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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.45 No.3 pp.77-85
DOI : https://doi.org/10.17779/KAOMP.2021.45.3.001

Amphiphilic Effects of Chlorhexidine Digluconate on Rotational Mobility in Bacterial Outer Membranes and Liposomes

Tae Young Kim1), Seong-Kweon Cha1), Seung-Il Eom1), Moon-Kyoung Bae2),3), Soo-Kyung Bae1),3), Ji-min Ju1), Il Yun1), Da-Sol Kim1),3)**, Hye-Ock Jang1)*
1)Department of Dental Pharmacology, Pusan National University School of Dentistry
2)Department of Oral Physiology, Pusan National University School of Dentistry
3)Periodontal Disease Signaling Network Research Center (MRC), School of Dentistry, Pusan National University
* Correspondence: *Hye Ock Jang, Dental pharmacology, School of Dentistry, Pusan National University, Yangsan, 50612, Korea Tel: +82-51-510-8232, Fax : +82-51-510-8233 Email: jho9612@pusan.ac.kr
** Da Sol Kim, Dental pharmacology, School of Dentistry, Pusan National University, Yangsan, 50612, Korea Tel : +82-51-510-8232, Fax : +82-51-510-8233 E-mail: k201797801@pusan.ac.kr
May 12, 2021 May 14, 2021 June 4, 2021

Abstract


Chlorhexidine is a cationic bisbiguanide with broad antibacterial activity, and diverse activity encompassing gram-positive and gram-negative bacteria, yeasts, dermatophytes, and some lipophilic viruses. Chlorhexidine alters the permeability of the bacterial membrane. They also neutralize periodontal pathogens such as Streptococcus aureus, Porphyromonas gingivalis, and Prevotella intermedia. Porphyromonas gingivalis were cultured to isolate the Porphyromonas gingivalis outer membranes (OPG). Also, OPG were used to prepare large unilamellar liposomes with total lipids (OPGTL) extracted and prepared with the phospholipids mixture (PL). The effect of chlorhexidine digluconate on cell membrane kinetic changes was investigated using fluorescence polarization of n-(9-anthroyloxy) stearic acid with different rotational mobility depending on the probe substitution position (n) in the membrane phospholipid aliphatic chain. Chlorhexidine digluconate increased the hydrocarbon rotational mobility interior of the OPG, OPGTL and PL, but native and model membranes interfacial mobility were decreased. The sensitivity to increasing chlorhexidine digluconate effect on rotational mobility was proportional to the depth of probe position in descending order of 16-AP, 12-AS, 6-AS and 9-AS. Chlorhexidine digluconate disordering or ordering the effects on the membrane lipids may cause its bacteriostatic and bacteriocidal actions.


Bacterial outer membranes and liposomes , Chlorhexidine digluconate , Fluorescence probe technique , Hydrocarbon interior , Membrane interface

Chlorhexidine digluconate가 세균외막과 인공세포막의 회전운동에 미치는 비대칭적 영향

김 태영1), 차 성권1), 엄 승일1), 배 문경2),3), 배 수경1),3), 주 지민1), 윤 일희1), 김 다솔1),3)**, 장 혜옥1)*
1)부산대학교 치의학전문대학원 치과약리학교실
2)부산대학교 치의학전문대학원 구강생리학교실
3)부산대학교 치의학전문대학원 치주질환네크워크신호전달센터 (MRC)

초록

    Ⅰ. INTRODUCTION

    Plaque is a resilient, yellowish material that firmly adheres to the tooth surface and restorations1). The key role of plaque is initiation of gingivitis or periodontal diseases. The complete elimination of plaque by mechanical therapy is often impossible, so antimicrobial agents are used as an adjunct to the mechanical approaches. Chlorhexidine (CHX) is the most effective antimicrobial agent, and the clinical effect of CHX is likely due to both its substantive and antibacterial properties2). It was reported that chlorhexidine is antiplaque, antigingivitis, fungicidal and effective in both the prevention and treatment of oral candidiasis3). Bacteriostatic action occurs at low concentrations against many gram-positive and gram-negative bacteria; at much higher concentrations, it exhibits bactericidal action4). CHX is a membrane-type antimicrobial agent and acts as the inner cytoplasmic membrane5). Tsuchiya6) reported that the chlorhexidine antiplaque effect is based on the membrane fluidity reduction of both hydrophilic and hydrophobic regions. However, such results were not the outcome of the tests using the membranes of gingivitis causative microorganisms and specifically, the results of Tsuchiya6)´s study were obtained through the measurements that used phospholipid liposomes as samples.

    With few exceptions6) the chlorhexidine digluconate effect analysis on the native fluidity and model membranes was focused on their lipid environments average change, not on the special domain of native and model membranes. Furthermore, no studies have been carried out about the effect of the chlorhexidine on the fluidity of bulk or special domain of membranes of Porphyromonas gingivalis (PG) of ginginvitis causative microorganisms. The fluorophores of anthroyloxy derivatives can also be used to differentiate whether the bilayer has a fluidity gradient across it, as the anthroyloxy group can be positioned at different positions of the stearic acid moiety7-9).

    It is intended to provide a basis for studying the chlorhexidine pharmacological action molecular mechanism. Using fluorescence polarization of n-(9-anthroyloxy)stearic acid (n-AS) or palmitic acid, we investigated the differential effects of the chlorhexidine digluconate on the differential rotational mobility of bacterial outer membranes and liposomes between a series of graded depths inside membranes, depending on its substitution position (n) of the membrane phospholipid aliphatic chain.

    Ⅱ. MATERIALS AND METHODS

    1. Matarials

    Fluorescent probes, 16-(9-anthroyloxy)palmitic acid (16-AP), 12-(9-anthroyloxy)stearic acid (12-AS), 9-(9-anthroyloxy)stearic acid (9-AS), 6-(9-anthroyloxy)stearic acid (6-AS) and 2-(9-anthroyloxy)stearic acid (2-AS), were purchased from Molecular Probes, Inc. (Junction City, OR.). The chlorhexidine digluconate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and bovine serum albumin (BSA) were obtained from Sigma Chemical Co.(St. Louis, MO.). All other reagents were purchased commercially and they were of the highest quality available. Water was deionized.

    2. Bacterial growth conditions

    ATCC 33277 (PG) was obtained from the American Type Culture Collection (Rockville, MD.) and cultured as described previously10). PG was maintained as frozen stock cultures and grown anaerobically (Bactron Ⅳ Anaerobic Chamber, Sheldon Manufacturing Inc., OR.) in trypticase soy broth(BBL Microbiology Systems, Cockeysville, MD.) supplemented with 1% (wt/vol) yeast extract, 0.0005% (wt/vol) hemin and 0.0001% (wt/vol) menadione at 37°C for 3 days in an atmosphere of 80 % N2, 10% CO2, and 10% H2.

    3. Isolation of outer membrane

    The procedure was essentially as described by Smalley et al.11). Briefly, freeze-dried whole cells (200 mg) were suspended in 60 ml of 0.14 M NaCl containing 10 mg of EDTA (pH 7.3) and the suspension was incubated with stirring for 30 min at 37°C. This serves to dissociate cultured Porphyromonas gingivalis outer membranes (OPG) and to inhibit any possible cell associated protease activity12) during preparation. After passing twice through a 25 gauge needle, the cells were pelleted by centrifugation (20,000 xg for 30 min, 4°C) leaving the supernatant which was crude OPG preparation. Residual EDTA and buffer salts were removed by dialysis against distilled water for 4 h at 4°C, and OPG fraction was then freeze-dried. Their protein concentration was determined by the method of Lowry et al.13), using BSA as a standard.

    4. Extraction of lipids from OPG

    Total lipids were extracted from OPG by the methods of Yun and Kang14). The OPG were homogenized with 2:1 chloroform-methanol mixture (v/v), and the homogenate was filtered. As a way to purify the crude extracts, the filtrate was mixed thoroughly with 0.2-fold its volume of either water or an adequate salt solution. The lower phase was saved, and evaporated to dryness under nitrogen at 37°C, and the residue was taken up in a small volume of chloroform.

    5. Liposome preparation

    Stock solutions of total lipids or the phospholipids (DPPE/DPPC 7:3, mol/mol) were made in chloroform. The concentration of the lipid stock solutions was 0.2 mg/ml. Giant unilamellar vesicles (GUVs:OPGTL or PL) with a mean diameter of 45 mm were prepared by the method developed by Angelova and Dimitrov15). A special temperature- controlled chamber, which was previously described, was used to grow the GUVs 9,16). The experiments were proceeded using an inverted microscope (Axiovert35: Zeiss, Thornwood, NY) in the same chamber after the vesicle formation.

    Ⅲ. RESULTS

    In this study, using the fluorescence probes n-AS (or 16-AP), we examined the amphiphilic and differential effects of the dicationic chlorhexidine digluconate on the rotational mobility among a graded series of depths inside membranes, depending on its substitution position (n) in the phospholipid aliphatic chain of OPG, OPGTL and PL. To determine the effect of the antimicrobial agent on the rotational mobility as mentioned earlier, it was first necessary to demonstrate that the drug did not directly interact with fluorescent probes, and thereby quenching their fluorescence. Quenching of absorbance-corrected fluorescence intensity of n-AS in OPG, OPGTL and PL by the chlorhexidine digluconate was not observed at all tested concentrations. Furthermore, if direct quenching of n-AS by the antimicrobial agent occurred, fluorescence lifetime would decrease.

    Table 1 lists the anisotropy (r) of n-AS for the intact OPG, OPGTL and PL. The degrees of rotational mobility of the intact five membrane components (16-AP, 12-AS, 9-AS, 6-AS and 2-AS) were dependent on the substitution positions (n) in the following descending order: 16-AP, 12-AS, 9-AS, 6-AS and 2-AS. The antimicrobial agent, chlorhexidine digluconate may cause disordering or ordering in their host lipids. Ordering occurs on the membrane interface, whereas disordering occurs deep within the acyl chains.

    1. Disordering effects of chlorhexidine digluconate on the rotational mobility of the hydrocarbon interior

    The effect of increasing concentrations of the chlorhexidine digluconate on the anisotropy (r) of 16-AP, 12-AS, 9-AS and 6-AS in the hydrocarbon interior of the OPG, OPGTL and PL is shown in Fig. 1-4. The chlorhexidine digluconate decreased the anisotropy (r) of the 16-AP and the 12-AS (increased range of rotational mobility) in the OPG, OPGTL and PL in a dose-dependent manner. The significant decreases in the anisotropy (r) values by the drug were observed at the 0.01, 0.10, 1.00, 2.00, 3.00, 4.00 and 5.00 mM (Fig. 1–4.). As to the differing degrees of increases of hydrocarbon interiors' rotational mobility by chlorhexidine digluconate depending on the membrane lipid with proteins or lipid without proteins, it was greater in both OPGTL and PL than in OPG (degrees of increases were calculated in percentage).

    The anisotropy (r) of 12-AS in hydrocarbon interior of OPG, OPGTL and PL are 0.110 ± 0.003, 0.093 ± 0.002 and 0.094 ± 0.001 at 37°C (pH 7.4), respectively. The anisotropy (r) of 12-AS in hydrocarbon interior of OPG, OPGTL and PL are 0.140 ± 0.005, 0.138 ± 0.006 and 0.140 ± 0.007 at 25°C (pH 7.4), respectively. Thus, the differences in the anisotropy (r) values of 12-AS in hydrocarbon interior of OPG, OPGTL and PL before and after adding 0.10 mM chlorhexidine digluconate were 0.009, 0.013 and 0.012, respectively, which were the same value differences as produced by temperature raises of approximate 3.6, 3.5 and 3.1°C, respectively.

    2. Ordering effects of chlorhexidine digluconate on the rotational mobility of the hydrocarbon interior

    The effect of increasing concentrations of chlorhexidine digluconate on the anisotropy (r) of 2-AS in surface region of OPG, OPGTL and PL are shown in Fig. 5. Chlorhexidine digluconate increased the anisotropy (r) of 2-AS (decreased range of rotational mobility) in a dose-dependent manner, but the degrees of increases differed depending on the membrane lipid components. The significant increases in the anisotropy (r) values by the drug were observed at the 0.01, 0.10, 1.00, 2.00, 3.00, 4.00 and 5.00 mM (Fig. 5). The anisotropy (r) of 2-AS in surface region of OPG, OPGTL and PL are 0.145 ± 0.004, 0.124 ± 0.002 and 0.126 ± 0.001 at 37°C (pH 7.4), respectively. The anisotropy (r) of 2-AS in surface region of OPG, OPGTL and PL are 0.172 ± 0.006, 0.167 ± 0.003 and 0.166 ± 0.004 at 25°C (pH 7.4), respectively. Thus, the differences in the anisotropy (r) values of 2-AS in surface region of OPG, OPGTL and PL before and after adding 0.10 mM chlorhexidine digluconate were 0.010, 0.014 and 0.012 respectively, which were the same value differences as produced by the temperature fall of approximate 4.4, 3.9 and 3.6°C, respectively.

    Ⅳ. DISCUSSION

    The precise location of the molecular mechanism of pharmacological action has continued to be a subject of controversy to the present day. The current consensus is that chlorhexidine has a site(s) of action, situated within the cell membrane, probably on the inner and outer membranes. The present research carried out only a part of those aforementioned essential studies. PG (causative germs for gingivitis and periodontitis) was cultured and OPG were separated to be used as samples. OPGTL and PL were also used as samples.

    It is known that while 2-AS is distributed in the surface region of the cell membrane lipid bilayer, 12-AS is distributed in the hydrophobic interior of the cell membranes. The anthroyloxy stearate (AS) probes can be utilized to distinguish whether the bilayer has a fluidity gradient across it, as the anthroyloxy group can be positioned at different positions of the stearic acid moiety7,8). These probes have been suggested to membrane primarily the dynamic component of membrane fluidity17). Since rates of rotational mobility of membrane lipid bilayers can be measured in a rather simple method without measuring life time of a fluorescent probe, it is economical. Our data presented herein have shown that, even at physiologically relevant concentrations 18,19), chlorhexidine digluconate increases or decreases the rate of hydrocarbon interior and surface region of OPG, OPGTL and PL. This is due to differences in the intrinsic component and/or the structure in surface and hydrocarbon region of bacterial outer membranes and liposomes. The clear mechanism of the action(s) of the chlorhexidine digluconate as to its ordering and disordering effects on bacterial outer membranes and liposomes is unknown. However, the following presumption might be possible.

    Chlorhexidine binds to anionic groups on the bacterial surface, probably the phosphate groups of teichoic acid in gram-positive bacteria and phosphate groups of lipopolysaccharides in gram-negative bacteria2). The chlorhexidine digluconate binds (the competitive binding of the chlorhexidine digluconate and water) strongly to the phosphate moiety of the phospholipids (in the case of bacterial outer membranes and liposomes) and lipopolysaccharides (in the case of bacterial outer membranes-OPG) in the surface, and weakly to the carbonyl group in competition with water in the surface region and effectively establish formation of hydrogen bonds with the carbonyl moiety, which is associated with a significant change in hydration of the chlorhexidine digluconate molecule itself. The incorporation of the chlorhexidine digluconate into the bacterial outer membranes and liposomes leads to alterations of the surface charge density of the membrane's surface and then a conformational change in the phospholipid head groups. Simultaneously, they may have a significant effect on hydration of the lipid bilayer. Fisher et al.20) and Fisher and Quintana21) have demonstrated that ion-ion interactions occur between the dicationic chlorhexidine molecules and anionic carboxylate groups in stearic acid monolayers with a pH of 5.0-6.0 (but not with pH 3), and that the hexamethylene hydrophobic chain of the biguanide is constrained at the cell surface. They further pointed out that their findings demonstrated the ability of chlorhexidine to anchor to the polar head groups of the film-forming molecules. These results help explain the hydrophobicity-increasing effect of chlorhexidine on the bacterial cell surface20,21). As a result, such competitive binding increases hydrophobicity and decreases rotational mobility. The interaction between the dicationic chlorhexidine digluconate and the hydrocarbon region will generate rearrangements of the intermolecular hydrogen- bonded network among phospholipid molecules and/or protein molecules that are associated with the liberation of hydrated water molecules on the bacterial outer membranes and liposomes. The interaction will also alter the orientation of the P-N dipole of phospholipid molecules. These alterations should give rise to disordering of the hydrocarbon interior. Consequently, disordering and ordering effects of the chlorhexidine digluconate could exert an influence on the transport of small molecules in bacterial outer membranes, causing the bacteriostatic or bactericidal action.

    The results of Tsuchiya´s study6) are not consistent with the results of this study. However, this study´s results coincide with the results of the study about the chlorhexidine effects on fluidity of DPPC hydrocarbon interior employing the florescent prove PNA. These differences cannot be fully explained.

    The sensitivities to the increasing effect of the rotational mobility of hydrocarbon interior and decreasing effect of the surface mobility by the chlorhexidine digluconate differed depending on the bacterial outer membranes and liposomes in the descending order of the bacterial outer membranes and liposomes. It is without a doubt that the chlorhexidine digluconate increases the rotational mobility of hydrocarbon interior of the membranes and decreases the mobility of surface of the membranes. These effects are not merely due to the influence of the chlorhexidine digluconate on lipids, but they are amplified by the interaction between lipids and proteins. It can be inferred that the disordering or ordering effects that chlorhexidine induces on the bacterial membranes may play an important role in bacteriostatic and bactericidal actions even though the role might be indirect rather than direct and exclusive.

    Opinions have been divided as to whether chlorhexidine digluconate interfered with membrane protein function by directly binding to the proteins, or whether the main modes of action occurred indirectly through a change in the physicochemical properties of the lipid membranes into which the chlorhexidine digluconate readily diffused. Since biological membranes are of highly complex composition, it has not been practicable to monitor changes in the local lipid environment and to verify its effect on the membrane protein function at the same time. As found out in this study, it is certain that chlorhexidine digluconate decreases the rotational mobility of the surface region of model mem branes without protein and increases the rotational mobility of acyl-chains of phospholipids. However, we need to take notice of the fact that the effect of chlorhexidine digluconate are greater on the protein-present OPG than those on the protein-absent liposomes. There is no doubt that chlorhexidine digluconate has direct effects on lipids regardless whether they are of bacterial membranes or liposomes. Then, why is the magnitude of disordering or ordering effects by chlorhexidine digluconate greater in bacterial outer membranes with protein than in liposomes without protein?

    It is possible to explain the multiplication effects citing the increased or decreased mobility of protein triggered by lipids, but the reverse case of protein triggering change in lipids is more likely. It is certain that the chlorhexidine digluconate increases or decreases the mobility of bacterial outer membranes but the direct effects of the chlorhexidine digluconate on protein appear to have magnified such effects on the lipid. In other words, before or during or even after the interaction between the chlorhexidine digluconate and proteins, the fluidization of membrane lipids may furnish an ideal microenvironment for optimum bacteriostatic or bactericidal effects. In conclusion, the present data suggest that chlorhexidine digluconate, in addition to its direct interaction with proteins, concurrently interact with membrane lipids, affects the fluidity of the membrane, and thus induces conformational changes of proteins which are known to be tightly associated with membrane lipids.

    It was demonstrated that chlorhexidine at low concentrations is a potent membrane-active agent against both gram-positive and gram-negative bacteria, including the release of K+, 260 nm-absorbing material and pentoses5). Chlorhexidine causes membrane damage to neutrophils and macrophages with release of intracellular enzymes21). A concentration dependent release of hemoglobin by chlorhexidine was observed in isotonic media22). Chlorhexidine is also an inhibitor of adenosine triphosphatase (ATPase) activity 4). Applicable chlorhexidine levels (3.2 mmol/liter, or 0.2 %) strongly activated (up to fourfold) the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (a typical chymotrypsin substrate) by whole cells of Treponema denticola ATCC 35405 and the purified chymotrypsin-like proteinase 23). CHX is the most potent chemical plaque control agent that has various clinical applications in periodontics. Its broad antimicrobial spectrum can be considered as boon for maintaining overall oral health24). At higher bactericidal concentrations, chlorhexidine induces precipitation of cytoplasmic protein and nucleic acids4).

    We think that it is essential to investigate whether such above actions of chlorhexidine have any relationship with chlorhexidine´s ordering or disordering effects on bacterial outer membranes and liposomes discovered through future studies. If there is any relation, it would be also vital to investigate through which process is displayed in order to study chlorhexidine´s pharmacological mechanisms.

    ACKNOWLEDGMENTS

    This work was supported by a 2-Year Research Grant of Pusan National University.

    Figure

    KAOMP-45-3-77_F1.gif

    Effects of chlorhexidine digluconate on the anisotropy (r) of 16-(9-anthroyloxy)palmitic acid (16-AP) in OPG, OPGTL and PL. Fluorescence measurements were performed at 37°C (pH 7.4). Each point represents the mean ± SEM of 5 sample determinations. The asterisk and double asterisk denote P < 0.05 and P < 0.01, respectively, compared to the control according to a Student's t-test.

    KAOMP-45-3-77_F2.gif

    Effects of chlorhexidine digluconate on the anisotropy (r) of 12-(9-anthroyloxy)stearic acid (12-AS) in OPG, OPGTL and PL. Fluorescence measurements were performed at 37°C (pH 7.4). Each point represents the mean ± SEM of 5 sample determinations. The asterisk and double asterisk denote P < 0.05 and P < 0.01, respectively, compared to the control according to a Student's t-test.

    KAOMP-45-3-77_F3.gif

    Effects of chlorhexidine digluconate on the anisotropy (r) of 9-(9-anthroyloxy)stearic acid (9-AS) in OPG, OPGTL and PL. Fluorescence measurements were performed at 37°C (pH 7.4). Each point represents the mean ± SEM of 5 sample determinations. The asterisk and double asterisk denote P < 0.05 and P < 0.01, respectively, compared to the control according to a Student's t-test.

    KAOMP-45-3-77_F4.gif

    Effects of chlorhexidine digluconate on the anisotropy (r) of 6-(9-anthroyloxy)stearic acid (6-AS) in OPG, OPGTL and PL. Fluorescence measurements were performed at 37°C (pH 7.4). Each point represents the mean ± SEM of 5 sample determinations. The asterisk and double asterisk denote P < 0.05 and P < 0.01, respectively, compared to the control according to a Student's t-test.

    KAOMP-45-3-77_F5.gif

    Effects of chlorhexidine digluconate on the anisotropy (r) of 2-(9-anthroyloxy)stearic acid (2-AS) in OPG, OPGTL and PL. Fluorescence measurements were performed at 37°C (pH 7.4). Each point represents the mean ± SEM of 5 sample determinations. The asterisk and double asterisk denote P < 0.05 and P < 0.01, respectively, compared to the control according to a Student's t-test.

    Table

    Fluorescence parameter of 16-(9-anthroyloxy)palmitic acid (16-AP), 12-(9-anthroyloxy)stearic acid (12-AS), 9-(9-anthroyloxy)stearic acid (9-AS), 6-(9-anthroyloxy)stearic acid (6-AS), 3-(9-anthroyloxy)stearic acid (3-AS) and 2-(9-anthroyloxy)stearic acid (2-AS) in model membranes (OPGTL) of total lipids extracted from cultured Porphyromonas gingivalis outer membranes (OPG)

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