Cyclodextrin-mediated enhancement of gastrointestinal drug delivery: unveiling mucoadhesive and mucopenetrating synergy

This study evaluates the in vivo mucoadhesive properties of thiolated cyclodextrins (CDs) with varying S-protection using polyethylene glycol (PEG) of different chain lengths. Free thiol groups of thiolated β-CDs (CD-SH) were S-protected with 1 kDa and 2 kDa PEG bearing a terminal thiol group, leading to third-generation of thiolated CDs (CD-SS-PEG). The structure of these thiolated CDs was confirmed and characterized by FT-IR, 1 H NMR, and colorimetric assays. Thiolated and S-protected CDs were evaluated regarding viscosity, cellular uptake and, in vitro and in vivo mucoadhesion. The viscosity of CD-SH, CD-SS-PEG 1 kDa, and CD-SS-PEG 2 kDa mixtures with mucus increased 9-, 7-, and 5.5-fold, respectively, compared to unmodified CD within 3 h. Cellular uptake on Caco-2 cells was 1.75 times higher for highly thiolated CDs than for unmodified CD. In vitro residence time on porcine intestine was prolonged 7-, 8.4-, and 7.9-fold for CD-SH, CD-SS-PEG 1 kDa, and CD-SS-PEG 2 kDa, respectively. In vivo results indicated CD-SS-PEG 1 kDa had the highest potential. Our comprehensive in vitro, ex vivo, and in vivo ffindings demonstrate that CD-SS-PEG 1 kDa is a highly promising candidate for mucoadhesive drug delivery systems.

Introduction

Cyclodextrins (CDs) and their derivatives serve as effective excipients with the potential to enhance the bioavailability of a wide array of active pharmaceutical ingredients (APIs) [1]. Due to their ability to enhance drug solubility, CDs are used in a variety of pharmaceutical products [2]. Because of their relatively less hydrophilic central cavity, CDs allow for the incorporation of lipophilic drugs, resulting in enhanced aqueous solubility, improved chemical stability, and masking undesirable tastes or odors associated with the APIs [3].

Because of their benefits in drug delivery, several cyclodextrins (CDs) and their derivatives have been officially recognized as pharmaceutical excipients. These include α-CD, β-CD, γ-CD, hydroxypropyl β-CD (HP-β-CD), hydroxypropyl γ-CD (HP-γ-CD), randomly methylated β-CD (RM-β-CD), and sulfobutylated β-CD (SB-β-CD) [2]. Nevertheless, their limited mucoadhesive characteristics constrain their suitability for administration through this pathway, given the relatively brief duration of contact with mucosal membranes [4]. To overcome this shortcoming, mucoadhesive thiolated CDs were introduced [5]. Evidence for their potential has been provided by numerous studies in recent years [4, 6,7,8,9]. These carriers can form disulfide bonds with cysteine-rich subdomains of mucus glycoproteins, resulting in robust mucoadhesive properties [10].

As the crucial factor for mucoadhesion is the degree of thiolation, the more hydroxyl groups are substituted by thiol groups, the stronger the mucoadhesion [11]. The main limitation of thiolated CDs is that the free thiol groups are prone to oxidation, making them susceptible to oxidation to disulfide bonds, decreasing the number of free thiol groups [12]. Furthermore, the thiol groups of these CDs readily interact with cysteines within the outer, loosely bound mucus gel layer, which is rapidly eliminated by the mucus turnover process. This underscores the clear requirement for enhanced interactions of thiolated CDs. Second and third generations of thiolated CDs, named as S-protected ones, received more interest recently [11, 13]. Free thiol groups can be partially and reversibly deactivated using various S-protecting agents. Depending on the reactivity of these agents, 2nd and 3rd generation thiomers can be synthesized.

The second generation is produced by highly reactive sulfhydryl ligands, for example, 2-mercaptonicotinic acid (MNA). In contrast, in case of the 3rd generation, sulfhydryl ligands with lower reactive, such as cysteine or glutathione, are used for S-protection [14]. Because of their low reactivity 3rd generation thiolated CDs can freely diffuse into deeper mucus regions as they do not immediately form new disulfide bonds with mucus glycoproteins [11], resulting in higher mucoadhesion [15]. To date, third-generation thiolated CDs have been subjected to in vitro assessments of their mucoadhesive properties, with no corresponding in vivo evaluations.

Hence, the objective of this study was to synthesize first and third-generation thiolated CDs featuring different degrees of thiolation (low and high), as well as distinct densities and lengths of S-protecting PEG chains. This synthesis aimed to comprehensively compare their mucoadhesive characteristics through both in vitro and in vivo assessments. Unprotected thiolated β-CD (1st generation) was synthesized by substituting hydroxyl groups with thiol moieties on the glucose subunits via direct conversion with phosphorous pentasulfide for high thiolations.

In the case of third generation thiolated CDs, thiol-terminated PEG chains of 1 and 2 kDa were employed to provide S-protection for the thiolated CDs. We evaluated how varying degrees of thiolation and distinct types of S-protection affect in vitro cell viability. Furthermore, the assessment involved membrane toxicity, cellular uptake, and mucoadhesion. Conclusively, the in vivo mucoadhesive characteristics of thiolated cyclodextrins loaded with the model drug Coumarin 6 (C6) were assessed using a rat model.

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Materials

Beta CD (β-CD, catalogue code: CY-2001; α-1,4 glycosidic bond connected cyclic anhydroglucose; fine chemical grade; purity > 95%; molecular weight: 1135.0 g/mol) and thiolated β-CD (catalogue code: CY-2224; heptakis (6-deoxy-6-thio)-beta-cyclodextrin; purity > 97%; molecular weight: 1247.4 g/mol) were obtained from CycloLab Ltd. 3-(2-Pyridyldithio) propionic acid methoxy polyethylene glycol (mPEG-OPSS) with PEG chains of 1 kDa and 2 kDa were sourced from Abbexa. Chemicals such as thiourea (CS(NH2)2, ≥ 99%), 2-mercaptonicotinic acid (2-MNA, ≥ 98%), and 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, ≥ 98%) were supplied by Sigma-Aldrich, Vienna, Austria. Additional materials from Sigma-Aldrich included Spectra/PorÒ dialysis membranes with molecular weight cut-offs (MWCO) of 1000 and 3500 Da, Hanks’ balanced salt solution (HBSS), resazurin sodium salt (7-hydroxy-3 H-phenoxazin-3-one 10-oxide, dye content ≥ 75%), cysteine (≥ 98%), sodium borohydride (NaBH4, ≥ 99%), dimethyl sulfoxide-d6 (DMSO-d6, ≥ 99.9%), Coumarin 6 (C6), minimum essential eagle medium (MEM), sodium chloride (NaCl), methanol (≥ 95–99%), phosphorus pentasulfide (P4S10, 99%), tetramethylene sulfone (sulfolane, 99%), and Triton-X 100. The cell culture medium was prepared in line with published protocols. Fetal bovine serum (FBS) and 100 mM phosphate-buffered saline (PBS) at pH 6.8 were obtained from Gibco, a part of Invitrogen, Lofer, Austria. All other reagents used were of analytical grade and sourced from commercial suppliers.