REVIEW ARTICLE PUBLISHED ONLINE: 23 JANUARY 2014 | DOI: 10.1038/NMAT3780

Imaging macrophages with nanoparticles Ralph Weissleder1,2,3*, Matthias Nahrendorf1,3 and Mikael J. Pittet1,3 Nanomaterials have much to offer, not only in deciphering innate immune cell biology and tracking cells, but also in advancing personalized clinical care by providing diagnostic and prognostic information, quantifying treatment efficacy and designing better therapeutics. This Review presents different types of nanomaterial, their biological properties and their applications for imaging macrophages in human diseases, including cancer, atherosclerosis, myocardial infarction, aortic aneurysm, diabetes and other conditions. We anticipate that future needs will include the development of nanomaterials that are specific for immune cell subsets and can be used as imaging surrogates for nanotherapeutics. New in vivo imaging clinical tools for noninvasive macrophage quantification are thus ultimately expected to become relevant to predicting patients’ clinical outcome, defining treatment options and monitoring responses to therapy.

T

he innate immune system (sometimes also called the non-specific immune system) is critically important to tissue injury response and represents the first line of defence against pathogens. Innate immune cells include mononuclear phagocytes (monocytes, macrophages, dendritic cells), granulocytes (neutrophils, eosinophils, basophils) and other cells. Mononuclear phagocytes, first identified by Metschnikoff over 100 years ago, are present in all adult mammalian tissues, albeit in different amounts. Except for the gut, most tissues in steady-state contain macrophages that originate from primitive macrophages; these cells are embryonically established before the appearance of haematopoietic stem and progenitor cells (HSPCs) and then maintained in tissue independently from circulating monocytes1 (Fig.  1). Diseased tissues, however, often accumulate macrophages that derive from circulating blood monocytes. Macrophage numbers in these tissues can increase profoundly by one or several of the following mechanisms: (1) monocyte release from the bone marrow 2, (2) monocyte release from the spleen3, (3) increased monocyte production by HSPCs4,5 or (4) local macrophage amplification in certain settings (for example, helminth infections)6. Macrophages play many key roles in host defence: (1) they remove dead cells, debris and pathogens by phagocytosis; (2) they shape the inflammatory response by secreting cytokines, enzymes and other factors; and (3) they modulate adaptive immunity (the acquired specific immune system) by presenting antigens to lymphocytes. Although many of these functions benefit the host, there is increasing evidence that macrophages can also be detrimental. For example, macrophages that infiltrate the tumour stroma, then called tumour-associated macrophages (TAMs), can operate as components of an inflammatory response that has been co-opted by carcinoma cells to construct a supportive stroma (Fig. 1). A tumour may exert its effects by secreting long-range factors that amplify macrophage progenitors away from the tumour stroma7, generating molecular gradients that attract circulating monocytes8, and producing immunoregulatory cytokines and other factors that educate TAMs in the local tumour microenvironment and foster the acquisition of tumour-promoting functions by these cells9. In turn, TAMs can promote cancer outgrowth by establishing tumour stroma conditions that stimulate angiogenesis, extracellular matrix remodelling, and tumour cell invasion and metastatic dissemination10,11. Many types of tumour with abundant TAMs have been shown to

progress faster and consequently decrease patient survival rates11. Macrophages also represent the largest leukocyte component of atherosclerotic plaques and contribute to lesion ruptures12 that result in stroke and myocardial infarction (Fig. 1). Uncontrolled inflammatory macrophage activity following myocardial infarction can cause ventricular remodelling and heart failure and death (Fig. 1). Other adverse functions have also been described in rheumatoid arthritis13, obesity 14 and diabetes15. The ability to image tissue macrophages (density, flux rates, function and subtypes) thus has important clinical and research applications. Nanomaterials have emerged as a favourite approach to imaging macrophages given their naturally high endocytosis activity (macrophages are ‘big eaters’). The goal of this Review is to summarize some of the nanomaterials and their applications in imaging macrophages in human disease. The approaches summarized here complement other advances in nanomaterial design for imaging, including targeted nanoparticles for imaging and theranostic agents16,17.

Types of nanomaterial for imaging

The following sections describe nanomaterials for different imaging modalities (Table 1). Magnetic nanomaterials. Magnetic resonance imaging (MRI) is a commonly used clinical imaging technology with submillimetre resolution and gives the ability to visualize magnetic nanomaterials in tissues. Biocompatible magnetic nanomaterials (Fig. 2) typically consist of a magnetic core and a hydrophilic surface coating 18,19. The magnetic core of most clinical and biomedical materials is composed of magnetite (Fe3O4) and maghemite (γ-Fe2O3) with a general formula of (FeO)1−n(Fe2O3)n (refs 20–22). More recently, elemental iron cores (cannonballs)23, metallic core–shell systems24,25 and doped magnetites have been described26,27 (Fig.  2). Some of these materials have higher magnetization, higher relaxivities or even different types of magnetism (for example, ferromagnetism rather than superparamagnetism) and are often designed for other magnetic applications, such as magnetic cell separation, diagnostic sensing or hyperthermia applications18,28. The magnetic core is also often solubilized with hydrophilic polymers, such as dextrans, carboxymethylated dextran, polyvinyl alcohol, starches, chitosan, polymethyl methacrylate, polyethylene glycol (PEG), polylactic-co-glycolic acid, polyvinylpyrrolidone and polyacrylic acid, among others29,30.

Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge Street, CPZN 5206, Boston, Massachusetts 02114, USA, 2Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA, 3Department of Radiology, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114, USA. *e-mail: [email protected] 1

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(i) Bone marrow 1

i

3

(ii) Splenic red pulp

HSPC

HSPC

Monocyte reservoir

Monocyte reservoir

ii Blood

Blood

Mø progenitor production and amplification sites Examples of Mø destinations and association with diseases 2. Atherosclerosis

1. Cancer

3. Myocardial infarction

Fibrin clot

Tumour cell

Mø Mø Mø Foam cells Cases in US: ~1.6 × 106 new cases per year Deaths in US: ~5.8 × 105 per year

~4.6 × 106 total cases Causes myocardial infarction and stroke

Dead cardiomyocytes

~8.5 × 105 new cases per year ~6.5 × 105 per year

Figure 1 | Macrophages and their progenitors in the body and their relevance to nanoparticles and imaging. Niches in the bone marrow and sometimes in the spleen contain self-renewing haematopoietic stem and progenitor cells (HSPC), which produce monocytes. These monocytes are released in circulation and can distribute to distant tissue where they differentiate into macrophages (Mø). 1. Tumour-associated Mø often infiltrate tumour sites, for example, in lung (depicted here), breast and pancreatic cancers, and can facilitate tumour progression by promoting angiogenesis, tissue remodelling, tumour cell invasion and metastasis. 2. Monocytes and Mø promote atherosclerotic plaque initiation and progression. They are a rich source of inflammatory mediators and proteases that destabilize the matrix and lead to plaque rupture, triggering myocardial infarction and stroke. 3. Mø orchestrate tissue inflammation and repair during the acute phase after myocardial infarction. Prolonged inflammation in the evolving scar hinders collagen lay-down and promotes heart failure. Mø in major diseases are shown in the lower panels.

The in vivo stability of magnetic nanoparticles can be improved by using dense packing and careful charge, size and shape design31, modified carbohydrate polymers32 or crosslinked polymeric coatings33 (Fig. 2). As an alternative to organic polymeric coating materials, several groups have investigated silica coatings27, monomeric coatings, including citric acid and gluconic acid34,35, phosphate and phosphonate surfactants36–38, high-density lipoproteins39,40, fatty acids41 and others. Table 1 summarizes some of the more common clinical preparations, which mostly consist of densely packed modified dextrans, slightly negative zeta potentials, average diameters of 20–50 nm and superparamagnetic properties for MRI. Library approaches have been used to modulate nanoparticles’ cell-specific tropism (Fig.  2). These approaches include modification of nanoparticles with small molecules42, use of libraries to create different coatings43,44, bionanotechnology approaches45, and peptide or nucleotide libraries46. The use of small-molecule modification is of particular interest, as it can yield unique materials based on the same backbone. Specific approaches explored in the past include using different chemistries, which can result in materials with unique tropism for specific innate immune cells47. Modelling such data can also facilitate the design of future materials48,49. One of the challenges with superparamagnetic agents has been the negative contrast on MRI, which is often hard to interpret, in particular when a haemorrhage is present near bone, lung or areas with blood flow. A number of positive contrast techniques relying on pulse sequence modifications have been described50. 126

Perfluorocarbons are an alternative 19F MRI technique51,52. Some of these agents have recently obtained clinical approval for MRI cell tracking. Nanomaterial-based PET agents. Positron emission tomography (PET) is an increasingly favoured imaging modality that allows rapid quantitation of positron-labelled nanomaterials in organs and tissues53. Several nanoparticle-based PET agents have been described in recent years. The materials most commonly include modified dextrans54,55, graft-copolymers56,57 and other scaffolds58,59. The most common PET isotopes used for labelling include 89Zr (half-life, t1/2: 78.4  hours; chelator: desferoxamine54), 64Cu (t1/2: 12.7 hours; chelator: DOTA, TETA), 124I (t1/2 = 4.18 days; direct conjugation)58,60,61, 76Br (t1/2: 16.2 hours; direct conjugation) and 18F (t1/2: 110 min; direct conjugation)62. The choice of radiotracer is primarily governed by the circulation time of the nanomaterial: long-circulating nanoparticles require longer half-life radioisotopes (89Zr, 64 Cu), whereas rapidly excreted materials can potentially be imaged with short half-life radioisotopes (18F). Fluorescent nanomaterials. Nanoparticles with macrophage affinity are used for microscopy, flow cytometry, endoscopy, optical tomography and intraoperative imaging 63–65. Even the development of MRI, PET, computed tomography and therapeutic nanoparticles often relies on fluorescent versions to optimize the design, validate targeting and analyse tissue sections. Indeed, fluorescent NATURE MATERIALS | VOL 13 | FEBRUARY 2014 | www.nature.com/naturematerials

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NATURE MATERIALS DOI: 10.1038/NMAT3780 Table 1 | Summary of commonly used nanomaterials for imaging of macrophages. Imaging type

Coating

Examples

Size (nm)

Comment

Magnetic

Carboxymethyldextran IO

Ferumoxytol (Feraheme™)

17–31

Food and Drug Administration approved for anaemia and in clinical use

Carboxydextran

IO

Ferucarbotran (SHU 555A, Resovist)

45–60

Food and Drug Administration approved. Discontinued from clinical use in 2009

Dextran

IO

Ferumoxstran (USPIO, AMI-227, NC100150, BMS-180549)

17–21

Discontinued first long-circulating, small iron oxide nanoparticle. Extensive clinical experience

Dextran

IO

Ferumoxides (Feridex™)

100

Discontinued first-generation magnetic resonance imaging agents (liver, spleen)

Crosslinked dextrans

IO

CLIO

30

Versatile experimental platform

Other sugars

IO

Starch, PEG-starch (Clariscan, NC100150), pullulan, chitosan, dextrins

Variable

Experimental

Synthetic

IO

PEG, silica, dendrimer, citrate, phosphates, catechols, monomers

Variable

Experimental

None

DNP

5–15

89

Synthetic

Synthetic

STAR, SCK, silica

Variable

Experimental

Polymers

CdSe, CdTe, ZnS

Qdot® Nanocrystal

1,000

For ultrasound imaging

Silica, PEG, others

Au, Os

Many different

Variable

Experimental Raman imaging

Silica, PEG, dextran

IO, Au, QD

Many different

Variable

Photoacoustic imaging

Positron emission Modified dextrans tomography Fluorescence

Core

Zr, 64Cu, 18F versions. In clinical development

For vascular and intravital imaging For imaging Mø related enzymes Variable

Experimental Clinical trials Experimental

Shown are representative examples with clinical, commercial and/or considerable experimental use (see text for detail on specific preparations). For additional information on some of the listed compounds see the National Institutes of Health Molecular Imaging and Contrast Agent Database (http://www.ncbi.nlm.nih.gov/books/NBK5330/). IO, iron oxides; PVA, polyvinyl alcohol; QD, quantum dots; CLIO, crosslinked dextran iron oxide nanoparticle; DNP, dextran nanoparticle; STAR, core–shell star copolymers; SCK, shell crosslinked knedel-like nanoparticles.

nanoparticles have yielded most of the present-day insights into how macrophages and other cells process nanomaterials. Fluorescent nanoparticles for macrophage imaging generally fall into two commonly used categories: (1) fluorochrome-labelled dextran nanoparticles66,67, PEG68,69, silica nanoparticles70,71 and other polymeric nanoparticles72, and (2) surface-stabilized quantum dots73–76 or upconverting nanoparticles77,78. A few of these preparations are well characterized and are now commercially available (Table 1), but the number of experimental agents abounds. Nanomaterial-based computed tomography agents. X-ray computed tomography is one of the most commonly used clinical imaging techniques. Computed tomography resolutions are typically in the submillimetre range for clinical systems and the micrometre range for preclinical systems. Unlike other imaging techniques, X-ray requires relatively high — even molar — concentrations of absorbent nanomaterials to delineate macrophages. Hence, only a few nanomaterials have emerged as macrophage computed tomography imaging agents, namely N1177 (refs 79–82) or PEGylated gold nanoparticles for experimental use83–85. N1177 is an emulsified suspension containing iodinated particles made up of ethyl3,5-bis(acetylamino)-2,4,6-triiodobenzoate, an esterified derivative of the X-ray contrast agent diatrizoic acid. Two biocompatible surfactants, a polyoxyethylene–polyoxypropylene block copolymer (poloxamer 338) and a PEG, are added to stabilize the particles and

prevent aggregation. Some other materials have also been explored, but their use has been more limited86–88. Higher atomic number materials that absorb X-rays more effectively can be theoretically used to create nanoparticles, but practically these materials are often toxic, placing practical limits on their design. Other modalities for nanomaterial imaging. A number of other imaging modalities exist and for which nanomaterials have been developed to image diverse cell populations. These modalities include ultrasound, photoacoustic imaging, optical coherence imaging, Raman imaging, terahertz imaging, thermography, singlephoton emission computed tomography imaging, magnetic particle imaging and electron microscopy, among others. These modalities are beyond the scope of this Review.

Biological properties of nanomaterials

A large number of nanomaterials have been described and they often have different biological properties. The following sections describe some of the in vivo properties of commonly used nanomaterials. Organ distribution. Pharmacokinetics and organ distribution of nanomaterials (Fig.  3) depend on numerous factors, such as size, surface properties (composition, density, charge), opsonization and particle shape. Recent studies have shown that the effects of opsonization and surface properties drive cell-specific uptake89. Future

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a

Core Cannonball

Doped metal oxide

c

O OH

n

OH O

Polyethylene glycol

O

n OH

OH

OH

Dextran

Carboxymethyl polyvinyl alcohol

O

O OH

OH

O OH

O OH

O

O OH

O

O OH

Carboxymethyl dextran

O

NH2 O O

O

OH

OH

OH

NP 261-16-6 NP 261-16-3 NP 261-16-7 NP 261-16-10 NP…

Nanoparticles with distinct surface modifications

HO

Surface modification Cell types

OH

NP 261-16-3

O

Crosslinked dextran

OH

20 nm

NP 261-16-7

Coating O

20 nm

NP 261-16-10

b

10 nm

NP 261-16-6

20 nm

Pomegranate

Activity profile Low High

NP…

MION

NH2

O COOH

N H

FITC

NH2

O COOH

N H

FITC

NH2

O

N H Cl

FITC

COOH Cl Cl Cl

NH2

O

COOH

N H

FITC

…

Figure 2 | Examples of magnetic nanoparticles for biomedical use. a, Different core materials in common use include monocrystalline iron oxide nanoparticles (MION), core–shell elemental iron nanoparticles (‘cannonballs’), iron oxide cores doped with metals, such as Mn, and multi-assembly cores in a silica coating (‘pomegranate’). b, Commonly used coatings include hydrophilic poly-ol polymers, which may or may not be crosslinked for additional stability. The coating is often further modified to (1) modulate charge, (2) add fluorochromes or other tags and/or (3) attach affinity ligands to achieve cell specificity. c, Small-molecule modified nanoparticles can be screened against different cell lines to determine which modifications alter uptake into subsets of cells. Using this approach, population-specific nanoparticles are being developed181. Panel c reproduced with permission from ref. 181, © 2009 RSC.

directions of nanoparticle development will thus likely focus on the development of more sensitive ‘pan-macrophage’ cell agents, as well as subset-specific agents capable of differentiating between ‘good’ and ‘bad’ macrophages in disease (see section ‘Future needs’). As a general rule, with exceptions of course, large nanomaterials (>1,000 nm in diameter) preferentially accumulate in the liver and lungs (larger materials can cause microemboli when stuck in capillaries), whereas tiny nanoparticles ( MDA-MB-436 triple negative breast cancer cell line).

the ligand for CCR2, monocyte chemotactic protein-1 (also called CCL2), for example, tumour beds, atherosclerotic plaques, ischemic myocardium and other sites. Ly-6Clo monocytes, by contrast, express the fractalkine receptor CX3CR1, patrol the endothelium of blood vessels, and may promote healing and resolution of inflammation107,108. Monocyte subset heterogeneity (and nanoparticle labelling) is largely conserved in mice and humans: human CD14hi monocytes resemble mouse Ly-6Chi cells, whereas human CD14loCD16+ monocytes resemble mouse Ly-6Clo cells109. Together these findings indicate that nanomaterial-based imaging is particularly well suited to assessing inflammatory macrophage activity in both mouse models and humans. Macrophage phenotype in tissue is further shaped by microenvironmental stimuli. Macrophages, for example, can acquire radically different activation states, including so-called inflammatory (M1) and alternatively activated (M2) phenotypes, although intermediary phenotypes prevail in vivo104,105. Nanomaterials have been reported to label efficiently both M1 (for example, in atherosclerotic plaques) and M2 macrophages (for example, in the tumour stroma). Future materials that selectively accumulate in functional subsets of mononuclear phagocytes may serve to dissect the complexity of macrophage biology and advance personalized clinical care. Elimination. The metabolism and excretion of macrophageinternalized nanoparticles has been studied for some preparations, primarily with the intent to have them approved for clinical use (for 130

example, Ferucarbotran, Ferumoxtran, Ferumoxytol)110. However, our understanding of many other materials is still incomplete and can be quite varied even for materials in the same class (Table 1)111. For example, there is limited knowledge of renal tubular effects on either endothelial cells that are at least transiently exposed to high nanoparticle concentrations or on other target cells. Dextran-coated iron oxide nanoparticles have been most closely studied111,112. On cellular uptake, materials are metabolized via the lysosomal pathway 114. The distribution and elimination of these materials have been evaluated using 59Fe and 14C-dextran preparations112,113. In essence, the iron in the core is incorporated into the body’s iron store and is progressively found in red blood cells (haemoglobin). Like endogenous iron, it is eliminated very slowly, predominantly via the faeces. The dextran coating undergoes progressive degradation and predominant renal elimination (~90%).

Imaging applications of nanomaterials

Macrophage-targeting nanoparticles have been mostly used for cancer, atherosclerosis, myocardial infarction and stroke imaging, as macrophage infiltrates are common in these diseases. Cancer imaging. Nanomaterials have been used to image both cancer cells as well as host cells. Imaging tumour-associated macrophages. Tumours are frequently infiltrated by various types of host cell, which regulate different stages NATURE MATERIALS | VOL 13 | FEBRUARY 2014 | www.nature.com/naturematerials

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NATURE MATERIALS DOI: 10.1038/NMAT3780 of cancer progression (positively and negatively) and contribute to tissue remodelling and angiogenesis9,11,115. Therefore, it is increasingly important to measure not only tumour cell fate (mitosis, apoptosis, senescence, molecular alterations), but also tumour stromal biology. TAMs were previously viewed as agents dispatched by the immune system to attack and eliminate tumours; however, extensive research over the past decade implicates these cells as tumoursupporting agents. The presence of TAMs in many human tumour types (breast, ovarian, prostate, thyroid, non-small cell lung cancers, lymphoma) seems to be associated with faster cancer progression and shorter patient survival11,116,117. Similarly, tumour overexpression of the chemokine CCL2, which attracts inflammatory monocytes44, and/or the cytokine macrophage colony-stimulating factor, which regulates monocyte and macrophage differentiation, growth and chemotaxis, is associated with poor prognosis118. Furthermore, the density of TAMs in patients with breast cancer may be used to predict chemotherapeutic efficacy 116. These clinical studies have been supported by a number of basic science studies10,11. Imaging TAMs could thus be used to (1) provide prognostic information, (2) visualize differences in TAM concentrations within a single tumour or across metastases, (3) guide biopsies, (4) define tumour margins101,119 and (5) quantitate responses to treatment by mapping apoptosis-initiated macrophage recruitment. The recognition that MRI-compatible nanomaterials can label TAMs dates back to the mid-1990s101,120,121 and has recently found renewed interest 122,123 (Fig.  5). Macrophage-specific PET imaging agents are also being developed, as quantification and whole-body imaging of TAMs is simpler 54. Interestingly, multiparameter imaging methods, which fuse PET, structural and optical data, integrate and visualize several cancer-related parameters simultaneously a

b

Optical imaging

in live mice. A recent study reported concomitant observation of tumour volume, TAM content, protease activity and integrin expression64, which may accelerate our understanding of tumour growth and response to therapy. Nodal staging by MRI. Lymph node cancer staging has been a major application of Food and Drug Administration-approved magnetic nanoparticles32,124–126 (Fig.  6), which were developed in the early 1990s127,128. Beyond depiction of all lymph nodes for sentinel marking and resection, how can imaging differentiate between benign and malignant nodes? Within all lymph nodes, either benign or malignant, nanoparticles are internalized by macrophages, and these nodal tissue distributions of particles are detectable by MRI. Disturbances in lymph flow or in nodal architecture caused by metastases lead to abnormal patterns of nanoparticle accumulation that present unique MRI features124,125 (Fig. 6). Results from recent clinical trials indicate that MR lymphography significantly enhances the ability to accurately stage lymph node status124,130,131. Cardiovascular imaging. The following section describes the different uses of nanomaterials in cardiovascular imaging. Macrophage imaging in atherosclerosis. Monocytes are recruited into nascent arterial plaques (primarily in the coronary and carotid arteries as well as the aorta) by chemokines and adhesion molecules. In the plaques, these cells ingest low-density lipoproteins, differentiate to macrophages and foam cells, and release pro-inflammatory molecules and proteases132. The end result is luminal narrowing that decreases blood and oxygen supply to dependent tissues, such

Optical–MR imaging

c

MR imaging

T2 MRI

Macrophages (CLIO-680) Tumour cells (H2B-GFP) Vasculature (Angiosense)

1 cm

FMT-MRI 10 µm

d

H&E

100 µm

PET imaging

CD11b

1 mm

1 cm

Figure 5 | Imaging tumour-associated macrophages (TAMs) with multimodal dextran nanoparticles. a, Distribution of TAMs (red; stained by intravenous administration of CLIO-VT680 before sacrifice) in subcutaneously grown flank tumours (H2B-GFP; green). Note the large amount of TAMs (red) inside (top panel) and around (bottom panel) the tumour. Vessels are stained in blue. b, Fusion optical–magnetic resonance (MR) imaging is used to image tumoural heterogeneity of TAMs. Top image shows T2-weighted MRI (T2-MRI) generated by the nanoparticle; middle image shows T2 MRI fused with fluorescence-mediated-tomography (FMT) information derived from the optical properties of the particle; finally, the bottom images show hematoxylin and eosin (H&E) and anti-CD11b staining in a nanoparticle-rich region of the tumour as identified by MRI and FMT. c, Nanoparticles accumulate in a ring-shaped fashion around a human glioblastoma, as indicated by the yellow arrow182. d, Positron emission tomography (PET) imaging of 89Zr-labelled nanoparticles shows extensive uptake in bilateral flank tumours, as indicated by the yellow arrows54. Figures reproduced with permission from: c, ref. 182, © 1999 Wiley; d, ref. 54, © 2011 ACS. NATURE MATERIALS | VOL 13 | FEBRUARY 2014 | www.nature.com/naturematerials

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as the myocardium and the brain. Macrophages destabilize lesions by digesting extracellular matrix, leading to sudden rupture of the plaque, acute thrombotic occlusion of the artery and myocardial infarction or stroke133. It has become clear that these catastrophic events are difficult to predict with anatomic imaging, which measures luminal narrowing rather than the composition of the arterial wall. Indeed, many infarcts originate from non-obstructive vulnerable plaques. For these reasons, macrophage imaging with nanoparticles is attractive and may be used to (1) identify high-risk patients and vascular regions, (2) predict risk of plaque rupture and (3) evaluate therapies. Inflammation in the arterial wall has been imaged with all major modalities in animal models of atherosclerosis83,134–137 and with magnetic nanoparticles in patients138–140 (Fig. 7, Table 1). Proof-of-concept studies have confirmed nanoparticle uptake in plaque macrophages by histology or flow cytometric analysis136,141. In most experimental imaging studies, in  vivo detection of macrophage-ingested nanoparticles has relied on MRI sensing of iron oxide cores142,143, gadolinium-containing particles144 or PET imaging 145 (Fig. 7). Fluorescence sensing of nanoparticles provides a facile tool for basic research in small animals, where tissue penetration is not an obstacle for optical imaging 146. Here, the ability to follow plaque macrophage content serially before and after therapeutic intervention and other perturbations of biology is a particularly interesting advantage136. Clinical imaging of plaque macrophages still remains challenging, because the volume of interest in coronaries and carotids is small and blood vessels move rapidly. Modalities with high sensitivity (PET) and high resolution (MRI) are the most likely to overcome these obstacles for clinical translation, possibly by using hybrid imaging methods. Macrophage imaging in aortic aneurysm. Macrophages also play a key role in aneurysmal dilatations of large arteries, primarily the aorta, and may facilitate rupture — a frequently fatal clinical event. Current clinical guidelines recommend surgical repair if a

aneurysms exceed certain size criteria. This is an imperfect metric as many stable aneurysms are repaired, exposing patients to unnecessary surgery-associated risks, while some smaller unstable aneurysms are left to rupture147. Detecting macrophages with nanoparticles may provide better risk indication than size criteria alone. In a murine model, the PET signal emanating from nanoparticles in macrophages residing in the aneurysm wall (Fig.  7) predicted growth followed by computed tomography 62. In a clinical pilot study 140, patients with distinct mural uptake of ferumoxtran detected by T2*-weighted MRI had a threefold higher growth rate. If this finding holds true in larger cohorts, and nanoparticle uptake also predicts aneurysm rupture risk, clinical decision-making could be based on macrophage infiltration of the aneurysm wall rather than aneurysm size. Macrophage imaging in infarction. Following an ischemic injury in the heart (myocardial infarction, MI) or the brain (stroke), innate immune cells are recruited to clear necrotic tissue and initiate the wound healing process. Dysfunctional resolution of inflammation in the heart can lead to irreversible heart failure; therefore, timely clearance of infarct macrophages is an interesting diagnostic and possibly also a future therapeutic target 148. Imaging myocardial macrophages was first accomplished using iron oxide core detection by T2*-weighted MRI148 (Fig.  7) or by detecting fluorochrome-labelled nanoparticles using optical tomography in mice after coronary ligation67. Gadolinium-loaded nanoparticles and 19F-labelled emulsions have since been used in a similar fashion150,151. Recently, the concept has been translated into patients with acute myocardial infarction (Fig.  7). In two clinical studies, Ferumoxytol enriched in the dyskinetic infarcted left ventricular wall and led to T2* hypoenhancement 152,153. Late gadolinium chelator enhancement, a routine clinical MR tool for infarct imaging, colocalized with the macrophage nanoparticle signal. As blood leukocyte levels after MI predict the development of heart failure

b

c

Macrometastasis

Micrometastasis

Efferent lymphatics yym m Intravenous injection

Metastasis

Aorta

Lymphocytes Iron particles

LN

LN

Before

Afferent lymphatics

Before

Bladder

LN

Bone Rectum

Marginal sinus

Spine

Psoas

Macrophage

Benign Macrometastasis

Blood vessel Lymph vessel

After ~24 hours

2 cm

After

After

Metastasis

Micrometastasis 1 cm

Figure 6 | Use of magnetic nanoparticles for nodal cancer staging. a, The systemically injected long-circulating magnetic particles gain access to the interstitium and are drained through lymphatic vessels. Disturbances in lymph flow or in nodal architecture caused by metastases lead to abnormal nanoparticle accumulation patterns, which are detectable by MRI124. b, Benign and malignant lymph nodes (LN) are not distinguishable by MRI based on their native signal intensity. However, following intravenous administration of magnetic nanoparticles, normal nodes decrease in signal due to accumulation, whereas metastases show heterogeneous or lower decreases that allow detection. c, Conventional MRI in the top panel shows a non-enlarged retroperitoneal node. Once nanoparticles are given intravenously, 2-mm metastases become obvious as hyperintense foci (arrow) within the node. Figure reproduced with permission from ref. 124, © 2003 Massachusetts Medical Society. 132

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b

PET/MRI atherosclerosis

c

PET/CT AAA

MRI atherosclerosis

1 mm

d

1 mm

e

PET/MRI acute MI

Pre injection Gd-HDL particle (mice)

Sinerem (human)

89

18

F-CLIO (mice)

Zr-DNP (mice)

Pre injection

Post injection

1 mm

Post injection

1 cm

f

T2* MRI acute MI

1 mm

T2 MRI heart transplant

1 mm

CLIO (mice)

Feraheme (human)

64

CLIO (mice)

Cu-CLIO (mice)

No rejection

1 mm

1 cm

Rejection

1 cm

1 cm

Figure 7 | Cardiovascular nanoparticle imaging. Acute and chronic inflammation of the cardiovascular system can be imaged with macrophage-avid nanomaterials. a, Positron emission tomography (PET) and magnetic resonance imaging (MRI) of inflamed atherosclerotic plaque in the aortic root of an ApoE–/– mouse with a 89Zr-labelled, core-free dextran nanoparticle (89Zr-DNP)145. b, PET and computed tomography (CT) imaging of an inflamed, expanding aneurysm in the ascending aorta (AAA) of an ApoE–/– mouse that receives angiotensin-2 (ref. 62). Crosslinked iron oxide nanoparticles were derivatized with 18F (18F-CLIO). The yellow dashed line indicates the aortic route. c, MRI of macrophages using iron oxide nanoparticles (ATHEROMA trial)138 and gadolinium-containing high-density lipoprotein (Gd-HDL) particles137. The arrowheads indicate atherosclerotic plaque. d, Acute inflammation detected in murine ischemic myocardium with 64Cu-labelled CLIO (own unpublished data). e, T2*-weighted cine MRI 24 hours after injection of CLIO detects iron content in macrophage-avid nanoparticles. Own unpublished image; please also see refs 66 and 148. This concept was recently translated to human patients with acute myocardial infarction153. In panels d,e, the arrows indicate myocardial infarct. f, Macrophage detection during heart transplant rejection after injection of CLIO156. The left image shows a greyscale MRI while T2 values are colour-coded in the right images (red indicates low T2 values). Figures reproduced with permission from: a, ref. 145, © 2013 Wolters Kluwer Health; b, ref. 62, © 2011 Wolters Kluwer Health; c, left, ref. 138, © 2009 Elsevier; right, ref. 137, © 2011 NPG.

and mortality 154, it is likely that nanoparticle-facilitated detection of protracted macrophage presence in the healing infarct can identify patients at risk with high individual precision. Quantification of infarct leukocytes with PET isotopes, possibly in a hybrid approach that uses delayed gadolinium enhancement MRI for infarct detection (Fig. 7), may have a quantitative edge for monitoring immunomodulatory therapy in future clinical studies. Macrophage imaging in myocarditis. Diffuse macrophage infiltration of the heart also occurs in myocarditis and heart transplants undergoing allograft rejection. The number of macrophages per gram of myocardium (or imaging voxel) is typically much lower when compared with acute inflammation after myocardial infarction. Nevertheless, it is possible to rely on nanoparticles for macrophage detection, at least in animal models. Most studies used MRI detection of iron oxide core nanoparticles in rats or mice after heart transplantation155,156 (Fig.  7). In these studies, the imaging signal correlated with the severity of myocardial inflammation157. If translated, the technique could help avoid transvenous myocardial biopsies, the current invasive clinical standard that is prone to sampling errors and carries risk of complications, such as heart valve damage. Macrophage imaging could potentially be applied to monitoring the acceptance and rejection of other grafted tissues. Macrophage imaging in other inflammatory diseases. Nanomaterials have also been used for macrophage imaging in diabetes158–159, arthritis13,162, asthma163 and neuroinflammation (multiple sclerosis, stroke, and so on)164–167. Perhaps one of the more clinically

advanced applications is imaging type 1 diabetes (T1D) with magnetic nanoparticles (Fig.  8). In a recent publication160, MRI was shown to effectively visualize the pancreas and distinguish patients with recent-onset diabetes from non-diabetic controls. The observation that magnetic nanoparticles accumulate in the pancreas of T1D patients opens the door to exploiting this noninvasive imaging method to follow T1D progression and monitor immunomodulatory agents’ ability to clear insulitis.

In vivo cell tracking

Nanomaterials have been used to exogenously label immune63, stem168,169 and other 170–172 cells. In these applications, the scientific question often concerns cell distribution and ultimate fate on systemic re-administration. Because the natural endocytic capacity of most immune or stem cells is low, compared with that of macrophages, additional manipulations are performed. These include using membrane permeabilizing peptide sequences173, comixtures174 and cell permeabilization175 procedures. Several excellent review articles summarize the state of the art in cell tracking and labelling 169,176.

Nanomaterials in cell biology

Quantum dots have been used to study macrophage uptake, distribution and — a particular theoretical concern — toxicity 177,178. Advantages of quantum dots include their strong fluorescence, lack of bleaching when compared with organic fluorochromes and detectability by electron microscopy. Perhaps an even broader application has been the use of antibody–quantum dot conjugates

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Control patient

Diabetic mouse Control mouse

Islets from control mouse

MRI (pancreatic T2 pseudocolouring)

Diabetic patient

b

IVM

Islet from diabetic mouse

a

NATURE MATERIALS DOI: 10.1038/NMAT3780

10 µm

1 cm

1 cm

Figure 8 | Inflammation imaging in diabetes using nanoparticles. a, During type 1 diabetes development, islets of Langerhans contain inflammatory cells (insulitis) and show increased microvascularity and permeability, shown here by intravital imaging (IVM) during the perfusion phase of a dextran nanoparticle (crosslinked iron oxide nanoparticles labelled with fluorescein isothiocyanate)158. At later phases, nanoparticles accumulate in macrophages in insulitis islets. b, Changes in T2, indicative of nanoparticle accumulation in insulitis, are pseudocoloured and superimposed onto T1 images. In both mice159 and human patients160, there is a clear difference between type 1 diabetes (top) and normal individuals (bottom).

to probe for protein interaction and other biological applications. Finally, a growing literature explores the emerging use of quantum dot-based sensors to study intracellular functions. Alternative materials have included fluorochrome-labelled organosilicates179 and dextran nanoparticles114.

Future needs

Despite considerable advances in nanotechnology over the past decade, some challenges remain. What are some of the opportunities ahead? Exciting opportunities lie ahead in using nanomaterials to image and quantitate the progeny, distribution, kinetics and function of diverse innate immune cells. We anticipate that such materials will be used much more extensively in the future, particularly in translational and clinical studies wherein macrophages play a key role. Examples include imaging innate immune activity in a host of inflammatory diseases, using imaging to gauge disease severity and symmetry and, importantly, quantitating the effects of therapeutic interventions. Initial proof-of-principle studies attest to the feasibility of these applications. Apart from these perhaps more obvious applications, we also foresee an expanded use of biocompatible materials for intraoperative imaging (for example, delineating tumour margins by targeting TAMs), endoscopic imaging and microscopic imaging (microendoscopy). Finally, we anticipate the emergence of newer materials that will target therapeutic drugs to subsets of innate immune cells in an effort to selectively modulate the immune response. For example, silencing the recruitment or function of ‘bad’ macrophages in atherosclerosis or rheumatoid arthritis represents a new approach to efficient therapy 44. Integrating drugs into nanomaterials has also been used to improve the drug safety profile180. What are some of the basic science challenges? We do not yet understand how nanomaterials are internalized and processed by different cells. For example, the detailed regulation of cellular uptake and intracellular processing of cores and coatings remains largely unexplored beyond a few select agents and cell types. This is not a consequence of disinterest, but rather because of limited understanding of macrophage biology, absence of optimized 134

biological reagents (for example, good antibodies against modified dextrans, PEGs) and model cells (for example, primary human cells from specific diseases). Mass kinetics and excretion are also mostly descriptive rather than quantitative at this time. Finally, a more thorough understanding of renal cell processing and toxicity is warranted. What are some of the future research directions? Reviewing recent literature reveals a disequilibrium: an exponentially growing number of papers describe the synthesis of new nanomaterials, but relatively few manuscripts comprehensively investigate the biological behaviour and/or advantages over existing materials. We clearly need more of the latter. Indeed, an argument could be made that all new nanomaterials should be accompanied by more comprehensive biological profiling, including cytometric analysis of cell distribution and other biological assays48,49. One critical need is higher sensitivity imaging that is able to follow individual cells. Although optical methods are certainly capable of following cells that travel close to the surface, there is a lack of methods capable of tracking single cells at depth and throughout the whole body. Newer imaging methods such as magnetic particle imaging and/or photoacoustic imaging might achieve that level of sensitivity. The design of more sensitive nanomaterials would go hand-in-hand with developments in imaging technology. Another area of need is the development of nanoparticle surrogates that can be used to gauge the efficacy of a growing list of nanotherapeutics. These drug carriers are often administered based on maximum tolerated doses and by measuring plasma or tissue levels of drug concentrations. However, these measurements often do not provide answers to questions such as: what is the temporal distribution of a given nanocarrier; which cell types does it accumulate in; what are the distributions to critical organs (for example, bone marrow); how efficient are specific preparations; how does size and/or manipulation of composition change distribution/efficacy in humans; can nanoparticle imaging be used to identify patients who are more likely to respond to nanotherapeutics (that is, those with high enhanced permeability and retention); is nanoparticle imaging predictive of therapeutics? An argument can thus be made to develop surrogate imaging agents for clinical use. NATURE MATERIALS | VOL 13 | FEBRUARY 2014 | www.nature.com/naturematerials

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NATURE MATERIALS DOI: 10.1038/NMAT3780 Finally, a key effort in our and other laboratories has been to determine how to specifically target nanomaterial payloads towards specific innate immune cell subsets. The ability to develop next-generation materials that target ‘bad’ macrophages without affecting ‘good’ ones will require better phenotyping, and perhaps library approaches42,43. Nanoparticle uptake studies conducted in vitro may not always predict nanoparticle behaviour in vivo; thus validation of agents identified in in  vitro screens, and evaluation of their true specificity for a given cell type, will require extensive validation in animal models and eventually in human patients. Alternatively, preclinical nanoparticle screens conducted directly in  vivo, may be envisaged. For example, nanomaterial libraries labelled with unique molecular identifiers may be transfused into mice to discover distinctive nanomaterials that selectively target ‘bad’ macrophages. Many of the materials will probably face regulatory hurdles and scrutiny despite the fact that a number of them are in clinical use and have proven relatively safe. As materials evolve, so will the regulatory process. It is hoped that these hurdles can be navigated more efficiently by defining regulation, setting expectations and canvassing rationales. Irrespective of the specifics, we believe that some of the developed and future materials will have unprecedented uses in basic science research, clinical practice and other scientific applications. Received 10 February 2013; accepted 17 September 2013; published online 23 January 2014

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Acknowledgements

We would like to thank the National Institutes of Health for supporting this research through the National Cancer Institute Centers of Cancer Nanotechnology Excellence consortia and the National Heart Lung and Blood Institute Program of Excellence in Nanotechnology consortia. We especially would like to thank our collaborators and members of Chemical Safety Board for many helpful discussions.

Additional information

Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to R.W.

Competing financial interests

The authors declare no competing financial interests.

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