BACKGROUND
tuberculosis  is a curable infectious disease affecting mostly the lungs which is caused by mycobacterium tuberculosis.
in  <dig>  an increase of  <dig>  million novel tb cases was reported, since the slow reduction in incidence rates per capita continues to be surpassed by increasing world population  <cit> .
control of tb remains a serious challenge for public health due to the emergence of mutated strains presenting resistance to at least the two major anti-tuberculosis drugs, isoniazid and rifampicin  <cit> .
treatment of multidrug resistant tb requires prolonged and expensive chemotherapy using highly toxic, second-line drugs  <cit> .
eventually, resistance to these second line drugs develops and the disease becomes untreatable  <cit> .
clearly, there is an urgent need to improve treatment by either enhancing the application of existing agents or introducing new drugs.
thus, it is important to develop not only new drug candidates and drug targets but also delivery systems.
cationic bilayers based on dioctadecyldimethylammonium bromide  have been useful as carriers in drug delivery  <cit> , immunoadjuvants for vaccines  <cit>  and as antimicrobial agents due to their quaternary ammonium moiety  <cit> .
in this work, the ability of such bilayers to incorporate and deliver antitubercular drugs such as rifampicin  and isoniazid  in vitro was investigated and assemblies tested against two mycobacterium species.
the results evidenced the micobactericidal activity in vitro of dodab alone or in combination with rifampicin.
methods
materials
dodab  <dig> % pure, dipalmitoylphosphatidylcholine  and asolecitin  were obtained from sigma  and used as such without further purification.
rif and iso were obtained from sigma-aldrich, usa.
anhydrous d-glucose was from merck  and water was milli-q quality.
chemical structures of dodab, rif and iso were shown in figure  <dig>
preparation of dodab dispersions
lipids were dispersed in water or  <dig>  m d- glucose solution.
dodab bilayer fragments  were prepared by sonication with tip above the gel-to-liquid-crystalline bilayer phase transition temperature  <cit> .
this procedure dispersed the amphiphile powder in water using a high-energy input, which not only produced bilayer vesicles but also disrupted these vesicles, thereby generating open bf.
dodab was alternatively dispersed by vortexing at 60°c to obtain lv.
dodab analytical concentration was determined by bromide microtitration  <cit> .
determination of size distribution, zeta-average diameter and zeta-potential for dispersions
the size distributions for dodab, rif and dodab/rif were determined by means of a zeta-potential analyser  equipped with a  <dig> nm laser and dynamic light scattering at 90° for particle sizing  <cit> .
the zeta-average diameter  referred to in this work should be understood as the mean hydrodynamic diameter.
zeta-potentials  were determined from the electrophoretic mobility μ and smoluchowski's equation, ζ = μη/ε, where η and ε are medium viscosity and dielectric constant, respectively.
all dz and ζ were obtained at 25°c,  <dig> h after mixing.
determination of rif or iso optical spectra in different media
rif or iso optical spectra were determined using a hitachi  <dig> spectrophotometer in the double-beam mode against a blank of lipid dispersions and solvent .
rif and iso stock solutions were prepared at  <dig> mg/ml in dimethylsulfoxide  and water, respectively.
rif spectra were obtained by adding rif stock solution to water, dmso, dodab bf or dodab lv.
iso spectra were obtained by adding the stock solution to water or dodab lv dispersions.
all optical spectra were obtained at 25° c just after mixing.
wavelengths of maximal absorption for each drug in different media plus drug molar absorptivity were determined from analysis of optical spectra.
determination of entrapment efficiency for rif in dodab dispersions
dodab lv or bf were obtained in a  <dig>  mm rif and  <dig>  m d-glucose solution.
aliquots of  <dig> ml of the rif solution  and dodab bf or lv  prepared in this rif solution were dialyzed against   <dig> ml of  <dig>  m d-glucose solution for  <dig> h. thereafter, aliquots of rif solution, dodab lv/rif or dodab bf/rif before and after dialysis were added of ethanol  in order to solubilize the dodab bilayers.rif concentration was determined spectrophotometrically from absorbance at  <dig> nm.
drug incorporation percentile  was given by equation :
where a <dig> and a <dig> are absorbances of rif/dodab lv or rif/dodab bf before and after dialysis, respectively; a 1c and a 2c are absorbances of rif solution before and after dialysis.
since % inc depends on dodab concentration, % inc was normalized to dodab concentration yielding entrapment efficiency  from equation :
ent in l/mol depends only on physical characteristics of the liposomal dispersion such as size and lamelarity but does not depend on lipid concentration.
determination of iso entrapment efficiency in dodab, asolecithin  or dodab/dppc 1: <dig> dispersions
dodab lv were prepared in a  <dig>  mm iso solution at  <dig> mm dodab.
large dodab/dppc vesicles were prepared by spreading and evaporating a chloroform solution of lipids on the bottom of a glass tube  <cit> .
briefly, films of dodab/dppc at  <dig> mm total lipid were prepared from stock chloroform solutions of the lipids by evaporating the solvent under n <dig> flux.
in order to remove the residual solvent, the films remained under vacuum overnight at room temperature.
vesicles were prepared by hydrating the films with  <dig>  mm iso water solution at 60° c and vortexing dispersions until they became homogeneous.
asolecitin lipids at  <dig> mm total lipid were dispersed by vortexing at room temperature to obtain multilamellar vesicles  in the same  <dig>  mm iso solution  <cit> .
the dialysis procedure employed to determine iso entrapment in the three different lipid dispersions was similar to the one described for rif in the previous item.
dialysis was carried out against water for  <dig> h. after dialysis, the iso contents inside each bag were evaluated at  <dig> nm after liposomes lysis either by adding ethanol  or triton x- <dig> .
% inc and ent were calculated as described in the previous section.
microrganisms and culture conditions
m. smegmatis  and m. tuberculosis  were obtained from mycobacterial laboratory culture collection of the universidade de são paulo.
m. smegmatis was cultured freshly on muller hinton agar  plates for  <dig> h at 30°c.
thereafter, one or two colonies were transferred to  <dig> ml middlebrook 7h <dig> broth and incubated in a shaker .
m. tuberculosis was cultured freshly on lowenstein- jensen  agar for  <dig> days at 37° c. thereafter, one or two colonies were transferred to  <dig> ml of middlebrook 7h <dig> culture medium supplemented with 10% oleic acid albumin dextrose complex  and  <dig> % glycerin and incubated at 37° c for 7- <dig> days to reach exponential growth.
cultures were then pelleted  and washed in a  <dig>  m d-glucose solution as an osmoprotectant  <cit> .
the washing procedure was repeated twice before adjusting turbidity to  <dig> of the mcfarland scale.
viability assays for m. smegmatis and m. tuberculosis in the presence of dodab dispersions
cell viability was determined after incubation of cells  with dodab dispersions over a range of concentrations  for  <dig> h in the case of m. smegmatis or  <dig> days for m. tuberculosis.
the positive control was performed at dodab concentration equal to zero.
after interaction between cells and dodab, serial dilution  of each tube using a solution of d-glucose  <dig>  m yielded  <dig> cfu/ml from which  <dig> μl were taken and plated in duplicate on mha plates, before incubation  for m. smegmatis.
plating for m. tuberculosis was made on middlebrook 7h <dig> agar supplemented with oadc and glycerol plates incubated in 5% co <dig>  10% humidity and 37°c for  <dig> -  <dig> weeks.
cfu counting was then performed and plotted as % cell viability as a function of dodab concentration.
determination of minimal bactericidal concentration  for rif or dodab against mycobacterium smegmatis and m. tuberculosis in  <dig>  d-glucose solution
the medium used for diluting cells was d-glucose  <dig>  m. usually, evaluation of combined action of antimicrobial drugs is performed from chequerboard experiments and presented as the standard fractional inhibitory concentration index .
unfortunately, the chequerboard experiment in the culture medium is not feasible for cationic dodab, which cannot remain in dispersion at usual ionic strengths of the culture medium.
dodab dispersions flocculated at very low ionic strength  and interacted with acidic components of the culture medium  <cit> .
this explained the use of the isotonic d-glucose solution for mic determination.
briefly, for m. smegmatis twofold serial dilutions of rif, and dodab bf were prepared at final volume of  <dig> ml per tube.
the final concentration of the antimicrobial agents ranged from  <dig> to  <dig> μg/ml for rif and from  <dig> to  <dig> μg/ml for dodab bf.
each tube was inoculated with bacterial cell suspension to a final concentration of  <dig> cells per tube and the plates were incubated at 30°c for  <dig> h. an aliquot  <dig>  ml was taken from each tube, diluted 1: <dig> and 1: <dig>  and cell viability was determined by plating  <dig>  ml of each dilution on mha plates in duplicate and incubating for  <dig> h at 30°c.
the mic endpoint was the lowest drug concentration that killed ≥  <dig> % of the inoculum.
for m. tuberculosis drugs were diluted in microplates in a solution of d-glucose in concentrations ranging from 1- <dig>  or 512- <dig> μg/ml for rif and dodab, respectively, in a volume of  <dig> μl per well.
after drugs dilution, each well was inoculated with  <dig> μl of the standardized bacterial suspension .
the plates were incubated at 37°c, 5% co <dig> and 10% humidity for  <dig> days.
an aliquot was removed from each well diluted 1: <dig> and 1: <dig> dilution and  <dig> μl were plated on 7h <dig> ágar.
the plates were then incubated at 37°c, 5% co <dig>  10% humidity, 3- <dig> weeks.
mbc was determined as the lowest drug concentration that killed ≥ 90% of the bacterial inoculum.
determination of fractional inhibitory concentration index  for combinations of dodab and rif dispersions against m. smegmatis and m. tuberculosis
twofold serial dilutions of rif solutions or dodab dispersions were distributed in tubes, in order to obtain final concentrations ranging from  <dig> to  <dig> μg/ml rif or dodab after interaction with cells.
thereafter, rif diluted solutions were added of dodab bf dispersion to yield a final dodab concentration of  <dig> μg/ml after interaction with cells.
similarly the dodab bf diluted dispersions were added of rif solution to yield a final rif concentration of  <dig> μg/ml after interaction with cells.
drug-lipid mixtures were incubated together for  <dig> h before adding the cell suspension.
each tube was inoculated with m. smegmatis cell suspension to yield  <dig> cells/tube and incubated at 30°c for  <dig> h. thereafter, an aliquot of  <dig>  ml was diluted 1: <dig> and 1: <dig>  and cell viability was determined by plating and incubation for  <dig> h, 30°c.
the lowest drug concentration that killed ≥  <dig> % of the inoculum was used to determine the fractional inhibitory concentration , defined as the ratio of the mbc of a drug used in combination with the mbc of the drug tested alone.
the fic index  was calculated as the sum of the fics for the most equally effective concentration of drugs.
according to odds, fici ≤  <dig>  corresponds to drug synergism, whereas an index >  <dig>  represents antagonism  <cit> .
each experiment was carried out in duplicate and no variation was obtained among them.
the microplate was used for the test of synergism between dodab/bf and rif against m. tuberculosis.
a serial dilution of rif was performed to yield final concentrations over the 1- <dig>  μg/ml range in  <dig> μl per well.
each well containing rif dilutions was added of  <dig> μl of a  <dig> μg/ml dodab dispersion.
furthermore, serial dilutions of dodab bf  in  <dig> μl per well were added of rif at  <dig>  μg/ml .
rif and dodab bf were mixed and allowed to interact for  <dig> hour before addition of bacterial cells.
in each tube  <dig> μl of the standardized bacterial suspension  was added.
the microplates were then incubated .
an aliquot of each well was diluted 1: <dig> and 1: <dig>   <dig> μl of the final dilution was plated on 7h <dig> agar and incubated .
mbc was determined as the lowest drug concentration that killed ≥ 90% of the bacterial inoculum.
RESULTS
physico-chemical characterization of drug/dodab assemblies
the solubility limit for rif in water is  <dig>  mg/ml or  <dig>  mm.
above this limit the drug aggregates in water solution.
optical spectra for the drug in different media such as water, dodab bf or dodab lv were similar to those for rif in its best organic solvent, dmso .
absorption maxima for the drug in water occurred at smaller wavelengths than those for rif in its best organic solvent dmso or in the dodab dispersions .
in the presence of dodab bilayers, the drug displayed absorption maxima at wavelengths practically equal to the one in its best organic solvent dmso.
the molar absorptivity  of the drug in dmso was  <dig>  m-1cm- <dig>  similar values occurred in dodab dispersions .
absorption maxima and molar absorptivities  for rif in different media.
optical spectra for iso in water were not affected by dodab lv or bf .
its single absorption peak around  <dig> nm in water remained unchanged in the presence of dodab bilayers.
the molar absorptivities in water or dodab lv were also similar,  <dig>  and  <dig>  m- <dig> cm- <dig>  respectively, again suggesting that iso was not incorporated in the dodab bilayer.
rifampicin incorporation in dodab dispersions from rif absorbance at  <dig> nm  before and after dialysis.
dialysis experiments designed to determine iso incorporation in dodab dispersions revealed the leaky character of the dodab lv towards iso.
after dialysis overnight iso incorporation fell to zero .
other liposomes prepared from asolecithin or dodab/dppc 1: <dig> also yielded very low incorporation percentiles: 0% iso incorporation in dodab/dppc large vesicles or  <dig> % incorporation in asolecithin multilamellar vesicles after 12- <dig> h dialysis.
isoniazid incorporation in dodab, dodab/dppc 1: <dig> or asolecithin  liposomal dispersions from iso absorbance at  <dig> nm  before and after dialysis.
final lipid concentration was  <dig>  mm.
micobactericidal activity of dodab alone or combined with rifampicin
the effect of dodab concentration for dodab dispersed as dodab bf or lv on m. smegmatis at  <dig>  ×  <dig> cfu/ml after  <dig> h interaction time is shown in figure  <dig>  micromolar dodab concentrations were effective against m. smegmatis.
dodab dispersed as lv or bf affected the cells similarly.
at  <dig>  mm dodab, cell viability was 50%.
after  <dig> h,  <dig>  mm dodab completely killed m. tuberculosis .
table <dig> showed mbc for rif alone or in combination with dodab.
the formulation dodab/rif yielded Σfic =  <dig>  evidencing synergism between dodab and rif action against m. smegmatis.
against m. tuberculosis, Σfic =  <dig> suggested independent action for dodab and rif in the combination.
micobactericidal activity of rif alone or in combination with dodab.
discussion
rif was partially anionic  at ph  <dig>  and electrostatically interacted with the dodab bilayer.
the insertion of rif in the dodab bilayer was supported by zeta-potential decrease and could be understood from the favourable electrostatic attraction between the partially anionic rif and the cationic bilayer which subsequently drove the hydrophobic effect.
this was consistent with literature for other lipid bilayers loaded with rif and ofloxacin  <cit>  or for rif interactions with neutral dimyristoil-l-α-phosphatidylcholine  liposomes  <cit> .
together eletrostatics and hydrophobic effect solubilized rif in the dodab bilayer yielding the very large entrapment efficiency determined both for dodab bf or dodab lv .
in fact, incorporation of rif was reported in various liposomal formulations such as dispersions of phosphatidylcholine ; dipalmitoyloglycero-pc , distearoylglycero-pc  with or without cholesterol  <cit> .
rif incorporation was evaluated in dppc, dspc or pc liposomes and reported at highest in dppc and dspc bilayers  <cit> .
similarly to dppc and dspc, dodab also bears saturated hydrocarbon chains, yielding optimal and high rif incorporation .
on the other hand, dialysis experiments showed that iso permeated freely through the dodab lv bilayer.
this high permeability was also observed for vesicles composed of other lipids such as dodab/dppc 1: <dig> or asolecithin  multilamellar vesicles, though the multilamellar assemblies yielded percentiles of incorporation slightly above zero .
iso solubility in water and permeability through the dodab bilayer hampered iso incorporation in dodab bf or dodab lv.
sustained release of iso from dppc multilamellar liposomes was observed over  <dig> h  <cit> .
this reconfirmed the high permeability of this drug across phospholipid bilayers.
dodab lv did not retain iso inside their aqueous compartment after the12- <dig> h dialysis procedure.
the slightly smaller rif entrapment efficiency for dodab bf in comparison to the one determined for dodab lv  might have been due to the extra amount of drug inside lv aqueous compartment which was not available for the open dodab bf.
reproducibility of entrapment experiments was checked by ultracentrifugation and results were shown to be very reproducible.
other studies have shown that the covalent combination of drugs with hydrophobic moieties restored or increased drug activity.
neomycin b had low activity against strains of methicillin resistant s. aureus  and p. aeruginosa, whereas kanamycin a had low activity against mrsa and methicillin resistant s. epidermidis  and p. aeruginosa.
recently, neomycin- or kanamycin a -cationic lipid conjugates were synthesized that displayed high activity against both mrsa and mrse, respectively  <cit> .
the conjugation of lipids increased the activity of both aminoglycosides against resistant strains: mic for neomycin b against mrsa was  <dig> μg/ml.
its conjugate bound to lipid had a mic of  <dig> μg/ml  <cit> .
our results showed that the noncovalent combination of rif with the cationic lipid dodab improved rif antibacterial activity, reducing mbc from  <dig> to  <dig> μg/ml .
this work shows for the first time that drug-dodab self-assembly results in enhanced micobactericidal activity at lower doses.
these novel formulations may find useful applications in clinics.
the use of low doses will possibly reduce drug toxicity in vivo.
CONCLUSIONS
electrostatic attraction and hydrophobic effect assembled rif and dodab bilayers yielding formulations with micobactericidal activity at reduced dodab and rif doses.
dodab by itself was also active against mycobacterium.
the present study in vitro is a necessary step before in vivo tests can be performed.
list of abbreviations used
rif: rifampicin; iso: isoniazid; dodab: dioctadecyldimethylammonium bromide; bf: bilayer fragments; lv: large unilamellar vesicles; dz: zeta-average diameter; ζ: zeta-potential; mbc: minimal bactericidal concentration; mic: minimal inhibitory concentration; tb: tuberculosis; dppc: dipalmitoylphosphatidylcholine; aso: asolecithin; dmso: dimethylsulfoxide; % inc: drug incorporation percentile; ent: entrapment efficiency; mha: mueller-hinton agar; fici: fractional inhibitory concentration index; oadc: oleic acid-albumin-dextrose complex; cfu: colony forming unit.
competing interests
the authors declare that they have no competing interests.
authors' contributions
lb collected the data and helped to write the manuscript.
emm participated in the conception of the study and provided the mycobacterium strains.
amc-r designed the study, interpreted the data and wrote the manuscript.
all authors read and approved the final manuscript.

 tuberculosis is a curable infectious disease affecting mostly the lungs which is caused by mycobacterium tuberculosis. in an increase of million novel tb cases was reported, since the slow reduction in incidence rates per capita continues to be surpassed by increasing world population. control of tb remains a serious challenge for public health due to the emergence of mutated strains presenting resistance to at least the two major anti-tuberculosis drugs, isoniazid and rifampicin. treatment of multidrug resistant tb requires prolonged and expensive chemotherapy using highly toxic, second-line drugs. eventually, resistance to these second line drugs develops and the disease becomes untreatable. clearly, there is an urgent need to improve treatment by either enhancing the application of existing agents or introducing new drugs. thus, it is important to develop not only new drug candidates and drug targets but also delivery systems. cationic bilayers based on dioctadecyldimethylammonium bromide have been useful as carriers in drug delivery, immunoadjuvants for vaccines and as antimicrobial agents due to their quaternary ammonium moiety. in this work, the ability of such bilayers to incorporate and deliver antitubercular drugs such as rifampicin and isoniazid in vitro was investigated and assemblies tested against two mycobacterium species. the results evidenced the micobactericidal activity in vitro of dodab alone or in combination with rifampicin. methods materials dodab % pure, dipalmitoylphosphatidylcholine and asolecitin were obtained from sigma and used as such without further purification. rif and iso were obtained from sigma-aldrich, usa. anhydrous d-glucose was from merck and water was milli-q quality. chemical structures of dodab, rif and iso were shown in figure preparation of dodab dispersions lipids were dispersed in water or m d- glucose solution. dodab bilayer fragments were prepared by sonication with tip above the gel-to-liquid-crystalline bilayer phase transition temperature. this procedure dispersed the amphiphile powder in water using a high-energy input, which not only produced bilayer vesicles but also disrupted these vesicles, thereby generating open bf. dodab was alternatively dispersed by vortexing at 60°c to obtain lv. dodab analytical concentration was determined by bromide microtitration. determination of size distribution, zeta-average diameter and zeta-potential for dispersions the size distributions for dodab, rif and dodab/rif were determined by means of a zeta-potential analyser equipped with a nm laser and dynamic light scattering at 90° for particle sizing. the zeta-average diameter referred to in this work should be understood as the mean hydrodynamic diameter. zeta-potentials were determined from the electrophoretic mobility μ and smoluchowski's equation, ζ = μη/ε, where η and ε are medium viscosity and dielectric constant, respectively. all dz and ζ were obtained at 25°c, h after mixing. determination of rif or iso optical spectra in different media rif or iso optical spectra were determined using a hitachi spectrophotometer in the double-beam mode against a blank of lipid dispersions and solvent. rif and iso stock solutions were prepared at mg/ml in dimethylsulfoxide and water, respectively. rif spectra were obtained by adding rif stock solution to water, dmso, dodab bf or dodab lv. iso spectra were obtained by adding the stock solution to water or dodab lv dispersions. all optical spectra were obtained at 25° c just after mixing. wavelengths of maximal absorption for each drug in different media plus drug molar absorptivity were determined from analysis of optical spectra. determination of entrapment efficiency for rif in dodab dispersions dodab lv or bf were obtained in a mm rif and m d-glucose solution. aliquots of ml of the rif solution and dodab bf or lv prepared in this rif solution were dialyzed against ml of m d-glucose solution for h. thereafter, aliquots of rif solution, dodab lv/rif or dodab bf/rif before and after dialysis were added of ethanol in order to solubilize the dodab bilayers.rif concentration was determined spectrophotometrically from absorbance at nm. drug incorporation percentile was given by equation: where a and a are absorbances of rif/dodab lv or rif/dodab bf before and after dialysis, respectively; a 1c and a 2c are absorbances of rif solution before and after dialysis. since % inc depends on dodab concentration, % inc was normalized to dodab concentration yielding entrapment efficiency from equation: ent in l/mol depends only on physical characteristics of the liposomal dispersion such as size and lamelarity but does not depend on lipid concentration. determination of iso entrapment efficiency in dodab, asolecithin or dodab/dppc 1: dispersions dodab lv were prepared in a mm iso solution at mm dodab. large dodab/dppc vesicles were prepared by spreading and evaporating a chloroform solution of lipids on the bottom of a glass tube. briefly, films of dodab/dppc at mm total lipid were prepared from stock chloroform solutions of the lipids by evaporating the solvent under n flux. in order to remove the residual solvent, the films remained under vacuum overnight at room temperature. vesicles were prepared by hydrating the films with mm iso water solution at 60° c and vortexing dispersions until they became homogeneous. asolecitin lipids at mm total lipid were dispersed by vortexing at room temperature to obtain multilamellar vesicles in the same mm iso solution. the dialysis procedure employed to determine iso entrapment in the three different lipid dispersions was similar to the one described for rif in the previous item. dialysis was carried out against water for h. after dialysis, the iso contents inside each bag were evaluated at nm after liposomes lysis either by adding ethanol or triton x-. % inc and ent were calculated as described in the previous section. microrganisms and culture conditions m. smegmatis and m. tuberculosis were obtained from mycobacterial laboratory culture collection of the universidade de são paulo. m. smegmatis was cultured freshly on muller hinton agar plates for h at 30°c. thereafter, one or two colonies were transferred to ml middlebrook 7h broth and incubated in a shaker. m. tuberculosis was cultured freshly on lowenstein- jensen agar for days at 37° c. thereafter, one or two colonies were transferred to ml of middlebrook 7h culture medium supplemented with 10% oleic acid albumin dextrose complex and % glycerin and incubated at 37° c for 7- days to reach exponential growth. cultures were then pelleted and washed in a m d-glucose solution as an osmoprotectant. the washing procedure was repeated twice before adjusting turbidity to of the mcfarland scale. viability assays for m. smegmatis and m. tuberculosis in the presence of dodab dispersions cell viability was determined after incubation of cells with dodab dispersions over a range of concentrations for h in the case of m. smegmatis or days for m. tuberculosis. the positive control was performed at dodab concentration equal to zero. after interaction between cells and dodab, serial dilution of each tube using a solution of d-glucose m yielded cfu/ml from which μl were taken and plated in duplicate on mha plates, before incubation for m. smegmatis. plating for m. tuberculosis was made on middlebrook 7h agar supplemented with oadc and glycerol plates incubated in 5% co 10% humidity and 37°c for - weeks. cfu counting was then performed and plotted as % cell viability as a function of dodab concentration. determination of minimal bactericidal concentration for rif or dodab against mycobacterium smegmatis and m. tuberculosis in d-glucose solution the medium used for diluting cells was d-glucose m. usually, evaluation of combined action of antimicrobial drugs is performed from chequerboard experiments and presented as the standard fractional inhibitory concentration index. unfortunately, the chequerboard experiment in the culture medium is not feasible for cationic dodab, which cannot remain in dispersion at usual ionic strengths of the culture medium. dodab dispersions flocculated at very low ionic strength and interacted with acidic components of the culture medium. this explained the use of the isotonic d-glucose solution for mic determination. briefly, for m. smegmatis twofold serial dilutions of rif, and dodab bf were prepared at final volume of ml per tube. the final concentration of the antimicrobial agents ranged from to μg/ml for rif and from to μg/ml for dodab bf. each tube was inoculated with bacterial cell suspension to a final concentration of cells per tube and the plates were incubated at 30°c for h. an aliquot ml was taken from each tube, diluted 1: and 1: and cell viability was determined by plating ml of each dilution on mha plates in duplicate and incubating for h at 30°c. the mic endpoint was the lowest drug concentration that killed ≥ % of the inoculum. for m. tuberculosis drugs were diluted in microplates in a solution of d-glucose in concentrations ranging from 1- or 512- μg/ml for rif and dodab, respectively, in a volume of μl per well. after drugs dilution, each well was inoculated with μl of the standardized bacterial suspension. the plates were incubated at 37°c, 5% co and 10% humidity for days. an aliquot was removed from each well diluted 1: and 1: dilution and μl were plated on 7h ágar. the plates were then incubated at 37°c, 5% co 10% humidity, 3- weeks. mbc was determined as the lowest drug concentration that killed ≥ 90% of the bacterial inoculum. determination of fractional inhibitory concentration index for combinations of dodab and rif dispersions against m. smegmatis and m. tuberculosis twofold serial dilutions of rif solutions or dodab dispersions were distributed in tubes, in order to obtain final concentrations ranging from to μg/ml rif or dodab after interaction with cells. thereafter, rif diluted solutions were added of dodab bf dispersion to yield a final dodab concentration of μg/ml after interaction with cells. similarly the dodab bf diluted dispersions were added of rif solution to yield a final rif concentration of μg/ml after interaction with cells. drug-lipid mixtures were incubated together for h before adding the cell suspension. each tube was inoculated with m. smegmatis cell suspension to yield cells/tube and incubated at 30°c for h. thereafter, an aliquot of ml was diluted 1: and 1: and cell viability was determined by plating and incubation for h, 30°c. the lowest drug concentration that killed ≥ % of the inoculum was used to determine the fractional inhibitory concentration, defined as the ratio of the mbc of a drug used in combination with the mbc of the drug tested alone. the fic index was calculated as the sum of the fics for the most equally effective concentration of drugs. according to odds, fici ≤ corresponds to drug synergism, whereas an index > represents antagonism. each experiment was carried out in duplicate and no variation was obtained among them. the microplate was used for the test of synergism between dodab/bf and rif against m. tuberculosis. a serial dilution of rif was performed to yield final concentrations over the 1- μg/ml range in μl per well. each well containing rif dilutions was added of μl of a μg/ml dodab dispersion. furthermore, serial dilutions of dodab bf in μl per well were added of rif at μg/ml. rif and dodab bf were mixed and allowed to interact for hour before addition of bacterial cells. in each tube μl of the standardized bacterial suspension was added. the microplates were then incubated. an aliquot of each well was diluted 1: and 1: μl of the final dilution was plated on 7h agar and incubated. mbc was determined as the lowest drug concentration that killed ≥ 90% of the bacterial inoculum.  physico-chemical characterization of drug/dodab assemblies the solubility limit for rif in water is mg/ml or mm. above this limit the drug aggregates in water solution. optical spectra for the drug in different media such as water, dodab bf or dodab lv were similar to those for rif in its best organic solvent, dmso. absorption maxima for the drug in water occurred at smaller wavelengths than those for rif in its best organic solvent dmso or in the dodab dispersions. in the presence of dodab bilayers, the drug displayed absorption maxima at wavelengths practically equal to the one in its best organic solvent dmso. the molar absorptivity of the drug in dmso was m-1cm- similar values occurred in dodab dispersions. absorption maxima and molar absorptivities for rif in different media. optical spectra for iso in water were not affected by dodab lv or bf. its single absorption peak around nm in water remained unchanged in the presence of dodab bilayers. the molar absorptivities in water or dodab lv were also similar, and m- cm- respectively, again suggesting that iso was not incorporated in the dodab bilayer. rifampicin incorporation in dodab dispersions from rif absorbance at nm before and after dialysis. dialysis experiments designed to determine iso incorporation in dodab dispersions revealed the leaky character of the dodab lv towards iso. after dialysis overnight iso incorporation fell to zero. other liposomes prepared from asolecithin or dodab/dppc 1: also yielded very low incorporation percentiles: 0% iso incorporation in dodab/dppc large vesicles or % incorporation in asolecithin multilamellar vesicles after 12- h dialysis. isoniazid incorporation in dodab, dodab/dppc 1: or asolecithin liposomal dispersions from iso absorbance at nm before and after dialysis. final lipid concentration was mm. micobactericidal activity of dodab alone or combined with rifampicin the effect of dodab concentration for dodab dispersed as dodab bf or lv on m. smegmatis at × cfu/ml after h interaction time is shown in figure micromolar dodab concentrations were effective against m. smegmatis. dodab dispersed as lv or bf affected the cells similarly. at mm dodab, cell viability was 50%. after h, mm dodab completely killed m. tuberculosis. table showed mbc for rif alone or in combination with dodab. the formulation dodab/rif yielded Σfic = evidencing synergism between dodab and rif action against m. smegmatis. against m. tuberculosis, Σfic = suggested independent action for dodab and rif in the combination. micobactericidal activity of rif alone or in combination with dodab. discussion rif was partially anionic at ph and electrostatically interacted with the dodab bilayer. the insertion of rif in the dodab bilayer was supported by zeta-potential decrease and could be understood from the favourable electrostatic attraction between the partially anionic rif and the cationic bilayer which subsequently drove the hydrophobic effect. this was consistent with literature for other lipid bilayers loaded with rif and ofloxacin or for rif interactions with neutral dimyristoil-l-α-phosphatidylcholine liposomes. together eletrostatics and hydrophobic effect solubilized rif in the dodab bilayer yielding the very large entrapment efficiency determined both for dodab bf or dodab lv. in fact, incorporation of rif was reported in various liposomal formulations such as dispersions of phosphatidylcholine; dipalmitoyloglycero-pc, distearoylglycero-pc with or without cholesterol. rif incorporation was evaluated in dppc, dspc or pc liposomes and reported at highest in dppc and dspc bilayers. similarly to dppc and dspc, dodab also bears saturated hydrocarbon chains, yielding optimal and high rif incorporation. on the other hand, dialysis experiments showed that iso permeated freely through the dodab lv bilayer. this high permeability was also observed for vesicles composed of other lipids such as dodab/dppc 1: or asolecithin multilamellar vesicles, though the multilamellar assemblies yielded percentiles of incorporation slightly above zero. iso solubility in water and permeability through the dodab bilayer hampered iso incorporation in dodab bf or dodab lv. sustained release of iso from dppc multilamellar liposomes was observed over h. this reconfirmed the high permeability of this drug across phospholipid bilayers. dodab lv did not retain iso inside their aqueous compartment after the12- h dialysis procedure. the slightly smaller rif entrapment efficiency for dodab bf in comparison to the one determined for dodab lv might have been due to the extra amount of drug inside lv aqueous compartment which was not available for the open dodab bf. reproducibility of entrapment experiments was checked by ultracentrifugation and results were shown to be very reproducible. other studies have shown that the covalent combination of drugs with hydrophobic moieties restored or increased drug activity. neomycin b had low activity against strains of methicillin resistant s. aureus and p. aeruginosa, whereas kanamycin a had low activity against mrsa and methicillin resistant s. epidermidis and p. aeruginosa. recently, neomycin- or kanamycin a -cationic lipid conjugates were synthesized that displayed high activity against both mrsa and mrse, respectively. the conjugation of lipids increased the activity of both aminoglycosides against resistant strains: mic for neomycin b against mrsa was μg/ml. its conjugate bound to lipid had a mic of μg/ml. our results showed that the noncovalent combination of rif with the cationic lipid dodab improved rif antibacterial activity, reducing mbc from to μg/ml. this work shows for the first time that drug-dodab self-assembly results in enhanced micobactericidal activity at lower doses. these novel formulations may find useful applications in clinics. the use of low doses will possibly reduce drug toxicity in vivo.  electrostatic attraction and hydrophobic effect assembled rif and dodab bilayers yielding formulations with micobactericidal activity at reduced dodab and rif doses. dodab by itself was also active against mycobacterium. the present study in vitro is a necessary step before in vivo tests can be performed. list of abbreviations used rif: rifampicin; iso: isoniazid; dodab: dioctadecyldimethylammonium bromide; bf: bilayer fragments; lv: large unilamellar vesicles; dz: zeta-average diameter; ζ: zeta-potential; mbc: minimal bactericidal concentration; mic: minimal inhibitory concentration; tb: tuberculosis; dppc: dipalmitoylphosphatidylcholine; aso: asolecithin; dmso: dimethylsulfoxide; % inc: drug incorporation percentile; ent: entrapment efficiency; mha: mueller-hinton agar; fici: fractional inhibitory concentration index; oadc: oleic acid-albumin-dextrose complex; cfu: colony forming unit. competing interests the authors declare that they have no competing interests. authors' contributions lb collected the data and helped to write the manuscript. emm participated in the conception of the study and provided the mycobacterium strains. amc-r designed the study, interpreted the data and wrote the manuscript. all authors read and approved the final manuscript. 
dispersions were evaluated by dynamic light-scattering for zeta-average diameter and zeta-potential analysis; dialysis for determination of drug entrapment efficiency; plating and cfu counting for determination of cell viability of mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration and synergism index for dodab/drug combinations. combination dodab/rif yielded mbcs of 2/ and 4/ μg/ml against mycobacterium smegmatis or mycobacterium tuberculosis, respectively. rif was incorporated in dodab bilayers at high percentiles in contrast to the leaky behavior of iso. cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents. rif aggregates in water solution were solubilised by dodab bf. synergism indexes equal to or, indicated synergism against the former and independent action, against the latter species. rifampicin or isoniazid interacted with dodab bilayer fragments or large vesicles.
 cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents. methods rifampicin or isoniazid interacted with dodab bilayer fragments or large vesicles. dispersions were evaluated by dynamic light-scattering for zeta-average diameter and zeta-potential analysis; dialysis for determination of drug entrapment efficiency; plating and cfu counting for determination of cell viability of mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration and synergism index for dodab/drug combinations.  dodab alone killed micobacteria over a range of micromolar concentrations. rif aggregates in water solution were solubilised by dodab bf. rif was incorporated in dodab bilayers at high percentiles in contrast to the leaky behavior of iso. combination dodab/rif yielded mbcs of 2/ and 4/ μg/ml against mycobacterium smegmatis or mycobacterium tuberculosis, respectively. synergism indexes equal to or, indicated synergism against the former and independent action, against the latter species.  in vitro, dodab acted effectively both as micobactericidal agent and carrier for rifampicin. the novel assemblies at reduced doses may become valuable against tuberculosis. 