Gadolinium(III)-Chelated Silica Nanospheres Integrating

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Gadolinium (III)-Chelated Silica Nanospheres Integrating Chemo- and Photothermal Therapy for Cancer Treatment and Magnetic Resonance Imaging Mingjing Cao, Pengyang Wang, Yu Kou, Jing Wang, Jing Liu, Yan-Hui Li, Jiayang Li, Liming Wang, and Chunying Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 29 Sep 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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ACS Applied Materials & Interfaces

Gadolinium (III)-Chelated Silica Nanospheres Integrating Chemo- and Photothermal Therapy for Cancer Treatment and Magnetic Resonance Imaging Mingjing Cao,a,b,§ Pengyang Wang,a,§ Yu Kou,a, c Jing Wang,a Jing Liu,a Yanhui Li,c Jiayang Li,a Liming Wanga,* and Chunying Chena,* a

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China and Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100190, P.R. China b Sino-Danish Center for Education and Research, Beijing, 100190, P.R. China c

Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266003, P.R. China ＊

Corresponding author: [email protected], [email protected].

§

These authors contributed equally.

Keywords: Photothermal therapy; Magnetic resonance imaging; Chemotherapy; Multimodal therapy; Drug delivery; Mesoporous silica nanoparticles; Gadolinium(III)-chelated silica nanospheres ABSTRACT The combination of therapy and diagnosis has been emerging as a promising strategy for cancer treatment. To realize chemotherapy, photothermal therapy, and magnetic resonance imaging (MRI) in one system, we have synthesized a new magnetic nanoparticle (Gd@SiO2-DOX/ICG-PDC) integrating doxorubicin (DOX), indocyanine green (ICG), and gadolinium (III)-chelated silica nanospheres (Gd@SiO2) with a Poly (diallyldimethylammonium chloride) (PDC) coating. PDC coating serves as a polymer layer to protect from quick release of drugs from the nanocarriers and increase cellular uptake. The DOX release from Gd@SiO2-DOX/ICG-PDC depends on pH and temperature. The process will be accelerated in the acidic condition than in a neutral pH 7.4. Meanwhile, upon laser irradiation, the photothermal effects promote DOX release and improve the therapeutic efficacy compared to either DOX-loaded Gd@SiO2 or ICG-loaded Gd@SiO2. Moreover, MRI results show that the Gd@SiO2-PDC nanoparticles are safe T1-type MRI contrast agents for imaging. The Gd@SiO2-PDC nanoparticles loaded with DOX and ICG can thus act as a promising theranostic platform for multimodal cancer treatment.

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INTRODUCTION Theranostics integrate diagnostic and therapeutic functions into one platform that has become a more precise mode for disease therapy and will achieve more powerful efficacy than conventional therapy.1 Nanocarriers possess unique physicochemical features such as large surface area for molecule adsorption, controllable shape and size, and tunable physical properties like optical, electronic, magnetic, ultrasound, and thermal effects.2 Nanocarriers can thus provide a structural platform to load both imaging agents and drugs in order to realize diagnostic and therapeutic functions simultaneously.2-4 Versatile nanoparticles (NPs) like gold, iron oxide, and polymeric NPs 5-7 are appropriate to deliver chemotherapeutic drugs and multimodal imaging agents in cancer therapy by achieving controllable cancer therapy with visible tumor details via MRI, CT, and photoacoustic imaging.8-11 Chemotherapy has some disadvantages including the internal toxicity of drugs, the damage in immune system, and the development of drug resistance.12,13 Comparatively, laser irradiation-assisted photothermal therapy (PTT) or photodynamic therapy (PDT) are non-invasive mode that can generate heats or free radicals at tumor site.14-16 Photothermal therapy agents are capable of converting light energy into heat that causes a rise in the local temperature beyond 42 °C that consequently kill cancer cells.17,18,19 As a FDA-approved drug, indocyanine green (ICG) is attractive for localized hyperthermia that absorbs near infrared light (700–1000 nm) even in deep tissue20,21 to generate heat.22,23 The multimodal therapies probably produce synergistic effects that eradicate residual tumor cells and prevent cancer recurrence.24,25 For example, hyperthermia makes cancer cells more sensitive to radiation and anticancer drugs like doxorubicin (DOX).15,26 Recent studies about the incorporation of photothermal therapy and chemotherapy into one single nanoplatform have been proved to be a great success in cancer treatment.1,9,27 By loading drugs into some light-responsive nanocarriers, the combination of physical therapy with chemotherapy will be a precise and local therapy mode. Upon light irradiation, this mode will realize controlled drug release and attain synergetic effects to improve the therapeutic efficacy and decrease the negative effects. MRI is a non-invasive modality for high resolution, precise, and threedimensional imaging in clinical diagnosis.28 The commercial MRI contrast agents include iron oxide particles (T2 agents) and gadolinium (III)-chelated complexes (T1 agents).29 Gadolinium possesses seven unpaired electrons that may alter the relaxation time of the surrounding water protons. As paramagnetic contrast agents, Gd (III) compounds are thus attractive to enhance the imaging sensitiveness and quality.30 Recently, the gadolinium-based T1 agents have achieved great progress in the development and applications, however, the toxicity of Gd (III) ions limits their use in biomedical fields.31 To resolve the problem, diethylenetriaminepentaacetic acid


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(DPTA) is used to stabilize Gd (III),32 which afford relatively inert and biocompatible clinical contrast agent of Gd-DTPA.33 To decrease the cytotoxicity and enhance the MRI properties, a highly conserved Gd NPs with silica shell encapsulation has been developed.34 Compared to Gd compounds, Gd-containing nanoparticles have more potential to be passively accumulated in tumor due to the enhanced permeability and retention effect (EPR) of NPs in cancer, and may achieve a longer circulation time, which would benefit MRI.35,36 It becomes necessary to modify Gd compounds, to chelate dissolved Gd3+ ions, or to encapsulate them in nanostructures for safe and high quality imaging. Herein, we design citric acid-stabilized mesoporous silica nanospheres containing Gd (III) complexes (Gd@SiO2) as a NIR laser-mediated multifunctional theranostic platform. The nanoparticles are expected to act as a multifunctional nanocarrier to achieve the goals for drug delivery, hyperthermia, and imaging for cancer therapy (Scheme 1). With respect to the integrated nanoparticles (Gd@SiO2-DOX/ICG-PDC), the core is composed of Gd compounds as MRI contrast agent and the mesoporous silica layer is used to load chemotherapeutic drugs (DOX) and photothermal chemicals (ICG). The positively charged poly (diallyldimethylammonium chloride) (PDC) serves as the surface coating to prevent the drug release and to improve cellular uptake. Transmission electron microscope and UV-vis-NIR spectra were used to characterize the shape and the stability of loaded drugs in the NPs. Then, X-ray absorption near edge structure (XANES) was used to study the chelated form of Gd within the NPs, while soft X-ray Scanning Transmission X-ray Microscope (STXM) images were employed to study locations of Gd element in a single cell. Furthermore, the release profile of DOX and Gd ions was measured with and without laser irradiation. Cell viability and live or dead cell ratio were obtained to evaluate the photothermal effects upon laser irradiation. Finally, MRI images were performed to show the MRI signal intensity for both Gd@SiO2-DOX/ICG-PDC NPs and NPs-internalized cells.



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Scheme 1. Schematic illustration for the synthesis of Gd@SiO2-PDC and Gd@SiO2-DOX/ICG-PDC. EXPERIMENTAL SECTION Materials. All reagents were used as received without further purification. Gadolinium (III) chloride (GdCl3·6H2O, 99%) was obtained from Aike, China. Gadolinium oxide (Gd2O3, 99%) and citric acid (C6H8O7, ≥99.5%) were purchased from Aladdin. Trisodium citrate (99%), ammonium hydroxide (NH4OH, 28-30 wt% NH3 in water), concentrated nitric acid (70%, metal-oxide-semiconductor grade), hydrogen peroxide (30%), hydrochloric acid (37%), ethanol (95%), sodium hydroxide, sodium chloride and methanol were purchased from Beijing Chemical Reagent Institute, China. Tetraethyl orthosilicate (TEOS, 98%) was obtained from Alfa Aesar, United States. Indocyanine green (ICG) and doxorubicin hydrochloride (DOX) were respectively obtained from Suzhou Bec Bio-Technique Co. Ltd and Beijing Huafeng United Technology Co. Ltd. Poly (diallyldimethylammonium chloride) solution (PDC, MW, 100,000-200,000, 20 wt% in H2O) and gadolinium hydroxide (Gd(OH)3, 99%) were commercially available in Sigma-Aldrich, United States. Phosphotungstic acid hydrate 44-hydrate (P2O5·24WO3·44H2O) was obtained from J&K Chemical, China. Cell Counting Kit-8 was purchased from Dojindo Laboratories in Japan. 1640 medium, trypsin and fetal bovine serum (FBS) were bought from Wisent, China. Ultrapure deionized water (D.I. H2O) with resistivity of 18.2 MΩ was generated using a Millipore Milli-Q plus system. Instruments. Morphology was visualized by a high-resolution transmission electron


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microscope (FEI, Tecnai G2-F20 U-TWIN). Hydrodynamic diameter and Zeta-potential measurements were obtained using a Malvern Zeta Sizer Nano ZS instrument (Malvern Instruments, United Kingdom) with a 1cm wide quartz cell in water solution at room temperature. The accurate Gd content in each sample was detected by an Elan DRC Ⅱ inductively coupled plasma mass spectrometer (ICP-MS, Perkin-Elmer, USA). A continuous wave laser (808 nm, 5 mm) was produced with GCSLS-05-7W00 fiber-coupled diode laser system (Daheng Science & Technology, China). The temperature was measured by a Flir E40 compact infrared (IR) thermal imaging camera (FLIR Systems, U.S.A.). UV-vis-NIR spectra were obtained using an Infinite M200 microplate reader (Tecan, Durham, USA). All magnetic resonance images were acquired at Center for Biomedical Imaging Research, Tsinghua University with a 3T scanner (3.0 T TX, Philips Achieva, Holland). The chromatogram and mass spectra of DOX and ICG were measured by liquid chromatography-mass spectroscopy (LCMS-8040, SHIMADZU, Japan). The Gd elemental distribution (Gd@SiO2-PDC) in MCF-7 cells was detected using Soft-X-ray STXM on beamline 08U in Shanghai Synchrotron Radiation Facility (SSRF) to detect. X-ray absorption near edge structure was obtained by using XAFS on beamline BL-14W1 in SSRF. Preparation of Gd@SiO2 and Gd@SiO2-PDC. The Gd@SiO2 NPs were synthesized by the previously reported Stöber method.34 Briefly, a Gd stock solution (0.17 M), which affords well dispersed nano-spherical core of Gd (III), was prepared by mixing 100 μL trisodium citrate (1 M), 50 μL GdCl3·6H2O aqueous solution (1 M), and 150 μL NH4OH (1.5 M) thoroughly. Subsequently, 100 μL the Gd stock solution was diluted into 7.5 mL D.I. H2O, and 50 μL TEOS was dissolved into 17.5 mL ethanol. The two solutions were mixed together and transferred into a round bottom flask. The above-mentioned mixture was then stirred for 5 min at 40 oC, followed by adding 700 μL aqueous ammonia solution (25 wt %). After another 24 h stirring at 40 o C, the core-shell colloid Gd@SiO2 were obtained by the purifications with methanol (4×20 mL) and D.I. H2O (1×20mL), and then the isolation by centrifugation. The obtained product was re-dispersed in 2.5 mL D.I. H2O for future use (7.7 mg/mL). In order to synthesize Gd@SiO2-PDC, 1.7 mL PDC aqueous solution (12.0 mg/mL) was first mixed with 300 μL of the above Gd@SiO2 aqueous solution. Subsequently, 40 μL aqueous NaCl (50 mM) was added to the mixture and stirred for 1.5 h, which was followed by centrifugation (9500 rpm, 5 min) and purification by D.I. H2O twice. Preparation of Drug-Loaded Gd@SiO2-PDC NPs. 100 μL DOX solution (1.0 mg/mL) mixed with 500 μL D.I. H2O was added dropwise to 300 μL the aqueous dispersion of Gd@SiO2 (7.7 mg/mL), which was followed by the addition of 100 μL



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ICG (1.0 mg/mL) after stirring for 5 min. The resulting solution was stirred overnight at room temperature to form Gd@SiO2-DOX/ICG. The product was centrifuged (9500 rpm, 5min) and dispersed into 300 μL D.I. H2O. Then 1.7 mL PDC (12.0 mg/mL) was added into the above aqueous solution, subsequent with the addition of 40 μL NaCl (50 mM). After stirring for 1.5 h, the Gd@SiO2-DOX/ICG-PDC NPs were obtained by centrifugation. Similarly, 100 μL 1.0 mg/mL DOX solution was added dropwise into 600 μL D.I. H2O, followed by mixing with 300 μL the Gd@SiO2 aqueous solution and stirring overnight to afford the Gd@SiO2-DOX. To prepare Gd@SiO2-ICG NPs, the Gd@SiO2-PDC NPs were firstly synthesized and re-dissolved in 300 μL D.I. H2O. Then 100 μL ICG solution (1.0 mg/mL) was added dropwise into 600 μL D.I. H2O and the solution was mixed with 300 μL Gd@SiO2-PDC for 1.5 h stirring at room temperature to form Gd@SiO2-PDC-ICG. After centrifugation with washing by D.I. H2O and re-dispersed into 300 μL D.I. H2O, 1.7 mL PDC aqueous solution (12.0 mg/mL) and 40 μL aqueous NaCl (50 mM) was added in order to prepare Gd@SiO2-PDC-ICG-PDC. All the dispersions were centrifuged with the speed of 9500 rpm for 5 min to collect nano-composites of Gd@SiO2-DOX/ICG-PDC, Gd@SiO2-DOX, or Gd@SiO2-PDC-ICG-PDC, respectively, followed by the re-dispersion of each product into D.I. H2O for future use. The drug content of the NPs was calculated by determining the concentrations of DOX or ICG in the above supernatants with UV-vis spectrophotometer at 480 nm or 780 nm. The drug encapsulation efficiency (EE) and drug loading efficiency (LE) of DOX or ICG in each kind of NPs was determined as follows: EE (%)=(mass of drug (ICG or DOX) in NPs)/(mass of initially added drug)×100 LE (%)=(mass of drug (ICG or DOX) in NPs)/(total weight of NPs)×100 Drug Loading for Gd@SiO2-DOX/ICG. 100 μL DOX solution (1.0 mg/mL) mixed with 500 μL D.I. H2O was added dropwise to 300 μL the aqueous dispersion of Gd@SiO2 (7.7 mg/mL), which was followed by the addition of 100 μL ICG (1.0 mg/mL) after stirring for 5 min to form sample 1. 100 μL DOX solution (2.0 mg/mL) mixed with 500 μL D.I. H2O was added dropwise to 300 μL the aqueous dispersion of Gd@SiO2 (7.7 mg/mL), which was followed by the addition of 100 μL ICG (2.0 mg/mL) after stirring for 5 min to afford Sample 2. Then Sample 3 (3.0 mg/mL DOX and 3.0 mg/mL ICG) and Sample 4 (4.0 mg/mL DOX and 4.0 mg/mL ICG) were synthesized in the same way. Laser-Controlled Drug Release. In the in vitro drug release experiment, 500 μL Gd@SiO2-DOX/ICG-PDC solutions (2.8 mg/mL Gd@SiO2-PDC) that contained 70 μg/mL DOX and 175 μg/mL ICG were prepared in different buffers (pH=5.0 and 7.4).


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At different intervals (0, 0.25, 0.5, 1, 2, 4, 6, 15, 24, 37, and 48 h), the solutions were treated without or with NIR laser irradiation for 5 min at 808 nm, then the mixtures were incubated in a water bath at 37 oC with continuous shaking. After centrifuged for 3 min, the supernatant after each interval was collected and replaced with an equal volume of fresh medium. The content of DOX in the supernatant was then determined by measuring the absorption at 480 nm using a UV-vis spectrophotometer. Meanwhile, the release profiles of DOX in different buffers supplemented with 10% FBS were tested with the same method. Temperature Rise Induced by Laser Irradiation. The solutions of Gd@SiO2-DOX/ICG-PDC (500 μL) containing various concentrations of ICG (1.0, 4.0, 6.0, 8.0, 10.0, and 20.0 μg/mL) were prepared by stepwise dilutions from the Gd@SiO2-DOX/ICG-PDC stock solution (0.4 mg/mL DOX, 1.0 mg/mL ICG, 15.8 mg/mL Gd@SiO2-PDC) in transparent plastic eppendorf tubes and then incubated in a water bath at 37 oC for 30 min before laser irradiation. All solutions were irradiated at the excitation wavelength of 808 nm (1.0 W/cm2) for 3 min with PBS buffer as a control. Meanwhile, temperatures were measured at 0, 15, 30, 45, 60, 90, 120, 150, and 180 s by an infrared thermal imaging. Four different laser irradiation powers (0.6, 1.0, 1.3, and 1.5 W/cm2) were used to irradiate 500 μL Gd@SiO2-DOX/ICG with concentration of ICG at 4.0 μg/mL for 3 min. Similarly, the Gd@SiO2-DOX/ICG-PDC solutions were incubated at 37oC for 30 min in the transparent plastic eppendorf tubes before laser irradiation. Temperatures were measured at 0, 15, 30, 45, 60, 90, 120, 150, and 180 s by an infrared thermal imaging. Influence of Laser Irradiation on Therapeutic Efficiency of DOX. The influence of laser irradiation on the therapeutic efficiency of DOX was determined by HPLC-MS. All the assays and identifications were performed on LCMS-8040 (Shimadzu, Kyoto, Japan). The instrument is equipped with one grade lowered components such as LC-20AD pump, DGU-20A3R degasser, SIL-20A autosampler and CTO-20AC column oven, coupled to a quadrupled type tandem mass spectrometer (LCMS-8040) via also an ESI interface. The LCMS-8040 system is controlled by LabSolutions LCMS Ver.5.6 (Shimadzu, Kyoto, Japan). Five solutions in D.I. water tested here were DOX (100 μM), ICG (100 μM), the mixture of DOX (100 μM) and ICG (100 μM), ICG (100 μM) irradiated with NIR laser and the mixture of DOX (100 μM) and ICG (100 μM) irradiated with NIR laser. In addition, several dilutions of DOX (0.5, 1, 5, 10, 15, and 25 μM) were prepared to obtain standard curve of DOX. All the laser-treated samples were irradiated by 808 nm laser at 0.6 W/cm2 for 5 min. A sample (1 μL) was injected into a reversed-phase packed



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column (C18, 50 mm×1.0 mm I.D., 3.5 μm, Waters) installed for LCMS-8040. It was eluted at a column temperature of 40 °C and flow rate of 0.3 mL/min, with a binary solvents of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at programed gradient from 95:5 A:B (v/v) to 0:100 A:B (v/v) over 7 min, and the chromatogram was detected via scan mode, while the quantification of DOX was completed via SIM mode. Positive ion species were detected by MS via MRM mode. The MS parameters for LCMS-8040 were set as follows: nebulizing gas flow at 3 L/min, drying gas flow at 15 L/min, DL temperature at 300 °C, heat block temperature at 450 °C and CID gas at 230 kPa. Study in Gadolinium Ions Release from Gd@SiO2-DOX/ICG-PDC. 500 μL Gd@SiO2-DOX/ICG-PDC solutions (0.063 mg/mL Gd@SiO2-PDC) that contained 1.5 μg/mL DOX and 4.0 μg/mL ICG were prepared in different buffers (pH=5.0 and 7.4). The solutions were treated with or without 1.0 W/cm2 NIR laser irradiation for 6 min at 808 nm, then the mixtures were incubated in a water bath at 37 oC with continuous shaking. At different intervals (0.5, 2, 24, 72, 120, 240, and 360 h), the supernatants were collected and replaced with an equal volume of fresh medium after the solutions were centrifuged for 5 min. The content of gadolinium ions in the supernatant was measured by ICP-MS. Meanwhile, the release profiles of gadolinium in different buffers supplemented with 10% FBS were measured with the same method. Characterization of Gadolinium Chemical Forms. Gadolinium-based compounds used as references are gadolinium citrate, GdCl3, Gd2O3, and Gd(OH)3. Before XANES measurement, all the samples (Gd@SiO2, Gd@SiO2-DOX/ICG-PDC, and Gd cores) were dried and pressed to be a uniform pellet adhering to a tape (3M). XANES spectra of Gadolinium L3-edge were mainly recorded on beamline BL-14W1 at SSRF in China. The transmission XANES mode was used to measure XANES spectra for references including Gd citrate, GdCl3, Gd2O3, and Gd(OH)3. Fluorescence XANES mode equipped with a Lytle detector was employed to collect the data for NPs and the Gd cores containing Gd. Data treatment and analysis based on our previous publications.37,38 In brief, XANES data were normalized in order to facilitate comparison of spectra from the varied samples, modes, or facilities. The preprocessed data were then analyzed with least-squares fitting (LSF) to calculate the ratio of gadolinium species by using IFEFFIT Athena software (CARS, the Consortium for Advanced Radiation Sources at University of Chicago). Magnetic Resonance Imaging Assays. Solutions of Gd@SiO2-DOX/ICG-PDC with various Gd@SiO2-PDC concentrations (0, 0.17, 0.33, 0.50, 0.80, and 1.00 mg/mL)


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were obtained by stepwise dilutions at room temperature and were placed in the transparent plastic eppendorf tubes. The corresponding Gd (III) concentrations were 0, 0.019, 0.038, 0.057, 0.092, and 0.115 mM, respectively. Subsequently, the T1 and T2 weighted MR imaging of Gd@SiO2-DOX/ICG-PDC solutions with gradient concentrations was taken. The corresponding relaxivity (r1 and r2) was then calculated by plotting 1/longitudinal or transverse relaxation time (Ti, i=1, 2) versus Gd concentrations. In addition, the magnetic resonance imaging assays for in vitro cells were performed with Gd@SiO2-PDC and Gd@SiO2-DOX/ICG-PDC. Michigan Cancer Foundation-7 (MCF-7) cells were seeded into a 10 cm culture dish with complete 1640 medium supplemented with 10% (v/v) FBS for 24 h, then the medium was replaced with the complete media, respectively, containing with Gd@SiO2-PDC and Gd@SiO2-DOX /ICG-PDC (0.02 mM Gd3+) for 24h. Afterwards, cells were gently washed three times with PBS, and digested with 0.25% trypsin containing 0.02% EDTA. The cells were then collected in a 0.5 mL eppendorf tube, followed by the centrifugation for 5 min at 1500 rpm, then the cells pellets at the bottom of the centrifuge tubes were used for MR imaging. Cytotoxicity Test. Cell viability was determined by a Cell Counting Kit-8. Firstly, 3×03 (cells/well) of MCF-7 cells were seeded into 96-well plates in 100 μL complete 1640 medium supplemented with 10% (v/v) FBS. After incubating (37 oC, 5% CO2 and 10% humidity) for 24 h, the culture medium was replaced with another 200 μL fresh complete media that contained serial diluents of Gd@SiO2-PDC, Gd@SiO2-DOX/ICG-PDC, Gd@SiO2-DOX, and Gd@SiO2-PDC-ICG-PDC, respectively. Except for Gd@SiO2-PDC, the concentrations of compounds were set according to their concentrations of DOX or ICG. Therefore, to provide gradient concentrations of DOX (0.5, 1, 2, 4, and 6 μg/mL) or ICG (1.25, 2.5, 5, 10, and 15 μg/mL), serial diluents of Gd@SiO2-DOX/ICG-PDC with Gd@SiO2-PDC concentration of 0.021, 0.042, 0.084, 0.168, and 0.252 mg/mL was prepared from the stock solution of Gd@SiO2-DOX/ICG-PDC (0.4 mg/mL DOX, 1.0 mg/mL ICG, 15.8 mg/mL Gd@SiO2-PDC). With untreated MCF-7 cells as control, we measured the cytotoxicity of various concentrations of NPs in triplicate. After 24 h incubation (37 o C, 5% CO2, and 10% humidity), a mixture of the tetrazolium reagent (from the Cell Counting Kit-8) and the complete medium (1:10) was added into each well. Finally, cell viability was calculated as the absorbance ratio between test and control wells. The absorbance at 450 nm was measured and referenced with that at 600 nm by Infinite M200 microplate reader. Cellular Uptake of Gd-Containing NPs. MCF-7 cells were seeded into 6-well plates



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with complete 1640 medium at a density of 2×105 cells/well for 24 h, then the medium was replaced respectively with fresh complete media containing Gd@SiO2 (0.063 mg/mL) and Gd@SiO2-PDC (0.063 mg/mL). The cells were incubated for different time intervals including 3 h, 6 h, 12 h, and 24 h, with four wells of cells at each time point. Cells were gently washed for three times with PBS, and digested with 0.25% trypsin containing 0.02% EDTA. After centrifuging for 5 min at 1,500 rpm, the cells were then collected and counted.In order to quantify the cellular uptake of our samples, ICP-MS was employed to detect the Gd content in each sample. The internalized was expressed as Gd content (ng) within 104 cells. STXM Imaging for Intracellular Gd Element. MCF-7 cells were seeded on Si3N4 film overnight and treated with 0.063 mg/mL Gd@SiO2-PDC NPs for 12 h. After twice rinse with PBS, cells were fixed and gradually dehydrated in 70% ethanol for 20 min, 85% ethanol for 15 min, 95% ethanol for 10 min, and 100% ethanol for 10 min. Soft-X-ray STXM on beamline 08U in SSRF was used to observe the distribution and accumulation of Gd inside a single cell. STXM images were captured at the absorption edge energy at 1187 eV and pre-edge one at 1181 eV, respectively. The difference or the contrast for two images represented the Gd distribution. In Vitro Chemo-Photothermal Treatment. In vitro Thermo-Chemotherapy treatment was performed with MCF-7 cell line. MCF-7 cells (2×105 cells per well) were seeded and cultured with 2 mL of complete 1640 medium in 6-well plates. After cultured for 24 h at 37 °C, 5% CO2 and 10% humidity, the culture medium was removed, and four cell groups were set to incubate with fresh complete media that contained Gd@SiO2-PDC-ICG-PDC (4.0 μg/mL ICG, 0.063 mg/mL Gd@SiO2-PDC), Gd@SiO2-DOX (1.5 μg/mL DOX), Gd@SiO2-DOX/ICG-PDC (4.0 μg/mL ICG, 1.5 μg/mL DOX, and 0.063 mg/mL Gd@SiO2-PDC), respectively. Divided into two sub-groups, each cell group was treated with or without an irradiation of an 808 nm laser with power density of 1.0 W/cm2 for 6 min per well, followed by another incubation for 24 h. Afterwards, cells were stained by LIVE-DEAD kits and observed under fluorescence microscope (10×) with live cells in green color and dead ones in red color, via which the number of live cells in each well was used to calculate cell viability. TEM Imaging. The morphologic and particle size examination of Gd complexes were performed by TEM with negative stain method. Before analysis, the samples were placed on copper grid with films and air-dried prior to dying. Then 40.0 mg of P2O5·24WO3·44H2O powder was dissolved in 960 μL D.I. H2O to prepare 4% (w/v) phosphotungstic acid. The samples were subsequently stained with phosphotungstic


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acid for 5 min, and then placed on copper grid with films and air-dried prior to imaging. The morphologic and particle size examination of Gd@SiO2, Gd@SiO2-PDC, and Gd@SiO2-DOX/ICG-PDC were prepared by placing the solution onto copper grid (10 µL, sufficient to cover the grid surface). After that the grid was placed to dry in air and examine the grid as soon as possible. Quantification of Gd by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). In order to quantify the cellular uptake of our samples and the content of Gd in NPs, ICP-MS was employed to detect the Gd content in each sample. The samples were pre-digested overnight with 5.0 mL of concentrated HNO3. Then, they were mixed with 3.0 mL of H2O2 (30%) and digested for 2 h in open vessels on a hot plate at 50-150 °C by gradually warming. When the residual volume decreased to about 1 mL, the remaining solution was cooled and diluted to 3.0 g on the balance with 2% HNO3 solution. For quantitative analysis, a series of Gd standard solutions (0.5, 1, 5, 10, and 50 ppb) were prepared with 2% HNO3 solution and tested to obtain the standard curve. Indium (20 ppb) in 2% HNO3 solution was used as an internal standard correction. Both the standard solutions and the samples were measured three times by ICP-MS. Triplicate samples were prepared for the experiment.

RESULTS AND DISCUSSION Synthesis of Gd-Containing Nanoparticles. To efficiently load DOX and ICG, we developed a core-shell structure of gadolinium-containing nanoparticles (NPs). As illuminated in Scheme 1, Gd (III)-containing nanospheroids (Gd cores) with the assist of chelation by citric acid are first prepared. A silica shell is subsequently developed onto the surface of Gd core by TEOS to prepare Gd@SiO2 NPs. With this silica shell as a protective barrier, the Gd (III) core would be protected in order to prevent the leakage of Gd3+ ions. In addition, the large special surface area of silica shell, providing plenty of nano-pores, would possibly allow easy transport of water across the shell and access to the Gd (III) in the core,34 which may contribute to enhanced T1-weighted contrast.39 Because the silanol groups on the surface will be deprotonated in D.I. H2O at neutral pH, the silica shell exhibits strong negative charges with Zeta potential of -40.3 mV. The property enables them to electrostatically adsorb some drugs with positive charges like DOX, which contributes to high drug loading efficacy of silica shell in Gd@SiO2 NPs. Furthermore, the adsorbed DOX in Gd@SiO2-DOX NPs affords a positively charged surface with a zeta potential of 17.1 mV, providing a proper environment to load ICG. The HPLC-MS results directly show the great loading of DOX and ICG in the Gd@SiO2 and Gd@SiO2-DOX/ICG. After the



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incorporation of ICG into Gd@SiO2-DOX/ICG NPs, the surface is coated by PDC which changes the surface charges from -11.9 mV to the positive 48.1 mV. Detailed information about the surface properties and the color during the preparation and durg loading is shown in Table S3 and Figure S4.

Figure 1. Characterization of Gd@SiO2 and Gd@SiO2-PDC NPs: (A) TEM image of Gd@SiO2-PDC NPs. (B) HAADF-STEM image and HAADF-STEM-EDS mappingbased on images of Gd@SiO2 core-shell structure. (C) Dynamic light scattering (DLS) measurements of the Gd@SiO2-PDC in water. (D) Elemental analysis for Gd@SiO2-PDC using TEM-equipped energy dispersive spectroscope analysis. Morphology of Gd-Containing Nanoparticles. Transmission electron microscopic (TEM) results show that Gd (III) cores exhibit uniform morphology and size with diameters around 40-60 nm (Figure S3). With respect to Gd@SiO2 NPs, they have a spherical shape and a well-defined core-shell structure with a ca. 30 nm thickness. For Gd@SiO2-PDC NPs, the core-shell structure was well remained (Figure 1A). After loading with DOX and ICG, the Gd@SiO2-PDC /ICG NPs show good dispersity (Figure S2). The average diameter of Gd@SiO2-PDC NPs was ca.120 nm, in which the thickness of PDC was around 20 nm, while their hydrodynamic diameter was


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214.8 nm with a polydispersity of 0.121 (Figure 1A, C, D). Because the size of Gd-containing NPs was around 100 nm, the EPR effect in tumor tissue would assist this nanoplatform to achieve tumor delivery.35,36,40 The high-angle annular dark-field scanning TEM (HAADF-STEM-EDS) was employed to iamge the elemental composition and distribution in Gd@SiO2 NPs (Figure 1B). Elemental mapping showed a core-shell structure in which a Gd core (green color) is concentrated in the center and a silica shell (yellow color) is outside. The relative content of multiple elements (e.g. Si, C, O, and Gd) within Gd@SiO2-PDC NPs was measured by energy dispersive X-ray spectroscopy (EDS, Figure 1D). The result indicated that the most abundant elements are O (30.38 wt %) and Si (28.16wt %), while the Gd content is 14.26 wt % (Table S1).

Gd@SiO2-DOX/ICG-PDC Gd citrate Fitting GdCl3 Gd@SiO 2

Fitting Gd core Fitting

Gd(OH)3 Gd2O3

7220 7230 7240 7250 7260 7270 Energy (eV)

B

Gd@SiO2-DOX/ICG-PDC

Derivative of XANES

A Gd L3-edge XANES

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7230

Gd@SiO2 Gd core Gd citrate GdCl3 Gd(OH)3 Gd2O3

7240

7250 7260 Energy (eV)

7270

Figure 2. Chemical species of gadolinium determined by Gd L3-edge XANES. (A) Chemical species of gadolinium in four reference samples (Gd citrate, GdCl3, Gd2O3, Gd(OH)3), the NPs containing Gd and Gd cores. The solid lines show the spectra of samples and the dashed lines represent their corresponding fitted results. (B) Structural fingerprints shown as inflection point energies (IPE) of the first derivative of normalized Gd L3-edge XANES. The IPE appears at the first peak of the spectra, which shows distinct characteristics in the IPEs of Gd(OH)3, Gd2O3, GdCl3, and Gd citrate. Characterization of the Form of Gd Present in the Nanoparticles. To understand the gadolinium chemical forms or coordination information for Gd atoms in the nanoparticles and the Gd cores, we used XANES to determine the ratio of Gd in the samples (Gd@SiO2, Gd@SiO2-DOX/ICG-PDC, and Gd cores) with different coordination information. Figure 2A showed the XANES results of references (Gd



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citrate, GdCl3, Gd2O3, and Gd(OH)3) and the samples. Each reference has its characteristic inflection point energies (IPE) according to the first derivative of normalized Gd L3-edge XANES (Figure 2B). GdCl3 and Gd citrate have two distinct IPEs and the test samples have partial similarity in the derivative spectra, which suggested a Gd composition in both GdCl3 and Gd citrate forms. The least squares fitting results of XANES showed that all of the NPs are composed of GdCl3 and Gd citrate (Figure 2 A and Table 1). Therefore, we concluded that gadolinium presented with trivalent state and the core mainly contained Gd citrate (67.5%~71.9%) and a minority of Gd c GdCl3. Table 1. Gd species within the Gd cores and other nanoparticles based on gadolinium L3-edge XANES.

Ratio of gadolinium species in the samples Gd citrate

GdCl3

Gd2O3

Gd(OH)3

Gd core

67.5%

32.5%

0

0

Gd@SiO2

68.9%

31.1%

0

0

71.9%

28.1%

0

0

Gd@SiO2-DOX /ICG-PDC

The Rise in Temperature and Drug Release Induced by NIR Irradiation. DOX and ICG were loaded into Gd@SiO2 via electrostatic interactions, which afforded Gd@SiO2-DOX/ICG NPs with a controllable drug loading (Table S2). Based on the loading efficacy (LE) of DOX or ICG into Gd@SiO2-DOX/ICG, we found that the most loading content was greater than 14.08 wt% for DOX and 13.86 wt% for ICG (Table S2). Sample 1 meets the requirements of diagnosis and therapy simultaneously, in which the encapsulation efficiency (EE) was 82.01% for DOX and 75.85% for ICG, respectively. After the coating by positively charged PDC, the repulsive-force between DOX and PDC decreases the loading content of DOX. The EE of DOX and ICG within Gd@SiO2-DOX/ICG-PDC NPs were 23.56% and 63.16%, meanwhile, the LE was 2.36 wt% for DOX and 6.32 wt% for ICG. As shown in Figure 3A, the UV-vis-NIR spectrum of Gd@SiO2-DOX/ICG-PDC NPs exhibit the characteristic absorption bands for both DOX (480 nm) and ICG (720 and 780 nm) (Figure S8). The red shift of ICG absorption is probably due to π-π stacking between the benzene rings of DOX and ICG as well as the interaction between ICG and PDC.


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Figure 3. (A) UV-vis-NIR spectra of Gd@SiO2-PDC and Gd@SiO2-DOX/ICG-PDC. (B) Release profile of DOX from Gd@SiO2-DOX/ICG-PDC in aqueous solution with/without laser at pH 5.0/7.4 at 37 oC. (C) Temperature change of Gd@SiO2-DOX/ICG-PDC at various concentrations of ICG (1, 4, 8, 10, and 20 μg/mL) as the function of photo-irradiation time (1.0 W/cm2). (D) Temperature change of Gd@SiO2-DOX/ICG-PDC (4.0 μg/mL ICG) irradiated by NIR laser with different photo-irradiation power (0.6, 1.0, 1.3, and 1.5 W/cm2) as the function of photo-irradiation time. The temperature curve indicated that Gd@SiO2-DOX/ICG-PDC NPs are capable of adsorbing near infrared light to generate heat (Figure 3). After encapsulating into the NPs, The absorption peak of ICG remains in NIR region (Figure 3A), which permits the relatively high transmissivity into biological tissues for Gd@SiO2-DOX/ICG-PDC NPs and hence benefits their clinic applications. Irradiated by 808 nm NIR laser, ICG converts the energy from light to heat (Figure 3C, D), which at the appropriate concentration and irradiation time would induce irreversible damage to tumor cells. The infrared thermal imaging camera recorded that the maximum temperature (Tmax) of PBS and ICG and DOX containing NPs at various concentrations of ICG (1, 4, 6, 8, 10, and 20 μg/mL) after laser irradiation for 180 s reached 37.3 °C and 38.7, 50.0, 54.1, 57.5, 58.7, and 63.1 °C, respectively. A quick increase of temperature from 37.0 oC to 50.0 oC was detected for



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Gd@SiO2-DOX/ICG-PDC with 4.0 μg/mL of ICG upon 1.0 W/cm2 photo-irradiation for 3 min (Figure 3D). It indicates that NPs contained even relatively low concentration of ICG could trigger a temperature increase within a short irradiation time. The temperature rising rate and the maximum temperature was proportional to the concentration of ICG and irradiation power (Figure 3C and 3D). Under NIR irradiation, Tmax result for Gd@SiO2-DOX/ICG-PDC reached 42.9, 50.0, 54.6, 55.5, 58.9, and 59.9 °C under NIR irradiation with different powers (0.6, 1.0, 1.3, and 1.5 W/cm2) for 180 s, respectively. As a result, we set the irradiation power to be 1.0 W/cm2. In addition, in vitro release profile of DOX from Gd@SiO2-DOX/ICG-PDC had been studied under several conditions with different pHs of the releasing buffers and with/without laser irradiation. To confirm the drug release in physiological environments, two different pH values (pH 5.0 similar to the lysosome, pH 7.4 for the blood) were chosen to carry out drug release experiments. As shown in Figure 3B, the released DOX from the NPs at pH 5.0 significantly accelerates, which benefits the DOX release inside cells. Without NIR irradiation at pH 5.0, 30.3% DOX was released for 15 h and 33.2% for 37 h, while at pH 7.4 the amount of released DOX was 28.5% and 31.0% for 15 h and 37 h, respectively. The reason is that the surface of silanols tend to be protonated at an acid condition, resulting in the decrease of electrostatic interaction and dissociation of DOX from the silica surface. Photothermal outcome due to NIR irradiation could also enhance DOX release. After 5 min irradiation, we studied the release process of DOX from Gd@SiO2-DOX/ICG-PDC NPs at two pHs, which presents dramatic increase behavior. Upon NIR irradiation at pH 5.0, DOX release reached 80.1% for 15 h and 89.1% for 37 h, while at pH 7.4 DOX release from NPs was 70.4% for 15 h and 82.0% by 37 h. The reason is that the heat weakens the electrostatic interactions between NP surface and DOX to help the release of DOX. The DOX release in buffers supplemented with 10%FBS shows the similar profile as that in buffers (Figure S5). It suggested the DOX release from Gd@SiO2-DOX/ICG NPs is remotely controlled by NIR irradiation and pHs, which makes the application clinically attractive. The influence of laser irradiation on the therapeutic efficiency of DOX with the presence of ICG was determined by HPLC-MS. DOX (100 μM), ICG (100 μM), the mixture of DOX (100 μM) and ICG (100 μM), ICG (100 μM) irradiated with NIR laser and the mixture of DOX (100 μM) and ICG (100 μM) irradiated with NIR laser were measured and their chromatogram traces were shown in Figure S6A, B, C, D, and E. The peaks in HPLC chromatograms are labelled as No. 1, 2, 3, 4, and 5, among which peak 1 and 2 were DOX and ICG with retention time of 2.86 min and 5.96 min, respectively. From the positive ion mass spectra of peak 1 and 2 (Figure S6G and H), corresponding molecular ion ([M+1]+) peaks of DOX and ICG were identified, which


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were m/z=544.2 and 753.3. The peaks 3, 4, and 5 appeared in the chromatogram of the samples with irradiated ICG (Figure S6D, E) that represented the decomposition products of ICG. The content of DOX in the mixture of DOX (100 μM) and ICG (100 μM) irradiated could be figured out according to the standard curve of DOX (Figure S6F) obtained by scanning and integrating the area of molecular ions peak of the several dilutions of DOX. Compared with the primal content of DOX in the mixture of DOX (100 μM) and ICG (100 μM) without laser irradiation, 88.66% DOX remained after irradiation. We concluded that the therapeutic efficiency of DOX with the presence of ICG did not weaken after laser irradiation.

Figure 4. MR relaxivity measurements of Gd@SiO2-DOX/ICG-PDC NPs in aqueous. (A) T1 weighted MR images with various Gd concentrations (0, 0.019, 0.038, 0.057, 0.091, and 0.115 mM). (B) T2 weighted MR images with various Gd concentrations (0, 0.019, 0.038, 0.057, 0.091, and 0.115 mM). (C) Plot of 1/Ti (i=1, 2) versus Gd concentration for corresponding relaxivity calculation, for the linear plot of 1/T 1 versus concentration, R2=0.995, for the linear plot of 1/T2 versus concentration, R2=0.988. (D) T1-weighted MRI signal intensity (1/T1) of MCF-7 cells incubated with Gd@SiO2-PDC and Gd@SiO2-DOX/ICG-PDC for 24 h, with insert as the MRI of MCF-7 cells at the bottom of centrifuge tubes (tube 1: control, tube 2: Gd@SiO2-PDC, tube 3: Gd@SiO2-DOX/ICG-PDC). Magnetic Resonance Property of Gd-Containing Nanoparticles. MRI has been widely used in diagnostics, such as tumor detection and vascular imaging.41 The brightness of Gd@SiO2-DOX/ICG-PDC NPs in T1-weighted MR imageing is proportional to the concentration of Gd (III) (Figure 4A), while that in T2-weighted



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MR images almost remained the same with the variation of the concentration of Gd (III) (Figure 4B). From the linear correlation between relaxivity and Gd concentration shown in Figure 4 C, the longitudinal relaxivity (r1) and transverse relaxivity (r2) of Gd@SiO2-DOX/ICG-PDC were 8.5 mM−1s−1 and 15.9 mM−1s−1, respectively. The r1 value of Gd@SiO2-DOX/ICG-PDC was much greater than that of Gd-DTPA (r1=3.50 mM−1s−1),42 one of the commonly used commercial MRI T1-contrast agent.43-46 The reason is that Gd (III) can be bound to citrate to produce Gd(III) core (as shown in Table 1 and Figure 2), which restricts the local motions of Gd. Additionally, the r2/r1 ratio is so low that is beneficial for high signal-to-noise ratio imaging. The relaxivity results suggest that Gd@SiO2-DOX/ICG-PDC may act as a potential T1-type MRI contrast agent. To further demonstrate the T1-weighted MRI capability for Gd-containing NPs, MCF-7 cells were used to perform T1-weighted images at cellular level. After cellular uptake of these NPs, the cells were centrifuged to the bottom of vial tubes before MR imaging and the results show that both the accumulated NPs inside cells have strong MR signals (Figures 4D). For Gd@SiO2-DOX/ICG-PDC NPs, the MR signal (1/T1) increased by 500% compared to untreated cells after 24h incubation.

Figure 5. Release profile of Gd3+ ions from Gd@SiO2-DOX/ICG-PDC in aqueous solution with/without laser at pH 5.0/7.4 with (B) and without (A) the addition of 10% fetal bovine serum at 37 oC. Stability of Gadolinium Chelate in Gd@SiO2-DOX/ICG-PDC NPs. It’s known that Gd3+ ions are toxic at micromolar concentrations.47, 48 The thermodynamic and kinetic stability of Gd core (Gd citrate and GdCl3) determines the potential toxicity of Gd@SiO2-DOX/ICG-PDC NPs.49 As shown in Figure 5, the release profile of Gd3+ ions measured by ICP-MS indicated that (1.05±0.001)% and (1.33±0.03)% Gd3+ were leaked from NPs at pH 7.4 and 5.0 buffers after 15 days. Upon NIR laser irradiation, the release increased to (1.06±0.04)% and (2.18±0.05)% , respectively. As shown in


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Figure 5B, the amount of released Gd3+ from NPs in pH 7.4 and 5.0 buffers with 10% FBS was (1.31 ±0.005)% and (1.47±0.08)% for 15 days. Under NIR laser irradiation, the release was (1.16±0.10)% and (1.39±0.11)%, respectively. It demonstrated that FBS reduced the leakage of Gd3+ ions slightly. Meanwhile, under irradiation, the release of Gd3+ ions in 10% FBS solution was less compared to non-irradiation. The reason may that the protein aggregates are induced by heat denaturation under NIR irradiation and these proteins are likely to block a part of the pores of silica to retard the leakage of Gd3+ ions.50,51 Compared with the release of Gd3+ ions from Gd-DTPA (1.9% Gd3+ released after 15 days in human serum, pH 7.4, 37 °C),48 the Gd3+leakage from Gd@SiO2-DOX/ICG-PDC was almost the same or less. Compared to the non-irradiation condition, the irradiated Gd@SiO2-DOX/ICG-PDC had a less release amount even after incubation for 15 days. The silica shell and PDC layer may act as protective barriers to retard the release of Gd3+ from the core of NPs to the PDC layer. Furthermore, the stable amorphous silica layer,52 PDC coating with good hydrolytic stability, and the protein corona absorbed on the NPs will attribute to the slower release rate of Gd3+ ions.



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Figure 6. Cytotoxicity and cellular uptake for MCF-7 cells. (A) Dose-dependent cytotoxicity for Gd@SiO2-PDC, Gd@SiO2-DOX, Gd@SiO2-PDC-ICG-PDC, and Gd@SiO2-DOX/ICG-PDC NPs, respectively. (B) Quantitative analysis of internalized Gd@SiO2-PDC and Gd@SiO2 in MCF-7 cells determined by ICP-MS. (C) Quantitative evaluation of cell survival of MCF-7 cells after chemo- or chemo-photothermal treatments. (D) Live–Dead staining of MCF-7 cells, which were incubated with Gd@SiO2-PDC, Gd@SiO2-DOX, Gd@SiO2-PDC-ICG-PDC, and Gd@SiO2-DOX/ICG-PDC for 24 h and then irradiated for 6 min at 1.0 W/cm2. The sign (**) indicates the significant differences between the groups (p

Gadolinium(III)-Chelated Silica Nanospheres Integrating

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