Galactose dialdehyde: the forgotten candidate for a protein cross-linker?

Chitosan derivatives with quaternary ammonium salt, such as N,N,N-trimethyl chitosan, N-N-propyl-N,N-dimethyl chitosan and N-furfuryl-N,N-dimethyl chitosan were prepared using different 96% deacetylated chitosan of M(v) 2.14x10(5), 1.9x10(4), 7.8x10(3). Amino groups on chitosan react with aldehydes to from a Schiff base intermediate. Quaternized chitosan were obtained by reaction of a Schiff base with methyl iodide. The yields, degree of quaternization and water-solubility of quaternized chitosan were influenced by the molecular weight of the chitosan sample. The antibacterial activities of quaternized chitosan against Escherichia coli were explored by calculation of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in water, 0.25 and 0.50% acetic acid medium. Results show the antibacterial activities of quaternized chitosan against E. coli is related to its molecular weight. Antibacterial activities of quaternized chitosan in acetic acid medium is stronger than that in water. Their antibacterial activities is increased as the concentration of acetic acid is increased. It was also found that the antibacterial activity of quaternized chitosan against E. coli is stronger than that of chitosan.     

Quaternization of N-aryl chitosan derivatives: synthesis, characterization, and antibacterial activity

Chemical modification of chitosan by introducing quaternary ammonium moieties into the polymer backbone renders excellent antimicrobial activity to the adducts. In the present study, we have synthesized 17 derivatives of chitosan consisting of a variety of N-aryl substituents bearing either electron-donating or electron-withdrawing groups. Selective N-arylation of chitosan was performed via Schiff bases formed by the reaction between the 2-amino groups of the glucosamine residue of chitosan with aromatic aldehydes under acidic conditions, followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Each of the derivatives was further quaternized using N-(3-chloro-2-hydroxypropyl)trimethylammonium chloride (Quat-188) as the quaternizing agent that reacted with either the primary amino or hydroxyl groups of the glucosamine residue of chitosan. The resulting quaternized materials were water soluble at neutral pH. Minimum inhibitory concentration (MIC) antimicrobial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria in order to explore the impact of the extent of N-substitution (ES) on their biological activities. At ES less than 10%, the presence of the hydrophobic substituent, such as benzyl and thiophenylmethyl, yielded derivatives with lower MIC values than chitosan Quat-188. Derivatives with higher ES exhibited reduced antibacterial activity due to low quaternary ammonium moiety content. At the same degree of quaternization, all quaternized N-aryl chitosan derivatives bearing either electron-donating or electron-withdrawing substituents did not contribute antibacterial activity relative to chitosan Quat-188. Neither the functional group nor its orientation impacted the MIC values significantly.     

Aggregation behavior of N-carboxyethylchitosan in aqueous solution: effects of pH, polymer concentration, and presence of a gemini surfactant

A kind of biocompatible derivative of chitosan, N-carboxyethylchitosan (CECh) with a degree of substitution of 0.21 (DS 0.21) was synthesized by a Michael addition reaction. The aggregation behavior of CECh in aqueous solution under the effects of pH, polymer concentration, as well as a gemini surfactant, was investigated by turbidity, zeta potential, fluorescence spectroscopy, viscosity, and surface tension measurements. In the pH range of 3-11, the macroscopic phase separation of CECh from water occurs near the isoelectric point (IEP) due to the intense electrostatic attraction, and the intermolecular interaction at pH 4 is stronger than that at pH 10 over the whole CECh concentration region. The critical aggregation concentration (CAC) of CECh/12-n-12 (n = 3, 6) in basic media is determined to be between 0.0010 and 0.0015 mmol/L, and the length of the surfactant spacer is found to play an important role in the interaction of 12-n-12 with CECh.     

High-efficient and synergetic antibacterial nanocomposite hydrogel with quaternized chitosan/Ag nanoparticles prepared by one-pot UV photochemical synthesis

Hydrogel dressings have significant advantages such as absorption of tissue exudate, maintenance of proper moist environment, and promotion of cell proliferation. However, facile preparation method and high-efficient antibacterial hydrogel dressings are still a great challenge. In this study, a facile approach to prepare antibacterial nanocomposite hydrogel dressing to accelerate healing was explored. The hydrogels consisted of quaternized chitosan and chemically cross-linked polyacrylamide, as well as silver nanoparticles (AgNPs) stabilized by chitosan. The synthesis of the hydrogels including the formation of AgNPs and polymerization of acrylamide was accomplished simultaneously under UV irradiation in 1 hour without adding initiator. The hydrogels showed favorable tensile strength of ∼100 kPa with elongation at break over 1000% and shear modulus of ∼10⁴ Pa as well as suitable swelling ratio, which were appropriate for wound dressing. The combination of quaternized chitosan and AgNPs exhibited high-efficient and synergetic antibacterial performance with low cytotoxicity. In vivo animal experiments showed that the hydrogel can effectively prevent wound infection and promote wound healing. This study provides a facile method to produce antibacterial hydrogel wound dressing materials.     

Delivery of dermatan sulfate from polyelectrolyte complex-containing alginate composite microspheres for tissue regeneration

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90-95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.     

Antialgal activity of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes against the marine alga Ulva

Marine biofouling has detrimental effects on the environment and economy, and current antifouling coatings research is aimed at environmentally benign, non-toxic materials. The possibility of using contact-active coatings is explored, by considering the antialgal activity of cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes. The antialgal activity was investigated via zoospore settlement and sporeling growth assays of the marine algae Ulva linza and U. lactuca. The assay results for PDMAEMA brushes were compared to those for anionic and neutral surfaces. It was found that only PDMAEMA could disrupt zoospores that come into contact with it, and that it also inhibits the subsequent growth of normally settled spores. Based on the spore membrane properties, and characterization of the PDMAEMA brushes over a wide pH range, it is hypothesized that the algicidal mechanisms are similar to the bactericidal mechanisms of cationic polymers, and that further development could lead to successful contact-active antialgal coatings.     

Antibacterial cellulose fiber via RAFT surface graft polymerization

2-(dimethylamino)ethyl methacrylate (DMAEMA) was polymerized from cellulosic filter paper via reversible addition-fragmentation chain transfer (RAFT) polymerization. The tertiary amino groups of the grafted PDMAEMA chains were subsequently quaternized with alkyl bromides of different chain lengths (C8-C16) to provide a large concentration of quaternary ammonium groups on the cellulose surface. The antibacterial activity of the quaternized and nonquaternized PDMAEMA-grafted cellulosic fibers was tested against Escherichia coli. The antibacterial activity was found to depend on the alkyl chain length and on the degree of quaternization, i.e., the amount of quaternary amino groups present in the cellulose graft copolymers. The PDMAEMA-grafted cellulose fiber with the highest degree of quaternization and quaternized with the shortest alkyl chains was found to exhibit particularly high activity against E. coli.     

Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films

Chitosan, a cationic biopolymer derived from chitin, has been described as having antibacterial activity. The modes of this activity, however, have not been established. One mode proposed is that chitosan perturbs bacterial cell membranes. To validate this proposal, in this study we investigated chitosan interactions with lipids in Langmuir monolayers as model membranes. The interactions were assessed by monitoring differences in the shape of the compression isotherms measured in the absence and presence of chitosan in the subphase (acetate buffer). To appraise the contribution of electrostatic interactions versus hydrogen bonding and hydrophobic interactions, three membrane lipids differing in charge were studied-anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and neutral cholesterol-and the pH of the subphase was varied between 3.5 and 6.0. In addition, the impact of the molecular weight of chitosan on the interactions was assessed at pH 3.5. It was found that while chitosan had a negligible effect on DPPC monolayers over the pH range studied, it distinctly affected DPPG and cholesterol monolayers. The effect on DPPG was found to decrease with increasing pH, that at pH 3.5 being ascribed to the charge-mediating action of chitosan on the local ionic environment and that at higher pHs to the intercalation of chitosan to the monolayers. Practically independent of pH, the effect of chitosan on cholesterol was accounted for by the formation of cholesterol-chitosan hydrogen bonds. Chitosan of lower molecular weight facilitated the interactions with all the three lipids studied. The results obtained may be helpful in identifying the mode of antibacterial activity of chitosan versus other modes that involve the disturbance of cell life cycles.     

Molecular weight and pH aspects of the efficacy of oligochitosan against methicillin-resistant Staphylococcus aureus (MRSA)

Oligochitosan samples varying in molecular weight (M_w) and having narrow polydispersities were prepared by means of depolymerization of chitosan in hydrochloric acid, and their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was measured at pH values 5.5-8.0. The antibacterial testing of oligochitosans obtained showed that oligochitosans having M_w in the range of 0.73-20.0 kDa could be used both at slightly acidic and neutral pH values, and that the activity against MRSA remained moderate for oligochitosan samples having M_w about 3-5 kDa even at slightly basic pH values. The self-assembling behavior of oligochitosan macromolecules in the dilute solution at various pH values as a function of chain length was investigated. At first it was shown that oligochitosans formed supramolecular aggregates in dilute solutions below the critical pH value 6.5. Despite the aggregation phenomenon, the formation of nano-sized aggregates did not prevent oligochitosan from demonstrating the bactiostatic activity.     

Mononuclear copper(II) complexes with quinolones and nitrogen-donor heterocyclic ligands: Synthesis, characterization, biological activity and interaction with DNA

The neutral mononuclear copper(II) complexes with the quinolone antibacterial drugs, pipemidic acid and N-propyl-norfloxacin, in the presence or absence of nitrogen-donor heterocyclic ligands, 2,2′-bipyridine, 1,10-phenanthroline or 2,2′-dipyridylamine, have been prepared and characterized spectroscopically. The interaction of copper(II) with the deprotonated quinolone ligand leads to the formation of the neutral mononuclear complexes of the type [Cu(quinolone)₂(H₂O)] (1)–(2) while the presence of the N-donor ligand leads to the formation of the neutral mononuclear complexes of the type [Cu(quinolone)(N-donor)Cl] (3)–(8). In all the complexes, copper(II) is pentacoordinate having a distorted square pyramidal geometry. The electron paramagnetic resonance spectra of 1 and 2 are typical of mononuclear Cu(II) complexes, while for the mixed-ligands complexes 3–8 a mixture of dimeric and monomeric species is indicated. The interaction of the complexes with calf-thymus DNA has been investigated with diverse spectroscopic techniques and has shown that the complexes can be bound to calf-thymus DNA by the intercalative mode. The antimicrobial activity of the complexes has been tested on three different microorganisms. All the complexes show an increased biological activity in comparison to the corresponding free quinolone ligand.     

pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).     

Neutral mononuclear dioxomolybdenum(VI) and dioxouranium(VI) complexes of oxolinic acid: Characterization and biological evaluation

The interaction of the first-generation quinolone antibacterial drug oxolinic acid (Hoxo) with the dioxomolybdenum(VI) and dioxouranim(VI) ions leads to the formation of the neutral mononuclear complexes MoO₂(oxo)₂ and UO₂(oxo)₂, respectively. The structure of the complexes has been characterized physicochemically and spectroscopically. The lowest energy model structure of the complexes has been determined with molecular modeling calculations. The antimicrobial activity of the complexes has been evaluated against three different microorganisms. The interaction of the complexes with calf-thymus DNA has been investigated with electronic and circular dichroism spectroscopies.     

About mechanism of chitosan cross-linking with glutaraldehyde

The regularities of the reaction of aminopolysaccharide chitosan with glutaraldehyde (GA) have been considered. The equilibrium forms of GA in water have been thoroughly studied by NMR spectroscopy. It has been established that at pH 5.6, the exchange of the protons of O=CHCH₂ groups for deuterium occurs, indicating the presence of an anion, a product of the first stage of the aldol reaction; at pH > 7.2, the formation of the products of an aldol reaction and aldol condensation takes place. The kinetics of the reaction between the amino groups of chitosan and GA, the kinetics of gel formation in chitosan solutions in the presence of GA, and the kinetics of changes in the rigidity of gels formed have been studied by UV spectroscopy. IR spectra of cross-linked chitosan have been obtained. It has been shown that chitosan catalyzes the polymerization of GA to form irregular products; in this process, the length of oligomeric chains in modified or cross-linked chitosan and the concentration of conjugated bonds increase with the GA concentration and pH of the reaction medium.     

Evaluation of a method for the determination of antibacterial activity of chitosan

A method for the determination of the antimicrobial activity of chitosan with the use of organic salts for the production of pH in the range of 5.5–8.2 was studied. The double-dilution method demonstrated the effectiveness of the determination of the antimicrobial activity of chitosan samples with different molecular weights and solubilities. It was found that the antibacterial activity increased at low pH values with increasing molecular weight, but chitosans with a molecular weight of 5–6 kDa showed higher activity at neutral and slightly alkaline pH levels. Determination of the antimicrobial activity of various chitosan samples at different pH values allowed a more reliable assessment of the potential biological activity of chitosan.     

The effect of the acidity of the medium on the magnitude of the light-dependent antibacterial activity of chlorophyllides]

The influence of medium acidity on the intensity of light-dependent antibacterial activity of chlorophyllides isolated from biomass of microscopic green alga Westella botryoides has been studied. This light-dependent antibacterial activity has been shown to be maximum at low pH values. The ability to suppress the bacterial growth under the action of light decreases in neutral and alkaline media. It has been concluded that the reason of increase of light-dependent antibacterial activity of chlorophyllides at low pH values is formation of pheophorbides out of them     

Neutral and cationic mononuclear copper(II) complexes with enrofloxacin: structure and biological activity

The mononuclear copper complexes with the quinolone antibacterial drug enrofloxacin (=Herx) in the presence or not of a nitrogen donor heterocyclic ligand 1,10-phenanthroline (=phen) and 2,2'-bipyridine (=bipy) have been prepared and characterized. Interaction of copper(II) with deprotonated enrofloxacin leads to the formation of the neutral complex Cu(erx)2(H2O), 1, while the presence of phen or bipy leads to the formation of a neutral or a cationic mononuclear complex, respectively. The crystal structures of (chloro)(1,10-phenanthroline)(enrofloxacinato)copper(II), 2, and (aqua)(2,2'-bipyridine)(enrofloxacinato)copper(II) chloride, 3, have been determined with X-ray crystallography. The complexes have been studied with X-band electron paramagnetic resonance in aqueous solutions at liquid helium temperature. The study of the interaction of the complexes with calf-thymus DNA has been performed with diverse spectroscopic techniques and has showed that all complexes are bound to DNA by the intercalative mode. The antimicrobial efficiency of the complexes has been tested on three different microorganisms and the available evidence supports that the best inhibition is provided by Cu(erx)2(H2O) (minimum inhibitory concentration=0.125 microg mL(-1)) against Escherichia coli and Pseudomonas aeruginosa.     

Antibacterial activity of quaternary ammonium chitosan containing mono or disaccharide moieties: Preparation and characterization

The 9 quaternary ammonium chitosans containing monosaccharides or disaccharides moieties were successfully synthesized by reductive N-alkylation then quaternized by N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat-188). The chemical structures of quaternary ammonium chitosan derivatives were characterized by ATR-FTIR and ¹H NMR spectroscopy. The degree of N-substitution (DS) and the degree of quaternization (DQ) were determined by ¹H NMR spectroscopic method. It was found that the DS was in the range of 12–40% while the DQ was in the range of 90–97%. The results indicated that the O-alkylation was occured in this condition. Moreover, all quaternary ammonium chitosan derivatives were highly water-soluble at acidic, basic, and neutral pH. Minimum inhibitory concentration (MIC) antibacterial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria compared to quaternary ammonium N-octyl and N-benzyl chitosan derivatives. The quaternary ammonium mono and disaccharide chitosan derivatives showed very high MIC values which were in the range of 32 to >256 μg/mL against both bacteria. Also it was found that the antibacterial activity decreased with increasing the DS. This was due to the increased hydrophilicity of mono and disaccharide moieties. On the other hand, the low MIC values (8–32 μg/mL) were obviously observed when the DS of quaternary ammonium N-octyl and N-benzyl chitosan derivatives was lower than 18%. The results showed that the presence of hydrophobic moiety such as the N-benzyl group enhanced the antibacterial activity compared to the hydrophilic moiety against both bacteria.     

Preparation and characterization of chitosan-based nanoparticles

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan for biomedical applications. Natural di- and tricarboxylic acids were used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. This method allows the formation of polycations, polyanions, and polyampholyte nanoparticles. The prepared nanosystems were stable in aqueous media at low pH, neutral, and mild alkaline conditions. The structure of products was determined by NMR spectroscopy, and the particle size was identified by laser light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that particle size depends on the pH, but at a given pH, it was independent of the ratio of cross-linking and the cross-linking agent. Particle size measured by TEM varied in the range 60-280 nm. In the swollen state, the average size of the particles measured by DLS was in the range 270-370 nm depending on the pH. The biodegradable cross-linked chitosan nanoparticles, as solutions or dispersions in aqueous media, might be useful for various biomedical applications.     

Effect of pH and the binding anion on In3+-transferrin interaction: spectroscopic study of the perturbed angular correlation of 172-245 keV gamma rays of indium 111

The formation of ternary complexes, transferrin-anion-In111 has been investigated by means of gamma-gamma coincidence spectrometry of the 172-245 keV rays. The angular correlation between the two gamma-rays emitted in cascade depends on the magnetic and electric fields gradients, consequently the chemical structure of metal holder. Any modification of this structure causes the variation of angular correlation. The study of G22 (infinity) as function of pH (G22(infinity): integrated perturbed angular correlation coefficient) has been performed to turn out the hydrolysis of In111 in aqueous solution, metal complex formation in presence of chelating agents (citric acid and sodium bicarbonate) and the formation of protein-metal complexes. The presence of complexing agents limits the domain of In111 colloid existence and allows fast transfer of ionised indium on the transferrin. Two types of metal-protein interactions has been turn out. The first in the weakly acidic range of pH is characterized by an affinity constant near to this of citric acid. The second lying in neutral and basic range of pH, where the formation rate of transferrin-In111 complex is fast (t less than 500 s). In citrate medium, for pH 6-7,5 the rate of metal transfer on the protein, studied by means of G22 (infinity) = f(t), is function of pH. The binding anion appears as an indispensable element for the formation of protein-metal complexes. The In111 previously chelated by 8-Hydroxyquinoline is fixed by the protein if only exits a binding anion in the solution. This mays bring in the formation of an intermediate active state, indispensable step for the ternary complex formation transferrin-anion-In111.     

Electrosprayed polyelectrolyte complexes between mucoadhesive N,N,N,-trimethylchitosan-homocysteine thiolactone and alginate/carrageenan for camptothecin delivery

Novel hydrogel polyelectrolyte complexes (PECs) between the N,N,N,-trimethylchitosan-homocysteine thiolactone (TM-HT-chitosan) and two anionic polymers were investigated. The particles of pure thiolated chitosan and its PECs with alginate and carrageenan were fabricated using the electrospray ionization technique. The hydrogel PEC particles were characterized by scanning electron microscopy, dynamic light scattering, Fourier transform infrared microscopy, thermogravimetric analysis, encapsulation efficiency (EE), mucoadhesive property and in vitro drug release behavior. TM-HT-chitosan/alginate particles could be loaded with camptothecin (CPT), employed as a model anti-cancer drug, at an over 70% EE, and revealed both a reduced burst effect and a prolonged release of CPT over 3 days. The resultant TM-HT-chitosan/alginate PEC particles displayed a 5.60-, 1.86- and 1.55-fold stronger mucoadhesive property compared to that of the unmodified chitosan/alginate PEC at pH 1.2, 4.0 and 6.4, respectively, and this was not affected by the CPT loading level.