Exploiting specific properties of squid pens for the preparation of oligochitosan hydrochloride.

Herein, we describe in first the application of squid pens for the preparation of pharmaceutical-grade oligochitosan hydrochloride with the physicochemical characteristics corresponding with the requirements of the European Pharmacopoeia. It is shown that the use of specific properties of squid pens as a source of parent chitosan allows preparing the product with a high yield at relatively moderate process conditions used for squid pens treatments and chitosan depolymerization.

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.

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.

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.