Radioisotope and anticancer agent incorporated layered double hydroxide for tumor targeting theranostic nanomedicine

Applied Clay Science 186 (2020) 105454

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Research paper

Radioisotope and anticancer agent incorporated layered double hydroxide for tumor targeting theranostic nanomedicine

T

Hyoung-Jun Kima,1, Jun Young Leeb,1, Tae-Hyun Kimc, Gyeong-Hyeon Gwakd, ⁎ ⁎ Jeong Hoon Parkb, , Jae-Min Oha, a

Department of Energy and Materials Engineering, Dongguk University-Seoul, Seoul 04620, Republic of Korea Radiation Utilization and Facilities Management Division, Korea Atomic Energy Research Institute, Jeongeup, Republic of Korea c Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense, Denmark d Beamline Research Division, Pohang Accelerator Laboratory, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Layered double hydroxide Theranosis Radioisotope Single-photon emission computed tomography

Theranostic nanomedicine was successfully prepared using layered double hydroxide (LDH). By means of stepby-step incorporation of an anticancer drug, methotrexate (MTX), and a radioisotope, Co-57, into the interlayer space and lattice of LDH, respectively, theranostic hybrid Nanomedicine could be prepared. X-ray diffractometry, scanning electron microscopy, X-ray adsorption spectroscopy, and Fourier-transform infrared spectroscopy systematically showed that incorporating the Co2+ into the MTX-LDH did not alter the crystalline phase, size, morphology, or intact structure of the hybrid. The labeled Co-57 in MTX-LDH was highly stable in human serum, showing almost 90% retention after 48 h. In vitro cellular uptake of Co-57-labeled MTX-LDH was very high in mouse colon carcinoma CT-26 cells, with ~60 ID% at 4 h. The cytotoxicity assay of MTX-LDH showed high cancer-cell suppression on CT-26 cells. To evaluate the diagnostic ability of Co-57-labeled MTXLDH, in vivo single-photon emission computed tomography (SPECT) images were investigated on CT-26 xenografted mouse model. The SPECT signal in tumor tissue began to appear within 1 h, and it increased for 3 h.

1. Introduction For decades, various materials, such as polymeric (Kumari et al., 2010), liposomal (Al-Jamal and Kostarelos, 2011), nanoporous (Zhang et al., 2012), and layered structures (Kim et al., 2014a), have been widely studied for use in drug delivery systems (DDS). In the early stage, drug delivery carriers were developed to accommodate as many drug moieties as possible while preserving drug efficacy and altering physical properties like solubility (Ahuja et al., 2007; Gabizon et al., 1982). Because of the recent advances in nanotechnology, each part of the carrier materials can be independently manipulated, and thus the research paradigm of DDS moved to more specific functionalities: (1) to improve uptake efficiency (Choy et al., 2004), (2) to by-pass drug resistance (Choi et al., 2010a), (3) to increase lesion specificity (Chertok et al., 2010), (4) to achieve stimulus-responsive drug release (Zhao et al., 2010). In addition to the above-mentioned functionalities in terms of therapy, many scientists currently try to introduce biologically traceable moieties into delivery carriers in order to take advantage of diagnosis as well as to comprehend the biological fate of carrier

materials (Kim et al., 2010). Fluorescent dyes are widely used for this purpose because of their easy labeling and high accessibility; however, they have limited applications for in vivo imaging because of their short penetration depth (Mishra, 2013; Ntziachristos et al., 2003; Thurn et al., 2009). In this regard, contrasting agents for X-ray imaging, magnetic resonance imaging (MRI) and radio imaging can be used to trace drug delivery nanocarriers. Among them, radioisotope (RI)-based imaging, such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET), are powerful because of their high sensitivity to RI, quantitative analysis, and trace amount required as contrasting agents (Xing et al., 2014). In this study, we chose layered double hydroxides (LDH) as a drug delivery carrier with RI labeling. Compared to other DDS carriers, LDH has various advantages, such as high drug incorporation efficiency (Rojas et al., 2012), selective cellular uptake (Oh et al., 2009), and biocompatibility (Choi et al., 2007). LDH is composed of two-dimensional metal hydroxide layers and interlayer anions. This characteristic layer-by-layer stacking structure guarantees preserving the drug molecules from harsh conditions and improving their controlled release

⁎

Corresponding author. E-mail addresses: [email protected] (J.H. Park), [email protected] (J.-M. Oh). 1 Authors equally contributed to the work. https://doi.org/10.1016/j.clay.2020.105454 Received 3 November 2019; Received in revised form 8 January 2020; Accepted 16 January 2020 Available online 21 January 2020 0169-1317/ © 2020 Published by Elsevier B.V.

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Korea). Radioisotope 57CoCl2 was purchased from Eckert & Ziegler Isotope Products Inc. (Valencia, USA). For cellular uptake, Hyclone™ RPMI (Roswell Park Memorial Institute)-1640 was purchased from GE healthcare (South Logan, USA); fetal bovine serum (FBS), phosphate buffered saline (PBS), and 0.05% trypsin-Ethylenediaminetetraacetic (EDTA) from Thermo Fisher Scientific, Inc. (Gibco®, Waltham, USA). All chemicals were used without further purification.

(Choy et al., 1999; Kang et al., 2015). The general chemical formula of 3+ n− an LDH is M2+ )x/n·mH2O (0 < x < 1; m and n are 1−xMx (OH)2(A integers), where M2+ and M3+ stand for divalent and trivalent metal cations, respectively, and An− represents the anionic species (Vaccari, 1998). The intercalated anions are stabilized with metal hydroxide layers by means of electrostatic interaction (Choy et al., 2000). There have been intensive studies on the intercalation of drug molecules, such as methotrexate (MTX), 5-fulorouracil, and ibuprofen, into interlayer spaces of LDH to improve anticancer activities (Ambrogi et al., 2001; Choi et al., 2010b; Oh et al., 2006b). In order to make LDH traceable, researchers have introduced fluorescent dyes and contrasting agents by tagging fluorophores at the surface (Musumeci et al., 2010; Oh et al., 2011) or by intercalating contrasting molecules in the interlayer space of an LDH (Kim et al., 2008; Xu et al., 2007). To exclude detachment of a tracing moiety from LDH, more systematic labeling was recently approached by directly introducing a tracing moiety into the framework of an LDH. For example, radioisotope Ga-68 (Musumeci et al., 2009) or MRI-contrasting Gd (Lee et al., 2011) could be directly incorporated into an LDH during synthesis. Our group suggested post-synthetic RI tagging into an LDH structure using hydrothermal substitution (Kim et al., 2016). The RI Co57-labeled LDH was stably retained in physiological solution for 24 h. The in vitro cellular uptake and in vivo biodistribution results indicated that Co-57-incorporated LDH nanoparticles show high cancer/tumor targeting efficiency, suggesting the diagnostic potential of RI-tagged LDH. Taking into account the above advantages of LDH in both DDS and diagnostics, we are going to investigate the theranostic aspect of LDH. First, we prepared pristine LDH nanoparticles with homogeneous particle sizes for effective cellular uptake and EPR effect (Li et al., 2017; Wang et al., 2017). Then anticancer MTX and RI Co-57 were step-bystep incorporated into LDHs' interlayer space and framework, respectively. The reaction was controlled topotactically so that the size and morphology of pristine LDH were preserved. In the cold experiment (non-radioactive Co2+ incorporation), we characterized physicochemical properties by using X-ray diffraction (XRD), Fourier-transform infrared (FT-IR), scanning electron microscopy (SEM), and X-ray adsorption spectroscopy (XAS). The Co-57-labeled MTX-LDH (Co-57@ MTX-LDH) was then investigated in terms of labeling efficiency, stability, in vitro cellular uptake, and in vivo SPECT/CT on a CT-26 xenografted mouse model.

2.2. Methods 2.2.1. Synthesis of pristine LDH with uniform size For the preparation of pristine LDH with a uniform particle size, we used coprecipitation and hydrothermal treatment (Oh et al., 2002). A mixed metal solution (0.2814 M of Mg(NO3)2·6H2O and 0.0938 M of Al (NO3)3·9H2O) was titrated by base solution (0.75 M of NaOH and NaNO3) until pH reached ~9.5. Then the white suspension was transferred to a Teflon-lined stainless-steel bomb for hydrothermal treatment at 150 °C for 24 h. The white precipitate was washed several times with decarbonated water and stored as a slurry after centrifugation.

2.2.2. Topotactic incorporation of MTX into pristine LDH (MTX-LDH) Powdered MTX was dispersed into decarbonated water and deprotonated with 1 M of NaOH until pH reached ~8.0. Pristine LDH slurry was dispersed with decarbonated water and a mixed MTX solution; the reactant was kept for 3 days at 70 °C under nitrogen atmosphere and gentle stirring. The yellowish precipitate was collected by centrifugation, washed several times with decarbonated water, and then lyophilized.

2.2.3. Topotactic co-incorporation into MTX-LDH (Co@MTX-LDH) in the cold In order to investigate the physicochemical changes upon RI labelling, we first carried out a cold experiment with non-radioactive Co2+substituted MTX-LDH. Following our previous report (Kim et al., 2016), MTX-LDH was dispersed in decarbonated water (~5 mg/mL), and a CoCl2 solution (0.02 M) was mixed (80 mL of suspension and 100 mL of solution). The mixture was hydrothermally treated at 150 °C for 2 h, and the Co@MTX-LDH was washed with decarbonated water and lyophilized.

2. Materials and methods

2.2.4. Characterization The XRD patterns of pristine LDH and MTX-LDH were obtained with a Bruker AXS D2 phaser (Bruker AXS GmbH, Karlsruhe, Germany) by using Ni-filtered Cu-Kα radiation (λ = 1.5406 Å). Data were collected from 3° to 30° (2θ) with time-step increments of 0.02° and 0.5 s per step, respectively. The local chemical environment around the Co atoms was analyzed by X-ray absorption spectroscopy (XAS) at 8C NanoXAFS beamline in the Pohang Accelerator Laboratory (PAL), Pohang, Republic of Korea. Each powder-type sample was measured in transmission mode at Co K-edge (7708.9 eV). Normalized X-ray absorption near-edge structure (XANES) spectra and Fourier-transform extended Xray absorption fine structure (FT-EXAFS) spectra were obtained by using Athena software. Multi-peak analysis of FT-EXAFS spectra was carried out by Gaussian function in OriginPro 8 software (OriginLab Corporation, Northampton, MA, USA). Fourier-transform infrared (FTIR; Perkin Elmer, spectrum one B.v5.0) spectroscopy was done with conventional KBr methods. The particle size and shape of the LDH was verified by scanning electron microscopy (SEM: Quanta 250 FEG (FEI Company, Hillsboro, OR, USA). For SEM measurement, the LDH suspension was prepared with a concentration ~0.4 mg/mL. Then a drop of suspension was placed on the pre-cleaned Si wafer and dried in vacuum. The surface of the specimen was sputtered with Pt/Pd plasma for 50 s, and images were collected by 30 kV of accelerated electron beam.

2.1. Materials Al(NO3)3·9H2O, NaHCO3, NaNO3, Mg(NO3)2·6H2O, C20H22N8O5·xH2O (methotrexate: MTX, Scheme 1) and human serum (from human male AB blood type plasma, Cat No. H4522) were purchased from Sigma-Aldrich Co. LLC. (St. Louis., USA). NaOH pellets were obtained from Daejung Chemicals & Metals Co., Ltd. (Siheung,

Scheme 1. Molecular structure of methotrexate. 2

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Fig. 1. (A) X-ray diffraction patterns of (a) pristine LDH, (b) MTX-LDH, and (c) Co@MTX-LDH, and (B) schematic diagram of pristine LDH, MTX-LDH, and Co@MTXLDH.

the cell pellet for re-culture in the plate. The cell line was seeded at 1 × 105 in 24-well plates at 1 mL per well and incubated at 37 °C in a 5% CO2 for 24 h for adherence and growth, and then 5 μCi of Co-57@ MTX-LDH was added to each well. At designated time points (1, 4, 24, and 48 h), cells were washed twice in cold 1×PBS to remove extracellular particles. Then cells were detached with 0.05% trypsin-EDTA and counted by gamma counter (1470 Wizard 2, Perkin-Elmer, Waltham, USA). Cellular uptake was presented as a percentage of the injected radioactivity dose (ID%).

2.2.5. Radioisotope Co-57 incorporation into MTX-LDH (Co-57@MTXLDH) and RI stability test All the radioisotope experiments were carried out at R&D Advanced Radiation Laboratory, Korean Atomic Energy Research Institute, under government standard regulation. In order to incorporate Co-57 into the framework of MTX-LDH, we used isomorphous substitution by modifying our previous work (Kim et al., 2016). Typically, 1 mL of MTX-LDH suspension (5 mg/mL) was mixed with 15.54 MBq (420 μCi) of Co-57 at room temperature, and the mixture was immediately placed in a 5 mL reaction vial for hydrothermal treatment at 150 °C for 2 h. Incorporated amount of Co-57 was evaluated by measuring the radioactivity of the supernatant and precipitate obtained by centrifugation of the reaction suspension (CRC15R; Capintec, Inc., Ramsey, USA). Then Co-57-incorporated MTX-LDH (precipitate) was shaken at 25 °C for 1 min and purified using 1.5 mL of 50 mM EDTA. The radiochemical yield (RCY) and radiochemical purity (RCP) were measured by radio-iTLC (mobile phase: 0.01 M citric acid). The labeling efficacy was calculated (non-decay corrected) by the following equation.

2.2.7. Anticancer activity Anticancer activity of MTX-LDH was checked in mouse colon-carcinoma CT-26 cells. First, cells were cultured as described in Section 2.2.6., and seeded as 5 × 103 cells in 96-well plates for further incubation at 37 °C in a 5% CO2 condition for 24 h, after which, cells were washed with 1×PBS before MTX-LDH treatment. After 24 h of incubation, extracellular particles were removed by means of 1× PBS washing, and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-treated cells were incubated in a CO2 incubator at 37 °C for 3 h. After incubation, supernatant was discarded, 100 μL of dimethyl sulfoxide was added in each plate, and absorbance was measured using a microplate reader at 540 nm after shaking for 15 min.

labeling efficacy(%) (radioactivity of solution before reaction) =

− (radioactivity of supernatant after reaction) × 100 (radioactivity before reaction)

2.2.8. In vivo SPECT/CT imaging The 5-weeks-old BALB/c female mice were purchased from Orient Bio, Inc. (Sungnam, Korea). The CT-26 cells (1 × 107 cell) were dispersed in 100 μL of Dulbecco's Modified Eagle's Medium (DMEM), which was injected subcutaneously into the right leg of mice (N = 3). The mice were acclimatized until tumor diameter reached 10–15 mm during 2–3 weeks. The mouse was anesthetized by exposing it to 2% isoflurane in oxygen (medical grade). Then, 100 μL (3.7 MBq, 100 μCi) of Co-57@MTX-LDH suspension was injected intravenously by means of the tail vein. At designed time points (1, 3, and 6 h), mice were scanned with SPECT/CT (Siemens Medical Solutions). All the procedures carried out involving human participants were in accordance with the ethical standards of the Gipuzkoa Clinical Research Ethics Committee, and with the principles of the 1964 Declaration of Helsinki and its later amendments, or comparable ethical standards.

The stability of incorporated Co-57 in MTX-LDH was evaluated with radio-instant thin-layered chromatography (Radio-iTLC; AR-2000, Bioscan, Poway, USA) in phosphate-buffered saline (PBS) and human serum, respectively. The Co-57@MTX-LDH having 100 μCi of radioactivity was dispersed in 1 mL of PBS or human serum at 37 °C and then incubated in a shaking incubator. The released Co-57 and the Co-57incorporated MTX-LDH were measured by radio-iTLC (mobile phase: 0.01 M citric acid) at time points 15, 30, 60, 120 min, 24 h, and 48 h. 2.2.6. Cellular uptake experiment Cellular uptake of Co-57@MTX-LDH was evaluated in a mouse colon-carcinoma CT-26 cell line. Cells were cultured in 6-cm Petri dishes at an initial density of 1 × 107 cells/mL in 10 mL Dulbecco's modified medium (DMEM) with 10% fetal bovine serum, 4 mM L-glutamine, 4500 mg/L glucose, and sodium pyruvate, then were incubated at 37 °C in a humidified 5% CO2 incubator. After 24 h of culture, cells grown in a monolayer were harvested by trypsinization using a 0.05% trypsin-EDTA solution, and the culture medium was removed by centrifugation (1000 rpm, 3 min) and 10 mL fresh medium was added to

3. Results and discussion The powder X-ray diffraction patterns and scanning electron microscopic results of pristine LDH and MTX-LDH are displayed in Fig. 1. The crystal structure of MgAl-LDH shows well-developed (00l) 3

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intensity because of the high X-ray attenuation effect of Co2+ compared to MTX; similar phenomena were often observed in super-lattice layered materials (Behera et al., 2018; Shunmugasundaram et al., 2016). In order to verify the location and chemical environment of Co2+ after incorporation into MTX-LDH, using a local structural analysis technique, we applied X-ray absorption spectroscopy (XAS) at the Co Kedge. For comparison, the CoAl-NO3-LDH and Co-MTX complex, in which Co2+ is located in the lattice of the LDH and in the coordinate molecule, were subjected to XAS. As shown in Fig. 3A, the sample and reference materials exhibited the same white line at ~7720 eV, which correspond to the 1 s → 4p transition of divalent cobalt ions (Kim et al., 2014b). They showed weak pre-edge at ~7709 eV (arrows in Fig. 3A), which stands for a partial transition of 1 s → 3 d that occurred because there was little distortion of octahedral symmetry (Yoon et al., 2002) in either the LDH lattice or the Co-MTX complex. A closer look at X-ray absorption near-edge structure (XANES) spectra (Fig. 3A) revealed that Co@MTX-LDH resembled CoAl-LDH rather than the Co-MTX complex in terms of edge position, white-line intensity, and overall spectral shape. This result implied that the primary position of Co2+ would be in the lattice. However, further analysis of XAS spectra, i.e., Fouriertransform extended X-ray absorption fine structure (FT-EXAFS) spectra showed a possible location of Co2+ among the interlayers of MTX-LDH. In Fig. 3B(b), the first shell that is attributed to the CoeO bond appeared near 1.60 Å (not phase-shift corrected) in all samples with a similar peak amplitude. The second shell of CoAl-LDH and Co@MTXLDH attributed to the Co-(Co, Mg, Al) bond appeared near 2.70 Å (not phase-shift corrected) because of the continuous connection of M(OH)6 octahedra in the LDH lattice. On the other hand, Co-MTX did not show a significant second shell, because the Co2+ ions were far apart from each other by the organic MTX moiety. Although the general spectral features of CoAl-LDH and Co@MTX-LDH were similar, we could not conclude that the local environment around Co was perfectly the same for both samples. The pattern in the range of 3–4 Å, which sometimes gives multiple information on local structure (Celorrio et al., 2018; Pokrovski et al., 2003), showed a slight similarity between Co@MTXLDH and the Co-MTX complex. In order to identify the bond in detail, we separated the 2.5–4 Å range in the spectrum of Co@MTX-LDH and then fitted it to the Gaussian model. For comparison, FT patterns of two references, CoAl-LDH and Co-MTX complex, were added up and analyzed similarly. As shown in Fig. S2 and Table S1, there were four peaks with high fitting reliability (R2 > 0.999). Considering all the FTEXAFS spectra and fitted results, we found that Co@MTX-LDH had properties of both CoAl-LDH (peaks 1, 4 in Table S1) and Co-MTX (peaks 2, 3 in Table S1) in terms of chemical environment around Co. Although it is difficult to quantitatively distinguish the location of Co2+ in Co@MTX-LDH, we could conclude that Co2+ was stably incorporated in MTX-LDH. The stability of Co2+ in physiological solution will be demonstrated in the RI experiment in the following paragraphs. Although there is no precise interpretation, at this stage, of how the

diffractions of (003) and (006) at 10.8° and 21.5°, respectively. The XRD pattern in the 2θ range 30°–80° (Fig. S1) showed typical lattice peaks, such as (012), (015), (018), (110), and (113) at 34.4°, 37.8°, 44.0°, 60.4° and 61.6°, respectively, suggesting the evolution of a hydrotalcite-like (JCPDS No.14-0191) LDH structure (Lee et al., 2016). After intercalation of MTX, both (003) and (006) peaks moved to a lowangle region, 4.9° and 9.1°, respectively, indicating lattice expansion of the LDH structure along the crystallographic c-axis. Calculated gallery height for pristine and MTX-LDH was 0.37 nm and 1.35 nm, respectively, showing that nitrate anions in the pristine LDH were successfully exchanged with the MTX moiety, as previously reported (Choy et al., 2004). The particle size and morphology are important factors for LDH in theranosis applications. Those parameters are reported to affect various biological behaviors, including cellular uptake, cellular retention (Oh et al., 2009), cytotoxicity (Choi et al., 2008), and intracellular compartment (Xu et al., 2008). Specifically, it was reported that LDH's cellular uptake and retention was best achieved in the size range of 100–200 nm taking advantage of targeted clathrin-mediated endocytosis (Oh et al., 2009). LDH with plate-like morphology is predominantly targeted to cytosol, whereas rod-like ones could penetrate the nucleus (Xu et al., 2008). As shown in Fig. 2, both pristine and MTXLDH showed the plate-like morphology with uniform lateral size. To statistically calculate the particle-size distribution, 100 particles in the images were randomly selected. The lateral sizes were found to be 147 ± 14 nm and 149 ± 16 nm for pristine and MTX-LDH, respectively. This result, along with XRD patterns (Fig. 1a, b), revealed topotactic incorporation of the MTX moiety between LDH layers, as has been demonstrated in the previous literature. Furthermore, the size and morphology was within the beneficial range of cytosolic cellular uptake and retention, which are essential in both drug delivery (therapy) and tumor accumulation (diagnosis). In our previous work (Kim et al., 2014b), we found that Co2+ in solution can replace lattice Mg2+ in MgAl-NO3-LDH under a controlled hydrothermal condition while preserving the crystal structure, particle size, and morphology. The MTX-LDH in this study is different from the previous MgAl-NO3-LDH, because it contains both organic and inorganic components in layer-by-layer manner. In order to confirm that labelling of Co-57 is possible in MTX-LDH, we carried out a cold experiment with non-radioactive Co2+ ions. From the XRD pattern (Fig. 1b, c), the (003) peak of MTX-LDH slightly shifted to a lower angle from 4.9° to 4.5° upon Co2+ incorporation, corresponding to the interlayer expansion from 1.35 nm to 1.51 nm. Also, we could observe that the peak ratio of area(006)/area(003) increased upon Co2+ incorporation. These results could be explained by the potential incorporation of Co2+ by the interlayer MTX moiety. Since MTX molecules have abundant base sites (primary amines and pyridine amines) in the pteridine part, their electron pairs could coordinate to Co2+ (Thomas et al., 1996; Wang et al., 2005). After incorporation among the interlayer MTX moiety, the Co2+ moiety could increase the (006) peak

Fig. 2. Scanning electron-microscopic image of (a) pristine LDH, (b) MTX-LDH, and (c) Co@MTX-LDH. 4

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Fig. 3. Co k-edge X-ray adsorption spectroscopy (XAS) spectra of (a) LDH, (b) Co@MTX-LDH hybrid, and (c) the Co2+-MTX complex. (A) X-ray absorption near-edge structure (XANES) and (B) Fourier-transform extended X-ray absorption fine structure (FT-EXAFS) spectra.

d-spacing and intensity of (00l) diffraction changes upon Co2+ labelling, it was clear that the particle size and morphology of MTX-LDH did not significantly change. According to the statistical analyses, the lateral size of Co@MTX-LDH of 150 ± 15 nm is not different from that of MTX-LDH under a 99.9% confidence level in the Student's t-test. In addition to the loading of the anticancer agent MTX, Co2+ incorporation was proven to occur topotactically, and the materials would have size and morphology benefits as theranostic nanomedicine. In order to elucidate the intact structure of MTX before and after Co2+ labelling, we obtained Fourier-transform infrared (FT-IR) spectra (Fig. 4). The MTX molecules themselves (Fig. 4a) show the characteristic stretching vibrations of COOH, C]C, and CeN at 1646 cm−1, 1450 cm−1 and 1099 cm−1, respectively. The IR spectra of MTX-LDH and Co@MTX-LDH both clearly show C]C and CeN stretching vibrations at the same wavenumber position as free MTX. The COOH stretching at 1646 cm−1 of MTX split into two peaks at 1610 cm−1 and 1401 cm−1, attributed to asymmetric (νas) and symmetric stretching vibration (νs) of COO−, showing that the MTX moiety existed as an anion for electrostatic stabilization with LDH layers (Choy et al., 2004). During MTX intercalation into LDH (MTX-LDH), the carboxylic group in MTX was deprotonated producing carboxylate in order to induce effective charge-charge attraction toward positive LDH layer. The disappearance of COOH stretching and the appearance of COO− vibration clearly showed the electrostatic interaction between LDH and MTX both in MTX-LDH and Co@MTX-LDH. It was worthwhile to note here that the wavenumber differences between νas and νs, which is sensitive to the coordination environment against carboxylate, were the same (209 cm−1) for both MTX-LDH and Co@MTX-LDH. Taking into account the XRD (Fig. 1) and XAS (Fig. 3) results together, we could conclude that the existence of interlayer Co2+ did not alter the intact structure of MTX or interrupt the electrostatic stabilization between the MTX and LDH layers.

Because we found that the hydrothermal reaction at 150 °C enabled topotactic Co2+ labeling to MTX-LDH globally preserving its physicochemical properties, we employed the condition for the RI Co-57 labeling using aqueous 15.54 MBq (420 μCi) of 57CoCl2 solution. The radio-labeling efficiency was found to be ~98% (white bar in Fig. 5(a)), which was higher than that of our previous Co-57 labeling into LDH (Kim et al., 2016). This value was also higher than Xu et al. reported, who suggested that layered double hydroxide labeled with Cu-64, Sc44, or Zr-89 have labeling efficiency of 59.0%, 41.4% or 9.5%, respectively (Shi et al., 2015). The interlayer expansion along the c-axis in MTX-LDH would facilitate access of Co-57 into the lattice; the complexation of Co-57 with interlayer MTX would be another reason for high labeling efficiency. After Co-57 labeling, we measured 1 mg of Co57@MTX-LDH particle as having 3.05 MBq (82.4 μCi)/mg of radioactivity (gray bar in Fig. 5(a)), which was sufficiently high to probe particles at either cellular or systemic level. High Co-57 labeling compared with our previous report (Kim et al., 2016) and other radio-labeling nanomedicine (Gawne et al., 2018; Shi et al., 2015) did not mean non-specific attachment of Co-57 on MTXLDH particles. In order to evaluate the stability of Co-57 in MTX-LDH, we did the release test of Co-57 from Co-57@MTX-LDH in phosphatebuffered saline (PBS) and human serum (from human male AB plasma), respectively, with radio-instant thin-layered chromatography during 48 h. The fractional release of Co-57 during the 48 h was ~0% and 12%, respectively, in PBS and human serum (Fig. 5(b)). Since the LDH lattice is not soluble in physiological solutions like PBS and serum, the Co-57 moiety stabilized in the lattice would not be released easily. Slight migration of Co-57 in human serum might result from the Co-57 complexed with the MTX moiety. Because MTX is known to make a stable conjugate with albumin (Taheri et al., 2011), the proteins in serum could detach some MTX moiety complexed with Co-57. Nevertheless, the 88% of Co-57 retention in human serum during 48 h makes Co-57@MTX-LDH attractive as theranostic nanomaterials. The stable retention of Co-57 can exclude false probing of theranostic nanomaterial by tracing released free Co-57. The time-dependent in vitro cellular uptake of Co-57@MTX-LDH was investigated CT-26 cell culture line. As shown in Fig. 6, cellular uptake was ~40 ID% at 1 h and increase during 4 h up to ~60 ID%. It is worthwhile to note that Co-57@MTX-LDH showed dramatically higher cellular uptake than did other nanomaterial labeled with radioisotopes. For example, lipid-polymer hybrid nanoparticles labeled with In-111 and Ga-6- labeled iron oxide nanoparticles showed cellular uptake of around 10% in cancer-cell lines (Cho et al., 2015; Wang et al., 2010). Our previous report on Co@MgAl-CO3-LDH showed a similar high cellular uptake at 1 h with ID% value ~45 in CT-26 cells. The high cellular uptake could be explained by the massive internalization of particles via endocytosis (Oh et al., 2009). The ID% value peaked at 4 h and gradually decreased afterwards, reaching 6.3 ID% at 48 h. The

Fig. 4. Fourier-transform infrared spectra for (a) MTX, (b) MTX-LDH, and (c) Co@MTX-LDH. 5

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Fig. 5. (a) Co-57 labeling efficacy (left white bar) and radioactivity per mass of Co-57/LDH (right gray bar) of Co-57@MTX-LDH. (b) Time-dependent stability of Co57 from Co-57@MTX-LDH in phosphate buffer saline (○) and human serum (□). Inset graphs indicate the stability of Co-57 in Co-57@MTX-LDH within 2 h.

et al., 2007), we carried out an MTT assay and microscopic observation in the MTX-LDH administration range of 5–500 μg/mL. As shown in Fig. 7(a), the MTX-LDH showed significant cytotoxicity (32.5 ± 9.17% cell viability) on CT-26 cell lines at very low concentration of 5 μg/mL. The cancer-cell suppression was fairly concentration-dependent, showing 18.3 ± 6.25% cell viability at 500 μg/mL MTX-LDH concentration. It has been reported that MTX has 50% anticancer activity on CT-26 cells at 209 μg/mL, which is lower anticancer activity than with using MTX-LDH in our research (Nagaj et al., 2015). The MTX loaded in LDH obviously showed increased anticancer activity on CT-26 thanks to the delivery function of the LDH carrier, as reported previously (Kim et al., 2014a; Oh et al., 2006b). Fig. 7(b) shows microscopic images of CT-26 cells before and after MTX-LDH treatment. As shown in the images, MTX-LDH-treated CT-26 cells clearly showed reduced confluency from low concentration. It is interesting that we could observe glassy morphology on the cells when a high concentration of MTX-LDH was administered, showing that the MTX-LDH particles were selectively attached on cancer cells to be internalized and to deliver payload drugs (Oh et al., 2006a; Oh et al., 2011). We further found that MTX-LDH-treated cells showed substantial apoptosis after 48 h of incubation (Fig. S3). These results strongly suggested that MTX-LDH is a suitable candidate for theranostic nanomedicine if Co-57 was properly labeled. The in vivo SPECT/CT images of the Co-57@MTX-LDH-injected mouse model are displayed in Fig. 8. The experiment was carried out

Fig. 6. Radioactivity delivery efficiency of Co-57@MTX-LDH in a CT-26 cellculture line.

tendency is attributed to the endocytosis-exocytosis behavior of LDH as reported before (Oh et al., 2011). In the systemic level, a once exocytosed particle can be re-endocytosed if the particles resides in the tissue. As the current Co-57@MTX-LDH has a suitable size for the EPR effect, the exocytosis is not considered critical in diagnosis or therapy. Although anticancer activity of MTX-LDH is well known in the literature, we investigated the anticancer potential of MTX-LDH in the CT26 cells which we used for in vitro and in vivo tests. Since biocompatibility of LDH itself has been extensively studied (Choi et al., 2008; Choi

Fig. 7. (a) In vitro cell viability test after MTX-LDH at 24 h. (b) Morphology of CT-26 cells upon the concentration of MTX-LDH. 6

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Fig. 8. SPECT/CT image of a tumor in mouse model at (a) 1 h, (b) 3 h and (c) 6 h post intravenous injection of the Co-57@MTX-LDH.

Declaration of competing interest

for the xenografted mouse model with a CT-26 tumor. At the early stage (after 1 h injection), a weak SPECT signal of Co-57 was detected in the tumor (arrows in Fig. 8(a)). The SPECT signal became definitely increased at 3 h of injection (arrows in Fig. 8(b)), suggesting the accumulation of Co-57@MTX-LDH in the tumor, possibly because of the EPR effect (Li et al., 2017). Then signal decrease was observed after 6 h of injection; this decrement could be attributed to the potential excretion of particles by metabolism (Han et al., 2017; Souris et al., 2010). To summarize, injected Co-57@MTX-LDH reached the tumor at an early stage (1 h) and accumulated in the tumor for 3 h; then it can be excreted within 6 h. Taking into account the high cellular uptake of Co57@MTX-LDH in cancer cells, the diagnostics is meaningful when imaging was obtained within 3 h.

The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.clay.2020.105454. References Ahuja, N., Katare, O.P., Singh, B., 2007. Studies on dissolution enhancement and mathematical modeling of drug release of a poorly water-soluble drug using water-soluble carriers. Eur. J. Pharm. Biopharm. 65, 26–38. Al-Jamal, W.T., Kostarelos, K., 2011. Liposomes: from a clinically established drug delivery system to a nanoparticle platform for theranostic nanomedicine. Acc. Chem. Res. 44, 1094–1104. Ambrogi, V., Fardella, G., Grandolini, G., Perioli, L., 2001. Intercalation compounds of hydrotalcite-like anionic clays with antiinflammatory agents—I. Intercalation and in vitro release of ibuprofen. Int. J. Pharm. 220, 23–32. Behera, J.K., Zhou, X., Ranjan, A., Simpson, R.E., 2018. Sb2Te3 and its superlattices: optimization by statistical design. ACS Appl. Mater. Interfaces 10, 15040–15050. Celorrio, V., Calvillo, L., van den Bosch, C.A.M., Granozzi, G., Aguadero, A., Russell, A.E., Fermín, D.J., 2018. Mean intrinsic activity of single Mn sites at LaMnO3 nanoparticles towards the oxygen reduction reaction. ChemElectroChem 5, 3044–3051. Chertok, B., David, A.E., Yang, V.C., 2010. Polyethyleneimine-modified iron oxide nanoparticles for brain tumor drug delivery using magnetic targeting and intra-carotid administration. Biomaterials 31, 6317–6324. Cho, B.-B., Park, J.H., Jung, S.J., Lee, J., Lee, J.H., Hur, M.G., Raj, C.J., Yu, K.-H., 2015. Synthesis and characterization of 68 Ga labeled Fe3O4 nanoparticles for positron emission tomography (PET) and magnetic resonance imaging (MRI). J. Radioanal. Nucl. Chem. 305, 169–178. Choi, S.-J., Oh, J.-M., Park, T., Choy, J.-H., 2007. Cellular toxicity of inorganic hydroxide nanoparticles. J. Nanosci. Nanotechnol. 7, 4017–4020. Choi, S.-J., Oh, J.-M., Choy, J.-H., 2008. Safety aspect of inorganic layered nanoparticles: size-dependency in vitro and in vivo. J. Nanosci. Nanotechnol. 8, 5297–5301. Choi, S.-J., Choi, G.E., Oh, J.-M., Oh, Y.-J., Park, M.-C., Choy, J.-H., 2010a. Anticancer drug encapsulated in inorganic lattice can overcome drug resistance. J. Mater. Chem. 20, 9463–9469. Choi, S.-J., Oh, J.-M., Choy, J.-H., 2010b. Biocompatible nanoparticles intercalated with anticancer drug for target delivery: pharmacokinetic and biodistribution study. J. Nanosci. Nanotechnol. 10, 2913–2916. Choy, J.-H., Kwak, S.-Y., Park, J.-S., Jeong, Y.-J., Portier, J., 1999. Intercalative nanohybrids of nucleoside monophosphates and DNA in layered metal hydroxide. J. Am. Chem. Soc. 121, 1399–1400. Choy, J.-H., Park, J.-S., Kwak, S.-Y., Jeong, Y.-J., Han, Y.-S., 2000. Layered double hydroxide as gene reservoir. Mol. Cry. Liq. Cryst. Sci. Technol. Sec. A Mol. Cryst. Liq. Cryst. 341, 425–429. Choy, J.-H., Jung, J.-S., Oh, J.-M., Park, M., Jeong, J., Kang, Y.-K., Han, O.-J., 2004. Layered double hydroxide as an efficient drug reservoir for folate derivatives. Biomaterials 25, 3059–3064. Gabizon, A., Dagan, A., Goren, D., Barenholz, Y., Fuks, Z., 1982. Liposomes as in vivo carriers of adriamycin: reduced cardiac uptake and preserved antitumor activity in mice. Cancer Res. 42, 4734–4739. Gawne, P., Man, F., Fonslet, J., Radia, R., Bordoloi, J., Cleveland, M., Jimenez-Royo, P., Gabizon, A., Blower, P.J., Long, N., de Rosales, R.T.M., 2018. Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine (8-hydroxyquinoline) as an ionophore. Dalton Trans. 47, 9283–9293. Han, Z., Wu, X., Roelle, S., Chen, C., Schiemann, W.P., Lu, Z.-R., 2017. Targeted gadofullerene for sensitive magnetic resonance imaging and risk-stratification of breast cancer. Nat. Commun. 8, 692. Kang, H., Kim, H.-J., Yang, J.-H., Kim, T.-H., Choi, G., Paek, S.-M., Choi, A.-J., Choy, J.-H., Oh, J.-M., 2015. Intracrystalline structure and release pattern of ferulic acid intercalated into layered double hydroxide through various synthesis routes. Appl. Clay

4. Conclusion We successfully prepared uniformly sized pristine LDH and incorporated anticancer MTX and radioisotope Co-57. We found that the reaction was well-controlled in a topotactic manner and thus the size and morphology of the pristine LDH-determining factor in the EPR effect and selective cellular uptake were successfully preserved. The Co2+ incorporation seemed to occur in both the LDH lattice and interlayer MTX moiety. However, the possible complexation between interlayer MTX and Co2+ did not alter the intact structure of the MTX. Furthermore, the retention of Co-57 in Co-57@MTX-LDH was almost 90% in human serum after 48 h, suggesting the high stability of labeled RI. These results revealed that, in current nanomedicine, the therapeutic moiety (MTX) and diagnostic component (Co-57) do not affect each other adversely. Rather, they generate a synergistic effect, while LDH serves as an excellent theranostic nanoplatform. The in vitro cellular uptake test indicated that Co-57@MTX-LDH showed a maximum ~60 ID% at 4 h on CT-26 cells. In vivo SPECT/CT images of Co/MTXLDH with a CT-26 xenografted mouse model showed significant SPECT signals in tumor tissue from 1 h, and accumulation of Co-57@MTX-LDH was observed for 3 h. As we found in this study that MTX-LDH had a high cancer-cell suppression effect on CT-26, we could conclude that radiolabeled MTX-LDH is a potential candidate for theranostic nanomaterial.

Funding This work was supported by the Radiation Technology R&D Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2017M2A2A6A05016600).

Ethics approval and consent to participate Animal studies were performed in compliance with the animal experimental guidelines and ethics approved by Korea Atomic Energy Research Institute (IACUC-2015-004). 7

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