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.     

Carboxyalkylation of chitosan in the gel state

This study presents a new approach for direct carboxyalkylation of chitosan in the gel state by using aza-Michael addition and substitution reactions. Various reagents were applied including acrylic and crotonic acids, and α-, β-, γ-, δ-, and ?-halocarboxylic acids. The reaction of chitosan with γ- and δ-halocarboxylic acids showed no target product formation either in solution or in the gel state. In the case of acrylic, crotonic, α- and β-halocarboxylic acids, the reaction performed in the gel state (concentration of chitosan 20-40%) shows higher degree of substitution at lower reaction time and temperature than in diluted solutions (concentration of chitosan 0.5-2%). The results were discussed in terms of kinetics of the target and side reactions. (1)H and (13)C NMR confirmed that in all cases the carboxyalkylation of chitosan proceeds exclusively at the amino groups.     

The study of gelation kinetics and chain-relaxation properties of glutaraldehyde-cross-linked chitosan gel and their effects on microspheres preparation and drug release

The effects of gelation kinetics and chain-relaxation properties of glutaraldehyde-cross-linked chitosan gel on microspheres preparation or drug release were studied. The rate of gelation is zero order corresponding to the chitosan concentration but non-zero order corresponding to the glutaraldehyde concentration. It was suggested that the cross-linking reaction was mainly dominated by the concentration of small molecule reactant, glutaraldehyde. The relaxation of an entangled polymer chain in a gel network as a result of the swelling of cross-linked chitosan hydrogel was investigated by the stress–strain determination. The higher the cross-linking density of chitosan hydrogel, the lower the swelling ability of chitosan hydrogel due to the slower relaxation rate of polymer chain, which then results in the decreased drug-release rate.     

Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent

A new process of formation of chitosan physical hydrogels in aqueous solution, without any organic solvent or cross-linking additive, was studied. The three conditions required for the physical gelation were an initial polymer concentration over C*, a critical value of the balance between hydrophilic and hydrophobic interactions, and a physicochemical perturbation responsible for a bidimensional percolating mechanism. The time necessary to reach the gel point was determined by rheometry, and gelations were compared according to different initial conditions. Thus, we investigated the influence of the polymer concentration and the degree of acetylation (DA) of chitosan on gelation. The number of junctions per unit volume at the gel point varied with the initial polymer concentration, i.e., the initial number of chain entanglements per unit volume or the number of gel precursors. The time to reach the gel point decreased with both higher DAs and concentrations. For a chitosan of DA = 36.7%, a second critical initial concentration close to 1.8% (w/w) was observed. Above this concentration, the decrease of the time to reach the gel point was higher and fewer additional junctions had to be formed to induce gelation. To optimize these physical hydrogels, to be used for cartilage regeneration, their final rheological properties were studied as a function of their degree of acetylation and their polymer concentration. Our results allowed us to define the most appropriate gel for the targeted application corresponding to a final concentration of chitosan in the gel of near 1.5% (w/w) and a DA close to 40%.     

Identification of mechanism of enzyme/transport process in the system of "enzyme/ transporter--substrate--effector" using the "ratio" method

Using equilibrium assumption the analysis of kinetics of enzyme/transport process has been developed. In the course of this process the enzyme can interact simultaneously with substrate S and effector E and as a result two products are generated: P1 (from substrate) and P2 (from effector) after catalytic effect of enzyme or transportation across the biological membrane. It has been demonstrated that the ratio (R = V0,1/V0,2) of initial rates of formation of reaction products P, (V0,1) and P2 (V0,2) represented in linearized form as a dependence on concentration of both substrate S0 and effector E0 allows to identify a specific mechanism of the present process. An algorithm of such identification has been developed.     

Formation kinetics of insulin-based amyloid gels and the effect of added metalloporphyrins

The kinetics of insulin-based amyloid gel formation has been studied using extinction and fluorescence detection. The process is treated as autocatalytic, and the kinetic profiles are fit using a nonconventional analysis involving a time-dependent rate constant (factor): k(t) = k(o) + k(c)(k(c)t)(n). The dependence of the kinetic parameters on initial solution conditions of concentration, pH, and ionic strength has been investigated. A mechanism is proposed in which the rate-determining step involves the activation of insulin solute species into partially unfolded, structurally modified monomers, which then aggregate. The influence of added metalloporphyrins on the rate and extent of gel formation is described. Metal derivatives of tetrakis(4-sulfonatophenyl)porphine prove effective at inhibiting the aggregation of insulin via pathways that depend on concentration and identity of the incorporated metal.     

Methacrylated glycol chitosan as a photopolymerizable biomaterial

Glycol chitosan is a derivative of chitosan that is soluble at neutral pH and possesses potentially useful biological properties. With the goal of obtaining biocompatible hydrogels for use as tissue engineering scaffolds or drug delivery depots, glycol chitosan was converted to a photopolymerizable prepolymer through graft methacrylation using glycidyl methacrylate in aqueous media at pH 9. N-Methacrylation was verified by both (1)H NMR and (13)C NMR. The degree of N-methacrylation, measured via (1)H NMR, was easily varied from 1.5% to approximately 25% by varying the molar ratio of glycidyl methacrylate to glycol chitosan and the reaction time. Using a chondrocyte cell line, the N-methacrylated glycol chitosan was found to be noncytotoxic up to a concentration of 1 mg/mL. The prepolymer was cross-linked in solution using UV light and Irgacure 2959 photoinitiator under various conditions to yield gels of low sol content ( approximately 5%), high equilibrium water content (85-95%), and thicknesses of up to 6 mm. Cross-polarization magic-angle spinning (13)C solid state NMR verified the complete conversion of the double bonds in the gel. Chondrocytes seeded directly onto the gel surface, populated the entirety of the gel and remained viable for up to one week. The hydrogels degraded slowly in vitro in the presence of lysozyme at a rate that increased as the cross-link density of the gels decreased.     

The kinetics of photooxidation of c-type cytochromes by Rhodospirillum rubrum reaction centers.

The kinetics of photooxidation of c-type cytochromes from horse heart, Rhodospirillum rubrum, and Rhodopseudomonas capsulata by purified reaction centers from R. rubrum have been investigated. The kinetic mechanism was found to be complex with a second-order step (complex formation) followed by a rate limiting first-order step. Based on studies of the reaction as a function of pH, ionic strength, and detergent concentration, it appears that the complex formation step is largely electrostatically controlled with only portions of the surfaces of the interacting molecules participating. Further, the first-order process observed at high cytochrome concentration appears to result from solvent reorganization and/or a conformational change following complex formation. Based on data analysis in terms of outersphere electron transfer, it is proposed that another first-order process exists which is not rate limiting and is the electron transfer step. Finally, it was found that the detergent concentration can have a profound effect on both the oxidation-reduction potential of the cytochromes and the kinetics of photooxidation. These results limit the detergent concentration range over which experiments can be conducted and interpreted.     

Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature

When adding beta-glycerophosphate (beta-GP), a weak base, to chitosan aqueous solutions, the polymer remains in solution at neutral pH and room temperature, while homogeneous gelation of this system can be triggered upon heating. It is therefore one of the rare true physical chitosan hydrogels. In this study, physicochemical and rheological properties of chitosan solutions in the presence of acetic acid and beta-GP were investigated as a function of temperature in order to gain a better understanding of the gelation mechanisms. The gel structure formed at high temperature was only partially thermoreversible upon cooling to 5 degrees C because of the existence of remaining associations, confirmed by the spontaneous recovery of the gel after breakup at low temperature. Increasing temperature had no effect on the pH values of this system, while conductivity (and calculated ionic strength) increased. Values from the pH measurements were used to estimate the degree of protonation of each species as a function of temperature. The decreasing ratio of -NH3+ in chitosan and -OPO(O-)2 in beta-GP suggested reduced chitosan solubility along with a diminution of ionic interactions such as ionic bridging with increasing temperature. On the other hand, the increased ionic strength as a function of temperature, in the presence of beta-GP, enhanced screening of electrostatic repulsion and increased hydrophobic effect, resulting in favorable conditions for gel formation. Therefore, our study suggests that hydrophobic interactions and reduced solubility are the main driving force for chitosan gelation at high temperature in the presence of beta-GP.     

Study of the near-neutral pH-sensitivity of chitosan/gelatin hydrogels by turbidimetry and microcantilever deflection

The fundamental properties and pH-sensitivity of chitosan/gelating hydrogels were investigated using spectroscopic and microelectro mechanical (MEMS) measurement approaches. Turbidimetric titration revealed that there were electrostatic attractive interactions between tripolyphosphate (TPP), chitosan, and gelatin in the acidic pH range, depending on their degree of ionization. The pH-sensitive swelling behavior of the hydrogels was investigated by monitoring the deflection of hydrogel-coated microcantilevers, which exhibited a sensitive and repeatable response to solution pH. The deflection of the microcantilever increased as the pH decreased, and the response speed of the system exhibited a nearly linear relationship with pH. The effects of the pH and concentration of TPP solution, as well as the ratio of chitosan to gelatin in gel precursor solutions, on the pH sensitivity of the hydrogels were also investigated. It was found that the swelling of the hydrogel is mainly a result of chain relaxation of chitosan-TPP complexes caused by protonation of free amino groups in chitosan, which depends on the crosslinking density set during the formation of the network. An increase in initial crosslink density induced a decrease in swelling and pH sensitivity. It can be concluded from this study that pH-sensitive chitosan gel properties can be tuned by preparatory conditions and inclusion of gelatin. Furthermore, microcantilevers can be used as a platform for gaining increased understanding of environmentally sensitive polymers.     

The kinetics and mechanism of the formation of crystalline phase of dipalmitoylphosphatidylethanolamine dispersed in aqueous dimethyl sulfoxide solutions

The phase transition kinetics and mechanism of formation of a lamellar-crystalline phase of dipalmitoylphosphatidylethanolamine (DPPE) dispersed in different concentrations of aqueous dimethyl sulfoxide (DMSO) during cooling have been examined by differential scanning calorimetry and synchrotron X-ray diffraction techniques. In dispersions containing mole fractions of DMSO (x<0.22), the phase transition sequence of the phospholipid is from lamellar liquid-crystal phase to lamellar-gel phase. Increasing the mole fraction of DMSO to 0.220.5 resulted in a direct transition from liquid-crystal phase to lamellar crystal phase with no detectable intermediate gel phase. A temperature versus DMSO concentration phase diagram was constructed based on calorimetric data with phase assignments made using synchrotron X-ray diffraction measurements. The non-isothermal formation kinetics of the lamellar crystal phase, which is expressed as the half time of the transformation process, was found to depend on DMSO concentration. The inducement of lamellar crystal phase in DPPE by DMSO is discussed in terms of the dehydration effect of DMSO and competitive molecular interactions between DMSO, water, and the phospholipid.     

Enhanced Healing of Rat Calvarial Defects with MSCs Loaded on BMP-2 Releasing Chitosan/Alginate/Hydroxyapatite Scaffolds

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy.     

Determination of the degree of acetylation of chitosan by UV spectrophotometry using dual standards

Determination of the degree of acetylation of chitosan by UV spectrophotometry using dual standards is investigated. The UV absorbance of a pure chitosan solution is contributed additively by the N-acetylglucosamine and glucosamine residues; the absorbance divided by the total molar concentration of the residues (A/c(t)) is linearly related to the degree of acetylation (DA). Using acetyl glucosamine and glucosamine hydrochloride as standards in 0.1M hydrochloric acid solution, the equation obtained by linear regression is A/c(t)=3.3615 DA+0.0218, R(2)=0.9958. The DA of the analytical sample (m milligram of chitosan in V liters solution) can be calculated by.     

Degradation of fully water-soluble, partially N-acetylated chitosans with lysozyme

Chitosans, prepared by homogeneous N-deacetylation of chitin, with degrees of N-acetylation ranging from 4 to 60% (F_A = 0·04 to 0·60) exhibiting full water solubility and known random distribution of acetyl groups, were degraded with lysozyme. Initial degradation rates (r) were determined from plots of the viscosity decrease (Δ1/[η]) against time of degradation. The time course of degradation of chitosans with lysozyme were non-linear, while the time course of degradation of chitosans with an oxidative-reductive depolymerization reaction (using H₂O₂) showed the expected linear relationship for a first-order, random depolymerization reaction, independent of the chemical composition of the chitosan.

The effect of lysozyme concentration and substrate concentration on the initial degradation rates were determined, showing that this lysozyme-chitosan system obeys Michaelis-Menten kinetics.

The initial degradation rates of chitosan with lysozyme increased strongly with increasing fraction of acetylated units (F_A). From a Michaelis-Menten analysis of the degradation data that assumes different catalytic activities of lysozyme for the different hexameric substrates in the polysaccharide chain, it is concluded that the hexameric substrates that contain three-four or more acetylated units contribute mostly to the initial degradation rate when lysozyme degrades partially N-acetylated chitosans.

A chitosan with a very low fraction of acetylated units (F_A = 0·010) was studied as an enzyme inhibitor. Initial degradation rates of chitosan (with different F_A values) decreased as the inhibitor concentration increased, while the relative rates stayed constant, indicating that the ratio between initial reaction rates for productive sites (hexamers containing three-four or more N-acetylated units) are unaffected by non-productive sites, as deduced from the theory of competing substrates.     

Chitosan cross-linking with a water-soluble,blocked diisocyanate. 2. Solvates and hydrogels

A water-soluble, blocked diisocyante was used to cross-link chitosan under various degrees of solvation, including hydration to form hydrogels. Thermal cross-linking of films cast from various amounts of organic cosolvents was found to increase with increased level of cosolvent up to a solvation level of 17% (w/w) and to be more efficient than for films prepared without cosolvent. Rheological studies revealed that gel modulus increased and gel time decreased with increasing cross-linker content and that gelation kinetics were consistent with a process having an activation energy of 103 kJ/mol. Swelling of hydrogels indicated that, even at high levels of hydration, the increased molecular mobility of reactants allowed for efficient network formation in a concentration-dependent manner. The extent of solvation via equilibrium swelling correlated well with degradative properties of chitosan networks in the presence of Chitinase (E. C. 3.2.1.14) from Streptomyces griseus with stability increasing with decreasing swelling (i.e., increased cross-linking).     

Rheological characterisation of a novel thermosensitive chitosan/poly(vinyl alcohol) blend hydrogel

Thermosensitive hydrogels that are triggered by changes in environmental temperature thus resulting in in situ hydrogel formation have recently attracted the attention of many investigators for biomedical applications. In the current work, the thermosensitive hydrogel was prepared through the mixture of chitosan (CS), poly(vinyl alcohol) (PVA) and sodium bicarbonate. The mixture was liquid aqueous solutions at low temperature (about 4 °C), but a gel under physiological conditions. The hydrogel was characterized by FTIR, swelling and rheological analysis. The effect of hydrogel composition and temperature on both the gel process and the gel strength was investigated from which possible hydrogel formation mechanisms were inferred. In addition, the hydrogel interior morphology as well as porosity of structure was evaluated by scanning electron microscopy (SEM). The potential of the hydrogels as vehicles for delivering bovine serum albumin (BSA) were also examined. In this study, the physically crosslinked chitosan/PVA gel was prepared under mild conditions without organic solvent, high temperature or harsh pH. The viscoelastic properties, as investigated rheologically, indicate that the gel had good mechanical strength. The gel formed implants in situ in response to temperature change, from low temperature (about 4 °C) to body temperature, which was very suitable for local and sustained delivery of proteins, cell encapsulation and tissue engineering.     

The kinetics of bovine growth hormone folding are consistent with a framework model

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.     

Two-stage optimization process for formulation of chitosan microspheres

The objective of the present study was to optimize the concentration of a chitosan solution, stirring speed, and concentration of drugs having different aqueous solubility for the formulation of chitosan microspheres. Chitosan microspheres (unloaded and drug loaded) were prepared by the chemical denaturation method and were subjected to measurement of morphology, mean particle size, particle size distribution, percentage drug entrapment (PDE), drug loading, and drug release (in vitro). Morphology of the microspheres was dependent on the level of independent process parameters. While mean particle size of unloaded microspheres was found to undergo significant change with each increase in concentration of chitosan solution, the stirring rate was found to have a significant effect only at the lower level (ie, 2000 to 3000 rpm). Of importance, spherical unloaded microspheres were also obtained with a chitosan solution of concentration less than 1 mg/mL. Segregated unloaded microspheres with particle size in the range of 7 to 15 microm and mean particle size of 12.68 microm were obtained in the batch prepared by using a chitosan solution of 2 mg/mL concentration and stirring speed of 3000 rpm. The highest drug load ( microg drug/mg microspheres) was 50.63 and 13.84 for microspheres containing 5-fluorouracil and methotrexate, respectively. While the release of 5-fluorouracil followed Higuchi's square-root model, methotrexate released more slowly with a combination of first-order kinetics and Higuchi's square-root model. The formation of chitosan microspheres is helped by the use of differential stirring. While an increase in the concentration of water-soluble drug may help to increase PDE and drug load over a large concentration range, the effect is limited in case of water-insoluble drugs.     

New aspects of the formation of physical hydrogels of chitosan in a hydroalcoholic medium

New aspects concerning the mechanism of formation of chitosan physical hydrogels without any cross-linking agent were studied. The gelation took place during the evaporation of a hydroalcoholic solution of chitosan. We first demonstrated that it was possible to form a physical hydrogel from a hydrochloride form of chitosan. Chromatographic methods showed that during the gel formation, when the initial concentration is over C, the critical concentration of chain entanglement, the water and acid used for the solubilization of the polymer were both eliminated. This particular situation contributed to decrease the dielectric constant of the medium and the apparent charge density of chitosan chains, thus inducing the formation of a three-dimensional network through hydrophobic interactions and hydrogen bonding. In the gelation process, this step was kinetically determining. The speed of evaporation of water and acid were determined and different initial conditions were compared. Thus, we investigated the influence of: the initial polymer concentration, the nature of the counterion and the alcohol, the temperature and the geometry of the reactor. Our results allowed us to confirm the existence of a second critical initial concentration C, from which the evaporation of water became more difficult. We suggested that C corresponded to a reorganization of the solution involving the presence of gel precursors. Then, a mechanism of formation of physical hydrogels of chitosan in a hydroalcoholic medium could be proposed. For the first time, we demonstrated that it was possible to generate physical hydrogels in the presence of various diols, which size of the carbonated chain appeared as a limiting factor for the gelation process. These physical hydrogels of chitosan are currently used in our laboratory for tissue engineering in the treatment of third degree burns with the possibility to adapt their mechanical properties from the choice of both the acid or the alcohol used.