Three-in-One Self-Assembled Nanocarrier for Dual-Drug Delivery

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Biological and Medical Applications of Materials and Interfaces

Three-in-One Self-assembled Nanocarrier for Dual-Drug Delivery, Two-Photon Imaging, and Chemo-Photodynamic Synergistic Therapy Gang-Gang Yang, Liang Hao, Qian Cao, Hang Zhang, Jing Yang, Liang-Nian Ji, and Zongwan Mao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07270 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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ACS Applied Materials & Interfaces

Three-in-One Self-assembled Nanocarrier for Dual-Drug Delivery, Two-Photon Imaging, and Chemo-Photodynamic Synergistic Therapy Gang-Gang Yang, Liang Hao, Qian Cao*, Hang Zhang, Jing Yang, Liang-Nian Ji and Zong-Wan Mao* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou 510275 (P. R. China).

KEYWORDS: Ruthenium complex, cyclodextrin monomer, lysosome damage; chemo-photodynamic synergistic therapy; two-photon imaging;

ABSTRACT: We herein present a three-in-one nanoplatform (named as Fu/LD@RuCD)

for

dual-drug

delivery,

two-photon

imaging,

and

chemo-photodynamic synergistic therapy, enabled by simple self-assembly between adamantine-functionalized ruthenium complexes ([Ru(phen-ad)3](PF6)2, Ru) and natural cyclodextrin (β-CD) monomers. By host-guest chemistry, nanocarrier RuCD 70-90 nm in diameter is fabricated through very simple mixing step in water at room temperature, in which the octahedral configuration of Ru complex provides a rigid skeleton and the hydrogen bonding of secondary hydroxyl groups formed between two adjacent β-CD monomers displays a bridging role allowing for three-dimensional architectures. The dual-drug loaded nanoparticle Fu/LD@RuCD (Fu: 5-fluorouracil; LD: lonidamine) effectively penetrates into cancer cells in 8 h and selectively accumulates in lysosomes, in which dual-drug release is promoted by the mildly acidic environment. Under visible light irradiation, nanocarrier RuCD exhibits excellent PDT capability by producing sufficient ROS and damaging lysosomes, accordingly 5-fluorouracil and lonidamine can escape from lysosomes and reach their sites of action, resulting in mitochondria dysfunction and cancer cell apoptosis. 1

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Simultaneously, the excellent photophysical properties of the nanocarrier enables the facile track of drug delivery under one-photon and two-photon excitation. Moreover, in vivo anti-cancer investigations show that Fu/LD@RuCD can effectively inhibit the tumor growth without systemic side effects by chemo-photodynamic synergistic therapy, and the therapeutic effect is better than the free anti-cancer drugs and the sole therapeutic modality.

2


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1. INTRODUCTION Conventional chemotherapy utilizing small molecular anticancer drugs are most widely applied in clinic use but usually suffers from serious deficiencies.1 To enhance the therapeutic efficacy and attenuate side effects of conventional chemotherapy, development of multifunctional nanocarriers is considered as a promising strategy, which can integrate various functions such as targeted delivery, stimuli response, bio-imaging, etc. into one single nanoplatform.2 Meanwhile, synergistic treatment is of much current interest, which combines two or more therapeutic modalities thus exhibiting substantially enhanced therapeutic efficacy than the sole chemotherapy.3-10 Photodynamic therapy (PDT) is a prominent therapeutic modality in cancer treatment because of its non-invasion properties and selective damage of tumor tissues without systemic toxicity under temporal-and-spatial control.11 It uses photosensitizers to transfer energy from light to oxygen molecules, resulting in reactive oxygen species (ROS) production to damage cancer cells efficiently. It has been found that combining PDT with other therapeutic modalities could enhance therapeutic efficacy by inducing different cytotoxic pathways.12 Therefore, utilizing photosensitizer as a skeleton element to build novel multifunctional nanocarrier appears to be a promising strategy in chemo-photodynamic synergistic therapy.13-18 Ruthenium complexes, as alternatives to platinum-based metallodrugs, also possess outstanding anticancer activity. Moreover, several Ru(II) polypyridyl complexes have been reported as efficient PDT sensitizers due to their abilities to generate ROS under visible light irradiation.19-20 Compared with the planar structure of platinum drugs, the octahedral configuration of ruthenium complexes provides a rigid framework for the construction of nanocarrier and their large planar ligands may provide hydrophobic cavity for drug loading. Meanwhile, Ru(II) complexes have been widely utilized for bio-imaging because of their high quantum yields, large Stoke shift, long-lived luminescence, and excellent photostability.21 Several Ru(II) complexes even possess high two-photon absorption cross-section, allowing near-infrared two-photon imaging, which is more promising with deeper tissue penetration, lower interference from the biological autofluorescence, and less photo-damage.22 Therefore 3



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Ru(II) complexes appear to be excellent skeleton element for constructing multifunctional nanocarriers, which integrate the drug encapsulation capability, PDT activity and two-photon imaging property into one single nanocarrier. Cyclodextrins (CDs), due to the natural availability, good water solubility, hydrophobic cavity, low toxicity and low immunogenicity, have been extensively utilized to construct drug delivery systems.23 However, almost all the reported cyclodextrins used for constructing nanocarriers must be functionalized in advance, such as CD-based cationic polymers,24-25 CD-capped MSN,26 CD-functionalized quantum dots,27 CD-modified metal complexes, 28 and so on.29-30 Nevertheless, reports on the direct use of non-functionalized, natural cyclodextrin monomers for building nanocarriers is still lacking. Stoddart and coworkers have reported a organic-inorganic hybrid sandwich-like complex, which lines up longitudinally by means of secondary face [O−H···O] interactions between β-CD molecules.31 Our earlier work has also reported an imidazolate-bridged dinuclear copper-β-CD supramolecular unit, which also lines up as a helical chiral chain through hydrogen bonding of secondary hydroxyl groups.32 These crystal structures indicate that secondary face [O−H···O] interactions between β-CD monomers may provide an opportunity mediating the self-assembly of three-dimensional architecture under mild conditions. Inspired by these, we herein constructed a three-in-one multifunctional nanocarrier through simply mixing the adamantine-functionalized Ru(II) complex and natural β-CD monomers in water at room temperature (Scheme 1). Each Ru(II) complex contains three adamantine (Ad) groups capable of

encapsulated in the

primary face of β-CD monomers through host-guest interactions, and two adjacent β-CD molecules is supposed to form a head-to-head dimer through hydrogen-bonding of secondary hydroxyl groups, thus realizing the self-assembly of a three-dimensional nanoparticle. Based on the excellent photophysical properties and the rigid configuration of Ru(II) complexes, this novel multifunctional nanocarrier RuCD integrates three promising functions, e.g. dual-drug loading, two-photon imaging, and chemo-photodynamic synergistic therapy, into one platform. 4


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Scheme 1. Schematic illustration to show (a) the fabrication process of the multifunctional nanocarrier RuCD and dual-drug loaded Fu/LD@RuCD; (b) the intracellular drug release and the chemo-photodynamic synergistic therapy.

5-fluorouracil (5-Fu) and lonidamine (LD) are chosen as the co-delivered drug candidates in this work, acting on different targets. 5-Fu has been known converted into different cytotoxic metabolites in cells that suppress the synthesis of nucleic acids, resulting in cell cycle arrest and apoptosis.33-34 Lonidamine is capable of inhibiting 5



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glycolysis under hypoxia conditions and triggering mitochondria-dependent apoptosis.35 Although 5-Fu and LD have been used in the first-line therapy of colorectal carcinoma and the phase III trials of benign prostate hypertrophy, respectively, their further clinical use is limited by their poor solubility, low therapeutic index and severe side-effects caused by continuous administration.36-38 Through simple ultrasonic mixing both drugs can be co-encapsulated in the new nanocarrier RuCD, and the drug loading efficiency is estimated to be 10.71 wt% and 6.62 wt% for 5-Fu and LD, respectively. Moreover, the drug release from RuCD is found promoted in mildly acidic solutions (pH 5.0-6.0) mimicking the tumor microenvironment.

The

drug-loaded

nanocarrier

Fu/LD@RuCD

effectively

penetrates into cancer cells in 8 h and selectively accumulates in lysosomes. Under visible light irradiation, nanocarrier RuCD exhibits excellent PDT capability by producing sufficient ROS and damaging lysosomes, accordingly 5-fluorouracil and lonidamine can escape from lysosomes and reach their sites of action, resulting in mitochondria

dysfunction

and

cancer

cell

apoptosis.

Simultaneously,

the

multifunctional nanoplatform enables the facile track of drug delivery under one-photon and two-photon imaging. In in vivo anti-cancer investigations, Fu/LD@RuCD can effectively inhibit the tumor growth without systemic side effects by chemo-photodynamic synergistic therapy, and the therapeutic effect is better than the free anti-cancer drugs and the sole therapeutic modality.

2. RESULTS AND DISCUSSION 2.1 Preparation of nanocarrier RuCD via self-assembly The fabrication process of the multifunctional nanocarrier RuCD was illustrated in Scheme 1. Initially, adamantane-tethered ruthenium polypyridyl compound [Ru(phen-ad)3](PF6)2

(Ru;

phen-ad=2-Adamantyl

carboxaldehyde

1H-imidazo[4,5-f][1,10]-phenanthroline) was obtained by reacting RuCl3 with phen-ad as reported procedures with slight modification,39 and characterized by using 1

H NMR, 13CNMR, and ESI-MS (Fig. S1-S4, ESI). With increasing concentrations of

β-CD, UV-Vis absorption spectra of Ru in aqueous solution display a substantial 6


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hyperchromic effect and reaches the plateau when the molar ratio of β-CD/Ru is 3 (Fig. 1a-b). This indicates the 1:3 host-guest interaction ratio between Ru and β-CD monomer, which is also confirmed by isothermal titration calorimetry (ITC) measurement (Fig. S5a-b, ESI). This is reliable that each Ru has three adamantine moieties thus interacting with three β-CD monomers. The binding constant of Ru and β-CD is determined to be 105 M-1 from ITC measurement, which is consistent with the reported binding constants (Ka. 104-105 M-1) of cyclodextrins and adamantine.40

Figure 1 (a) UV-Vis absorption spectra of Ru (50 µM) upon the titration of β-CD monomers in aqueous solution. (b) Plot of the absorbance changes of Ru at 253 and 488 nm as a function of the increased β-CD amounts. (c) The hydrodynamic diameter distribution of as-prepared nanocarrier RuCD in ultra pure water determined by DLS, inset is the TEM image. (d) AFM image of the nanocarrier RuCD.

The nanocarrier RuCD was readily obtained by ultrasonic mixing Ru and 3-fold excess amounts of naturally available β-CD monomers in aqueous solution (4 h at 4 o

C) and purified by centrifuge (20000 r/min, 8 min). As shown in transmission

electron microscopy (TEM), the as-prepared RuCD has a uniform and well-distributed spherical morphology with average particle sizes of 70-90 nm (inset 7



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in Fig. 1c). Dynamic laser scattering (DLS) shows that the hydrodynamic diameter of RuCD is ca. 110 nm, which is relatively larger due to hydrated surroundings (Fig. 1c). In atomic force microscopy (AFM), the measured diameter (the width of half peak) and height is 70-90 nm and 20 nm, respectively, suggesting a hemispherical morphology of RuCD nanoparticles. It was found that RuCD was very stable and well dispersed in ultra pure water for at least two weeks without aggregation or precipitation. As monitored by DLS and UV-Vis spectroscopy, RuCD in PBS exhibits little change in particle size or absorption spectrum for over 48 h, further confirming its good stability (Fig. S5c-d, ESI). This novel nanocarrier is supposed to be formed by simple self-assembly: one Ru molecule possesses three adamantine (Ad) groups and each Ad group is encapsulated in the cavity of one β-CD monomer via host-guest interactions, simultaneously head-to-head β-CD dimers can be formed through hydrogen-bonding of secondary hydroxyl groups,31-32 accordingly a three-dimensional nanoparticle with the rigid octahedral Ru(II) complexes as the skeleton has been readily formed (as shown in Scheme 1). To provide solid evidence for the intermolecular hydrogen bonding together with host-guest interactions, nuclear overhauser enhancement spectroscopy (NOESY) of RuCD and natural β-CD have been investigated in d6-DMSO, respectively (25 mg/mL). As shown in Fig. 2, cross-correlation peaks corresponding to the adamantyl protons (between 1.70 and 2.25 ppm) and the inner CD protons (between 3.20 and 3.80 ppm) are clearly observed in the NOESY spectrum of RuCD, indicating the close proximity of these two sets of protons and the formation of the inclusion complex between adamantine group of Ru complexes and β-CD41-42. In addition, another set of cross peak correlation is observed between OH-6 protons at 4.45 ppm and OH-2 / OH-3 protons at 5.71 and 5.66 ppm (marked by red circles in Fig. 2), which is not appearing in the NOESY spectrum of natural β-CD (Fig. S6, ESI). It has been reported that the secondary hydroxyl groups OH-3 and OH-2 of β-CD form intramolecular bonds whereas OH-6 is not participating in intramolecular bonding,43-44 therefore, the new emerging cross-correlation peaks of OH-6 suggests the formation of a large supramolecular structure in which OH-6 is 8


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participating intermolecular H bonding. Moreover, although NOESY spectrum cannot discriminate the intra- or intermolecular nature of the secondary hydroxyl H bonding, it is still obvious that cross peak correlation between OH-2 and OH-3 in RuCD is much more intensive than that in natural β-CD (marked by arrows in Fig. 2), indicating the presence of secondary hydroxyl H bonding as well. These results accompanied with literatures indicate that intermolecular [O−H···O] interactions between β-CD monomers together with host-guest interactions direct the the self-assembly of supramolecular RuCD under mild conditions.

Figure 2 Section of the NOESY NMR spectrum of RuCD recorded in d6-DMSO at room temperature.

2.2 Dual-drug loading and release in vitro To assess the drug loading capacity of RuCD, two widely used anticancer drugs 5-Fu and LD were selected. It is supposed that RuCD has positive charges due to the positively charged Ru center, and an hydrophobic interior due to the phenanthroline-like ligands of Ru complexes and a hydrophilic surface due to the 9



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β-CD. This allows drug loading through hydrophobic/hydrophilic interactions. Moreover, 5-Fu is small enough to be encapsulated in the pores of RuCD and the negatively charged aromatic structure of LD is suitable for surface loading by electrostatic attraction and π−π interactions. After ultrasonic mixing two drugs and RuCD in deionized water for 6 h, drug-loaded nanocarrier Fu/LD@RuCD was obtained and separated from free drugs by centrifugation. In UV-Vis absorption spectra, Fu/LD@RuCD shows more intensive absorption than RuCD at 265 and 298 nm, which is the characteristic band of 5-Fu and LD, respectively, indicating the successful dual-drug loading (Fig. S7a-b, ESI). The synergistic loading capacity is 10.71 wt% for 5-Fu (drug/RuCD molar ratio is 4) and 6.62 wt% for LD (drug/RuCD molar ratio is 1), respectively. The morphology and particle size of Fu/LD@RuCD is similar to blank RuCD, as observed by AFM, TEM and DLS(Fig. S7c-d, ESI). Zeta potentials of RuCD and Fu/LD@RuCD have also been determined to be 22.3 and 7.48 mV, respectively (Fig S8a, ESI).

Figure 3 Cumulative release of (a) 5-Fu and (b) LD from Fu/LD@RuCD measured in PBS at different pH values.

Drug release profiles were investigated at various pH values in PBS at room temperature by using UV/Vis spectroscopy. As shown in Fig. 3, about 40% of 5-Fu and 48% of LD are released from Fu/LD@RuCD in 10 h at pH 7.4. When the pH value decreases to 5.0 mimicking the mildly acidic tumor microenvironment, the cumulative release of 5-Fu and LD increases to 72%, suggesting the promoted drug release at the tumor site. We speculate that acidic solution destabilizes the hydrogen 10


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bonding between β-CD molecules and causes protonation of imidazole moiety in Ru, leading to the collapse of the nanocarrier and weakening the π−π interactions between drugs and nanocarrier, thus promoting drug release. This speculation is supported by the DLS results, showing that the particle size of RuCD is unchangeable at pH 7.4 but gradually increases in acidic solutions (pH 5.0–5.8), in agreement with the gradual collapse of nanocarrier and the formation of Ru aggregates (Fig. S8b, ESI).

2.3 Two-photon imaging capability of RuCD The absorption spectrum of RuCD is similar to that of Ru molecule, showing an intense absorption at ca. 250-300 nm (intraligand ππ* transition) and a relatively weak absorption at ca. 420-500 nm (metal-to-ligand charge-transfer, MLCT), respectively (Fig. S9, ESI). Upon excitation at 488 nm, RuCD exhibits intensive fluorescence with maxima at ca. 610 nm in aqueous solution, although its fluorescence is partially quenched as compared with Ru. Moreover, the fluorescence of RuCD is pH independent (Fig. S10, ESI). The feasibility of RuCD for one-photon and two-photon imaging was then investigated in living A549 cells. Monolayer cell experiment shows that Fu/LD@RuCD can effectively penetrate into A549 cells in 8 hours, exhibiting red fluorescence under one-photon (λex= 488 nm) and two-photon (λex= 810 nm) excitation, which displays high colocalization with a commercially available lysosome-specific stain (Fig. S11, ESI). Two-photon imaging of RuCD was further investigated in A549 multicellular tumor spheroid (MCTS), which was accepted as a valid 3D tumor model that lied between a cell monolayer and a solid tumor. After incubation with Fu/LD@RuCD (20 µM) for 12 h, the fluorescence images were captured every 4.8-4.9 µm along the Z-axis to a depth of ∼140 µm (Fig. 4). The spheroids under two-photon excitation exhibit a much stronger fluorescence in the deeper layer cells than that under one-photon excitation, indicating deeper penetration of the two-photon imaging. On the other hand, cellular uptake of Ru(II) contents can be determined quantitatively by using ICP-MS, which is 250.38 ± 32.54 ng and 296.81 ± 44.81 ng 11



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per million cells, respectively, for RuCD and Fu/LD@RuCD (10 µM, 24 h). Based on the drug loading capacity of the nanocarrier, the intracellular contents of 5-Fu and LD can be determined accordingly.

Figure 4 (a) One-photon (λex= 488 nm) and two-photon (λex= 810 nm) CLSM images of the 3D tumor spheroids incubated with Fu/LD@RuCD (20 µM, 12 h); (b) The one-photon and two-photon Z-stack images were taken of every 4.8-4.9 µm section from the top to bottom. λem= 600 ± 20 nm. Inset scale bars: 200 µm.

2.4 PDT activity of RuCD and ROS-induced Lysosome Damage Since singlet oxygen (1O2) is the main toxic factor in PDT,45 the capability of RuCD and Fu/LD@RuCD to produce 1O2 under 488 nm irradiation was investigated by monitoring the absorption decay of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA, used as a 1O2 scavenger) at 380 nm. As shown in Fig. 5a-b, neither 5-Fu nor LD exhibits effective 1O2 production. The 1O2 production capacity of RuCD and Fu/LD@RuCD is slightly higher than that of Ru molecule, which also increases significantly in acidic solution (pH=5.0). By using [Ru(bpy)3]Cl2 as the reference, the 12


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1

O2 quantum yeilds (Φ∆) of RuCD is determined to be 0.27 at pH 5.0 and 0.21 at pH

7.4, respectively, similar to that of Fu/LD@RuCD (Table S1, ESI). In living A549 cells, the intracellular ROS levels induced by RuCD or Fu/LD@RuCD was evaluated by using DCF fluorescence assay, in which the naturally non-fluorescent 2’,7’-dichlorofluorescein diacetate (DCFH-DA) would be converted into highly fluorescent 2’,7’-dichlorofluorescein (DCF) by intracellular ROS. Confocal microscopy shows that ROS levels in RuCD or Fu/LD@RuCD treated cells are negligible in the absence of light but substantially increase upon irradiation (450 nm, 20 mW/cm2), as indicated by the bright green fluorescence from DCF. Similar results were also obtained and quasi-quantified by flow cytometry. Under irradiation, both RuCD and Fu/LD@RuCD induce the similar increase in the intracellular ROS levels in a concentration-dependent manner, which is ca. 300-folds higher than those under dark conditions (Fig. 5c). These results indicate the potential of Fu/LD@RuCD for chemo-PDT synergistic therapy, and the nanocarrier RuCD is the main contributor for PDT activity. Because Fu/LD@RuCD has been found selectively accumulating in the lysosome (Fig. S11, ESI), PDT induced lysosome damage is very likely to occur. An indicator of lysosomal membrane permeabilization is the release of cathepsin B from lysosomes to cytosol, which can be detected using fluorogenic substrate Magic Red MR-(RR)2 assay.46 As shown in Fig. 5d, control cells display red dot-like fluorescence indicating that cathepsin B is mostly localized in lysosomes. After incubating with Fu/LD@RuCD (5 µM) for 12 h in the dark, cathepsin B is still localized in lysosomes as indicated by the red-dot like fluorescence. However, upon 450 nm irradiation (20 mW/cm2) the red fluorescence shows different levels of diffusion, indicating the release of cathepsin B from lysosomes to the cytosol. These results indicate that the Fu/LD@RuCD can induce lysosomal damage upon PDT treatment, accordingly the chemodrugs can escape from lysosomes and reach their site of action.

13



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Figure 5 (a, b) Singlet oxygen generation of Ru, RuCD, Fu/LD@RuCD, free drugs 5-Fu and LD under 450 nm irradiation (20 mW/cm2) at different pH values. (c) Intracellular ROS levels detected by DCFH-DA staining. Scale bar: 20 µm. (d) Release of cathepsin B from lysosomes to the cytosol in Fu/LD@RuCD (5 µM, 12 h) treated A549 cells by fluorogenic substrate Magic Red-(RR)2 assay. Scale bar: 20 µm. Cells were incubated in the dark or irradiated with a 450 nm laser (20 mW/cm2).

2.5 In Vitro Cytotoxicity and Phototoxicity The anti-cancer activity of our nanoparticles against A549 cells was evaluated by 48 h MTT assays. As shown in Fig. 6a, under dark conditions blank RuCD as high concentration as 100 µM is practically non-toxic against A549 cells, indicating the good biocompatibility of the nanocarrier in vitro. However, all the drug loaded 14


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nanocarriers exhibit dose-dependent anti-cancer activities following the order LD@RuCD < Fu@RuCD < Fu/LD@RuCD, supporting the efficient drug release in A549 cells. The half-maximal inhibitory concentration (darkIC50) of Fu/LD@RuCD is determined to be 13.6 µM at Ru equivalent concentration. According to the drug loading efficacy of the nanocarrier, this

dark

IC50 value is equivalent to 54.4 µM 5-Fu

and 13.6 µM LD, respectively. By comparison, free drugs under the same conditions exhibit much less anti-proliferation activity (Table S2, ESI). Moreover, The

dark

IC50

value of Fu/LD@RuCD at 5-Fu equivalent concentration is comparable to other reported 5-Fu delivery systems,37 further confirming the good chemotherapeutic efficacy of our nanoparticles. The photodynamic activity of our nanoparticles was also evaluated by 48 h MTT assay as shown in Fig. 6b. Upon 450 nm irradiation (20 mW/cm2, 10 min), blank RuCD exhibits a high anti-tumor activity in A549 cells with

light

IC50 values of ca. 5

µM (at Ru equiv concentrations), indicating the efficient photodynamic activity of the nanocarrier. Accordingly, irradiation of drug-loaded nanocarrier would activate PDT at the same time of chemotherapy thus further enhancing the therapeutic efficacy. It is found that all LD@RuCD, Fu@RuCD and Fu/LD@RuCD display a substantially increased toxicity upon irradiation compared with their dark toxicity, moreover, the photo-toxicity of drug loaded RuCD is much higher than that of blank RuCD at every same concentration, confirming an eventually improved therapeutic efficacy due to the chemo-photodynamic synergistic therapy. The anti-cancer activity of our nanoparticles against other cancer cell lines (HeLa, human pulmonary carcinoma; A549R, cisplatin-resistant cell line) was also investigated (Table S2, ESI), showing similar results.

15



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Figure 6 Relative viability of A549 cells after incubation with free drugs, blank nanocarrier and drug-loaded nanocarrier at different Ru equivalent concentrations for 48 h in the (a) absence and (b) presence of irradiation (450 nm, 20 mW/cm2, 10 min).

2.6 Induction of Cellular Apoptosis and Mitochondria Dysfunction Apoptosis is a common cell death pathway in many photodynamic therapy, and 5-Fu and LD have also been reported capable of inducing cell apoptosis.35-36 We investigated cell apoptosis by Annexin V-FITC labeling, which detected the phosphatidylserine externalization. A549 cells were incubated with RuCD and Fu/LD@RuCD for 36 h in the absence and presence of light. As shown in flow cytotmetry (Fig. 7), under dark conditions RuCD does not induce cancer cell apoptosis whereas a significant increase in the percentage of apoptotic cells (27.9 ± 2.5%, 20 µM) is observed in the Fu/LD@RuCD treated cells, indicating the efficient chemotherapy of Fu/LD@RuCD via inducing apoptosis. Upon irradiation (450 nm, 20 mW/cm2, 10 min) both RuCD and Fu/LD@RuCD significantly increase the percentage of apoptotic cells, as observed in the Annexin V-FITC assay. It is worth mentioning that the single use of chemotherapy or PDT induces a lower apoptosis rate in A549 cells than that of chemo-PDT combined therapy, supporting the decent chemo-photodynamic synergistic effect of Fu/LD@RuCD.

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Figure 7 Flow cytometry of Annexin V labeled A549 cells treated with RuCD and Fu/LD@RuCD for 36 h in the (a) absence and (b) presence of 450 nm irradiation.

Mitochondrial dysfunction often accompanies with cell apoptosis. Thus the capability of RuCD and Fu/LD@RuCD to induce mitochondrial dysfunction was also investigated. 5,5’,6,6’-tetrachloro-1,’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) staining was used to monitor the changes in the mitochondrial membrane potential (MMP, ∆Ψm). The loss of MMP is characterized by and a decrease in red fluorescence (JC-1 aggregates) and an increase in green fluorescence (JC-1 monomers). As shown in Fig. 8a, under dark conditions red fluorescence of JC-1 is mainly observed in control cells and RuCD treated cells, indicating the high MMP; After treating cells with Fu/LD@RuCD for 18 h in the absence of light, decreased MMP is observed indicated by a red-to-green color shift. Upon irradiation, both RuCD and Fu/LD@RuCD at relatively lower concentrations induce more significant MMP loss, supporting the good synergistic effect of chemo-photodynamic therapy. The intracellular ATP levels were further investigated to assess the impact on mitochondrial bioenergetics. As shown in Fig. 8b, under dark conditions the impact of RuCD is negligible whereas Fu/LD@RuCD (18 h) induces a significant decrease in the intracellular ATP levels in a concentration-dependent 17



manner,

indicating

the

efficient

chemotherapy

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of

Fu/LD@RuCD

via

mitochondria dysfunction. Upon irradiation, both RuCD and Fu/LD@RuCD further decrease the ATP levels, the latter of which exhibits the strongest impact, indicating the enhanced therapeutic efficacy of chemo-photodynamic synergistic therapy compared to the single use of chemotherapy or PDT.

Figure 8 Induction of mitochondrial dysfunction by blank RuCD and drug-loaded nanoparticles in the absence and presence of irradiation. (a) Flow cytometric quantification of MMP in treated (18 h) A549 cells (JC-1: λex = 488 nm; green λem= 530 ± 30 nm and red 585 ± 30 nm); (b) Intracellular ATP levels in treated (18 h) A549 cells.

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2.7 In Vivo Treatment Efficacy The efficacy of chemo-photodynamic synergistic therapy was further evaluated in the nude mice bearing A549 tumors. When the tumor grew to ca. 100 mm3, nude mice were intratumorally injected with PBS (control), RuCD (25 mg/kg) and Fu/LD@RuCD (25 mg/kg), respectively, and irradiated with a 450 nm laser. As shown in Fig. 9a, under dark conditions blank RuCD only shows small influence on the tumor growth whereas Fu/LD@RuCD effectively arrests the tumor growth and decreases the tumor volume by 60%. The inhibitory effect of Fu/LD@RuCD is much better than the free drugs mixture applied under the same conditions, indicating the excellent chemotherapeutic efficacy of Fu/LD@RuCD mainly due to the successful dual-drug delivery. Moreover, Fu/LD@RuCD exhibits more prominent anti-tumor efficacy upon light irradiation than the sole chemotherapy at the same doses, which decreases the tumor volume by 85% compared with control group (Fig. 9c), indicating the excellent PDT activity and the good chemo-photodynamic synergistic effect. All the drug-treated nude mice do not show obvious body weight loss (Fig. 9b), and histological analysis shows that no obvious pathological abnormalities are observed in the heart, spleen, lung, liver, and kidney during the treatment (Fig. S12, ESI), indicating that neither RuCD nor Fu/LD@RuCD has serious side effects under the in vivo experimental conditions.

19



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Figure 9 Antitumor activities of PBS, free drugs, blank RuCD and drug loaded RuCD intratumoral injected at a dose of 25 mg/kg in the first day in the absence and presence of irradiation (450 nm, 20 mW/cm2). (a) Changes in A549 tumor volumes and (b) Body weight changes during the course of treatment; (c) Images of resected tumors after mice sacrificed .

3. CONCLUSIONS We herein fabricate a three-in-one nanoplatform, Fu/LD@RuCD, by very simple mixing of adamantine-functionalized ruthenium complexes and natural cyclodextrin (β-CD) monomers in aqueous solution at room temperature. The octahedral configuration of Ru complex provides a rigid skeleton and the hydroxyl hydrogen bonding formed between two adjacent β-CD monomers displays a bridging role allowing for three-dimensional architectures. The intrinsic fluorescence from nanocarrier RuCD enabled one-photon (450 nm) and near-infrared two-photon (810 nm) imaging, the latter of which was more prominent for in vivo imaging. The ability of RuCD to produce ROS efficiently under visible light irradiation also enabled its feasibility for PDT. Drug loading efficiency of this nanocarrier is estimated to be 10.71 wt% and 6.62 wt% for 5-fluorouracil and lonidamine, respectively. Cellular 20


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experiments show that Fu/LD@RuCD effectively penetrates into cancer cells in 8 h and selectively accumulates in lysosomes, the mildly acidic environment of which can promote the dual-drug release. Under visible light irradiation, RuCD produce sufficient ROS to damage lysosomes, accordingly 5-fluorouracil and lonidamine can escape from lysosomes and reach their sites of action, resulting in mitochondria dysfunction and cancer cell apoptosis. Both in vitro and in vivo investigations indicate that Fu/LD@RuCD possess excellent chemo-photodynamic synergistic therapy, the anti-cancer efficacy of which is much better than the free anti-cancer drugs and the sole therapeutic modality. Overall, this newly synthesized three-in-one nanoplatform implies potential applications in dual-drug delivery, as well as in vivo near-infrared two-photon imaging, and chemo-photodynamic synergistic therapy in the future.

4. EXPERIMENTAL SECTION 4.1 Materials Ruthenium(III) Trichloride (Sigma Aldrich), 1,10-phenanthroline-5,6-dione (Alfa

Aesar),

1-Adamantyl

carboxaldehyde

(Sigma-Aldrich),

β-cyclodextrin

(Sigma-Aldrich) was used as purchased. Anticancer active ingredient 5-Fluoraciaoul (5-Fu) and lonidamine (LD) was supplied by Sigma USA. Cisplatin (Sigma Aldrich), PBS

(phosphate

buffered

saline,

Sigma

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide,

Aldrich),

MTT

Sigma

Aldrich),

LTDR (Life Technologies, USA), DCFH-DA and Annexin V-FITC apoptosis detection kit was purchased from Sigma Aldrich, Magic Red MR-(RR)2 was purchased from Immunochemistry Tech. Cisplatin was dissolved in saline. 4.2 General Instruments 1

H NMR spectra were recorded on Varian Mercury Plus 400 Nuclear Magnetic

Resonance Spectrometer. ESI-MS was carried out on an LCQ system (Finnigan MAT, USA). Microanalyses (C, H, and N) were measured by using an Elemental Vario EL CHNS analyzer (Germany). UV/Vis spectrophotometer (Varian Cary 100, USA). Fluorescence spectra were obtained on a FLS 980 steady state spectrometer (Edinburgh Instrument, UK). Inductively Coupled Plasma Mass Spectrometer 21



Page 22 of 33

(Thermo X Series 2, Thermo Fisher Corp., USA). Morphology of the supramolecular self-assembly nanoparticles was characterized by a Bruker Multimode 8 AFM under ScanAsyst mode (Bruker) and a JEOL JEM-2010HR transmission electron microscope (TEM). Isothermal titration calorimetry (ITC) were carried out on the MicrolCal ITC200 (Malvern, UK). Confocal microscopy (Leica TCS SP8, Wetzlar, Germany). Flow cytometry (Tree Star, USA, FlowJo 7.6 software for data analysis). 4.3 Synthesis and Characterization Synthesis

of

ligand

Phen-ad:1,10-phenanthroline-5,6-dione

(0.315 g,

1.5 mmol), 1-Adamantylcarboxaldehyde (0.345 g, 2.1 mmol) and ammonium acetate (2.31 g, 30 mmol) was stirred and refluxed in glacial acetic acid (20 mL) for 4 h. The reaction fluid was diluted with deionized water and neutralized with ammonia water. The pink deposition was granted and washed by water, ethyl alcohol or diethyl ether three times to give the compound as a pink powder. Yield:76%.1H NMR (400 MHz, DMSO-d6, δ): 12.94 (s 1H; NH), 9.0 (d, J = 4 Hz, 2H; phen-H), 8.88 (q, J = 48 Hz, 2H; phen-H), 7.80 (s 2H; phen-H), 2.19 (s 6H; ad-H), 2.14 (s 3H; ad-H), 1.82 (d, J = 40 Hz 6H; ad-H). ESI-MS [CH3OH, m/z]: 355.4 ([M+H]+) , 731.2 ([2M+Na]+). Synthesis of complex [Ru(phen-ad)2Cl2]·2H2O: Cis-[Ru(phen-ad)2Cl2]·2H2O was synthesized following the procedure of Meyer et al.39 for the synthesis of [Ru(bpy) Cl 2] except that the phen-ad was used in place of 2,2’-bipyridine Synthetic procedure of Ru(Phen-ad)3(PF6)2 (Ru) : Phen-ad (0.070 g, 0.2 mmol) was dissolved in 30 mL EtOH/H2O (2:1, v/v) and heated at 70 °C under argon atmosphere, and the Ru(Phen-ad)2Cl2•2H2O (0.2 g, 0.22 mmol) was added into the reaction solution by several times. After the 8 h of reaction, it was evaporated and the black powder was purified by alumina column chromatography containing CH2Cl2-acetonitrile (1:10, v/v) as eluent, giving pure product as reddish brown powder. Yield: 184 mg (68%).1H NMR (400 MHz, DMSO-d6, δ): 13.51 (s 3H; NH), 9.06 (s 3H; phen-H), 8.98 (s 3H; phen-H), 7.97 (s 6H; phen-H), 7.73 (s 6H; phen-H) 2.20 (d, J = 32 Hz, 18H; ad-H), 2.17 (m 9H; ad-H), 1.85 (s 18H; ad-H).

13

C NMR

(400 MHz, DMSO-d6, δ):176.54, 165.22, 153.52, 151.57, 146.69, 131.90, 127.67, 42.77, 37.81, 37.24, 29.42. ESI-MS [CH3OH, m/z]: 582.2 ([M-2PF6]2+). Elemental 22


Page 23 of 33 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


analysis: calcd (%) for Ru C69H66N12F12P2:C,56.98; H, 4.57; N, 11.56; found: C, 56.94; H, 4.60; N, 11.54. Synthetic procedure of RuCD nanoparticle: For the preparation of RuCD nanoparticles, Ru was puted into the DMSO liquor with a ratio of β-CD aqueous solution [Ru]:[β-CD]=1:3 under ultrasonic agitation at 4 °C for 20 min, and the mixed solution was further stirred overnight to ensure complete self-assembly. The nanoparticles were stored at 4 °C. Synthetic procedure of Fu/LD@RuCD nanoparticle: In order to preparation of Fu/LD@RuCD, 5-Fu (15 mg) was added into the RuCD (5 mmol/L, 5 mL) aqueous solution under ultrasonic agitation at 4 °C for 60 min and the mixed solution was by centrifugal separation 5 min (4000 r/min). Then collected the supernatant added into LD with ultrasonic agitation at 4 °C for 60 min, and the mixed solution was by centrifugal separation 5 min (4000 r/min) in order to collect supernatant. The mixture was stored at 4 °C before use. The standard plot of 5-Fu or LD concentrations in PBS was implemented (0–200 µM; R2 = 0.999) and the concentration of loaded drugs were determined by UV absorption at 265 nm and 298 nm. The drug loading capacity (wt%) of 5-Fu of LD in Fu/LD@RuCD was counted according to the following formula: (wt%)=[Amount of loaded 5-Fu] / [Amount of RuCD]×100% UV-Vis titration: Ru and β-CD were dissolved in distilled water to prepare stock solutions. Ru (50 µM) was titrated into β-CD (2 mM) in 600 µL cuvette to achieve different ratios. After each titration, the mixture solution was stabilized for 5 min before recording the spectra were write down. The absorbance changes at 254 nm (∆A 254) were plotted as a function of the Ru / β-CD ratios. Isothermal titration calorimetry: ITC was experimented to characterize the stoichiometry value for the binding of Ru (Host) with β-CD (Guest). The titration experiments were carried out on the MicrolCal ITC200 at 298 ± 0.02 K. The guest molecules Ru (0.025 mM) were loaded into the 200 µL sample cell and the host molecules β-CD (0.75 mM) were loaded into the continuously rotating (750 rpm) 40 µL syringe. The titration was conducted by adding 1.5 µL aliquots of the host solution 23



Page 24 of 33

into the guest solution at an interval of 2.5 min (19 injections in total). The titration data were fitted using a MicroCal-enabled Origin program. The value of stoichiometry N, the molar enthalpy changes (∆H), entropic changes (∆S) and the inclusion complex association constants Ka were calculated accordingly. Atomic force microscopy (AFM) measurement: The aqueous solution of Ru and β-CD was mixed together to initial the supramolecular self-assembly process ([RuCD] = 50 µM). Then the solution was dropped onto newly clipped mica and air-dried before measurement. Fu/LD@RuCD sample was prepared the same. Transmission electron microscopy (TEM): Samples were prepared by adding 10 µL of RuCD or Fu/LD@RuCD (50 µM) solution onto a 300-mesh copper grid, and air-dried prior to collecting images. Dynamic light scattering (DLS) measurements: The solution of RuCD or Fu/LD@RuCD (50 µM) was prepared as described above. The hydrodynamic size was measured using a Brookhaven EliteSizer Nanoparticle size-Zeta potential and molecular weight analyzer at a wavelength of 677 nm with a constant angle of 90° at 298 K. 4.4 Photophysical Properties and Stability of RuCD: To investigate their Photophysical Properties, Ru, RuCD, Fu/LD@RuCD (10 uM) in PBS ,Fluorescence spectra were taken with FLS 980 and UV spectra were measurements by Varian Cary 100

UV/vis

spectrophotometer.

Stabilities

of

RuCD

were

dispersed

in

FBS(10%)+PBS, then taken with UV/vis spectrophotometer several times. 4.5 Detection of Singlet Oxygen (1O2): The 1O2 quantum yields (Ф∆) of ruthenium complexes were detected according to the literature procedure.47 The Ru, RuCD, Fu/LD@RuCD, 5-Fu, LD and ABDA (100 µM) solutions were aerated for 10 min, then photoirradiation at 450 nm (20 mW/cm2). The absorbance of ABDA at 380 nm was recorded every 2 s. Ru(bpy)3Cl2 was used as the reference (Ф∆ = 0.18). 4.6 Release of 5-Fu or LD from Fu/LD@RuCD: Fu/LD@RuCD were prepared with a 5-Fu or LD concentration of 100 µM. 3 mL of the sample solution was placed in a dialysis bag (MWCO 1000), which was immersed in 30 mL PBS (pH 7.4) under mild agitation (200 rpm) at 37 °C. Then, 1.0 mL of the dissolution medium was 24


Page 25 of 33 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


withdrawn for detection at certain time intervals up to 12 h and replaced with the same volume of fresh PBS. The cumulative 5-Fu or LD release was determined by measuring the absorbance at 265 nm and 298 nm using the calibration curve. Release was calculated as Eq: (%)=[Amount of release 5-Fu] / [Amount of 5-Fu]×100% 4.7 Cellular uptake and distribution: Confocal microscopy: For cellular distribution measurements, A549 cells (1 × 105 cells) were seeded in 35 mm culture dishes (Corning) for 18 h. Then cells were incubated with RuCD (10 µM) or Fu/LD@RuCD (10 µM) at 37 °C for 24 h and stained with LTDR (500 nM) for another 30 min. Cells were washed three times with PBS and visualized by confocal microscopy immediately.. ICP-MS measurement: A549 cells were seeded in 10 cm tissue culture dishes and incubated for 36 h. After the medium was refreshed, cells were incubated with RuCD and Fu/LD@RuCD (10 µM) for 12 h incubation, followed by PBS wash and trypsinized, and digestion (HNO3, 65%, 0.2 mL, 24 h). The solution was then diluted to a final volume of 10 mL with Milli-Q water for ICP-MS. 4.8 Measurement of intracellular ROS levels: A549 cells were incubated with RuCD, Fu/LD@RuCD for 12 h and then stained with 10 µM DCFH-DA in serum-free 1640 at 37 °C for 20 min. After twice wash and 450 nm irradiation (20 mW/cm2 , 10 min), the fluorescence intensity of intracellular DCFH-DA (excitation at 488 nm, emission at 530 nm) was measured immediately by confocal microscopy and flow cytometry. 4.9 Detection of Cathepsin B Release: Cathepsin B activity was detected using the fluorogenic susbtrate Magic Red MR-(RR)2 according to the manufacturer’s instructions. Briefly, A549 cells were treated with RuCD (5 µM) for 12 h in the absence and presence of irradiation for 5 or 10 min (450 nm , 20 mW/cm2). Then cells were washed twice with PBS and treated with fluorogenic susbtrate Magic Red MR-(RR)2 for another 1 h. Confocal microscopic images were recorded after PBS wash with excitation at 543 nm and emission at 630 ± 20 nm. 4.10 Cytotoxicity assay: In 96-well tissue culture plates cells were treated with varies concentrations of RuCD, Fu/LD@RuCD, Fu@RuCD, LD@RuCD 5-Fu and LD for 25



Page 26 of 33

44 h. Then, 20 µL MTT (5 mg/mL) was added to each well for another 4 h of incubation before evaluating the cell viability by measuring the absorbance at 595 nm (Infinite F200, Tecan, Switzerland). Each experiment was repeated at least for three times. 4.11 Detection of apoptosis: A549 cells were treated with different concentrations of RuCD, Fu/LD@RuCD for 36 h and then harvested and washed twice with PBS. These cells were re-suspended in 500 µL Annexin V-FITC for 15 min before flow cytometry. 4.12 Analysis of MMP and ATP level: A549 cells were treated with RuCD, Fu@RuCD, Fu/LD@Ru@CD at various concentrations for 18 h in the absence and presence of light (450 nm, 20 mW/cm2 , 10 min) followed by continuing incubation for 6 h. For MMP analysis, cells were collected and resuspended at 1 × 106/mL in pre-warmed PBS containing 5 µg/mL JC- 1, incubated at 37 °C for 20 min, washed twice with PBS, and then analyzed by flow cytometer immediately. For ATP analysis, CellTiter-Glo kit (Promega) was used following the manufacturer’s instructions and the relative luminescent units were detected with a microplate reader (Infinite F200, Tecan, Switzerland). 3 replicates were conducted for obtaining average values and untreated cells were used for normalization. 4.13 In vivo antitumor efficacy: 4-5 weeks old female BALB/c-(nu/nu) nude mice were purchased and bred in the Center of Experiment Animals at Sun Yat-Sen University. A549 cells (2×106) were suspended in PBS and subcutaneously injected to establish xenografts. The mice were randomly allocated into six groups (n = 4) and treated with PBS, PBS + light, Fu/LD, RuCD, Fu/LD@RuCD and Fu/LD@RuCD + light, respectively, via the intratumoral injection at a dose of 25 mg/kg. For photodynamic therapy, 450 nm irradiation (20 mW/cm2, 600 s) was conducted 2 h after intratumoral injection. The tumor sizes and body weights were measured every 3 days. The tumor sizes was calculated by the formula V=a×b2×0.5, where a and b was the tumor’s longest and shortest diameters, respectively. In the 18th day, the mice were sacrificed for tumor separation and histological analysis. 26


Page 27 of 33 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


ASSOCIATED CONTENT Corresponding Author * E-mail: [email protected]; [email protected]. Notes The authors have no competing financial interests to declare. Supporting Information ESI-MS, 1H and 13C NMR spectra of Ru complex; NOESY NMR spectrum of natural β-CD; ITC measurement of Ru toward β-CD; Morphological characterization, zeta potential, drug loading capacity, stability, and photophysical properties of nanoparticles; singlet oxygen quantum yields of Ru complex and nanoparticles; Cellular uptake and lysosomal localization;

50

IC values of nanoparticles and free

drugs against various cancer cell lines; histological analysis. Acknowledgements We are grateful for financial support from the National Natural Science Foundation of China [21231007, 21572282 and 21401217], 973 Program [2014CB845604, 2015CB856301], Natural Science Foundation of Guangdong [2017A030313041],

Science

and

Technology

Program

of

Guangzhou

[201607010379], Fundamental Research Funds for the Central Universities.

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