development-characterization-evaluation-cage-based-ionic-liquid

Development, Characterization, and Evaluation of CAGE-based Ionic Liquid Systems for Transdermal Delivery

Ionic liquids have recently gained remarkable recognition for their applications within drug delivery, in particular their potential to enable cutaneous ...

University of Copenhagen

The absence of a surface-lining mucus layer is a major pitfall for the Caco-2 epithelial model. However, this limitation can be alleviated by applying biosimilar mucus (BM) to the apical surface of the cell monolayer, thereby constructing a mucosa mimicking in vivo conditions. This study aims to elucidate the influence of BM as a barrier towards exogenic compounds such as permeation enhancers, and components of fed state simulated intestinal fluid (FeSSIF). Caco-2 cell monolayers surface-lined with BM were exposed to several compounds with distinct physicochemical properties, and the cell viability and permeability of the cell monolayer was compared to that of cell monolayers without BM and well-established mucus-secreting epithelial models (HT29-MTX-E12 cell monolayers and HT29-MTX-E12/Caco-2 cell co-culture monolayers). Exposure of BM-covered cells to constituents from FeSSIF revealed that it comprised a strong, hydrophilic barrier effect as 90% of BM-covered cells remained viable for >4 h, and the permeation rate of hydrophobic drugs was reduced. In contrast, the permeation rate of hydrophilic drugs was largely unaffected. Control monolayers displayed a loss of barrier function and <10% viable cells. The efficacy of fatty acid permeation enhancers were altered when investigated in BM-covered cells as compared to all the other studied epithelial models. Thus, Caco-2 cell monolayers surface-lined with BM constitute a valuable in vitro model that makes it possible to mimic intestinal fed state conditions when studying drug permeation.

Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium.

Delivery of therapeutic peptides upon oral administration is highly desired and investigations report that the cell-penetrating peptide (CPP) penetratin and its analogues shuffle and penetramax show potential as carriers to enhance insulin delivery. Exploring this, the specific aim of the present study was to understand the impact that their complexation with a lipidated or non-lipidated therapeutic cargo would have on the delivery, to evaluate the effect of differences in membrane interactions in vitro and in vivo, as well as to deduce the mode of action leading to enhanced delivery. Fundamental biophysical aspects were studied by a range of orthogonal methods. Transepithelial permeation of therapeutic peptide was evaluated using the Caco-2 cell culture model supplemented with epithelial integrity measurements, real-time assessment of the carrier peptide effects on cell viability and on mode of action. Pharmacokinetic and pharmacodynamic (PK/PD) parameters were evaluated following intestinal administration to rats and tissue effects were investigated by histology. The biophysical studies revealed complexation of insulin with shuffle and penetramax, but not with penetratin. This corresponded to enhanced transepithelial permeation of insulin, but not of lipidated insulin, when in physical mixture with shuffle or penetramax. The addition of shuffle and penetramax was associated with a lowering of Caco-2 cell monolayer integrity and viability, where the lowering of cell viability was immediate, but reversible. Insulin delivery in rats was enhanced by shuffle and penetramax and accompanied by a 10-20-fold decrease in blood glucose with immediate effect on the intestinal mucosa. In conclusion, shuffle and penetramax, but not penetratin, demonstrated to be potential candidates as carriers for transmucosal delivery of insulin upon oral administration, and their effect depended on association with both cargo and cell membrane. Interestingly, the present study provides novel mechanistic insight that peptide carrier-induced cargo permeation points towards enhancement via the paracellular route in the tight epithelium. This is different from the anticipated belief being that it is the cell-penetrating capability that facilitate transepithelial cargo permeation via a transcellular route.

Revealing the importance of carrier-cargo association in delivery of insulin and lipidated insulin.

This chapter describes the use of cell-penetrating peptides (CPPs) as carriers for transepithelial delivery of therapeutic peptides. Assessment of transepithelial peptide permeation and the mechanisms of action that permeability enhancing drug carriers exert on the epithelium requires subtle sample preparation and analysis by orthogonal methods. Here, the preparation and use of CPP-insulin physical mixture samples including the quantification of insulin by enzyme-linked immunosorbent assay (ELISA) is described. In addition, effects of CPPs on the epithelium and its barrier properties immediately upon exposure and after a recovery period are evaluated by epithelial cell viability, transepithelial electrical resistance, immunostaining of the tight junction associated zonula occludens (ZO-1) protein, and actin cytoskeleton staining.

Cell-Penetrating Peptides as Carriers for Transepithelial Drug Delivery.

The inherent low oral bioavailability of therapeutic peptides can be enhanced by the cell-penetrating peptide penetratin and its analogues shuffle and penetramax applied as carriers for delivery of insulin. In this study, the objective was to gain mechanistic insights on the effect of the carrier peptide stereochemistry on their interactions with insulin and on insulin delivery. Insulin-carrier peptide interactions were investigated using small-angle X-ray scattering and cryogenic transmission electron microscopy, while the insulin and peptide stability and transepithelial insulin permeation were evaluated in the Caco-2 cell culture model along with the carrier peptide-induced effects on epithelial integrity and cellular metabolic activity. Interestingly, the insulin transepithelial permeation was influenced by the degree of insulin-carrier peptide complexation and depended on the stereochemistry of penetramax but not of penetratin and shuffle. The l-form of the peptides initially decreased the epithelial integrity comparable to that induced by the d-peptides, suggesting a comparable mechanism of action. The immediate decrease was reversible during exposure of the Caco-2 epithelium to the l-peptides but not during exposure to the d-peptides, likely a result of their higher stability. Overall, exploration of the stereochemistry showed to be an interesting strategy for carrier peptide-mediated insulin delivery.

Stereochemistry and Intermolecular Interactions Influence Carrier Peptide-Mediated Insulin Delivery.

The cell-penetrating peptide penetratin and its analogues shuffle and penetramax have been used as carrier peptides for oral delivery of therapeutic peptides such as insulin. Their mechanism of action for this purpose is not fully understood but is believed to depend on the interactions of the peptide with the cell membrane. In the present study, peptide-liposome interactions were investigated using advanced biophysical techniques including small-angle neutron scattering and fluorescence lifetime imaging microscopy. Liposomes were used as a model system for the cell membrane. All the investigated carrier peptides induced liposome clustering at a specific peptide/lipid ratio. However, distinctively different types of membrane interactions were observed, as the liposome clustering was irreversible for penetratin, but fully or partly reversible for shuffle and penetramax, respectively. All three peptides were found to adsorb to the surface of the lipid bilayers, while only shuffle and penetramax led to shape deformation of the liposomes. Importantly, the peptide interactions did not disrupt the liposomes under any of the investigated conditions, which is advantageous for their application in drug delivery. This detailed insight on peptide-membrane interactions is important for understanding the mechanism of peptide-based excipients and the influence of peptide sequence modifications.

Carrier peptide interactions with liposome membranes induce reversible clustering by surface adsorption and shape deformation.

In this study, we report the degradation of smooth and rough lipopolysaccharides (LPS) from Gram-negative bacteria and of lipoteichoic acid (LTA) from Gram-positive bacteria by peptide-coated TiO nanoparticles (TiO NPs). While bare TiO NPs displayed minor binding to both LPS and LTA, coating TiO NPs with the antimicrobial peptide LL-37 dramatically increased the level of binding to both LPS and LTA, decorating these uniformly. Importantly, peptide coating did not suppress reactive oxygen species generation of TiO NPs; hence, UV illumination triggered pronounced degradation of LPS and LTA by peptide-coated TiO NPs. Structural consequences of oxidative degradation were examined by neutron reflectometry for smooth LPS, showing that degradation occurred preferentially in its outer O-antigen tails. Furthermore, cryo-TEM and light scattering showed lipopolysaccharide fragments resulting from degradation to be captured by the NP/lipopolysaccharide coaggregates. The capacity of LL-37-TiO NPs to capture and degrade LPS and LTA was demonstrated to be of importance for their ability to suppress lipopolysaccharide-induced activation in human monocytes at simultaneously low toxicity. Together, these results suggest that peptide-coated photocatalytic NPs offer opportunities for the confinement of infection and inflammation.

Photocatalytic Degradation of Bacterial Lipopolysaccharides by Peptide-Coated TiO Nanoparticles.

Oral delivery of biopharmaceuticals can be enhanced by some cell-penetrating peptides applied as carriers. The objective here was to improve the permeability enhancing properties of such two peptides by branching into dimer and trimer variants. The transepithelial permeation of cargos including insulin, dextran, mannitol, and metoprolol mediated by penetratin and its analogue penetramax in their linear and branched versions was evaluated in the Caco-2 cell culture model. The enhancing properties of penetramax and particularly penetratin were significantly increased in accordance with the degree of branching, overall increasing the potency of the molecule. The enhanced cargo permeation was associated with peptide-induced effects on the Caco-2 cell monolayers including immediate and reversible decrease in integrity and alterations of the cell cytoskeleton. Overall, the results of the in vitro studies display that the paracellular pathway is involved in the enhanced cargo permeation and that peptide modification by branching indeed increases the permeation of cargos. In vivo pharmacokinetic and histological assessment with insulin as a cargo confirmed the potential of dimeric penetramax as a carrier in comparison to its linear counterpart.

Article	Year
Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences\| PubMed ID: 29524592	2018
Revealing the importance of carrier-cargo association in delivery of insulin and lipidated insulin. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 34298056	2021
Cell-Penetrating Peptides as Carriers for Transepithelial Drug Delivery. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34766302	2022
Stereochemistry and Intermolecular Interactions Influence Carrier Peptide-Mediated Insulin Delivery. Molecular pharmaceutics\| PubMed ID: 36607603	2023
Carrier peptide interactions with liposome membranes induce reversible clustering by surface adsorption and shape deformation. Journal of colloid and interface science\| PubMed ID: 37515972	2023
Photocatalytic Degradation of Bacterial Lipopolysaccharides by Peptide-Coated TiO Nanoparticles. ACS applied materials & interfaces\| PubMed ID: 39443826	2024
Branched penetratin and penetramax display enhanced intestinal insulin delivery potency compared to their linear counterparts. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 39983923	2025

Ragna G. Diedrichsen

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