Cucurbit[8]uril-Based Giant Supramolecular Vesicles: Highly Stable

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Cucurbit[8]uril-based Giant Supramolecular Vesicles: Highly Stable, Versatile Carriers for Photoresponsive and Targeted Drug Delivery Cuihua Hu, Ningning Ma, Fei Li, Yu Fang, Yao Liu, Linlu Zhao, Shanpeng Qiao, Xiumei Li, Xiaojia Jiang, Tiezhu Li, Fangzhong Shen, Yibing Huang, Quan Luo, and Junqiu Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00297 • Publication Date (Web): 15 Jan 2018 Downloaded from http://pubs.acs.org on January 15, 2018

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ACS Applied Materials & Interfaces

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cucurbit[8]uril-based

Giant

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Supramolecular

Vesicles: Highly Stable, Versatile Carriers for Photoresponsive and Targeted Drug Delivery Cuihua Hu,†, ‡ Ningning Ma,† Fei Li,† Yu Fang,† Yao Liu,† Linlu Zhao,† Shanpeng Qiao,† Xiumei Li,† Xiaojia Jiang,† Tiezhu Li, † Fangzhong Shen,† Yibing Huang,*,‡ Quan Luo,*,† Junqiu Liu† †

State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical

Chemistry, Jilin University, 2699 Qianjin Road, Changchun 130012, China. ‡

Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education,

College of Life Sciences, Jilin University, 2699 Qianjin Road, Changchun 130012, China. KEYWORDS: cucurbit[8]uril, supra-amphiphile, supramolecular vesicle, photo-controlled drug delivery, targeting specificity

ABSTRACT: Highly stable giant supramolecular vesicles were constructed by hierarchical selfassembly of cucurbit[8]uril (CB[8])-based supra-amphiphiles for photoresponsive and targeted intracellular drug delivery. These smart vesicles can encapsulate the model drugs with high loading efficiencies, and then release them by manipulating photoswitchable CB[8] heteroternary complexation to regulate the formation and dissociation of supra-amphiphiles that cause dramatic morphological changes of the assemblies to achieve remote optically-controlled drug


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delivery. More importantly, confocal microscopy analysis, cellular uptake experiment, and cell viability assay have showed that the giant vesicles are able to maintain the structural integrity and stability within actual cellular environments and exhibit obvious advantages for intracellular drug delivery such as low toxicity, easy surface modification for tumor-targeting selectivity, and rapid internalization into different human cancer cell lines. A synergistic mechanism that integrates multiple pathways including energy-dependent, macropinocytosis, cholesteroldenpendent, and microtubule-related endocytosis was determined to facilitate the internalization process. Moreover, cytotoxicity experiments and flow cytometric analysis have demonstrated that the doxorubicin hydrochloride (DOX)-loaded vesicles exhibited a significant therapeutic effect for tumor cells upon UV light irradiation, which makes the photoresponsive system more promising for potential applications in pharmaceutically relevant fields.

INTRODUCTION The development of drug delivery systems with both stimuli-responsiveness and targeting ability to tumor cells for minimized side effects has attracted increasing attention in cancer treatment.1 Since phospholipid-based biomembranes have inspired chemists to create biomimetic nanocarriers, a variety of chemically synthesized molecules have been explored for selfassembly into well-defined nanostructures to promote their applications for drug delivery, including the encapsulation efficiency, specific targeting, controlled release, and cellular toxicity.2-5 Among them, macrocycle-based supra-amphiphiles such as calixarene, cyclodextrin, cucurbituril, and pillararene complexes that build on noncovalent interactions between macrocyclic host and guest molecules have become a promising building block for designing new generation of smart delivery systems due to their simplicity, biocompatibility, versatility,


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and especially reversibility.6-9 This dynamic nature endows the supramolecular architectures with stimuli-responsibility to induce their morphological changes for controlled release of the encapsulated compounds. In some cases, functional groups such as cell-specific ligands can be non-covalently introduced onto vesicle surface by host-guest complexation to improve the specificity of anticancer drugs to tumor cells, thus making it more suitable for the facile fabrication of multifunctional nanocarriers without tedious synthesis processes.10 High tumor selectivity is realized and allows for a combinatorial flexibility through multivalent ligand display for various targeting delivery applications,11,12 which may represent a new strategy for therapeutic innovation in oncology. Cucurbit[n]urils (CB[n]) are a family of barrel-shaped macrocyclic hosts that contain 5-10 glycoluril units and differ in the diameter of two identical carbonyl-fringed portals and a hydrophobic cavity.13 They are capable of forming 1:1 binary complexes and even 1:1:1 heteroternary complexes with aromatic ligands on the basis of complementary shape and size through a combination of the hydrophobic effect and ion-dipole interactions with the carbonyl oxygens on the portals.14 Many previous studies have shown that CB[n] can act as molecular receptors to construct large supra-amphiphiles by linking the hydrophobic and hydrophilic segments for self-assembly into highly ordered nanostructures with desired sizes, shape, and properties in drug delivery studies.15 These supramolecular systems were reported to have several remarkable features: (i) their ability to form giant vesicles with high drug-loading capacity;16 (ii) allowing for modular surface modification by multivalent noncovalent interactions for targeted delivery and immunization;17 (iii) low intrinsic cytotoxicity and bioadaptability in compliance with the pharmaceutical applications.18 In particular, CB[8] exhibits distinct advantages because it has a bigger cavity size and can simultaneously bind two


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ligands with extraordinary high association constants (typically Ka = 1011-1012 M-2),19,20 which provides feasibilities to construct more stable, versatile delivery systems for preventing in vivo premature drug release caused by noncovalent assembly. On the basis of CB[8] ternary complexation, new strategies have been developed to fabricate smart delivery carriers for the release of drugs in response to different stimuli. For example, Scherman et al. constructed multi-triggered peptide amphiphile vesicles through pyrenemethylviologen (MV) complexation with CB[8] for chemoresponsive drug delivery, which was also confirmed to be effective in the encapsulation and release of bioactive cytokines.21,22 Ji and co-workers

employed

(3-indolepropionic-acid(IPA)·MV)⊂CB[8]

and

(naphthalene(Np)·MV)⊂CB[8] heteroternary complexation as driving forces to prepare reduction-responsive and pH-responsive nanocarriers by using the redox chemistry of MV and an acid-sensitive hydrazone linkage, respectively.23,24 However, the design of photoresponsive delivery vehicles was motivated since CB[8]-mediated heteroternary complexation with MV and azobenzene (Azo) was discovered in 2012.25 Incorporation of the specific recognition component into amphiphilic or polymeric systems has successfully led to the formation of supramolecular vesicles and colloidal microcapsules for photo-triggered drug release.26,27 Nevertheless, how to realize their dynamic and responsive behaviors within actual cellular environments remains a great challenge that needs to be addressed by rational design of supra-amphiphile molecules to improve the self-assembled morphology, stability, surface properties, internalization efficiency, etc. Herein, a relatively non-cytotoxic, efficient, and targeted intracellular delivery system was constructed by hierarchical self-assembly of CB[8]-based supra-amphiphiles into highly stable giant supramolecular vesicles for remote optically-controlled drug release. The hydrophilic part


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is the complex of CB[8] and maleimide-modified methylviologen (MMV), while the hydrophobic part is 3,4,5-tris(n-dodecyloxy)benzoylamide with an Azo moiety (TBA-Azo), whose trans to cis isomerization under UV light irradiation leads to photo-triggered reconfiguration of assemblies to achieve the controlled drug release. Moreover, supramolecular vesicles with the maleimide groups on external surface can be easily functionalized with various thiol-containing ligands, ranging from iRGD peptide to BSA protein, by “click” reactions to promote their targeting specificity and cell internalization. We also demonstrated the rapid cellular uptake of the vesicles into three models of human cancer cell lines, as well as their excellent structural integrity, stability, drug-loading capacity, and photoswitchable drug release in a complex cellular environment. Cell viability assay, cellular uptake experiment, confocal microscopy, and flow cytometric analysis were further performed to explore their toxicology, internalization mechanism, tumor selectivity, and anticancer pharmacological efficacy at the cellular level, which provide insights for the design of preclinical models toward stimuliresponsive drug delivery in real-life applications. RESULTS AND DISCUSSION Construction of Supramolecular Heteroternary Vesicles via CB[8]-Based Host-Guest Complexation. The construction of supramolecular vesicles by supra-amphiphiles based on photoresponsive CB[8] heteroternary complexation is shown in Figure 1a and the synthesis details of MMV, TBA-Azo, and CB[8] are described in the Supporting Information (Figure S1S7). Due to the poor solubility of TBA-Azo in water, 4-Aminoazobenzene hydrochloride (AAH) was used as a substitute for studying CB[8]-mediated heteroternary complexation with MV and TBA-Azo by 1H NMR spectroscopy. Figure S8 shows the 1H NMR results upon addition of MV and AAH into CB[8] solution. Compared with the 1H NMR spectrum of free MV and AAH,


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significant upfield chemical shifts were observed for their aromatic protons in the presence of CB[8], which is consistent with the previously reported results for (MV·Azo)⊂CB[8] heteroternary complexation in D2O.25 This finding can be ascribed to the shielding effect of the stacked aromatic rings in the cavity of CB[8]. In addition, electrospray ionization mass spectrometry (ESI-MS) spectrum of an aqueous solution of the heteroternary complex shows the peaks at m/z 1267.5 and 840.5 corresponding to doubly charged (TBA-Azo·MMV2+)⊂CB[8] and MMV2+⊂CB[8] complexes, respectively (Figure S9). All experimental results demonstrate the formation of a ternary inclusion complex, in which the binding between TBA-Azo and MMV2+⊂CB[8] relies on a relatively weak non-covalent interaction that can be disrupted upon ionization during ESI-MS analysis.


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Figure 1. (a) Schematic representation of the assembly strategy to construct supramolecular vesicles by supra-amphiphiles based on photoresponsive CB[8] heteroternary complexation. (b) Schematic representation of targeted and photo-controlled intracellular drug delivery through surface modification and UV light irradiation of supramolecular vesicles. The inclusion of MV and Azo moiety into CB[8] could endow the ternary complex with amphiphilic nature for self-assembly into nanostructures in water. As expected, the solution exhibits a typical Tyndall effect (Figure S10), revealing the existence of abundant nanoparticles. Light microscope (LM), scanning electron microscope (SEM), and transmission electron microscope (TEM) were employed to investigate the self-assembled morphologies. The molar ratio between CB[8] and two ligands to construct giant supramolecular aggregates is determined to be 1:1:1, while the aggregation concentration of CB[8] with MMV and TBA-Azo is 10-4 M (Figure S11 and Figure S12). As shown in Figure 2a and b, a large area of monodispersed spherical aggregates was formed with a fairly uniform size in the range of 0.8-1 µm. These assemblies were further validated by the TEM image (Figure 2c), where the hollow spherical nanoparticles with a high contrast between the bright center and dark periphery indicate the formation of vesicular structure. In addition, the outer and inner diameters of supramolecular vesicles are measured to be 860 nm and 820 nm, respectively, and the wall thickness is about 20 nm. Considering that the extended length of (TBA-Azo·MV2+)⊂CB[8] inclusion complex calculated by GaussView 5.0 is ~4 nm, the vesicle is deduced to be composed of three bilayers of the supra-amphiphiles (Figure 1a). Dynamic light scattering (DLS) data also indicates a narrow size distribution of the vesicles with an average hydrodynamic diameter of 878 nm (Figure 2d), which agrees well with those obtained by SEM and TEM.


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Figure 2. Microscopic analysis of the vesicular structures self-assembled from CB[8]-based supra-amphiphiles. (a) LM; (b) SEM; (c) TEM; (d) a narrow size distribution of the vesicles determined by DLS. Molecular Encapsulation and Photoresponsive Release Behavior of the Supramolecular Vesicles. Supramolecular vesicles formed by photoresponsive supra-amphiphiles provide an opportunity for developing smart drug delivery system, whose photolability is a very important property to promote their application potential. The encapsulation and release of a fluorescent dye, 5(6)-carboxyfluorescein (CF), was directly evaluated by in situ and real-time analysis using confocal laser scanning microscopy (CLSM). As shown in Figure 3a, uniformly distributed bright green fluorescence dots with no observable leakage in a dark background was observed, suggesting the formation of vesicular structures without defect. After irradiation at 365 nm for 20 min, the fluorescent dots underwent budding and fission due to the light-induced isomerization


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of the Azo moiety of supra-amphiphiles to drive the deformation of vesicular membranes. An increasing green fluorescent background caused by a continuous membrane permeation was observed, which confirms the photo-triggered release of CF from the interior of the vesicles to the aqueous solutions. Moreover, these giant supramolecular vesicles with an approximately micrometer-sized internal volume can guarantee a large drug-loading capacity and excellent encapsulation efficiency, which were further quantified by UV-Vis spectroscopy. The hydrophilic rhodamine B (RhB) was first selected as a model drug to explore the entrapment properties of this delivery vehicle. RhB-loaded vesicles were prepared by mixing RhB solution with an aqueous solution of CB[8] and MMV followed by the injection of a solution of TBAAzo in dimethylformamide (DMF), and then removing the non-encapsulated RhB by dialysis against water. As expected, the drug-loading and encapsulation efficiency of the vesicles are calculated to be about 9.88% and 65.0%, respectively. Similarly, the drug-loading and encapsulation efficiency of CF (6.34% and 84.21%) and DOX (5.57% and 62.15%) are also estimated. The release profile of DOX from the vesicles upon UV irradiation was recorded in Figure S13. These findings reveal that CB[8]-based supramolecular vesicles could serve as an ideal nanocarrier for effective encapsulation and controlled release of different small molecules for drug delivery application.


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Figure 3. (a) In situ and real-time analysis of photo-controlled CF release behavior of CB[8]based supramolecular vesicles by CLSM upon UV light irradiation for 60 min. (b) In vitro release profile of CF-loaded vesicles. The blue line represents the vesicles exposed to UV light irradiation, while the black line represents the system without any triggers. (c) UV/Vis spectral changes of CB[8]-based supra-amphiphiles in water under UV light irradiation for 70 min. In Vitro Release of Model Drugs upon UV Light Irradiation. Photo-controlled release capability of the vesicles was further investigated by monitoring the fluorescence intensities of the solution and the release percentage of CF versus time was calculated using a previously described method.26 Figure 3b shows in vitro release profile of CF-loaded vesicles. Without UV light irradiation, the cumulative leakage of CF from supramolecular vesicles is less than 6%, suggesting that the vesicular membrane is relatively stable and impermeable to the dye. In contrast, a rapid release behavior was observed for the irradiated sample and over 50% of the loaded fluorescent dye was released into the water within 80 min, revealing that UV light


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irradiation results in the destabilization of the vesicles to achieve the release process. After adding Triton X-100, a well-known vesicle destroyer, a complete release of CF was detected, which is also an evidence for the encapsulation of the CF dye by supramolecular vesicles. To confirm the enhanced release of CF coupled to the azobenzene photoisomerization, the UV-Vis spectra of a water suspension of supramolecular vesicles were recorded at different time intervals in the presence of UV light. As shown in Figure 3c, exposure of the vesicles to UV light irradiation caused significant spectral changes. A gradual decrease of π-π* absorbance at 362 nm and a concomitant increase of n-π* absorbance at 450 nm that represent the typical absorption spectrum of the trans-to-cis isomerization of the Azo unit were observed.28 In addition, the isomerization time is > 70 min, which is obviously slower than that of the Azo in (MV·Azo)⊂CB[8] heteroternary complex (~ 64 s) and agrees well with the release process.25 This indicates that densely packed self-assembled vesicular structures may hinder the dissociation of CB[8]-based supra-amphiphiles to affect the photo-induced isomerization of Azo moiety. Figure S14 show the TEM images of CF-loaded supramolecular vesicles before and after 20 min UV light irradiation. The irradiated vesicles still maintain an unbroken ellipsoid structure with apparently distorted membranes and protrusions as compared with the non-irradiated samples. We can clearly observe that the entrapped fluorescent dye molecules were released from the ruptured site of supramolecular vesicles. Surface Functionalization of the Vesicles with Bioactive Molecules. Based on the proposed self-assembly mechanism, supramolecular vesicles should be covered with highly reactive maleimide groups, thereby making it easy to be functionalized with various thiol-containing ligands via the thiol-maleimide ‘‘click’’ reaction (Figure 1a and 1b). This conjugation procedure was demonstrated to be efficient for coupling cyclic iRGD-C peptide (CRGDKGPDCC) (Figure


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S15 and S16), hyaluronic acid (HA), and bovine serum albumin (BSA). Among them, iRGD and HA are extensively used as targeting molecules that can selectively recognize cell surface receptors to improve the targeting specificity of supramolecular vesicles. Figure 4a shows the 1H NMR spectrum of MMV reacted with the cysteine residue of iRGD. The characteristic peak of the maleimide group at 6.89 ppm was found to be completely disappeared after the reaction, which indicates that the final MMV-iRGD conjugate was formed.29 In addition, a comparison of the Fourier transform infrared spectroscopy (FTIR) spectra of HA and its product after EDC crosslinking reaction (Figure S17) reveals the successful synthesis of thiol-modified HA when the appearance of the thiol US-H band at 2538.18 cm−1 and the decrease in peak intensities of asymmetric (C-O) and symmetric (C-O) stretching bands of the planar carboxyl groups in HA at 1616.30 cm−1 and 1413.52 cm−1 were observed (Figure 4b).30 The subsequent disappearance of the thiol band after “click” reaction further confirms the conjugation of thiol-modified HA on MMV. Supramolecular vesicles decorated with fluorescein isothiocyanate (FITC)-labeled BSA were prepared in the same way as described above. As shown in Figure 4c, the formation of monodisperse vesicles with green fluorescence on the spherical periphery further confirms that surface modification has virtually no impact on their self-assembly process. Based on the noncovalent interactions between the accessible CB[8] units on the surface and MMV derivatives, multifunctional nanodelivery systems that combine targeting, diagnostic, and therapeutic actions could also be designed by the introduction of different ligand-modified MMV into the self-assembled system for biomedical applications.


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Figure 4 (a) 1H NMR spectrum of MMV before (I) and after (II) “click” reaction; (b) FTIR spectra of HA (I) and its products thiol-modified HA (II) and MMV-HA conjugate (III) (c) Fluorescence microscopy images of the vesicles that were coated with FITC-labeled BSA. Tumor Cell Selectivity of Ligand-modified Vesicles. The multifunctional properties of supramolecular vesicles provide unique advantages for tumor-specific delivery. Three cancer cell lines (e.g. lung carcinoma cells (A549), breast cancer cells (MDA-MB-231), and prostate cancer cells (PC-3)) and two normal cell lines (umbilical vein endothelial cells (HUVEC) and liver cells (L-O2)) were chosen for CLSM analysis to track the unmodified vesicles and iRGD-/HA-coated vesicles with the encapsulated RhB dye in living cells. As shown in Figure 5, the differences of red fluorescence intensity in the cytoplasm provide a direct assessment of the selectivity of these


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supramolecular vesicles towards different cell lines. After adding the unmodified vesicles into the five cells, almost no red fluorescence was observed within 28 s in both cancer cells and normal cells. In comparison, the cells mixed with iRGD- and HA-coated vesicles for the same incubation time and condition exhibit obvious fluorescence difference. In the former case, red fluorescence was apparently observed in the MDA-MB-231 cells and relatively weak fluorescence signals were detected in A549 and PC-3 cells, while the fluorescence was invisible in HUVEC and L-O2 cells. This indicates that iRGD-coated vesicles are more easily internalized by cancer cells than normal cells due to their specific binding ability to neuropilin-1 (NRP-1) receptor-overexpressing cancer cells when iRGD was cleaved into the CRGDK fragment.31 Selective cellular uptake by cancer cells can be attributed to the different expression level of NRP-1 receptor on MDA-MB-231, A549, and PC-3 cells. For the latter delivery system, most of the HA-coated vesicles were internalized into PC-3 cells with relatively bright fluorescence. Only light red fluorescence and very weak red fluorescence were observed in A549 and MDAMB-231 cells, respectively. The HA-coated vesicles exhibited high selectivity to PC-3 cells that overexpress cluster of differentiation 44 (CD44) for specific targeting of HA molecules, thereby enabling to distinguish cancer cells from normal cells based on CD44 expression density.32 These results demonstrated that the differential uptake efficiency of supramolecular vesicles mainly relies on the surface-attached ligands to achieve targeted intracellular drug delivery. By prolonging the incubation time until the fluorescence intensity reached a stable level, we also determined the time corresponding to maximal uptake by MDA-MB-231 cells for the unmodified vesicles and iRGD-coated vesicles, which are 8 min and 2.5 min respectively. Figure S18 shows a significantly higher intracellular accumulation of iRGD-coated vesicles as


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compared with the unmodified vesicles, revealing the enhanced cellular uptake of targeted vesicles via ligand-mediated internalization pathway.

Figure 5. Fluorescent confocal microscopic analysis of the selective cellular uptake of nontargeted and targeted supramolecular vesicles. Three tumor cells (A549, MDA-MB-231, and PC3 cells) and two normal cells (HUVEC and L-O2 cells) were incubated with 25 µM RhB-loaded vesicles, including the unmodified vesicles and iRGD-/HA-coated vesicles, for 28 s. Red is RhBloaded vesicles and blue is the nucleus stained by Hoechst 33258. Scale bar, 20 µm. Cellular Uptake and the Internalization Mechanism of the Vesicles. The internalization mechanism responsible for the rapid cellular uptake of supramolecular vesicles was investigated by flow cytometric analysis. Generally, highly charged amphiphilic molecules may translocate across eukaryotic cell membranes in a disruptive way through interaction with cell membranes to permeabilize and even lyse living cells. Thus, supramolecular vesicles with positively charged MV2+ group on the outer surface were first cultured with A549 cells for 1 h at different


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concentrations, then the cells were stained with the membrane impermeant dye propidium iodide (PI) to evaluate the cell permeabilization affected by the internalization process. For control experiments, the A549 cells were cultured with the subsequently added Triton X-100 as positive control or without the sample as negative control. As shown in Figure 6a, the increase of vesicle concentration from 25 µm to 50 µm had almost no effect on the fluorescence intensity, indicating that the vesicles enter the A549 cells in a non-dose-dependent manner without membrane damage. Furthermore, the effect of temperature on internalization was also analyzed in A549, PC-3 cell and HUVEC cell lines. 25 µM RhB-loaded vesicles, including the unmodified vesicles and iRGD-/HA-coated vesicles, were cultured on these cells under 4 °C and 37 °C for 1 h. Flow cytometry histograms (Figure 6b and Figure S19) show that significant reductions were observed in the fluorescence intensity levels at 4 °C compared with 37 °C for all the above cells with the internalized targeted and non-targeted vesicles. This finding indicates that supramolecular vesicles might be transported into the cytoplasm via an energy-dependent endocytosis. The detailed endocytic pathways were further elucidated by studying the effects of several membrane entry inhibitors on the cellular internalization. A549 cells were pretreated with different inhibitors, including chlorpromazine (CPZ), cytochalasin-D (Cyto-D), nystatin, nocodazole, methyl-β-cyclodextrin (M-β-CD), and NaN3 with deoxyglucose (DG + NaN3), which can competitively inhibit the chathrin-mediated endocytosis, macropinocytosis, caveolin-dependent endocytosis, microtubule-associated endocytosis, cholesterol-dependent endocytosis, and energy-dependent endocytosis, respectively.33 As shown in Figure 6c, DG + NaN3, Cyto-D, M-βCD, and nocodazole have a significant inhibitory effect on the uptake of supramolecular vesicles, as they can greatly reduce the cellular uptake to 35.77 ± 0.81%, 43.17 ± 1.21%, 38.3 ± 0.98%, 41.82 ± 1.84% respectively. On the contrary, the uptake inhibition by CPZ and nystatin was


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considerably weak, with the relative uptake rate of 57.60 ± 1.81% and 72.78 ± 0.97% that are closed to or higher than that without any inhibitor treatment (62.74 ± 0.96%), These results suggest that at least four uptake pathways are involved in the endocytosis process, which allows rapid entry of the supramolecular vesicles into the cytoplasm.


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Figure 6. Flow cytometry histograms illustrating the internalization mechanism of supramolecular vesicles. (a) Effect of vesicle concentration on cellular uptake of the vesicles. A549 cells were cultured with 25 µM/50 µM vesicles and Triton X-100 (positive control) respectively or without the sample (negative control) for 1 h at 37 °C, 5% CO2; (b) Effect of temperature on cellular uptake of the vesicles. A549 cells were cultured with 25 µM RhB-loaded vesicles for 1 h at 4 °C and 37 °C; Cells treated in the absence of vesicles were used as controls. (c) Effect of endocytosis inhibitors on cellular uptake of the vesicles. A549 cells were pretreated with various inhibitors for 1 h, and then incubated with 50 µM RhB-loaded vesicles for 1 h at 37 °C, 5% CO2. Cells treated in the absence of inhibitors were used as controls. Cellular Localization of RhB-Loaded Vesicles. In order to determine the intracellular localization and distribution of supramolecular vesicles, A549 cells were incubated with RhBloaded vesicles to monitor the internalization process using CLSM. After seconds of incubation, a few spots with bright red fluorescence were clearly visualized in the cells (Figure 7a and Figure S20), which confirms that the self-assembled vesicular structure is stable enough to retain high structural integrity and stability in a complex cellular environment. However, a significant change that the entire cytoplasm is stained with red fluorescence dye was observed upon UV light exposure (Figure 7b). This indicates that the vesicle membrane became permeable to the loaded RhB dye when photo-induced isomerization of azobenzene occurs and subsequently cause the deformation of the vesicular membrane. Actually, we also found that the release of the encapsulated dye is only partial and some of the vesicles located in the vicinity of the cell surface are still unaffected by UV light irradiation. The existence of red fluorescent dots after 20 min irradiation provides an additional evidence for the stability of supramolecular vesicles. Given that the endocytosis pathway is known to deliver the cargo from early to late endosomes and


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eventually to lysosomes for degradation, A549 cells with LysoTracker® Green-labelled lysosomes were incubated with RhB-loaded vesicles to study co-localization between the vesicles and lysosomes. From the CLSM images (Figure 7c-e), most of the red and green spots are separated from each other. A low level of co-localization between RhB-loaded vesicles (red) and lysosomes (green) confirms that the vesicles mainly localize in the cytoplasm at the early stages of internalization, which is beneficial for the enhanced release of cargo via remotely photo-triggered changes in the vesicular structure.

Figure 7. CLSM images of the internalized vesicles in A549 cells before (a) and after (b) UV light irradiation; The intracellular localization and distribution of the vesicles at the early stages of internalization (c-e). Cells were cultured with RhB-loaded vesicles at 37 °C, 5% CO2. Lysosomes were labelled by LysoTracker® Green. Cellular Toxicity and Anticancer Efficiency of DOX-Loaded Vesicle. The cytotoxicity of CB[8]-based supramolecular vesicles and its components MMV, CB[8], TBA-Azo, CB[8] plus MMV were measured by MTT assay. Each of the above agents was added to the A549 cells at


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the concentrations of 1 to 32 µM. As shown in Figure S21a, there was no obvious difference in the relative survival rate (%) of tumor cells when incubated with the vesicles and its components. The highest vesicle concentration of 32 µM still did not suppress cell proliferation and the cell viability was kept above 90%, indicating that the vesicles have no significant cytotoxic activity in vitro within the tested concentration range. In order to study light-responsive release behavior, the cytotoxic effect of DOX-loaded vesicles at the concentrations of 1 to 25 µM with or without UV irradiation were also evaluated (Figure S21b). Under the dark condition, the survival rate of A549 cells were approximately 70%, which is lower than that treated with empty vesicles. The observed drop in cell viability may be explained by two factors: (1) the residual nonencapsulated drugs; (2) a low drug permeability due to some deformed vesicles. By contrast, DOX-loaded vesicles caused a further increased cytotoxicity in A549 cells after 20 min of UV light exposure and only 50% of cells survived, suggesting that the release of DOX from the vesicles to the cytosol can be controlled through UV light intervention. Photo-induced cytotoxicity of DOX-loaded vesicles was further assessed by measuring their induction effect on the cell apoptosis. Flow cytometry was performed to determine the apoptosis rates that are correlated with the intracellular DOX level, because DOX can intercalate into DNA and interact with topoisomerase II to inhibit DNA replication and macromolecular biosynthesis for the induction of cell apoptosis. The A549 cells were first incubated with 1.5 µg/mL DOX and 50 µM DOX-loaded vesicles for 24 h at different irradiation time, 0 min, 10 min, and 20 min, and then subjected to Annexin V-FITC/PI staining. Compared with the blank control with cell culture medium alone (1.38 ± 0.14%), free DOX induced a high cell apoptosis rate in A549 cells (90.75 ± 3.57%) and exhibited an obvious effect on the induction of late apoptosis (Figure 8 and Figure S22). For the cells treated with DOX-loaded vesicles, neither 0 min nor 10 min UV


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irradiation was able to promote the cell apoptosis when compared to untreated cells. The corresponding apoptosis rate was calculated to be 1.20 ± 0.21% and 1.30 ± 0.15%, respectively. However, DOX-loaded vesicles upon 20 min UV irradiation led to a significant increase in apoptosis rate (41.06 ± 6.29%) (P < 0.001), which was > 31.5-fold higher than that of the same system upon 0 and 10 min UV irradiation. All the apoptotic cells were detected to enter the late apoptosis stage and the percentage of early apoptotic cells was decreased to 0, which is consistent with the result of free DOX-induced cell apoptosis. These findings confirm that tumor cell apoptosis is due to the DOX released from CB[8]-based supramolecular vesicles inside cells through “remote control” upon photoexcitation. In addition, we also evaluated the apoptosis rate of the cells treated with 10.6 µM vesicles that contain the same drug concentration as 1.5 µg/mL DOX. As shown in Figure S23, the vesicles at this concentration upon 20 min UV irradiation led to an apoptosis rate of 10.51%. By prolonging the UV irradiation time to 80 min, the apoptosis rate increased to 23.35%. The rate of apoptosis is much lower than that induced by 1.5 µg/mL DOX, but agrees well with that treated with 0.69 µg/mL DOX (23.95%), which happens to be the drug concentration released from 10.6 µM vesicles according to the release profile in Figure S13.


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Figure 8. Apoptosis assay of the A549 cells treated with 50 µM DOX-loaded vesicles upon 0 min, 10 min and 20 min UV irradiation. The untreated cells and the cells treated with 1.5 µg/mL DOX were used as control. The apoptosis rates were measured using Annexin V-FITC/PI kit by flow cytometry. (***P < 0.001; **P < 0.01; *P < 0.05) CONCLUSION A facile strategy to construct supramolecular heteroternary vesicles that were assembled by host-guest complexation of CB[8] with MMV and TBA-Azo has been developed. These approximately micro-sized vesicles provide a stable and versatile platform for designing


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multifunctional nanocarriers to achieve the targeted and controlled delivery of the entrapped drugs upon photoexcitation. Drug loading and in vitro release experiments show that the vesicles exhibit an excellent encapsulation efficiency to water-soluble molecules such as CF and RhB, and their photoresponsiveness allows for a rapid release of the model drugs through a remotely triggered process. Furthermore, specific targeting to tumor cells was easily realized by surface functionalization of the vesicles with biologically active ligands via a “click” reaction and the cell selectivity varies with the change of the modified molecules. Investigation of the internalization mechanism and intracellular trafficking of vesicles indicates that several endocytosis pathways, including energy-dependent, macropinocytosis, cholesterol-dependent, and microtubule-related endocytosis contribute to the cellular uptake process and most of the vesicles located in the cytoplasm can maintain outer membrane integrity at the early internalization stage to facilitate the photoresponsive drug release. The cytotoxicity assessment further confirms that unloaded vesicles and its components have low toxicity within the tested concentration range while DOX-loaded vesicles show significantly enhanced cytotoxic effect and apoptosis rate in tumor cells under UV light irradiation. Together, these data demonstrate the potential of this nanocarrier as a new vesicular delivery system for remote optically-controlled drug release in cancer treatment. EXPERIMENTAL SECTION Materials

and

Instruments:

N-Boc-ethylenediamine,

bromoacetyl

bromide,

p-

aminoazobenzene, 1-bromododecane, methyl 3,4,5-trihydroxybenzoate, and glycoluril were purchased from Aladdin Reagent Co., Ltd. while 4,4-bipyridine and maleic anhydride were purchased from Macklin Biochemical Co., Ltd. DOX was purchased from Changchun Third Party Pharmaceutical Technology Co., Ltd.

Among them, glycoluril was further purified by


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recrystallization and used for CB[8] synthesis based on the previously reported procedures.34,35 Formaldehyde solution (37 wt %), thionyl chloride, triethylamine, dichloromethane, and dimethylformamide were purchased from Beijing Chemical Works. Human A549 cells, PC-3 cells, and normal cell live including HUVEC cells and L-O2 cells were purchased from American Type Culture Collection, while MDA-MB-231 cells were obtained from Shanghai Institute for Biological Sciences, Chinese Academy of Science. The morphologies of the vesicles were examined by LM (OLYMPUS BX61), SEM (JEOL JSM-6700F), TEM (JEOL JEM2100F). UV light irradiations were performed by using a handheld UV lamp (λex = 365 nm, 8W, 0.34 mW cm-2) encased in a box. UV/Vis spectral changes were monitored by Shimadzu UV 2450 spectrophotometer. The hydrodynamic diameter and size distribution of the vesicles were determined by a ZetaSizer Nano-ZS (Malvern Instruments, Malvern, UK). 1HNMR spectra were recorded on a Bruker AVANCE III 500 NMR spectrometer using tetramethylsilane (TMS) as internal standard. ESI-MS analyses were performed at a Thermo Finnigan LCQ Advantage mass spectrometer. FT-IR spectroscopy was recorded on Bruker IFS-FT66V FT-IR spectrometer. The fluorescence intensity was analyzed using a Shimadzu RF-5301 PC spectrophotometer. All confocal images were acquired using a Zeiss LSM 710 (Hoechst 33258: λex = 405 nm, LysoTracker® Green: λex = 488 nm, RhB: λex = 540 nm). The stained cells were analyzed by BD FACSCalibur flow cytometer. Preparation of Dye/Drug-loaded Vesicles: MMV was added to the solution of CB[8] and dyes/drugs (e.g. RhB, CF, and DOX) in Milli-Q and sonicated for 30 min at 323 K. TBA-Azo was dissolved in DMSO and then added to the above solution. The mixture was sonicated for another 2 h under the same conditions. The solution was placed at room temperature overnight


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until the vesicles reach thermodynamic steady state for further experiments. Dye/drug-loaded vesicles were purified by dialysis in distilled water for several times. The encapsulation and loading efficiency of vesicles with dye/drug were calculated as follow, Encapsulation efficiency (%) = (We/Wt) × 100 % where We is the dye/drug encapsulated in vesicles and Wt is the total amount of the added dye/drug. Loading efficiency (%) = (Wi/Wv) × 100 % where Wi is the amount of the loaded dye/drug in vesicles and Wv is the weight of vesicles. The fluorescence intensity of dye/drug (RhB: λem = 577 nm and λex = 540 nm; CF: λem = 517 nm and λex = 492 nm; DOX: λem = 558 nm and λex = 488 nm) was measured by fluorescence spectrometer and calculated as relative to a standard calibration curve in water. Photocontrolled Release of CF Molecules: The release of entrapped CF molecules from the vesicles was investigated using the previously reported method.26 The vesicle solution was placed in a quartz cuvette and exposed to UV light for different periods of time. The initial fluorescence intensity (I0) of CF-loaded vesicles was measured by fluorescence spectrometer at 512 nm after exciting the solution at 492 nm, then the fluorescence intensity (It) was recorded as a function of time. The CF release (%) was calculated according to the following equation: CF release (%) = (It − I0)/(Iα − I0) × 100 %


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where Iα is the fluorescence intensity when all the CF molecules were released from the vesicles, which was realized by the addition of Triton X-100 for 10 min after 90 min UV irradiation. Peptide Synthesis and Surface Modification of Vesicles: Homing peptide iRGD is a cyclic peptide (CRGDKGPDC) with a disulfide bridge between C1 and C9. iRGD-C was used as a tumor-targeting motif for surface modification of the vesicles via the “click” reaction between its maleimide group and the cysteine (C) of iRGD-C. Firstly, the linear peptide of Cys(Acm)Arg(Pdf)-Gly-Asp(OtBu)-Lys(Boc)-Gly-Pro-Asp(OtBu)-Cys(Acm)-C(Trt) was synthesized on Rink amide MBHA resin by Fmoc method, and HBTU, HOBt, and DIEA were used as coupling agents.36 Secondly, the disulfide bridge was formed by cyclization on resin with thallium trifluoroacetate.37 Finally, a cysteine was synthesized on C-terminal and the peptide was cleaved from resin by trifluoroacetic acid (TFA) / triisopropylsilane (TIS)/H2O = (95/2.5/2.5). The crude peptide was purified on a LC-6A preparative reversed-phase high performance liquid chromatography (RP-HPLC) using a Zorbax 300 SB-C8 column as described previously.38 The purity of the peptide was characterized by analytical RP-HPLC, mass spectrometry, and amino acid analysis, respectively. Cell Culture and Cellular Uptake Experiment: A549, PC-3, HUVEC and L-O2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 U/mL Streptomycin, while MDA-MB-231 cells were cultured with L15 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL Streptomycin. Then, all the cells were incubated at 37 °C in a humid atmosphere with 5% CO2. To test the cell selectivity of vesicles, the above cells were cultured in glass-bottomed dishes for 24 h and then stained with Hoechst 33258 for 30 min at 37 °C. 25 µM


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RhB-loaded unmodified vesicles and iRGD/HA-decorated vesicles were added to the cells, respectively. The images were taken at 7 s intervals by LSCM. The effect of vesicle concentration on cellular uptake was studied with A549 cells, which were plated in 6-well plates at the density of 1 × 105 cells per well. After 24 h incubation, the completed medium was replaced by fresh medium containing 25 µM or 50 µM empty vesicles for 1 h at 37 °C, 5% CO2. The cells were collected, washed with ice-cold PBS for 3 times and stained with 5 µg/mL PI at 37 °C for 30 min. The cell permeabilization was detected by flow cytometry. The effect of temperature on cellular uptake of RhB-loaded vesicles was studied in A549, PC3, and HUVEC cells, which were seeded in 6-well plates at the density of 1 × 105 cells per well and incubated for 24 h. The cells were prechilled at 4 °C for 1 h to consume endogenous ATP and then incubated with the unmodified vesicles and iRGD/HA-decorated vesicles for 1 h at 4 °C and 37 °C, respectively. Then, the cells were collected, washed with ice-cold PBS for 3 times, and stained with 5 µg/mL PI at 37 °C for 30 min. The cell permeabilization was detected by flow cytometry. The detailed endocytosis pathways were further investigated by using different membrane entry inhibitors.33 Before adding RhB-loaded vesicles, the cells were pre-incubated with the inhibitors, including Cyto-D (10 µg/mL), nystatin (25 µM), Cpz (10 µg/mL), nocodazole (20 µM), M-β-CD (50 mM), and NaN3 (10 µM) with DG (50 mM) for 1 h, respectively. The cells with PBS were prepared as the control. Finally, the cells were collected and washed with icecold PBS for 3 times for the detection of the internalized fluorescence using flow cytometry analysis.


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Intracellular Tracking and Co-localization: A549 cells were cultured in glass-bottomed dishes for 24 h and then stained with Hoechst 33258 for 30 min at 37 °C. Cell imaging by CLSM was performed after the cells were treated with RhB-loaded vesicles. The images were taken every 7 s from 0 s to 600 s. A549 cell were cultured with lysosome-specific probe lysoTracker® Green (Life technologies) and then washed with PBS for 3 times. The co-localization between the vesicles and lysosomes was measured by CLSM after the cells were placed in the medium containing RhB-loaded vesicles and cultured at 37 °C, 5% CO2 for 10 min. Cell Viability and Apoptosis Assay: Cell viability was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A549 cells were plated in triplicate in a 96-well pate at 5000 cells/well for 24 h at 37 °C. Then, the complete medium was replaced by 100 µL fresh medium containing DOX-loaded vesicles with or without UV light for 20 min. After a further 24 h incubation, the cells were treated with 20 µL MTT, which was reduced to an insoluble formazan crystal for an additional 4 h. Finally, 150 µL DMSO was added to dissolve the formazan product in each well and cell viability was photometrically quantified by measuring the absorbance of formazan at 492 nm. The apoptosis rates of the cells were measured using flow cytometry. A549 cells (1×105) in the logarithmic growth phase were plated in triplicate in 6-well plate. After 24 h, the fresh culture medium (negative control), 50 µM DOX-loaded vesicle with UV light 0 min, 10 min and 20 min (test group), 1.5 µg/mL DOX (positive control) were added to 6-well plates respectively and cultured for another 24 h at 37 °C, 5% CO2. Then, the cells were washed with ice-cold PBS for 3 times, collected by centrifugation and stained by Annexin V-FITC/PI kit.


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ASSOCIATED CONTENT Supporting Information The details about the synthesis and characterization of MMV, TBA-Azo, and their CB[8] heteroternary complex, including ESI-MS spectrum, MALDI-TOF mass spectrum, and 1H NMR spectrum. DLS data of the vesicles with different concentrations and molar ratios. TEM images of CF-loaded vesicles before and after UV light irradiation. The release profile of DOX-loaded vesicles. MALDI-TOF mass spectrum and HPLC chromatogram of cyclic iRGD-C. The synthetic details of thiol-modified HA. Cellular uptake experiment of iRGD-coated vesicles and the unmodified vesicles. Effect of temperature on cellular uptake of the vesicles. Confocal image of the internalized vesicles under bright field. MTT cell viability assay of the vesicles and its components. Apoptosis assay of the untreated cells, the cells treated with UV light and empty vesicles upon UV irradiation AUTHOR INFORMATION Corresponding Authors *Email: [email protected]. *Email: [email protected]. Author Contributions Cuihua Hu and Ningning Ma contributed equally to this work. Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (nos. 21474038, 21004028, 21234004, 21420102007, 21574056, and 91527302), the Chang Jiang Scholars Program of China, the Science Development Program of Jilin Province (nos. 20160520005JH and 20140101047JC), and the Open Project of State Key Laboratory of Supramolecular Structure and Materials (no: sklssm201708)


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2007, 13, 833-838. 38. Huang, Y. B.; Wang, X. F.; Wang, H. Y.; Liu, Y.; Chen, Y. X. Studies on Mechanism of Action of Anticancer Peptides by Modulation of hydrophobicity within a Defined Structural Framework. Mol. Cancer Ther. 2011, 10, 416-426.


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Cucurbit[8]uril-Based Giant Supramolecular Vesicles: Highly Stable

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