In vivo nanoparticle-mediated radiopharmaceutical-excited fluorescence molecular imaging

Abstract

Cerenkov luminescence imaging utilizes visible photons emitted from radiopharmaceuticals to achieve in vivo optical molecular-derived signals. Since Cerenkov radiation is weak, non-optimum for tissue penetration and continuous regardless of biological interactions, it is challenging to detect this signal with a diagnostic dose. Therefore, it is challenging to achieve useful activated optical imaging for the acquisition of direct molecular information. Here we introduce a novel imaging strategy, which converts γ and Cerenkov radiation from radioisotopes into fluorescence through europium oxide nanoparticles. After a series of imaging studies, we demonstrate that this approach provides strong optical signals with high signal-to-background ratios, an ideal tissue penetration spectrum and activatable imaging ability. In comparison with present imaging techniques, it detects tumour lesions with low radioactive tracer uptake or small tumour lesions more effectively. We believe it will facilitate the development of nuclear and optical molecular imaging for new, highly sensitive imaging applications.

Figure 1. EO nanoparticle morphology and fluorescence characterization.

(a) SEM visualization of EO nanoparticles with mean size of 85 nm. Scale bar, 500 nm. (b) The optical excitation spectrum with a 620 nm filter displaying the characteristic absorption peaks of EO at 301, 323, 365, 384, 396, 467 and 536 nm. (c,d) The emission spectra of EO excited by 400- and 535-nm lasers displaying the peak emission with both excitation wavelengths at 620 nm.

Figure 2. The study of the radiopharmaceutical excitation mechanism.

(a,b) EO is excited by ¹⁸F-FDG (a) and ^99mTc-MDP (b) with different radiation blocking conditions. From row I to V: normal view, black box blocking CL, aluminium foil blocking β⁺, lead torus blocking γ, normal view. (c,d) The emission intensity of each condition is plotted for using ¹⁸F-FDG (c) and ^99mTc-MDP (d), respectively. Both cases reveal obvious intensity decrease in Condition IV. The slight intensity decrease from Condition I to V in (c) indicates the relatively shorter half life of ¹⁸F.

Figure 3. The investigation of different factors influencing the excitation.

(a) The emission intensity of EO depends on the radioactivity and the radiotracers. (b) The linear relationship between emission intensity and radioactivity. ¹⁸F-FDG and ^99mTc-MDP shows different excitation efficiencies. (c) The emission intensity of EO depends on its mass. (d) The exponential relationship between emission intensity and EO mass. (e) The emission intensity of EO depends on the excitation distance. (f) The inverse relationship between emission intensity and excitation distance. (g,h) Using mirrors and a lead partition to achieve γ and CL dual excitation (g) and pure CL excitation (h), respectively, to measure the fractions of γ- and Cerenkov excitation. (i) The quantification reveals that CL contributed a small portion of the dual-radiation excitation.

Figure 4. The emission profiles and the tissue penetration assessment.

(a) The emission spectra of ¹⁸F-FDG, ¹⁸F-FDG+EO, ¹³¹I-NaI, ¹³¹I-NaI+EO, ^99mTc-MDP+EO and PBS (each radiotracer, 140 μCi; EO, 10 mg). The spectra of ¹⁸F-FDG and ¹³¹I-NaI indicate the Cerenkov light is weighted towards blue (< 500 nm), and the spectra of ¹⁸F-FDG+EO, ¹³¹I-NaI+EO and ^99mTc-MDP+EO show a similar profile with peak emission at 620 nm. The EO absorbance of part of the Cerenkov light is indicated by the difference between the emission spectra of radiotracer with and without EO from the wavelength of 500–570 nm. (b,c) The comparison between Cerenkov luminescence (Control, 50 μCi ¹⁸F-FDG) and ¹⁸F-FDG radiation-excited fluorescence (well 1–4) before (b) and after (c) covering with a piece of 1-mm thick porcine gastric mucosa tissue. (d) The quantification of the optical intensity of each well after covering the tissue.

Figure 5. The in vitro and in vivo phantom comparison between different imaging techniques.

(a) A schematic illustration of the in vitro tissue-mimicking phantom. (b) FMI of the 100 μCi ¹⁸F-FDG+1 mg EO mixture. (c,d) With no filtering, CLI using ¹⁸F-FDG only (c) and REFI using the mixture of EO and ¹⁸F-FDG (d) show obvious different signal intensity. (e,f) With 620-nm filtering, CLI (e) and REFI (f) also demonstrate intensity differences. (g) The optical intensity comparison between CLI and REFI with no filtering and 620-nm filtering. (h) The signal-to-background ratio comparison between FMI and REFI. (i) The PET image of the in vivo phantom shows no significant difference between the two implanted glass tubes (left tube: 50 μCi of ¹⁸F-FDG+0.15 mg of EO mixture, right tube: ¹⁸F-FDG, 50 μCi). (j,k) REFI and CLI show significant differences with no filtering (j) or 620-nm filtering (k). (l) FMI of the in vivo phantom. (m) The quantitative comparison of CLI and REFI in optical intensity with no filtering and 620 nm filtering. (n) The comparison of FMI and REFI in signal-to-background ratio.

Figure 6. The comparison between different optical techniques for in vivo imaging xenografts.

(a) After direct intratumoural injection with EO (0.05 mg) and tail-vein injection of ¹⁸F-FDG (800 μCi) into the Bcap-37 xenografts, REFI shows the best tumour to normal tissue contrast among all three imaging modalities. (b) After tail-vein injection with EO (0.1 mg, 24 h prior) and ¹⁸F-FDG (500 μCi) in the U87MG-xenografted mice, REFI shows significantly greater signal than CLI did with both no filtering and 620-nm filtering. This demonstrates the passive accumulation of the EO nanoparticle in the tumour tissue. (c) The longitudinal observation comparing CLI and REFI in Bcap-37 xenografts. EO (0.1 mg) and 480 μCi of ¹⁸F-FDG are mixed and injected via the tail-vein. Both tumour and bladder show greater optical signal compared with that in the control mice at all time points.

Figure 7. The multimodality comparison for in vivo imaging the dual-tumour xenografts.

(a) Six days after the subcutaneous injection of 4T1-luc2 tumour cells, two tumour lesions are visible on the back of a mouse model (red arrows). (b) Three slices of PET (280 μCi ¹⁸F-FDG) in sagittal and axial directions show clear ¹⁸F-FDG uptake in the lower tumour (red arrows) but no significant uptake in the upper one (white arrows). The position of the three PET slices are indicated in (a) with black dotted lines. (c) Without filtering, REFI (280 μCi ¹⁸F-FDG with 0.1 ml, 1 mg ml⁻¹ EO) shows optical signal of both tumours and brown adipose tissue (left mouse), but CLI does not visualize the upper back tumour (right mouse). (d) With 620 nm filtering, two tumours are visualized in REFI (left mouse), but Cerenkov signal are nearly vanished. (e) FMI of QD620 (0.1 ml, 10 mg ml⁻¹) does not show great tumour to normal tissue contrast due to the non-specificity of the fluorescent probe. The black circle indicates the regions of interest (ROI) of the background for calculating signal-to-background ratio.

Figure 8. The multimodality comparison for in vivo early detection of small tumour lesions.

(a) Sixty-five hours after tumour cell injection, a white light image shows a small tumour lesion (red arrow). (b) BLI confirms the location of the small tumour lesion. (c) Axial PET shows false-negative scan. (d,e) With no filtering and 620 nm filtering, CLI (right mouse) shows false-negative detection (black arrows), but REFI (left mouse) shows true-positive detection (red arrows). (f) FMI of untargetted QD620 shows multiple suspected lesions, and targeted RJ2-DG750 shows overestimation of the tumour region. The signal-to-background ratios of both fluorescent probes are significant lower than that of REFI.

Figure 9. Biodistrubition of EO and toxicity evaluation using MTT assays and H&E microscopy.

(a) In vitro cytotoxicity assay indicated no significant cell morphologic changes and no obvious cell aggregation. Scale bar, 200 μm. (b) The optical signal difference of the EO-injected group and control group (left image, upper row and bottom row) indicated the different concentrations of EO in tissues, blood and urine samples. The quantitative measurements showed that liver, kidneys and urine had a similar EO concentration, which proved the reason of signal enhancement in the bladder during in vivo REFI imaging. The high EO concentration in the spleen was probably because of phagocytosis by macrophages. (c) Compared with the control tissues, the kidneys, liver, lungs, spleen, heart and tumour of the EO tail-vein-injected group (Bcap-37 xenografts) did not show obvious structural changes.