Ghost Membranes Research Articles

Cryo-EM structures of the human band 3 transporter indicate a transport mechanism involving the coupled movement of chloride and bicarbonate ions.

The band 3 transporter is a critical integral membrane protein of the red blood cell (RBC), as it is responsible for catalyzing the exchange of bicarbonate and chloride anions across the plasma membrane. To elucidate the structural mechanism of the band 3 transporter, detergent solubilized human ghost membrane reconstituted in nanodiscs was applied to a cryo-EM holey carbon grid to define its composition. With this approach, we identified and determined structural information of the human band 3 transporter. Here, we present 5 different cryo-EM structures of the transmembrane domain of dimeric band 3, either alone or bound with chloride or bicarbonate. Interestingly, we observed that human band 3 can form both symmetric and asymmetric dimers with a different combination of outward-facing (OF) and inward-facing (IF) states. These structures also allow us to obtain the first model of a human band 3 molecule at the IF conformation. Based on the structural data of these dimers, we propose a model of ion transport that is in favor of the elevator-type mechanism.

Assessment of silver nanoparticles' antitumor effects: Insights into cell number, viability, and morphology of glioblastoma and prostate cancer cells

Silver nanoparticles (AgNPs) hold promise for cancer therapy. This study aimed to evaluate their impact on tumor and non-tumor cell number, viability, and morphology. Antitumor activity was tested on U-87MG (glioblastoma) and DU-145 (prostate cancer) cell lines. Treatment with AgNPs notably reached a reduction of U-87MG and DU-145 cell growth by 89.30% and 79.74%, respectively, resulting in slower growth rates. AgNPs induced DNA damage, evidenced by reduced nuclear area and DNA content via fluorescent image-based analyses. Conversely, HFF-1 non-tumor cells displayed no significant changes post-AgNPs exposure. Viability assays revealed substantial reductions in U-87MG and DU-145 cells (79% and 63% in MTT assays, 30% and 52.2% in high-content analyses), while HFF-1 cells exhibited lower sensitivity. Tumor cells had notably lower IC50 values than non-tumor cells, indicating selective susceptibility. Transmission electron microscopy (TEM) showed morphological changes post-AgNPs administration, including increased vacuoles, myelin figures, membrane ghosts, cellular extravasation, and membrane projections. The findings suggest the potential of AgNPs against glioblastoma and prostate cancer, necessitating further exploration across other cancer cell lines.

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.

Assignment of molecular origins of NOE signal at -3.5ppm in the brain.

Nuclear Overhauser enhancemen mediated saturation transfer effect, termed NOE (-3.5ppm), is a major source of CEST MRI contrasts at 3.5ppm in the brain. Previous phantom experiments have demonstrated that both proteins and lipids, two major components in tissues, have substantial contributions to NOE (-3.5ppm) signals. Their relative contributions in tissues are informative for the interpretation of NOE (-3.5ppm) contrasts that could provide potential imaging biomarkers for relevant diseases, which remain incompletely understood. Experiments on homogenates and supernatants of brain tissues collected from healthy rats, that could isolate proteins from lipids, were performed to evaluate the relative contribution of lipids to NOE (-3.5ppm) signals. On the other hand, experiments on ghost membranes with varied pH, and reconstituted phospholipids with different chemical compositions were conducted to study the dependence of NOE (-3.5ppm) on physiological conditions. Besides, CEST imaging on rat brains bearing 9L tumors and healthy rat brains was performed to analyze the causes of the NOE (-3.5ppm) contrast variations between tumors and normal tissues, and between gray matter and white matter. Our experiments reveal that lipids have dominant contributions to the NOE (-3.5 ppm) signals. Further analysis suggests that decreased NOE (-3.5ppm) signals in tumors and higher NOE (-3.5ppm) signals in white matter than in gray matter are mainly explained by changes in membrane lipids, rather than proteins. NOE (-3.5ppm) could be exploited as a highly sensitive MRI contrast for imaging membrane lipids in the brain.

CSL112 Increases Plasma Cholesterol Efflux Capacity and Reduces Erythrocyte Membrane Cholesterol Content in Blood Samples from Patients with Sickle Cell Disease

Background: Lipid homeostasis is abnormal in sickle cell disease (SCD) and contributes to elevated erythrocyte cholesterol content which has been implicated in sickling. The highly oxidative environment in blood from patients with SCD alters high-density lipoprotein (HDL) composition and impairs HDL particle remodeling. Specifically, it negatively impacts on the capacity of HDL particles to mediate apolipoprotein A-I (apoA-I) exchange and protect against inflammation. Low apoA-I levels and altered composition of HDL have been well-documented at steady state, associated with high risk features of SCD, including endothelial dysfunction and risk of pulmonary hypertension. The low levels and activity of apoA-I fall even further during vaso-occlusive events in SCD. This diminished pool of apoA-I activity may mediate reduced capacity of HDL to extract cholesterol from erythrocyte membranes and increase risk of vascular dysfunction. Purpose: In this study we aimed to determine whether plasma derived apoA-I (CSL112; apolipoprotein A-I (human)) could elevate the cholesterol efflux capacity (CEC) of plasma from SCD patients and thereby reduce erythrocyte membrane cholesterol content. Methods: All SCD subjects (n=14) had confirmed HbSS genotype except one who was with HbS/β0-thalassemia, healthy control subjects (n=13) were not race-matched. CSL112 was incubated with fresh blood derived from control subjects and SCD patients for 6 hours at 37°C. The cholesterol content of ghost membranes of isolated erythrocytes was measured. HDL particle size distribution was assessed by native gel electrophoresis followed by western blotting. The ex vivo capacity of apoB-depleted plasma to efflux cholesterol was determined using [3H]cholesterol-loaded RAW264.7 macrophages. Results: CEC of SCD plasma was attenuated (75.9 ± 7.4%, n=14) compared to controls (100%, n=13), with a predominant decrease of ATP binding cassette transporter A1 (ABCA1)-dependent CEC (77.7 ± 9.2% vs 100%, respectively). Consistently, SCD plasma showed a trend towards lower levels of pre-β1 HDL (78.5 ± 6.5%) as compared to controls (100 %). Incubation with CSL112 raised plasma levels of small HDL species and pre-β1 HDL, resulting in a more than 3.5 fold increase of CEC in both patients and controls. Treatment of SCD blood with CSL112 reduced erythrocyte cholesterol content to a greater extent compared to control blood (% reduction: 12.8% ± 3.1% in SCD, 6.3% ± 5.4% in controls). Conclusion: Our data suggest that reduced levels of lipid-poor pre-β1 HDL in SCD plasma reflect impaired HDL particle remodeling and account for a decreased CEC. CSL112 may acutely generate HDL species which optimally support ABCA1-dependent cholesterol efflux and thereby improve HDL function, normalize erythrocyte membrane cholesterol content and correct the imbalance in cellular cholesterol homeostasis in SCD patients. ApoA-I as a potential therapeutic could have implications in the pathobiologic understanding and future clinical management of vaso-occlusive crisis and of chronic vascualopathy such as pulmonary hypertension.

S299: ROLE OF RED BLOOD CELL MEMBRANE-DERIVED PARTICLES ON ENDOTHELIAL DAMAGES DURING ONSET STAGE OF DELAYED HEMOLYTIC TRANSFUSION REACTION

Background: Delayed hemolytic transfusion reaction (DHTR) is a life-threatening complication of red blood cell (RBC) transfusion in sickle cell disease (SCD) patients, leading to the increase of plasma hemoglobin (Hb) and heme which are responsible for endothelial damage and organs failure. By the development of an in vitro model reproducing endothelial damages at the early phase of DHTR (Nguyen et al, ASH 2018), our previous study suggested that RBC membrane-derived particles released during the onset stage of DHTR could be also involved in DHTR physiopathology. Aims: In this study, we aim to determine the mechanism of endothelial damages induced by these RBC particles. Methods: Human Umbilical Vein Endothelial Cells (HUVECs) cultured in flow condition for 24 hours were perfused by either serum only (decomplemented or not) or serum containing either whole hemolysate (sonicated RBCs) or different hemolysate components or RBCs membranes (ghosts) under shear stress 1 dyne/cm2. Annexin V and Eculizumab were used for complement studies. Endothelial activation and damages were assessed by membrane CD54 (PECAM-1, Platelet endothelial cell adhesion molecule 1), CD106 (VCAM-1, Vascular Cell Adhesion Molecule-1) and actin network staining. The whole blood adhesion assays and platelets aggregation study on hemolysate-preconditioned HUVECs were performed in continuous flow. Confocal microscopy was used to study interaction between HUVECs and hemoglobin (Hb) and RBC membrane-derived particles. Results: Our fluidic model reproduced a hemolysate-induced pro-inflammatory phenotype and damages of HUVECs via NFkB signaling pathway in a TLR-4 independent manner. The most deleterious effects were observed in whole hemolysate conditions in which, an important quantity of RBC large-size particles (> 2 μm) mostly positive for C3 was detected. These effects on HUVECs were significantly reduced after their elimination by 14000 g centrifugation (Figure 1). Noteworthy, the suspension of ghosts induced a similar activation on HUVECs compared to whole hemolysate suggesting the major role of RBC membrane-derived particles in early hemolysis-induced endothelial damages. Confocal microscopy for intracellular compartment of hemolysate-treated HUVECs demonstrated that while the CD235a+ Hb+ particles were only found in whole hemolysate conditions, the Hb was detected similarly in all conditions tested. Selective blocking of RBCs particles and Hb endocytosis did not affect hemolysate-induced HUVECs activation. These observations demonstrate that in the very early stage (≤4H) of intravascular hemolysis, erythrocytes large-size particles can be internalized in HUVECs, but their internalization is not necessary for endothelial activation effect. Complement inactivation of serum or treatment of RBC particles by either Annexin V or Eculizumab could reduced their effects on endothelial activation suggesting a complement-dependent mechanism Image:Summary/Conclusion: This study suggested for the first time the major role of RBCs large-size particles on endothelial damages at the onset of DHTR in a complement-dependent manner justifying the complement blocking treatment in certain DHTR patients. Furthermore, we have also demonstrated the endocytosis of free Hb by HUVECs, but its pathological impact is still unclear. This endocytosis would facilitate the Hb clearance during a massive hemolysis? These new understandings will allow a better and earlier management to preserve endothelial functions.

Reprint of: Is Vitamin E the Only Lipid-Soluble, Chain-Breaking Antioxidant in Human Blood Plasma and Erythrocyte Membranes?

The concentrations of lipid-soluble, chain-breaking antioxidants in human plasma and in erythrocyte ghosts have been determined for the first time by an inhibited autoxidation method. The results are very similar to the concentrations of vitamin E measured for the same blood components by the HPLC method. It is concluded that vitamin E, which is largely present as α-tocopherol, is the only significant lipid-soluble, chain-breaking type of antioxidant present in human blood. The concentration of vitamin E in the plasma lipids divided by the concentration of vitamin E in the ghost membrane lipids is approximately a constant despite the large differences in vitamin E-intake and in plasma lipid concentrations in different individuals. Vitamin E/lipid ratios for plasma and ghosts were larger for subjects taking a supplement of α-to- copherol acetate of 100 IU per week, compared to nonsupplemented subjects (based on data from a limited number of subjects). A larger supplement of 2800 IU per week did not significantly increase the vitamin E/lipid ratios.

In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes

Antimicrobial peptides are promising therapeutic agents to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane and are thus less likely to induce resistance. Because they are membrane-active molecules, it is critical to verify and understand their potential action toward eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ 31P and 2H solid-state NMR (SS-NMR). We established a protocol to integrate up to 25% of deuterated fatty acids in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. Fatty acid incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the antimicrobial peptides. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by 31P and 2H SS-NMR is consistent with membrane perturbation through a carpet mechanism for aurein 1.2, whereas caerin 1.1 acts on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, highlighting the importance of hydrophobicity when determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity toward eukaryotic cells.

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.

Metallobiochemistry of ultratrace levels of bismuth in the rat I. Metabolic patterns of 205+206Bi3+ in the blood

BackgroundThe number of the applications of bismuth (Bi) is rapidly and remarkably increasing, enhancing the chance to increase the levels to which humans are normally daily exposed. The interest to Bi comes also from the potential of Bi-based nanoparticles (BiNPs) for industrial and biomedical purposes. Like other metal-based NPs used in nanomedicine, BiNPs may release ultratrace amounts of Bi ions when injected. The metabolic fate and toxicity of these ions still needs to be evaluated. At present, knowledge of Bi metabolism in laboratory animals refers almost solely to studies under unnatural “extreme” exposures, i.e. pharmacologically relevant high-doses (up to thousand mg kg−1) in relation to its medical use, or infinitesimal-doses (pg kg−1 as non-carrier-added Bi radioisotopes) for radiobiology protection, diagnostic and radiotherapeutic purposes. No specific study exists on the “metabolic patterns” in animal models exposed to levels of Bi, i.e. at “environmental dose exposure” that reflect the human daily exposure (μg kg−1). MethodologyRats were intraperitoneally injected with 0.8 μg Bi kg−1 bw as 205+206Bi(NO)3 alone or in combination with 59Fe for radiolabelling of iron proteins. The use of 205+206Bi radiotracers allowed the detection and measurement down to pg fg−1 of the element in the blood biochemical compartments and protein fractions as isolated by differential centrifugation, size exclusion- and ion exchange chromatography, electrophoresis, solvent extraction, precipitation and dialysis. Results24 h after the administration, the blood concentration of Bi was 0.18 ng mL−1, with a repartition plasma/red blod cells (RBC) in a ratio of 2:1. Elution profiles of plasma from gel filtration on Sephadex G-150 showed four pools of Bi-binder proteins with different molecular sizes (> 300 kDa, 160 kDa, 70 kDa and < 6.5 kDa). In the 70 kDa fraction transferrin and albumin were identified as biomolecule carriers for Bi. In red blood cells, Bi was distributed between cytosol and membranes (ghosts) in a ratio of about 5:1. In the cytosol, low molecular components (LMWC) and the hemoglobin associated the Bi in a ratio of about 1.8:1. In the hemoglobin molecule, Bi was bound to the beta polypeptide chain of the globin. In the ghosts, Bi was detected at more than one site of the protein fraction, with no binding with lipids. Dialysis experiments and the consistently high recovery (80–90 %) of 206Bi from chromatography of 206Bi-containing biocomponents suggest that Bi was firmly complexed at physiological pH with a low degree of breaking during the applications of experimental protocols for the isolation of the 206Bi-biocomplexes. These latter were sensitive to acid buffer pH 5, and to the presence of complexing agents in the dialysis fluid. ConclusionsOn the basis of an environmental biochemical toxicology approach, we have undertaken a study on the metabolic patterns of Bi3+ ions in rats at tissue, subcellular and molecular level with the identification of cellular Bi-binding components. As a first part of the study the present work reports the results concerned with the metabolic fate of ultratrace levels of 205+206Bi(NO)3 in the blood.

Hemolysis-associated phosphatidylserine exposure promotes polyclonal plasmablast differentiation.

Antimalarial antibody responses are essential for mediating the clearance of Plasmodium parasite-infected RBCs from infected hosts. However, the rapid appearance of large numbers of plasmablasts in Plasmodium-infected hosts can suppress the development and function of durable humoral immunity. Here, we identify that the formation of plasmablast populations in Plasmodium-infected mice is mechanistically linked to both hemolysis-induced exposure of phosphatidylserine on damaged RBCs and inflammatory cues. We also show that virus and Trypanosoma infections known to trigger hemolytic anemia and high-grade inflammation also induce exuberant plasmablast responses. The induction of hemolysis or administration of RBC membrane ghosts increases plasmablast differentiation. The phosphatidylserine receptor Axl is critical for optimal plasmablast formation, and blocking phosphatidylserine limits plasmablast expansions and reduces Plasmodium parasite burden in vivo. Our findings support that strategies aimed at modulating polyclonal B cell activation and phosphatidylserine exposure may improve immune responses against Plasmodium parasites and potentially other infectious diseases that are associated with anemia.

Differential dielectroscopic data on the relation of erythrocyte membrane skeleton to erythrocyte deformability and flicker.

Two dielectric relaxations, βsp (1.5MHz) and γ1sp (7MHz), have been detected on spectrin-based membrane skeleton (MS) of red blood cells (RBCs) using the plot of admittance changes at the spectrin denaturation temperature (Ivanov and Paarvanova in Bioelectrochemistry 110: 59-68, 2016, Electrochim Acta 317: 289-300, 2019a). In this study, we treated RBCs and RBC ghost membranes with agents that make membranes rigid and suppress membrane flicker, and studied the effect on βsp and γ1sp relaxations. Diamide (diazene dicarboxylic acid bis-(N,N-dimethylamide)) (up to 0.85mM), taurine mustard (tris(2-chloroethyl)amine) (up to 2mM), known to specifically cross-link and stiffen spectrin, and glutaraldehyde (up to 0.044%) all inhibited the relaxations in RBC ghost membranes. Similar inhibition was obtained resealing RBC ghost membranes with 2,3-diphosphoglicerate (up to 15mM), binding WGA (wheat germ agglutinin) (up to 0.025mg/ml) to exofacial aspect of RBCs, incubating RBCs in hypotonic (200mOsm) and hypertonic (600-900mOsm) media and depleting RBCs of ATP. By contrast, concanavalin A (1mg/ml) and DIDS (4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid) (75μM, pH 8.2), both known to bind specifically band 3 integral protein of RBCs without effect on RBC membrane rigidity, did not affect the relaxations. We conclude there might be a relation between the strength of dielectric relaxations on MS spectrin and the deformability and flicker of RBC membrane.

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.

Preparation and Characterization of Dog Erythrocytic Membrane Antigen

Dog erythrocytic membrane antigen plays a major role for determining blood group. Structural and molecular characterization of erythrocytic membrane antigen improves the production of blood typing antisera, to study the auto antibody production in canine autoimmune haemolyticanemia. The proteins in the lipid domain arranged from the inside of the erythrocyte to the outside. The integral membrane proteins include membrane protein 3 visible in coomassie Brilliant blue-stained polyacrylamide gels. The erythrocyte cytoskeleton consists of spectrin, ankyrin, actin, and protein 4.1 form a filamentous network under the lipid bilayer of erythroctic membrane. The cytoskeletal proteins interact with integral proteins and lipids of the bilayer forms network for maintaining membrane integrity. The external clustering of membrane protein 3 creates a recognition site for auto antibodies. The auto antibodies from the erythrocytes of dogs with acute immune haemolytic anemia have similar electrophoretic mobility to the human erythrocyte Rh related proteins and glycoproteins and able to immunoprecipitate membrane proteins that on SDS-PAGE and western blotting. The DEA 1 blood group system consists of antigens 1.1, 1.2, 1.3 which are most significant in terms of transfusion reactions and alloantibodies produced against DEA 3, 5 and 7 are not much clinical significance. Blood samples collected from registered donor dogs and RBC membrane were isolated by hypotonic freeze thaw method. The ghost membrane and cytoplasmic marker were identified in western blot.

Bacteria-Inspired Nanomedicine.

The natural world has provided a host of materials and inspiration for the field of nanomedicine. By taking design cues from naturally occurring systems, the nanoengineering of advanced biomimetic platforms has significantly accelerated over the past decade. In particular, the biomimicry of bacteria, with their motility, taxis, immunomodulation, and overall dynamic host interactions, has elicited substantial interest and opened up exciting avenues of research. More recently, advancements in genetic engineering have given way to more complex and elegant systems with tunable control characteristics. Furthermore, bacterial derivatives such as membrane ghosts, extracellular vesicles, spores, and toxins have proven advantageous for use in nanotherapeutic applications, as they preserve many of the features from the original bacteria while also offering distinct advantages. Overall, bacteria-inspired nanomedicines can be employed in a range of therapeutic settings, from payload delivery to immunotherapy, and have proven successful in combatting both cancer and infectious disease.

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.

Biomimetic Mesoporous Silica Nanoparticles for Enhanced Blood Circulation and Cancer Therapy.

The biomimetic cell membrane camouflaged approach provides numerous opportunities in designing therapeutic platforms for various biomedical applications. It is necessary to understand the engineering of physicochemical properties on materials' surfaces for target biological functions to develop the next-generation anticancer nanomedicines. Herein, we envelope mesoporous silica nanoparticles (MSNs) with red blood cell (RBC) membrane ghosts to obtain MSN@RBC, which possesses significantly stronger physiological stability and longer circulation time than bare MSNs. The surface functionalization of the core material is a critical design parameter for RBC membrane coating efficiency. Therefore, various surface functionalization (-COOH, -SH, -NH2) modifications were performed on MSNs. Compared with other groups, MSN-COOH possessed a better RBC membrane coating efficiency. Then, MSN-COOH of different particle sizes were coated by RBC-derived vesicle membranes. The results indicated that smaller types were able to last longer in blood circulation and accumulate more in target sites than the larger ones. Overall, MSN-ICG@RBC with surface functionalization of -COOH and optimized particle size of 60 nm led to efficient imaging-guided photothermal cancer treatment and could be potentially appealing to actual clinical applications in the future.

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.

Biomimetic Rebuilding of Multifunctional Red Blood Cells: Modular Design Using Functional Components.

The design and synthesis of artificial materials that mimic the structures, mechanical properties, and ultimately functionalities of biological cells remains a current holy grail of materials science. Here, based on a silica cell bioreplication approach, we report the design and construction of synthetic rebuilt red blood cells (RRBCs) that fully mimic the broad properties of native RBCs: size, biconcave shape, deformability, oxygen-carrying capacity, and long circulation time. Four successive nanoscale processing steps (RBC bioreplication, layer-by-layer polymer deposition, and precision silica etching, followed by RBC ghost membrane vesicle fusion) are employed for RRBC construction. A panel of physicochemical analyses including zeta-potential measurement, fluorescence microscopy, and antibody-mediated agglutination assay proved the recapitulation of RBC shape, size, and membrane structure. Flow-based deformation studies carried out in a microfluidic blood capillary model confirmed the ability of RRBCs to deform and pass through small slits and reconstitute themselves in a manner comparable to native RBCs. Circulation studies of RRBCs conducted ex ovo in a chick embryo and in vivo in a mouse model demonstrated the requirement of both deformability and native cell membrane surface to achieve long-term circulation. To confer additional non-native functionalities to RRBCs, we developed modular procedures with which to load functional cargos such as hemoglobin, drugs, magnetic nanoparticles, and ATP biosensors within the RRBC interior to enable various functions, including oxygen delivery, therapeutic drug delivery, magnetic manipulation, and toxin biosensing and detection. Taken together, RRBCs represent a class of long-circulating RBC-inspired artificial hybrid materials with a broad range of potential applications.