BMC Cancer 2004, 4:63 http://www.biomedcentral.com/1471-2407/4/63 Background At present, the prognosis of anaplastic thyroid carcinomas is very poor [1,2]. Since they are usually unable to concen- trate radioiodine, the therapeutical approach is based on combination of aggressive surgery, external beam radia- tions and chemotherapy. Only rarely, however, this treat- ment results effective especially for treating metastatic disease [1,2]. A major limit of the chemotherapeutic agents presently used in these tumours, including doxoru- bicin, paclitaxel and various drug combinations [1,2], is represented by their low therapeutic index. Thus, the iden- tification of systemic antineoplastic drugs effective against these carcinomas has particularly relevant implications. Gemcitabine is a new fluorinated nucleoside analogue provided with a potent anti-tumour activity, tested in vitro and in vivo against a variety of solid malignancies [3- 6]. In addition, gemcitabine is a potent radiosensitizing agent [7]. In a preclinical study, gemcitabine showed a marked cytotoxic activity against poorly differentiated human thyroid carcinoma cell lines [8]. In contrast, no appreciable response was observed in four patients with aggressive thyroid anaplastic carcinoma treated with com- bination of gemcitabine and vinorelbine [9]. Since hema- tological and other toxicities have been reported when effective anti-tumour doses of gemcitabine were tested [10], the aim of this study was to verify the possibility of maintaining the drug cytotoxicity at lower doses, by using a liposomal drug delivery system. Our data demonstrate that incorporation of gemcitabine in liposomes enhances the cytotoxic effect of the drug against a well-established human thyroid cancer cell line, as compared to the free drug. Methods Chemicals Gemcitabine (2,21-difluorodeoxycytidine) hydrochloride (HPLC purity >99%) was a kind gift of Eli-Lilly Italia S.p.A. (Sesto Fiorentino, Firenze, Italy) and was used with- out further purification. Cholesterol was obtained from Sigma Chemicals Co. (St. Louis, USA). 1,2-dipalmitoyl- sn-glycero-3-phosphocholine monohydrate and N-(carb- onyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine sodium salt (MPEG- 2000-DSPE) were purchased from Genzyme products (Suffolk, United Kingdom). Double-distilled pyrogen-free water from Sifra S.p.A. (Verona, Italy) was used. Sterile saline was a product of Frekenius Kabi Potenza S.r.l. (Verona, Italy). All other materials and solvents were of analytical grade (Carlo Erba, Milan, Italy). Liposome preparation Liposome formulations were made up of DPPC/Chol/ MPEG-2000-DSPE (8:3:1 molar ratio). To maximize the entrapment efficiency of a hydrophilic molecule such as gemcitabine, various liposome preparation procedures [11,12] were applied together. Namely, liposome colloi- dal suspensions were prepared by dissolving the lipid mixture (40 mg) in chloroform-methanol (3:1 v/v). Organic solvent was removed by a rotavapor thus allow- ing the formation of a thin lipid film on the inner surface of a pyrex glass vial. To remove any trace of organic sol- vent, lipid films were stored overnight in a Büchi T-50 under high vacuum at 30°C in the dark. Lipid films were hydrated with a solution (2 ml) of 250 mM ammonium sulphate by vortexing at 45°C for 15 min. The resulting colloidal suspension of multilamellar vesicles was sub- mitted to ten cycles of freeze in liquid nitrogen and thaw in warm (35°C) water in order to allow the homogenous distribution of the ionic species. The entrapped ammo- nium sulphate solution was removed by centrifugation at 14000 rpm (IEC, International Equipment Company, mod MP4R, equipped with a mod. 851 rotor) for 1 h at a temperature of 4°C. The pellet was re-suspended in 400 µl of a 1 mM gemcitabine aqueous solution and stored at room temperature for 3 h. Double-distilled pyrogen-free water (1.6 ml) was added to the liposome suspension and then a freeze-drying procedure was carried out (Edwards freeze-dryer Modulayo). Then, the non-incorporated gem- citabine was removed and the freeze-dried liposomes were re-suspended just before the experiments with 2 ml of the culture medium. Scalar dilutions were prepared in the same medium to obtain the final concentrations used in the experiments. Liposome loading capacity The loading capacity of liposomes was evaluated after removing the free gemcitabine (centrifugation at 14000 × g for 1 h at 4°C) from the vesicular colloidal suspension coming from the re-suspension of freeze-dried liposomes. The amount of gemcitabine in the supernatant was spec- trophotometrically determined at 268.8 nm (Shimadzu UV-1601). UV calibration straight-line (y = 6.958 × 10-3 + 0.3971x, where y is the UV adsorbance and x is the drug concentration) presented a r2 value of 0.9993. The amount of gemcitabine entrapped in liposomes was determined as a difference between the amount of drug added during liposome preparation and the amount of the entrapped drug present in the supernatant. The encap- sulation efficiency was expressed as percentage of the total amount of gemcitabine that became entrapped. Cell cultures and cell viability The ARO cells, from a human anaplastic thyroid carci- noma, were grown as previously described [13]. All exper- iments were performed in 12-well culture dishes, when cells reached approximately 50% confluence. Cell viabil- ity was evaluated by trypan bleu dye exclusion assay. Briefly, after incubation with gemcitabine or liposomes containing the drug at different doses, cells were Page 2 of 5 (page number not for citation purposes)