BMC Cancer 2004, 4:63 http://www.biomedcentral.com/1471-2407/4/63 trypsinized and the pellet re-suspended in a 0.4% trypan bleu buffer and counted in the hemocytometric chamber. Cell mortality was calculated as the percentage of stained cells over the total and expressed as the ratio between treated and untreated (control) cells. DNA cell content was assayed by using a fluorimetric DNA assay kit (Bio- rad laboratories, Segrate, Milano, Italy). Results We first analysed the effects of different concentrations of gemcitabine on the viability of ARO thyroid cancer cells. As shown in Figure 1 the cytotoxic effect was observed only after 48 or 72 h incubation of the drug. After 72 h, the cell mortality increased 4.6 and 7.9 fold over control using 1 and 100 µM of gemcitabine. In accordance with previous reports [8], no significant effect was visible after 24 h exposure (Fig. 1). In order to improve the drug entry into the cells, gemcit- abine entrapment in a liposome capsule was next per- formed, using a combination of various liposome preparation procedures. In particular, a pH gradient method was used [14]. Namely, the presence of ammo- nium sulphate in the internal compartments of liposomes provide an acidic environment that elicit the protonation of gemcitabine in order to drastically reduce the drug back-diffusion (leakage) from liposomes (Figure 2). The combination of various liposome preparation procedures together with the application of a pH gradient provided an encapsulation efficiency of ~90%. The gemcitabine-loaded liposomes were then tested for their cytotoxic activity against ARO cell, and the results compared with those of gemcitabine alone. As shown in Figure 3, a cytotoxic effect appeared after 24 h incubation, with an initial increased cell mortality at 0.3 µM (2.4 fold over control), and a maximum at 10 µM (death of almost all the cells) (Figure 3). Similar results were obtained by analysing the DNA content of ARO cells treated with free or liposome-encapsulated gemcitabine (data not shown). Discussion Despite an intense application of aggressive multimodal therapeutic regimens, no standardized protocol has shown success in the treatment of anaplastic thyroid car- cinoma [2]. A critical need for new approaches is therefore vital for the systemic therapy of metastatic anaplastic thy- roid carcinoma. At present one promising option is pro- vided by the gene therapy. Studies are currently in progress attempting to: a. reintroducing iodine uptake by viral-driven Iodide/sodium symporter gene expression [15,16]; b, inducing tumour cell death by expressing p53 or by suicide gene prodrug systems [17,18]. An alternative approach is represented by increasing the effectiveness of FCiygtuortoex1ic effect of gemcitabine Cytotoxic effect of gemcitabine. ARO cells were grown to 50% confluence and treated with increasing concentrations of gemcitabine for different incubation time. Cell mortality was evaluated by trypan bleu dye exclusion assay (see Meth- ods). In control cells, mortality was always lower than 5%. The values, expressed as the ratio over control, represent the mean ± SEM of at least three different experiments in triplicates. chemotherapy, using novel agents, combination therapy or enhancing drug delivery inside the tumour cell. The latter option has been taken advantages in the progress related to liposome research. Traditional dosage forms of anticancer drugs have the limitation of many potential obstacles (barriers) before they reach their target site, one of these is the large volume of distribution after i.v. administration. This situation elicits a low therapeutic index and a high toxicity in healthy tissues [19]. After encapsulation into liposomes similar to those used in our investigation the volume of distribution of a drug is reduced, and its concentration in the tumour tissue is increased [20]. In fact, liposomes can protect drugs from metabolic inactivation, and, due to size limitation, they are not able to transport encapsulated molecules across healthy endothelium, thus avoiding anticancer drug accu- mulation in a large extent in healthy tissues and hence a decrease of side-effects [21]. On the other hand, the increased permeability of the tumour endothelium allows Page 3 of 5 (page number not for citation purposes)