Stereochemistry and Intermolecular Interactions Influence Carrier Peptide-Mediated Insulin Delivery

Abstract

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.

Epithelium dynamics differ in time and space when exposed to the permeation enhancers penetramax and EGTA. A head-to-head mechanistic comparison

“…Here, the penetramax-mediated modulation of the actin cytoskeleton and TJ proteins, resulting in size-selective permeation of the paracellular markers of FD4 and FD10 (Figure 3), as the P app values for FD10 at all tested penetramax concentrations were lower than those for FD4. Previous work display that the enhanced permeation of FD4 by penetramax to a reasonable extent resembles the effect on insulin permeation, although the latter is slightly lower (Diedrichsen et al, 2021;Diedrichsen et al, 2023), likely due to differences in molecular weight, structure, and net charge. The observed permeation enhancement can be attributed to penetramax-induced TJ remodeling towards the leak pathway since cldn-2 is upregulated while cldn-4 and -7 is downregulated.…”

“…Here, we investigate the effects of penetramax, a membraneinteracting peptide-based enhancer, and analog of penetratin, a cellpenetrating peptide, which we and others previously reported to enhance peptide delivery in vitro and in vivo (Kamei et al, 2013a;Diedrichsen et al, 2021). In vitro, effective concentrations of penetramax are in the two-digit µM range (Diedrichsen et al, 2023). We compare the findings head-to-head to those observed with EGTA with the aim to analyze the underlying cellular mechanisms that regulate TJ and cytoskeleton dynamics for both enhancers.…”

Stereoisomer-Dependent Membrane Association and Capacity for Insulin Delivery Facilitated by Penetratin

“…The advantage of endocytosis is that it does not compromise the cell membrane’s integrity, thereby avoiding concurrent cytotoxicity that is related to membrane damage [ 6 , 20 ] as opposed to direct translocation, where this is a risk [ 21 ]. In addition to aiming at cargo delivery to intracellular targets, CPPs may be applied as carriers for the delivery of cargo across the epithelia, either by transcytosis, cell membrane perturbation [ 22 ], or by affecting the epithelial integrity [ 3 , 23 ].…”

“…Penetratin is a very well-studied CPP, and both l - and d -enantiomers ( l -PEN and d -PEN, respectively) of this peptide have been examined for their potential to deliver a variety of cargoes to intracellular [ 6 , 24 , 25 , 26 ] or transepithelial [ 5 , 23 , 27 , 28 , 29 , 30 , 31 ] targets in vitro, as well as in situ [ 4 , 32 ] and in vivo [ 3 , 33 , 34 ] Nevertheless, their detailed mode of action for enhancing deliveries remains unresolved. While initially believed to translocate across cell membranes by direct translocation [ 35 , 36 ], l -PEN was later, at lower concentrations, demonstrated to internalize by endocytosis [ 36 , 37 ] without compromising the membrane integrity of the examined cells [ 37 ].…”