Biosurfactant complexed with arginine has antibiofilm activity against methicillin-resistant Staphylococcus aureus.

Aim: The present study investigated the antimicrobial effectiveness of a rhamnolipid complexed with arginine (RLMIX_Arg) against planktonic cells and biofilms of methicillin-resistant Staphylococcus aureus (MRSA). Methodology: Susceptibility testing was performed using the Clinical & Laboratory Standards Institute protocol: M07-A10, checkerboard test, biofilm in plates and catheters and flow cytometry were used. Result: RLMIX_Arg has bactericidal and synergistic activity with oxacillin. RLMIX_Arg inhibits the formation of MRSA biofilms on plates at sub-inhibitory concentrations and has antibiofilm action against MRSA in peripheral venous catheters. Catheters impregnated with RLMIX_Arg reduce the formation of MRSA biofilms. Conclusion: RLMIX_Arg exhibits potential for application in preventing infections related to methicillin-resistant S. aureus biofilms.

[Box: see text].

Arginine Gemini-Based Surfactants for Antimicrobial and Antibiofilm Applications: Molecular Interactions, Skin-Related Anti-Enzymatic Activity and Cytotoxicity.

The antimicrobial and antibiofilm properties of arginine-based surfactants have been evaluated. These two biological properties depend on both the alkyl chain length and the spacer chain nature. These gemini surfactants exhibit good activity against a wide range of bacteria, including some problematic resistant microorganisms such us methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Moreover, surfactants with a C10 alkyl chain and C3 spacer inhibit the (MRSA) and Pseudomonas aeruginosa biofilm formation at concentrations as low as 8 µg/mL and are able to eradicate established biofilms of these two bacteria at 32 µg/mL. The inhibitory activities of the surfactants over key enzymes enrolled in the skin repairing processes (collagenase, elastase and hyaluronidase) were evaluated. They exhibited moderate anti-collagenase activity while the activity of hyaluronidase was boosted by the presence of these surfactants. These biological properties render these gemini arginine-based surfactants as perfect promising candidates for pharmaceutical and biological properties.

Zein Nanoparticles Containing Arginine-Based Surfactants: Physicochemical Characterization and Effect on the Biological Properties.

Cationic surfactants carry antimicrobial activity, based on their interaction and disruption of cell membranes. Nonetheless, their intrinsic toxicity limits their applicability. To overcome this issue, a feasible strategy consists of using solid nanoparticles to improve their delivery. The zein nanoparticles were loaded with four cationic arginine-based surfactants: one single chain Nα-lauroyl-arginine (LAM) and three Gemini surfactants Nα Nω-Bis (Nα-lauroyl-arginine) α, ω-diamide) (C3(LA)2, C6(LA)2 and C9(LA)2). Blank and loaded zein nanoparticles were characterized in terms of size, polydispersity and zeta potential. Furthermore, the antimicrobial activity against bacteria and yeasts and the hemolytic activity were investigated and compared to the surfactants in a solution. Nanoparticles were found to be monodisperse, presenting a size of between 180-341 nm, a pdI of <0.2 and a positive zeta potential of between +13 and +53 mV, remaining stable over 365 days. The nanoencapsulation maintained the antimicrobial activity as unaltered, while the extensive hemolytic activity found for the surfactants in a solution was reduced drastically. Nuclear Magnetic Ressonance (NMR), molecular docking and monolayer findings indicated that zein entraps the surfactants, interfering in the surfactant-membrane interactions. Accordingly, the nanoepcasulation of arginine surfactants improved their selectivity, while the cationic charges were free to attack and destroy bacteria and fungi; the aliphatic chains were not available to disrupt the cellular membranes.

Zein Nanoparticles Containing Arginine-Phenylalanine-Based Surfactants: Stability, Antimicrobial and Hemolytic Activity.

Although cationic surfactants have a remarkable antimicrobial activity, they present an intrinsic toxicity that discourages their usage. In this work novel zein nanoparticles loaded with arginine-phenylalanine-based surfactants are presented. The nanoparticles were loaded with two single polar head (LAM and PNHC12) and two with double amino acid polar head surfactants, arginine-phenylalanine (C12PAM, PANHC12). The formulations were characterized and their stability checked up to 365 days. Furthermore, the antimicrobial and hemolytic activities were investigated. Finally, NMR and molecular docking studies were carried out to elucidate the possible interaction mechanisms of surfactant-zein. The nanoparticles were obtained with satisfactory size, zeta potential and dispersibility. The surfactants containing arginine-phenylalanine residues were found to be more stable. The nanoencapsulation maintained the antimicrobial activities unaltered in comparison to the surfactants' solutions. These results are in agreement with the NMR and docking findings, suggesting that zein interacts with the surfactants by the aromatic rings of phenylalanine. As a result, the cationic charges and part of the aliphatic chains are freely available to attack the bacteria and fungi, while not available to disrupt the cellular membranes. This approach opens new possibilities for using cationic surfactants and benefits from their extraordinary antimicrobial responses for several applications.

Cationic Surfactants Based on Arginine-Phenylalanine and Arginine-Tryptophan: Synthesis, Aggregation Behavior, Antimicrobial Activity, and Biodegradation.

Cationic surfactants have great potential as drug vehicles and for use in gene therapy (cationic vesicles made from cationic surfactants can encapsulate RNA or DNA for cellular transfer). They can also be used as antimicrobial and antifungal agents to treat human infections. In an era of increasing antimicrobial resistance, the development of new biocompatible surfactants suitable for application as antimicrobial agents is of high interest. In this work, a library of amino acid-based surfactants was synthesized, characterized and tested for antimicrobial activity. The head group architecture (number and type of amino acids, density of cationic charge, ionic character) and the hydrophobic moiety (alkyl chain length and position of the hydrophobic group) were systematically modified, and the effect on the surfactant biological and aggregation behavior was studied. Thus, the pKa values, micellization process, antimicrobial efficiency and biodegradability were evaluated. The critical micelle concentration values of the surfactants depended on their hydrophobic character, but changes in the polar head as well as the position and length of the alkyl chain also significantly affected activity against some of the tested microorganisms. Moreover, biodegradability was closely related to the hydrophobic character of the surfactant and attachment of the alkyl chain to the polar head. The structure-activity relationships established here may open perspectives for the design of effective biodegradable antimicrobial materials that can overcome emerging resistance.

Arginine-phenylalanine and arginine-tryptophan-based surfactants as new biocompatible antifungal agents and their synergistic effect with Amphotericin B against fluconazole-resistant Candida strains.

In the past two decades, the increase in microbial resistance to conventional antimicrobials has spurred scientists around the world to search tirelessly for new treatments. Synthetic amino acid-based surfactants constitute a promising alternative to conventional antimicrobial compounds. In this work, two new cationic amino acid-based surfactants were synthesized and their physicochemical, antifungal and antibiofilm properties evaluated. The surfactants were based on phenylalanine-arginine (LPAM) and tryptophan-arginine (LTAM) and prepared from renewable raw materials using a simple chemical procedure. The critical micelle concentrations of the new surfactants were determined by conductivity and fluorescence. Micellization of LPAM and LTAM took place at 1.05 and 0.54 mM, respectively. Both exhibited good antifungal activity against fluconazole-resistant Candida spp. strains, with a low minimum inhibitory concentration (8.2 µg/mL). Their mechanism of action involves alterations in cell membrane permeability and mitochondrial damage, leading to death by apoptosis. Furthermore, when LPAM and LTAM were applied with Amphotericin B, a significant synergistic effect was observed against all the studied Candida strains. These new cationic surfactants are also able to disperse biofilms of Candida spp. at low concentrations. The results indicate that LPAM and LTAM have potential application to combat the advance of fungal resistance as well as microbial biofilms.

Aggregation Behavior, Antibacterial Activity and Biocompatibility of Catanionic Assemblies Based on Amino Acid-Derived Surfactants.

The surface activity, aggregates morphology, size and charge characteristics of binary catanionic mixtures containing a cationic amino acid-derived surfactant N(π), N(τ)-bis(methyl)-L-Histidine tetradecyl amide (DMHNHC14) and an anionic surfactant (the lysine-based surfactant Nα-lauroyl-Nεacetyl lysine (C12C3L) or sodium myristate) were investigated for the first time. The cationic surfactant has an acid proton which shows a strong pKa shift irrespective of aggregation. The resulting catanionic mixtures exhibited high surface activity and low critical aggregation concentration as compared with the pure constituents. Catanionic vesicles based on DMHNHC14/sodium myristate showed a monodisperse population of medium-size aggregates and good storage stability. According to Small-Angle X-Ray Scattering (SAXS), the characteristics of the bilayers did not depend strongly on the system composition for the positively charged vesicles. Negatively charged vesicles (cationic surfactant:myristate ratio below 1:2) had similar bilayer composition but tended to aggregate. The DMHNHC14-rich vesicles exhibited good antibacterial activity against Gram-positive bacteria and their bactericidal effectivity declined with the decrease of the cationic surfactant content in the mixtures. The hemolytic activity and cytotoxicity of these catanionic formulations against non-tumoral (3T3, HaCaT) and tumoral (HeLa, A431) cell lines also improved by increasing the ratio of cationic surfactant in the mixture. These results indicate that the biological activity of these systems is mainly governed by the cationic charge density, which can be modulated by changing the cationic/anionic surfactant ratio in the mixtures. Remarkably, the incorporation of cholesterol in those catanionic vesicles reduces their cytotoxicity and increases the safety of future biomedical applications of these systems.

Biocompatible Catanionic Vesicles from Arginine-Based Surfactants: A New Strategy to Tune the Antimicrobial Activity and Cytotoxicity of Vesicular Systems.

Their stability and low cost make catanionic vesicles suitable for application as drug delivery systems. In this work we prepared catanionic vesicles using biocompatible surfactants: two cationic arginine-based surfactants (the monocatenary Nα-lauroyl-arginine methyl ester-LAM and the gemini Nα,Nϖ-bis(Nα-lauroylarginine) α, ϖ-propylendiamide-C3(CA)2) and three anionic amphiphiles (the single chain sodium dodecanoate, sodium myristate, and the double chain 8-SH). The critical aggregation concentration, colloidal stability, size, and charge density of these systems were comprehensively studied for the first time. These catanionic vesicles, which form spontaneously after mixing two aqueous solutions of oppositely charged surfactants, exhibited a monodisperse population of medium-size aggregates and good stability. The antimicrobial and hemolytic activity of the vesicles can be modulated by changing the cationic/anionic surfactant ratio. Vesicles with a positive charge efficiently killed Gram-negative and Gram-positive bacteria as well as yeasts; the antibacterial activity declined with the decrease of the cationic charge density. The catanionic systems also effectively eradicated MRSA (Methicillin-resistant Staphylococcus Aureus) and Pseudomonas aeruginosa biofilms. Interestingly, the incorporation of cholesterol in the catanionic mixtures improved the stability of these colloidal systems and considerably reduced their cytotoxicity without affecting their antimicrobial activity. Additionally, these catanionic vesicles showed good DNA affinity. Their antimicrobial efficiency and low hemolytic activity render these catanionic vesicles very promising candidates for biomedical applications.

Antifungal and antiprotozoal green amino acid-based rhamnolipids: Mode of action, antibiofilm efficiency and selective activity against resistant Candida spp. strains and Acanthamoeba castellanii.

Nowadays, infections caused by fungi and protists constitute a serious problem for public health services. The limited number of treatment options coupled with the increasing number of resistant microorganisms makes necessary the development of new non-toxic antifungal and antiprotozoal agents. Cationic amino acid-based rhamnolipids have been recently prepared by our group and exhibited good antibacterial activity. In this work, the antifungal, antibiofilm and antiprotozoal activity of these new rhamnolipids was investigated against a collection of fluconazole-resistant strains of different Candida species and Acanthamoeba castellanii, respectively. The arginine-RLs exhibited good antifungal activity against all fluconazole-resistant Candida spp. strains tested at MICs ranging from 6.5 to 20.7 mg/L. Their mechanism of action involves alterations in the permeability of the cell membranes that provoke death by apoptosis. The Arginine based-RLs also disperse Candida biofilms at low concentrations, similar to the MICs. All RLs tested (anionic and cationic) showed antiprotozoal activity, the arginine derivatives had the best activity killing the Acanthamoeba castellanii at concentrations of 4 mg/L. Interestingly, these surfactants have a wide range of action against yeast and A. castellanii in which they do not show toxicity against keratinocytes and fibroblasts. These results indicate that these new rhamnolipids have a sufficiently wide safety margin to be considered good candidates for several pharmaceutical applications such as combating fungal resistance and microbial biofilms and the formulation of antiprotozoal drugs.

Rhamnolipids functionalized with basic amino acids: Synthesis, aggregation behavior, antibacterial activity and biodegradation studies.

Rhamnolipids have been intensively studied due to their remarkable properties; however, the biosynthesis of RLs cannot compete commercially with the production of synthetic surfactants. Here, novel cationic rhamnolipids (RLs) derivatives containing arginine and lysine were prepared for the first time using a straightforward synthetic procedure. The RLs used to prepare these new cationic derivatives were produced by Pseudomonas aeruginosa using waste frying oil as carbon source. It was found that the amino acid-based RLs form aggregates at very low concentrations, even below the CMC. Biodegradation studies indicate that these cationic RLs can be classified as readily biodegradable. Interestingly, the RL arginine conjugates exhibited notable DNA binding affinity and good antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, which increases the potential applications of these compounds. Consequently, the use of low-cost substrates and the added value of the final product constitute a more cost-effective rhamnolipid production.

A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study.

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120⁻290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.

Pharmaceutical versatility of cationic niosomes derived from amino acid-based surfactants: Skin penetration behavior and controlled drug release.

The natural capability shown by cationic vesicles in interacting with negatively charged surfaces or biomolecules has recently attracted increased interest. Important pharmacological advantages include the selective targeting of the tumour vasculature, the promotion of permeation across cell membranes, as well as the influence of cationic vesicles on drug delivery. Accordingly, cationic amphiphiles derived from amino acids may represent an alternative to traditional synthetic cationic surfactants due to their lower cytotoxicity. The importance of a synthesized lysine-based gemini surfactant (labelledC6(LL)2) was evaluated in drug delivery by designing cationic niosomes as usable pharmaceutical tools of chemotherapeutics and antibiotics, respectively like methotrexate and tetracycline. The influence of formulation factors on the vesicles' physical-chemical properties, drug entrapment efficiency, in vitro release and ex-vivo skin permeation were investigated. A niosomal gel containing the gemini surfactant was also tested as a viable multi-component topical formulation. Results indicate that in the presence of cholesterol, C6(LL)2 was able to form stable and nanosized niosomes, loading hydrophilic or hydrophobic molecules. Furthermore, in vitro release studies and ex-vivo permeation profiles showed that C6(LL)2-based vesicles behave as sustained and controlled delivery systems in the case of parenteral administration, and as drug percutaneous permeation enhancers after topical application. Finally, cationic C6(LL)2 acts as a carrier constituent, conferring peculiar and interesting functionality to the final formulation.

Green Catanionic Gemini Surfactant-Lichenysin Mixture: Improved Surface, Antimicrobial, and Physiological Properties.

Catanionic surfactant mixtures form a wide variety of organized assemblies and aggregates with improved physicochemical and biological properties. The green catanionic mixture NαNω-Bis(Nαcaproylarginine) α,ω-propyldiamide (C3(CA)2):Lichenysin (molar ratio 8:2) showed antimicrobial synergies against Yersinia enterocolitica, Bacillus subtilis, Escherichia coli O157:H7, and Candida albicans. Flow cytometry and viability studies indicated that this catanionic mixture increases the probability of Y. enterocolitica (38.2%) and B. subtilis (17.1%) cells entering a viable but nonculturable state. Zeta potential showed that one of the cationic charges of C3(CA)2 is neutralized by Lichenysin. An isotherm study demonstrated the formation of a stable aggregate between the two surfactants. The catanionic aggregate was able to interact with bacterial phospholipids. The lowest hemolysis (22.1 µM) was obtained with the catanionic mixture, although an irritant potential (0.70) was characterized. According to the therapeutic index, the C3(CA)2:Lichenysin mixture was the formulation least toxic to eukaryotic cells. Partial neutralization of C3(CA)2 by Lichenysin modified the mode of action that enhances the transition of bacterial cells into a viable but nonculturable state (VBNC) and improved the cell selectivity.

Lichenysin-geminal amino acid-based surfactants: Synergistic action of an unconventional antimicrobial mixture.

Recently it has been demonstrated that catanionic mixtures of oppositely charged surfactants have improved physicochemical-biological properties compared to the individual components. Isotherms of mixtures of an anionic biosurfactant (lichenysin) and a cationic aminoacid surfactant (C3(LA)2) indicate a strong interaction suggesting the formation of a new "pseudo-surfactant". The antimicrobial properties of the mixture lichenysin and C3(LA)2 M80:20, indicate a synergistic effect of the components. The mechanism of action on the bacterial envelope was assessed by flow cytometry and Transmission Electron Microscopy.

Interfacial chiral selection by bulk species.

A chiral selection process in a self-assembled soft monolayer of an achiral amphiphile as a consequence of its interaction with chiral species dissolved in the aqueous subphase, is reported. The extent of the chiral selection is statistically measured in terms of the enantiomorphic excess of self-assembled submillimeter domains endowed with well-defined orientational chirality that is unambiguously resolved using optical microscopy. Our results show that the emergence of chirality is mediated by electrostatic interactions and significantly enhanced by hydrophobic effects. This chiral chemical effect can be suppressed and even reversed by opposing a macroscopic physical influence, such as vortical stirring. This result gives evidence for the crucial role of hydrodynamic effects in supramolecular aggregation.

Gemini surfactants from natural amino acids.

In this review, we report the most important contributions in the structure, synthesis, physicochemical (surface adsorption, aggregation and phase behaviour) and biological properties (toxicity, antimicrobial activity and biodegradation) of Gemini natural amino acid-based surfactants, and some potential applications, with an emphasis on the use of these surfactants as non-viral delivery system agents. Gemini surfactants derived from basic (Arg, Lys), neutral (Ser, Ala, Sar), acid (Asp) and sulphur containing amino acids (Cys) as polar head groups, and Geminis with amino acids/peptides in the spacer chain are reviewed.

Mixed monolayer of DPPC and lysine-based cationic surfactants: an investigation into the antimicrobial activity.

In this paper, we report studies which aim to elucidate the mechanisms involved in the antimicrobial activity of three cationic lysine-based surfactants: LLM, LALM, and C6 (LL)2. To this end, a simple membrane model (i.e., 1,2-dipalmitoyl-sn-phosphatidylcholine, DPPC) was used to explore the monolayer properties at the air/liquid interface. Compression π-A isotherms of mixtures of DPPC/lysine surfactants at different pH showed an expansion of the DPPC monolayer, suggesting cationic lysine surfactant/DPPC interactions, which strongly depend on surfactant structure and hydrophobic interactions. Antimicrobial activity of the three surfactants has also been assessed with transmission electron microscopy, observing the effects on Staphylococcus aureus and Escherichia coli . The three surfactants caused various kinds of damage to the bacteria tested, such as structural alterations, leakage of internal material, and cell destruction.

Self assembly of pH-sensitive cationic lysine based surfactants.

Three cationic surfactants of the type N(ε)-acyl lysine methyl ester hydrochloride have been studied with respect to solution behavior and adsorption on the air/water interface, as well as the thermolyotropic behavior. The self-assembly of these surfactants, which have the cationic charge on amine protonated groups, was assessed by different physicochemical methods. Depending on the pH value, these surfactants can dissociate in aqueous solutions, losing the cationic charge. Therefore, knowledge of the pK(a) of these compounds is essential to explain their behavior in aqueous solutions. The bulk techniques, conductivity, and nuclear magnetic resonance diffusion (NMR) obtained similar critical micellar concentration (CMC) values, which were well above those obtained from surface tension. Surface tension measurements were strongly dependent on the technique used, namely, Wilhelmy plate and pendant drop. The phase behavior at medium to high concentrations has been studied by optical polarizing microscopy and small angle x-ray scattering (SAXS). The X-ray studies showed that the lysine-based surfactants at low hydration have rich thermotropic liquid crystalline behavior. The results are discussed in terms of the structure of the compounds and the cationic charge of the molecule. We will show how apparently small changes in molecule structure have a large influence on phase behavior.

Thermoresponsive hydrogels with low toxicity from mixtures of ethyl(hydroxyethyl) cellulose and arginine-based surfactants.

Ethyl(hydroxyethyl) cellulose (EHEC) is known to form hydrogels in water at elevated temperatures in the presence of an ionic surfactant. In this paper, the potential use of arginine-based surfactants is explored considering the production of a low toxicity thermoresponsive hydrogel for pharmaceutical and biomedical applications. The interactions between EHEC and the monomeric surfactant N(α)-lauroyl-L-arginine methyl ester (LAM) and two gemini surfactants N(α),N(ω)-bis(N(α)-acylarginine) α,ω-dialkyl amides were evaluated by Rheo-Small Angle Light Scattering measurements. The complex viscosity of the systems was dependent on surfactant concentration and temperature. Under specific conditions, soft gels of homogeneous structure were produced. The cloud point (CP) of the EHEC-LAM system varied significantly with surfactant concentration, while only moderate CP changes were found in the presence of the gemini surfactants. Finally, the effect of the surfactants on the viability of a human cell line was evaluated. Despite the lower toxicity of LAM, the superior gel forming efficiency of the gemini surfactants at lower concentrations revealed their advantageous suitability as components of a biocompatible thermoresponsive gel system.

pH-Sensitive surfactants from lysine: assessment of their cytotoxicity and environmental behavior.

The toxicity and environmental behavior of new pH-sensitive surfactants from lysine are presented. Three different chemical structures are studied: surfactants with one amino acid and one alkyl chain, surfactants with two amino acids on the polar head and one alkyl chain, and gemini surfactants. The pH sensitivity of these compounds can be tuned by modifying their chemical structures. Cytotoxicity has been evaluated using erythrocytes and fibroblast cells. The toxic effects against these cells depend on the hydrophobicity of the molecules as well as their cationic charge density. The effect of hydrophobicity and cationic charge density on toxicity is different for each type of cells. For erythrocytes, the toxicity increases as hydrophobicity and charge density increases. Nevertheless, for fibroblasts cationic charge density affects cytotoxicity in the opposite way: the higher charge density, the lower the toxicity. The effect of the pH on hemolysis has been evaluated in detail. The aquatic toxicity was established using Daphnia magna . All surfactants yielded EC(50) values considerably higher than that reported for cationic surfactants based on quaternary ammonium groups. Finally, their biodegradability was evaluated using the CO(2) headspace test (ISO 14593). These lysine derivatives showed high levels of biodegradation under aerobic conditions and can be classified as "readily biodegradable compounds".