Intestinal enzyme delivery: Chitosan/tripolyphosphate nanoparticles providing a targeted release behind the mucus gel barrier

Aim: The aim of this study was to evaluate the potential of chitosan/tripolyphosphate (TPP) nanoparticles to provide a targeted release of beta-galactosidase behind the intestinal mucus gel barrier. Methods: Nanoparticles were prepared by ionic gelation of chitosan and TPP in the presence of beta-galactosidase. Particles were characterized regarding size, polydispersity index and drug load. Target mediated hydrolysis of the TPP cross-linker followed by particle degradation and release of beta-galactosidase was investigated during incubation with isolated as well as cell and tissue associated intestinal alkaline phosphatase (IAP). Phosphate content in the media was quantified via malachite assay, whereas particle disintegration was monitored in parallel by measuring the decrease in particle size as well as in optical density at 600 nm. The released amount of beta-galactosidase was either determined utilizing bicinchoninic acid (BCA) protein detection or via an enzymatic activity assay with 2-nitrophenyl beta-D-galactopyranoside (ONPG) as substrate. Protection towards tryptic degradation was verified by ONPG assay. Results: The size of nanoparticles was 573 +/- 34 nm and a payload of 376 +/- 18 mu g beta-galactosidase per mg particles was achieved. Degradation studies with isolated IAP revealed a maximum phosphate cleavage of 118 +/- 1 mu g/mg particles, a size decrease up to 38 +/- 7 % and a release of 58 +/- 0.5 % beta-galactosidase. Release of 94 +/- 6 % of the incorporated initial amount of beta-galactosidase was proven after 3 h incubation on porcine mucosa. Furthermore a protection against tryptic degradation was attained resulting in a 3-fold higher residual enzymatic activity of encapsulated beta-galactosidase compared to a control of free enzyme. Conclusion: Chitosan/TPP nanoparticles seem to be qualified as a suitable carrier for a targeted delivery of active ingredients to mucosal tissues expressing alkaline phosphatase.