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The purpose of the present study was to synthesize and evaluate mucoadhesive polymers, exhibiting a high capacity to bind bivalent cations which are essential co-factors for intestinal proteolytic enzymes. Under the formation of amide bonds, the complexing agent EDTA was covalently bound to the primary amino groups of chitosan. One gram of the resulting conjugate with the lowest amount of remaining free amino groups (0.1 +/- 0.03%; mean +/- SD, n = 3) based on free chitosan as 1.0 was capable of binding 1.4 +/- 0.1 mM calcium, 2.0 +/- 0.1 mM zinc and 1.9 +/- 0.03 mM cobalt (mean +/- SD, n = 3) under intestinal pH-conditions, respectively. Whereas proteolytic activity of the serine proteases trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) could not be inhibited, proteolytic activity of the zinc proteases carboxypeptidase A (EC 3.4.17.1) and aminopeptidase N (EC 3.4.11.2) was strongly inhibited by the chitosan-EDTA conjugate. Moreover, it displays quick swelling properties in water and basic aqueous solutions. The adhesive force of the conjugate was even higher than of chitosan HCl. However, lowering the percentage of covalently attached EDTA on the polymer, leads to a significantly reduced adhesive force. According to these results, chitosan-EDTA conjugates exhibiting the lowest amount of remaining free amino groups, seem to be a useful tool in overcoming the enzymatic barrier for perorally administered therapeutic peptides.

A novel method for the preparation of N-acylchitosan fiber and N-acylchitosan-cellulose fiber is described. Each aqueous solution of sodium N-acetyl and N-propionylchitosan salts in aqueous 14% NaOH was spun through a viscose-type spinneret at 10-15 degrees C into a coagulating bath containing aqueous 10%, H2SO4. 25% Na2SO4 and 1.3% ZnSO4 to afford the corresponding white fiber. By the same method, a clear solution of sodium N-acetyl and N-propionyl chitosan salts were respectively mixed with sodium cellulose xanthate in aqueous 14% NaOH and spun to afford the corresponding white N-acylchitosan-cellulose fiber. Their filament tenacity and elongation values were 0.4-0.7 times as large as cellulose. These fibers were digestible in reactions by chitinase and lysozyme, and the digestibility was controlled by the structure of the N-acyl group.

In this study a (poly)peptide drug delivery system providing a protective effect towards serine pancreatic proteases was generated. Tablets containing insulin (3.3%), chitosan-EDTA (56.7%), chitosan-EDTA Bowman Birk Inhibitor (= BBI) conjugate (10%) and mannitol (30%) were homogenised in a mortar and compressed to tablets. The protective effect of this dosage form for the incorporated model drug was evaluated in vitro. Tablets were therefore incubated with an artificial intestinal fluid containing trypsin (1350 spectrophotometric BAEE units/ml), chymotrypsin (3.6 BTEE units/ml) and elastase (0.14 succinyl-Ala-Ala-Ala-p-nitroanilide units/ml) for 4.5 h at 37 degrees C. Following analysis of the dosage form demonstrated that 58.6+/-26.8% (mean +/- SD; n = 3) insulin in lateral parts and 44.4+/-12.4% (mean +/- SD: n = 3) insulin in inner parts of the swollen carrier-matrix were degraded, whereas insulin was completely metabolised in lateral parts and by 90.3+/-12.5% (mean +/- SD: n = 3) in inner parts of tablets without the chitosan-EDTA BBI conjugate. As chitosan-EDTA also provides a protective effect towards zinc-dependent proteases, the delivery system described in this study should therefore guarantee a protection towards the most abundant intestinal proteases. It might be a promising formulation for the peroral administration of peptide and protein drugs.

Chitosan, a hydrophilic biopolymer, is obtained industrially by hydrolysing the aminoacetyl groups of chitin. It is a natural, non-toxic, biodegradable polysaccharide available as solution, flake, fine powder, bead and fibre. The sources, biochemical aspects, structure and chemical modification, physico-chemical and functional properties, and applications of chitosan have been investigated extensively in the literature. In this paper, the attractive properties and broad applications of chitosan-based microparticles, their versatile properties, different preparation methods, and pharmaceutical and biopharmaceutical applications are reviewed.

Chitosans having degrees of acetylation (da) of 0.04, 0.10, and 0.22, respectively, were N-sulfated under a variety of reaction conditions. The derivatives obtained ranged in degree of sulfation (ds) from 0.4 to 0.86 (+/-0.05). All were soluble in water, and the rheological properties of their solutions varied markedly with da and ds values. Both ionic strength and pH had an effect on their solubility properties, and also on interactions that they exhibited with O-(carboxymethyl)cellulose, xanthan gum, and heparin. Being compatible with other polyelectrolytes such as these, the chitosan derivatives may be useful in some aqueous formulations.

Chitosans which are substituted with alkyl chains having a minimum of six carbon atoms demonstrate hydrophobic interactions in solution. The chemical structure of synthetized polymers is determined from NMR spectroscopy and microanalysis and the distribution between the different types of substituted units is obtained. In relation to the rheological behaviour different parameters are studied such as the nature of the hydrophobic chain and the substitution degree, the polymer concentration, the temperature and the ionic content of the polymeric solution. All the observations tend to prove the hydrophobic nature of the interaction mechanism.

The use of chitin as a support for solid-phase peptide synthesis is described and illustrated by synthesis of four peptides, varying in length from 10 to 29 residues. Syntheses were performed in a continuous-flow peptide synthesizer, using Fmoc chemistry. A cleavable linker, p-[(R,S)-alpha-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-di methoxybenzyl]- phenoxyacetic acid, was attached to chitosan at the desired substitution level, and the complex acetylated to yield a linker substituted chitin. The effects of temperature, solvents and degree of linker substitution on the syntheses were studied. Acyl carrier peptide (ACP) synthesis studies indicated that temperature was the single most important parameter. Increasing the temperature of the synthesis from 20 to 55 degrees C resulted in an enormous improvement of this synthesis, with about 90% of the crude product being the correct peptide. Denaturing solvents, such as DMSO, could be used without significant effect on the flow properties of the support. The synthesis of one peptide was mainly improved by lowering the degree of substitution from 0.3 to 0.1 mmol/g, suggesting peptide aggregation was a problem in this case. The results of three syntheses on chitin were comparable with those obtained with a commonly used commercial support. This work shows that, under appropriate conditions, chitin can be utilized directly as a support for peptide synthesis.

N,N',N"-Tri(monochloro)acetylchitotriose prepared by N-monochloroacetylation of chitotriose trihydrochloride was successfully polymerized into higher-molecular-weight oligomers by a lysozyme-catalyzed transglycosylation reaction, and a following base-catalyzed N-demonochloroacetylation gave a chitosan oligomer mixture mainly composed of oligomers with dp > 6. Partially N-deacetylated chitin oligomers (DAC oligomers) with dp 4-12 were synthesized by the enzyme reaction using N,N',N"-tri(monochloro)acetylchitotriose and N,N',N"-triacetylchitotriose (chitin trimer) as initial substrates followed by N-demonochloroacetylation. The structures of synthetic oligomers were analyzed by 1H NMR spectroscopy, enzymatic hydrolysis and nitrous acid deamination-NaBH4 reduction treatment. The dp of synthetic oligomers was measured by MALDI TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) using per-N-acetylated derivatives. The synthetic chitosan and DAC oligomers were strong elicitors for phytoalexin induction in Pisum sativum and Phaseolus vulgaris. This chemo-enzymatic method utilizing N-acylated chitotrioses as substrates is a novel approach to the synthesis of high-molecular-weight chitosan oligomers and DAC oligomers of biological importance.

Chitosan-amino acid conjugates were prepared by coupling amino acid esters to the carboxyl group of glyoxylic acid-substituted chitosan. The removal of heavy metals (Cu, Ni, Co and Mn) was increased by introduction of amino acids to chitosan, especially for Mn. Heavy metals were almost completely removed by chitosan-amino acid conjugates from solutions at an initial concentration of 100 parts per million.

This work deals with the synthesis and blood compatibility studies of Heparin immobilized chitosan--polyethyleneglycol (Chit-PEG) hydrogels for various biomedical applications. Chit-PEG interpenetrating net work (IPN) had been synthesised by crosslinking different ratios of chitosan with glutaraldehyde using schiffs base reaction mechanism and interpenetrating polyethyleneglycol (PEG) to form hydrogen bonding between the amino hydrogen in chitosan and polyether oxygen. An optimum gel combination was selected from the IPN of Chit-PEG and used for bonding heparin. This modified gel had dramatically improved its blood compatibility. The antithrombotic function of this gel and the release profile of heparin had been investigated using coagulation assays, and spectrophotometric quantitation. Recalcification times of plasma exposed to heparin immobilized Chit-PEG hydrogel were markedly increased as compared to heparin free gels. The anticoagulant function of this gel matrix may be due to partially released heparin and bonded heparin.

6-O-[(2-Hydroxyethyl)poly(2-oxyethyl)]chitosan ("glycolchitosan") was oxidatively cleaved with nitrous acid and then partly acetylated with acetic anhydride, reacted with bromoacetyl-N-hydroxysuccinimide, and reacted further with acetic anhydride. Conditions were selected, including fractionation by size-exclusion chromatography, so that the resulting "Chitin Leash" had an estimated, average molecular weight of 10,000 (dextran standards), corresponding to a length of approximately 40 sugar residues. It possessed 0.9 terminal aldehyde and 2.6 random (presumably) side-chain bromoacetyl reactive groups per chain (average values). As a model system, the Chitin Leash was used to crosslink staphylococcal nuclease (SNase) to ribonuclease A (RNase) with retention of 75 and 78%, respectively, of the starting enzyme activities. For this coupling, the Nase was first converted to a sulfhydryl SNase derivative which retained 74% of the activity of starting enzyme. The yields in this synthesis were: 13% Chitin Leash from glycolchitosan, 24% Chitin Leash-RNase from Chitin Leash and 45% SNase-Chitin Leash-RNase from the latter conjugate. The ratio of SNase to RNase in this conjugate was 1.0:0.94. In a second preparation, in which [14C]acetic anhydride was used, a longer reaction time was employed for the coupling of Chitin Leash to RNase. This gave a 1.0:1.8:0.95 molar ratio of Nase: [14C]Chitin Leash: RNase, revealing multiple attachment of the [14C]Chitin Leash to RNase. The activity of the RNase in the final conjugate was 20%. The latter conjugate was approximately 70% hydrolyzed by diaminooctyl-succinyl-lysozyme, disconnecting the two enzymes while not affecting their activities.

The conjugate of N4-(4-carboxybutyryl)-1-beta-D-arabinofuranosylcytosine (glu-ara-C) with chitosan, named chi-glu-ara-C, was prepared and evaluated on its utility as a macromolecular prodrug. 1-beta-D-Arabinofuranosylcytosine (ara-C) was predominantly regenerated gradually from chi-glu-ara-C in 1/15 M phosphate buffer, pH 7.4, at 37 degrees C, and its amount after 7 d incubation was 56% (w/w). The antitumor effect was evaluated by an increase in the lifespan (ILS) of the mice bearing P388 leukemia at the single administration at 24 h after inoculation. Ara-C exhibited an ILS of 3.4% at a dose of 100 mg/kg, and the survival time was not significantly different from that of the control. However, chi-glu-ara-C showed an ILS of 60.7% at a dose of 88 mg eq ara-C/kg, and the survival time was significantly different from that of the control. Chitosan and chi-glu-ara-C were observed to cause the side effects judged from the body weight loss to a certain extent. However, chi-glu-ara-C was recognized to be useful because of its effectiveness.

In order to provide the water-soluble and biodegradable macromolecular prodrug of 5-fluorouracil (5FU), the fixation of 5FUs to 6-O-carboxymethyl chitin(CM-chitin) through pentamethylene, monomethylene spacer groups via amide, ester bonds was carried out. The obtained CM-chitin/5FU conjugate showed the slow release of 5FU and exhibited remarkable antitumor activity against P388 lymphocytic leukemia in mice by intraperitoneal(i.p.) implantation/i.p. injection.

N-carboxymethylchitosan-N-O-sulfate (NCMCS), a sulfated polysaccharide derivative of chitin, inhibited the propagation of the human immunodeficiency virus type 1 (HIV-1) in human CD4+ cells and that of Rauscher murine leukemia virus (RLV) in murine fibroblasts. A dose-dependent inhibition of both viruses was observed without significant cytotoxicity. NCMCS blocked the binding of HIV-1 to human CD4+ target cells and competitively inhibited HIV-1 reverse transcriptase. Thus, NCMCS may prevent HIV-1 infection by inhibiting viral adsorption to the CD4 receptor and reverse transcription of the viral genome.

In view of the desirability to increase the survival time of the lipsome-based artificial red blood cells in vivo, bovine hemoglobin-loaded liposomes (LEHb) are incorporated with a polyanionic polymer, a highly substituted type of carboxymethylchitin (CMC). The liposomes are prepared by a modified Reverse Phase Evaporation technique and then purified using a Sepharose 4B column. A comparative study between experimental techniques for the determination of adsorption efficiency suggests that FT-IR spectroscopy gives a more accurate quantitative adsorption index while the chitinase-based enzymatic assay should be used as a qualitative detection tool. The overall composition of the bilayer(s) can be approximated by that of the natural (RBC) membrane in terms of total lipids and carbohydrates at 87.8% (phospholipids and cholesterol) and 12.2% CMC respectively.

The food industry is interested in polyelectrolytic coagulants of natural origin for the clarification of food beverages and the recovery of colloidal and dispersed particles from processing waste streams. This paper discusses potential industrial applications of recent findings on polymer complex formation obtained with a chitosan-poly(acrylic acid) model system. Process recommendations could be made on the basis of the ionic strength, pH, and charged group concentration of the fluid to be treated. Ionic strength does not affect the complex formation process. The amount of chitosan in the complex formed is controlled by the solution pH. The mechanism of complex formation indicates that pH measurements could be used to monitor the coagulation process.

The effect of O-(carboxymethyl)chitins (CM-chitins) on the activation of mouse-peritoneal macrophages in vivo and their mitogenic activity on mouse spleen-cells were investigated. The induction of cytotoxic macrophages is enhanced by an increase of negative charge at O-6 and decreased by further modification at O-3 of the GlcNAc residue. CM-Chitins had a minor effect on mitogenic activity that was independent of the site of modification; partially N-deacetylated chitins had little activity. Although there was remarkable enhancement of accessibility to lysozyme upon modification at O-6 of the GlcNAc residue, the accessibility was decreased by further substitution at O-3.

Liposome-type artificial red blood cells stabilized with carboxymethylchitin (mean diameter 310 nm) were prepared by a two-step emulsification technique. Sheep haemolysate was dispersed as fine droplets in a lecithin solution in dichloromethane to yield a W/O-type emulsion. The W/O emulsion thus obtained was then dispersed in an aqueous carboxymethylchitin solution to give a W/O/W-type complex emulsion. Removal of the organic solvent by evaporation from the complex emulsion left an aqueous suspension of the artificial red blood cells. The haemoglobin-trapping efficiency of the cells was found strongly dependent on the pH of the carboxymethylchitin solution used. The artificial red blood cells underwent disintegration by the action of surfactants. When a comparison was made among those surfactants which have the same alkyl chain length, the degree of cell disintegration was in the increasing order, anionic greater than cationic greater than nonionic. Globulin and fibrinogen produced no disintegration of the cells while albumin disrupted the cells to a slight extent.

The effect of multi-porous beads prepared from 80% deacetylated chitin on the activation of mouse peritoneal macrophages was examined. Deacetylated chitin bead (DAC-bead) preparations were shown to activate macrophages for tumoricidal activity depending on the increasing concentration of acetic acid used for the pretreatment of beads. The large DAC-bead was more susceptible to treatment with acetic acid than small DAC-bead, and showed more potent capacity for the activation of macrophages under the same pretreatment conditions with acetic acid. Deacetylated bead preparations, on the other hand, showed less activities. In addition, DAC-bead pretreated with acetic acid stimulated macrophages to produce interleukin 1. The possibilities of multi-porous beads as cancer chemotherapeutic-carrier were examined by the method of column chromatography and of in vitro antitumor experiment. Forty-four percent of adriamycin adsorbed on the surface of and in bead was released within the first 60 min. of elution, and then adriamycin was released more slowly in proportion to the elution time. Antitumor activity of adriamycin-adsorbed bead was less effective than that of free adriamycin if they were compared on the basis of total content of adriamycin.

N-(Carboxymethyl)chitosan was subjected to sulfation in a mixture of concentrated sulfuric acid (oleum) and N,N-dimethylformamide, under anhydrous conditions. The resulting product contained 11% of sulfur and degree of substitution: N-acetyl, 42%; N-carboxymethyl, 58%; and sulfate, 100%. Sonication of the sulfated N-(carboxymethyl)chitosan gave two main fractions whose molecular weights were 39,000 and 80,000. In human blood, complexes of sulfated N-(carboxymethyl)chitosan and antithrombin inhibited both thrombin and factor Xa, and produced neither hemolysis nor alterations in erythrocytes and lymphocytes. Sulfated N-(carboxymethyl)chitosan is therefore proposed as a blood anticoagulant.

Measurements are reported on the kinetics of the lysozyme-catalysed hydrolysis of several beta-aryl di-N-acetylchitobiosides, some of which have been synthesized for the first time. The catalytic rate constants (k(cat.)) at 45 degrees yield a curved Hammett plot (concave up) and the plot of DeltaH(double dagger) versus DeltaS(double dagger) has a sharp break. Substrates with electron-withdrawing groups exhibit a kinetic deuterium isotope effect (k(H) (cat.)/k(D) (cat.)), whereas those with electron-donating groups show no such isotope effect. The results suggest the operation of different mechanisms for the two types of substrates.