Mixed Langmuir Monolayers Research Articles

Impact of cholesterol on the structure and phase separation of DPPC Langmuir monolayers: Experiments and simulations

Cholesterol is a lipid that plays a key role mainly in the plasma membrane of animal cells, determining their structural and mechanical properties. While Langmuir monolayers have been a traditional model for studying these membranes, they may not fully capture microscopic phenomena. We studied mixed Langmuir monolayers of DPPC and cholesterol in various proportions, using a Langmuir trough for surface compression isotherms and micro-Brewster Angle Microscopy (MicroBAM) for imaging. Furthermore, atomistic Molecular Dynamics (MD) simulations were used to examine molecular distributions, geometric conformations, and emergence of a gas phase in the monolayers. Both experimental and simulation results were compared to obtain a comprehensive understanding. The results reveal strong attraction between DPPC tails and cholesterol, increasing monolayer stiffness and density. These interactions align the lipid tails more perpendicularly to the water surface, effectively minimizing their projected area and preventing tail entanglement. Consequently, the reduced flexibility triggers a phase separation in the monolayer at smaller areas per lipid, with low-density voids and denser regions, as observed in both MicroBAM and simulations. These outcomes emphasize the critical role of cholesterol in biological membranes. Finally, the consistency of MD simulations with experiments validate them as a valuable tool for investigating lipid monolayer behavior.

Surface miscibility of Gemini surfactants and DOPE in binary mixed monolayers

AbstractSurface pressure (π)–molecular area (A) isotherms were gathered to characterize the packing of binary mixed Langmuir monolayers of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE) and one of two Gemini surfactants (GS), N,N‐bis(dimethyloctadecyl)‐1,7‐nonanediammonium dibromide (18‐7‐18) or 1,9‐bis(octadecyl)‐1,1,9,9‐tetramethyl‐5‐amino‐1,9‐nonanediammonium dibromide (18‐7NH‐18) of varying molar fractions. Information about miscibility behavior was derived from the π–A curves by examining the excess free energy of mixing (ΔGexc) that was calculated through the surface area additivity rule. Surface compressibility modulus (Cs−1) was also used to characterize intermolecular interactions. Mutual interactions between GS and DOPE were analyzed in terms of excess Gibbs energy of mixing and the value of this parameter depended strongly on the composition of the mixed film. GS and DOPE are generally miscible as DOPE reduces intermolecular repulsion between highly charged GS molecules leading to the formation of more densely packed mixed monolayers. However, GS and DOPE are immiscible in equimolar mixtures due to tail group packing mismatch between saturated and unsaturated alkyl chains. The prevalence of attractive synergistic interactions in the monolayers studied differs from a previous finding of antagonistic mixing behavior in GS/DOPE micelles. These results contribute to the understanding of GS‐lipid interactions and packing that are critical to the in vitro and in vivo stability of liposomes composed of these molecules used for non‐viral gene therapy applications.

Effect of γ-Oryzanol on the LE-LC Phase Coexistence Region of DPPC Langmuir Monolayer.

We have studied the effect of relative composition of γ-Oryzanol (γ-Or) on the liquid expanded-liquid condensed phase coexistence region in the mixed Langmuir monolayer of γ-Or and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) molecules at air-water interface. The surface manometry studies at a fixed temperature show that the mixture of γ-Or and DPPC forms a stable monolayer at air-water interface. As the relative composition of γ-Or increases the range of area per molecule over which the coexistence of liquid expanded (LE)-liquid condensed (LC) phases exists reduces. Although the LE-LC phase coexistence corresponds to the first-order phase transition, the slope of the surface pressure-area per molecule isotherm is non-zero. Earlier studies have attributed the non-zero slope in LE-LC phase coexistence region to the influence of the strain between the ordered LC phase and disordered LE phase. The effect of strain on the coexistence of LE-LC phases can be studied in terms of molecular density-strain coupling. Our analysis of the liquid condensed-liquid expanded coexistence region in the isotherms of mixed monolayers of DPPC and γ-Or shows that with the increase in the mole fraction of sterol in the mixed monolayer the molecular lateral density-strain coupling increases. However, at 0.6 mole fraction of γ-Or in the mixed monolayer the coupling decreases. This is corroborated by the observation of minimum Gibb's free energy of the mixed monolayer at this relative composition of γ-Or indicating better packing of molecules.

Regulation of calcium ions on the interaction between amphotericin B and cholesterol-rich phospholipid monolayer in LE phase and LC phase

Amphotericin B, as a “gold standard”, is used to treat invasive fungal infections. The AmB molecule can bind easily to cholesterol and damage cell membranes, so it produces the toxicity on cell membrane, which limits its clinical dose. However, the interaction between AmB and cholesterol-rich membrane is unclear now. The phase state of the membrane and the metal cation outside cell membrane may affect the interaction between AmB and the membrane. In this work, the effects of amphotericin B on the mean molecular area, elastic modulus and stability of mammalian cell membrane rich in cholesterol in the presence of Ca2+ ions were studied using DPPC/Chol mixed Langmuir monolayer as a model. The Langmuir-Blodgett method and AFM test were used to study the effects of this drug on the morphology and height of cholesterol-rich phospholipid membrane in the presence of Ca2+ ions. The influence of calcium ions on the mean molecular area and the limiting molecular area was similar in LE phase and in LC phase. The calcium ions made the monolayer more condensed. However, calcium ions can weaken the shortening effect of AmB on the relaxation time of the DPPC/Chol mixed monolayer in LE phase but enhance it in LC phase. Interestingly, calcium ions caused a LE-LC coexistence phase to occur in the DPPC/Chol/AmB mixed monolayers at 35mN/m, which was confirmed by atomic force microscopy. The results can help to understand the interaction between amphotericin B and cell membrane rich in cholesterol in the calcium ions environment.

Milk sphingosomes as lipid carriers for tocopherols in aqueous foods: Thermotropic phase behaviour and morphology

Foods containing polyunsaturated lipids are prone to oxidation. Designing food-grade hydrocolloidal encapsulation systems able to load lipophilic antioxidant molecules, such as tocopherols (vitamin E), is necessary to prevent oxidation and its deleterous consequences. In this study, we hypothesised that α-tocopherol molecules could incorporate in a host membrane composed of milk sphingomyelin (milk-SM) and performed a multi-scale biophysical study. The thermal properties of milk-SM bilayers with various molar proportions of α-tocopherol were characterised by differential scanning calorimetry (DSC), their structural properties were examined by X-ray diffraction (XRD). The miscibility between milk-SM and α-tocopherol was investigated in mixed Langmuir monolayers. The morphology of milk-SM sphingosomes was observed by confocal laser scanning microscopy (CLSM). We found that molecules of α-tocopherol inserted into the milk-SM bilayers and induced a physical desorganisation in the membrane packing, both in the ordered and fluid states. In the presence of α-tocopherol, the bilayers were no longer in a gel phase below the phase transition temperature Tm, but in the liquid ordered Lo phase. Furthermore, the sphingosomes formed elongated structures in presence of α-tocopherol as a result of membrane softening and changes in the bilayer curvature associated to membrane fusion. The findings of this work contribute in a better understanding of the capacity of milk-SM bilayers to incorporate guest molecules. Milk-SM sphingosomes loaded with tocopherols could be used to prevent oxidation in aqueous foods containing polyunsaturated lipids such as oil-in-water emulsions.

Dominant hydrophobic interactions with β-glucan in nanoarchitectonics with mixed Langmuir monolayers of cholesterol/dipalmitoyl phosphatidyl choline.

The polysaccharide β-glucan, found in the cell wall of cereals such as wheat, oats, and barley, is believed to lower the concentration of bad cholesterol in humans, but the molecular-level mechanisms responsible for such an action are unknown. In this study, we use Langmuir monolayers of cholesterol and dipalmitoyl phosphatidyl choline (DPPC) as cell membrane models that are made to interact with β-glucan. Neat cholesterol and mixed cholesterol/DPPC monolayers were expanded upon incorporating β-glucan from the aqueous subphase. This incorporation was found to induce ordering in mixed monolayers and dehydration of the carbonyl group at higher cholesterol concentrations. These effects are attributed to hydrophobic interactions as identified with polarization-modulated infrared reflection-absorption spectroscopy. They correlate well with the hypothesis that cholesterol levels can be lowered by the formation of soluble fibers with β-glucan through hydrophobic interactions, blocking cholesterol absorption by the organism.

Two-dimensional synthesis of silver nanoparticle in situ Langmuir films from the reduction of silver sulfadiazine

Two–dimensional (2D) arrangement of monodisperse metallic nanoparticles (NPs) has attracted attention because of the unique properties of this assembly. These properties offer new perspectives for several applications. Thin film preparation techniques involve the synthesis of NPs through chemical methods and their subsequent attachment to various substrates based on different interactions. Langmuir–Blodgett (LB) technique is a promising method, which offers possibilities of in-situ preparation of metallic NP-structured and ordered monolayer films. In this study, 2D AgNPs were synthesized in situ through the reduction of a Langmuir monolayer consisting of a mixture of silver sulfadiazine (AgSD) molecules and stearic acid (SA) surfactant at the air-water interface. Two different Ag reduction methods were used. First, a NaBH4 aqueous solution was used as the subphase to react with the mixed Langmuir monolayer films. Second, the mixed films were irradiated by an ultraviolet (UV) light source (λ = 254 nm) for different time intervals. Scanning electron microscope images, energy-dispersive X-ray spectra, and contact angle measurements show that AgSD was an efficient precursor for the synthesis of Ag NPs LB films with long-range order. A small amount of SA is sufficient to cause the arrangement of the NPs, prevent their aggregation, and provide a hydrophobic characteristic to the Ag NPs LB films. Furthermore, the chemical reduction method using NaBH4 is better than the reduction under UV light for the synthesis of Ag NPs arrangement.

Maze Pattern at Nanometer-Scale in a Mixed Langmuir Monolayer of Fatty Acids

Abstract We investigated the morphology of binary monolayers of palmitic acid and behenic acid using atomic force microscopic observations. The monolayers exhibited a phase-separated morphology composed of meandering domains with a width of nanometer order, which is probably due to fixation of the monolayer morphology at a stage on the way to phase separation.

Surface Chemistry Studies on the Formation of Mixed Stearic Acid/Phenylalanine Dehydrogenase Langmuir and Langmuir-Blodgett Films.

This work investigates the physicochemical properties of mixed stearic acid (HSt)/phenylalanine dehydrogenase enzyme (PheDH) Langmuir films and their immobilization onto solid supports as Langmuir-Blodgett (LB) films. PheDH from the aqueous subphase enters the surfactant matrix up to an exclusion surface pressure of 25.3 mN/m, leading to the formation of stable and highly condensed mixed Langmuir monolayers. Hydrophobic interactions between the enzyme and HSt nonpolar groups tuned the secondary structure of PheDH, evidenced by the presence of β-sheet structures as demonstrated by infrared and circular dichroism spectra. The floating monolayers were successfully transferred to solid quartz supports, yielding Y-type LB films, and then characterized employing fluorescence, circular dichroism, and microscopic techniques, which indicated that PheDH was co-immobilized with HSt proportionally to the number of transferred layers. The enzyme fluidized the HSt monolayers, reducing their maximum dipoles when condensed to their maximum, and disorganized the alkyl chains of the fatty acid, as detected with infrared spectroscopy. The stability of the mixed floating monolayers enabled their transfer to solid supports as LB films, which is important for producing optical and electrochemical sensors for phenylalanine whose molecular architecture can be controlled with precision.

Peptidoglycans modulating the interaction of a bactericide compound with lipids at the air-water interface

A known monoterpene, named γ-terpineol, was incorporated in mixed Langmuir monolayers composed of dipalmitoyl-phosphoethanolamine (DPPE) and peptidoglycans as a model of microbial membranes. Surface pressure and surface potential isotherms, dynamical surface rheology, Brewster angle microscopy (BAM), and infrared spectroscopy were employed to characterize the compound-membrane interactions. The compound expanded the monolayers denoting repulsive interactions. At 30 mN/m, the monolayer presented lower viscoelastic and in-plane elasticity parameters and an increased all-trans/gauche conformers ratio for the alkyl chains, confirming molecular order. The morphology of the monolayer was analyzed by BAM, which revealed a heterogeneous distribution of γ-terpineol along the mixed monolayer, which tends to segregate. In conclusion, the compound changes the thermodynamic, electric, rheological, morphological, and structural properties of the peptidoglycan-DPPE monolayer, which may be essential to understand, at the molecular level, the action of bioactives in selected membrane models.

Thermodynamic quantification of sodium dodecyl sulfate penetration in cholesterol and phospholipid monolayers

Sodium dodecyl sulfate (SDS) is one of the extensively used surfactants in bioprocesses. Present work analyzes the penetration of SDS in pure and mixed Langmuir monolayers consisting of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol. A series of isotherms are obtained by compressing the monolayers over subphase containing varying concentrations of SDS. The extent of penetration of SDS is quantified in terms of its mole percentage in the monolayer by applying various thermodynamic equations derived from Gibbs adsorption equation. Analysis of pure monolayers shows that SDS penetration is directly correlated with the monolayer fluidity and the lipid packing density. We find that POPC monolayers, which do not pack closely on compression due to a kink in one of the acyl chains, exhibit higher SDS penetration than DPPC monolayers which are more rigid due to two saturated acyl chains that pack very efficiently. The SDS penetration is also analyzed in mixed lipid monolayers in which the lipids are present in molar proportions similar to that in the plasma membranes of mammalian cells. Thermodynamic quantification shows that mixed monolayers of DPPC with POPC (in 2:1 molar ratio) exhibit penetration similar to pure POPC monolayers indicating dominating effect of unsaturated phospholipids on membrane packing. We find lower SDS penetration in mixed cholesterol-POPC monolayers than pure POPC monolayers at all surface pressures and for all SDS subphase concentrations used. This confirms with the well-known condensation effect of cholesterol on POPC monolayers. Cholesterol is known to have a rigidifying effect on DPPC monolayers at low surface pressures and a fluidizing effect at high surface pressures. Consistent with these experimental observations, our thermodynamic quantification predicts lower SDS penetration at low pressures and higher penetration at high pressures when compared to pure DPPC monolayers. Thus, thermodynamic theories are able to accurately predict the experimentally observed trends for penetration in mixed monolayers of cholesterol with POPC and DPPC. Our work demonstrates that thermodynamic quantification is a reliable technique to estimate extent of penetration of various additives in pure and mixed lipid monolayers.

Mixed Langmuir Monolayers of C18 Fatty Acids: Effect of Degree of Saturation

The degree of unsaturation of hydrocarbon chains in the lipid diversity of biological membrane will greatly influence the membrane protein function. Lipid-lipid interactions in the membrane domain are crucial in the development of novel liposomal nanocarrier as the stability and function of liposomes are important points to be considered. The embedded therapeutic proteins are dependent on lipid membrane composition and their physical properties. In this study, we elucidated lipid-lipid interactions in the biological membrane domain with energetic quantitative data by mixing any two C18 fatty acids namely, stearic acid (SA), oleic acid (L1), linoleic acid (L2), and linolenic acid (L3) in Langmuir-Blodgett trough with water subphase. In general, SA has a saturated hydrocarbon chain; meanwhile, L1, L2 and L3 have increasing degree of unsaturation in their respective hydrocarbon chain. The tail-to-tail interactions were studied by the combinations of SA/L1, SA/L2, SA/L3, L1/L2, L1/L3 and L2/L3 at various mole ratios ranging from 9:1, 8:2, 7:3 ... to 1:9. Among the mixtures of C18 fatty acids, L2/L3 is considered as the energetically stable at the optimum amount of XL2 and XL3 = 0.5 respectively, at all discrete surface pressure.

Mechanical Properties of DPPC-POPE Mixed Langmuir Monolayers.

The mechanical properties of lipid monolayers and their responses to shear and compression stresses play an important role in processes such as breathing and eye blinking. We studied the mechanical properties of Langmuir monolayers of a model mixture, composed of an unsaturated lipid, 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE), and a saturated lipid, 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC). We performed isothermal compressions and sinusoidal shear deformations of these mixed monolayers. Also, the different phases were observed with Brewster angle microscopy. We found that the mechanical behavior is affected by the miscibility of both lipids. In the two-phase region, the compression elastic modulus increases with the amount of the LC phase but does not follow the predictions of a simple effective medium model. The discrepancies arise from the fact that, upon compression, the domains grow at a rate faster than the compression rate but not fast enough to reach thermodynamic equilibrium. Before reaching the LC phase, domain percolation is observed and compression and shear moduli become equal to those of the pure LC phase. Most of the monolayers behave as viscoelastic fluids at the frequencies investigated. A minimum in the compression modulus and shear viscosity was observed for mixtures close to equimolar composition, with the minimum being accompanied by a change in domain shapes.

Nonradiative energy transfer in mixed Langmuir monolayers and Langmuir–Blodgett films of compounds of different chemical composition and structure

The efficiency of the Forster resonance energy transfer (FRET) in a monolayer film containing the energy donor and energy acceptor fluorophores is low since the energy transfer process occurs in one plane only. As the number of layers increases to five, the energy transfer efficiency increases due to the interlayer energy transfer occurring in three dimensions. Further increase in the number of layers (up to 10) causes no significant increase in the efficiency since in this case the total film thickness exceeds an optimum value for the FRET process.

Study of aggregation behavior of water insoluble metalloporphyrin (Zn) in LB film

In the present communication, a water insoluble metalloporphyrin, 5,10,15,20-Tetraphenyl-21H,23H-porphine zinc (abbreviated as ZnTPP) has been used to prepare stable Langmuir monolayer at the air–water interface by allowing it to adsorb from the aqueous subphase, into the preformed Langmuir monolayer of stearic acid (SA). Surface pressure versus area per molecule (π-A) isotherm studies gave the thermodynamical behavior of the ZnTPP-SA mixed Langmuir monolayer formed at the air water interface. Access area per molecule (AE) vs molefraction curve indicated the repulsive interaction between the two binary components in the mixed Langmuir monolayer. BAM images gave the visual evidence of the aggregation pattern of the mixed molecules at the air-water interface. UV-vis spectroscopic studies confirmed the formation of prominent J-band in the ultra-thin films.

Emerging investigator series: exploring the surface properties of aqueous aerosols coated with mixed surfactants.

Mixed Langmuir monolayers of cholesterol with both saturated and unsaturated fatty acids, stearic acid (SA), and oleic acid (OA) spread at the air-seawater surface were studied. The phase behavior, molecular interaction, and conformational order of the monolayers were investigated by surface pressure-area (π-A) isotherms and infrared reflection-absorption spectroscopy (IRRAS) measurements. The thermodynamic parameters of the mixed films, including excess molecular area and excess Gibbs free energy were calculated by using the isotherm data. The interaction between SA (or OA) and cholesterol varied with the molar fraction of the fatty acids and surface pressure. OA/chol monolayers showed the characteristics of miscibility, but they acted as nonideal systems. Cholesterol has been observed to have a stabilizing effect on OA monolayers. The negative values of the excess Gibbs free energy in the entire composition range demonstrated that mixed OA/chol monolayers were thermodynamically stable. IRRAS spectra showed that mixing with cholesterol changes the ordering of fatty acid monolayers at the air-seawater surface. The findings provide general information regarding the structural changes in the monolayer induced by lateral packing. These results help in the understanding of the mixing behavior of fatty acids and cholesterol and provide insights into the fate of the mixed-monolayer-coated sea salt aerosol in the ocean environment.

Mixing Behavior in Binary Anionic Gemini Surfactant-Perfluorinated Fatty Acid Langmuir Monolayers.

The miscibility and film structure of mixed Langmuir monolayer films composed of an anionic gemini N,N,N',N'-dialkyl-N,N'-diacetate ethylenediamine surfactant (Ace(12)-2-Ace(12)) with perfluorotetradecanoic acid (C13F27COOH; PF) have been investigated using a variety of thermodynamic and structural characterization methods. The two film components were found to be miscible in monolayers at the air-water interface over a range of compositions and at all but the lowest surface pressures, with attractive interactions occurring between the two components. While pure PF monolayers formed crystalline lattices with hexagonal symmetry and with the surfactant tails oriented normal to the underlying water subphase, the pure gemini surfactant formed amorphous films with little tendency to orient at the subphase. In mixed films with mole ratios of PF:Ace(12)-2-Ace(12) < 2.5, the miscibility of the two components resulted in a nearly complete loss of crystallinity of the PF, though films at higher mole fractions of PF showed some residual crystallinity, albeit with lattice structures that were significantly different from that of pure PF. Miscibility and film structure in this mixed system are discussed in comparison with other mixed gemini surfactant systems in the literature as well as binary mixtures of phospholipids or monomeric fatty acids with PF.

Organization and structure of mixed Langmuir films composed of polydiacetylene and hemicyanine

Mixed Langmuir monolayers of 10,12-Pentacosadiynoic acid (DA) monomer and an amphiphilic Hemicyanine dye derivative have been formed at the air/water interface. Two derivatives of docosylpyridinium have been used, with either one included in the monolayer in 1:1molar ratio. The DA monomers within the mixed monolayers have been polymerized in situ at the air/water interface. The crystalline structure of the monolayer and the kinetics of polymerization have been probed by grazing incidence X-ray diffraction (GIXD). The polymerization of DA proceeds with no phase segregation, exclusively leading to the red polydiacetylene form. The kinetics of polymerization at the air/water interface has been monitored in situ by GIXD. The experimental results have been combined with Molecular Mechanics computer simulations, revealing that DA molecules are sequentially arranged with molecules of Hemicyanine dye in alternating rows. The hydrophobic chains of the dye molecules act as spacers between the DA monomers. Surprisingly, such molecular arrangement does not hinder the in situ photopolymerization of DA. The mechanism of polymerization of DA within the mixed Langmuir monolayers has been convincingly described in molecular detail. This approach for interfacial polymerization of DA holds great potential for optically active devices and nanostructures comprising self-assembled thin films based in polydiacetylene.

Mixed Langmuir monolayers of perfluorostearic acid and stearic acid studied by epifluorescence microscopy using fluorinated rhodamines and infrared reflection absorption spectroscopy (IRRAS)

The fluorophilicity of fluorinated rhodamine-based fluorescence dyes (F-rhodamines) functionalized with CH2-C3F7 (Rh-CH2-C3F7) or C2H4-C10F21 (Rh-C2H4-C10F21) at both amine groups was tested in mixed monolayers of stearic acid (SA) and perfluorostearic acid (FC18) on a Langmuir trough coupled with epifluorescence microscopy. The composition of the Langmuir monolayers was systematically changed from 100% FC18 to 100% SA in order to investigate the fluorophilicity and lipophilicity of the two F-rhodamines by observing their partitioning behavior within the different monolayers. The F-rhodamine bearing the longer perfluoroalkyl group and alkyl spacer (Rh-C2H4-C10F21) showed a substantially higher affinity to the FC18 rich phases in contrast to the F-rhodamine having the shorter perfluoroalkyl chain and alkyl spacer (Rh-CH2-C3F7) which was excluded from FC18 as well as from SA domains. The FC18, SA, and FC18/SA 50:50 (mol%) monolayers were further investigated by infrared reflection absorption spectroscopy (IRRAS) in order to investigate the phase behavior of FC18 and SA. It was observed that the orientations of FC18 and SA molecules in the pure and mixed monolayers do not change upon compression. Furthermore, a decrease in the order of the FC18 molecules in the mixed monolayer is observed upon the addition of SA. The opposite is not valid in a sense that no loss in order of the SA molecules was observed upon the addition of FC18. This shows that SA partitions more into FC18 phases than FC18 into SA domains. This is due to the weaker van der Waals forces present between the perfluoroalkyl chains in comparison with stronger ones existing between the alkyl chains.

Variable and low-toxic polyampholytes: complexation with biological membranes

In this paper, we describe three series of polyampholytes synthesized via quaternization of poly(4-vinylpyridin) by ω-bromocarboxylic acids and alkyl bromides: (1) with cationic and anionic groups in each unit (polybetaines), (2) with betaine and cationic groups, and (3) with betaine and pendant alkyl groups. The polymers were complexed with anionic mixed lipid membranes, liposomes, and Langmuir monolayers. By varying a length of –(CH2)n– spacer in the betaine group, different behaviors of polybetaines in a suspension of anionic liposomes can be realized: from no interaction to complexation followed by significant structural reorganization in the liposomal membrane. Cytotoxicities of polyampholytes are one to two orders of magnitude less than the cytotoxicity of a pure polycationic polymer with the same degree of polymerization. These results are of importance in designing polyelectrolytes with a higher affinity to the biolodical (cell) membrane and minimum cytotoxicity and demonstrate the potential of polyampholytes in developing biocompatible polymeric structures.