Radiolabeling, whole-body single photon emission computed tomography/computed tomography imaging, and pharmacokinetics of carbon nanohorns in mice

Abstract

In this work, we report that the biodistribution and excretion of carbon nanohorns (CNHs) in mice are dependent on their size and functionalization. Small-sized CNHs (30–50 nm; S-CNHs) and large-sized CNHs (80–100 nm; L-CNHs) were chemically functionalized and radiolabeled with [¹¹¹In]-diethylenetriaminepentaacetic acid and intravenously injected into mice. Their tissue distribution profiles at different time points were determined by single photon emission computed tomography/computed tomography. The results showed that the S-CNHs circulated longer in blood, while the L-CNHs accumulated faster in major organs like the liver and spleen. Small amounts of S-CNHs- and L-CNHs were excreted in urine within the first few hours postinjection, followed by excretion of smaller quantities within the next 48 hours in both urine and feces. The kinetics of excretion for S-CNHs were more rapid than for L-CNHs. Both S-CNH and L-CNH material accumulated mainly in the liver and spleen; however, S-CNH accumulation in the spleen was more prominent than in the liver.

Keywords:

• biodistribution
• excretion
• functionalization
• nano-carbon
• nanomedicine

Supplementary materials

Materials and methods

All reagents and solvents were purchased from different commercial suppliers and used as received. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300 MHz instrument (Bruker Corporation, Billerica, MA, USA). The peak values were obtained as ppm (δ) and referenced to the solvent. The resonance multiplicity is indicated as s (singlet), t (triplet), m (multiplet), and br (broad). Liquid chromatography–mass spectrometry analyses were performed on a Thermo Fisher Finnigan LCQ Advantage Max system (Thermo Fisher Scientific, Waltham, MA, USA). Thermogravimetric analysis was performed using a Thermogravimetric analysis 1 (Mettler Toledo, Schwerzenbach, Switzerland) apparatus from 100°C to 900°C with a ramp of 10°C min⁻¹ under N₂ (flow rate of 50 mL⋅min⁻¹) and platinum pans.

The amino-diethylenetriaminepentaacetic acid derivative was synthesized from the t-Bu-protected diethylenetriaminepentaacetic acid ligand reported in Marangon et al.¹ The latter (665 mg) was stirred in trifluoroacetic acid (TFA) (8 mL) and deionized water (0.89 mL) for 17 hours.

Then, the reaction mixture was evaporated under vacuum to give a beige solid in a quantitative yield. ¹H NMR (300 MHz, dimethyl sulfoxide-d₆ as a TFA salt, the signal of TFA was not included) δ=1.42–1.64 (m, 4 H), 1.74–1.92 (m, 2 H), 2.72–2.85 (m, 2 H), 2.98–3.15 (m, 4 H), 3.25–3.40 (m, 4 H), 3.60 (s, 8 H), 4.45 ppm (t, J=6.5 Hz, 1 H), 7.84 (br s, 3 H). ¹³C NMR (75 MHz, dimethyl sulfoxide-d₆ as a TFA salt, the signal of TFA was not included) δ=23.1, 26.3, 26.9, 38.3, 49.9, 50.1, 54.1, 63.7, 170.5, 172.2 ppm. Liquid chromatography–mass spectrometry [molecular weight (ammonium) 465]: m/z 465 [M]⁺.

Figure S1 Preparation of amino-DTPA.

Abbreviations: DTPA, diethylenetriaminepentaacetic acid; h, hours.

Figure S2 Radiolabeling efficiency of [¹¹¹In]DTPA-S-CNHs and [¹¹¹In]DTPA-L-CNHs compared to control [¹¹¹In]DTPA, measured directly after radiolabeling reaction.

Abbreviations: DTPA, diethylenetriaminepentaacetic acid; CNHs, carbon nanohorns; S-CNHs, small-sized CNHs; L-CNHs, large-sized CNHs.

Figure S3 TLC papers showing the radiolabeling stability of [¹¹¹In]DTPA-S-CNHs and [¹¹¹In]DTPA-L-CNHs in mouse serum (left column) and PBS (right column) over 24 hours (h).

Abbreviations: TLC, thin-layer chromatography; DTPA, diethylenetriaminepentaacetic acid; CNHs, carbon nanohorns; S-CNHs, small-sized CNHs; L-CNHs, large-sized CNHs; PBS, phosphate-buffered saline.

Figure S4 Radiolabeling stability of [¹¹¹In]DTPA-S-CNHs and [¹¹¹In]DTPA-L-CNHs in mouse serum (left) and PBS (right) over 24 hours (h).

Abbreviations: DTPA, diethylenetriaminepentaacetic acid; CNHs, carbon nanohorns; S-CNHs, small-sized CNHs; L-CNHs, large-sized CNHs; PBS, phosphate-buffered saline.

Figure S5 SPECT/CT images of [¹¹¹In]DTPA control injected mouse at 0.5, 3.5, and 24 hours (h) postinjection.

Notes: The bright areas indicate the accumulation of [¹¹¹In]DTPA. Images from left to right show whole-body, sagittal, frontal, and transverse views. Cross-sectional images of the spleen, liver, lung, kidney, and urinary bladder are shown in the right column.

Abbreviations: SPECT/CT, single photon emission computed tomography/computed tomography; DTPA, diethylenetriaminepentaacetic acid.

Figure S6 Quantities of [¹¹¹In]DTPA control injected intravenously in mice detected by gamma scintigraphy up to 24 hours (h) postinjection.

Notes: (A) Detected in whole blood by concentration (µg/mL). (B) Percentage ID per whole organ, and (C) expressed by % ID per gram of tissues. Data are represented as mean ± standard error, n=4 mice per group.

Abbreviations: DTPA, diethylenetriaminepentaacetic acid; conc, concentration.

Figure S7 Metabolic profile represented by amounts of ¹¹¹In labeled S- or L-CNHs (% ID) detected in urine and feces 24 hours postinjection.

Notes: (A) [¹¹¹In]DTPA-S-CNHs, (B) [¹¹¹In]DTPA-L-CNHs, and (C) [¹¹¹In]DTPA control.

Abbreviations: DTPA, diethylenetriaminepentaacetic acid; CNHs, carbon nanohorns; S-CNHs, small-sized CNHs; L-CNHs, large-sized CNHs; h, hours.

Table S1 Percentage ID detected in bladder by single-photon emission computed tomography/computed tomography semi-quantification

Reference

• MarangonIMénard-MoyonCKolosnjaj-TabiJCovalent functionalization of multi-walled carbon nanotubes with a gadolinium chelate for efficient T1-weighted magnetic resonance imagingAdv Funct Mater2014244571737186
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Acknowledgments

The authors thank Dr Kasuya and Dr Azami for synthesis of CNHs. MZ thanks Grants-in-Aid for Scientific Research C (22510119) from the Japan Society for the Promotion of Science. This work was supported by the Centre National de la Recherche Scientifique (CNRS), the International Center for Frontier Research in Chemistry (icFRC). AB wishes to thank JSPS (Japanese Society for the Promotion of Science) for the Invitation Fellowship in Japan (ID number L15526). The authors would like to also thank Dr Jane Sosabowski and Professor Stephen Mather from the Barts Cancer Institute, Queen Mary University of London, for their advice with SPECT/CT imaging and analysis.

Disclosure

The authors report no conflicts of interest in this work.