Radioisotope Co-57 incorporated layered double hydroxide nanoparticles as a cancer imaging agent

Abstract

Radioisotope Co-57 was stably incorporated into a framework of layered double hydroxide nanoparticles through isomorphous substitution. An in vitro cellular uptake test resulted in ∼45 ID% high radioactivity delivering efficiency. In vivo biodistribution demonstrated time-dependent tumor accumulation of Co-57 by LDH showing high tumor-to-organ ratios.

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Revolutionary advances in nanotechnology over the past few decades have heralded a new era of biomedical nanomaterials, or so-called nanomedicines. Nanomaterials such as liposome,^1,2 polymeric,^3–5 ceramic,^6,7 nanoporous^8,9 and layered nanomaterials^10–13 have been extensively studied as smart drug delivery nanocarriers that possess functionalities like organ targeting, high cellular delivery and controlled release. More recently, the research paradigm of nanomedicine shifted to nano-theranosis which enables simultaneous therapeutic drug delivery and diagnostic function.^14,15 Typically, diagnostic function in nanomedicine can be achieved when certain nanomaterials are designed to be traceable in biological systems^16,17 as well as targetable to specific lesions.^18,19 Tracing of nanomedicine is achieved by attaching chromophores like fluorescence or quantum dots to the nanomaterial.^20–22 Furthermore, imaging of nanomedicine through methods like computed tomography (CT), magnetic resonance imaging (MRI) and by nuclear medicine imaging such as single-photon emission computed tomography (SPECT) or positron emission tomography (PET), can be achieved by introducing contrasting moieties to the nanomaterials.^23,24 However, most of diagnostic nanomedicine has potential drawbacks in stability. Contrasting moieties are generally introduced on the outer surface of nanomedicine,^25,26 and thus they can be detached inside the biological system, resulting in false-tracing of nanomedicine.

In order to overcome this difficulty, layered double hydroxide (LDH) could be used as a potential nanomedicine platform. LDHs have a layer-by-layer stacking structure between positively charged metal hydroxide nanolayers (∼0.5 nm in thickness, tens to hundreds of nm in diameter) and interlayer anions. LDHs have various advantages as drug delivery carriers, especially for anticancer therapy. They can accommodate massive amount of anionic drug molecules in their interlayer space (upto 40 w/w%).¹⁰ Controlling the particle diameter to ∼150 nm was considered to aid in both passive tumor targeting through enhanced permeation and retention (EPR)²⁷ and cellular uptake efficiency through clathrin-mediated endocytosis.²⁸ Payload drug molecules can be released into the cytoplasm through partial dissolution of the LDH lattice and by ion exchange reactions.²⁹ In terms of diagnosis, direct incorporation of radioactive metal ion into hydroxide framework of LDH is expected to guarantee stable and robust imaging function.^23,30 They will allow not only bioimaging independent of tissue-depth due to gamma radiation but also accurate quantification of nanoparticles in various organs in vivo.³¹ As a proof-of-concept research for radionuclide Co-57 incorporation into LDH framework, we previously reported isomorphous incorporation of nonradioactive Co into LDH preserving particle size, morphology and crystallinity.³² In the current paper, we isomorphously incorporated radioisotope Co-57 into the LDH nanomaterial, which was synthesized to have a uniformly controlled particle diameter of ∼150 nm in order to take advantage of EPR and efficient endocytosis. Furthermore, both in vitro cellular uptake and in vivo biodistribution tests were performed on cultured cell lines and tumor xenografted mouse model, respectively, to elucidate radioactivity delivery efficiency of Co-57 incorporated into LDHs at both the cellular and systemic levels.

First, we synthesized LDH nanoparticles consisting of Mg and Al hydroxide framework with a homogenous particle diameter of ∼150 nm through coprecipitation-hydrothermal treatment, as previously reported.^33,34 By using powder X-ray diffraction pattern and microscopic studies (Fig. 1), we confirmed that the synthesized LDHs had a well-crystallized hydrotalcite phase (JCPDS no. 14-0191) and an average particle diameter of ∼150 nm, as they were designed for EPR and endocytosis.

Fig. 1 (a) Powder X-ray diffraction pattern, (b) scanning electron microscopic image and (c) atomic force microscopic image with line profile for LDH before Co-57 incorporation.

The scanning electron microscopic (SEM) image in Fig. 1(b) revealed that LDH particles had a fairly homogeneous distribution of diameter and thickness. Particle diameter and thickness were computed from 200 randomly selected particles in SEM images and were determined to be 161.6 ± 17 nm and 61.0 ± 8 nm, respectively, suggesting a uniform size among the LDH particles. A three-dimensional atomic force microscopic (AFM) image and the corresponding line profile for a single LDH particle (Fig. 1(c)) showed a diameter and thickness of 164 nm and 60 nm, respectively, which corresponded well with the SEM results.

The surface charge of nanomaterials was reported to be important for their interaction with cellular membranes,^35–37 as the plasma membrane is negatively charged.³⁸ Positive surface charge on nanoparticles were excreted rapidly by the hepatobiliary route because of charge dependent serum protein adsorption without any organ toxicity.³⁹ Therefore, we evaluated the surface charge of the prepared LDH particles in either deionized water (pH ∼ 7) or in cell culture media (Hyclone RPMI-1640 with 10% fetal bovine serum (FBS)) by measuring the zeta potential with light scattering electrophoresis. The zeta potential of LDH in deionized water was in the positive region of 0–+60 mV, with highly positive average values of +33.6 mV (closed circles in Fig. 2(a)). The high positive surface charge is one of LDHs' advantages, as it enables their efficient cellular interaction.^40–42 Although the zeta potential of LDH shifted in a negative direction in the culture media due to adsorption of electrolytes and proteins,⁴³ positive domains still remained in the zeta potential distribution (open circles in Fig. 2(a)), suggesting efficient cellular interaction of the LDHs.

Fig. 2 (a) Zeta potential distributions and (b) hydrodynamic diameter distributions of LDH (before Co-57 incorporation) in deionized water (pH ∼ 7) (–●–) and cell culture media (RPMI 1640) with 10% FBS (–○–), respectively.

Because LDH nanoparticles may agglomerated into lumps in suspension due to strong inter-particle interactions,⁴⁴ we examined their hydrodynamic diameter in both deionized water and in cell culture media. LDHs in a deionized water suspension exhibited an average hydrodynamic diameter of ∼190 nm (closed circles in Fig. 2(b)), suggesting that LDH particles were well separated in aqueous suspension. LDHs in cell culture media were determined to have average hydrodynamic diameter of ∼310 nm (open circles in Fig. 2(b)), suggesting that only two or three particles gathered due to adsorbed electrolytes.⁴³

Radionuclide Co-57 was isomorphously incorporated into the LDH framework by treating LDHs with 880 μCi of ⁵⁷CoCl₂ solution per 5 mg of LDH under 150 °C hydrothermal condition for 2 h. The resulting mixture was cooled down at room temperature, and then centrifuged to remove free Co-57 under 10 000 rpm for 5 min. The centrifuged precipitate, Co-57 incorporated LDH (Co-57/LDH), was subjected to γ-counter to check the radio-labeling efficiency, which was determined ∼27% (white bar in Fig. 3(A)). Radioactivity per mass of Co-57 incorporated LDH was 48 μCi mg⁻¹ (gray bar in Fig. 3(A)). Taking into account the radioactivity of Co-57, 0.001% of Mg in LDH was replaced by Co-57, and this substitution stoichiometry was in a range that was similar to our previous result.³² That amount of Co-57 incorporation was considered to be advantageous as SPECT imaging agent. Furthermore, the radioactivity of Co-57/LDH (48 μCi mg⁻¹) was sufficient to be detected, and a small amount of Co-57 substitution did not seriously alter the crystal structure, size, morphology or colloidal properties of the LDHs. In order to prove that the Co-57 stably and robustly existed in the LDH, time-dependent Co-57 release from the Co-57/LDH in human serum (from human male AB plasma) was evaluated with radio-thin layered chromatography for 24 h. The Co-57/LDH exhibited only a 20% release of Co-57 over 24 h. This result indicated that the Co-57 was well stabilized in the LDH lattice through the substitution reaction and that the Co-57/LDH could be utilized for nuclear tracing in biological system.

Fig. 3 (A) Co-57 incorporation efficiency (white bar with left y-axis) and radioactivity per mass of Co-57/LDH (grey bar with right y-axis) of Co-57/LDH. (B) Time dependent stability of Co-57 from Co-57/LDH in human serum. Inset graphs indicate the radio-thin layered chromatogram at (a) 15 min and (b) 24 h (◆: Co-57/LDH and ◇: Free Co-57), respectively.

The partition coefficient (log P) of the Co-57/LDH was monitored in a mixture of 1-octanol and phosphate buffered saline (pH 7.4). Radioactivity was then measured in a dosimeter and log P values were estimated. log P was measured as −0.94 ± 0.02 (n = 3), which is an indication of the hydrophilic nature of Co-57/LDH.

We checked the time dependent in vitro cellular uptake of Co-57/LDH in a mouse colon carcinoma cell line (CT-26) and human hepatocellular carcinoma cell line (HepG2). Each cell line (1 × 10⁵) were seeded in 24 well plates and incubated for 24 h. Then 5 μCi of Co-57/LDH was administered to each well. At designed time points (15, 30, 60 and 120 min), cells were collected and the radioactivity was measured with γ-counter. The cellular uptake was presented as a percentage of the injected radioactivity dose (ID%). As displayed in Fig. 4, Co-57 ions in ⁵⁷CoCl₂ solution was not effectively taken up by the cell lines, showing almost 0 ID% even after 2 h. On the other hand, Co-57/LDH showed cellular uptake of ∼13 ID% at early stages (15 min) in both CT-26 and HepG2 cell lines, and uptake finally reached ∼45 ID% after 2 h. The cellular radioactivity delivery of the present Co-57/LDH is relatively high compared to other reported nanoparticle radio-delivery systems. Lipid-polymer hybrid nanoparticles (ChemRad NPs) incorporated with In-111 had ∼2% uptake in prostate adenocarcinoma LNCaP cells over 45 min, and their uptake increased to only ∼15% with targeting aptamer moiety.⁴⁵ Ga-68 labelled iron oxide nanoparticles were reported to have ∼8 ID% of radio-delivery efficiency in CT-26 cells⁴⁶ and ∼4 ID% in human colorectal adenocarcinoma (HT-29) cells⁴⁷ over 2 h, respectively. Therefore, we suggest that Co-57/LDH is a powerful radioactivity delivery carrier for CT-26 or HepG2 cells.

Fig. 4 Radioactivity delivery efficiency of Co-57/LDH in CT-26 (–■–: Co-57/LDH and –□–: Co-57 solution) and HepG2 (–●–: Co-57/LDH and –○–: Co-57 solution) cell culture lines.

Although radioactivity could be effectively delivered to cancer cells by LDH nanoparticles, there remained various concerns regarding biodistribution at a systemic level of radioactivity delivery. Administered nanoparticles can be removed by clearance organs like liver, kidney and spleen,^48,49 which hinders efficient delivery of incorporated radioactivity to the target tumor. Fig. 5(a) shows the time dependent accumulation of Co-57/LDH in main clearance organs (liver, kidney and spleen) as well as tumor on CT-26 xenografted BALB/c mice model at time points 0.25, 0.5, 1, 2 and 24 h. The injected dose per g% (ID per g%) in liver increased to ∼2 ID per g% during 1 h, and deceased afterward. Kidney showed increasing radioactivity upto ∼3 ID per g% over 24 h. In case of spleen, it showed ∼1.5 ID per g% at 0.25 h and rapidly decreased to ∼0.7 ID per g% afterward, with slight increase to ∼1 ID per g% at 24 h. The radioactivity in tumor was relatively low at early stage but significantly increased and reached ∼2.6 ID per g% at 2 h. In order to evaluate tumor targeting efficiency of Co-57/LDH, we calculated tumor-to-organ (T/O) ID per g% ratios at each time point. The T/O ratios were the highest at 2 h (1.6, 1.2 and 4.5 for liver (L), kidney (K) and spleen (S), respectively), while the ratio slightly decreased to 1.0, 0.5, and 1.3 for T/L, T/K and T/S, respectively, at 24 h (Fig. 5(b)). The ratios at 2 and 24 h were similar or higher compared to other reports on radioisotope tagged nanoparticles and nuclear medicines. For instance, nano-graphene oxide with Ga-66 showed T/L ratio approximately 0.3 in 2 h;⁵⁰ Ga-68 labeled N-(2-diethylaminoethyl)benzamide⁵¹ and (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-benzamide⁵² derivative respectively showed T/L = ∼1.5, ∼0.92, T/K = ∼0.28, ∼0.27 and T/S ∼4.5, ∼2.2 at 2 h. Hollow mesoporous silica nanoparticles with Cu-64 showed T/L ratio of ∼1 at 24 h;⁵³ Ga-66 tagged nano-graphene oxide had T/L ratio of ∼0.6 at 24 h;⁵⁰ thiolated gelatin nanoparticle tagged with In-111 showed T/L, T/K and T/S ratio of ∼0.2, ∼0.5 and ∼0.1, respectively, at 24 h.⁵⁴ It should be noted that the surface of current Co-57/LDH had no functional moieties to enhance biological selectivity such as specific ligands, antibodies and etc. The results suggested Co-57/LDH itself as a potential cancer imaging nanoparticle having high tumor specificity.

Fig. 5 (a) Biodistribution of Co-57/LDH in CT-26 xenografted mouse after 24 h (the five bars indicate (from left to right) 0.25, 0.5, 1, 2 and 24 h). (b) Time dependent radioactivity ratio of tumor-to-organs (–□–: liver, –○–: kidney and –△–: spleen) after i.v. injection of Co-57/LDH.

In this work, we successfully incorporated radioisotope Co-57 into LDH framework through hydrothermal assisted substitution reaction, preserving uniform size and morphology of LDH nanoparticles. We found that Co-57/LDH exhibited significantly high cellular uptake in CT-26 and HepG2 cell lines, suggesting that the LDH nanoparticles have the capacity for efficient radioactivity delivery. In vivo biodistribution results of Co-57/LDH with CT-26 xenografted BALB/c mice showed that LDH nanoparticles could selectively deliver radioactivity to tumors with relatively high tumor-to-organ ratios for clearance organs like liver, kidney and spleen. In conclusion, LDH could be utilized to deliver radioactivity to tumors, while avoiding serious clearance and biological removal. Successful demonstration of in vitro/in vivo tumor targeting with Co-57/LDH suggested them suitable nano-platforms for cancer imaging agents.

Acknowledgements

This research was supported by Nuclear R&D Program through National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2015M2B2A4031430, 2015M2A2A4A02043261/5 and 2010M2A2A7035182).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Materials, characterization and experimental procedures. See DOI: 10.1039/c6ra06256c

‡ Equally contributed to the work reported.

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