Surface-Bioengineered Extracellular Vesicles Seeking Molecular Biotargets in Lung Cancer Cells.

Personalized medicine is a new approach to modern oncology. Here, to facilitate the application of extracellular vesicles (EVs) derived from lung cancer cells as potent advanced therapy medicinal products in lung cancer, the EV membrane was functionalized with a specific ligand for targeting purposes. In this role, the most effective heptapeptide in binding to lung cancer cells (PTHTRWA) was used. The functionalization process of EV surface was performed through the C- or N-terminal end of the heptapeptide. To prove the activity of the EVs functionalized with PTHTRWA, both a model of lipid membrane mimicking normal and cancerous cell membranes as well as human adenocarcinomic alveolar basal epithelial cells (A549) and human normal bronchial epithelial cells (BEAS-2B) have been exposed to these bioconstructs. Magnetic resonance imaging (MRI) showed that the as-bioengineered PTHTRWA-EVs loaded with superparamagnetic iron oxide nanoparticle (SPIO) cargos reach the growing tumor when dosed intravenously in NUDE Balb/c mice bearing A549 cancer. Molecular dynamics (MD) in silico studies elucidated a high affinity of the synthesized peptide to the α5ß1 integrin. Preclinical safety assays did not evidence any cytotoxic or genotoxic effects of the PTHTRWA-bioengineered EVs.

Effect of Distigmasterol-Modified Acylglycerols on the Fluidity and Phase Transition of Lipid Model Membranes.

Plant sterols are known for their health-promoting effects, lowering blood cholesterol levels and alleviating cardiovascular disease. In this work, we continue our research on the asymmetric acylglycerols in which fatty acid residues are replaced by two stigmasterol residues in sn-1 and sn-2 or sn-2 and sn-3 positions as new thermostable carriers of phytosterols for their potential application in foods or as components of new liposomes in the pharmaceutical industry. The aim of this manuscript was to compare and analyze the effects of four distigmasterol-modified acylglycerols (dStigMAs) on the fluidity and the main phase transition temperature of the model phospholipid membrane. Their properties were determined using differential scanning calorimetry (DSC), steady-state fluorimetry and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR). The determination of the effect of the tested compounds on the mentioned physicochemical parameters of the model membranes will allow for the determination of their properties and stability, which is essential for their practical application. The results indicated that all compounds effect on the physicochemical properties of the model membrane. The degree of these changes depends on the structure of the compound, especially the type of linker by which stigmasterol is attached to the glycerol backbone, as well as on the type of hydrocarbon chain.

Influence of lipophilicity of anthracyclines on the interactions with cholesterol in the model cell membranes - Langmuir monolayer and SEIRAS studies.

The interactions of anthracyclines with biological membranes strongly depend on the drug lipophilicity, which might also determine the specific affinity to cholesterol molecules. Therefore, in this work we show the studies concerning the effect of two selected anthracyclines, daunorubicin (DNR) and idarubicin (IDA) on simple models of healthy (DMPC:Chol 7:3) and cancer cells membranes with increased level of cholesterol (DMPC:Chol 3:7) as well as pure cholesterol monolayers prepared at the air-water interface and supported on gold surface. It has been shown that more lipophilic IDA is able to penetrate cholesterol monolayers more effectively than DNR due to the formation of IDA-cholesterol arrangements at the interface, as proved by the thermodynamic analysis of compression-expansion cycles. The increased interactions of IDA were also confirmed by the time measurements of pre-compressed monolayers exposed to drug solutions as well as grazing incidence X-ray diffraction studies demonstrating differences in the 2D organization of cholesterol monolayers. Langmuir studies of mixed DMPC:Chol membranes revealed the reorganization of molecules in the cancer cell models at the air-water interface at higher surface pressures due to the removal of DNR, while increased affinity of IDA towards cholesterol allowed this drug to penetrate the layer more efficiently without its removal. The SEIRAS spectra obtained for supported DMPC:Chol bilayers proved that IDA locates both in the ester group and in the acyl chain region of the bilayer, while DNR does not penetrate the membranes as deeply as IDA. The increased penetration of the mixed phospholipid layers by idarubicin might be attributed to the higher lipophilicity caused by the lack of methoxy group and resulting in a specific affinity towards cholesterol.

Lipid membranes exposed to dispersions of phytantriol and monoolein cubosomes: Langmuir monolayer and HeLa cell membrane studies.

The interactions of liquid-crystalline nanoparticles based on lipid-like surfactants, glyceryl monooleate, monoolein (GMO) and 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane, phytantriol (PT) with selected model lipid membranes prepared by Langmuir technique were compared. Monolayers of DPPC, DMPS and their mixture DPPC:DMPS 87:13 mol% were used as simple models of one leaflet of a cell membrane. The incorporation of cubosomes into the lipid layers spread at the air-water interface was followed by surface-pressure measurements and Brewster angle microscopy. The cubosome - membrane interactions lead to the fluidization of the model membranes but this effect depended on the composition of the model membrane and on the type of cubosomes. The interactions of PT cubosomes with lipid layers, especially DMPS-based monolayer were stronger compared with those of GMO-based nanoparticles. The kinetics of incorporation of cubosomal material into the lipid layer was influenced by the extent of hydration of the polar headgroups of the lipid: faster in the case of smaller, less hydrated polar groups of DMPS than for strongly hydrated uncharged choline of DPPC. The membrane disrupting effect of cubosomes increased at longer times of the lipid membrane exposure to the cubosome solution and at larger carrier concentrations. Langmuir monolayer observations correspond well to results of studies of HeLa cells exposed to cubosomes. The larger toxicity of PT cubosomes was confirmed by MTS. Their ability to disrupt lipid membranes was imaged by confocal microscopy. On the other hand, PT cubosomes easily penetrated cellular membranes and released cargo into various cellular compartments more effectively than GMO-based nanocarriers. Therefore, at low concentrations, they may be further investigated as a promising drug delivery tool.

How do lipid nanocarriers - Cubosomes affect electrochemical properties of DMPC bilayers deposited on gold (111) electrodes?

Cubosome nanocarriers are promising biomimetic drug delivery systems used in particular for highly toxic drugs in cases where decreasing unwanted side effects is especially important. The properties of electrode supported lipid bilayer prepared by the combined Langmuir-Blodgett and Langmuir-Schaefer techniques were studied using electrochemical techniques following exposure of the film - covered electrode to a solution containing phytantriol - based cubosomes. The inclusion of the carrier in the model membrane under different experimental conditions was probed and the modifications induced in the lipid organization were for the first time inferred by quantitative analysis of the responses of cyclic voltammetry (CV), AC voltammetry and Electrochemical Impedance Spectroscopy (EIS) as well as blocking assays using a redox probe in the solution. Exposure of a preformed DMPC bilayer to cubosome solution resulted in the improved barrier properties of the film reflecting disintegration of cubosomes and formation of additional phytantriol/Pluronic F-108 polymer layer on the top of the DMPC bilayer. On the other hand, formation of the layer in the presence of cubosomes in the subphase lead to an increased capacitance of the film since penetration of the lipid layers by the cubosomal phytantriol increased the porosity of the film.

The influence of charge and lipophilicity of daunorubicin and idarubicin on their penetration of model biological membranes - Langmuir monolayer and electrochemical studies.

The interactions of two selected anthracyclines, daunorubicin (DNR) and idarubicin (IDA), with phospholipid monolayers used as simple models of cell membranes, were investigated. The results of Langmuir experiments together with Brewster angle microscopy showed that both drugs strongly affect cancer cell membranes composed of 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS). Electrostatic interactions allow positively charged DNR and IDA to interact with negatively charged DMPS polar heads but increased lipophilicity of IDA allows it to penetrate the layer more effectively than DNR and prevents from its expulsion at higher surface pressures. The analysis of the thermodynamical functions of hysteresis proves the presence of the enthalpically favorable interactions within the monolayer during its compression in the presence of idarubicin, which may form aggregates with DMPS molecules. The influence of the drugs was significantly less pronounced for a healthy cell model composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) due to the lack of strong electrostatic attractions. The interactions of drugs with pre-compressed phospholipid monolayers were also examined. The physical state of the monolayer and its packing determined only to some extent the penetration of anthracyclines. Since drug molecules first approach the polar region of the monolayer, the increase in surface pressure in time was more pronounced for negatively charged DMPS monolayers than for zwitterionic DMPC. Additionally, idarubicin was able to penetrate the precompressed DMPS monolayers more effectively than daunorubicin due to increased lipophilicity. This property of the drug was also responsible for IDA better penetration of hydrocarbon chains of supported DMPS monolayers compared to DNR, as shown by electrochemical studies.

Phytosphingosine, sphingosine and dihydrosphingosine ceramides in model skin lipid membranes: permeability and biophysics.

Ceramides based on phytosphingosine, sphingosine and dihydrosphingosine are essential constituents of the skin lipid barrier that protects the body from excessive water loss. The roles of the individual ceramide subclasses in regulating skin permeability and the reasons for C4-hydroxylation of these sphingolipids are not completely understood. We investigated the chain length-dependent effects of dihydroceramides, sphingosine ceramides (with C4-unsaturation) and phytoceramides (with C4-hydroxyl) on the permeability, lipid organization and thermotropic behavior of model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesteryl sulfate. Phytoceramides with very long C24 acyl chains increased the permeability of the model lipid membranes compared to dihydroceramides or sphingosine ceramides with the same chain lengths. Either unsaturation or C4-hydroxylation of dihydroceramides induced chain length-dependent increases in membrane permeability. Infrared spectroscopy showed that C4-hydroxylation of the sphingoid base decreased the relative ratio of orthorhombic chain packing in the membrane and lowered the miscibility of C24 phytoceramide with lignoceric acid. The phase separation in phytoceramide membranes was confirmed by X-ray diffraction. In contrast, phytoceramides formed strong hydrogen bonds and highly thermostable domains. Thus, the large heterogeneity in ceramide structures and in their aggregation mechanisms may confer resistance towards the heterogeneous external stressors that are constantly faced by the skin barrier.

Recent developments in methodology employed to study the interactions between nanomaterials and model lipid membranes.

With the boom of nanotechnology, nanomaterials (NMs) have been widely utilized in diverse applications, especially in biological and biomedical fields. Understanding how NMs interact with biomolecules, including proteins, DNA, and lipids, is of great importance for revealing the limitations posed and opportunities offered. Model lipid membrane, as a simplified cell membrane model, has been widely used to study the nanomaterial-lipid membrane interactions. In this article, current and emerging techniques, both experimental and theoretical, to investigate the interactions between NMs and model lipid membrane are summarized with each tool's capacities and limitations, along with future directions and challenges in this exciting area. This critical information will provide methodological guidance for researchers in this field.

pH dependence of daunorubicin interactions with model DMPC:Cholesterol membranes.

Mixed monolayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC):Cholesterol 7:3 prepared by the Langmuir-Blodgett method were used as model membranes to investigate the influence of the anticancer drug daunorubicin (DNR) on the properties of the lipid membrane. The Langmuir monolayer experiments revealed that drug - membrane interactions are pH-dependent. The changes in monolayer organization at subphases of pH 7.4 containing daunorubicin visualized by Brewster Angle Microscopy showed that in the presence of the drug the typical morphology observed for phospholipid layers containing cholesterol was no longer seen. It supports the explanation of the mechanism of the drug incorporation into the layers in terms of the competition between DNR molecules and cholesterol in the layer. Increasing surface pressure with time and increasing value of limiting surface pressure with increasing drug concentration in the subphase confirmed incorporation of the drug into the membranes. The interactions between the lipid monolayer and the drug at pH 5.4 were of electrostatic nature between the negative part of the DMPC molecule and positively charged drug, while at pH 7.4 contribution of interactions of daunorubicin with cholesterol was observed. Large differences of the surface-pressure vs. time plots were observed at both pH values when the DMPC:Cholesterol monolayer was not well organized yet. The voltammograms recorded for DMPC:Cholesterol monolayers transferred from the air-water interface onto gold electrode confirmed the presence of the drug in the lipid layer. Based on the charge of the oxidation-reduction peaks corresponding to the redox processes of quinone-hydroquinone group in daunorubicin, the initial surface concentration of the drug in the membrane and the drug release profile to the solution were evaluated.