Efficient Cellular Labeling by CD44 Receptor‐Mediated Uptake of Cationic Liposomes Functionalized with Hyaluronic Acid and Loaded with MRI Contrast Agents

Cell tracking is now a well-established technique in magnetic resonance imaging (MRI) because of its three-dimensional capabilities, its good safety profile, and the superb anatomical resolution (near the cellular level, ~100 mm). For these applications, cells must be labeled with MRI contrast agents in order to decrease the relaxation times of the H atoms of water molecules. The most common contrast agents in current use are iron oxide particles (USPIO, SPIO, MPIO) because of their high sensitivity. They act as negative contrast agents because of their dominant effect on the magnetic susceptibility term. 6] Typically the R2 value of monocytes labeled with SPIO at a Fe concentration of 1.0 mg mL 1 is 13.1 s 1 mm . It has been shown that this leads to a detection limit of 58 labeled monocytes per voxel volume of 0.05 mL. These magnetic particles enable the visualization of labeled cells as dark spots in the MR images as a consequence of their largely dominant T2* effects. However, there is some concern about their long-term cellular toxicity related to the metabolic fate of the iron payload. 9] Other problems with superparamagnetic particles include difficulty in the quantification of cell number due to susceptibility artifacts and the potential transfer of contrast among cells (macrophages) after cell death, giving false-positive results. The use of positive contrast agents is a viable alternative to overcome these problems, but to attain sufficient contrast for the MRI of the labeled cells, it is necessary to entrap a number of Gd complexes on the order of 10–10 per cell. Various routes have been explored in pursuit of effecting the internalization of such large quantities of paramagnetic complexes. Pinocytosis is sometimes a useful route, but the cells must be kept in the presence of high concentrations (~50 mm) of highly stable, hydrophilic agents for many hours (12–24 h). 13] Rapid entrapment of the required Gd complexes may be attained by electroporation of the cell suspension; however, this treatment results in extensive cell death and serious damage to surviving cells. Finally, for cells endowed with phagocytic activity, it has been shown that cellular labeling can be achieved by using particles made of insoluble Gd complexes. 15] These particles are designed to be solubilized upon release of insolubilizing moieties effected by a specific enzyme present in the cellular compartment in which these particles have been entrapped. This procedure is particularly efficient, as it allows the entrapment of a large number of Gd complexes through a single internalization step. Herein we report a cellular labeling methodology that allows the rapid internalization of large amounts of soluble Gd complexes though exploitation of the receptor-mediated endocytotic route. The Gd payload is contained in the inner cavity of liposomes, which are appropriately functionalized at their external surface to bind a given receptor, thus starting the uptake process. As a proof of concept, hyaluronan (hyaluronic acid, HA) was used as a vector for the targeting procedure because HA receptors (CD44) are expressed in a variety of tumors such as breast, colon, intestinal, and brain, as well as melanoma, basal cell carcinoma, and stem cells. HA is a high-molecular-weight glycosaminoglycan polymer (Mr : 10 –10 Da) composed of repeating units of the disaccharide b1,3-N-acetylglucosaminyl-b1,4-glucuronide. Thus each diACHTUNGTRENNUNGsaccharide moiety contains a free carboxylate group, leading to highly negatively charged polymer. For the purpose of this application, HA can be bound to the surface of Gd-loaded liposomes in either a covalent or noncovalent manner. The formation of a noncovalent supramolecular adduct appeared a straightforward route, as cationic liposomes have been widely used in transfection experiments as carriers of negatively charged DNA macromolecules. 21] Cationic liposomes were prepared by using a mixture of phospholipids (POPC 52 % and DOTAP 25 %) and cholesterol (23 %). The lipid film was hydrated with an aqueous solution of Gd-HPDO3A (100 mm). The latter is a commercially available, highly hydrophilic, neutral MRI contrast agent largely used in clinical practice. The liposomes thus formed were extruded through progressively smaller pore sizes to select a population with an average diameter of 127 5 nm and a z potential of + 35 5 mV. The Gd complexes not entrapped in the liposomes were removed by dialysis. The supramolecular HA-functionalized Gd-liposome (HA–L) adducts were obtained by adding aliquots of a solution of HA to the liposome suspension. Interestingly, a dramatic increase in the supramolecular adduct size takes place (Figure 1 a) at the isoelectric point— the point at which the number of positive charges on the liposome equals the number of negative charges on the added HA polymer. Light scattering measurements showed that the particle diameter nearly triples (~350 nm) under these conditions, suggesting the formation of an aggregate that consists of three to four liposomes and several HA molecules (Scheme 1). At higher HA/liposome stoichiometric ratios, the prevalence of a monomeric liposome population was again detected. It appears that in the presence of an excess in positive [a] Dr. G. Esposito, Dr. S. Geninatti Crich, Prof. S. Aime Dipartimento di Chimica IFM and Molecular Imaging Center Via Nizza 52, 10125 Torino (Italy) Fax: (+ 39) 011-670-6487 E-mail : silvio.unito@unito.it