Chargebased Targeting Of Liposomes

Inclusion of lipids that bear a net positive charge into the liposome lipid composition has been shown to result in preferential accumulation of liposomes in neovasculature (59,60). One of the first studies, performed by McLean et al., focused on liposomes composed of cationic 1-[2-[9-(Z)-octadecenoyloxy]]-2-[8](Z)-heptadecenyl]-3-[hydroxyethyl] imidazolinium chloride:cholesterol complexed to DNA in healthy mice (61). After IV administration, the cationic lipid complexes showed aggregation in the circulation through interactions with blood components leading to a rapid clearance by the mononuclear phagocyte system. In addition to this rapid uptake by macrophages in liver and spleen, certain ECs exhibited pronounced uptake of the cationic liposomes as well. It was shown that the endothelium in capillaries of lungs, ovaries, anterior pituitary, and in the high venules of lymph nodes were primarily responsible for liposome uptake, whereas ECs in other organs only took up few liposomes. Binding to ECs was already observed at five minutes postinjection. Bound liposomes were subsequently internalized and processed through the endosomal/lysosomal pathway within four hours. The authors concluded from the specific distribution pattern of liposomal uptake by the endothelium that a heterogeneously distributed endothelial membrane receptor was responsible for the uptake of cationic complexes. Nevertheless, earlier studies have demonstrated that the EC can be an important cell type for clearance of foreign particles, like polystyrene beads and bacteria, from the blood stream. As such, the distribution pattern of liposome uptake may reflect activity of the endothelium in the clearance of foreign material rather than distribution of a theoretical receptor (62). These studies showed that normal endothelium could bind and internalize liposomes bearing a cationic charge. This is an important finding when employing cationic liposomes for targeting neovasculature, as specific uptake of these liposomes by normal endothelium can occur and can produce side effects.

Cationic albumin-functionalized sterically stabilized liposomes have been studied for targeting brain ECs. The presence of cationic albumin indeed induced binding and internalization by these cells; however, for in vitro systems, cationic charge usually promotes interaction with any cell type. As in vivo observations are lacking, it is uncertain whether targeting of brain endothelium would occur in animals (63).

In murine models of pancreatic islet cell carcinoma or chronic Mycoplasma pulmonis-induced airway inflammation, cationic liposomes were shown to be preferentially taken up by the activated (angiogenic) endothe-lium. Degree of uptake in these areas was approximately 15- to 30-fold higher than by quiescent endothelium in disease-free animals (64). The majority of EC-associated liposomes were already internalized at 20 minutes postinjection. Within the angiogenic endothelium, uptake was not homogenous. Certain areas displayed pronounced uptake, whereas uptake in other regions was much lower. This heterogeneity in angiogenic EC uptake may reflect differences in phase of angiogenesis and EC activity.

Campbell et al. investigated intratumoral distribution of EC-targeted cationically charged PEG-coated liposomes (65). Tumor uptake was similar for liposomes with or without the cationic charge. However, intravital microscopy revealed that by increasing the cationic lipid content from 10 to 50 mol% the degree of liposomal uptake by the tumor vasculature increased two-fold (66). The authors suggest that cationic liposomes will interact preferentially with tumor endothelium because of the slow and irregular tumor blood flow. In addition, tumor vessels are tortuous and leaky (65,67). As a result, cationic liposomes have more opportunities to interact with anionic structures, like proteoglycans, on the angiogenic endothelium than with these structures in normal blood vessels where blood velocity is higher.

Cationic liposomes loaded with drugs have been investigated for therapeutic efficacy in preclinical models. In a model of amelanotic hamster melanoma grown in a dorsal skinfold window chamber, cationic liposome-encapsulated paclitaxel effectuated strong inhibition of tumor growth (66). At a dose of 5mg/kg body weight of paclitaxel in cationic liposomes, tumor volume was approximately 6-fold, 6-fold, and 10-fold lower as compared to Taxol, empty cationic liposomes, or buffer-treated animals, respectively, and significantly delayed local lymph node metastasis. The observation that control treatments (paclitaxel and empty cationic liposomes) provide a modest therapeutic effect on their own may suggest that the antitumor effects for paclitaxel-loaded cationic liposomes represent a merely additive effect. Nevertheless, it may also be the result of angiogenic EC delivery of pacli-taxel by cationic liposomes. The focus of a subsequent study was the mechanism of action of paclitaxel-loaded cationic liposomes in this model. Analysis of microvessels in the tumor showed that functional vessel density was reduced by liposomal paclitaxel. After treatment, vessel diameters were smaller, leading to reduced blood flow resulting in a reduced microcircula-tory perfusion index. Staining for apoptosis revealed that the lower index was mainly associated with microvessels in treated tumors, indicating that the changes in microcirculation are the result of cytotoxic effects on tumor endothelium (68).

Kunstfeld et al. used a humanized SCID mouse melanoma model, in which human melanoma cells grow on human dermis and are partly supported by human microvasculature (69). In this model, cationic liposomal paclitaxel reduced tumor growth and tumor invasiveness and improved the lifespan of the mice. Interestingly, by measuring the mitotic index of endothelium in vivo, it was demonstrated that cationic liposome-encapsulated paclitaxel particularly reduced EC proliferation.

Similar observations were made in a model of Meth-A sarcoma where porphyrins were delivered by cationic liposomes to the mouse tumor vascu-lature. After laser irradiation of the tumor, neovascular destruction was seen with concomitant reduced tumor growth along with a prolonged survival time of the mice. Immunohistochemistry was used to confirm that antitumor effects were related to the destruction of angiogenic endothelium resulting in tumor cell apoptosis (70).

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