Flavonoid-Labeled Biopolymer in the Structure of Lipid Membranes to Improve the Applicability of Antioxidant Nanovesicles.

Nanovesicles produced with lipids and polymers are promising devices for drug and bioactive delivery and are of great interest in pharmaceutical applications. These nanovesicles can be engineered for improvement in bioavailability, patient compliance or to provide modified release or enhanced delivery. However, their applicability strongly depends on the safety and low immunogenicity of the components. Despite this, the use of unsaturated lipids in nanovesicles, which degrade following oxidation processes during storage and especially during the proper routes of administration in the human body, may yield toxic degradation products. In this study, we used a biopolymer (chitosan) labeled with flavonoid (catechin) as a component over a lipid bilayer for micro- and nanovesicles and characterized the structure of these vesicles in oxidation media. The purpose of this was to evaluate the in situ effect of the antioxidant in three different vesicular systems of medium, low and high membrane curvature. Liposomes and giant vesicles were produced with the phospholipids DOPC and POPC, and crystalline cubic phase with monoolein/DOPC. Concentrations of chitosan-catechin (CHCa) were included in all the vesicles and they were challenged in oxidant media. The cytotoxicity analysis using the MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) revealed that concentrations of CHCa below 6.67 µM are non-toxic to HeLa cells. The size and zeta potential of the liposomes evidenced the degradation of their structures, which was minimized by CHCa. Similarly, the membrane of the giant vesicle, which rapidly deteriorated in oxidative solution, was protected in the presence of CHCa. The production of a lipid/CHCa composite cubic phase revealed a specific cubic topology in small-angle X-ray scattering, which was preserved in strong oxidative media. This study demonstrates the specific physicochemical characteristics introduced in the vesicular systems related to the antioxidant CHCa biopolymer, representing a platform for the improvement of composite nanovesicle applicability.

Unveiling Sticholysin II and plasmid DNA interaction: Implications for developing non-viral vectors.

Non-viral gene delivery systems offer significant potential for gene therapy due to their versatility, safety, and cost advantages over viral vectors. However, their effectiveness can be hindered by the challenge of efficiently releasing the genetic cargo from endosomes to prevent degradation in lysosomes. To overcome this obstacle, functional components can be incorporated into these systems. Sticholysin II (StII) is one of the pore-forming proteins derived from the sea anemone Stichodactyla helianthus, known for its high ability to permeabilize cellular and model membranes. In this study, we aimed to investigate the interaction between StII, and a model plasmid (pDNA) as an initial step towards designing an improved vector with enhanced endosomal escape capability. The electrophoretic mobility shift assay (EMSA) confirmed the formation of complexes between StII and pDNA. Computational predictions identified specific residues involved in the StII-DNA interaction interface, highlighting the importance of electrostatic interactions and hydrogen bonds in mediating the binding. Atomic force microscopy (AFM) of StII-pDNA complexes revealed the presence of nodular fiber and toroid shapes. These complexes were found to have a predominantly micrometer size, as confirmed by dynamic light scattering (DLS) measurements. Despite increase in the overall charge, the complexes formed at the evaluated nitrogen-to-phosphorus (N/P) ratios still maintained a negative charge. Moreover, StII retained its pore-forming capacity regardless of its binding to the complexes. These findings suggest that the potential ability of StII to permeabilize endosomal membranes could be largely maintained when combined with nucleic acid delivery systems. Additionally, the still remaining negative charge of the complexes would enable the association of another positively charged component to compact pDNA. However, to minimize non-specific cytotoxic effects, it is advisable to explore methods to regulate the protein's activity in response to the microenvironment.

Small-angle scattering from flat bilayers containing correlated scattering length density inhomogeneities.

Model lipid bilayers have been widely employed as a minimal system to investigate the structural properties of biological membranes by small-angle X-ray (SAXS) and neutron scattering (SANS) techniques. These have nanometre resolution and can give information regarding membrane thickness and scattering length densities (SLDs) of polar and apolar regions. However, biological membranes are complex systems containing different lipids and protein species, in which lipid domains can be dynamically assembled and disassembled. Therefore, SLD variations can occur within the biomembrane. In this work, a novel method has been developed to simulate SAXS and SANS profiles obtained from large unilamellar vesicles containing SLD inhomogeneities that are spatially correlated over the membrane surface. Such inhomogeneities are represented by cylindrical entities with equivalent SLDs. Stacking of bilayers is also included in the model, with no correlation between horizontal and vertical order. The model is applied to a lipid bilayer containing SLD inhomogeneities representing pores, lipid domains, and transmembrane, partially immersed and anchored proteins. It is demonstrated that all the structural information from the host lipid bilayer and from the SLD inhomogeneity can be consistently retrieved by a combined analysis of experimental SAXS and SANS data through the methodology proposed here.

The Latin American Federation of Biophysical Societies (LAFeBS).

The Latin American Federation of Biophysical Societies (LAFeBS) was constituted in 2007 in Montevideo, Uruguay, as a collaborative effort among the Biophysical Societies of Argentina, Brazil, and Uruguay. This visionary collaboration foresees the future of Biophysics in Latin America. In this commentary, we will briefly review the history of LAFeBS, the remarkable path undertaken since its foundation 16 years ago, and its key initiative, the Latin American Postgraduate Program in Biophysics (POSLATAM).

Self-oriented anisotropic structure of G-hydrogels as a delicate balance between attractive and repulsive forces.

Guanine (G) hydrogels are very attractive materials made by the supramolecular organization of G-derivatives in water. In this paper, hydrogels composed of guanosine 5'-monophosphate (GMP) and guanosine (Gua), that make long, flexible and knotted G-quadruplexes, were investigated by small- and wide-angle X-ray scattering (SAXS and WAXS) to comprehend the origin of their unique orientational properties. The SAXS intensity, analysed at a fixed scattering vector modulus Q as a function of polar angle, allowed us to derive the Maier-Saupe orientation parameter m. The strong dependence of m on hydrogel composition and temperature demonstrated that the preferred orientation is controlled by the quadruplex surface charge and flexibility. Indeed, a possible correlation between the orientation parameter m and the quadruplex-to-quadruplex lateral interactions was explored. Results confirmed that the balance between attractive and repulsive interactions plays a main role in the orientational anisotropy: quadruplex clusters lose their orientational properties when attractive interactions decrease. The key role of the number of negative charges per unit length of the G-quadruplex filaments was confirmed by Atomic Force Microscopy (AFM) observations. Indeed, directionality histograms showed that in the presence of a large amount of Gua, G-quadruplexes follow preferential orientations other than those related to the strong interactions with the K+ pattern on the mica surface. The fact that lateral quadruplex-to-quadruplex interactions, even in the presence of external (opposing) forces, can tune the hydrogel alignment in a given preferred direction provides novel possibilities for scaffold/3D printing applications.

Unveiling protein-protein interaction potential through Monte Carlo simulation combined with small-angle X-ray scattering.

Protein interactions are investigated under different conditions of lysozyme concentration, temperature and ionic strength by means of in-solution small angle X-Ray scattering (SAXS) experiments and Monte Carlo (MC) simulations. Initially, experimental data were analysed through a Hard-Sphere Double Yukawa (HSDY) model combined with Random Phase Approximation (RPA), a closure relationship commonly used in the literature for monodisperse systems. We realized by means of MC that the HSDY/RPA modelling fails to describe the protein-protein pair potential for moderated and dense systems at low ionic strength, mainly due to inherent distortions of the RPA approximation. Our SAXS/MC results thus show that lysozyme concentrations between 2 (diluted) and 20 mg/mL (not crowded) present similar protein-protein pair potential preserving the values of surface net charge around 7 e, protein diameter of 28 Å, decay range of attractive well potential of 3 Å and a depth of the well potential varying from 1 to 5 kBT depending on temperature and salt addition. Noteworthy, we here propose a novel method to analyse the SAXS data from interacting proteins through MC simulations, which overcomes the deficiencies presented by the use of a closure relationship. Furthermore, this new methodology of combining SAXS with MC simulations gives a step forward to investigate more complex systems as those composed of a mixture of proteins of distinct species presenting different molecular weights (and hence sizes) and surface net charges at low, moderate and very dense systems.

Dynamic photodamage of red blood cell induced by CisDiMPyP porphyrin.

It is well-known that oxidative damage in red blood cell (RBC) usually causes morphological changes and increased membrane rigidity. Although many studies have focused on investigating how RBC responds to a photodynamic stimulus, the intermediate steps between membrane damage and hemolysis are not reported. To give a comprehensive insight into changes of RBC membrane property under different oxidative damage levels, we employed the photoactivation of CisDiMPyP porphyrin that primarily generates singlet oxygen 1O2 as oxidant species. We found that there were distinguishable characteristic damages depending on the 1O2 flux over the membrane, in a way that each impact of photooxidative damage was categorized under three damage levels: mild (maintaining the membrane morphology and elasticity), moderate (membrane elongation and increased membrane elasticity) and severe (wrinkle-like deformation and hemolysis). When sodium azide (NaN3) was used as a singlet oxygen quencher, delayed cell membrane alterations and hemolysis were detected. The delay times showed that 1O2 indeed plays a key role that causes RBC photooxidation by CisDiMPyP. We suggest that the sequence of morphological changes (RBC discoid area expansion, wrinkle-like patterns, and hemolysis) under photooxidative damage occurs due to damage to the lipid membrane and cytoskeletal network proteins.

Rheological and mechanical properties of hydroxypropyl methylcellulose-based hydrogels and cryogels controlled by AOT and SDS micelles.

HYPOTHESIS: The type and concentration of surfactants affect the rheological behavior of hydroxypropyl methylcellulose (HPMC) chains in hydrogels, influencing the microstructure and mechanical properties of HPMC cryogels. EXPERIMENTS: Hydrogels and cryogels containing HPMC, AOT (bis (2-ethylhexyl) sodium sulfosuccinate or dioctyl sulfosuccinate salt sodium, two C8 chains and sulfosuccinate head group), SDS (sodium dodecyl sulfate, one C12 chain and sulfate head group), and sodium sulfate (salt, no hydrophobic chain) at different concentrations were investigated using small-angle X-ray scattering (SAXS), scanning electron microscopy (SEM), rheological measurements, and compressive tests. FINDINGS: SDS micelles bound to the HPMC chains building "bead necklaces", increasing considerably the storage modulus G' values of the hydrogels and the compressive modulus E values of the corresponding cryogels. The dangling SDS micelles promoted multiple junction points among the HPMC chains. AOT micelles and HPMC chains did not form "bead necklaces". Although AOT increased the G' values of the hydrogels, the resulting cryogels were softer than pure HPMC cryogels. The AOT micelles are probably embedded between HPMC chains. The AOT short double chains rendered softness and low friction to the cryogel cell walls. Therefore, this work demonstrated that the structure of the surfactant tail can tune the rheological behavior of HPMC hydrogels and hence the microstructure of the resulting cryogels.

Biophysical Reviews special issue call: LAFeBS-highlighting biophysics in Latin America.

This Commentary describes a call for contributions to an upcoming Special Issue (SI) of Biophysical Reviews on the Latin American Federation of Biophysical Societies (LAFeBS). It details the reason for the SI, the SI Editors contact information and the relevant submission details for those wishing to contribute.

Unveiling the mono-rhamnolipid and di-rhamnolipid mechanisms of action upon plasma membrane models.

Rhamnolipids (RLs) are biosurfactants with significant tensioactive and emulsifying properties. They are mainly composed by mono-RL and di-RL components. Although there are numerous studies concerning their molecular properties, information is scarce regarding the mechanisms by which each of the two components interacts with cell membranes. Herein, we performed phase-contrast and fluorescence microscopy experiments on plasma membrane models represented by giant-unilamellar-vesicles (GUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-[[(E,2S,3R)-1,3-dihydroxy-2-(octadecanoylamino) octadec-4-enyl]peroxy-hydroxyphosphoryl]oxyethyl-trimethylazanium (sphingomyelin, SM) and (3ß)-cholest-5-en-3-ol (cholesterol, CHOL) (1:1:1 M ratio), which present liquid-order (Lo) liquid-disorder (Ld) phase coexistence, in the presence of either mono-RL or di-RL in 0.06-0.25 mM concentration range. A new method has been developed to determine area and volume of GUVs with asymmetrical shape and a kinetic model describing GUV-RL interaction in terms of two mechanisms, RL-insertion and pore formation, has been worked out. Results show that the insertion of mono-RL in the membrane outer leaflet is the dominant process with no pore formation and a negligible effect in modifying membrane permeability, but induces lipid mixing. Conversely, the di-RL-GUV interaction begins with the insertion mechanism and, as the time passes by, the pore formation process occurs. The analyses of di-RL show that the whole process is only relevant in the Ld phase with a higher extent to 0.25 mM than to 0.06 mM.

The pore-forming activity of sticholysin I is enhanced by the presence of a phospholipid hydroperoxide in membrane.

Sticholysin I (StI) is a pore-forming toxin (PFT) belonging to the actinoporin protein family characterized by high permeabilizing activity in membranes. StI readily associates with sphingomyelin (SM)-containing membranes originating pores that can lead to cell death. Binding and pore-formation are critically dependent on the physicochemical properties of membrane. 1-palmitoyl-2-oleoylphosphatidylcholine hydroperoxide (POPC-OOH) is an oxidized phospholipid (OxPL) containing an -OOH moiety in the unsaturated hydrocarbon chain which orientates towards the bilayer interface. This orientation causes an increase in the lipid molecular area, lateral expansion and decrease in bilayer thickness, elastic and bending modulus, as well as modification of lipid packing. Taking advantage of membrane structural changes promoted by POPC-OOH, we investigated its influence on the permeabilizing ability of StI. Here we report the action of StI on Giant Unilamellar Vesicles (GUVs) made of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and SM containing increasing amount of POPC-OOH to assess vesicle permeability changes when compared to OxPL-lacking membranes. Inclusion of POPC-OOH in membranes did not promote spontaneous vesicle leaking but resulted in increased membrane permeability due to StI action. StI activity did not modify the fluid-gel phase coexistence boundaries neither in POPC:SM or POPC-OOH:SM membranes. However, the StI insertion mechanism in membrane seems to differ between POPC:SM and POPC-OOH:SM mixtures as suggested by changes in the time course of monolayer surface tension measurements, even though a preferable binding of the toxin to OxPL-containing systems could not be here demonstrated. In summary, modifications in the membrane imposed by lipid hydroperoxidation favor StI permeabilizing activity.

A special issue of Biophysical Reviews dedicated to the 20th IUPAB (virtual) Congress "in" Foz do Iguaçu.

The 20th IUPAB Congress took place online, together with the annual meetings of the Brazilian Biophysical Society and the Brazilian Society for Biochemistry and Molecular Biology, from the 4th to the 8th of October, 2021. The ten keynote lectures, 24 symposia, two poster sessions, and a series of technical seminars covered the full diversity of current biophysical research and its interfaces with other fields. The event had over 1000 attendees, with an excellent gender balance. Although the Americas dominated, there were also significant numbers of participants from Europe, Asia, and Africa.

Lipid Hydroperoxide Compromises the Membrane Structure Organization and Softens Bending Rigidity.

Lipid hydroperoxides are key mediators of diseases and cell death. In this work, the structural and dynamic perturbations induced by the hydroperoxidized POPC lipid (POPC-OOH) in fluid POPC membranes, at both 23 and 37 °C, were addressed using advanced small-angle X-ray scattering (SAXS) and fluorescence methodologies. Notably, SAXS reveals that the hydroperoxide group decreases the lipid bilayer bending rigidity. This alteration disfavors the bilayer stacking and increases the swelling in-between stacked bilayers. We further investigated the changes in the apolar/polar interface of hydroperoxide-containing membranes through time-resolved fluorescence/anisotropy experiments of the probe TMA-DPH and time-dependent fluorescence shifts of Laurdan. A shorter mean fluorescence lifetime for TMA-DPH was obtained in enriched POPC-OOH membranes, revealing a higher degree of hydration near the membrane interface. Moreover, a higher microviscosity near TMA-DPH and lower order are predicted for these oxidized membranes, at variance with the usual trend of variation of these two parameters. Finally, the complex relaxation process of Laurdan in pure POPC-OOH membranes also indicates a higher membrane hydration and viscosity in the close vicinity of the -OOH moiety. Altogether, our combined approach reveals that the hydroperoxide group promotes alterations in the membrane structure organization, namely, at the level of membrane order, viscosity, and bending rigidity.

Announcing the call for the Special Issue on the 20th International Congress of the International Union of Pure and Applied Biophysics (IUPAB)-virtual meeting, October 2021.

This Commentary describes a call for submissions for the upcoming Special Issue focused on the science presented at the 20th IUPAB Congress to be held in conjunction with the 45th Annual Meeting of the Brazilian Biophysical Society and the 49th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology.

Self-assembled guanosine-hydrogels for drug-delivery application: Structural and mechanical characterization, methylene blue loading and controlled release.

It is known that guanosine derivatives (G) self-assemble in water forming long, flexible, and interacting aggregates (the so-called G-quadruplexes): by modulating the quadruplex charges, e.g. simply using a mixture of guanosine 5'-monophosphate (GMP) and guanosine (Gua), multi-responsive, self-healing hydrogels can be obtained. In this paper, the potential application of G-hydrogels as drug delivery systems has been assessed. Hydrogels were prepared at different Gua:GMP molar ratios. The photosensitizer Methylene Blue and the pro-apoptotic protein cytochrome C were used as cargo molecules. Small angle x-ray scattering and atomic force microscopy experiments confirmed the presence of G-quadruplexes disposed in swollen matrices with different mesh-sizes. Rheology measurements showed that the Gua:GMP molar ratio leads to specific drug release mechanisms, as the gel strength is finely tuned by electrostatic repulsion and van der Waals attraction between G-quadruplexes. Noteworthy, the gel cohesion and the drug release were pH responsive. Swelling, self-healing and cell viability features were also investigated: the results qualify the Gua:GMP hydrogel as an excellent biomaterial that can entrap and deliver key biomolecules in a sustained and responsive release manner.

The intriguing role of rhamnolipids on plasma membrane remodelling: From lipid rafts to membrane budding.

Rhamnolipids (RLs) comprise a class of glycolipids produced by Pseudomonas aeruginosa under appropriate culture medium. They act as biosurfactants being composed by a hydrophilic head of either one (mono-RL) or two (di-RL) rhamnose moieties coupled to hydroxyaliphatic chains. It is well accepted that RLs present low biolitic activity as compared to other synthetic surfactants. However, their mechanisms of action in biological systems are not well defined yet. The interaction of RLs with lipid bilayers are here investigated to address how they impact on plasma membrane at molecular level. Our experimental approach was based on a deep analysis of optical microscopy data from giant unilamellar vesicles (GUVs) dispersed in aqueous solutions containing up to 0.5 mM of commercially available RLs (a mixture of mono-RL, 33-37 mol%, and di-RL, 63-67 mol%, cmc of 0.068±0.005 mM). GUVs were made up of a single lipid POPC and a ternary system containing DOPC, sphingomyelin and cholesterol, which mimic lipid raft platforms. Our results demonstrate that RLs have a low partition in the lipid bilayer in respect to the total molecules in solution. We suppose that RLs insert in the outer leaflet with low propensity to flip-flop. In the case of POPC GUVs, the insertion of RL molecules in the outer leaflet impairs changes in spontaneous membrane curvature with incubation time. Then, small buds are formed that remain linked to the original membrane. No changes in membrane permeability have been detected. A remarkable result refers to the insertion of RLs in membranes containing liquid ordered (Lo) - liquid disordered (Ld) phase coexistence. The rate of interaction has been observed to be higher for Ld phase than for Lo phase (0.12·10-6 s-1 and 0.023·10-6 s-1 for Ld and Lo, respectively, at RL concentration of 0.5 mM). As a consequence, the preferential RL insertion in Ld phase may also alter the membrane spontaneous curvature which, coupled to the change in the line tension associated to the domains boundary, conducted to Lo domain protrusion. Even if it has been observed on a model system, such membrane remodelling might correlate to endocytic processes activated in cell membranes, regardless of the participation of specific proteins. Further, changes imposed by RLs in lipid rafts may affect the association of key proteins enrolled in cell signaling, which may perturb cell homeostasis.

Cellular compartments challenged by membrane photo-oxidation.

The lipid composition impacts directly on the structure and function of the cytoplasmic as well as organelle membranes. Depending on the type of membrane, specific lipids are required to accommodate, intercalate, or pack membrane proteins to the proper functioning of the cells/organelles. Rather than being only a physical barrier that separates the inner from the outer spaces, membranes are responsible for many biochemical events such as cell-to-cell communication, protein-lipid interaction, intracellular signaling, and energy storage. Photochemical reactions occur naturally in many biological membranes and are responsible for diverse processes such as photosynthesis and vision/phototaxis. However, excessive exposure to light in the presence of absorbing molecules produces excited states and other oxidant species that may cause cell aging/death, mutations and innumerable diseases including cancer. At the same time, targeting key compartments of diseased cells with light can be a promising strategy to treat many diseases in a clinical procedure called Photodynamic Therapy. Here we analyze the relationships between membrane alterations induced by photo-oxidation and the biochemical responses in mammalian cells. We specifically address the impact of photosensitization reactions in membranes of different organelles such as mitochondria, lysosome, endoplasmic reticulum, and plasma membrane, and the subsequent responses of eukaryotic cells.

Mapping the underlying mechanisms of fibrinogen benzothiazole drug interactions using computational and experimental approaches.

Three-dimensional conformational crystallographic binding-modes are of paramount importance to understand the docking mechanism of protein-ligand interactions and to identify potential "leading drugs" conformers towards rational drugs-design. Herein, we present an integrated computational-experimental study tackling the problem of multiple binding modes among the ligand 3-(2-Benzothiazolylthio)-propane sulfonic acid (BTS) and the fibrinogen receptor (E-region). Based on molecular docking simulations, we found that the free energy of binding values for nine of different BTS-docking complexes (i.e., BTS-pose_1-9) were very close. We have also identified a docking-mechanism of BTS-interaction mainly based on non-covalent hydrophobic interactions with H-bond contacts stabilizing the fibrinogen-BTS docking complexes. Interestingly, the different BTS-poses_1-9 were found to be able to block the fibrinogen binding site (E-region) by inducing local perturbations in effector and allosteric residues, reducing the degree of collectivity in its flexibility normal modes. As such, we theoretically suggest that the BTS-binding modes can significantly affect the physiological condition of the unoccupied fibrinogen protein structure by bringing global and local perturbations in the frequency domain spectra. The proposed theoretical mechanisms, the interactions involved and the conformational changes suggested, were further corroborated by different experimental techniques such as isothermal titration calorimetry (ITC), zeta potential, UV-vis, fluorescence and small angle X-ray scattering (SAXS). The combined results shall open new avenues towards the application of complex supra-molecular information in rational drugs-design.