Abreu et al. Nanoscale Research Letters 2011, 6:482 http://www.nanoscalereslett.com/content/6/1/482 through the dialysis membrane in this situation, this will be even easier with the lipids that are in the fluid phase. The NBD-labelled lipids were either labelled at head group (NBD-PE), at position 6 of the fatty acid chain (NBD-C6-HPC) or at position 12 of the fatty acid chain (NBD-C12-HPC). Figure 3 displays (as examples) the emission spectra of compound 1 in DPPC/DPPG donor liposomes and of the NBD-PE/DPPC/DPPG acceptor nanoliposomes, before (t = 0 s) and after (t = 36 h) dif- fusion of compound 1 through the two dialysis mem- branes used in the study. After the dialysis assay, the fluorescence of compound 1 in the donor liposomes is notably reduced (Figure 3), and its emission can be detected in the acceptor liposomes solution, showing the diffusion of compound 1 through the dialysis mem- brane. Besides, due to the energy transfer from com- pound 1 to NBD, the fluorescence intensity of the latter notably increases (Figure 3). The effect is stronger for the membrane of 12 to 14 KDa. The percentage of energy transfer from compound 1 to NBD is higher when the acceptor nanoliposomes are labelled with NBD-PE (NBD linked at lipid head group) (Figure 4). In this case, it can be observed that energy transfer is higher for the 12- to 14-KDa dialysis mem- brane. It can also be concluded that, after 36 h of dialy- sis, compound 1 is located mainly near the polar head groups of the phospholipids in the acceptor nanolipo- somes, as energy transfer to NBD is less efficient when this energy acceptor is located deeper in the lipid chain (NBD-C12 or NBD-C6) (Figure 4). Conclusions The fluorescent methyl 6-methoxy-3-(4-methoxyphenyl)- 1H-indole-2-carboxylate (1) exhibits excellent antitu- moral properties, with very low GI50 values in the three Figure 3 Fluorescence spectra of compound 1 in DPPC/DPPG liposomes and NBD-PE labelled DPPC/DPPG liposomes before and after dialysis. Page 5 of 6 Figure 4 Percentage of drug transfer in dialysis between DPPC/DPPG liposomes and NBD-labelled lipid/DPPC/DPPG liposomes. human tumor cell lines tested. Several nanoliposome for- mulations containing the fluorescent drug were success- fully prepared by an injection/extrusion combined method, with particle sizes lower than 120 nm, low poly- dispersity index, and good stability after 2 weeks. The Egg-PC/Ch/DPPG (6.25:3:0.75) and Egg-PC/DPPG/ DSPE-PEG (5:5:1) showed to be the best formulations for encapsulation of this compound, considering their low hydrodynamic diameter, high negative zeta potential, and very high encapsulation efficiency. Dialysis experiments allowed to follow compound diffusion from DPPC/DPPG donor liposomes to NBD-labelled lipid/DPPC/DPPG acceptor liposomes, through dialysis membranes of 6 to 8 KDa and 12 to 14 KDa. These results may be important for future drug delivery applications using nanoliposomes for the encapsulation and transport of this promising antitumoral compound. Further developments of the pre- sent study will involve assays of liposome cell internaliza- tion and mechanism of action, keeping in mind the application of this compound as an antitumoral drug. Abbreviations A375-C5: melanoma cell line; Ch: cholesterol; DCP: dihexadecyl phosphate; DLS: dynamic light scattering; DPPC: dipalmitoyl phosphatidylcholine; DPPG: dipalmitoyl phosphatidylglycerol; DSPC: distearoyl phosphatidylcholine; DSPE: PEG: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]; DTS: dispersion technology software; Egg-PC: egg yolk phosphatidylcholine; FRET: Förster resonance energy transfer; MCF-7: breast adenocarcinoma cell line; NBD-C6-HPC: 2-(6-(7-nitrobenz-2-oxa-1,3- diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine; NBD-C12-HPC: 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1- hexadecanoyl-sn-glycero-3-phosphocholine; NBD-PE: N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; NCI-H460: non-small cell lung cancer line. Acknowledgements Thanks are due to the Foundation for Science and Technology (FCT, Portugal) for financial support through the research centers (CFUM and CQ- UM) and project PTDC/QUI/81238/2006 (cofinanced by FEDER/COMPETE, ref. FCOMP-01-0124-FEDER-007467). A.S. Abreu (SFRH/BPD/24548/2005) and