Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The antimycobacterial activity of 3-(4'-bromo[1,1'-bipheny1-4-yl)-3-(4-bromo-phenyl-N,N-dimethyl-2-propen-1-amine (BBAP), free or incorporated in preformed liposomes, on extracellular M. tuberculosis H37Rv was 8 and 25 mu M (MIC), respectively. Extracellular antimycobacterial activity was not significantly improved by entrapment of BBAP in liposomes, but there was a 6.1-fold reduction of BBAP cytotoxicity on J774 macrophages. Liposomal BBAP or its free form showed IC(50) values of 165 and 27 mu M, resulting in a selectivity index (SI=IC(50)/MIC) of 3.4 and 6.6, respectively. Free BBAP in concentrations from 10 to 80 mu M were quite effective in eliminating intracellular M. tuberculosis while liposomal formulation was less effective at these concentrations.334871874Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Andrade-Santana, MH

de Souza, AO

Duran, N

Silva, CL

Química Nova

Repositorio da Producao Cientifica e Intelectual da Unicamp

Quim. Nova, Vol. 33, No. 4, 871-874, 2010
Ar
ti
go
*e-mail: olivia@butantan.gov.br
#Current address: Laboratório de Bioquímica e Biofísica, Instituto Butantan, 
Av. Vital Brasil, 1500, 05503-900 São Paulo - SP, Brasil
ANTIMYCOBACTERIAL AND CYTOTOXICITY ACTIVITIES OF FREE AND LIPOSOME-ENCAPSULATED 
3-(4’-BROMO[1,1’-BIPHENYL-4-YL)-3-(4-BROMO-PHENYL)-N,N-DIMETHYL-2-PROPEN-1-AMINE
Ana O. de Souza*# e Célio L. Silva
Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. dos Bandeirantes, 3900, 14049-900 Ribeirão Preto - SP, Brasil
Nelson Durán
Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas - SP, Brasil
Maria H. Andrade-Santana
Faculdade de Engenharia Química, Universidade Estadual de Campinas, CP 6066, 13083-970 Campinas - SP, Brasil
Recebido em 17/6/09; aceito em 2/12/09; publicado na web em 26/3/10
The antimycobacterial activity of 3-(4’-bromo[1,1’-biphenyl-4-yl)-3-(4-bromo-phenyl)-N,N-dimethyl-2-propen-1-amine (BBAP), free 
or incorporated in preformed liposomes, on extracellular M. tuberculosis H37Rv was 8 and 25 mM (MIC), respectively. Extracellular 
antimycobacterial activity was not significantly improved by entrapment of BBAP in liposomes, but there was a 6.1-fold reduction 
of BBAP cytotoxicity on J774 macrophages. Liposomal BBAP or its free form showed IC
50
 values of 165 and 27 µM, resulting in a 
selectivity index (SI=IC
50
/MIC) of 3.4 and 6.6, respectively. Free BBAP in concentrations from 10 to 80 mM were quite effective in 
eliminating intracellular M. tuberculosis while liposomal formulation was less effective at these concentrations.
Keywords: liposome; tuberculosis; 3-(4’-bromo[1,1’-biphenyl-4-yl)-3-(4-bromo-phenyl)-N,N-dimethyl-2-propen-1-amine.
INTRODUCTION
Tuberculosis still ranks among the world’s most deadly infectious 
diseases, killing around 2-3 million people per year.1 A third of the 
world’s population may carry a latent tuberculosis infection, and 
the lifetime risk of developing tuberculosis ranges between 10 and 
20%.2 Although tuberculosis is a preventable and treatable disease, 
chemotherapy fails to reach its aim due to several reasons that include 
daily multiple drug administrations for several months, poor patient 
compliance, drug toxicity and emergence of drug resistance. Thus, 
one of the current strategies to enhance therapeutic activity, while 
minimizing toxicity, is to entrap drugs in a delivery system, assuring 
slow release over extended time periods. Liposome-encapsulated 
drugs often exhibit reduced toxicity and have also been shown to 
enhance retention of drugs in tissues, resulting in an improved overall 
therapeutic efficacy.3 Macrophage-specific delivery systems are cur-
rently a subject of much interest, since macrophages act as host cells 
for many parasites and bacteria, which give rise to the outbreak of 
many human deadly diseases, such as leishmaniasis and tuberculosis.
It has been previously shown that 3-(4’-bromo[1,1’-biphenyl-4-
yl)-3-(4-bromo-phenyl)-N,N-dimethyl-2-propen-1-amine (BBAP) 
is a drug with good antimycobacterial activity in vitro4-8 but also 
cytotoxic to mammalian cells. Our group recently prepared inclusion 
complexes of BBAP in b-cyclodextrin8,9 in order to reduce cytotoxi-
city and to improve the antimycobacterial effect. Several combined 
spectroscopy techniques indicated the formation of a complex of 
BBAP/b-CD in the molar proportion of 1:1 and 1:2.
Biological assays regarding cytotoxicity towards mammalian 
cells (J774) and towards a permanent lung fibroblast cell line (V79) 
indicated no significant toxic effects with the BBAP/b-CD complexes. 
The complexes were more effective than the free compound on several 
mycobacteria species. Similar behavior of the inclusion complexes 
BBAP/b-CD (1:1 and 1:2) and rifampicin, a front-line antitubercular 
drug, was observed for M. tuberculosis H37Rv growing inside J774 
macrophages.
These previous results indicate that the entrapment of BBAP is 
a promising strategy for reduction of cytotoxicity and enhancement 
of its effectiveness against mycobacteria. In this study, the efficacy 
of a formulation containing liposome -entrapped BBAP was evalu-
ated in vitro regarding cytotoxicity, extracellular and intracellular 
antimycobacterial activities.
EXPERIMENTAL
Preparation of liposomes
BBAP was synthesized as previously described.10 Liposomes 
were composed by phospholipid:cholesterol (60:40 molar), a known 
composition which provides stability of the liposomal membrane 
for drug encapsulation. The phospholipid used in this study was 
the hydrogenated soybean phosphatidylcholine Epikuron 200SH 
(Lipoid), a commercial soy lecithin useful for liposome preparations 
due to high percentage of the phosphatidylcholine structural lipids 
(distearoylphosphatidylcholine (DSPC:0-15%) and dipalmitoylphos-
phatidylcholine (DPPC:85-100%).
Small unilamellar vesicles containing BBAP were prepared by 
the classical Bangham method as described previously.11 Briefly, the 
lipids (phospholipid: cholesterol 60:40%) and BBAP were dissolved 
in chloroform:methanol (9:1 v/v) in BBAP/phospholipid ratios of 0 
(empty liposomes); 0.1; 0.2; 0.3; 0.4; 0.5; and 0.6 and a dried film 
was formed by rotatory evaporation at 55 °C. The drug-lipid film was 
hydrated with 5 mL of Hepes buffer (10 mM, pH=7.4 containing NaCl 
120 mM), forming a 7.5 mM lipid concentration and the suspension 
sonicated for 30 min (Ultrasonic Cleaner Unique) to produce small 
liposomes and maintained in repose during 2 h and 30 min.
Preformed liposomes were sized by multiple extrusions (15 
times) through a drain disk (Millipore C32WP02500) and two 
stacked polycarbonate membranes (pore sizes 100 nm - Osmomics 
K01CP02500) by a high-pressure extruder (10 kgf/cm2) (Lipex Bio-
membranes, Vancouver, BC). Non-entrapped BBAP was removed 
de Souza et al.872 Quim. Nova
from the liposome suspension by ultrafiltration through a membrane 
(YM 10 NMWL 10.000 - Amicon 13622) in an Amicon cell (Model 
850) under nitrogen pressure (2.5 kgf/cm2).
Characterization of liposomes
Phospholipid content in liposomes was characterized by quanti-
fication of total phosphate in samples, according to the methodology 
developed by Chen et al..12
To measure BBAP concentration, the liposomes were lysed with 
ethanol and the absorbance of the solution measured spectrophoto-
metrically at 266 nm (Spectrophotometer, Hitachi U-200). BBAP 
concentrations in the liposomes were determined by comparison 
with a standard curve prepared with BBAP from 0 to 20 µg/mL and 
the trapping efficiency was calculated as follows:
% E = 100*(mols BBAP/mols lipids)
final
/(mols BBAP/mols lipids)
initial
% E =Percent of encapsulation
Liposome mean diameter and particle size distribution were de-
termined by quasi elastic light scattering at 90º with a He-Ne laser, 
633 nm and at 25 °C (QLS - Light Scatter - Malvern Instruments 
Autosizer model 4700, UK).
Biological assays
Bacteria
M. tuberculosis H37Rv ATCC 27294, was grown in Loweinstein-
Jensen medium at 37 °C for 3 weeks and subcultured in Middlebrook 
7H9 broth medium, supplemented with OADC at 37 °C for 10 d. 
Cell concentration in the mycobacterial suspension was adjusted by 
optical absorbance in comparison with a standard curve and diluted 
in Middlebrook 7H9 broth medium to 4.0x105 mycobacteria/mL to 
determine the minimal inhibitory concentration (MIC) or in RPMI 
1640 (5.0x106 mycobacteria/mL) to detect macrophage infection in 
intracellular antimycobacterial activity.13
J774 Macrophages
J774 cells were grown as monolayers in RPMI 1640 medium 
supplemented with 10% heat inactivated fetal calf serum (FCS), 
100 IU/mL penicillin and 100 µg/mL streptomycin in a humidified 
incubator with a 5% CO
2
 atmosphere at 37 °C.
Extracellular antimicrobial susceptibility testing – minimal 
inhibitory concentration (MIC)
Free BBAP dissolved in dimethylsulphoxide (DMSO), empty 
and BBAP-containing liposomes were sterilized by passage through 
a 0.22 mm PFTE filter and diluted to the range of 10 to 160 mM in 
Middlebrook 7H9. Rifampicin was dissolved in DMSO, diluted in 
Middlebrook 7H9 broth and used as a reference drug at 12 µM.
Tests were performed by the microplate Alamar Blue assay, as 
previously described.14 M. tuberculosis H37Rv (100 mL) were seeded 
in a 96-well microplate (4.0x105 mycobacteria/mL) containing 100 mL 
of serial dilutions of BBAP - entrapped liposomes, empty liposomes 
or free BBAP. Plates were incubated at 37 °C for 6 d, followed by the 
addition of 25 mL mixed(1/1) Alamar Blue reagent and 10% Tween 
80 (v/v) to each well. Plates were reincubated at 37 °C and after 24 h, 
a change in color from blue to pink, indicated mycobacterial growth.
Control wells consisted of either bacterium only, drugs with me-
dium or only medium. The visual MICs were defined as the lowest 
drug concentration that prevented a color change from blue to pink.
Cytotoxicity to mammalian cells – J774 macrophages
The BBAP cytotoxic effect was assayed on J774 macrophages 
by measuring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) according to Denizot and 
Lang15 and expressed as IC
50
 values (concentration in which at least 
50% of the cells are viable). Stock solutions of BBAP were prepared 
in DMSO and diluted in RPMI 1640 without phenol red. The final 
solvent concentrations in the assay were less than 0.3% and each 
sample concentration was tested in six replicates, and repeated three 
times in separate experiments. Cells (2.0x106 cells/mL) added to 
96-well plates had the medium removed after 12 h and replaced by 
a medium containing free or liposomal BBAP in concentrations 
ranging from 10 to 160 mM. As controls, macrophages were trea-
ted with empty liposomes and with RPMI 1640, and considered as 
100% viable. Cells were exposed for 24 h to test medium with or 
without drugs (controls).
After exposure to the test compounds, the medium in the wells 
was removed and replaced by 0.1 mL of serum-free medium contai-
ning MTT (0.5 mg/mL). Following 4 h incubation, the supernatant 
was removed and the blue formazan product obtained was dissolved 
in 0.1 mL of isopropanol in 0.1 M HCl. Plates were stirred for 15 
min on a microtiter plate shaker and absorbance was read at 570 nm.
Intracellular antimicrobial susceptibility testing
J774 macrophages were harvested and plated at a concentra-
tion of 5.0x105 cells per well in 24-well tissue culture plates. 
After overnight incubation, the cells were overlaid with 1 mL of a 
suspension of M. tuberculosis H37Rv (5.0x106 mycobacteria/mL) 
adjusted to yield a multiplicity of infection of 10 mycobacteria 
per macrophage. Cells were infected for 3-4 h and washed with 
phosphate-buffered saline (PBS), pH=7.4, to eliminate unbound 
mycobacteria. M. tuberculosis H37Rv-infected cells were fed 
again with culture medium containing free or liposomal BBAP 
in concentrations ranging from 10 to 160 mM.8,13
Control experiments included, RPMI 1640 medium, empty 
liposomes and rifampicin at 12 mM. Rifampicin and the empty 
liposomes were administered diluted in Middlebrook 7H9 broth. 
After 72 h, cells were washed with PBS to eliminate extracellular 
bacteria and lysed by addition of 500 µL of 0.25% (wt/vol) sodium 
dodecyl-sulphate in phosphate-buffered saline (PBS - pH=7.4). 
The lysates were serially diluted and 100 mL aliquots were dis-
persed onto 7H10 agar plates. The colony formation units (CFU) 
of M. tuberculosis H37Rv were counted 2-4 weeks after incubation 
at 37 °C with 5% CO
2
.
Selectivity index
Selectivity index (SI) was calculated for free and liposomal BBAP 
formulations taking into account the MIC against M. tuberculosis 
H37Rv and the IC
50
 on J774 cells (SI=IC
50
/MIC) by the MTT assay.8
Statistical analysis
Results of cytotoxicity assays were expressed as cellular viabi-
lity and displayed graphically using the computer software package 
Origin-Data Analysis and Technical Graphics, version 6.0 (Copyri-
ght Software, Inc.). The IC
50
 values, concentrations that produced 
a 50% inhibitory effect on the evaluated parameter, were obtained 
by data interpolation.
Statistical analysis of the intracellular antimycobacterial activity 
was performed by the graph Prisma Software.
Antimycobacterial and cytotoxicity activities 873Vol. 33, No. 4
RESULTS AND DISCUSSION
Liposomes characterization
Figure 1A shows the profile relating the initial and final drug/lipid 
ratios. The BBAP encapsulation increased from 0.1 to 0.3, remaining 
constant at higher drug/lipid ratios. These results indicate the satu-
ration of the lipid bilayer, from which the encapsulation efficiencies 
decreased strongly (Figure 1B). The final drug/lipid ratios were 10 
times lesser than the initial ones, characterizing a low capability of 
the lipid matrix for BBAP entrapment. Further optimization of the 
formulation in terms of phospholipids and cholesterol concentration 
could enhance the packing of lipids in the matrix, providing higher 
entrapment efficiencies.
The average diameter of liposomes sizes was 130 nm and the 
polydispersity index was 0.3. The distributions were monodisperse 
in the range of 100 to 150 nm. 
Biological assays
Antimycobacterial activity of free BBAP and of liposomes car-
rying BBAP was assayed on extracellular M. tuberculosis H37Rv and 
the MIC values obtained were, respectively, 8 and 25 mM (Table 1).
As shown in Figure 2 liposomes entrapment of BBAP succes-
sfully decreased the cytotoxicity of BBAP. The IC
50
 of liposomes 
with free and entrapped BBAP were 27 and 165 µM, respective-
ly. The cytotoxicity of the compound was reduced by 6.1 fold, 
showing that this pharmaceutical formulation was important in 
reducing the toxicity of the drug, although the increase of the 
extracellular antimycobacterial activity was modest. Empty 
liposomes were not toxic to the cells (Figure 2). Table 1 shows 
the selectivity indexes (SI=IC
50
/MIC) of free BBAP and BBAP 
entrapped in liposomes and the values are 3.4 and 6.6, respectively. 
For a drug to be considered a good candidate against tuberculosis 
its SI should be higher than 10.
Although lower efficacy of liposomal BBAP compared to free 
BBAP, these results indicate that BBAP liposomal formulation is 
promising for antimycobacterial activity as demonstrated by decre-
asing of BBAP cytotoxicity and the selectivity index. However, an 
optimization of liposome composition, drug encapsulation and release 
as well as in vitro and in vivo studies are required for a complete 
evaluation of antimycobacterial efficacy of liposomal BBAP.
Free BBAP in concentrations ranging from 10 to 80 mM was 
significantly effective in eliminating intracellular M. tuberculosis 
H37Rv while BBAP liposomal formulation was not so efficient at 
the same concentrations (Figure 3) in comparison to control cells, 
which were infected and treated only with culture medium or empty 
liposomes (data not shown). The effects of the BBAP liposomal 
formulation on the biological results could be related to the slow 
release of drug and liposome phagocytosis. Enhancements on these 
Figure 1. Profile relating the initial and final drug/lipid ratios (A) and en-
capsulation efficiencies of BBAP in liposomes (B). D/L = drug/lipid ratios. 
Bars represent mean ± SEM of two independent experiments
Figure 2. Cytotoxicity to J774 macrophages of free BBAP (), empty liposo-
mes () and liposome-entrapped BBAP () determined by the MTT assay. 
Bars represent mean ± SEM of three independent experiments
Table 1. Antimycobacterial parameters of free and liposome-entrapped BBAP
Samples MIC* (mM) IC
50
** (mM) SI=IC
50
/MIC
Free BBAP 8 27 3.4
Liposomal BBAP 25 165 6.6
*MIC = corresponds to the MIC against M. tuberculosis H37Rv; **IC
50
 = cor-
responds to the IC
50
 on J774 macrophages by the MTT assay; SI=IC
50
/MIC.
Figure 3. Effect of free and liposomal BBAP, and rifampicin (RFP) against 
M. tuberculosis H37Rv growth inside J774 macrophages. Macrophages were 
lysed to determine CFU of M. tuberculosis H37Rv following the procedure 
described in the text. Bars represent mean ± SEM of three independent ex-
periments. RPMI 1640 and rifampicin (RFP - 12 mM) were used as controls
de Souza et al.874 Quim. Nova
aspects could be provided by adding lipids of low phase transition 
temperature and cationic lipids to the formulation. 
As expected rifampicin at 12 mM was effective and eliminated 
more than 2 log in the bacillus number.
CONCLUSION
The encapsulation of BBAP in liposomes and the in vitro evalu-
ations of the cytotoxicity, extracellular and intracellular antimyco-
bacterial activities were characterized. The liposomal formulation 
is promising for the treatment of M. tuberculosis infections due to 
its reduced citotoxicity and higher selectivity index, compared to 
free BBAP. 
ACKNOWLEDGMENTS
The authors thank Conselho Nacional de Desenvolvimento 
Científico e Tecnológico (CNPq) for financial support to A. O. de 
Souza (Profix-CNPq) and Brazilian Tuberculosis Research Network 
(REDE TB - Instituto do Milênio).
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ANTIMYCOBACTERIAL AND CYTOTOXICITY ACTIVITIES OF FREE AND LIPOSOME-ENCAPSULATED 3-(4 '-BROMO[1,1 '-BIPHENYL-4-YL)-3-(4-BROMO-PHENYL)-N,N-DIMETHYL-2-PROPEN-1-AMINE

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ANTIMYCOBACTERIAL AND CYTOTOXICITY ACTIVITIES OF FREE AND LIPOSOME-ENCAPSULATED 3-(4 '-BROMO[1,1 '-BIPHENYL-4-YL)-3-(4-BROMO-PHENYL)-N,N-DIMETHYL-2-PROPEN-1-AMINE

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