89Zr-labeled dextran nanoparticles allow in vivo macrophage imaging

Abstract

Tissue macrophages play a critical role both in normal physiology and in disease states. However, because of a lack of specific imaging agents, we continue to have a poor understanding of their absolute numbers, flux rates, and functional states in different tissues. Here, we describe a new macrophage specific positron emission tomography imaging agent, labeled with zirconium-89 ((89)Zr), that was based on a cross-linked, short chain dextran nanoparticle (13 nm). Following systemic administration, the particle demonstrated a vascular half-life of 3.9 h and was found to be located primarily in tissue resident macrophages rather than other white blood cells. Subsequent imaging of the probe using a xenograft mouse model of cancer allowed for quantitation of tumor-associated macrophage numbers, which are of major interest in emerging molecular targeting strategies. It is likely that the material described, which allows the visualization of macrophage biology in vivo, will likewise be useful for a multitude of human applications.

Figure 1

(A) General synthesis and radiolabeling of dextran nanoparticles (DNPs). (B) Dynamic laser light scattering data for the 13 nm DNP preparation. C. Transmission electron microscopy image of several individual DNPs measuring 13 nm in diameter on average (arrows).

Figure 2

Pharmacokinetics of different DNP. (A) Effect of size on blood half-life. (B) Effect of surface modification on blood half-life. (C) Biodistribution plotted as tissue concentration 24 h after intravenous administration of succinylated 13 nm DNP. Insert: Distribution to major organs.

Figure 3

PET imaging of ⁸⁹Zr-DNP in mice bearing bilateral flank tumors (24 h after administration). (A and B) two different animals showing similar distribution to all 4 tumors (coronal stacks). (C) Three-dimensional rendering of animal presented in A.

Figure 4

Autoradiography and histology of DNP distribution. (A) Autoradiography of 1 mm tumor and other tissue sections show predominant accumulation in tumors. (B) Co-localization of representative autoradiography sections with adjacent Mac3 immunohistology sections. (C) Distribution of fluorescent DNP to Mac3 positive cells.

Figure 5

Microscopy of DNP distribution in tumor microenvironment in live mouse. (A) 3 h after injection; (B) 24 h after injection. Initially the DNP is primarily seen in vasculature whereas it is almost exclusively co-localized (yellow color) with TAM at 24 h.

Figure 6

(A) Flow cytometric identification of mononuclear phagocytes (CD11b⁺ Lin^low), neutrophils (CD11b⁺ Lin^high (Ly-6G⁺)), and other cells (CD11b⁻) in the tumor stroma (left). The three histograms on the top right show the percent of each population with detectable DNP-680 particles. Nearly all fluorescent DNP (DNP-680) is associated with mononuclear phagocytes; (B) Flow cytometric identification of mononuclear phagocyte subsets. CD11b⁺ Lin^low cells were further divided into Ly6C^hi (i) and Ly6C^int F4/80^int (ii) monocyte-like cells and Ly-6C^int F4/80^hi macrophages (left). The three histograms on the right show the percent of each population with detectable DNP-680 particles. DNP is associated with both monocyte subsets (Ly6C^hi and Ly6C^lo) as well as with macrophages (F4/80^hi).