Willem J. M. Mulder

Multifunctional gold nanoparticles for diagnosis and therapy of disease

Gold nanoparticles (AuNPs) have a number of physical properties that make them appealing for medical applications. For example, the attenuation of X-rays by gold nanoparticles has led to their use in computed tomography imaging and as adjuvants for radiotherapy. AuNPs have numerous other applications in imaging, therapy and diagnostic systems. The advanced state of synthetic chemistry of gold nanoparticles offers precise control over physicochemical and optical properties. Furthermore gold cores are inert and are considered to be biocompatible and nontoxic. The surface of gold nanoparticles can easily be modified for a specific application, and ligands for targeting, drugs or biocompatible coatings can be introduced. AuNPs can be incorporated into larger structures such as polymeric nanoparticles or liposomes that deliver large payloads for enhanced diagnostic applications, efficiently encapsulate drugs for concurrent therapy or add additional imaging labels. This array of features has led to the aforementioned applications in biomedical fields, but more recently in approaches where multifunctional gold nanoparticles are used for multiple methods, such as concurrent diagnosis and therapy, so-called theranostics. This review covers basic principles and recent findings in gold nanoparticle applications for imaging, therapy and diagnostics, with a focus on reports of multifunctional AuNPs.

Atherosclerotic plaque composition: analysis with multicolor CT and targeted gold nanoparticles.

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.

MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle

In oncological research, there is a great need for imaging techniques that specifically identify angiogenic blood vessels in tumors on the basis of differences in the expression level of biomolecular markers. In the angiogenic cascade, different cell surface receptors, including the αvβ3‐integrin, are strongly expressed on activated endothelial cells. In the present study, we aimed to image angiogenesis by detecting the expression of αvβ3 in tumor bearing mice with a combination of magnetic resonance imaging (MRI) and fluorescence microscopy. To that end, we prepared MR‐detectable and fluorescent liposomes, which carry ∼700 αvβ3‐specific RGD peptides per liposome. RGD competition experiments and RAD‐conjugated liposomes were used as controls for specificity. In vivo, both RAD liposomes and RGD liposomes gave rise to signal increase on T1‐weighted MR images. It was established by the use of ex vivo fluorescence microscopy that RGD liposomes and RAD liposomes accumulated in the tumor by different mechanisms. RGD liposomes were specifically associated with activated tumor endothelium, while RAD liposomes were located in the extravascular compartment. This study demonstrates that MR molecular imaging of angiogenesis is feasible by using a targeted contrast agent specific for the αvβ3‐integrin, and that the multimodality imaging approach gave insight into the exact mechanism of accumulation in the tumor.

Nanocrystal core high-density lipoproteins: a multimodality contrast agent platform

High density lipoprotein (HDL) is an important natural nanoparticle that may be modified for biomedical imaging purposes. Here we developed a novel technique to create unique multimodality HDL mimicking nanoparticles by incorporation of gold, iron oxide, or quantum dot nanocrystals for computed tomography, magnetic resonance, and fluorescence imaging, respectively. By including additional labels in the corona of the particles, they were made multifunctional. The characteristics of these nanoparticles, as well as their in vitro and in vivo behavior, revealed that they closely mimic native HDL.

Nanoparticulate assemblies of amphiphiles and diagnostically active materials for multimodality imaging

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.

Perspectives and opportunities for nanomedicine in the management of atherosclerosis

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 Administrations 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.

MRI contrast agents : current status and future perspectives

Magnetic Resonance Imaging (MRI) is increasingly used in clinical diagnostics, for a rapidly growing number of indications. The MRI technique is non-invasive and can provide information on the anatomy, function and metabolism of tissues in vivo. MRI scans of tissue anatomy and function make use of the two hydrogen atoms in water to generate the image. Apart from differences in the local water content, the basic contrast in the MR image mainly results from regional differences in the intrinsic relaxation times T(1) and T(2), each of which can be independently chosen to dominate image contrast. However, the intrinsic contrast provided by the water T(1) and T(2) and changes in their values brought about by tissue pathology are often too limited to enable a sensitive and specific diagnosis. For that reason increasing use is made of MRI contrast agents that alter the image contrast following intravenous injection. The degree and location of the contrast changes provide substantial diagnostic information. Certain contrast agents are predominantly used to shorten the T(1) relaxation time and these are mainly based on low-molecular weight chelates of the gadolinium ion (Gd(3+)). The most widely used T(2) shortening agents are based on iron oxide (FeO) particles. Depending on their chemical composition, molecular structure and overall size, the in vivo distribution volume and pharmacokinetic properties vary widely between different contrast agents and these largely determine their use in specific diagnostic tests. This review describes the current status, as well as recent and future developments of MRI contrast agents with focus on applications in oncology. First the basis of MR image contrast and how it is altered by contrast agents will be discussed. After some considerations on bioavailability and pharmacokinetics, specific applications of contrast agents will be presented according to their specific purposes, starting with non-specific contrast agents used in classical contrast enhanced magnetic resonance angiography (MRA) and dynamic contrast enhanced MRI. Next targeted contrast agents, which are actively directed towards a specific molecular target using an appropriate ligand, functional contrast agents, mainly used for functional brain and heart imaging, smart contrast agents, which generate contrast as a response to a change in their physical environment as a consequence of some biological process, and finally cell labeling agents will be presented. To conclude some future perspectives are discussed.

Improved biocompatibility and pharmacokinetics of silica nanoparticles by means of a lipid coating: a multimodality investigation

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.

Nanotechnology in medical imaging: probe design and applications.

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 αvβ3-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 Molecular Probes for Imaging Atherosclerotic Lesions With Magnetic Resonance Using Antibodies That Recognize Oxidation-Specific Epitopes

Background— Oxidized low-density lipoprotein plays a key role in the initiation, progression, and destabilization of atherosclerotic plaques and is present in macrophages and the lipid pool. The aim of this study was to assess the feasibility of magnetic resonance imaging of atherosclerotic lesions in mice using micelles containing gadolinium and murine (MDA2 and E06) or human (IK17) antibodies that bind unique oxidation-specific epitopes. Methods and Results— MDA2 micelles, E06 micelles, IK17 micelles, nonspecific IgG micelles, and untargeted micelles (no antibody) were prepared and characterized with respect to pharmacokinetics and biodistribution in wild-type and atherosclerotic apolipoprotein E–deficient (apoE−/−) mice. Magnetic resonance imaging was performed at 9.4 T over a 96-hour time interval after the administration of 0.075–mmol Gd/kg micelles. MDA2, E06, and IK17 micelles exhibited a longer plasma half-life than IgG or untargeted micelles in apoE−/− but not wild-type mice. In apoE−/− mice, MDA2 and IK17 micelles showed maximal arterial wall uptake at 72 hours and E06 micelles at 96 hours, manifested by 125% to 231% enhancement in magnetic resonance signal compared with adjacent muscle. Confocal microscopy revealed that MDA2, IK17, and E06 micelles accumulated within atherosclerotic lesions and specifically within macrophages. Intravenous injection of free MDA2 before imaging with MDA2 micelles resulted in significantly diminished magnetic resonance signal enhancement. IgG micelles and untargeted micelles showed minimal enhancement in apoE−/− mice. There was no significant signal enhancement with all micelles in wild-type mice. Conclusions— Magnetic resonance imaging with micelles containing gadolinium and oxidation-specific antibodies demonstrates specific targeting and excellent image quality of oxidation-rich atherosclerotic lesions.