Abreu et al. Nanoscale Research Letters 2011, 6:482 http://www.nanoscalereslett.com/content/6/1/482 Page 2 of 6 Figure 1 Structure of methyl 6-methoxy-3-(4-methoxyphenyl)- 1H-indole-2-carboxylate. distribution, zeta potential, and stability were evaluated, keeping in mind future drug delivery applications using this compound as an anticancer drug. The intrinsic fluorescence of compound 1 was used to obtain relevant information about its location in nanolipo- somes and its diffusion across the membrane in dialysis experiments. For the latter, Förster resonance energy transfer (FRET) between compound 1 (energy donor) and nitrobenzoxadiazole (NBD)-labelled lipids in different positions (at head group or fatty acid), acting as energy acceptor, was used to monitor compound behavior, as this photophysical process strongly depends on the donor- acceptor distance [11]. These studies are important, not only to evaluate the best liposome formulations to encap- sulate this promising antitumoral agent, but also to con- firm the possibility of compound 1 to permeate the lipid bilayer (cell membrane model). Experimental Nanoliposome preparation Dipalmitoyl phosphatidylcholine (DPPC), egg yolk phos- phatidylcholine (Egg-PC), dipalmitoyl phosphatidylglycerol (DPPG), Ch, and dihexadecyl phosphate (DCP) were obtained from Sigma-Aldrich (St. Louis, MI, USA). Dis- tearoyl phosphatidylcholine (DSPC) and distearoyl phos- phatidylethanolamine-N-[methoxy(polyethylene glycol)- 2000] (ammonium salt) (DSPE-PEG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Fluorescent- labelled lipids N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2- dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (NBD-PE), 2-(6-(7-nitrobenz-2- oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn- glycero-3-phosphocholine (NBD-C6-HPC), and 2-(12-(7- nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1- hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C12- HPC) were obtained from Invitrogen (Carlsbad, CA, USA). Nanoliposomes were prepared by injection of an etha- nolic solution of lipids/compound 1 mixture in an aqu- eous buffer solution under vigorous stirring, above the lipid melting transition temperature (ca. 41°C for DPPC [12] and 39.6°C for DPPG [13]), followed by three extru- sion cycles through 100 nm polycarbonate membranes. The final lipid concentration was 1 mM, with a com- pound/lipid molar ratio of 1:333. Encapsulation efficiency (percent) The encapsulation efficiency (EE) was determined through fluorescence emission measurements. After pre- paration, liposomes were subjected to centrifugation in an Eppendorf 5804 R centrifuge (Hamburg, Germany) at 11,000 rpm for 60 min. The supernatant was pipetted out, and its fluorescence was measured, allowing to cal- culate the compound concentration using a calibration curve previously obtained. The encapsulation efficiency of compound 1 was determined using the following equation: EE (%)=(Amountoftotalcompound1intheliposomepreparation−Amountofnon−encapsulatedcompound)/(Amountoftotalcompound1intheliposomepreparation)×100. DLS and zeta potential measurements The liposomes’ mean diameter, size distribution (poly- dispersity index), and zeta potential were measured with dynamic light scattering (DLS) NANO ZS Malvern Zetasizer equipment (Worcestershire, UK), at 25°C, using a He-Ne laser of 633 nm and a detector angle of 173°. Five independent measurements were performed for each sample. Malvern dispersion technology software (DTS) (Worcestershire, UK) was used with multiple nar- row mode (high-resolution) data processing, and mean size (nanometer), and error values were considered. Dialysis Permeability studies of compound 1 between DPPC/ DPPG mixed liposomes (donor liposomes) and NBD- labelled DPPC/DPPG liposomes (acceptor liposomes) were performed using two different sizes of dialysis membranes (6 to 8 KDa and 12 to 14 KDa). Three fluorescent NBD-labelled lipids were used, either labelled at head group (NBD-PE) or labelled at fatty acid (NBD-C6-HPC and NBD-C12-HPC). The experiments were carried out using a reusable 96-well micro-equili- brium dialysis device HTC 96 (Gales Ferry, CT, USA) and left in an incubator at 25°C (80 rpm) for 36 h. Spectroscopic measurements Fluorescence measurements were obtained in a Fluoro- log 3 spectrofluorimeter (HORIBA Scientific, Kyoto,