“…Deuterated cholesterol (average 80% D as determined by mass spectrometry) was produced using previously reported methods (Sebastiani et al, 2021;Recsei et al, 2023) (ca. 90% D 2 O in the growth medium).…”
Peptide-based liquid droplets (coacervates) produced by spontaneous liquid-liquid phase separation (LLPS), have emerged as a promising class of drug delivery systems due to their high entrapping efficiency and the simplicity of their formulation. However, the detailed mechanisms governing their interaction with cell membranes and cellular uptake remain poorly understood. In this study, we investigated the interactions of peptide coacervates composed of HBpep—peptide derived from the histidine-rich beak proteins (HBPs) of the Humboldt squid—with model cellular membranes in the form of supported lipid bilayers (SLBs). We employed quartz crystal microbalance with dissipation monitoring (QCM-D), neutron reflectometry (NR) and atomistic molecular dynamics (MD) simulations to reveal the nature of these interactions in the absence of fluorescent labels or tags. HBpep forms small oligomers at pH 6 whereas it forms µm-sized coacervates at physiological pH. Our findings reveal that both HBpep oligomers and HBpep-coacervates adsorb onto SLBs at pH 6 and 7.4, respectively. At pH 6, when the peptide carries a net positive charge, HBpep oligomers insert into the SLB, facilitated by the peptide’s interactions with the charged lipids and cholesterol. Importantly, however, HBpep coacervate adsorption at physiological pH, when it is largely uncharged, is fully reversible, suggesting no significant lipid bilayer rearrangement. HBpep coacervates, previously identified as efficient drug delivery vehicles, do not interact with the lipid membrane in the same manner as traditional cationic drug delivery systems or cell-penetrating peptides. Based on our findings, HBpep coacervates at physiological pH cannot cross the cell membrane by a simple passive mechanism and are thus likely to adopt a non-canonical cell entry pathway.
“…Deuterated cholesterol (average 80% D as determined by mass spectrometry) was produced using previously reported methods (Sebastiani et al, 2021;Recsei et al, 2023) (ca. 90% D 2 O in the growth medium).…”
Peptide-based liquid droplets (coacervates) produced by spontaneous liquid-liquid phase separation (LLPS), have emerged as a promising class of drug delivery systems due to their high entrapping efficiency and the simplicity of their formulation. However, the detailed mechanisms governing their interaction with cell membranes and cellular uptake remain poorly understood. In this study, we investigated the interactions of peptide coacervates composed of HBpep—peptide derived from the histidine-rich beak proteins (HBPs) of the Humboldt squid—with model cellular membranes in the form of supported lipid bilayers (SLBs). We employed quartz crystal microbalance with dissipation monitoring (QCM-D), neutron reflectometry (NR) and atomistic molecular dynamics (MD) simulations to reveal the nature of these interactions in the absence of fluorescent labels or tags. HBpep forms small oligomers at pH 6 whereas it forms µm-sized coacervates at physiological pH. Our findings reveal that both HBpep oligomers and HBpep-coacervates adsorb onto SLBs at pH 6 and 7.4, respectively. At pH 6, when the peptide carries a net positive charge, HBpep oligomers insert into the SLB, facilitated by the peptide’s interactions with the charged lipids and cholesterol. Importantly, however, HBpep coacervate adsorption at physiological pH, when it is largely uncharged, is fully reversible, suggesting no significant lipid bilayer rearrangement. HBpep coacervates, previously identified as efficient drug delivery vehicles, do not interact with the lipid membrane in the same manner as traditional cationic drug delivery systems or cell-penetrating peptides. Based on our findings, HBpep coacervates at physiological pH cannot cross the cell membrane by a simple passive mechanism and are thus likely to adopt a non-canonical cell entry pathway.
“…Alternatively, deuterium can be incorporated using direct H/D exchange of protonated derivatives in D 2 O using metal catalysts [ 14 , 18 – 23 ], or by biosynthesis using biological organisms grown in D 2 O (Fig. 1 ) [ 13 , 24 – 26 ]. Fig.…”
DGet! is an open-source analysis package written in Python for calculating the degree of deuterium enrichment in isotopically labelled molecules using mass spectrometric data. The nuclear properties of deuterium make it a valuable tracer in metabolic studies and an excellent contrast agent in nuclear spectroscopies. Determination of molecular deuteration levels is typically performed using mass spectrometry, however software options to perform these calculations are scarce. The in-house scripts and spreadsheets currently used rarely account for isotopic interferences from 13C or multi-isotopic elements that impact deuteration calculations. DGet! removes isotopic interferences using de-convolution and both the isotopological makeup and overall deuteration level can be accurately recovered. The software is available as command line and web applications that take a molecular formula and mass spectrometry data and output a graphical representation of the degree of deuteration as well as the distribution of partially deuterated analogues. These applications are designed to be easy to use and enable superior characterisation of deuterated molecules for users of all levels of expertise, without the limitations of techniques currently used by the majority of deuteration laboratories and researchers.
Graphical Abstract
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