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Polymeric Prodrug Grafted Hollow Mesoporous Silica Nanoparticles Encapsulating NIR Absorbing Dye for Potent Combined Photothermal-Chemotherapy Yuanyuan Zhang, Chung Yen Ang, Menghuan Li, Si Yu Tan, Qiuyu Qu, and Yanli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00376 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 7, 2016

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ACS Applied Materials & Interfaces

Polymeric Prodrug Grafted Hollow Mesoporous Silica Nanoparticles Encapsulating NIR Absorbing Dye for Potent Combined Photothermal-Chemotherapy Yuanyuan Zhang,† Chung Yen Ang,† Menghuan Li,† Si Yu Tan,† Qiuyu Qu,† Yanli Zhao*,†,‡ †

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences,

Nanyang Technological University, 21 Nanyang Link, 637371 (Singapore) ‡

School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang

Avenue, 639798 (Singapore)

Keywords: Chemotherapy; Hollow mesoporous silica nanoparticles; NIR absorbing dye; Photothermal therapy; Polymeric prodrug

Abstract: In this study, polymeric prodrug coated hollow mesoporous silica nanoparticles (HMSNs) with encapsulated near-infrared (NIR) absorbing dye were prepared and explored for combined photothermal-chemotherapy. A copolymer integrated with tert-butoxycarbonyl protected hydrazide groups and oligoethylene glycols was initially grafted on the surface of HMSNs via reversible addition-fragmentation chain-transfer (RAFT) polymerization, followed by the deprotection to reactivate the hydrazide groups for the conjugation of anticancer drug doxorubicin (DOX). DOX is covalently bound onto the polymer substrate by acid-labile hydrazone bond, and released quickly in weak acidic environment for chemotherapy. The hollow cavity of HMSNs was loaded with an NIR absorbing dye IR825 to form the final multifunctional hybrid denoted as HMSNs-DOX/IR825. The hybrid exhibits good dispersity and stability as well as high light-to-heat conversion efficiency. As 1

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revealed by confocal microscopy and flow cytometry analysis, the hybrid was efficiently taken up by cancer cells and the conjugated DOX could be released under the cellular environment. In vitro cytotoxicity study demonstrates that anticancer activity of HMSN-DOX/IR825 could be significantly improved by the NIR irradiation, leading to a satisfactory therapeutic efficacy through the combination treatment. Thus, the developed hybrid could be a promising candidate for the combined photothermal-chemotherapy of cancer.

INTRODUCTION

Mesoporous silica nanoparticles (MSNs) have become a versatile platform for various biomedical applications such as drug/gene delivery, cancer diagnosis and bioimaging.1-3 Compared with other self-assembled nanomaterials like polymeric micelles, polymersomes and liposomes, MSNs offer unique advantages including high mechanical strength, good stability, controllable uniform particle size, favorable biocompatibility and easy surface modification. More importantly, the well-defined mesopores in the MSNs can be designed to have suitable pore volume capable of loading therapeutic agents for delivery to the pathological sites.4 In the field of chemotherapy research, using MSNs as the carriers for controlled drug release is a hot topic.5 Various gated MSNs have been developed, in which the drugs can be retained within the mesopore channels under physiological conditions, and quickly released upon the exposure to specific stimuli such as pH change, temperature variation, treatment of reducing agents, and light irradiation.4,6 Despite numerous conventional delivery methods of physically encapsulating the drugs inside, there is also an alternative tactic, which is to couple drugs with MSNs through biologically cleavable covalent bonds.7-9 The covalent conjugation of drugs with MSNs could minimize the undesired leakage and reduce side effects during the blood circulation. To date, there were only limited reports showing that 2


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anticancer drugs were directly anchored onto the surface of MSNs, including doxorubicin (DOX),10,11 cisplatin derivatives,12 methotrexate13,14 and chlorambucil.15 However, this strategy suffers from low drug coupling efficiency due to limited specific surface area of MSNs and steric hindrance between the drug molecules attached. Among many capping agents, coating MSNs with polymers is of special significance, since the grafted polymers can provide tailored properties including better biocompatibility, high stability, multiple functionality, and good release control of the loaded cargoes to meet various requirements.16-18 As compared to other strategies, the integrated polymers offer far more surface functional groups for the drug conjugation on account of the repeating units in the polymer chain. This design may thus enhance the drug loading capacity along with improved therapeutic efficacy of MSN-based prodrugs. As the polymers only cover the surface of MSNs with the availability of mesopore channels, such systems also allow for the encapsulation of a second therapeutic agent into mesopores. To our best knowledge, no anticancer drugs have yet been reported to be conjugated onto the polymer modified MSNs. Current studies show increasing evidence that chemotherapy alone is not ideal for successful anticancer treatment due to several factors such as the development of drug resistance, severe toxicity to normal cells and individual therapy differences in patients.19 To overcome these issues in conventional chemotherapy, the combination therapy has been proven to be an promising strategy. Photothermal therapy is one of commonly used anticancer treatments that have been easily employed in combination with chemotherapy, which utilizes photo absorbing agents to convert the absorbed light energy into heat, leading to prompt cell death and subsequent ablation of tumor tissue.20,21 With rapid development of nanoscience, various near-infrared (NIR) absorbing inorganic nanomaterials such as gold nanorods,22 nanographene,23 carbon nanotubes,24 and copper sulfide nanoparticles25 have 3



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been prepared and exploited as photothermal agents. Compared with the inorganic nanomaterials, NIR absorbing organic dyes are more promising for clinical translation, due to their reduced long-term toxicity and excellent biodegradability. Thus, NIR absorbing organic dyes including indocyanine green (ICG),26,27 cypate,28 IR780,29,30 and IR82531,32 have already been used to achieve encouraging photothermal therapeutic outcome. Due to their limited aqueous solubility and stability, these organic dyes are commonly encapsulated in amphiphilic polymer micelles or liposomes to improve the therapeutic efficiency. However, poor colloidal stability and relatively low loading percentage of these self-assembled nanocarriers resulted in a limitation for further utilization of photothermal dyes. The development of novel drug delivery systems with remarkable circulation stability, high loading efficiency, favorable biocompatibility as well as multiple functionalities for combination therapy may be of great importance for future clinical use.33 According to this concept, a multifunctional therapeutic nanoplatform based on hollow MSNs (HMSNs) was successfully developed for both photothermal therapy and chemotherapy (Scheme 1). After the grafting of DOX-conjugated polymer onto HMSNs, IR825, an NIR absorbing dye with excellent photothermal conversion efficiency and high photostability, was encapsulated in the HMSNs to fulfill the combination therapy. In this work, HMSNs not only were used to accommodate the therapeutic agents,34-37 but also served as an effective support for the conjugation of different kinds of therapeutic agents on the surface. There are several important features associated with this therapeutic nanoplatform. (1) In contrast with conventional MSNs, the loading capacity of HMSNs is rather high on account of the central hollow cavities used for the cargo storage. (2) Unlike previously reported MSN-based prodrug systems where the drug molecules were attached to the surface of MSNs directly, the anticancer drug DOX was integrated in the polymer shell coated on the surface of HMSNs to increase the drug conjugation amount in this study. Acid-labile hydrazone bond was used to connect DOX with the coating polymer, and triggered 4


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cleavage of the hydrazone linkage under intracellular acidic conditions would readily initiate controllable release of DOX. (3) Organic photothermal dye IR825 was loaded inside HMSNs with good stability, which could be used for subsequent photothermal treatment of cancer cells. By using simultaneous photothermal-chemotherapy, an improved therapeutic efficiency was achieved for enhanced cancer treatment. Scheme 1. Schematic illustration for polymeric prodrug coated HMSNs encapsulating NIR absorbing dye for potent combined photothermal-chemotherapy.

EXPERIMENTAL SECTION

Materials and reagents. Monomethoxy oilgo(ethylene glycol) methacrylate (OEGMA, MW = 450 g mol-1, Sigma-Aldrich) was passed through a basic alumina column to remove any inhibitors prior

to

use.

3-Aminopropyltriethoxysilane

(APTS),

azobisisobutyronitrile

(AIBN),

2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DMPA), DOX•HCl and trifluoroacetic acid

were

purchased

from

Sigma-Aldrich

and

used

without

any

purification.

Diisopropylcarbodiimide (DIC), diisopropylethylamine (DIPEA) and 4-(dimethylamino)pyridine (DMAP) were purchased from Alfa and used as received. All solvents were purchased from 5



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Sigma-Aldrich and used directly. Methacrylamide tert-butyl carbazate (MABH) was synthesized according to procedures reported in literature.38 Instruments. 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker BBFO-400 spectrometer and deuterated dimethyl sulfoxide (DMSO-d6) was used as the solvent. Transmission electron microscopy (TEM) images were obtained on JEM-1400 (JEOL) at an acceleration voltage of 100 kV. The UV-Vis-NIR absorption and fluorescence emission spectra were performed on a Shimadzu UV-3600 and Shimadzu RF5301PC spectrophotometer respectively. Hydrodynamic diameters and zeta potential values were determined by a Malvern Zetasizer Nano-S dynamic light scattering (DLS) system at 25 oC. X-Ray photoelectric spectroscopy (XPS) was carried out on a Phoibos 100 spectrometer. N2 adsorption/desorption analyses were conducted using Quantachrome Instruments Autosorb-iQ (Boynton Beach, Florida USA) with extra-high pure gases. Specific surface areas were calculated from the adsorption data in low-pressure range using the Brunauer-Emmett-Teller (BET) model, and pore size was determined by following the Barret-Joyner-Halenda (BJH) method. Thermogravimetric analysis (TGA) was carried out for powder samples using a TGA Q500 recorded from 100 to 1000 °C in an air flow at a heating rate of 10 °C min-1. Powder X-ray diffraction (PXRD) patterns were collected using Shimadzu XRD-6000e quipped with Cu-Ka radiation (λ = 1.5418 Å). Confocal laser microscopy (CLSM) images were acquired by a Leica TCS confocal microscope with a Nikon Eclipse TE2000-S objective (60 × oil). Flow cytometry experiments were conducted on BD LSRFortessa X-20 cell analyzer. Synthesis of HMSNs-NH2. HMSNs filled with cetyltrimethylammonium bromide (CTAB) inside mesopores were prepared according to the literature reports.39,40 For the amino group functionalization (HMSNs-NH2), the HMSNs containing CTAB (1.0 g) were redispersed in anhydrous toluene (100 mL) containing APTS (1.0 mL), and the suspension was stirred gently at 80 oC for 24 h. Then, the 6


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unconjugated APTS were removed by extensively washing with ethanol. To remove the CTAB surfactant inside the mesopore channels, the resultant hollow nanoparticles (0.8 g) were dispersed in methanol (200 mL), to which a concentrated hydrochloric acid (37%, 16 mL) was added, and the suspension was refluxed at 80 oC for 48 h. CTAB-removed HMSNs-NH2 was obtained by centrifugation, washed thoroughly with methanol, and then dried under vacuum for future use. Synthesis of HMSNs-RAFT. Reversible addition-fragmentation chain transfer (RAFT) functionalities were immobilized on surface of HMSNs before the polymerization. Typically, the RAFT agents DMPA (0.5 g, 1.38 mmol), DIC (0.320 mL, 2.76 mmol) and DIPEA (0.360 mL, 2.76 mmol) were added into CH2Cl2 (3 mL) at 0 oC. The resulting solution was stirred gently for 3 h, and subsequently HMSNs-NH2 (0.5 g) suspension in dimethylformamide (5 mL) was added dropwise. After 48 h of reaction at room temperature in the dark, the resultant nanoparticles HMSNs-RAFT were collected through centrifugation, washed thoroughly with methanol and then dried under vacuum. Polymerization to form HMSNs-Polymer. Briefly, HMSNs-RAFT (0.1 g) was dispersed in dioxane (3 mL) assisted by sonication, and the formed suspension was added with OEGMA (0.3 mL), MABH (0.5 g, 2.5 mmol) and AIBN (1.6 mg, 0.01 mmol). The mixture was degassed by freeze-pump-thaw for three cycles. After stirring at 70 °C for 48 h under nitrogen atmosphere, the polymerization was stopped upon air exposure. The polymer coated nanoparticles were then centrifuged and washed 4 times with tetrahydrofuran to remove the ungrafted polymer or unreacted reagents. After being washed with ethanol for additional 2 times, the product was dried under vacuum and denoted as HMSNs-Polymer. Synthesis of HMSNs-NHNH2. To remove the protection group of tert-butoxycarbonyl unit (BOC), trifluoroacetic acid (3.0 mL) was added to the suspension of HMSNs-Polymer (0.1 g) in dichloromethane (3.0 mL). After stirring at room temperature for 3 h, the nanoparticles were collected 7



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by centrifugation, washed extensively with methanol, dried under vacuum, and denoted as HMSNs-NHNH2. Synthesis of HMSNs-DOX prodrug. DOX•HCl (20 mg) was dissolved in anhydrous methanol suspension (6 mL) of HMSNs-NHNH2 (50 mg) at room temperature. The mixture was added with trifluoroacetic acid (2 µL), which was stirred at room temperature away from light for 48 h. The resultant prodrug was isolated by centrifugation and washed thoroughly with methanol to remove unconjugated DOX. After drying under vacuum, the product was obtained and denoted as HMSNs-DOX. To evaluate the DOX conjugation efficiency of the prodrug, HMSNs-DOX (2.0 mg) was treated with hydrochloride acid (1.0 M) at 25 oC for 24 h, and the supernatant was collected after the centrifugation. The remaining nanoparticles were repeatedly washed with deionized water until the supernatant was nearly colorless. The collected supernatant solution was combined and the total amount of the hydrolyzed DOX was determined by using a UV-Vis spectrometer at a wavelength of 490 nm. Preparation of HMSNs-DOX/IR825. The loading of photothermal organic dye IR825 inside HMSNs-DOX prodrug was achieved through the procedures introduced as follows. Typically, HMSNs-DOX (50 mg) and IR825 (30 mg) were added into methanol (0.6 mL), and the mixture was stirred at room temperature in the dark overnight. The nanoparticles were collected by the centrifugation, washed extensively with 10% (v/v) methanol/water solution, dried under vacuum, and denoted as HMSNs-DOX/IR825. To determine the loading capacity of IR825 in the therapeutic nanosystem, HMSNs-DOX/IR825 (2.0 mg) was washed with methanol by repeated centrifugation/redispersion cycles until the supernatant became colorless. The supernatants were combined together and the amount of the 8


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extracted IR825 was determined by using a UV-Vis spectrometer at a wavelength of 825 nm. In vitro drug release. The acid-triggered release of conjugated DOX from prodrug was evaluated when exposed to buffer medium at pH 5.0 (acetate), pH 6.0 (phosphate) and pH 7.4 (phosphate), respectively. Briefly, HMSNs-DOX suspensions in phosphate buffer (PB, 0.01M) at pH 7.4 (2.0 mL) were firstly placed into dialysis bags (molecular weight cut-off = 3,500), and dialyzed against buffer medium (40 mL, 0.05M) of pH 5.0, 6.0 or 7.4 at 37 oC in the dark. At the preset time intervals, a small portion of medium (2.0 mL) was taken out, and the intensity of its fluorescence emission at 560 nm under the excitation wavelength of 490 nm was monitored using the fluorescence spectrometer, while the same amount of fresh buffer solution was added back to compensate the volume loss. The concentration of DOX presented in the dialysate was calculated. After conducting the release experiments for three independent times, the mean value was obtained as the final result. Cellular uptake study. CLSM was employed to observe the intracellular release behavior of HMSNs-DOX/IR825 within DOX-sensitive HeLa cells and DOX-resistant A2780/DOXR cells. HeLa cells were incubated in Dulbecco's modified eagle medium, while A2780/DOXR cells were seeded in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U mL-1) and streptomycin (100 µg mL-1). Cells were seeded in a 6-well tissue culture plate (2 mL medium) at a density of 2.0 × 105 cells per well. After culturing at 37 oC and 5% CO2 for 24 h, an appropriate amount of HMSNs-DOX/IR825 suspension with an equivalent DOX concentration (5 µg mL-1) was added into the cell culture medium. After further incubation for 4, 12 and 24 h respectively, the culture medium was removed and the cells on microscope plates were washed three times with phosphate buffered saline (PBS). In addition, cells treated with free DOX were incubated as a control. Finally, the samples were stained with 4',6-diamidino-2-phenylindole (DAPI, blue color) for 5 min and then mounted for observation with CLSM. 9



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In flow cytometry studies, HeLa cells were seeded in a 6-well tissue culture plate (2 mL medium) at a density of 2.0 × 105 cells per well. After culturing for 24 h, free DOX or HMSNs-DOX/IR825 was added in the culture medium, all of which were maintained at the equivalent DOX concentration (1 µg mL-1) for every well. After incubation for 1, 3 or 24 h, the cells were washed with PBS for several times and treated with trypsin (0.5 mL) for 2 min. Then, fresh medium (0.5 mL) was added to each culture well, and the cells were collected via centrifugation at 2000 rpm for 3 min. After one more round of washing with PBS through centrifugation, the cells were re-suspended in PBS (1 mL), and subjected to the flow cytometry analysis. In vitro cytotoxicity study. The anticancer efficacy of HMSNs-DOX/IR825 on the proliferations of HeLa cells and A2780/DOXR cells with or without NIR irradiation was quantitatively investigated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the cells were seeded in 96-well plates (100 µL medium) and incubated. When the cell confluence reached around 60-70%, the medium was replaced with fresh one (90 µL), and then HMSNs-DOX/IR825 suspensions (10 µL each) with various concentrations were added. After 4 h of incubation, the cultural medium was replaced and the cells were illuminated by 808 laser at certain power densities for 5 min. After incubation for 24 h, the medium was again replaced with fresh one, followed by the addition of MTT solution (10 µL, 5 mg L-1). The cells were incubated for another 4 h, and then the medium was replaced by DMSO (150 µL) to dissolve the resulted purple crystals. Finally, optical densities of the samples were measured using a microplate reader (infinite M200, TECAN) at 490 nm. The cell viability (%) was calculated based on the following equation: (Asample/Acontrol) × 100%, where Asample and Acontrol represent the absorbance of the sample and control groups, respectively.

Scheme 2. Synthetic route of HMSNs-DOX prodrug.

10


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RESULTS AND DISCUSSION

The synthetic procedure of HMSNs-DOX prodrug was shown in Scheme 2. Initially, highly dispersed HMSNs filled with CTAB in the mesopores were prepared, and then functionalized with amino groups by silylation with an amino silane coupling agent in anhydrous toluene. Subsequently, CTAB was removed by refluxing the nanoparticles in methanol solution of hydrochloric acid. The morphology of resulted surfactant-free HMSNs-NH2 was characterized by TEM (Figure 1a and Figure S1 in the Supporting Information), and uniform hollow structure and well-defined mesoporous shell with an average thickness of 20 nm were displayed. The average size of the nanoparticles was observed to be around 120 nm, which was close to the results determined by DLS (Figure 1c). The mesopores were homogeneously arranged, which could serve as efficient channels for the dye diffusion and loading. 11



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Figure 1. TEM images of (a) HMSNs-NH2 and (b) HMSNs-DOX. (c) Size distributions of HMSNs-NH2 and HMSNs-DOX in PB (0.01 M, pH = 7.4) determined by DLS, where hydrodynamic diameter and polydispersity index of HMSNs-NH2 and HMSNs-DOX were 175 nm and 0.308 as well as 225 nm and 0.110, respectively. Prior to the coating of polymer on the nanoparticles by surface-initiated RAFT polymerization, RAFT agents should be first anchored on the surface of HMSNs.41 After the attachment of RAFT agents by amidation to form HMSNs-RAFT, a new peak from HMSNs-RAFT around 1535 cm-1 appeared in the FT-IR spectra (Figure 2a), ascribing to the N-H bending vibration of the formed secondary amine on HMSNs-RAFT. Consistently, the increased peak intensities at 2926 and 2855 cm-1 were also observed, which could be due to the stretching vibration of additional C-H bond from the introduced RAFT agents. In addition, it was observed that the zeta potential at pH 7.4 became more negative after the introduction of RAFT agents (Figure 2b), suggesting that some of the amino groups on the nanoparticle surface were consumed during the reaction. XPS spectra were also used to monitor the process of modification. As compared with XPS spectrum of HMSNs-NH2 (Figure S2, Supporting Information), the S 2s peak of HMSNs-RAFT appeared, confirming the attachment of RAFT agents.

12


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Figure 2. (a) FT-IR spectra of HMSNs-NH2, HMSNs-RAFT, HMSNs-Polymer and HMSNs-NHNH2. (b) Zeta-potential values of HMSNs-NH2, HMSNs-RAFT, HMSNs-NHNH2 and HMSNs-DOX. All the samples were dispersed in PB (pH 7.4, 10 mM) for the measurements. (c) UV–Vis–NIR absorption spectra of HMSNs-NHNH2, HMSNs-DOX and HMSNs-DOX/IR825 dispersed in PB (0.01M, pH = 7.4). The absorption spectra of free DOX (in distilled water) and free IR825 (in methanol) were presented as the control. (d) TGA curves of HMSNs-NH2, HMSNs-RAFT, HMSNs-NHNH2 and HMSNs-DOX. (e) N2 adsorption/desorption isotherms and corresponding BJH pore size distribution (inset) of HMSNs-NH2, HMSNs-RAFT, HMSNs-NHNH2 and HMSNs-DOX. Two monomers were chosen for the RAFT copolymerization on the surface of HMSNs. One was the OEGMA macromonomer, of which the hydrophilic oligo(ethylene glycol) side chain could improve the water dispersibility and biocompatibility of polymer-coated nanocarriers, whereas the other monomer MABH can provide the acylhydrazine groups for further DOX conjugation after the deprotection. As shown from the FT-IR spectrum of HMSNs-Polymer (Figure 2a), the appearance of strong peak around 1720 cm−1 was attributed to the C=O stretching vibration from the tert-butyloxycarbonyl (BOC) protection group, indicating the formation of the grafted polymer shell. After the as-prepared polymer coated HMSNs were treated with trifluoroacetic acid in dichloromethane solution, it was observed that the stretching vibration peak of C=O at 1720 cm−1 13



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disappeared, which could be explained by the fact that the BOC protection group was cleaved and thus HMSNs-NHNH2 was formed. Consistently, after the deprotection of the BOC group, the zeta potential of HMSNs-NHNH2 increased significantly (Figure 2b), indicating the formation of positively charged hydrazide group. To further confirm the chemical composition of the copolymer shell immobilized on the HMSNs, the silica content of the hybrid was etched by hydrofluoric acid, and typical proton signals of both hydrazide group and oligo(ethylene glycol) chain could be found in the 1H NMR spectrum of the extracted polymer (Figure S3, Supporting Information), again verifying successful immobilization of the polymer on the surface of HMSNs. DOX, one of the most widely used chemotherapeutic anticancer drugs, contains the carbonyl group, which allows for easy conjugation of this drug molecule with other substrates through hydrazone linkage.42 In this step, the deprotected HMSNs-NHNH2 was treated with DOX in anhydrous methanol in the presence of trace trifluoroacetic acid. Initially, the methanol suspension of HMSNs-NHNH2 was opaque white in color. After the conjugation of DOX, it turned light red as shown in Figure S4 (Supporting Information). Consistently, the zeta potential increased from -14 mV of HMSNs-NHNH2 to 13 mV of HMSNs-DOX (Figure 2b). In addition, the presence of conjugated DOX was further confirmed by UV-Vis-NIR absorption spectrum. As compared with the absorption spectrum of HMSNs-NHNH2 (Figure 2c), HMSNs-DOX had a wide absorption peak in the range of 600-400 nm, assigning to the conjugated DOX. It is well known that the silica structure is stable and could endure high temperature, while the organic component would decompose thermally to result in the weight loss of the sample. TGA was used to study the relative ratio of different components in the hybrid. The results shown in Figure 2d reveal that the weight losses of HMSNs-NH2, HMSNs-RAFT, HMSNs-NHNH2 and HMSNs-DOX were 14.1, 24.3, 32.6, and 40.0 % respectively, when the temperature increased eventually to 1000 oC. 14


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The upward trend of weight loss could be explained by the increasing content of organic component in the hybrid as the modification proceeded step by step. Thus, all the characterizations verify the successful grafting of polymeric drug on HMSNs. Since the introduction of amino groups was taken place before the removal of the surfactant filled in the mesopores, the grafted polymer prepared via the RAFT polymerization should not occupy the mesopore channels and thus would not compromise the drug loading and release capability.43,44 To investigate the mesopore structure parameters, N2 adsorption/desorption measurements were carried out and the results are summarized in Figure 2e and Table S1 (Supporting Information). The isotherms of the samples afforded characteristic type IV adsorption/desorption patterns, indicating the presence of cylindrical channel-like mesopores in the nanoparticles. After the grafting of DOX conjugated polymer via multi-step modifications, pore volume of HMSNs decreased from 0.917 cm3/g to 0.490 cm3/g, while the surface area dropped from 1267 m2/g to 514 m2/g, suggesting that the mesopore channel entrances were partially blocked by the grafted DOX-polymer conjugate during the drying process before the measurement. On the other hand, the relevant pore size was not changed obviously after the surface modifications, confirming that only negligible amount of the polymer was attached onto the internal surface of mesopore channels. Therefore, the as-prepared HMSNs-DOX with vacant mesoporous nanostructure is capable of subsequent loading of photothermal agents. The pore structure was also investigated by the PXRD. As shown in Figure S5 (Supporting Information), the PXRD pattern of HMSNs-NH2 shows one strong diffraction peak corresponding to the (100) plane of hexagonally packed pores. However, no obvious peak shift was observed after the covalent attachment of the polymer and drug, indicating that the mesoporous structure of the HMSNs was unaltered during the polymer coating and DOX conjugation on the surface.44 The polymer coating on the surface of HMSNs is amphiphilic. The oligo(ethylene glycol) side chain of the grafted polymer 15



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is hydrophilic, thereby improving the water dispersibility of the system. The conjugated DOX unit and the methyl methacrylate main chain of the grafted polymer are hydrophobic (see Scheme 1 for the structural illustration). DOX was conjugated onto the grafted polymer via acid-cleavable hydrazone bond and released through its cleavage under endo/lysosomal acid environment. The DOX conjugation capacity was determined by UV-Vis absorption measurements after the treatment of HMSNs-DOX with 1.0 M HCl. The result reveals that the DOX conjugation content of HMSNs-DOX was about 12 wt%, higher than conventional directly attached MSN prodrugs where the DOX conjugation amount was less than 5%.10,11 The increased drug conjugation content could improve the therapeutic efficacy as well as reduce the side effects, since the high content of drug could be used to scale down to a necessary amount of therapeutic systems for the treatment.

Figure 3. Drug release and photothermal effects. (a) pH-Dependent release of DOX from the HMSNs-DOX prodrug at 37 °C. (b) Heating curves of PB solution (pH = 7.4, 10 mmol), HMSNs-DOX (1 mg mL-1), and different concentrations of HMSNs-DOX/IR825 (0.2, 0.5 and 1 mg mL-1) suspended in PB under 808 nm laser irradiation at a power density of 0.8 W cm-2. (c) Temperature increments of HMSNs-DOX/IR825 (1.0 mg mL-1) suspension under the NIR laser irradiation at the power density of 0.8 W cm-2 for four cycles (5 min of irradiation for each cycle). The acid-triggered release of DOX from the HMSNs-DOX prodrug in vitro was investigated at 37 o

C in buffer medium at different pH values. As shown in Figure 3a, only 18% of conjugated DOX was

released from the prodrug after continuous incubation at pH 7.4 for 58 h, suggesting that most DOX 16


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molecules were still covalently conjugated on the nanoparticles. In contrast, the released amount of DOX drastically increased to 69% and 76% at pH 6.0 and 5.0 during the same incubation time, respectively. These results indicate that HMSNs-DOX has good stability under physiological conditions, and the release of DOX would be significantly boosted upon decreasing pH values. Thus, HMSNs-DOX shows a great potential for intracellular delivery of anticancer drug DOX in weak acidic endo/lysosomal environment. The hollow cavity of HMSNs was used for the accommodation of hydrophobic photothermal dye IR825. The successful loading of the dye was implied by the color change of nanoparticles from red to dark brown after the dye loading (Figure S4, Supporting Information). In addition, the resultant HMSNs-DOX/IR825 suspension displayed a new strong absorption band from 700 nm to 900 nm with a sharp absorption peak at 828 nm (Figure 2c), assigning to the loaded dye. The HMSNs-DOX/IR825 also demonstrated good dispersion in aqueous medium without obvious aggregation, which could be attributed by the amphiphilic polymer shell. The loading content of IR825 was determined to be 13 wt%. The high dye loading capacity was ascribed to the hollow cavity inside HMSNs that provide additional room for the storage. Due to the hydrophobicity, the IR825 dye was expected to be retained inside HMSNs without obvious release during the blood circulation, and thus the dye-loaded nanoparticles should be stable enough for the photothermal agent delivery. Figure S6 (Supporting Information) shows fluorescence spectra of HMSNs-DOX/IR825 upon the excitation at 480 nm and 808 nm, respectively. Under the excitation at 480 nm, a significant fluorescent signal in the range of 640-530 nm was detected, which was attributed to the conjugated DOX. However, upon the excitation at 808 nm, no obvious fluorescent signal can be observed. The low quantum yield of HMSNs-DOX/IR825 confers it with high photothermal conversion efficiency for effective treatment of cancer. 17



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Figure 4. (a) CLSM images of HeLa and A2780/DOXR cancer cells incubated with free DOX and HMSNs-DOX/IR825 for different periods of time. For each panel, the images from left to right show cell nuclei stained by DAPI (blue), DOX fluorescence in cells (red), and overlay of the two images. Scale bars are 20 µm in all images. Flow cytometric profiles against HeLa cells incubated with (b) free DOX and (c) HMSNs-DOX/IR825 prodrug for 1 h, 3 h and 24 h, and the DOX intensity was recorded through the propidium iodide (PI) channel. The relatively high IR825 loading capacity enabled possible use of the hybrid as photothermal agents. In order to investigate the photothermal heating capability of IR825 integrated system, HMSNs-DOX/IR825 with different concentrations of 0.2, 0.5 and 1.0 mg mL-1 was illuminated by 808 nm NIR laser at a power density of 0.8 W cm-1 for 5 min, respectively. As shown in Figure 3b, 18


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more rapid heating rate and greater temperature increment were observed upon increasing the concentration of HMSNs-DOX/IR825 under the same laser irradiation. By contrast, no obvious temperature fluctuations were observed for HMSNs-DOX suspension or the PB solution under the same experiment conditions. To evaluate the photostability of HMSNs-DOX/IR825, it was irradiated by 808 nm laser for four on/off cycles (Figure 3c). In each cycle, the sample was firstly illuminated by NIR laser for 5 min, and then cooled down to room temperature. It was found that a temperature increment of 37 oC was achieved in the first cycle of irradiation, and no significant loss of the photothermal capability was observed in the following cycles. Thus, HMSNs-DOX/IR825 could be implemented as a robust photothermal heater on account of its excellent photostability and photothermal capability, which is preferable for successful photothermal therapy. Motivated by the successful DOX release in weak-acidic condition, we further investigated the cellular uptake of HMSNs-DOX/IR825 and its DOX release inside cancer cells using CLSM. As seen from Figure 4a, the fluorescence of DOX (shown in red) and cell-labeling dye DAPI (shown in blue) from the cells was observed at different incubation times. For HeLa cell samples that were incubated with free DOX for 4 h, nearly all the red fluorescence focused in the cell nuclei, suggesting that free DOX could easily diffuse into HeLa cells and rapidly interact with the DNA in the nuclei. When HeLa cells were treated with HMSNs-DOX/IR825, however, only slight red fluorescence was observed in the perinucleus region of cells after 4 h incubation. The reason may be that the uptake of HMSNs-DOX/IR825 is relatively slow as compared with free drug, and the fluorescence of DOX could be self-quenched when combined into the delivery system.45 The intracellular microenvironment of cancer cells was slightly acidic in the endosomal (pH 5.0-6.0) and lysosomal (pH 4.0-5.0) compartments.46 After the prolonged incubations of 24 h, strong DOX fluorescence was observed in the cell nucleus region, which was attributed to the sustained acid-induced DOX release 19



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after the internalization of HMSNs-DOX/IR825 by HeLa cells. The drug-resistant cancer cells are often associated with reduced cellular drug accumulation due to the drug efflux or the activation of coordinately regulated detoxifying systems.47,48 It was observed that DOX resistant A2780/DOXR cells exhibited negligible intracellular DOX fluorescence after incubation with free DOX for 4 h. In contrast, the DOX fluorescence was stronger in the A2780/DOXR cells incubated with HMSNs-DOX/IR825 under an equivalent DOX amount for the same incubation time, indicating that HMSNs-DOX/IR825 could be more easily endocytosed by the drug-resistant cancer cells as compared with the free drug. The DOX fluorescence intensity in the case of HMSNs-DOX/IR825 was more pronounced with the increasing incubation time. This experiment not only confirms that HMSNs-DOX/IR825 could enhance the DOX uptake by drug-resistant cells, but also indicates that the loaded DOX is persistently released inside these cancer cells. The quantitative measurement of cellular uptake by flow cytometry showed that the uptake of free DOX and HMSNs-DOX/IR825 was all enhanced evidently over time. After 1 h incubation (Figure 4b,c), the DOX fluorescence intensity in the case of free DOX was stronger than that of the HMSNs-DOX/IR825 prodrug. However, this trend was reversed when the incubation time was prolonged to 24 h, and the DOX fluorescence intensity of the prodrug exceeded free DOX. The rapidly increased fluorescence intensity in the case of HMSNs-DOX/IR825 under longer incubation time could be explained by the fact that the conjugated DOX was gradually released from the HMSNs-DOX/IR825 prodrug after cellular uptake, which is consistent with the observation by CLSM for the intracellular release of DOX.

20


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Figure 5. In vitro anticancer activity. (a) Relative viabilities of HeLa and A2780/DOXR cells after being incubated with various concentrations of HMSNs-NHNH2 for 24 h. (b) Relative viabilities of HeLa and A2780/DOXR cells incubated with HMSNs-NHNH2/IR825 (0.1 mg mL-1) after 5 min of 808 nm laser irradiation at different power densities for 24 h. (c) Fluorescence images of calcein AM/PI co-stained HeLa cells with HMSNs-NHNH2/IR825 (0.1 mg mL-1) incubation for 24 with the 808 nm laser irradiation for 5 min at different power densities. Live and dead cells were stained by calcein AM and PI, which displayed green and red colors, respectively. Relative viabilities of (d) HeLa and (e) A2780/DOXR cells after incubation with different concentrations (1, 2, 5, 10, and 20 µg mL-1 based on DOX) of free DOX and HMSNs-DOX/IR825 for 24 h with or without laser irradiation (808 nm irradiation, 2 W cm-2, 5 min). Encouraged by the above results, the cancer cell killing efficiency of HMSNs-DOX/IR825 in vitro 21



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was evaluated. The cytotoxicity of HMSNs-NHNH2 nanocarriers without DOX conjugation and dye loading were first investigated against HeLa cells and A2780/DOXR cells by using MTT assay (Figure 5a). It was found that HMSNs-NHNH2 nanocarriers have favorable biocompatibility, since the viabilities of two types of cells were still above 80% even when the concentration of HMSNs-NHNH2 increased to 0.2 mg mL-1 after 24 h incubation, indicating that the nanocarriers were suitable for anticancer therapy. To eliminate the interference of conjugated DOX during the investigation of the photothermal ablation efficiency, IR825 directly loaded HMNS-NHNH2 nanoparticles (HMSNs-NHNH2/IR825) were used as the photothermal agent in the study. Enhanced cancer cell killing rate was observed upon increasing the laser power densities. Notably, when the laser power density was above 1.2 W cm-2, the light irradiation conferred significant photothermal damage against HeLa as well as A2780/DOXR cells (Figure 5b). It was demonstrated that the laser power was a crucial factor for efficient photothermal therapy using this therapeutic system. In order to visualize the cancer cell killing efficiency of the photothermal treatment, the HeLa cells were co-stained by calcein AM and PI (Figure 5c). As expected, the fluorescent images confirm that HeLa cells treated with HMSNs-NHNH2/IR825 could exhibit enhanced cell death rate upon increasing the laser power density. The HeLa cells were eradicated when the laser power density was increased to 2 W cm-2. These data collectively demonstrate the excellent photothermal therapeutic efficacy of the IR825 loaded nanocarriers in vitro. In vitro anticancer study revealed a concentration-dependent cytotoxicity of DOX and HMSNs-DOX/IR825 (Figure 5d,e). For the treatment of HeLa cells, it was shown that the chemotherapeutic efficacy of the HMSNs-DOX/IR825 prodrug was lower than free DOX at equivalent DOX concentrations. A possible reason might lie in the fact that time/pH-dependent drug release characteristics of HMSNs-DOX/IR825 result in a delay of therapeutic efficacy, leading to 22


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lower cytotoxicity as compared to free DOX. On the other hand, the anticancer efficacy of HMSNs-DOX/IR825 drastically enhanced after 5 min NIR irradiation (2 W cm-2), and the cancer cells were almost completely destroyed at high concentrations of HMSNs-DOX/IR825 (equivalent DOX concentrations were above 5 µg mL-1). Under the irradiation of NIR laser, the half-maximal inhibitory concentration (IC50) of HMSNs-DOX/IR825 for HeLa cells through the combination therapy was 2.8 µg mL−1, while the IC50 for the chemotherapy alone was 8.4 µg mL−1. In a parallel experiment for the treatment of A2780/DOXR cells, DOX and HMSNs-DOX/IR825 showed much lower cytotoxicity without NIR irradiation and their IC50 could not be determined under the same experimental conditions. The IC50 of HMSNs-DOX/IR825 for the A2780/DOXR cells under NIR irradiation was determined to be 2.0 µg mL−1, lower than the value for HeLa cells under the same irradiation. These results indicate that DOX resistant A2780/DOXR cells are more vulnerable to the temperature enhancement.

By

combining

the

photothermal

treatment

with

chemotherapy

using

HMSNs-DOX/IR825, the therapeutic outcome against the drug-resistant cancer cells was further improved. HEK 293 normal cells were also employed as the control studies. Free DOX and HMSNs-DOX/IR825 showed a similar cytotoxicity against HEK 293 normal cells, which were comparable to the case of HeLa cells with or without the NIR irradiation (Figure S7, Supporting Information). Thus, the present system has no obvious selectivity between cancerous cells and normal cells studied. Since the nanocarriers could be accumulated at tumor sites via the enhanced permeability and retention (EPR) effect, the present therapeutic system is expected to reach tumor to express cancer cell-killing efficiency by remote-controlled light irradiation, while causing minimal side effects to normal tissues. Another strategy is to introduce targeting ligands on the nanoparticle system to enhance its selectivity and specificity toward tumor, which will be our follow-up work. 23



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CONCLUSION

In summary, a surface-initiated RAFT polymerization strategy has been applied to graft HMSNs with a polymer layer. By the conjugation of DOX to the polymer layer via an acid-cleavable hydrazone linkage, the obtained HMSNs-DOX with excellent stability and dispersibility was used to load photothermal dye IR825. The conjugated DOX from HMSNs-DOX could be released at weak acidic conditions for chemotherapy, while the loaded IR825 dye could convert the NIR irradiation into heat energy for photothermal therapy. By combining these two treatments, the HMSNs-DOX/IR825 showed higher therapeutic efficacy on both DOX-sensitive HeLa cells and DOX-resistant A2780/DOXR cells. Overall, HMSNs-DOX/IR825 with grafted polymeric drug and loaded photothermal dye may provide a promising strategy for efficient combinational cancer therapy.

ASSOCIATED CONTENT

Supporting Information:

Additional analytical data and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Email: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 24


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Acknowledgements

This research is supported by the National Research Foundation (NRF), Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) Programme-Singapore Peking University Research Centre for a Sustainable Low-Carbon Future, the NTU-A*Star Silicon Technologies Centre of Excellence under program no. 11235100003, as well as the NTU-Northwestern Institute for Nanomedicine.

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