UNASSIGNED: Lipid-based nanoparticles (LNPs) is increasingly recognized for their potential in drug delivery, offering protection to hydrophobic drugs from degradation. Industrial synthesis of LNPs, exemplified by Pfizer-BioNTech and Moderna mRNA vaccines, utilizes flow chemistry or microfluidics, showcasing its scalability. This study explores the utilization of a novel design reactor, the vortex tube reactor, within flow chemistry for LNPs synthesis, aiming to optimize its conditions and compare them with batch synthesis.
UNASSIGNED: LNPs were synthesized using the vortex tube reactor, incorporating bovine serum albumin (BSA) as a model drug in the aqueous phase, alongside 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol in the organic phase. Design of experiments (DoE), specifically Box-Behnken design, was employed to optimize parameters, including X1: the flow rate ratio (10-100 mL/min), X2: the aqueous-to-organic volumetric ratio (1:1-10:1), and X3: the number of reactor units (1-5 units). Responses evaluated encompassed physical properties and productivity. Optimized conditions were determined by minimizing particle size (Y1), polydispersity index (Y2), and zeta potential (Y3), while maximizing entrapment efficiency (Y4), drug loading (Y5), and productivity (Y5).
UNASSIGNED: Results indicated that optimal conditions were achieved at X1 of 100 mL/min, X2 of 5.278, and X3 of 1 unit. LNPs synthesized under these conditions exhibited favorable physical properties and productivity, with uniformity maintained across batches. The vortex tube reactor demonstrated superiority over batch synthesis, yielding smaller particles (166.23 ± 0.98 nm), more uniform nanoparticles (PDI 0.17 ± 0.01), and higher entrapment (67.75 ± 1.55%) and loading capacities (36.39 ± 0.83%), indicative of enhanced productivity (313.4 ± 12.88 mg/min).
UNASSIGNED: This study elucidates the potential of flow chemistry, particularly utilizing the vortex tube reactor, for large-scale LNPs formulation, offering insights into parameter relationships and advancing nanoparticle synthesis for drug delivery applications.

Gramicidin S (GS), one of the first discovered antimicrobial peptides, still shows strong antibiotic activity after decades of clinical use, with no evidence of resistance. The relatively high hemolytic activity and narrow therapeutic window of GS limit its use in topical applications. Encapsulation and targeted delivery may be the way to develop the internal administration of this drug. The lipid composition of membranes and non-covalent interactions affect GS\'s affinity for and partitioning into lipid bilayers as monomers or oligomers, which are crucial for GS activity. Using both differential scanning calorimetry (DSC) and FTIR methods, the impact of GS on dipalmitoylphosphatidylcholine (DPPC) membranes was tested. Additionally, the combined effect of GS and cholesterol on membrane characteristics was observed; while dipalmitoylphosphatydylglycerol (DPPG) and cerebrosides did not affect GS binding to DPPC membranes, cholesterol significantly altered the membrane, with 30% mol concentration being most effective in enhancing GS binding. The effect of star-like dextran-polyacrylamide D-g-PAA(PE) on GS binding to the membrane was tested, revealing that it interacted with GS in the membrane and significantly increased the proportion of GS oligomers. Instead, calcium ions affected GS binding to the membrane differently, with independent binding of calcium and GS and no interaction between them. This study shows how GS interactions with lipid membranes can be effectively modulated, potentially leading to new formulations for internal GS administration. Modified liposomes or polymer nanocarriers for targeted GS delivery could be used to treat protein misfolding disorders and inflammatory conditions associated with free-radical processes in cell membranes.

Fluorescent lipid probes such as 1-palmitoyl-2-(6-[7-nitro-2-1,3-benzoxadiazol-4-yl]amino-hexanoyl)-sn-glycero-3-phosphocholine (C6 NBD-PC) have been used extensively to study the kinetics of lipid flip-flop. However, the efficacy of these probes as reliable reporters of native lipid translocation has never been tested. In this study, sum-frequency vibrational spectroscopy (SFVS) was used to measure the kinetics of C6 NBD-PC lipid flip-flop and the flip-flop of native lipids in planar supported lipid bilayers. C6 NBD-PC was investigated at concentrations of 1 and 3 mol. % in both chain-matched 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chain-mismatched 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) to assess the ability of C6 NBD-PC to mimic the behavior of the surrounding matrix lipids. It was observed that C6 NBD-PC exhibited faster flip-flop kinetics compared to the native lipids in both DPPC and DSPC matrices, with notably accelerated rates in the chain-mismatched DSPC system. SFVS was also used to measure the acyl chain orientation and gauche content of C6 NBD-PC in both DPPC and DSPC membranes. In the DSPC matrix (chain mismatched), C6 NBD-PC was more disordered in terms of both gauche content and acyl tilt, whereas it maintained an orientation similar to that of the native lipids in the DPPC matrix (chain matched). In addition, the flip-flop kinetics of C6 NBD-PC were also measured using second-harmonic generation (SHG) spectroscopy, by probing the motion of the NBD chromophore directly. The flip-flop kinetics measured by SHG were consistent with those obtained from SFVS. This study also marks the first instance of phospholipid flip-flop kinetics being measured via SHG. The results of this study clearly demonstrate that C6 NBD-PC does not adequately mimic the behavior of native lipids within a membrane. These findings also highlight the significant impact of the lipid matrix on the flip-flop behavior of the fluorescently labeled lipid, C6 NBD-PC.

The crucial role of zwitterionic phosphatidylcholines (PC) within mucus gel is essential for maintaining intestinal homeostasis, while the underlying mechanism remains incompletely understood. Herein, we compared the dynamic interfacial adsorption behavior of saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated dioleoylphosphatidylcholine (DOPC) to intestinal mucin and their impact on the intestinal mucus barrier function. Results of quartz crystal microbalance with dissipation showed that the highly surface-hydrated DPPC vesicles exhibited significantly faster and more extensive adsorption to purified intestinal mucin than the slightly surface-hydrated DOPC vesicles. Utilizing an intestinal Caco-2/HT29-MTX coculture model, we observed that DPPC vesicles adsorbed much more to the mucus gel compared to DOPC vesicles. Additionally, DPPC vesicle adsorption displayed increased wetting, and converse for DOPC vesicles. Interestingly, both of them exhibited nearly the same protective effects against cell injury induced by peptic-tryptic digests of gliadin (PTG). The partial mechanism involved the binding of PTG to DPPC and DOPC within the mucus gel, thereby restricting PTG contact with the underlying epithelial cells. These findings shed light on the intricate interfacial dynamics of PC adsorption to mucin and their implications for maintaining the integrity of the intestinal mucus barrier.

The widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers\' optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations.

A widely known property of lipid membranes is their tendency to undergo a separation into disordered (Ld) and ordered (Lo) domains. This impacts the local structure of the membrane relevant for the physical (e.g., enhanced electroporation) and biological (e.g., protein sorting) significance of these regions. The increase in computing power, advancements in simulation software, and more detailed information about the composition of biological membranes shifts the study of these domains into the focus of classical molecular dynamics simulations. In this chapter, we present a versatile yet robust analysis pipeline that can be easily implemented and adapted for a wide range of lipid compositions. It employs Gaussian-based Hidden Markov Models to predict the hidden order states of individual lipids by describing their structure through the area per lipid and the average SCC order parameters per acyl chain. Regions of the membrane with a high correlation between ordered lipids are identified by employing the Getis-Ord local spatial autocorrelation statistic on a Voronoi tessellation of the lipids. As an example, the approach is applied to two distinct systems at a coarse-grained resolution, demonstrating either a strong tendency towards phase separation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DIPC), cholesterol) or a weak tendency toward phase separation (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PUPC), cholesterol). Explanations of the steps are complemented by coding examples written in Python, providing both a comprehensive understanding and practical guidance for a seamless integration of the workflow into individual projects.

The synergy between hyaluronic acid (HA) and lipid molecules plays a crucial role in synovial fluids, cell coatings, etc. Diseased cells in cancer and arthritis show changes in HA concentration and chain size, impacting the viscoelastic and mechanical properties of the cells. Although the solution behavior of HA is known in experiments, a molecular-level understanding of the role of HA in the dynamics at the interface of HA-water and the cellular boundary is lacking. Here, we perform atomistic molecular dynamics simulation of short HA chains in an explicit water solvent in the presence of a DPPC bilayer, relevant in pathological cases. We identify a stable interface between HA-water and the bilayer where the water molecules are in contact with the bilayer and the HA chains are located away without any direct contact. Both translation and rotation of the interfacial waters in contact with the lipid bilayer and translation of the HA chains exhibit subdiffusive behavior. The diffusive behavior sets in slightly away from the bilayer, where the diffusion coefficients of water and HA decrease monotonically with increase in HA concentration. On the contrary, the dependence on HA chain size is only marginal due to enhanced chain flexibility as their size increases.

We have studied the effect of calcium ions (Ca2+) at various concentrations on the structure of lipid vesicles in the presence of amyloid-beta peptide Aβ(25-35). In particular, we have investigated the influence of calcium ions on the formation of recently documented bicelle-like structures (BLSs) emerged as a result of Aβ(25-35) triggered membrane disintegration. First, we have shown by using small-angle X-ray and neutron scattering that peptide molecules rigidify the lipid bilayer of gel phase DPPC unilamellar vesicles (ULVs), while addition of the calcium ions to the system hinders this effect of Aβ(25-35). Secondly, the Aβ(25-35) demonstrates a critical peptide concentration at which the BLSs reorganize from ULVs due to heating and cooling the samples through the lipid main phase transition temperature (Tm). However, addition of calcium ions does not affect noticeably the Aβ-induced formation of BLSs and their structural parameters, though the changes in peptide\'s secondary structure, e.g. the increased α-helix fraction, has been registered by circular dichroism spectroscopy. Finally, according to 31P nuclear magnetic resonance (NMR) measurements, calcium ions do not affect the lipid-peptide arrangement in BLSs and their ability to align in the magnetic field of NMR spectrometer. The influences of various concentrations of calcium ions on the lipid-peptide interactions may prove biologically important because their local concentrations vary widely in in-vivo conditions. In the present work, calcium ions were investigated as a possible tool aimed at regulating the lipid-peptide interactions that demonstrated the disruptive effect of Aβ(25-35) on lipid membranes.

The transformation of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers from the gel (Lβ\') to the fluid (Lα) phase involves an intermediate ripple (Pβ\') phase forming a few degrees below the main transition temperature (Tm). While the exact cause of bilayer rippling is still debated, the presence of amphiphilic molecules, pH, and lipid bilayer architecture are all known to influence (pre)transition behavior. In particular, fatty acid chains interact with hydrophobic lipid tails, while the carboxylic groups simultaneously participate in proton transfer with interfacial water in the polar lipid region which is controlled by the pH of the surrounding aqueous medium. The molecular-level variations in the DPPC ripple phase in the presence of 2% palmitic acid (PA) were studied at pH levels 4.0, 7.3, and 9.1, where PA is fully protonated, partially protonated, or fully deprotonated. Bilayer thermotropic behavior was investigated by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy which agreed in their characterization of (pre)transition at pH of 9.1, but not at pH 4.0 and especially not at 7.3. Owing to the different insertion depths of protonated and deprotonated PA, along with the ability of protonated PA to undergo flip-flop in the bilayer, these two forms of PA show a different hydration pattern in the interfacial water layer. Finally, these results demonstrated the hitherto undiscovered potential of FTIR spectroscopy in the detection of the events occurring at the surface of lipid bilayers that obscure the low-cooperativity phase transition explored in this work.

Pulmonary surfactant forms a thin film on the liquid that lines the alveolar air-sacks. When compressed by the decreasing alveolar surface area during exhalation, the films avoid collapse from the air/water interface and reduce surface tension to exceptionally low levels. To define better the structure of compressed films that determines their susceptibility to collapse, we measured how cholesterol affects the structure and collapse of dipalmitoyl phosphatidylcholine (DPPC) monolayers at physiological temperatures. Grazing incidence X-ray diffraction (GIXD) and grazing incidence X-ray off-specular scattering (GIXOS) established the lateral and transverse structures of films on a Langmuir trough at a surface pressure of 45 mN m-1, just below the equilibrium spreading pressure at which collapse begins. Experiments with captive bubbles at a surface pressure of 51 mN m-1 measured how the steroid affects isobaric collapse. Mol fractions of the steroid (Xchol) at 0.05 removed the tilt by the acyl chains of DPPC, shifted the unit cell from centered rectangular to hexagonal, and dramatically decreased the long-range order. Higher Xchol produced no further change in diffraction, suggesting that cholesterol partitions into a coexisting disordered phase. Cholesterol had minimal effect on rates of collapse until Xchol reached 0.20. Our results demonstrate that the decreased coherence length, indicating conversion of positional order to short-range, is insufficient to make a condensed monolayer susceptible to collapse. Our findings suggest a two-step process by which cholesterol induces disorder. The steroid would first convert the film with crystalline chains to a hexatic phase before generating a fully disordered structure that is susceptible to collapse. These results lead to far-reaching consequences for formulation of animal-derived therapeutic surfactants. Our results suggest that removal of cholesterol from these preparations should be unnecessary below Xchol = 0.20.