Impact Of Lipid Composition Research Articles

Impact of Lipid Composition on Vesicle Protein Adsorption: A BSA Case Study.

Investigating the interaction between liposomes and proteins is of paramount importance in the development of liposomal formulations with real potential for bench-to-bedside transfer. Upon entering the body, proteins are immediately adsorbed on the liposomal surface, changing the nanovehicles' biological identity, which has a significant impact on their biodistribution and pharmacokinetics and ultimately on their therapeutic effect. Albumin is the most abundant plasma protein and thus usually adsorbs immediately on the liposomal surface. We herein report a comprehensive investigation on the adsorption of model protein bovine serum albumin (BSA) onto liposomal vesicles containing the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), in combination with either cholesterol (CHOL) or the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). While many studies regarding protein adsorption on the surface of liposomes with different compositions have been performed, to the best of our knowledge, the differential responses of CHOL and DOTAP upon albumin adsorption on vesicles have not yet been investigated. UV-vis spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a strong influence of the phospholipid membrane composition on protein adsorption. Hence, it was found that DOTAP-containing vesicles adsorb proteins more robustly but also aggregate in the presence of BSA, as confirmed by DLS and TEM. Separation of liposome-protein complexes from unadsorbed proteins performed by means of centrifugation and size exclusion chromatography (SEC) was also investigated. Our results show that neither method can be regarded as a golden experimental setup to study the protein corona of liposomes. Yet, SEC proved to be more successful in the separation of unbound proteins, although the amount of lipid loss upon liposome elution was higher than expected. In addition, coarse-grained molecular dynamics simulations were employed to ascertain key membrane parameters, such as the membrane thickness and area per lipid. Overall, this study highlights the importance of surface charge and membrane fluidity in influencing the extent of protein adsorption. We hope that our investigation will be a valuable contribution to better understanding protein-vesicle interactions for improved nanocarrier design.

Changes in lipid composition of host-derived extracellular vesicles following Salmonella infection.

This study delves into the previously unexplored territory of extracellular vesicle (EV) cargo and composition, specifically focusing on lipid composition changes in EVs following Salmonella infection. EVs play crucial roles in intercellular communication, carrying a variety of biomolecules. Investigating how these EV cargo lipids change post-infection with Salmonella is significant for understanding the molecular mechanisms underlying lipid trafficking during infection. Given the impact of lipid composition on EV function, this research uncovers distinct differences in the lipid profiles of EVs at different time points post-infection and between infected and uninfected macrophages. This study identified lipids that are differentially abundant in EVs produced by the host during infection, offering novel insights into the dynamics of lipid profiles in EVs during cellular processes and infections. This work advances our understanding of host-pathogen interactions, specifically lipid-mediated EV functions during infection.

Specific Tumor Localization of Immunogenic Lipid-Coated Mesoporous Silica Nanoparticles following Intraperitoneal Administration in a Mouse Model of Serous Epithelial Ovarian Cancer.

Immunogenic lipid-coated mesoporous silica nanoparticles (ILM) present pathogen-associated molecular patterns (PAMPs) on the nanoparticle surface to engage pathogen-associated receptors on immune cells. The mesoporous core is capable of loading additional immunogens, antigens or drugs. In this study, the impact of lipid composition, surface potential and intercalation of lipophilic monophosphoryl lipid A (MPL-A) in the lipid coat on nanoparticle properties and cellular interactions is presented. Loading and retention of the model antigen ovalbumin into the mesoporous silica core were found to be similar for all nanoparticle formulations, with presentation of ova peptide (SIINFEKL) by major histocompatibility complex (MHC) evaluated to facilitate the selection of an anionic nanoparticle composition. ILM were able to induce lysosomal tubulation and streaming of lysosomes towards the cell surface in dendritic cells, leading to an enhanced surface presentation of MHC. Myeloid cells robustly internalized all ILM formulations; however, non-myeloid cells selectively internalized cationic ILM in vitro in the presence of 20% serum. Interestingly, ILM administration to the peritoneal cavity of mice with disseminated ovarian cancer resulted in selective accumulation of ILM in tumor-associated tissues (>80%), regardless of nanoparticle surface charge or the presence of MPL-A. Immunofluorescence analysis of the omental tumor showed that ILMs, regardless of surface charge, were localized within clusters of CD11b+ myeloid cells 24 h post administration. Selective uptake of ILMs by myeloid cells in vivo indicates that these cells outcompete other cell populations in the ovarian tumor microenvironment, making them a strong target for therapeutic interventions.

Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Lipid-Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells: Impact of Lipid Composition on Performance in Different Media.

To combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid-polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20-40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)-LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles' performance in supplemented media.

How bilayer properties influence membrane protein folding.

The question of how proteins manage to organize into a unique three-dimensional structure has been a major field of study since the first protein structures were determined. For membrane proteins, the question is made more complex because, unlike water-soluble proteins, the solvent is not homogenous or even unique. Each cell and organelle has a distinct lipid composition that can change in response to environmental stimuli. Thus, the study of membrane protein folding requires not only understanding how the unfolded chain navigates its way to the folded state, but also how changes in bilayer properties can affect that search. Here we review what we know so far about the impact of lipid composition on bilayer physical properties and how those properties can affect folding. A better understanding of the lipid bilayer and its effects on membrane protein folding is not only important for a theoretical understanding of the folding process, but can also have a practical impact on our ability to work with and design membrane proteins.

Impact of lipid composition and muscle microstructure on myoglobin-mediated lipid oxidation in washed cod and pig muscle

The roles of lipid oxidation substrates and muscle microstructure in lipid oxidation were investigated in two muscle models (cod and pig). Added myoglobin (Mb) promoted lipid oxidation in washed cod muscle (WCM) but not in washed pig muscle (WPM). The differing microstructure of WCM e.g. more exposed fat cells or membrane of muscle cells compared to the “denseness” or “wrapped” structure of WPM, may have contributed to the better ability of Mb to facilitate lipid oxidation in the WCM. Added phospholipids with polyenoic indexes of 282 and 24 activated Mb as an oxidant similarly in WPM while added neutral lipids and added free fatty acids had little effect. It is suggested that muscle microstructure and accessibility of Mb to phospholipids play critical roles in relation to Mb-mediated lipid oxidation while the degree of unsaturation in the phospholipids was less important.

Impact of lipid composition and photosensitizer hydrophobicity on the efficiency of light-triggered liposomal release.

Photo-triggerable liposomes are considered nowadays as promising drug delivery devices due to their potential to release encapsulated drugs in a spatial and temporal manner. In this work, we have investigated the photopermeation efficiency of three photosensitizers (PSs), namely verteporfin, pheophorbide a and m-THPP when incorporated into liposomes with well-defined lipid compositions (SOPC, DOPC or SLPC). By changing the nature of phospholipids and PSs, the illumination of the studied systems was shown to significantly alter their lipid bilayer properties via the formation of lipid peroxides. The system efficiency depends on the PS/phospholipid association, and the ability of the PS to peroxidize acyl chains. Our results demonstrated the possible use of these three clinically approved (or under investigation) PSs as potential candidates for photo-triggerable liposome conception.

Impact of Lipid Composition and Receptor Conformation on the Spatio-temporal Organization of μ-Opioid Receptors in a Multi-component Plasma Membrane Model.

The lipid composition of cell membranes has increasingly been recognized as playing an important role in the function of various membrane proteins, including G Protein-Coupled Receptors (GPCRs). For instance, experimental and computational evidence has pointed to lipids influencing receptor oligomerization directly, by physically interacting with the receptor, and/or indirectly, by altering the bulk properties of the membrane. While the exact role of oligomerization in the function of class A GPCRs such as the μ-opioid receptor (MOR) is still unclear, insight as to how these receptors oligomerize and the relevance of the lipid environment to this phenomenon is crucial to our understanding of receptor function. To examine the effect of lipids and different MOR conformations on receptor oligomerization we carried out extensive coarse-grained molecular dynamics simulations of crystal structures of inactive and/or activated MOR embedded in an idealized mammalian plasma membrane composed of 63 lipid types asymmetrically distributed across the two leaflets. The results of these simulations point, for the first time, to specific direct and indirect effects of the lipids, as well as the receptor conformation, on the spatio-temporal organization of MOR in the plasma membrane. While sphingomyelin-rich, high-order lipid regions near certain transmembrane (TM) helices of MOR induce an effective long-range attractive force on individual protomers, both long-range lipid order and interface formation are found to be conformation dependent, with a larger number of different interfaces formed by inactive MOR compared to active MOR.

In vitro performance of lipid-PLGA hybrid nanoparticles as an antigen delivery system: lipid composition matters.

Due to the many beneficial properties combined from both poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and liposomes, lipid-PLGA hybrid NPs have been intensively studied as cancer drug delivery systems, bio-imaging agent carriers, as well as antigen delivery vehicles. However, the impact of lipid composition on the performance of lipid-PLGA hybrid NPs as a delivery system has not been well investigated. In this study, the influence of lipid composition on the stability of the hybrid NPs and in vitro antigen release from NPs under different conditions was examined. The uptake of hybrid NPs with various surface charges by dendritic cells (DCs) was carefully studied. The results showed that PLGA NPs enveloped by a lipid shell with more positive surface charges could improve the stability of the hybrid NPs, enable better controlled release of antigens encapsulated in PLGA NPs, as well as enhance uptake of NPs by DC.

Thermodynamics of Membrane Partitioning and Self-association of Transmembrane Helices: Impact of Lipid Composition

Membrane partitioning and self-association of transmembrane helices (TMHs) have been extensively investigated using model TMHs to elucidate the driving forces of membrane protein folding. We designed the inert hydrophobic model TMH (AALALAA)3 to obtain thermodynamic parameters related to the steps. The formation of the antiparallel dimer was detected by fluorescence resonance energy transfer between fluorescent labeled peptides [1]. Stronger dimerization was observed in thicker membranes and at lower temperatures (ΔG = −9 – −26 kJ mol−1), driven by large negative ΔH values (−18 – −80 kJ mol−1). The enthalpy changes for helix-helix interaction can be well explained by electrostatic interaction between helix macrodipoles in different dielectric environments. The incorporation of cholesterol and PE also facilitated the dimerization by large negative ΔH values. The partitioning process was also investigated based on the transfer of the helix between vesicles [2]. Under hydrophobic mismatch conditions up to ∼7 Å, the helix partitioning became unfavorable up to +7 kJ mol−1, hampered by an increase in entropic (up to +20 kJ mol−1) and enthalpic (up to +66 kJ mol−1) terms in thinner and thicker membranes, respectively. The obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, respectively. I also present a design of a water-soluble TMH for the direct measurement of the partitioning process. Furthermore, I will introduce a novel method for quick fluorescent labeling of membrane proteins to detect TMH interactions in living cell membranes [3]. [1] Y. Yano and K. Matsuzaki, Biochemistry 45, 3370 (2006). [2] Y. Yano et al., Biochemistry 45, 3379 (2006). [3] Y. Yano et al., ACS Chem. Biol. 6, 341 (2008).