Puncturing lipid membranes: onset of pore formation and the role of hydrogen bonding in the presence of flavonoids.

Products of lipid peroxidation induce detrimental structural changes in cell membranes, such as the formation of water pores, which occur in the presence of lipids with partially oxidized chains. However, the influence of another class of products, dicarboxylic acids, is still unclear. These products have greater mobility in the lipid bilayer, which enables their aggregation and the formation of favorable sites for the appearance of pores. Therefore, dodecanedioic acid (DDA) was selected as a model product. Additionally, the influence of several structurally different flavonoids on DDA aggregation via formation of hydrogen bonds with carboxyl groups was investigated. The molecular dynamics of DDA in DOPC lipid bilayer revealed the formation of aggregates extending over the hydrophobic region of the bilayer and increasing its polarity. Consequently, water penetration and the appearance of water wires was observed, representing a new step in the mechanism of pore formation. Furthermore, DDA molecules were found to interact with lipid polar groups, causing them to be buried in the bilayer. The addition of flavonoids to the system disrupted aggregate formation, resulting in the displacement of DDA molecules from the center of the bilayer. The placement of DDA and flavonoids in the lipid bilayer was confirmed by small-angle X-ray scattering. Atomic force microscopy and electron paramagnetic resonance were used to characterize the structural properties. The presence of DDA increased bilayer roughness and decreased the ordering of lipid chains, confirming its detrimental effects on the membrane surface, while flavonoids were found to reduce or reverse these changes.

Kinetics of Flavonoid Degradation and Controlled Release from Functionalized Magnetic Nanoparticles.

Flavonoids are naturally occurring antioxidants that have been shown to protect cell membranes from oxidative stress and have a potential use in photodynamic cancer treatment. However, they degrade at physiological pH values, which is often neglected in drug release studies. Kinetic study of flavonoid oxidation can help to understand the mechanism of degradation and to correctly analyze flavonoid release data. Additionally, the incorporation of flavonoids into magnetic nanocarriers can be utilized to mitigate degradation and overcome their low solubility, while the release can be controlled using magnetic fields (MFs). An approach that combines alternating least squares (ALS) and multilinear regression to consider flavonoid autoxidation in release studies is presented. This approach can be used in general cases to account for the degradation of unstable drugs released from nanoparticles. The oxidation of quercetin, myricetin (MCE), and myricitrin (MCI) was studied in PBS buffer (pH = 7.4) using UV-vis spectrophotometry. ALS was used to determine the kinetic profiles and characteristic spectra, which were used to analyze UV-vis data of release from functionalized magnetic nanoparticles (MNPs). MNPs were selected for their unique magnetic properties, which can be exploited for both targeted drug delivery and control over the drug release. MNPs were prepared and characterized by X-ray diffraction, infrared spectroscopy, scanning electron microscopy, superconducting quantum interference device magnetometer, and electrophoretic mobility measurements. Autoxidation of all three flavonoids follows a two-step first-order kinetic model. MCE showed the fastest degradation, while the oxidation of MCI was the slowest. The flavonoids were successfully loaded into the prepared MNPs, and the drug release was described by the first-order and Korsmeyer-Peppas models. External MFs were utilized to control the release mechanism and the cumulative mass of the flavonoids released.

Anti-Oxidative, Anti-Inflammatory and Anti-Apoptotic Effects of Flavonols: Targeting Nrf2, NF-κB and p53 Pathways in Neurodegeneration.

Neurodegenerative diseases are one of the leading causes of disability and death worldwide. Intracellular transduction pathways that end in the activation of specific transcription factors are highly implicated in the onset and progression of pathological changes related to neurodegeneration, of which those related to oxidative stress (OS) and neuroinflammation are particularly important. Here, we provide a brief overview of the key concepts related to OS- and neuroinflammation-mediated neuropathological changes in neurodegeneration, together with the role of transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-κB (NF-κB). This review is focused on the transcription factor p53 that coordinates the cellular response to diverse genotoxic stimuli, determining neuronal death or survival. As current pharmacological options in the treatment of neurodegenerative disease are only symptomatic, many research efforts are aimed at uncovering efficient disease-modifying agents. Natural polyphenolic compounds demonstrate powerful anti-oxidative, anti-inflammatory and anti-apoptotic effects, partially acting as modulators of signaling pathways. Herein, we review the current understanding of the therapeutic potential and limitations of flavonols in neuroprotection, with emphasis on their anti-oxidative, anti-inflammatory and anti-apoptotic effects along the Nrf2, NF-κB and p53 pathways. A better understanding of cellular and molecular mechanisms of their action may pave the way toward new treatments.

The Fine-Tuned Release of Antioxidant from Superparamagnetic Nanocarriers under the Combination of Stationary and Alternating Magnetic Fields.

Superparamagnetic magnetite nanoparticles (MNPs) with excellent biocompatibility and negligible toxicity were prepared by solvothermal method and stabilized by widely used and biocompatible polymer poly(ethylene glycol) PEG-4000 Da. The unique properties of the synthesized MNPs enable them to host the unstable and water-insoluble quercetin as well as deliver and localize quercetin directly to the desired site. The chemical and physical properties were validated by X-ray powder diffraction (XRPD), field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), superconducting quantum interference device (SQUID) magnetometer, FTIR spectroscopy and dynamic light scattering (DLS). The kinetics of in vitro quercetin release from MNPs followed by UV/VIS spectroscopy was controlled by employing combined stationary and alternating magnetic fields. The obtained results have shown an increased response of quercetin from superparamagnetic MNPs under a lower stationary magnetic field and s higher frequency of alternating magnetic field. The achieved findings suggested that we designed promising targeted quercetin delivery with fine-tuning drug release from magnetic MNPs.

Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper.

Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration.

Flavonol clustering in model lipid membranes: DSC, AFM, force spectroscopy and MD simulations study.

We here report on flavonols (myricetin (MCE) and its glycoside myricitrin (MCI)) - 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane interactions focusing on the effects of flavonol clustering on the membrane thermotropic and nanomechanical properties. Atomic force microscopy (AFM), force spectroscopy (FS) and differential scanning calorimetry (DSC) together with molecular dynamics (MD) simulations provided a consistent picture of flavonol - DMPC membrane interactions. DMPC membrane as a supported lipid bilayer preserved its integrity even at higher flavonol molar fraction x. When present at x = 0.1 - 0.3, MCE and MCI both slightly improve DMPC bilayer fluidity which is evidenced by the decrease in the main phase transition temperature Tm. MCE is found within the interior of the bilayer, while MCI incorporates in the head group-water interface region. AFM and FS confirmed clusters as protrusions with an average height of 0.012 µm and average diameters of 0.60 and 0.24 µm for MCE and MCI clusters, respectively. The average membrane thickness in DMPC fluid phase decreases for 7% at xMCE = 0.30, while only 4% at xMCI = 0.27. The induced membrane changes are dependent on the chemical and physical properties of inserted flavonols. The hypothesis regarding the tendency of flavonol to clustering in membranes by increasing flavonol molar fraction has been confirmed.

The Structural Integrity of the Model Lipid Membrane during Induced Lipid Peroxidation: The Role of Flavonols in the Inhibition of Lipid Peroxidation.

The structural integrity, elasticity, and fluidity of lipid membranes are critical for cellular activities such as communication between cells, exocytosis, and endocytosis. Unsaturated lipids, the main components of biological membranes, are particularly susceptible to the oxidative attack of reactive oxygen species. The peroxidation of unsaturated lipids, in our case 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), induces the structural reorganization of the membrane. We have employed a multi-technique approach to analyze typical properties of lipid bilayers, i.e., roughness, thickness, elasticity, and fluidity. We compared the alteration of the membrane properties upon initiated lipid peroxidation and examined the ability of flavonols, namely quercetin (QUE), myricetin (MCE), and myricitrin (MCI) at different molar fractions, to inhibit this change. Using Mass Spectrometry (MS) and Fourier Transform Infrared Spectroscopy (FTIR), we identified various carbonyl products and examined the extent of the reaction. From Atomic Force Microscopy (AFM), Force Spectroscopy (FS), Small Angle X-Ray Scattering (SAXS), and Electron Paramagnetic Resonance (EPR) experiments, we concluded that the membranes with inserted flavonols exhibit resistance against the structural changes induced by the oxidative attack, which is a finding with multiple biological implications. Our approach reveals the interplay between the flavonol molecular structure and the crucial membrane properties under oxidative attack and provides insight into the pathophysiology of cellular oxidative injury.

Enhanced Protection of Biological Membranes during Lipid Peroxidation: Study of the Interactions between Flavonoid Loaded Mesoporous Silica Nanoparticles and Model Cell Membranes.

Flavonoids, polyphenols with anti-oxidative activity have high potential as novel therapeutics for neurodegenerative disease, but their applicability is rendered by their poor water solubility and chemical instability under physiological conditions. In this study, this is overcome by delivering flavonoids to model cell membranes (unsaturated DOPC) using prepared and characterized biodegradable mesoporous silica nanoparticles, MSNs. Quercetin, myricetin and myricitrin have been investigated in order to determine the relationship between flavonoid structure and protective activity towards oxidative stress, i.e., lipid peroxidation induced by the addition of hydrogen peroxide and/or Cu2+ ions. Among investigated flavonoids, quercetin showed the most enhanced and prolonged protective anti-oxidative activity. The nanomechanical (Young modulus) measurement of the MSNs treated DOPC membranes during lipid peroxidation confirmed attenuated membrane damage. By applying a combination of experimental techniques (atomic force microscopy-AFM, force spectroscopy, electrophoretic light scattering-ES and dynamic light scattering-DLS), this work generated detailed knowledge about the effects of flavonoid loaded MSNs on the elasticity of model membranes, especially under oxidative stress conditions. Results from this study will pave the way towards the development of innovative and improved markers for oxidative stress-associated neurological disorders. In addition, the obtained could be extended to designing effective delivery systems of other high potential bioactive molecules with an aim to improve human health in general.