Hypothesis: Permeation of macromolecular drugs across biological plasma membranes is a major chal-lenge in drug delivery. Cationic cell-penetrating peptides (CPPs) are attractive functional excipient can-didates for the delivery of macromolecules across membrane barriers, due to their membrane translocating ability. The properties of CPPs can be tailored by lipidation, a promising approach to facil-itate enhanced membrane insertion, potentially promoting increased translocation of the CPP and cargo. Experiments: To explore the impact that site and degree of lipidation have on the membrane interaction of a cationic CPP, we designed and investigated CPP conjugates with one or two fatty acid chains. Findings: Compared to the parent CPP and the single-lipidated conjugates, the double-lipidated conjugate exhibited the most pronounced membrane perturbation effects, as measured by several biophysical tech-niques. The experimental findings were supported by molecular dynamics (MD) simulations, demon-strating that all CPP conjugates interacted with the membrane by insertion of the lipid chain(s) into the core of the bilayer. Moreover, membrane-thinning effects and induced membrane curvature were dis-played upon CPP interaction. Our results demonstrate that the impact exerted by the CPP on the mem-brane is notably affected by positioning and especially the degree of lipidation, which might influence the properties of CPPs as functional excipients. (c) 2020 Elsevier Inc. All rights reserved.

The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.

Lipidation of proteins is used in the pharmaceutical field to increase the therapeutic efficacy of proteins. In this study, we investigate the effect of a 14-carbon fatty acid modification on the adsorption behavior of human insulin to a hydrophobic solid surface and the subsequent fibrillation development under highly acidic conditions and elevated temperature by comparing to the fibrillation of human insulin. At these stressed conditions, the lipid modification accelerates the rate of fibrillation in bulk solution. With the use of several complementary surface-sensitive techniques, including quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and neutron reflectivity (NR), we show that there are two levels of structurally different protein organization at a hydrophobic surface for both human insulin and the lipidated analogue: a dense protein layer formed within minutes on the surface and a diffuse outer layer of fibrillar structures which took hours to form. The two layers may only be weakly connected, and proteins from both layers are able to desorb from the surface. The lipid modification increases the protein surface coverage and the thickness of both layer organizations. Upon lipidation not only the fibrillation extent but also the morphology of the fibrillar structures changes from fibril clusters on the surface to a more homogeneous network of fibrils covering the entire hydrophobic surface.

We report on the formation of nanostructured aqueous dispersions based on the negatively charged food-grade emulsifier citrem (citric acid esters of mono- and diglycerides). To our knowledge, this is the first report in the literature on the spontaneous formation of aqueous PEGylated and non-PEGylated dispersions of citrem. Citrem can be easily dispersed in water in the absence of other surfactant-like lipids without applying high-energy input. However, to investigate the stability and to characterize the internal structure of dispersions with a narrow size distribution, ultrasonication was applied for the emulsification of citrem or its binary mixture with PEGylated lipids of poly (ethylene glycol) monooleate with Mn ∼ 460 (MO-PEG460) or Mn ∼ 860 (MO-PEG860) and non-PEGylated lipids (monoolein (MO) or phytantriol (PHYT)). We obtained stable dispersions (>4 months) consisting of particles with highly negative zeta potentials (in the range of −41–−31 mV) and a mean particle size range of 139–172 nm. A decrease in the particle mean size by 13–19% was observed when dispersing citrem in the presence of a PEGylated monoolein (either MO-PEG860 or MO-PEG460). The structural characterization was studied by synchrotron small angle X-ray scattering (SAXS), where the effect of partial replacement of citrem by two PEGylated lipids or other well-known lipids (MO and PHYT) was investigated. In addition, cryogenic transmission electron microscopy (cryo-TEM) was applied to characterize the morphology and the internal structure of different PEGylated dispersions of citrem. The cryo-TEM revealed particles enveloping internal structures in co-existence with vesicles. No long-range order was observed, in correlation with the results obtained by SAXS, suggesting that the particles were comprised of the internal reverse micellar (L2) phase. Based on the SAXS results, the partial replacement of citrem by high amount of MO or PHYT induced the formation of hexosomes. The investigated dispersions of citrem could be attractive as nanocarriers of poorly water-soluble drugs and functional foods.