- Email: [email protected]
photothermal therapy
Accepted Manuscript Interfacial engineered gadolinium oxide nanoparticles for magnetic resonance imaging guided microenvironment-mediated synergetic chemodynamic/photothermal therapy Zhenghuan Zhao, Kai Xu, Chen Fu, Heng Liu, Ming Lei, Jianfeng Bao, Ailing Fu, Yang Yu, Weiguo Zhang PII:
S0142-9612(19)30478-8
DOI:
https://doi.org/10.1016/j.biomaterials.2019.119379
Article Number: 119379 Reference:
JBMT 119379
To appear in:
Biomaterials
Received Date: 26 January 2019 Revised Date:
16 July 2019
Accepted Date: 24 July 2019
Please cite this article as: Zhao Z, Xu K, Fu C, Liu H, Lei M, Bao J, Fu A, Yu Y, Zhang W, Interfacial engineered gadolinium oxide nanoparticles for magnetic resonance imaging guided microenvironmentmediated synergetic chemodynamic/photothermal therapy, Biomaterials (2019), doi: https:// doi.org/10.1016/j.biomaterials.2019.119379. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interfacial Engineered Gadolinium Oxide Nanoparticles for Magnetic ACCEPTED MANUSCRIPT Resonance Imaging Guided Microenvironment-Mediated Synergetic Chemodynamic/Photothermal Therapy
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Zhenghuan Zhao1*, Kai Xu2,3, Chen Fu1, Heng Liu2,3, Ming Lei1, Jianfeng Bao5, Ailing Fu1, Yang Yu1, and Weiguo Zhang2,3*
College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China.
2
Department of Radiology, Daping Hospital, Army Medical Center of PLA, Army Medical
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University, Chongqing 400010, China. 3
Chongqing Clinical Research Center for Imaging and Nuclear Medicine, Chongqing 400010, China.
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College of Medical Technology and Engineering, Henan University of Science and Technology,
*email: [email protected]
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Luoyang 471000, China.
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[email protected]
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Abstract
Engineering interfacial structure of biomaterials have drawn much attention due to it can improve the diagnostic accuracy and therapy efficacy of nanomedicine, even introducing new moiety to construct theranostic agents. Nanosized magnetic resonance imaging contrast agent holds great
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promise for the clinical diagnosis of disease, especially tumor and brain disease. Thus, engineering its interfacial structure can form new theranostic platform to achieve effective disease diagnosis and therapy. In this study, we engineered the interfacial structure of typical MRI contrast agent, Gd2O3,
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to form a new theranostic agent with improved relaxivity for MRI guided synergetic
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chemodynamic/photothermal therapy. The synthesized Mn doped gadolinium oxide nanoplate exhibit improved T1 contrast ability due to large amount of efficient paramagnetic metal ions and synergistic enhancement caused by the exposed Mn and Gd cluster. Besides, the introduced Mn element endow this nanomedicine with the Fenton-like ability to generate ·OH from excess H2O2 in
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tumor site to achieve chemodynamic therapy (CDT). Furthermore, polydopamine engineered surface allow this nanomedicine with effective photothermal conversion ability to rise local temperature and accelerate the intratumoral Fenton process to achieve synergetic CDT/photothermal therapy (PTT).
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This work provides new guidance for designing magnetic resonance imaging guided synergetic
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CDT/PTT to achieve tumor detection and therapy.
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1. Introduction
Developments of multifunctional nanomaterials that integrate diagnosis and therapy into a single system may offer great advantages in disease treatment and have received significant interest in fields of biomedicine.[1-3] Engineering interfacial structure of biomaterials can improve the
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diagnostic accuracy or therapy efficacy of nanomedicine, and introduce new moiety to construct theranostic agents.[4, 5] Nanosized magnetic resonance imaging (MRI) contrast agent holds great promise for the clinical diagnosis of disease, especially tumor and brain disease, as the high spatial
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and temporal resolution of MRI. Thus, engineering its interfacial structure can form new theranostic
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platform to achieve effective disease diagnosis and therapy. Various T1 MRI contrast agents has been developed, including gadolinium based,[6, 7] iron based,[8-10] and manganese based agents.[11, 12] Because of its large amounts of unpaired electrons and long electronic relaxation time, Gd-based nanomaterials, especially gadolinium oxide (Gd2O3) nanoparticles, have been extensively explored
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as T1 contrast agent through controlling over the morphology and size. Based on the classical Solomon-Bloembergen-Morgan (SBM) theory, number of coordinating (q), proton residence lifetime (τM), and molecular tumbling time (τR) determine the contrast capacity of nano-scale Gd2O3.
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Ultra-small Gd2O3 nanoparticles with high surface-to-volume ratio have been proved to be
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high-performance T1 contrast agent due to the improvement on q value caused by the increase of efficient paramagnetic metal ions on surface.[13, 14] Therefore, optimization the crystal structure of Gd2O3 nanoparticles with high surface-to-volume ratio by morphology controllable synthesis may be the effective strategy to improve T1 relaxivity of Gd based T1 contrast agent.[15] Besides, expose two different paramagnetic ions may synergistically enhance protons T1 relaxation by improve the proton coordination and chemical exchange.[5] Therefore, further introducing another paramagnetic element with therapy effect may construct high-performance MRI-guided theranositc agent.
Mn-based materials, includingACCEPTED MnCl2, manganese (II) chelates,[16] manganese oxide (MnO) MANUSCRIPT nanoparticles,[17, 18] and MnO2 nanostructure[19] have been used to achieve T1 contrast imaging as its labile water exchange, high spin number, and long electronic relaxation time. Since manganese ion is an essential element in cell biology and often acts the cofactor for some crucial enzymes or
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receptors, optimization the crystal structure of Gd2O3 nanoparticles by partially replacement of Gd ions with Mn ions may endow it with high biocompatibility to tumor diagnosis in vivo. Furthermore, manganese ions show Fenton-like activity in acidic environment, endowing manganese ions to
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convert overproduced H2O2 in tumor site into reactive oxygen species (ROS).[20-23] Since ROS
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could damage the cancer cells and suppress the solid tumor growth, many ROS-based cancer treatment strategies have been developed, especially chemodynamic therapy (CDT).[24-26] Based on the specific tumor environment, CDT could in situ generate ROS through intratumoral Fenton reaction, which can reduce the harmful effect to the normal tissue. Besides, ROS could be cleared
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out by the antioxidation systems, further reducing side effect of CDT.[27] Therefore, introducing manganese ions into Gd2O3 nanocrystals may ensure it with the ability to achieve tumor therapy through CDT. Unfortunately, the CDT efficacy is hampered by the catalytic efficiency in vivo, thus
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application.
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improve the amount of hydroxyl radical (·OH) produced by CDT in vivo is important to its
The catalytic efficiency is determined by many physical parameter, especially temperature. Generally, rise of the temperature could improve the efficiency of Fenton-like reaction and increase the amount of ·OH.[28] Photothermal therapy (PTT) agent can convert the energy of near-infrared light into heat and increase the local temperature of tumor. Therefore, one can introduce a PTT moiety to improve CDT efficacy and achieve synergetic CDT/PTT. Polydopamine (PDA) generated by the self-polymerization of dopamine, one of the natural neurotransmitters, has been widely used as the PTT agent due to its easy to synthesis, high biocompatibility, and high photothermal
conversion efficiency.[29-32] Besides, dopamineMANUSCRIPT have recently evolved as preferred components to ACCEPTED efficiently transfer metal oxide nanoparticles into aqueous media in mild condition to form high hydrophilicity and loose structure on crystal surface, which are benefit to the chemical exchange between proton and T1 magnetic resonance imaging (MRI) contrast system.[11, 33] Thus, PDA is the
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good candidate as the PTT motif to develop MRI-guided synergetic CDT/PTT theranostic agent. Herein, we present a novel strategy to engineer the interfacial structure of Gd2O3 nanocrystals and construct PDA and polyethylene glycol coated Mn doped Gd2O3 nanoplate (MnGdOP@PDA-PEG)
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to achieve MRI-guided synergetic CDT/PTT. The prepared MnGdOP@PDA-PEG exhibits relatively
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higher T1 relaxivity than commercial T1 contrast agent (Gd-DTPA) and traditional Gd2O3 nanoplate. Besides, MnGdOP@PDA-PEG can catalyze H2O2 convert into ·OH, ensuring it as a potential CDT agent to kill tumor cells. Furthermore, the PDA coated surface endow it with the ability to effectively transform NIR energy into heat to rise the local temperature of tumor to accelerate the intratumoral
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Fenton process and achieve PTT. In vivo study indicates that MnGdOP@PDA-PEG exhibits sensitive solid tumor detection and can effectively suppress the tumor growth through synergetic CDT/PTT, thus showing that combine MRI contrast imaging, CDT, and PTT into one materials may
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provide clues to develop efficient anticancer theranostic agent.
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2. Experiment section 2.1 Chemical.
Oleylamine (tech 90%), Manganese(II) chloride tetrahydrate (tech 90%), Gadolinium (III) chloride hexahydrate (99.99%), and 1-octadecene (90%) were purchased from Alfa Aesar. Dopamine hydrochloride, L-Dopa, hexane, tetrahydrofuran, sodium oleate, sodium hydroxide, isopropanol, and ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methylene blue, 2’,7’-dichlorofluorescein diacetate, propidium iodide, and calcein
acetoxymethyl ester were purchased from Dalian MANUSCRIPT Meilun Biotechnology Co. Ltd. All chemicals were ACCEPTED used as received without further purification. 2.2 Synthesis of manganese doped gadolinium oxide nanoplate (MnGdOP). In a typical experiment, 0.18 g Gd-oleate and 0.07 g Mn-oleate was dissolved in 10 mL
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1-octadecene containing 1 mL oleylamine at room temperature. The mixture was degassed in vacuum for 30 min and backfilled with argon to remove any low volatile impurities and oxygen at room temperature. And then, we heated the reaction solution to reflux and kept at that temperature
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for 1.5 h. The resultant solution was then cooled to room temperature and mixed with 30 mL
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isopropanol to precipitate the nanoparticles. The nanoparticles were separated by centrifugation and washed 3 times with ethanol.
2.3 Preparation of polydopamine coated MnGdOP (MnGdOP@PDA) nanostructures. Typically, 30 mg dopamine, 10 mg L-dopa, and ~0.1 mmol nanoparticles were mixed in a mixture
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containing 10 mL of sodium hydroxide solution and 20 mL of tetrahydrofuran in air at room temperature. After stirring for 2 h, the PDA coated nanoparticles were collected by centrifugation and re-dispersed in distilled water for long-term storage at 4 °C.
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nanostructures.
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2.4 Preparation of polydopamine coated MnGdOP polyethylene glycol (MnGdOP@PDA-PEG)
MnGdOP@PDA-PEG were prepared by simply mixing PDA with OH-PEG-NH2 (M.W 2000) at a mass ratio of 1:2 at pH with the value of 9 under magnetic stirring at room temperature. After stirring for 24 h, the products were collected by centrifugation and re-dispersed in distilled water for long-term storage at 4 °C. 2.5 ·OH generation through MnGdOP@PDA-PEG based Fenton-like reaction. MnGdOP@PDA-PEG 0.2 mg were added in 10 µL H2O2 (1 M) and 1 mL MB solution (10 µM) with the pH value of 6.5. After incubation for 20 min at room temperature or 320 K, the mixture
solution was centrifuged to remove MnGdOP@PDA-PEG and measured the absorbance at 661 nm ACCEPTED MANUSCRIPT by UV-vis spectroscopy. 2.6 In vitro DCFH-DA assay. The generated ROS in tumor cell was investigated by intracellular ROS fluorescence probe
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2’,7’-dichlorofluorescein diacetate (DCFH-DA) at pH 6.5. The cells were seeded on a 6-well plate with a density of 1 × 105 cells per well, and incubated in the atmosphere of 5% CO2 at 37 °C for 24 h. The cells were then incubated with PBS or MnGdOP@PDA-PEG with the concentration of 50 or
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100 ppm for 6 h, and then treated each well without or with an 808 nm laser irradiation at a power
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density of 1 W/cm2 for 2 min. The cells were stained by DCFH-DA (10 µM) for 30 min. The fluorescence images were capture by fluorescence microscope (Nikon eclipse Ti-S). 2.7 In vitro synergistic CDT/PTT therapy.
The synergistic CDT/PTT therapy was evaluated through propidium iodide (PI) and calcein
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acetoxymethyl ester (calcein-AM) double staining. Cells were seeded on a 6-well plate with a density of 1 × 105 cells per well, and incubated in the atmosphere of 5% CO2 at 37 °C for 24 h. The cells were then incubated with MnGdOP@PDA-PEG with the concentration of 50 or 100 ppm for 6
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h at pH 6.5, and then irradiated on each well with an 808 nm laser at a power density of 2 W/cm2 for
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5 min. After incubation for another 6 h, PI and calcein-AM double staining were conducted. The fluorescence images were capture by fluorescence microscope (Nikon eclipse Ti-S). 2.8 In vivo synergistic CDT/PTT. Animal experiments were executed according to the protocol approved by institutional Animal Care and Use Committee of Southwest University. The subcutaneous tumor model was established by injection of U-87 MG cells into the subcutaneous tissue of nude mice (18-22 g). When the tumor reached ~ 0.5 cm in diameter, the mice bearing tumor were intravenously injected MnGdOP@PDA-PEG with the amount of 15 mg/kg of mice weight and irradiated with 808 nm laser
for 5 min with the power of 1.5 ACCEPTED W/cm2. Meanwhile, three control groups were used to assess the MANUSCRIPT therapy efficiency: (a) intravenously injection of 150 µL saline; (b) intravenously injection of 150 µL saline and irradiation with 808 nm laser for 5 min with the power of 1.5 W/cm2; and (c) intravenously injection of MnGdOP@PDA-PEG with the amount of 15 mg/kg of mice weight. The
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mice weights and tumor sizes were monitored during the treatment period. The tumor volumes were calculated by the equation of Vtumor = (a2 × b)/2 (a and b represent the maximum and minimum diameter of the tumor). Relative tumor volumes were calculated as V/Vo (V and Vo represent the
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tumor volume after treatment and initial tumor volume).
3. Results and discussion 3.1 Synthesis and characterization. We synthesized manganese dope Gd2O3 nanoplate (MnGdOP) by thermal decomposition of the mixture of manganese-oleate and gadolinium-oleate in 1-octadecene (ODE) containing oleylamine as surfactant. The transmission electron microscopy (TEM) images show that some as-prepared nanoparticles assemble into the stacked ribbons (Fig. 1a). This phenomenon clearly indicates that the morphology of as-prepared nanoparticles is plate.[34] Based on the face-to-face stacking, the
thickness of this plate was measured with the value of approximately 1.8 ± 0.3 nm (Fig. S1). Further ACCEPTED MANUSCRIPT high-resolution TEM (HRTEM) images of the perpendicular nanoplate shows the uniform lattice fringes cross the entire nanoplate with the lattice spacing of 2.7 Å, corresponding to the (400) planes of Gd2O3, revealing its good crystallinity (Fig. 1a). Additionally, plate morphology endues it with
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high surface-to-volume ratio and large amount of paramagnetic metal ions on the surface to achieve high T1 contrast ability. To confirm the co-existence of Gd and Mn species in this nanoplate, we conducted energy-dispersive X-ray element mapping (EDX mapping). EDX mapping images show
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that the Gd and Mn signals merge well, revealing the successful Mn dopant in the MnGdOP (Fig.
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1b). The molar percentage of Mn ions with respect to total metal ions for MnGdOP is approximately 16.7 %, determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). We then analyzed the crystallographic structure of the MnGdOP by X-ray powder diffraction (XRD) pattern. MnGdOP show typical mixed Gd2O3 (JCPDS no. 00-012-0797) and MnO (00-075-0625)
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diffractogram patterns (Fig. 1c). It should be noted that MnGdOP show a sharp and enhanced diffraction peak at 33.1 degree corresponding to (400) plane of Gd2O3, which could be ascribed to the face-to-face assembly of MnGdOP and further suggest the plate morphology of MnGdOP.[35, 36]
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Further X-ray photoelectron spectroscopy (XPS) analysis in Gd 3d5/2 shows a typical peak at binding
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energy of 1187 eV, which agrees with the value of Gd2O3 nanocrystals (Fig. 1d).[37] Compared to Gd 3d5/2 peak, the Mn 2p3/2 peak is measured at the binding energy of 640.5 eV, suggesting the oxidation of doped Mn ions is +2 (Fig. 1e).[38] The magnetic properties of MnGdOP nanostructure was investigated by the superconducting quantum interference device (SQUID) magnetometer at room temperature (300 K). Field-dependent magnetization curve (M-H) of MnGdOP is a straight smooth line without any coercivities and remanences, indicating its paramagnetic behavior at room temperature (Fig. 1f). The unsaturated magnetic moments of MnGdOP reach to 2.1 emu/g under magnetic field of 5 T at 300 K. Consistent
with the M-H curve, standard zero-field-cooling (ZFC) and field-cooling (FC) curve shows no ACCEPTED MANUSCRIPT magnetic transition below 300 K, further confirming its paramagnetic behavior at room temperature (Fig. 1g). Based on its paramagnetic behavior, MnGdOP may efficiently reduce the longitudinal relaxation of proton and achieve T1 contrast imaging.
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3.2 Formation of MnGdOP nanostructure. Synthesis of doped metal oxide nanostructure by introducing another metal oleate could result in the formation of anisotropic nanostructure, which may be caused by the ionizing and dissociating of
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the precursor into oleate (OL-) at high temperature.[39] To investigate the effect of different amount
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Mn-oleate (MnOL) on the formation of MnGdOP, we conducted a series of experiment at a fixed reaction temperature, time, and surfactant but systematically varied Mn-oleate amount. We obtained the nearly spherical Mn doped Gd2O3 by supplying the amount of Mn-oleate with the molar ratio of MnOL/Gd-oleate (GdOL) to 1:20 (Fig. S2a). This result indicates that the negligible amount of
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MnOL is insufficient to affect the equal absorption of oleylamine on various crystal planes, resulting in the formation of spherical nanocrystals. Surprisingly, nanostructures with the morphology of plate can be obtained by increasing the molar ratio of MnOL/GdOL (Fig. S2b,c). In addition, the yield of
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nanostructures with anisotropic morphology improved along with the increase of the MnOL/GdOL
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molar ratio. These results indicate that the free ionic OL- dissociated from MnOL may be able to selectively bind to Gd ions and induce the anisotropic growth of nanocrystals, leading to the formation of MnGdOP nanostructures. This mechanism can be further supported by the observation of the formation of nanoplate assembly, flower-liked nanostructure (Fig. S2d). To investigate the morphology evolution of this nanoplate, we carefully took aliquots from the reaction solution and analyzed the dynamic growth process of MnGdOP (Fig. S3). Irregular crystal nuclei are first formed after 15 min reaction, indicating that the monomer concentration is saturated and could overcome the energy barrier to accomplish the nucleation process under this reaction condition. With continuous
consumption of the monomer, small tripodal nanoplate formed with the thickness of about 1.5 nm. ACCEPTED MANUSCRIPT After aging at the same temperature, Ostwald ripening occurs and causes improvement of size and uniformity of nanoplate. Interestingly, the thickness of the nanoplate maintains the same during its growth process, demonstrating the formation of MnGdOP is based on the capping agent assistant
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process. 3.3 T1 MRI contrast ability.
To transfer MnGdOP into aqueous and accelerate manganese base Fenton-like reaction, we
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engineered MnGdOP nanostructure surface with dopamine in air and form polydopamine (PDA)
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shell. Further PEG modification was applied to improve its behavior in vivo and form PDA and PEG coated MnGdOP (MnGdOP@PDA-PEG).[40, 41] TEM image clearly shows the PDA shell on the surface of MnGdOP nanostructures, indicating the successful formation of MnGdOP@PDA structure (Fig. S4a). The surface charge of MnGdOP-PDA-PEG was ~-13.4 mV, which is benefit to
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decrease the protein absorption on the MnGdOP@PDA-PEG surface (Fig. S5). Dynamic light scattering (DLS) analysis indicated that MnGdOP@PDA-PEG was monodisperse in aqueous media without any clustering or aggregation (Fig. S4). To confirm the successful PDA coating, we
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performed fouriertransform infrared spectroscopy (FT-IR) and NIR spectra analyses. Compare to
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MnGdOP, MnGdOP@PDA and MnGdOP@PDA-PEG presented absorption peaks at around 1600 cm-1 assigned to the benzene skeleton vibrations of PDA (Fig. S6).[42] Besides, MnGdOP and MnGdOP@PDA-PEG revealed a broad absorption at NIR region in absorption spectra. More importantly, the absorption value of MnGdOP@PDA-PEG was higher than that of MnGdOP (Fig. S7). These results suggested the successful coating of PDA shell on MnGdOP. The amount of PDA coated on the surface of MnGdOP was approximately 12 %, which was assessed by thermogravimetric analysis (Fig. S8). To verify the MnGdOP@PDA-PEG nanostructure can be used as a contrast agent in T1-weighted contrast imaging, we utilized Gd-DTPA and gadolinium oxide
nanoplate (GdOP) as controls toACCEPTED investigate itsMANUSCRIPT T1 contrast ability (Fig. 2 and Fig. S9,10). We assessed the T1 contrast performance of all samples at 0.5 T and 1.5 T magnetic fields. The r1 values of MnGdOP@PDA-PEG, Gd-DTPA, and GdOP are 6.7, 4.1, and 4.2 mM-1s-1 at 0.5 T, respectively (Fig. 2a and Fig. S10). The r1 value of MnGdOP@PDA-PEG is higher than that of Gd-DTPA and
ions and synergistic enhancement caused
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GdOP. The elevated relaxivity could be attributed to large amount of efficient paramagnetic metal by the exposed Mn and Gd cluster on
MnGdOP@PDA-PEG surface. [5, 43-45] Consistent with the results at 0.5 T, MnGdOP@PDA-PEG
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exhibits obviously higher r1 value (5.3 mM-1s-1) than Gd-DTPA (3.8 mM-1s-1) and GdOP (3.3
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mM-1s-1) at 1.5 T as well (Fig. 2c and Fig. S10). These results prove that interfacial engineering of gadolinium oxide nanoparticles is an effective strategy to construct T1 contrast agent with high performance. Due to the relative high T1 relaxivity and low r2/r1 ratio, T1-weighted phantom analyses reveal that MnGdOP@PDA-PEG exhibits clearly brightness gradients along with the increase of
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metal concentration (Fig. 2b,d and Fig. S11). More importantly, the MnGdOP@PDA-PEG offers high quality contrast imaging with brighter signal than Gd-DTPA and GdOP at the same
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concentration, revealing the high sensitivity of MnGdOP@PDA-PEG in T1-weighted contrast
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The stability of nanomaterials can affect its performance and behavior in vivo, which is the crucial issue to determine its efficiency in biomedical imaging field.[46, 47] We analyzed the stability of MnGdOP@PDA-PEG in long term. The hydrodynamic diameter of MnGdOP@PDA-PEG remained unaltered after storage in phosphate buffer saline (PBS) for one month (Fig. S12). Moreover, the leakage ratio of Gd and Mn are blow 0.2 % after storage in PBS for one month. Since the main toxicity of Gd based contrast agent is caused by the released Gd ions, the negligible leakage of Gd ions in one month suggests the high biocompatibility of MnGdOP@PDA-PEG and low side effect.
Relaxivities analyses indicate that the r1 valuesMANUSCRIPT of MnGdOP@PDA-PEG stored in PBS for one ACCEPTED month are highly preserved at 0.5 and 1.5 T (Fig. S12) 3.4 In vivo liver MRI. Based on the high stability, we utilized MnGdOP@PDA-PEG to assist liver MR imaging to
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evaluate its contrast capacity in vivo. Accordingly, GdOP was used for comparison. T1-weighted contrast images were acquired before and after intravenous injection of MnGdOP@PDA-PEG or GdOP with the dosage of 2 mg magnetic ions/kg body weight of mice on a 7 T MRI scanner. After
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intravenous injection, we indeed observed signal increase in liver region. More importantly, the T1
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signal enhancement in liver region of mice treated by MnGdOP@PDA-PEG is much stronger than that of the mice treated by GdOP (Fig. 2e). Signal to noise ratio (SNR) is calculated by choosing liver as the region of interests (ROIs) to quantify the contrast enhancement (Fig. 2f and Table S1). It appears that MnGdOP@PDA-PEG exhibit significantly higher contrast than GdOP, suggesting
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MnGdOP@PDA-PEG could be a candidate to assist to obtain comprehensive information in T1-weighted MRI imaging and achieve tumor diagnosis. 3.5 ·OH generation capacity under laser irradiation.
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To investigate the MnGdOP@PDA-PEG activity to convert H2O2 into ·OH in acidic environment,
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we detect ·OH through the degradation of methylene blue (MB).[20] The UV-Vis spectra analyses indicated that the H2O2 do not affect the absorption of MB at acidic environment (Fig. 3a). However, a decrease of maximum absorbance peak for MB was observed after incubating MB with H2O2 and MnGdOP@PDA-PEG for 20 min at pH 6.5, which could be ascribed to the ·OH generation through Fenton-like reaction (Fig. 3b). These results clearly indicate that MnGdOP@PDA-PEG could catalyze H2O2 convert into ·OH in acidic environment, ensure it as a potential chemodynamic therapy (CDT) agent to kill tumor cells. It worthwhile to note that the leakage of Mn ions from MnGdOP@PDA-PEG at pH 6.5 increased compare to that at pH 7.4, which may be ascribed to the
Fenton-like reaction (Fig. S13). ACCEPTED To determine MANUSCRIPT whether the rise of temperature can improve the Fenton-like reaction activity of MnGdOP@PDA-PEG, we incubated MB with H2O2 and MnGdOP@PDA-PEG at 320 K in acidic environment for 20 min. As expected, we observed a more remarkable decrease of maximum absorbance peak for MB compare to incubation with
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MnGdOP@PDA-PEG at room temperature, indicating temperature rising could accelerate the MnGdOP@PDA-PEG based Fenton-like reaction (Fig. 3c). Further quantitative analyses indicated that the maximum absorption peak for MB reduced to 99.3%, 92.7%, and 83.7% after incubation
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with only H2O2, H2O2 and MnGdOP@PDA-PEG, and H2O2 and MnGdOP@PDA-PEG at 320 K,
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respectively (Fig. 3d). These results demonstrate that rising temperature could improve the efficiency of MnGdOP@PDA-PEG based Fenton-like reaction and enhance ·OH generation, which is benefit to the further CDT in vitro and in vivo.
We investigated the photothermal conversion capacity of MnGdOP@PDA-PEG to evaluate its
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capacity on temperature rise under near-infrared (NIR) irradiation. The absorption spectra analyses indicate that MnGdOP@PDA-PEG show the broad absorption from visible to NIR, indicating the potential of MnGdOP@PDA-PEG to be used as a photothermal conversion agent to rise local
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temperature in CDT (Fig. S7). Consequently, we assessed the photothermal conversion capacity of
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MnGdOP@PDA-PEG through exposure its aqueous solution with different concentrations to 808 nm laser with different power density (Fig. 4). The heating curves and infrared thermal images indicate that the solution containing MnGdOP@PDA-PEG show obviously larger temperature increase than pure water after irradiation (Fig. 4a,b). The temperature rising of aqueous solution containing MnGdOP@PDA-PEG with the concentration of 200 and 400 ppm can reach 11.0 and 15.1 °C under the laser with the power density of 1 W/cm2. These results prove that MnGdOP@PDA-PEG can effectively absorb 808 nm laser energy and cause substantial overheating. In addition, the heating effect of aqueous solution containing MnGdOP@PDA-PEG increased with
the increase of laser power density (Fig. 4c,d). Along with the rise of the power density, the ACCEPTED MANUSCRIPT temperatures increasement of the aqueous solution containing MnGdOP@PDA-PEG with the concentration of 400 ppm increased to 20.0 °C. To quantitative the photothermal conversion capacity, we calculated the photothermal conversion efficiency (η) of MnGdOP@PDA-PEG
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according to the previous work.[48, 49] The η value of MnGdOP@PDA-PEG was determined to be ~21.5 %, which is comparable to other PTT agent (Fig. S14 and Table S2). These results further confirm that the heating effect is mainly caused by the photothermal conversion agent,
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MnGdOP@PDA-PEG, and show the potential of MnGdOP@PDA-PEG as photothermal conversion
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agent to rise local temperature in CDT and act as a PTT agent in tumor therapy. Since the photostability of photothermal agent can affect its performance and behavior in vivo, we analyzed the photostability of MnGdOP@PDA-PEG during several irradiation cycles.[32] The absorption spectra shows that the MnGdOP@PDA-PEG exhibits the similar curves in NIR region before and
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after three irradiation cycles (Fig. 4e). Moreover, we observed that the solution temperature can reach to the same level on the assist by MnGdOP@PDA-PEG, even after three irradiation cycles (Fig. 4f). These results indicate the high photostability of MnGdOP@PDA-PEG and show the
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potential of MnGdOP@PDA-PEG to efficiently convert NIR laser power into heat reproducibly.
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Since MnGdOP@PDA-PEG exhibit good photothermal conversion ability, we evaluated the Fenton-like reaction activity of MnGdOP@PDA-PEG under laser irradiation. We incubated MB with MnGdOP@PDA-PEG and H2O2 with or without laser irradiation. UV-Vis spectra analyses indicated that the maximum absorption peak for MB gradually decrease with the extension of time, indicating the Fenton-like reaction activity of MnGdOP@PDA-PEG (Fig. 5a). Interestingly, the decrease of maximum absorption peak for MB significantly increased under 1 W/cm2 laser irradiation, indicating more ·OH generation compare to without laser irradiation group (Fig. 5b). These results could be ascribed to that MnGdOP@PDA-PEG convert the energy of NIR into heat to
increase the solution temperatureACCEPTED and MnGdOP@PDA-PEG based Fenton-like reaction activity. MANUSCRIPT Moreover, the maximum absorption peak of MB could further decrease with the increase of laser power density (Fig. 5c). Compared to the solution without laser irradiation, the maximum absorption peak for MB reduction decrease from 92.7 % to 86.0 % (1 W/cm2) and 82.2 % (2 W/cm2) (Fig. 5d).
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These results demonstrate that the laser irradiation could accelerate the MnGdOP@PDA-PEG based Fenton-like reaction and enhance ·OH generation, which may improve the CDT efficacy in vivo. To exclude the activity improvement is caused by laser, we assessed the MB degradation under laser
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maintains the same after laser irradiation (Fig. S15).
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irradiation without MnGdOP@PDA-PEG. It appears that the absorption curve of MB almost
3.6 In vitro cell assay analyses.
The synergistic CDT/PTT therapy effect at cellular level was investigated by incubation tumor cells with MnGdOP@PDA-PEG and exposure to 808 nm NIR laser at 2 W/cm2 for 5 min. We did
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not observe apparent cytotoxicity after incubation tumor cells with MnGdOP@PDA-PEG without laser irradiation, demonstrating the acceptable biocompatibility of MnGdOP@PDA-PEG (Fig. S16). Whereas, the cell viability remarkably decreased when the laser was introduced (Fig. 6a). These
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the cancer cell. It should be noted that MnGdOP@PDA-PEG show negligible CDT effect, which could be ascribe that the activity of Fenton-like reaction at pH 7.4 is low.[50] Thus, we incubated U-87 MG with MnGdOP@PDA-PEG with or without laser at pH with the value of 6.5, which was used to mimic the extracellular environment of tumor. It appears that the cell viability reduced to 75.8 % when incubation it with MnGdOP@PDA-PEG with the concentration of 100 ppm (Fig. 6b). Since the slight acidic environment could not affect the viability of tumor cells, this result indicates that MnGdOP@PDA-PEG could be used as a potential CDT agent (Fig. S17).[28] The generated ROS
in
tumor
cell
was
investigated
by
intracellular
ROS
fluorescence
probe
2’,7’-dichlorofluorescein diacetateACCEPTED (DCFH-DA) MANUSCRIPT at pH 6.5 (Fig. 6c).[51, 52] Compared to the PBS treated group, slight green emission could be observed after adding MnGdOP@PDA-PEG into media. It should be noted that the intensity of green emission increased with the amount elevation of MnGdOP@PDA-PEG, which could be ascribed to the increase of generated ·OH along caused by
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the rising of MnGdOP@PDA-PEG. Furthermore, cells treated with MnGdOP@PDA-PEG and laser irradiation showed obvious green emission, suggesting sufficient ·OH were produced to achieve CDT on the assistant of laser induced temperature increase. Additionally, the further in vitro
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synergistic CDT/PTT effect was elevated through propidium iodide (PI) and calcein acetoxymethyl
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ester (calcein-AM) double staining (Fig. 6d).[53, 54] Florescence images show that the MnGdOP@PDA-PEG with NIR laser irradiation could kill the cancer cells more effectively compare to treat cancer cell with only laser or MnGdOP@PDA-PEG. These results clearly reveal the excellent synergistic CDT/PTT effect of MnGdOP@PDA-PEG to kill cancer cells.
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3.7 Tumor diagnosis and therapy.
To further assess the in vivo diagnostic and therapeutic effect of MnGdOP@PDA-PEG, we performed the in vivo study. We firstly injected MnGdOP@PDA-PEG intravenously into mice
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bearing tumors and conducted T1-weighted MRI. The T1-weighted MR images show that T1 signal of
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tumor region gradually increase over time and show sufficient signal to detect tumor (Fig. 7a). To quantify the contrast enhancement, we calculated SNR according to the T1-weighted images. It appears that the ∆SNR values of T1-weighted images at tumor are 5.23, 8.37, and 15.14 % at 0.5, 1, and 2 h post injection, respectively (Fig. 7b). The T1 signal enhancement demonstrates that MnGdOP@PDA-PEG is an effective contrast agent for accurate tumor detection. We further investigate the biodistribution of MnGdOP@PDA-PEG to confirm the signal change is caused by the accumulation of MnGdOP@PDA-PEG in tumor. It appears that the tumor uptake of MnGdOP@PDA-PEG is about 4.60 % ID/g, suggesting that the T1 signal change is caused by the
accumulation of MnGdOP@PDA-PEG (Fig. 7c).MANUSCRIPT We then performed in vivo study to evaluate the ACCEPTED therapeutic effect by intravenous injection of MnGdOP@PDA-PEG into tumor with 808 nm laser irradiation (denoted as MnGdOP@PDA-PEG+Laser). Meanwhile, we chose the mice treated by injection of Saline (denoted as Saline), injection of MnGdOP@PDA-PEG without laser irradiation
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(denoted as MnGdOP@PDA-PEG), and irradiation with laser (denoted as Laser only) as controls. To analyze the in vivo photothermal conversion capacity of MnGdOP@PDA-PEG, we recorded the temperature change at tumor area by infrared thermal mapping apparatus. The thermographic images
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reveal that the temperatures at tumor site raise rapidly for the MnGdOP@PDA-PEG+Laser group
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(Fig. 7d). After irradiation for 5 min, the tumor temperature of MnGdOP@PDA-PEG+Laser group can reach to 49.8 °C, which suggest MnGdOP@PDA-PEG can irreversibly damage tumor tissues.[55, 56] However, the maximum temperature of Laser only group is just about 39 °C under the same laser power density, demonstrating the safety of NIR laser for normal tissue (Fig. 7e).
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During the treatment period, the body weights of all mice nearly maintained at the same level, indicating the minimal side effect of MnGdOP@PDA-PEG (Fig. 8a). Tumor growth curves and digital photographs indicate that indicated that MnGdOP@PDA-PEG can inhibit tumor growth due
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to mild MnGdOP@PDA-PEG based CDT (Fig. 8b,c). Notably, the tumor growth rate of
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MnGdOP@PDA-PEG+Laser group was almost inhibited, revealing the high synergistic CDT/PTT therapy efficiency. On the contrary, the tumors in mice of Saline and Laser only groups grew rapidly (Fig. 8b,c). Further hematoxylin and eosin (H&E) analyses indicate that the tumor of MnGdOP@PDA-PEG group show slight necrotic response after treatment due to the CDT effect. However, tumor of Saline and Laser group show their typical structure. Notably, the tumor of MnGdOP@PDA-PEG+laser group show more obvious nuclear pyknosis and cancer necrosis compare to MnGdOP@PDA-PEG group, indicating its effective synergistic CDT/PTT under laser irradiation (Fig. 8d). Consistent with the H&E staining, TUNEL staining images of the tumor from
MnGdOP@PDA-PEG and MnGdOP@PDA-PEG+laser group show obvious green florescence, ACCEPTED MANUSCRIPT indicating typical apoptosis response. Whereas, Saline and Laser only group show negligible green florescence signal (Fig. 8e). These results reveal that MnGdOP@PDA-PEG is suitable to be used as a synergistic CDT/PTT agent to achieve tumor therapy. Clearance and systemic toxicity are
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important to the nanoparticles for their biomedical application. Compare to the T1 signal of liver and tumor at 2 h after intravenous injection, the T1 signal of liver and tumor at 4 h after intravenous injection showed an obviously decrease (Fig. S18). This signal decrease indicate the clearance of
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MnGdOP@PDA-PEG over time. To further assess systemic toxicity in vivo, we sacrificed all mice
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and conducted H&E staining after treatment. It appears that the main organs of all mice show their typical structure phenotypes without any obvious pathological lesion, including necrosis and inflammation (Fig. S19).[57] These results reveal the clinic potential of MnGdOP@PDA-PEG on
4. Conclusions
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tumor diagnosis and therapy.
In summary, a novel strategy to engineer the interfacial structure of Gd2O3 to construct
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MRI-guided CDT/PTT theranostic agent, MnGdOP@PDA-PEG, has been developed. Due to the
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unique surface structure, it exhibits relatively higher T1 relaxivity than commercial T1 contrast agent and traditional nanoscale T1 contrast agent and achieve sensitive tumor detection in vivo. Due to the introducing of manganese ions, MnGdOP@PDA-PEG shows Fenton-like reaction activity, endowing it to catalyze H2O2 convert into ·OH to accomplish CDT to kill tumor cells. Furthermore, its PDA surface endow it can effectively transform NIR energy into heat to rise the local temperature of tumor, which can accelerate the intratumoral Fenton process and even ensure it with the PTT capacity. Based on the synergetic CDT/PTT, MnGdOP@PDA-PEG can effectively cause the death of tumor cells and suppress the tumor growth in vivo. The present work may not only provide
theranostic agent to assist tumor imaging and therapy, but also give an avenue to develop theranostic ACCEPTED MANUSCRIPT
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agent combined MRI, CDT, and PTT in single platform to treat cancer.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (81601607, 81871421, and 81601470), Research Funds for the Central Universities (XDJK2016C182), Open Research Fund of State Key Laboratory of Molecular Vaccinology and Molecular Diagnosticsh
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(2016KF02), and Postdoctoral Science Special Foundation of Chongqing (Xm2017018).
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[57] Z. Zhao, X. Wang, Z. Zhang, H. Zhang, H. Liu, X. Zhu, H. Li, X. Chi, Z. Yin, J. Gao, Real-Time Monitoring ACCEPTED of Arsenic Trioxide Release and Delivery by ActivatableMANUSCRIPT T1 Imaging, ACS Nano 9 (2015) 2749-2759.
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Scheme 1. Schematic cartoon illustrates the MRI-guided synergetic CDT/PTT tumor therapy by
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injection of MnGdOP@PDA-PEG nanotheranostic agent.
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Fig 1. Characterization of MnGdOP nanostructures. (a) TEM and HRTEM (insets) images of MnGdOP, with the lattice spacing distance of 2.7 Å, corresponding to the (400) planes of Gd2O3. (b) EDX mapping analysis. EDX mapping images of the MnGdOP, indicating that the homogeneous Gd
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and Mn signals merge well and reveal the successful Mn dopant in the MnGdOP. (c) XRD patterns of MnGdOP. The XRD pattern show the typical mixed Gd2O3 (JCPDS no. 00-012-0797) and MnO (00-075-0625) diffractogram patterns. XPS analyses of MnGdOP in (d) Gd 3d5/2 and (e) Mn 2p3/2,
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respectively. (f) The smooth M-H curve of MnGdOP measured at 300 K using a superconducting
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quantum interference device magnetometer. The M-H curve of MnGdOP is a straight smooth line without any coercivities and remanences, indicating its paramagnetic behavior at room temperature. (g) ZFC/FC curve of MnGdOP measured under an applied magnetic field at 50 Oe, indicating its paramagnetic behavior at room temperature.
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Fig 2. T1 MRI contrast of MnGdOP@PDA-PEG. (a) The analysis of relaxation rate R1 (1/T1) vs magnetic ions concentration for MnGdOP@PDA-PEG, Gd-DTPA, and GdOP nanoparticles and (b) relative T1-weighted MR images at 0.5 T. (c) The analysis of relaxation rate R1 (1/T1) vs magnetic
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ions concentration for MnGdOP@PDA-PEG, Gd-DTPA, and GdOP nanoparticles and (d) relative T1-weighted MR images at 1.5 T. (e) T1-weighted MR images of mice at 0, 0.5, 1, and 2 h after
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intravenous injection of MnGdOP@PDA-PEG (upper) and GdOP (lower). (f) Quantification of
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relative liver contrast collected at different time after administration of MnGdOP@PDA-PEG and
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Fig 3. ·OH generation capacity of MnGdOP@PDA-PEG. Uv/Vis absorption curves of MB solution after treatment by (a) H2O2, (b) MnGdOP@PDA-PEG plus H2O2 at room temperature, and (c) MnGdOP@PDA-PEG plus H2O2 at 320 K. (d) Histogram analysis of the degradation of MB after
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different treatment (*p<0.05, **p < 0.01, n = 3/group).
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Fig 4. Photothermal performance of MnGdOP@PDA-PEG. (a) Temperature elevation curves and (b) relative infrared thermal images of MnGdOP@PDA-PEG solution with the concentration of 200 and 400 ppm under the irradiation of 808 nm laser with the power of 1 W/cm2 for 10 min, respectively. (c) Temperature elevation curves and (d) relative infrared thermal images of MnGdOP@PDA-PEG
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solution with the concentration of 200 and 400 ppm under the irradiation of 808 nm laser with the power of 2 W/cm2 for 10 min, respectively. (e) The absorption curves of MnGdOP@PDA-PEG solution with the concentration of 400 ppm before and after laser irradiation over three on/off cycles.
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(f) Photothermal heating curves of MnGdOP@PDA-PEG solution with the concentration of 400
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Fig 5. ·OH generation capacity of MnGdOP@PDA-PEG under laser irradiation. Uv/Vis absorption analyses of MB after degradation by MnGdOP@PDA-PEG mediated Fenton-like reaction (a) without laser, with (b) 1 W/cm2, and (c) 2 W/cm2 laser irradiation. (d) Degradation of MB due to
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the ·OH generated by different treatments for different times.
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Fig 6. In vitro cell assay analyses. Cell viability of U-87 MG cells treated with PBS, laser, free MnGdOP@PDA-PEG, and MnGdOP@PDA-PEG+laser at (a) pH 7.4 and (b) pH 6.5. (c) Fluorescence
images
of
cells
incubated
with
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fluorescence
probe
DCFH-DA
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(2’,7’-dichlorofluorescein diacetate) and different amount of MnGdOP@PDA-PEG with or without
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laser irradiation at pH 6.5. Scale bar is 200 µm. (d) Live/dead cell staining assays were analyzed by the fluorescence microscopy. Red, dead cells; green, live cells. Scale bar is 200 µm.
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Fig 7. Tumor Diagnosis in vivo and IR thermal imaging of tumor-bearing mice. (a) T1-weighted MR images of mice bearing subcutaneous tumor at 0, 0.5, 1, and 2 h after intravenous injection of
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MnGdOP@PDA-PEG (2 mg (Mn+Gd)/kg per mice weight), respectively. (b) Quantification of relative tumor contrast collected at different time after administration of MnGdOP@PDA-PEG. (c) Biodistribution of Gd ions in mice after intravenous injection of MnGdOP@PDA-PEG (n = 3/group).
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(d) IR thermographs of mice tumor under laser irradiation with the power of 1.5 W/cm2 for 0, 1, 2, 3,
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4, and 5 min with intravenously injection of saline or MnGdOP@PDA-PEG. (e) The heating curves of tumor treated with saline or MnGdOP@PDA-PEG with NIR laser irradiation (1.5 W/cm2, 5 min).
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Fig 8. In vivo therapeutic study. (a) Body weight change curves of the mice during treatment by saline, laser, free MnGdOP@PDA-PEG, and MnGdOP@PDA-PEG + laser, respectively. (b) Tumor growth curves after treated with saline, laser, free MnGdOP@PDA-PEG, and MnGdOP@PDA-PEG + laser, respectively. (c) Digital photographs of mice taken at 0 day before different treatment and 15 days after different treatment. Red arrows indicate the tumor. (d) Tumor H&E staining images of the mice with different treatment. Red arrows indicate the necrotic cells in tumor. Scale bar, 100 µm. (e) TUNEL staining of tumor tissue after different treatment. The TUNEL positive cells were monitored by staining the cells with fluorescein isothiocyanate. Scar bar, 100 µm.
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ACCEPTEDTOC MANUSCRIPT
S0142-9612(19)30478-8
DOI:
https://doi.org/10.1016/j.biomaterials.2019.119379
Article Number: 119379 Reference:
JBMT 119379
To appear in:
Biomaterials
Received Date: 26 January 2019 Revised Date:
16 July 2019
Accepted Date: 24 July 2019
Please cite this article as: Zhao Z, Xu K, Fu C, Liu H, Lei M, Bao J, Fu A, Yu Y, Zhang W, Interfacial engineered gadolinium oxide nanoparticles for magnetic resonance imaging guided microenvironmentmediated synergetic chemodynamic/photothermal therapy, Biomaterials (2019), doi: https:// doi.org/10.1016/j.biomaterials.2019.119379. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interfacial Engineered Gadolinium Oxide Nanoparticles for Magnetic ACCEPTED MANUSCRIPT Resonance Imaging Guided Microenvironment-Mediated Synergetic Chemodynamic/Photothermal Therapy
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Zhenghuan Zhao1*, Kai Xu2,3, Chen Fu1, Heng Liu2,3, Ming Lei1, Jianfeng Bao5, Ailing Fu1, Yang Yu1, and Weiguo Zhang2,3*
College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China.
2
Department of Radiology, Daping Hospital, Army Medical Center of PLA, Army Medical
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University, Chongqing 400010, China. 3
Chongqing Clinical Research Center for Imaging and Nuclear Medicine, Chongqing 400010, China.
4
College of Medical Technology and Engineering, Henan University of Science and Technology,
*email: [email protected]
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Luoyang 471000, China.
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[email protected]
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Abstract
Engineering interfacial structure of biomaterials have drawn much attention due to it can improve the diagnostic accuracy and therapy efficacy of nanomedicine, even introducing new moiety to construct theranostic agents. Nanosized magnetic resonance imaging contrast agent holds great
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promise for the clinical diagnosis of disease, especially tumor and brain disease. Thus, engineering its interfacial structure can form new theranostic platform to achieve effective disease diagnosis and therapy. In this study, we engineered the interfacial structure of typical MRI contrast agent, Gd2O3,
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to form a new theranostic agent with improved relaxivity for MRI guided synergetic
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chemodynamic/photothermal therapy. The synthesized Mn doped gadolinium oxide nanoplate exhibit improved T1 contrast ability due to large amount of efficient paramagnetic metal ions and synergistic enhancement caused by the exposed Mn and Gd cluster. Besides, the introduced Mn element endow this nanomedicine with the Fenton-like ability to generate ·OH from excess H2O2 in
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tumor site to achieve chemodynamic therapy (CDT). Furthermore, polydopamine engineered surface allow this nanomedicine with effective photothermal conversion ability to rise local temperature and accelerate the intratumoral Fenton process to achieve synergetic CDT/photothermal therapy (PTT).
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This work provides new guidance for designing magnetic resonance imaging guided synergetic
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CDT/PTT to achieve tumor detection and therapy.
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1. Introduction
Developments of multifunctional nanomaterials that integrate diagnosis and therapy into a single system may offer great advantages in disease treatment and have received significant interest in fields of biomedicine.[1-3] Engineering interfacial structure of biomaterials can improve the
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diagnostic accuracy or therapy efficacy of nanomedicine, and introduce new moiety to construct theranostic agents.[4, 5] Nanosized magnetic resonance imaging (MRI) contrast agent holds great promise for the clinical diagnosis of disease, especially tumor and brain disease, as the high spatial
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and temporal resolution of MRI. Thus, engineering its interfacial structure can form new theranostic
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platform to achieve effective disease diagnosis and therapy. Various T1 MRI contrast agents has been developed, including gadolinium based,[6, 7] iron based,[8-10] and manganese based agents.[11, 12] Because of its large amounts of unpaired electrons and long electronic relaxation time, Gd-based nanomaterials, especially gadolinium oxide (Gd2O3) nanoparticles, have been extensively explored
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as T1 contrast agent through controlling over the morphology and size. Based on the classical Solomon-Bloembergen-Morgan (SBM) theory, number of coordinating (q), proton residence lifetime (τM), and molecular tumbling time (τR) determine the contrast capacity of nano-scale Gd2O3.
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Ultra-small Gd2O3 nanoparticles with high surface-to-volume ratio have been proved to be
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high-performance T1 contrast agent due to the improvement on q value caused by the increase of efficient paramagnetic metal ions on surface.[13, 14] Therefore, optimization the crystal structure of Gd2O3 nanoparticles with high surface-to-volume ratio by morphology controllable synthesis may be the effective strategy to improve T1 relaxivity of Gd based T1 contrast agent.[15] Besides, expose two different paramagnetic ions may synergistically enhance protons T1 relaxation by improve the proton coordination and chemical exchange.[5] Therefore, further introducing another paramagnetic element with therapy effect may construct high-performance MRI-guided theranositc agent.
Mn-based materials, includingACCEPTED MnCl2, manganese (II) chelates,[16] manganese oxide (MnO) MANUSCRIPT nanoparticles,[17, 18] and MnO2 nanostructure[19] have been used to achieve T1 contrast imaging as its labile water exchange, high spin number, and long electronic relaxation time. Since manganese ion is an essential element in cell biology and often acts the cofactor for some crucial enzymes or
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receptors, optimization the crystal structure of Gd2O3 nanoparticles by partially replacement of Gd ions with Mn ions may endow it with high biocompatibility to tumor diagnosis in vivo. Furthermore, manganese ions show Fenton-like activity in acidic environment, endowing manganese ions to
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convert overproduced H2O2 in tumor site into reactive oxygen species (ROS).[20-23] Since ROS
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could damage the cancer cells and suppress the solid tumor growth, many ROS-based cancer treatment strategies have been developed, especially chemodynamic therapy (CDT).[24-26] Based on the specific tumor environment, CDT could in situ generate ROS through intratumoral Fenton reaction, which can reduce the harmful effect to the normal tissue. Besides, ROS could be cleared
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out by the antioxidation systems, further reducing side effect of CDT.[27] Therefore, introducing manganese ions into Gd2O3 nanocrystals may ensure it with the ability to achieve tumor therapy through CDT. Unfortunately, the CDT efficacy is hampered by the catalytic efficiency in vivo, thus
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application.
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improve the amount of hydroxyl radical (·OH) produced by CDT in vivo is important to its
The catalytic efficiency is determined by many physical parameter, especially temperature. Generally, rise of the temperature could improve the efficiency of Fenton-like reaction and increase the amount of ·OH.[28] Photothermal therapy (PTT) agent can convert the energy of near-infrared light into heat and increase the local temperature of tumor. Therefore, one can introduce a PTT moiety to improve CDT efficacy and achieve synergetic CDT/PTT. Polydopamine (PDA) generated by the self-polymerization of dopamine, one of the natural neurotransmitters, has been widely used as the PTT agent due to its easy to synthesis, high biocompatibility, and high photothermal
conversion efficiency.[29-32] Besides, dopamineMANUSCRIPT have recently evolved as preferred components to ACCEPTED efficiently transfer metal oxide nanoparticles into aqueous media in mild condition to form high hydrophilicity and loose structure on crystal surface, which are benefit to the chemical exchange between proton and T1 magnetic resonance imaging (MRI) contrast system.[11, 33] Thus, PDA is the
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good candidate as the PTT motif to develop MRI-guided synergetic CDT/PTT theranostic agent. Herein, we present a novel strategy to engineer the interfacial structure of Gd2O3 nanocrystals and construct PDA and polyethylene glycol coated Mn doped Gd2O3 nanoplate (MnGdOP@PDA-PEG)
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to achieve MRI-guided synergetic CDT/PTT. The prepared MnGdOP@PDA-PEG exhibits relatively
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higher T1 relaxivity than commercial T1 contrast agent (Gd-DTPA) and traditional Gd2O3 nanoplate. Besides, MnGdOP@PDA-PEG can catalyze H2O2 convert into ·OH, ensuring it as a potential CDT agent to kill tumor cells. Furthermore, the PDA coated surface endow it with the ability to effectively transform NIR energy into heat to rise the local temperature of tumor to accelerate the intratumoral
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Fenton process and achieve PTT. In vivo study indicates that MnGdOP@PDA-PEG exhibits sensitive solid tumor detection and can effectively suppress the tumor growth through synergetic CDT/PTT, thus showing that combine MRI contrast imaging, CDT, and PTT into one materials may
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provide clues to develop efficient anticancer theranostic agent.
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2. Experiment section 2.1 Chemical.
Oleylamine (tech 90%), Manganese(II) chloride tetrahydrate (tech 90%), Gadolinium (III) chloride hexahydrate (99.99%), and 1-octadecene (90%) were purchased from Alfa Aesar. Dopamine hydrochloride, L-Dopa, hexane, tetrahydrofuran, sodium oleate, sodium hydroxide, isopropanol, and ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methylene blue, 2’,7’-dichlorofluorescein diacetate, propidium iodide, and calcein
acetoxymethyl ester were purchased from Dalian MANUSCRIPT Meilun Biotechnology Co. Ltd. All chemicals were ACCEPTED used as received without further purification. 2.2 Synthesis of manganese doped gadolinium oxide nanoplate (MnGdOP). In a typical experiment, 0.18 g Gd-oleate and 0.07 g Mn-oleate was dissolved in 10 mL
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1-octadecene containing 1 mL oleylamine at room temperature. The mixture was degassed in vacuum for 30 min and backfilled with argon to remove any low volatile impurities and oxygen at room temperature. And then, we heated the reaction solution to reflux and kept at that temperature
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for 1.5 h. The resultant solution was then cooled to room temperature and mixed with 30 mL
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isopropanol to precipitate the nanoparticles. The nanoparticles were separated by centrifugation and washed 3 times with ethanol.
2.3 Preparation of polydopamine coated MnGdOP (MnGdOP@PDA) nanostructures. Typically, 30 mg dopamine, 10 mg L-dopa, and ~0.1 mmol nanoparticles were mixed in a mixture
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containing 10 mL of sodium hydroxide solution and 20 mL of tetrahydrofuran in air at room temperature. After stirring for 2 h, the PDA coated nanoparticles were collected by centrifugation and re-dispersed in distilled water for long-term storage at 4 °C.
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nanostructures.
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2.4 Preparation of polydopamine coated MnGdOP polyethylene glycol (MnGdOP@PDA-PEG)
MnGdOP@PDA-PEG were prepared by simply mixing PDA with OH-PEG-NH2 (M.W 2000) at a mass ratio of 1:2 at pH with the value of 9 under magnetic stirring at room temperature. After stirring for 24 h, the products were collected by centrifugation and re-dispersed in distilled water for long-term storage at 4 °C. 2.5 ·OH generation through MnGdOP@PDA-PEG based Fenton-like reaction. MnGdOP@PDA-PEG 0.2 mg were added in 10 µL H2O2 (1 M) and 1 mL MB solution (10 µM) with the pH value of 6.5. After incubation for 20 min at room temperature or 320 K, the mixture
solution was centrifuged to remove MnGdOP@PDA-PEG and measured the absorbance at 661 nm ACCEPTED MANUSCRIPT by UV-vis spectroscopy. 2.6 In vitro DCFH-DA assay. The generated ROS in tumor cell was investigated by intracellular ROS fluorescence probe
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2’,7’-dichlorofluorescein diacetate (DCFH-DA) at pH 6.5. The cells were seeded on a 6-well plate with a density of 1 × 105 cells per well, and incubated in the atmosphere of 5% CO2 at 37 °C for 24 h. The cells were then incubated with PBS or MnGdOP@PDA-PEG with the concentration of 50 or
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100 ppm for 6 h, and then treated each well without or with an 808 nm laser irradiation at a power
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density of 1 W/cm2 for 2 min. The cells were stained by DCFH-DA (10 µM) for 30 min. The fluorescence images were capture by fluorescence microscope (Nikon eclipse Ti-S). 2.7 In vitro synergistic CDT/PTT therapy.
The synergistic CDT/PTT therapy was evaluated through propidium iodide (PI) and calcein
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acetoxymethyl ester (calcein-AM) double staining. Cells were seeded on a 6-well plate with a density of 1 × 105 cells per well, and incubated in the atmosphere of 5% CO2 at 37 °C for 24 h. The cells were then incubated with MnGdOP@PDA-PEG with the concentration of 50 or 100 ppm for 6
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h at pH 6.5, and then irradiated on each well with an 808 nm laser at a power density of 2 W/cm2 for
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5 min. After incubation for another 6 h, PI and calcein-AM double staining were conducted. The fluorescence images were capture by fluorescence microscope (Nikon eclipse Ti-S). 2.8 In vivo synergistic CDT/PTT. Animal experiments were executed according to the protocol approved by institutional Animal Care and Use Committee of Southwest University. The subcutaneous tumor model was established by injection of U-87 MG cells into the subcutaneous tissue of nude mice (18-22 g). When the tumor reached ~ 0.5 cm in diameter, the mice bearing tumor were intravenously injected MnGdOP@PDA-PEG with the amount of 15 mg/kg of mice weight and irradiated with 808 nm laser
for 5 min with the power of 1.5 ACCEPTED W/cm2. Meanwhile, three control groups were used to assess the MANUSCRIPT therapy efficiency: (a) intravenously injection of 150 µL saline; (b) intravenously injection of 150 µL saline and irradiation with 808 nm laser for 5 min with the power of 1.5 W/cm2; and (c) intravenously injection of MnGdOP@PDA-PEG with the amount of 15 mg/kg of mice weight. The
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mice weights and tumor sizes were monitored during the treatment period. The tumor volumes were calculated by the equation of Vtumor = (a2 × b)/2 (a and b represent the maximum and minimum diameter of the tumor). Relative tumor volumes were calculated as V/Vo (V and Vo represent the
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tumor volume after treatment and initial tumor volume).
3. Results and discussion 3.1 Synthesis and characterization. We synthesized manganese dope Gd2O3 nanoplate (MnGdOP) by thermal decomposition of the mixture of manganese-oleate and gadolinium-oleate in 1-octadecene (ODE) containing oleylamine as surfactant. The transmission electron microscopy (TEM) images show that some as-prepared nanoparticles assemble into the stacked ribbons (Fig. 1a). This phenomenon clearly indicates that the morphology of as-prepared nanoparticles is plate.[34] Based on the face-to-face stacking, the
thickness of this plate was measured with the value of approximately 1.8 ± 0.3 nm (Fig. S1). Further ACCEPTED MANUSCRIPT high-resolution TEM (HRTEM) images of the perpendicular nanoplate shows the uniform lattice fringes cross the entire nanoplate with the lattice spacing of 2.7 Å, corresponding to the (400) planes of Gd2O3, revealing its good crystallinity (Fig. 1a). Additionally, plate morphology endues it with
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high surface-to-volume ratio and large amount of paramagnetic metal ions on the surface to achieve high T1 contrast ability. To confirm the co-existence of Gd and Mn species in this nanoplate, we conducted energy-dispersive X-ray element mapping (EDX mapping). EDX mapping images show
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that the Gd and Mn signals merge well, revealing the successful Mn dopant in the MnGdOP (Fig.
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1b). The molar percentage of Mn ions with respect to total metal ions for MnGdOP is approximately 16.7 %, determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). We then analyzed the crystallographic structure of the MnGdOP by X-ray powder diffraction (XRD) pattern. MnGdOP show typical mixed Gd2O3 (JCPDS no. 00-012-0797) and MnO (00-075-0625)
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diffractogram patterns (Fig. 1c). It should be noted that MnGdOP show a sharp and enhanced diffraction peak at 33.1 degree corresponding to (400) plane of Gd2O3, which could be ascribed to the face-to-face assembly of MnGdOP and further suggest the plate morphology of MnGdOP.[35, 36]
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Further X-ray photoelectron spectroscopy (XPS) analysis in Gd 3d5/2 shows a typical peak at binding
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energy of 1187 eV, which agrees with the value of Gd2O3 nanocrystals (Fig. 1d).[37] Compared to Gd 3d5/2 peak, the Mn 2p3/2 peak is measured at the binding energy of 640.5 eV, suggesting the oxidation of doped Mn ions is +2 (Fig. 1e).[38] The magnetic properties of MnGdOP nanostructure was investigated by the superconducting quantum interference device (SQUID) magnetometer at room temperature (300 K). Field-dependent magnetization curve (M-H) of MnGdOP is a straight smooth line without any coercivities and remanences, indicating its paramagnetic behavior at room temperature (Fig. 1f). The unsaturated magnetic moments of MnGdOP reach to 2.1 emu/g under magnetic field of 5 T at 300 K. Consistent
with the M-H curve, standard zero-field-cooling (ZFC) and field-cooling (FC) curve shows no ACCEPTED MANUSCRIPT magnetic transition below 300 K, further confirming its paramagnetic behavior at room temperature (Fig. 1g). Based on its paramagnetic behavior, MnGdOP may efficiently reduce the longitudinal relaxation of proton and achieve T1 contrast imaging.
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3.2 Formation of MnGdOP nanostructure. Synthesis of doped metal oxide nanostructure by introducing another metal oleate could result in the formation of anisotropic nanostructure, which may be caused by the ionizing and dissociating of
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the precursor into oleate (OL-) at high temperature.[39] To investigate the effect of different amount
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Mn-oleate (MnOL) on the formation of MnGdOP, we conducted a series of experiment at a fixed reaction temperature, time, and surfactant but systematically varied Mn-oleate amount. We obtained the nearly spherical Mn doped Gd2O3 by supplying the amount of Mn-oleate with the molar ratio of MnOL/Gd-oleate (GdOL) to 1:20 (Fig. S2a). This result indicates that the negligible amount of
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MnOL is insufficient to affect the equal absorption of oleylamine on various crystal planes, resulting in the formation of spherical nanocrystals. Surprisingly, nanostructures with the morphology of plate can be obtained by increasing the molar ratio of MnOL/GdOL (Fig. S2b,c). In addition, the yield of
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nanostructures with anisotropic morphology improved along with the increase of the MnOL/GdOL
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molar ratio. These results indicate that the free ionic OL- dissociated from MnOL may be able to selectively bind to Gd ions and induce the anisotropic growth of nanocrystals, leading to the formation of MnGdOP nanostructures. This mechanism can be further supported by the observation of the formation of nanoplate assembly, flower-liked nanostructure (Fig. S2d). To investigate the morphology evolution of this nanoplate, we carefully took aliquots from the reaction solution and analyzed the dynamic growth process of MnGdOP (Fig. S3). Irregular crystal nuclei are first formed after 15 min reaction, indicating that the monomer concentration is saturated and could overcome the energy barrier to accomplish the nucleation process under this reaction condition. With continuous
consumption of the monomer, small tripodal nanoplate formed with the thickness of about 1.5 nm. ACCEPTED MANUSCRIPT After aging at the same temperature, Ostwald ripening occurs and causes improvement of size and uniformity of nanoplate. Interestingly, the thickness of the nanoplate maintains the same during its growth process, demonstrating the formation of MnGdOP is based on the capping agent assistant
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process. 3.3 T1 MRI contrast ability.
To transfer MnGdOP into aqueous and accelerate manganese base Fenton-like reaction, we
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engineered MnGdOP nanostructure surface with dopamine in air and form polydopamine (PDA)
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shell. Further PEG modification was applied to improve its behavior in vivo and form PDA and PEG coated MnGdOP (MnGdOP@PDA-PEG).[40, 41] TEM image clearly shows the PDA shell on the surface of MnGdOP nanostructures, indicating the successful formation of MnGdOP@PDA structure (Fig. S4a). The surface charge of MnGdOP-PDA-PEG was ~-13.4 mV, which is benefit to
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decrease the protein absorption on the MnGdOP@PDA-PEG surface (Fig. S5). Dynamic light scattering (DLS) analysis indicated that MnGdOP@PDA-PEG was monodisperse in aqueous media without any clustering or aggregation (Fig. S4). To confirm the successful PDA coating, we
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performed fouriertransform infrared spectroscopy (FT-IR) and NIR spectra analyses. Compare to
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MnGdOP, MnGdOP@PDA and MnGdOP@PDA-PEG presented absorption peaks at around 1600 cm-1 assigned to the benzene skeleton vibrations of PDA (Fig. S6).[42] Besides, MnGdOP and MnGdOP@PDA-PEG revealed a broad absorption at NIR region in absorption spectra. More importantly, the absorption value of MnGdOP@PDA-PEG was higher than that of MnGdOP (Fig. S7). These results suggested the successful coating of PDA shell on MnGdOP. The amount of PDA coated on the surface of MnGdOP was approximately 12 %, which was assessed by thermogravimetric analysis (Fig. S8). To verify the MnGdOP@PDA-PEG nanostructure can be used as a contrast agent in T1-weighted contrast imaging, we utilized Gd-DTPA and gadolinium oxide
nanoplate (GdOP) as controls toACCEPTED investigate itsMANUSCRIPT T1 contrast ability (Fig. 2 and Fig. S9,10). We assessed the T1 contrast performance of all samples at 0.5 T and 1.5 T magnetic fields. The r1 values of MnGdOP@PDA-PEG, Gd-DTPA, and GdOP are 6.7, 4.1, and 4.2 mM-1s-1 at 0.5 T, respectively (Fig. 2a and Fig. S10). The r1 value of MnGdOP@PDA-PEG is higher than that of Gd-DTPA and
ions and synergistic enhancement caused
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GdOP. The elevated relaxivity could be attributed to large amount of efficient paramagnetic metal by the exposed Mn and Gd cluster on
MnGdOP@PDA-PEG surface. [5, 43-45] Consistent with the results at 0.5 T, MnGdOP@PDA-PEG
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exhibits obviously higher r1 value (5.3 mM-1s-1) than Gd-DTPA (3.8 mM-1s-1) and GdOP (3.3
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mM-1s-1) at 1.5 T as well (Fig. 2c and Fig. S10). These results prove that interfacial engineering of gadolinium oxide nanoparticles is an effective strategy to construct T1 contrast agent with high performance. Due to the relative high T1 relaxivity and low r2/r1 ratio, T1-weighted phantom analyses reveal that MnGdOP@PDA-PEG exhibits clearly brightness gradients along with the increase of
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metal concentration (Fig. 2b,d and Fig. S11). More importantly, the MnGdOP@PDA-PEG offers high quality contrast imaging with brighter signal than Gd-DTPA and GdOP at the same
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concentration, revealing the high sensitivity of MnGdOP@PDA-PEG in T1-weighted contrast
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The stability of nanomaterials can affect its performance and behavior in vivo, which is the crucial issue to determine its efficiency in biomedical imaging field.[46, 47] We analyzed the stability of MnGdOP@PDA-PEG in long term. The hydrodynamic diameter of MnGdOP@PDA-PEG remained unaltered after storage in phosphate buffer saline (PBS) for one month (Fig. S12). Moreover, the leakage ratio of Gd and Mn are blow 0.2 % after storage in PBS for one month. Since the main toxicity of Gd based contrast agent is caused by the released Gd ions, the negligible leakage of Gd ions in one month suggests the high biocompatibility of MnGdOP@PDA-PEG and low side effect.
Relaxivities analyses indicate that the r1 valuesMANUSCRIPT of MnGdOP@PDA-PEG stored in PBS for one ACCEPTED month are highly preserved at 0.5 and 1.5 T (Fig. S12) 3.4 In vivo liver MRI. Based on the high stability, we utilized MnGdOP@PDA-PEG to assist liver MR imaging to
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evaluate its contrast capacity in vivo. Accordingly, GdOP was used for comparison. T1-weighted contrast images were acquired before and after intravenous injection of MnGdOP@PDA-PEG or GdOP with the dosage of 2 mg magnetic ions/kg body weight of mice on a 7 T MRI scanner. After
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intravenous injection, we indeed observed signal increase in liver region. More importantly, the T1
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signal enhancement in liver region of mice treated by MnGdOP@PDA-PEG is much stronger than that of the mice treated by GdOP (Fig. 2e). Signal to noise ratio (SNR) is calculated by choosing liver as the region of interests (ROIs) to quantify the contrast enhancement (Fig. 2f and Table S1). It appears that MnGdOP@PDA-PEG exhibit significantly higher contrast than GdOP, suggesting
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MnGdOP@PDA-PEG could be a candidate to assist to obtain comprehensive information in T1-weighted MRI imaging and achieve tumor diagnosis. 3.5 ·OH generation capacity under laser irradiation.
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To investigate the MnGdOP@PDA-PEG activity to convert H2O2 into ·OH in acidic environment,
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we detect ·OH through the degradation of methylene blue (MB).[20] The UV-Vis spectra analyses indicated that the H2O2 do not affect the absorption of MB at acidic environment (Fig. 3a). However, a decrease of maximum absorbance peak for MB was observed after incubating MB with H2O2 and MnGdOP@PDA-PEG for 20 min at pH 6.5, which could be ascribed to the ·OH generation through Fenton-like reaction (Fig. 3b). These results clearly indicate that MnGdOP@PDA-PEG could catalyze H2O2 convert into ·OH in acidic environment, ensure it as a potential chemodynamic therapy (CDT) agent to kill tumor cells. It worthwhile to note that the leakage of Mn ions from MnGdOP@PDA-PEG at pH 6.5 increased compare to that at pH 7.4, which may be ascribed to the
Fenton-like reaction (Fig. S13). ACCEPTED To determine MANUSCRIPT whether the rise of temperature can improve the Fenton-like reaction activity of MnGdOP@PDA-PEG, we incubated MB with H2O2 and MnGdOP@PDA-PEG at 320 K in acidic environment for 20 min. As expected, we observed a more remarkable decrease of maximum absorbance peak for MB compare to incubation with
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MnGdOP@PDA-PEG at room temperature, indicating temperature rising could accelerate the MnGdOP@PDA-PEG based Fenton-like reaction (Fig. 3c). Further quantitative analyses indicated that the maximum absorption peak for MB reduced to 99.3%, 92.7%, and 83.7% after incubation
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with only H2O2, H2O2 and MnGdOP@PDA-PEG, and H2O2 and MnGdOP@PDA-PEG at 320 K,
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respectively (Fig. 3d). These results demonstrate that rising temperature could improve the efficiency of MnGdOP@PDA-PEG based Fenton-like reaction and enhance ·OH generation, which is benefit to the further CDT in vitro and in vivo.
We investigated the photothermal conversion capacity of MnGdOP@PDA-PEG to evaluate its
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capacity on temperature rise under near-infrared (NIR) irradiation. The absorption spectra analyses indicate that MnGdOP@PDA-PEG show the broad absorption from visible to NIR, indicating the potential of MnGdOP@PDA-PEG to be used as a photothermal conversion agent to rise local
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temperature in CDT (Fig. S7). Consequently, we assessed the photothermal conversion capacity of
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MnGdOP@PDA-PEG through exposure its aqueous solution with different concentrations to 808 nm laser with different power density (Fig. 4). The heating curves and infrared thermal images indicate that the solution containing MnGdOP@PDA-PEG show obviously larger temperature increase than pure water after irradiation (Fig. 4a,b). The temperature rising of aqueous solution containing MnGdOP@PDA-PEG with the concentration of 200 and 400 ppm can reach 11.0 and 15.1 °C under the laser with the power density of 1 W/cm2. These results prove that MnGdOP@PDA-PEG can effectively absorb 808 nm laser energy and cause substantial overheating. In addition, the heating effect of aqueous solution containing MnGdOP@PDA-PEG increased with
the increase of laser power density (Fig. 4c,d). Along with the rise of the power density, the ACCEPTED MANUSCRIPT temperatures increasement of the aqueous solution containing MnGdOP@PDA-PEG with the concentration of 400 ppm increased to 20.0 °C. To quantitative the photothermal conversion capacity, we calculated the photothermal conversion efficiency (η) of MnGdOP@PDA-PEG
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according to the previous work.[48, 49] The η value of MnGdOP@PDA-PEG was determined to be ~21.5 %, which is comparable to other PTT agent (Fig. S14 and Table S2). These results further confirm that the heating effect is mainly caused by the photothermal conversion agent,
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MnGdOP@PDA-PEG, and show the potential of MnGdOP@PDA-PEG as photothermal conversion
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agent to rise local temperature in CDT and act as a PTT agent in tumor therapy. Since the photostability of photothermal agent can affect its performance and behavior in vivo, we analyzed the photostability of MnGdOP@PDA-PEG during several irradiation cycles.[32] The absorption spectra shows that the MnGdOP@PDA-PEG exhibits the similar curves in NIR region before and
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after three irradiation cycles (Fig. 4e). Moreover, we observed that the solution temperature can reach to the same level on the assist by MnGdOP@PDA-PEG, even after three irradiation cycles (Fig. 4f). These results indicate the high photostability of MnGdOP@PDA-PEG and show the
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potential of MnGdOP@PDA-PEG to efficiently convert NIR laser power into heat reproducibly.
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Since MnGdOP@PDA-PEG exhibit good photothermal conversion ability, we evaluated the Fenton-like reaction activity of MnGdOP@PDA-PEG under laser irradiation. We incubated MB with MnGdOP@PDA-PEG and H2O2 with or without laser irradiation. UV-Vis spectra analyses indicated that the maximum absorption peak for MB gradually decrease with the extension of time, indicating the Fenton-like reaction activity of MnGdOP@PDA-PEG (Fig. 5a). Interestingly, the decrease of maximum absorption peak for MB significantly increased under 1 W/cm2 laser irradiation, indicating more ·OH generation compare to without laser irradiation group (Fig. 5b). These results could be ascribed to that MnGdOP@PDA-PEG convert the energy of NIR into heat to
increase the solution temperatureACCEPTED and MnGdOP@PDA-PEG based Fenton-like reaction activity. MANUSCRIPT Moreover, the maximum absorption peak of MB could further decrease with the increase of laser power density (Fig. 5c). Compared to the solution without laser irradiation, the maximum absorption peak for MB reduction decrease from 92.7 % to 86.0 % (1 W/cm2) and 82.2 % (2 W/cm2) (Fig. 5d).
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These results demonstrate that the laser irradiation could accelerate the MnGdOP@PDA-PEG based Fenton-like reaction and enhance ·OH generation, which may improve the CDT efficacy in vivo. To exclude the activity improvement is caused by laser, we assessed the MB degradation under laser
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maintains the same after laser irradiation (Fig. S15).
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irradiation without MnGdOP@PDA-PEG. It appears that the absorption curve of MB almost
3.6 In vitro cell assay analyses.
The synergistic CDT/PTT therapy effect at cellular level was investigated by incubation tumor cells with MnGdOP@PDA-PEG and exposure to 808 nm NIR laser at 2 W/cm2 for 5 min. We did
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not observe apparent cytotoxicity after incubation tumor cells with MnGdOP@PDA-PEG without laser irradiation, demonstrating the acceptable biocompatibility of MnGdOP@PDA-PEG (Fig. S16). Whereas, the cell viability remarkably decreased when the laser was introduced (Fig. 6a). These
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results suggest that MnGdOP@PDA-PEG can efficiently convert NIR laser power into heat and kill
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the cancer cell. It should be noted that MnGdOP@PDA-PEG show negligible CDT effect, which could be ascribe that the activity of Fenton-like reaction at pH 7.4 is low.[50] Thus, we incubated U-87 MG with MnGdOP@PDA-PEG with or without laser at pH with the value of 6.5, which was used to mimic the extracellular environment of tumor. It appears that the cell viability reduced to 75.8 % when incubation it with MnGdOP@PDA-PEG with the concentration of 100 ppm (Fig. 6b). Since the slight acidic environment could not affect the viability of tumor cells, this result indicates that MnGdOP@PDA-PEG could be used as a potential CDT agent (Fig. S17).[28] The generated ROS
in
tumor
cell
was
investigated
by
intracellular
ROS
fluorescence
probe
2’,7’-dichlorofluorescein diacetateACCEPTED (DCFH-DA) MANUSCRIPT at pH 6.5 (Fig. 6c).[51, 52] Compared to the PBS treated group, slight green emission could be observed after adding MnGdOP@PDA-PEG into media. It should be noted that the intensity of green emission increased with the amount elevation of MnGdOP@PDA-PEG, which could be ascribed to the increase of generated ·OH along caused by
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the rising of MnGdOP@PDA-PEG. Furthermore, cells treated with MnGdOP@PDA-PEG and laser irradiation showed obvious green emission, suggesting sufficient ·OH were produced to achieve CDT on the assistant of laser induced temperature increase. Additionally, the further in vitro
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synergistic CDT/PTT effect was elevated through propidium iodide (PI) and calcein acetoxymethyl
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ester (calcein-AM) double staining (Fig. 6d).[53, 54] Florescence images show that the MnGdOP@PDA-PEG with NIR laser irradiation could kill the cancer cells more effectively compare to treat cancer cell with only laser or MnGdOP@PDA-PEG. These results clearly reveal the excellent synergistic CDT/PTT effect of MnGdOP@PDA-PEG to kill cancer cells.
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3.7 Tumor diagnosis and therapy.
To further assess the in vivo diagnostic and therapeutic effect of MnGdOP@PDA-PEG, we performed the in vivo study. We firstly injected MnGdOP@PDA-PEG intravenously into mice
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bearing tumors and conducted T1-weighted MRI. The T1-weighted MR images show that T1 signal of
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tumor region gradually increase over time and show sufficient signal to detect tumor (Fig. 7a). To quantify the contrast enhancement, we calculated SNR according to the T1-weighted images. It appears that the ∆SNR values of T1-weighted images at tumor are 5.23, 8.37, and 15.14 % at 0.5, 1, and 2 h post injection, respectively (Fig. 7b). The T1 signal enhancement demonstrates that MnGdOP@PDA-PEG is an effective contrast agent for accurate tumor detection. We further investigate the biodistribution of MnGdOP@PDA-PEG to confirm the signal change is caused by the accumulation of MnGdOP@PDA-PEG in tumor. It appears that the tumor uptake of MnGdOP@PDA-PEG is about 4.60 % ID/g, suggesting that the T1 signal change is caused by the
accumulation of MnGdOP@PDA-PEG (Fig. 7c).MANUSCRIPT We then performed in vivo study to evaluate the ACCEPTED therapeutic effect by intravenous injection of MnGdOP@PDA-PEG into tumor with 808 nm laser irradiation (denoted as MnGdOP@PDA-PEG+Laser). Meanwhile, we chose the mice treated by injection of Saline (denoted as Saline), injection of MnGdOP@PDA-PEG without laser irradiation
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(denoted as MnGdOP@PDA-PEG), and irradiation with laser (denoted as Laser only) as controls. To analyze the in vivo photothermal conversion capacity of MnGdOP@PDA-PEG, we recorded the temperature change at tumor area by infrared thermal mapping apparatus. The thermographic images
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reveal that the temperatures at tumor site raise rapidly for the MnGdOP@PDA-PEG+Laser group
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(Fig. 7d). After irradiation for 5 min, the tumor temperature of MnGdOP@PDA-PEG+Laser group can reach to 49.8 °C, which suggest MnGdOP@PDA-PEG can irreversibly damage tumor tissues.[55, 56] However, the maximum temperature of Laser only group is just about 39 °C under the same laser power density, demonstrating the safety of NIR laser for normal tissue (Fig. 7e).
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During the treatment period, the body weights of all mice nearly maintained at the same level, indicating the minimal side effect of MnGdOP@PDA-PEG (Fig. 8a). Tumor growth curves and digital photographs indicate that indicated that MnGdOP@PDA-PEG can inhibit tumor growth due
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to mild MnGdOP@PDA-PEG based CDT (Fig. 8b,c). Notably, the tumor growth rate of
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MnGdOP@PDA-PEG+Laser group was almost inhibited, revealing the high synergistic CDT/PTT therapy efficiency. On the contrary, the tumors in mice of Saline and Laser only groups grew rapidly (Fig. 8b,c). Further hematoxylin and eosin (H&E) analyses indicate that the tumor of MnGdOP@PDA-PEG group show slight necrotic response after treatment due to the CDT effect. However, tumor of Saline and Laser group show their typical structure. Notably, the tumor of MnGdOP@PDA-PEG+laser group show more obvious nuclear pyknosis and cancer necrosis compare to MnGdOP@PDA-PEG group, indicating its effective synergistic CDT/PTT under laser irradiation (Fig. 8d). Consistent with the H&E staining, TUNEL staining images of the tumor from
MnGdOP@PDA-PEG and MnGdOP@PDA-PEG+laser group show obvious green florescence, ACCEPTED MANUSCRIPT indicating typical apoptosis response. Whereas, Saline and Laser only group show negligible green florescence signal (Fig. 8e). These results reveal that MnGdOP@PDA-PEG is suitable to be used as a synergistic CDT/PTT agent to achieve tumor therapy. Clearance and systemic toxicity are
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important to the nanoparticles for their biomedical application. Compare to the T1 signal of liver and tumor at 2 h after intravenous injection, the T1 signal of liver and tumor at 4 h after intravenous injection showed an obviously decrease (Fig. S18). This signal decrease indicate the clearance of
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MnGdOP@PDA-PEG over time. To further assess systemic toxicity in vivo, we sacrificed all mice
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and conducted H&E staining after treatment. It appears that the main organs of all mice show their typical structure phenotypes without any obvious pathological lesion, including necrosis and inflammation (Fig. S19).[57] These results reveal the clinic potential of MnGdOP@PDA-PEG on
4. Conclusions
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tumor diagnosis and therapy.
In summary, a novel strategy to engineer the interfacial structure of Gd2O3 to construct
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MRI-guided CDT/PTT theranostic agent, MnGdOP@PDA-PEG, has been developed. Due to the
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unique surface structure, it exhibits relatively higher T1 relaxivity than commercial T1 contrast agent and traditional nanoscale T1 contrast agent and achieve sensitive tumor detection in vivo. Due to the introducing of manganese ions, MnGdOP@PDA-PEG shows Fenton-like reaction activity, endowing it to catalyze H2O2 convert into ·OH to accomplish CDT to kill tumor cells. Furthermore, its PDA surface endow it can effectively transform NIR energy into heat to rise the local temperature of tumor, which can accelerate the intratumoral Fenton process and even ensure it with the PTT capacity. Based on the synergetic CDT/PTT, MnGdOP@PDA-PEG can effectively cause the death of tumor cells and suppress the tumor growth in vivo. The present work may not only provide
theranostic agent to assist tumor imaging and therapy, but also give an avenue to develop theranostic ACCEPTED MANUSCRIPT
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agent combined MRI, CDT, and PTT in single platform to treat cancer.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (81601607, 81871421, and 81601470), Research Funds for the Central Universities (XDJK2016C182), Open Research Fund of State Key Laboratory of Molecular Vaccinology and Molecular Diagnosticsh
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(2016KF02), and Postdoctoral Science Special Foundation of Chongqing (Xm2017018).
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[57] Z. Zhao, X. Wang, Z. Zhang, H. Zhang, H. Liu, X. Zhu, H. Li, X. Chi, Z. Yin, J. Gao, Real-Time Monitoring ACCEPTED of Arsenic Trioxide Release and Delivery by ActivatableMANUSCRIPT T1 Imaging, ACS Nano 9 (2015) 2749-2759.
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Scheme 1. Schematic cartoon illustrates the MRI-guided synergetic CDT/PTT tumor therapy by
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injection of MnGdOP@PDA-PEG nanotheranostic agent.
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Fig 1. Characterization of MnGdOP nanostructures. (a) TEM and HRTEM (insets) images of MnGdOP, with the lattice spacing distance of 2.7 Å, corresponding to the (400) planes of Gd2O3. (b) EDX mapping analysis. EDX mapping images of the MnGdOP, indicating that the homogeneous Gd
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and Mn signals merge well and reveal the successful Mn dopant in the MnGdOP. (c) XRD patterns of MnGdOP. The XRD pattern show the typical mixed Gd2O3 (JCPDS no. 00-012-0797) and MnO (00-075-0625) diffractogram patterns. XPS analyses of MnGdOP in (d) Gd 3d5/2 and (e) Mn 2p3/2,
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respectively. (f) The smooth M-H curve of MnGdOP measured at 300 K using a superconducting
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quantum interference device magnetometer. The M-H curve of MnGdOP is a straight smooth line without any coercivities and remanences, indicating its paramagnetic behavior at room temperature. (g) ZFC/FC curve of MnGdOP measured under an applied magnetic field at 50 Oe, indicating its paramagnetic behavior at room temperature.
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Fig 2. T1 MRI contrast of MnGdOP@PDA-PEG. (a) The analysis of relaxation rate R1 (1/T1) vs magnetic ions concentration for MnGdOP@PDA-PEG, Gd-DTPA, and GdOP nanoparticles and (b) relative T1-weighted MR images at 0.5 T. (c) The analysis of relaxation rate R1 (1/T1) vs magnetic
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ions concentration for MnGdOP@PDA-PEG, Gd-DTPA, and GdOP nanoparticles and (d) relative T1-weighted MR images at 1.5 T. (e) T1-weighted MR images of mice at 0, 0.5, 1, and 2 h after
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intravenous injection of MnGdOP@PDA-PEG (upper) and GdOP (lower). (f) Quantification of
GdOP.
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relative liver contrast collected at different time after administration of MnGdOP@PDA-PEG and
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Fig 3. ·OH generation capacity of MnGdOP@PDA-PEG. Uv/Vis absorption curves of MB solution after treatment by (a) H2O2, (b) MnGdOP@PDA-PEG plus H2O2 at room temperature, and (c) MnGdOP@PDA-PEG plus H2O2 at 320 K. (d) Histogram analysis of the degradation of MB after
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different treatment (*p<0.05, **p < 0.01, n = 3/group).
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Fig 4. Photothermal performance of MnGdOP@PDA-PEG. (a) Temperature elevation curves and (b) relative infrared thermal images of MnGdOP@PDA-PEG solution with the concentration of 200 and 400 ppm under the irradiation of 808 nm laser with the power of 1 W/cm2 for 10 min, respectively. (c) Temperature elevation curves and (d) relative infrared thermal images of MnGdOP@PDA-PEG
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solution with the concentration of 200 and 400 ppm under the irradiation of 808 nm laser with the power of 2 W/cm2 for 10 min, respectively. (e) The absorption curves of MnGdOP@PDA-PEG solution with the concentration of 400 ppm before and after laser irradiation over three on/off cycles.
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(f) Photothermal heating curves of MnGdOP@PDA-PEG solution with the concentration of 400
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Fig 5. ·OH generation capacity of MnGdOP@PDA-PEG under laser irradiation. Uv/Vis absorption analyses of MB after degradation by MnGdOP@PDA-PEG mediated Fenton-like reaction (a) without laser, with (b) 1 W/cm2, and (c) 2 W/cm2 laser irradiation. (d) Degradation of MB due to
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the ·OH generated by different treatments for different times.
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Fig 6. In vitro cell assay analyses. Cell viability of U-87 MG cells treated with PBS, laser, free MnGdOP@PDA-PEG, and MnGdOP@PDA-PEG+laser at (a) pH 7.4 and (b) pH 6.5. (c) Fluorescence
images
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with
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fluorescence
probe
DCFH-DA
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(2’,7’-dichlorofluorescein diacetate) and different amount of MnGdOP@PDA-PEG with or without
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laser irradiation at pH 6.5. Scale bar is 200 µm. (d) Live/dead cell staining assays were analyzed by the fluorescence microscopy. Red, dead cells; green, live cells. Scale bar is 200 µm.
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Fig 7. Tumor Diagnosis in vivo and IR thermal imaging of tumor-bearing mice. (a) T1-weighted MR images of mice bearing subcutaneous tumor at 0, 0.5, 1, and 2 h after intravenous injection of
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MnGdOP@PDA-PEG (2 mg (Mn+Gd)/kg per mice weight), respectively. (b) Quantification of relative tumor contrast collected at different time after administration of MnGdOP@PDA-PEG. (c) Biodistribution of Gd ions in mice after intravenous injection of MnGdOP@PDA-PEG (n = 3/group).
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(d) IR thermographs of mice tumor under laser irradiation with the power of 1.5 W/cm2 for 0, 1, 2, 3,
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4, and 5 min with intravenously injection of saline or MnGdOP@PDA-PEG. (e) The heating curves of tumor treated with saline or MnGdOP@PDA-PEG with NIR laser irradiation (1.5 W/cm2, 5 min).
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Fig 8. In vivo therapeutic study. (a) Body weight change curves of the mice during treatment by saline, laser, free MnGdOP@PDA-PEG, and MnGdOP@PDA-PEG + laser, respectively. (b) Tumor growth curves after treated with saline, laser, free MnGdOP@PDA-PEG, and MnGdOP@PDA-PEG + laser, respectively. (c) Digital photographs of mice taken at 0 day before different treatment and 15 days after different treatment. Red arrows indicate the tumor. (d) Tumor H&E staining images of the mice with different treatment. Red arrows indicate the necrotic cells in tumor. Scale bar, 100 µm. (e) TUNEL staining of tumor tissue after different treatment. The TUNEL positive cells were monitored by staining the cells with fluorescein isothiocyanate. Scar bar, 100 µm.
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