Spinning of hydroalcoholic chitosan solutions

We investigated the spinning of hydroalcoholic chitosan solutions. The dope composition was optimized in order to obtain a continuous alcogel fiber by water evaporation on heating the extruded hydroalcoholic solution. This alcogel fiber was then neutralized in aqueous alkali baths and washed in water to eliminate the residual alcohol and salts before final drying. Depending on the alcohol content in the filament at the neutralization step, on specific alcohol–chitosan interactions and on the nature and concentration of the coagulation base, the process yielded semicrystalline chitosan fibers with different proportions of anhydrous and hydrated allomorphs. Contrarily to the classical annealing method, the formation of mainly anhydrous crystals was obtained without significant molecular weight decrease by neutralizing the polymer in hydrophobic conditions. The control of allomorph content was shown to be related to the hydrophobicity of the solvent (alcohol fraction) at the neutralization step.     

5-methyl-pyrrolidinone chitosan films as carriers for buccal administration of proteins

The purpose of this research was to investigate 5-methyl-pyrrolidinone chitosan (MPC) films as carriers for buccal delivery of protein drugs. Placebo and protein-loaded MPC films were prepared by casting and were then cross-linked with tripolyphosphate at different pH conditions. Myoglobin (MHb) was chosen as the model protein because its molecular weight is under the permeability limit of the buccal mucosa. The observed characteristics like bioadhesiveness, swelling behavior, and in vitro release of MHb from loaded films furnish information on the functional behavior of these films. The results obtained show that the modulation of Mhb release was achieved only through chitosan cross-linking; the best results in release rate control were obtained by cross-linking performed at pH 6.5. Good bioadhesion properties were maintained even with high cross-linking degrees; the swelling index of MHb-loaded films at different cross-linking degrees evaluated at pH 7.4 and pH 6.4 were comparable to those of placebo films. By setting suitable tripolyphosphate cross-linking conditions for MPC films, one can control protein release without affecting bioadhesion. Published: September 1, 2006     

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%.     

Thermo-sensitive chitosan based semi-IPN hydrogels for high loading and sustained release of anionic drugs

Thermo-sensitive poly(N-isopropyl acrylamide-co-vinyl pyrrolidone)/chitosan [P(NIPAM-co-NVP)/CS] semi-IPN hydrogels with improved loading capacity and sustained release for anionic drugs NAP were prepared by free-radical polymerization. The LCST of hydrogels was adjusted to the vicinity of body temperature by introducing hydrophilic NVP. The presence of CS in semi-IPN networks improves the swelling behavior and provides a high affinity for anionic drug NAP due to the strong interactions between NAP molecules and CS chains. Release of NAP was suppressed at pH 2.2 and 5.0 and accelerated at pH 7.4 due to the deprotonation of amino groups in CS. Increasing temperature above LCST, hydrogels showed a continuous release of NAP without burst diffusion due to the shrinkage of PNIPAM restraining the drug release.     

Typical physicochemical behaviors of chitosan in aqueous solution

Physicochemical properties of a homogeneous series of chitosans with different degrees of acetylation and almost the same degree of polymerization were investigated in an ammonium acetate buffer. Techniques such as interferometry, static light scattering (in batch or coupled on line with a chromatographic system), and viscometry were processed. All of the results agree with a unique law of behavior only depending on the degree of acetylation of the polymer. Indeed, values of the refractive index increment, radius of gyration, second viral coefficient, and intrinsic viscosity are decreasing in the same way as DA is increasing. Three distinct domains of DA were defined and correlated to the different behaviors of chitosans: (i) a polyeletrolyte domain for DA below 20%; (ii) a transition domain between DA = 20% and 50% where chitosan loses its hydrophilicity; (iii) a hydrophobic domain for DAs over 50% where polymer associations can arise. Conformations of chitosan chains were studied by the calculations of the persistence lengths (L(p)). The average value was found to be close to 5 nm, in agreement with the wormlike chain model, but no significant variation of L(p) with the degree of acetylation was noticed.     

Dependence on the Preparation Procedure of the Polymorphism and Crystallinity of Chitosan Membranes

Differences in the polymorphism and crystallinity of chitosan were found in membranes prepared by different procedures when examined by X-ray diffraction measurements for four samples of chitosan differing in the degree of polymerization. When an acetic acid solution of chitosan was dried in air and then soaked in an alkaline solution (method A), both hydrated and anhydrous polymorphs of chitosan were present in the resulting membranes; the latter polymorph made chitosan insoluble in common solvents of chitosan, and its crystallinity increased with decreasing chitosan molecular weight. When a highly concentrated chitosan solution in aqueous acetic acid was neutralized with an alkaline solution (method B), no anhydrous polymorphs were detected in the membrane because of incomplete drying. When aqueous formic acid was used as the solvent, behavior basically similar to that in aqueous acetic acid was observed. In contrast, even with method A, aqueous hydrochloric acid gave a chitosan membrane having very little anhydrous crystallinity. The crystalline polymorph called “1–2”, which has been proposed to be one of four chitosan polymorphs, is considered to be a mixture of hydrated and anhydrous crystals.     

Surface modification of chitosan films. Effects of hydrophobicity on protein adsorption

The surface of chitosan films was modified using acid chloride and acid anhydrides. Chemical composition at the film surface was analyzed by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). ATR-FTIR data verified that the substitution took place at the amino groups of chitosan, thus forming amide linkages, and the modification proceeded to the depth at least 1 microm. Choices of molecules substituted at the amino groups of the glucosamine units did affect the hydrophobicity of the film surface, as indicated by air-water contact angle analysis. The surface became more hydrophobic than that of non-modified film when a stearoyl group (C(17)H(35)CO-) was attached to the films. The reaction of chitosan films with succinic anhydride or phthalic anhydride, however, produced more hydrophilic films. Selected modified films were subjected to protein adsorption study. The amount of protein adsorbed, determined by bicinchoninic acid (BCA) assay, related to the types of attached molecules. The improved surface hydrophobicity affected by the stearoyl groups promoted protein adsorption. In contrast, selective adsorption behavior was observed in the case of the chitosan films modified with anhydride derivatives. Lysozyme adsorption was enhanced by H-bonding and charge attraction with the hydrophilic surface. While the amount of albumin adsorbed was decreased possibly due to negative charges that gave rise to repulsion between the modified surface and albumin. This study has demonstrated that it is conceivable to fine-tune surface properties which influence its response to bio-macromolecules by heterogeneous chemical modification.     

Preparation and characterization of sodium hexameta phosphate cross-linked chitosan microspheres for controlled and sustained delivery of centchroman

The cross-linked microspheres using chitosan with different molecular weights and degree of deacetylation have been prepared in presence of sodium hexameta polyphosphate (SHMP) as physical cross-linker. The degree of cross-linking through electrostatic interactions in chitosan microspheres has been evaluated by varying the charge density on chitosan and varying degree of dissociation of sodium hexameta polyphosphate by solution pH. The degree of deacetylation and molecular weight of chitosan has controlled electrostatic interactions between hexameta polyphosphate anions and chitosan, which played significant role in swelling, loading and release characteristics of chitosan microspheres for centchroman. The microspheres prepared by hexameta polyphosphate anions cross-linker were compact and more hydrophobic than covalently cross-linked microspheres, which has been attributed to the participation of all amino groups of chitosan in physical cross-linking with added hexameta polyphosphate anions. The microspheres prepared under different experimental conditions have shown an initial step of burst release, which was followed by a step of controlled release for centchroman. The extent of drug release in these steps has shown dependence on properties of chitosan and degree of cross-linking between chitosan and added polyanions. The degree of swelling and release characteristics of microspheres was also studied in presence of organic and inorganic salts, which shown significant effect on controlled characteristics of microspheres due to variations in ionic strength of the medium. The initial step of drug release has followed first order kinetics and become zero order after attaining an equilibrium degree of swelling in these microspheres. The microspheres prepared using chitosan with 62% (w/w) degree of deacetylation and molecular weight of 1134 kg mol⁻¹ have shown a sustained release for centchroman for 50 h at 4% (w/w) degree of cross-linking with SHMP.     

Design and Evaluation of Hydrophilic Matrix System Containing Polyethylene Oxides for the Zero-Order Controlled Delivery of Water-Insoluble Drugs

The aim of this study was to design a polyethylene oxide (PEO) binary hydrophilic matrix controlled system and investigate the most important influence(s) on the in vitro water-insoluble drug release behavior of this controlled system. Direct-compressed PEO binary matrix tablets were obtained from a variety of low viscosity hydrophilic materials as a sustained agent, using anhydrous drugs as a model drug. Water uptake rate, swelling rate, and erosion rate of matrices were investigated for the evaluation of the PEO hydrophilic matrix systems. The effect of the dose, the solubility of water-insoluble drug, and the rheology of polymers on in vitro release were also discussed. Based on the in vitro release kinetics study, three optimized PEO binary matrices were selected for further research. And, these PEO binary matrices had shown the similar release behavior that had been evaluated by the similarity factor f ₂. Further study indicated that they had identical hydration, swelling, and erosion rate. Moreover, rheology study exhibited the similar rheological equation of Herschel–Bulkley and their viscosity was also within the same magnitude. Therefore, viscosity plays the most important role to control drug release compared to other factors in PEO binary matrix system. This research provides fundamental understanding of in vitro drug release of PEO binary hydrophilic matrix tablets and helps pharmaceutical workers to develop a hydrophilic controlled system, which will effectively shorten the process of formulation development by reducing trial-and-error.     

Sulfasalazine release from alginate-N,O-carboxymethyl chitosan gel beads coated by chitosan

In the present study, spherical beads were prepared from a water-soluble chitosan (N,O-carboxymethyl chitosan, NOCC) and alginate with ionic gelation method. Then, swollen calcium–alginate–NOCC beads were coated with chitosan. To prepare drug loaded beads, sulfasalazine (SA) was added to the initial aqueous polymer solution. The effect of coating, as well as drying procedure, on the swelling behavior of unloaded beads and SA release of drug loaded ones were evaluated in simulated gastrointestinal tract fluid. The rate of swelling and drug release were decreased for air-dried and coated beads in comparison with freeze-dried and uncoated ones, respectively. No burst release of drug was observed from whole tested beads. Chitosan coated beads released approximately 40% of encapsulated drug in simulated gastric and small intestine tract fluid. Based on these results, the chitosan coated alginate–NOCC hydrogel may be used as potential polymeric carrier for colon-specific delivery of sulfasalazine.     

Hydrophilic excipients modulate the time lag of time-controlled disintegrating press-coated tablets

An oral press-coated tablet was developed by means of direct compression to achieve the time-controlled disintegrating or rupturing function with a distinct predetermined lag time. This press-coated tablet containing sodium diclofenac in the inner core was formulated with an outer shell by different weight ratios of hydrophobic polymer of micronized ethylcellulose (EC) powder and hydrophilic excipients such as spray-dried lactose (SDL) or hydroxypropyl methylcellulose (HPMC). The effect of the formulation of an outer shell comprising both hydrophobic polymer and hydrophilic excipients on the time lag of drug release was investigated. The release profile of the press-coated tablet exhibited a time period without drug release (time lag) followed by a rapid and complete release phase, in which the outer shell ruptured or broke into 2 halves. The lag phase was markedly dependent on the weight ratios of EC/SDL or EC/HPMC in the outer shell. Different time lags of the press-coated tablets from 1.0 to 16.3 hours could be modulated by changing the type and amount of the excipients. A semilogarithmic plot of the time lag of the tablet against the weight ratios of EC/SDL or EC/HPMC in the outer shell demonstrated a good linear relationship, withr=0.976 andr=0.982, respectively. The predetermined time lag prior to the drug release from a press-coated tablet prepared by using a micronized EC as a retarding coating shell can be adequately scheduled with the addition of hydrophilic excipients according to the time or site requirements.     

Immobilization of Bacillus licheniformis α-amylase onto reactive polymer films

Alpha-amylase was covalently immobilized onto maleic anhydride copolymer films preserving activity. The initial activity of the immobilized layers strongly depended on the immobilization solution, and on the physicochemical properties of the copolymer film. Higher enzyme loading (quantified by amino acid analysis using HPLC) and activity (measured by following starch hydrolysis) were attainable onto hydrophilic, highly swelling 3-D poly(ethylene-alt-maleic anhydride) (PEMA) copolymer films, while immobilization onto hydrophobic poly(octadecene-alt-maleic anhydride) (POMA) copolymer films resulted in low content enzyme layers and lower activity. No significant activity was lost upon dehydration/re-hydration or storage of enzyme containing PEMA copolymer layers in deionised water for up to 48 h. In contrast, α-amylase decorated POMA films suffered a significant activity loss under those conditions. The distinct behaviours may be attributed to the different intrinsic physicochemical properties of the copolymer films. The compact, hydrophobic POMA films possibly favours hydrophobic interactions between the hydrophobic moieties of the protein and the surface, which may result in conformational changes, and consequent loss of activity. Surprisingly, residual activity was found after harsh treatments of active α-amylase PEMA based layers revealing that immobilization onto the hydrophilic polymer films improved the stability of the enzyme.     

The Inclusion of Chitosan in Poly-ε-caprolactone Nanoparticles: Impact on the Delivery System Characteristics and on the Adsorbed Ovalbumin Secondary Structure

This report extensively explores the benefits of including chitosan into poly-ε-caprolactone (PCL) nanoparticles (NPs) to obtain an improved protein/antigen delivery system. Blend NPs (PCL/chitosan NPs) showed improved protein adsorption efficacy (84%) in low shear stress and aqueous environment, suggesting that a synergistic effect between PCL hydrophobic nature and the positive charges of chitosan present at the particle surface was responsible for protein interaction. Additionally, thermal analysis suggested the blend NPs were more stable than the isolated polymers and cytotoxicity assays in a primary cell culture revealed chitosan inclusion in PCL NPs reduced the toxicity of the delivery system. A quantitative 6-month stability study showed that the inclusion of chitosan in PCL NPs did not induce a change in adsorbed ovalbumin (OVA) secondary structure characterized by the increase in the unordered conformation (random coil), as it was observed for OVA adsorbed to chitosan NPs. Additionally, the slight conformational changes occurred, are not expected to compromise ovalbumin secondary structure and activity, during a 6-month storage even at high temperatures (45°C). In simulated biological fluids, PCL/chitosan NPs showed an advantageous release profile for oral delivery. Overall, the combination of PCL and chitosan characteristics provide PCL/chitosan NPs valuable features particularly important to the development of vaccines for developing countries, where it is difficult to ensure cold chain transportation and non-parenteral formulations would be preferred.     

Inflammation responsive logic gate nanoparticles for the delivery of proteins

Oxidative stress and reduced pH are important stimuli targets for intracellular delivery and for delivery to diseased tissue. However, there is a dearth of materials able to deliver bioactive agents selectively under these conditions. We employed our recently developed dual response strategy to build a polymeric nanoparticle that degrades upon exposure to two stimuli in tandem. Our polythioether ketal based nanoparticles undergo two chemical transformations; the first is the oxidation of the thioether groups along the polymer backbone of the nanoparticles upon exposure to reactive oxygen species (ROS). This transformation switches the polymeric backbone from hydrophobic to hydrophilic and thus allows, in mildly acidic environments, the rapid acid-catalyzed degradation of the ketal groups also along the polymer backbone. Dynamic light scattering and payload release studies showed full particle degradation only in conditions that combined both oxidative stress and acidity, and these conditions led to higher release of encapsulated protein within 24 h. Nanoparticles in neutral pH and under oxidative conditions showed small molecule release and swelling of otherwise intact nanparticles. Notably, cellular studies show absence of toxicity and efficient uptake of nanoparticles by macrophages followed by cytoplasmic release of ovalbumin. Future work will apply this system to inflammatory diseases.     

Glutaraldehyde and glyoxal cross-linked chitosan microspheres for controlled delivery of centchroman

Glutaraldehyde and glyoxal cross-linked microspheres were prepared using chitosan with different molecular weights (MWs) and degrees of deacetylation (DDAs) for sustained release of centchroman under physiological conditions. The DDA in chitosan was determined by different methods, and the samples were categorized as chitosan with low (48%), medium (62%), and high (75%) DDA. The size and shape of the microspheres were determined by scanning electron microscopy (SEM), and hydrophobicity was determined by adsorption of Rose Bengal dye on microspheres cross-linked with glutaraldehyde or glyoxal. The effect of MW, DDA, and degree of cross-linking in microspheres was studied on the degree of swelling, as well as by the loading and release of centchroman. The glyoxal cross-linked microspheres were more compact and hydrophobic and showed better sustained release in companion to chitosan microspheres and glutaraldehyde cross-linked microspheres. The linear fractional release of centchroman with the square root of time indicated a Fickian behavior of centchroman, and the microspheres also showed zero-order release kinetics for centchroman.     

Development and Evaluation of Biodegradable Chitosan Films of Metronidazole and Levofloxacin for the Management of Periodontitis

Metronidazole (MZ) and levofloxacin (LF) are widely employed for treatment of periodontitis, but high oral dose and resistance development after long-term oral administration limit their use. The aim of this study was to alleviate shortcomings in the treatment of periodontitis by fabrication of intrapocket, biodegradable films of chitosan (CS) loaded with MZ and LF meant for inserting into periodontal pockets to treat infections. The films were developed by solvent casting technique using propylene glycol as plasticizer and glutaraldehyde as crosslinking agent. Their physical characteristics, such as drug content, surface pH, swelling index, and folding endurance, exhibited results within limit. Further, FTIR and DSC studies revealed stability of films and compatibility between drugs and excipients. SEM images of films showed the presence of free drug particles on the surface causing burst effect. In vitro release in McIlvaine buffer pH 6.6 was of sustained nature assisted by the burst effect. CS and crosslinking agent concentrations negatively affected drug release and positively affected T₉₀ (time for releasing 90% of the drug) due to altered matrix density. In contrast, the plasticizer concentration increases membrane permeability and hence increased drug release, lowering T₉₀. Crosslinked films demonstrated sustained release up to 7 days. The antibacterial efficacy of films was tested on Staphylococcus aureus and Escherichia coli, indicating good antibacterial activity. Clinical trials on patients proved the therapeutic efficacy of the films by a significant (p?<?0.05) decrease in the clinical markers of periodontitis, i.e. gingival index, plaque index and pocket depth. Conclusively, the films of MZ and LF were successful tools for the management of periodontitis.     

Elaboration of biodegradable polymer substrate for cultivation of human dermal fibroblasts

The influence of polylactic acid (PLA) surface films on the pattern of cell behavior was studied. The human dermal fibroblasts were cultivated on PLA covered glasses. The hydrophobic nature of PLA films depends on the availability of polymer solvent in the film preparation. PLA films obtained from a more polar solvent--aceton--appeared to be more hydrophilic than those obtained from methylene chloride. More hydrophilic polymer films also appeared to be more preferable for cell cultivation, and human dermal fibroblasts demonstrated a better adhesion and proliferation on hydrophilic rather than on hydrophobic PLA films.     

Physicochemical behavior of homogeneous series of acetylated chitosans in aqueous solution: role of various structural parameters

Physicochemical properties of four different homogeneous series of chitosans with degrees of acetylation (DA) and weight-average degrees of polymerization (DP(w)) ranging from 0 to 70% and 650 to 2600, respectively, were characterized in an ammonium acetate buffer (pH 4.5). Then, the intrinsic viscosity ([eta](0)), the root-mean-square z-average of the gyration radius (R(G,z)), and the second virial coefficient (A(2)) were studied by viscometry and static light scattering. The conformation of chitosan, according to DA and DP(w), was highlighted through the variations of alpha and nu parameters, deduced from the scale laws [eta](0) = K(w)and R(G,z) = K', respectively, and the total persistence length (L(p,tot)). In relation with the different behaviors of chitosan in solution, the conformation varied according to two distinct domains versus DA with a transition range in between. Then, (i) for DA < 25%, chitosan exhibited a flexible conformation; (ii) a transition domain for 25 < DA < 50%, where the chitosan conformation became slightly stiffer and, (iii) for DA > 50%, on increasing DP(w) and DA, the participation of the excluded volume effect became preponderant and counterbalanced the depletion of the chains by steric effects and long-distance interactions. It was also highlighted that below and beyond a critical DP(w,c) (ranging from 1 300 to 1 800 for DAs from 70 to 0%, respectively) the flexibility of chitosan chains markedly increased then decreased (for DA > 50%) or became more or less constant (DA < 50%). All the conformations of chitosan with regards to DA and DP(w) were described in terms of short-distance interactions and excluded volume effect.     

Porous bone morphogenetic protein-2 microspheres: Polymer binding and in vitro release

This research compared the binding and release of recombinant human bone morphogenetic protein 2 (rhBMP-2) with a series of hydrophobic and hydrophilic poly-lactide-co-glycolide (PLGA) copolymers. Porous microspheres were produced via a double emulsion process. Binding and incorporation of protein were achieved by soaking microspheres in buffered protein solutions, filtering, and comparing protein concentration remaining to nonmicrosphere-containing samples. Protein release was determined by soaking bound microspheres in a physiological buffer and measuring protein concentration (by reversed-phase high-performance liquid chromatography) in solution over time. Normalized for specific surface area and paired by polymer molecular weight. microspheres made from hydrophilic 50∶50 or 75∶25 PLGA bound significantly more protein than microspheres made from the corresponding hydrophobic PLGA. Increased binding capacity correlated with higher polymer acid values. With certain polymers, rhBMP-2 adsorption was decreased or inhibited at high protein concentration, but protein loading could be enhanced by increasing the protein solution:PLGA (volume:mass) ratio or by repetitive soaking. Microspheres of various PLGAs released unbound protein in 3 days, whereas the subsequent bound protein release corresponded to mass loss. RhBMP-2 binding to PLGA was controlled by the acid value, protein concentration, and adsorption technique. The protein released in 2 phases: the first occurred over 3 days regardless of PLGA used and emanated from unbound, incorporated protein, while the second was controlled by mass loss and therefore was dependent on the polymer molecular weight. Overall, control of rhBMP-2 delivery is achievable by selection of PLGA microsphere carriers. Published: October, 7, 2001.