Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal caco-2 cell monolayers.

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.

Effects of N-trimethyl chitosan chloride, a novel absorption enhancer, on caco-2 intestinal epithelia and the ciliary beat frequency of chicken embryo trachea.

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.

Effect of the degree of quaternization of N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2).

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.

Enhancement of paracellular drug transport with highly quaternized N-trimethyl chitosan chloride in neutral environments: in vitro evaluation in intestinal epithelial cells (Caco-2).

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.