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20BIOLOGICAL SCIENCES
МОЖЛИВ1 МЕХАН1ЗМИ БЮЛОГ1ЧНО1 АКТИВНОСТ1 ПОЛ1ГЕКСАМЕТИЛЕНГУАН1ДИНУ
ЩОДО МЕМБРАН КЛ1ТИН
Лисиця А.В.
Ргвненський державний гуматтарний унгверситет
Мандигра Ю.М.
Досл1дна станщя епгзоотологИ 1нституту ветеринарной медицини НААН, м. Ргвне, Украша
Кривошия П.Ю.
Досл1дна станщя епгзоотологИ 1нституту ветеринарной медицини НААН, м. Ргвне, Украша
Нечипорук Б.Д.
Ргвненський державний гуматтарний унгверситет
POSSIBLE MECHANISMS OF THE BIOLOGICAL ACTIVITY OF POLYHEXAMETHYLENE
GUANIDINE ON CELL MEMBRANES
Lysytsya A.,
Rivne State University of Humanities Mandygra J.,
Research Station of Epizootology, Institute of Veterinary Medicine NAAS, Rivne, Ukraine
Kryvoshyya P.,
Research Station of Epizootology, Institute of Veterinary Medicine NAAS, Rivne, Ukraine
Nechyporuk B.
Rivne State University of Humanities
АНОТАЦ1Я
Мета дослвджень: запропонувати i обгрунтувати можлив1 мехашзми ди дезшфектанпв яш мютять по-л1мерш похвдт гуашдину, зокрема полп-ексаметиленгуашдин, на мембрани клгган. Методи: мас-спектро-метри, культивування культур клiтин, мшробюлоги, штучних бiшарових лiпiдних мембран. Наведено результата бiофiзичного i бiохiмiчного анатзу можливих механiзмiв взаемодп полiмерних похщних гуань дину з цитоплазматичними мембранами клгган прокарiот та еукарiот. Встановлено, що основною мiшенню для цих сполук е фосфолiпiди цитоплазматично! мембрани. Ввдмшносл в дИ препарату на кль тиннi мембрани залежать, перш за все, вщ !х лшвдного складу. Запропоновано можливi теоретичнi модел^ як1 пояснюють специфiку бiоцидного ефекту дезiнфектантiв, виготовлених на основi полгексаметиленгу-анiдину, наприклад Епiдез для ветеринарно! медицини. При ввдносно низьких концентрацгях препарату (10-4%) i дозованому чай експозицп вiдбуваеться змiна лiпiдного складу мембрани (через видалення час-тини фосфолшщв або полiгексаметиленгуанiдин-лiпiдних везикул). З цим пов'язаш неогенез лiпiдiв i ви-явленi нами ростостимулюючi та цитопротекторш ефекти. Бактерiостатичнi дози полтексаметиленгуаш-дину (10-3-10-2%) гальмують пролiферацiю клгган еукарiот (фiбробласти курячого ембрiону), бактерицидш дози (10-2-10-1%) викликають значнi порушення структури i функцiй цитоплазматичних мембран. Мембрани досить швидко пошкоджуються, найбiльш ймовiрним е килимовий мехашзм ди. В результатi лiзису клггана гине. Отриманi результати дозволяють краще зрозумiти механiзми високо! бактерицидно! актив-ностi полiгексаметиленгуанiдину щодо бiльшостi мiкроорганiзмiв i, в той же час, його ввдносну безпеч-нiсть для людини, тварин та вищих рослин. Цi данi сприятимуть розробцi як нових ефективних i безпечних засобiв для дезшфекци, так i стимуляторiв або засобiв захисту рослин.
ABSTRACT
To propose and substantiate possible mechanisms of action of disinfectants, containing polymer derivatives of guanidine, in particular, polyhexamethylene guanidine (PHMG), onto cell membranes. We used the following methods: mass spectrometry, cell culture cultivation, microbiology, artificial bilayer lipid membranes. The results of biophysical and biochemical analysis of possible mechanisms of interaction between polymeric guanidine derivatives and cytoplasmic membranes of prokaryotic and eukaryotic cells have been presented. It has been established that the main targets for these compounds are phospholipids of the cytoplasmic membrane. The differences in the action of the drug on different kind of the cell membranes depend, above all, on their lipid composition. Possible theoretical models have been proposed to explain the specificity of biocide effect of disinfectants, made on the basis of PHMG, for example it is Epidez for veterinary medicine. At relatively low concentrations (10-4%) of the drug and the metered exposure time (1-2 min) there is a change in the lipid composition of the membrane (via the removal of some phospholipids or PHMG-lipid vesicles), which is associated with neogenesis of the phospholipids and the growth-stimulating and cytoprotective effects from viruses, detected by us. Bacteriostatic or sublethal concentrations (10-3-10-2%) of PHMG inhibit the proliferation of eukaryotic cells (chicken embryo fi-
broblasts), and bactericidal doses (10-2-10-1<%) result in considerable perturbations which of the structure and functions of its cytoplasmic membranes. The membranes are rather rapidly damaged via, most probably, the carpet mechanism. It is the most common cause of cell death. The results obtained by us explain the high bactericidal activity of PHMG regarding most microorganisms and, at the same time, its relative safety for humans, animals and higher plants. These data will facilitate the development of new effective and safe means of disinfection, and stimulants or plant protection products.
Ключовi слова: полп-ексаметиленгуашцин, цезшфектанти, загибель клтгин, фосфолшци, бiофiзичнi модель
Keywords: polyhexamethylene guanidine, disinfectants, cell death, phospholipids, biophysical models.
Introduction.
Polyhexamethylene guanidine (PHMG) is known since the 1950s as a cationic biocide with a wide spectrum of action, impacting the cell membrane and its metabolism [1, 2]. Due to the specific structure of the molecule, containing hydrophobic hexamethylene areas (spacers) and positively charged guanidine groups, it has antibacterial, antiviral and antifungal activity [3, 4, 5]. It is proved that PHMG may be capable of impairing the stability of cytoplasmic membrane (CPM) of the cell via electrostatic interaction with acid phospholipids [2, 6]. At present, veterinary medicine uses many guan-idine-based preparations for disinfection, including Ep-idez [7, 8]. Its main active substance is polyhexamethylene guanidine hydrochloride, whose characteristics and advantages were frequently discussed already [9, 10, 11, 12, 13]. At the same time, biochemical and biophysical specificities of PHMG impact on CPM of pro-karyotic and eukaryotic cells are not fully understood [14, 15]. This issue is urgent for the elaboration of new disinfectants, which would be highly efficient and at the same time have low toxicity for humans and animals. Mass spectrometry research of PHMG [16, 17, 18] requires proper interpretation and continuation.
The aim of research is to investigate and analyse possible mechanisms of action of polyhexamethylene guanidine on cytoplasmic membranes of different cells.
Materials and methods
The results of our own experimental studies, obtained via mass-spectrometry methods, were used in the work [19, 20]. Mass spectrometry research was made by time-of-flight plasma desorption (TOF-PDMS) and matrix assisted (by 2.5-dihydroxybenzoic acid or 3,5-dimethoxy-4-hydroxy-cinnamic acid) laser desorp-tion/ionization time-of-flight (MALDI-TOF). PDMS mass spectra of PHMG samples were acquired by MSBC-01 spectrometer (SELMI, Ukraine) with 252Cf nuclei fragments ionization. MALDI-TOF mass spectra of samples were acquired by Voyager DE PRO spectrometer (Applied Biosystems, USA) with H+-matrix ionization. The results were analysed by MSBC program, version 4.0/m, and Data Explorer 4.0 software systems, respectively. Hereafter, m/z values of the mono-isotopic peaks of the ion distribution have been reported. The molar concentration of PHMG was calculated by the molecular weight of hexamethylene guanidine monomer residue, 141 Da.
The method of cell cultures [21, 22, 23] was applied at the Research Station of Epizootology, the Institute of Veterinary Medicine of the NAAS (Rivne, Ukraine), using the primary culture of fibroblasts of the chicken embryo and interweaved culture of the tracheal
cells of calf. The cells were grown in the solution, containing a mixture of 199 medium (45 %), a minimum Eagle medium or MEM (45 %) and blood serum of cattle (10 %). The monolayer was grown after seeding cell suspensions in 96-well plastic plates at 0.1 ml per well.
The method of artificial bilayer lipid membranes (BLM) formed of different lipid composition involved the application of lipid bilayers [21, 24]. The membrane washing solution contained 10mM Tris-HCl (pH 7.4) and the required quantities of potassium chloride, sodium chloride (USB, Cleveland, OH, USA), lithium chloride and cesium chloride. The membrane separated chambers were stirred when required.
The biological test objects were the cultures of Escherichia coli (strain ATCC 055 K59 No. 3912/41), Staphylococcus aureus (strain ATCC No. 25923 F 49), Bacillus cereus (reference strain DNKIBSHM, Kyiv), Mycobacterium bovis (strain Vallee), field strains of Leptospira interrogans, vegetative forms and spores of American foulbrood Paenibacillus larvae subsp. larvae, micromycetes of the fungal species Aspergillus fu-migatus, A. flavus, A. niger (field strains), herpes viruses of equine rhinopneumonitis Equine herpesvirus type 1 (strain SV-69, Moscow, RF) NB: No literature reference to this strain, no culture collection number and bovine rhinotracheitis Rhinotracheitis infectiosa bovine (strain TK-A, Kharkiv), retrovirus - equine infectious anaemia virus (field strain).
Polyhexamethylenebiguidined hydrochloride was synthesized in PE "Termite" (Rivne, Ukraine) by poly-condensation of hexamethylenediamine and dicyandi-amide with the addition of ammonium chloride (Si-nopharm Chemical Reagents Co. Ltd., Shanghai, China). The molecular weights of PHMG polymers determined by the viscosity of PHMG-containing solutions exhibited their distribution within the range of about 1000-2000 Da (8-16 repeat units). The estimates of kinematic and reduced viscosities were carried out by Ostwald viscometer (VPZH-2) with a capillary diameter of 0.56 mm.
Research results
We used the mass-spectrometry to determine the oligomeric composition of PHMG preparations [19]. It was established that in most cases it was rather inho-mogeneous. For instance, when the composition of four most typical oligomers of linear structure was determined, they were shown to differ both in the number of monomer parts and the content of terminal groups. The mass-spectra clearly demonstrate the difference A m/z = 141, which corresponds to the mass of one monomer. The work [20] analysed the interaction between the preparation of PHMG with such lipids as lecithin and cholesterol, which are main components of
cytoplasmic membranes of mammalian eukaryotes. The analysis of mass-spectra demonstrated that no stable intermolecular complexes of PHMG oligomers with lipids were formed. Based on this fact, an assumption was made that during the adsorption of PHMG on the negatively charged bacterial membrane there may be either electrostatic interaction or the formation of looplike structures. Such a stereochemical mechanism ensures adsorption stability on the membrane, related to the plurality of the bonds, formed with phospholipids, and enhances along with the increase in the molecular mass of the polymer. The biocide activity of the preparation decreases in case of poor availability of membrane phospholipids. In particular, this is true about bacterial spores and mycobacteria with wax-like envelopes.
This fact is also confirmed with microbiological investigations on differential sensitivity of microorganisms to different PHMG salts. The experiments, conducted with test objects being E. coli, S. aureus, B. ce-reus, M. bovis, L. interrogans, P. larvae, A. fumigatus, A. flavus, A. niger demonstrated that the sensitivity of microorganisms to the preparation is firstly defined by the total share of lipids in the membrane and the availability of their phosphate groups [7]. There is also a remarkable regularity: the increase in the relative share of acidic lipids in the external layer of CPM and thus a higher value of the negative external superficial electric potential of the membrane and the decrease in the length of fatty acid tails of phospholipids is in clear correlation with the increase in the sensitivity of microorganisms to PHMG. It is also relevant what type of anion is present in PHMG salts, for instance, the biocidal activity of PHMG chloride is generally higher compared against PHMG salts with organic acids, such as PHMG valerate, PHMG maleate and PHMG succinate.
In the context of studying possible mechanisms of PHMG connecting to CPM the adsorption of the former is practically irreversible; we have studied the dynamics of stereochemical changes in polycation molecule depending on pH. During titrating of aqueous PHMG solutions, there are considerable changes in the optic density and viscosity, in the degree of polycation molecule ionization and its conformation. As there are considerable gradients of pH and concentrations of cations, like Ca2+, Mg2+, when a PHMG molecule binds to the lipids of the external CPM monolayer, there are local changes in pH and conformation of polycation molecules. The change in conformation of polycation molecule during adsorption promotes strong fixation of PHMG on the membrane, its penetration into the lipid bilayer, the change in the position of phospholipids in CPM (segregation of anionic and zwitterionic phospho-lipids), including the facilitation via their lateral diffusion.
Another direction was the investigation on growth-stimulating and cytoprotective effects of PHMG. In particular, the cell cultures of bovine trachea (calves) and fibroblasts of chicken embryo were used to determine the toxicity of PHMG salts and their stimulating and protective effect. PHMG salts impact the rate of cell monolayer formation, in particular, PHMG hydrochloride concentrations in the growth medium,
equalling and exceeding 10-6-10-5 %, inhibit the formation of the monolayer culture of fibroblasts. But PHMG in nanomolar concentrations (0.07-7.0 nM or 10-8-10-7 %) stimulates the proliferative activity of eu-karyotic cells and accelerates the formation of the monolayer. In addition, it was first discovered that preliminary treatment of eukaryotic cells with PHMG salts in the concentrations of 10-5-10-2 % for 10-15 min prevents their being damaged with retroviruses (RNA-viruses) and herpes viruses (DNA-viruses). The cyto-protective effect depended on the anionic composition of PHMG salts, the presence of lipids in the viral envelope, the stage of the cellular cycle [25]. The experiments with seeds of several species of agricultural crops demonstrated that PHMG salts both disinfect the seeds and may stimulate the germination and energy of sprouting. The highest growth-stimulating effect was manifested for pre-sowing treatment of the seeds of beets and legumes (peas, kidney beans, soybeans.) For instance, in some experiments the maximal values of germination and the energy of sprouting exceeded the control more than twice. PHMG succinate was found to be more efficient than PHMG chloride, the optimal concentrations of the former for the seeds of fodder beet were 0.1-0.5 %, and for peas - 0.001-0.01 %. The energy of sprouting increased by 50 % for kidney beans, and the germination - by 30-35 % at the preparation concentration of 0.01 %.
As for the study of PHMG effect on bilayer phospholipids membranes [21], it was determined that after a long-term period of membrane stability, when its conductivity had almost no changes, there is a sharp increase in the ionic current a few seconds prior to BLM breakage. The time, required for PHMG polycation adsorption on BLM and the rate of membrane breakage depend on the polarity of electrode charge in cis-chamber and phospholipids composition of the membrane. The active concentration of PHMG from the external side of the membrane was 0.0001 %, or ~ 7 ^M; the potential from the cis-side of BLM changed from +100 mV to -100 mV; the solution, surrounding the membrane from both sides, was 100 mM KCl. The difference in the rate of BLM breakage on condition of different electric potentials on the electrode demonstrated that the electrostatic interaction of the poly-cation and the membrane is relevant in the general mechanism of adsorption and destruction of CPM, but this relevance is not decisive. Even on condition of negative potential of the electrode (-100 mV), the adsorption of PHMG on BLM and its destruction occur, albeit at a slower rate.
The investigation on the possible negative impact of PHMG preparations (disinfectants, plant protection products or stimulators of seed germination) on zoo-and phyto-constituents of biocenoses demonstrated the results, presented below [26, 27]. For insects (bees), when coming with sugar syrup, the toxic action was manifested at PHMG concentration of > 0.66 %, LD50 per os for mammals (white laboratory mice) -2000±100 mg/kg of bodyweight. The mentioned concentrations are practically unavailable in normal conditions. The minimal toxic concentration of PHMG hydrochloride for hydrobionts (fish, shellfish, flatworms,
crustaceans) is 0.0001 % (or 1 mg/l), that for ciliates -0.001 %. The concentrations, starting with 0.00001 % or 0.1 mg/l and below, are safe for the formed mono-layers of eukaryotic cells. The toxicity of the preparation depends considerably on its chemical purity, availability and amount of low molecular admixtures of hex-amethylenimine, hexamethylendiamine,
methylenimine, etc. The plant components of bioceno-ses are more tolerant to the effect of PHMG, higher plants are resistant to the treatment with 0.1-0.3 % aqueous solution of the preparation. The biocide or inhibiting effects for algae are manifested at the concentration of > 0.0001 %. The transfer coefficient in the "soil-plant" system is < 0.01 %, "water-plant" (algae) -< 0.1 %. In general, the potential threats for ecosystems from PHMG preparations, penetrating therein, are minimal - they are quickly adsorbed onto organic and inorganic components of soil, and in water they bind particulate matters, organic substances, surface active substances etc. The migration along food chains is almost absent due to the polymer structure of PHMG and its fast decomposition. No negative consequences were determined if chronic exposure was absent. The preparation has no considerable impact on the ability of bio-cenoses to self-purify, self-regulate and self-restore.
At the same time, taking into consideration the fact that bactericidal concentrations of PHMG hydrochlo-ride for most gram-positive and gram-negative bacteria are 0.005-0.1 %, and the bacteriostatic ones - 0.00010.005, one may not state categorically that eukaryotic cells should be more resistant to PHMG preparations.
Discussion of research results
Therefore, the generalization and analysis of the results obtained by us and other authors led us to the conclusion that the main target for PHMG molecules is a cell membrane, and the specificity of the interaction between the preparation and cytoplasmic membranes of cells is related to several key issues.
Firstly, the main target for PHMG in the cell membrane is its phospholipids, although the interaction with negatively charged groups of membrane proteins, glycolipids, glyco- and lipoproteins is also possible. The share of glycoproteins and glycolipids is known to take about 25 % of the surface potential of CPM. The mass content of lipids in cell membranes usually fluctuates from 25 to 70 %. The effect of PHMG polycation on other membrane components may be considered secondary and auxiliary, or indirect. Surely, all the models, presented below, are intentional simplifications because the specificities of functioning of the living systems cannot be reproduced exactly in any of the simplified model systems [28].
Modern methods of molecular modelling demonstrate that the surface of even a simple single-component lipid membrane (for instance, with 1,2-diphyt-anoyl-sn-glycero-3-phosphocholine or di-oleoylphosphatidylcholine) is not polar homogeneous as it could be assumed judging by the schematic presentation of lipids in the form of balloons with tails. Some of these tails surface on the water-membrane boundary and form hydrophobic areas, i.e. there is an emerging mosaic-like, mostly polar surface with some hydropho-bic isles with the size of several square nanometers.
Somewhat more complicated multicomponent models demonstrate the presence of more liquid lamellar phases or Ld-phases in the membranes (with prevailing phospholipids with unsaturated acid tails) and solid, or Lo-phases (with saturated fatty acids). If such a model membrane is added transmembrane spiral peptides, they are distributed between phases, getting mostly located in the liquid Ld-phase and avoiding the orderly Lo-phase. Thus, the areas from Lo-phase are more accessible for the adsorption of PHMG molecules.
The absence of cholesterol in the membranes of bacteria does not allow for confident assertions about the formation of rafts, as in case of CPM of eukaryotes, but bacterial membranes are also laterally inhomogene-ous. The lateral heterogeneity of the membrane structure of pro- and eukaryotes, their different transmembrane, dipole and surface potentials have implicit effect on the specificities of adsorption of PHMG molecules.
Secondly, PHMG polycations may get adsorbed on any phospholipid membrane. A sufficient prerequisite is the availability of negatively charged phosphate groups of phospholipids. In our experiments on BLM, PHMG salts are quickly and irreversibly adsorbed on comparatively "neutral" membrane with PC± (phosphatidylcholine) and cholesterol [19, 21]. Contrary to Ca2+ or Mg2+ ions, PHMG interacts both with charged and zwitterionic phospholipids. Obviously, while contacting even acidic phospholipids like PC- (phosphatidyl-serine), polycation iminogroups bind not only carboxyl groups of serine, but also phosphate groups, similarly to Ca2+. However, the ratio of acidic and neutral lipids in the external layer of CPM is relevant. For instance, some authors believe that bacteria, whose external layer of CPM contains a higher percentage of acidic lipids, are more sensitive to the effect of the preparation than eukaryotic cells [29]. In addition, prokaryotic membranes differ considerably from plasmatic membranes of eukaryotes both in their lipid composition, for instance, the presence of cardiolipin (CL2-) in CPM, and superficial potential, non-lipid components, etc., thus, the rate of adsorption and its consequences should be also different. The adsorption of PHMG on CPM occurs unevenly, the preparation gets mostly concentrated in the areas, enriched with lipids, especially anion ones [1].
Thirdly, the properties of the lipid membrane change after PHMG adsorption. During the interaction of guanidine groups and polar heads of phospholipids, there may be the re-distribution of ions in the outer submembrane layer, for instance, forcing out counter ions Ca2+ and Mg2+, which usually stabilize CPM. This is accompanied with local changes in pH. Similar to other polycation antimicrobial preparations, there is possible segregation of acidic and neutral phospholipids and formation of membrane domains with different superficial electric potential. It is known that both the charge of phospholipid heads and ions, bound to them (in this case, it is polycation PHMG), define the value of transmembrane potential.
The example of the effect of acellisine oligomers on the membranes can be used as an analogue [30]. It is also a polycation, binding anion phospholipids mostly. In this case, the membrane should contain both
anion and zwitterionic lipids. After the adsorption of domains, enriched with anion phospholipids in the ar-polycation, there is segregation with the formation of eas of the highest accumulation of acellisine oligomers
(Fig. 1).
Fig. 1. The theoretical scheme ofpossible segregation of anion (in a darker colour) and zwitterionic phospholipids in the membrane after polycation adsorption (Source of Figure: Epand et al., 2008)
However, the reason for segregation and accumulation of anion lipids on the internal side of the membrane is not quite clear in the abovementioned scheme for acyllysine. Probably, there is correlation between the interaction of lipids of the external and internal monolayers of the membrane or excessive positive charge in the areas of polycation adsorption. For instance, in the model membranes, where the separation of Ld and Lo phases can be observed, the clusters of these phases coincide for both monolayers. As for PHMG, it was noted [1, 31, 32] that after its adsorption there is segregation of phospholipids, along with their removal from the CPM. A similar segregation of phos-pholipids takes place after adsorption of antimicrobial peptides (AMP) on the membranes. For instance, according to the data of computer simulation of a bacterial membrane, containing 70 % PE± (phosphatidyleth-anolamine) and 30 % PG- (phosphatidylglycerol), there is possible formation of nanodomains (molecules of PE± have efficient interaction and force out the "unfavourable" partner PG-), and the adsorption of AMP leads to the increase in PG- domains and the occurrence of phase separation of lipids.
Thus, the adsorption of a considerable number of PHMG molecules on CPM promotes global separation of Lo and Ld phases of lipids and segregation of neutral and acidic phospholipids. This is a thermodynamically favourable process. It is generally known [28] that the composition of the lipid matrix of native membranes has evolutionarily been formed so as to be always near the phase transition in physiological conditions. This is a condition for the formation of a mesophase (rafts) in the membranes, and the adsorption of PHMG poly-cations shifts natural equilibrium and "pushes" the process of phase transition.
The step, following the segregation of lipids, is the transition of the membrane in some areas from the lamellar L into hexagonal (cylindrical) Hnphase. Generally, most purified membrane phospholipids in aqueous medium are known not to form bilayers, but be situated predominantly in the hexagonal phase Hn. As for the membranes, first of all this is notable for those, containing a considerable percentage of lipids, asymmetrical in
their form. For instance, the form of PE± or CL2- molecules resembles a cone-type form rather than the cylin-dric one, similar to PC± or PS-. Noteworthy is the fact that, compared to eukaryotic membranes, the bacterial membranes contain both a higher percentage of negatively charged lipids and a higher amount of such lipids of negative curvature. The places of their accumulation in CPM may serve as sources of formation for Hn phase and impairment to the integrity of the external lipid monolayer. PHMG-lipid hexagonal or vesicle-like structures are formed and are likely to leave CPM surface rather fast (detach themselves).
In case of BLM, after the adsorption of PHMG thereon, in about 5-10 min there is rather fast polarization of a conditionally neutral PC± membrane. As for the membrane, containing acidic PG- or CL2- in its external layer, there may be depolarization from "-" to "+" and a local positive surface potential may be formed. The changes in the dipole potential of the membrane promote the hydration and decompression of lipids.
Re-charging (depolarization) of the external lipid monolayer of the membrane is accompanied with the increase in flip-flop transitions, thus the internal lipid layer of CPM undergoes changes as well. Similarly to the interaction between synthetic polyampholytes and anionic liposomes, when the lipids of both layers of the membrane participate due to flip-flops in the microphase distribution or in case of the interaction with liposomes of synthetic polycation, its adsorption leads to the migration of anionic CL2- from the internal liposome layer into the external one. Flip-flop transition is especially relevant for eukaryotic CPM, as the highest number of acidic lipids therein is located on the internal side of the membrane. Wilfully flip-flops occur rather slowly (hours, days), so they are not significant for artificial BLM, whereas in the native cells there are a number of enzymes, ensuring the structural asymmetry of CPM, and these transitions may occur in a matter of minutes. It is possible that growth-stimulating effect of low PGHM doses [25, 32] is related to the neogenesis of acidic lipids proper, the number of which on the internal side of CPM decreases due to flip-flops, irreversible binding to polycation and further removal.
Due to the depolarization of the membrane during the adsorption of polycation, there is an impairment of CPM asymmetry and the physical properties of the external and internal monolayers of the membrane. In eu-karyotes, the release of anionic lipids usually is known to occur only in specific functional states of the cell (apoptosis, activation of platelets).
It is clear that the abovementioned changes in CPM depend on the dose of the preparation, the duration of the exposure and concentration of cells in the sample.
Fourthly, during the adsorption and binding of PHMG molecules with polar phospholipids heads there is a change in both the conformation of lipids and the polycations molecule. There are changes in the value of the charge of guanidine groups and, as a result, the form of the whole molecule. It conditions the changes in the position of some phospholipid molecules in CPM (segregation). Similarly to some AMP, which are usually also polycations, PHMG molecules in the aqueous solution have mostly an unorganized structure, and while interacting with lipid membranes they become more protruded, their form approximates the linear one. For instance, the molecules of latarcin in the solution have unorganized globule-like structure, and, while adsorbing onto lipid structures (micelles, liposomes, bilayer membranes) acquire the form of a-helices [34].
One may assume that the perturbation of the lipid bilayer of the membrane is somewhat conditioned by conformational transformations of the very PHMG molecules. It is possible that the form of the polymer molecule depends on the anion considerably [29]. In practice, there is the widest application of PHMG chloride, less frequently - PHMG phosphate, even less frequently - PHMG succinate or salts with other organic acids. In the first case, the anion is Cl-. The positive charge of the guanidine is delocalized on three nitrogen atoms and is additionally delocalized in the system of c-bonds of carbohydrate (hexamethylene) area. Pulling electrons onto itself, Cl- promotes the increase in "+" potential on guanidine cation, due to the internal electrostatic forces of repulsion a polymer molecule acquires a more linear form. On the contrary, the electron density in salts with anions of phosphoric, succinate or other organic acids shifts towards the guanidine group. Here its positive charge decreases. Due to his fact, the redistribution of electron density on guanidine groups
PHMG
spreads along the whole polymer chain, the intramolecular interactions of functional groups, distant in the chain, are enhanced. As a result, the form of the macro-molecule approximates the globule-like one. In the aqueous solution it is energetically more favourable and is stabilized with hydrogen bonds and van der Waals' interactions of hexamethylene areas.
Thus, the type of the anion affects the degree of delocalization of the positive charge and the hexameth-ylene area promotes the redistribution of electron density in the macromolecule. During the adsorption of PHMG on CPM there is a change in the anionic composition, pH, localization of charges along the polymer molecule and thus its form. In their essence, PHMG salts are supramolecular complexes where the involved anions of acids (guests) affect the properties of the whole substance.
The fact that the conformational changes in PHMG molecules have relevance during the impairment of CPM functions is also confirmed by the increase in the antimicrobial activity of polycation along with the increase in the molecular mass [29]. The efficient antimicrobial effect is inherent to PHMG oligomers, whose molecular mass is at least 800 Da [35]. These are oligomers with the polymerization degree of n > 6, the molecules with smaller mass have low activity. It is clear that the larger a polycation molecule is, the more places of its binding to phospholipids there are, and the higher perturbation of the membrane is.
Fifthly, what ensures fast, irreversible binding of PHMG to the phospholipid membrane? First of all, this is the plurality of forming non-covalent bonds of poly-cation iminogroups and phospholipid polar heads. In addition, it is probable that the fixation of PHMG molecules on comparatively electro-neutral lecithin-cholesterol lipid bilayer and on CPM may be related to the formation of peculiar "loops". This mechanism of adsorption on the surface of liposomes has been described for some polycations. In our opinion, it may look for PHMG in the way, schematically presented in Fig. 2. This process is promoted by flip-flop transitions of phospholipids [11, 36], local changes in the flow (fluidity) of the membrane in the places of polycation adsorption, the change in the form of PHMG molecule during adsorption. These loops may be one of the factors, conditioning the perturbation in the lipid bilayer of CPM, and cell death effect causes of the disinfectant.
PHMG
PHMG
Fig. 2. The scheme of one of the possible ways of PHMG molecule fixation on the phospholipid bilayer,
1-3 - the stages (own model)
The results, obtained by us on BLM, may serve as indirect evidence, proving this scheme [21]. In particular, the adsorption of the polycation and the breakage of the membrane occur much faster on the evener and thinner flat surface of BLM from synthetic 1,2-diphyt-anoyl-sn-glycero-3-phosphocholine compared against the application of a thicker and uneven (inhomogene-ous in its phospholipids composition) BLM, prepared from lecithin or phosphatidylcholine of the yoke.
It is also known that PHMG has comparatively weak effect on Mycobacteria tuberculosis [29]. This is explained not only by the fact that it is more poorly adsorbed on the cell surface due to its wax-like envelope, and there are many mycolic acids with long hydropho-bic tails (C ~ 78-95), but also by the fact that aliphatic chains of membrane lipids of mycobacteria are longer (C ~ 22-24) compared to most other microorganisms. Therefore, the formation of "loops" after PHMG adsorption and the perturbation of lipids get complicated. In addition, it is known that the longer carbohydrate chains of fatty acids are the denser and more compact layer, formed by such phospholipids, is.
Sixly, the mechanism of PHMG action is most likely to be multiple-factor and to depend on the preparation concentration. It seems that different PHMG concentrations have different effect on the membrane. For instance, as for E. coli, low concentrations damage the external envelope and change the permeability of the internal membrane, whereas high concentrations cause the disorganization of the membrane in some local areas and the formation of through pores [37]. At the same time, the authors are not sure that the only reason of bactericide effect of PHMG was the interaction
with CPM, they assume that the effect of polycation on cellular DNA and proteins may also be relevant.
At comparatively low concentrations of PHMG the membrane is still capable of "self-treatment", though its structure and permeability change. The example may be found in the fact that at the inhibiting concentrations of PHMG chloride of 2* 10-5 %, A. niger have a smaller general number of lipids, especially polar phospholipids, required to build CPM. The composition of fatty acids of these polar lipids has an increasing number of the saturated ones, probably, to increase the "rigidity" of the membrane under breakage. At a twice lower "growth-stimulating" concentration of 1*10-5 %, A. niger has a contrary effect of the increasing percentage of non-saturated fatty acids in both polar and neutral lipids. Here the membrane becomes more "liquid" and permeable. The proliferative activity of the fungus increases (growth stimulation).
At "growth-stimulating" and subbacteriostatic concentrations there may start the mechanism of the cell "pushing-out" or rejecting the phospholipids, which bound to PHMG and lost their functionality. Contrary to a bilayer lipid membrane (BLM), in case of CPM this is promoted by high intracellular osmotic pressure, typical for any bacteria. There is rejection proper, not pulling-out of lipids, like in case of bactericide or bacteriostatic concentrations [1]. During the ionic interaction of PHMG iminogroups and hydro-philic heads of phospholipids, their amphiphilic properties decrease, and the hydration and decompression of the lipid bilayer, occurring due to the penetration of hydrophobic areas of the polymer thereto, promote the removal of "defective" lipids from the membrane (Fig. 3).
5
Fig. 3. The scheme of PHMG molecules removing phospholipids from the bilayer, not containing steroids, and imitating the bacterial CPM, acidic lipids are indicated with dark heads; 1-5 - the stages of destruction of bilayer phospholipid membrane, acidic lipids are indicated with dark heads (own model)
Due to the presence of cholesterol in eukaryotic CPM, the latter are stronger and more resistant to PHMG effect, here the removal of phospholipids is somewhat more complicated. Probably, therefore the stimulating effect of the preparation was noted only for eukaryotic cells including micromycetes (whose CPM contained ergosterol).
In addition, PHMG binding to phosphoglycerides, which actually are organic anions, causes both the change in polymer conformation and folding of the polymer chain, and the aggregation of anions of the formed complex. Mostly, acidic phospholipids are removed from CPM along with PHMG molecules. This reaction occurs on the edge of phase division, and many factors, which are sometimes impossible to consider, impact the rate of such heterogeneous processes. PHMG-phospholipid vesicles leave the surface of the cell.
Noteworthy is the fact, that the transition of PHMG concentrations from bacteriostatic to bactericide (or from stimulating to inhibiting for eukaryotic cell) is often rather abrupt and similar to phase transition. It is known that in case of destroying the surface of phase division there is a jump-like change in the properties of CPM. For instance, it has been described that at the effect of the preparation on E. coli in the concentration of 1.3*10-3 % there are only slightly visible impairments in the external membrane structure of bacteria, the permeability of CPM increases and the morphology of cells remains without any obvious changes. When the concentration got slightly higher, 2.3*10-3 %, the authors noted complete destruction of the external membrane structure, the local through membrane pore got formed, considerable damage of the internal structure of the cells became evident, and the intracellular components came out, the bacterium perished [37].
1
2
3
4
Thus, the transition from the bacteriostatic to bactericide concentration occurs rather quickly. There may be a need for some minimal additional critical number of polycation molecules for the energy, released during their adsorption on CPM to be sufficient to ensure the endothermic process of their penetration (or pushing through) into the depth of the lipid bilayer. The cooperative transition occurs due to the fact that a hydropho-bic mechanism (PHMG penetration into the hydropho-bic part of the bilayer) gets involved to substitute the ionic mechanism (the interaction of PHMG imino-groups with phospholipid heads on the first stage of adsorption). From the standpoint of thermodynamics, it looks profitable because due to the binding of hydro-phobic alkyl areas of PHMG to fatty acid tails of phospholipids, there is a release of counter ions and solvent molecules (with the increase in entropy) [38].
In case of further increase in PHMG concentration it may affect the membrane according to the "carpet" mechanism, it is similar to AMP of latarcins [34]. It depends on the phospholipid composition of CPM to some extent. The matter is that the formation of hexagonal structures of phospholipids or vesicles for PS-, for
instance, is remarkable only when it is in the acidic form, and as for the neutralized form (whether it is neutralization with metal cations or with PHMG poly-cation) lamellar structures are also rather stable. Global impairment of the membrane structure occurs due to multiple superficial binding of polymer molecules. As a result of neutralizing guanidine groups of PHMG with phosphate groups of lipids, the general hydrophobicity of the formed PHMG-lipid supramolecular complex increases and the properties of the polycation change. Hy-drophobic forces push (or press) PHMG-lipid formations inside the lipid bilayer, and the hexamethylene areas of PHMG promote it. The hydrophobic areas of PHMG interact with cholesterol (or ergosterol in yeast). The increase in the partial pressure of polycations due to the increase in their concentration from cis-side of PCM does not promote the removal of PHMG-lipid vesicles. There is phase layering of lipids in the membrane plane with the formation of structurally rigid clusters, forming hexagonal lipid or PHMG-lipid structures in the middle hydrophobic part of the bilayer (Fig. 4).
1 2 Fig. 4. The scheme of PHMG molecules destroying the lipid bilayer, which imitates bacterial CPM; 1, 2 -
consecutive stages of the process (own model)
hydrophobic part of the membrane along with them.
In addition, not all the iminogroups of PHMG get "neutralized" by phospholipids, some of them may remain non-involved during adsorption and preserve its charge. Taking high hydration enthalpy of polymer derivatives of guanidine [39], which is also confirmed by their good solubility in water (for instance, the solubility of PHMG exceeds 40 %), the intramolecular forces counteract entropy and direct the polycation molecule to "folding" in the aqueous medium. It also promotes its immersion into the hydrophobic part of the lipid bi-layer and the destruction of the latter. The PHMG pol-ycation transforms into an amphiphilic compound with considerable detergent properties. Thus, there is a theoretical possibility that some iminogroups in the globule-like area of PHMG molecule remains non-involved, and some water molecules may penetrate the
The ability of the membrane to "self-cure" decreases rapidly. It loses its integrity and relative homogeneity.
As for CPM, it undergoes depression (carpet mechanism) and fast formation of one (or several) transmembrane pores, while the cell goes through lysis. In addition, in case of BLM, the breakage occurs relatively quickly (1-2 sec), without the long period of gradual increase in the transmembrane ionic current [21], compared against a complicatedly organized and non-homogeneous CPM.
As for the superficial tension (o), it is clear that the adsorption is positive, if PHMG concentration in the near-surface layer is higher than in the volume. With the increase in the volume of the preparation concentration (c), the excess of concentration on the surface (Ac) decreases and is according to the Gibbs formula:
Ac = c/RT x (do/dc)T. Thus, the superficial tension decreases considerably as well, which also promotes the implementation of the "carpet" mechanism.
Therefore, while agreeing in many respects with other authors [1, 3, 29, 30, 31, 32, 37, 38, 39, 40, 41, 42], we think that in dynamics the process of damaging CPM of the cell may look as follows below. After the adsorption of the first PHMG molecules on the membrane due to lateral diffusion of lipids in the external monolayer (besides, the bactericidal activity of PHMG increases with the rise in temperature), mostly acidic phospholipids start getting concentrated (or held) near the polycation molecule of PHMG. Here mostly the lipids of liquid Ld are moving, not Lo of the CPM phase or those, composing rafts. The changes of the form of disinfectant molecules and their peculiar folding also promote the segregation of lipids. The areas with different value of electric potential are formed in the external layer of the membrane. Due to the effect of electrostatic forces, the places of accumulation of acidic li-pids accumulate more and more polycation molecules. The hydrophobicity and curvature of the membrane increase in the adsorption areas for PHMG molecules. The depression or bulging of some membrane areas occurs according to the "carpet" mechanism (for high concentrations of the preparation). The hydration and decompression of lipids lead to the transition of some areas of the external layer of the membrane from the lamellar into the hexagonal phase. This is promoted by the "loop-like" mechanism of fixing a PHMG molecule on the membrane. Phase transition of lipids starts affecting the work of membrane proteins. The internal layer of CPM is getting damaged due to depolarization of the membrane, there is a change in dipole potential and flip-flop transitions of lipids. PHMG also belongs to the group of comparatively weak cation surface active substances, but the detergent properties of the formed amphiphilic PHMG-lipid complexes are enhanced. The vesicles, leaving CPM, are formed (more frequently for comparatively low concentrations of the preparation). The cell gets rid of some phospholipids, which have become dysfunctional. Further fate of the cell depends on the ability of the lipid layer of its CPM to "self-cure", on the rate of including the neogenesis (synthesis) of new lipids, the number of PHMG molecules and the duration of its effect. During further increase in the preparation concentration due to the dilatation of the lipid bilayer the cell loses K+ ions, there are changes in the transmembrane gradients of other ions, the increase in the sizes of hydrophobic areas on CPM surface, and there is partial plasmolysis. The functioning of proteins, bound to the membrane, is impaired. The continued increase in PHMG concentration or prolongation of exposure time leads to irreversible changes in the membrane. The depression of a rather dilated membrane occurs further according to the "carpet" mechanism, one or several transmembrane pores are formed therein, and CPM loses its barrier, transportation and other functions completely. The first pore is likely to form in the part of CPM, where the sizes of the hydrophobic area in the external monolayer
are maximal. Some authors also believe that direct contact between PHMG and cell membranes is necessary for PHMG-induced toxicity [43].
Conclusions. Therefore, the results of our investigations and the analysis of the data, obtained by other authors, allow for the assumption that the main targets for PHMG are phospholipids of the cytoplasmic membrane. With comparatively low concentrations of the preparation and metered exposure time, there is a change in the lipid composition of CPM (via the removal of some phospholipids or PHMG-lipid vesicles), this is related to neogenesis of lipids and growth-stimulating and cytoprotective effects, observed by us. In case of bacteriostatic or sublethal doses for eukaryotes, the growth cells is inhibited, in case of bactericide or lethal doses - the lipid bilayer of the membrane undergoes considerable perturbation, is damaged quickly (carpet mechanism) and the cell perishes due to lysis. It should be also added that a biological membrane is first and foremost an anisotropic structure in all three dimensions. This is an unbalanced structure, where concentration gradients are created and constantly maintained. Due to this fact and the inclusion of various protein and non-protein components, the external and internal monolayers of this bilayer structure differ significantly in their composition, in the electrostatic potential of the surface and the binding of ions. The plas-matic membrane interacts with the cytoskeleton. In addition to the transmembrane transfer of molecules, the transfer of functionally important signals is carried out through coordinated structural changes in the membrane itself. The adsorption of some PHMG molecules on the membrane may have a relevant effect on its integrity and functioning.
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