Modulation of bisquaternary ammonium agents effect on model biomembranes by complex formation with an organic anion

Aim. To study membranotropic activity modulation of bisquaternary ammonium compounds (BQAC) decamethoxinum and aethonium determined by their interaction with dihydroxybenzoic acid (DHB) organic anion. Methods. Differential scanning calorimetry, mass spectrometry. Results. Doping phospholipid membranes with individual BQAC or DHB leads to a considerable decrease in the membrane melting temperature. At the same time, when BQAC and DHB are introduced together, a certain increase in the membrane melting temperature is observed, implying non-additivity of their action and incorporation of their complexes into the membranes. Conclusions. DHB decreases the efficiency of BQAC destabilizing action on the membranes, i. e. DHB is a modulator of their membranotropic activity. A possible molecular mechanism of the modulation consists in the compensation of charges of the BQAC dications by organic DHB anions on the complex formation; parameters of the complex interaction with the membrane structures differ from those of individual ionic compounds.


Introduction.
It is known that the efficiency of pharmacological preparations is determined by their main active compound and the modulation of activity by other substances present in the pharmaceutical form.The present investigation was aimed at modulating influence of antimicrobial preparations based on bisquarternary ammonium compounds (BQAC) by their binding to organic anions.One of the main mechanisms of activity of membranotropic BQAC, which are the cation surface active compounds (surfactants), is believed to be their binding to the cytoplasmic membranes of microorganisms, resulting in their malfunction [1].In the series of our previous publications [2][3][4], dedicated to systematic study on the molecular mechanisms of activity of the BQAC-based antimicrobial preparations, decamethoxinum and aethonium (Fig. 1, a, b), we showed that these preparations interacted with model phospholipid membranes and formed stable non-covalent complexes with phospholipids.The further mass-spectrometry experiments revealed that an anion of organic 2, 5-dihydroxybenzoic acid (DHB) is able to replace a counter-ion of chloride, thus forming a stable complex with decamethoxinum dication.This fact allowed us to suppose a possibility of formation of such complexes as a result of joint action of these two substances on phospholipid membranes, as well as to assume an effect of binding BQAC dications to DHB organic anions on their membranotropic activity.To check this assumption, a model system was selected as water dispersions of dipalmitoylphosphatidylcholine (DPPC) ?multilayer lamellar structures consisting of phospholipid bilayers separated by water layers, which imitate phospholipid membranes.BQAC decamethoxinum and aethonium were introduced separately or in combination with DHB.It should be noted that DHB was chosen as a compound modelling the acid and aromatic groups present in a number of amino acids [5].The main method of investigation was differential scanning calorimetry (DSC) allowing determining changes in the calorimetric parameters of phase transitions in the model membranes when they are affected by membranotropic agents (MTA).
Materials and Methods.The DSC studies were carried out using the Mettler TA 3000 thermoanalytic system (Switzerland).Samples of 15-25 mg were placed into aluminium crucibles with half-closed lids.Programmed scheme of temperature scanning consisted of consecutive cycles of heating and cooling with the rate of 2 K/min.
Crystalline DPPC and BQAC were mixed and water dispersions of this composition with mass ratio DPPC: water of 50:50 [were prepared] using the standard method, described in [6].While introducing BQAC and DHB simultaneously, we chose the concentrations to ensure the ratio between quaternary groups and DHB as 2:1, 1:1, and 1:2, corresponding to the molar ratios of 1:1, 1:2, and 1:4.The investigation of all the systems was performed at pH 7, the total amount of MTA, introduced into phospholipid matrix, was 5% (mass).
The investigation by matrix-assisted laser desorption/ionization (MALDI) mass-spectrometry was performed using a time-of-flight mass spectrometer MALDI-TOF AutoFlex (Bruker Daltonics, Germany).In these experiments, one of the components of investigated systems, namely, DHB, functioned also as a UV-absorbing matrix for MALDI.
Preparations of DPPC (5.45% humidity) of Alexis Biochemicals (Switzerland) and 2, 5-dihydroxybenzoic acid of Sigma (Germany) were Results and Discussion.The formation of stable non-covalent complex of BQAC decamethoxinum dication with DHB anion was registered in the conditions of MALDI mass-spectrometry experiment using solid samples obtained from dried water solution of decamethoxinum and DHB (Fig. 2).Along with the dication associate with one chloride anion Cat +2 •Cl -, m/z 657, characteristic ion in mass-spectra of pure decamethoxinum [2], the dication associate with DHB anion Cat 2+ •(DHB-H) -, m/z 775 is formed.At equimolar ratio of components in initial solution, the intensity of associate Cat 2+ •(DHB-H) in MALDI mass-spectra is considerably higher than that of associate Cat 2+ •Cl -, which indicates the competition between anions and predominate binding of organic anion to dication (DHB-H) -.
DSC method was used to determine the calorimetric parameters of phase transitions in model membranes in the range of physiological temperatures, namely, transition from gel phase into liquid crystalline state.The study was performed on water dispersions of DPPC alone, DPPC with addition of BQAC of decamethoxinum, aethonium, and DHB, and mixtures of BQAC: and DHB .Table 1 presents the data of DSC: temperature (T m ) and enthalpy (DH m ) of the main phase transition, determined in the regimes of heating and cooling, as well as hysteresis (DT).
To solve the question about stoichiometry of possible complexes, we analyzed the initial data using the method of quasibinary systems [7,8].In this method, the phospholipid medium is considered as a matrix, in which two dissolved components interact.In the absence of interaction, any thermodynamic feature of the system, expressed in corresponding units, is additive with respect to the relative concentrations of components and vice versa, specific interaction results in a deviation from the additivity.The analysis of the data obtained gives grounds for the following conclusions.The change in thermodynamic parameters of model membranes at introducing individual MTA into the water dispersion of DPPC proves the interaction of all investigated MTA with phospholipid bilayers.The introduction of MTA considerably decreases the temperature of the main phase transition, i.e. it results in disorder of phospholipid bilayers.As quaternary ammonium compounds and DHB acid dissociate in water solution, it is possible to suppose that these MTA are built into the membrane in the form of ions: dications of decamethoxinum and aethonium Cat 2+ and anion (DHB-H) -.
Simultaneous introduction of BQAC and DHB into DPPC dispersion also changes its thermodynamic parameters, but there is no observed additivity of BQAC and DHB effects related to the concentration of introduced components.A considerable deviation from additivity in systems (DPPC + BQAC + DHB) is an evidence of specific interaction between the additives introduced into phospholipid matrix.The complexes of BQAC and DHB are likely to be formed similarly to the complexes of Cat 2+ • (DHB-H) -registered in the mass-spectrometry experiment ([see] Fig. 2) due to Coulomb interaction between corresponding cations and anions.
Plotting of quasibinary diagrams allowed determining the stoichiometry of complexes BQAC: DHB 1:2, which is notable for the maximal deviation from additivity ([see] Fig. 3).In these complexes one DHB anion corresponds to each ammonium group in the composition of BQAC.
The degree of effect of simultaneously introduced BQAC and DHB on the system calorimetric parameters significantly depends on the ratio between components.If molar ratio BQAC to DHB is 1:1, T m values for the three-component system (DPPC + BQAC + DHB) do not differ much from those for the binary system (DPPC + BQAC).If BQAC ratio to DHB is 1:2 and 1:4, there are qualitative changes: a shift of the main transition temperature changes the sign with respect to T m of non-doped DPPC; however, an increase in T m absolute value is insignificant.This effect may be considered as elimination of disordering influence of BQAC on membranes in the presence of definite DHB concentrations.Therefore, it is possible to regard DHB as a modulator of BQAC activity.On the other hand, complex formation with BQAC decreases the DHB activity.
Neutralization of the charge of BQAC dication while forming its complex with DHB seems to be the most probable molecular mechanism of the abovementioned effect of modulation of the BQAC activity.This assumption is based on the fact that the transition from decrease to increase in melting temperature T m in the system (DPPC + BQAC + DHB) occurs at the DHB content that either equals or exceeds the amount of positively charged ammonium groups (two groups per one BQAC molecule), which is sufficient for their neutralization at the complex formation.If molecular ratio BQAC to DHB is 1:1, one of the dication positive centres on average is not "compensated" by DHB anion and the calorimetric parameters still change in accordance with the effect of ion MTA on the membrane.
It should be noted that previously we described the dependence of membranotropic activity of the BQAC-based surfactants on their structure, i.e. the nature of hydrophobic "tail" and a distance between positively charged atoms of quaternary nitrogen, on the example of decamethoxinum and aethonium [2,3].The structure of aethonium dication, including a polar "head" of two closely located ammonium groups and two hydrophobic carbohydrate chains, is generally similar to the structure of DPPC molecules, which provides the building of aethonium dication into the membrane due to the substitution of DPPC molecule.A more complicated structure of hydrophobic "tails" of decamethoxinum, containing methyl residues, as well as rather large distance between positively charged ammonium groups result in greater destabilizing effect of decamethoxinum on the membranes in comparison to aethonium.
As for enthalpy val ues of the main phase tran si tion in model mem branes de ter mined in the re gime of sample heat ing, they dem on strate a gen eral ten dency to de creas ing when MTA is in tro duced into the sys tem.At first sight, this fact shows that dis or der ing of membranes re quires much less en ergy in the pres ence of destabilizing agents in their com po si tion.The processes oc cur ring dur ing cool ing of the sys tem are re -lated to the pe cu liar i ties of MTA in clu sion into the liquid crys tal struc ture of mem branes, which is a sub ject of fur ther studies beyond the framework of this review.
Conclusions.The possibility of modulation of the activity of MTA, based on BQAC salts, was considered for the case of their simultaneous with organic acid DHB introduction into the model phospholipid membranes.
The data of DSC research demonstrated the absence of additivity of calorimetric parameters, which is a characteristic of the model membranes containing individual MTA, in the case of simultaneous introduction of the mixture of DHB with BQAC decamethoxinum and aethonium into the water dispersion of hydrated DPPC.This experimental fact evidences the insertion of MTA complexes instead of individual MTA into the membranes.
The data of mass-spectrometry showed the possibility of formation of stable complexes of cations of quaternary ammonium compounds with DHB anion.
The analysis of calorimetric parameters of the systems investigated revealed that the insertion of dissociated in solution individual components in ionic state into the membranes, results in a decrease in the temperature of main transition and corresponding disordering of the membrane structure.The similar effect of decrease in the membrane melting temperature is observed if components of the MTA mixtures added are in the ratio sustaining a charged state of their complexes.In case of the formation of neutral complexes of BQAC dications with DHB anion, an increase in the temperature of main transition is revealed along with ordering the membrane structure.This effect may be considered as BQAC deactivation, i.e. modulation of the BQAC activity by organic acid.Neutralization of ionic MTA due to the formation of cation-anion complexes was suggested as a molecular mechanism of such modulation.
The possibility of weakening membranotropic activity of the BQAC-based antimicrobial agents, revealed at their introduction together with organic acid, should be taken into account while developing polycomponent drugs of antimicrobial activity.The approach proposed in this work may be used on a broader scale for testing the combined activity of MTA of various nature.