Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery

Journal of Alloys and Compounds xxx (xxxx) xxx

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Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery Junwei Zhao a, c, 1, Zhiping Zhang c, 1, Xin Wang b, c, * a

Materials Science and Engineering School & Henan Key Laboratory of Special Protective Materials, Luoyang Institute of Science and Technology, Luoyang, 471023, PR China b Henan Key Laboratory of Photovoltaic Materials, Henan University, Kaifeng, 475004, PR China c Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 January 2019 Received in revised form 17 November 2019 Accepted 21 November 2019 Available online xxx

The combination of diagnosis and treatment is considered as a promising strategy to improve the efficiency of cancer treatment. Here, the authors report an intelligent theranostic nanoplatform that combines diagnosis and chemotherapy based on the NaMnF3 nanoparticles (NPs). Poly (acrylic acid) (PAA) modified NaMnF3 NPs (PAA-NaMnF3 NPs) were synthesized by a facile and low cost reaction method. Doxorubicin hydrochloride (DOX)-loaded PAA-NaMnF3 (PAA-NaMnF3@DOX) hybrid nanostructures have been successfully developed via electrostatic interaction for dual-functional magnetic resonance imaging (MRI) and pH-responsive drug delivery. The obtained PAA-NaMnF3@DOX hybrid nanostructures could not only act as T1 and T2-weighted contrast agents for MRI as a result of the existence of Mn2þ ions but also exhibit a pH-responsive drug releasing behavior, which brings an obvious cytotoxicity to cancer cells via MTT assay. The endocytosis process of PAA-NaMnF3@DOX hybrid nanostructures was further evaluated using optical microscopy. These results demonstrate that the developed PAA-NaMnF3@DOX hybrid nanostructures as a multifunctional theranostic nanoplatform could serve as imaging contrast agents and drug delivery system for cancer imaging and therapy. © 2019 Elsevier B.V. All rights reserved.

Keywords: NaMnF3 Hybrid nanostructure DOX MRI Drug delivery

1. Introduction With the rapid development of materials science and nanotechnology, various hybrid nanostructures have been developed based on functionalized nanoparticles (NPs) due to their many advantages, such as small size, stable chemical properties, low toxicity and side effects, improved cell specific recognition ability, and easy surface modification [1e6]. In the past decades, nanotechnology has been extensively applied to improve the cure rate of cancers [2,5]. Nowadays, many functional nanomaterials have been used as novel drug carriers for the study of drug controlled release, including liposomes [3], polymer nanomaterials [7], gold NPs [8], magnetic NPs [9], mesoporous silica NPs [10] and upconversion NPs [11]. The construction of drug carriers for external stimulus

* Corresponding author. Henan Key Laboratory of Photovoltaic Materials, Henan University, Kaifeng, 475004, PR China. E-mail addresses: [email protected] (J. Zhao), [email protected] (X. Wang). 1 These authors contributed equally.

response is one aspect of the application of nanomaterials. Stimuliresponsive NPs for controlled drug delivery system have the ability to respond to external stimuli such as temperature, light irradiation, redox reagents, pH, enzymes, and ionic strength [12e21]. Among these drug carriers, pH-responsive drug delivery systems have shown great advantages and attracted much attention because the pH values in tumors and inflammatory tissues are significantly lower than those in blood and normal tissues [22e24]. In the past years, many strategies for constructing multifunctional drug carriers based on nanomaterials and organic polymers have been developed [25e28]. The functionalization of NPs with some polyelectrolyte layers enables pH-responsive encapsulation and release of drug molecules [29e31]. Selecting suitable polymers is the key factor to construct controlled drug delivery system based on functionalized NPs [32]. As a member of synthetic polypeptide family, Poly (acrylic acid) (PAA) that is completely biodegradable seems to have considerable advantages over other polymers due to its protein-like structure and has become an attractive candidate for drug carriers due to its reduced toxicity, antigenicity and immunogenicity [26,27]. PAA is usually used to replace the original hydrophobic ligands on the surface of nanocrystals to achieve the

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Please cite this article as: J. Zhao et al., Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery, Journal of Alloys and Compounds, https://doi.org/10.1016/j.jallcom.2019.153142

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phase transfer for water-soluble nanocrystals [33,34]. Therefore, the PAA-modified NPs can be uniformly dispersed in aqueous solutions [35]. After grafting onto the NPs, the PAA molecules with the abundant carboxyl groups not only make the NPs hydrophilic, but also could effectively load some drugs through electrostatic interaction. Because PAA is a pH responsive hydrophilic polymer, the release of DOX from drug delivery system can be triggered under acidic conditions [35]. As a pH-sensitive nanovalve, the PAAmodified NaYF4:Yb,Er upconversion NPs with dual functions of drug delivery and release have been developed for loading drug molecules [33]. Wang et al. have reported a multifunctional theranostic agent composed of ultrasmall PAA-functionalized Ni0.85Se NPs [35]. The multifunctional PAA-modified lanthanidedoped GdVO4 nanocomposites have also been constructed by filling PAA hydrogel into GdVO4 hollow spheres via photoinduced polymerization [36]. To develop controlled drug delivery system based on NPs with dual capability of simultaneous magnetic resonance imaging (MRI) and drug delivery, the co-encapsulation or conjugating magnetic NPs into carriers along with drugs become an important strategy. The PAA-functionalized Fe3O4 NPs have been developed for multimodal cancer imaging (magnetic resonance, computed X-ray tomography and fluorescence imaging) and targeted chemotherapy or chemo-thermal therapy [37,38]. Recently, the Mn2þ-based nanocrystals have been considered as ideal magnetic materials to achieve MRI because Mn ion has five unpaired d electrons [39e41]. Up to now, various Mn2þ-based nanocrystals have been reported, such as MnF2, KMnF3 and NaMnF3 [42e45]. In our previous work, we have developed a smart ‘all-in-one’ platform that conveniently combined chemo- and photothermal therapies based on ultrasmall CuS-PAA NPs [46], and constructed pHtriggered drug delivery systems based on upconversion NPs and magnetic NPs [25,47]. To our knowledge, the functionalized NaMnF3 NPs have been hardly reported for simultaneous pHresponsive drug nanocarries and MRI contrast agents. In this study, we reported a pH-responsive drug delivery system based on NaMnF3 NPs in combination with PAA polymer and doxorubicin hydrochloride (DOX). The well-known DOX was selected as anticancer drug, which is a hydrophilic molecule and can pass through biological barriers [48]. The PAA polymers were first coupled to the surface of NaMnF3 NPs by a ligand exchange process. DOX molecules were then absorbed on the surface of PAAmodified NaMnF3 (PAA-NaMnF3) NPs via electrostatic interaction. Moreover, the DOX-loaded PAA-NaMnF3 (PAA-NaMnF3@DOX) NPs exhibited a pH-triggering drug release behavior. The in vitro cellular cytotoxicity assay was performed to assess the biocompatibility of PAA-NaMnF3 NPs and the cytotoxic effect of PAA-NaMnF3@DOX hybrid nanostructures on cancer cells. The obtained PAANaMnF3@DOX hybrid nanostructures could act as T1 and T2weighted contrast agents for MRI. Our work highlights a facile fabrication for a drug delivery system based on NaMnF3 NPs, which provides a new insight into the effective diagnosis and treatment of cancers. 2. Materials and methods 2.1. Materials and reagents Ammonium fluoride (NH4F, 98%), sodium hydroxide and manganese acetate tetrahydrate (Mn(Ac)2$4H2O) were purchased from the Sinopharm Chemicals Reagent Co. (Shanghai, China). Poly (acrylic acid) (PAA, Mw ¼ 1800), 1-octadecene (ODE, 90%), oleic acid (OA, 90%), 3-[4, 5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide and anhydrous ethanol were purchased from Sigma-Aldrich. Doxorubicin hydrochloride (DOX, 99%) was obtained from Shanghai Sangon Biotech Co. Ltd. Fetal bovine

serum (FBS), RPMI-1640 cell culture medium, and Penicillinstreptomycin solution were supplied by Gibco Life Technologies. All other reagents were purchased from the Sinopharm Chemicals Reagent Co. (Shanghai, China). Deionized water (DI water, Milli-Q 18.2 MU cm, Millipore System Inc.) was used in the preparation of all aqueous solutions. A human cervical cell line (HeLa cell) was provided by American Type Culture Collection (ATCC). The HeLa cells were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum (FBS) and 100 IU/mL penicillin-streptomycin and incubated at 37  C in a humidified incubator containing 5% CO2. All the chemicals were of analytical grade and used without further purification. 2.2. Synthesis of OA-capped NaMnF3 nanocrystals Mn(Ac)2$4H2O (1.0 mmol) were added to a flask containing the mixture of ODE (15 mL) and OA (6 mL) under vigorous stirring at room temperature. The resulting mixture was degassed by a vacuum pump and then heated to 160  C for 0.5 h. After the mixed solution was cooled to room temperature, a methanolic solution (10 mL) of NaOH (1.0 mmol) and NH4F (3.0 mmol) was injected into the flask. The mixture was stirred at 60  C for 30 min and then purged by N2 at 100  C for 20 min. Subsequently, the temperature was raised to 300  C and kept for 45 min under nitrogen atmosphere. Finally, the reaction system was cooled to room temperature. The as-prepared OA-capped NaMnF3 (OA-NaMnF3) NPs were collected by centrifugation, washed with ethanol and hexane for three times, and finally re-dispersed in chloroform or toluene for further modification. 2.3. Modification of NaMnF3 nanocrystals by PAA The prepared NaMnF3 NPs were modified using PAA in diethylene glycol (DEG) solvent by a robust and versatile ligand exchange method, in which the hydrophilic PAA molecules replace the original hydrophobic OA ligands at elevated temperatures. Briefly, a solution of PAA (3 mL, 1.0%) in DEG (30 mL) was heated to 110  C to form a clear solution in the flask. The 100 mg of OANaMnF3 NPs dispersed in toluene (20 mL) was mixed with the above PAA solution, and the temperature was maintained at 110  C for 1 h under a nitrogen atmosphere. The mixture was then heated to 240  C for 1.5 h. The resulting solution was cooled to room temperature, and the NaMnF3 NPs was precipitated by the addition of ethanol. The prepared PAA-NaMnF3 NPs were centrifuged and washed three times with ethanol and water (volume ratio by 1:1). 2.4. Standard curve of DOX An appropriate amount of DOX was dissolved in water by ultrasonic oscillation. Then, a series of different concentrations of DOX aqueous solution (0e0.03 mg/mL) were prepared. The integral fluorescence intensity of the different concentrations of DOX solution (lex ¼ 490 nm) was measured. Finally, the standard curve of DOX was determined through the curve fitting of the fluorescence intensity vs the DOX concentration. The area standard curve: Y ¼ 447.44 þ 69745.08X. Precision rate of standard curve: R [2] ¼ 0.9992. 2.5. DOX loading and release The drug loading and release behavior of the PAA-NaMnF3 NPs was investigated using DOX as a model drug. Briefly, a certain amount solution of DOX with different concentration was mixed with the PAA-NaMnF3 NPs (3 mL, 2 mg/mL) in phosphate buffered saline (PBS) and stirred at room temperature for 24 h. The positively

Please cite this article as: J. Zhao et al., Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery, Journal of Alloys and Compounds, https://doi.org/10.1016/j.jallcom.2019.153142

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charged DOX combined with the negatively charged PAA to form PAA-NaMnF3 @DOX hybrid nanostructures by electrostatic interaction. To remove free DOX, the dispersion was collected by centrifugation and washed three times with PBS. To measure the loading efficiency of the PAA-NaMnF3 @DOX hybrid nanostructures, the supernatant was collected after centrifugation of the prepared hybrid nanostructures. The fluorescence spectra of the DOX molecules in the supernatant were examined, and the concentration of DOX in the supernatant was calculated by the DOX standard curve. The percentages of DOX remaining in the PAA-NaMnF3@DOX NPs were calculated according to the following equation: Loading efficiency (%) ¼ (W0 -Ws)/W0 100% where W0 and Ws represent the initial DOX mass and the DOX mass in the supernatants, respectively. For the cumulative DOX release studies in PBS buffer solutions (pH 5.0 and 7.4) with the same NaCl concentration of 0.15 M, the PAA-NaMnF3@DOX hybrid nanostructures were dispersed in 1.0 mL buffer solution and then transferred to a dialysis bag. Then it was kept in buffer solution and gently shaken at 37  C in a dark room. At the selected time intervals, 100 mL of solution was taken and analyzed by fluorescence spectroscopy. To maintain a constant volume, 100 mL of the solution was reinjected into the buffer solution after each test. 2.6. MRI measurements The MRI measurements were performed in an 11.7 T Bruker AVANCE 500WB spectrometer (Bruker NMR, Germany). The different amount of the PAA-NaMnF3@DOX hybrid nanostructures were dispersed in 1.2 mL agarose aqueous solution and then transfered into the microtubes for MRI measurements. The final Mn2þ ion concentration were 0, 0.018, 0.036, 0.071, 0.142, 0.284 and 0.568 mM, respectively. The measurement parameters of T1 and T2 relaxation time are as follows: repetition time (TR) ¼ 3000 ms, echo time (TE) ¼ 40 ms, imaging matrix ¼ 128  128, slice thickness ¼ 1.2 mm, field of view (FOV) ¼ 12.0  12.0 cm, and number of averages (NA) ¼ 2. 2.7. Cellular uptake and MR imaging To demonstrate efficient cellular uptake, the HeLa cells were seeded on the coverslip in the confocal dish and incubated in a humidified 5% CO2 atmosphere for 4 h at 37  C. Then, the PAANaMnF3@DOX hybrid nanostructures were added into the incubation medium at the different concentration and incubated for 2 h. The final Mn2þ ion concentrations were 4.26, 8.52, 16.79, 31.21 mM, respectively. After the medium was removed, the cells were washed twice with PBS (pH ¼ 7.4, 20 mM) and directly used for MR Imaging. 2.8. In vitro cytotoxicity of PAA-NaMnF3@DOX hybrid nanostructures In vitro cytotoxicity of the PAA-NaMnF3@DOX hybrid nanostructures was assessed against HeLa cells based on the methyl thiazolyl tetrazolium (MTT) assay. HeLa cells were cultured in APMI 1640 growth medium complemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin and 100 mg/mL penicillin. The cells were cultivated at 37  C in a humidified 95% air and 5% CO2 atmosphere. The assay was performed in triplicate with the same manner. Briefly, HeLa cells were seeded into 96-well plates at a density of 104 cells per well in 100 mL of media. After overnight growth, the cells were then incubated with five parallel wells at

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various concentrations of PAA-NaMnF3 NPs, DOX, and PAANaMnF3@DOX hybrid nanostructures (0.085, 0.17, 0.34, 0.68, 1.36 mg/mL). The material contents were calculated according to the concentration of DOX. That is, the free DOX concentration was the same as the DOX concentration in PAA-NaMnF3@DOX hybrid nanostructures and the PAA-NaMnF3 NP concentration was the same as that of the PAA-NaMnF3@DOX hybrid nanostructures. After being incubated for 24 h, the 10 mL MTT solution (5 mg/mL) was then added each well and the cells were further incubated for 4 h at 37  C. After the MTT solution was removed, 100 mL of dimethyl sulphoxide (DMSO) was added to each well and the plate was gently shaken for 10 min to dissolve the precipitated violet crystals. The optical density (OD) was measured at 490 nm using microplate reader (Perkin Elmer, Victor X4). Cell viability was evaluated as a percentage compared to control cells. 2.9. Characterization The sizes and morphologies of NaMnF3 NPs were examined by a FEI Tecnai G2-F20 transmission electron microscope (TEM) at an accelerating voltage of 200 kV. Fourier transform infrared spectra (FTIR) of the samples were recorded by FTIR instrument (Bruker Tensor 27). The UVevis absorption spectra were acquired by a Perkin Elmer Lambda-25 UVevis spectrometer. The fluorescence spectra were recorded using a Hitachi F-4600 fluorescence spectrophotometer with a photomultiplier tube operating at 400 V and a 150 W Xe lamp used as the excitation source. The slits of excitation and emission spectra are 5 nm. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Agilent 5100) was used to analyze the element Mn concentrations in the samples. 3. Results and discussion To obtain the PAA-NaMnF3@DOX hybrid nanostructures, PAA polymer with hydrophilic and rich carboxyl groups was selected as pH-sensitive nanovalves for drug molecular loading. Therefore, the positively charged drug molecules can be effectively loaded via electrostatic interaction. In this study, DOX was used as a model drug to evaluate drug loading and release behavior. The synthesis schematic of multifunction PAA-NaMnF3@DOX hybrid nanostructures was illustrated in Fig. 1. Firstly, the water-soluble PAANaMnF3 NPs were prepared by replacing the OA groups on the surface of NaMnF3 NPs by PAA molecules. Then, the negatively charged PAA on the surface of NaMnF3 NPs would attract the positively charged DOX molecules by the electrostatic interaction, thus to form the PAA-NaMnF3@DOX hybrid nanostructures. Interestingly, in acidic conditions (such as in the lysosomes of cells), DOX would be released from the surface of the PAA-NaMnF3@DOX hybrid nanostructures due to the protonation reaction of carboxyl group of PAA, which would break down the electrostatic interaction between PAA and DOX. Therefore, the PAA-NaMnF3@DOX hybrid nanostructures not only would be used as imaging contrast agents for MRI and fluorescent imaging but also would act as pH-triggered drug delivery system. Hydrophobic OA-NaMnF3 NPs were initially produced by a modified solvothermal route in organic solvent. As shown in the TEM images (Fig. 2a), the as-prepared OA-NaMnF3 NPs are spherical and uniform with an average diameter of about 30 ± 3 nm. The OA-NaMnF3 NPs were then modified by PAA molecules through a robust and general ligand exchange. After surface modification by PAA, the size and shape of the NaMnF3 NPs is still maintained (Fig. 2b). Fig. 2c displays the XRD patterns of the synthesized OANaMnF3 and PAA-NaMnF3 NPs. It can be seen that all the diffraction peaks of the samples correspond to the NaMnF3 crystal (JCPDS standard card no. 18e1224). The crystal structure remains

Please cite this article as: J. Zhao et al., Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery, Journal of Alloys and Compounds, https://doi.org/10.1016/j.jallcom.2019.153142

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Fig. 1. Schematic illustration of the PAA-NaMnF3@DOX hybrid nanostructures.

Fig. 2. Characterization of the as-prepared NaMnF3 NPs. (a) TEM image of OA- NaMnF3 NPs; (b) TEM image of PAA-NaMnF3 NPs; (c) The XRD patterns of the OA-NaMnF3 (red), PAANaMnF3 (green) NPs, and the standard card of NaMnF3 crystal (JCPDS: 18-1224, black); (d) FTIR spectra of OA-NaMnF3 and PAA-NaMnF3 NPs.

unchanged after modification. FTIR spectra of the NaMnF3 NPs before and after ligand exchange were shown to confirm the successful functionalization of the NaMnF3 NP surface (Fig. 2d). The bands at 2930 and 2850 cm1 were obviously found in the spectrum of the OA-NaMnF3 NPs, associated with the asymmetric (Vas) and symmetric (Vs) stretching vibrations of the eCH2 group of OA. The peak at 1710 cm1 for C¼O stretch of OA was also observed. However, after ligand exchange by PAA, two new bands at 1560 and 1410 cm1 are observed corresponding to the carboxylic group, which are assigned to the shift of C¼O bands caused by coordination of -COO- groups and metal cations. Additionally, the shoulder at 2930 cm1 associated with the asymmetrical stretching mode of eCH2 groups became inconspicuous and shoulder at 3450 cm1 corresponding to O-H stretching vibration became conspicuous after ligand exchange. The results confirm the successful ligand

exchange between PAA and OA on the surface of NaMnF3 NPs. To evaluate the potential of PAA-NaMnF3 NPs as imaging contrast agents, the T1-and T2-weighted MR images of the PAANaMnF3 NPs were investigated using an 11.7 T MR system and the longitudinal (r1) and transversal relaxivity (r2) of different concentrations were measured, respectively. The Mn2þ concentrations in the PAA-NaMnF3 NPs have a significant effect on both the longitudinal and transversal relaxation times. As shown in Fig. 3a, the brightness of T1-weighted MRI gradually brightens while the brightness of T2-weighted MRI gradually decreases with the increase of Mn ion concentration. The functional relationship between the inverse relaxation time (1/T1 and 1/T2) and Mn2þ concentration can be fitted by a straight line, as shown in Fig. 3b. The r1 longitudinal relaxivity of the PAA-NaMnF3 NPs are found to be 6.80 (mM)1 s1, which is higher than that of the clinically used

Please cite this article as: J. Zhao et al., Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery, Journal of Alloys and Compounds, https://doi.org/10.1016/j.jallcom.2019.153142

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Fig. 4. The solution photographs of PAA-NaMnF3 NPs (a), free DOX (b), and PAANaMnF3@DOX hybrid nanostructures (c); The fluorescent spectra of free DOX and the PAA-NaMnF3@DOX hybrid nanostructures loaded with different concentrations of DOX after centrifugation; Inset: The loading efficiency of the PAA-NaMnF3@DOX hybrid nanostructures based on the standard curve of DOX (d). Fig. 3. (a) T1-and T2-weighted MR images of PAA-NaMnF3 NPs. The upper part of panel (a) shows the T1-weighted images, and the lower part of panel (a) shows the T2weighted images. (b) T1 and T2 relaxivity plot of aqueous suspension of PAA-NaMnF3 NPs.

T1-contrast agent [Dotarem, 3.74 (mM)1 s1 at 11.7 T and MnDPDP, 2.0 (mM)1 s1] and that of hollow MnO NPs coated with mesoporous silica shells [0.99 (mM)1 s1 at 11.7 T] [49e51]. In the presence of contrast agents, the r1 values is related to the relaxation rate change of the protons of water [52]. The proportion of atoms on the surface of nanomaterials increases with the decrease of size. Therefore, most of the Mn2þ in NaMnF3 NPs will be located on the surface when the size of NPs decreases. The PAA-NaMnF3 NPs with small size show a higher r1 value, which is contributed to the high hydrophilicity of the PAA polymer, thus increasing the accessibility of paramagnetic ions (Mn2þ) with water molecules. Further research is required to fully understand the relaxation enhancement mechanism. Fig. 3b shows that the r2 transversal relaxivity of the PAA-NaMnF3 NPs are calculated to be 139.35 (mM)1 s1, which is higher than that of Mn2þ-doped NaNdF4 nanocrystals [41]. Furthermore, the r2/r1 ratio is 20.49, which indicates that the PAANaMnF3 NPs has great potential as a T1/T2 dual-mode MRI contrast agent. As drug delivery carriers, the drug loading capacity of PAANaMnF3@DOX hybrid nanostructures was also investigated. The storage of DOX in the hybrid nanostructures was revealed by the color change of the solution. The obtained PAA-NaMnF3 NPs can be stably dispersed in PBS for more than half a year due to the presence of sufficient hydrophilic groups on the NP surface. The colloid solution of PAA-NaMnF3 NPs is transparent (Fig. 4a). The color of the pure DOX solution is orange (Fig. 4b). After the interaction between DOX and the PAA-NaMnF3 NPs, the color of the solution of PAA-NaMnF3@DOX hybrid nanostructures became light orange (Fig. 4c). Fig. 4d shows the photoluminescence spectra of free DOX and the PAA-NaMnF3@DOX hybrid nanostructures with different concentrations of DOX added after centrifugation. Compared with free DOX, the PAA-NaMnF3@DOX hybrid nanostructures showed a weak fluorescence emission, which suggests the strong binding of DOX to the PAA-NaMnF3 NPs and effective fluorescence quenching by the PAA-NaMnF3 NPs [53]. A slight redshift of emission peak was observed as the concentration increasing, which has also been

observed in the previous work [27]. We consider that the phenomenon would be contributed to the existence of interaction between PAA-NaMnF3 NPs and DOX or the interaction between DOX molecules due to the compression of space. Further verifications will be confirmed in future work. The photoluminescence spectra of the PAA-NaMnF3@DOX hybrid nanostructures showed a characteristic DOX emission from 520 to 700 nm, which verified the successful loading of DOX onto the PAA-NaMnF3 NPs. The loading efficiency of the DOX adsorbed on the surface of PAA-NaMnF3 NPs is highly important parameter for the bio-application. As a widely adapted method, the loading efficiency was calculated using the integral fluorescence intensity of DOX in supernatant based on the standard curve of DOX [25]. The loading efficiency of DOX on the PAA-NaMnF3 NPs was shown in a DOX concentration-dependent manner (Inset of Fig. 4d). There is an optimum concentration of DOX absorbed on the surface of PAA-NaMnF3 NPs. When the concentration of DOX was 5 mg/mL, the highest loading efficiency was found to be 5.49%. The drug loading efficiency is slightly lower than that of PLGA(UCNPs/DOX) nanocapsules because of the different ways of loading drugs [25]. The DOX loading content of PAA-NaYF4: Yb, Er NPs is about 8% when applying a different method of calculation [33]. Drug loading efficiency of DOX is dependent closely on DOX concentration, carrier concentration and properties [35,36]. The adsorption of DOX onto the PAA-NaMnF3 NPs is attributed to the electrostatic interactions between DOX and PAA. Consequently, the antitumor drug DOX can be effectively absorbed onto the surface of PAA-NaMnF3 NPs. To build up a therapeutic platform, the corresponding PAA-NaMnF3@DOX hybrid nanostructures would further apply to perform biomedical experiment. The in vitro drug-releasing profiles of the PAA-NaMnF3@DOX hybrid nanostructures under two different environmental pH values are demonstrated in Fig. 5. The PAA-NaMnF3@DOX hybrid nanostructures were dialyzed in pH 5.0 and 7.4 phosphate buffers at a temperature of 37  C, respectively. The released DOX from the hybrid nanostructures was collected and then the release amount of DOX was calculated by fluorescence intensity of the supernatant. It can be seen that the release rate of the PAA-NaMnF3@DOX hybrid nanostructures at pH 5.0 (simulated in vivo and lysosome microenvironment) is higher than that at pH 7.4 (physiological pH of blood flow). The low pH is beneficial to the release of DOX. At pH

Please cite this article as: J. Zhao et al., Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery, Journal of Alloys and Compounds, https://doi.org/10.1016/j.jallcom.2019.153142

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Fig. 5. DOX release profile of PAA-NaMnF3@DOX hybrid nanostructures in different pH values solution at 37  C.

5.0, about 80 wt% of DOX was released gradually from the PAANaMnF3@DOX hybrid nanostructures at initial 25 h. However, the drug release observed at physiological pH 7.4 was a slow process. About 22 wt% of DOX was released at initial 10 h, and then entered the stable stage of slow release. The plateau percentages of DOX release observed over a period of 30 h was 81 ± 3 wt% and 23 ± 3 wt % at pH 5.0 and 7.4, respectively, which means that the cytotoxic concentration of the PAA-NaMnF3@DOX in normal cells is about three times higher than that in cancer cells. This phenomenon is attributed to a weak electrostatic interaction between DOX and PAA-NaMnF3@DOX hybrid nanostructures at low pH values because most of the PAA was protonated with the decrease of pH [33]. The drug release studies indicate good stability of electrostatically bound drug molecules in PAA-NaMnF3@DOX hybrid nanostructures in physiological environment and pH-triggered release at acidic conditions. Therefore, the PAA-NaMnF3@DOX hybrid nanostructures are pH-responsive systems for DOX delivery and suitable for the specific treatment of solid tumors. To evaluate the pharmacological activity of the DOX-loaded hybrid nanostructures, the in vitro cytotoxic effect of the PAANaMnF3@DOX hybrid nanostructures on HeLa cells was assessed via standard MTT assay. Fig. 6 shows the cell viability against PAANaMnF3 NPs, free DOX and PAA-NaMnF3@DOX hybrid nanostructures at different concentrations after incubation with Hela cells for 24 h. When incubated with PAA-NaMnF3 NPs for 24 h, the cell viability of 88% maintained with the increase of PAA-NaMnF3 concentration from 0.085 to 1.36 mg/mL. It was revealed that the PAA-NaMnF3 NPs had no obvious cytotoxic effect on cancer cells

Fig. 6. The cell viability rate of HeLa cells incubated with the PAA-NaMnF3 NPs, free DOX, and the PAA-NaMnF3@DOX hybrid nanostructures, respectively.

after 24 h treatment, even if the concentration was as high as 1.36 mg/mL. This result shows that the PAA-NaMnF3 NPs are highly biocompatible. To demonstrate that the intracellular delivery of DOX is pharmacologically active, HeLa cells were treated with free DOX and PAA-NaMnF3@DOX hybrid nanostructures, respectively. When the free DOX concentration was set to be the same as that in the PAA-NaMnF3@DOX hybrid nanostructures, the cellular viability progressively decreased with increasing effective DOX concentration. As shown in Fig. 6, free DOX and PAA-NaMnF3@DOX hybrid nanostructures exhibited noticeable cytotoxicity after incubating with cells for 24 h. As the concentration of the PAA-NaMnF3@DOX hybrid nanostructures was increased from 0.085 up to 1.36 mg/mL, the relative cell viability decreased to about 37% for free DOX, and to about 45% for PAA-NaMnF3@DOX hybrid nanostructures, respectively. Moreover, the value of the half maximal inhibitory concentration (IC50) of free DOX and the PAA-NaMnF3@DOX hybrid nanostructures was calculated to be 1.01 and 1.40 mg/mL, respectively. This result implies that both free DOX and PAA-NaMnF3@DOX hybrid nanostructures have dose-dependent cytotoxicity to cancer cells. The cytotoxicity originates mainly from the loaded DOX rather than the PAA-NaMnF3 NPs. At the cellular level, most of the internalization of hybrid nanostructures is achieved through endocytosis. Small DOX molecules can quickly diffuse into cells, while PAA-NaMnF3@DOX hybrid nanostructures must be endocytosed to enter cancer cells. Therefore, cells uptake of free DOX is faster than that of PAA-NaMnF3@DOX hybrid nanostructures. As the concentration increases, more and more DOX-loaded hybrid nanostructures can be endocytosed to enter the cancer cells and then release DOX molecules, which lead to the cancer cell death. Because of the hypoxia-induced coordinated upregulation of glycolysis, the acidic extracellular environment of solid tumors was stronger than that of normal tissues [54]. After being engulfed by cells, the released DOX usually enter the early endosomes, then enter the late endosomes/lysosomes, and finally fused with lysosomes. Both endosomes (pH 5.0e6.0) and lysosomes (pH 4.5e5.0) have an acidic microenvironment. Therefore, the pH-responsive PAA-NaMnF3@DOX hybrid nanostructures would release drugs in acidic environments, thereby enhancing pH-response antitumor therapy efficacy. The released DOX molecules are located in the cell nucleus that is the main target of DOX [48]. Furthermore, DOX can bind to double-stranded DNA to form DNA adducts, which inhibit the activity of topoisomerase and induce cell death (apoptosis) [48]. Therefore, the pH-responsive PAA-NaMnF3@DOX hybrid nanostructures could act as an excellent therapeutic platform for cancer chemotherapy. The cellular uptake and cytotoxicity are the important factors in evaluating the potential of new drug delivery system. The intercellular uptake of the PAA-NaMnF3@DOX hybrid nanostructures was further investigated by monitoring the fluorescence of DOX using optical and fluorescence microscopy, as shown in Fig. 7. After incubation for 0.5 h, the obvious red fluorescence was observed around the cells, indicating the existence of the PAA-NaMnF3@DOX hybrid nanostructures around the HeLa cells. As is well known, non-functional NPs can be internalized by cancer cells through endocytosis [55]. In fact, some PAA-NaMnF3@DOX hybrid nanostructures should have been in the cytoplasm of individual cancer cells by endocytosis after incubation for 0.5 h (Fig. 7a). When the incubation time increased to 24 h, the intercellular fluorescence intensity in HeLa cells increased, revealing that the PAANaMnF3@DOX hybrid nanostructures had been internalized (Fig. 7b). Free DOX can diffuse into cells and accumulate in cells, while the PAA-NaMnF3@DOX hybrid nanostructures were mainly internalized by endocytosis [56]. After being internalized by cells, DOX molecules were released from the PAA-NaMnF3@DOX hybrid nanostructures in the acidic environment around the endosome/

Please cite this article as: J. Zhao et al., Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery, Journal of Alloys and Compounds, https://doi.org/10.1016/j.jallcom.2019.153142

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the surface of the PAA-NaMnF3@DOX hybrid nanostructures to specially target a specific site of the animals in the future work. 4. Conclusion The multifunctional PAA-NaMnF3@DOX hybrid nanostructures were developed as imaging contrast agents for MRI and a pHtriggered drug delivery system for effective cancer chemotherapy. The DOX can be released from the PAA-NaMnF3@DOX hybrid nanostructures at acidic environment and exhibit an excellent cellular cytotoxic effect on HeLa cells. Furthermore, the PAANaMnF3@DOX hybrid nanostructures as multifunctional theranostic platform have great potential for biomedical application, including MRI, bioimaging and stimuli-responsive drug delivery nanocarries. Author contribution statement

Fig. 7. Optical microscope images (left), fluorescence images (middle) and merge images (right) of PAA-NaMnF3@DOX hybrid nanostructures incubated with HeLa cells for different hours. (a) 0.5 h; (b) 24 h; (c) 48 h. The scale bar is 100 mm.

lysosome, where a low pH value could trigger an effective DOX release (pH 5.0, Fig. 5). The released DOX in the cytoplasm eventually accumulated in the cell nucleus through the nuclear membrane and then killed the cancer cells by changing the DNA conformation [57]. After incubation for 48 h, most of HeLa cells died. Moreover, the fluorescence brightness was increased and the spot became smaller under dark field condition (Fig. 7c). These results are consistent with DOX release results (Fig. 5). These results indicate that the PAA-NaMnF3@DOX hybrid nanostructures have been engulfed by HeLa cells and the DOX molecules have been released in cancer cells. To further confirm the internalization of the PAA-NaMnF3@DOX hybrid nanostructures in cells, the dual-mode T1-and T2-weighted MR images were collected with different concentrations of the PAA-NaMnF3@DOX hybrid nanostructures. The in vitro MRI was performed on cell pallets packed in capillaries. Fig. 8 shows the changes of in vitro T1-and T2-weighted MR images with increasing the concentrations of the PAA-NaMnF3@DOX hybrid nanostructures. The T1-weighted MR images were significantly enhanced with the increase of the hybrid nanostructure concentration, while the T2-weighted MR images showed significant signal weakening. These results further demonstrate that the PAANaMnF3@DOX hybrid nanostructures can be easily incorporated into cancer cells through endocytosis and show the great potential for in vivo MRI. Despite the current research is limited to the molecular level, further in vivo studies will be performed by modifying

Fig. 8. In vitro T1-and T2-weighted MR images of HeLa cells. The cells in each tube were incubated with PAA-NaMnF3@DOX hybrid nanostructures (4.26e31.21 mM) by electroporation.

Junwei Zhao: Curation and analysis of the data, Software, Investigation, Writing, Original draft preparation. Zhiping Zhang: Synthesis and characterizations of samples. Xin Wang: Conceptualization, Methodology, Supervision, Writing, Reviewing. Author contributions Both Junwei Zhao and Xin Wang were contributed to analysis of the data and writing the manuscript. Zhiping Zhang carried out the synthesis of materials, the characterizations of the as-synthesized samples. Xin Wang was contributed to the conception and design of the experiment. All authors reviewed the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos.11774384, 11174324 and 11204122), the Youth Innovation Promotion Association of Chinese Academy of Sciences (No.2011235), and the Opening Foundation of Henan Key Laboratory of Special Protective Materials, Institute of Defense Engineering, AMS, PLA (Grant No. SZKFJJ201903). References [1] Q. Chen, et al., Functionalization of upconverted luminescent NaYF4: Yb/Er nanocrystals by folic acidechitosan conjugates for targeted lung cancer cell imaging, J. Mater. Chem. 21 (2011) 7661e7667. [2] D. Peer, et al., Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (2007) 751e760, https://doi.org/10.1038/nnano.2007.387. [3] K. Kostarelos, W.T. Al-Jamal, Liposomes: from a clinically established drug delivery system to a nanoparticle platform for theranostic nanomedicine, Accounts Chem. Res. 44 (2011) 1094e1104, https://doi.org/10.1021/ ar200105p. [4] J.E. Lee, N. Lee, T. Kim, J. Kim, T. Hyeon, Multifunctional mesoporous silica nanocomposite nanoparticles for theranostic applications, Accounts Chem. Res. 44 (2011) 893e902, https://doi.org/10.1021/ar2000259. [5] L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancer therapy, Adv. Drug Deliv. Rev. 64 (2012) 206e212, https://doi.org/ 10.1016/j.addr.2012.09.033. [6] X. Wang, Q. Zhang, J. Zhao, J. Dai, One-step self-assembly of ZnPc/NaGdF4: Yb, Er nanoclusters for simultaneous fluorescence imaging and photodynamic effects on cancer cells, J. Mater. Chem. B 1 (2013) 4637e4643, https://doi.org/ 10.1039/c3tb20533a. [7] H. Koo, et al., In vivo targeted delivery of nanoparticles for theranosis, Accounts Chem. Res. 44 (2011) 1018e1028, https://doi.org/10.1021/ar2000138. [8] F. Wang, et al., Doxorubicin-tethered responsive gold nanoparticles facilitate

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Please cite this article as: J. Zhao et al., Fabrication of pH-responsive PAA-NaMnF3@DOX hybrid nanostructures for magnetic resonance imaging and drug delivery, Journal of Alloys and Compounds, https://doi.org/10.1016/j.jallcom.2019.153142