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

https://doi.org/10.1016/j.ejpb.2019.09.012Get rights and content

Abstract

Aim

The aim of this study was to evaluate the potential of chitosan/tripolyphosphate (TPP) nanoparticles to provide a targeted release of β-galactosidase behind the intestinal mucus gel barrier.

Methods

Nanoparticles were prepared by ionic gelation of chitosan and TPP in the presence of β-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 β-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 β-galactosidase was either determined utilizing bicinchoninic acid (BCA) protein detection or via an enzymatic activity assay with 2-nitrophenyl β-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 µg β-galactosidase per mg particles was achieved. Degradation studies with isolated IAP revealed a maximum phosphate cleavage of 118 ± 1 µg/mg particles, a size decrease up to 38 ± 7 % and a release of 58 ± 0.5 % β-galactosidase. Release of 94 ± 6 % of the incorporated initial amount of β-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 β-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.

Introduction

The development of oral delivery systems for therapeutic biomacromolecules such as enzymes, peptides or antibodies guaranteeing their stability in the intestine is still challenging. Especially in the case of enzymes, activity has to be preserved in this hostile environment but it is from a formulator’s point of view difficult to avoid premature degradation by intestinal proteases. Strategies to face these issues are focusing on the co-administration of protease inhibitors, physicochemical modifications of the macromolecules or the design of nanoparticulate-based carriers to increase proteolytic stability [1], [2].

In this context, chitosan nanoparticles cross-linked with tripolyphosphate (TPP) have been widely investigated for the delivery of biologics until now [3], [4], [5], [6]. Nevertheless the applicability of this carrier system was not examined yet in respect of a targeted release at the brush border membrane in the small intestine involving alkaline phosphatase. Intestinal alkaline phosphatase (IAP) is present in the brush border membrane of the enterocyte of the small intestine and catalyzes the nonspecific hydrolysis of phosphate esters into inorganic phosphate and alcohol. Besides, polyphosphates including tripolyphosphate are prone to degradation by IAP [7], [8], [9]. As a result of this, the TPP cross-links within the particle network should be hydrolyzed if the delivery system reaches the absorption membrane, particles should lose their stability and release their active ingredient.

Particularly within the field of local enzyme substitution therapy of β-galactosidase this novel approach might be promising, because the nanoparticles could provide shelter from the metabolizing intestinal environment combined with a local release behind the mucus gel barrier directly at the target location. β-Galactosidase, better known under the name lactase, is involved in the basic process in digestion of lactose. Absence or malfunction of this enzyme causes malabsorption up to lactose intolerance. Lactase replacement is an option for therapy and a variety of preparations is available over the counter [10], [11]. Limitations of the existing therapy option are changes in taste, because it is usually consumed together with dairy goods and the susceptibility to enzymatic metabolism in the lumen of the gastro intestinal tract (GIT). Facing these issues the incorporation of β-galactosidase in chitosan/TPP nanoparticles could be a purposeful alternative to improve its delivery.

Section snippets

Materials

Chitoscience Chitosan 85/10 (average molecular weight ~50 kDa) was obtained from Heppe Medical Chitosan GmbH (Halle, Germany). Pierce™ BCA Protein Assay Kit was purchased from Thermo Scientific (Vienna, Austria). Sodium tripolyphosphate, phosphatase (alkaline from bovine intestinal mucosa, lyophilized powder, ≥10 DEA units/mg solid), β-galactosidase from Kluyveromyces lactis (≥2600 units/g), 2-nitrophenyl β-D-galactopyranoside, phosphatase inhibitor cocktail 2 and all other substances and

Nanoparticle preparation and characterization

Nanoparticles were prepared via ionic gelation method between positively charged chitosan and negatively charged TPP as cross-linking agent. Dropwise addition of TPP to the chitosan solution resulted in a turbid mixture, indicating the formation of nanoparticles. β-Galactosidase was selected as active ingredient because of two main reasons, first it might be expected that it underlies a pre-systemic enzymatic metabolism in the lumen of the small intestine [20], [21] and therefore shielding by

Conclusion

Within the scope of this study the suitability of chitosan/TPP nanoparticles in order to achieve a targeted release system was proven. Using ionic gelation, the model drug β-galactosidase could be successfully encapsulated. The results obtained from different degradation tests with isolated as well as cell-associated IAP showed a good correlation. Additionally a release study of β-galactosidase in a tissue based experiment gave a hint that the system might be able to pass the mucus barrier and

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