Liposomal formulations of DOX have been studied for a number

Liposomal formulations of DOX have been studied for a number of years as a means of reducing the cardiac toxicity of the drug because liposomal encapsulation would be expected to shift DOX away from myocardial tissue (with a normal endothelial barrier), while maintaining tumor exposure (Rahman et al., 2007). Furthermore, encapsulation of DOX within long-circulating liposomes decorated with poly(ethylene glycol) (PEG liposomes), reduces its uptake by the reticulo-endothelial system and prolongs its serum half-life to around 50h compared with 10min for the free drug (Berry et al., 1998). As a result, DOX in PEG liposomal has been shown to have an increased therapeutic efficacy and reduced cardiotoxicity compared to free DOX (Gabizon et al., 2003) and a commercial formulation is marketed as Doxil or Caelyx (Janssen Pharmaceuticals).
In order to achieve a high yield of DOX encapsulation in liposomes a remote loading approach is used (Ogawara et al., 2009). The method involves the addition of drug to preformed liposomes through a pH gradient or an ion gradient capable of generating a pH gradient (Mayer et al., 1990). Due to their high payload of drug, liposomal systems are the best method to passively target anthracyclines to tumors, compared with microspheres or nanoparticles (Minotti et al., 2004).

Material and methods

Results

Discussion
For many years, drug delivery strategies have been developed to improve the effectiveness and reduce the cardiotoxicity of anthracyclines. In a first approach, association with colloidal nanosystems such as liposomes and nanoparticles allows the distribution to be modified avoiding the heart. In a second step, carriers specifically directed toward cancer Apoptosis Compound Library have been developed (Barenholz, 2001).
For the first strategy, liposomes have been the most frequently studied carrier system and were therefore chosen for the present work. Our results for DOX encapsulation are in agreement with previous studies, showing that the use of a transmembrane pH gradient yields high trapping efficiency (Woodle, 1995; Fenske and Cullis, 2007; Ogawara et al., 2009) and encapsulation close to 100% (Fenske and Cullis, 2007; Ogawara et al., 2009). In our case more than 90% of added DOX was incorporated. This slightly lower value compared with the literature can be explained by the difference in composition of lipids entering in the structure of the liposomal membrane as well as by the difference salts used for the generation of the gradient of pH (Abraham et al., 2002; Fritze et al., 2006).
Several studies have shown that the organ distribution of liposome-encapsulated DOX is different from that of the free drug, and this contributes to a reduction in toxicity to normal tissues (Berry et al., 1998; Gabizon et al., 2003; Batist, 2007). Our work focussed on the interactions between free and encapsulated drug and cardiac cells. Observations made by confocal microscopy and flow cytometry suggest that DOX-liposomes can be taken up by these cells by endocytosis, despite the presence of PEG on their surface. H9c2 cells treated with empty fluorescent liposomes displayed rhodamine fluorescence in a punctuate pattern in the cytoplasm (Fig. 5) and flow cytometry showed that the peak of fluorescence shifted toward higher values with increasing time of incubation (Fig. 3A3 and B3). We were also able to use the intrinsic fluorescence of DOX to follow its uptake by cells. In confocal microscopy the fluorescence of the DOX, localized mainly in the nucleus, was more intense in cells treated with the free DOX compared with cells exposed for the same time to the same concentration in liposomal form. This result was corroborated by the flow cytometry results and is in accordance with earlier observations (Morisco et al., 2011). The high affinity of DOX for DNA appears to be the major driving force for the drug to enter cells by diffusion (Frezard and Garnier-Siullerot, 1991). Confocal microscopy revealed that the fluorescence of DOX is mainly in the nuclear compartment while the cytoplasm shows a weak intensity of fluorescence (Xiong et al., 2005). Administration of DOX to a sensitive cell line results in rapid nuclear uptake, with >99.8% of nuclear drug becoming directly associated with DNA (Cutts and Phillips, 1995). It appears that endocytosis of the liposomes is slower and therefore limits accumulation within the cells. However, DOX fluorescence inside these liposomes may be greatly quenched (Lee and Low, 1995), and the cytoplasm may contain significantly more Lipo-DOX than indicated by their relative fluorescence intensity.