Paramagnetic and fluorescent liposomes for target-specific imaging and therapy of tumor angiogenesis
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Angiogenesis is essential for tumor growth and metastatic potential and for that reason considered an important target for tumor treatment. Noninvasive imaging technologies, capable of visualizing tumor angiogenesis and evaluating the efficacy of angiostatic therapies, are therefore becoming increasingly important. Among the various imaging modalities, magnetic resonance imaging (MRI) is characterized by a superb spatial resolution and anatomical soft-tissue contrast. Revolutionary advances in contrast agent chemistry have delivered versatile angiogenesis-specific molecular MRI contrast agents. In this paper, we review recent advances in the preclinical application of paramagnetic and fluorescent liposomes for noninvasive visualization of the molecular processes involved in tumor angiogenesis. This liposomal contrast agent platform can be prepared with a high payload of contrast generating material, thereby facilitating its detection, and is equipped with one or more types of targeting ligands for binding to specific molecules expressed at the angiogenic site. Multimodal liposomes endowed with contrast material for complementary imaging technologies, e.g., MRI and optical, can be exploited to gain important preclinical insights into the mechanisms of binding and accumulation at angiogenic vascular endothelium and to corroborate the in vivo findings. Interestingly, liposomes can be designed to contain angiostatic therapeutics, allowing for image-supervised drug delivery and subsequent monitoring of therapeutic efficacy.
KeywordsAngiogenesis Angiostatic therapy Magnetic resonance imaging Molecular imaging Liposomes Synergistic targeting Tumor
Inhibition of tumor angiogenesis is a modern and popular approach to fight tumor progression [1, 2]. Therapeutic interventions aimed at reducing tumor growth by angiogenesis inhibition are rapidly finding their way into clinical practice . The origin of this approach is that angiogenesis inhibitors target cells that support tumor growth, i.e., endothelial cells, instead of targeting the tumor cells directly. Endothelial cells are genetically more stable than tumor cells and thus less likely to develop drug resistance. Moreover, the endothelial cells are directly accessible from the bloodstream, enabling nanocarriers with a high payload of angiostatic drugs to reach their target site efficiently. There are several classes of angiostatic drugs, e.g., growth factor blockers , growth factor signaling inhibitors, extracellular matrix modulators , and endothelial cell proliferation and migration inhibitors [5, 6, 7]. In 2004, the Food and Drug Administration approved the first angiostatic drug (Avastin) for human application .
These developments call for new noninvasive readouts to test the efficacy of novel angiostatic drugs. In a preclinical setting, different methods are available to monitor changes in angiogenic activity during the course of treatment. Typically, new agents are first evaluated in vitro before further assessment in animal models. Inhibition or regression of the volume of a subcutaneously implanted tumor can be estimated by an external morphological measurement, e.g., using a caliper. Such measurements are often inaccurate, as they do not account for inward growth, irregular tumor shape with multiple lobes and development of metastases. Moreover, morphological changes often occur late after initiation of the angiostatic treatment and are therefore a poor readout of early drug efficacy. The effects of the new drug on the tumor vasculature can be assessed by ex vivo analysis of the tumor microvessel density (MVD). Several angiogenesis inhibitors, including endostatin and anginex (Anx), have shown to decrease the tumor MVD, which is therefore considered a parameter that may be used to predict therapeutic effect [5, 9]. A major drawback of the MVD readout is that—as with all histological analyses—it can only be determined with invasive and laborious procedures following staining of ex vivo tumor tissue sections, making it difficult to assess tumor vessel density heterogeneity and precluding longitudinal assessment during the course of treatment. Therefore, noninvasive imaging methods that report on tumor angiogenesis are highly desired, since methods to visualize and quantify angiogenic activity in vivo would allow longitudinal monitoring and thus facilitate the early evaluation of a given angiostatic therapy. For this purpose, magnetic resonance imaging (MRI) is a prime candidate.
MRI is a powerful noninvasive imaging modality capable of generating high-resolution images with superb soft tissue contrast. Already, MRI offers a broad spectrum of techniques to localize, visualize and characterize tumors. For example, anatomical T2-weighted images help to define anatomical borders of solid tumors with outstanding resolution and contrast, and aid in distinguishing and classifying healthy from diseased tissue . Also MR spectroscopy can be used to assess tumor pH and metabolic activity . MR image contrast reflecting endogenous cellular protein and peptide content in the tumor can be produced by selective radiofrequency labeling of the protein and peptide amide protons and exploiting the magnetization transfer to water via hydrogen exchange . Recent advances in whole-body diffusion-weighted imaging show promise for diagnosing lesions throughout the entire human body as well as for assessing lymph node metastases, with superior spatial resolution, and sensitivity and specificity that rival FDG-PET imaging . In addition, hemodynamic parameters such as the fractional blood volume, vascular permeability, tumor blood perfusion, and lymphatic drainage can be assessed using contrast-enhanced MRI [14, 15, 16].
Recent advances in molecular imaging—the field that aims to noninvasively visualize processes at the molecular and cellular level—have broadened the use of MRI in oncology [16, 17, 18]. Sophisticated contrast agents have been developed, equipped with targeting ligands directed toward angiogenesis-specific markers, which allow for noninvasive in vivo visualization of the angiogenic activity in the tumor. A variety of targeted Gd-, ironoxide-, and fluorine-based MRI contrast agents have been developed for this purpose, ranging from low molecular weight agents to nanoparticulate agents of different size and composition [19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30].
In this paper, we review our recent work on the use of multimodal liposomes for target-specific preclinical imaging of tumor angiogenesis and therapy. Over the last decades, liposomes were extensively studied as drug carriers for cancer therapy . Encapsulation of the drug in a liposome improves the pharmacokinetic profile, thereby increasing the amount of drug effectively delivered to the tumor, while preventing rapid clearance, drug degradation, and inactivation. When equipped with a high payload of contrast generating material and conjugated with one or more types of targeting ligands, liposomes turn into a potent contrast agent for tumor angiogenesis imaging. Often, the liposomes are endowed with a contrast agent for a complementary imaging modality, e.g., fluorescence imaging, for validation purposes and to investigate binding and accumulation at the cellular level. Combined with angiostatic drugs, these multimodal liposomes allow for image-supervised drug delivery and monitoring of angiostatic therapy.
Multimodal liposomes for visualization of tumor angiogenesis
A liposome is a spherical vesicle composed of a bilayer of naturally occurring phospholipids or closely related synthetic amphiphiles enclosing an aqueous interior [32, 33]. For in vivo application purposes, the liposome should be biocompatible, stable, and possess favorable pharmacokinetic properties. Therefore, liposomes are usually comprised of components that energetically favor a bilayer organization, such as phospholipids comprising a polar head group and two fatty acyl chains . For additional stabilization, cholesterol is commonly included . To reduce interactions with blood circulating proteins and cells polyethyleneglycol-lipids (PEG-lipids) may be incorporated, which increases the liposome blood circulation half-life and ensures optimal pharmacokinetics [34, 35].
Liposomes are commonly prepared by a lipid film hydration technique. The preparation involves several steps. First, the appropriate amounts of lipids are dissolved and mixed in an organic solvent, e.g., chloroform or chloroform/methanol. The organic solvent is then removed by rotary evaporation yielding a thin lipid film on the sides of a round bottom flask. Next, the lipid film is hydrated by addition of an aqueous medium at a temperature above the lipid gel-liquid crystal transition temperature, resulting in a solution of multilamellar lipid vesicles. Finally, the multilamellar vesicles are downsized by extrusion of the lipid suspension through a polycarbonate filter of well-defined pore size at elevated temperature or by sonication to yield a suspension of liposomes with a narrow and well-defined size distribution.
Specificity for tumor angiogenesis can be introduced by decorating the exterior of the liposomes with suitable targeting ligands, e.g., antibodies or peptides, as schematically illustrated in Fig. 1. Covalent binding of the targeting ligands to the liposome is achieved by coupling to lipids with a functional reactive group, such as maleimide, thiol, amine, or carboxylic acid. Targeted liposomal MRI contrast agent can be used to follow the effects of therapeutic interventions, as will be discussed in detail further on. Imaging and therapy may be combined by inclusion of drugs in the liposomal formulation. Water-soluble drug can be enclosed in the liposomal interior. Drugs may also be enclosed in the liposomal bilayer or attached to the distal end of the PEG chains (Fig. 1).
Multimodal imaging of tumor angiogenesis and angiostatic therapy
Many studies, which aimed at in vivo visualization of the molecular hallmarks of tumor angiogenesis, have focused on the αvβ3 integrin [19, 20, 29, 30, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63]. The αvβ3 integrin is strongly expressed on the activated endothelium of angiogenic blood vessels and is involved in many tumor related processes. Importantly, the integrin is only weakly expressed on resting endothelial cells in nondiseased tissue, making it an attractive specific target for tumor angiogenesis imaging . MR imaging of tumor angiogenesis using a targeted paramagnetic vesicles was pioneered by Sipkins et al. . They demonstrated enhanced and detailed imaging of the αvβ3 expressing angiogenic vasculature in rabbit carcinomas after intravenous administration of the aforementioned agent, providing a noninvasive means to assess the growth and malignant phenotype of tumors. The αvβ3 integrin binds to a wide variety of extracellular matrix proteins, which expose the tripeptide sequence Arg-Gly-Asp (RGD) as a common receptor recognition signal . This peptide is used as a targeting moiety for angiogenesis targeting and several isoforms that include the tripeptide sequence have been developed. A cyclic conformation (cyclic RGD) has favorable binding properties when compared to a linear peptide and multimers of the RGD peptide have been shown to increase the affinity and specificity [56, 57, 66].
A bimodal, MRI and fluorescence, approach to tumor angiogenesis imaging was applied by Mulder et al. . As a bimodal contrast agent, the above-described liposomes were used, which have a high payload of Gd-containing lipid for MRI purposes and rhodamine fluorescent lipids for optical imaging. The 150-nm-diameter liposomes were conjugated with on average about 700 cyclic RGD peptides per particle to introduce specificity for the αvβ3 integrin. The high number of RGD moieties attached to the liposomes ensures a high affinity for the target on the basis of multivalency.
The targeting and multimodal-imaging concept was first tested in vitro using human endothelial cells (human umbilical vein endothelial cells). Cells incubated with specific RGD-conjugated liposomes displayed bright rhodamine fluorescence, which was lacking in case of control incubations with nonspecific RAD-conjugated liposomes. Detailed analysis showed that the RGD-conjugated liposomes were internalized in the perinuclear region of the cells. MR imaging of cell pellets revealed a large contrast between specific and control incubations. Application of RGD-conjugated liposomes resulted in a substantial T1-relaxation time shortening of the cell pellets. These results proved specificity and high affinity for endothelial cells. The mechanism of RGD-conjugated liposome uptake and the effect of internalization on the relaxation properties were further investigated in two recent studies [67, 68]. A dramatic lowering of the longitudinal relaxation rate r1 (relaxivity quenching) for cell-internalized targeted liposomes was observed that warrants caution in the quantitative interpretation of the observed MRI signal.
Improving target specificity by avidin-induced clearance of circulating liposomes
The RGD-conjugated liposomes for imaging angiogenesis, as described in the previous section, were designed to exhibit a long blood circulation half-life, which was achieved by including PEG-lipids in the liposomal bilayer. On the one hand, a long circulation half-life is potentially advantageous for molecular imaging as it may increase the amount of contrast agent that accumulates at the angiogenic site enabled by prolonged time for interaction with the targets. This is particularly necessary for MRI, since this is a relatively insensitive technique for the detection of low concentrations of contrast agent. On the other hand, the MR images will suffer from undesired background signal because of circulating liposomes, resulting in a suboptimal target-to-background ratio. The use of flow suppression techniques potentially can solve this problem. However, low flow velocity, reduction of the blood T1-relaxation time by the liposomes, and the complex and unstructured vasculature of the tumor blood vessels makes flow-suppression unreliable in tumors. Additionally, control liposomes generally display different blood-clearance rates, complicating a straightforward comparison between targeted and control particles. Ideally, the nonbound fraction should be cleared from the blood stream as soon as sufficient liposomes have associated with the target site. This would significantly enhance the specificity of the targeted liposomes for molecular imaging of angiogenesis. Furthermore, this approach would enable investigations into the accumulation kinetics of targeted liposomes and the optimization of MR imaging protocols for their detection.
A strategy to achieve rapid clearance of nonbound liposomes from the vasculature was developed by van Tilborg et al. [70, 71]. The technology is based on a so-called avidin chase experiment. Avidin is a 66-kDa glycoprotein, which can bind four biotin molecules with high affinity (Kd < 10−15 M) . Avidin alone displays a very short intrinsic blood-circulation half-life, accumulates in the liver, spleen and kidneys, and is subsequently cleared from all organs within a couple of days [72, 73]. The basic idea is to equip the liposomal contrast agent with a targeting ligand, as well as with biotin. After initial intravenous injection of the agent, the liposomes are allowed to specifically bind and accumulate at the desired target site. In a second step, avidin is injected which then binds with high affinity to the biotin on the liposomes. As a consequence, the avidin triggers fast clearance of the circulating, nontarget-associated liposomes from the blood. This clearance strategy has been previously successfully applied to radiolabeled antibodies [74, 75] and liposomes  for nuclear imaging, as well as to dendrimer-based  and albumin-based  agents for MR imaging.
This clearance strategy can be used to study relative contributions of target-associated, extravasated, as well as circulating nonbound liposomes to the MRI contrast enhancement in the tumor tissue. The avidin-chase technology can be applied to remove the blood pool component in the contrast-enhanced MRI experiment where flow suppression is not sufficient, which opens exciting possibilities for studying detection limits and angiogenesis targeting kinetics of the liposomal contrast agents.
Synergistic targeting for combined imaging and treatment
Despite the major advances with the introduction of multimodal targeted liposomes, the modest amounts of target-associated contrast material still remains an obstacle for widespread application of molecular imaging of tumor angiogenesis. Recently, Kluza et al.  introduced a novel concept which may, at least partly, help to alleviate the sensitivity and specificity problems. The idea was to involve several different molecular markers specific for angiogenesis in the targeting process. The activated endothelium of angiogenic blood vessels seems to be an excellent candidate for this synergistic targeting concept as it over expresses a diverse set of molecules [79, 80]. Targeting of multiple receptors is relatively new in the field of molecular imaging and has been investigated before in a few studies only concerning drug delivery to cells [81, 82], for MR imaging of atherosclerosis , and for ultrasound imaging of tumor angiogenesis .
Moreover, for the first time Kluza et al.  showed that synergistic targeting with liposomes could be used to combine imaging and treatment of angiogenesis. This was achieved by using a unique combination of two targeting ligands, RGD and anginex (Anx), which bind specifically to two important angiogenic markers, αvβ3 and galectin-1, respectively, and exert angiostatic activity as well [7, 85]. Similar to aforementioned studies, the liposomes were of a bimodal nature containing a high payload of Gd-containing lipid for MR imaging and fluorescent lipids for fluorescent imaging, which enabled extensive evaluation and validation of the applied strategy.
Angiostatic potential of the RGD and Anx dual-conjugated liposomes was assessed by performing cell cycle analyses to investigate the antiproliferative effects induced by the synergistic effects of RGD and Anx. As readout to assess the therapeutic outcome, the percentages of cells in the S (DNA replication phase) and the G2/M (interphase/mitosis) phases were used. A decrease in S and G2/M phase is generally considered a hallmark of impaired proliferation. A considerable reduction was found for treatments with the dual-targeted liposomes, which was stronger, however, not significantly, when compared to the single-conjugated liposomes. The synergistic effects of these dual-targeted liposomes create a great opportunity for their use in combined imaging and therapy of angiogenesis.
Summary and perspective
This paper reviewed our recent strategies on the design and application of multimodal targeted-liposomes for in vivo visualization, quantification, and treatment of tumor angiogenesis. As an angiogenesis-specific targeting ligand, we mainly used the cyclic RGD peptide, which binds with high specificity to the αvβ3 integrins expressed on angiogenic endothelium. Liposomes conjugated with a few hundred of these RGD peptides were shown to bind with great specificity to mouse tumor vascular endothelium. This enabled specific in vivo MRI visualization of angiogenic activity in tumors. Multimodality imaging properties were introduced by addition of fluorescent labels in the liposomes. This proved an essential tool for validation purposes, as it combined the best of both worlds—in vivo MR imaging of tumor angiogenesis with ex vivo fluorescence imaging of the liposomal accumulation and fate at the cellular level. Furthermore, the RGD-conjugated liposomes were applied for in vivo visualization of changes in tumor angiogenic activity after treatment with several angiostatic compounds.
The PEG-coated liposomes exhibit long in vivo blood circulation times. This can be exploited to increase the amount of contrast agent that accumulates at the angiogenic site. However, background MR signal from circulating liposomes in the blood pool reduces the target-to-background ratio. Incorporation of biotin-PEG-lipids in the bilayer was used for fast clearance of the liposomes from the blood pool using an avidin chase. This strategy improved the specificity of the MR readout and increased the target-to-background ratio dramatically. Finally, liposomes were equipped with two types of angiogenesis-specific ligands, i.e., RGD and anginex. First of all, this improved targeting efficacy by synergistic effects of the two targeting ligands. Second, the dual-targeted liposomes exhibited considerable angiostatic effects and therefore can be used for combined tumor angiogenesis imaging and therapy.
The reviewed work convincingly demonstrated that molecular MRI of angiogenesis and therapy monitoring in mouse models of cancer are feasible using the multimodal targeted liposomes. In recent years, several competitive platforms have been introduced in the field of nanoparticle-based molecular imaging. Some examples are iron-oxides , perfluorocarbon nanoparticles , polymeric nanocarriers , viruses , and high-density lipoprotein-based nanoparticles [89, 90]. In our work, we have mainly focused on small peptides as targeting ligand for angiogenesis. Alternative ligands include antibodies and antibody fragments , small molecules , peptidomimetics , aptamers , and nanobodies . This multi-disciplinary field of molecular imaging is evolving very fast and whether a given nanoparticle-ligand combination will be successful eventually remains to be evaluated for each particle separately.
Therefore, there is still a long way to go before this technology can be translated to human cancer. In particular, more knowledge is needed on the long-term fate of the bound and cell-internalized nanoparticles and on the organ-clearance kinetics and pathways in view of possible toxicity issues. Nevertheless, a lot of knowledge can be gained from preclinical studies in mouse models of cancer and for this the in vivo angiogenesis imaging technology proves a valuable tool already.
This study was funded in part by the Integrated EU Project MEDITRANS (FP6-2004-NMP-NI-4/IP 026668-2), the European Community EC-FP6-project DiMI, LSHB-CT-2005-512146 and by the BSIK program entitled Molecular Imaging of Ischemic Heart Disease (project number BSIK03033). This study was performed in the framework of the European Cooperation in the field of Scientific and Technical Research (COST) D38 Action Metal-Based Systems for Molecular Imaging Applications.
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