Erythrocyte Ghost Membranes Research Articles

Berberine mediated fluorescent gold nanoclusters in biomimetic erythrocyte ghosts as a nanocarrier for enhanced photodynamic treatment.

Photodynamic therapy (PDT) is a well-established cancer treatment method that employs light to generate reactive oxygen species (ROS) causing oxidative damage to cancer cells. Nevertheless, PDT encounters challenges due to its oxygen-dependent nature, which makes it less effective in hypoxic tumor environments. To address this issue, we have developed a novel nanocomposite known as AuNC@BBR@Ghost. This nanocomposite combines the advantageous features of erythrocyte ghost membranes, the photoresponsive properties of gold nanoclusters (AuNC) and the anticancer characteristics of Berberine (BBR) for cancer treatment. Our synthesized AuNC efficiently produce ROS, with a 25% increase in efficiency when exposed to near-infrared (NIR) irradiation. By harnessing the oxygen-carrying capacity of erythrocyte ghost cells, AuNC@BBR@Ghost demonstrates a significant improvement in ROS generation, achieving an 80% efficiency. Furthermore, the AuNC exhibit tunable emission wavelengths due to their excellent fluorescent properties. In normoxic conditions, treatment of A549 lung carcinoma cells with AuNC@BBR@Ghost followed by exposure to 808 nm NIR irradiation results in a notable increase in intracellular ROS levels, accelerating cell death. In hypoxic conditions, when A549 cells were treated with AuNC@BBR@Ghost, the erythrocyte ghost acted as an oxygen supplement due to the residual hemoglobin, alleviating hypoxia and enhancing the nanocomposite's sensitivity to PDT treatment. Thus, the AuNC@BBR@Ghost nanocomposite achieves an improved effect by combining the advantageous properties of its individual components, resulting in enhanced ROS generation and adaptability to hypoxic conditions. This innovative approach successfully overcomes PDT's limitations, making AuNC@BBR@Ghost a promising nanotheranostic agent with significant potential for advanced cancer therapy.

Role of Plasma Membrane at Dielectric Relaxations and Intermembrane Interaction in Human Erythrocytes.

Dielectric relaxations at 1.4 MHz (βsp) and 9 MHz (γ1sp) on the erythrocyte spectrin network were studied by dielectric spectroscopy using dense suspensions of erythrocytes and erythrocyte ghost membranes, subjected to extraction with up to 0.2% volume Triton-X-100. The step-wise extraction of up to 60% of membrane lipids preserved γ1sp and gradually removed βsp-relaxation. On increasing the concentration up to 100 mM of NaCl at either side of erythrocyte plasma membranes, the βsp-relaxation was linearly enhanced, while the strength of γ1sp-relaxation remained unchanged. In media with NaCl between 100 and 150 mM βsp-relaxation became slightly inhibited, while γ1sp-relaxation almost disappeared, possibly due to the decreased electrostatic repulsion allowing erythrocytes to come into closer contact. When these media contained, at concentrations 10-30 mg/mL dextran (MW 7 kDa), polyethylene glycol or polyvinylpyrrolidone (40 kDa), or albumin or homologous plasma with equivalent concentration of albumin, the γ1sp-relaxation was about tenfold enhanced, while βsp-relaxation was strengthened or preserved. The results suggest the Maxwell-Vagner accumulation of ions on the lipid bilayer as an energy source for βsp-relaxation. While βsp-relaxation appears sensitive to erythrocyte membrane deformability, γ1sp-relaxation could be a sensitive marker for the inter-membrane interactions between erythrocytes.

Biointeraction of Erythrocyte Ghost Membranes with Gold Nanoparticles Fluorescents.

The application of new technologies for treatments against different diseases is increasingly innovative and effective. In the case of nanomedicine, the combination of nanoparticles with biological membranes consists of a “camouflage” technique, which improves biological interaction and minimizes the secondary effects caused by these remedies. In this work, gold nanoparticles synthesized by chemical reduction (Turkevich ≈13 nm) were conjugated with fluorescein isothiocyanate to amplify their optical properties. Fluorescent nanoparticles were deposited onto the surface of hemoglobin-free erythrocytes. Ghost erythrocytes were obtained from red blood cells by density gradient separation in a hypotonic medium and characterized with fluorescence, optical, and electron microscopy; the average size of erythrocyte ghosts was 9 µm. Results show that the functional groups of sodium citrate (COO-) and fluorophore (-N=C=S) adhere by electrostatic attraction to the surface of the hemoglobin-free erythrocyte membrane, forming the membrane–particle–fluorophore. These interactions can contribute to imaging applications, by increasing the sensitivity of measurement caused by surface plasmon resonance and fluorescence, in the context of biological membranes.

The Synergy of Membranotropic Effect of the Two Pla2 Fractions of Macrovipera lebetina obtusa Venom on the Model Membranes.

It is known that snake venoms are a complex of enzymes and proteins and the interaction of different venom components with the membranes could be significantly enhanced in course of their action in an orchestra. The aim of the proposed investigation is to obtain detailed information about the mechanism and topology of two snake venom PLA2 isoforms from the Macrovipera lebetina obtusa venom in the membrane-binding process. We investigated the impact of the interaction on the properties of the model membrane (namely, GUVs and erythrocytes ghost) for each of these isoforms, as well as their synergetic action if they act simultaneously. The 6-lauroyl-2-dimethylaminonaphthalene and 6-propionyl-2-dimethylaminonaphthalene fluorescence probes were used to allow us to determine the membrane polarity more accurately via a generalized polarization function. Our results show that two types of PLA2 bring viscosity reduction in GUVs membrane and the effect became more potent when these PLA2 acts together. Intriguingly, we have not observed any significant difference in the case of the erythrocytes ghost membrane.

Novel Fast and Reliable Method for Nano-Erythrosome Production Using Shear Force.

PurposeThe production of nano-erythrosomes (NEs) by extrusion, which is considered the “gold standard”, has several disadvantages such as difficult equipment assembly, long procedure time, variable pressure, and problems with sterility. An alternative approach, using ultrasound probe, has been shown to overheat the sample and have suboptimal results compared to the extrusion method. In our study, we propose, develop, and test a new method for the fabrication of NEs based on shear force and then compare it to the “gold standard” extrusion approach.MethodsThe new method consists of mechanical shear force disruption of the hemoglobin-depleted erythrocyte ghost membranes, with the aid of a rotor stator based tissue homogenizer. Using the same batches of erythrocyte ghost membranes, we compared NEs produced by shear force to NEs produced by the well-established extrusion approach. NEs were characterized for yield, size, encapsulation efficiency, morphology, and stability by flow cytometry (FC), transmission electron microscopy (TEM), and zeta potential analysis.ResultsThe shear force based process was easier to set up, significantly faster, had better sterility control, and decreased variability between batches. The shear force method generated NEs with the desired size distribution (particles diameter ~125 nm), which were morphologically and functionally equivalent to the NEs produced by extrusion. NEs produced by shear force were stable in terms of counts, size, and fluorescence intensity for 3 weeks at +4°C. Moreover, they showed colloidal stability and minimal influence to centrifugal stress, turbulence shock, and hemolytic potential.ConclusionThe newly proposed shear force method allows faster, easier, and highly reproducible NEs production when compared to the conventional extrusion approach. The new setup allows simultaneous production of sterile batches of NEs, which have homogenous size distribution, good stability, and improved shelf life storage. The ability of the shear force method to process also high concentration samples indicates a future potential development of large-scale NEs production and industrial application, which has been a challenge for the extrusion method.

Endocytosis and exocytosis processes of gold nanoparticle with erythrocyte ghosts

AbstractThe interaction of spherical gold nanoparticles (AuNPs) of 20nm elaborated by Turkevich method with the erythrocytes ghosts (7-8 μm) cell membrane was evaluated. The AuNPs-Membrane interaction was determined using confocal microscope, Uv-Vis spectroscopy and SEM analysis. The result show that nanoparticles larger than 20nm are adhered to the erythrocyte ghost membrane due their size and surface modification. Smaller AuNPs enter onto the cell by simple diffusion through the plasmatic membrane voids, these data may favor the best design and application in the treatments applied in biomedicine.

Interactions of amphipathic α-helical MEG proteins from Schistosomamansoni with membranes

Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole–mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane–active peptides.

Probing lipids in biological membranes using SERS

A selective and sensitive experimental approach to the examination of lipids as well as lipid–protein interactions in biological membranes using surface-enhanced Raman spectroscopy has been proposed. This approach has been used for probing the intact mitochondria, membranes of erythrocyte ghosts and liposomes.

Thermal sensitivity and haemolysis of erythrocytes with membranopathy

Measuring the impedance of heated suspensions of erythrocytes and erythrocyte ghost membranes, two thermally-induced alterations are registered in the plasma membrane at TA (denaturation of spectrin with inducing temperature at 49,5 °C) and TG (hyperthermic activation of basal ion permeability with inducing temperature at 60.7 °C). In this study erythrocytes from 9 healthy patients and 15 patients with hemolytic anemia were studied and divided into four groups depending on their TA and TG top temperatures. The TA and TG of erythrocytes with hemoglobinopathy were the same as those of control erythrocytes while those of erythrocytes with membranopathy were significantly reduced. In erythrocytes with severe membranopathy, the TG was decreased by about 5 °C. In latter cells the normal value of TG was restored and the resistance to thermal haemolysis was increased by 90% after the specific stabilization of band 3 protein by 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS). Obtained results indicate the involvement of band 3 in the membrane alteration at TG and in the heat target responsible for thermal haemolysis.

Effects of heat and freeze on isolated erythrocyte submembrane skeletons.

In this study we heated insoluble residues, obtained after Triton-X-100 (0.1 v/v%) extraction of erythrocyte ghost membranes (EGMs). Specific heat capacity, electric capacitance and resistance, and optical transmittance (280 nm) sustained sharp changes at 49°C (TA) and 66°C (TC), the known denaturation temperatures of spectrin and band 3, respectively. The change at TA was selectively inhibited by diamide (1 mM) and taurine mustard (1 mM) while its inducing temperature was selectively decreased by formamide in full concert with the assumed involvement of spectrin denaturation. In the residues of EGMs, pretreated with 4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid (DIDS), the change at TC was shifted from 66 to 78°C which indicated the involvement of band 3 denaturation. The freeze and rapid thaw of EGM residues resulted in a strong reduction of cooperativity of band 3 denaturation while the slow thaw completely eliminated the peak of this denaturation. These effects of freeze-thaw were prevented in residues obtained from DIDS-treated EGMs. The freeze-thaw of residues slightly affected spectrin denaturation at 49°C although an additional denaturation appeared at 55°C. The results indicate preserved molecular structure and dynamics of the membrane skeleton in Triton-X-100 extracts of EGMs. The freeze-thaw inflicted strong damage on band 3 and spectrin-actin skeleton of EGM extracts which is relevant to cryobiology, cryosurgery and cryopreservation of cells.

MOLECULAR EVENTS ASSOCIATED WITH VIPERA LATIFI VENOM EFFECT ON CONDITION OF HUMAN RED BLOOD CELLS

Viper venom as a hemolytic biochemical “cocktail” of toxins, primarily cause to the systemic alteration of blood cells. In the sixties and seventies, human erythrocytes were extensively studied, but the mechanical and chemical stresses commonly exerted on red blood cells continue to attract interest of scientists for the study of membrane structure and function. Here we monitor the effect of Vipera latifi venom on human erythrocytes ghost membranes using phase contrast microscopy and changes in ATPase activity under snake venom influence in vitro. The ion pumps (Na+, K+)-ATPase and (Ca2+, Mg2+)-ATPase plays a pivotal role in the active transport of certain cations and maintenance of intracellular electrolyte homeostasis. We also describe the action of Vipera latifi venom on the freeradical processes in the membrane of erythrocyte ghosts and changes of activity of superoxide dismutase in course of envenomation.

Dielectric relaxations on erythrocyte membrane as revealed by spectrin denaturation

We studied the effect of spectrin denaturation at 49.5°C (TA) on the dielectric relaxations and related changes in the complex impedance, Z*, complex capacitance, C*, and dielectric loss curve of suspensions containing human erythrocytes, erythrocyte ghost membranes (EMs) and Triton-X-100 residues of EMs. The loss curve prior to, minus the loss curve after TA, resulted in a bell-shaped peak at 1.5MHz. The changes in the real and imaginary components of Z* and C* at TA, i.e., ΔZre, ΔZim, ΔCre and ΔCim, calculated in the same way, strongly varied with frequency. Between 1.0 and 12MHz the −ΔZim vs ΔZre, and ΔCim vs ΔCre plots depicted semicircles with critical frequencies, fcr, at 2.5MHz expressing recently reported relaxation of spectrin dipoles. Between 0.02 and 1.0MHz the −ΔZim vs ΔZre plot exhibited another relaxation whose fcr mirrored that of beta relaxation. This relaxation was absent on Triton-X-shells, while on erythrocytes and EMs it was inhibited by selective dissociation of either attachment sites between spectrin and bilayer. Considering above findings and inaccessibility of cytosole to outside field at such frequencies, the latter relaxation was assumed originating from a piezoelectric effect on the highly deformable spectrin filaments.

The Influence of Fatty Acid Methyl Esters (FAMEs) in the Biochemistry and the Na(+)/K(+)-ATPase Activity of Culex quinquefasciatus Larvae.

Culex quinquefasciatus is the main vector of lymphatic filariasis and combating this insect is of great importance to public health. There are reports of insects that are resistant to the products currently used to control this vector, and therefore, the search for new products has increased. In the present study, we have evaluated the effects of fatty acid methyl esters (FAMEs) that showed larvicidal activity against C. quinquefasciatus, on glucose, total protein, and triacylglycerol contents and Na(+)/K(+)-ATPase activity in mosquito larvae. The exposure of the fourth instar larvae to the compounds caused a decrease in the total protein content and an increase in the activity of the Na(+)/K(+)-ATPase. Furthermore, the direct effect of FAMEs on cell membrane was assessed on purified pig kidney Na(+)/K(+)-ATPase membranes, erythrocyte ghost membranes, and larvae membrane preparation. No modifications on total phospholipids and cholesterol content were found after FAMEs 20min treatment on larvae membrane preparation, but only 360µg/mL FAME 2 was able to decrease total phospholipid of erythrocyte ghost membrane. Moreover, only 60 and 360µg/mL FAME 3 caused an activation of purified Na(+)/K(+)-ATPase, that was an opposite effect of FAMEs treatment in larvae membrane preparation, and caused an inhibition of the pump activity. These data together suggest that maybe FAMEs can modulate the Na(+)/K(+)-ATPase on intact larvae for such mechanisms and not for a direct effect, one time that the direct effect of FAMEs in membrane preparation decreased the activity of Na(+)/K(+)-ATPase. The biochemical changes caused by the compounds were significant and may negatively influence the development and survival of C. quinquefasciatus larvae.

Antioxidant Properties of ADJ6 and ITS Effect on Isolated Human Erythrocyte Ghost Membrane

Introduction: Oxidative stress is one of the major causes for many adverse complications especially in disorders such as diabetes, cardiovascular diseases, cancer, etc. Many studies have proven that erythrocytes are affected during these conditions and oxidative stress plays a critical role in damaging the membrane integrity of erythrocytes by inducing lipid peroxidation causing instability to membrane in turn affecting the ion transport mechanisms. Objective and methods: The present study focuses to study the ameliorative effect of ADJ6 against oxidative stress on human erythrocytes. The membrane stabilization activity, Inhibition of lipid peroxidation and the effect against Ouabain induced Na+ K+ ATPase activity was assessed. Further, in vitro antioxidant activity of ADJ6 was also determined. Results and Discussion: DPPH radical scavenging activity, metal chelating and H2O2 radical scavenging activity were IC50=37.9 ± 0.22 μg/ml, IC50=53.95 ± 0.337 μg/ml and IC50=69.5 ± 0.628 μg/ml respectively. ADJ6 showed an EC50=90.83 ± 1.905 μg/ml against hypotonicity induced lysis of erythrocytes. H2O2 induced Lipid peroxidation of ghost membrane was also inhibited by ADJ6 (IC50=84.5 ± 2.613 μg/ml). Ouabain induced sodium potassium ATPase activity increased with the treatment of ADJ6 in a dose dependent manner. This proves that ADJ6 is effective in stabilizing the membrane potential of erythrocyte membrane. The scavenging activity in vitro also proves that ADJ6 is effective against free radicals. Conclusion: Though exact mechanism hitherto unknown, it is evident that ADJ6 stabilizes erythrocyte membrane activity. Further studies will be conducted in order to prove the mechanism of action of ADJ6 against of free radicals.

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca(2+)-ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein-undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l(-1), respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) activity, Ca(2+)/CaM activated EGM Ca(2+) ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca(2+)/CaM to activate EGM Ca(2+)-ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition.

Physicochemical characterization of artificial nanoerythrosomes derived from erythrocyte ghost membranes

Colloidal stabile nanoerythrosomes with 200nm average diameter were formed from hemoglobin-free erythrocyte ghost membrane via sonication and membrane extrusion. The incorporation of extra lipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), added to the sonicated ghosts, caused significant changes in the thermotropic character of the original membranes. As a result of the increased DPPC ratio the chain melting of the hydrated DPPC system and the characteristic small angle X-ray scattering (SAXS) of the lipid bilayers appeared. Significant morphological changes were followed by transmission electron microscopy combined with freeze fracture method (FF-TEM). After the ultrasonic treatment the large entities of erythrocyte ghosts transformed into nearly spherical nanoerythrosomes with diameters between 100 and 300nm and at the same time a great number of 10–30nm large membrane proteins or protein clusters were dispersed in the aqueous medium. The infrared spectroscopy (FT-IR) pointed out, that the sonication did not cause changes in the secondary structures of the membrane proteins under our preparation conditions. About fivefold of extra lipid – compared to the lipid content of the original membrane – caused homogeneous dispersion of nanoerythrosomes however the shape of the vesicles was not uniform. After the addition of about tenfold of DPPC, monoform and monodisperse nanoerythrosomes became typical. The outer surfaces of these roughly spherical objects were frequently polygonal, consisting of a net of pentagons and hexagons.

Label-free spectroscopic detection of membrane potential using stimulated Raman scattering

Hyperspectral stimulated Raman scattering microscopy is deployed to measure single-membrane vibrational spectrum as a function of membrane potential. Using erythrocyte ghost as a model, quantitative correlation between transmembrane potential and Raman spectral profile was found. Specifically, the ratio between the area under Raman band at ∼2930 cm−1 and that at ∼2850 cm−1 increased by ∼2.6 times when the potential across the erythrocyte ghost membrane varied from +10 mV to −10 mV. Our results show the feasibility of employing stimulated Raman scattering microscopy to probe the membrane potential without labeling.

Interaction of shungite carbon nanoparticles with blood protein and cell components

We have analyzed and compared the effects of aqueous dispersion of shungite carbon nanoparticles, fullerene C60, and nanodiamonds on structure, dynamics, and thermodynamic and redox properties of blood proteins (serum albumin and hemoglobin), proteins of erythrocyte ghost membranes as well as on erythrocyte integrity and aggregation.. All the nanomaterials dispersions have induced similar effects; however, nanodiamonds have not influences the redox properties. Basing on the results, the experimental and theoretical approaches presented can be employed to estimate the effects of biological structures contact with the nanoparticles on the bioreactivity.

Synthesis and Evaluation of Antioxidant Properties of Novel 1,2,4‐Triazole‐Based Schiff Base Heterocycles

A series of 1,2,4-triazole-based Schiff base heterocyclic compounds (5a-f and 8a-i) and phenethylamines (7a-h) were synthesized and evaluated for antioxidant properties by free-radical scavenging, anti-hemolytic activity, lipid peroxidation, and their protective effects against DNA oxidative damage. Compounds 7c, 7d, 7h, 8b, and 8i showed promising DPPH(•) radical scavenging activity with the level of inhibition between 86.8% and 94%. Compounds 8a, 8b, 8d, 8g, and 8i were effective against the oxidative hemolysis of human erythrocytes and lipid peroxidation, in a dose-dependent manner, with IC50 values in the range of 55.7-80.7 and 53.2-81.2 µg/mL, respectively. Compounds 8a and 8b were effective against oxidative damage on erythrocyte ghost membrane proteins, and 8g and 8i were able to protect against DNA oxidative damage.

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.