 °C for 24 h to allow the cells to attach before they were exposed to serial concentrations of compound 1 and FA−Rh6G−1 HNS. After 24 or 48 h of incubation, the media containing compound 1 and FA−Rh6G−1 HNS were removed, and 200 μL of MTT solution, diluted in a culture medium with a final concentration of 1 mg mL−1, was added and incubated for an additional 4 h. The medium 3225

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3. RESULTS AND DISCUSSION 3.1. Synthesis of Compounds 1 and 2. The nonconjugated multiaryl molecules 1 and 2 have been successfully prepared by modifying the reported procedures.27 Reacting exifone with 2,6-dimethyaniline or 2,6-diisopropylaniline in the presence of CF3SO3H in DMF easily afforded compound 1 or 2 in reasonable yields. These tetrahedral aromatic compounds have four conjugated aryl rings connected by a typical sp3hybridized carbon. The aryl rings are substituted with six hydroxyl groups, two amine groups, and four methyl or isopropyl groups. The two structures are different in that compound 2 is sterically bulkier than 1. The PP module in Materials Studio can be used as an ab initio prediction in a sophisticated structural analysis with other modules that make it possible to examine the packing arrangement and the hydrogen bonding for each structure. If there is no explicit formulation of thermal effects in the force field model, empirically derived parameters can be expected to incorporate the average effect of thermal motion on the crystal structure.35 Typically, unit cell edges in organic crystals may increase around 3% over a temperature range of several hundred degrees. The present force field is intended for use primarily at room temperature. However, because we included in the training data set crystal structures determined at below liquid nitrogen temperature up to room temperature, we generally did not expect the model to predict cell edge lengths to greater accuracy than 3%. Another way of stating this is to say that, if the force field is able to predict cell edge lengths within 3%, it probably cannot be further improved without elaboration of the model to include thermal effects. The PP module explores and ranks polymorphs of fairly rigid, nonionic or ionic molecules.40 Using energy-minimized molecular structures given in Figure 1A, the geometry of each unique structure is optimized with respect to all degrees of freedom or with rigid body constraints, where the relative distance between a group of atoms is fixed. The final polymorph structures are ranked according to lattice energy. The top 30 ranked structures were examined for each conformer in all 10 space groups, producing a total of 5000 theoretical structures. A lattice energy/density plot of the output revealed a good spread of the data, indicating that a well-distributed search of packing space was achieved. Both compounds 1 and 2 are best fitted with a P21 monoclinic space group (Figure S6, Supporting Information). The density of the predicted crystal structures of 1 and 2 is 1.352 (with a unit cell volume of 1.23 × 103 Å3) and 1.161 (with a unit cell volume of 1.76 × 103 Å3), respectively. The lengths a, b, and c for 1 are 8.992, 16.594, and 8.281 Å, respectively, and those for 2, 15.103, 12.013, and 9.769 Å, respectively. The angles α, β, and γ for 1 are 90, 92.66, and 90°, respectively, and those for 2, 90, 82.84, and 90°, respectively. The molecular arrangement for compounds 1 and 2 was solved using the powder XRD technique. The XRD patterns indicate that compound 1 displays good crystalline behavior, whereas compound 2 has a more amorphous structure (Figure S7, Supporting Information). Intramolecular hydrogen bonding among hydroxyl groups and intermolecular hydrogen bonding interactions among hydroxyl and amine groups are observed in all the crystal structures. The most common pattern is the formation of hydrogen bonding chains, which propagate infinitely in specific directions throughout the crystal. These selective, directional,

and strongly attractive hydrogen bonding interactions can induce the self-assembly of the predictable supramolecular aggregates.41 The strong tendency of compounds 1 and 2 to form hydrogen-bonded dimers allows them to function as sticky sites that compel molecules to associate, thereby driving the self-assembly of aggregates joined by extensive networks of hydrogen bonds. Accordingly, the creative incorporation of multiple sticky sites into rigid frameworks, together with arylring-derived π−π stacking and van der Waals forces, might induce the self-assembly of 3D networks. 3.2. Fabrication of Nano- and Microstructures by SelfAssembly. In general, reprecipitation is the most widely adopted method for the production of 0D organic nanoparticles, although sometimes, laser ablation,42 microwaves,43 microemulsion,44 and a membrane reactor45 are used to assist the formation of nanoparticles in specific surroundings. Compared with their 0D counterparts, 1D nanomaterials are more suitable for the construction of active nanodevices and interconnects. There is an agreement that the development of facile, mild, and widely applicable methods for the construction of organic 1D nanostructures, such as self-assembly in the liquid phase, self-assembly through organogelation, selfassembly with solvent evaporation, the soft and hard template method, and vapor deposition, is of great scientific and technical significance.1 Controllable synthesis is a key task for nanoscale science, and the morphologies of inorganic nanostructures have been successfully controlled by choosing appropriate materials and changing the synthetic methods.46 However, the morphology control of organic nanomaterials has had limited success. The commonly used strategy to modulate the organic nanostructures is to slightly alter the molecular structures in a series of derivatives. For example, Zang and coworkers have prepared either nanobelts or nanospheres by using two different derivatives of perylene diimide.47 The self-assembly behavior of compounds 1 and 2 with similar structures was investigated in various organic solvents such as EtOH, THF, DMF, DCM, DCE, and mixed organic− inorganic solvents like THF/water, DMF/water, and EtOH/ water (Figures S8 and S9, Supporting Information). The results revealed that THF/water was the best solvent system to induce self-assembly of the tetraaryl derivatives. Figure 2 shows the SEM and TEM images of the self-assembled structure of the tetraaryl derivatives in THF/water (9/1, v/v). Compound 1 forms HNSs at a concentration of 2.0 mg mL−1 (Figure 2a,b), with an outer diameter of ∼200 nm and an inner diameter of ∼100 nm. Similar structures have been reported for the selfassembly of stearamide48 and α-diimine compounds.49 Interestingly, compound 2 yields short and round hollow microtubules with a diameter of ∼1 μm and a length of several micrometers (Figure 2c) under the same self-assembly conditions, demonstrating that a simple modification in the steric bulk from methyl to isopropyl induces completely different self-assembly behavior. Self-assembly of 2 at a slightly higher concentration (2.5 mg mL−1) yields microrods (Figure 2d) instead of microtubules. The resulting nano- and microstructures were stable in the air and in aqueous environments for an extended period of time. In order to investigate the effect of compound concentration on the self-assembly behavior, the self-assembly of 1 was performed at various concentrations, 1.0, 1.5, 2.0, and 2.5 mg mL−1. In all cases, highly uniform HNSs were obtained; the average diameters of the HNSs measured by DLS are 150 ± 20, 295 ± 40, 342 ± 50, and 458 ± 60 nm, respectively (Figure 3226

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will be stable in vivo, where considerable dilution takes place. The CMC values of compounds 1 and 2 are 6.5 × 10−2 and 13.0 × 10−2 mg mL−1, respectively. Compound 1 exhibits a 2fold lower CMC value than compound 2, and it is thermodynamically stable even after severe dilution. This is clearly an advantage for compound 1 micelles with regard to their application as drug carriers, because the micelles will be stable and will not dump their drug content upon in vivo administration. 3.3. Spectroscopic Investigation of Self-Assembled Micelles. FT-IR spectroscopy is a powerful tool for investigating hydrogen bonding interactions and has been reliably applied in the analysis of the morphology of selfassembled organic compounds. Figure S14 (Supporting Information) shows the FT-IR spectra of compounds 1 and 2 before and after self-assembly, and Table 1 summarizes the Figure 2. (a) SEM and (b) TEM images of hollow nanospheres fabricated using 1 at the concentration of 2.0 mg mL−1 in THF/water (9/1, v/v). (c) SEM image of microtubules fabricated using 2 at a concentration of 2.0 mg mL−1 in THF/water (9/1, v/v). (d) SEM image of microrods with 2 at a concentration of 2.5 mg mL−1 in THF/ water (9/1, v/v).

Table 1. FT-IR Signal Assignments for Compounds 1 and 2 before (Neat Powder) and after (Hollow Spheres and Microtubes) Self-Assembly in THF/Water (9:1, v/v) absorption peak of sample (cm−1)

S10, Supporting Information). The TEM images (Figure S11, Supporting Information) also indicate that the wall thickness of the HNSs is about 24 nm at a concentration of 1.0 mg mL−1, and it increases to 30, 50, and 60 nm at 1.5, 2.0, and 2.5 mg mL−1, respectively. Therefore, the diameter and wall thickness of these HNSs can be tuned by simply changing the selfassembly concentration. Since compound 1 has multiple hydroxyl and amine groups on the aryl rings, it might show pH-dependent self-assembly. Thus, the pH-dependent micellization of compound 1 was carried out in THF/H2O (9:1, v/v) at a concentration of 2.0 mg mL−1. Surprisingly, under acidic conditions (pH 4), compound 1 self-assembled to yield nanotubules, while, under basic conditions (pH 11), it yielded solid nanospheres (see Figure S12, Supporting Information). Note that it yielded HNS under neutral conditions. In general, hydrogen bonding plays an important role in the self-assembly of the morphology, and together with π−π stacking, they are deemed to be important in the cationic system.50 To some extent, compound 1 behaves like a surfactant. The amine and hydroxyl groups separate from the hydrophobic phenyl and methyl groups to form bilayers. When compound 1 self-assembles at pH 4.0, the amine and hydroxyl groups on the aryl rings are protonated and the coarsely stacked vesicle-like structure changes to a more compactly stacked structure to form nanotubes rapidly by reassembly. In addition, the FTIR spectra indicative of compound 1 aggregation change with pH (Figure S13, Supporting Information). As the pH increases from 4.0 to 11.0, the peaks assigned to the hydroxyl and amine groups become narrower owing to the decrease in protonation and in the hydrogen-bond strength. This also indicates that hydrogen bonding and π−π stacking govern the self-assembly process. Compounds 1 and 2 possess an amphiphilic structure consisting of hydrophobic and hydrophilic segments, leading to aggregate formation in the THF/water solution. The CMC is a very important parameter for determining whether the molecule forms aggregates or exists as a monomer. A low CMC is an important feature for the application of these micelles in drug delivery, because it ensures that the micelles

assignments

microtube

neat powder 1

HNS

neat powder 2

vas (OH) vas (NH2) vas (CH) v (CC, Ar) v (CN) δas (CH3) δ (Ar)

3425 3239 3003, 2889 1665 1542 1388 729

3445 3223 2982, 2867 1652 1531 1407 739

3438 3223 2941 1644 1267 1374 697

3237 3170 2881 1662 1229 1376 705

results of assignments. All IR signals related to the formation of the hydrogen bonds show a blue shift after self-assembly. The O−H and N−H signals at 3237 and 3170 cm−1 for the neat sample are shifted to 3438 and 3223 cm−1, respectively. The unsaturated C−H stretching vibration of the neat sample at 2881 cm−1 is also shifted to 2941 cm−1 for the self-assembled HNSs. For compound 2, O−H and N−H signals at 3445 and 3223 cm−1 for the neat sample are shifted to 3425 and 3239 cm−1, respectively. The unsaturated C−H (methyl and methylene) stretching vibrations of the neat sample at 2982 and 2867 cm−1 are also shifted to 3003 and 2889 cm−1, with peak broadening for the self-assembled microtube. The spectral variations before and after self-assembly suggest that the morphologies of compounds 1 and 2 are mediated mainly by hydrogen bonds among multiple hydroxyl and amine groups. Nonetheless, the hydrophobic interactions between the phenyl/methyl or phenyl/isopropyl groups mediated by van der Waals, π−π stacking, and other weak interactions cannot be completely neglected. 3.4. Formation Mechanism. On the basis of the data collected for the self-assembly of compounds 1 and 2 in a THF/water mixture, we propose a possible mechanism of formation of the different nano- and microstructures, as illustrated in Figure 3. The hydrogen bond between the hydroxyl and amine groups of compound 1, together with the π−π stacking interactions, might be the driving force for the self-assembly of HNSs.46 First, emulsion droplets of THF are formed by the self-emulsification process, and then, the added water molecules slowly diffuse into the droplets.51 The count and size of the internal water droplets increase with time, with the internal tiny droplets coalescing into bigger ones. Once the double emulsion droplets are formed via coalescence of the 3227

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The reconstruction of nanostructures releases even more curvature energy. The THF molecules are essential in this process, because they erode the membrane and help in the tube formation. The etching or dissolution of compound 2 by THF starts at the membrane defects of the neighboring vesicles, gradually eroding the membranes, then connecting the vesicles, and finally completing the tubular formation. This can be proved by the fact that no morphological transition would happen if the vesicles are collected from the suspension or if the solvents are allowed to evaporate. In order to get further insight into the inter- and intramolecular interactions between the atoms, we have constructed an amorphous state model, as given by the force field model used. It is important to emphasize that our constructed models are only valid for an entirely amorphous, isotropic, and nonoriented phase. Figure 4 shows the optimized amorphous cell structure comprising 5, 10, and 90 molecules of compound 1 or 2, water, and THF. Even though the systems are oversimplified compared to the real scenario, based on these MD calculations, some intuitive information about the distribution of each molecule in an amorphous cell can be obtained. For example, the main factor controlling the probability for water uptake in the amorphous phase of the studied compounds is the number of hydrophilic groups, which affects both the solubility parameter and the pair correlation functions when water and THF molecules are present in the cells. In both amorphous cells, the water molecules are either located close to or embedded inside compounds 1 and 2. This is probably due to the effect of the hydrophobic methyl and aryl groups located in the periphery of the compounds, which force more water molecules closer to the hydroxyl and amine groups. In addition to the hydrophilic/hydrophobic balance, the local flexibility of the central sp3 carbon affects the water uptake. As the water molecules diffuse into the compound molecules dissolved in the THF droplets, the solubility of the compounds decreases, solidifying into spherical (for 1) and tubular (for 2) polymer shells. The difference in the concentration of water significantly increases during this self-assembly process, leading to internal coalescence among the water droplets. The coalescence process originates from the deformation of the

Figure 3. Illustration of the proposed microstructure formation steps: (a) THF droplets bearing compound 1 (1.0−2.5 mg mL−1), formed by dropwise addition of water with vigorous stirring; (b) the count and size of the internal water droplets increased with time because of fast water molecule diffusion; (c) internal tiny water droplets coalesced into bigger ones; (d) double emulsion droplets formed via coalescence of the internal water droplets and the self-assembly of compound 1; (e) microcapsules obtained after the complete evaporation of THF and water molecules; (f) an array of THF droplets bearing compound 2 with a thin ellipse section; (g) vesicles in a necklace-like structure with the widened ellipse; (h) cylindrical structure formed by the maximum ellipse obtained when the internal water droplets coalesced into bigger ones; (i) microtubules obtained after the complete evaporation of THF and water molecules; and (j) microrods obtained at a concentration of compound 2 above 2.0 mg mL−1.

internal water droplets, HNSs are obtained after the complete evaporation of THF and water. In the combined solvent system, amphiphilic compound 2 might first self-assemble to form layered structures and then finally assemble to form vesicles.52,53 The process of tube formation is believed to be similar to the “curvature strain releasing” process proposed by Rowan and Nolte to explain the fusion of polystyrene− poly[(isocyano-L-alanyl-amino-ethyl)-thiophene)] from small vesicles to larger ones.54 The initially formed vesicles have a high curvature and a large number of membrane defects. The fusion of vesicles releases curvature energy, leading to a thermodynamically more stable tubular structure (Figure 3g).

Figure 4. Energy-minimized amorphous cell structures comprised of 10 and 90 molecules of water and THF inserted in the presence of five molecules of (a) compound 1 and (b) compound 2. Compounds 1 and 2 are represented by the ball model, and water and THF molecules are represented by the stick, space filling (CPK) model. 3228

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THF/water interface and the breaking of the interfacial films, since the compounds lower the interfacial tension and modify the interfacial spontaneous curvature. The internal instabilities (coalescence) lead to a gradual reduction in the internal water droplet count and an increase in diameter. Evidently, the capsule and tubule formation processes reflect the dynamic competition between the diffusion of the water molecules into the THF droplets, and the solubility of the compounds in water. Microcapsules and microtubules were thus obtained through the evaporation of the organic solvent from the selfassembled microstructures containing the compounds. 3.5. Fabrication of DOX@FA−Rh6G−1 HNS. An HNS fabricated with compound 1 was used to test the encapsulation of DOX for targeted drug release. A one-pot protocol was employed to fabricate compound 1 HNS conjugated with FA and Rh6G−COOH during the micellization, in the presence of DOX molecules.55 The resulting mixture was dialyzed by membrane (MWCO 3500) against distilled water to remove the organic solvent and the unreacted organic compounds. Figure 5 shows a TEM image of DOX@FA−Rh6G−1 HNS,

Rh6G−COOH, FA−Rh6G−1, and DOX@FA−Rh6G−1 HNS. The characteristic UV−vis absorption peaks of FA at 282 and 359 nm and Rh6G−COOH at 520 nm are observed for FA−Rh6G−1 without a noticeable shift, suggesting that FA has been successfully grafted onto compound 1. All the corresponding peaks are also observed in DOX@FA−Rh6G−1 HNS with decreased intensity. The graft of FA and Rh6G− COOH on compound 1 followed by encapsulation of DOX resulted in the decrease of the fluorescence intensity of the nascent Rh6G−COOH as expected (Figure 6b). Figure 7 shows the FTIR spectra of DOX@FA−Rh6G−1, free DOX, FA−Rh6G−1, compound 1, FA, and Rh6G−

Figure 7. FTIR spectra of (i) Rh6G−COOH, (ii) FA, (iii) compound 1, (iv) FA−Rh6G−1, (v) free DOX, and (vi) DOX@FA−Rh6G−1.

COOH. After grafting FA and Rh6G−COOH onto compound 1, the well-resolved vibrational peak at 1607 cm−1 assigned to the N−H bending vibration of the amide was present in the spectrum of FA−Rh6G−1. In addition, the vibrational peaks at 3023 and 2982 cm−1 attributed to the C−H stretching of the methyl groups from the Rh6G−COOH molecule are also observed. Compared to nascent FA and Rh6G−COOH, a bathochromic shift is noticed for FA−Rh6G−1, revealing a modification in the environment of the FA and Rh6G−COOH by grafting them onto compound 1.50 Therefore, the FTIR analysis further confirmed that FA and Rh6G−COOH were

Figure 5. TEM image of DOX@FA−Rh6G−1 HNSs obtained by reacting compound 1, FA, and Rh6G−COOH in the mole ratio 40:5:7. The mass ratio of DOX/FA−Rh6G−1 is 1/10.

which is stable enough to retain its structural integrity after modification. Figure 6a shows the UV−vis spectra of FA,

Figure 6. (a) UV−vis spectra of (i) FA, (ii) Rh6G−COOH, (iii) FA−Rh6G−1, and (iv) DOX@FA−Rh6G−1 in water at room temperature and (b) fluorescence spectra of (i) Rh6G−COOH and (ii) DOX@FA−Rh6G−1. 3229

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Figure 8. Cytotoxicity of compound 1 and FA−Rh6G−1 HNS at various concentrations to (a) 293T and (b) CT26 cells measured by the MTT assay. The percent cell viability is an average absorbance of the HNS group at different concentrations divided by that of the corresponding control (cell only) group.

Figure 9. Flow cytometer analysis of CT26 cells showing a receptor-mediated endocytosis of FA−Rh6G−1 HNS measured with pretreating the HNSs with FA (2 mM) for 1 h (FA+) to block FA receptor or with no FA pretreatment (FA−). Independent analysis was done in the serum-free RPMI1640 media as a control.

the number of living cells. The absorbance is directly proportional to the number of cells within a reasonable linear range. The percent cell viability was obtained from the average absorbance of the HNS group at different concentrations divided by that of the corresponding control group. In this study, the cell-only group was used as the control in each experiment, and therefore, its absorbance was similar for all eight parallel experiments. Compared to the control, the viability of both types of cells against compound 1 HNS was higher than 80% in a concentration range from 1 to 100 μg/mL (Figure 8). The HNSs fabricated with FA−Rh6G−1 show slightly lower cell viability. The conjugation of FA and Rh6G has little influence on the cytotoxicity of compound 1 at a concentration of HNS

successfully grafted onto compound 1. Two peaks at 2921 and 2860 cm−1 attributed to the C−H stretching of the methyl groups in free DOX were also observed on the spectrum of DOX@FA−Rh6G−1, suggesting that the DOX molecules have been encapsulated successfully into the FA−Rh6G−1 HNS. 3.6. In Vitro Cytotoxicity and Cell Uptake of FA− Rh6G−1 HNS. The biocompatibility of the HNSs is a very important factor for their utilization in biomedical applications. To investigate the biocompatibility of FA−Rh6G−1 micelles on the cellular level, FA−Rh6G−1 and compound 1 HNS were incubated with 293T and CT26 cells. The cytotoxicity of the nanosized micelles at various concentrations was determined by the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay. The absorbance of a formazan crystal at 560 nm reflects 3230

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lower than 20 μg/mL, demonstrating that the FA−Rh6G−1 HNS might be utilized for drug delivery vehicles. To investigate the receptor-mediated endocytosis of FA− Rh6G−1, the FA receptors of CT26 cells were blocked by pretreatment with FA and compared with cells treated with nonblocking treatment. Figure S15 (Supporting Information) shows the CT26 cells treated with FA−Rh6G−1 in the absence (FA−) and presence (FA+) of FA pretreatment. When FA− Rh6G−1 is added to the cells in the absence of FA pretreatment (FA−), the CT26 cells reveal a strong red color because of the Rh6G dye of the nanoparticles. However, red color is significantly decreased when the FA receptor is blocked by pretreatment with FA (FA+). These results indicate that the uptake of FA−Rh6G−1 HNSs into cells is caused by FA receptor-mediated endocytosis. Results of flow cytometric analysis of CT26 cells support these data, as shown in Figure 9. The fluorescence intensity of CT26 cells in the absence of FA receptor blocking (FA−) is stronger than in the presence of FA receptor blocking (FA+). Interestingly, the CT26 cells with FA receptor blocking (FA+) also show fluorescence intensity compared to the control, indicating that a small portion of nanoparticles is also endocytosed via a non-receptor-mediated pathway. Even though the endocytosis of nanoparticles is not fully inhibited by blocking of the FA receptor, the fluorescence intensity of CT26 tumor cells decreases when the FA receptors of tumor cells are blocked (FA+). 3.7. Loading and Controlled Release of DOX. Drug loading is influenced by the compatibility between the drug and the micelle material, as well as the solubility of the materials.56 The compatibility is highly dependent on the 3D arrangement and conformation of the different groups in the structure of the drug and the micelle core. For example, the attachment of cholesterol to the poly(caprolactone) block of a poly(ethylene glycol)-b-poly(caprolactone) copolymer significantly improved the loading of cucurbitacin I, which is similar to cholesterol in molecular structure.57 Note that the molecular structure of compound 1 is similar to that of DOX. Thus, the DOX molecules are successfully loaded into compound 1 HNSs with 57 wt % efficiency, with a drug loading capacity of 9.6 wt %. In the case of using nanotubes fabricated at low pH as drug nanocarriers, owing to their hollow cylindrical nanochannels with open ends (Figure S16, Supporting Information), DOX molecules can be encapsulated and/or adsorbed by capillary force or electrostatic interactions. However, the low drug loading efficiency and the high aspect ratio made them inappropriate to use as a drug carrier. In this sense, HNSs were chosen to study the loading and controlled release of DOX. The size and the wall thickness of the HNSs are approximately 250 and 60 nm, respectively. The time-dependent release of DOX from the DOX@FA− Rh6G−1 HNSs was measured over a 70 h period in buffered solutions at pHs of 5.0, 6.5, and 7.4, to mimick the physiological pH in normal tissue and blood, the tumor extracellular environment, and the subcellular endosome, respectively. Note that, if the same release tests were performed at a pH lower than 5.0, the self-assembled structure becomes nanotubes. Figure 10 shows the profiles under different pH conditions, revealing that DOX release is sensitive to the pH of the solution. The initial release rate is fastest at the lowest pH of 5.0, and the total amount of DOX molecules released is more than 80% after 70 h, while, at pH 7.4, less than 55% of the DOX molecules loaded into the FA−Rh6G−1 HNS are released over

Figure 10. Profiles for the time-dependent release of DOX from compound 1 HNSs in PBS solution at 37 °C at different pHs.

a 70 h period. The release amount and rate decrease in the following order: pH 5.0 > pH 6.5 > pH 7.4, demonstrating that the FA−Rh6G−1 HNSs are quite stable at pH 7.4, while significant destabilization in the micelle core takes place under acidic conditions. At acidic pH, compound 1 may be easily ionized, resulting in repulsion between the charged groups, which leads to high swelling. The degree of ionization of the amine groups increases with decreasing pH, resulting in weaker hydrogen bonds, which in turn favor drug release. Furthermore, the solubility of compound 1 also increases under acidic conditions, which also helps to diffuse the drug to the external medium. Therefore, FA−Rh6G−1 HNSs, as anticancer drug delivery micelles, can effectively target and enhance the anticancer drug delivery efficacy. Considering that the extracellular pH of tumor tissue is acidic, pH-sensitive FA−Rh6G−1 HNS micelles, specifically at acidic pH, have advantages for tumor targeting. Because the pH of the tissue and bloodstream is approximately 7.4, DOXloaded micelles can selectively kill the tumor cells because of the delivery of the anticancer drug at low pH. Thus, combining the receptor-mediated endocytosis of FA−Rh6G−1 HNSs into CT26 cells with their pH-sensitive DOX releasing behavior, tumor targeting might be greatly enhanced. This is a fascinating topic that has been studied in detail, and the related in vitro and in vivo studies are ongoing.

4. CONCLUSIONS Tetraarylmethane derivatives comprising two pyrogallol and two alkyl substituted aniline connected by a typical sp3hybridized carbon were synthesized and investigated for their self-assembly behavior. The self-assembly of compounds 1 and 2 showed that the concentration of the compound, the type of solvent, and the pH of the solvent play critical roles in determining the final morphology. Compound 2 yielded nanotubules and microrods from the self-assembly at low and high concentrations, respectively. The self-assembly of 1 at different concentrations and solvent mixtures gave highly uniform HNSs with tunable size. By varying the pH of the solution from 4.0 to 11.0, the self-assembled morphology was diversified from nanotubules to solid nanospheres. As a means of designing a targeted anticancer drug delivery system, FA and Rh6G were coordinatively conjugated to compound 1, giving highly uniform HNSs that are reasonably biocompatible. The receptor-mediated endocytosis of FA−Rh6G−1 in CT26 colon carcinoma cells showed that the uptake of FA−Rh6G−1 HNS 3231

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(11) Li, H. Q.; Bahuleyan, B. K.; Johnson, R. P.; Shchipunov, Y. A.; Suh, H.; Ha, C. S.; Kim, I. Morphology-Tunable Architectures Constructed by Supramolecular Assemblies of α-Diimine Compound: Fabrication and Application as Multifunctional Host Systems. J. Mater. Chem. 2011, 21, 17938−17945. (12) Li, Y. J.; Li, X. F.; Li, Y. L.; Liu, H. B.; Wang, S.; Gan, H. Y.; Li, J. B.; Wang, N.; He, X. R.; Zhu, D. B. Controlled Self-Assembly Behavior of an Amphiphilic Bisporphyrin−Bipyridinium−Palladium Complex: From Multibilayer Vesicles to Hollow Capsules. Angew. Chem., Int. Ed 2006, 45, 3639−3643. (13) Wu, G.; Verwilst, P.; Xu, J.; Xu, H. P.; Wang, R.; Smet, M.; Dehaen, W.; Faul, C. F. J.; Wang, Z. Q.; Zhang, X. Bolaamphiphiles Bearing Bipyridine as Mesogenic Core: Rational Exploitation of Molecular Architectures for Controlled Self-Assembly. Langmuir 2012, 28, 5023−5030. (14) Gao, J.; Yan, J.; Beeg, S.; Long, D.-L.; Cronin, L. Assembly of Molecular “Layered” Heteropolyoxometalate Architectures. Angew. Chem. 2012, 124, 3429−3432. (15) Lin, Z.-Q.; Sun, P.-J.; Tay, Y.-Y.; Liang, J.; Liu, Y.; Shi, N.-E.; Xie, L.-H.; Yi, M.-D.; Qian, Y.; Fan, Q.-L.; Zhang, H.; Hng, H. H.; Ma, J.; Zhang, Q.; Huang, W. Kinetically Controlled Assembly of a Spirocyclic Aromatic Hydrocarbon into Polyhedral Micro/Nanocrystals. ACS Nano 2012, 6, 5309−5319. (16) Hasell, T.; Chong, S. Y.; Jelfs, K, E.; Adams, D. J.; Cooper, A. I. Porous Organic Cage Nanocrystals by Solution Mixing. J. Am. Chem. Soc. 2012, 134, 588−598. (17) Zhang, L. D.; Jeong, Y.-I.; Zheng, S. D.; Suh, H. S.; Kang, D. H.; Kim, I. Fabrication of Microspheres via Solvent Volatization Induced Aggregation of Self-Assembled Nanomicellar Structures and Their Use as a pH-Dependent Drug Release System. Langmuir 2013, 29, 65−74. (18) Ikeda, S.; Ishino, S.; Harada, T.; Okamoto, N.; Sakata, T.; Mori, H.; Kuwabata, S.; Torimoto, T.; Matsumura, M. Ligand-Free Platinum Nanoparticles Encapsulated in a Hollow Porous Carbon Shell as a Highly Active Heterogeneous Hydrogenation Catalyst. Angew. Chem., Int. Ed. 2006, 45, 7063−7066. (19) Su, J. F.; Wang, L. X.; Ren, L.; Huang, Z. Mechanical Properties and Thermal Stability of Double-Shell Thermal-Energy-Storage Microcapsules. J. Appl. Polym. Sci. 2007, 103, 1295−1302. (20) Yu, A. M.; Wang, Y. J.; Barlow, E.; Caruso, F. Mesoporous Silica Particles as Templates for Preparing Enzyme-Loaded Biocompatible Microcapsules. Adv. Mater. 2005, 17, 1737−1741. (21) Emami, J.; Hamishehkar, H.; Najafabadi, A. R.; Gilani, K.; Minaiyan, M.; Mahdavi, H.; Nokhodchi, A. A Novel Approach to Prepare Insulin-Loaded Poly(Lactic-Co-Glycolic Acid) Microcapsules and the Protein Stability Study. J. Pharm. Sci. 2009, 98, 1712−1731. (22) Poe, S. L.; Kobaslija, M.; McQuade, D. T. Mechanism and Application of a Microcapsule Enabled Multicatalyst Reaction. J. Am. Chem. Soc. 2007, 129, 9216−9221. (23) Sakai, S.; Hashimoto, I.; Kawakami, K. Production of CellEnclosing Hollow-Core Agarose Microcapsules via Jetting in WaterImmiscible Liquid Paraffin and Formation of Embryoid Body-Like Spherical Tissues From Mouse ES Cells Enclosed within These Microcapsules. Biotechnol. Bioeng. 2008, 99, 235−243. (24) Liu, H.; Xu, J.; Li, Y.; Li, Y. Aggregate Nanostructures of Organic Molecular Materials. Acc. Chem. Res. 2010, 43, 1496−1508. (25) Zhao, Y. S.; Fu, H.; Peng, A.; Ma, Y.; Xiao, D.; Yao, J. LowDimensional Nanomaterials Based on Small Organic Molecules: Preparation and Optoelectronic Properties. Adv. Mater. 2008, 20, 2859−2876. (26) Gazave, J.-M.; Rancurel, A.; Grenier, G. Certain Biphenyl Derivatives Used to Treat Disorders Caused by Increased Capillary Permeability. US Patent 4015017, 1977. (27) Okubo, H.; Feng, F.; Nakano, D.; Hirata, T.; Yamaguchi, M. Miyashita, Synthesis and Monolayer Behaviors of Optically Active 1,12-Dimethylbenzo[c]phenanthrene-5,8-diamides and the Formation of Chiral Langmuir-Blodgett Films. Tetrahedron 1999, 55, 14855− 14864.

into cells was caused by the FA receptor-mediated endocytosis, which was further demonstrated by flow cytometer analysis. The DOX molecules were successfully loaded into the FA− Rh6G−1 HNSs with 9.6 wt % efficiency. In vitro release tests at different pH values showed that the FA−Rh6G−1 HNS system bearing DOX molecules gave pH-dependent release behavior, with the highest rate under acidic conditions. Thus, the resulting FA−Rh6G−1 HNS with reasonable receptor-mediated endocytosis might be a promising candidate for a targeted drug delivery system for cancer therapy.

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ASSOCIATED CONTENT

* Supporting Information S

Additional experimental details and results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+82) 51-510-2466. Fax: (+82) 51-513-7720. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants-in-aid for the World Class University Program (No. R32−2008−000−10174−0) and the Basic Research Program through the National Research Foundation of Korea, funded by the MEST (2012R1A1A2041315).

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REFERENCES

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dx.doi.org/10.1021/la305069e | Langmuir 2013, 29, 3223−3233