Increased Carrier Peptide Stability through pH Adjustment Improves Insulin and PTH(1-34) Delivery In Vitro and In Vivo Rather than by Enforced Carrier Peptide-Cargo Complexation

Abstract

Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34).

Stereochemistry and Intermolecular Interactions Influence Carrier Peptide-Mediated Insulin Delivery

“…Moreover, L-penetratin has previously been observed to associate with insulin at the investigated pH although at lower concentrations. 24 In relation to the higher insulin P app values when in the presence of shuffle and penetramax compared to when in the presence of penetratin (Figure 3C), insulin complexation appeared to influence the enhancing effect of the carrier peptides. Insulin was used as a model cargo in the present study, and naturally, the degree of peptide interactions with the cargo will depend on their physiochemical properties such as charge of the cargo, and thus, the enhancing effect will depend on the cargo as demonstrated with other therapeutic peptides.…”

“…This is consistent with the lack of a linear combination for mixtures with l -penetratin (Figure S3), suggesting some complex formation despite the lack of a distinct increase at low q (<0.03 Å –1 ), indicative of aggregation (Figure A). Moreover, l -penetratin has previously been observed to associate with insulin at the investigated pH although at lower concentrations …”

“…The unchanged levels of insulin when applied alone, that is, without the peptides, are consistent with findings in another previous study from our group. 24 The decreased insulin concentration could be speculated to be a result of complexation between insulin and the D-carrier peptides and subsequent precipitation. However, this speculation seems unlikely as the D-peptide concentrations were similar when alone and in the presence of insulin (Figures 2E,F and S6B,C) and as the carrier peptide− insulin interactions were found to be stereochemistry independent (Figure 1).…”

Screening for effective cell-penetrating peptides with minimal impact on epithelial cells and gut commensals in vitro

“…Tat and Penetratin are among the most studied CPPs, which have been employed as a vector to improve intracellular delivery of various drug compounds, including oligonucleotides ( Brown & Graham, 2010 ) and proteins ( Harada et al, 2002 ; Liang & Yang, 2005 ; Kamei et al, 2008 ; Khafagy et al, 2009 ). Besides that, both Tat and Penetratin have been shown to promote permeation through tight junction modulation ( Bai et al, 2008 ; Kristensen et al, 2020 ; Diedrichsen et al, 2021 ). However, results from our study showed that Tat and Penetratin had no permeabilizing effect in the Caco-2 monolayer setup at 100 µM.…”

“…Paracellular spaces are aqueous filled channels through which these drugs prefer to diffuse ( Salamat-Miller and Johnston, 2005 ; Patel and Misra, 2011 ). Several studies have reported that some CPPs can also promote permeation of various cellular barriers through paracellular route both in vitro and in vivo ( Ohtake et al, 2002 ; Bai et al, 2008 ; Kristensen and Nielsen, 2016a ; Kristensen and Nielsen, 2016b ; Kristensen et al, 2020 ; Diedrichsen et al, 2021 ; Ragupathy et al, 2021 ; Frøslev et al, 2022 ). This could be either due to the high local concentration of the CPPs, influencing the dynamics of the tight junction proteins or due to cell-penetrating tendency of CPPs, targeting intracellular proteins which are involved in regulation of opening and closing of tight junctions ( Taverner et al, 2015 ; Kristensen and Nielsen, 2016a ).…”