Biomembranes evolved to protect cells and regulate exchange, forming a powerful barrier to large, charged macromolecules such as nucleic acids. In recent years, this paradigm has been competently overturned by soft biomaterials based on cell-penetrating peptides (CPPs). Herein, we investigate and compare the structural dynamics of peptiplexes formed between DNA and two cationic CPPs, TAT-HIV and NLS-SV40T. Combining experimental approaches and molecular dynamics (MD) simulations, we examined peptiplexes across mesoscopic scales to elucidate their supramolecular assembly and correlate these features with cellular uptake. We found that peptiplexes based on TAT-HIV exhibit greater structural flexibility, adopting ordered secondary structures and self-assembling into clusters and nanofibrils. In contrast, NLS-SV40T/DNA complexes retain random coil configurations, forming globule-studded coiled nanoassemblies with internal 2D hexagonal columnar phases. Calorimetry data indicated that TAT-HIV/DNA complexation is more favorable and exothermic, whereas NLS-SV40T binding to DNA is weaker and endothermic. MD simulations supported the experiments by showing that NLS-SV40T moves across DNA strands, settling into major grooves, whereas TAT-HIV bridges major and minor grooves via persistent arginine-mediated H-bonds and stronger energetics. Cell uptake assays showed that NLS-SV40T/DNA peptiplexes are internalized comparatively more efficiently, likely due to their more compact organization and lower lytic potential. Conversely, TAT-HIV induces membrane damage, as observed by atomic force microscopy, suggesting that its stronger electrostatics and enhanced H-bonding capacity may contribute to lytic activity. The findings presented here bring mechanistic insights into the structural landscape of peptiplexes, improving the rationale that supports the design of peptide-mediated gene delivery materials.

Membrane fusion is essential for signaling, cargo delivery, and synthetic cell growth, yet its mechanical consequences remain poorly defined. How fusion-driven membrane growth can be sustained without compromising compartment stability remains an unresolved challenge. Here, we established a minimal reconstituted system where content-loaded small liposomes fuse with single cell-sized giant unilamellar vesicles (GUVs), combining micropipette delivery, electrodeformation, and live imaging. Fusion outcomes were quantified through lipid and content mixing assays, GUV electrodeformation to track area and tension, and phase contrast imaging to monitor leakage. GUVs incorporated lipids and cargo from hundreds of thousands of vesicles at unprecedented efficiency rates, enabling substantial growth. However, accumulation of leaflet asymmetries induced curvature and tension, driving budding, rupture and leakage. Hemifusion amplified these destabilizing effects. Lipid number asymmetries emerge as a dominant mechanical cost of fusion, highlighting how cells may regulate these processes and guiding the design of therapeutic delivery systems and synthetic cells capable of robust and stable growth.

This chapter summarizes a spectrum of phenomena observed on model membranes exposed to electric fields. The considered model membrane system is giant unilamellar vesicles with sizes in the range of tens of microns. Because of their large size, the response of the membrane to electric fields can be directly visualized under the microscope. The membrane behavior is exemplified by several types of responses: First, the vesicles undergo morphological changes and adopt prolate, oblate, or spherocylindrical shapes. In general, the vesicle morphology depends on the conductivity conditions of the immersion and encapsulated solutions, and in the case of alternating fields – on the field frequency. Second, after switching the electric field off, these shapes can relax back to a sphere. The relaxation times depend on the initial membrane tension and on the reached transmembrane potential. Third, the vesicles can undergo topological changes such as formation of pores and, in the case of vesicles in contact, fusion. All these processes depend on the material characteristics of the membrane such as mechanical (bending rigidity and stretching elasticity), rheological (membrane shear surface viscosity), and electrical (capacitance) properties of the lipid bilayer. This chapter gives an overview of these properties and their dependence on the membrane phase state, and presents approaches for directly assessing them using giant unilamellar vesicles.

This study investigates how membrane composition governs the stability and fusogenic behavior of biomimetic hybrid liposomes composed of phosphatidylcholine (PC) and red blood cell (RBC) membrane fragments. While prior work on RBC-derived hybrid systems has largely focused on biological performance metrics such as circulation time, cellular uptake, and immune evasion, the biophysical and thermodynamic consequences of membrane hybridization at the molecular level remain poorly explored. Here, we address this gap through a systematic, multitechnique physicochemical characterization of a series of RBC-hybrid liposome formulations spanning a range of lipid:protein ratios. A comprehensive physicochemical characterization was performed using electron spin resonance (ESR) to probe membrane dynamics, microdifferential scanning calorimetry (microDSC) to assess thermotropic behavior, and isothermal titration calorimetry (ITC) to examine membrane energetics, solubilization, and intervesicular interactions. Incorporation of RBC membrane fragments increased membrane rigidity, as indicated by ESR and microDSC, and was associated with enhanced resistance to Triton X-100-induced solubilization, a finding attributed to compositional heterogeneity and protein-lipid organization introduced by membrane hybridization. ITC-based interaction assays further showed that cationic hybrid liposomes exhibited stronger fusion-associated signatures than their conventional counterparts, indicating that RBC membrane incorporation significantly alters interfacial membrane behavior in ways that cannot be explained by lipid composition alone. Together, these results show that RBC membrane content modulates both the structural stability and interaction profile of liposomes, establishing a biophysical framework for understanding how biomembrane-derived components reshape vesicle behavior and providing insights and useful design parameters for the development of robust biomimetic lipid-based delivery systems.

Abstract Biological membranes continuously experience leaflet lipid imbalances during growth, lipid synthesis, and vesicle fusion. To alleviate such imbalances and prevent these asymmetries from compromising membrane integrity, cells rely on lipid scramblases – fast and non-specific lipid channel proteins. Here we show that lipid number asymmetry alone is sufficient to drive spontaneous formation of transient hydrophilic pores that function as self-resealing lipid scramblases. Using giant unilamellar vesicles, living cells, and coarse-grained molecular dynamics simulations, we demonstrate that fusion-induced excess lipids in one leaflet lowers membrane edge tension, generating size-selective pores whose size and lifetime scale with the magnitude of asymmetry. Below a critical threshold, these pores reseal spontaneously; above it, membranes collapse. Strikingly, pore opening enables rapid, non-selective lipid translocation between leaflets, dissipating the asymmetry that nucleates the pore and thereby promoting their own closure. Cholesterol buffers moderate imbalances through spontaneous flip flop before pore formation, whereas pore-mediated lipid scrambling relieves the remaining asymmetry and restores cholesterol’s initial distribution. Our findings identify transient lipid pores as an intrinsic, protein-independent mechanism that couples membrane destabilization to self-repair, providing a universal physical principle for membrane homeostasis during growth, remodelling, and early cellular evolution.

Membrane solubilization by detergents is routinely performed to separate membrane components, and to extract and purify membrane proteins. This process depends both on the characteristics of the detergent and properties of the membrane. Here we investigate the interaction of eight detergents with very distinct physicochemical features with model membranes in different biologically relevant phases. The detergents chosen were the non-ionic Triton X-100, Triton X-165, C10E5, octyl glucopyranoside (OG) and dodecyl maltoside (DDM) and the ionic sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Chaps. Three lipid compositions were explored: pure palmitoyl oleoyl phosphatidylcholine (POPC), in the liquid-disordered (Ld) phase, sphingomyelin (SM)/cholesterol 7:3 (chol) in the liquid-ordered (Lo) phase and the biomimetic POPC/SM/chol 2:1:2, which might exhibit Lo/Ld phase separation. Turbidity measurements of small liposomes were performed along the titration with the detergents to obtain the overall solubilization profiles and optical microscopy of giant unilamellar vesicles (GUVs) was used to reveal the mechanism of interaction of the detergents. The presence of cholesterol renders the membranes partly/fully insoluble in all detergents, and the charged detergents are the least effective to solubilize POPC. The non-ionic detergents, with exception of DDM, with the bulkiest headgroup, caused a substantial increase in surface area of POPC, which was quantified directly on single GUVs. The other detergents induced mainly vesicle burst. Detergents that caused some increase in area induced Lo/Ld phase separation in the ternary mixture, with preferential solubilization of the latter. The insoluble area fraction left intact was quantified. Overall, the non-ionic detergents were the most effective in solubilizing lipid membranes.

Antimicrobial peptides (AMPs) are a primary defense against pathogens. Here, we examined the interaction of two BP100 analogs, R2R5-BP100 (where Arg substitutes Lys 2 and 5) and R2R5-BP100-A-NH-C16 (where an Ala and a C16 hydrocarbon chain are added to the R2R5-BP100 C-terminus), with membrane models. Large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs) were prepared with the major lipids in Gram-positive (GP) and Gram-negative (GN) bacteria, as well as red blood cells (RBCs). Fluorescence data, dynamic light scattering (DLS), and zeta potential measurements revealed that upon achieving electroneutrality through peptide binding, vesicle aggregation occurred. Circular dichroism (CD) spectra corroborated these observations, and upon vesicle binding, the peptides acquired α-helical conformation. The peptide concentration, producing a 50% release of carboxyfluorescein (C50) from LUVs, was similar for GP-LUVs. With GN and RBC-LUVs, C50 decreased in the following order: BP100 > R2R5-BP100 > R2R5BP100-A-NH-C16. Optical microscopy of GP-, GN-, and RBC-GUVs revealed the rupture or bursting of the two former membranes, consistent with a carpet mechanism of action. Using GUVs, we confirmed RBC aggregation by BP100 and R2R5-BP100. We determined the minimal inhibitory concentrations (MICs) of peptides for a GN bacterium (Escherichia coli (E. coli)) and two GP bacteria (two strains of Staphylococcus aureus (S. aureus) and one strain of Bacillus subtilis (B. subtilis)). The MICs for S. aureus were strain-dependent. These results demonstrate that Lys/Arg replacement can improve the parent peptide’s antimicrobial activity while increasing hydrophobicity renders the peptide less effective and more hemolytic.

Membrane solubilization by detergents is routinely used to separate and extract membrane components, especially membrane proteins. This process depends on the characteristics of the detergent and properties of the membrane. Here we study the interaction of eight detergents with distinct physicochemical features with model membranes in different biologically relevant phases. Among the selected detergents are some PEG-and sugar-based non-ionic detergents and one anionic, one cationic and one zwitterionic.

Transmembrane asymmetry is ubiquitous in cells, particularly with respect to lipids, where charged lipids are mainly restricted to one monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply the fluorescence quenching assay, which is often difficult to reproduce, and caution in handling the quencher is generally underestimated. We first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol by using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers but with ∼20% interleaflet mixing. To probe the stability of asymmetric versus symmetric membranes, we expose the GUVs to porating electric pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover or exhibit leakage or bursting/collapse. Residual oil destabilizes porated membranes, and destabilization is even more pronounced in asymmetrically charged membranes. This is corroborated by the measured pore edge tension, which is also found to decrease with increasing charge asymmetry. Using GUVs with imposed transmembrane pH asymmetry, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

Membrane fusion is a ubiquitous process associated with a multitude of biological events. Although it has long been appreciated that membrane mechanics plays an important role in membrane fusion, the molecular interplay between mechanics and fusion has remained elusive. For example, although different lipids modulate membrane mechanics differently, depending on their composition, molar ratio, and complex interactions, differing lipid compositions may lead to similar mechanical properties. This raises the question of whether (i) the specific lipid composition or (ii) the average mesoscale mechanics of membranes acts as the determining factor for cellular function. Furthermore, little is known about the potential consequences of fusion on membrane disruption. Here, we use a combination of confocal microscopy, time-resolved imaging, and electroporation to shed light onto the underlying mechanical properties of membranes that regulate membrane fusion. Fusion efficiency follows a nearly universal behavior that depends on membrane fluidity parameters, such as membrane viscosity and bending rigidity, rather than on specific lipid composition. This helps explaining why the charged and fluid membranes of the inner leaflet of the plasma membrane are more fusogenic than their outer counterparts. Importantly, we show that physiological levels of cholesterol, a key component of biological membranes, has a mild effect on fusion but significantly enhances membrane mechanical stability against pore formation, suggesting that its high cellular levels buffer the membrane against disruption. The ability of membranes to efficiently fuse while preserving their integrity may have given evolutionary advantages to cells by enabling their function while preserving membrane stability.

Nano-structured and functionalized materials for encapsulation, transport, targeting and controlled release of drugs are of high interest to overcome low bioavailability in oral administration. We develop lipid-based cubosomes, which are surface-functionalized with biocompatible chitosan-N-arginine and alginate, displaying internal liquid crystalline structures. Polyelectrolyte-shell (PS) cubosomes have pH-responsive characteristics profitable for oral delivery. The obtained PScubosomes can strongly interact with serum albumin, a protein which is released in the stomach under gastric cancer conditions. An effective thermodynamic PScubosome-protein interaction was characterized at pH 2.0 and 7.4 by isothermal titration calorimetry at 37 °C. A high increment of the albumin conformation transition temperature was evidenced by differential scanning calorimetry upon incubation with PScubosomes. The performed structural studies by synchrotron small-angle X-ray scattering (SAXS) revealed essential alterations in the internal liquid crystalline topology of the nanocarriers including an Im3m to Pn3m transition and a reduction of the cubic lattice parameters. The PScubosome nanoparticle interaction with serum albumin, leading to inner structural changes in a range of temperatures, promoted the release of water from the cubosomal nanochannels. Altogether, the results revealed effective interactions of the PScubosomes with albumin under simulated gastrointestinal pH conditions and suggested promising nanocarrier characteristics for triggered oral drug release.

Polymers are options as antimicrobials for skin protection, antifouling surfaces, and fabrics. Here we analyzed the interaction of polymers based on poly(methacrylate) (PMMA) and poly((dimethylamino ethyl) methacrylate) (PDMAEMA) with model membranes and bacteria. We used the homopolymers PMMA, PDMAEMA, and the diblock copolymer(s) prepared with different PMMAm:PDMAEMAn ratios (m/n). The interactions of PDMAEMA and PMMAm-b-PDMAEMAn with large unilamellar vesicles (LUVs) prepared with phosphatidylcholine and phosphatidylglycerol at different pHs, were analyzed by nuclear magnetic resonance (NMR), dynamic light scattering, and zeta potential. These polymers promoted LUVs leakage of a fluorescent probe (5,6-carboxyfluorescein) localized exclusively in the internal aqueous compartment. Interestingly, all copolymers exhibit a bell-shaped pH dependence for the polymer-induced LUVs leakage. The interaction of the positively charged polymers and the pH effect was also demonstrated using giant unilamellar vesicles. These copolymers inhibited bacterial growth in the micromolar range and can be used to prevent bacterial growth on surfaces.

Lateral phase heterogeneity in biomembranes can govern cellular functions and may serve as a platform for enrichment or depletion of membrane-anchored molecules. In this work, we address the question of how the process of mem-brane fusion is affected by the membrane phase state (fluid or gel) and by phase coexistence, as well as the effects of fusion -mediated incorporation of exogeneous lipids on phase separation. Our system is based on the fusion of cationic fluid large uni-lamellar vesicles (LUVs) composed of dioleoyl trimethylammonium propane (DOTAP) and dioleoyl phosphoethanolamine (DOPE) with neutral and anionic giant unilamellar vesicles (GUVs) composed of phosphatidylcholine and phosphatidylglycerol. By changing the lipid composition of the GUVs, we modulated the phase state and charge of the different phases (charged or neutral, fluid or gel) and identified systems in which we can target fusion to specific domains on phase-separated membranes. Fusion efficiency was quantified using fluorescence microscopy-based lipid and content mixing assays, and flow chamber de-vices were used to assess the real-time sequence of events of the fusion process. To investigate the bilayer thermal behavior, differential scanning calorimetry (DSC) experiments were performed on LUVs. The results show that fusion is extensive in sin-gle-component GUVs only for fluid and negatively charged acceptor membranes. On the other hand, in phase-separated GUVs, high fusion efficiency was observed even when the gel phase was anionic and phase separation somewhat increased the fusion efficiency. Extensive fusion led to dissolution of the gel domains as a result of extensive incorporation of lipids in the fluid state from the fusogenic liposomes. Altogether, these findings have the potential to unravel the important role of membrane phase state, phase separation, charge, and the effects of extensive fusion on membrane organization and may give insights in the regulation of the interactions between cells and liposomes that are used in drug delivery systems.

ADVERTISEMENT RETURN TO ISSUEEditorialNEXTVirtual Issue: Interfacial Science Developments in Latin AmericaKarin A. Riske*Karin A. RiskeMore by Karin A. Riskehttps://orcid.org/0000-0003-4080-1358, Greco González MieraGreco González MieraMore by Greco González Mierahttps://orcid.org/0000-0002-2856-5295, and Gilbert C. WalkerGilbert C. WalkerMore by Gilbert C. Walkerhttps://orcid.org/0000-0002-5248-5498Cite this: Langmuir 2023, 39, 51, 18673–18677Publication Date (Web):December 26, 2023Publication History Received5 December 2023Published online26 December 2023Published inissue 26 December 2023https://doi.org/10.1021/acs.langmuir.3c03761Copyright © Published 2023 by American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views266Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (969 KB) Get e-AlertscloseSUBJECTS:Hydrogels,Interfaces,Lipids,Nanoparticles,Thin films Get e-Alerts

Detergents are amphiphilic molecules often used to solubilize biological membranes and separate their components. Here we investigate the solubilization of lipid vesicles by the commonly used non-ionic detergents polyoxyethylene (20) oleyl ether (Brij 98), n-octyl-β-D-glucoside (OG), and n-dodecyl β-D maltoside (DDM) and compare the results with the standard detergent Triton X-100 (TX-100). The vesicles were composed of palmitoyl oleoyl phosphatidylcholine (POPC) or of a biomimetic ternary mixture of POPC, egg sphingomyelin (SM) and cholesterol (2:1:2 molar ratio). To follow the solubilization profile of large unilamellar vesicles (LUVs), 90° light scattering measurements were done along the titration of LUVs with the detergents. Then, giant unilamellar vesicles (GUVs) were observed with optical microscopy during exposure to the detergents, to allow direct visualization of the solubilization process. Isothermal titration calorimetry (ITC) was used to assess the binding constant of the detergents in POPC bilayers. The results show that the incorporation of TX-100, Brij 98 and, to a lesser extent, OG in the pure POPC liposomes leads to an increase in the vesicle area, which indicates their ability to redistribute between the two leaflets of the membrane in a short scale of time. On the other hand, DDM incorporates mainly in the external leaflet causing an increase in vesicle curvature/tension leading ultimately to vesicle burst. Only TX-100 and OG were able to completely solubilize the POPC vesicles, whereas the biomimetic ternary mixture was partially insoluble in all detergents tested. TX-100 and OG were able to incorporate in the bilayer of the ternary mixture and induce macroscopic phase separation of liquid-ordered (Lo) and liquid-disordered (Ld) domains, with selective solubilization of the latter. Combination of ITC data with turbidity results showed that TX-100 and OG can be incorporated up to almost 0.3 detergent/lipid, significantly more than Brij 98 and DDM. This fact seems to be directly related to their higher capacity to solubilize POPC membranes and their ability to induce macroscopic phase separation in the biomimetic lipid mixture.

Membrane fusion is a ubiquitous process in biology and potentially an alternative to endocytic internalization pathways in drug delivery. Due to its high complexity, assays have been developed to disentangle its key aspects, although many of its molecular mechanisms remain elusive. Here, we use a combination of multicolour and time-resolved confocal fluorescence microscopy to study the effects of membrane mechanics on fusion and conversely, how fusion alters membrane mechanics. As a minimal fusion system, we used cationic large unilamellar fusogenic liposomes (LUVs) that rapidly fuse with giant unilamellar vesicles (GUVs) as evidenced by the probes being delivered into the GUVs. Fusion is favoured in fluid membranes and hampered upon an increase in packing. Extensive fusion of a large number of LUVs leads to the creation of area leaflet asymmetry in GUVs, resulting in budding formation driven by spontaneous curvature. In these conditions, the membrane becomes perforated as a result of a decrease in edge tension and the size of the formed pores seems to scale with LUV concentration. Here, the GUVs also display a range of permeability states. In live human embryonic kidney (HEK) cells, lipid mixing and intracellular delivery of LUV encapsulated water-soluble probes are observed, consistent with fusion of the liposomes with the plasma membrane. Furthermore, when exposed to liposomes, cell membrane permeabilization and cell death is observed at liposomal concentrations an order of magnitude higher than pore formation in the GUVs, suggesting an increased resilience of cells compared to GUVs. Fusion also seems to fluidize the membrane and effects are observed within minutes. Overall, the results shed new light onto the effects of liposome-membrane interactions.

The influence of hydrophilic surfactants acting on the membrane elasticity of liposomes on the skin absorption of vitamin C is investigated. The purpose of encapsulation inside cationic liposomes is to improve the skin delivery of vitamin C. The properties of elastic liposomes (ELs) are compared to that of conventional liposomes (CLs). ELs are formed by the addition of the "edge activator" Polysorbate 80 to the CLs composed of soybean lecithin, cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammoniopropane chloride), and cholesterol. The liposomes are characterized by dynamic light scattering and electron microscopy. No toxicity is detected in human keratinocyte cells. Evidences of Polysorbate 80 incorporation into liposome bilayers and of the higher flexibility of ELs are given by isothermal titration calorimetry and pore edge tension measurements in giant unilamellar vesicles. The presence of a positive charge in the liposomal membrane increases the encapsulation efficacy by approximately 30% for both CLs and ELs. Skin absorption of vitamin C from CLs, ELs and a control aqueous solution measured in Franz cells shows a high delivery of vitamin C into each skin layer and the acceptor fluid from both liposome types. These results suggest that another mechanism drives skin diffusion, involving interactions between cationic lipids and vitamin C depending on the skin pH.

Knowledge of the material properties of membranes is crucial to understanding cell viability and physiology. A number of methods have been developed to probe membranes in vitro, utilizing the response of minimal biomimetic membrane models to an external perturbation. In this review, we focus on techniques employing giant unilamellar vesicles (GUVs), model membrane systems, often referred to as minimal artificial cells because of the potential they offer to mimick certain cellular features. When exposed to electric fields, GUV deformation, dynamic response and poration can be used to deduce properties such as bending rigidity, pore edge tension, membrane capacitance, surface shear viscosity, excess area and membrane stability. We present a succinct overview of these techniques, which require only simple instrumentation, available in many labs, as well as reasonably facile experimental implementation and analysis.

Liposomes represent important drug carrier vehicles in biological systems. A fusogenic liposomal system composed of equimolar mixtures of the cationic lipid DOTAP and the phospholipid DOPE showed high fusion and delivery efficiencies with cells and lipid vesicles. However, aspects of the thermodynamics involving the interaction of these fusogenic liposomes and biomimetic systems remain unclear. Here, we investigate the fusion of this system with large unilamellar vesicles (LUVs) composed of the zwitterionic lipid POPC and increasing fractions of the anionic lipid POPG and up to 30 mol % cholesterol. The focus here is to concomitantly follow changes in size, zeta-potential, and enthalpy binding upon membrane interaction and fusion. Isothermal titration calorimetry (ITC) data showed that membrane fusion in our system is an exothermic process in the absence of cholesterol, suggesting that electrostatic attraction is the driving force for fusion. An endothermic component appeared and eventually dominated the titration at 30 mol % cholesterol, which we propose is caused by membrane fluidification when cholesterol is diluted upon fusion. The inflection points of the ITC data occurred around 0.5-0.7 POPG/DOTAP for all systems, the same stoichiometry for which zeta-potential and dynamic light scattering measurements showed an increase in size coupled with charge neutralization of the system, which is consistent with the fact that fusion in our system is charge-mediated. Microscopy observations of the final mixtures revealed the presence of giant vesicles, which is a clear indication of fusion, coexisting with intermediate-sized objects that could be the result of both fusion and/or aggregation. The results show that the fusion efficiency of the DOTAP:DOPE fusogenic system is modulated by the charge and membrane packing of the acceptor membrane and explain why the system fuses very efficiently with cells.

The protein adsorption onto poly(acrylic acid)-block-polystyrene (PAA22-b-PS144) polymersomes has been investigated with regard to structural features, thermodynamic aspects and biological consequences. The light scattering measurements revealed the formation of protein coronas enveloping the polymeric capsules regardless of the chemical nature of the biomacromolecules. The experiments were conducted by using lysozyme, immunoglobulin G - IgG and bovine serum albumin - BSA as model proteins due to their differences concerning size and residual surface charge at physiological pH. The protein adsorption was further confirmed by isothermal titration calorimetry, and the experimental data suggest that the phenomenon is mainly governed by hydrogen bonding and van der Waals interactions. The pre-existing protein layer via the pre-incubation in protein environments notably attenuates the cytotoxicity of the nanomaterial compared to the pristine counterparts. This approach can possibly be extended to different types of assemblies when intermolecular interactions are able to induce protein adsorption and the development of protein coronas around nanoparticles. Such fairly simple method may be convenient to engineer safer nanomaterials towards a variety of biomedical applications when the nanotoxicity is an issue. Additionally, the strategy can possibly be used to tailor the surface properties of nanoparticles by adsorbing specific proteins for targeting purposes.