Non-enhanced imaging techniques are useful only when relatively large tissue areas are involved in the pathological process. In addition, though attenuations (i.e., the ability of a tissue to absorb a certain signal, such as X-rays, sound waves, radiation, or radiofrequencies) of different tissues differ; however, in the majority of cases this difference is not sufficient for clear discrimination between normal and pathological areas. To solve this problem and to achieve a sufficient attenuation, contrast agents are used able to absorb certain types of signal (irradiation) much stronger than surrounding tissues. The main task in this case is to accumulate a sufficient quantity of a contrast agent in the area of interest and to keep its presence in normal tissues and organs on a minimum level. No matter which imaging modality is used, precise medical diagnostic imaging requires that the sufficient intensity of a corresponding signal from an area of interest be achieved in order to differentiate the target area from surrounding tissues. Currently used medical imaging modalities include (i) gamma scintigraphy (based on the application of gamma-emitting radioactive materials; either single-photon emission computed tomography [SPECT] or dual-photon annilation image detection, positron emission tomography [PET]); (ii) magnetic resonance (MR, phenomenon involving the transition between different energy levels of atomic nuclei under the action of radiofrequency signal); (iii) computed tomography (CT, the modality utilizing ionizing radiation-X-rays-with the aid of computers to acquire cross-images of the body and three-dimensional images of areas of interest); (iv) ultrasonography (US, the modality utilizes the irradiation with ultrasound and is based on the different passage rate of ultrasound through different tissues); (v) near-infrared (NIR, relies on differentiating long-wavelength fluorescent emissions of diagnostic fluorophores from normal tissue auto-fluorescence via whole-body CCD cameras with specific high cut-off filters). See also Table 5.1 for diagnostic moieties (reporter groups) used in different imaging modalities. Table 5.1 Diagnostic Moiety Required for Appropriate Tissue Attenuation in Different Imaging Modalitites
Modality Reporter group Required tissue concentrationGamma-scintigraphy Radionuclide, e.g. 111In, 99mTc, 67Ga 10-10MPET imaging Radionuclide (with short half-life), e.g. 11C, 18F, 15O 10-8MMR Imaging Paramagnetic metal, e.g. Gd, Mn, and iron oxide 10-4MCT Imaging Heavy elements, e.g. I, Br, Ba 10-2 MNear IR imaging Fluorescent probe / Quantum dots 10-2 MUltrasonography Gas (air, argon, nitrogen)
The combination of newer imaging techniques providing high sensitivity and spatial resolution and use of nanoscale devices to deliver diagnostic agents with high target specificity would indeed enable more accurate detection and staging, leading to more accurate therapy planning of the disease state. 5.2 LIPOSOMES AND MICELLES AS CARRIERS OF CONTRAST
AGENTS FOR VARIOUS IMAGING MODALITIESAmong particulate drug carriers, micro/nano-reservoir-type systems, such as liposomes (mainly, for water-soluble drugs) and micelles (mainly, for water-insoluble drugs) are the most extensively studied and have certain advantages over other delivery systems and possess the most suitable characteristics for encapsulation of many drugs, genes, and diagnostic (imaging) agents [64, 65]. Simply, the possibility to easily control composition, size, and in vivo stability of a nanoreservoir is a major advantage. More importantly, these nanovesicular systems retain remarkable loading capacity of various imaging and contrast agents, of either hydrophilic or lipophilic nature, or both . Liposomes are artificial phospholipid vesicles of size varying from 50 to 1000 nm and even more, which can be loaded with a variety of water-soluble drugs (into their inner aqueous compartment) and water-insoluble drugs (into the hydrophobic compartment of the phospholipid bilayer), and are considered as promising drug carriers for well over two decades . The use of targeted liposomes, i.e., liposomes
selectively accumulating inside the affected organ or tissue, may increase the efficacy of the liposome incorporated drug and decrease the loss of liposomes and their contents in reticuloendothelial system [RES] . Several successful protocols have been employed to actively and passively target liposomal nanocarierrs to various pathological tissues and into target cells in particular, via various targeting ligands and homing moieties . In a similar fashion, micelles, including polymeric micelles, represent another promising type of pharmaceutical carrier. Micelles are amphiphilic compound-formed colloidal particles with hydrophobic core and hydrophilic corona with size range between 5 and 100 nm. An important property of micelles is their ability to increase the solubility and bioavailability of poorly soluble pharmaceuticals . The use of certain special amphiphilic molecules as micelle building blocks can extended blood half-life of the micellar carrier, in combination with different targeting ligands, strategically incorporated into the micelle structure. There are several key micelle properties (size, critical micelle concentration or CMC, and loading capacity of the hydrophobic core of the micelle) important for the preparation of micelle-incorporated pharmaceuticals and contrast agents . Usually, these parameters are as follows: the size of a pharmaceutical micelle is between 10 and 80 nm, an optimal CMC value is in a low micromolar region, and the loading efficiency toward a
hydrophobic drug between 5 and 25 wt.%. In case of targeted micelles, a local release of a free drug from micelles in the target organ should lead to the increased efficacy of the drug, while the stability of the micelles en route to the target organ or tissue should contribute to drug solubility and toxicity reduction due to less interaction with nontargeted organs [63, 65]. 5.3 FORMULATION OF LIPOSOMAL AND MICELLAR CARRIERS FOR
IMAGING APPLICATIONSPursuing different in vivo delivery purposes, one can easily change the size, charge and surface properties of these carriers simply by adding new ingredients to the lipid or amphiphilic compound mixture before liposome or micelles preparation and/or by variation of preparation methods . As follows from Fig. 5.1, while micelles may be loaded with a contrast agent only into the micelle core in the process of micelle assembly, liposomes may incorporate contrast agents in both internal water compartment and membrane. Gamma-scintigraphy and MR imaging both require the sufficient quantity of radionuclide or paramagnetic metal to be associated with liposome. Independently on the supposed imaging modality, there exist several general approaches to liposome/micelle loading with a contrast agent (reporter group, label). The contrast agent can be: (i) added and incorporated into the aqueous interior of liposome or into the liposome membrane during the manufacturing process to liposomes; (ii) adsorbed onto the surface of preformed liposomes; (iii) incorporated into the lipid bilayer of preformed liposomes; (iv) loaded into preformed liposomes using membrane-incorporated transporters or ion channels. Two very general approaches are the most often used and efficient to prepare liposomes/micelles for gamma-and MR-imaging. First, metal is chelated into a soluble chelate (such as diethylene triamine pentaacetic acid [DTPA]) and then entrapped into the water interior of a liposome . Alternatively, DTPA or a similar chelating compound may be chemically derivatized by the incorporation of a hydrophobic group, which can anchor the chelating moiety on the liposome or micelle surface during or after liposome preparation . Different chelators and different hydrophobic anchors were tried for the preparation of indium-111 (111In), technetium -99m (99mTc), manganese (Mn-), and gadolinium (Gd) Gd-liposomes [1, 47]. Several detailed reviews describing the various methods and chelators for radiolabeling liposomes were recently published [15, 36, 43] .Taking into account that most clinically relevant radioisotopes have rather short half-life (under 5 days), the last step of the preparation of contrast liposomes for gamma-imaging takes place directly before the application moment. With this in mind, liposomes have to be prepared which can be sufficiently loaded with a contrast label by applying simple and fast labeling protocol. Stability issues and remedies for
gamma-imaging with liposomes will be discussed in another chapter in this book by Phillips et al. In the case of MR imaging, for a better MR signal, all reporter atoms should be freely exposed for interaction with water. This requirement makes metal encapsulation into the liposome less attractive than metal coupled with chelators exposed into the water space. In addition, low-molecular-weight water-soluble paramagnetic probes may leak from liposomes upon contact with body fluids, which destabilizes most liposomal membranes. Moreover, it has been shown that when too high concentrations of Gd-DTPA are encapsulated inside liposomes in an attempt to achieve better enhancement, the relaxivity of the compound might be even lower than for non-encapsulated Gd-DTPA complex, probably because of decreased residence lifetime of water molecules inside vesicles . Membranotropic chelating agents, such as DTPA-stearylamine or DTPA-phosphatidyl ethanolamine , consist of the polar head containing a chelated paramagnetic atom, and a lipid moiety which anchors the metal-chelate complex in the liposome membrane . This approach has been shown to be far more superior in terms of the relaxivity of the final preparation when compared with liposome-encapsulated paramagnetic ions due to the decrease in the rotational correlation times of the paramagnetic moiety rigidly connected with relatively large particle. Liposomes with membrane-bound paramagnetic ion also demonstrate a reduced risk of leakage in the body . Membranotropic chelates are suitable for micelle incorporation (they anchor in the hydrophobic micelle core), and may also serve to load micelles with heavy radiometals (see Fig. 5.1) .