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Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres (size 0.2 microm) and chitosan microparticles (size 1.7 microm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.

Quaternized modifications of chitosan present characteristics that might be useful in DNA condensing and efficient gene delivery. Trimethylated chitosan (TMO) was synthesized from oligomeric chitosan (<20 monomer units). TMOs spontaneously formed complexes (chitoplexes) with RSV-alpha3 luciferase plasmid DNA. These complexes were characterized by photon correlation spectroscopy and were investigated for their ability to transfect COS-1 and Caco-2 cell lines in the presence and absence of fetal calf serum and compared with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulphate) lipoplexes. Additionally, their effect on the viability of the respective cell cultures was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results showed that quaternized chitosan oligomers were able to condense DNA and form complexes with a size ranging from 200 to 500 nm. Chitoplexes proved to transfect COS-1 cells, however, to a lesser extent than DOTAP-DNA lipoplexes. The quaternized oligomer derivatives appeared to be superior to oligomeric chitosan. The presence of fetal calf serum (FCS) did not affect the transfection efficiency of the chitoplexes, whereas the transfection efficiency of DOTAP DNA complexes was decreased. Cells remained 100% viable in the presence of chitosan oligomers whereas viability of DOTAP treated cells decreased to approximately 50% in both cell lines. Both DOTAP-DNA lipoplexes and chitoplexes resulted in less transfection efficiency in Caco-2 cell cultures than in COS-1 cells; however quaternized chitosan oligomers proved to be superior to DOTAP. Effects on the viability of Caco-2 cells were similar to the effects observed in COS-1 cells. We conclude that trimethylated chitosan-DNA complexes present suitable characteristics and the potential to be used as gene delivery vectors.

Chitosan is a non-toxic, biocompatible polymer that has found a number of applications in drug delivery including that of absorption enhancer of hydrophilic macromolecular drugs. Chitosan, when protonated (pH<6.5), is able to increase the paracellular permeability of peptide drugs across mucosal epithelia. Chitosan derivatives have been evaluated to overcome chitosan's limited solubility and effectiveness as absorption enhancer at neutral pH values such as those found in the intestinal tract. Trimethyl chitosan chloride (TMC) has been synthesized at different degrees of quaternization. This quaternized polymer forms complexes with anionic macromolecules and gels or solutions with cationic or neutral compounds in aqueous environments and neutral pH values. TMC has been shown to considerably increase the permeation and/or absorption of neutral and cationic peptide analogs across intestinal epithelia. The mechanism by which TMC enhances intestinal permeability is similar to that of protonated chitosan. It reversibly interacts with components of the tight junctions, leading to widening of the paracellular routes. Mono-carboxymethylated chitosan (MCC) is a polyampholytic polymer, able to form visco-elastic gels in aqueous environments or with anionic macromolecules at neutral pH values. MCC appears to be less potent compared to the quaternized derivative. Nevertheless, MCC was found to increase the permeation and absorption of low molecular weight heparin (LMWH; an anionic polysaccharide) across intestinal epithelia. Neither chitosan derivative provokes damage of the cell membrane, and therefore they do not alter the viability of intestinal epithelial cells.

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyer's patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.

Numerous studies have demonstrated that chitosan and their derivatives (N-trimethyl chitosan, mono-N-carboxymethyl chitosan) are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophylic macromolecules such as peptide and protein drugs and heparins. This absorption enhancing effect of chitosans is caused by opening of the intercellular tight junctions, thereby favouring the paracellular transport of macromolecular drugs. Chitosan nano- and microparticles are also suitable for controlled drug release. Association of vaccines to some of these particulate systems has shown to enhance the antigen uptake by mucosal lymphoid tissues, thereby inducing strong systemtic and mucosal immune responses against the antigens. The aspecific adjuvant activity of chitosans seems to be dependent on the degree of deacetylation and the type of formulation. From the studies reviewed it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery.

Chitosan is a non-toxic, biocompatible polymer that has found a number of applications in drug delivery including that of absorption enhancer of hydrophilic macromolecular drugs. Chitosan, when protonated (pH<6.5), is able to increase the paracellular permeability of peptide drugs across mucosal epithelia. Chitosan derivatives have been evaluated to overcome chitosan's limited solubility and effectiveness as absorption enhancer at neutral pH values such as those found in the intestinal tract. Trimethyl chitosan chloride (TMC) has been synthesized at different degrees of quaternization. This quaternized polymer forms complexes with anionic macromolecules and gels or solutions with cationic or neutral compounds in aqueous environments and neutral pH values. TMC has been shown to considerably increase the permeation of neutral and cationic peptide analogs across Caco-2 intestinal epithelia. The mechanism by which TMC is enhancing the intestinal permeability is similar to that of protonated chitosan. It reversibly interacts with components of the tight junctions, leading to widening of the paracellular routes. This chitosan derivative does not provoke damage of the cell membrane, and does not alter the viability of intestinal epithelial cells. Co-administrations of TMC with peptide drugs were found to substantially increase the bioavailability of the peptide in both rats and juvenile pigs compared with administrations without the polymer.

Purpose: To investigate the enhancing effect of trimethyl chitosan chloride (TMC) on the enteral absorption of octreotide and to delineate the required doses of both TMC and peptide in vivo in juvenile pigs., Methods: Six female pigs (body weight, 25 kg) were operated to induce a stoma at the beginning of their jejunum and to insert an in-dwelling fistula for intrajejunal (IJ) administration of the formulations. A silicone cannula was inserted at the jugular vein for blood sampling. One week after surgery the pigs received IJ octreotide solution administrations with or without TMC at pH 7.4 or chitosan HCl at pH 5.5. For determining bioavailability (F) values, the pigs also received an octreotide solution intravenously (IV). Blood samples were taken from the cannulated jugular vein and subsequently analyzed by radioimmunoassay., Results: Intrajejunal administration of 10 mg octreotide without any polymer (control solution) resulted in F values of 1.7 +/- 1.1% (mean +/- SE). Chitosan HCl 1.5% (w/v) at pH 5.5 led to a 3-fold increase in F compared to the control (non-polymer containing) formulations. Co-administration of octreotide with 5 and 10% (w/v) TMC at pH 7.4 resulted in 7.7- and 14.5-fold increase of octreotide absorption, respectively (F of 13.9 +/- 1.3% and 24.8 +/- 1.8%). IJ administration of 5 mg octreotide solutions resulted in low F values of 0.5 +/- 0.6%, whereas co-administration with 5% (w/v) TMC increased the intestinal octreotide bioavailability to 8.2 +/- 1.5%., Conclusions: Cationic polymers of the chitosan type are able to enhance the intestinal absorption of the peptide drug octreotide in pigs. In this respect, TMC at neutral pH values of 7.4 appears to be more potent than chitosan HCl at a weak acidic pH of 5.5.

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine in the gut and low uptake in the lymphoid tissue of the gastrointestinal tract still complicate the development of oral vaccines. In this study chitosan microparticles were prepared and characterized with respect to size, zeta potential, morphology and ovalbumin-loading and -release. Furthermore, the in vivo uptake of chitosan microparticles by murine Peyer's patches was studied using confocal laser scanning microscopy (CLSM). Chitosan microparticles were made according to a precipitation/coacervation method, which was found to be reproducible for different batches of chitosan. The chitosan microparticles were 4.3+/-0.7 microm in size and positively charged (20+/-1 mV). Since only microparticles smaller than 10 microm can be taken up by M-cells of Peyer's patches, these microparticles are suitable to serve as vaccination systems. CLSM visualization studies showed that the model antigen ovalbumin was entrapped within the chitosan microparticles and not only associated to their outer surface. These results were verified using field emission scanning electron microscopy, which demonstrated the porous structure of the chitosan microparticles, thus facilitating the entrapment of ovalbumin in the microparticles. Loading studies of the chitosan microparticles with the model compound ovalbumin resulted in loading capacities of about 40%. Subsequent release studies showed only a very low release of ovalbumin within 4 h and most of the ovalbumin (about 90%) remained entrapped in the microparticles. Because the prepared chitosan microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. Initial in vivo studies demonstrated that fluorescently labeled chitosan microparticles can be taken up by the epithelium of the murine Peyer's patches. Since uptake by Peyer's patches is an essential step in oral vaccination, these results show that the presently developed porous chitosan microparticles are a very promising vaccine delivery system.

The synthesis and evaluation of mono-N-carboxymethyl chitosan (MCC) as an intestinal permeation enhancer for macromolecular therapeutics is presented. MCCs were synthesized from two different viscosity grade chitosans to yield both high and low viscosity grade products. These MCCs were tested on Caco-2 cells for their efficiency to decrease the transepithelial electrical resistance (TEER) and to increase the paracellular permeability of the anionic macromolecular anticoagulant low molecular weight heparin (LMWH). For in vivo studies, LMWH was administered intraduodenally with or without MCC to rats. Both types of experiments were performed at pH 7.4. Results show that both viscosity grade MCCs managed to significantly decrease the TEER of Caco-2 cell monolayers when they were applied apically at concentrations of 3-5% (w/v). Transport studies with Caco-2 cells revealed substantial increases of LMWH permeation in the presence of both viscosity grade MCCs compared with controls. In rats, 3% (w/v) low viscosity MCC significantly increased the intestinal absorption of LMWH, reaching the therapeutic anticoagulant blood levels of LMWH. Both in vitro and in vivo results indicate that the polyampholytic chitosan modification MCC is a suitable and functional polymer for the delivery and intestinal absorption of anionic macromolecular therapeutics like LMWH., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90: 38-46, 2001)

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine and low uptake by the gut associated lymphoid tissue (GALT) still complicate the development of efficient oral vaccines. However, previous studies in our laboratory demonstrated that chitosan microparticles can have suitable size, charge, loading and release characteristics for oral vaccination using ovalbumin as model vaccine. In this study, two different approaches were used to investigate the in vivo uptake of chitosan microparticles by murine Peyer's patches. Firstly, a confocal laser scanning microscopy (CLSM) study was performed to visualize the uptake of fluorescent-labeled chitosan microparticles in the Peyer's patches after intragastrical feeding. Subsequently, the intestinal epithelial uptake of ovalbumin loaded chitosan microparticles was visualized using immunohistochemical staining of ovalbumin. Because the microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. CLSM visualization demonstrated that chitosan microparticles enhance the uptake of fluorescent-labeled ovalbumin by the epithelium of the Peyer's patches. No ovalbumin uptake by the intestinal epithelium was observed when the protein was administered without microparticles. Moreover, immunohistochemical visualization studies revealed that ovalbumin could only be transported into the Peyer's patches after association to chitosan microparticles. Since uptake by Peyer's patches is an essential step in oral vaccination, these in vivo experiments demonstrate that chitosan microparticles are very promising vaccine delivery systems.

N-Trimethyl chitosan chloride (TMC) is a permanently quaternized chitosan derivative with improved aqueous solubility compared to native chitosan. TMC is able to open the tight junctions of intestinal epithelia at physiological pH values, where chitosan is insoluble and therefore ineffective. TMCs with degrees of substitution of 40 and 60% were synthesized according to a novel synthesis procedure and their effect on the permeability of the tight junctions of the intestinal Caco-2 monolayers was studied, measuring the transepithelial electrical resistance and the transport of a mainly paracellularly transported compound, [14C]-mannitol. Toxicity studies using nucleic stains were done to establish the transport as a cause of opening of the tight junctions and not of possible cytotoxicity. TMC60 showed higher transport enhancement ratios than TMC40 in all concentrations tested (0.05-1. 0%, w/v). Both derivatives did not affect the viability of the Caco-2 cell monolayers. These results suggest that high charge density is necessary for TMC to substantially improve the paracellular permeability of intestinal epithelia. It is expected that TMC40 and TMC60 will enhance the intestinal permeation of hydrophilic macromolecular drugs such as peptides and proteins.

Purpose: To evaluate N-trimethyl chitosan chloride (TMC) of high degrees of substitution as intestinal permeation enhancers for the peptide drug buserelin in vitro using Caco-2 cell monolayers, and to investigate TMCs as enhancers of the intestinal absorption of buserelin in vivo, in rats., Methods: TMCs were tested on Caco-2 cells for their efficiency to increase the paracellular permeability of the peptide buserelin. For the in vivo studies male Wistar rats were used and buserelin was administered with or without the polymers intraduodenally. Both types of experiments were performed at pH 7.2., Results: Transport studies with Caco-2 cell monolayers confirmed that the increase in buserelin permeation is dependent on the degree of trimethylation of TMC. In agreement with the in vitro results, in vivo data revealed highly increased bioavailability of buserelin following intraduodenal co-administration with 1.0% (w/v) TMCs. Intraduodenally applied buserelin resulted in 0.8% absolute bioavailability, whereas co-administrations with TMCs resulted in mean bioavailability values between 6 and 13 %. Chitosan HCl (1.0%; pH = 7.2) did not significantly increase the intestinal absorption of buserelin., Conclusions: Both the in vitro and in vivo results indicate that TMCs are potent mucosal permeation enhancers of the peptide drug buserelin at neutral pH values.

N-trimethyl chitosan (TMC) polymers are quaternized chitosans in different degrees of trimethylation. These polymers enhance the absorption of macromolecules through mucosal epithelia by triggering the reversible opening of tight junctions and only allow for paracellular transport. To investigate the safety of these novel absorption enhancers cytotoxicity and ciliotoxicity studies have been performed. Intestinal Caco-2 cell monolayers were chosen to study possible membrane damaging effects of these polymers, using confocal laser scanning microscopy visualization of nuclear staining by a membrane impermeable fluorescent probe during transport of the paracellular marker Texas red dextran (MW 10 000). Ciliated chicken embryo trachea tissue was used to study the effect of the polymers on the ciliary beat frequency (CBF) in vitro. In both studies the TMC polymers of different degrees of substitution (20, 40 and 60%) were tested at a concentration of 1.0% (w/v). No substantial cell membrane damage could be detected on the Caco-2 cells treated with TMCs, while the effect on the CBF in vitro was found to be marginal. TMC60 and TMC40 enhance paracellular transport of Texas red dextran in Caco-2 cell monolayers, whereas TMC20 is ineffective. In conclusion, TMCs of high degrees of substitution may be effective and safe absorption enhancers for peptide and protein drug delivery.

N-trimethyl chitosan chloride (TMC), a partially quaternized derivative of chitosan with superior water solubility, was synthesized with different degrees of quaternization [12.6% quaternized (TMC-L) and 19.9% quaternized (TMC-H)] and the effects of these novel polymers on the permeability of intestinal epithelial cells were investigated in Caco-2 cell monolayers. Transepithelial electrical resistance (TEER) measurements showed that both polymers in 1.5-2.5% w/v concentrations caused a pronounced, concentration dependent lowering in TEER values, but that TMC-H was more effective than TMC-L at similar concentrations (36 +/- 3% reduction with TMC-L and 53 +/- 6% reduction with TMC-H at 2.0% concentrations). Similar results were obtained in transport studies with the hydrophilic radioactive markers [14C]mannitol (MW 182.2) and [14C]polyethylene glycol 4000 ([14C]PEG 4000, MW 4000). The transport of [14C]mannitol was increased 51-fold (TMC-L) and 97-fold (TMC-H) at 2.5% concentrations. No deleterious effects to the cells could be demonstrated with trypan blue exclusion studies. The results show that TMC is able to open the tight junctions of intestinal epithelial cells to allow for paracellular transport of hydrophilic molecules. It is concluded that the charge density of TMC, as determined by the degree of quaternization, is an important factor determining its potential use as an absorption enhancer across intestinal epithelia.

Previous studies have established that a partially quaternized derivative of chitosan, N-trimethyl chitosan chloride (TMC), can be used as an absorption enhancer for large hydrophilic compounds across mucosal surfaces. This study evaluates and compares the effects of the degree of quaternization of TMC, in a neutral environment, on the permeability of intestinal epithelial cells in vitro, where normal chitosan salts are ineffective as absorption enhancers. The effects of TMC-H [61.2% quaternized, (0.05-1.5% w/v)], TMC-L [12.3% quaternized, (0.5-1.5% w/v)], and chitosan hydrochloride [0.5-1.5% w/v] on the transepithelial electrical resistance (TEER) and permeability, for the hydrophilic model compound [14C]mannitol, of intestinal epithelial Caco-2 cell monolayers, were investigated at pH values of 6.20 and 7.40. The viability of the monolayers was checked with the trypan blue exclusion technique. At a pH of 6.20, all the polymers caused a pronounced reduction (37-67% at 0.5% w/v concentrations) in the TEER of Caco-2 cells. On the contrary, at a pH of 7.40, only TMC-H was able to decrease the TEER values, even in a concentration as low as 0.05% w/v (35% reduction). Comparable results were obtained with the permeation of [14C]mannitol. Large increases in the transport rate (18-23-fold at 0.5% w/v concentrations) were found at pH 6.20, whereas only TMC-H was able to increase the permeation of [14C]mannitol at pH 7.40 (31-48-fold at 0.05-1.5% w/v concentrations of TMC-H). For all the polymers studied, no deleterious effects to the cells could be demonstrated with the trypan blue exclusion technique. It is concluded that highly quaternized TMC is a potent absorption enhancer and the potential use of this polymer, especially in neutral and basic environments where normal chitosan salts are not effective, is expected to be an important contribution to the development of effective delivery systems for hydrophilic compounds such as peptide drugs.

In this study the effects of two chitosan salts, namely chitosan hydrochloride and chitosan glutamate (0.5 and 1.5% w/v), on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cell monolayers, using the radioactive marker [14C]-mannitol, were investigated in a slightly acidic (pH 6.2) and neutral (pH 7.4) environment. Both salts are soluble in acidic conditions up to a concentration of 1.5% w/v and solutions of this strength, at a pH of 6.2, caused a pronounced lowering in the TEER of Caco-2 cell monolayers in the order of 70+/-1% (chitosan glutamate) and 77+/-3% (chitosan hydrochloride), 20 min after incubation started. In agreement with the TEER results the transport of the radioactive marker, [14C]-mannitol, was increased 25-fold (chitosan glutamate) and 36-fold (chitosan hydrochloride), respectively, at this pH. However, at a pH of 7.4 both salts are insoluble and prove to be ineffective since no reduction in the TEER values or increase in the transport of [14C]-mannitol were found. The results show that these chitosan salts are potent absorption enhancers in acidic environments. We conclude that there is a need for chitosan derivatives with increased solubility, especially at neutral and basic pH values, for use as absorption enhancers aimed at the delivery of therapeutic compounds in the more basic environment of the large intestine and colon.

A partially quaternized chitosan derivative, N-trimethyl chitosan chloride (TMC) (degree of quaternization 12.28%), was synthesized and the effects of this novel polymer on the permeability of intestinal epithelial cells, using Caco-2 cell monolayers, were investigated and compared with those of chitosan hydrochloride and chitosan glutamate. Transepithelial electrical resistance (TEER) measurements at pH 6.20 revealed that all these polymers (0.25-1.5% w/v) caused an immediate and pronounced lowering in TEER values in the order chitosan hydrochloride (84% reduction after 2 h incubation) > chitosan glutamate (60% reduction) > TMC (24% reduction) at 0.25% w/v concentrations. At higher concentrations (up to 2.5% w/v), TMC was able to decrease the TEER further. Similar results were obtained in transport studies, using the hydrophilic radioactive markers, [14C]-mannitol (MW 182.2) and [14C]-polyethylene glycol 4000 (PEG-4000, MW 4000). Large increases in the permeation of these markers were found. The transport of [14C]-mannitol was increased 34-fold (chitosan hydrochloride), 25-fold (chitosan glutamate) and 11-fold (TMC) at 0.25% w/v concentrations. Further increases in the permeation of both markers were found at higher concentrations of TMC. Due to its quaternary structure, TMC is better soluble than the other chitosan salts, and its higher solubility may compensate for its lesser effectivity at similar concentrations. It is also soluble at pH 7.40, where the chitosan salts are insoluble and therefore ineffective. No deleterious effects to the cells could be demonstrated with trypan blue exclusion studies and confocal laser scanning microscopy (CLSM). CLSM confirmed that these polymers increase the transport of large hydrophilic compounds (using the fluorescent markers FD-4, MW 4400 and FD-20, MW 19,600) through opening of tight junctions to allow for paracellular transport. It is concluded from this study that the charge, charge density and the structural features of chitosans and chitosan derivatives are important factors determining their potential use as absorption enhancers.

Purpose: Previous studies have established that chitosan hydrochloride and glutamate are potent absorption enhancers for large hydrophilic compounds across mucosal surfaces. However, these compounds lack solubility at neutral pH values. A partially quaternized and well-soluble derivative of chitosan, N-trimethyl chitosan chloride, was synthesized and the effects of this polymer on the transepithelial electrical resistance and permeability of intestinal epithelial cells were investigated in vitro., Methods: N-trimethyl chitosan chloride was synthesized by reductive methylation and characterized with NMR. The effect of this polymer (1.0-2.5% w/v) on the transepithelial electrical resistance of intestinal epithelial cells, using Caco-2 cell monolayers, was investigated. Permeation of the hydrophilic model compounds [14C]-mannitol (MW 182.2), FITC-Dextran (MW 4400) and the peptide drug buserelin (MW 1299.5), in the presence of N-trimethyl chitosan chloride (1.5-2.5% w/v), was followed for 3 hours. The transport process of the fluorescent marker, FITC-Dextran 4400, across the cell monolayers was visualised with confocal laser scanning microscopy. Viability of the cells was checked with the trypan blue exclusion technique., Results: N-trimethyl chitosan chloride was found to be a perfectly water-soluble, partially quaternized (about 12%) derivative of chitosan. This polymer (1.5-2.5% w/v) caused a pronounced and immediate reduction (25-85%) in the transepithelial electrical resistance of Caco-2 cells. Large increases in the transport rate of [14C]-mannitol (32-60 fold), FITC-Dextran 4400 (167-373 fold) and buserelin (28-73 fold) were demonstrated. Confocal laser scanning microscopy confirmed that N-trimethyl chitosan chloride opens the tight junctions of intestinal epithelial cells to allow increased transport of hydrophilic compounds through the paracellular transport pathway. No deleterious effects to the cells could be demonstrated with trypan blue., Conclusions: The potential use of N-trimethyl chitosan chloride as an absorption enhancer across mucosal surfaces could be an important contribution towards the development of effective delivery systems for hydrophilic drugs.

Purpose: To evaluate the effect of the crosslinked poly(acrylate) carbomer 934P (C934P) and its freeze-dried neutralized sodium salt (FNaC934P) as well as chitosan hydrochloride on the intestinal absorption of the peptide drug buserelin., Methods: Buserelin was applied intraduodenally in control buffer, 0.5% (w/v) C934P, 0.5% (w/v) FNaC934P, 1.5% (w/v) chitosan hydrochloride or FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture in rats., Results: All polymer preparation showed a statistically significant improvement of buserelin absorption compared to the control solution. The absolute bioavailabilities for the different polymer preparations were: control, 0.1%; 0.5% FNaC934P, 0.6%; 0.5% C934P, 2.0%; chitosan hydrochloride, 5.1% and FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture, 1.0%. The higher bioavailability with chitosan hydrochloride compared to C934P and FNaC934P indicates that for buserelin the intestinal transmucosal transport enhancing effect of the polymer plays a more dominant role than the protection against proteases such as alpha-chymotrypsin., Conclusions: The mucoadhesive polymers carbomer 934P and chitosan hydrochloride are able to enhance the intestinal absorption of buserelin in vivo in rats, and may therefore be promising excipients in peroral delivery systems for peptide drugs.

Chitosan, a biodegradable and biocompatible polymer, has attracted great attention in the pharmaceutical and biomedical fields especially due to its properties to reversibly open the tight junctions of the epithelial tissues and to allow for paracellular transport of hydrophilic macromolecules. However, chitosan exhibits low solubility at pH values above 6 that may prevent its enhancing effects at the sites of intestinal absorption. Hence, a number of alkylated chitosan salts have been synthesized and characterized. These derivatives have been shown to have good solubility at neutral pH and act as effective permeation enhancers. In this study, two new derivatives of chitosan, C2-C6 trimethyl 6-amino-6-deoxy chitosan and C2-C6 triethyl 6-amino-6-deoxy chitosan were synthesized and characterized using 1H-NMR and FTIR spectra. Moreover, the zeta potential and the antibacterial properties of these polymers were compared to chitosan, trimethyl chitosan (TMC) and triethyl chitosan (TEC). Our results suggest that both C2-C6 trimethyl and triethyl 6-amino- 6-deoxy chitosan, as highly water soluble polymers, have higher positive surface charge than chitosan, TMC, and TEC. Moreover, the new synthesized polymers show higher antibacterial activity against Gram-positive Staphylococcus aureus bacteria. Consequently, these polymers with substitution degrees of 50-60% may be good candidates for the enhancement of peptides in mucosal drug delivery. [ABSTRACT FROM AUTHOR]