Protein-Liposome Interactions Research Articles

Protein-liposome interactions: the impact of surface charge and fluidisation effect on protein binding

At the dawn of a new nanotechnological era in the pharmaceutical field, it is very important to examine and understand all the aspects that influence in vivo behaviour of nanoparticles. In this point of view, the interactions between serum proteins and liposomes with incorporated anionic, cationic, and/or PEGylated lipids were investigated to elucidate the role of surface charge and bilayer fluidity in protein corona’s formation. 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) liposomes with the presence or absence of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (chloride salt) (DOTAP), and/or 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DPPE-PEG 5000) lipids were prepared by the thin-film hydration method. The evaluation of their biophysical characteristics was enabled by differential scanning calorimetry and dynamic and electrophoretic light scattering. The physicochemical characteristics of mixed liposomes were compared before and after exposure to foetal bovine serum (FBS) and were correlated to calorimetric data. Our results indicate protein binding to all liposomal formulations. However, it is highlighted the importance of surface charge and fluidisation effect to the extent of protein adsorption. Additionally, considering the extensive use of cationic lipids for innovative delivery platforms, we deem PEGylation a key parameter, because even in a small proportion can reduce protein binding, and thus fast clearance and extreme toxicity without affecting positive charge. This study is a continuation of our previous work about protein-liposome interactions and fraction of stealthiness (Fs) parameter, and hopefully a design road map for drug and gene delivery.

Evaluating the interaction of human serum albumin (HSA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes in different aqueous environments using anisotropy resolved multi-dimensional emission spectroscopy (ARMES)

Studying the interaction between plasma proteins and liposomes is critical, particularly for their use as drug delivery systems. Here, the efficacy of anisotropy resolved multidimensional emission spectroscopy (ARMES) for investigating the interaction of human serum albumin (HSA) with liposomes was explored and compared to conventional spectroscopic techniques. Dynamic Light Scattering (DLS) and absorbance spectroscopy (with Multivariate Curve Resolution (MCR) modeling) indicated that the highest degree of liposome rupturing, and aggregation occurred in water, with less in ammonium bicarbonate buffer (ABC) and phosphate buffered saline (PBS). Fluorescence emission spectra of HSA-liposome mixtures revealed significant hypsochromic shifts for water and ABC, but much less in PBS, where the data suggests a non-penetrating protein layer was formed. Average fluorescence lifetimes decreased upon liposome interaction in water (6.2→5.2 ns) and ABC buffer (6.3→5.6 ns) but increased slightly for PBS (5.6→5.8 ns). ARMES using polarized Total Synchronous Fluorescence Scan measurements with parallel factor (PARAFAC) analysis resolved intrinsic HSA fluorescence into two components for interactions in water and ABC buffer, but only one component for PBS. These components, in water and ABC buffer, corresponded to two different HSA populations, one blue-shifted and penetrating the liposomes (λex/em = ~ 280/320 nm) and a second, similar to free HSA in solution (λex/em = ~ 282/356 nm). PARAFAC scores for water and ABC buffer suggested that a large proportion of HSA interacted in an end on configuration. ARMES provides a new way for investigating protein-liposome interactions that exploits the full intrinsic emission space of the protein and thus avoids the use of extrinsic labels. The use of multivariate data analysis provided a comprehensive and structured framework to extract a variety of useful information (resolving different fluorescent species, quantifying their signal contribution, and extracting light scatter signals) all of which can be used to discriminate between interaction mechanisms.

DMPC Phospholipid Bilayer as a Potential Interface for Human Cystatin C Oligomerization: Analysis of Protein-Liposome Interactions Using NMR Spectroscopy.

Studies revolving around mechanisms responsible for the development of amyloid-based diseases lay the foundations for the recognition of molecular targets of future to-be-developed treatments. However, the vast number of peptides and proteins known to be responsible for fibril formation, combined with their complexity and complexity of their interactions with various cellular components, renders this task extremely difficult and time-consuming. One of these proteins, human cystatin C (hCC), is a well-known and studied cysteine-protease inhibitor. While being a monomer in physiological conditions, under the necessary stimulus—usually a mutation, it tends to form fibrils, which later participate in the disease development. This process can potentially be regulated (in several ways) by many cellular components and it is being hypothesized that the cell membrane might play a key role in the oligomerization pathway. Studies involving cell membranes pose several difficulties; therefore, an alternative in the form of membrane mimetics is a very attractive solution. Here, we would like to present the first study on hCC oligomerization under the influence of phospholipid liposomes, acting as a membrane mimetic. The protein–mimetic interactions are studied utilizing circular dichroism, nuclear magnetic resonance, and size exclusion chromatography.

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (Fs), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when Fs is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.

Membrane-Protein-Hydration Interaction of α-Synuclein with Anionic Vesicles Probed via Angle-Resolved Second-Harmonic Scattering.

Amyloid formation of the protein α-synuclein promotes neurodegeneration in Parkinson's disease. The normal function of α-synuclein includes synaptic vesicle transport and fusion, and the protein binds strongly to negatively charged vesicles in vitro. Here, we demonstrate that nonresonant angle-resolved second-harmonic scattering detects α-synuclein binding to liposomes through changes in water orientational correlations and can thus be used as a high-accuracy and high-throughput label-free probe of protein-liposome interactions. The obtained results support a binding model in which the N-terminus of α-synuclein adopts an α-helical conformation that lies flat on the vesicle surface while the negatively charged C-terminus remains in solution.

ProLIF - quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes.

Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719).

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes.

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.

Protein-liposome interactions and their relevance to the structure and function of cell membranes

Recent studies on the interactions of soluble proteins, membrane proteins and enzymes with phospholipid model membranes are reviewed. Similarities between the properties of such systems and the behavior of biomembranes, such as alterations in the redox potential of cytochrome c after binding to membranes and effects of phospholipid fluidity on (Na+K) ATPase activity, are emphasized. The degree of correspondence between the behavior of model systems and natural membranes encourages the continuing use of model membranes in studies on protein-lipid interactions. However, some of the data on the increase of surface pressure of phospholipid monolayers by proteins and increases in the permeability of liposomes indicate that many soluble proteins also have a capability to interact hydrophobically with phospholipids. Thus a sharp distinction between both peripheral and integral membrane proteins and non-membrane proteins are not seen by these techniques. Cautious use of such studies, however, should lead to greater understanding of the molecular basis of cell membrane structure and function in normal and pathological states. Studies implicating protein-lipid interactions and (Na+K) ATPase activity in membrane alterations in disease states are also briefly discussed.