Tuning supramolecular structuring at the nanoscale level: nonstoichiometric soluble complexes in dilute mixed solutions of alginate and lactose-modified chitosan (chitlac)

Two oppositely charged polysaccharides, alginate and a lactose-modified chitosan (chitlac), have been used to prepare dilute binary polymer mixtures at physiological pH (7.4). Because of the negative charge on the former polysaccharide and the positive charge on the latter, polyanion-polycation complex formation occurred. A complete miscibility between the two polysaccharides was attained in the presence of both high (0.15 M) and low (0.015 M) concentrations of simple 1:1 supporting salt (NaCl), as confirmed by turbidity measurements; phase separation occurred for intermediate values of the ionic strength (I). The binary solutions were further characterized by means of light scattering, specific viscosity, and fluorescence quenching measurements. All of these techniques pointed out the fundamental role of the electrostatic interactions between the two oppositely charged polysaccharides in the formation of nonstoichiometric polyelectrolyte soluble complexes in dilute solution. Fluorescence depolarization (P) experiments showed that the alginate chain rotational mobility was impaired by the presence of the cationic polysaccharide when 0.015 M NaCl was used. Moreover, upon addition of calcium, the P values of the binary polymer mixture in 0.015 M NaCl increased more rapidly than that of an alginate solution without chitlac, suggesting an efficient crowding of the negatively charged alginate chains caused by the polycation.     

Polysaccharide-based polyanion--polycation--polyanion ternary systems. A preliminary analysis of interpolyelectrolyte interactions in dilute solutions

The present contribution deals with the preparation and characterization of ternary mixtures of polysaccharides with potential applications in the field of tissue engineering. Two natural polyanions, i.e., alginate and hyaluronic acid, and a polycation, a lactose-modified chitosan (chitlac), were mixed in dilute conditions. The miscibility between the three components was explored in the presence of different amounts of supporting simple salt. These analyses allowed to identify the experimental conditions avoiding polymer phase separation and leading to either solution of independent polymers or soluble nonstoichiometric interpolyelectrolyte complexes. The characterization of the interpolyelectrolyte complexes was tackled by means of viscometry, light scattering, fluorescence quenching, and energy transfer. The electrostatic interactions taking place among the different polyelectrolytes led to synergistic effects on the viscosity of the polymer mixtures which strongly depend on the ionic strength. It has been found that, starting from binary soluble complexes of alginate and chitlac, the addition of hyaluronan led to the dissolution of the complexes. At variance, the addition of alginate to a phase-separated binary mixture of hyaluronan and chitlac led to the formation of soluble complexes composed of all three polysaccharides and, eventually, to their dissolution. In addition, the results showed that at low ionic strength the overall properties of the ternary mixtures depend on their order of mixing.     

Synthesis and rheological properties of responsive thickeners based on polysaccharide architectures

New thermothickening copolymers were synthesized by grafting responsive poly(ethylene oxide-co-propylene oxide) [PEPO] onto three different polysaccharide backbones: carboxymethylcellulose [CMC], alginate [ALG], and carboxylated dextran [DEX]. The coupling reaction between carboxylic groups of biopolymers and the terminal amine of PEPO was activated at low temperature ( T < 10 degrees C) in water by using carbodiimide and N-hydroxysuccinimide. In these conditions it was shown that the formation of amide bonds strongly depends on the concentration of reactive groups, which is limited by the viscosity of the polymer sample. While a full conversion was obtained for the low molecular weight dextran, the efficiency of grafting remains low (between 30 to 40%) for CMC and alginate, which give a solution of high viscosity even at low concentration. When studied in the semidilute regime, all the copolymer solutions clearly exhibit thermothickening behavior with a large and reversible increase of viscosity upon heating. The association temperature and the gelation threshold were shown to depend on polymer concentration as it is expected from the phase diagram of PEPO precursor. Similarly, the influence of added salt on PEPO solubility in water has been used to control the self-assembling behavior of copolymer formulations. The relative comparison between the three copolymers reveals that the amplitude of the viscosity jump induced by heating mainly depends on the proportion of responsive material inside the macromolecular architecture rather than the dimensions of the main chain. The high increase of viscosity, which can reach several orders of magnitude between 20 degrees C and body temperature, clearly demonstrates the potentiality of these copolymers in biomedical applications like injectable gels for tissue engineering.     

DNA-polycation complexation and polyplex stability in the presence of competing polyanions

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.     

Rheological and structural characterization of the interactions between cyclodextrin compounds and hydrophobically modified alginate

Interactions in semidilute solutions of a hydrophobically modified alginate (HM-alginate) in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) monomer or a beta-cyclodextrin polymer (poly(beta-CD)) have been characterized at different temperatures with the aid of rheology and small-angle neutron scattering (SANS). The viscosity results for the HM-alginate/HP-beta-CD system reveal progressive deactivation of the hydrophobic associations as the concentration of HP-beta-CD increases. For the HM-alginate/poly(beta-CD) system, on the other hand, addition of poly(beta-CD) sets up bridges between adjacent polymer chains and thereby strengthens the associative network. A novel shear-thickening effect is observed at fairly high shear rates for the HM-alginate/poly(beta-CD) system, and this feature is influenced by temperature. Elevated temperature induces higher chain mobility and the formation of weaker network associations. Analyses of the SANS data disclosed that the association strength in HM-alginate/poly(beta-CD) mixtures increases strongly with increasing cosolute concentration, whereas no effect or a moderate weakening of the strength can be traced in HM-alginate/HP-beta-CD solutions upon addition of HP-beta-CD. The value of the correlation length xi is virtually not affected by the addition of cosolute for the HM-alginate/poly(beta-CD) system, whereas the decoupling of hydrophobic moieties of the polymer upon addition of HP-beta-CD gives rise to a smaller value of xi, suggesting that the size of the heterogeneity patches is reduced. The SANS results suggest that compact association structures are formed in the HM-alginate/poly(beta-CD) solutions.     

Viscoelastic properties of an exopolysaccharide: Aeromonas gum, produced by Aeromonas nichidenii 5797

The viscoelastic properties of Aeromonas (A) gum in water were investigated by using the Rheometric Scientific ARES controlled strain rheometer. An intrinsic viscosity of 8336 ml/g was obtained according to the Fuoss-Straus equation. The effect of salt concentration on intrinsic viscosity revealed that the A gum exists as semiflexible chain. Typical shear-thinning (pseudoplastic) behavior was observed at concentrations higher than 0.52%. The zero shear viscosity (eta(0)) increased with increasing polysaccharide concentration (c) showing a gradient of approximately 1.0, 2.9 and 4.8 in different concentration domains. The critical concentrations c* and c**, at which the transitions from a dilute solution of independently moving chains to semidilute and then concentrated domains occurred, were determined roughly to be 1.2% and 3.5%. The results from dynamic experiments revealed that the A gum solution shows characteristics of polymer solutions without any evidence of gel-like character. All the results from steady and dynamic tests suggest that the A gum is a non-gelling polysaccharide. The temperature dependence of apparent viscosity was described by Arrhenius equation and the flow activation energy was estimated to be 45.2 kJ/mol, which is independent on polymer concentration.     

Pullulan-sodium alginate based edible films: Rheological properties of film forming solutions

Rheological properties of pullulan, sodium alginate and blend solutions were studied at 20 °C, using steady shear and dynamic oscillatory measurements. The intrinsic viscosity of pure sodium alginate solution was 7.340 dl/g, which was much higher than that of pure pullulan (0.436 dl/g). Pure pullulan solution showed Newtonian behavior between 0.1 and 100 s⁻¹ shear rate range. However, increasing sodium alginate concentration in pullulan-alginate blend solution led to a shear-thinning behavior. The effect of temperature on viscosities of all solutions was well-described by Arrhenius equation. Results from dynamical frequency sweep showed that pure sodium alginate and blend solutions at 4% (w/w) polymer concentration were viscoelastic liquid, whereas the pure pullulan exhibited Newtonian behavior. The mechanical properties of pure sodium alginate and pullulan-alginate mixture were analyzed using the generalized Maxwell model and their relaxation spectra were determined. Correlation between dynamic and steady-shear viscosity was analyzed with the empirical Cox-Merz rule.     

Electrochemical assay of protease activities based on polycation/polyanion complex as substrate and polyion sensitive membrane electrode detection

A novel electrochemical method to detect protease activities is demonstrated. The assay is based on the use of a macromolecular polycation/polyanion substrate; specifically, a complex of the arginine-rich peptide protamine and pentosan polysulfate (PPS), a highly sulfated polysaccharide. As the protease of interest cleaves the protamine within the complex into smaller fragments, free PPS is generated and detected potentiometrically via a polyanion sensitive membrane electrode. Thus, the rate of free PPS generation is proportional to the activity of the protease in the assay solution. The effect of the substrate concentration is examined, as is the influence of the protamine/PPS stoichiometry on the assay performance. Using the optimized composition and concentration of the complex, the determination of trypsin at levels down to 5 U/ml and plasmin at levels approaching 0.002 U/ml can be achieved in a 10 min period. The prospects of further adapting this scheme to determine clot-busting plasminogen activators (e.g. streptokinase, tissue plasminogen activator, etc.) in samples as complex in whole blood are discussed.     

Salt effects on the cross-linking mechanism of cupric-induced sol—gel transition in alginate solutions

The viscosity behaviour of alginate-Cu²⁺-NaCl systems has been experimentally examined at various concentrations of cupric and sodium salts. Dependence of the intrinsic viscosity of alginate as a function of NaCl concentration is discussed to supplement the previous study which shows a similar behaviour to that found for other polyelectrolytes in aqueous solution in the presence of an added salt. The effects of sodium ions on the cupric association in cupric-induced alginate solutions were investigated by means of viscosity measurements. The mechanisms of complex formation in the presence of the simple added salt were studied. It was found that, at a given NaCl concentration, the viscosity of the mixture will pass through a maximum with increasing cupric concentration. The amounts of cupric cations corresponding to the maximum depends on the concentration of NaCl in the solution. Comparison of salt effects on the viscosity behaviour of alginate solutions during sol—gel transition reveals that an optimum NaCl concentration of 10⁻² mol 1⁻¹ exists where the viscosity of the mixture gives a maximum value at a certain cupric amount. This result indicates that salt effects play an important role in the sol—gel transition of the polyelectrolyte solutions. The observed phenomenon was interpreted in terms of conformational change of polyelectrolyte chain due to the addition of salt resulting in a different cross-linking mode in the system.     

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.     

Shear and extensional rheology of cellulose/ionic liquid solutions

In this study, we characterize the shear and extensional rheology of dilute to semidilute solutions of cellulose in the ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIAc). In steady shear flow, the semidilute solutions exhibit shear thinning, and the high-frequency complex modulus measured in small amplitude oscillatory shear flow exhibits the characteristic scaling expected for solutions of semiflexible chains. Flow curves of the steady shear viscosity plotted against shear rate closely follow the frequency dependence of the complex viscosity acquired using oscillatory shear, thus satisfying the empirical Cox-Merz rule. We use capillary thinning rheometry (CaBER) to characterize the relaxation times and apparent extensional viscosities of the semidilute cellulose solutions in a uniaxial extensional flow that mimics the dynamics encountered in the spin-line during fiber spinning processes. The apparent extensional viscosity and characteristic relaxation times of the semidilute cellulose/EMIAc solutions increase dramatically as the solutions enter the entangled concentration regime at which fiber spinning becomes viable.     

Triple interpolyelectrolyte complexes DNA-polycation-polyanion: a new approach to optimization of mixtures for transfection in eukaryotic cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M 30-40 kDa as polycation and polyacrylic acid (PA) with M 20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA-PEG incorporation into a ternary complex (by itself or as a 1:1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA-PEG (as a 1:9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a beta-galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1:1 mixture of PA and PA-PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.     

Protein-protein association in polymer solutions: from dilute to semidilute to concentrated

In a typical cell, proteins function in the crowded cytoplasmic environment where 30% of the space is occupied by macromolecules of varying size and nature. This environment may be simulated in vitro using synthetic polymers. Here, we followed the association and diffusion rates of TEM1-beta-lactamase (TEM) and the beta-lactamase inhibitor protein (BLIP) in the presence of crowding agents of varying molecular mass, from monomers (ethylene glycol, glycerol, or sucrose) to polymeric agents such as different polyethylene glycols (PEGs, 0.2-8 kDa) and Ficoll. An inverse linear relation was found between translational diffusion of the proteins and viscosity in all solutions tested, in accordance with the Stokes-Einstein (SE) relation. Conversely, no simple relation was found between either rotational diffusion rates or association rates (k(on)) and viscosity. To assess the translational diffusion-independent steps along the association pathway, we introduced a new factor, alpha, which corrects the relative change in k(on) by the relative change in solution viscosity, thus measuring the deviations of the association rates from SE behavior. We found that these deviations were related to the three regimes of polymer solutions: dilute, semidilute, and concentrated. In the dilute regime PEGs interfere with TEM-BLIP association by introducing a repulsive force due to solvophobic preferential hydration, which results in slower association than predicted by the SE relation. Crossing over from the dilute to the semidilute regime results in positive deviations from SE behavior, i.e., relatively faster association rates. These can be attributed to the depletion interaction, which results in an effective attraction between the two proteins, winning over the repulsive force. In the concentrated regime, PEGs again dramatically slow down the association between TEM and BLIP, an effect that does not depend on the physical dimensions of PEGs, but rather on their mass concentration. This is probably a manifestation of the monomer-like repulsive depletion effect known to occur in concentrated polymer solutions. As a transition from moderate to high crowding agent concentration can occur in the cellular milieu, this behavior may modulate protein association in vivo, thereby modulating biological function.     

Extensional properties of hydroxypropyl ether guar gum solutions

The extensional properties of 2-hydroxypropyl ether guar gum solutions were investigated using a capillary breakup extensional rheometer (CaBER). Optimization of the geometric parameters of this device allowed for the measurement of the characteristic relaxation times and the apparent extensional viscosities of a series of dilute to semidilute guar gum solutions. The measured relaxation times were compared with predicted Zimm relaxation times, assuming that the hydrophobically modified guar was in a good solvent. Good agreement was found at low concentrations (0.01 wt % approximately 0.17 c*, where c* is the polymer overlap concentration), and this technique allowed for relaxation times on the order of 1 ms to be measured for solutions with shear viscosities of 2 mPa.s. Both the shear and (apparent) steady-state extensional viscosities of this set of industrially relevant fluids exhibited two regions of dependency on polymer concentration: linear up to concentrations of 0.2 wt % ( c/ c* approximately 3) and power law thereafter, where interchain interactions became significant. The extracted relaxation times followed the same trend (i.e., having a near linear dependency on concentration up to 0.2 wt % and a power-law dependency on concentration up to 9 c*). The results indicate that the transition from dilute to semidilute behavior occurs at a nominal concentration of approximately 3 c* instead of c*. The results presented suggest that interchain interactions for this modified guar are weak overall, and the solutions investigated are absent of entanglements over the whole range of frequencies and concentrations explored ((0.17-9) c*).     

Inter-Grade and Inter-Batch Variability of Sodium Alginate Used in Alginate-Based Matrix Tablets

The purpose of this study is to characterize the inter-grade and inter-batch variability of sodium alginate used in the formulation of matrix tablets. Four different grades and three batches of one grade of sodium alginate were used to prepare matrix tablets. Swelling, erosion, and drug release tests of sodium alginate matrix tablets were conducted in a USP dissolution apparatus. Substantial differences in swelling and erosion behavior of sodium alginate matrix tablets were evident among different viscosity grades. Even different batches of the same grade exhibit substantial differences in the swelling and erosion behavior of their matrix tablets. The erosion behavior of sodium alginate matrix tablets can be partly explained by their rheological properties (both apparent viscosity and viscoelasticity) in solution. Sodium alginate with higher apparent viscosity and viscoelasticity in solution show slower erosion rate and higher swelling rate. Compacts prepared from grades or batches with higher viscosity and higher viscoelasticity show slower drug release. For grades or batches with similar apparent viscosities, apparent viscosities of sodium alginate solution at low concentration alone are not sufficient to predict the functionality of sodium alginate in matrix tablets. Viscoelastic properties of sodium alginate solutions at one high concentration corresponding to the polymer gel state, may be suitable indicia of the extended release behavior of sodium alginate matrix tablets.     

Mechanical spectroscopy and relaxometry on alginate hydrogels: a comparative analysis for structural characterization and network mesh size determination

The structure of calcium-saturated alginate hydrogels has been studied by combining rheological determinations and relaxometry measurements. The mechanical spectroscopy analyses performed on alginate gel cylinders at different polysaccharide concentration allowed estimating their main structural features such as the average mesh size. The calculation was based on the introduction of a front factor in the classical rubber elasticity approach which was correlated to the average length of the Guluronic acid blocks along the polysaccharide chain. Transverse relaxation time (T(2)) determinations performed on the cylinders revealed the presence of two relaxation rates of the water entrapped within the hydrogel network. The cross-correlation of the latter data with the rheological measurements allowed estimating the mesh size distribution of the hydrogel network. The results obtained for the hydrogel cylinders were found to be consistent with the relaxometric analysis performed on the alginate microbeads where, however, only one type of water bound into the network structure was detected. A good correlation was found in the average mesh size determined by means of relaxometric measurements on alginate microbeads and by a statistical analysis performed on TEM micrographs. Finally, the addition of a solution containing calcium ions allowed investigating further the different water relaxation modes within alginate hydrogels.     

Ternary Interpolyelectrolyte Complexes DNA–Polycation–Polyanion: A New Approach to Optimization of Mixtures for Transfection of Eukaryotic Cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M30–40 kDa as polycation and polyacrylic acid (PA) with M20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA–PEG incorporation into a ternary complex (by itself or as a 1 : 1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA–PEG (as a 1 : 9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a -galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1 : 1 mixture of PA and PA–PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.     

Effects of beta-cyclodextrin addition and temperature on the modulation of hydrophobic interactions in aqueous solutions of an associative alginate

Novel information about the effects of beta-cyclodextrin (beta-CD) addition and temperature on structural and rheological features of semidilute solutions of alginate and its hydrophobically modified analogue (HM-alginate) is given. Enhanced turbidity is observed for the HM-alginate solutions at high levels of beta-CD addition and low temperatures. The viscosity results revealed cross-linking of the alginate chains at high beta-CD concentrations and low temperatures. Rheological results for the HM-alginate solutions demonstrated that high levels of beta-CD addition and elevated temperatures promoted decoupling of the hydrophobic polymer-polymer associations via inclusion complex formation between beta-CD cavities and the hydrophobic side chains of the polymer. Analysis of small-angle neutron scattering (SANS) results from HM-alginate solutions in the presence of beta-CD suggested that the polymer chains are locally stretched at all of the considered levels of beta-CD and temperatures. The SANS results revealed association structures. The general picture that emerges is that beta-CD addition and temperature can be combined to tune the intensity of the hydrophobic interactions and to cross-link the unmodified alginate.     

Characterization of aggregate structure in mercerized cellulose/LiCl.DMAc solution using light scattering and rheological measurements

The structure of a semidilute solution of mercerized cellulose (CC1m) in 8% (w/w) LiCl.DMAc, which contained some aggregates, was investigated using static and dynamic light scattering measurements. The static scattering function of the polymer solution containing a small amount of aggregates can be separated into fast- and slow-mode components by combining static and dynamic light scattering measurements. The osmotic modulus was identical for the fast-mode component of the CC1m solutions and the native cellulose (CC1) solutions, in which cellulose is dispersed molecularly. This indicates that the molecularly dispersed component of the CC1m solutions has an identical conformation with the cellulose molecules in the CC1 solutions. The correlation length was also identical for the fast-mode components of CC1m solutions and the CC1 solutions, indicating that these solutions have the same mesh size of the polymer entanglement. These observations for the fast-mode components are consistent with the concentration dependence of the zero shear rate viscosity and the plateau modulus estimated in the rheological measurements. The slow-mode component, on the other hand, gave information on the aggregate structure in the CC1m solution. The radius of gyration of the aggregate structure estimated from the slow-mode component was about 70 nm, which is independent of the concentration of the solution. The plots for particle scattering factor of the slow-mode component lay between the theoretical curve of a sphere and a Gaussian chain, implying that the structure of the aggregate in the CC1m solution is like a multiarm polymer. A characteristic time of the slow-mode component calculated with the translational diffusion coefficient and the radius of gyration were almost identical with the relaxation time of the long-time relaxation observed in the rheological measurements. This indicates that the long-time relaxation of CC1m solutions originates in the translational diffusion of the aggregate structure in the solution.     

Polycation induced actin bundles

Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4 Å) MTS-1 (1,1-Methanedyl Bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.