Controllable Construction of Biocompatible Supramolecular Micelles

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Controllable Construction of Biocompatible Supramolecular Micelles and Vesicles by Water-Soluble Phosphate Pillar[5,6]arenes for Selective Anti-Cancer Drug Delivery Xiao-Yu Hu, Xin Liu, Wenyi Zhang, Shan Qin, Chenhao Yao, Yan Li, Derong Cao, Luming Peng, and Leyong Wang Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b00691 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Chemistry of Materials

Controllable Construction of Biocompatible Supramolecular Micelles and Vesicles by Water-Soluble Phosphate Pillar[5,6]arenes for Selective Anti-Cancer Drug Delivery Xiao-Yu Hu,† Xin Liu,† Wenyi Zhang,‡ Shan Qin,† Chenhao Yao,† Yan Li,‡ Derong Cao,§ Luming Peng,† and Leyong Wang*† †

Key Laboratory of Mesoscopic Chemistry of MOE and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China. ‡ State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory of Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China. § School of Chemistry and Chemical Engineering, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, 510641, China. ABSTRACT: The controllable construction of biocompatible supramolecular nanocarriers with different morphologies based on dynamic non-covalent interactions to achieve selective delivery of drugs with different properties remains highly challenging. We herein report controllable construction of two types of supramolecular nanocarriers based on biocompatible water-soluble phosphate-based pillar[5]arene (WP5P) or pillar[6]arene (WP6P) with pyridinium bromide guest G for the selective anticancer drug delivery. Solid supramolecular micelles could be obtained by the amphiphilic host−guest inclusion complex formed from WP5P and G, whereas hollow supramolecular vesicles were acquired from WP6P and G. Both of them showed pH- and Zn2+responsiveness. Furthermore, the resulting solid micelles were able to encapsulate hydrophobic anticancer drug doxorubicin (DOX) to achieve DOX-loaded micelles, while hydrophilic anticancer drug mitoxantrone (MTZ) could be successfully loaded into the hollow vesicles. And the encapsulated anticancer drugs could be efficiently released at low-pH environment or with the introduction of Zn2+. More importantly, cytotoxicity experiments indicated that these water-soluble phosphate-based pillar[5,6]arenes showed excellent biocompatibility, and the drug-loaded nanoparticles exhibited comparable therapeutic effect for cancer cells as free anticancer drugs and remarkably reduced damage for normal cells as well. Cellular uptake and intracellular localization experiments further confirmed that these two types of nanocarriers, taken up by cancer cells via endocytosis, could lead to efficient drug accumulation in cancer cells. Therefore, this strategy of controllable construction of different types of stimuli-responsive supramolecular nanocarriers based on biocompatible phosphate-based pillar[5,6]arenes have great potential applications in the field of controlled drug delivery.

1. INTRODUCTION Currently, advanced supramolecular drug delivery platforms for chemotherapy are urgently needed and have attracted considerable attention owing to their fascinating ability to significantly enhance the therapeutic efficacy and obviously reduce the side effects and even drug resistance compared with free drugs.1-3 To construct functional and smart supramolecular nanostructures including micelles,4-6 vesicles,7-9 and other novel nano-assemblies,10-13 supramolecular amphiphiles (SAs), whose hydrophilic and hydrophobic components are connected by non-covalent interactions, provide an attractive approach in the fabrication of sophisticated supramolecular nanocarriers, which is associated with richer amphiphile topologies and simpler chemical synthesis than traditional covalent amphiphiles.14-17 More importantly, supramolecular nanostructures constructed by reversible non-covalent interactions have the abilities to undergo dynamic switching of structure, morphology, and function in response to various external stimuli,18-26 which endow them outstanding potential applications in

drug delivery. Therefore, the self-assembly of stimuliresponsive SAs provides a flexible and robust platform for designing functional supramolecular drug delivery systems (DDSs), which may well satisfy the urgent demands of contemporary nanomedicine development. On the other hand, nanocarriers with different morphorphological structures can encapsulate drug molecules with different solubilities and properties. For example, hydrophobic anticancer drugs such as doxorubicin (DOX), camptothecin (CPT), paclitaxel (PTX) are usually loaded within the solid micellar structures, while the hollow vesicles tend to encapsulate hydrophilic drugs such as mitoxantrone (MTZ) in their hydrophilic internal cavities. Thus, controllable construction of nanocarriers with desired nanostructure can endow them specific functions for selective drug delivery. However, controllably constructing biocompatible supramolecular nanocarriers with different morphologies based on host−guest interactions to achieve selective delivery of drugs with different properties remains highly challenging. Therefore, to address this problem, it is highly desirable to develop controllable construction of smart drug nanocarriers 1

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with different morphologies based on the supramolecular approach. Recently, research on pillararene-based supramolecular materials has received burgeoning attention and advanced rapidly. 27-46 Our group has also developed a series of supramolecular amphiphiles based on water-soluble pillar[5,6]arenes, which can further assemble to form higher-ordered aggregates such as vesicles, micelles, and nanoparticles to achieve controllable anticancer drug delivery.47-52 In most of these achievements, water-soluble pillar[5,6]arenes with carboxylate anions were used as host molecules due to their good solubility and acidresponsive properties in aqueous media (Scheme 1). To achieve controllable construction of smart drug nanocarriers for selective drug delivery, we intend to develop more biocompatible water-soluble pillararenes for their potential applications in drug delivery. It is well known that phosphonic acid and phosphate motifs are biocompatible groups, which are usually associated with many biological functions.53 Consequently, we expect that the modification of pillararenes with phosphate groups will not only increase their water-solubility but also greatly improve their biocompatibility. Meanwhile, we envision that we can design an alkyl chain-functionalized pyridinium guest molecule, in which the alkyl chain and pyridinium moiety could be used as recognition motifs to selectively bond with water-soluble phosphate-based pillar[5]arene and pillar[6]arene, respectively, to form supramolecular amphiphiles. Since the alkyl chain-functionalized pyridinium guest could bond with pillar[5]arene or pillar[6]arene host in different binding sites, the formed supramolecular amphiphiles would have different hydrophilic and hydrophobic portions, which would lead to the formation of higher-ordered aggregates with different morphological structures54 for their potential applications in selective drug delivery. Herein, we report the successfully controllable construction of two different types of pillararene-based stimuli-responsive supramolecular nanocarriers for selective anticancer drug delivery, which were formed from biocompatible phosphatebased pillar[5]arene (WP5P) or pillar[6]arene (WP6P) with alkyl chain-modified pyridinium guest G as schematically depicted in Scheme 2. The obtained amphiphilic WP5PG Scheme 1. Structures of Previous Reported CarboxylatedBased Pillararenes (WP5C, WP6C) and the PhosphateBased Pillar[5,6]arenes (WP5P, WP6P) in This Work.

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Scheme 2. Schematic Illustration of the Controllable Construction of Supramolecular Micelles (WP5PG) and Vesicles (WP6PG) and Their Applications in Selective Drug Delivery.

inclusion complex could self-assemble into solid supramolecular micelles, which were able to efficiently encapsulate the hydrophobic anticancer drug doxorubicin (DOX); whereas, hollow supramolecular vesicles were constructed by WP6PG complex, which were demonstrated to be efficient carriers for the encapsulation of hydrophilic anticancer drug mitoxantrone (MTZ). And the loaded anticancer drugs could be efficiently released at acidic environment or with the introduction of Zn2+. Notably, cytotoxicity experiments indicated that these phosphate-based pillar[5,6]arenes exhibited better biocompatibility than those carboxylate-based counterparts, and the resulting drug-loaded nanoparticles exhibited remarkably reduced damage for normal cells. Furthermore, these two types of supramolecular nanocarriers entered cancer cells mainly via endocytosis and could lead to significant drug accumulation in cancer cells, which exhibited multiple advantages and comparable therapeutic effects for cancer cells relative to free drugs, implying their promising future for cancer therapy. To the best of our knowledge, there is the first example of controllable construction of smart supramolecular nanovehicles with different morphological structures based on host−guest interactions to achieve selective anticancer drug delivery.

2. RESULTS AND DISCUSSION Synthesis of Phosphate-Based Pillararenes. Watersoluble phosphate-based pillar[5]arene (WP5P) and pillar[6]arene (WP6P) were synthesized by means of the classic Arbuzov reaction between triethyl phosphite and bromosubstituted pillararene derivative 2 (or 5),55 and followed by the Mckenna reaction56 by using bromotrimethylsilane (TMSBr) to react with the generated pillararene-based phosphonate ester 3 (or 6), which is one of the most widely used methods in the synthesis of organophosphorus acids and the target molecule WP5P and WP6P were generated in good yields (Scheme 3). Scheme 3. Synthetic Route for Water-Soluble PhosphateBased Pillar[5,6]arenes (WP5P and WP6P).


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Host-Guest Complexation Study. The host–guest complexation between guest G and pillar[5]arene-based phosphate (WP5P) was firstly studied by 1H NMR spectroscopy. As shown in Figure 1, upon addition of WP5P to the aqueous solution of G, remarkable upfield shifts of the pyridine protons (Hc) and methylene protons (H1, H2, H3, and H4) of G could be observed due to the shielding effect of the electron-rich cavities of WP5P toward G. However, the pyridine protons (Hb) only showed slightly upfield shifts and almost no change could be observed for the methyl proton Ha and methylene protons (H5, H6, H7, and H8) on G. These results revealed that the host WP5P (the „wheel‟) was fully threaded by the guest G (the „axle‟) with the protons Hb, Hc, and H1-4 in the cavity of WP5P and other protons Ha, H5-10, and Hd-f out of its cavity. Moreover, 2D NOESY experiment further indicated the above conformation of such inclusion complex (Figure S20, Supporting Information), from which NOE correlation signals could be observed between protons H1′, H2′, and H3′ of WP5P and H1-4 of G, confirming that the alkyl chain moiety (H1-4) was located in the cavity of WP5P.

Figure 1. 1H NMR (300 MHz, D2O, 298 K) spectra: (a) G (6.0 mM), (b) G (6.0 mM) and WP5P (6.0 mM), and (c) WP5P (6.0 mM).

However, the binding mode of G with pillar[6]arene-based phosphate (WP6P) was quite different from that of WP5PG. It was found that when WP6P was added into the solution of G, resonances of the pyridine protons (Ha, Hb, and Hc) and methylene protons (H1, H2, H3, and H4) of G showed obviously upfield shift, whereas almost no change could be observed for the methylene protons (H5-9) on G. These results implied that the methyl-pyridinium moiety of G (Ha, Hb, Hc) and adjacent methylene groups (H1-4) were encapsulated into the cavity of WP6P and other protons H5-10 and Hd-f were out of its cavity (Figure 2). Furthermore, NOE correlations could be observed between the signals related to protons H5′ of WP6P and Ha, Hb, Hc of G, as well as protons H7′ of WP6P and Ha, indicating the methyl-pyridinium moiety was located in the cavity of WP6P (Figure S22, Supporting Information).

Figure 2. 1H NMR (300 MHz, D2O, 298 K) spectra: (a) G (6.0 mM), (b) G (6.0 mM) and WP6P (6.0 mM), and (c) WP6P (6.0 mM).

Subsequently, the stoichiometry of complexation for both WP5PG and WP6PG was investigated by Job‟s plots method by using 1H NMR titration experiments, in which a model compound G′, an analogue of G, was used for measurement (since undesirable intermolecular aggregation of WP5PG or WP6PG drastically broadens the signals, making the spectra undistinguishable), and the results indicated 1:1 binding stoichiometry for both WP5PG′ and WP6PG′ (Figure S24 and S27, Supporting Information). After validating the complex stoichiometry, the association constants (Ka) for WP5PG′ and WP6PG′ were calculated to be (1.01  0. 28) × 105 M-1 and (3.35  0.87) × 103 M-1, respectively (Figure S25 and S28, Supporting Information). The main driven force for these host–guest systems should be attributed to the cooperative hydrophobic and multiple electrostatic interactions, which lead to the formation of stable 1:1 amphiphilic inclusion complexes (WP5PG and WP6PG) with different binding sites. Construction of Supramolecular Nanocarriers Based on the Host−Guest Complexation of WP5PG and WP6PG. With the above WP5PG and WP6PG supramolecular amphiphilic inclusion complexes in hand, we further investigated their abilities to form high-ordered aggregates in water. Since G is also an amphiphilic molecule, we first investigated its self-assembly behavior in water. Dynamic light scattering (DLS) measurements of the aqueous G solution (8 × 10-5 M) showed no measurable signal, indicating that no aggregate assembled by G existed under the examined concentration. However, when the aqueous solution of WP5P or WP6P was added to the above G solution (8 × 10-5 M), a light opalescence and clear Tyndall effect could be observed (Figure 3a and 4a), which suggested that supramolecular microaggregates were formed based on their host–guest interactions. Moreover, since the intensity of opalescence changed gradually upon adding different amounts of WP5P or WP6P, we further determined the best molar ratio between WP5P/WP6P and G for constructing such supramolecular aggregates by using UVVis titration experiments. As shown in Figure S29, upon gradually increasing the adding amount of WP5P, the absorbance at 450 nm first increased gradually until reaching a maximum at a G/WP5P ratio of 10:1, and then it showed an inverse decrease upon further addition of WP5P. The rapid increase of the absorbance intensity indicated that WP5P and G formed a higher-order complex with a tendency toward amphiphilic


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aggregation, whereas it underwent disassembly upon further addition of WP5P, generating a simple 1:1 supramolecular inclusion complex. Thus, the best molar ratio 10:1 ([G]/[WP5P]) for the formation of supramolecular aggregates was determined at the inflection point. Similarly, the best molar ratio of WP6P and G for such amphiphilic aggregation was also determined ([G]/[WP6P] = 10:1) (Figure S31, Supporting Information).

Figure 3. (a) DLS data of the WP5PG aggregates, inset: Tyndall effect of G (left) and WP5PG (right); TEM images: (b) WP5PG aggregates; (c) enlarged image of b. [WP5P] = 2.0 × 10-5 M and [G] = 8.0 × 10-5 M.

Subsequently, the size distribution and morphology of the nano-aggregates formed by WP5PG or WP6PG supramolecular amphiphiles were identified by DLS and transmission electron microscopy (TEM) measurements (Figure 3 and 4). DLS results suggested that the obtained WP5PG complex formed spectacular nano-aggregates with a narrow size distribution, giving an average hydrodynamic diameter of 70 nm (Figure 3a), and TEM images indicated the formation of solid spherical micellar structure with a diameter ranging from 60 to 80 nm (Figure 3b and 3c). Whereas, with respect to the aggregates formed from WP6PG, TEM images and DLS results showed the spherical morphology with an average diameter of 202 nm (Figure 4a−4c), indicating the formation of spherical nanoparticles with a well-defined smooth surface. Meanwhile, a simple staining technique for TEM with uranyl acetate was used to further characterize the morphology of the formed WP6PG nanoparticles, which showed that each nanoparticle had a black wall with distinct contrast to the inner part (Figure 4d), indicating the formation of hollow supramolecular vesicular structure. Moreover, the wall thickness of the vesicles was calculated to be about 4 nm from the TEM images, which was close to the extended length of two molecules of WP6PG complex calculated by Chem 3D (Figure S33, Supporting Information). The above results revealed that the obtained WP6PG vesicles should possess a bilayer structure. In addition, zeta-potential measurements showed that both the WP5PG micelles and WP6PG vesicles formed at the best molar fraction possessed negative zeta-potentials (−12.1 mV and −17.6 mV, respectively), primarily due to the excessive negative charges on phosphate host upon its complexation with pyridinium guest. Considering the repulsive-forceinduced increasing-stability of the nanoparticles, the molar ratios of [G]/[WP5P] = 4:1 (ζ-potential = −34.1 mV) and [G]/[WP6P] = 4:1 (ζ-potential = −35.6 mV) were chosen for further investigating their stimuli-responsive behaviors before drug-loading (Figure S30 and S32, Supporting Information); whereas, after loading with drugs, the molar ratios of [G]/[WP5P] = 3:1 and [G]/[WP6P] = 5:1 were used for constructing the DOX-loaded micelles and MTZ-loaded vesicles, respectively, since under these conditions more stable drug-loaded nanoparticles with high drug-loading efficiency could be obtained for further investigating their applications in drug delivery.

Figure 4. (a) DLS data of the WP6PG aggregates, inset: Tyndall effect of G (left) and WP6PG (right); TEM images: (b) WP6PG aggregates; (c) enlarged image of b; (d) TEM images of WP6PG aggregates staining by uranyl acetate. [WP6P] = 2.0 × 10-5 M and [G] = 8.0 × 10-5 M.

pH- and Zn2+- Dual-Responsiveness of the WP5PG Micelles and WP6PG Vesicles. Supramolecular nanocarrier systems usually incorporated with stimuli-responsive properties, which would be amenable to address some of the systemic and intracellular delivery barriers. It is well known that phosphate exhibits pH-responsiveness in aqueous solution, and Zn2+ usually has strong bonding affinity with phosphate groups.57-60 Therefore, we envisioned that the obtained supramolecular micelles and vesicles are expected to have pH- and Zn2+-responsiveness. As expected, Tyndall effect for the WP5PG micellar solution disappeared after adjusting the solution pH to 5.0; meanwhile, no micellar structure could be observed in the TEM image (Figure 5a and Figure S34) due to the precipitation of WP5P as its acid form out of the acidic aqueous solution (since the generated WP5P as its acid form is insoluble in water). However, when the solution pH was adjusted back to about 7.4, micelles were reformed as shown in Figure 5b. Similar phenomenon could also be observed for the WP6PG vesicular solution (Figure S34). These results confirmed the pH-responsive assembly and disassembly behaviors of the obtained supramolecular nanocarriers.

Figure 5. TEM images: (a) WP5PG aggregates after the solution pH was adjusted to 5.0; (b) WP5PG aggregates after the solution pH was adjusted back to 7.4. (c) Tyndall effect of WP5PG aggregates (right) and WP5PG aggregates in the presence of Zn2+ (left), [Zn2+] = 5 mM. [WP5P] = 2.0 × 10-5 M and [G] = 8.0 × 10-5 M.

Besides the pH-responsiveness, these two types of nanocarriers also exhibited good Zn2+-responsive properties due to the strong complexation ability of phosphate groups with Zn2+. As shown in Figure 5c, when ZnCl2 solution was added into the


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WP5PG micellar or WP6PG vesicular solution, opalescence of the solution gradually disappeared within 30 min, accompanied by the disappearance of Tyndall effect and the formation of precipitated large aggregates, meanwhile, no micelles or vesicles could be observed in the TEM images (Figure S34, Supporting Information). The disassembly process of these two types of nanoparticles might be understood as follows: firstly, Zn2+ binding to the phosphate headgroups of WP5P or WP6P, since the outside surface of the nanoparticles was covered with hydrophilic phosphate groups which could be chelated by Zn2+ ions; and then, such Zn2+WP5P/Zn2+-WP6P binding further induced the serious aggregation of the micelles or vesicles; finally, the aggregation process resulted in the collapse of the nanostructures, accompanying with the contents release. All the above results demonstrated that such supramolecular micelles and vesicles exhibited good pH- and Zn2+-responsiveness, which endows them with the ability to encapsulate substrates at physiological condition and release them in response to acidic conditions or upon the addition of Zn2+ (Schematic illustration was provided in Scheme S5, Supporting Information).

micelles, while MTZ was encapsulated into the hydrophilic cavity of vesicles. In comparison with the unloaded WP5PG micellar solution, DOX-loaded micellar solution turned from colorless to light red (Figure 6a). Meanwhile, fluorescence intensity of DOX-loaded micelles became stronger from 550 to 650 nm, which represents the characteristic emission of DOX (Figure 6c). For MTZ-loaded vesicles, the solution turned to light blue compared with the colorless unloaded vesicular solution (Figure 7a), and the UV-vis absorption became stronger from 610 to 660 nm, which represents the characteristic absorption of MTZ in aqueous solution (Figure 7c). All of the above results demonstrated that DOX and MTZ were successfully encapsulated into the supramolecular micelles and vesicles, respectively. Furthermore, DLS and TEM experiments showed that both the DOX-loaded micelles and MTZ-loaded vesicles became larger in size than those of the unloaded micelles or vesicles (Figure 6b, 6d, 7b, and 7d). Based on the fluorescence and UV-absorption spectra, the DOX and MTZ loading efficiency were calculated to be 7.4% and 76%, respectively, indicating that such supramolecular nanocarriers were good and selective drug-loading systems.

Drug Loading and Its Controllable Release. Combining all of the aforementioned results, we have controllably constructed two different types of pH- and Zn2+-responsive supramolecular nanocarriers: one is solid micelles formed by WP5PG complex and the other is hollow vesicles selfassembled from WP6PG complex, which might have potential applications in selective drug delivery. The solid micellar structures prefer to load hydrophobic drugs, whereas hollow vesicles are often used as carriers for hydrophilic drug delivery. Therefore, in the following study, hydrophobic anticancer drug DOX and hydrophilic anticancer drug MTZ were used as model drugs to investigate the encapsulation efficiency and drug release behavior of the formed WP5PG micelles and WP6PG vesicles, respectively. After purification by dialysis, DOX was successfully loaded in the hydrophobic core of

Figure 7. (a) Color changes of MTZ-loaded vesicles (left) and unloaded vesicles (right); (b) TEM images of MTZ-loaded vesicles; (c) UV-Vis spectra of the solutions of free MTZ, unloaded vesicles, and MTZ-loaded vesicles; (d) DLS data of MTZ-loaded vesicles. [WP6P] = 2.5 × 10-5 M and [G] = 1.25 × 10-4 M.

Figure 6. (a) Color changes of unloaded micelles (left) and DOXloaded micelles (right); (b) TEM images of DOX-loaded micelles; (c) Fluorescence spectra of the solutions of unloaded micelles, DOX-loaded micelles, and DOX-loaded micelles after the solution pH was adjusted to 5.0; (d) DLS data of DOX-loaded micelles. [WP5P] = 2.5 × 10-5 M and [G] = 7.5 × 10-5 M.

Subsequently, the release behaviors of drug-loaded supramolecular nanocarriers were first investigated under acidic condition (pH 5.0 and 7.4, respectively), by simulating the endolysosomal environment. As shown in Figure 8a, up to 80 % of DOX was released from the DOX-loaded micelles at pH 5.0 within 12 h, however the cumulative leakage of DOX was less than 5% within same time interval under the physiological conditions (pH 7.4). For MTZ-loaded vesicles, the cumulative release of MTZ was up to 67 % at pH 5.0 within 12 h, but under physiological conditions, almost no release of MTZ could be detected (Figure 8b). Due to the microenvironments of tumor tissues is more acidic than in blood or normal tissues, increasing the drug release rate with a relatively low pH- stimulus could be a positive factor for efficient therapy.61 Moreover, the excellent stability of drug-loaded nanoparticles at physiological environment could remarkably reduce the side effects of drugs for normal tissues.


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se drug nanocarriers were further evaluated by the methyl thiazole tetrazolium (MTT) cell survival assay. Initially, free anticancer drug and drug-loaded nanoparticles were incubated with L929 cells (normal cells), respectively, to evaluate the cytotoxicity for their future potential clinical applications. The relative cell viability after 24 h incubation at different concentrations was shown in Figure 9. It was found that the relative cell viability in drug-loaded nanoparticle groups were always much higher than that in the free drug groups (p < 0.05), indicating that the systemic toxicity of free drugs (DOX·HCl and MTZ) was remarkably reduced upon its encapsulation by WP5PG micelles and/or WP6PG vesicles.

Figure 8. (a) pH-Responsive DOX release profiles of the DOXloaded micelles in the release media of different pH values; (b) pH-Responsive MTZ release profiles of the MTZ-loaded vesicles in the release media with different pH values; (c) Zn2+-induced DOX release profiles from DOX-loaded micelles in the presence of different concentrations of Zn2+; (d) Zn2+-induced MTZ release profiles from MTZ-loaded micelles in the presence of Zn2+ (2 mM).

Furthermore, when different amounts of ZnCl2 were added to the drug-loaded micellar or vesicular solution, Zn2+-induced drug release behavior could be clearly observed (Figure 8c and 8d). The addition of Zn2+ from 1 to 5 mM could trigger a gradual enhancement of DOX release from 24% to 53% within 1 h (Figure 8c). Meanwhile, Zn2+-induced efficient MTZ release could be realized for the MTZ-loaded vesicles (Figure 8d). It is well-known that zinc is one kind of important trace element in the human body, so such Zn2+-responsive behavior of the obtained supramolecular nanocarriers will be considerably attractive in the field of biotechnology, especially diagnostics and drug delivery. In addition, since the content of Zn2+ in blood is relatively low (about 0.014 − 0.015 mM in serum and plasma, and 0.15 − 0.2 mM in red blood cells),62 the premature release of drugs from the drug-loaded nanoparticles might be avoided, which is particularly important for developing efficient drug delivery systems. Biocompatibility of the Water-Soluble Phosphate Pillar[5,6]arenes. As DDS materials, the biocompatibility of nanocarriers is an important index for their biomedical applications. Therefore, the biocompatibility of WP5P and WP6P was evaluated by MTT assay, where L929 cell line was treated with different concentrations of WP5P and WP6P, respectively; meanwhile, carboxylate-based pillar[5,6]arenes (WP5C and WP6C)63-64 were used as references since most of the reported pillararene-based DDSs usually use water-soluble pillararenes as their carboxylate forms. It was found that the relative viability of L929 cells incubated with WP5P or WP6P was always higher than that in the WP5C or WP6C group at the same concentration, and the biocompatibility of WP6P was better than WP5P (For details, see Figure S35). Furthermore, the cytotoxicity of guest G for L929 cells could be obviously reduced once the formation of host–guest inclusion complex (Figure S36, Supporting Information). Consequently, these phosphate-based pillar[5,6]arenes (WP5P and WP6P) are more suitable supramolecular host molecules for drug delivery. Cytotoxicity and Anticancer Efficiency of Drug-Loaded Nanoparticles. Cytotoxicity and anticancer efficiency of the-

Figure 9. In vitro cytotoxicities of (a) DOX-loaded micelles and free DOX·HCl; (b) MTZ-loaded vesicles and free MTZ against L929 cells after 24 h incubation (p < 0.05).

Following the above results, another key point is whether these supramolecular nanoparticles could effectively kill cancer cells, so the drug-loaded micelles and vesicles were then incubated with A549 and Hela cells (cancer cells), respectively to investigate their anticancer efficiency, for which the unloaded nanoparticles and free drugs were used as controls, and the relative cell viability in different groups were recorded after 24 h. As shown in Figure 10 and Figure S37, the relative cell viability in drug-loaded groups were always much lower than that in the unloaded groups, moreover, the drug-loaded groups showed similar therapeutic effects to the free drug groups at a wide range of concentrations. The above results indicated that the loading of drugs by supramolecular nanoparticles did not affect the therapeutic effect of drugs for cancer cells, but its damage for normal cells was obviously reduced. A reasonable explanation is that compared with normal cells, tumor cells have strong phagocytosis and a relatively acidic microenvironment which can cause the disassembly of drug-

Figure 10. In vitro cytotoxicities of free DOX·HCl, free MTZ, DOX-loaded micelles, and MTZ-loaded micelles against A549 cells (a-b) and HeLa cells (c-d), respectively, after 24 h incubation.


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loaded nanoparticles accompanying with the efficient release of the encapsulated drugs. Therefore, such supramolecular nanoparticles based on controllable host–guest complexation are promising drug nanocarriers. Celluar Uptake and Intracelluar Localization. The celluar uptake and intracellular release behaviors of DOX-loaded micelles and MTZ-loaded vesicles were then investigated by confocal laser scanning microscopy (CLSM) and flow cytometry toward A549 cancer cells. Since free DOX·HCl and MTZ have inherent fluorescence, they can be directly observed with CLSM (DOX shown in orange, MTZ shown in red), and the fluorescence intensity detected in cells treated with WP5PG/WP6PG nanoparticles or free drugs can be considered to be consistent with the concentration of drug internalized into the cells. Thus, the fluorescence intensity detected in cells by CLSM can indicate the concentration of these systems internalized into the cells. Meanwhile, LysoTracker Red and Hochest were used to label lysosomes and cell nucleus, respectively for co-localizing these nanocarriers (LysoTracker shown in green, Hochest shown in blue). Firstly, A549 cells were incubated with DOX-loaded micelles, MTZ-loaded vesicles, free DOX·HCl and MTZ at 37 °C for 1 h and 5 h, respectively. As is shown in Figure 11, after incubation with DOX-loaded micelles or MTZ-loaded vesicles for 1 h, only weak fluorescence could be detected, whereas, the cells showed intense intracellular orange or red fluorescence after incubation for 5 h, suggesting the essential cellular uptake of DOX or MTZ. Moreover, the cells showed strong yellow-green fluorescence that overlapped well with the labeled the orange/red and green fluorescence after incubation for 5 h, indicating the co-localization of DOX-loaded micelles or MTZ-loaded vesicles with lysosomes (Figure S38A, Supporting Information). Meanwhile, after incubating for 5 h, the orange/red fluorescence was not only accumulated in the lysosomes but also in the nuclei (Figure S38B, Supporting Information), suggesting that DOX and MTZ had been effectively released from the nanoparticles, escaped from the endosomes, and entered into the nuclei. The above results implied that these two types of drug-loaded nanoparticles entered cancer cells mainly through endocytosis and first localized in lysosomes, where a low-pH microenvironment triggered the disassembly of these nanoparticles to release the encapsulated

Figure 11. Cellular uptake and intracellular localization of: (a) DOX-loaded micelles, (b) MTZ-loaded vesicles in A549 cancer cells examined by confocal laser scanning microscopy at 37 °C for 1 h and 5 h, respectively. The scale bars correspond to 20 μm.

drugs into the cytosol, finally entering the cell nucleus for cancer therapy. The quantitative results obtained by flow cytometry further confirmed the relatively higher cellular uptake activity of nanoparticles compared with free drugs, especially for the DOXloaded supramolecular micelles (Figure 12). As shown in the flow cytometric profiles, the fluorescence intensity of DOXloaded group obviously surpassed the free DOX·HCl group whatever for 1 h or 5 h of incubation at the same DOX concentration, whereas, for the MTZ group, the drug-loaded vesicle group exhibited similar fluorescence intensity with the free MTZ group. We speculated that the size of nanoparticles might influence cell uptake and result in the relatively slower cellular uptake rates of MTZ-loaded large sized vesicles than DOX-loaded smaller micelles. Thus, all above results confirmed that these two types of smart supramolecular nanocarriers could efficiently and selectively delivery hydrophobic or hydrophilic anticancer drugs into the nuclei of cancer cells.

Figure 12. Flow cytometric profiles of A549 cells incubated with MTZ, DOX·HCl, DOX-loaded micelles, and MTZ-loaded vesicles at 37 °C for 1 h (a, c) and 5 h (b, d).

3. CONCLUSIONS In summary, a smart stimuli-responsive drug delivery system based on water-soluble phosphate-based pillar[5]arene (WP5P) or pillar[6]arene (WP6P) with alkyl chain-modified pyridinium guest G was successfully fabricated, in which two kinds of different pillararene-based recognition motif: one is pillar[5]arene-alkyl chain and the other is pillar[6]arenepyridinium, were used to controllably construct supramolecular aggregates with different morphologies. The amphiphilic WP5PG inclusion complex could self-assemble into solid micelles with an average diameter of ~70 nm, which were demonstrated to be efficient carriers for the encapsulation of hydrophobic anticancer drug DOX; whereas, hollow vesicles with an average diameter of ~200 nm were constructed by WP6PG complex, which could efficiently encapsulate hydrophilic anticancer drug MTZ. And the loaded anticancer drugs could be efficiently released at low-pH environment or with the introduction of Zn2+. More importantly, cytotoxicity experiments indicated that these phosphate-based watersoluble pillararenes showed better biocompatibility than those carboxylate-based counterparts, and the resulting drug-loaded nanoparticles exhibited lower cytotoxicity for normal cells than free drugs. Moreover, loading of drugs by supramolecular nanoparticles did not affect the therapeutic effect of free drugs, and the resulting drug-loaded nanoparticles exhibited multiple


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advantages and comparable curative effects for cancer cells relative to free drugs. Cellular uptaking and intracellular localization experiments further suggested that these two types of supramolecular nanocarriers entered cancer cells mainly via endocytosis, which could lead to significant drug accumulation in A549 cancer cells. This work thus provides a novel and effective strategy for the controllable construction of smart supramolecular nanovehicles with different morphological structures. Further studies on the active targeting effects of this promising supramolecular drug delivery platform are ongoing in our lab.

4. EXPERIMENTAL SECTION General Procedure for the Preparation of Desalted Doxorubicin (DOX). DOX·HCl (5 mg, 0.0086 mM) was dissolved in

CHCl3 (3 mL), and then triethylamine (TEA, 10 μL) was added, the mixture was stirred in the dark at 25 ˚C for 12 h. Finally, the solvent was removed under vacuum in the dark, and the desalted doxorubicin was obtained almost in 100% yield, which was directly used without further purification, and the contained TEA·HCl was removed during the following dialysis process. Fabrication of the DOX-Loaded Micelles. DOX-loaded micelles were prepared as follows: a certain amount of DOX was added to a solution containing WP5P and G (1% EtOH was added to improve the solubility of G). The ultimate concentrations of DOX, G, and WP5P were 0.025, 0.075, and 0.025 mM, respectively. After standing overnight, the prepared DOX-loaded vesicles were purified by dialysis (molecular weight cutoff 10 000) in distilled water for several times until the water outside the dialysis tube exhibited negligible DOX fluorescence. The DOX encapsulation efficiency was calculated by the following equation. Encapsulation efficiency (%) = (mDOX-loaded/mDOX) × 100 where mDOX-loaded and mDOX are mass of DOX encapsulated in vesicles and mass of DOX added, respectively. The mass of DOX was measured by a fluorescence spectrophotometer at 550 nm and calculated as relative to a standard calibration curve in the concentrations from 1.0 to 10.5 μg/mL in water (Figure S39, Supporting Information). Fabrication of the MTZ-Loaded Vesicles. MTZ-loaded vesicles were prepared as follows. A certain amount of MTZ was added to an aqueous solution of G (1% EtOH was added to improve the solubility of G). The ultimate concentrations of MTZ, G, and WP6P were 0.025, 0.125, and 0.025 mM, respectively. After standing overnight, the prepared MTZ-loaded vesicles were purified by dialysis (molecular weight cutoff 10000) in distilled water for several times until the water outside the dialysis tube exhibited negligible MTZ absorbance. The MTZ encapsulation efficiency was calculated by the following equation. Encapsulation efficiency (%) = (mMTZ-loaded/mMTZ) × 100 where mMTZ-loaded and mMTZ are mass of MTZ encapsulated in vesicles and mass of MTZ added, respectively. The mass of MTZ was measured by a UV spectrophotometer at 660 nm and calculated as relative to a standard calibration curve in the concentrations from 0.37 to 6.3 μg/mL in water (Figure S39, Supporting Information). Stimuli-Responsive Behaviors of the Drug-Loaded Nanoparticles.

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1) pH-Responsive Drug Release in vitro. 0.05 M tris-HCl buffer solutions (pH = 7.4) and 0.1 M citrate buffer solutions (pH = 5.0) were used as drug release media to simulate normal physiological conditions and the intracellular conditions of tumor. In a typical release experiment, 8.4 mL of DOX-loaded micelles or MTZ-loaded vesicles was added into 1.6 mL of appropriate release medium at 37 °C. At selected time intervals, 2 mL of the release media was taken out for measuring the released drug concentrations (DOX by the fluorescence technique and MTZ by the UV-Vis absorption technique, respectively), and then was returned to the original release media. A nearly 100% release of drug from drug-loaded nanoparticles was obtained by using a very low pH medium (solution of HCl, pH = 3), in which most of the nanoparticles collapsed. 2+ 2+ 2) Zn -Responsive Drug Release in vitro. In a typical Zn induced release experiment, a certain amount of ZnCl2 solution was added into 10 mL of drug-loaded micellar or vesicular solution, the final Zn2+ concentration was 1 mM, 2 mM, and 5 mM, respectively in the micellar or vesicular solution. At selected time intervals, 2 mL of the release media was taken out for measuring the released drug concentrations (DOX by the fluorescence technique and MTZ by the UV-Vis absorption technique, respectively), and then was returned to the original release media. A nearly 100% release of drug from drug-loaded nanoparticles was obtained by using a very low pH medium (solution of HCl, pH = 3), in which most of the nanoparticles collapsed. In vitro Cell Assay. The relative in vitro cytotoxicities of guest G, hosts WP5P and WP6P, DOX·HCl, MTZ, unloaded micelles and vesicles, DOX-loaded micelles, and MTZ-loaded vesicles against L929 normal cell, A549 cancer cell, and HeLa cancer cell were assessed by using the MTT assay. Briefly, the cells were seeded in 96-well plates at a density of 104 cells per well in 200 uL of complete DMEM containing 10% fetal bovine serum, supplemented with 50 U·mL−1 penicillin and 50 U·mL−1 streptomycin, and cultured in 5% CO2 at 37 °C for 24 h. Then the three kinds of cells were exposed to serial dilutions of G, WP5P, WP6P, DOX·HCl, MTZ, unloaded micelles, unloaded vesicles, DOX-loaded micelles, and MTZloaded vesicles, respectively and further incubated for 24 h. The cells were washed and replenished with fresh culture medium and further incubated for 2 h. Subsequently, 20 μL of MTT solution was added into each cell and incubated for another 4 h. After that, the medium containing MTT was removed, and dimethyl sulfoxide (DMSO, 150 μL) was added to each well to dissolve the MTT formazan crystals. Finally, the plates were shaken for 10 min, and the absorbance of formazan product was measured at 490 nm by a microplate reader (BioTek ELx808). Untreated cells in media were used as the blank control. All experiments were carried out with six replicates. The cytotoxicity was expressed as the percentage of the cell viability as compared with the blank control. Cellular Uptake and Intracellular Localization Observed by Fluorescence Microscope. The cellular uptake and intracellular

localization of DOX and MTZ were examined in A549 cacer cell lines. Briefly, tumor cells were plated onto glass-bottomed Petri dishes in 1.5 mL of complete culture medium for 24 h before treatment. Then cells were treated with free DOX·HCl solution (1.0 μg·mL−1) or DOX-loaded micellar solution (equivalent to 1.0 μg·mL−1 DOX) and free MTZ solution (3.0 μg·mL−1) or MTZ-loaded vesicular solution (equivalent to 3.0 μg·mL−1 MTZ) for designated time periods. For A549 cells, LysoTracker and Hochest (Molecular Probes, USA) were di-


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rectly added to the medium at a final concentration of 100 nM for 1 h to label lysosomes and nucleus, respectively. The tumor cells were washed three times with fresh medium and investigated by fluorescence microscopy (LSM710, CarlZeiss). The fluorescence characteristics of DOX·HCl, MTZ, DOXloaded micelles, and MTZ-loaded vesicles were used to directly monitor the localization of these drugs without utilizing additional dye. Flow Cytometric Analysis. A549 cells were seeded in sixwell plates at a density of 5 × 105 cells per well in 3 mL of complete DMEM and cultured at 37 °C in a 5% CO2 humidified atmosphere for 24 h. After treating with DOX·HCl, MTZ, DOX-loaded micelles, and MTZ-loaded vesicles, respectively for determined intervals at 37 °C, the cells were then rinsed three times with cold PBS. After trypsinizing, the cells were washed with cold PBS, centrifuged, and dispersed in cold PBS. And then, the cells were subjected to flow cytometric analysis using a BD FACSCalibur flow cytometer, and 10 5 cells were tested for each sample. Statistical Analysis. Differences between treatment groups were statistically analyzed using the paired Student‟s test. A statistically significant difference was reported if p < 0.05 or less. The data were expressed as mean ± standard deviation from at least three separate experiments.

(3) Cabral, H.; Nishiyama, N.; Kataoka, K. Supramolecular Nanodevices: From Design Validation to Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44, 999-1008. (4) Moughton, A. O.; O‟Reilly, R. K. Noncovalently Connected Micelles, Nanoparticles, and Metal-Functionalized Nanocages using Supramolecular Self-Assembly. J. Am. Chem. Soc. 2008, 130, 87148725. (5) Chen, C. J.; Li, D. D.; Wang, H. B.; Zhao, J.; Ji, J. Fabrication of Dual-Responsive Micelles Based on the Supramolecular Interaction of Cucurbit[8]uril. Polym. Chem. 2013, 4, 242-245. (6) Guo, M.; Jiang, M. Non-Covalently Connected Micelles (NCCMs): The Origins and Development of A New Concept. Soft Matter. 2009, 5, 495-500. (7) Xing, P.; Sun, T.; Hao, A. Vesicles from Supramolecular Amphiphiles. RSC Adv. 2013, 3, 24776-24793.

ASSOCIATED CONTENT

(8) Jiao, D.; Geng, J.; Loh, X. J.; Das, D.; Lee, T. C.; Scherman, O.

Supporting Information Experimental procedures and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.

A. Supramolecular Peptide Amphiphile Vesicles through Host–Guest Complexation. Angew. Chem., Int. Ed. 2012, 51, 9633-9637.

AUTHOR INFORMATION

(9) Zhao, Q.; Wang, Y.; Yan, Y.; Huang, J. Smart Nanocarrier:

Corresponding Author *[email protected]

Self-Assembly of Bacteria-Like Vesicles with Photoswitchable Cilia.

Author Contributions

ACS Nano 2014, 8, 11341-11349.

†

Xiao-Yu Hu and † Xin Liu contributed equally. (10) Lim, Y.; Moon, K. S.; Lee, M. Recent Advances in Functional

ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (2014CB846004), National Natural Science Foundation of China (No. 21572101, 21472089, 21572069), and Jiangsu Provincial Natural Science Foundation of China (BK20140595). We also thank Dr. Wei Xia and Mr. Xuan Wu in our group for their kind help in drawing. The referees are acknowledged for helpful comments.

Supramolecular Nanostructures Assembled from Bioactive Building Blocks. Chem. Soc. Rev. 2009, 38, 925-934. (11) Cheetham, A. G.; Zhang, P.; Lin, Y.; Lock, L. L.; Cui, H. Supramolecular Nanostructures Formed by Anticancer Drug Assembly. J. Am. Chem. Soc. 2013, 135, 2907-2910.

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Controllable Construction of Biocompatible Supramolecular Micelles and Vesicles by Water-Soluble Phosphate Pillar[5,6]arenes for Selective Anti-Cancer Drug Delivery Xiao-Yu Hu,† Xin Liu,† Wenyi Zhang,‡ Shan Qin,† Chenhao Yao,† Yan Li,‡ Derong Cao,§ Luming Peng,† and Leyong Wang*†


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Controllable Construction of Biocompatible Supramolecular Micelles

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