Abreu et al. Nanoscale Research Letters 2011, 6:482 http://www.nanoscalereslett.com/content/6/1/482 Japan), equipped with double monochromators in both excitation and emission and a temperature controlled cuvette holder. Fluorescence spectra were corrected for the instrumental response of the system. Nanoliposomes containing only the fluorescent compound 1 (energy donor) served as negative (no FRET) control. The per- centage of energy transfer, ET (percent), was calculated from the fluorescence emission intensities, IDA ET (%) = 1 − IDi − IDf × 100, where IDA is the donor emission intensity after the dialysis experiment in NBD-labelled lipid/DPPC/DPPG liposomes, IDi is the initial donor emission intensity in DPPC/DPPG liposomes and IDf is the final donor emis- sion intensity in DPPC/DPPG liposomes. Biological activity Fetal bovine serum, L-glutamine, phosphate-buffered saline, trypsin, and RPMI-1640 medium were purchased from Invitrogen (Carlsbad, CA, USA). Acetic acid, dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin, ethylenediaminetetraacetic acid, sulforho- damine B, and trypan blue were from Sigma-Aldrich (St. Louis, MI, USA). A stock solution of 1 was prepared in DMSO and was kept at -70°C. Appropriate dilutions of the compound were freshly prepared in the test medium just prior to the assays. The vehicle solvent had no influence on the growth of the cell lines. Human tumor cell lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and A375-C5 (melanoma) were tested. MCF-7 and A375-C5 were obtained from the European Collection of Cell Cultures (Salisbury, UK), and NCI-H460 was kindly provided by National Cancer Institute (NCI) (Bethesda, MD, USA). The pro- cedure followed was described elsewhere [14]. The in vitro effect on the growth of human tumor cell lines was evaluated according to the procedure adopted by the NCI in their “In vitro Anticancer Drug Discovery Screen,” using the protein-binding dye sulforhodamine B to assess cell growth [15,16]. Doxorubicin was tested following the same protocol and was used as positive control. Results and discussion Antitumoral evaluation The in vitro growth inhibitory activity of compound 1 was evaluated using three human tumor cell lines, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460), and a melanoma cell line (A375-C5), after 48 h of continuous exposure to compound 1. Results given in concentrations that were able to cause 50% of cell growth inhibition (GI50) are summarized in Table 1. Page 3 of 6 Table 1 Values of compound 1 concentration needed for 50% of cell growth inhibition (GI50) 1 MCF-7 0.37 ± 0.02 GI50 (μM) NCI-H460 0.33 ± 0.03 A375-C5 0.25 ± 0.02 Results represent means ± SEM of three independent experiments performed in duplicate. Doxorubicin was used as positive control (GI50: MCF-7 = 43.3 ± 2.6 nM; NCI-H460 = 35.6 ± 1.6 nM; and A375-C5 = 130.2 ± 10.1 nM). It can be observed that compound 1 inhibited the growth of the three tumor cell lines with very low GI50 values. These inhibitory concentrations are significantly lower than those obtained with other potential antitumoral compounds recently tested [17-19], some of them also containing the indole nucleus [17-21], and point to a pro- mising utility of this compound as an antitumoral agent. Doxorubicin, used as positive control, presents a very high cytotoxicity because the planar aromatic moiety effi- ciently intercalates into DNA base pairs, while the six- membered daunosamine sugar binds to the minor groove, interacting with flanking base pairs adjacent to the intercalation site [22]. Nevertheless, doxorubicin pre- sents also a high toxicity for the human body, and the search for other antitumoral compounds, even less active but also less toxic, is still an active domain of interest. Nanoliposomes characterization Selected liposome formulations [23-25] with encapsulated compound 1 were prepared. All the formulations have mean hydrodynamic diameters lower than 120 nm, gener- ally low polydispersity and very good encapsulation effi- ciency (Table 2). Pegylation of nanoliposomes surface with DSPE-PEG generally leads to the increase of the hydrody- namic diameter that, however, remains close to 100 nm. The mean diameter of the Egg-PC/DCP/Ch (7:2:1) lipo- some is considerably smaller than the others (Table 2), but with a higher polydispersity index. Formulations including egg phosphatidylcholine show a tendency to a lower parti- cle size. All the different nanoliposomes prepared are gen- erally monodisperse and stable after 2 weeks, with no evidence of aggregation (Table 2). Zeta potential measurements were used to evaluate the relationship between surface charge and stability. All the nanoliposome formulations have negative zeta potential (Table 2). The higher colloidal stability was obtained for Egg-PC/Ch/DPPG (6.25:3:0.75) formulation (ζ value more negative), while the lower stability (higher aggrega- tion tendency) is observed for Egg-PC/Ch/DSPE-PEG (5:5:1) liposomes, which exhibit a ζ-potential value clearly less negative than -30 mV. Dialysis Previous fluorescence experiments showed the possibi- lity of FRET between the excited compound 1 and the