Search Results (92)

Curcumin, a natural polyphenol derived from Curcuma longa, is widely recognized for its therapeutic properties. However, its clinical utility is limited because of poor solubility, rapid degradation and hence low bioavailability. To overcome these issues, nanoformulation approaches, especially PEGylated liposomes, have been explored as advanced delivery systems. PEGylation, which involves attaching polyethylene glycol (PEG) to the liposomal surface, enhances circulation time by creating a steric shield that reduces protein interactions and clearance by the mononuclear phagocyte system (MPS). However, PEG can alter lipid membrane properties, which may in turn affect curcumin’s solubility and distribution within the liposomal bilayer, ultimately reducing its loading efficiency. To ensure that PEG-modified liposomes can be effectively loaded with curcumin, we investigated curcumin–membrane interactions in saturated (DMPC) and unsaturated (POPC) liposomes, both in the presence and absence of PEG. Based on dissociation constants (K_d) obtained from fluorescence spectroscopy measurements, we found that PEGylated DMPC liposomes exhibit the strongest binding affinity for curcumin. Fluorescence quenching experiments showed that curcumin adopts a transbilayer orientation in all membranes examined. Curcumin’s location within PEGylated and non-PEGylated liposomal membranes was further confirmed by examining its effects on membrane properties, including fluidity, polarity, and oxygen transport. These effects were investigated using electron paramagnetic resonance (EPR) spectroscopy with spin labels. The results indicate that PEG does not impose major changes on membrane properties. Curcumin, however, was found to reinforce the liposomal membranes, increase their polarity, and reduce oxygen availability. Overall, the findings suggest that liposomes, particularly those composed of PEGylated DMPC, are effective vehicles for curcumin delivery. Full article

Ionotropic alginate-based hydrogelation by divalent metal interaction has been employed to study the effect that different types of ions might have on gel formation. In this regard, EPR and IR spectroscopies, as well as rheology techniques, have been used to evaluate the influence of divalent cations on gel formation, and at the same time to assess host–guest interactions. Alginate was functionalized with TEMPO moieties; therefore, TEMPO-alginate system was taken as a reference. The novelty of this study consists of using a mixture of adamantyl-TEMPO-functionalized alginate and β-cyclodextrin linked through 1,3-diaminopropane to assess the host–guest interactions in functionalized gels. The properties of divalent cations considered in this study (Ba²⁺, Ca²⁺, Sr²⁺, Zn²⁺) were proven by changes in spectral parameters of paramagnetic moieties, while the viscoelastic moduli as functions of shear strain and frequency were evaluated through rheology measurements. Overall, the information obtained from these investigations has shown that the properties of the alginate gels are influenced both by the type of divalent cation used for complexation and by the host–guest interactions. The results show that the type of the cation significantly affects gel strength; therefore, Ba²⁺ forms the strongest gel, while Zn²⁺ the least resistant. Additionally, a high immobilization of the spin-labeled probes has been obtained by the addition of tosylated β-cyclodextrin in the alginate gel network containing Ba²⁺ ions. Full article

The cycloaddition reaction is one of the most common reactions in organic chemistry. It has been applied in various fields. Herein, we focus on the application of cycloaddition reactions in investigating biological molecules and materials using magnetic resonance techniques. To facilitate magnetic resonance studies such as electron paramagnetic resonance (EPR) spectroscopy and paramagnetic nuclear magnetic resonance (NMR) spectroscopy, there is often a requirement to attach spin labels and paramagnetic tags to the system of interest. The cycloaddition reaction is one of the ways to tether these spin labels and paramagnetic tags. In this review, we highlight the applications of various cycloaddition reactions such as the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction, the strain-promoted azide–alkyne cycloaddition (SPAAC) reaction and the Diels–Alder reaction in the interdisciplinary field of magnetic resonance studies of biomolecules, including proteins, nucleic acids, carbohydrates, lipids and glycans, as well as materials. Full article

We report a systematic investigation on the physicochemical properties of polymer systems consisting of polyvinyl alcohol (PVA) and hyaluronic acid (HA) mixed in various volume ratios (1/4, 2/3, 1/1, 3/2, and 4/1). At PVA/HA ratios above 1/1, in the presence of glutaraldehyde and divinyl sulfone as crosslinking agents, hydrogels are formed. Their swelling behavior is dependent on the polymer ratio, with the highest water uptake determined for PVA/HA 4/1. The in situ generation of reactive oxygen species (HO^• radicals) under UV-A irradiation, in the presence of riboflavin as a photoinitiator, is evidenced by electron paramagnetic resonance (EPR) spectroscopy. The diffusion of small paramagnetic molecules across the interface of two PVA/HA 4/1 gel pieces placed in direct contact reveals the occurrence of molecular exchange, which could indicate some degree of self-repair of the hydrogel network. When the paramagnetic moiety is attached to the HA polymer by spin labeling, the absence of diffusion demonstrates the stability of the crosslinked HA chains within the PVA/HA network. The structural modifications induced by crosslinking, by the presence of riboflavin, and by exposure to UV-A light, and the resulting alterations in the mechanical behavior of the hydrogels are monitored by infrared spectroscopy and rheology. Only a slight decrease in the viscoelastic moduli values is noted, indicating that the formation of HO^• radicals has minimal impact on the macroscopic properties of the hydrogels. Full article

Electron paramagnetic resonance (EPR) spectroscopy is gaining increasing recognition in research on various nanostructures. In the case of iron oxide nanoparticles, EPR measurements offer the possibility of determining the magnetic phase and the exact type (Fe₃O₄, γ-Fe₂O₃, α-Fe₂O₃, or a combination) of the core material. Furthermore, the EPR technique enables the study of relaxation processes, estimation of the effective and surface anisotropy constants, and assessment of the influence of sample aging on the magnetic properties of nanoparticles. The scope of the information obtained can be further expanded by utilizing spin labeling of polymer-coated nanoparticles. By analyzing the signals from the attached nitroxide, one can determine certain properties of the coating and its interactions with the environment (e.g., body fluids, cells, tissues) and also perform imaging of nanoparticles in various media. In some cases, EPR can help monitor the encapsulation of active substances and their release processes. Unfortunately, despite the enormous potential, not all of the possibilities offered by EPR are routinely used in nanoscience. Therefore, the present article aims not only to present the current applications and existing trends but also to indicate directions for future EPR research, constituting a road map. Full article

Nitroxides are stable organic free radicals with a wide range of applications. They have found applications in chemistry, biochemistry, biophysics, molecular biology, and biomedicine as EPR/NMR imaging techniques. As spin labels and probes, they are used in electron paramagnetic resonance (EPR) spectroscopy in the study of proteins, lipids, nucleic acids, and enzymes, as well as for measuring oxygen concentration in cells and cellular organelles, as well as tissues and intracellular pH. Their unique redox properties have allowed them to be used as exogenous antioxidants. In this review, we have discussed the chemical properties of nitroxides and their antioxidant properties. Furthermore, we have considered their use as radioprotectors and protective agents in ischemia/reperfusion in vivo and in vitro. We also presented other applications of nitroxides in protecting cells and tissues from oxidative stress and in protein studies and discussed their use in EPR/MRI. Full article

Studying membrane proteins in a native environment is crucial to understanding their structural and/or functional studies. Often, widely accepted mimetic systems have limitations that prevent the study of some membrane proteins. Micelles, bicelles, and liposomes are common biomimetic systems but have problems with membrane compatibility, limited lipid composition, and heterogeneity. To overcome these limitations, polymersomes and hybrid vesicles have become popular alternatives. Polymersomes form from amphiphilic triblock or diblock copolymers and are considered more robust than liposomes. Hybrid vesicles are a combination of lipids and block copolymers that form vesicles composed of a mixture of the two. These hybrid vesicles are appealing because they have the native lipid environment of bilayers but also the stability and customizability of polymersomes. Gramicidin A was incorporated into these polymersomes and characterized using continuous wave electron paramagnetic resonance (CW-EPR) and transmission electron microscopy (TEM). EPR spectroscopy is a powerful biophysical technique used to study the structure and dynamic properties of membrane proteins in their native environment. Spectroscopic studies of gramicidin A have been limited to liposomes; in this study, the membrane peptide is studied in both polymersomes and hybrid vesicles using CW-EPR spectroscopy. Lineshape analysis of spin-labeled gramicidin A revealed linewidth broadening, suggesting that the thicker polymersome membranes restrict the motion of the spin label more when compared to liposome membranes. Statement of Significance: Understanding membrane proteins’ structures and functions is critical in the study of many diseases. In order to study them in a native environment, membrane mimetics must be developed that can be suitable for obtaining superior biophysical data quality to characterize structural dynamics while maintaining their native functions and structures. Many currently widely accepted methods have limitations, such as a loss of native structure and function, heterogeneous vesicle formation, restricted lipid types for the vesicle formation for many proteins, and experimental artifacts, which leaves rooms for the development of new biomembrane mimetics. The triblock and diblock polymersomes and hybrid versicles utilized in this study may overcome these limitations and provide the stability and customizability of polymersomes, keeping the biocompatibility and functionality of liposomes for EPR studies of membrane proteins. Full article

The N-terminal ectodomain of the influenza A M2 protein is a target for universal influenza vaccine development and novel antiviral strategies. Despite the significance of this domain, it is poorly understood and most structural studies of the M2 protein have disregarded the N-terminal ectodomain in their analyses. Here, we report conformational properties and describe insights into the membrane topology of sites along the N-terminal ectodomain. Full-length M2 protein is embedded in lipid bilayer nanodiscs and studied using site-directed spin labeling electron paramagnetic resonance spectroscopy. Results are consistent with a turn in the middle of the ectodomain that changes in proximity to the membrane surface upon the addition of cholesterol or the antiviral drug rimantadine. Similarly to other domains of M2 protein, lineshape analysis suggests that the N-terminal ectodomain can adopt multiple conformations. Full article

In this study, we comparatively analyzed the variable-temperature crystal structures for two isomorphous salts, [1-benzyl-4-aminopyridinium][M(mnt)₂] (M = Ni or Cu; mnt²⁻ = maleonitriledithiolate; labeled as APy-Ni or APy-Cu). Both salts crystallize in the triclinic P–1 space group at 296 K, comprising linear [M(mnt)₂]⁻ (M = Ni or Cu) tetramers. A magnetostructural phase transition occurs at T_C~190 K in S = ½ APy-Ni at ambient pressure, with a conversion of paramagnetic tetramers into nonmagnetic spin-paired dimers. The discontinuous alteration of cell parameters at T_C signifies the characteristic of first-order phase transition in APy-Ni. No such transition appears in the nonmagnetic APy-Cu within the same temperature vicinity, demonstrating the magnetic interactions promoting the structural phase transition in APy-Ni, which is further reinforced through a comparison of the lattice formation energy between APy-Ni and APy-Cu. The phase transition may bear a resemblance to the mechanisms typically observed in spin-Peierls systems. We further explored the magnetic and phase transition properties of APy-Ni under varying pressures. Significantly, T_C shows a linear increase with rising pressure within the range of 0.003–0.88 GPa, with a rate of 90 K GPa⁻¹, manifesting that the applied pressure promotes the transition from tetramer to dimer. Full article

The investigation and clarification of the properties of surface-functionalized superparamagnetic nanoparticles in a biological environment are key challenges prior to their medical applications. In the present work, electron paramagnetic resonance spectroscopy (EPR) combined with the spin labeling technique was utilized to better understand the behavior of nitroxides attached to magnetite nanoparticles dispersed in body fluid. EPR spectra of spin-labeled, silane-coated Fe₃O₄ nanoparticles in human serum and whole blood were recorded and analyzed for both room- and low-temperature values. In all cases, the obtained EPR signal consisted of a broad line from magnetite cores and a characteristic signal from the attached 4-Amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO). Even for liquid samples, the anisotropic components of magnetic tensors did not fully average out, which was reflected in the differences in the intensity of three narrow hyperfine lines from nitroxide. At 230 K the irregular slow-motion signal from the attached radical was also simulated using the EasySpin toolbox, which allowed to determine the parameters related to magnetic tensors and the dynamics of the spin label. The study showed that the anisotropy of the motion of the spin label 4-amino-TEMPO reflects its interactions with the surrounding medium and the manner of the attachment of the nitroxide to the surface of nanoparticles. Full article

The influence of bovine serum albumin (BSA) on collapsing poly(N-isopropylacrylamide) (PNIPAM) chains was studied with turbidimetry and spin probe and spin label electron paramagnetic resonance spectroscopy. An increased ratio of collapsed chains in aqueous solutions in the narrow temperature region near the LCST appeared in the presence of 2.5–10 wt% BSA. The spin probe EPR data indicate that the inner cavities of the BSA dimers are probably responsive to the capture of small hydrophobic or amphiphilic molecules, such as TEMPO nitroxyl radical. The observed features of the structure and dynamics of inhomogeneities of aqueous PNIPAM-BSA solutions, including their mutual influence on the behavior of the polymer and protein below the LCST, should be considered when developing and investigating PNIPAM-based drug delivery systems. Full article

Eye lens α-crystallin has been shown to become increasingly membrane-bound with age and cataract formation; however, to our knowledge, no studies have investigated the membrane interactions of α-crystallin throughout the development of cataracts in separated cortical membrane (CM) and nuclear membrane (NM) from single human lenses. In this study, four pairs of human lenses from age-matched male and female donors and one pair of male lenses ranging in age from 64 to 73 years old (yo) were obtained to investigate the interactions of α-crystallin with the NM and CM throughout the progression of cortical cataract (CC) and nuclear cataract (NC) using the electron paramagnetic resonance spin-labeling method. Donor health history information (diabetes, smoker, hypertension, radiation treatment), sex, and race were included in the data analysis. The right eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 1, NC: 2), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Similarly, left eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 2, NC: 3), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Analysis of α-crystallin binding to male and female eye lens CM and NM revealed that the percentage of membrane surface occupied (MSO) by α-crystallin increases with increasing grade of CC and NC. The binding of α-crystallin resulted in decreased mobility, increased order, and increased hydrophobicity on the membrane surface in male and female eye lens CM and NM. CM mobility decreased with an increase in cataracts for both males and females, whereas the male lens NM mobility showed no significant change, while female lens NM showed increased mobility with an increase in cataract grade. Our data shows that a 68 yo female donor (long-term smoker, pre-diabetic, and hypertension; grade 3 CC) showed the largest MSO by α-crystallin in CM from both the left and right lens and had the most pronounced mobility changes relative to all other analyzed samples. The variation in cholesterol (Chol) content, size and amount of cholesterol bilayer domains (CBDs), and lipid composition in the CM and NM with age and cataract might result in a variation of membrane surface mobility, membrane surface hydrophobicity, and the interactions of α-crystallin at the surface of each CM and NM. These findings provide insight into the effect of decreased Chol content and the reduced size and amount of CBDs in the cataractous CM and NM with an increased binding of α-crystallin with increased CC and NC grade, which suggests that Chol and CBDs might be a key component in maintaining lens transparency. Full article

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The K_a of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: K_a (αBc) ≈ K_a (αABc) > K_a (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation. Full article

The accumulation of proteins in filter membranes limits the efficiency of filtering technologies for cleaning wastewater. Efforts are ongoing to coat commercial filters with different materials (such as titanium dioxide, TiO₂) to reduce the fouling of the membrane. Beyond monitoring the desired effect of the retention of biomolecules, it is necessary to understand what the biophysical changes are in water-soluble proteins caused by their interaction with the new coated filter membranes, an aspect that has received little attention so far. Using spin-label electron paramagnetic resonance (EPR), aided with native fluorescence spectroscopy and dynamic light scattering (DLS), here, we report the changes in the structure and dynamics of bovine serum albumin (BSA) exposed to TiO₂ (P25) nanoparticles or passing through commercial polyvinylidene fluoride (PVDF) membranes coated with the same nanoparticles. We have found that the filtering process and prolonged exposure to TiO₂ nanoparticles had significant effects on different regions of BSA, and denaturation of the protein was not observed, neither with the TiO₂ nanoparticles nor when passing through the TiO₂-coated filter membranes. Full article

Several discoveries show that with age and cataract formation, β-crystallin binds with the lens membrane or associates with other lens proteins, which bind with the fiber cell plasma membrane, accompanied by light scattering and cataract formation. However, how lipids (phospholipids and sphingolipids) and cholesterol (Chol) influence β-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of β-crystallin to the membrane and the membrane’s physical properties (mobility, order, and hydrophobicity) with β-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of β_L-crystallin with a model of porcine lens-lipid (MPLL), model of mouse lens-lipid (MMLL), and model of human lens-lipid (MHLL) membrane with and without Chol. Our results show that β_L-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by β_L-crystallin and the binding affinity (K_a) of β_L-crystallin to the membranes followed trends: MMSO (MPLL) > MMSO (MMLL) > MMSO (MHLL) and K_a (MHLL) > K_a (MMLL) ≈ K_a (MPLL), respectively, in which the presence of Chol reduces the MMSO and K_a for all membranes. The mobility near the headgroup regions of the membranes decreases with an increase in the binding of β_L-crystallin; however, the decrease is more pronounced in the MPLL and MMLL membranes than the MHLL membrane. In the MPLL and MMLL membranes, the membranes become slightly ordered near the headgroup with an increase in β_L-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with β_L-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that β_L-crystallin binding creates a hydrophobic barrier for the passage of polar molecules, which supports the barrier hypothesis in cataract formation. However, in the presence of Chol, there is no significant increase in hydrophobicity with β_L-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation. Full article