Biodegradability of end-groups of the biocide polyhexamethylene biguanide (PHMB) assessed using model compounds

Polyhexamethylene biguanide (PHMB), a biocide used in a wide variety of disinfection and preservation applications, is a polydisperse mixture in which the end-groups may be any combination of amine, guanidine and cyanoguanidine. Using PHMB model compounds (1,6-diaminohexane; 1,6-diguanidinohexane; 1,6-di(cyanoguanidino)hexane; 4-guanidinobutyric acid), we have determined the biodegradation characteristics of each end-group in several strains of bacteria isolated for their ability to utilise PHMB as a sole source of nitrogen. Bacteria were screened for growth at the expense of each model compound (at non-inhibitory concentrations) as sole nitrogen source. None of the isolated bacteria was capable of utilising a cyanoguanidine end-group as growth substrate, whereas several bacteria were shown to utilise amine or guanidine end-groups. In particular, a strain of Pseudomonas putida was capable of extensive growth with 1,6-diguanidinohexane as a sole nitrogen source, with complete removal of guanidine groups from culture medium within 2 days, and with concomitant formation of unsubstituted urea, which in turn was also utilised by the organism. We conclude that whilst amine and guanidine end-groups in PHMB are likely to be susceptible to biodegradation, cyanoguanidine end-groups are likely to be recalcitrant.     

Surface acetylation of polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane permeability

Improving the oral bioavailability of therapeutic compounds remains a challenging area of research. Polyamidoamine (PAMAM) dendrimers are promising candidates for oral drug delivery due to their well-defined compact structure, versatility of surface functionalities, low polydispersity, and ability to enhance transepithelial transport. However, potential cytotoxicity has hampered the development of PAMAM dendrimers for in vivo applications. In this article, we have systematically modified the surface groups of amine-terminated PAMAM dendrimers with acetyl groups. The effect of this modification on cytotoxicity, permeability, and cellular uptake was investigated on Caco-2 cell monolayers. Cytotoxicity was reduced by more than 10-fold as the number of surface acetyl groups increased while maintaining permeability across the cell monolayers. Furthermore, a decrease in nonspecific binding was evident for surface-modified dendrimers compared to their unmodified counterparts. These studies point to novel strategies for minimizing PAMAM dendrimer toxicity while maximizing their transepithelial permeability.     

Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB)

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed.

Methodology/Principal Findings

Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB’s DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB.

Conclusions

Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.     

The effect of polyhexamethylene guanidine hydrochloride (PHMG) derivatives introduced into polylactide (PLA) on the activity of bacterial enzymes

The present study was aimed at investigating bactericidal properties of polylactide (PLA) films containing three different polyhexamethylene guanidine hydrochloride (PHMG) derivatives and effect of the derivatives on extracellular hydrolytic enzymes and intracellular dehydrogenases. All PHMG derivatives had a slightly stronger bactericidal effect on Staphylococcus aureus than on E. coli but only PHMG granular polyethylene wax (at the concentration of at least 0.6 %) has a bactericidal effect. PHMG derivatives introduced into PLA affected the activity of microbial hydrolases to a small extent. This means that the introduction of PHMG derivatives into PLA will not reduce its enzymatic biodegradation significantly. On the other hand, PHMG derivatives introduced into PLA strongly affected dehydrogenases activity in S. aureus than in E. coli.     

Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences

Aims: The purpose of this study was to provide micrographic evidences for the damaged membrane structure and intracellular structure change of Escherichia coli strain 8099, induced by polyhexamethylene guanidine hydrochloride (PHMG). Methods and Results: The bactericidal effect of PHMG on E. coli was investigated based on β‐galactosidase activity assay, fluorescein‐5‐isothiocyanate confocal laser scanning microscopy, field emission scanning electron microscopy and transmission electron microscopy. The results revealed that a low dose (13 μg ml^?1) of PHMG slightly damaged the outer membrane structure of the treated bacteria and increased the permeability of the cytoplasmic membrane, while no significant damage was observed to the morphological structure of the cells. A high dose (23 μg ml^?1) of PHMG collapsed the outer membrane structure, led to the formation of a local membrane pore across the membrane and badly damaged the internal structure of the cells. Subsequently, intracellular components were leaked followed by cell inactivation. Conclusions: Dose‐dependent membrane disruption was the main bactericidal mechanism of PHMG. The formation of the local membrane pores was probable after exposure to a high dose (23 μg ml^?1) of PHMG. Micrographic evidences were provided about the damaged membrane structure and intracellular structure change of E. coli. Significance and Impact of the Study: The presented information helps understand the bactericidal mechanism of PHMG by membrane damage.     

Lysis of egg phosphatidylcholine vesicles by tricyclic carboxamide antitumor agents

The stability of small unilamellar vesicles formed by egg phosphatidylcholine has been examined in the presence of 38 tricyclic carboxamide DNA-intercalating agents (19 phenylquinolines, 17 phenylbenzimidazoles, an acridine and an anthracene). Lysis of the vesicular membrane is time-dependent and also dependent on the concentration of the cytotoxic agent. The relative concentration of agent to cause a fixed degree of lysis in a fixed time, as measured by the release of encapsulated 6-carboxyfluorescein, is directly related to the relative hydrophobicity of the agents.     

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin was investigated by measuring the amount released of a marker dye from the vesicles. In solutions of pH around 7, anionic and nonionic surfactants caused vesicle disintegration at very low concentrations, while cationic surfactants produced a breakdown of the vesicles at rather high concentrations. Increase in the alkyl chain-length of surfactant molecules brought about decrease in the surfactant concentration at which vesicle disintegration starts. As the length of the polyoxyethylene chain in nonionic surfactant molecules increased, the tendency of vesicle disintegration to occur decreased. Both anionic and cationic surfactants gave clear solutions above their critical micelle concentrations when they acted on the phospholipid vesicles, whereas nonionic surfactants left ghost cell-like debris consisting of carboxymethylchitin molecules in their micellar solutions. The effect of pH on vesicle disintegration was notable for ionic surfactants but not for nonionic surfactants. Thus, anionic surfactants increased the degree of disintegration as pH increased, while cationic surfactants produced an identical vesicle disintegration curve below pH 8 above which the curve started to shift toward the lower concentration region of the agents. These findings were explained in terms of surfactant penetration into phospholipid bilayers and solubilization of phospholipid molecules by surfactant micelles.     

Influence of immobilized quaternary ammonium group surface density on antimicrobial efficacy and cytotoxicity

Bacterial colonization of medical devices causes infections and is a significant problem in healthcare. The use of antibacterial coatings is considered as a potential solution to this problem and has attracted a great deal of attention. Using concentration density gradients of immobilized quaternary ammonium compounds it was demonstrated that a specific threshold of surface concentration is required to induce significant bacterial death. It was determined that this threshold was 4.18% NR₄⁺ bonded nitrogen with a surface potential of + 120.4 mV. Furthermore, it is shown for the first time that adhesion of constituents of the culture medium to the quaternary ammonium modified surface eliminated any cytotoxicity towards eukaryotic cells such as primary human fibroblasts. The implications of this type of surface fouling on the antimicrobial efficacy of surface coatings are also discussed.     

Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

Interaction kinetics of salmeterol with egg phosphatidylcholine liposomes by surface plasmon resonance

Surface plasmon resonance (SPR) Biacore™ and equilibrium dialysis were applied to investigate the membrane affinities of salmeterol and propranolol and the kinetic interactions of salmeterol with egg phosphatidylcholine liposomes. The two methods revealed similar affinity values; however, they were dependent on the investigated drug concentrations. The kinetic experiments with salmeterol were optimized to obtain pseudo-first-order kinetics that were independent of the drug concentration. The adsorption and desorption phases followed biexponential functions up to pH 8.8 and mono or biexponential functions at higher pH values (i.e., between the two pK_a values). The apparent rate constants of the faster phases of the biexponential functions were beyond the time resolution of the instrument in most measurements. The apparent rate constants of the slower phases ranged from 0.01 to 0.03 s⁻¹ and were pH independent between pH 5.0 and pH 8.0. The rates of the monoexponential kinetics were between 0.08 and 0.12 s⁻¹. We conclude that the biexponential kinetics at physiological pH reflect the partitioning into the outer lipid leaflet and “flip-flop,” respectively, of the cationic species.     

Protective effect of phenolic antioxidants on the cytotoxicity induced by phosphatidylcholine hydroperoxide

Phosphatidylcholine hydroperoxides show weak but distinct toxicity toward cultured human umbilical vein endothelial cells. The protective effect of phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides was examined. Probucol depressed the toxicity most effectively among the antioxidants studied under both pretreatment and concurrent treatment conditions. alpha-Tocopherol showed a protective effect in the case of concurrent treatment. Protection by phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides seems to depend on their incorporation rate into cells, their affinity for phospholipids, their antioxidative activity, and their orientation in membranes.     

Microbial degradation of the biocide polyhexamethylene biguanide: isolation and characterization of enrichment consortia and determination of degradation by measurement of stable isotope incorporation into DNA

AIMS: To isolate micro-organisms capable of utilizing polyhexamethylene biguanide (PHMB) as a sole source of nitrogen, and to demonstrate biodegradation of the biocide. METHODS AND RESULTS: Two consortia of bacteria were successfully enriched at the expense of PHMB, using sand from PHMB-treated swimming pools as inoculum. Both consortia were shown to contain bacteria belonging to the genera Sphingomonas, Azospirillum and Mesorhizobium. It was shown that the presence of both Sphingomonas and Azospirillum spp. was required for extensive growth of the consortia. In addition, the Sphingomonads were the only isolates capable of growth in axenic cultures dosed with PHMB. Using a stable isotope (15N)-labelled PHMB, metabolism of the biocide by both consortia was demonstrated. By comparing the level of 15N atom incorporation into bacterial DNA after growth on either 15N-PHMB or 15N-labelled NH4Cl, it was possible to estimate the percentage of PHMB biodegradation. CONCLUSIONS: The microbial metabolism of nitrogen from the biguanide moiety of PHMB has been demonstrated. It was revealed that Sphingomonas and Azospirillum spp. are the principal organisms responsible for growth at the expense of PHMB. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the microbial metabolism of PHMB.     

Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins)

Ceragenins are cationic bile salt derivatives having antimicrobial activity. The interactions of several ceragenins with phospholipid bilayers were tested in different systems. The ceragenins are capable of forming specific associations with several phospholipid species that may be involved with their antimicrobial action. Their antimicrobial activity is lower in bacteria that have a high content of phosphatidylethanolamine. Gram negative bacteria with a high content of phosphatidylethanolamine exhibit sensitivity to different ceragenins that corresponds to the extent of interaction of these compounds with phospholipids, including the ability of different ceragenins to induce leakage of aqueous contents from phosphatidylethanolamine-rich liposomes. A second class of bacteria having cell membranes composed largely of anionic lipids and having a low content of phosphatidylethanolamine are very sensitive to the action of the ceragenins but they exhibit similar minimal inhibitory concentrations with most of the ceragenins and for different strains of bacteria. Although Gram negative bacteria generally have a high content of phosphatidylethanolamine, there are a few exceptions. In addition, a mutant strain of Escherichia coli has been made that is essentially devoid of phophatidylethanolamine, although 80% of the lipid of the wild-type strain is phosphatidylethanolamine. Furthermore, certain Gram positive bacteria are also exceptions in that they can have a high content of phosphatidylethanolamine. We find that the antimicrobial action of the ceragenins correlates better with the content of phosphatidylethanolamine in the bacterial membrane than whether or not the bacteria has an outer membrane. Thus, the bacterial lipid composition can be an important factor in determining the sensitivity of bacteria to antimicrobial agents.     

Perturbation of egg phosphatidylcholine and dipalmitoylphosphatidylcholine multilamellar vesicles by n-alkanols. A fluorescent probe study

The perturbing effects of n-alkanols (pentanol, decanol and tetradecanol) in egg phosphatidylcholine and dipalmitoylphosphatidylcholine multilamellar vesicles were studied with five fluorescent probes, 1-(4'-trimethylaminophenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene, and 2-, 7-, and 12-(9-anthroxyloxy)stearic acid (2-, 7-, and 12-AS). These probes localize at various depths in the membrane, enabling study of the membrane-order gradient. Phase-modulation fluorescence spectroscopy was used to measure steady-state anisotropies, excited-state lifetimes and differential polarized lifetimes from which the limiting hindered anisotropies (r infinity) and the logarithm of the rotational rate (log R) were calculated. The probes that localize at about the same depth in the membrane (TMA-DPH and 2-AS, diphenylhexatriene and 12-AS) generally, but not always, showed similar changes in r infinity and log R with added alkanols. However, the absolute values of r infinity and log R were usually different. The inconsistencies are attributed to differences in the probes' sizes, structures, photophysical properties and perturbing abilities. The perturbation of membranes by alkanols is chain-length-dependent. Pentanol disorders the membrane at all depths but is more effective in the membrane center than nearer to the polar headgroups of the phospholipids, tetradecanol can be accommodated into the membrane without effect or with increased order and the effects of decanol are intermediate between pentanol and tetradecanol. Our results with alkanols indicate that: a single perturber can have different effects on membrane order at different depths in the bilayer; the perturbation is observed at and distant from the perturbers' location in the membrane, and the bilayer center is more susceptible to perturbation by alkanols than the region of the bilayer near the phospholipid headgroups.     

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.     

Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins)

Ceragenins are cationic bile salt derivatives having antimicrobial activity. The interactions of several ceragenins with phospholipid bilayers were tested in different systems. The ceragenins are capable of forming specific associations with several phospholipid species that may be involved with their antimicrobial action. Their antimicrobial activity is lower in bacteria that have a high content of phosphatidylethanolamine. Gram negative bacteria with a high content of phosphatidylethanolamine exhibit sensitivity to different ceragenins that corresponds to the extent of interaction of these compounds with phospholipids, including the ability of different ceragenins to induce leakage of aqueous contents from phosphatidylethanolamine-rich liposomes. A second class of bacteria having cell membranes composed largely of anionic lipids and having a low content of phosphatidylethanolamine are very sensitive to the action of the ceragenins but they exhibit similar minimal inhibitory concentrations with most of the ceragenins and for different strains of bacteria. Although Gram negative bacteria generally have a high content of phosphatidylethanolamine, there are a few exceptions. In addition, a mutant strain of Escherichia coli has been made that is essentially devoid of phophatidylethanolamine, although 80% of the lipid of the wild-type strain is phosphatidylethanolamine. Furthermore, certain Gram positive bacteria are also exceptions in that they can have a high content of phosphatidylethanolamine. We find that the antimicrobial action of the ceragenins correlates better with the content of phosphatidylethanolamine in the bacterial membrane than whether or not the bacteria has an outer membrane. Thus, the bacterial lipid composition can be an important factor in determining the sensitivity of bacteria to antimicrobial agents.     

Detoxification of lipopolysaccharide (LPS) by egg white lysozyme

Abstract Recent studies carried out by our group suggest that lysozyme binds to bacterial lipopolysaccharide with a high affinity to produce a complex, and inhibits various biological activities of lipopolysaccharide. Although the basic structure of lipopolysaccharide is independent of the species and strains of Gram-negative bacteria, many structural factors such as O-antigenic polysaccharide, lipid A, substituted groups, and associated molecules, affect the biological activities of lipopolysaccharide. In this study, we prepared lysozyme/lipopolysaccharide complexes using various structures of lipopolysaccharide and compared the activity and physiochemical properties. Native and dansylated lysozyme were found to bind to all tested lipopolysaccharides. The mitogenic activity and TNF production by all tested lipopolysaccharides were significantly reduced by complex formation in vitro. Administration of the complex prepared by various lipopolysaccharides produced significantly less quantities of TNF in the septic shock model. These results suggested that binding of lysozyme to lipopolysaccharide is important for the host both in pathophysiological responses to lipopolysaccharides and in the modification of lipopolysaccharide biological activity.     

Inhibition of autoxidation of egg yolk phosphatidylcholine in homogenous solution and in liposomes by oxidized ubiquinone

The aim of this study was to obtain a quantification of the antioxidant activity of ubiquinone. To this purpose the oxidation of egg yolk phosphatidylcholine both in solvent and in liposomes initiated by an azocompound has been studied either in the absence or in the presence of ubiquinone-3, using alpha-tocopherol as a reference antioxidant. The two experimental systems gave similar results. In the presence of ubiquinone-3 the oxidation rate was reduced with respect to control experiments but was faster than that in the presence of alpha-tocopherol. The amount of ubiquinone required to decrease the autoxidation rate was so high as to prevent detection of the induction period. The stoichiometric factor was greater than 2 and the rate constant of inhibition was two orders of magnitude lower than that of alpha-tocopherol. It is concluded that high concentrations of ubiquinone are required to exhibit significant antioxidant activity. A possible mechanism compatible with the stoichiometric factor larger than 2 for the inhibiting effect of ubiquinone is also suggested.     

Antagonism of xylazine hydrochloride-ketamine hydrochloride immobilization in guineafowl (Numida meleagris) by yohimbine hydrochloride

The mean time to arousal (MTA), the mean time to sternal recumbency (MTSR) and the mean time to walking (MTW) were measured in 10 adult guineafowl (Numida meleagris) immobilized with a combination of xylazine hydrochloride (1 mg/kg) and ketamine hydrochloride (25 mg/kg). Yohimbine hydrochloride, given intravenously (1 mg/kg) at 40 min after the injection of the xylazine-ketamine, significantly shortened the MTA, the MTSR and the MTW compared to saline controls. Increasing the dosage of yohimbine to 2.5 mg/kg did not shorten recovery when compared to the lower dosage. No adverse effects were noted at either dosage of yohimbine. Yohimbine appeared to be a safe and effective antagonist of xylazine-ketamine immobilization in guineafowl and may prove useful in other avian species to produce more rapid recovery from xylazine-ketamine immobilization, xylazine sedation or xylazine overdosage.