Paclitaxel Dimer Assembling Vesicles: Reversible

Research Article www.acsami.org

Cyclodextrin/Paclitaxel Dimer Assembling Vesicles: Reversible Morphology Transition and Cargo Delivery Qing Pei,†,‡ Xiuli Hu,*,† Lei Wang,† Shi Liu,† Xiabin Jing,† and Zhigang Xie*,† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China ‡ University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China S Supporting Information *

ABSTRACT: Here, we developed stable supramolecular binary vesicles on the basis of the host−guest interaction between β-cyclodextrins (β-CDs) and paclitaxel (PTX) dimer. The inclusion complexation between PTX dimer and β-CDs in water was studied by proton nuclear magnetic resonance spectroscopy and two-dimensional rotating-frame Overhauser effect spectroscopy. The resulting inclusion complex was amphiphilic and could self-assemble into vesicles with average diameter of 230 nm. The vesicles could evolve to nanoparticles (NPs) by adding competitive binding guest amantadine hydrochloride or by digesting β-CDs through α-amylase. Moreover, this process was reversible, and the NPs could also transform to vesicles by adding enough β-CDs again. The obtained hollow supramolecular vesicles were further explored to load hydrophilic dye indocyanine green molecule or hydrophobic anticancer drug doxorobicin for their controlled release under external stimulus. This work provides a new strategy for the design of supramolecular systems by using prodrug as building blocks. KEYWORDS: supramolecular, vesicles, host−guest complexation, reversible structure transition, drug delivery

1. INTRODUCTION Inspired by the self-assembly of amphiphiles in nature, scientists have devoted considerable efforts to the design and construction of biomimetic nanostructures1,2 from the selfassembly of surfactants,3 phospholipids,4 amphiphilic block polymers,5−9 and macrocyclic amphiphiles.10,11 Among them, supramolecular self-assembly from macrocyclic amphiphiles has attracted significant attention in biomedical fields.12−14 Compared with traditional covalent amphiphiles, supraamphiphiles are mediated by noncovalent forces,15−20 which reduce the need of tedious chemical synthesis and are more environmentally friendly.5,21 More importantly, desired architectures can be exquisitely designed and easily controlled through intelligent design of the building blocks.22−25 Macrocyclic compounds, such as crown ethers,26,27 cyclodextrins (CDs),15,28 calixarenes,29 cucurbiturils,30,31 pillararenes, and their analogues,32−35 can all act as supramolecular hosts to encapsulate guest molecules into their cavities and have been extensively designed and synthesized in the past decades.36−38 CDs, as one of the most widely used host molecules in supramolecular chemistry, have been widely investigated for their complexation with a series of guest molecules, including azobenzene compounds, ferrocene derivatives, anthraquinone derivates, and asymmetric viologen.28,39 The natural availability from starch endows CDs with low cost, water solubility, and good biocompatibility, which has further fostered extensive studies on using CDs for drug/gene delivery.40 In the early study, bare CDs or CD derivatives were used to form inclusion complexes with specific drugs that could serve as guest © XXXX American Chemical Society

molecules, including camptothecin, paclitaxel, curcumin, doxorubicin (DOX), and anti-inflammatory drugs, thus enhancing their bioavailability.41,42 However, it had been reported that the introduction of simple CDs had fairly limited improvement on the solubility of drugs. For better pharmaceutical uses, chemical modifications on CDs were carried out to further improve drug solubility, encapsulation ability, and controlled drug release.43−45 More advanced CDbased drug-delivery systems need to be developed. Paclitaxel (PTX) is an anticancer drug used in clinic for wide range of solid tumors, but its strong hydrophobicity drastically limits its applications. Nanoscale formulation has been proved to be an effective approach to increase the solubility and enhance drug accumulation in tumor site, and various polymers have been designed and used to solubilize PTX for better clinical effects.46−49 In pursue of an easy, effective, and environmentally friendly nanoparticle (NP) formulation, alternative carriers, such as CDs and CD derivatives, have been considered as promising candidates for PTX delivery.50,51 The inclusion complexes of PTX with various CDs and CD analogues have been investigated. However, due to the bulky structure of PTX, the supramolecular complexation between CDs and PTX is weak.52 Various mono-CD derivatives53,54 and β-CD dimers bridged with different spacers55 have been designed and showed obvious enhancement of PTX solubility Received: June 7, 2017 Accepted: July 25, 2017

A

DOI: 10.1021/acsami.7b08110 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of the Complexation between PTX Dimer and CD, Formation of Supramolecular Vesicles, and Their Reversible Structure Transformation Between Nanoparticles and Vesicles Induced by Dual Stimulus (α-Amylase and Adamantanamine Hydrochloride (AD))

Figure 1. (A) 1H NMR spectra (D2O, 298 K, 400 MHz) of CD/C6 and CD/NP (C6) complexes in comparison with CD. (B) Possible recognition binding mode of the CD/C6 complex. B


Research Article


Figure 2. (A) Size distribution and Tyndall effect of CD/C6 NVs. (B) TEM image and (C) enlarged TEM image of CD/C6 NVs. (D) SEM image and enlarged SEM image (inset) of CD/C6 NVs. (E) AFM image and (F) the corresponding height profile of CD/C6 NVs.

and antitumor activity compared to those included in natural βCD. Another viable strategy is conjugating CD, drug, or both CD and drug to different polymers, where multivalent polymer−polymer complexation is achieved by virtue of the host−guest interactions.56 However, PTX is reported to be prone to self-aggregation because of the π−π interaction of its planar aromatic rings and the high crystallinity,57 which challenges the stability of the present nanoformulations. Instead of modifying the CDs for CD/PTX complexation, in this study, we try to prepare a novel inclusion complex between β-cyclodextrins (β-CDs) and PTX dimer, in which two PTX molecules are conjugated together via a cleavable ester linkage. Recently, we have found that organic dimer is prone to selfassemble into nanoparticles in aqueous solution,58,59 and one of the most glaring examples is that nanoparticles obtained from the self-assembly of PTX dimers show a 2500-fold higher aqueous solubility compared to that of free PTX.60 Such dimeric prodrug strategy has been proved to be an effective approach to obtain high-drug-loading or self-delivery nanoparticles.61,62 Therefore, we investigate the host−guest interaction between β-CD (abbreviated as “CD”) and PTX dimer and their formed supramolecular structure, which combines the favorable features of supramolecular chemistry and dimeric prodrug strategy. It is quite different from the reported binding behavior of PTX with CD, the PTX dimer binds two water-soluble CD molecules to achieve a stable inclusion complex with PTX dimer as the bridge. Furthermore, the resulting inclusion complex is amphiphilic and can selfassemble into supramolecular vesicles. To the best of our knowledge, supramolecular amphiphilic inclusion complex based on dimeric prodrug has not been reported so far. The obtained supramolecular vesicles are further explored to load hydrophilic and hydrophobic cargos and to release them upon external stimulus. A schematic illustration of the formation of supramolecular vesicles and their reversible structure transition are shown in Scheme 1.

2. RESULTS AND DISCUSSION 2.1. Host−Guest Complexation Study. PTX dimer bridged with adipic acid (abbreviated as “C6”) was first synthesized according to our previous work with high yields (>80%).60 Its chemical structure was confirmed by proton nuclear magnetic resonance (1H NMR) spectroscopy and a linear ion trap mass spectrometer. The host−guest complexation between CD and C6 was studied by 1H NMR spectroscopy and two-dimensional (2D) rotating-frame Overhauser effect spectroscopy (ROESY). As shown in Figure 1A, C6 induced obvious upfield chemical shifts for the signals related to H3 and H5 protons (Δδ = 0.022 and 0.017, respectively) in the internal cavities of CD, which demonstrated the formation of inclusion complex between C6 and CD. In addition, the comparable slight chemical shifts of H4 and H6 protons (Δδ = −0.005 and 0.002, respectively) of CD suggested the C6 had entered the CD cavities from the top of the truncated cone structure.53 Figure 1A showed similar chemical shifts of CD after complexation with PTX dimer nanoparticles, which will be illustrated in Section 2.3. Twodimensional ROESY was carried out to further investigate the complex pattern between C6 and CD. As illustrated in Figures S1 and S2, H5 proton in CD cavity and H4 proton in external of CD exhibited obvious associations with the A and C phenyl rings of C6, respectively, which further indicated the successful entrance of PTX dimer into CD internal cavities. On the basis of the above 1H NMR and 2D ROESY spectra, a possible binding model was given in Figure 1B. Phenyl ring A of C6 entered the internal cavities of CD, whereas phenyl ring C interacted with H4 protons on the surface of CD (Figure S2). The complexation stoichiometry ratio of C6 to CD was determined to be 1:2 in aqueous solution according to the Job plot tested by ultraviolet−visible (UV−vis) spectroscopy (Figure S3). Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) were further employed to investigate the molecular interactions between C6 and CD and elucidate their complexation mechanism. As shown in Figure S4, the C


Research Article


Figure 3. (A) TEM image and (B) size distribution of CD/C6 NVs after α-amylase treatment. (C) TEM image and (D) size distribution and Tyndall effect (inset) of CD/NP(C6) NVs obtained by complexation between CD and C6 NPs. (E) TEM image and (F) size distribution of CD/C6 NVs after AD treatment.

characteristic absorption peaks (νOH = 3373 cm−1, νCH = 2923 cm−1, and δC−O−C = 1030 cm−1) of CD and ester bond absorption (νCO = 1737 cm−1 and νC−O = 1247 cm−1) of C6 could be found in both curves of physical mixtures and inclusion complexes, demonstrating the coexistence of CD and C6 in the former and the latter. However, the curve of the inclusion complex was distinctly different from that of the physical mixture. The absorption peaks (δAr−H = 798 cm−1, νCH3 = 2969 cm−1, and νCH2 = 2853 cm−1) belonging to C6 existed in the physical mixture curve but disappeared in the inclusion complex. Some shifts of the peaks corresponding to hydrogenbonded OH groups of CD (from 3373 to 3396 cm−1) were also observed, suggesting part of the hydrogen bonds were broken. All of the above information implied that inclusion complex was formed between C6 and CD. XRD patterns provided the status of C6 and CD in the complex. As shown in Figure S5, CD showed a diffractogram consistent with its typical crystal state, whereas C6 was amorphous due to the introduction of adipic acid. The physical mixture pattern contained some sharp peaks from CD and could be regarded as a superimposition of crystalline CD and amorphous C6, indicating their initial states in the physical mixture. On the contrary, the sharp peaks of CD almost disappeared in the inclusion complex, which displayed an amorphous nature, suggesting the new formulation between C6 and CD. 2.2. Construction of Supramolecular Binary Vesicles. The formed CD/C6 inclusion complex was a supramolecular amphiphile and expected to self-assemble into higher-order aggregates. The self-assembly of the CD/C6 inclusion complex was prepared using the nanoprecipitation method. As shown in Figure 2A, the obtained CD/C6 complex exhibited a tinge of blue opalescence and typical Tyndall effect (Figure 2A, inset), indicating the formation of abundant nanoaggregates in the solution. Dynamic light scattering (DLS) results showed that the average hydrodynamic size of the nanoaggregates was about

230 nm with a narrow size distribution (PDI = 0.082) (Figure 2A). Under the same condition, obvious white precipitates were formed for CD and free PTX (Figure S6), suggesting the selfassembly between CD and C6. The morphology and size of the resulted nanoaggregates were further characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The TEM and enlarged TEM images showed spherical morphology with distinct color contrast between the center and the periphery of the assemblies, indicating the formation of vesicular structure (Figure 2B,C). The partially collapsed morphologies in Figure 2B also validated their vesicular structure. The average diameter of the nanovesicles (NVs) was about 230 nm, according to TEM results. The hollow vesicular morphology was further confirmed by SEM (Figure 2D), as evidenced by spherical morphology with a dark core and a light periphery. AFM results shown in Figure 2E illustrated similar spherical morphology and average diameter to those of TEM and SEM. According to the height image and height profile in AFM (Figure 2F), the widths of the assemblies were about 250 nm, whereas the heights were only 25−40 nm, implying that these assemblies were oblate and sufficiently flattened on the surface of silicon disk, which further confirmed the vesicular structure.63,64 Notably, the obtained vesicles (abbreviated as CD/C6 NVs) were quite stable and the uniform vesicular morphology kept intact after storing for 3 weeks without external stimuli, as evidenced by TEM and DLS results (Figure S7). 2.3. Responsiveness of the Supramolecular Vesicles and Reversibility. The dynamic nature of the supramolecular interactions endows the obtained CD/C6 NVs amylase- and amantadine-responsive property.19,65 As shown in Figure 3A, after adding α-amylase to the solution of CD/C6 NVs, the previous vesicular morphology (Figure 2) turned to solid spheres according to the TEM images. Considering the digestion of CD host molecules by the α-amylase, we inferred D


Research Article


Figure 4. (A) UV−vis spectra of free ICG, CD/C6 NVs, and ICG@CD/C6 NVs. (B) Size distribution and schematic view (inset) of ICG@CD/C6 NVs. (C) TEM images and (D) enlarged TEM images of ICG@CD/C6 NVs. (E, F) Fluorescence intensity changes of ICG@CD/C6 NVs before and after treatment with α-amylase and AD, respectively.

the skeleton of the vesicles but also serves as antitumor agent after cleavage into its authentic form by intracellular specific enzyme. The cell cytotoxicity experiment was conducted via a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The blank CD showed no cytotoxicity against HepG2 cells even at the concentration of 1000 μg/mL (Figure S9A). The cellular proliferation inhibition abilities of Taxol, C6 NPs, CD/C6 NVs, and CD/NP(C6) NVs were evaluated against HepG2 cells at different concentrations. As shown in Figure S9B, all samples exhibited efficient cell growth suppression and comparable cytotoxicity against HepG2 cells in a concentration-dependent manner for 48 h. The images of cells after incubation with different formulations as shown in Figure S9C were in accordance with the MTT results. The incubation of HepG2 cells with CD displayed vigorous vitality, similar to that of control groups, whereas all of the PTXcontaining groups, including Taxol, C6 NPs, CD/C6 NVs, and CD/NP(C6) NVs, showed obvious cancer-cell-killing efficacy after 48 h incubation (Figure S9C). These results indicated that PTX dimer in the supramolecular nanovesicles showed efficient therapeutic effect after entering tumor cells. 2.5. Loading and Release of Indocyanine Green (ICG) and DOX. Supramolecular chemistry provides a facile way to construct assemblies with stimuli responsiveness by virtue of host−guest interactions. The hollow structure of CD/C6 NVs allows for encapsulation of both hydrophilic and hydrophobic agents. ICG, a near-infrared hydrophilic dye approved by the U.S. Food and Drug Administration for clinic, was selected as model molecule to assess the ability of encapsulating hydrophilic molecule into the supra-amphiphilic vesicles, and the corresponding stimuli-triggered release was characterized. ICG-encapsulated supra-amphiphilic vesicles were prepared similarly to CD/C6 NVs by adding ICG to CD aqueous solution, and the obtained nanovesicles were referred as ICG@ CD/C6 NVs. The ICG loading content and loading efficiency

that the solid nanoparticles were corresponding to C6 nanoparticles (abbreviated as C6 NPs), according to our previous report.60 So the nanoparticles directly self-assembled from C6 were obtained by injecting tetrahydrofuran (THF) solution of C6 into water. The morphology and size distribution were characterized by TEM and DLS, as shown in Figure S8A, similar to that in Figure 3A,B. This verified that the solid spheres obtained after adding α-amylase to CD/C6 NV solution were corresponding to C6 NPs. Next, we investigated its reversibility. CD was added to the C6 NP solution, and vesicles (abbreviated as CD/NP(C6) NVs) reoccurred as shown in Figure 3C, suggesting that supramolecular complex could also be formed between C6 NPs and CD. In addition, similar typical Tyndall effect (Figure 3D) and upfield chemical shifts of CD (Figure 1A) were both observed, and the average size was about 230 nm tested by DLS (Figure 3D). On the other hand, the morphology and size distribution of C6 NPs kept intact after incubating with equal concentration of α-amylase (Figure S8B), indicating α-amylase could digest the CD layer but had no effect on C6 NPs. Taken together, we concluded that CD/C6 NVs could evolve to C6 NPs by adding α-amylase through the degradation of hydrophilic CD layer. In the meantime, this process was reversible, and CD complexation could also direct C6 NPs reassemble and transition to supramolecular vesicles. The reversible structure transition could also be tuned by adding competitive amantadine hydrochloride (AD) guests. As shown in Figure 3E,F, similar solid C6 NPs were also observed by adding AD to CD/C6 NV solution. It is well known that AD has a higher binding constant with CD than PTX. Therefore, as a competitive guest to C6, AD is prone to enter the cavity of CD to replace C6 and thus expose it, which induces the reassembly to solid nanoparticles. 2.4. Anticancer Efficiency of Supra-Amphiphilic Vesicles. The C6 in the CD/C6 NVs not only functions as E


Research Article


Figure 5. (A) UV−vis spectra of DOX, CD/C6 NVs, and DOX@CD/C6 NVs. (B) Size distribution and schematic view (inset) of DOX@CD/C6 NVs. (C) TEM image and (D) enlarged TEM image of DOX@CD/C6 NVs. (E, F) Fluorescence intensity changes of DOX@CD/C6 NVs induced by α-amylase and AD, respectively. (G) Confocal laser scanning microscopy (CLSM) images of HepG2 cells incubated with DOX@CD/C6 NVs at 37 °C for 1.5 h. From left to right: cell images dyed with 4′,6-diamidino-2-phenylindole (blue), DOX@CD/C6 NVs (green), Lyso-Tracker green (red), and their merged images. The scale bar is 20 μm.

were 15.1 wt % and 93%, respectively. After encapsulating ICG, the color of ICG@CD/C6 NVs in aqueous solution changed from slight pale to light green (Figure 4A, inset). Obvious UV− vis absorption spectra of ICG were observed (Figure 4A), and the red shift of the maximum absorption wavelength of ICG was due to its aggregation, indicating the successful loading of ICG into the vesicles. After loading with ICG, the mean diameters of ICG@CD/C6 NVs slightly increased from 230 to 239 nm determined by DLS, as shown in Figure 4B. TEM and enlarged TEM images indicated their intact vesicular structure after loading with ICG (Figure 4C,D). On-demand ICG release could be realized by the introduction of external stimulus (αamylase and AD). As shown in Figure 4E, fluorescence intensity of ICG in ICG@CD/C6 NVs increased after adding α-amylase, which was attributed to the destruction of vesicular structure by α-amylase, and the remaining solid C6 NPs could not accommodate hydrophilic ICG molecule. A similar fluorescence intensity increase was also observed after treating with competitive guest molecules (AD) (Figure 4F).

Anticancer drug DOX, desalted by triethylamine (TEA), was selected as hydrophobic model cargo to be loaded into the hydrophobic membranes of CD/C6 NVs. The obtained DOXloaded vesicles were referred as DOX@CD/C6 NVs. The DOX loading content and loading efficiency were 6 wt % and 81%, respectively. The apparent light pink in DOX@CD/C6 NVs and the appearance of DOX characteristic absorption from 420 to 550 nm in the UV−vis absorption spectra indicated the successful encapsulation of DOX into the supra-amphiphilic vesicles (Figure 5A). TEM and DLS were used to characterize the morphology and size distribution of obtained DOX@CD/ C6 NVs. TEM images showed similar spherical vesicles to those of CD/C6 NVs, and DLS results indicated that the average diameter of DOX@CD/C6 NVs was 239 nm (Figure 5B−D). Different from the ICG release from ICG@CD/C6 NVs, the release of DOX from the DOX@CD/C6 NVs under external stimulus (α-amylase and AD) was indistinctive according to the variation of the fluorescence intensity of DOX (Figure 5E,F). The possible reason should be that the F


Research Article


water (10 mL) at room temperature with vigorous stirring. After removing THF by evaporation and the unincorporated C6 by centrifugation (5000 rpm, 5 min), C6 NPs were obtained and mixed with CD with stirring at room temperature overnight to obtain CD/ NP(C6) NVs. 4.3. Detection of the Complexation Stoichiometry Ratio between CD and C6. Mother solutions of CD and C6 (3 × 10−4 mol/L) were prepared by directly dissolving certain molar quantities of powder samples in a water−ethanol solution (1:1, v/v). Then, a series of solutions with host and guest molecule molar ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 were prepared. UV−vis absorption of the mixture of CD and C6 in water at different molar ratios was tested. 4.4. Preparation of ICG- or DOX-Loaded CD/C6 NVs. ICGencapsulated supra-amphiphilic vesicles (ICG@CD/C6 NVs) were prepared similarly to CD/C6 NVs by adding ICG to CD aqueous solution. Briefly, 4 mL of THF solution of C6 (1 mg) was injected into distilled water (10 mL) containing CD (1.25 mg) and ICG (0.5 mg) at room temperature with vigorous stirring. After removing THF by evaporation and the unincorporated C6 by centrifugation (5000 rpm, 5 min), the mixture was dialyzed for 48 h to remove the unencapsulated ICG. ICG@CD/C6 NVs were obtained after freeze drying. DOX-encapsulated supra-amphiphilic vesicles (DOX@CD/C6 NVs) were prepared similarly by injecting the mixture of DOX solution in N,N-dimethylformamide and TEA and C6 solution in THF into 10 mL of CD aqueous solution. 4.5. In Vitro Stability of CD/C6 NVs. The stability of CD/C6 NVs was evaluated by detecting the change of their size and size distribution at room temperature for different time periods. 4.6. Evaluation of Crystalline Status of CD, C6, Their Physical Mixture, and Inclusion Complex. Powder X-ray diffraction (PXRD) was employed to test the status of the four samples, including C6, CD, their physical mixture, and inclusion complex. After being fully mulled, they were determined by a Bruker D8 Focus power X-ray diffractometer using Cu Kα radiation, and the incidence angle 2θ was monitored from 8 to 30°. 4.7. α-Amylase- and AD-Triggered Morphology Change and Corresponding Drug Release. Due to the dynamic nature of the supramolecular system, the CD/C6 NVs could inherit the amylaseand AD-responsive property of CD. α-Amylase (59.3 units/mg) or AD was added to aqueous solution of CD/C6 NVs under moderate shaking at 37 °C for 48 h. Then, the TEM images of reconstructed dispersion were evaluated. For the amylase- or AD-induced drug release of ICG@CD/C6 NVs and DOX@CD/C6 NVs, an aqueous dispersion of ICG@CD/C6 NVs or DOX@CD/C6 NVs was incubated with α-amylase or AD, respectively, at 37 °C air bath under shaking for 48 h. The reaction mixture was monitored by fluorescence spectroscopy.

hydrophobic DOX was encapsulated in the C6 layer. Different from the ICG locating in the cavity of the vesicles, the morphology transition from vesicles to C6 NPs induced by αamylase or AD had little effect on its release from the hydrophobic layer due to the stability of C6 NPs. CLSM was then used to investigate the cellular uptake of supramolecular vesicles by virtue of the inherent fluorescence of DOX. As shown in Figure S10, red fluorescence signals uniformly distributed in the cytoplasm of HepG2 cells after being cultured with DOX@CD/C6 NVs, and the fluorescence intensity increased as a function of incubation time from 0.5 to 4 h, indicating efficient internalization by cancer cells and the endocytosis continued in a time-dependent manner. Furthermore, the perfect superimposing signal between the green fluorescence DOX in DOX@CD/C6 NVs and the red fluorescence of lysosome tracker in Figure 5G indicated their active endocytosis through endolysosomal pathway. The cell cytotoxicity of DOX-loaded vesicles (DOX@CD/C6 NVs) was also evaluated. As shown in Figure S11, DOX@CD/C6 NVs exhibited much higher cytotoxicity than CD/C6 NVs, indicating the synergistic capability of DOX and the PTXbased vesicles. However, the lower cytotoxicity of DOX@CD/ C6 NVs compared to free DOX was ascribed to the delayed release of DOX from the vesicles. From all of these results, the obtained supra-amphiphilic vesicles showed promising application as nanovesicles for both hydrophilic and hydrophobic cargoes with controlled release behavior.

3. CONCLUSIONS In summary, we have successfully constructed stable supramolecular vesicles (CD/C6 NVs) on the basis of the host− guest interaction between paclitaxel dimer (C6) and β-CDs. Benefiting from the intrinsic advantages of supramolecular interaction, the obtained vesicles are transformable. Adding the competitive binding guest, AD, or α-amylase, to CD/C6 NV aqueous solution could induce C6 exposing from the inclusion complex, and the morphology evolves from vesicles to C6 nanoparticles. Significantly, the process is reversible, and C6 nanoparticles could also transform to vesicles by adding enough β-CDs. Hydrophilic ICG molecule and hydrophobic anticancer drug DOX are successfully encapsulated into the inner cavities and the hydrophobic membrane of CD/C6 NVs, respectively. The external stimulus α-amylase- and AD-induced drug release is evaluated. The present study provides a novel method for the construction of responsive and transformable vesicles, which have great potential applications for drug delivery.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08110. Materials; experimental details and analytic data; NMR and ROESY spectra; UV−vis absorption spectra; FT-IR spectra; XRD spectra; TEM, DLS, and CLSM images; and cellular uptake (PDF)

4. EXPERIMENTAL SECTION 4.1. Preparation of Supramolecular Vesicles with PTX Dimer and β-CD. The inclusion complexes between C6 and CD were prepared using the nanoprecipitation method. Briefly, 4 mL of C6 (1 mg) solution in THF was injected into the solution of CD (1.25 mg) in distilled water (10 mL) at room temperature with vigorous stirring. The mixture was stirred at room temperature until complete evaporation of the organic solvent. The residue was centrifuged (5000 rpm, 5 min) to remove the unincorporated C6 and lyophilized to yield 1.91 mg of white solid (CD/C6 NVs) (yield, 85%). The content of C6 in the inclusion complexes was determined by highperformance liquid chromatography (Shimadzu, CBM-20A) with a UV−vis detector. 4.2. Complexation of PTX Dimer Nanoparticles with β-CDs. C6 nanoparticles (abbreviated as C6 NPs) without CD were first prepared using the above-mentioned nanoprecipitation method. Briefly, 4 mL of THF solution of C6 was injected into distilled

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.H.). *E-mail: [email protected] (Z.X.). ORCID

Lei Wang: 0000-0003-4395-5002 Zhigang Xie: 0000-0003-2974-1825 Notes

The authors declare no competing financial interest. G


Research Article


■

(21) Jeon, Y. J.; Bharadwaj, P. K.; Choi, S.; Lee, J. W.; Kim, K. Supramolecular Amphiphiles: Spontaneous Formation of Vesicles Triggered by Formation of A Charge-Transfer Complex in A Host. Angew. Chem. 2002, 114, 4654−4656. (22) Jin, H.; Liu, Y.; Zheng, Y.; Huang, W.; Zhou, Y.; Yan, D. Cytomimetic Large-Scale Vesicle Aggregation and Fusion Based on Host−Guest Interaction. Langmuir 2012, 28, 2066−2072. (23) Liu, Y.; Yu, C.; Jin, H.; Jiang, B.; Zhu, X.; Zhou, Y.; Lu, Z.; Yan, D. A Supramolecular Janus Hyperbranched Polymer and Its Photoresponsive Self-Assembly of Vesicles with Narrow Size Distribution. J. Am. Chem. Soc. 2013, 135, 4765−4770. (24) Chakrabarty, R.; Mukherjee, P. S.; Stang, P. J. Supramolecular Coordination: Self-Assembly of Finite Two-and Three-Dimensional Ensembles. Chem. Rev. 2011, 111, 6810−6918. (25) Rangadurai, P.; Molla, M. R.; Prasad, P.; Caissy, M.; Thayumanavan, S. Temporal and Triggered Evolution of Host-Guest Characteristics in Amphiphilic Polymer Assemblies. J. Am. Chem. Soc. 2016, 138, 7508−7511. (26) Dong, S.; Luo, Y.; Yan, X.; Zheng, B.; Ding, X.; Yu, Y.; Ma, Z.; Zhao, Q.; Huang, F. A Dual-Responsive Supramolecular Polymer Gel Formed by Crown Ether Based Molecular Recognition. Angew. Chem., Int. Ed. 2011, 50, 1905−1909. (27) Zheng, B.; Wang, F.; Dong, S.; Huang, F. Supramolecular Polymers Constructed by Crown Ether-Based Molecular Recognition. Chem. Soc. Rev. 2012, 41, 1621−1636. (28) Del Valle, E. M. Cyclodextrins and Their Uses: A Review. Process Biochem. 2004, 39, 1033−1046. (29) Chi, X.; Zhang, H.; Vargas-Zúñiga, G. I.; Peters, G. M.; Sessler, J. L. A Dual-Responsive Bola-Type Supra-Amphiphile Constructed from A Water-Soluble Calix [4] Pyrrole and A TetraphenyletheneContaining Pyridine bis-N-oxide. J. Am. Chem. Soc. 2016, 138, 5829− 5832. (30) Chen, K.; Kang, Y. S.; Zhao, Y.; Yang, J. M.; Lu, Y.; Sun, W. Y. Cucurbit [6] Uril-Based Supramolecular Assemblies: Possible Application In Radioactive Cesium Cation Capture. J. Am. Chem. Soc. 2014, 136, 16744−16747. (31) Kim, K. Mechanically Interlocked Molecules Incorporating Cucurbituril and Their Supramolecular Assemblies. Chem. Soc. Rev. 2002, 31, 96−107. (32) Liu, X.; Jia, K.; Wang, Y.; Shao, W.; Yao, C.; Peng, L.; et al. Dual-Responsive Bola-Type Supra-Amphiphile Constructed from Water-Soluble Pillar [5] arene and Naphthalimide-Containing Amphiphile for Intracellular Drug Delivery. ACS Appl. Mater. Interfaces 2017, 9, 4843−4850. (33) Zhou, J.; Chen, M.; Diao, G. Magnetic-Responsive Supramolecular Vesicles From Self-Organization of Amphiphilic Pillar [5] arene and Application in Controlled Release. ACS Appl. Mater. Interfaces 2014, 6, 18538−18542. (34) Cao, Y.; Hu, X. Y.; Li, Y.; Zou, X.; Xiong, S.; Lin, C.; Shen, Y. Z.; Wang, L. Multistimuli-Responsive Supramolecular Vesicles Based on Water-Soluble Pillar[6]arene and SAINT Complexation for Controllable Drug Release. J. Am. Chem. Soc. 2014, 136, 10762−10769. (35) Meng, L. B.; Zhang, W.; Li, D.; Li, Y.; Hu, X. Y.; Wang, L.; Li, G. pH-Responsive Supramolecular Vesicles Assembled by Water-Soluble Pillar[5]arene and a BODIPY Photosensitizer for Chemo-Photodynamic Dual Therapy. Chem. Commun. 2015, 51, 14381−14384. (36) Peters, R. J.; Marguet, M.; Marais, S.; Fraaije, M. W.; van Hest, J.; Lecommandoux, S. Cascade Reactions in Multicompartmentalized Polymersomes. Angew. Chem., Int. Ed. Engl. 2014, 53, 146−150. (37) Liao, X.; Chen, G.; Liu, X.; Chen, W.; Chen, F.; Jiang, M. Photoresponsive Pseudopolyrotaxane Hydrogels Based on Competition of Host-Guest Interactions. Angew. Chem., Int. Ed. 2010, 49, 4409−4413. (38) Guo, D. S.; Liu, Y. Supramolecular Chemistry of pSulfonatocalix [n] arenes and Its Biological Applications. Acc. Chem. Res. 2014, 47, 1925−1934. (39) Zhang, J.; Ma, P. X. Cyclodextrin-Based Supramolecular Systems for Drug Delivery: Recent Progress and Future Perspective. Adv. Drug Delivery Rev. 2013, 65, 1215−1233.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Project Nos. 51373167 and 51522307).

■

REFERENCES

(1) Marguet, M.; Bonduelle, C.; Lecommandoux, S. Multicompartmentalized Polymeric Systems: Towards Biomimetic Cellular Structure and Function. Chem. Soc. Rev. 2013, 42, 512−529. (2) Seo, S. H.; Chang, J. Y.; Tew, G. N. Self-Assembled Vesicles from An Amphiphilic Ortho-Phenylene Ethynylene Macrocycle. Angew. Chem., Int. Ed. 2006, 45, 7526−7530. (3) Davies, T. S.; Ketner, A. M.; Raghavan, S. R. Self-Assembly of Surfactant Vesicles that Transform into Viscoelastic Wormlike Micelles upon Heating. J. Am. Chem. Soc. 2006, 128, 6669−6675. (4) Torchilin, V. P. Recent Advances with Liposomes as Pharmaceutical Carriers. Nat. Rev. Drug Discovery 2005, 4, 145−160. (5) Wang, C.; Wang, Z.; Zhang, X. Amphiphilic Building Blocks for Self-Assembly: from Amphiphiles to Supra-Amphiphiles. Acc. Chem. Res. 2012, 45, 608−618. (6) Klaikherd, A.; Nagamani, C.; Thayumanavan, S. Multi-Stimuli Sensitive Amphiphilic Block Copolymer Assemblies. J. Am. Chem. Soc. 2009, 131, 4830−4838. (7) Mai, Y.; Eisenberg, A. Self-Assembly of Block Copolymers. Chem. Soc. Rev. 2012, 41, 5969−5985. (8) Miao, X.; Cao, W.; Zheng, W.; Wang, J.; Zhang, X.; Gao, J.; Yang, C.; Kong, D.; Xu, H.; Wang, L.; Yang, Z. Switchable Catalytic Activity: Selenium-Containing Peptides with Redox-Controllable Self-Assembly Properties. Angew. Chem., Int. Ed. 2013, 52, 7781−7785. (9) Cao, W.; Gu, Y.; Meineck, M.; Li, T.; Xu, H. TelluriumContaining Polymer Micelles: Competitive-Ligand-Regulated Coordination Responsive Systems. J. Am. Chem. Soc. 2014, 136, 5132−5137. (10) Jie, K.; Zhou, Y.; Yao, Y.; Huang, F. Macrocyclic Amphiphiles. Chem. Soc. Rev. 2015, 44, 3568−3587. (11) Yu, G.; Jie, K.; Huang, F. Supramolecular Amphiphiles Based on Host-Guest Molecular Recognition Motifs. Chem. Rev. 2015, 115, 7240−7303. (12) Yang, B.; Dong, X.; Lei, Q.; Zhuo, R.; Feng, J.; Zhang, X. Host− Guest Interaction-Based Self-Engineering of Nano-Sized Vesicles for Co-Delivery of Genes and Anticancer Drugs. ACS Appl. Mater. Interfaces 2015, 7, 22084−22094. (13) Xu, X. D.; Zhao, L.; Qu, Q.; Wang, J. G.; Shi, H.; Zhao, Y. Imaging-Guided Drug Release from Glutathione-Responsive Supramolecular Porphysome Nanovesicles. ACS Appl. Mater. Interfaces 2015, 7, 17371−17380. (14) Ma, X.; Zhao, Y. Biomedical Applications of Supramolecular Systems Based on Host-Guest Interactions. Chem. Rev. 2015, 115, 7794−7839. (15) Yan, Q.; Yuan, J.; Cai, Z.; Xin, Y.; Kang, Y.; Yin, Y. VoltageResponsive Vesicles Based on Orthogonal Assembly of Two Homopolymers. J. Am. Chem. Soc. 2010, 132, 9268−9270. (16) Tao, W.; Liu, Y.; Jiang, B.; Yu, S.; Huang, W.; Zhou, Y.; Yan, D. A Linear-Hyperbranched Supramolecular Amphiphile and Its SelfAssembly into Vesicles with Great Ductility. J. Am. Chem. Soc. 2012, 134, 762−764. (17) Wang, L.; Li, L.-l.; Fan, Y.-s.; Wang, H. Host-guest supramolecular nanosystems for cancer diagnostics and therapeutics. Adv. Mater. 2013, 25, 3888−3898. (18) Yu, G.; Ma, Y.; Han, C.; Yao, Y.; Tang, G.; Mao, Z.; Gao, C.; Huang, F. A Sugar-Functionalized Amphiphilic Pillar [5] arene: Synthesis, Self-assembly in Water, and Application in Bacterial Cell Agglutination. J. Am. Chem. Soc. 2013, 135, 10310−10313. (19) Jiao, D.; Geng, J.; Loh, X. J.; Das, D.; Lee, T. C.; Scherman, O. A. Supramolecular Peptide Amphiphile Vesicles Through Host−Guest Complexation. Angew. Chem., Int. Ed. 2012, 51, 9633−9637. (20) Zimmerman, S. C.; Wendland, M. S.; Rakow, N. A.; Zharov, I.; Suslick, K. S. Synthetic Hosts By Monomolecular Imprinting Inside Dendrimers. Nature 2002, 418, 399−403. H


Research Article


(60) Pei, Q.; Hu, X.; Liu, S.; Li, Y.; Xie, Z.; Jing, X. Paclitaxel Dimers Assembling Nanomedicines for Treatment of Cervix Carcinoma. J. Controlled Release 2017, 254, 23−33. (61) Cai, K.; He, X.; Song, Z.; Yin, Q.; Zhang, Y.; Uckun, F. M.; Jiang, C.; Cheng, J. Dimeric Drug Polymeric Nanoparticles with Exceptionally High Drug Loading and Quantitative Loading Efficiency. J. Am. Chem. Soc. 2015, 137, 3458−3461. (62) Ma, Y.; Mou, Q.; Zhu, X.; Yan, D. Small Molecule Nanodrugs for Cancer Therapy. Mater. Today Chem. 2017, 4, 26−39. (63) Sakaino, H.; Sawayama, J.; Kabashima, S. I.; Yoshikawa, I.; Araki, K. Dry Micromanipulation of Supramolecular Giant Vesicles on a Silicon Substrate: Highly Stable Hydrogen-Bond-Directed Nanosheet Membrane. J. Am. Chem. Soc. 2012, 134, 15684−15687. (64) Nayak, N.; Gopidas, K. R. Unusual Self-Assembly of A Hydrophilic β-Cyclodextrin Inclusion Complex into Vesicles Capable of Drug Encapsulation and Release. J. Mater. Chem. B 2015, 3, 3425− 3428. (65) Inoue, Y.; Miyauchi, M.; Nakajima, H.; Takashima, Y.; Yamaguchi, H.; Harada, A. Self-Threading of A Poly(Ethylene Glycol) Chain in A Cyclodextrin-Ring: Control of the Exchange Dynamics by Chain Length. J. Am. Chem. Soc. 2006, 128, 8994−8995.

(40) Laza-Knoerr, A.L.; Gref, R.; Couvreur, P. Cyclodextrins for Drug Delivery. J. Drug Targeting 2010, 18, 645−656. (41) Okamatsu, A.; Motoyama, K.; Onodera, R.; Higashi, T.; Koshigoe, T.; Shimada, Y.; Hattori, K.; Takeuchi, T.; Arima, H. FolateAppended Beta-Cyclodextrin as A Promising Tumor Targeting Carrier for Antitumor Drugs in Vitro and in Vivo. Bioconjugate Chem. 2013, 24, 724−733. (42) Swiech, O.; Mieczkowska, A.; Chmurski, K.; Bilewicz, R. Intermolecular Interactions Between Doxorubicin and Beta-Cyclodextrin 4-Methoxyphenol Conjugates. J. Phys. Chem. B 2012, 116, 1765−1771. (43) Harada, T.; Giorgio, L.; Harris, T. J.; Pham, D. T.; Ngo, H. T.; Need, E. F.; Coventry, B. J.; Lincoln, S. F.; Easton, C. J.; Buchanan, G.; Kee, T. W. Diamide Linked γ-Cyclodextrin Dimers as Molecular-Scale Delivery Systems for the Medicinal Pigment Curcumin to Prostate Cancer Cells. Mol. Pharmaceutics 2013, 10, 4481−4490. (44) Ma, M.; Xu, S.; Xing, P.; Li, S.; Chu, X.; Hao, A. A MultistimuliResponsive Supramolecular Vesicle Constructed by Cyclodextrins and Tyrosine. Colloid Polym. Sci. 2015, 293, 891−900. (45) Tong, R.; Cheng, J. Paclitaxel-Initiated, Controlled Polymerization of Lactide for the Formulation of Polymeric Nanoparticulate Delivery Vehicles. Angew. Chem. 2008, 120, 4908−4912. (46) Hu, X.; Jing, X. Biodegradable Amphiphilic Polymer−Drug Conjugate Micelles. Expert Opin. Drug Delivery 2009, 6, 1079−1090. (47) Hu, X.; Li, J.; Lin, W.; Huang, Y.; Jing, X.; Xie, Z. Paclitaxel Prodrug Nanoparticles Combining Chemical Conjugation and Physical Entrapment for Enhanced Antitumor Efficacy. RSC Adv. 2014, 4, 38405−38411. (48) Tong, R.; Cheng, J. Paclitaxel-Initiated, Controlled Polymerization of Lactide for the Formulation of Polymeric Nanoparticulate Delivery Vehicles. Angew. Chem. 2008, 120, 4908−4912. (49) Lin, R.; Cheetham, A. G.; Zhang, P.; Lin, Y. A.; Cui, H. Supramolecular Filaments Containing A Fixed 41% Paclitaxel Loading. Chem. Commun. 2013, 49, 4968−4970. (50) Alcaro, S.; Ventura, C. A.; Paolino, D.; Battaglia, D.; Ortuso, F.; Cattel, L.; Puglisi, G.; Fresta, M. Preparation, Characterization, Molecular Modeling and in Vitro Activity of Paclitaxel−Cyclodextrin Complexes. Bioorg. Med. Chem. Lett. 2002, 12, 1637−1641. (51) Sun, T.; Yan, H.; Liu, G.; Hao, J.; Su, J.; Li, S.; Xing, P.; Hao, A. Strategy of Directly Employing Paclitaxel to Construct Vesicles. J. Phys. Chem. B 2012, 116, 14628−14636. (52) Hamada, H.; Ishihara, K.; Masuoka, N.; Mikuni, K.; Nakajima, N. Enhancement of Water-Solubility and Bioactivity of Paclitaxel Using Modified Cyclodextrins. J. Biosci. Bioeng. 2006, 102, 369−371. (53) Jing, J.; Szarpak-Jankowska, A.; Guillot, R.; Pignot-Paintrand, I.; Picart, C.; Auzély-Velty, R. Cyclodextrin/Paclitaxel Complex in Biodegradable Capsules for Breast Cancer Treatment. Chem. Mater. 2013, 25, 3867−3873. (54) Sun, T.; Guo, Q.; Zhang, C.; Hao, J.; Xing, P.; Su, J.; Li, S.; Hao, A.; Liu, G. Self-Assembled Vesicles Prepared From Amphiphilic Cyclodextrins as Drug Carriers. Langmuir 2012, 28, 8625−8636. (55) Liu, Y.; Chen, G. S.; Chen, Y.; Cao, D. X.; Ge, Z. Q.; Yuan, Y. J. Inclusion Complexes of Paclitaxel and Oligo (ethylenediamino) Bridged Bis(β-Cyclodextrin)s: Solubilization and Antitumor Activity. Bioorg. Med. Chem. 2004, 12, 5767−5775. (56) Namgung, R.; Mi Lee, Y.; Kim, J.; Jang, Y.; Lee, B. H.; Kim, I. S.; Sokkar, P.; Rhee, Y. M.; Hoffman, A. S.; Kim, W. J. Poly-Cyclodextrin and Poly-Paclitaxel Nano-Assembly for Anticancer Therapy. Nat. Commun. 2014, 5, No. 3702. (57) Lorenz, S. A.; Bigwarfe, P. M., Jr.; Balasubramanian, S. V.; Fetterly, G. J.; Straubinger, R. M.; Wood, T. D. Noncovalent Dimerization of Paclitaxel in Solution: Evidence From Electrospray Ionization Mass Spectrometry. J. Pharm. Sci. 2002, 91, 2057−2066. (58) Zhang, W.; Lin, W.; Pei, Q.; Hu, X.; Xie, Z.; Jing, X. RedoxHypersensitive Organic Nanoparticles for Selective Treatment of Cancer Cells. Chem. Mater. 2016, 28, 4440−4446. (59) Zhang, W.; Lin, W.; Zheng, X.; He, S.; Xie, Z. Comparing Effects of Redox Sensitivity of Organic Nanoparticles to Photodynamic Activity. Chem. Mater. 2017, 29, 1856−1863. I


Paclitaxel Dimer Assembling Vesicles: Reversible

Recommend Documents