Sjoerd Hak

The Effect of Nanoparticle Polyethylene Glycol Surface Density on Ligand-directed Tumor Targeting Studied in vivo by Dual Modality Imaging

The development and application of nanoparticles as in vivo delivery vehicles for therapeutic and/or diagnostic agents has seen a drastic growth over the last decades. Novel imaging techniques allow real-time in vivo study of nanoparticle accumulation kinetics at the level of the cell and targeted tissue. Successful intravenous application of such nanocarriers requires a hydrophilic particle surface coating, of which polyethylene glycol (PEG) has become the most widely studied and applied. In the current study, the effect of nanoparticle PEG surface density on the targeting efficiency of ligand-functionalized nanoemulsions was investigated. We synthesized 100 nm nanoemulsions with a PEG surface density varying from 5 to 50 mol %. Fluorescent and paramagnetic lipids were included to allow their multimodal detection, while RGD peptides were conjugated to the PEG coating to obtain specificity for the α(v)β(3)-integrin. The development of a unique experimental imaging setup allowed us to study, in real time, nanoparticle accumulation kinetics at (sub)-cellular resolution in tumors that were grown in a window chamber model with confocal microscopy imaging, and at the macroscopic tumor level in subcutaneously grown xenografts with magnetic resonance imaging. Accumulation in the tumor occurred more rapidly for the targeted nanoemulsions than for the nontargeted versions, and the PEG surface density had a strong effect on nanoparticle targeting efficiency. Counterintuitively, yet consistent with the PEG density conformation models, the highest specificity and targeting efficiency was observed at a low PEG surface density.

A high relaxivity Gd(III)DOTA-DSPE-based liposomal contrast agent for magnetic resonance imaging.

The field of molecular imaging aims to visualize and quantify (patho)physiological processes at the cellular and molecular level. Sensitive and site-targeted contrast agents are employed to visualize molecular constituents of processes of interest. The principal aim of this study was to develop a magnetic resonance imaging (MRI) detectable liposome with high relaxivity and stability. To this end, Gd(III)DOTA-DSPE was synthesized and incorporated in a liposomal formulation. The resulting liposomes were extensively characterized in vitro in terms of contrast agent efficiency and structural properties. The liposomes were shown to have a high longitudinal relaxivity, which is crucial for the detection of low concentration molecular markers in molecular imaging studies. We also demonstrated that Gd(III)DOTA-DSPE exhibits no detectable transmetallation upon incubation with Zn(II). This is important as it significantly contributes to the biocompatibility of the contrast agent. The present liposome preparation will serve as versatile and well characterized platform for molecular imaging and targeted drug delivery studies.

Cellular compartmentalization of internalized paramagnetic liposomes strongly influences both T1 and T2 relaxivity

In recent years, numerous Gd3+‐based contrast agents have been developed to enable target‐specific MR imaging of in vivo processes at the molecular level. The combination of powerful contrast agents and amplification strategies, aimed at increasing the contrast agent dose at the target site, is an often‐used strategy to improve the sensitivity of biomarker detection. One such amplification mechanism is to target a disease‐specific cell membrane receptor that can undergo multiple rounds of internalization following ligand binding and thus shuttle a sizeable amount of contrast agent into the target cell. An example of such a membrane receptor is the ανβ3 integrin. The goal of this study was to investigate the consequences of this amplification approach for the T1‐ and T2‐shortening efficacy of a paramagnetic contrast agent. Cultured endothelial cells were incubated with paramagnetic liposomes that were conjugated with a cyclic RGD‐peptide to enable internalization by means of the ανβ3 integrin receptor. Non‐targeted liposomes served as a control. This study showed that ανβ3 targeting dramatically increased the uptake of paramagnetic liposomes. This targeting strategy, however, strongly influenced both the longitudinal and transverse relaxivity of the internalized paramagnetic liposomes. Magn Reson Med, 2009.

Three-compartment T1 relaxation model for intracellular paramagnetic contrast agents

The goal of this work was to elaborate a model describing the effective longitudinal relaxation rate constant R1 for 1H2O in three cellular compartments experiencing possible equilibrium water exchange, and to apply this model to explain the effective R1 dependence on the overall concentration of a cell‐internalized Gd3+‐based contrast agent (CA). The model voxel comprises three compartments representing extracellular, cytoplasmic, and vesicular (e.g., endosomal, lysosomal) subcellular spaces. Relaxation parameters were simulated using a modified Bloch–McConnell equation including magnetization exchange between the three compartments. With the model, several possible scenarios for internalized CA distribution were evaluated. Relaxation parameters were calculated for contrast agent restricted to the cytoplasmic or vesicular compartments. The size or the number of CA‐loaded vesicles was varied. The simulated data were then separately fitted with empirical mono‐ and biexponential inversion recovery expressions. The voxel CA‐concentration dependencies of R1 can be used to qualitatively and quantitatively understand a number of different experimental observations reported in the literature. Most important, the simulations reproduced the relaxivity “quenching” for cell‐internalized contrast agent that has been observed. Magn Reson Med, 2009.

Multimodality nanotracers for cardiovascular applications

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 αvβ3 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.

Intravital microscopy in window chambers: a unique tool to study tumor angiogenesis and delivery of nanoparticles

Solid tumor growth is heavily dependant on angiogenesis. Tumor angiogenesis is the result of a complex interplay between tumor cells, endothelial cells, and other stromal cells. It has been found to be under strict control of a plethora of molecular factors that function as angiogenic up- and down-regulators; nevertheless, the identification of molecular and cellular players and their roles in angiogenesis is still ongoing. The microvasculature resulting from tumor angiogenesis lacks hierarchy and has a high permeability for macromolecules and nanoparticles, which offers significant potential for nanoparticulate tumor imaging and drug delivery platforms. However, improvements in the delivery to poorly vascularized regions and the distribution throughout the tumor interstitium are critical for nanoparticles to become more effective in the battle against cancer. A tool that has proven extremely valuable in both unraveling angiogenic pathways and characterizing in vivo nanoparticle behavior in solid tumors is intravital microscopy of tumors grown in window chamber preparations. In this review this technique is explained, several exciting examples illustrating its value in elucidating tumor angiogenesis are presented and the study of nanoparticle behavior in solid tumors using this approach is described. We conclude with a discussion of the potential value of intravital microscopy in window chambers in multimodality studies of tumor pathophysiology and nanoparticle dynamics.

L‐DOPA‐Coated Manganese Oxide Nanoparticles as Dual MRI Contrast Agents and Drug‐Delivery Vehicles

Manganese oxide nanoparticles (MONPs) are capable of time-dependent magnetic resonance imaging contrast switching as well as releasing a surface-bound drug. MONPs give T2/T2* contrast, but dissolve and release T1-active Mn(2+) and L-3,4-dihydroxyphenylalanine. Complementary images are acquired with a single contrast agent, and applications toward Parkinsons disease are suggested.

Nanoparticle delivery to the brain--By focused ultrasound and self-assembled nanoparticle-stabilized microbubbles.

The blood-brain barrier (BBB) constitutes a significant obstacle for the delivery of drugs into the central nervous system (CNS). Nanoparticles have been able to partly overcome this obstacle and can thus improve drug delivery across the BBB. Furthermore, focused ultrasound in combination with gas filled microbubbles has opened the BBB in a temporospatial manner in animal models, thus facilitating drug delivery across the BBB. In the current study we combine these two approaches in our quest to develop a novel, generic method for drug delivery across the BBB and into the CNS. Nanoparticles were synthesized using the polymer poly(butyl cyanoacrylate) (PBCA), and such nanoparticles have been reported to cross the BBB to some extent. Together with proteins, these nanoparticles self-assemble into microbubbles. Using these novel microbubbles in combination with focused ultrasound, we successfully and safely opened the BBB transiently in healthy rats. Furthermore, we also demonstrated that the nanoparticles could cross the BBB and deliver a model drug into the CNS.

Near-Infrared Fluorescence Energy Transfer Imaging of Nanoparticle Accumulation and Dissociation Kinetics in Tumor-Bearing Mice

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.

Nanoparticle-stabilized microbubbles for multimodal imaging and drug delivery

Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery. We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG)-coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air-liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NP-MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue, providing improved therapy and imaging contrast compared with freely distributed NPs.