Long-term multimodal imaging of tumor draining sentinel lymph nodes using mesoporous silica-based nanoprobes

Abstract

The imaging of sentinel lymph nodes (SLNs), the first defense against primary tumor metastasis, has been considered as an important strategy for noninvasive tracking tumor metastasis in clinics. In this study, we report the development and application of mesoporous silica-based triple-modal nanoprobes that integrate multiple functional moieties to facilitate near-infrared optical, magnetic resonance (MR) and positron emission tomography (PET) imaging. After embedding near-infrared dye ZW800, the nanoprobe was labeled with T(1) contrast agent Gd(3+) and radionuclide (64)Cu through chelating reactions. High stability and long intracellular retention time of the nanoprobes was confirmed by in vitro characterization, which facilitate long-term in vivo imaging. Longitudinal multimodal imaging was subsequently achieved to visualize tumor draining SLNs up to 3 weeks in a 4T1 tumor metastatic model. Obvious differences in uptake rate, amount of particles, and contrast between metastatic and contra-lateral sentinel lymph nodes were observed. These findings provide very helpful guidance for the design of robust multifunctional nanomaterials in SLNs' mapping and tumor metastasis diagnosis.

Fig. 1

Schematic illustration of tri-modal imaging MSN-probes. (A) Diagram of ZW800 doped MSN fabrication; (B) Diagram of Gd³⁺ and ⁶⁴Cu integration.

Fig. 2

Characterization of MSN-nanoprobes. (A) TEM of nanoprobes, and (B) excitation and emission of MSN-nanoprobes. Fluorescence imaging of particles was directly acquired before (left) and after (right) excitation by Maestro imaging system. (C) Phantom images and quantitative analysis of MSN-nanoprobes by MRI. (D) ⁶⁴Cu labeling efficiency at different ratios between ⁶⁴Cu and particles. The images were shown before (left) and after (right) PET imaging.

Fig. 3

Long-term imaging ability and biodistribution of MSN-nanoprobes in vivo. (A) In vivo optical imaging of nanoprobes over time. The images were normalized.(B) Ex vivo imaging of MSN-nanoprobe biodistribution in different organs after injection for 7 days (left) and 14 days (right).

Fig. 4

Cellular uptake of MSN-probes in macrophage. MSN-nanoprobes were incubated with macrophages for 2 h and free MSN-nanoprobes in the medium were removed by washing. The nucleus and cytoskeleton of cells were stained and subsequently imaged by a confocal microscopy. (A) Nucleus staining of cells with DAPI. (B) Imaging of MSN-FITC. (C) F-actin staining with phallotoxins. (D) The merged imaging of nucleus, particles and F-actin.

Fig. 5

Optical imaging of sentinel lymph nodes in a 4T1 tumor metastatic model. (A) Tumor metastasis was tracked before (left) and after (right) injection with substrate of luciferase by IVIS imaging system. The tumor metastasis model was established by right hock injection of 4T1 cells. Right sentinel lymph node: tumor metastasis (T-SLN). Left opposite sentinel lymph node: normal lymph node (N-SLN) as control. (B) Ex vivo imaging of T-SLN (left) and N-SLN (right) by IVIS imaging system. (C) Optical imaging of tumor metastatic lymph nodes after injection of MSN-nanoprobes at different time points (1 h, 6 h, 1 day, 5 days, 10 days, 15 days and 21 days). Square dot, T-SLN; solid line, N-SLN. (D) Quantitative analysis of the fluorescence signal of T-SLN and N-SLN at different time point. (E) The co-localization of particles and nucleus (DAPI) in T-SLN (E) and N-SLN (F) were imaged by microscopy.

Fig. 6

MRI of sentinel lymph nodes in a 4T1 tumor metastatic model. MRI of lymph nodes were shown (A) before and after injection of MSN-nanoprobes for (B) 1 h, (C) 6 h, (D) 1 day, (E) 5 days and (F) 15 days. Arrow, the accumulation area of particles.

Fig. 7

PET imaging of sentinel lymph nodes in a 4T1 tumor metastatic model. (A) PET imaging of T-SLN (square dot) and N-SLN (solid line) after injection of particles for 1 h, 6 h, 1 day and 2 days. Arrow denotes bladder. Top, cross section; bottom, transverse section. (B) Quantitative comparison of signal between T-SLN and N-SLN.