Nanoparticulate assemblies of amphiphiles and diagnostically active materials for multimodality imaging

Abstract

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.

Figure 1. Paramagnetic and fluorescent liposomes for target-specific imaging

(a) Schematic of liposomes composed of the amphiphilic molecules indicated left. Thiol containing targeting ligands can be covalently linked to the maleimide containing liposomes. (b) Cryo-TEM reveals unilamellar vesicular structures. (c) NMRD-profiles of GD-DTPA (starts high and drops at higher field strengths) and paramagnetic liposomes that show a typical peak at clinically relevant field strengths. (d) Confocal microscopy of cultured endothelial cells (HUVEC) that had been incubated with αvβ3-specific RGD-liposomes and non-specific RAD-liposomes. Reproduced with permission from ref and ref.

Figure 2. MR molecular imaging of tumor angiogenesis

(a) Tumor bearing mice were intravenously injected with the dual modality liposomes. (b) MR image of a slice through the tumor of an animal that was injected with paramagnetic RGD-liposomes reveals pronounced signal increase in the tumor periphery (c) Fluorescence microscopy of DAPI colored ten-μm sections from dissected tumors of mice that were injected with RGD-liposomes. (d) Endothelial cell and liposome co-registration. Vessel staining was done with an endothelial cell specific CD31 antibody (green), while the red fluorescence represents the RGD-liposomes. RGD-liposomes were exclusively found within the vessel lumen or associated with vessel endothelial cells, indicative of a specific association with the angiogenic endothelium. (e, f) Groups of tumor bearing mice that were treated with the anti-angiogenic agents anginex and endostatin underwent molecular MRI, which was followed by quantitative histology of the microvessel density (MVD). (e) Quantification of MVD as mean number of vessels per 0.25 mm². (f) The percentage of the tumor with significant MRI signal enhancement after intravenous injection with RGD-liposomes. Reproduced with permission from ref.

Figure 3. Avidin induced clearance of biotinylated liposomes

Cryo-TEM images of biotinylated paramagnetic liposomes (a) prior to and (b) after overnight incubation with avidin. (c, d) Maximum intensity projections of three-dimensional MRI data sets. Mice that had been injected with paramagnetic biotin-liposomes (pre), followed by the infusion of (c) saline or (d) avidin. (e) Blood Gd levels following intravenous injection of biotinylated liposomes. Avidin (open symbols) or saline infusion (closed symbols) was started 26 minutes after liposome injection. Data points for mice that received avidin are interpolated to guide the eye (dotted line). Reproduced with permission from ref.

Figure 4. Paramagentic and fluorescent PEGylated micelles

(a) Schematic representation of the micellar contrast agent. (b) Cryo-TEM reveals small structures. (c) High resolution T₁-weighted MR images before and 24 hours after administration of macrophage scavenger receptor specific micelles revealed pronounced signal enhancement in the lesioned aortic wall of an apoE-KO mouse. (d) Fluorescence microscopy of a DAPI colored (to stain nuclei) plaque section showed uptake of the rhodamine labeled micelles by macrophages (green). Reproduced with permission from ref.

Figure 5. Imaging apoptosis with micellar iron oxide

(a) Schematic representation of the micellar iron oxide that contained fluorescent amphiphiles (red) and was functionalized with the apoptosis-specific protein Annexin A5. (b) TEM and negative staining TEM of micellar iron oxide. (c) On T₂-weighted images, the uptake of the contrast agent resulted in a signal decrease of the pellet of cells that were incubated with Annexin A5 functionalized micellar iron oxide (right), as compared to control cells (left). (d) Quantitative analysis revealed a significant R₂ increase for the Annexin A5 conjugated nanoparticles, while non-conjugated micelles had no such effect. (e) The specific binding of the agent with apoptotic cells was visualized with fluorescence microscopy. Reproduced with permission from ref.

Figure 6. Paramagnetic QD-micelles for multimodality imaging

(a) Schematic depiction of αvβ3-specific and paramagnetic QD-micelles. (b) Photograph of the QD-micelles in normal light (left) and under UV illumination (right). (c) Fluorescence microscopy of HUVEC incubated with αvβ3-specific and paramagnetic QD-micelles. (d) Intravital microscopy of microvessels in tumor-bearing mice after intravenous injection of RGD-pQDs. The brightfield (left) and the fluorescence image (right) revealed labeling of endothelial cells in tumor blood vessels. (e) Fluorescence image of a tumor bearing mouse following intravenous administration of the contrast agent, revealed tumor accumulation. (f) T₁-weighted MR images before and 45 minutes after the injection of the αvβ3-specific and paramagnetic QD-micelles. Reproduced with permission from ref.

Figure 7. Pharmacokinetics of lipid-coated silica nanoparticles investigated with multimodality imaging

Schematic depiction of quantum dot containing silica particle with (top, left) and without (top, right) a lipid coating. (a) Determination of blood circulation half-life values by fluorescence imaging (photographs, blue graphs) and ICP-MS (red graphs). The values were normalized to the maximum photon count (I_max) or maximum Cd concentration (Cd_max). (b) Confocal microscopy of sections from different organs collected from mice at 24 hrs after injection and stained for endothelial cells to visualize blood vessels (green) and nuclei (blue), shows particle accumulation in red. (c) TEM was used to show particle uptake by cells in the liver, spleen and lung. The lipid-coated silica particles were individually dispersed, while the bare silica particles were found to be aggregated and trapped in the capillary bed of the lungs (bottom, right). Reproduced with permission from ref.

Figure 8. Nanocrystal core high density lipoprotein

Schematic depiction of (a) Au-HDL, (b) FeO-HDL and (c) QD-HDL. (d) Different (functional) molecules that are employed in the HDL coating. (e) Negative stain TEM images of FeO-HDL. (f) Summary of the (physical) properties of nanocrystal HDL and PEG-coated control particles. Values are given as the mean ± the standard deviation. Reproduced with permission from ref.

Figure 9. Uptake of nanocrystal HDL by macrophage cells in vitro

(a) Confocal microscopy of cells incubated for 2 hours with FeO-HDL, which appear red (rhodamine) and the nuclei were stained with DAPI (blue). (b) T₁-weighted MR image of pellets of cells incubated for 4 hrs with QD-HDL, QD-PEG, or cells that were left untreated. (c) TEM image where black circles indicate areas of Au-HDL uptake. (d) CT images of cell pellets incubated with Au-HDL and Au-PEG. (e) Photographs of pellets of cells incubated with QD-HDL, QD-PEG or control cells taken under UV irradiation. (f) Chart of fluorescence intensity of the cell pellets from (e), while the inset shows typical fluorescence images. Reproduced with permission from ref.

Figure 10. Multimodality imaging of atherosclerosis using nanocrystal HDL

T₁-weighted MR images of the aorta of apoE-KO mice 24 hours post-injection with paramagnetic (a) Au-HDL or (b) QD-HDL particles. Arrows indicate areas enhanced in the post images. (c) T₂*-weighted image of an apoE-KO mouse 24 hours post-injection with FeO-HDL. The arrow indicates a vessel wall region with hypo-intense signal. (d, e, f) Confocal microscopy images of aortic sections of mice injected with different nanocrystal HDL preparations. Red is nanocrystal HDL, macrophages are green and nuclei are shown in blue. Yellow indicates co-localization of nanocrystal HDL with macrophages and is indicated by arrowheads. (g) Ex vivo