Small Lipid Vesicles Research Articles

Exploring the functionality of fluorescent liposomes in cancer: diagnosis and therapy

Abstract Fluorescent liposomes are pivotal in cancer research, serving as adaptable vehicles for imaging and therapeutics. These small lipid vesicles, capable of encapsulating fluorescent dyes, offer precise visualization and monitoring of their targeted delivery to cancer cells. This review delves into the critical role fluorescent liposomes play in enhancing both cancer diagnosis and treatment. It provides an in-depth analysis of their structural features, fluorescent labeling techniques, targeting strategies, and the challenges and opportunities they present. In the domain of cancer diagnosis, the article sheds light on various imaging modalities enabled by fluorescent liposomes, including fluorescence imaging and multimodal techniques. Emphasis is placed on early detection strategies, exhibiting the utility of targeted contrast agents and biomarker recognition for enhanced diagnostic precision. Moving on to cancer treatment, the review discusses the sophisticated drug delivery mechanisms facilitated by fluorescent liposomes, focusing on chemotherapy and photodynamic therapy. Moreover, the exploration extends to targeted therapy, explaining the applications of fluorescent liposomes in gene delivery and RNA interference. In a nutshell, his article comprehensively explores the multifaceted impact of fluorescent liposomes on advancing cancer diagnosis and treatment, combining existing knowledge with emerging trends.

Research progress in the treatment of cardiovascular diseases with extracellular vesicles

Extracellular vehicles (EVs) are small lipid vesicles that can release signaling substances containing proteins, nucleic acids, and other bioactive molecules. Recent research shows that EVs have potential application prospects in the treatment of cardiovascular diseases. EVs can alter the function of damaged cells and promote the repair and regeneration of cardiovascular tissue by transmitting bioactive molecules. In addition, EVs have the characteristics of low immunogenicity and high stability, making it a potential therapeutic strategy for cardiovascular diseases. Although the current research is still in the pre-clinical stage, with the further revelation of the mechanism of cardiovascular disease treatment using EVs, the treatment of cardiovascular diseases by EVs is bound to enter the clinical stage. This review expounds the advantages of EVs in cardiovascular diseases, and systematically lists the current application progress of EVs in a variety of cardiovascular diseases.

How Microbubble-Enhanced Shock Waves Promote the Delivery of Lipid-siRNA across Neuronal Plasma Membrane: A Computational Study.

In this computational study, we examine the potential of microbubble-enhanced shock waves to improve the delivery of lipid-siRNA nanoparticles across neuronal plasma membranes with the ultimate aim of enhancing brain tumor treatment. We critically evaluate several variables related to experiments, including the bubble size, the shock speed and action time, and the amount of siRNA encapsulated in the liposome. Our findings reveal that microbubble-enhanced shock waves are essential for the high delivery of small lipid vesicles (under 30 nm diameter); its corresponding variables significantly impact drug penetration and absorption rates and influence the overall efficacy of the drug delivery system. Long-time recovery simulations further provide valuable insights into the self-healing ability of the plasma membrane following shock wave exposure and the subsequent absorption dynamics of siRNA. This work provides the dynamic process of siRNA released from lipid vesicles with shock wave and nanobubbles, thereby serving as a molecular mechanism support for developing tunable delivery systems for RNA-based therapy in brain tumors.

Biologically Representative Lipid-Coated Gold Nanoparticles and Phospholipid Vesicles for the Study of Alpha-Synuclein/Membrane Interactions.

Alpha-synuclein is an intrinsically disordered protein whose formation of beta-sheet-rich protein aggregates in the brain is implicated in the development of Parkinson's disease. Due to its believed role in synaptic vesicle trafficking and neurotransmission, many studies have employed simple, synthetic model systems to investigate alpha-synuclein/membrane interactions in an attempt to gain a better understanding of the protein's native and pathogenic functions. Interestingly, these studies seem to suggest that alpha-synuclein interacts differently with rigid vesicle mimics in comparison to malleable vesicle mimics. However, the use of different mimic sizes and surface chemistries across existing studies makes it challenging to directly compare the effects of membrane mechanical properties on protein behavior observed thus far. In this work, we developed a synaptic vesicle mimic library comprising a range of both malleable and rigid synaptic vesicle mimics possessing the same size and biologically representative lipid surface chemistry. Limited proteolysis mass spectrometry experiments revealed distinct fragmentation patterns between rigid and malleable synaptic vesicle mimics. The N-terminal and C-terminal regions of alpha-synuclein were found to become less solvent-accessible upon binding to all synaptic vesicle mimics. Nevertheless, minor variations in digestion pattern were observed in the central region of the protein dependent upon mimic size, rigidity, and lipid composition. Higher binding affinities were observed for alpha-synuclein binding to rigid synaptic vesicle mimics compared to malleable synaptic vesicle mimics. Additionally, the binding affinity of alpha-synuclein toward small lipid vesicles and small lipid-coated gold nanoparticles without cholesterol was found to be lower than that of their respective malleable and rigid counterparts. Interestingly, the binding curves for the rigid synaptic vesicle mimics demonstrated a nontraditional peak and dip shape believed to arise from differences in alpha-synuclein orientation on the particle surface at different protein-to-particle incubation ratios.

Cellular communication through extracellular vesicles and lipid droplets

AbstractCellular communication is essential for effective coordination of biological processes. One major form of intercellular communication occurs via the release of extracellular vesicles (EVs). These vesicles mediate intercellular communication through the transfer of their cargo and are actively explored for their role in various diseases and their potential therapeutic and diagnostic applications. Conversely, lipid droplets (LDs) are vesicles that transfer cargo within cells. Lipid droplets play roles in various diseases and evidence for their ability to transfer cargo between cells is emerging. To date, there has been little interdisciplinary research looking at the similarities and interactions between these two classes of small lipid vesicles. This review will compare the commonalities and differences between EVs and LDs including their biogenesis and secretion, isolation and characterisation methodologies, composition, and general heterogeneity and discuss challenges and opportunities in both fields.

Confinement effect on hydrolysis in small lipid vesicles.

In living organisms most chemical reactions take place within the confines of lipid-membrane bound compartments, while confinement within the bounds of a lipid membrane is thought to be a key step in abiogenesis. In previous work we demonstrated that confinement in the aqueous cavity of a lipid vesicle affords protection against hydrolysis, a phenomenon that we term here confinement effect (C e) and that we attributed to the interaction with the lipid membrane. Here, we show that both the size and the shape of the cavity of the vesicle modulate the C e. We link this observation to the packing of the lipid following changes in membrane curvature, and formulate a mathematical model that relates the C e to the radius of a spherical vesicle and the packing parameter of the lipids. These results suggest that the shape of the compartment where a molecule is located plays a major role in controlling the chemical reactivity of non-enzymatic reactions. Moreover, the mathematical treatment we propose offers a useful tool for the design of vesicles with predictable reaction rates of the confined molecules, e.g., drug delivery vesicles with confined prodrugs. The results also show that a crude form of signal transduction, devoid of complex biological machinery, can be achieved by any external stimuli that drastically changes the structure of the membrane, like the osmotic shocks used in the present work.

The effect of cholesterol on highly curved membranes measured by nanosecond Fluorescence Correlation Spectroscopy

Small lipid vesicles (with diameter ≤100 nm) with their highly curved membranes comprise a special class of biological lipid bilayers. The mechanical properties of such membranes are critical for their function, e.g. exocytosis. Cholesterol is a near-universal regulator of membrane properties in animal cells. Yet measurements of the effect of cholesterol on the mechanical properties of membranes have remained challenging, and the interpretation of such measurements has remained a matter of debate. Here we show that nanosecond fluorescence correlation spectroscopy (FCS) can directly measure the ns-microsecond rotational correlation time (τ r) of a lipid probe in high curvature vesicles with extraordinary sensitivity. Using a home-built 4-Pi fluorescence cross-correlation spectrometer containing polarization-modulating elements, we measure the rotational correlation time (τ r) of Nile Red in neurotransmitter vesicle mimics. As the cholesterol mole fraction increases from 0 to 50%, τ r increases from 17 ± 1 to 112 ± 12 ns, indicating a viscosity change of nearly a factor of 7. These measurements are corroborated by solid-state NMR results, which show that the order parameter of the lipid acyl chains increases by about 50% for the same change in cholesterol concentration. Additionally, we measured the spectral parameters of polarity-sensitive fluorescence dyes, which provide an indirect measure of viscosity. The green/red ratio of Nile Red and the generalized polarization of Laurdan show consistent increases of 1.3× and 2.6×, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and a wider dynamic range compared to other conventional techniques. Significantly, we observe that the viscosity of neurotransmitter vesicle mimics is remarkably sensitive to their cholesterol content.

Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo.

Simple SummaryExtracellular vesicles are vesicles produced by cells and organisms, capable of developing different functions. In this sense, these vesicles have been involved in communication, in the production of damage during an infection and in the modulation of different cell pathways. These vesicles have also been proposed as key players for the diagnosis and treatment of several diseases. In this study, we report for the first time the production of extracellular vesicles by Naegleria fowleri, the free-living amoeba that causes primary amoebic meningoencephalitis, a severe infection of the central nervous system that is fatal in more than 95% of cases. The vesicles were isolated and size, surface charge and protein content were analyzed. The report of the production of extracellular vesicles by this amoeba is relevant for different reasons: i. these vesicles could be implicated during the processes of invasion and damage to the brain; ii. they could influence the environment to trigger specific responses by cells and, iii. specific vesicles secreted by the amoeba could be employed for diagnostic purposes. Several studies to reveal the role of extracellular vesicles of this pathogen are being performed in our laboratory.Extracellular vesicles (EVs) are small lipid vesicles released by both prokaryotic and eukaryotic cells, involved in intercellular communication, immunomodulation and pathogenesis. In this study, we performed a characterization of the EVs produced by trophozoites of a clinical isolate of the free-living amoeba Naegleria fowleri (N. fowleri). Size distribution, zeta potential, protein profile and protease activity were analyzed. Under our incubation conditions, EVs of different sizes were observed, with a predominant population ranging from 206 to 227 nm. SDS-PAGE revealed protein bands of 25 to 260 KDa. The presence of antigenic proteins was confirmed by Western blot, which evidenced strongest recognition by rat polyclonal antibodies raised against N. fowleri in the region close to 80 KDa and included peptidases, as revealed by zymography. Proteins in selected immunorecognized bands were further identified using nano-ESI-MS/MS. A preliminary proteomic profile of the EVs identified at least 184 proteins as part of the vesicles’ cargo. Protease activity assays, in combination with the use of inhibitors, revealed the predominance of serine proteases. The present characterization uncovers the complexity of EVs produced by N. fowleri, suggesting their potential relevance in the release of virulence factors involved in pathogenicity. Owing to their cargo’s diversity, further research on EVs could reveal new therapeutic targets or biomarkers for developing rapid and accurate diagnostic tools for lethal infections such as the one caused by this amoeba.

Analysis of the Interaction between Giant Liposomes and Graphene Oxide

Recently, the interaction between graphene and biological membranes has been under intensive research because graphene and its related materials exhibit distinctive characteristics. For example, water-soluble graphene oxide (GO) is expected to be used as a carrier of drug delivery systems (DDS), but it is also known to have cytotoxicity. Thus, it is important to understand the interaction between lipid bilayers and graphene/GO in order to design molecular systems. Furthermore, these interactions have been studied mostly using small lipid vesicles (< 1 μm), although the size of biological cells is much greater. In this study, we studied the interaction between GO and cell-sized giant unilamellar vesicles (GUVs). We especially examined the effect of the lipid compositions, including neutral, negatively charged, and pyrene-grafted lipids. Pyrene is known to adhere to graphene with the non-covalent π-π stacking interaction. Microscope observations and flow cytometry measurement revealed that GUVs composed of lipids which have attractive interaction with GO rupture at a high probability.

Fibrotic extracellular matrix induces release of extracellular vesicles with pro-fibrotic miRNA from fibrocytes

RationaleExtracellular vesicles (EVs) are small lipid vesicles, and EV-coupled microRNAs (miRNAs) are important modulators of biological processes. Fibrocytes are circulating bone marrow-derived cells that migrate into the injured lungs and...

Interactions between Phase-Separated Liquids and Membrane Surfaces.

Liquid-liquid phase separation has recently emerged as an important fundamental organizational phenomenon in biological settings. Most studies of biological phase separation have focused on droplets that “condense” from solution above a critical concentration, forming so-called “membraneless organelles” suspended in solution. However, membranes are ubiquitous throughout cells, and many biomolecular condensates interact with membrane surfaces. Such membrane-associated phase-separated systems range from clusters of integral or peripheral membrane proteins in the plane of the membrane to free, spherical droplets wetting membrane surfaces to droplets containing small lipid vesicles. In this review, we consider phase-separated liquids that interact with membrane surfaces and we discuss the consequences of those interactions. The physical properties of distinct liquid phases in contact with bilayers can reshape the membrane, and liquid-liquid phase separation can construct membrane-associated protein structures, modulate their function, and organize collections of lipid vesicles dynamically. We summarize the common phenomena that arise in these systems of liquid phases and membranes.

An intact membrane is essential for small extracellular vesicle‐induced modulation of α‐synuclein fibrillization

The misfolding and fibrillization of the protein, α‐synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on αsyn fibrillization using biologically‐derived vesicles. In this study, we comprehensively characterised the ability of lipid‐rich small extracellular vesicles (sEVs) to alter the misfolding of αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in αsyn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to αsyn misfolding in the synucleinopathy disorders.

Action of antimicrobial peptides and cell-penetrating peptides on membrane potential revealed by the single GUV method.

Membrane potential plays various key roles in live bacterial and eukaryotic cells. So far, the effects of membrane potential on action of antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) have been examined using cells and small lipid vesicles. However, due to the technical drawbacks of these experiments, the effect of membrane potential on the actions of AMPs and CPPs and the elementary processes of interactions of these peptides with cell membranes and vesicle membranes are not well understood. In this short review, we summarize the results of the effect of membrane potential on the action of an AMP, lactoferricin B (LfcinB), and a CPP, transportan 10 (TP10), in vesicle membranes revealed by the single giant unilamellar vesicle (GUV) method. Parts of the actions and their elementary steps of AMPs and CPPs interacting vesicle membranes under membrane potential are clearly revealed using the single GUV method. The experimental methods and their analysis described here can be used to elucidate the effects of membrane potential on various activities of peptides such as AMPs, CPPs, and proteins. Moreover, GUVs with membrane potential are more suitable as a model of cells or artificial cells, as well as GUVs containing small vesicles.

Exosomes from dairy cows of divergent fertility; Action on endometrial cells

Abnormalities in endometrial function contribute to poor fertility and reproductive failure. Exosomes are small lipid vesicles that contain transferable bioactive substances; they participate in intercellular signaling and may have critical roles in reproductive mechanisms, including endometrial remodeling in preparation for pregnancy. In this study, we evaluated the effects of exosomes from heifers with high and low genetic merit for fertility on inflammatory mediator expression by bovine endometrial epithelial and stromal cell lines. Co-incubation of exosomes from low, compared with high, fertility heifers upregulated the gene expression of pro-inflammatory IL1A and IL8 (CXCL8) but downregulated IL4 gene expression in epithelial cells. In contrast, stromal cells co-incubated with exosomes from low, compared with high, fertility heifers downregulated the gene expression of CXCL9, CXCL10, and CX3CL1. Our findings demonstrated that circulating exosomes from high fertility heifers did not alter endometrial inflammatory mediator gene expression. In contrast, circulating exosomes from low fertility heifers enhanced endometrial expression of inflammatory mediators, which may contribute to aberrant inflammation, leading to a reduced fertility in low fertility heifers. However, an in-depth investigation is required to elucidate the role of exosomes in regulating endometrial remodeling events required for enhanced reproductive performance and fertility in dairy cows.

Phase Separation of Synaptic Vesicles at the Nerve Terminal

Neuronal transmission relies on the sustained release of neurotransmitters from synaptic vesicles (SVs) upon depolarization of neurons. Nerve terminals contain hundreds of SVs that form tight clusters. Despite being held together, vesicles are highly mobile within clusters, so that they can be randomly recruited to the surface of the cell to release their content upon activation of the neuron. How this compact, yet the dynamic organization is achieved remained elusive. Several studies in the past decade showed that macromolecules may assemble into distinct liquid compartments not-limited by a membrane, a process known as liquid-liquid phase separation. We have discussed (Milovanovic and De Camilli, Neuron, 2017) how several features of SV clusters suggest that they may be part of a distinct liquid phase in the cytosol. For example, SV clusters have sharp boundaries, exclude other organelles, vesicles in these clusters are mobile, and SVs can be exchanged with vesicles outside the cluster. Thus, SV cluster may represent a distinct liquid phase in which one component of the phase are synaptic vesicles and the other component are proteins of the intervening matrix. Recently we showed that synapsin 1, a highly abundant synaptic protein, forms a distinct liquid phase in an aqueous environment (Milovanovic et al., Science, 2018). Synapsin 1 exchanges readily between the phase where is enriched and the surrounding medium. Additional synapsin 1 binding scaffolding proteins further modulate this phase but are not necessary for its formation. Importantly, synapsin 1 can capture small lipid vesicles into its phase. The phase of synapsin 1 rapidly disassembles upon phosphorylation by CaMKII, mimicking the dispersion of synapsin 1 that occurs at presynaptic boutons upon simulation. Thus, a minimal system of synapsin (with or without binding partners) may sequester lipid vesicles, forming a distinct liquid phase.

Imaging of human cells exposed to an antifungal antibiotic amphotericin B reveals the mechanisms associated with the drug toxicity and cell defence

Amphotericin B is an antibiotic used in pharmacotherapy of life-threatening mycotic infections. Unfortunately, the applicability of this antibiotic is associated with highly toxic side effects. In order to understand molecular mechanisms underlying toxicity of amphotericin B to patients, two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of the drug and imaged with the application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. The results of the cell viability assays confirm high toxicity of amphotericin B towards human cells. The images recorded demonstrate effective binding of amphotericin B to biomembranes. Analysis of the images reveals the operation of a defence mechanism based upon the elimination of molecules of the drug from living cells via formation of small amphotericin B-containing lipid vesicles. The fact that exosomes formed are devoid of cholesterol, as concluded on the basis of the results of Raman analysis, suggests that sequestration of sterols from the lipid phase of biomembranes is not a sole mechanism responsible for the toxic side effects of amphotericin B. Alternatively, the results imply that molecules of the drug present directly within the hydrophobic membrane core disturb the lipid membrane structure and affect their biological functions.

Extracellular Vesicles in Chagas Disease: A New Passenger for an Old Disease.

Extracellular vesicles (EVs) are small lipid vesicles released by prokaryotic and eukaryotic cells containing nucleic acids, proteins, and small metabolites essential for cellular communication. Depending on the targeted cell, EVs can act either locally or in distant tissues in a paracrine or endocrine cell signaling manner. Released EVs from virus-infected cells, bacteria, fungi, or parasites have been demonstrated to perform a pivotal role in a myriad of biochemical changes occurring in the host and pathogen, including the modulation the immune system. In the past few years, the biology of Trypanosoma cruzi EVs, as well as their role in innate immunity evasion, has been started to be unveiled. This review article will present findings on and provide a coherent understanding of the currently known mechanisms of action of T. cruzi-EVs and hypothesize the implication of these parasite components during the acute and chronic phases of Chagas disease.

Trefoil Factor 3 Deficiency Affects Liver Lipid Metabolism

Background/Aims: Tff3 protein plays a well recognized role in the protection of gastrointestinal mucosa. The role of Tff3 in the metabolism is a new aspect of its function. Tff3 is one of the most affected liver genes in early diabetes and fatty liver rodent models. The aim of this study was to investigate the effect of Tff3 deficiency on lipid and carbohydrate metabolism and on markers of oxidative stress that accompanies metabolic deregulation. Methods: Specific markers of health status were determined in sera of Tff3 deficient mice, including glucose level, functional glucose and insulin tolerance. Composition of fatty acids (FAs) was determined in liver and blood serum by using gas chromatography. Oxidative stress parameters were determined: lipid peroxidation level via determination of lipid hydroperoxide and thiobarbituric acid reactive substances (TBARS), antioxidative capacity (FRAP) and specific antioxidative enzyme activity. The expression of several genes and proteins related to the metabolism of lipids, carbohydrates and oxidative stress (CAT, GPx1, SOD2, PPARα, PPARγ, PPARδ, HNF4α and SIRT1) was determined. Results: Tff3 deficient mice showed better glucose utilization in the glucose and insulin test. Liver lipid metabolism is affected and increased formation of small lipid vesicles is noticed. Formation of lipid droplets is not accompanied by increased liver oxidative stress, although expression/activity of monitored enzymes is deregulated when compared with wild type mice. Tff3 deficient mice exhibit reduced expression of metabolism relevant SIRT1 and PPARγ genes. Conclusion: Tff3 deficiency affects the profile and accumulation of FAs in the liver, with no obvious oxidative stress increase, although expression/activity of monitored enzymes is changed as well as the level of SIRT1 and PPARγ protein. Considering the strong downregulation of liver Tff3 in diabetic/obese mice, presence in circulation and regulation by food/insulin, Tff3 is an interesting novel candidate in metabolism relevant conditions.

Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons.

Amyloid beta peptide (Aβ), the main component of senile plaques of Alzheimer's disease brains, is produced by sequential cleavage of amyloid precursor protein (APP) and of its C-terminal fragments (CTFs). An unanswered question is how amyloidogenic peptides spread throughout the brain during the course of the disease. Here, we show that small lipid vesicles called exosomes, secreted in the extracellular milieu by cortical neurons, carry endogenous APP and are strikingly enriched in CTF-α and the newly characterized CTF-η. Exosomes from N2a cells expressing human APP with the autosomal dominant Swedish mutation contain Aβ peptides as well as CTF-α and CTF-η, while those from cells expressing the non-mutated form of APP only contain CTF-α and CTF-η. APP and CTFs are sorted into a subset of exosomes which lack the tetraspanin CD63 and specifically bind to dendrites of neurons, unlike exosomes carrying CD63 which bind to both neurons and glial cells. Thus, neuroblastoma cells secrete distinct populations of exosomes carrying different cargoes and targeting specific cell types. APP-carrying exosomes can be endocytosed by receiving cells, allowing the processing of APP acquired by exosomes to give rise to the APP intracellular domain (AICD). Thus, our results show for the first time that neuronal exosomes may indeed act as vehicles for the intercellular transport of APP and its catabolites.

Preparation of liposomes: A comparative study between the double solvent displacement and the conventional ethanol injection—From laboratory scale to large scale

Here, we propose a new method for lipid vesicles preparation: double solvent displacement. Influence of various parameters on colloidal properties was studied to assess scale-up performances in comparison with ethanol injection. Lipid vesicles were prepared by double solvent displacement (DSD). The first one was performed in non-aqueous media, only with an ethanol phase containing phospholipids dispersed in PEG400. The second one consisted of a dispersion of the previous mixture in an aqueous phase containing glycerin. Our results showed that double solvent displacement could be able to produce large amount of small and homogeneous lipid vesicles. With such method, we could use higher phospholipids concentrations without significantly affecting vesicle colloidal properties compared to ethanol injection. Indeed, for concentrations higher than 12.5mg/ml, we observed a continuous size increase (from 129±54nm to 318±220nm) associated with a higher heterogeneity of the population whereas results remained stable for the DSD technique (size between 123±57nm and 141±67nm, respectively). DSD technique allowed preserving colloidal properties over a large range of solvent/non solvent (S/NS) ratios. Scale-up studies showed more pronounced impact with ethanol injection technique for which colloidal properties were more altered. Indeed, size decreased significantly by 11.8% compared to DSD for which the increase was just about 3.5%. The latter appeared to be more easily scalable than ethanol injection. The simplicity and the rapidity of this new technique enable us to produce liposomes with good proficiency of the process offering high yield while keeping economically attractive.