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Understanding the formation process of nanoparticles is of the utmost importance to improve their design and production. This especially holds true for self-assembled nanoparticles whose formation processes have been largely overlooked. Herein, we present a new technology that integrates a microfluidic-based nanoparticle synthesis method and Förster resonance energy transfer (FRET) microscopy imaging to visualize nanoparticle self-assembly in real time. Applied to different nanoparticle systems, for example, nanoemulsions, drug-loaded block-copolymer micelles, and nanocrystal-core reconstituted high-density lipoproteins, we have shown the approach's unique ability to investigate key parameters affecting nanoparticle formation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cerenkov luminescence (CL) is an emerging imaging modality that utilizes the light generated during the radioactive decay of many clinical used isotopes. Although it is increasingly used for background-free imaging and deep tissue photodynamic therapy, in vivo applications of CL suffer from limited tissue penetration. Here, we propose to use quantum dots (QDs) as spectral converters that can transfer the CL UV-blue emissions to near-infrared light that is less scattered or absorbed in vivo. Experiments on tissue phantoms showed enhanced penetration depth and increased transmitted intensity for CL in the presence of near-infrared (NIR) QDs. To realize this concept for in vivo imaging applications, we developed three types of NIR QDs and 89 Zr dual-labeled nanoparticles based on lipid micelles, nanoemulsions, and polymeric nanoplatforms, which enable codelivery of the radionuclide and the QDs for maximized spectral conversion efficiency. We finally demonstrated the application of these self-illuminating nanoparticles for imaging of lymph nodes and tumors in a prostate cancer mouse model.

Tissue macrophage numbers vary during health versus disease. Abundant inflammatory macrophages destruct tissues, leading to atherosclerosis, myocardial infarction and heart failure. Emerging therapeutic options create interest in monitoring macrophages in patients. Here we describe positron emission tomography (PET) imaging with 18 F-Macroflor, a modified polyglucose nanoparticle with high avidity for macrophages. Due to its small size, Macroflor is excreted renally, a prerequisite for imaging with the isotope flourine-18. The particle's short blood half-life, measured in three species, including a primate, enables macrophage imaging in inflamed cardiovascular tissues. Macroflor enriches in cardiac and plaque macrophages, thereby increasing PET signal in murine infarcts and both mouse and rabbit atherosclerotic plaques. In PET/magnetic resonance imaging (MRI) experiments, Macroflor PET imaging detects changes in macrophage population size while molecular MRI reports on increasing or resolving inflammation. These data suggest that Macroflor PET/MRI could be a clinical tool to non-invasively monitor macrophage biology., Competing Interests: R.W., N.K. and M.N. are inventors on a related patent application. The remaining authors declare no competing financial interests.

Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. We here propose a radically different approach: implementing and evaluating in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library's nanoparticles are based on endogenous high-density lipoprotein, which can preferentially deliver therapeutic compounds to pathological macrophages in atherosclerosis. Using the apolipoprotein E-deficient (Apoe -/- ) mouse model of atherosclerosis, we quantitatively evaluated the library's immune cell specificity by combining immunological techniques and in vivo positron emission tomography imaging. Based on this screen, we formulated a liver X receptor agonist (GW3965) and abolished its liver toxicity while still preserving its therapeutic function. Screening the immune cell specificity of nanoparticles can be used to develop tailored therapies for atherosclerosis and other inflammatory diseases., Competing Interests: The authors declare no conflict of interest.

Unlabelled: Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)-facilitated TAM PET imaging in a breast cancer model., Methods: Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide (89)Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. (89)Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate (89)Zr-PL-HDL and (89)Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of (89)Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry., Results: The phospholipid- and apoA-I-labeled rHDL were produced at 79% ± 13% (n = 6) and 94% ± 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 ± 2.8 and 8.6 ± 1.3 percentage injected dose per gram for apoA-I- and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively., Conclusion: We have developed (89)Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our (89)Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

High-density lipoprotein (HDL) is a natural nanoparticle that exhibits an intrinsic affinity for atherosclerotic plaque macrophages. Its natural targeting capability as well as the option to incorporate lipophilic payloads, e.g., imaging or therapeutic components, in both the hydrophobic core and the phospholipid corona make the HDL platform an attractive nanocarrier. To realize controlled release properties, we developed a hybrid polymer/HDL nanoparticle composed of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core. This novel HDL-like nanoparticle (PLGA-HDL) displayed natural HDL characteristics, including preferential uptake by macrophages and a good cholesterol efflux capacity, combined with a typical PLGA nanoparticle slow release profile. In vivo studies carried out with an ApoE knockout mouse model of atherosclerosis showed clear accumulation of PLGA-HDL nanoparticles in atherosclerotic plaques, which colocalized with plaque macrophages. This biomimetic platform integrates the targeting capacity of HDL biomimetic nanoparticles with the characteristic versatility of PLGA-based nanocarriers.

Atherosclerosis is a major cause of global morbidity and mortality that could benefit from novel targeted therapeutics. Recent studies have shown efficient and local drug delivery with nanoparticles, although the nanoparticle targeting mechanism for atherosclerosis has not yet been fully elucidated. Here we used in vivo and ex vivo multimodal imaging to examine permeability of the vessel wall and atherosclerotic plaque accumulation of fluorescently labeled liposomal nanoparticles in a rabbit model. We found a strong correlation between permeability as established by in vivo dynamic contrast enhanced magnetic resonance imaging and nanoparticle plaque accumulation with subsequent nanoparticle distribution throughout the vessel wall. These key observations will enable the development of nanotherapeutic strategies for atherosclerosis.

Introduction: The application of intravenously administered nanoparticle (NP) therapies is increasingly being explored for a variety of diseases. The key to their success lies in a thorough understanding of nanoparticle pharmacological behavior, specificity and elimination kinetics. Fluorescent imaging techniques provide exciting opportunities to gain insight into such NP behavior in complex biological systems, at macroscopic as well as microscopic levels., Areas Covered: In this review, we will summarize NP labeling methods in relation to their established and emerging fluorescent imaging modalities for in vitro, in vivo, and ex vivo studies of NP behavior. We will highlight novel applications and discuss recent developments in techniques such as fluorescence molecular tomography (FMT), Förster resonance energy transfer (FRET), and Raman imaging. Finally, we will provide a perspective on the challenges and future directions of NP-based fluorescent labeling and imaging in nanotherapeutics., Expert Opinion: Commonly used in preclinical applications, fluorescent imaging of NPs can be achieved with minimal invasiveness and low toxicity in a multiplex fashion. Increasingly applied in the study of NP biodistribution, dissociation, and elimination behavior, fluorescent imaging allows fluid longitudinal tracking and visualization of NP interactions at the level of the whole animal, target organs/tissues, and individual cells.

Unlabelled: Advances in preclinical molecular imaging have generated new opportunities to noninvasively visualize the biodistribution and tumor targeting of nanoparticle therapeutics. Capitalizing on recent achievements in this area, we sought to develop an (89)Zr-based labeling strategy for liposomal nanoparticles that accumulate in tumors via passive targeting mechanisms., Methods: (89)Zr-labeled liposomes were prepared using 2 different approaches: click labeling and surface chelation. Pharmacokinetic and biodistribution studies, as well as PET/CT imaging of the radiolabeled nanoparticles, were performed on a mouse model of breast cancer. In addition, a dual PET/optical probe was prepared by incorporation of a near-infrared fluorophore and tested in vivo by PET and near-infrared fluorescence imaging., Results: The surface chelation approach proved to be superior in terms of radiochemical yield and stability, as well as in vivo performance. Accumulation of these liposomes in tumor peaked at 24 h after injection and was measured to be 13.7 ± 1.8 percentage injected dose per gram. The in vivo performance of this probe was not essentially perturbed by the incorporation of a near-infrared fluorophore., Conclusion: We have developed a highly modular and efficient strategy for the labeling of liposomal nanoparticles with (89)Zr. In xenograft and orthotopic mouse models of breast cancer, we demonstrated that the biodistribution of these nanoparticles can be visualized by PET imaging. In combination with a near-infrared dye, these liposomal nanoparticles can serve as bimodal PET/optical imaging agents. The liposomes target malignant growth, and their bimodal features may be useful for simultaneous PET and intraoperative imaging., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

In the past two decades advances in the development of targeted nanoparticles have facilitated their application as molecular imaging agents and targeted drug delivery vehicles. Nanoparticle-enhanced molecular imaging of the angiogenic tumor vasculature has been of particular interest. Not only because angiogenesis plays an important role in various pathologies, but also since endothelial cell surface receptors are directly accessible for relatively large circulating nanoparticles. Typically, nanoparticle targeting towards these receptors is studied by analyzing the contrast distribution on tumor images acquired before and at set time points after administration. Although several exciting proof-of-concept studies demonstrated qualitative assessment of relative target concentration and distribution, these studies did not provide quantitative information on the nanoparticle targeting kinetics. These kinetics will not only depend on nanoparticle characteristics, but also on receptor binding and recycling. In this study, we monitored the in vivo targeting kinetics of αvβ3-integrin specific nanoparticles with intravital microscopy and dynamic contrast enhanced magnetic resonance imaging, and using compartment modeling we were able to quantify nanoparticle targeting rates. As such, this approach can facilitate optimization of targeted nanoparticle design and it holds promise for providing more quantitative information on in vivo receptor levels. Interestingly, we also observed a periodicity in the accumulation kinetics of αvβ3-integrin targeted nanoparticles and hypothesize that this periodicity is caused by receptor binding, internalization and recycling dynamics. Taken together, this demonstrates that our experimental approach provides new insights in in vivo nanoparticle targeting, which may proof useful for vascular targeting in general.

Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show that this effect is mediated through the inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show that they accumulate in atherosclerotic lesions in which they directly affect plaque macrophages. Finally, we demonstrate that a 3-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a 1-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

In the current study we show the dissociation and tumor accumulation dynamics of dual-labeled near-infrared quantum dot core self-assembled lipidic nanoparticles (SALNPs) in a mouse model upon intravenous administration. Using advanced in vivo fluorescence energy transfer imaging techniques, we observed swift exchange with plasma protein components in the blood and progressive SALNP dissociation and subsequent trafficking of individual SALNP components following tumor accumulation. Our results suggest that upon intravenous administration SALNPs quickly transform, which may affect their functionality. The presented technology provides a modular in vivo tool to visualize SALNP behavior in real time and may contribute to improving the therapeutic outcome or molecular imaging signature of SALNPs.

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail.

We present a multifunctional nanoparticle platform that has targeting moieties shielded by a matrix metalloproteinase-2 (MMP2) cleavable PEG coating. Upon incubation with MMP2 this surface-switchable coating is removed and the targeting ligands become available for binding. The concept was evaluated in vitro using biotin and αvβ3-integrin-specific RGD-peptide functionalized nanoparticles.

Inorganic nanocrystals have myriad applications in medicine, including their use as drug or gene delivery complexes, therapeutic hyperthermia agents, in diagnostic systems and as contrast agents in a wide range of medical imaging techniques. In MRI, nanocrystals can produce contrast themselves, with iron oxides having been the most extensively explored, or can be given a coating that generates MR contrast, for example gold nanoparticles coated with gadolinium chelates. These MR-active nanocrystals can be used for imaging of the vasculature, liver and other organs, as well as molecular imaging, cell tracking and theranostics. As a result of these exciting applications, the synthesis and rendering of these nanocrystals as water soluble and biocompatible are therefore highly desirable. We discuss aqueous phase and organic phase methods for the synthesis of inorganic nanocrystals, such as gold, iron oxides and quantum dots. The pros and cons of the various methods are highlighted. We explore various methods for making nanocrystals biocompatible, i.e. direct synthesis of nanocrystals coated with biocompatible coatings, ligand substitution, amphiphile coating and embedding in carrier matrices that can be made biocompatible. Various examples are highlighted and their applications explained. These examples signify that the synthesis of biocompatible nanocrystals with controlled properties has been achieved by numerous research groups and can be applied to a wide range of applications. Therefore, we expect to see reports of preclinical applications of ever more complex MRI-active nanoparticles and their wider exploitation, as well as in novel clinical settings., (Copyright © 2013 John Wiley & Sons, Ltd.)

Objectives: This study sought to develop magnetic resonance contrast agents based on high-density lipoprotein (HDL) nanoparticles to noninvasively visualize intraplaque macrophages and collagen content in mouse atherosclerotic plaques., Background: Macrophages and collagen are important intraplaque components that play central roles in plaque progression and/or regression. In a Reversa mouse model, plaque regression with compositional changes (from high macrophage, low collagen to low macrophage, high collagen) can be induced., Methods: This study labeled HDL nanoparticles with amphiphilic gadolinium chelates to enable target-specific imaging of intraplaque macrophages. To render HDL nanoparticles specific for the extracellular matrix, labeled HDL nanoparticles were functionalized with collagen-specific EP3533 peptides (EP3533-HDL) via poly(ethylene glycol) spacers embedded in the HDL lipid layers. The association of nanoparticles with collagen was examined in vitro by optical methods. The in vivo magnetic resonance efficacy of these nanoparticles was evaluated in a Reversa mouse model of atherosclerosis regression. Ex vivo confocal microscopy was applied to corroborate the in vivo findings and to evaluate the fate of the different HDL nanoparticles., Results: All nanoparticles had similar sizes (10 ± 2 nm) and longitudinal relaxivity r1 (9 ± 1 s(-1) mmol/l(-1)). EP3533-HDL showed strong association with collagen in vitro. After 28 days of plaque regression in Reversa mice, EP3533-HDL showed significantly increased (p < 0.05) in vivo magnetic resonance signal in aortic vessel walls (normalized enhancement ratio [NERw] = 85 ± 25%; change of contrast-to-noise ratio [ΔCNRw] = 17 ± 5) compared with HDL (NERw = -7 ± 23%; ΔCNRw = -2 ± 4) and nonspecific control EP3612-HDL (NERw = 4 ± 24%; ΔCNRw = 1 ± 6) at 24 h after injection. Ex vivo confocal images revealed the colocalization of EP3533-HDL with collagen. Immunohistostaining analysis confirmed the changes of collagen and macrophage contents in the aortic vessel walls after regression., Conclusions: This study shows that the HDL nanoparticle platform can be modified to monitor in vivo plaque compositional changes in a regression environment, which will facilitate understanding plaque regression and the search for therapeutic interventions., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

One of the challenges of tailored antiangiogenic therapy is the ability to adequately monitor the angiogenic activity of a malignancy in response to treatment. The α(v)β(3) integrin, highly overexpressed on newly formed tumor vessels, has been successfully used as a target for Arg-Gly-Asp (RGD)-functionalized nanoparticle contrast agents. In the present study, an RGD-functionalized nanocarrier was used to image ongoing angiogenesis in two different xenograft tumor models with varying intensities of angiogenesis (LS174T > EW7). To that end, iron oxide nanocrystals were included in the core of the nanoparticles to provide contrast for T(2)*-weighted magnetic resonance imaging (MRI), whereas the fluorophore Cy7 was attached to the surface to enable near-infrared fluorescence (NIRF) imaging. The mouse tumor models were used to test the potential of the nanoparticle probe in combination with dual modality imaging for in vivo detection of tumor angiogenesis. Pre-contrast and post-contrast images (4 hours) were acquired at a 9.4-T MRI system and revealed significant differences in the nanoparticle accumulation patterns between the two tumor models. In the case of the highly vascularized LS174T tumors, the accumulation was more confined to the periphery of the tumors, where angiogenesis is predominantly occurring. NIRF imaging revealed significant differences in accumulation kinetics between the models. In conclusion, this technology can serve as an in vivo biomarker for antiangiogenesis treatment and angiogenesis phenotyping.

Lipid-polymer hybrid (LPH) nanoparticles can deliver a wide range of therapeutic compounds in a controlled manner. LPH nanoparticle syntheses using microfluidics improve the mixing process but are restricted by a low throughput. In this study, we present a pattern-tunable microvortex platform that allows mass production and size control of LPH nanoparticles with superior reproducibility and homogeneity. We demonstrate that by varying flow rates (i.e., Reynolds number (30-150)) we can control the nanoparticle size (30-170 nm) with high productivity (∼3 g/hour) and low polydispersity (∼0.1). Our approach may contribute to efficient development and optimization of a wide range of multicomponent nanoparticles for medical imaging and drug delivery.

Inorganic nanocrystals have a variety of applications in medicine. They may serve as contrast agents, therapeutics, and for in vitro diagnostics. Frequently, the synthesis route yields hydrophobically capped nanocrystals, which necessitates their subsequent coating to render a water-soluble and biocompatible probe. Biocompatibility is crucial for cellular imaging applications, which require large quantities of diagnostically active nanoparticles to be loaded into cells. We have previously reported the design and synthesis of a fluorescent and magnetic resonance imaging-detectable core-shell nanoparticle that encapsulates hydrophobically coated iron oxide nanocrystals. The core of soybean oil and iron oxide is covered by a shell mixture of phospholipids, some of which contained polyethylene glycol. Despite the biocompatibility of these components, we hypothesize that we can improve this formulation with respect to in vitro toxicity. To this aim, we measured the effect of six different core compositions on nanoparticle structure, cell labeling efficacy, and cell viability, as well as cell tracking potential. We methodically investigated the causes of toxicity and conclude that, even when combining biocompatible materials, the resulting formulation is not guaranteed to be biocompatible.

The use of nanotechnology for medical purposes--nanomedicine--has grown exponentially over the past few decades. This is exemplified by the US Food and Drug Administration's approval of several nanotherapies for various conditions, as well as the funding of nanomedical programmes worldwide. Although originally the domain of anticancer therapy, recent advances have illustrated the considerable potential of nanomedicine in the diagnosis and treatment of atherosclerosis. This Review elaborates on nanoparticle-targeting concepts in atherosclerotic disease, provides an overview of the use of nanomedicine in atherosclerosis, and discusses potential future applications and clinical benefits.

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.

The study of lipoproteins, natural nanoparticles comprised of lipids and apolipoproteins that transport fats throughout the body, is of key importance to better understand, treat, and prevent cardiovascular disease. In the current study, we have developed a lipoprotein-based nanoparticle that consists of a quantum dot (QD) core and Cy5.5 labeled lipidic coating. The methodology allows judicious tuning of the QD/Cy5.5 ratio, which enabled us to optimize Förster resonance energy transfer (FRET) between the QD core and the Cy5.5-labeled coating. This phenomenon allowed us to study lipoprotein-lipoprotein interactions, lipid exchange dynamics, and the influence of apolipoproteins on these processes. Moreover, we were able to study HDL-cell interactions and exploit FRET to visualize HDL association with live macrophage cells.

Purpose: To investigate the potential of spectral computed tomography (CT) (popularly referred to as multicolor CT), used in combination with a gold high-density lipoprotein nanoparticle contrast agent (Au-HDL), for characterization of macrophage burden, calcification, and stenosis of atherosclerotic plaques., Materials and Methods: The local animal care committee approved all animal experiments. A preclinical spectral CT system in which incident x-rays are divided into six different energy bins was used for multicolor imaging. Au-HDL, an iodine-based contrast agent, and calcium phosphate were imaged in a variety of phantoms. Apolipoprotein E knockout (apo E-KO) mice were used as the model for atherosclerosis. Gold nanoparticles targeted to atherosclerosis (Au-HDL) were intravenously injected at a dose of 500 mg per kilogram of body weight. Iodine-based contrast material was injected 24 hours later, after which the mice were imaged. Wild-type mice were used as controls. Macrophage targeting by Au-HDL was further evaluated by using transmission electron microscopy and confocal microscopy of aorta sections., Results: Multicolor CT enabled differentiation of Au-HDL, iodine-based contrast material, and calcium phosphate in the phantoms. Accumulations of Au-HDL were detected in the aortas of the apo E-KO mice, while the iodine-based contrast agent and the calcium-rich tissue could also be detected and thus facilitated visualization of the vasculature and bones (skeleton), respectively, during a single scanning examination. Microscopy revealed Au-HDL to be primarily localized in the macrophages on the aorta sections; hence, the multicolor CT images provided information about the macrophage burden., Conclusion: Spectral CT used with carefully chosen contrast agents may yield valuable information about atherosclerotic plaque composition., ((c) RSNA 2010.)

The development of hybrid organic-inorganic nanoparticles is of interest for applications such as drug delivery, DNA and protein recognition, and medical diagnostics. However, the characterization of such nanoparticles remains a significant challenge due to the heterogeneous nature of these particles. Here, we report the direct visualization and quantification of the organic and inorganic components of a lipid-coated silica particle that contains a smaller semiconductor quantum dot. High-angle annular dark-field scanning transmission electron microscopy combined with electron energy loss spectroscopy was used to determine the thickness and chemical signature of molecular coating layers, the element atomic ratios, and the exact positions of different elements in single nanoparticles. Moreover, the lipid ratio and lipid phase segregation were also quantified.

An important challenge in medical diagnostics is to design all-in-one contrast agents that can be detected with multiple techniques such as magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single photon emission tomography (SPECT) or fluorescence imaging (FI). Although many dual labeled agents have been proposed, mainly for combined MRI/FI, constructs for three imaging modalities are scarce. Here gold/silica nanoparticles with a poly(ethylene glycol), paramagnetic and fluorescent lipid coating were synthesized, characterized and applied as trimodal contrast agents to allow for nanoparticle-enhanced imaging of macrophage cells in vitro via MRI, CT and FI, and mice livers in vivo via MRI and CT. This agent can be a useful tool in a multitude of applications, including cell tracking and target-specific molecular imaging, and is a step in the direction of truly multi-modal imaging.

High density lipoprotein (HDL), an endogenous nanoparticle, transports fat throughout the body and is capable of transferring cholesterol from atheroma in the vessel wall to the liver. In the present study, we utilized HDL as a multimodal nanoparticle platform for tumor targeting and imaging via nonspecific accumulation and specific binding to angiogenically activated blood vessels. We reconstituted HDL (rHDL) with amphiphilic gadolinium chelates and fluorescent dyes. To target angiogenic endothelial cells, rHDL was functionalized with alphavbeta3-integrin-specific RGD peptides (rHDL-RGD). Nonspecific RAD peptides were conjugated to rHDL nanoparticles as a control (rHDL-RAD). It was observed in vitro that all 3 nanoparticles were phagocytosed by macrophages, while alphavbeta3-integrin-specific rHDL-RGD nanoparticles were preferentially taken up by endothelial cells. The uptake of nanoparticles in mouse tumors was evaluated in vivo using near infrared (NIR) and MR imaging. All nanoparticles accumulated in tumors but with very different accumulation/binding kinetics as observed by NIR imaging. Moreover, confocal microscopy revealed rHDL-RGD to be associated with tumor endothelial cells, while rHDL and rHDL-RAD nanoparticles were mainly found in the interstitial space. This study demonstrates the ability to reroute HDL from its natural targets to tumor blood vessels and its potential for multimodal imaging of tumor-associated processes.

The development of novel and effective contrast agents is one of the drivers of the ongoing improvement in medical imaging. Many of the new agents reported are nanoparticle-based. There are a variety of natural nanoparticles known, e.g. lipoproteins, viruses or ferritin. Natural nanoparticles have advantages as delivery platforms such as biodegradability. In addition, our understanding of natural nanoparticles is quite advanced, allowing their adaptation as contrast agents. They can be labeled with small molecules or ions such as Gd(3+) to act as contrast agents for magnetic resonance imaging, (18)F to act as positron emission tomography contrast agents or fluorophores to act as contrast agents for fluorescence techniques. Additionally, inorganic nanoparticles such as iron oxide, gold nanoparticles or quantum dots can be incorporated to add further contrast functionality. Furthermore, these natural nanoparticle contrast agents can be re-routed from their natural targets via the attachment of targeting molecules. In this review, we discuss the various modified natural nanoparticles that have been exploited as contrast agents., (Copyright 2009 Elsevier B.V. All rights reserved.)

Multifunctional imaging nanoprobes have proven to be of great value in the research of pathological processes, as well as the assessment of the delivery, fate, and therapeutic potential of encapsulated drugs. Moreover, such probes may potentially support therapy schemes by the exploitation of their own physical properties, e.g., through thermal ablation. This review will present four classes of nanoparticulate imaging probes used in this area: multifunctional probes (1) that can be tracked with at least three different and complementary imaging techniques, (2) that carry a drug and have bimodal imaging properties, (3) that are employed for nucleic acid delivery and imaging, and (4) imaging probes with capabilities that can be used for thermal ablation. We will highlight several examples where the suitable combination of different (bio)materials like polymers, inorganic nanocrystals, fluorophores, proteins/peptides, and lipids can be tailored to manufacture multifunctional probes to accomplish nanomaterials of each of the aforementioned classes. Moreover, it will be demonstrated how multimodality imaging approaches improve our understanding of in vivo nanoparticle behavior and efficacy at different levels, ranging from the subcellular level to the whole body.

Nanoemulsions are increasingly investigated for the delivery of hydrophobic drugs to improve their bioavailability or make their administration possible. In the current study, oil-in-water emulsions with three different mean diameters (30, 60, and 95 nm) were developed as a new multimodality nanoparticle platform for tumor targeting and imaging. To that aim, hydrophobically coated iron oxide particles were included in the soybean oil core of the nanoemulsions to enable their detection with magnetic resonance imaging (MRI), while the conjugation of a near infrared fluorophore allowed optical imaging. The accumulation of this novel nanocomposite in subcutaneous human tumors in nude mice was demonstrated with MRI and fluorescence imaging in vivo, and with Perl's staining of histological tumor sections ex vivo.

Modern medicine has greatly benefited from recent dramatic improvements in imaging techniques. The observation of physiological events through interactions manipulated at the molecular level offers unique insight into the function (and dysfunction) of the living organism. The tremendous advances in the development of nanoparticulate molecular imaging agents over the past decade have made it possible to noninvasively image the specificity, pharmacokinetic profiles, biodistribution, and therapeutic efficacy of many novel compounds. Several types of nanoparticles have demonstrated utility for biomedical purposes, including inorganic nanocrystals, such as iron oxide, gold, and quantum dots. Moreover, natural nanoparticles, such as viruses, lipoproteins, or apoferritin, as well as hybrid nanostructures composed of inorganic and natural nanoparticles, have been applied broadly. However, among the most investigated nanoparticle platforms for biomedical purposes are lipidic aggregates, such as liposomal nanoparticles, micelles, and microemulsions. Their relative ease of preparation and functionalization, as well as the ready synthetic ability to combine multiple amphiphilic moieties, are the most important reasons for their popularity. Lipid-based nanoparticle platforms allow the inclusion of a variety of imaging agents, ranging from fluorescent molecules to chelated metals and nanocrystals. In recent years, we have created a variety of multifunctional lipid-based nanoparticles for molecular imaging; many are capable of being used with more than one imaging technique (that is, with multimodal imaging ability). These nanoparticles differ in size, morphology, and specificity for biological markers. In this Account, we discuss the development and characterization of five different particles: liposomes, micelles, nanocrystal micelles, lipid-coated silica, and nanocrystal high-density lipoprotein (HDL). We also demonstrate their application for multimodal molecular imaging, with the main focus on magnetic resonance imaging (MRI), optical techniques, and transmission electron microscopy (TEM). The functionalization of the nanoparticles and the modulation of their pharmacokinetics are discussed. Their application for molecular imaging of key processes in cancer and cardiovascular disease are shown. Finally, we discuss a recent development in which the endogenous nanoparticle HDL was modified to carry different diagnostically active nanocrystal cores to enable multimodal imaging of macrophages in experimental atherosclerosis. The multimodal characteristics of the different contrast agent platforms have proven to be extremely valuable for validation purposes and for understanding mechanisms of particle-target interaction at different levels, ranging from the entire organism down to cellular organelles.

Nanoparticles have become more and more prevalent in reports of novel contrast agents, especially for molecular imaging, the detection of cellular processes. The advantages of nanoparticles include their potency to generate contrast, the ease of integrating multiple properties, lengthy circulation times, and the possibility to include high payloads. As the chemistry of nanoparticles has improved over the past years, more sophisticated examples of nano-sized contrast agents have been reported, such as paramagnetic, macrophage targeted quantum dots or alpha(v)beta(3)-targeted, MRI visible microemulsions that also carry a drug to suppress angiogenesis. The use of these particles is producing greater knowledge of disease processes and the effects of therapy. Along with their excellent properties, nanoparticles may produce significant toxicity, which must be minimized for (clinical) application. In this review we discuss the different factors that are considered when designing a nanoparticle probe and highlight some of the most advanced examples.

Targeted imaging and therapeutics is becoming a field of prime importance in the study and treatment of cardiovascular disease; it promises to enable early diagnosis, promote improved understanding of pathology, and offer a way to improve therapeutic efficacy. Agents, particularly for cardiovascular disease, have been reported to permit the in vivo imaging, by multiple modalities, of macrophages, vascular targets such as vascular cell adhesion molecule 1, and markers for angiogenesis such as alpha(v)beta(3) integrin. In this Article, we first discuss the general concept of multimodality nanoparticles and then focus in greater depth on their clinical application for molecular imaging and therapy. Lastly, several examples of cardiovascular applications are discussed, including combined imaging and therapy approaches.

Silica is a promising carrier material for nanoparticle-facilitated drug delivery, gene therapy, and molecular imaging. Understanding of their pharmacokinetics is important to resolve bioapplicability issues. Here we report an extensive study on bare and lipid-coated silica nanoparticles in mice. Results obtained by use of a wide variety of techniques (fluorescence imaging, inductively coupled plasma mass spectrometry, magnetic resonance imaging, confocal laser scanning microscopy, and transmission electron microscopy) showed that the lipid coating, which enables straightforward functionalization and introduction of multiple properties, increases bioapplicability and improves pharmacokinetics.

The development of nanoparticulate contrast agents is providing an increasing contribution to the field of diagnostic and molecular imaging. Such agents provide several advantages over traditional compounds. First, they may contain a high payload of the contrast-generating material, which greatly improves their detectability. Second, multiple properties may be easily integrated within one nanoparticle to allow its detection with several imaging techniques or to include therapeutic qualities. Finally, the surface of such nanoparticles may be modified to improve circulation half-lives or to attach targeting groups. Magnetic resonance imaging and optical techniques are highly complementary imaging methods. Combining these techniques would therefore have significant advantages and may be realized through the use of nanoparticulate contrast agents. This review gives a survey of the different types of fluorescent and magnetic nanoparticles that have been employed for both magnetic resonance and optical imaging studies.