Jorge Augusto de Moura Delezuk

Extensively deacetylated high molecular weight chitosan from the multistep ultrasound-assisted deacetylation of beta-chitin

High intensity ultrasound irradiation was used to convert beta-chitin (BCHt) into chitosan (CHs). Typically, beta-chitin was suspended in 40% (w/w) aqueous sodium hydroxide at a ratio 1/10 (gmL(-1)) and then submitted to ultrasound-assisted deacetylation (USAD) during 50min at 60°C and a fixed irradiation surface intensity (52.6Wcm(-2)). Hydrogen nuclear magnetic resonance spectroscopy and capillary viscometry were used to determine the average degree of acetylation (DA‾) and viscosity average degree of polymerization (DPv‾), respectively, of the parent beta-chitin (DA‾=80.7%; DPv‾=6865) and USAD chitosans. A first USAD reaction resulted in chitosan CHs1 (DA‾=36.7%; DPv‾=5838). Chitosans CHs2 (DA‾=15.0%; DPv‾=5128) and CHs3 (DA‾=4.3%; DPv‾=4889) resulted after repeating the USAD procedure to CHs1 consecutively once and twice, respectively. Size-exclusion chromatography analyzes allowed the determination of the weight average molecular weight (Mw‾) and dispersity (Ð) of CHs1 (Mw‾=1,260,000gmol(-1); Ð=1.4), CHs2 (Mw‾=1,137,000gmol(-1); Ð=1.4) and CHs3 (Mw‾=912,000gmol(-1); Ð=1.3). Such results revealed that, thanks to the action of high intensity ultrasound irradiation, the USAD process allowed the preparation of unusually high molecular weight, randomly deacetylated chitosan, an important breakthrough to the development of new high grade chitosan-based materials displaying superior mechanical properties.

Low molecular-weight chitosans are stronger biomembrane model perturbants

The influence from the chitosan molecular weight on its interaction with cell membrane models has been studied. A low molecular weight chitosan (LMWChi) adsorbed from the subphase expanded the surface pressure-area and surface potential-area isotherms of dimyristoyl phosphatidic acid (DMPA) monolayers and decreased the compressional modulus. The expansion in the monolayers and the decrease in the compressional modulus were larger for LMWChi than for a high molecular weight chitosan (Chi). The polymeric nature is still essential for the interaction though, which was demonstrated by measuring negligible changes in the mechanical properties of the DMPA monolayer when the subphase contained glucosamine and acetyl-glucosamine. The results were rationalized in a model through which chitosan interacted with the membrane via electrostatic and hydrophobic interactions, with the smaller chains of LMWChi having less steric hindrance to be accommodated in the membrane. In summary, the activity based on membrane interactions depends on the distribution of molar mass, with lower molecular weight chitosan more likely to have stronger effects.

Experimental evidence for the mode of action based on electrostatic and hydrophobic forces to explain interaction between chitosans and phospholipid Langmuir monolayers.

The interaction between chitosans and Langmuir monolayers mimicking cell membranes has been explained with an empirical scheme based on electrostatic and hydrophobic forces, but so far this has been tested only for dimyristoyl phosphatidic acid (DMPA). In this paper, we show that the mode of action in such a scheme is also valid for dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG), whose monolayers were expanded and their compressibility modulus decreased by interacting with chitosans. In general, the effects were stronger for the negatively charged DPPG in comparison to DPPC, and for the low molecular weight chitosan (LMWChi) which was better able to penetrate into the hydrophobic chains than the high molecular weight chitosan (Chi). Penetration into the hydrophobic chains was confirmed with polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum frequency generation (SFG) spectroscopy. A slight reduction in conformational order of the lipid chains induced by the chitosans was quantitatively estimated by measuring the ratio between the intensities of the methyl (r(+)) and methylene (d(+)) peaks in the SFG spectra for DPPG. The ratio decreased from 35.6 for the closely packed DPPG monolayer to 7.0 and 6.6 for monolayers containing Chi and LMWChi, respectively. Since in both cases there was a significant phospholipid monolayer expansion, the incorporation of chitosans led to chitosan-rich and lipid-rich condensed domains, which mantained conformational order for their hydrophobic tails. The stronger effects from LMWChi are ascribed to an easier access to the hydrophobic tails, as corroborated by measuring aggregation in solution with dynamic light scattering, where the hydrodynamic radius for LMWChi was close to half of that for Chi. Taken together, the results presented here confirm that the same mode of action applies to different phospholipids that are important constituents of mammalian (DPPC) and bacterial (DPPG) cell membranes.

Silk fibroin organization induced by chitosan in layer-by-layer films: Application as a matrix in a biosensor.

In this paper, we show that chitosan may induce conformation changes in silk fibroin (SF) in layer-by-layer (LbL) films, which were used as matrix for immobilization of the enzyme phytase to detect phytic acid. Three chitosan (CH) samples possessing distinct molecular weights were used to build CH/SF LbL films, and a larger change in conformation from random coils to β-sheets for SF was observed for high molecular weight chitosan (CHH). The CHH/SF LbL films deposited onto interdigitated gold electrodes were coated with a layer of phytase, with which phytic acid could be detected down to 10-9M using impedance spectroscopy as the principle of detection and treating the data with a multidimensional projection technique. This high sensitivity may be ascribed to the suitability of the CHH/SF matrix, thus indicating that the molecular-level interactions between chitosan and SF may be exploited in other biosensors and biodevices.