Cross-Modal Imaging Reveals Nanoparticle Uptake Dynamics in Hematopoietic Bone Marrow during Inflammation

Abstract

Nanoparticles have been employed to elucidate the innate immune cell biology and trace cells accumulating at inflammation sites. Inflammation prompts innate immune cells, the initial responders, to undergo rapid turnover and replenishment within the hematopoietic bone marrow. Yet, we currently lack a precise understanding of how inflammation affects cellular nanoparticle uptake at the level of progenitors of innate immune cells in the hematopoietic marrow. To bridge this gap, we aimed to develop imaging tools to explore the uptake dynamics of fluorescently labeled cross-linked iron oxide nanoparticles in the bone marrow niche under varying degrees of inflammation. The inflammatory models included mice that received intramuscular lipopolysaccharide injections to induce moderate inflammation and streptozotocin-induced diabetic mice with additional intramuscular lipopolysaccharide injections to intensify inflammation. In vivo magnetic resonance imaging (MRI) and fluorescence imaging revealed an elevated level of nanoparticle uptake at the bone marrow as the levels of inflammation increased. The heightened uptake of nanoparticles within the inflamed marrow was attributed to enhanced permeability and retention with increased nanoparticle intake by hematopoietic progenitor cells. Moreover, intravital microscopy showed increased colocalization of nanoparticles within slowly patrolling monocytes in these inflamed hematopoietic marrow niches. Our discoveries unveil a previously unknown role of the inflamed hematopoietic marrow in enhanced storage and rapid deployment of nanoparticles, which can specifically target innate immune cells at their production site during inflammation. These insights underscore the critical function of the hematopoietic bone marrow in distributing iron nanoparticles to innate immune cells during inflammation. Our findings offer diagnostic and prognostic value, identifying the hematopoietic bone marrow as an imaging biomarker for early detection in inflammation imaging, advancing personalized clinical care.

Keywords: MRI; inflammation; intravital microscopy; iron nanoparticle; myelopoiesis.

Figure 1

CLIO-AF647 nanoparticles are taken up by cultured macrophages. (A) RAW264.7 macrophages or bone marrow-derived macrophages (BMDMs) were incubated with and without CLIO-AF647 (CLIO) for 2h at 37 °C. (B) Overview microscopic images of RAW264.7 macrophages incubated with or without CLIO. (C) Target-to-background (TBR) fluorescent ratio of CLIO in RAW264.7 macrophages with or without CLIO-AF647 incubation. (D) Confocal microscopy images of RAW264.7 macrophages after incubation with CLIO nanoparticles, showing two representative cells. (E) Histograms and (F) ImageStream flow cytometric quantification of mean pixel of CLIO per BMDM incubated without (gray; n = 314) and with (red; n = 249) CLIO. (G) ImageStream cytometric visuals display BMDMs in the absence of CLIO (upper panel) and with CLIO nanoparticle internalization (lower panels depict two representative images), with the nanoparticles localized within the cytoplasm of F4/80-positive BMDMs. (H) Confocal micrographs reveal RAW264.7 macrophages post Lysotracker and CLIO nanoparticle treatment. Arrows highlight the colocalization of Lysotracker with CLIO, indicating where nanoparticles fuse with lysosomes (***<0.001, ****<0.0001).

Figure 2

MRI and IVIS fluorescence quantification of signal variations by CLIO-AF647 concentration and by macrophage number incubated with CLIO-AF647. (A) T₂-weighted (T₂w; using Rapid Acquisition with Relaxation Enhancement (RARE) sequence) scout MR image with regions of interest (ROIs) in green dotted line. The rows below are R₁ (by RARE sequence with variable TRs), R₂ (by Multi-Slice Multi-Echo (MSME) sequence with variable TEs), and R₂* (Multi Gradient Echo (MGE) sequence with variable TEs) with parametric maps with increasing concentrations of CLIO-AF647, showing that the lowest number of cells that can be detected with R₂* (yellow arrow). (B) Fluorescence in vivo imaging system (IVIS) images with increasing concentrations of CLIO-AF647 (ROIs in green dotted line). (C, D) Quantification of (C) MRI and (D) IVIS showing an increasing signal with increasing particle concentrations. (E) Phantom T₂w (by RARE sequence), R₂* (by MGE sequence with variable TEs), and IVIS fluorescence images with various numbers of RAW264.7 macrophages after incubation with CLIO-AF647 (ROIs in green dotted line). (F, G) Quantification of signal from (F) R₂* and (G) IVIS fluorescence images in various numbers of macrophages. (MRI was done on a preclinical 9.4T Bruker Biospin; each dot represents one replicate; ns = not significant, *P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 3

In vivo uptake of CLIO-AF647 nanoparticle by blood myeloid cells in healthy mice. (A) Flow cytometric gating strategy employed to quantify the immune response and nanoparticle uptake 48 h post CLIO-AF647 administration, highlighting CD45⁺CD11b⁺ myeloid cells originating from the bone marrow. (B) Gating strategies to identify distinct myeloid subsets in healthy mice, both with and without CLIO-AF647 administration 48h prior. (C) Numbers of Ly6C^{intermediate/high} (Ly6C^int/high) monocytes, Ly6C^low monocytes, and neutrophils remained unchanged regardless of CLIO-AF647 treatment, indicating no overt immune response elicited by the nanoparticles in healthy mice. (D) Quantitative analysis of CLIO-AF647 uptake within each myeloid subset, with (E, F) representative flow cytometric plots for C^int/high monocytes, Ly6C^low monocytes, and neutrophils, (E) with and (F) without prior CLIO-AF647 administration.

Figure 4

In vivo imaging demonstrates increased uptake of CLIO-AF647 nanoparticles in inflamed muscle and bone marrow. (A) In vivo experimental groups depicted progressive increments in inflammation. All mice, including the controls (Con), were subjected to MRI R₂* imaging before and after MRI/IVIS fluorescence imaging 2 days following the intravenous (i.v.) injection of CLIO-AF647 nanoparticles. Lipopolysaccharide was administered intramuscularly (i.m.) 1 day before and 1 day after the i.v. administration of CLIO-AF647 nanoparticles in normal mice (LPS) and in mice with streptozotocin-induced diabetes (STZ+LPS). (B, C) Representative MRI images with an overlay of R₂* map of (B) muscle and (C) femoral bone marrow (using MGE sequence with variable TEs; scale bar = 1 cm). (D, E) Quantification of pre-CLIO R₂*, post-CLIO R₂*, and ΔR₂* (post-CLIO R₂* minus pre-CLIO R₂*) values in muscle and femoral bone marrow, showing increased signal from CLIO-AF647 nanoparticles with increasing inflammation in muscle and bone marrow. (F) Correlation of ΔR₂* in muscle with ΔR₂* in femoral marrow. (G) In vivo whole-body IVIS fluorescence imaging and quantification of i.v. injected CLIO-AF647 nanoparticles. (H) Ex vivo fluorescence imaging and (I) quantification of CLIO-AF647 nanoparticles in inflamed muscle and bone marrow. (J) Correlation of fluorescence in muscle with fluorescence in femoral marrow. (K) Correlation of ΔR₂* in femoral marrow with fluorescence in femoral marrow. (MRI was done on a preclinical 9.4T Bruker Biospin; Each dot represents one mouse; *P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 5

Ex vivo microscopy confirms higher levels of CLIO-AF647 nanoparticles in inflamed muscle and bone marrow. (A) Histological hematoxylin and eosin (H&E) images of muscle tissue, with muscle damage and inflammatory infiltrates indicated by yellow arrows. (B) Fluorescence microscopic images of adjacent muscle tissue showing CLIO-AF647 infiltration (4 μm slice shown). (C) Quantification of CLIO-AF647 particles in muscle per field of view (FOV = 250 × 250 × 4 μm). (D) Confocal maximal intensity projection (MIP) images displaying CLIO-AF647 in Cx3cr1^GFP/+ femoral bone marrow localizing the FOV below the growth plate (GP) in the metaphysis. (E) Quantification of CLIO-AF647 particles in femoral bone marrow per FOV (250 × 250 × 40 μm). (F) Confocal microscopy images, covering 40 μm depth displaying femoral bone marrow uptake of CLIO-AF647, colocalizing with Cx3cr1^GFP/+ monocytes and macrophages. (G) Quantification of overlap coefficient between CLIO and Cx3cr1^GFP/+ monocytes and macrophages in the femoral bone marrow. (H) Perls’ Prussian Blue staining of femoral bone marrow on 4 μm histological sections. (I) Quantification of iron in femoral bone marrow per FOV(250 × 250 × 4 μm). (J) Blue channel of Perls’ Prussian Blue staining to visualize iron deposits from CLIO-AF647. (K) Correlation of CLIO-AF647 fluorescence on 40 μm confocal microscopy images with blue iron deposits of CLIO-AF647 on Perls’ Prussian Blue staining of contralateral femoral bone marrow on 4 μm histological sections (each dot represents one mouse; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 6

Enhanced permeability at inflamed bone marrow. (A) Confocal microscopy images display femoral bone marrow uptake of CLIO-AF647, colocalizing with albumin-RhoB, a blood pool agent 5 min after injection, which quantifies permeability*surface area of vessels. (B) Percentage (%) area of albumin-RhoB at the femoral bone marrow. (C) Representative R₁ parametric maps of pre-CLIO R₁ after injection of Gadolinium (Gd)-DOTA (using a RARE sequence with variable TRs), showing enhanced permeability and retention of Gd in LPS and STZ+LPS femoral marrow. (D) Quantification of pre-CLIO R₁. (MRI was done on a preclinical 9.4T Bruker Biospin; each dot represents one mouse; *P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 7

Elevated CLIO-AF647 uptake in hematopoietic stem and progenitor cells at inflamed bone marrow. (A) Gating strategy for quantification CLIO-AF647 particle uptake by Lineage⁺ (Lin⁺). From Lineage^– cells (Lin^–), LSKs were identified as Lin^–Sca1⁺cKit⁺ and LK cells were identified as Lin^–cKit⁺. (B–D) Flow cytometry and flow cytometric quantification of CLIO⁺ uptake in bone marrow cells: (B) nonhematopoietic lineage⁺ (Lin⁺) cells, (C) Lin^–cKit⁺ (LK), which are erythro-myeloid progenitor cells, and (D) Lin^–Sca1⁺cKit⁺ (LSK) hematopoietic stem cells (each dot represents one mouse; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 8

Enhanced trafficking of CLIO-AF647 nanoparticles at the bone marrow under cumulative inflammatory conditions. (A) Experimental setup illustrating that intravital microscopy (IVM) was conducted either 10 min (day 0) or on day 2 following the intravenous (i.v.) injection of CLIO-AF647 in control mice (con), mice after intramuscular (i.m.) injections of lipopolysaccharide (LPS), or in diabetic mice that also received i.m. injections of lipopolysaccharide (streptozotocin; STZ+LPS). (B) Intravital images displaying a maximum intensity projection (MIP) of a Z-stack from the calvaria 10 min post CLIO-AF647 injection i.v. Cx3cr1^GFP/+ monocytes (green), CD31⁺ vessels (red), and CLIO-AF647 particles (white; dotted rings) are visualized. Time-lapse image processing reveals color-coded maps indicating the maximum speed of each CLIO-AF647 particle. (C) Quantification of the maximum speed of CLIO-AF647 particles 10 min postinjection. (D) Intravital images from the calvaria on day 2 post i.v. injection of CLIO-AF647 showing Cx3cr1^GFP/+ monocytes (green), CD31⁺ vessels (red), and CLIO-AF647 particles (white), with time-lapse series of CLIO-AF647 particles and color-coded maps indicating each particle’s maximum speed. (E) Quantification of CLIO-AF647 particles in calvaria (skull marrow) per field of view (FOV). (F) Quantification of the overlap coefficient between CLIO and Cx3cr1^GFP/+ monocytes in the calvaria. (G) Quantification of the maximum speed while tracking each CLIO-AF647 particle 2 days after injection.