Nanoliposomes for encapsulation and delivery of the potential antitumoral methyl 6-methoxy-3-(4-methoxyphenyl)-1H-indole-2-carboxylate
© Abreu et al; licensee Springer. 2011
Received: 28 October 2010
Accepted: 3 August 2011
Published: 3 August 2011
A potential antitumoral fluorescent indole derivative, methyl 6-methoxy-3-(4-methoxyphenyl)-1H-indole-2-carboxylate, was evaluated for the in vitro cell growth inhibition on three human tumor cell lines, MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), and NCI-H460 (non-small cell lung cancer), after a continuous exposure of 48 h, exhibiting very low GI50 values for all the cell lines tested (0.25 to 0.33 μM). This compound was encapsulated in different nanosized liposome formulations, containing egg lecithin (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG), DSPC, cholesterol, dihexadecyl phosphate, and DSPE-PEG. Dynamic light scattering measurements showed that nanoliposomes with the encapsulated compound are generally monodisperse and with hydrodynamic diameters lower than 120 nm, good stability and zeta potential values lower than -18 mV. Dialysis experiments allowed to monitor compound diffusion through the lipid membrane, from DPPC/DPPG donor liposomes to NBD-labelled lipid/DPPC/DPPG acceptor liposomes.
Anticancer drugs are crucial agents in the global approach to fight cancer. Drug-loaded nanoparticles provide a perfect solution to afford higher therapeutic efficacy and/or reducing toxicity and the possibility of targeting cancer tissues. Nanoliposomes are one of the best drug delivery systems for low molecular weight drugs, imaging agents, peptides, proteins, and nucleic acids. Nanoliposomes are able to enhance the performance of bioactive agents by improving their bioavailability, in vitro and in vivo stability, as well as preventing their unwanted interactions with other molecules [1–3]. It is believed that the efficient antitumor activity can be attributed to the selective delivery and the preferential accumulation of the liposome nanocarrier in the tumor tissue via the enhanced permeability and retention effect [4–6].
Nanoliposomes may contain, in addition to phospholipids, other molecules such as cholesterol (Ch) which is an important component of most natural membranes. The incorporation of Ch can increase stability by modulating the fluidity of the lipid bilayer preventing crystallization of the phospholipid acyl chains and providing steric hindrance to their movement. Further advances in liposome research found that surface modification with polyethylene glycol (PEG), which is inert in the body, generally reduces the clearance of liposome by RES, and therefore allows longer circulatory life of the drug delivery system in the blood . Pegylated liposomal doxorubicin has shown great prolonged circulation and substantial efficacy in breast cancer treatment . The net charge of nanoliposomes is also an important factor and generally anionic and neutral liposomes survive longer than cationic liposomes in the blood circulation after intravenous injection [8, 9].
The intrinsic fluorescence of compound 1 was used to obtain relevant information about its location in nanoliposomes 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 . These studies are important, not only to evaluate the best liposome formulations to encapsulate this promising antitumoral agent, but also to confirm the possibility of compound 1 to permeate the lipid bilayer (cell membrane model).
Dipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC), dipalmitoyl phosphatidylglycerol (DPPG), Ch, and dihexadecyl phosphate (DCP) were obtained from Sigma-Aldrich (St. Louis, MI, USA). Distearoyl phosphatidylcholine (DSPC) and distearoyl phosphatidylethanolamine-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 ethanolic solution of lipids/compound 1 mixture in an aqueous buffer solution under vigorous stirring, above the lipid melting transition temperature (ca. 41°C for DPPC  and 39.6°C for DPPG ), followed by three extrusion cycles through 100 nm polycarbonate membranes. The final lipid concentration was 1 mM, with a compound/lipid molar ratio of 1:333.
Encapsulation efficiency (percent)
DLS and zeta potential measurements
The liposomes' mean diameter, size distribution (polydispersity 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 narrow mode (high-resolution) data processing, and mean size (nanometer), and error values were considered.
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-equilibrium dialysis device HTC 96 (Gales Ferry, CT, USA) and left in an incubator at 25°C (80 rpm) for 36 h.
where I DA is the donor emission intensity after the dialysis experiment in NBD-labelled lipid/DPPC/DPPG liposomes, is the initial donor emission intensity in DPPC/DPPG liposomes and is the final donor emission intensity in DPPC/DPPG liposomes.
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, sulforhodamine 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 procedure followed was described elsewhere . 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
Values of compound 1 concentration needed for 50% of cell growth inhibition (GI50)
0.37 ± 0.02
0.33 ± 0.03
0.25 ± 0.02
Hydrodynamic diameter, polydispersity, zeta potential, and encapsulation efficiency of several drug-loaded liposomes
Hydrodynamic diameter (nm) (mean ± SD)
Polydispersity (mean ± SD)
Zeta potential (mV) (mean ± SD)
115.4 ± 0.5
0.15 ± 0.01
-30 ± 1
1 week after
116 ± 2
0.15 ± 0.01
2 weeks after
116.0 ± 0.8
0.15 ± 0.01
120 ± 2
0.19 ± 0.01
-27 ± 4
104.3 ± 0.6
0.25 ± 0.01
-19 ± 2
79.3 ± 0.8
0.37 ± 0.01
-39 ± 3
103.5 ± 0.9
0.12 ± 0.01
-52 ± 6
2 weeks after
95.4 ± 0.5
0.14 ± 0.01
104 ± 3
0.27 ± 0.01
-43 ± 3
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 aggregation 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.
Previous fluorescence experiments showed the possibility of FRET between the excited compound 1 and the widely used fluorescence probe nitrobenzoxadiazole, NBD. The FRET mechanism involves a donor fluorophore in an excited electronic state (here compound 1), which may transfer its excitation energy to a nearby acceptor chromophore (NBD) in a nonradiative way through long-range dipole-dipole interactions. Because the range over which the energy transfer can occur is limited to approximately 100 Å and the efficiency of transfer is extremely sensitive to the donor-acceptor separation distance, resonance energy transfer measurements can be a valuable tool for probing molecular interactions .
The fluorescent methyl 6-methoxy-3-(4-methoxyphenyl)-1H-indole-2-carboxylate (1) exhibits excellent antitumoral properties, with very low GI50 values in the three human tumor cell lines tested. Several nanoliposome formulations containing the fluorescent drug were successfully prepared by an injection/extrusion combined method, with particle sizes lower than 120 nm, low polydispersity 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 present study will involve assays of liposome cell internalization and mechanism of action, keeping in mind the application of this compound as an antitumoral drug.
melanoma cell line
dynamic light scattering
PEG: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]
dispersion technology software
egg yolk phosphatidylcholine
Förster resonance energy transfer
breast adenocarcinoma cell line
non-small cell lung cancer line.
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 L. Vale-Silva (SFRH/BPD/29112/2006) acknowledge FCT for their postdoctoral grants.
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