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An investigation is carried out about biophysical basis for the membrane formation and thermal stability by using electron spin resonance (ESR) of membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon sulfolobus solfataricus. It showed that at a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes.

The article studies polarity of the environment, librational dynamics and conformational exchange, of the spin-labeled group with pulsed electron paramagnetic resonance (EPR) by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy. Conventional EPR lineshapes showcases conformational microheterogeneity at low temperatures.

Hydration of water affects the dynamics and in turn the activity of biomacromolecules. We investigated the dependence of the librational oscillations and the dynamical transition on the hydrating conditions of two globular proteins with different structure and size, namely β-lactoglobulin (βLG) and human serum albumin (HSA), by spin-label electron paramagnetic resonance (EPR) in the temperature range of 120–270 K. The proteins were spin-labeled with 5-maleimide spin-label on free cysteins and prepared in the lyophilized state, at low (h = 0.12) and full (h = 2) hydration levels in buffer. The angular amplitudes of librations are small and almost temperature independent for both lyophilized proteins. Therefore, in these samples, the librational dynamics is restricted and the dynamical transition is absent. In the small and compact beta-structured βLG, the angular librational amplitudes increase with temperature and hydrating conditions, whereas hydration-independent librational oscillations whose amplitudes rise with temperature are recorded in the large and flexible alpha-structured HSA. Both βLG and HSA at low and fully hydration levels undergo the dynamical transition at about 230 K. The overall results indicate that protein librational dynamics is activated at the low hydration level h = 0.12 and highlight biophysical properties that are common to other biosamples at cryogenic temperatures.

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu2+ or Zn2+ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein. [ABSTRACT FROM AUTHOR]

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A letter to the editor is presented about the use of trimipramine in treating patients with obsessive-compulsive disorder.

Human serum albumin (HSA) is the most abundant plasma protein of the circulatory system. It is a multidomain, multifunctional protein that, combining diverse affinities and wide specificity, binds, stores, and transports a variety of biological compounds, pharmacores, and fatty acids. HSA is finding increasing uses in drug-delivery due to its ability to carry functionalized ligands and prodrugs. All this raises the question of competition for binding sites occupancy in case of multiple ligands, which in turn influences the protein structure/dynamic/function relationship and also has an impact on the biomedical applications. In this work, the effects of interactive binding of palmitic acid (PA), warfarin (War) and ibuprofen (Ibu) on the thermal stability of HSA were studied using DSC, ATR-FTIR, and EPR. PA is a high-affinity physiological ligand, while the two drugs are widely used for their anticoagulant (War) and anti-inflammatory (Ibu) efficacy, and are exogenous compounds that accommodate in the deputed drug site DS1 and DS2, respectively overlapping with some of the fatty acid binding sites. The results indicate that HSA acquires the highest thermal stability when it is fully saturated with PA. The binding of this physiological ligand does not hamper the binding of War or Ibu to the native state of the protein. In addition, the three ligands bind simultaneously, suggesting a synergic cooperative influence due to allosteric effects. The increased thermal stability subsequent to binary and multiple ligands binding moderates protein aggregation propensity and restricts protein dynamics. The biophysics findings provide interesting features about protein stability, aggregation, and dynamics in interaction with multiple ligands and are relevant in drug-delivery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding how cells process nanoparticles is crucial to optimize nanomedicine efficacy. However, characterizing cellular pathways is challenging, especially if non-canonical mechanisms are involved. In this Article a genome-wide forward genetic screening based on insertional mutagenesis is applied to discover receptors and proteins involved in the intracellular accumulation (uptake and intracellular processing) of silica nanoparticles. The nanoparticles are covered by a human serum corona known to target the low-density lipoprotein receptor (LDLR). By sorting cells with reduced nanoparticle accumulation and deep sequencing after each sorting, 80 enriched genes are identified. We find that, as well as LDLR, the scavenger receptor SCARB1 also mediates nanoparticle accumulation. Additionally, heparan sulfate acts as a specific nanoparticle receptor, and its role varies depending on cell and nanoparticle type. Furthermore, some of the identified targets affect nanoparticle trafficking to the lysosomes. These results show the potential of genetic screening to characterize nanoparticle pathways. Additionally, they indicate that corona-coated nanoparticles are internalized via multiple receptors., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The interaction between chiral drugs and biomimetic membranes is of interest in biophysical research and biotechnological applications. There is a belief that the membrane composition, particularly the presence of cholesterol, could play a pivotal role in determining enantiospecific effects of pharmaceuticals. Our study explores this topic focusing on the interaction of ibuprofen enantiomers (S- and R-IBP) with cholesterol-containing model membranes. The effects of S- and R-IBP at 20 mol% on bilayer mixtures of dipalmitoylphosphatidylcholine (DPPC) with 0, 10, 20 and 50 mol% cholesterol were investigated using circular dichroism and spin-label electron spin resonance. Morphological changes due to IBP enantiomers were studied with atomic force microscopy on supported cholesterol-containing DPPC monolayers. The results reveal that IBP isoforms significantly and equally interact with pure DPPC lipid assemblies. Cholesterol content, besides modifying the structure and the morphology of the membranes, triggers the drug enantioselectivity at 10 and 20 mol%, with the enantiomers differently adsorbing on membranes and perturbing them. The spectroscopic and the microscopic data indicate that IBP stereospecificity is markedly reduced at equimolar content of Chol mixed with DPPC. This study provides new insights into the role of cholesterol in modulating enantiospecific effects of IBP in lipid membranes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Curcumin, a plant polyphenol extracted from the Chinese herb turmeric, has gained widespread attention in recent years because of its multifunctional properties as antioxidant, antinflammatory, antimicrobial, and anticancer agent. Effects of the molecule on mitochondrial membranes properties have also been evidenced. In this work, the interaction of curcumin with models of mitochondrial membranes composed of dimyristoylphosphatidylcholine (DMPC) or mixtures of DMPC and 4 mol% tetramyristoylcardiolipin (TMCL) has been investigated by using biophysical techniques. Spectrophotometry and fluorescence allowed to determine the association constant and the binding energy of curcumin with pure DMPC and mixed DMPC/TMCL aqueous bilayers. The molecular organization of pure DMPC and cardiolipin-containing Langmuir monolayers at the air-water interface were investigated and the morphology of the monolayers transferred into mica substrates were characterized through atomic force microscopy (AFM). It is found that curcumin associates at the polar/apolar interface of the lipid bilayers and the binding is favored in the presence of cardiolipin. At 2 mol%, curcumin is well miscible with lipid monolayers, particularly with mixed DMPC/TMCL ones, where compact terraces formation characterized by a reduction of the surface roughness is observed in the AFM topographic images. At 10 mol%, curcumin perturbs the stability of DMPC monolayers and morphologically are evident terraces surrounded by cur aggregates. In the presence of TMCL, very few curcumin aggregates and larger compact terraces are observed. The overall results indicate that cardiolipin augments the incorporation of curcumin in model membranes highlighting the mutual interplay cardiolipin-curcumin in mitochondrial membranes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The mutual influence of chiral bioactive molecules and supramolecular assemblies is currently being studied in many research fields, including medical-pharmaceutical applications. Model membranes of phospholipids, such as the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the anionic dipalmitoylphosphatidylglycerol (DPPG), interact with a variety of chiral compounds that include amino acids. In this work, the interaction of tryptophan enantiomers, L-Trp and D-Trp, on DPPC and DPPG bilayers was investigated by using differential scanning calorimetry, attenuated total reflectance-Fourier transform infrared and spin-label electron spin resonance spectroscopies as well as molecular docking simulations. The results show that Trp enantiomers slightly perturb the bilayer thermotropic phase transitions. For both membranes, O atoms in the carbonyl groups have a propensity to act as acceptors of a (weak) hydrogen bond. The Trp chiral forms also promote formation of hydrogen bonds and/or hydration in the PO 2 - moiety of the phosphate group, especially for the DPPC bilayer. In contrast, they interact more closely with the glycerol group of DPPG polar head. Only for DPPC bilayers, both enantiomers increase the packing of the first hydrocarbon chain segments for temperatures through the gel state, whereas they do not affect the lipid chain order and mobility in the fluid state. The results are consistent with a Trp association in the upper region of the bilayers without permeation in the innermost hydrophobic region. The findings suggest that neutral and anionic lipid bilayers are differently sensitive to amino acid chirality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Methods of electron spin echo of pulse electron paramagnetic resonance (EPR) spectroscopy are increasingly employed to investigate biophysical properties of nitroxide-labeled biosystems at cryogenic temperatures. Two-pulse echo-detected ED-spectra have proven to be valuable tools to describe the librational dynamics in the low-temperature phases of both lipids and proteins in membranes. The motional parameter, α 2 τ C , given by the product of the mean-square angular amplitude, α 2 , and the rotational correlation time, τ C , of the motion, is readily determined from the nitroxide ED-spectra as well as from the W -relaxation rate curves. An independent evaluation of α 2 is obtained from the motionally averaged 14 N-hyperfine splitting separation in the continuous wave cw-EPR spectra. Finally, the rotational correlation time τ C can be estimated by combining ED- and cw-EPR data. In this mini-review, results on the librational dynamics in model and natural membranes are illustrated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bartucci and Aloi.)

Continuous wave electron paramagnetic resonance spectroscopy of chain-labeled phospholipids is used to investigate the effects of hydration on the librational oscillations and the dynamical transition of phospholipid membranes in the low-temperature range 120-270 K. Bilayers of dipalmitoylphostatidiycholine (DPPC) spin-labeled at the first acyl chain segments and at the methyl ends and prepared at full, low, and very low hydration are considered. The segmental mean-square angular amplitudes of librations, 〈α 2 〉, are larger in the bilayer interior than at the polar/apolar interface and larger in the fully and low hydrated than in the very low hydrated membranes. For chain segments at the beginning of the hydrocarbon region, 〈α 2 〉-values are markedly restricted and temperature independent in DPPC with the lowest water content, whereas they increase with temperature in the low and fully hydrated bilayers, particularly at the highest temperatures. For chain segments at the chain termini, the librational amplitudes increase progressively, first slowly and then more rapidly with temperature in bilayers at any level of hydration. From the temperature dependence of the mean-square librational amplitude, the dynamical transition is detected around 240 K at the polar/apolar interface in fully and low hydrated DPPC and at around 225 K at the inner hydrocarbon region for bilayers at any hydration condition. At the dynamical transition the bilayers cross low energy barriers of activation energy in the range 10-20 kJ/mol. The results highlight biophysical properties of DPPC bilayers at low-temperature and provide evidence of the effects of the hydration on the dynamical transition in bilayers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human serum albumin binds a wide variety of drugs with different structure and affinity to two main binding sites, drug site 1 (DS1) and drug site 2 (DS2), which partially or totally overlap with fatty acid (FA) sites. Although multiple binding sites are available for endogenous compounds, FAs are the primary physiological ligands of albumin and their competition in the occupancy of DS1 and DS2 affects the binding of exogenous molecules, with a possible impact on drug delivery. In this work, we have investigated the simultaneous binding of oleic acid, warfarin and ibuprofen to albumin using differential scanning calorimetry and fluorescence to evaluate the impact on the conformational stability of the protein. The two drugs are widely used for their anticoagulant (warfarin) and anti-inflammatory (ibuprofen) properties, and can be also considered as site markers to probe DS1 and DS2, respectively. Oleic acid is one of the most important fatty acids from a physiological point of view for its role as a source of energy for cells, and also it binds albumin with the highest association constant. When complexed with oleic acid the calorimetric profile of albumin shows a biphasic trend whose line shape depends on the ligand concentration. The binding capacity of either warfarin or ibuprofen to albumin is modulated by oleate molecules in a concentration-dependent mode being synergic cooperative (warfarin) or competitive-like (ibuprofen). The overall results provide insights on the dynamics of albumin/ligands complex, which in turn may have important pharmacokinetic and pharmacodynamic implications., (© 2021. European Biophysical Societies' Association.)

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D 2 O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Chronic exposure and accumulation of persistent nanomaterials by cells have led to safety concerns on potential long-term effects induced by nanoparticles, including chronic inflammation and fibrosis. With this in mind, we used murine precision-cut liver tissue slices to test potential induction of inflammation and onset of fibrosis upon 72 h exposure to different nanomaterials (0-200 µg/ml). Tissue slices were chosen as an advanced ex vivo 3D model to better resemble the complexity of the in vivo tissue environment, with a focus on the liver where most nanomaterials accumulate. Effects on the onset of fibrosis and inflammation were investigated, with particular care in optimizing nanoparticle exposure conditions to tissue. Thus, we compared the effects induced on slices exposed to nanoparticles in the presence of excess free proteins (in situ), or after corona isolation. Slices exposed to daily-refreshed nanoparticle dispersions were used to test additional effects due to ageing of the dispersions. Exposure to amino-modified polystyrene nanoparticles in serum-free conditions led to strong inflammation, with stronger effects with daily-refreshed dispersions. Instead, no inflammation was observed when slices were exposed to the same nanoparticles in medium supplemented with serum to allow corona formation. Similarly, no clear signs of inflammation nor of onset of fibrosis were detected after exposure to silica, titania or carboxylated polystyrene in all conditions tested. Overall, these results show that liver slices can be used to test nanoparticle-induced inflammation in real tissue, and that the exposure conditions and ageing of the dispersions can strongly affect tissue responses to nanoparticles.

Warfarin is a coumarin derivative drug widely used for its anticoagulant properties. The interaction of warfarin with fully hydrated lipid bilayers has been studied by combining differential scanning calorimetry, spectrophotometry, electron spin resonance of chain-labelled lipids and molecular docking. Bilayers formed by lipids with different chemico-physical properties were considered, namely dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), and dioleoyltrimethyl-ammoniumpropane (DOTAP). We observed in all cases the binding of warfarin in proximity of the surface of the bilayers, leading to a variety of distinct effects on key molecular properties of the membranes. The drug associates with the lipid bilayers in the deprotonated open chain form, with an association constant similar for DMPC and DMPG (1.27·10 4 and 2.82·10 4  M -1 , respectively) and lower for DOTAP (0.46·10 4  M -1 ). In DMPC bilayers, which are zwitterionic and with saturated symmetrical chains, warfarin at 10 mol% suppresses the pre-transition, slightly stabilizes the fluid state and reduces the cooperativity of the main transition. Moreover, it alters the lateral packing density of the chain segments close to the polar/apolar interface at any temperature through the gel phase. In anionic DMPG bilayers, the drug slightly perturbs the thermotropic phase behavior, and at 10 mol% markedly loosens the compact gel phase packing of the first chain segments. In cationic DOTAP bilayers, possessing unsaturated acyl chains, the drug induces a slightly higher degree of order and motional restriction in the outer hydrocarbon region in the frozen state. In all cases, as a surface adsorbed molecule, warfarin does not affect the segmental chain order and dynamics for temperatures in the fluid phase. The overall results provide an outline of the action of warfarin on membranes formed by lipids of different types., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Poly(ethylene glycol) (PEG)-grafted lipid dispersions are widely investigated in fundamental and biotechnological research for their successful use in drug-delivery. Here, we consider mixtures of the bilayer-forming lipid dipalmitoylphosphatidylcholine (DPPC) with the micelle-forming lipid PEG:2000-phosphatidilethanolamine (PEG:2000-DPPE) fully hydrated in D 2 O and measured at 77 K. Electron Spin Echo Envelope Modulation and continuous wave Electron Paramagnetic Resonance of chain-labelled lipids are employed to detect the extent of solvent permeation and the environmental polarity, respectively, across the hydrocarbon regions of the lipid assemblies. Sigmoidal water penetration and polarity profiles are described in sterically stabilized liposomes (SSL) formed at submicellar content of PEG:2000-DPPE incorporated in DPPC. Compared to DPPC bilayers, SSL show increased hydrophobicity at both the polar/apolar interface and the chain termini, and a broader transition that is shifted toward the interface. Solvent exposure and polarity decrease on going down the chain in PEG:2000-DPPE micelles. However, compared to SSL, polymer-lipid micelles show higher solvent permeation at any chain segment and the chain termini are accessible to water. In any sample, heterogeneity is found in H-bond formation between the spin-label nitroxide groups and the solvent molecules. The results at cryogenic temperature add new insights into the biophysico-chemical characterization of PEGylated lipid dispersions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To determine responses to nanoparticles in a more comprehensive way, current efforts in nanosafety aim at combining the analysis of multiple endpoints and comparing outcomes in different models. To this end, here we used tissue slices from mice as 3D ex vivo models and performed for the first time a comparative study of uptake and impact in liver, lung, and kidney slices exposed under the same conditions to silica, carboxylated and amino-modified polystyrene. In all organs, only exposure to amino-modified polystyrene induced toxicity, with stronger effects in kidneys and lungs. Uptake and distribution studies by confocal microscopy confirmed nanoparticle uptake in all slices, and, in line with what observed in vivo, preferential accumulation in the macrophages. However, uptake levels in kidneys were minimal, despite the strong impact observed when exposed to the amino-modified polystyrene. On the contrary, nanoparticle uptake and accumulation in macrophages were particularly evident in lung slices. Thus, tissue digestion was used to recover all cells from lung slices at different exposure times and to determine by flow cytometry detailed uptake kinetics in lung macrophages and all other cells, confirming higher uptake by the macrophages. Finally, the expression levels of a panel of targets involved in inflammation and macrophage polarization were measured to determine potential effects induced in lung and liver tissue. Overall, this comparative study allowed us to determine uptake and impact of nanoparticles in real tissue and identify important differences in outcomes in the organs in which nanoparticles distribute.

Much effort within the nanosafety field is currently focused on the use of advanced in vitro models to reduce the gap between in vitro and in vivo studies. Within this context, precision-cut tissue slices are a unique ex vivo model to investigate nanoparticle impact using live tissue from laboratory animals and even humans. However, several aspects of the basic mechanisms of nanoparticle interactions with tissue have not yet been elucidated. To this end, liver slices are exposed to carboxylated and amino-modified polystyrene known to have a different impact on cells. As observed in standard cell cultures, amino-modified polystyrene nanoparticles induce apoptosis, and their impact is affected by the corona forming on their surface in biological fluids. Subsequently, a detailed time-resolved study of nanoparticle uptake and distribution in the tissue is performed, combining fluorescence imaging and flow cytometry on cells recovered after tissue digestion. As observed in vivo, the Kupffer cells accumulate high nanoparticle amounts and, interestingly, they move within the tissue towards the slice borders. Similar observations are reproduced in liver slices from human tissue. Thus, tissue slices can be used to reproduce ex vivo important features of nanoparticle outcomes in the liver and study nanoparticle impact on real tissue., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)