An insight into the effect of interaction with protein on antibacterial activity of chitosan derivatives.

Antimicrobial activity of chitosan in protein-rich media is of a particular interest for various protein-based drug delivery and other systems. For the first time, bacteriostatic activity of chitosan derivatives in the presence of caseinate sodium (CAS) was studied and discussed. Complexation of chitosan derivatives soluble in acidic (CH and RCH) or alkalescent (RCH) media with CAS was confirmed by fluorescent spectroscopy, turbodimetry, light scattering data and measurement of electrical potentials of CAS/chitosan derivative complexes. An addition of CH and RCH caused a static quenching of CAS. Binding constants Kb determined for CH/CAS and RCH/CAS complexes at pH 6.0 were equal to 29.8 × 106 M-1 and 8.9 × 106 M-1, respectively. Kb value of RCH/CAS complex at pH 7.4 was equal to 1.1 × 105'M-1. The poisoned food method was used for counting the number and the direct measurement of the size of bacterial colonies on the surfaces of turbid agar media containing CAS/chitosan derivative complexex. Complete suppression of E. coli cells growth and restriction of S. aureus cells growth were observed on the surface of acidic media. A high concentration of CAS reduced the activity. The activity of RCH in alkalescent media is low or absent. These results can be promising for preparation of microbiologically stable protein-based drug delivery systems.

Formation Kinetics and Antimicrobial Activity of Silver Nanoparticle Dispersions Based on N-Reacetylated Oligochitosan Solutions for Biomedical Applications.

The paper presents the results of the synthesis, a detailed kinetics study, and an investigation of the biological activity of silver nanoparticles (AgNPs) in aqueous solutions of N-reacetylated oligochitosan hydrochloride. UV-visible spectrophotometry and dynamic light scattering were employed to control silver ion reduction. The process was observed to follow a pseudo-first-order law. Transmission and scanning electron microscopy demonstrated that AgNPs ranging in size from 10 to 25 nm formed aggregates measuring 60 to 90 nm, with the aggregate surface coated by a 2-4 nm chitosan shell. X-ray microanalysis and powder X-ray diffractometry were used to study the phase composition, identifying two crystalline phases, nanocrystalline silver and AgCl, present in the dispersions. The antibacterial effect was assessed using the serial dilution method for dispersions with varying degrees of Ag+ conversion. Nanodispersions exhibited significant activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus. Interestingly, the activity did not appear to be heavily influenced by the presence of the AgCl phase or the concentration of Ag+ ions. These synthesized dispersions hold promise for the development of materials tailored for biomedical applications.

Chitosan polyplexes: Energetics of formation and conformational changes in DNA upon binding and release.

Energetics of chitosan (CS) polyplexes and conformational stability of bound DNA were studied at pH 5.0 by ITC and HS-DSC, respectively. The CS-DNA binding isotherm was well approximated by the McGhee-von Hippel model suggesting the binding mechanism to be a cooperative attachment of interacting CS ligands to the DNA matrix. Melting thermograms of polyplexes revealed the transformation of different conformational forms of bound DNA in dependence on the CS/DNA weight ratio rw. At 0

Complexation between chitosan and carboxymethyl cellulose in weakly acidic, neutral, and weakly alcaline media.

The interaction between carboxymethyl cellulose and partially reacetylated chitosan soluble in acidic and alkaline aqueous media is studied by light scattering and isothermal titration calorimetry in a wide pH range. It is shown that the formation of polyelectrolyte complexes (PEC) can occur in the pH range of 6-8, while this pair of polyelectrolytes loses the ability to complexation upon transition to a more alkaline medium. The revealed dependence of the observed enthalpy of interaction on the ionization enthalpy of the buffer indicates the participation of proton transfer from the buffer substance to chitosan and its additional ionization in the binding process. This phenomenon is first observed in a mixture of a weak polybase chitosan and a weak polyacid. The possibility to obtain soluble nonstoichiometric PEC by a direct mixing of the components in a weakly alkaline medium is shown. The resulting PECs are polymolecular particles in shape close to homogeneous spheres with a radius of about 100 nm. The obtained results are promising for creating of biocompatible and biodegradable drug delivery systems.

Effect of Soft Preheating of Bovine Serum Albumin on the Complexation with Oligochitosan: Structure and Conformation of BSA in the Complex.

Phase analysis, spectroscopic, and light scattering methods are applied to investigate the peculiarities of the interaction of oligochitosan (OCHI) with native and preheated bovine serum albumin (BSA) as well as the conformational and structural changes of BSA in BSA/OCHI complex. As shown, untreated BSA binds with OCHI mainly forming soluble electrostatic nanocomplexes, with the binding causing an increase in BSA helicity without a change in the local tertiary structure and thermal stability of BSA. In contrast, soft preheating at 56 °C enhances the complexation of BSA with OCHI and slightly destabilizes the secondary and local tertiary structures of BSA within the complex particles. Preheating at 64 °C (below the irreversible stage of BSA thermodenaturation) leads to further enhancement in the complexation and formation of insoluble complexes stabilized by both Coulomb forces and hydrophobic interactions. The finding can be promising for the preparation of biodegradable BSA/chitosan-based drug delivery systems.

Complexation of oligochitosan with sodium caseinate in alkalescent and weakly acidic media.

Сomplexation of oligochitosan (OCHI) having the degree of acetylation (DA 26 %) with sodium caseinate (SC) at pH 5.8 and 7.2 is described and compared with the complexation of OCHI (DA 2 %) at pH 5.8. In the alkalescent medium, the complexation of OCHI (DA 26 %) is weaker and dualistic depending on SC concentration in the system. In the diluted alkalescent system, the formation of only soluble complexes is observed at OCHI/SC ratio ≤0.9. In the semi diluted one, the complexation results in the formation of insoluble complexes those composition changes symbatically with the OCHI/SC ratio in the system. At pH 5.8, OCHI/SC ratio in insoluble complexes remains the same regardless of OCHI/SC ratio in the solution. At pH 5.8, the electrostatic complexation weakens with an increase in DA and is completely suppressed at a high ionic strength. These results can be promising for construction of biodegradable protein/chitosan drug delivery systems.

A feasible approach to tune the interaction of chitosan with sodium dodecyl sulfate.

Interaction of binary chitosan/nonionic surfactant (NIS) system with sodium dodecyl sulfate (SDS) in aqueous solution is described using turbodimetry, light scattering, electophoretic mobility and cryogenic electron microscopy. The formation of insoluble CHI/SDS complexes is weakened with a decrease in molecular weight of chitosan and critical micelle concentration of NIS as well as with an increase in NIS concentration. Soluble chitosan/NIS complexes absorb SDS molecules until the charge of mixed chitosan/NIS/SDS complexes reaches a critical value that depends on chitosan molecular weight followed by aggregation of primary electrostatic complexes via hydrogen bonding to complex nanoparticles. In contrast to formation of asymmetric swarm-like structures in the binary chitosan/SDS system, the aggregation of complex nanoparticles in the ternary chitosan/NIS/SDS system occurs by a head-to-tail binding mechanism with formation of elongated filamentous microstructures. The finding can be promising for preparation of microbiologically stable pharmaceutical and cosmetic compositions and drug delivery systems containing mixed surfactants.

Peculiarities of the interaction of sodium dodecyl sulfate with chitosan in acidic and alkaline media.

In this work, the interaction between the negatively charged surfactant sodium dodecyl sulfate (SDS) and partially N-reacetylated chitosan (RA-CHI), which is soluble at pH range up to pH 12, is studied in a wide pH range including alkaline media by light scattering (LS) and isothermic titration calorimetry (ITC). It is shown that in the weakly alkaline medium (pH 7.4), RA-CHI/SDS interaction is exothermic and cooperative. This interaction is found to be coupled with proton transfer from the buffer substance to chitosan as it is revealed by the dependence of the measured heat release on the ionization enthalpy of the buffer. At higher pH values (pH > 8), another mechanism of interaction is observed that include SDS micellization induced by hydrophobic interactions with polymer segments, so that no phase separation occurred in these mixtures. The results obtained can contribute to expand the knowledge about application of chitosan for preparation of pharmaceutical and cosmetic compositions containing anionic surfactants.

Molecular features of the interaction and antimicrobial activity of chitosan in a solution containing sodium dodecyl sulfate.

Molecular interaction of chitosan with sodium dodecyl sulfate (SDS) is a more complicated process than it has been imagined so far. For the first time it has been shown that the shorter chitosan chains are, the more preferably they interact with the SDS and the larger-in-size microparticles they form. The influence of ionic strength, urea and temperature on microparticles formation allows interpreting the mechanism of microparticles formation as a cooperative electrostatic interaction between SDS and chitosan with simultaneous decrease in the surface charge of the complexes initiating the aggregation of microparticles. It is shown that hydrogen bonding is mainly responsible for the aggregation while hydrophobic interaction has a lesser effect. Chitosan demonstrates a high bacteriostatic activity in the presence of SDS in solution and can be promising for preparation of microbiologically stable pharmaceutical hydrocolloids, cosmetic products and chitosan-based Pickering emulsions containing strong anionic surfactants.

Biodegradable thermoresponsive oligochitosan nanoparticles: Mechanisms of phase transition and drug binding-release.

Oligochitosan, a low molecular weight derivative of the cationic biopolymer, chitosan, currently shows a great potential of application as a biodegradable non-toxic stimuli-sensitive drug carrier. This paper aimed to elucidate the thermoresponsive potential of oligochitosan and the temperature-controlled drug binding and release to shed light on oligochitosan potential in stimuli-responsive drug delivery. Mechanisms of thermoresponsive behavior of oligochitosan induced by ß-glycerophosphate (GP) were investigated using ITC, DSC, and DLS. Upon heating, the aqueous oligochitosan solution underwent a cooperative transition of the microphase separation type resulting in the formation of stable nano-sized particles. Energetics of the GP-oligochitosan interaction (evaluated by ITC) revealed a positive enthalpy of the GP binding to oligochitosan, which pointed to a notable contribution of dehydration and the related rearrangement of the polysaccharide hydration shell. Energetics of the thermal phase transition of oligochitosan was investigated by DSC upon variation of the solvent dielectric constant and GP concentration. The dependences of the transition parameters on these variables were determined and used for the analysis of the oligochitosan thermoresponsivity mechanism. The binding of ibuprofen to the thermotropic oligochitosan nanogel particles and its release from them were evaluated under near-physiological conditions. Relevantly, the oligochitosan nanoparticles surpassed some reference macromolecular adsorbers by the affinity for the drug and by the delayed release kinetics.

Residual heavy metals in industrial chitosan: State of distribution.

A series of industrial chitosans were analyzed on the presence of residual heavy metals. For the first time, optical microscopy data showed that chitosan solution retained a huge number of insoluble microparticles while transmittance electron microscopy revealed that insoluble fibrous microparticles were incrusted by crystalline nanoparticles with the sizes 5-50 nm. A series of filters used for chitosan solution filtration was analyzed on the presence of retained heavy metal and other residuals by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDXS) and mass-spectrometry with inductively coupled plasma (ICP-MS) methods. The SEM-EDXS analysis revealed the presence of Fe residuals together with Si, Al, N and S in the particles found on the filters. ICP-MS analysis found the presence of heavy metals (mainly Fe, Cr and Ni) both on the filter surfaces and in the effluent chitosan solution passed though the filters. This study draws attention to the necessity of a careful selection of industrially manufactured chitosan in order to avoid hidden undesirable effects of chitosan on pharmaceuticals and biomaterials and gives a warning of inapplicability of a stainless-steel made apparatus as a reactor susceptible to caustic soda corrosion for chitin deacetylation and production of medical and food grade chitosan.

Extraction of residual heavy metals from commercial chitosan and approach to preparation of oligochitosan hydrochloride.

For biomedical applications, chitosan and oligochitosan must be appropriately characterized and meet pharmacological requirements in terms of contamination by residual heavy metals. In this work, a series of commercial chitosans was analyzed by ICP-MS method, and high concentration of Fe (44-382 ppm), Cr (3.1-35.5 ppm) and Ni (0.33-7.91 ppm) exceeding pharmacologically acceptable level was found. It was shown that as a chelating agent EDTA was an ineffective remedy for solid-phase extraction of residual heavy metals from chitosan. It was proposed that corrosion of stainless steel apparatus in the process of chitin deacetylation contributed to chitosan contamination by heavy metals. A two-step treatment of chitosan with hydrochloric acid allowed remediation of chitosan and preparation of oligochitosan hydrochloride with molecular weight 5-16 kDa and acceptable level of Fe<10, Cr<1 and Ni<1 ppm.

Consequences of chitosan decomposition by nitrous acid: Approach to non-branched oligochitosan oxime.

It is well known that chitosan degradation by nitrous acid leads to oligochitosan (oligoCHIt-ahm) bearing reactive 2,5-anhydromannose (3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-aldehyde) units at the new reducing ends of macromolecules. Standard protocol requires reduction of oligoCHIt-ahm with NaBH4 to corresponding oligoCHIt-hml bearing unreactive hydroxymethyl group instead of reactive aldehyde group. For the first time, HP SEC as well as UV and CD spectroscopy methods have revealed that the reduction leads to an indefinite side modification and the formation of a branched oligoCHIt-hml with increased molecular weight. Here, it is shown that the branching and modification can be prevented by means of the simple and reproducible reaction of oligoCHIt-ahm with hydroxylamine that allows preparation of a stable linear oligochitosan oxime, oligoCHIt-oxm. Cytotoxicity tests show that oligoCHIt-ahm, oligoCHIt-hml and oligoCHIt-oxm are non-toxic at concentration below 2.5 mg/ml, and the cytotoxicity is concentration dependent and decreases in the order oligoCHIt-ahm > oligoCHIt-hml > oligoCHIt-oxm at higher concentrations both before and after long shelf-storage. The elaborated approach and cytotoxicity data give an opportunity to use the non-branched oligoCHIt-oxm for biomedical applications.

N-Reacetylated Oligochitosan: pH Dependence of Self-Assembly Properties and Antibacterial Activity.

Oligochitosan (short chain chitosan) is more soluble in acidic aqueous media than a high molecular weight (MW) chitosan, but its antimicrobial activity decreases with increase in degree of acetylation (DA) and increase in pH above a critical pH threshold point. In the present study, oligochitosans varying in MW were additionally N-acetylated and their self-assembly properties and antibacterial activity toward Staphylococcus aureus and Escherichia coli were investigated in a wide pH range as a function of MW and DA. Light scattering studies reveals that reacetyleted oligochitosan with Mw ≤ 11 kDa is completely soluble in alkaline media (up to pH 12.5), if its DA is not less than 16%. Reacetylated chitosans with DA ∼ 30% are solubile in the entire pH range up to 12.5, if their Mw is not higher than 25 kDa, but they aggregate and precipitate from the solution at pH ≥ 8 when their Mw is above 25 kDa. Considering the influence of DA and MW, the antibacterial activity of reacetylated oligochitosans is maximal in the short interval of DA 16-28% at pH 7.4. These results are promising for expanding practical application of oligochitosan in pharmaceutical, cosmetic, and food compositions.

Antifungal activity of oligochitosans (short chain chitosans) against some Candida species and clinical isolates of Candida albicans: molecular weight-activity relationship.

A series of oligochitosans (short chain chitosans) prepared by acidic hydrolysis of chitosan and characterized by their molecular weight, polydispersity and degree of deacetylation were used to determine their anticandidal activities. This study has demonstrated that oligochitosans show a high fungistatic activity (MIC 8-512 µg/ml) against Candida species and clinical isolates of Candida albicans, which are resistant to a series of classic antibiotics. Flow cytometry analysis showed that oligochitosan possessed a high fungicidal activity as well. For the first time it was shown that even sub-MIC oligochitosan concentration suppressed the formation of C. albicans hyphal structures, cause severe cell wall alterations, and altered internal cell structure. These results indicate that oligochitosan should be considered as a possible alternative/additive to known anti-yeast agents in pharmaceutical compositions.

Influence of glucosamine on oligochitosan solubility and antibacterial activity.

Light scattering studies indicate that oligochitosan (short-chain chitosan) solutions contain aggregates at pH values below the critical pH of phase separation, while at or above this point the gel phase coexists with the aggregate solution. This work demonstrates for the first time that the presence of D-glucosamine in an oligochitosan solution shifts the critical pH to a higher value and improves the oligochitosan antibacterial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermis in neutral and slightly alkaline aqueous media. By comparing the results of light scattering studies and antimicrobial assays one can conclude that the antimicrobial activity of oligochitosan is dependent on its unimolecular form, not its supramolecular structures. The widening of the homogeneity region of an oligochitosan solution could lead to promising biomedical applications.

Endochitinase activity determination using N-fluorescein-labeled chitin.

A fluorimetric method for the determination of endochitinolytic activity using N-fluorescein-labeled chitin (FITC-Chitin) is proposed, and a procedure for FITC-Chitin preparation with a degree of FITC content of 2.2 mol% (one FITC molecule per 45 glucosamine residues) is described. FITC-Chitin is capable to distinguish endochitinase and exochitinase (beta-N-acetylglucosaminidase) activities.

Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium.

Culture filtrates of the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium growing on colloidal chitin showed increasing chitinolytic activity and production of two (32- and 43-kDa) main proteins. Maximum activity was found 18-20 days after inoculation, but V. suchlasporium always displayed higher activity. Zymography of such filtrates on carboxymethyl-chitin-Remazol brilliant violet 5R/acrylamide gels showed five bands of substrate degradation for V. suchlasporium and three for V. chlamydosporium. Filtrates with maximum activity were chromatographed on macroporous cross-linked chitin affinity matrix, showing a peak of main (50-60%) activity, which only contained a 43-kDa protein for both fungi. Zymography and colloidal chitin degradation showed that it was a single endochitinase (CHI43) with optimum pH range of 5.2-5.7. The main isoforms had pIs of 7.6 for V. suchlasporium and 7.9 for V. chlamydosporium. Eggs of the nematode Globodera pallida treated with CHI43 and the serine protease P32 from V. suchlasporium alone or in combination showed surface damage in comparison with controls when examined by scanning electron microscopy.