European Journal of Pharmaceutics and Biopharmaceutics
Research paperSurface-functionalized polymethacrylic acid based hydrogel microparticles for oral drug delivery
Introduction
Advanced delivery systems capable of improving drug permeability across biological barriers are of prime significance in modern pharmaceutical research [1]. Intestinal epithelium represents the major barrier for the absorption of orally administered therapeutic macromolecules into the systemic circulation. It is formed by a high-resistance epithelial cell barrier which restricts the diffusion of various hydrophilic compounds across the small intestine. Between cells, tight junctions form a paracellular barrier which limits the passage of molecules through the intercellular spaces of the intestinal epithelium [2]. The paracellular permeability depends mainly on the regulation of the intercellular tight junctions, which in turn is governed mostly by a group of proteins often referred as tight junction proteins [3], [4]. The utility of the paracellular route for oral drug delivery has remained unexplored mainly due to the lack of substances capable of modulating reversible opening of the tight junctions, hence enhancing drug transport without irreversibly compromising the integrity of the gut epithelium [5].
Polymeric absorption enhancer systems based on poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMAA) and chitosan (CS) seem to be a promising approach in this aspect [6], [7]. They received good attention in recent years as transmucosal penetration enhancers improving absorption of hydrophilic drugs [8], [9]. Unlike small molecular weight permeation enhancers, polymeric systems can improve the intestinal permeability by imparting less or no toxicity on the biological membrane [10]. Most of these systems enhance drug transport by a completely reversible mechanism, which exerts minimal damage on the integrity of tight junctions [10]. The mechanism by which permeation enhancement occurs seems to be slightly different for each polymer. PAA-based polymers are believed to be responsible for permeation-enhancing effects due to its capacity to bind calcium ions. Indeed, calcium chelators can disturb cell–cell adhesion phenomena by depleting the concentration in the extracellular calcium ions, which play a major role in maintaining the integrity of the epithelial tight junctions. Chelation of calcium further activates protein tyrosine kinases (PTK), which subsequently leads to the phosphorylation of the tyrosine moieties in the transmembrane protein – occludin [11], [12]. Although phosphorylation of tyrosine groups lead to the opening of the tight junctions, protein tyrosine phosphatases (PTP) have the role of closing the tight junctions by dephosphorylating the tyrosine groups. The second polymer, chitosan, is polycationic in nature. Its protonated form interacts with the epithelial tight junctions inducing a redistribution of actin filaments and of the tight junction protein ZO-1 [13], [14]. This ultimately leads to the opening of tight junctions across the intestinal epithelium.
A novel class of polymers called “thiomers” was introduced to improve the bioadhesion of polymeric drug delivery systems [15]. Thiomers are thiolated polymers which display thiol groups. They are expected to form disulfide bonds, thanks to thiol/disulfide exchange reactions and/or simple oxidation processes between thiomers and cysteine-rich subdomains of the mucus glycoproteins [16]. Polymers such as poly(acrylic acid), alginate and chitosan were modified with thiol-containing molecules to yield thiomers. Drug delivery systems formulated with such polymers showed higher mucoadhesion capacity compared with the corresponding non-thiolated systems. Interestingly, the introduction of thiol groups has also improved the permeation-enhancing capability of these polymers. However, the exact mechanism at the origin of the permeation-enhancing effect still remains unclear. It was postulated that thiolated systems have the ability to inhibit PTP and regulate the opening of the tight junctions in a reversible manner. PTP has a cysteine residue in its active site, which is largely involved in the activity of this protein. Thus, it is believed that thiomers can form disulfide linkage with these cysteine residues and hence modifies the activity of the PTP. This could be the major mechanism involved for the permeation-enhancing effect of thiol-containing system [17]. However, the leading factor to the activation of PTK in such a case still remains unclear.
Surface modification of polymeric nano- and microparticles seems to be a promising approach in advanced drug delivery. A novel core–shell type nanoparticulate delivery system, including poly(iso-butyl cyanoacrylate) (PIBCA) core with chitosan/thiolated chitosan brushes at the surface were developed in our laboratory [18], [19]. Presence of polysaccharide brushes on the particle surface significantly enhanced the adhesion behavior of the PIBCA nanoparticles to the mucosa and also improved the permeation of hydrophilic markers across the intestinal membrane through the intercellular pathway [20], [21]. Surface thiolation may be an interesting approach to be applied to hydrogel systems. It may be expected that thiol groups may increase permeability of the gut epithelium to hydrophilic macromolecules and help in anchoring the delivery system onto the mucus layer, whereas the hydrogel system with optimum chain flexibility and mobility may help in the diffusion of the delivery system across the mucus layer to access the underlying epithelium.
The objective of the present work was to study the effect of surface thiolation of hydrogel microparticles on the mucoadhesion and paracellular permeability through the gut epithelium. We suggest that the specific surface modification of the particles may increase the amount of thiol groups available on the surface of the microparticles. The microparticles used in this work were obtained by a modified ionic gelation method, and surface thiolation was achieved by the activation of surface carboxylic acid groups followed by the coupling with amino groups of cysteine. The characteristics of these novel surface-modified particles were evaluated in terms of their amount of thiol groups, size and ability to enhance the paracellular permeability of hydrophilic macromolecular marker with Caco 2 cell-culture model and Ussing chamber facility.
Section snippets
Materials and methods
Methacrylic acid (MAA), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), ethylene glycol dimethacrylate (EDMA), 5,5′-dithiobis (nitrobenzoic acid) (Ellman’s reagent), sodium borohydride, l-cysteine, 2-[N-morpholno]ethane sulfonic acid (MES), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), N-acetyl cysteine, fluorescein isothiocyanate–dextran, MW 4400 (FD4), polyethylene glycol (PEG) MW 20,000, potassium persulfate and sodium borohydride were from
Preparation and characterization of the PCP and Cys-PCP microparticles
Poly(methacrylic acid) (PMAA)-based hydrogel microparticles were prepared by ionic gelation method, and thiol groups were introduced by grafting cysteine on the microparticle by a surface modification strategy. Thiol content of the microparticles was determined by Ellman’s assay, and Ellman’s test did not show any reactivity towards non-thiolated-PCP microparticles. Table 1 gives the results of the thiol concentration found in microparticles obtained by using different initial concentrations of
Discussion
The ionic gelation process was used to prepare hydrogel microparticles made of PMAA–PEG–Chitosan (PCP microparticles). The main advantage of the technique is that the microparticles are formed spontaneously during polymerization of methacrylic acid without the addition of any surfactants and/or steric stabilizers. The aim of the present work was to introduce new chemical groups, namely thiol groups, on the surface of the PCP microparticle to improve both microparticle mucoadhesion and to
Conclusions
Thiolation of PCP microparticles improved the intercellular permeability of a hydrophilic macromolecule through Caco 2 cell monolayers. The improvement of the paracellular permeability was due to the combination of two mechanisms. The depletion of calcium concentration in the vicinity of the cell epithelium can be due to the presence of PMAA in the composition of the microparticles. Indeed, this effect can be responsible for a loss of integrity in the tight junction functionality. An additional
Acknowledgements
S.S. thanks French Embassy in India for the award of a Sandwich PhD Fellowship. Financial support from FADDS (DST, New Delhi) is also acknowledged. Authors thank Dr. Véronique Marsaud for the help in cell-culture studies and Prof. Gilles Ponchel for his creative suggestions.
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