pH- and Temperature-Responsive Theranostics for ... - ACS Publications

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Triple Hit with Drug Carriers: pH- and Temperature-Responsive Theranostics for Multimodal Chemo- and Photothermal Therapy and Diagnostic Applications Seonmi Baek,†,# Rajendra K. Singh,‡,§,# Tae-Hyun Kim,‡,§,# Jae-won Seo,§ Ueon Sang Shin,∥,○ Wojciech Chrzanowski,*,†,⊥,△ and Hae-Won Kim*,‡,§,∥ †

Faculty of Pharmacy, University of Sydney, NSW 2006, Australia Institute of Tissue Regenerative Engineering (ITREN) and ∥Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan 330-714, Republic of Korea § Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 330-714, Republic of Korea ⊥ Charles Perkins Centre, The University of Sydney, NSW 2006, Australia △ Australian Institute of Nanoscale Science and Technology, The University of Sydney, NSW 2006, Australia ○ Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan 330-714, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Currently there is a strong need for new drug delivery systems, which enable targeted and controlled function in delivering drugs while satisfying highly sensitive imaging modality for early detection of the disease symptoms and damaged sites. To meet these criteria we develop a system that integrates therapeutic and diagnostic capabilities (theranostics). Importantly, therapeutic efficacy of the system is enhanced by exploiting synergies between nanoparticles, drug, and hyperthermia. At the core of our innovation is nearinfrared (NIR) responsive gold nanorods (Au) coated with drug reservoirsmesoporous silica shell (mSi)that is capped with thermoresponsive polymer. Such design of theranostics allows the detection of the system using computed tomography (CT), while finely controlled release of the drug is achieved by external trigger, NIR light irradiationON/OFF switch. Doxorubicin (DOX) was loaded into mSi formed on the gold core (Au@mSi-DOX). Pores were then capped with the temperature-sensitive poly(N-isopropylacrylamide)-based N-butyl imidazolium copolymer (poly(NIPAAm-co-BVIm)) resulting in a hybrid systemAu@mSi-DOX@P. A 5 min exposure to NIR induces polymer transition, which triggers the drug release (pores opening), increases local temperature above 43 °C (hyperthermia), and upregulates particle uptake (polymer becomes hydrophilic). The DOX release is also triggered by drop in pH enabling localized drug release when particles are taken up by cancer cells. Importantly, the synergies between chemo- and photothermal therapy for DOX-loaded theranostics were confirmed. Furthermore, higher X-ray attenuation value of the theranostics was confirmed via X-ray CT test indicating that the nanoparticles act as contrast agent and can be detected by CT. KEYWORDS: theranostics, gold nanorods, mesoporous silica, thermoresponsive polymer, controlled drug delivery, CT-scannable, biocompatible materials, drug carriers

1. INTRODUCTION A demand for “smart” drug delivery systems, which are capable of enhancing therapeutic efficacy of many remedies and which drastically improve patient satisfaction and offer effective and long-lasting remission, is steeply increasing. These goals can be achieved by combining different modes of treatment to generate synergies that are likely to reduce the possibility of therapy-related complications. An example of such solution is nanoparticles, which are “catalysts” that offer multiple functions, for example, hyperthermia + delivery of cytotoxic drug. Currently, it is believed that therapeutic function can be © 2016 American Chemical Society

further enhanced by providing the system with diagnostic capabilitytheranostics. This could allow more precise targeting of diseased tissues and monitoring of the therapy progress. Theranostics integrate minimum two functional domains of materials to provide therapeutic and diagnostic capability in a single system.1,2 In cancer therapy, the detection (imaging) of tumor, with simultaneous delivery of drug in a Received: January 25, 2016 Accepted: February 29, 2016 Published: February 29, 2016 8967

DOI: 10.1021/acsami.6b00963 ACS Appl. Mater. Interfaces 2016, 8, 8967−8979

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ACS Applied Materials & Interfaces

Figure 1. (A) Schematic illustration of theranostics nanohybrid platform, composed of gold nanorod core coated with mesoporous silica and then capped with thermoresponsive polymer (Au@mSi@P), designed for stimuli−responsive controlled delivery of anticancer drug simultaneously with X-ray CT scanning ability for cancer treatment. (B−D) Characteristics of gold nanorods: (B) TEM image of the gold nanorods; (C) high-resolution TEM-FFT analysis of gold nanorods; and (D) UV−vis extinction spectra for gold nanorods with corresponding image. (E, F) Characteristics of Au@mSi: (E) TEM image of the mSi@Au; and (F) mesoporous characteristics of Au@mSi, with N2 adsorption/desorption isotherm and mesopore size (inset).

localized and finely controlled manner, is of prime interest because it increases the efficacy of the treatment and minimizes side effects.2−5 Recent research indicated that hybrid mesoporous silica nanoparticles are highly promising candidates to circumvent the side effect of chemotherapy.6−9 Mesoporous silica nanoparticles are classified as a hybrid nanodevice due to their properties, including unique porous architecture, modifiable surface, chemical stability, and biocompatibility.10,11 The pores of mesoporous silica (mSi) can be loaded with drug and then capped with various materials (gatekeepers) that can be opened and closed via different internal or external triggers to achieve a stimulus−responsive release system.11−14 Herein, we develop a theranostics system that allows detection of diseases using computed tomography (CT) and finely controlled release of a drug upon irradiation with nearinfrared (NIR) light. At the core of our innovation is NIR-

responsive gold nanorods (Au) coated with drug reservoir mSithat is capped with newly developed thermoresponsive polymer (P). The single use of Au has few disadvantages, including lack of drug loading capacity due to their relatively small surface area15,16 and their cytotoxicity resulting from the hexadecyltrimethylammonium bromide (CTAB) on the surface of Au.17 CTAB is an indispensable element in controlling the shape and size of Au.17,18 CTAB is highly toxic to human cell; thus, Au often requires intensive washing or coating with biocompatible compounds including mSi.17,18 To minimize preleakage of drug inside the pores of mSi, several strategies of pore sealing have been developed.12−14,19−21 For example, photothermally sensitive gatekeeper poly(N-isopropylacrylamide) (poly(NIPAAm) is one of the typical pore-sealing materials.19−21 Below the lower critical solution temperature (LCST) of poly(NIPAAm), the cargo in the voids of mSi is gradually released due to predomination of the hydrogen bonds 8968


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allow the seed solution to grow as rod-shaped gold nanoparticles, was prepared by adding (dropwise) a mild reducing agent (Au3+ to Au+) of ascorbic acid (100 μL, 0.057 M) into a solution that consisted of 150 mL of 0.1 M CTAB, 7.5 mL of 0.01 M HAuCl4, and 12 μL of 0.1 M AgNO3 until the color of mixture turned from bright yellow to colorless. Finally, 240 μL of the presynthesized seed solution was mixed with the growth solution and resulted in the second reduction of gold from Au+ to Au0, which assembled on the surface of the seeds. In the mixture, CTAB was considered as a rod-shaped micellar template because CTAB can strongly adsorb along the long axis face of the Au, form a bilayer, and lead to dominant, longitudinal growth on the ends of gold nanoparticles. 2.1.2. Mesoporous Silica Coating on Gold Nanorods (Au@mSi). mSi shell was formed on Au using a previously reported method with some modification.35 First, the Au were washed by centrifugation (12 500 rpm, 25 min) followed by dilution with 10 mL of DW. Next, 100 μL of 0.1 M NaOH solution was added to adjust pH into the 9− 10 range to induce the electrostatic interaction between Au and mSi. In the next step, tetraethyl orthosilicate in methanol (200 μL/5 mL) was added dropwise, while the solution was stirred (1200 rpm). Next, the resultant mixture was stirred at 500 rpm for 48 h at room temperature (25 °C). To remove excess CTAB molecules from mSi surface and inner pores, an intensive washing procedure was performed. After this step, the mixture was dispersed in the solution containing 500 mg of ammonium nitrate dissolved in 60 mL of pure ethanol and stirred at 60 °C overnight. Next, the mixture was centrifuged at 12 000 rpm for 5 min and redispersed in DW. The Au@ mSi was collected and dried at 45 °C for 24 h. 2.1.3. Drug (DOX) Loading into Au@mSi (Au@mSi-DOX). For DOX loading test, 20 mg of the nanoparticles (Au@mSi) was added into 20 mL of DOX (0.2 mg/mL) in phosphate-buffered saline (PBS; pH 7.4) and sonicated for 5 min to obtain uniform dispersion. Then, the mixture was kept at the water bath (37 °C) for 24 h to allow electrostatic interaction between the positively charged amino groups of DOX and the negatively charged silanol groups of mSi, followed by centrifugation and drying at room temperature (25 °C) overnight. 2.1.4. Synthesis of Poly(NIPAAm-co-BVIm) Capping Agent. To synthesize capping component N-isopropylacrylamide (NIPAAM)based and partially cationic copolymer with N-butyl imidazolium moieties, poly(NIPAAm-co-BVIm) was synthesized.26 NIPAAm (0.22 g, 2.0 mmol) and 0.18 g (0.6 mmol) of 1-butyl-3-vinylimidazolium bromide ([BVIm]Br) were dissolved in 8 mL of DW, and then 2 μL (0.1 mmol) of ammonium persulfate solution (10%(w/v)) as an initiator and 3 μL (0.02 mmol) of tetramethylethylenediamine as an activator were added to the solution. Prior to polymerization, the reaction solution was purged with nitrogen for at least 30 min to remove oxygen. Polymerizations were performed for 24 h at 80 °C. The reaction solution became slightly yellowish and viscous during the reaction. After completion of the reaction, all possible impurities were removed by extraction with methylene chloride from the aqueous solution, following dialysis (membrane tubing, molecular weight cutoff 12 000−14 000 Da, Spectrum Laboratories, Savannah, GA, USA) against DW and then freeze-drying. To obtain the copolymer product as a pure solid mass, the polymer product clearly dissolved in 5.0 mL of cold DW was incubated in 60 °C for 1 h, and then the precipitated white solid was separated by centrifugation from the aqueous solution. The process was repeated three more times for further purification. Finally the purified polymer product dissolved in 5.0 mL of cold DW was freeze-dried, and ∼0.32 g of the polymer product that looks like white cotton wool was obtained. 2.1.5. Capping Poly(NIPAAm-co-BVIm) onto the Au@mSi (Au@ mSi@P). For smart polymer coating, 20 mg of the dried DOX-loaded Au@mSi or Au@mSi and 40 mg of poly(NIPAAm-co-BVIm) were added into the 10 mL of DW and sonicated for 10 s in an ultrasonic bath then stirred (500 rpm) at room temperature for 1 h. The mixture Au@mSi-DOX@P or Au@mSi@P was centrifugated (13 200 rpm, 2.5 min) and dried in vacuum oven to evaporate the solvent. 2.2. Material Characterizations. 2.2.1. Structural and Chemical Analyses. Crystallinity: to examine the crystallinity of the nanoparticles, X-ray diffraction (XRD; Rigaku) with Cu Kα radiation (λ =

between poly(NIPAAm) chain and solvent water, while the polymer chains are shrunken above the LCST of poly(NIPAAm), causing a faster release of the agent.22,23 Yang et al. synthesized the hybrid Au@mSi core−shell structure capped with pure poly(NIPAAm);21 however, the hydrophobicity of the system at the body temperature is more likely to induce cellular uptake of the poly(NIPAAm) capped Au@mSi and interact with proteins in the blood circulation causing aggregation of the particles.24 This is due to the LCST of poly(NIPAAm), which is lower than the body temperature (37 °C).24 To avoid such unspecific and undesired uptake, LCST should be adjusted via copolymerization of poly(NIPAAm) with a hydrophilic monomer, such as acrylamide (AAm)24 and 1-vinylimidazole,25 exhibiting their LCST at the ranges of 38− 42 °C and 35−45 °C, respectively. Our group has already reported that the newly developed p-NIPAAm-based copolymer (BVIm) was highly effective in drug-entrapping and -releasing at 38−42 °C.26 Thus, the poly(N-isopropylacrylamide)-based N-butyl imidazolium copolymer, (poly(NIPAAmco-BVIm)) = (P), was employed as a gatekeeper in this study. Gold nanoparticles are able to generate a thermal energy by converting a light source, a phenomenon called localized surface plasmon resonance (LSPR), and is commonly used to obtain hyperthermia in cancer therapy.27,28 The light source NIR, located in the transparent window (wavelength of 650− 900 nm), can penetrate soft tissues deeply and thus can be used as a trigger ffor therapeutic application.29 The wavelength of surface plasmon resonance is dependent on Au shape and dimensions.30 Furthermore, Au can induce a strong X-ray attenuation. Since CT is a standard tool for cancer diagnosis due to its wide field of view, ability to provide cross sectional images, and detect subtle differences between body tissues,31 it makes Au an ideal candidate for a X-ray CT contrast agent.32,33 The main side effects of conventional doxorubicin (DOX) chemotherapy are related to the lack of target specificity and uncontrolled release.34 To overcome these problems, DOX can be loaded into mSi, which is subsequently coated with thermoresponsive polymer. So the release can be controlled with NIR irradiation. Herein, we report a theranostics platform, which comprises three major components (as depicted in Figure 1A), to enable a “triple hit”: (i) enhance drug loading and control its release using external and internal triggers (ON/OFF temperature switch and pH), (ii) combine synergistically chemo (DOX drug) and photothermal (NIR-LED) therapies in a single system to reduce required dose of the drug thus minimizing possible side effects, and (iii) provide imaging capability with Xray CTcontrast agent. The current system that characterizes with diagnostic capability combines chemotherapy with hyperthermia and enables on-demand drug release by the temperature and pH, and represents a leap forward in the quest for the effective advanced theranostics nanoplatforms.

2. EXPERIMENTAL SECTION 2.1. Development of Hybrid Nanoparticles. 2.1.1. Synthesis of Au. Au were synthesized using the seed-mediated method with some modifications of a reported procedure.15 First, gold seeds were prepared mixing 7.5 mL of 0.1 M CTAB with 250 μL of 10 mM HAuCl4 as a surfactant followed by adding 1.65 mL of deionized water (DW). Next, 0.6 mL of 0.01 M ice-cold NaBH4 was added in the mixture as a strong reducing agent, leading to the color change from yellow to yellowish-brown, indicating the reduction of Au3+ to Au0. This solution was kept at room temperature for 2 h to provide enough time for the formation of seeds. The growth solution, which is used to 8969


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placed in the incubators at 25 and 41 °C. The amount of drug released into the medium was measured at different time points, and the medium was refreshed at each time point. To demonstrate the photothermal and pH-triggered DOX release, the dried Au@mSi-DOX@P were dispersed in 1 mL of PBS (pH 7.4 and 5.0) and exposed to NIR-LED (850 nm, 100 mW) for 5 min at different time points (0, 60, 120, 180, 240, and 300 min). The supernatants were collected at each time point to analyze the cumulative amount of DOX released from the nanoparticles. The samples were then redispersed in PBS (pH 7.4 and 5.0). 2.2.5. Characterization of the Attenuation of X-ray−Diagnostic Applications. To demonstrate the ability to induce X-ray attenuation, Au@mSi@P samples were dispersed in DW at different concentrations in the range of 0.5 to to 4.0 mg/mL and visualized using X-ray CT (Sky CT 1176; Micro Photonics Inc., USA). The attenuation values were obtained by processing the CT imaging using built-in software. 2.3. Characterization of the Nanohybrids in In Vitro Environment. 2.3.1. Cell Culture. Human cervical cancer cell line HeLa cells were purchased from the American Type Culture Collection (Rockville, USA) and maintained at 37 °C in an atmosphere of 5% CO2 in α-minimum essential medium (αMEM; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin. The medium was replaced three times per week, and cells were passaged at sub-confluency. 2.3.2. In Vitro Cytotoxicity of Nanoparticles. HeLa cells were seeded in 96-well plates with a density of 1 × 104 cells per well and allowed to attach for 24 h. The attached cells were then treated with Au@mSi@P, or Au@mSi-DOX@P (0, 0.25, 0.5, 1, 5, 10, 20, 40, 80, 160, 320, and 640 μg/mL) in culture media for 24 h. DOX only at corresponding concentrations (0, 0.0175, 0.035, 0.07, 0.35, 0.7, 1.4, 2.8, 5.6, 11.2, 22.4, and 44.8 μg/mL) were used as a reference. Cytotoxicity was measure using CCK-8 assay (Dojindo, Japan) by incubating samples with the CCK-8 reagent for 3 h in dark condition at 37 °C. OD values at 450 nm were measure using the iMark microplate reader (BioRad, USA) and converted into cell viability. 2.3.3. Characterization of Cell Morphology. HeLa cells were seeded at 1 × 104/well density in 96-well culture plates and treated with Au@mSi@P, DOX, and Au@mSi-DOX@P at different concentrations (see above) for 24 h. Next, cells were fixed with 4% paraformaldehyde solution for 1 h. Fixed cells were washed with cold PBS (4 °C) and stained with AlexFluor 488 Phalloidin (Molecular Probes, USA) for F-actin. Cells were counterstained with 4′,6diamidino-2-phenylindole (DAPI; Invitrogen, USA) to observe the nucleus and imaged using Zeiss LSM 510 laser-scanning confocal microscope (Zeiss, Germany). 2.3.4. Characterization of Drug−Nanoparticle Uptake. HeLa cells were seeded at the density of 1 × 104/well in 96-well culture plates and treated with 10 μg/mL of Au@mSi@P, Au@mSi-DOX@P, or 0.7 μg/mL of DOX for 4 h. Then the cells were washed with cold PBS and harvested using 0.05% trypsin−ethylenediaminetetraacetic acid (Gibco, USA). DAPI solution was added to stain the nuclei of the cells. The presence of autofluorescent (red) DOX and nuclei were visualized using scanning confocal microscope (Zeiss LSM 510). To quantify the drug localization in the cells flow cytometry was used. HeLa cells (1 × 105 cells/well) were seeded in six-well culture plates and allowed to attach for 24 h. Then cells were treated with 10 μg/mL of Au@mSi@P, Au@mSi-DOX@P, or 0.7 μg/mL of DOX for 4 h and harvested. The harvested cells were fixed with 4% paraformaldehyde solution. Fixed cells were washed with cold PBS, and the fluorescence of DOX in the HeLa cells was measured by FACSCalibur flow cytometer (BD Biosciences, USA). The data acquired for 10 000 cells in each sample were analyzed using the CellQuest Pro software. 2.3.5. Characterization of Drug−Nanoparticle Induced Cell Apoptosis. FITC-Annexin V and PI double stain was used to detect the apoptosis induced by Au@mSi@P. The HeLa cells treated with 10 μg/mL of Au@mSi@P, Au@mSi-DOX@P, and 0.7 μg/mL of DOX for 24 h were harvested and washed with cold PBS. The washed cells were then stained using an FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, USA). Briefly, the cells were resuspended in 1 mL of

1.5418 Å) operated at 40 kV and 40 mA was used. X-ray diffractograms were collected at diffraction angles of 2θ = 10−60°, scanning rate of 2θ/min, and step size of 0.02°. Chemical analysis: Fourier transform infrared spectroscopy (FTIR; Varian 640-IR) was used to characterize the functional groups and chemical structure of the samples. For each spectrum, 20 scans in the range of 400−1800 cm−1 wave numbers were recorded in transmission mode, using the KBr pellet method. Elemental composition of samples was analyzed by energy dispersive spectroscopy (EDS; Bruker). Charge, size, and morphology of nanoparticles: nanoparticle size and morphology were determined using a transmission electron microscope (TEM; JEOL-7100). The samples were prepared by dispersing the nanoparticles in ethanol and placing a drop of the suspension onto a carbon-coated copper grid. Zeta (ζ) potential of the samples was measured using a Malvern Zetasizer (ZEN3600; Mlavern). ζ-potential was measured at 25 °C in DW in triplicate. Specific surface area, pore volume, and pore size were determined by nitrogen gas adsorption/desorption isotherm at 77 K using a Quadrasorb SI automated surface area and pore size analyzer (2SIMP-9 Quantachrome). Hydrodynamic particle size was measured using dyanic light scattering, Zetasizer (ZEN3600; Malvern). Nanoparticles were suspended in DW (200 μg/mL); the measurements were conducted at pH 7 and 25 °C. Polymer transition temperature: the LCST of polymers was determined using a differential scanning calorimetry (DSC 131 evo; Bonsai advanced technologies, Spain) with heating and cooling rate 1 °C/min in the range from 8 to 60 °C. Five mg/mL in DW was used for each of the measurements. Weight % of the capping polymer was quantified using thermogravimetric analysis (TGA N-1500 SCINCO, Korea). Samples were heated at rate of 10 °C/min, and temperature range was 22−700 °C. The thermally induced reversible phase transition was investigated using the dispersion stability analyzer (Turbiscan Lab Expert with Cooler; lesmat, S.A.). Twenty mL of 0.1 mg/mL aqueous Au@mSi@P solution was used to measure the per cent optical transmittance with the temperature range of 25−50 °C. The initial temperature was kept at 25 °C, increased to 50 °C at a rate of 1 °C/min, and subsequently decreased to 25 °C at the rate of 1 °C/min after reaching 50 °C. The change of temperature repeated three times, observing the per cent transmittance of the mixture. The plasmon resonance peaks of Au, Au@mSi, and Au@mSi@P were investigated via ultraviolet−visible (UV−vis) spectrophotometry (Cary-100, Australia) at a wavelength of 400−900 nm. 2.2.2. Characterization of Photothermal Effects. Aqueous solutions of Au@mSi@P with different concentration and negative control sample (H2O) were placed in 96-well plate and irradiated with the near-infrared light emitted diode (NIR-LED; 850 nm, 100 mW) at 30 s time intervals for 6 min, while the temperature was continuously measured. This experiment enabled to establish minimum required exposure time to obtain the desired heating (43 °C) to induce hyperthermia but also phase transition of capping component (poly(NIPAAm-co-BVIm)). 2.2.3. DOX Loading Capacity. DOX was dissolved in PBS (pH 7.4) to produce stock solution with a concentration of 0.2 mg/mL. Final concentrations were obtained by serial dilutions of the stock solution (20, 40, 60, 80, 100, and 200 μg/mL). Au@mSi (1 mg) was dispersed in DOX solutions and sonicated in the ultrasonic bath. The absorbance of the solution was measured at 484 nm using UV−vis. Solutions were kept at 37 °C for 24 h to allow penetration of DOX into mSi structure. To quantify the drug-loading capacity, samples were centrifuged at 12 000 rpm for 5 min and washed once with PBS, and absorbance of the supernatant was measured with UV−vis. Loading capacity was calculated from the standard curve. 2.2.4. Characterization of Temperature and pH-Triggered DOX Release. To investigate temperature and pH-triggered DOX release, Au@mSi-DOX@P (200 μg/mL of initial DOX concentration) was suspended in 1 mL of PBS solution (pH 7.4 and 5.0) and incubated at 25 and 41 °C for up to 216 h. Next, the solutions were centrifuged, and the supernatants were collected for the DOX quantification. Nanoparticles were redispersed with fresh PBS (pH 7.4 and 5.0) and 8970


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Figure 2. (A, B) Analyses of Au@mSi: (A) XRD pattern; (B) FTIR spectrum. (C−F) Characteristics of Au@mSi@P: (C) optical transmittance as a function of temperature increase (red) and decrease (blue), including optical images of aqueous solutions at different temperatures, demonstrating the reversible phase transition; (D) FTIR spectra (also included P(NIPAAm-co-BVIm) (P) and Au@mSi; (E) thermogravimetric analysis showing the weight loss due to the polymer combustion; (F) UV−vis extinction spectra, displaying the maintenance of optical properties of Au and a slight red-shift of the longitudinal peak due to surface coatings. 1X binding buffer at a concentration of 1 × 106 cells/ml. Next, 5 μL of FITC Annexin V and 5 μL of PI were added per 100 μL of the cell suspension (1 × 105 cells). After they were gently vortexed, the cells were incubated for 15 min at room temperature in the dark. Subsequently, 400 μL of 1X binding buffer was added to each tube and analyzed using a FACSCalibur flow cytometer. The data acquired for 10 000 cells in each sample were analyzed using the CellQuest Pro software (BD Biosciences). 2.3.6. Characterization of Synergies between Photothermal- and Chemotherapy. HeLa cells (1 × 104 cells/well) were seeded in 96well culture plates and allowed to attach for 24 h. After cells attachment, cells were treated with 10 μg/mL of Au@mSi@P, Au@ mSi-DOX@P, and 0.7 μg/mL of DOX for 4 h and then irradiated with NIR-LED (850 nm, 100 mW) for 1, 3, and 5 min. The irradiated cells were cultured for 24 h, and then the cytotoxicity was assessed using CCK-8 assay.

therapy. However, gold nanoparticles bear two disadvantages: (i) relatively low specific surface area limits the amount of drugs that can be tethered, and (ii) the LSPR peaks often shift from desirable NIR window to the visible spectral region diminishing the ability to penetrate deep tissue, which is due to the aggregation of nanoparticles within cells. To overcome these drawbacks, we herein developed Au@mSi. The large specific surface area of mSi shell enables a high drug payload within its structure. Thirty nm thin protective/shield-like silica shell preserves also the LSPR maxima (peaks) in the optimum lighttransparent window. The theranostics design that synergizes the unique properties of the Au@mSi@P is illustrated in Figure 1 (A). NIR laser irradiation is used to induce plasmon resonance that converts into heat. Here we used heat to simultaneously control drug release from Au@mSi-DOX@Pchemotherapy, and to enable hyperthermia, which causes cell death. Such design combines synergistically both modes, and leads to higher efficacy of therapy with minimized side effects, due to reduced amount of the drug required in combined therapy to achieve the same effects as in traditional approaches. In terms of imaging modality, the hybrid Au@mSi@P can induce a strong X-ray attenuation, thus it acts as a contrast agent and enables CT visualisation (diagnostic). CT is a standard tool in cancer diagnostics that provides detailed cross-sectional images of tissues with subtle differences. 3.1. Au@mSi Hybrid System Has Structure and Properties Suitable for Drug Delivery. First, the physicochemical properties of Au@mSi were examined. The TEM image revealed that the synthesized Au had an average length,

3. RESULTS AND DISCUSSION Development of multifunctional materials that enable simultaneous drug delivery, combination therapy (e.g., hyperthermia + chemotherapy), and diagnosis, in particular, if multifaceted functions are catalyzed by a single system, is a necessary step to transform current therapies into highly efficacious, costeffective, and more comfortable for patients. These systems offer further benefits such as on-demand drug delivery, which accelerates progress in the quest for personalized therapies precision medicine. Gold characterizes with a prominent spectroscopic feature called LSPR, which gives rise to a sharp and intense absorption band in the visible range (650 and 950 nm). This property of gold has an immense potential to be used for various biomedical applications including bioimaging and photo8971


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ACS Applied Materials & Interfaces width, and aspect ratio of 45 ± 5.0 nm, 10 ± 2.0 nm, and 4.5 ± 0.5, respectively (Figure 1B). The high-resolution fast Fourier transform (FFT) images confirmed crystalline structure of Au with an interplanar spacing (d-spacing) of ∼0.20 nm (Figure 1C). The analysis of optical properties by UV−vis spectroscopy confirmed the presence of two transverse and longitudinal LSPR bands of Au (Figure 1D). The transverse peak (516 nm) corresponds to light absorption and scattering along the short axis of the nanorods, while the longitudinal peak centered at 760 nm corresponds to the interaction of light along the long axis. The mSi layer assembled at the Au core was shown to be uniform with a thickness of ∼30 nm (Figure 1E). The N2 adsorption/desorption curve of the Au@mSi showed a hysteresis loop, which is typical for mesoporous nanomaterials, and evidenced mesoporous structure of silica coating (Figure 1F). The specific surface area and the pore volume were 602.9 m2/g and 0.46 cm3/g, respectively, and confirmed a high level of mesoporosity. Furthermore, the mesopore size distribution (in inset image) showed a sharp peak at ∼3.3 nm, which can be effective in loading drug molecules.9 Further analysis of Au@mSi using EDS evidenced the presence of Si, O, and Au (Figure S1A). The XRD pattern of mSi@Au evidenced a broad peak at 2θ ≈ 22°, which corresponds to the amorphous mSi, and two sharp peaks at 2θ = 38° and 44° are associated with (111) and (200) diffractions at the face-centered cubic gold lattice (Figure 2A).16 Furthermore, the presence of silanol groups Si−O−Si at 1068, 794, and 447 cm−1 and Si−O−H at 962 cm−1 was confirmed by FTIR analysis (Figure 2B). The ζ-potential of Au@mSi and Au were −11.7 mV and +29.8 mV, respectively (Figure S1B). This drop of the ζ-potential after the formation of the coating suggests the presence of the abundant negatively charged silanol groups on the surface. Collectively, the mSi was assembled uniformly at the Au and the resultant Au@mSi nanohybrid system had a high level of mesoporosity and was negatively charged, which allows for effective loading of small drug molecules. 3.2. Temperature-Responsive Polymer Capping Enables On-Demand Drug Release. In the next step, to provide the Au@mSi with on-demand drug delivery capacity, we capped the surface with temperature-responsive polymer (gatekeeper). The surface modification with polymers may also improve the colloidal stability of the nanoparticles in the bloodstream.36,37 To enable controlled release of the drug cargo at the target site (cancer tissue) via temperature-induced shrinking of the polymer, the LCST of the polymer must be higher than the body temperature (37 °C).24,25 Therefore, we selected well-known temperature-responsive polymer NIPAAm and then copolymerized it with a hydrophilic polymer, 1vinylimidazole, to optimize the LCST. The low LCST of poly(NIPAAm) (32.3 °C) increased substantially to 40.85 °C for the copolymer poly(NIPAAm-co-BVIm), as evidenced by thermal analysis (DSC, Figure S2A-B). This increase in LCST was due to the repulsion force between the positively charged N-butyl imidazolium moieties contained in the copolymer chain, which is likely to impede the arrangement of hydrophilic (or ionic) parts of N-butyl imidazolium and hydrophobic moieties of NIPAAm. The poly(NIPAAm-co-BVIm) was then tethered to the surface of Au@mSi (Au@mSi@P) via electrostatic interaction between silanol groups of mSi and amino groups of poly(NIPAAm-co-BVIm).

The temperature-responsiveness of the three-component nanohybrid (Au@mSi@P) was then examined by measuring the solution turbidity, which is a well-established method to assess the phase transition of temperature-responsive polymers. The optical transmittance of the Au@mSi@P solution was ∼89% at 25 °C, and it remained at this level up to ∼37 °C. When the temperature increased above 37 °C the optical transmittance decreased sharply and dropped to ∼0.2% at 41 °C (Figure 2C). When temperature was reversed and dropped to 25 °C, the solution became transparent again, showing that the temperature-related phase transition is reversible. These results indicated that developed hybrid system (Au@mSi@P) may enable on-demand (ON/OFF-switchable) release of drug molecules that are incorporated into the mesopores, which is controlled by the temperature. Increase in local temperature can be achieved using external trigger, that is, NIR. The presence of the temperature-responsive polymer on the surface was further confirmed by FTIR (Figure 2D). FTIR spectra showed two main peaks at 1635 and at 1540 cm−1 that correspond to CO and N−H stretches of poly(NIPAAm-coBVIm), respectively. Moreover, the TGA showed that the weight loss of Au@mSi@P, when heated to 500 °C, was ∼67%, indicating the lost amount was equivalent to the capped polymer (Figure 2E). This dramatic weight loss of Au@mSi@P is associated with the combustion of poly(NIPAAm-co-BVIm) layer. Hydrodynamic particle size distribution of the hybrid Au@mSi@P was measured in DW by DLS, as shown in Figure S3. The nanoparticles were monodispersed showing a single narrow DLS peak with an average of 220 nm. The optical characteristics of Au after the surface modification with mSi and temperature-responsive polymer were then analyzed. The UV−vis experiment demonstrated that after the surface was coated, the main peak shifted slightly to 832 nm for the Au@mSi and to 850 nm for Au@mSi@P (Figure 2F). This shift could be explained because local refractive index of the silica shell (1.45) and polymer was larger than that of water (1.33).23,38 Importantly, the Plasmon band remained narrow, well-defined, and stayed in the NIR window,39,40 implying the Au@mSi@P nanohybrids preserve the optical properties of Au. 3.3. Near-Infrared Enables “On-Demand” Drug Release from Nanohybrids. Synthesized gold-cored nanohybrids have generally strong light absorbance in the “‘water window”’ (650−900 nm).23,38,41 This phenomenon implies that when the nanorods are irradiated with light, the light energy will be directly transformed into a thermal energy producing heat. Therefore, the exposure of the hybrid nanoparticles to NIR will result in the increase of the local temperature. It is important to note that the use of NIR is a significant advantage because NIR penetrates deep tissue and can be precisely spatially focused to a very small area with millimeter accuracy. Furthermore, no associated significant damage to the surrounding tissues is expected by the beam due to the lowenergy absorption of NIR light by the tissues.42,43 Subsequently, we examined whether the Au@mSi@P nanohybrids can induce temperature increase when exposed to NIR irradiation. In fact, the amount of heat induced by NIR irradiation depends on the NIR-LED power, the concentration of nanoparticles, and the irradiation time. By fixing the NIRLED power at 100 mW it was possible to interrogate the influence of the nanohybrid concentration and exposure time on the temperature change. Thus, to investigate the photothermal effect, different concentrations of Au@mSi@P ranging 8972


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Figure 3. (A) Induction of photothermal effects with NIR; temperature increase as a function of Au@mSi@P nanohybrids concentrations (5 to 60 μg/mL) and NIR-LED irradiation time (up to 5 min; 850 nm, 100 mW). (B) Temperature- and pH-dependent DOX release from the nanohybrids (25 °C vs 41 °C; pH 7.4 vs 5.0) recorded over a long-time period up to 216 h. (C) DOX release from the nanohybrids with and without exposure to external stimuli (NIR-LED, 850 nm, 100 mW, 5 min). The stimulus was applied in intervals (ON/OFF of NIR-LED), and the drug release was measured up to 360 min at different pH values (7.4 and 5.0). (D) The attenuation of X-ray signal as a function of Au@mSi@P nanohybrids concentration. The in vitro CT attenuation (HU) has a linear relationship, which indicates the suitability of the nanohybrids as a contrast agent.

from 5 to 60 μg/mL were exposed to NIR-LED. As expected the NIR-LED irradiation raised temperature gradually with exposure time. The stepwise increase in the nanohybrid concentration (5 to 60 μg/mL) proportionally increased temperature (Figure 3A). This result is vital to establish exposure time and concentration and tailor them for specific application. For example, to achieve the temperature of 42−44 °C, which is generally a target range for photothermal therapy used for cancer treatment, a minimum concentration of 10 μg/ mL with an irradiation time of 5 min might be required. Changes in the local temperature can be also used to induce the phase transition of temperature-responsive polymerstheir use as gatekeepers to enable on-demand release of drug molecules. First, we put together the thermal effects with chemotherapy (for multimodal therapy) to achieve greater therapeutic efficacy. The anticancer drug DOX was loaded into the nanohybrids, and the release kinetics of the drug was investigated as a function of the temperature. The DOX drug-loading efficiency was first measured (Figure S4). Different concentrations of DOX (up to 200 μg/mL) were used to load into 1 mg of Au@ mSi@P nanohybrids, and the amount of loaded drug was recorded. The amount of DOX loaded into the hybrids increased linearly for the concentrations up to 100 μg/mL. Further increase in DOX concentration resulted in saturation, and loading curve plateaued. Maximum DOX concentration that was effectively loaded was ∼70 μg. Therefore, the loading

efficiency of the nanohybrids for DOX is considered to be ∼7% (70 μg DOX per 1 mg of nanohybrid). Loading efficiency achieved here is significant when compared with other systems where DOX molecules were loaded into the mesoporous silica nanoparticles.44,45 3.4. DOX Release Is Regulated by Both Internal (pH) and External (Near-Infrared) Stimuli. Next, the release kinetics of DOX was investigated. In particular, DOX release at different temperatures (25 and 41 °C) and pH (5.0 and 7.4) was recorded (Figure 3B). Variations in pH were to mimic the in vivo environment: cancerous tissue (acidic pH) versus physiological tissue (neutral pH).46,47 The DOX release rate was shown to be both temperature- and pH-dependent. DOX was released faster at elevated temperatures and acidic conditions. The higher DOX release at pH 5.0 (vs pH 7.4, at both temperatures) may be explained because −NH2 groups in DOX molecules in acidic conditions become protonated, which subsequently weakens the electrostatic interactions with mSi and accelerates dissociation.9,48 Because tumor extracellular space is relatively acidic compared to normal tissue environment the pH can be an effective internal trigger to induce cancer-targeted drug release11,49−51 Not only pH sensitivity, but the DOX release was also temperature-responsive. This was due to the phase transition of the poly(NIPAAm-co-BVIm), which was capped on nanohybrids. The DOX release from the 8973


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Figure 4. (A) The effects of Au@mSi@P, DOX, and Au@mSi-DOX@P on HeLa cell viability. HeLa cells were treated with various concentrations of Au@mSi@P and Au@mSi-DOX@P (0, 0.25, 0.5, 1, 5, 10, 20, 40, 80, 160, 320, and 640 μg/mL) for 24 h and assessed the cell viability by CCK-8 assay. The corresponding concentrations of DOX were 0, 0.0175, 0.035, 0.07, 0.35, 0.7, 1.4, 2.8, 5.6, 11.2, 22.4, and 44.8 μg/mL. (B) The confocal microscopic images of the HeLa cells incubated with various doses of Au@mSi@P, DOX, and Au@mSi-DOX@P for 24 h. The cytoskeletons of the HeLa cells were stained with Alexa Fluor 488-Phalloidin, and the nucleus was stained with DAPI. Scale bars indicate 100 μm.

aqueous solutions of Au@mSi@P at different concentrations (from 0.5 to 4.0 mg/mL) were visualized using X-ray CT. With the increase of the nanohybrid concentration, the intensity of the CT signal increased, resulting in brighter images (Figure 3D). To quantify the attenuation of the signal and thus the detectability of the nanohybrids in CT, Hounsfield unit (HU) scale was used. We found that the relationship between HU and nanohybrid concentration is linear (R2 = 0.997). The attenuation HU unit of Au@mSi@P was also above the unit of physiological tissue, indicating their visibility (detectability) by CT. These results suggest that the Au@mSi@P nanohybrids can act as effective contrast agents. 3.6. Nanohybrids are Biocompatible and Potentiate Anticancer Drug-Actioned Cell Apoptosis. Next, we examined the effects of the DOX-loaded nanohybrids (Au@ mSi-DOX@P) on the cell viability using representative tumor cell line (HeLa). Free drug (DOX) at the concentration equal to the concentration loaded to nanohybrids was used as a control treatment. HeLa cell viability treated with DOX for 24 h was substantially reduced in a DOX dose-dependent manner, well-illustrating the cytotoxic effects of DOX (Figure 4A). Furthermore, the fluorescent images of cells reflected well the dose-dependent decrease of cell viability (Figure 4B). However, the cells treated with Au@mSi@P nanohybrids (without DOX) were highly viable over a wide range of concentrations (viability well above 90% at 320 μg/mL). The results confirm the cellular compatibility of the nanohybrids, and this result is highly beneficial, and can be considered as a prerequisite for the applications of the nanohybrids as intracellular delivery vehicles. Next DOX was loaded into the nanohybrids and then treated to

Au@mSi@P nanohybrids was regulated by both temperature and pH. To further confirm the ability of external stimulus (NIRLED) to switch the drug release “ON” and “OFF”, the nanohybrids were exposed to NIR-LED for 5 min with time intervals at both pH 5.0 and 7.4 (Figure 3C). In the absence of NIR-LED, only 10.6% (pH 7.4) and 14.5% (pH 5) of DOX was released during 6 h. However, when the nanohybrids were exposed to NIR-LED the cumulative release of DOX reached as high as 49% (pH 5) and 25% (pH 7.4) after 6 h. Below the LCST, the polymer chains form hydrogen bonds with water molecules through hydrophilic sites and swell, resisting gate opening of the mesopores. Above the LCST, the polymer corona collapses, due to the loss of hydrogen bonding to hydrophilic sites (−C = O, −NH) in the polymer shell; thus, the mesopore gate opens. Therefore, the temperature-dependent swollen/collapsed behavior of the polymer shell is the On/ OFF switch of the on-demand drug delivery of the hybrid nanocarrier system. More importantly, this ON/OFF DOX release pattern was observed periodically and for entire time of experiment, implying the continuous photothermal-responsive capacity of the nanohybrids. DOX loading and release results confirmed: (i) high DOX loading capacity of the nanohybrids, (ii) the ability to release DOX on-demand using extremal stimuli NIR-LED (ON/OFF mechanism), and (iii) the synergistic effects of pH and temperature on the regulation of the drug release. 3.5. Nanohybrids with Gold Nanorod Core May Be Used As Contrast Agent for Diagnostic Applications. To demonstrate the ability of the nanohybrids to attenuate X-rays, 8974


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Figure 5. (A) The confocal microscopic images of the HeLa cells incubated with various doses of Au@mSi@P, DOX, and Au@mSi-DOX@P for 4 h. The fluorescence signals of DOX are in red, while those of nuclei with DAPI are in blue. Images reveal difference in red signals, that is, mostly diffused form in free DOX vs some dotlike discrete pattern also visible in DOX-nanocarrier. Scale bars indicate 20 μm. (B) FACS analysis for the efficiency of cell uptakes of Au@mSi@P, DOX, and Au@mSi-DOX@P.

Figure 6. Quantification of the cell apoptosis induced by the treatment with Au@mSi@P, DOX, and Au@mSi-DOX@P: (A) representative dot plots of FITC-Annexin V and PI double stained cells: untreated (Ctrl), treated with 10 μg/mL of Au@mSi@P, 10 μg/mL Au@mSi-DOX@P and 0.7 μg/mL of DOX for 24 h. (lower left quadrant) The vital (double negative) population. (lower right quadrant) The early apoptotic (Annexin V positive/PI negative) population. (upper left quadrant) The necrotic cell (Annexin V negative/PI positive) population. (upper right) Lately apoptotic population. (B) Percentage of apoptotic cells including those in early and late apoptosis stage. Significantly increased amount of late apoptotic cells after treatment with hybrid nanoparticles.

the cells. The DOX-loaded nanohybrids exhibited cellular toxicity in a DOX dose-dependent manner, a similar trend to

that observed in free DOX treatment. However, on closer examination the reduction in the first 24 h caused by free drug 8975


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Figure 7. Viability of cells treated with Au@mSi@P (10 μg/mL), Au@mSi-DOX@P(10 μg/mL), and DOX (0.7 μg/mL) and exposed to NIR-LED (850 nm, 100 mW) irradiation for 1, 3, and 5 min. The drop of cell viability was statistically significant between different groups for each exposure time; columns a, b, and c represent significant differences (P < 0.05, ANOVA by Bonferroni) between different treatments: Au@mSi@P vs DOX vs Au@mSi-DOX@P.

was slightly higher than that caused by the DOX-loaded nanohybrids. It is likely that the DOX might not have been fully released from the nanohybrids during this short time period. On the basis of aforementioned result in Figure 3B, it can be deduced that the amount of DOX released from the nanohybrids in the first 24 h is between 15% and 65% (depending on the conditions); then the effective dose of DOX delivered from nanohybrids is substantially lower. In such situation it can be assumed that the viability of cells that were treated with 20 μg/mL of DOX-loaded nanohybrids should be comparable to the viability observed for cell treated with 5−13 μg/mL of free DOX. Although here results confirm such theory, the mechanisms of drug and nanohybrid transport are very different, and direct comparison may not be fully justified. In fact, the nanoparticle-based drug delivery is likely to bypass primary transporters and localize within cells more rapidly.52,53 This could suggest that lower concentration of drug can achieve greater efficacy when delivered intracellularly using nanoparticles and release sustainably using mesoporous silica carrier as demonstrated here. After confirming high cell viability of the nanohybrids (without drug) and the possible cellular toxicity incurred by DOX loading, we next interrogated the localization of the drug within the cells. The intracellular uptake of Au@mSi-DOX@P in HeLa cells for 4 h was investigated using confocal laser scanning microscopy and flow cytometry (Figure 5A,B). The red signals are from the DOX, while the blue signals are DAPIstained nuclei. For both cases of delivering DOX (free DOX and DOX-loaded nanocarriers), the red signals were observed in most cells (∼98%). On closer examination, when DOX was delivered freely, most red signals were found to be in diffused form; however, when DOX was delivered through nanocarriers, some parts of the red signals were spotlike, although some are also in diffused form. The DOX localized in the nanocarriers should exhibit more confined fluorescence signals. On the while, ∼15−20% of released DOX during ∼4 h (as deduced from the DOX release profile in Figure 3B) is considered to take part in the diffused DOX signals. It is envisaged that the internalized nanocarriers will release DOX with time slowly and exert therapeutic functions for long-term periods without the continual dosage treatment of DOX, which is one of the merits of the nanohybrid delivery system. Next, the anticancer biological functions of the DOX delivered from the nanocarriers were assessed. Apoptosis is a

well-known effect of DOX in cancerous cells. For this, the cells treated with DOX and DOX-loaded nanocarriers were doublestained with Annexin V-FITC, and PI and analyzed the populations by flow cytometry (Figure 6A,B). The externalization of phosphatidylserine (PS) from the inner to outer cell membrane is an early indicator of apoptosis. Therefore, Annexin V, a phospholipid binding protein with a high affinity for PS, can be used as an effective apoptosis marker. However, PI, a nonspecific DNA intercalating agent, can enter necrotic or damaged cells but is excluded by membranes of living cells or early apoptotic cells. On the basis of the plots, negligible quantities of apoptotic cells were detected in both control (untreated) and nanocarrier only (no drug) treated cells. However, a substantial fraction of apoptotic cells (Annexin V positive) was observed in both groups treated with free DOX (7.29%) and nanocarrier DOX (55.18%). Notably, the apoptotic cell fraction was more significant in the nanocarrier DOX than in the free DOX.54 The nonapoptotic cell fraction is considered to be necrotic, another type of cell death, different from the apoptosis (a programmed cell death), and for the free DOX this fraction was substantially high (56.55%; Annexin V negative but PI positive). These results demonstrated that the drug delivered by nanocarriers yielded higher efficacy to induce programmed cell death “apoptosis” than the case treated with free DOX. The cell death mechanism between free DOX and nanocarrier DOX is thus considered different. Although the reduction in cell viability is comparable for both free DOX and nanocarrier DOX, detailed tests confirmed that nanocarriers are more effective in inducing apoptosis. It is thought that the slow DOX release within cells by the nanocarriers can enhance the drug retention and activity, allowing for the appropriate/ effective biological role of DOX in the apoptosis of cancer cells, such as prolonged interactions with intracellular machineries like higher intercalation of DNA by the DOX molecules. Some previous studies have also reported that the internalization and cancer cell behaviors were different between nanoparticledelivered DOX and free DOX.54,55 3.7. Nanohybrids Synergize Chemo- and Photothermal-Therapeutic Effects. The focal point of this study was the examination of synergistic effects of chemo- and photothermal therapy. HeLa cells were treated with either 10 μg/mL of Au@mSi@P, 10 μg/mL of DOX(0.7 μg)-loaded Au@mSi@P, or 0.7 μg of free DOX and then exposed to LED8976


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release of DOX rapidly increases. In addition, the increase in local temperature (hyperthermia) induces cell death too. It has also been previously shown that the cytotoxicity of DOX increases at elevated temperatures,57 which might have been an additional factor that achieved very high efficacy of the treatment with nanohybrids. Therefore, it can be concluded that nanohybrids catalyze synergies between chemo- and photothermal therapies. By exploiting these synergies it may be possible to further reduce the effective dose of drug, localize and externally control the treatment, and subsequently achieve higher therapeutic efficacy. The findings in the present study indicate that the synergistic combination of phototherapy, light-activated chemotherapy, and selective drug delivery to tumor cells results in improved cancer cell-killing performance, compared to chemotherapy alone. It is well-known that DOX is highly toxic and can cause severe cardiac damage;58 thus, a good trapping, to avoid premature drug release achieved in our study, might be mandatory. Herein, the introduction of poly(NIPAAm-coBVIm) as gating moieties effectively regulates the release behaviors of DOX from the mesoporous cavities, with a prominent nearly zero premature drug release under physiological conditions. Therefore, the toxicity of the chemotherapeutics can be highly quenched before external trigger is applied. However, triggered release can be achieved under external heating or internal heating generated upon NIR irradiation. Remarkably enhanced apoptosis induction for the synergistic therapy in the HeLa cell line can be attributed to combined three effects: (i) photothermal contribution, by which NIR light energy is converted into thermal energy thanks to Au@mSi@P, (ii) increased intracellular concentration of DOX and Au@mSi-DOX@P with the aid of targeting moieties, with which lower NIR power density is needed to reach an optimal temperature and subsequent triggered drug release, and (iii) a synergistic combination between the photothermal effect and DOX’s anticancer function, as well-documented in previous reports and also proved to be useful in those clinical practices of tumor treatment.15,52,59 Moreover, the synergistic contribution from Au-assisted phototherapy and chemotherapy could overcome those deficiencies, which are encountered in the regional or over body hyperthermia treatments, such as discomfort to the patient, high risk of damage to normal tissues.60 However, the Au modality has been well-documented in some previous reports as a photoacoustic imaging agent,21,61 developed for in vivo and/or in vitro biomedical imaging or tracking of drug delivery systems. Herein, in the case of Au@ mSi-DOX@P, apart from local heat generation when exposed to NIR irradiation, the Au cores could also impart the integrity with some supplementary functions, such as aforementioned biomedical photoacoustic imaging or photoacoustic-assisted in vivo DDS tracking, which could be of great interest for followup research. Although here we demonstrated well the in vitro performance of the current nanocarrier system, that is, the controlled delivery of DOX with sufficient cancer cell toxicity, and the possible CT-imaging modality, further in vivo study needs to confirm the potential applications for tumor theranostics. Moreover, further modifications of the nanocarriers, that is, link with cancer cell receptors for cancer targeting action and the PEGylation chemistry to enable intravenous delivery, will improve the applicability to cancer treatment, which remains as further meaningful study.

NIR light (850 nm, 100 mW) for 1, 3, or 5 min. The cells exposed to NIR-LED irradiation without any treatment with nanoparticles or drug maintained high viability regardless of the exposure time (Figure 7). In contrast, the viability of cells treated with the nanohybrids, with or without DOX (Au@ mSi@P and Au@mSi-DOX@P), and exposed to NIR-LED dropped significantly. The drop was proportional to the exposure time and confirmed photoinduction of hyperthermia by nanohybrids. For the maximum exposure time (5 min) the viability of cells treated with Au@mSi@P dropped to ∼51%. This reduction in cell viability was primarily due to the increase in local temperature caused by the photothermal response of the nanohybrids. Interestingly, the group treated with DOX and irradiated with NIR-LED showed some increased cytotoxicity as well; after 5 min of irradiation, the cell viability dropped to ∼65%. This drop was however less than that observed for cells treated with nanohybrids (∼51%). Of special note was that the most substantial drop of cell viability was observed when cells were treated with DOX-loaded nanohybrids and exposed to NIR-LED. After the 5 min irradiation the cell viability dropped to as low as 11%. By calculating synergy factor SF = [(cell viability after treatment with DOX) × (cell viability after treatment with nanohybrids and irradiated with NIR-LED)/ (cell viability after treatment with DOX-loaded nanohybrids and irradiated with NIR)], the synergistic effects of chemo- and photothermal therapies can be defined. If the factor is ∼1, only cumulative effects are observed, while numbers above 1 suggest synergies. Calculated synergy factor here was as high as 3.02, which confirmed the nanohybrid-driven synergies of chemoand photothermal therapies. Relatively high synergy factors suggest that the effectiveness of hybrid system to eradicate cancer cells is significantly improved when compared with chemo- and photothermal therapy only. Importantly, the amount of late apoptotic cells was significantly higher than observed for chemotherapy only, which could be related to the release of the drug directly within the cell compartment. We could speculate that hybrid system, which “hits” cells simultaneously with chemotherapeutic and “high” temperature (hyperthermia) that both act locally will provide higher efficacy than traditional photochemotherapy. In conventional photochemotherapy the drug is activated with light and exerts an antiproliferative effect.56 However, the drug is transported to cells via active transporter mechanisms, which could be less effective than uptake of nanoparticles. Since both the drug and light alone are ineffective at doses used for the treatment, synergistic effects are not expected and could potentially suggest it lower efficacy than observed for hybrid systems. Hybrid system is also likely to have greater efficacy than combined photothermal and chemotherapy where drug and temperature-inducing nanoparticles are delivered separately. Drug could also be tethered to these nanoparticles. However, these approaches are not practical due to significant challenges related to the purification of nanoparticles (removal of toxic chemicals used for their synthesis) and limited amount of the drug that can be immobilized on the surface. Moreover, when comparing such system there is a remaining question, which nanoparticles will be internalized more effectively: Au or spherical hybrids? The synergistic effects observed here can be explained as follows: at the physiological temperature only a small amount of DOX is released from the nanohybrids; then, when the nanohybrids are exposed to NIR-LED, a local temperature increases, which triggers the polymer phase transition, and the 8977


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Au@Sio2 Nanocapsules for Drug Delivery and Fluorescence Imaging Of Cancer Cells. J. Colloid Interface Sci. 2011, 358, 109−115. (8) Kwon, S.; Singh, R. K.; Perez, R. A.; Abou Neel, E. A.; Kim, H.W.; Chrzanowski, W. Silica-Based Mesoporous Nanoparticles for Controlled Drug Delivery. J. Tissue Eng. 2013, 4, 10.1177/ 2041731413503357. (9) Kwon, S.; Singh, R. K.; Kim, T.-H.; Patel, K. D.; Kim, J.-J.; Chrzanowski, W.; Kim, H.-W. Luminescent Mesoporous Nanoreservoirs for The Effective Loading and Intracellular Delivery Of Therapeutic Drugs. Acta Biomater. 2014, 10, 1431−1442. (10) Vivero-Escoto, J. L.; Slowing, I. I.; Trewyn, B. G.; Lin, V. S. Y. Mesoporous Silica Nanoparticles for Intracellular Controlled Drug Delivery. Small 2010, 6, 1952−1967. (11) Baek, S.; Singh, R. K.; Khanal, D.; Patel, K. D.; Lee, E.-J.; Leong, K. W.; Chrzanowski, W.; Kim, H.-W. Smart Multifunctional Drug Delivery Towards Anticancer Therapy Harmonized in Mesoporous Nanoparticles. Nanoscale 2015, 7, 14191−14216. (12) Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C.-W.; Lin, V. S. Y. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers. Adv. Drug Delivery Rev. 2008, 60, 1278−1288. (13) Manzano, M.; Vallet-Regi, M. New Developments in Ordered Mesoporous Materials for Drug Delivery. J. Mater. Chem. 2010, 20, 5593−5604. (14) Chan, A.; Orme, R. P.; Fricker, R. A.; Roach, P. Remote and Local Control of Stimuli Responsive Materials for Therapeutic Applications. Adv. Drug Delivery Rev. 2013, 65, 497−514. (15) Zhang, Z.; Wang, L.; Wang, J.; Jiang, X.; Li, X.; Hu, Z.; Ji, Y.; Wu, X.; Chen, C. Mesoporous Silica-Coated Gold Nanorods as a Light-Mediated Multifunctional Theranostic Platform for Cancer Treatment. Adv. Mater. 2012, 24, 1418−1423. (16) Liu, W.; Zhu, Z.; Deng, K.; Li, Z.; Zhou, Y.; Qiu, H.; Gao, Y.; Che, S.; Tang, Z. Gold Nanorod@Chiral Mesoporous Silica Core− shell Nanoparticles with Unique Optical Properties. J. Am. Chem. Soc. 2013, 135, 9659−9664. (17) Zhang, J.-J.; Liu, Y.-G.; Jiang, L.-P.; Zhu, J.-J. Synthesis, Characterizations of Silica-Coated Gold Nanorods and its Applications in Electroanalysis of Hemoglobin. Electrochem. Commun. 2008, 10, 355−358. (18) Zhang, Y.; Xu, D.; Li, W.; Yu, J.; Chen, Y. Effect of Size, Shape, and Surface Modification on Cytotoxicity of Gold Nanoparticles to Human Hep-2 and Canine MDCK Cells. J. Nanomater. 2012, 2012, 1−7. (19) Zhu, S.; Zhou, Z.; Zhang, D.; Jin, C.; Li, Z. Design and Synthesis of Delivery System Based on Sba-15 With Magnetic Particles Formed in Situ And Thermo-Sensitive Pnipa as Controlled Switch. Microporous Mesoporous Mater. 2007, 106, 56−61. (20) You, Y.-Z.; Kalebaila, K. K.; Brock, S. L.; Oupický, D. Temperature-Controlled Uptake and Release in PNIPAM-Modified Porous Silica Nanoparticles. Chem. Mater. 2008, 20, 3354−3359. (21) Yang, J.; Shen, D.; Zhou, L.; Li, W.; Li, X.; Yao, C.; Wang, R.; El-Toni, A. M.; Zhang, F.; Zhao, D. Spatially Confined Fabrication of Core−Shell Gold Nanocages@Mesoporous Silica for Near-Infrared Controlled Photothermal Drug Release. Chem. Mater. 2013, 25, 3030−3037. (22) Chen, Z.; Cui, Z.-M.; Cao, C.-Y.; He, W.-D.; Jiang, L.; Song, W.G. Temperature-Responsive Smart Nanoreactors: Poly(N-isopropylacrylamide)-Coated Au@Mesoporous-SiO2 Hollow Nanospheres. Langmuir 2012, 28, 13452−13458. (23) Byeon, J. H.; Roberts, J. T. Aerosol-Based Fabrication of Biocompatible Organic−Inorganic Nanocomposites. ACS Appl. Mater. Interfaces 2012, 4, 2693−2698. (24) Shiotani, A.; Akiyama, Y.; Kawano, T.; Niidome, Y.; Mori, T.; Katayama, Y.; Niidome, T. Active Accumulation of Gold Nanorods in Tumor in Response to Near-Infrared Laser Irradiation. Bioconjugate Chem. 2010, 21, 2049−2054. (25) Muratalin, M.; Luckham, P. F. Preparation and Characterization of Microgels Sensitive Toward Copper II Ions. J. Colloid Interface Sci. 2013, 396, 1−8.

4. CONCLUSIONS Our results demonstrated that hybrid nanoparticles may be used effectively to enhance drug therapies. By combining different modes of treatment (e.g., chemo and hyperthermia) and the temperature and pH-repressiveness of the nanoparticles it is possible: (i) to enhance the drug uptake, (ii) to trigger the drug release locally using internal and external stimuli, and (iv) to combine synergistically drug and temperature effects to induce cell death in desired location. Furthermore, the attenuation of the X-ray signal achieved with the nanohybrids suggests their suitability as a contrast agent (CT).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00963. Figures illustrating TEM-EDS elemental signal, zeta potential of gold and Au@mSi, DSC thermograms that demonstrate the phase transition temperature of LCST, the DLS hydrodynamic diameter of Au@mSi-DOX@P, loading capacity of mesoporous nanohybrids for DOX. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (W.C.) *E-mail: [email protected]. (H.W.K.) Author Contributions #

These authors (S.B., R.K.S., and T.-H.K.) contributed equally to this work Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors acknowledge the support from the Global Research Laboratory Program (GRL; 2015032163) and Priority Research Centers Program (2009-0093829), funded by National Research Foundation, Republic of Korea.

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REFERENCES

(1) Zheng, M.; Liu, S.; Li, J.; Qu, D.; Zhao, H.; Guan, X.; Hu, X.; Xie, Z.; Jing, X.; Sun, Z. Integrating Oxaliplatin with Highly Luminescent Carbon Dots: An Unprecedented Theranostic Agent for Personalized Medicine. Adv. Mater. 2014, 26, 3554−3560. (2) Wang, Y.; Yang, T.; Ke, H.; Zhu, A.; Wang, Y.; Wang, J.; Shen, J.; Liu, G.; Chen, C.; Zhao, Y.; Chen, H. Smart Albumin-Biomineralized Nanocomposites for Multimodal Imaging and Photothermal Tumor Ablation. Adv. Mater. 2015, 27, 3874−3882. (3) Nie, S. Understanding and Overcoming Major Barriers in Cancer Nanomedicine. Nanomedicine (London, U. K.) 2010, 5, 523−528. (4) Basuki, J. S.; Duong, H. T. T.; Macmillan, A.; Erlich, R. B.; Esser, L.; Akerfeldt, M. C.; Whan, R. M.; Kavallaris, M.; Boyer, C.; Davis, T. P. Using Fluorescence Lifetime Imaging Microscopy to Monitor Theranostic Nanoparticle Uptake and Intracellular Doxorubicin Release. ACS Nano 2013, 7, 10175−10189. (5) Jin, Y. Multifunctional Compact Hybrid Au Nanoshells: A New Generation of Nanoplasmonic Probes for Biosensing, Imaging, and Controlled Release. Acc. Chem. Res. 2014, 47, 138−148. (6) Kim, C.; Kim, S.; Oh, W.-K.; Choi, M.; Jang, J. Efficient Intracellular Delivery of Camptothecin by Silica/Titania Hollow Nanoparticles. Chem. - Eur. J. 2012, 18, 4902−4908. (7) Wang, T.-T.; Chai, F.; Wang, C.-G.; Li, L.; Liu, H.-Y.; Zhang, L.Y.; Su, Z.-M.; Liao, Y. Fluorescent Hollow/Rattle-Type Mesoporous 8978


Research Article


(45) Meng, H.; Liong, M.; Xia, T.; Li, Z.; Ji, Z.; Zink, J. I.; Nel, A. E. Engineered Design of Mesoporous Silica Nanoparticles to Deliver Doxorubicin and P-Glycoprotein siRNA to Overcome Drug Resistance in a Cancer Cell Line. ACS Nano 2010, 4, 4539−4550. (46) Silva, A. S.; Yunes, J. A.; Gillies, R. J.; Gatenby, R. A. The Potential Role of Systemic Buffers in Reducing Intratumoral Extracellular pH and Acid-Mediated Invasion. Cancer Res. 2009, 69, 2677−2684. (47) Helmlinger, G.; Yuan, F.; Dellian, M.; Jain, R. K. Interstitial pH and pO2 Gradients in Solid Tumors In Vivo: High-Resolution Measurements Reveal a Lack of Correlation. Nat. Med. 1997, 3, 177− 182. (48) Singh, R. K.; Kim, T.-H.; Mahapatra, C.; Patel, K. D.; Kim, H.W. Preparation of Self-Activated Fluorescence Mesoporous Silica Hollow Nanoellipsoids for Theranostics. Langmuir 2015, 31, 11344− 11352. (49) Liu, Y.; Wang, W.; Yang, J.; Zhou, C.; Sun, J. pH-Sensitive Polymeric Micelles Triggered Drug Release for Extracellular and Intracellular Drug Targeting Delivery. Asian J. Pharm. Sci. (Amsterdam, Neth.) 2013, 8, 159−167. (50) Liu, Z.; Zhang, N. pH-Sensitive Polymeric Micelles for Programmable Drug and Gene Delivery. Curr. Pharm. Des. 2012, 18, 3442−3451. (51) Lee, E. S.; Gao, Z.; Bae, Y. H. Recent Progress in Tumor pH Targeting Nanotechnology. J. Controlled Release 2008, 132, 164−170. (52) Shen, S.; Tang, H.; Zhang, X.; Ren, J.; Pang, Z.; Wang, D.; Gao, H.; Qian, Y.; Jiang, X.; Yang, W. Targeting Mesoporous SilicaEncapsulated Gold Nanorods for Chemo-Photothermal Therapy with Near-Infrared Radiation. Biomaterials 2013, 34, 3150−3158. (53) Tang, H.; Shen, S.; Guo, J.; Chang, B.; Jiang, X.; Yang, W. Gold nanorods@mSiO2 with a Smart Polymer Shell Responsive to Heat/ Near-Infrared Light for Chemo-Photothermal Therapy. J. Mater. Chem. 2012, 22, 16095−16103. (54) Shi, M.; Ho, K.; Keating, A.; Shoichet, M. S. DoxorubicinConjugated Immuno-Nanoparticles for Intracellular Anticancer Drug Delivery. Adv. Funct. Mater. 2009, 19, 1689−1696. (55) Liu, Z.; Tang, S.; Xu, Z.; Wang, Y.; Zhu, X.; Li, L.-c.; Hong, W.; Wang, X. Preparation and In Vitro Evaluation of a Multifunctional Iron Silicate@Liposome Nanohybrid for Ph-Sensitive Doxorubicin Delivery and Photoacoustic Imaging. J. Nanomater. 2015, 2015, 1−13. (56) Dalla Via, L.; Marciani Magno, S. Photochemotherapy in the Treatment of Cancer. Curr. Med. Chem. 2001, 8, 1405−1418. (57) Lee, S.-M.; Park, H.; Yoo, K.-H. Synergistic Cancer Therapeutic Effects of Locally Delivered Drug and Heat Using Multifunctional Nanoparticles. Adv. Mater. 2010, 22, 4049−4053. (58) Tacar, O.; Sriamornsak, P.; Dass, C. R. Doxorubicin: an Update on Anticancer Molecular Action, Toxicity And Novel Drug Delivery Systems. J. Pharm. Pharmacol. 2013, 65, 157−170. (59) You, J.; Zhang, R.; Zhang, G.; Zhong, M.; Liu, Y.; Van Pelt, C. S.; Liang, D.; Wei, W.; Sood, A. K.; Li, C. PhotothermalChemotherapy with Doxorubicin-Loaded Hollow Gold Nanospheres: a Platform for Near-Infrared Light-Trigged Drug Release. J. Controlled Release 2012, 158, 319−328. (60) Fomina, N.; Sankaranarayanan, J.; Almutairi, A. Photochemical Mechanisms of Light-Triggered Release from Nanocarriers. Adv. Drug Delivery Rev. 2012, 64, 1005−1020. (61) Chen, Y.-S.; Frey, W.; Kim, S.; Kruizinga, P.; Homan, K.; Emelianov, S. Silica-Coated Gold Nanorods as Photoacoustic Signal Nanoamplifiers. Nano Lett. 2011, 11, 348−354.

(26) Seo, J.-W.; Hwang, J.-Y.; Shin, U. S. Ionic Liquid-Doped and PNipaam-Based Copolymer (P-Nibim): Extraordinary Drug-Entrapping and -Releasing Behaviors at 38−42 [degree]C. RSC Adv. 2014, 4, 26738−26747. (27) Bi, N.; Hu, M.; Zhu, H.; Qi, H.; Tian, Y.; Zhang, H. Determination of 6-thioguanine Based on Localized Surface Plasmon Resonance of Gold Nanoparticle. Spectrochim. Acta, Part A 2013, 107, 24−30. (28) You, J.; Zhang, R.; Xiong, C.; Zhong, M.; Melancon, M.; Gupta, S.; Nick, A. M.; Sood, A. K.; Li, C. Effective Photothermal Chemotherapy Using Doxorubicin-Loaded Gold Nanospheres That Target EphB4 Receptors in Tumors. Cancer Res. 2012, 72, 4777− 4786. (29) Sharma, P.; Brown, S.; Walter, G.; Santra, S.; Moudgil, B. Nanoparticles for Bioimaging. Adv. Colloid Interface Sci. 2006, 123− 126, 471−485. (30) El-Brolossy, T. A.; Abdallah, T.; Mohamed, M. B.; Abdallah, S.; Easawi, K.; Negm, S.; Talaat, H. Shape And Size Dependence of The Surface Plasmon Resonance of Gold Nanoparticles Studied by Photoacoustic Technique. Eur. Phys. J.: Spec. Top. 2008, 153, 361−364. (31) Mody, V. V.; Siwale, R.; Singh, A.; Mody, H. R. Introduction to Metallic Nanoparticles. J. Pharm. BioAllied Sci. 2010, 2, 282−289. (32) Popovtzer, R.; Agrawal, A.; Kotov, N. A.; Popovtzer, A.; Balter, J.; Carey, T. E.; Kopelman, R. Targeted Gold Nanoparticles enable Molecular CT Imaging of Cancer. Nano Lett. 2008, 8, 4593−4596. (33) Huang, P.; Bao, L.; Zhang, C.; Lin, J.; Luo, T.; Yang, D.; He, M.; Li, Z.; Gao, G.; Gao, B.; Fu, S.; Cui, D. Folic Acid-Conjugated SilicaModified Gold Nanorods for X-ray/CT Imaging-Guided Dual-Mode Radiation and Photo-Thermal Therapy. Biomaterials 2011, 32, 9796− 9809. (34) Maeng, J. H.; Lee, D.-H.; Jung, K. H.; Bae, Y.-H.; Park, I.-S.; Jeong, S.; Jeon, Y.-S.; Shim, C.-K.; Kim, W.; Kim, J.; Lee, J.; Lee, Y.-M.; Kim, J.-H.; Kim, W.-H.; Hong, S.-S. Multifunctional Doxorubicin Loaded Superparamagnetic Iron Oxide Nanoparticles for Chemotherapy and Magnetic Resonance Imaging in Liver Cancer. Biomaterials 2010, 31, 4995−5006. (35) Singh, R. K.; Patel, K. D.; Kim, J.-J.; Kim, T.-H.; Kim, J.-H.; Shin, U. S.; Lee, E.-J.; Knowles, J. C.; Kim, H.-W. Multifunctional Hybrid Nanocarrier: Magnetic CNTs Ensheathed with Mesoporous Silica for Drug Delivery and Imaging System. ACS Appl. Mater. Interfaces 2014, 6, 2201−2208. (36) Sperling, R. A.; Parak, W. J. Surface Modification, Functionalization and Bioconjugation of Colloidal Inorganic Nanoparticles. Philos. Trans. R. Soc., A 2010, 368, 1333−1383. (37) Mahapatro, A.; Singh, D. K. Biodegradable Nanoparticles are Excellent Vehicle for Site Directed In-Vivo Delivery of Drugs and Vaccines. J. Nanobiotechnol. 2011, 9, 55−55. (38) Li, H.; Tan, L.-L.; Jia, P.; Li, Q.-L.; Sun, Y.-L.; Zhang, J.; Ning, Y.-Q.; Yu, J.; Yang, Y.-W. Near-Infrared Light-Responsive Supramolecular Nanovalve Based on Mesoporous Silica-Coated Gold Nanorods. Chem. Sci. 2014, 5, 2804−2808. (39) Vigderman, L.; Khanal, B. P.; Zubarev, E. R. Functional Gold Nanorods: Synthesis, Self-Assembly, and Sensing Applications. Adv. Mater. 2012, 24, 4811−4841. (40) Jiang, R.; Li, B.; Fang, C.; Wang, J. Metal/Semiconductor Hybrid Nanostructures for Plasmon-Enhanced Applications. Adv. Mater. 2014, 26, 5274−5309. (41) Heo, C.-J.; Kim, S.-H.; Jang, S. G.; Lee, S. Y.; Yang, S.-M. Gold “Nanograils” with Tunable Dipolar Multiple Plasmon Resonances. Adv. Mater. 2009, 21, 1726−1731. (42) Kuo, W.-S.; Chang, C.-N.; Chang, Y.-T.; Yang, M.-H.; Chien, Y.H.; Chen, S.-J.; Yeh, C.-S. Gold Nanorods in Photodynamic Therapy, as Hyperthermia Agents, and in Near-Infrared Optical Imaging. Angew. Chem., Int. Ed. 2010, 49, 2711−2715. (43) Johnston, A. P. R.; Such, G. K.; Caruso, F. Triggering Release of Encapsulated Cargo. Angew. Chem., Int. Ed. 2010, 49, 2664−2666. (44) Shen, J.; He, Q.; Gao, Y.; Shi, J.; Li, Y. Mesoporous Silica Nanoparticles Loading Doxorubicin Reverse Multidrug Resistance: Performance and Mechanism. Nanoscale 2011, 3, 4314−4322. 8979


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