MRI detection of free-contrast agent nanoparticles

Abstract

Purpose: The integration of nanotechnology into biomedical imaging has significantly advanced diagnostic and theranostic capabilities. However, nanoparticle detection in imaging relies on functionalization with appropriate probes. In this work, a new approach to visualize free-label nanoparticles using MRI and MRS techniques is described, consisting of detecting by ¹H CSI specific proton signals belonging to the components naturally present in most of the nanosystems used in preclinical and clinical research.

Methods: Three different nanosystems, namely lipid-based micelles, liposomes, and perfluorocarbon-based nanoemulsions, were synthesized, characterized by high resolution NMR and then visualized by ¹H CSI at 300 MHz. Subsequently the best ¹H CSI performing system was administered to murine models of cancer to evaluate the possibility of tracking the nanosystem by looking at its proton associated signal. Furthermore, an in vitro comparison between ¹H CSI and ¹⁹F MRI was carried out.

Results: The study successfully demonstrates the feasibility of detecting nanoparticles using MRI/MRS without probe functionalization, employing ¹H CSI. Among the nanosystems tested, the perfluorocarbon-based nanoemulsion exhibited the highest SNR. Consequently, it was evaluated in vivo, where its detection was achievable within tumors and inflamed regions via ¹H CSI, and in lymph nodes via PRESS.

Conclusions: These findings present a promising avenue for nanoparticle imaging in biomedical applications, offering potential enhancements to diagnostic and theranostic procedures. This non-invasive approach has the capacity to advance imaging techniques and expand the scope of nanoparticle-based biomedical research. Further exploration is necessary to fully explore the implications and applications of this method.

Keywords: CSI; MRI; liposomes; micelles; nanoemulsions; nanoparticles.

FIGURE 1

High‐resolution NMR spectra of (PEG‐LIPO), PEG‐MIC, and PFCE‐NE (diluted 1:4). High‐resolution NMR spectra of PEG‐LIPO (black line), PEG‐MIC (red line), and PFCE‐NE (orange line) samples acquired at 14T (298 K), without water suppression. A magnification of the 3.5–3.9 ppm spectral window is displayed in the dashed frame, to better appreciate the main peak of DSPE‐PEG2000 and Kolliphor® P188 (3.71 ppm). The peak at 0 ppm corresponds to TSP‐d₄, used for proton quantification. The assignment of the other visible peaks is reported in Figures S1 and S2. The PFCE‐NE spectrum intensity has been reduced to the 25% of the original value, for comparison purposes.

FIGURE 2

In vitro ¹ H‐CSI of PEG‐LIPO, PEG‐MIC, and PFCE‐NE. In vitro MRI of phantoms containing PEG‐LIPO, PEG‐MIC, and PFCE‐NE samples diluted at different concentrations in low‐gelling agar (sample 1 corresponds to the highest concentration of each nanosystem: 0.240 M ¹H and 7.7 x 10¹³ liposomes/mL for PEG‐LIPO, 0.410 M ¹H and 1.6 x 10¹⁵ micelles/mL for PEG‐MIC, 2.640 M ¹H and 3.2 x 10¹⁴ NPs/mL for PFCE‐NE, sample 6 to the lowest one: 0.005 M ¹H and 1.5 x 10¹² liposomes/mL for PEG‐LIPO, 0.008 M ¹H and 3.2 x 10¹³ micelles/mL for PEG‐MIC, 0.050 M ¹H and 6.5 x 10¹² NPs/mL for PFCE‐NE, sample 7 to pure low‐gelling agar). MRI was performed at 7T (Bruker Avance) with a quadrature coil. The mean SNR values calculated over the external noise (N) are displayed for each nanosystem. The error bars correspond to the SD of the SNR in each ROI.

FIGURE 3

In vivo CSI after intratumoral injection of PFCE‐NE. In vivo MRI (7T, Bruker Pharmascan) of a nude mouse bearing a subcutaneous ovarian cancer (A2780 cell line) in the right flank. Anatomical T₂ weighted images of the tumor are displayed before (A) and 10 min after (D) the intratumoral injection of the PFCE‐NE (100 μL, corresponding to around 30 mmol protons/kg body weight). The CSI of the same slices, at 3.71 ppm, was performed before (B) and 30 min after (E) the intratumoral injection of the PFCE‐NE. In (C) and (F) the merging of the T₂ weighted and CSI images is reported. The two circles placed in front of the mouse correspond to two different standard reference tubes: The left tube contains the PFCE‐NE in agar (proton concentration 2.64 M), and the right tube corresponds to pure low‐gelling agar.

FIGURE 4

In vivo PRESS of PLNs acquired 24 h post PFCE‐NE injection in the footpad. In vivo MRI and PRESS of the PLNs of a 4T1 tumor‐bearing mouse performed at 7T (Bruker Pharmascan). Left: Anatomical T₂ weighted image of the PLNs. The two circles placed in front of the mouse correspond to the two standard reference tubes. The squares pictured in the image correspond to the voxels acquired by PRESS: Black, PLN of the left leg; red, PLN of the right leg (tumor‐bearing side); orange, pure low‐gelling agar; yellow, PFCE‐NE in low‐gelling agar (0.66 M proton concentration). Right: Spectra obtained by PRESS in the 0 to 10 ppm spectral region are reported. The water signal was suppressed. The peak corresponding to PFCE‐NE is highlighted with a dashed line.

FIGURE 5

Ex vivo ¹H‐CSI and confocal fluorescence microscopy of PLNs after PFCE‐NE injection in the footpad. Ex vivo MRI of the PLNs of a 4T1 tumor‐bearing mouse performed at 7T. In (A) the anatomical T₂ weighted image of the PLNs is reported. In (B) the CSI‐related signal, acquired at 3.7 ppm, is displayed. In (C) merging of (A) and (B) is performed to perfectly localize the signal. R corresponds to the right PLN, L to the left PLN, G to a standard reference tube containing pure low‐gelling agar, P to a standard reference tube containing PFCE‐NE in low‐gelling agar (proton concentration 0.66 M), W to a water containing tube. In (D, E) confocal microscopy images of (D) the left PLN and (E) the right one are displayed: Blue corresponds to nuclear staining (DAPI), while red corresponds to the rhodamine signal (PFCE‐NE). The scale bar corresponds to 200 μm.

FIGURE 6

In vivo ¹H CSI after intravenous injection of PFCE‐NE in a murine model of inflammation. In vivo MRI (7T, Bruker Pharmascan) images of a murine model of inflammation. Anatomical T₂‐weighted images overlaid with CSI images at 3.71 ppm are shown in the top part of the figure: (A) before, (B) 4, and (C) 24 h post iv injection of PFCE‐NE (50 mmol protons/kg bw). The bottom part of the figure displays graphs showing the (D) quantitative results of PFCE‐NE proton concentration in the two regions of the striatum, both inflamed and non‐inflamed, as well as in the surgery region, and (E) the inflammation index, indicating the asymmetric accumulation of the nanosystem between the inflamed and non‐inflamed striatum.

FIGURE 7

Comparison between ¹H CSI and ¹⁹F MRI detection modes for PFCE‐NE. Comparison between ¹H CSI and ¹⁹F MRI carried out at 7T (Bruker Avance 300 MHz) with a 40 mm ¹H/¹⁹F volume transmit‐receive probe. (A) T₂ weighted image of the phantom, containing different dilutions of PFCE‐NE in low‐gelling agar (sample 1 corresponds to the highest concentration of PFCE‐NE: 6.35 M ¹H, 3.2 M ¹⁹F, and 7.77 x 10¹⁴ NPs/mL, sample 6 to the lowest one: 0.130 M ¹H, 0.064 M ¹⁹F, and 1.55 x 10¹³ NPs/mL, sample 7 to pure low‐gelling agar). (B) Comparison between the SNR values calculated over the external noise (N) for ¹H CSI acquired with a quadrature coil (red triangles), ¹H CSI acquired with a linear ¹H/¹⁹F coil (orange diamonds), or ¹⁹F MRI (black circles). On the x‐axis, the % dilution of PFCE‐NE in low‐gelling agar is reported to better compare the two imaging techniques. In (C), the ¹H CSI of the phantom acquired with the 40 mm ¹H/¹⁹F volume transmit‐receive probe, at 3.71 ppm is displayed. In (D), the ¹⁹F MRI of the phantom is reported.