A mucoadhesive nanoparticulate system for the simultaneous delivery of macromolecules and permeation enhancers to the intestinal mucosa

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Abstract

The feasibility of combining safe permeation enhancers in a mucoadhesive particulate system for the oral delivery of peptide drugs was investigated in this study. Polyelectrolyte complex nanoparticles (NPs) were prepared by ionic interaction of spermine (SPM) with polyacrylic acid (PAA) polymer. Cytotoxicity studies in Caco-2 monolayers revealed the safety of the delivery system in the concentration range used for permeation enhancement. The cellular transport of fluorescein isothiocyanate dextran (FD4) showed higher permeation enhancing profiles of SPM–PAA NPs, as compared to SPM solution or PAA NPs prepared by ionic gelation with MgCl2 (Mg-PAA NPs). These permeation enhancing effects were associated with a reversible decrease in TEER values, suggesting a paracellular permeation pathway by reversible opening of the tight junctions. Furthermore, confocal microscopy results revealed strong association of the NPs prepared using fluorescence labeled PAA to Caco-2 cells. The permeation enhancing properties of SPM–PAA NPs were further evaluated in vivo after oral administration to rats, using FD4 and calcitonin as models of poorly permeating drugs. Confocal microscopy images of rats' small intestine confirmed previous findings in Caco-2 cells and revealed a strong and prolonged penetration of FD4 from the mucosal to the basolateral side of the intestinal wall. In addition, the proposed NPs were efficient in improving the oral absorption of calcitonin, as evidenced by the significant and prolonged reduction of the blood calcemia in rats.

Graphical abstract

Efficacy of SPM–PAA NPs in enhancing the in vitro transport of FD4 across Caco-2 cell monolayers (A), and the in vivo oral absorption of calcitonin in rats (B).

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Introduction

The oral route offers a convenient alternative for the needle-free systemic delivery of protein drugs. Unfortunately, the intestinal absorption of these drugs is challenged by their poor membrane permeability as a result of high molecular weight, hydophilicity, and surface charge. Furthermore, they are highly susceptible for enzymatic degradation in the GI tract after oral administration. Over the last few decades, researchers have investigated various approaches for the efficient oral delivery of peptides. Entrapment into particulate systems [1], [2], [3], [4], mucoadhesive polymer formulations [5], [6], [7], [8], and use of permeation enhancer [9], [10], [11] and protease inhibitor [12], [13], [14] adjuvants are among the most commonly utilized strategies.

Entrapment of peptide drugs in nanoparticulate systems protects them against the harsh environment of the GI tract until they absorbed in released or intact particulate form. Moreover, the use of mucoadhesive particulate systems can extend the residence time of the drug at the site of absorption, achieve higher local drug concentrations in the mucous gel layer, minimize drug dilution and degradation by the luminal contents, and consequently, enhance drug absorption into systemic circulation.

The use of absorption enhancers, that improve the mucosal permeation of macromolecules without causing serious tissue damage, has been the focus of many research groups. Various permeation enhancers have been investigated for the improvement of peptide absorption through the intestinal membrane. Common examples of non-specific permeation enhancers are surfactants, chelating agents, bile salts, and fatty acids [15]. However, efficient permeation enhancement achieved by many of these compounds is usually associated with membrane damage and toxicity [16]. In addition, the simultaneous delivery of the drug and sufficient concentration of the permeation enhancer at the site of absorption after in vivo oral administration is a challenging process.

Polyamines, including spermine (SPM) and spermidine (SPD), are a group of naturally occurring components of living cells, and are essential for cell growth and differentiation [17], [18]. They are simple aliphatic cations with two to four amine groups that are fully protonated at physiological pH. Previous reports showed that polyamines are efficient permeation enhancers through epithelial membranes without causing any tissue damage [19], [20], [21], [22], [23]. Studies revealed that SPM, among all the evaluated polyamine derivatives, has the highest permeation enhancing effects for fluorescein isothiocyanate dextrans, insulin, and other drug candidates of low permeability [20], [21], [22], [23]. In addition, a concentration-dependent reduction of TEER values through the rat jejunal membranes caused by SPM was observed, indicating a paracellular transport by opening of the tight junctions [23]. However, the absorption enhancing effect of polyamines in solution with drugs after oral administration was usually lower than that achieved in the in vitro or in situ permeation studies [20], [21]. The reason might be attributed to the dilution and dispersion of the permeation enhancer within the GI fluid over a large surface area [16]. In addition, polyamines are reported to be well absorbed from the GI tract after oral administration [24], which might be responsible for the rapid decrease in their concentration available for permeation enhancement on the luminal side. Another limitation for the use of polyamines as efficient permeation enhancers for the oral delivery of peptide and protein drugs is the presence of GI degradation enzymes.

In the current study, polyelectrolyte complex NPs of SPM and PAA were prepared as an alternative approach that precludes the permeation enhancer from being diluted by the luminal contents or rapidly absorbed through the mucosal surface of the GI tract after oral administration. The nanoparticulate system was evaluated as an oral delivery carrier for peptide and macromolecular drugs. The cytotoxicity, permeation enhancing properties, and cellular association of the NPs were characterized in vitro, in Caco-2 cell monolayers. In addition, the mucosal penetration enhancing effects and the therapeutic efficacy of the prepared NPs were evaluated in vivo after oral administration to rats, using FD4 and calcitonin as models of poorly absorbable macromolecules.

Section snippets

Materials

Spermine (SPM, 202.35 Da), polyacrylic acid (PAA, 450 kDa), fluorescein isothiocyanate (FITC), and FD4 (4400 Da) were purchased from Sigma-Aldrich, USA. Calcitonin was kindly supplied by Asahi Kasei, Japan. Hank's balanced salt solution (HBSS), Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), l-glutamine, non essential amino acids (NEAA), penicillin–streptomycin, and Trypsin–EDTA were obtained from Invitrogen, USA. The MTS reagent was purchased from Promega, USA. All other

Nanoparticles preparation and characterization

Electrostatic interaction between positively charged SPM and negatively charged PAA resulted in the formation of polyelectrolyte complexes in the nanometer scale. The formation of NPs was investigated using various concentrations and weight ratios of the two agents. As shown in Table 1, the NPs with optimum properties were formed at a weight ratio of 1.0:1.2 (w/w ratio) of SPM to PAA. At relatively higher or lower SPM/PAA ratios, either clear solutions or particle aggregates were obtained,

Discussion

The need for the conversion of injectable protein therapy to oral formulations has been recognized already more than 20 years ago. Researchers of various scientific disciplines are developing technologies to make feasible the uptake of protein therapeutics after oral administration. Generally speaking, particulate systems offer various advantages with respect to oral delivery of peptide and protein drugs. They can protect the therapeutic agent from GI enzymes and adhere to a large surface area

Acknowledgements

A. Makhlof acknowledges the Egyptian Ministry of High Education for financial support. This study was also supported by a grant from the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) to M. Werle.

References (39)

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