Impact of Cyclization and Methylation on Peptide Penetration through Droplet Interface Bilayers.

Cell-penetrating peptides enter cells via diverse mechanisms, such as endocytosis, active transport, or direct translocation. For the design of orally delivered cell-penetrating peptides, it is crucial to know the contribution of these different mechanisms. In particular, the ability of a peptide to translocate through a lipid bilayer remains a key parameter for the delivery of cargos. However, existing approaches used to assess translocation often provide discrepant results probably because they have different sensitivities to the distinct translocation mechanisms. Here, we focus on the passive permeation of a range of hydrophobic cyclic peptides inspired by somatostatin, a somatotropin release-inhibiting factor. Using droplet interface bilayers (DIB), we assess the passive membrane permeability of these peptides and study the impact of the peptide cyclization and backbone methylation on translocation rates. Cyclization systematically improved the permeability of the tested peptides while methylation did not. By studying the interaction of the peptides with the DIB interfaces, we found membrane insertion and peptide intrinsic diffusion to be two independent factors of permeability. Compared to the industrial gold standard Caco-2 and parallel artificial membrane permeability assay (PAMPA) models, DIBs provide intermediate membrane permeability values, closer to Caco-2. Even for conditions where Caco-2 and PAMPA are discrepant, the DIB approach also gives results closer to Caco-2. Thereupon, DIBs represent a robust alternative to the PAMPA approach for predicting the permeability of peptides, even if the latter present extremely small structural differences.

Membrane determinants for the passive translocation of analytes through droplet interface bilayers.

Understanding how small molecules cross cell membranes is crucial to pharmaceutics. Several methods have been developed to evaluate such a process, but they need improvement since many false-positive candidates are often selected. Robust tools enabling rapid and reproducible screening can increase confidence on hits, and artificial membranes based on droplet interface bilayers (DIBs) offer this possibility. DIBs consist in the adhesion of two phospholipid-covered water-in-oil droplets which reproduce a bilayer. By having donor and acceptor droplets, the permeability of an analyte can be studied. However, the relevance of this system relies on the comprehension of how well the physical chemistry of the produced bilayer recapitulates the behavior of cell membranes. This information is missing, and we address it here. Taking small fluorophores as model analytes, we studied their permeation through DIBs made of a wide range of phospholipids. We found that both the phospholipid acyl chain and polar head affect permeability. Overall, these parameters impact the phospholipid shape and thereupon the membrane lateral pressure, which is a major factor modulating with permeability in our system. These results depend on the nature of the chosen oil. We thereupon identified relevant physical chemistry conditions that best mimic the compactness and subsequent permeability of biological membranes.

Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands.

INTRODUCTION: Primary aldosteronism accounts for 6-15% of hypertension cases, the single biggest contributor to global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas, only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11 radiolabelled metomidate ([11C]MTO) is a positron emission tomography (PET) radiotracer able to selectively identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [11C]MTO is limited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position ([18F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-cyclotron-based imaging centres. METHODS: Two strategies for the one-step radio-synthesis of [18F]FAMTO were developed. [18F]FAMTO was obtained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [18F]FAMTO was evaluated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents. Rat plasma and urine were analysed to determine [18F]FAMTO metabolites. RESULTS: [18F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-isolated radiochemical yield (RCY), respectively. Formulated [18F]FAMTO was obtained with >99% radiochemical and enantiomeric purity with a synthesis time of 140 min from the trapping of [18F]fluoride ion on an anion-exchange resin (QMA cartridge). In vitro autoradiography of [18F]FAMTO demonstrated exquisite specific binding in CYP11B-rich pig adrenal glands. In vivo [18F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was about 34% of that in the adrenals and all other organs were <12% of the adrenal uptake at 60 min post-injection. Metabolite analysis showed 13% unchanged [18F]FAMTO in blood at 10 min post-administration and rapid urinary excretion. In vitro assays in human blood showed a free fraction of 37.5%. CONCLUSIONS: [18F]FAMTO, a new 18F-labelled analogue of metomidate, was successfully synthesised. In vitro and in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and CYP11B2), supporting the potential of this radiotracer for human investigation.

Comb-Like Fluorophilic-Lipophilic-Hydrophilic Polymers for Nanocapsules as Ultrasound Contrast Agents.

Imaging the enhanced permeation and retention effect by ultrasound is hindered by the large size of commercial ultrasound contrast agents (UCAs). To obtain nanosized UCAs, triblock copolymers of poly(ethylene glycol)-polylactide-poly(1 H,1 H,2 H,2 H-heptadecafluorodecyl methacrylate) (PEG-PLA-PFMA) with distinct numbers of perfluorinated pendant chains (5, 10, or 20) are synthesized by a combination of ring-opening polymerization and atom transfer radical polymerization. Nanocapsules (NCs) containing perfluorooctyl bromide (PFOB) intended as UCAs are obtained with a 2-fold increase in PFOB encapsulation efficiency in fluorinated NCs as compared with plain PEG-PLA NCs thanks to fluorous interactions. NC morphology is strongly influenced by the number of perfluorinated chains and the amount of polymer used for formulation, leading to peculiar capsules with several PFOB cores at high PEG-PLA-PFMA20 amount and single-cored NCs with a thinner shell at low fluorinated polymer amount, as confirmed by small-angle neutron scattering. Finally, fluorinated NCs yield higher in vitro ultrasound signal compared with PEG-PLA NCs, and no in vitro cytotoxicity is induced by fluorinated polymers and their degradation products. Our results highlight the benefit of adding comb-like fluorinated blocks in PEG-PLA polymers to modify the nanostructure and enhance the echogenicity of nanocapsules intended as UCAs.