Co-Delivery of Cisplatin Prodrug and Chlorin e6 by Mesoporous Silica

May 10, 2016 - Department of Nanomedicine, Houston Methodist Research Institute, Houston, Texas 77030, United States ... ESI-MS spectrum of c,c,t-[Pt(...
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Co-Delivery of Cisplatin Prodrug and Chlorin e6 by Mesoporous Silica Nanoparticles for Chemo-Photodynamic Combination Therapy to Combat Drug Resistance Wei Zhang,†,‡ Jianliang Shen,§ Hua Su,† Ge Mu,† Jing-Hua Sun,† Cai-Ping Tan,† Xing-Jie Liang,*,‡ Liang-Nian Ji,† and Zong-Wan Mao*,† †

MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, P. R. China ‡ CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, P. R. China § Department of Nanomedicine, Houston Methodist Research Institute, Houston, Texas 77030, United States S Supporting Information *

ABSTRACT: Combination therapy shows great promise in circumventing cisplatin resistance. We report herein the development of a novel nanoscale drug delivery system (nDDS) based nanotherapeutic that combines chemotherapy and photodynamic therapy (PDT) into one single platform to achieve synergistic anticancer capacity to conquer cisplatin resistance. Mesoporous silica nanoparticle (MSNs) was used as the drug delivery vector to conjugate cisplatin prodrug and to load photosensitizer chlorin e6 (Ce6) to afford the dual drug loaded delivery system MSNs/Ce6/Pt. The hybrid nanoparticles have an average diameter of about 100 nm and slightly positive surface charge of about 18.2 mV. The MSNs/Ce6/Pt nanoparticles can be efficiently internalized by cells through endocytosis, thereby achieving much higher cellular Pt uptake than cisplatin in cisplatin-resistant A549R lung cancer cells. After 660 nm light irradiation (10 mW/cm2), the cellular reactive oxygen species (ROS) level in MSNs/Ce6/Pt treated cells was elevated dramatically. As a result of these properties, MSNs/Ce6/ Pt exhibited very potent anticancer activity against A549R cells, giving a half-maximal inhibitory concentration (IC50) value for the combination therapy of 0.53 μM, much lower than that of cisplatin (25.1 μM). This study suggests the great potential of nDDS-based nanotherapeutic for combined chemo-photodynamic therapy to circumvent cisplatin resistance. KEYWORDS: cisplatin, photodynamic therapy, combination therapy, mesoporous silica nanoparticles, drug resistance

1. INTRODUCTION

shown to be down-regulated in cancer cells with cisplatin resistance.10−12 Recent years, nanoscale drug delivery systems (nDDS) have emerged as an innovative and promising strategy to circumvent cisplatin resistance.13−15 nDDS can act as a Trojan horse to shuttle drugs into cells through endocytosis, which results in higher intracellular accumulation.7 Platinum(IV) prodrugs based nDDS garner especially tremendous research attention owing to their superiorities compared to the platinum(II) analogues.16−21 Platinum(IV) complexes typically have lowspin d6 election configurations and octahedral geometry, endowing them high kinetic inertness to substitution.22 Therefore, they exhibit very high stability in the biological

Chemotherapy is one of the most important modalities of cancer treatment. Among all the chemotherapeutic agents, platinum anticancer drugs play a crucial role. About half of all the cancer patients accept treatment regimens containing platinum drugs.1 Cisplatin is the first platinum-based anticancer drug and has been proved to be one of the most potent anticancer drugs since it was first approved to clinical use by U.S. Food and Drug Administration (FDA) in 1978.2,3 However, the efficacy of cisplatin is badly hindered by either inherent (as observed in lung or prostate cancers) or acquired (as often seen in ovarian cancer) resistance of cancer cells.4 The underlying mechanisms of cisplatin resistance have been extensively studied and appear to be multifactorial.5,6 Reduced intracellular cisplatin accumulation is revealed to be a key factor of cisplatin resistance.7−9 Human copper transport protein 1 (hCTR1), the major influx transporter of cisplatin, has been © XXXX American Chemical Society

Received: March 31, 2016 Accepted: May 10, 2016

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ACS Applied Materials & Interfaces fluid and diminished side effects. Upon entry into the cell, platinum(IV) complexes can be reduced to release the cytotoxic platinum(II) species.23,24 Moreover, platinum(IV) complexes are more flexible of functionalization via the axial ligands to adjust their pharmacological properties, such as redox potential, aqueous solubility, and lipophilicity. Combination therapy is a widely employed strategy in the clinic to combat chemoresistance.25,26 Administering a cocktail of different anticancer drugs or combining chemotherapeutic agents with other conventional cancer treatment modalities can achieve synergetic anticancer efficacy through a multipronged assault to overcome drug resistance.27,28 Although nDDS mediated codelivery of cisplatin and other kinds of anticancer chemotherapeutic agents (e.g., doxorubicin) has been extensively explored,29−32 nDDs-based combination therapy of cisplatin with other modalities is still a burgeoning research area.33,34 Previously, for the first time we reported the combinatorial chemo-photodynamic dual therapy against cisplatin resistant cancer cells by a supramolecular self-assembly system.35 The sensitization effect of photodynamic therapy (PDT) greatly boosts the activity of cisplatin against resistant human lung carcinoma (A549R) cells. PDT is an effective and noninvasive cancer treatment procedure, which utilizes a light source to activate tumor localized photosensitizer to generate highly toxic reactive oxygen species (ROS), particularly, singlet oxygen (1O2), thus eradicating tumor tissues with higher specificity and fewer side effects.36,37 In the present study, we construct a novel nDDS based on mesoporous silica nanoparticles (MSNs) for cisplatin prodrug and chlorin e6 (Ce6) codelivery to enable combination chemophotodynamic dual therapy against cisplatin resistant cancer cells. The cisplatin prodrug was conjugated to the surface of MSNs by utilizing β-cyclodextrin-grafted polyethylenimine (CD-PEI) as the linker, while the photosensitizer Ce6 was loaded into the pores of MSNs through the hydrophobic effect. The schematic presentation of the fabrication process of the dual drug loaded nDDS MSNs/Ce6/Pt is shown in Scheme 1. Subsequently, the anticancer activity of the multipronged MSNs/Ce6/Pt nanoparticles was evaluated against cisplatinresistant A549R lung cancer cells. The combinatorial actions of chemotherapy from cisplatin and PDT from Ce6 were

investigated by antiproliferation assay, cellular localization, Pt content determination, ROS level evaluation, transmission electron microscopy cell morphological and ultrastructural observation, and so on, which revealed the underlying mechanisms of the synergistically enhanced anticancer efficacy of chemo-photodynamic combination therapy to combat cisplatin resistances.

2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. Branched polyethylenimine (MW 1.8 kDa), β-cyclodextrin, N-cetyltrimethylammonium bromide (CTAB), tetraethoxysilane (TEOS), 3-isocyanatopropyltriethoxysilane (TES-ICP), and methoxypolyethylene glycol amine (NH2-PEG, MW 5 kDa) were obtained from Aladdin Reagent (China). 1-Adamantaneacetic acid, Ce6, cisplatin, N,N-dicyclohexylcarbodiimide (DCC), and 1-hydroxybenzotrizole (HOBt) were purchased from J&K Chemical (China). RPMI 1640 medium, fetal bovine serum (FBS), trypsinEDTA solution, Penicillin-streptomycin solution, Lyso-tracker Green (LTG), Calcein-AM, and propidium diiodide (PI) were purchased from Gibco Life Technologies. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), Hoechst 33342, and N,N-dimethyl-4-nitrosoaniline (RNO) were obtained from Sigma-Aldrich. All the other chemicals were of analytical grade, and Millipore water (18.2 MΩ) was used throughout the experiments. 2.2. General Instruments. UV−vis spectra were monitored with a Varian Cary 300 UV−vis spectrophotometer. The fluorescence emission spectra were obtained using a Shimadzu RF-5301PC spectrofluorophotometer. 1H NMR spectra were recorded on a Bruker AVANCE-400 NMR spectrometer. The transmission electron microscope (TEM) images and energy-dispersive spectrometry (EDS) were taken on a JEOL JEM-200CX transmission electron microscope. The X-ray diffraction (XRD) pattern was collected on a Bruker D8 Advance diffractometer. Pt content was measured on a Thermo X series 2 inductively coupled plasma-mass spectrometer (ICP-MS). Confocal laser scanning fluorescence microscopy images were obtained with a Zeiss LSM-710 microscope. 2.3. Synthesis of CD-PEI and Ad-PEG. Beta-cyclodextrin-grafted branched polyethylenimine (CD-PEI) was synthesized according to a previous report,38 the synthesis route is shown in Scheme S1. Briefly, 6-monotosylated cyclodextrin (450 mg, 0.35 mmol) was added dropby-drop into a solution containing PEI (90 mg, 0.5 mmol) in 6 mL of anhydrous dimethyl sulfoxide. The resulting mixture was stirred at 70 °C in a nitrogen atmosphere for 5 days. The solution was dialyzed against water in a Spectra/Por MWCO 1000 bag and then lyophilized to afford a slightly colored solid. The 1H NMR spectrum of CD-PEI was shown in Figure S1. Adamantyl moiety grafted poly(ethylene glycol) (Ad-PEG) was synthesized by amide coupling of 1-adamantaneacetic acid with NH2PEG (Scheme S1). To a solution of 1-adamantaneacetic acid (0.0291 g, 0.15 mmol) in chloroform was added DCC (0.0618 g, 0.3 mmol), HOBt (0.0405 g, 0.3 mmol), and triethylamine (50 μL). After stirring for 10 min, NH2-PEG (0.5 g, 0.1 mmol) was added. The solution was stirred at room temperature for 24 h. The white precipitate was filtered off and the solvent was removed under vacuum. Then water was added and the solution was dialyzed against water in a Spectra/Por MWCO 2000 bag and lyophilized to afford a white powder. The 1H NMR spectrum of Ad-PEG was shown in Figure S2. 2.4. MSNs Preparation and Drug Loading. The synthesis of MCM-41 type MSNs (MSNs-OH) and 3-isocyanatopropyl group modified MSNs (MSNs-ICP) was based on a previously reported protocol.39 Then MSNs-ICP (20 mg) was suspended in ethanol (4 mL), and Ce6 (2.0 mg) in DMSO was added. After 5 min sonication, the mixture was stirred at room temperature for 24 h. Then CD-PEI (10 mg) and Ad-PEG (20 mg) were added. After another 24 h, Ce6 loaded MSNs (MSNs/Ce6) was purified by repeated centrifugation and washing with PBS 7.4, then redispersed in PBS and stored at 4 °C for use.

Scheme 1. Schematic Illustration on the Fabrication of the MSNs Drug Delivery System for Cisplatin Prodrug and Photosensitizer Ce6

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ACS Applied Materials & Interfaces For cisplatin prodrug conjugation, cisplatin prodrug c,c,t-[Pt(NH3 ) 2 Cl 2(OH)(O 2 CCH 2 CH 2 CO 2 H)] 40 was first synthesized (Scheme S1) and its ESI-MS spectrum was shown in Figure S3. To the aqueous solution of c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CO2H)] (2 mg) was added EDC (10 mg) and NHS (5 mg) and stirred for 10 min. Then this mixture was added to the solution of MSNs/Ce6 (2 mL, 5 mg/mL) and stirred at room temperature for 24 h. Finally, Ce6 loaded and cisplatin prodrug conjugated MSNs (MSNs/Ce6/Pt) was obtained by repeated centrifugation and washing, then redispersed in PBS and stored at 4 °C for use. 2.5. Cell Lines and Culture Conditions. A549, A549R cells were obtained from Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Cells were routinely maintained in RPMI 1640 (Roswell Park Memorial Institute 1640) medium containing 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin at under 5% CO2 and 95% humidity at 37 °C. Cisplatin-resistant A549R cells were cultured in medium containing cisplatin to maintain the resistance. 2.6. MTT Cell Proliferation Assay. A549R cells were seeded into 96 well tissue culture plates at a confluence of 2000 cells per well and incubated for 24 h. Then cells were treated with various concentrations of cisplatin, MSNs/Ce6, or MSNs/Ce6/Pt. The plates were incubated for 68 h at 37 °C and then 20 μL of MTT (5 mg/mL in PBS) was added and incubated for 4 h. The medium was removed, cells were lysed by adding 150 μL of DMSO, and the absorbance of the purple formazan was recorded at 570 nm using a BioRad iMark plate reader. For Chemo-Photodynamic combination therapy, 24 h after the addition of the compounds, the plates were irradiated with 660 nm LED light (HTLD-4II, Height-LED) for 5 min (10 mW/cm2). Then the plates were incubated for another 48 h and treated with the same procedure as described above. 2.7. Cellular Platinum Uptake. To measure the cellular uptake of the platinum complexes, A549R cells were seeded in 10 cm tissue culture dishes and incubated for 24 h. The medium was removed and replaced with fresh medium containing cisplatin or MSNs/Ce6/Pt ([Pt] = 0.5, 1 μM). After 3 h incubation, the cells were washed with PBS, trypsinized, and collected. The cells were counted and digested with HNO3 (65%, 0.5 mL). The platinum content in cells was determined by ICP-MS. 2.8. Detection of Singlet Oxygen. Singlet oxygen in aqueous was determined following the Kraljic procedure.41 Solution of pnitrosodimethylaniline (RNO, 30 μM), imidazole (0.5 mM) in PBS (10 mM, pH = 7.4) was added MSNs/Ce6/Pt ([Ce6] = 2 μM) and then irradiated with 660 nm light at the power density of 10 mW/cm2. The absorbance spectra of the solution were recorded at 20 s interval. The intracellular ROS can be detected by ROS probe DCFH-DA, because cell-permeable nonfluorescent DCFH-DA can be de-esterified intracellularly and turns to highly fluorescent DCF upon oxidation. A549R cells were seeded and incubated for 24 h. Then the medium was replaced with fresh medium containing MSNs/Ce6 or MSNs/ Ce6/Pt ([Ce6] = 0.5 μM) for 24 h. The cells were washed with serum-free DMEM and then incubated with 10 μM DCFH-DA in serum-free DMEM at 37 °C for 20 min. After washing twice with serum-free DMEM, cells were irradiated with 660 nm LED light for 5 min. Then the cells were immediately examined under a confocal microscope (LSM 710) with excitation at 488 nm and emission at 530 ± 20 nm or the cells were harvested and the fluorescence intensity of cells was measured immediately by flow cytometry (FACSCaliburTM, Becton Dickinson). Green mean fluorescence intensities were analyzed using FlowJo 7.6 software. 2.9. Cell Morphological and Ultrastructural Observation by Bio-TEM. A549R cells were seeded into 6 well tissue culture plates and incubated for 24 h. Then the cells were treated with cisplatin, MSNs/Ce6 or MSNs/Ce6/Pt ([Pt] = 1 μM) for 24 h. For PDT treatment, the plates were irradiated with 660 nm light for 5 min and incubated for another 24 h. Then the cells were harvested and fixed overnight at 4 °C in PBS (pH 7.4) containing 2.5% glutaraldehyde. Subsequently, the cells were treated with osmium tetroxide, stained with uranyl acetate and lead citrate, and visualized under a transmission electron microscope (JEM 100 CX, JEOL).

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization. MSNs were widely used in biomedical applications, such as drug delivery and

Figure 1. Characterization of MSNs nanoparticles. TEM image (a) and SAXRD pattern (b) of MSNs-ICP and TEM image (c) and EDS spectrum (d) of MSNs/Ce6/Pt.

biosensing,42−44 because silica is considered as “Generally Recognized As Safe” by FDA.45 MSNs were synthesized according to a previously reported method.39 Subsequently, the surface of MSNs was modified with TES-ICP to afford MSNsICP. The ICP group has very high reactivity with amino group, thus MSNs-ICP can be easily conjugated with CD-PEI, which facilitates the surface modification procedure. The morphology of MSNs-ICP was characterized by TEM (Figure 1a), which showed the spherical particles with diameter around 100 nm and clearly the MCM-41 type of highly ordered hexagonally packed mesoporous structure. Meanwhile, the result of smallangle X-ray powder diffraction (SAXRD) of MSNs-ICP also showed the typical pattern of MCM-41 type MSNs (Figure 1b). In order to confirm the successful extraction of surfactant CTAB and surface modification with ICP groups, FT-IR spectra were collected. As shown in Figure S4, the removal of CTAB was verified by the disappearance of peaks around 2850 and 2924 cm−1, which are contributed to the vibration of the long carbon chains of CTAB. Introduction of the ICP groups on MSNs-ICP was certified by the appearance of the minor peak around 1582 cm−1. The TEM image of drug loaded hybrid system MSNs/Ce6/ Pt showed well-dispersed uniform nanoparticles, while the hexagonal mesoporous channels were not as clear as MSNsICP, which indicated the successful surface coating with CDPEI and Ad-PEG (Figure 1c). CD-PEI was chosen to be the linker to conjugate cisplatin prodrug, it is because modification with β-CD could greatly reduce the cytotoxicity of unmodified PEI.46 Moreover, the grafted β-CD can serve as a platform to assemble other functional moiety. Therefore, Ad-PEG was introduced by supramolecular self-assembly to enhance colloid stability and to reduce protein adsorption.47 EDX spectrum of MSNs/Ce6/Pt was collected, as shown in Figure 1d, the typical Pt peaks were clearly observed, which confirmed the successful conjugation of cisplatin prodrug. Subsequently, the amount of C

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Figure 2. UV−vis spectra (a) and fluorescence spectra (b) of free Ce6 and MSNs/Ce6/Pt in PBS; zeta potentials of MSNs-ICP, MSNs/Ce6, and MSNs/Ce6/Pt (c).

Figure 3. Intracellular trafficking of MSNs/Ce6/Pt nanoparticles in A549R cells. (a) Confocal microscopy images of A549R cells labeled with LysoTracker Green (LTG). A549R cells were incubated with MSNs/Ce6/Pt nanoparticles (50 μg/mL) for indicated times and then stained with LTG (150 nM, 30 min). The red fluorescence is from Ce6 inside MSNs/Ce6/Pt under 405 nm excitation, the green fluorescence is from LTG under 488 nm excitation. (b) Bio-TEM images of A549R cells after incubated with MSNs/Ce6/Pt nanoparticles. The red arrows indicate the nanoparticles inside the lysosome vesicles.

Pt on MSNs/Ce6/Pt was determined by ICP-MS to be ∼4.0% (w/w). The absorption spectrum of Ce6 shows a strong peak between 600 and 700 nm (Figure 2a), which makes it a promising photosensitizer for PDT, because the light at this range could penetrate relatively deeper tissue to activate the photosensitizer.48 Mono-L-aspartyl Ce6 (talaporfin) has been approved in Japan to treat centrally located early stage lung cancer in 2004.49 The Soret-band absorption of Ce6 at 404 nm was used to determine the loading efficiency, which was calculated to be 2.4% (w/w). Fluorescence spectra showed that when excited at 400 nm, strong red emission from MSNs/Ce6/ Pt aqueous solution could be detected (Figure 2b), which can be used to track the intracellular distribution of the delivery system. In the zeta potential measurements (Figure 2c), MSNsICP offers a negative zeta potential (−17.9 ± 0.6 mV), which was reversed to a positive value (+21.8 ± 1.4 mV) after CD-PEI was covalently linked onto the nanoparticles. The zeta potential value remains to be positively charged (+18.2 ± 1.5 mV) after cisplatin prodrug conjugation, which could facilitate the cellular internalization of the nanoparticles.50

3.2. Cellular Uptake of MSNs/Ce6/Pt Nanoparticles. The cellular internalization and intracellular distribution of MSNs/Ce6/Pt in A549R cells was evaluated by confocal laser scanning microscopy (CLSM) to monitor the time-dependent intracellular behavior of the drug delivery system. An Invitrogen probe LysoTracker Green (LTG) was used to stain the acidic organelles in A549R cells. We found that MSNs/Ce6/Pt was dominantly localized in LTG-labeled acidic organelles after 2 h of incubation, which displayed as the yellow dots in the cytoplasm arising from the well-matched overlay of the red and green fluorescence. After 6 h incubation, the red fluorescence of MSNs/Ce6/Pt mostly separated with the green fluorescence of LTG and located in the cytoplasm, indicating the successful escape of the nanoparticles from the lysosome (Figure 3a). As shown in the zeta potential measurement results, the surface charge of MSNs/Ce6/Pt is slightly positive, which may contribute to the efficient cellular internalization and accumulation of the nanoparticles in A549R cells. Furthermore, the CD-PEI polyamine moiety on the surface of nanoparticles exhibits a strong “proton sponge effect” upon protonated inside lysosome, which facilitates the rupture of lysosome to release D

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Figure 6. Intracellular Pt content in A549R cells determined by ICPMS.

therapy is extensively explored in the clinic. PDT, as an emerging modality of cancer treatment, triggers both apoptosis and necrosis of cancer cells, thus exhibiting great potential to be combined with chemotherapeutics agents to achieve enhanced anticancer efficacy. To examine the potential synergistic anticancer effect by codelivery of cisplatin prodrug and Ce6 together. The in vitro cytotoxicity of MSNs/Ce6/Pt mediated chemo-photodynamic dual therapy was evaluated against A549R cisplatin-resistant lung cancer cells by MTT assay. First of all, the resistant factor of A549R cells was determined by comparing with cisplatin-sensitive A549 cells. The IC50 values of cisplatin to A549 and A549R cells are 5.2 ± 0.5 μM and 25.1 ± 0.8 μM, respectively, giving a resistant factor of 4.8 (Figure S4 and Figure 4a). Subsequently, A549R cells were treated with cisplatin, MSNs/Ce6, or MSNs/Ce6/Pt at different concentrations for 72 h. For chemo-photodynamic dual therapy, cells were irradiated with 660 nm LED light for 5 min (10 mW/cm2) after 24 h incubation. The results revealed that in the absence of photosensitizer the light irradiation does not affect the viability of cells. MSNs/Ce6 in the dark is almost nontoxic to the cells, and after light irradiation MSNs/Ce6 showed moderate toxicity. Notably, MSNs/Ce6/Pt showed very strong cytotoxicity in the dark, giving an IC50 value of 1.16 ± 0.2 μM, which is 21.6-fold lower than that of cisplatin. Upon

Figure 4. MTT profiles of (a) cisplatin to A549R cells, (b) MSNs/ Ce6/Pt and equal amount of MSNs/Ce6 to A549R cells in the dark or with light irradiation. Light dose: 660 nm, 10 mW/cm2, 5 min.

the MSNS/Ce6/Pt nanoparticles.46 Moreover, the internalization of MSNS/Ce6/Pt nanoparticles by A549R cells was investigated by Bio-TEM. As shown in Figure 3b, after incubation with MSNs/Ce6/Pt nanoparticles, some vesicles appeared in the cytoplasm. Also a lot of nanoparticles could be found in these vesicles, indicating that the hybrid nanoparticles could be phagocytized by the A549R cells through endocytosis, well consistent with the fluorescence labeling results. 3.3. Anticancer Efficacy of Chemo-Photodynamic Dual Therapy. To circumvent chemoresistance, combination

Figure 5. Fluorescence microscopy images of A549R cells stained by Calcein-AM/PI dyes after MSNs/Ce6/Pt treatment: (a) control, (b) MSNs/ Ce6/Pt group, and (c) MSNs/Ce6/Pt plus light irradiation group. MSNs/Ce6/Pt concentration: 25 μg/mL. Light dose: 660 nm, 10 mW/cm2, 5 min. E

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Figure 7. Intracellular ROS levels evaluation by DCFH-DA: (a) confocal microscopy images of A549R cells after incubation with MSNs/Ce6/Pt in the dark or with light irradiation, (b) flow cytometric quantification, and (c) the corresponding histograms of the mean fluorescence intensity (MFI).

light irradiation, the IC50 value of MSNs/Ce6/Pt further decreased to 0.53 ± 0.1 μM, which confirmed our hypothesis that combined PDT effect of Ce6 would promote the anticancer efficacy of cisplatin. In addition, live/dead cell staining was used to visualize the cell viability more directly by fluorescence microscope after different treatments. Live and dead cells were stained with Calcein-AM and propidium iodide (PI), respectively. As shown in Figure 5, compared with the control group, the proliferation of A549R cells treated with MSNs/Ce6/Pt was greatly inhibited and a lot of dead cells can be stain by PI. After 660 nm light irradiation, much more superior cytotoxicity of MSNs/Ce6/Pt was achieved as evidenced by significantly reduced viable cells stained with calcein-AM, meanwhile most cells could be stained by PI. These results indicate that MSNs/Ce6/Pt nanoparticles mediated delivery of cisplatin prodrug combined with PDT could achieve synergistically enhanced anticancer efficacy to circumvent cisplatin resistance. To further illustrate the underlined mechanisms of the antiproliferation actions of MSNs/Ce6/Pt drug delivery system, more details were obtained by cellular Pt content determination, ROS level evaluation, and bio-TEM cell structural study.

Figure 8. Bio-TEM images of A549R cells with different treatment: (a) vehicle-treated control, (b) individual PDT treatment by incubating with MSNs/Ce6 plus light irradiation, (c) individual chemotherapy by incubating with MSNs/Ce6/Pt, and (d) chemoPDT combination therapy by incubating with MSNs/Ce6/Pt plus light irradiation.

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ACS Applied Materials & Interfaces 3.4. Cellular Pt Content Determination by ICP-MS. The anticancer mechanism studies of cisplatin have strongly demonstrated that cross-linking of DNA backbone by cisplatin leads to cell apoptosis.51 Therefore, the amount of cisplatin accumulated inside cells is the key factor to decide the fate of cancer cells. The intracellular Pt content can be easily and precisely determined by ICP-MS because platinum is an exogenous element of cells. After incubation of A549R cells with cisplatin or MSNs/Ce6/Pt for 3 h, the cells were harvested and digested and the intracellular Pt contents were determined by ICP-MS. As shown in Figure 6, the Pt uptake derived from MSNs/Ce6/Pt was about 7−9 times higher than that derived from cisplatin at both incubation conditions ([Pt] = 0.5 or 1.0 μM), which may explain the fact that MSNs/Ce6/Pt showed much higher cytotoxicity to A549R cells than cisplatin in the absence of light irradiation. Reduced cisplatin uptake was considered to be the main manner of cancer cells to gain drug resistance, which is associated with the down-regulation of cell membrane transporters (such as, Ctr1). While as shown in the cellular uptake study, MSNs/Ce6/Pt nanoparticles can be internalized by the cells through efficient endocytosis, therefore this nDDS could bypass the pathway of traditional cisplatin to achieve higher Pt uptake. 3.5. Cellular ROS Evaluation. The basic principle of PDT is the generation of highly cytotoxic ROS, especially singlet oxygen (1O2), under light activation. The capacity of MSNs/ Ce6/Pt to generate 1O2 in aqueous solution under 660 nm light irradiation was assessed following the Kraljic procedure. As shown in Figure S6, the absorbance of RNO at 440 nm decreased remarkably owning to the photooxidation bleaching effect after irradiation for 200 s in the presence of MSNs/Ce6/ Pt, which indicated the significant production of 1O2 from MSNs/Ce6/Pt when exposed to 660 nm light. Subsequently, the intracellular ROS levels of A549R cells after MSNs/Ce6/Pt treatment was examined by fluorescent probe DCFH-DA using confocal microscopy and flow cytometry. The CLSM images showed that much stronger green fluorescence was detected from the cells exposed to 660 nm light than those in the dark (Figure 7a). Meanwhile, the flow cytometry analysis indicated that the mean fluorescence intensity (MFI) of the cells treated with MSNs/Ce6/Pt under light irradiation is ∼4-times higher than that obtained in the dark (Figure 7b). It has been widely proved that these highly toxic ROS could damage the cellular proteins, lipids and nucleic acids. Therefore, elevated intracellular ROS level causes oxidative stress and a series of signal transduction pathway changes, which are highly related to transcription factors, cell cycle regulation, inflammation, and so on.52 The cellular metabolism changes following PDT treatment make the cells more fragile to chemotherapeutic treatment and result in apoptosis or necrosis. 3.6. Ultrastructural and Morphological Features of Cells by Bio-TEM. To get more insights into the detailed antiproliferative actions of the hybrid nDDS, ultrathin cell section analysis was carried out by Bio-TEM to investigate the ultrastructural and morphological alternations. The morphology of vehicle-treated control cells is clear and complete, with stretched and polygonal shape as well as abundant microvilli on the surface (Figure 8a). When monochemotherapy or monoPDT treatment was introduced by MSNs/Ce6 plus light irradiation or MSNs/Ce6/Pt alone, respectively, the cells exhibit some characteristics of apoptosis, the disappearance of microvilli, distorted or lysed organelles (Figure 8b,c). Finally, when chemo-PDT combination therapy was applied by treating

the cells with MSNs/Ce6/Pt plus light irradiation, cells started to lyse and lose structural integrity, suggesting severe necrosis (Figure 8d). These results showed clearly that the hybrid nDDS could efficiently transport cargos into cells and induce cell apoptosis and the synergism of chemotherapy and PDT exhibited very potent anticancer capacity against cisplatinresistant A549R lung cancer cells.

4. CONCLUSION In summary, we present herein a simple and efficient strategy to coadministrate cisplatin prodrug and photosensitizer Ce6 with a single nDDS by taking advantage of the versatile properties of MSNs to combat cisplatin-resistant cancer cells. The asestablished nDDS MSNs/Ce6/Pt nanoparticles were fully characterized. Cell experiments showed that MSNs/Ce6/Pt nanoparticles can be rapidly internalized through endocytosis and released into cytoplasm owing to the slightly positive surface charge and “proton sponge effect” derived from the surface ligand CD-PEI. Much higher cellular Pt uptake was achieved compared with cisplatin, meanwhile cellular ROS level was remarkably elevated after 660 nm light irradiation. The combined chemotherapy and PDT achieved very efficient anticancer activity against cisplatin-resistant A549R lung cancer cells. In conclusion, our study provides a novel solution to construct nDDS-based nanotherapeutic that combines two treatment modalities, chemotherapy and PDT, into one single platform to achieve very potent and synergistically enhanced anticancer capacity to combat drug resistance. This simple solution can be easily expanded to other drugs, treatment modalities, and drug delivery vectors to screen out the most appropriate combination entities to construct specific nDDS to a certain disease.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b03881. Synthesis routes of Ad-PEG, CD-PEI, and cisplatin prodrug (Scheme S1), 1H NMR spectra of CD-PEI and Ad-PEG (Figures S1 and S2), ESI-MS spectrum of c,c,t[Pt(NH3)2Cl2(OH)(O2CCH2CH2CO2H)] (Figure S3), FT-IR spectra (Figure S4), cytotoxicity profile of cisplatin in A549 cells determined by MTT (Figure S5), and photooxidation of RNO under light irradiation (Figure S6) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grants 21231007, 21572282, and 21201183), the 973 Program (Grants 2014CB845604 and 2015CB856301), the Ministry of Education of China (Grant IRT1298), Guangdong Provincial Department of Science and Technology, Guangdong Provincial Department of Human Resources and Social Security, and the Fundamental Research Funds for the Central Universities. G

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DOI: 10.1021/acsami.6b03881 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX