Preclinical developments of enzyme-loaded red blood cells

Erythrocyte-cancer hybrid membrane-camouflaged melanin nanoparticles for enhancing photothermal therapy efficacy in tumors

Introduction Red blood cells (RBCs) express anionic phospholipids such as phosphatidylserine (PS) in their inner cell membrane layer. As RBCs age, physiochemical changes in their cell membranes, particularly exposure of PS on the outer membrane layer, lead to phagocytosis and clearance from circulation. PS in RBCs induces activation of the complement system, increasing binding of C3 to RBCs. Thus, quantifying PS-expressing (PS+) RBCs and complement deposition on RBCs (C3+) is a potential measure of RBC injury and may improve transfusion quality when used for predicting post-transfusion RBC recovery. Our goal was to develop a flow cytometry assay that quantifies PS+ and C3+RBCs and to assess how this assay correlates with the direct antiglobulin test for C3 (C3-DAT). Methods Using the Cytoflex S flow cytometer, we measured PS+ and C3+ RBCs using anti-annexin-PE (PS+RBC) and biotin-labeled anti-C3 with streptravidin-PE-Cy5 (C3+RBC). RBCs from healthy donors (NL-RBCs) with negative direct antiglobulin tests for C3 (C3-DAT) (n = 4) were analyzed. To assess the sensitivity of the assay and correlation with C3-DATs, complement control cells (CCC; Immucor) were used as a positive control for both PS+RBCs and C3+RBCs and diluted to different concentrations by mixing CCCs with NL-RBCs. Aliquots of diluted RBCs were analyzed blinded for C3-DAT. Differences between means for PS+RBC and C3+RBC were compared using two-sample t-test, and one-way ANOVA and Tukey’s HSD test were used to determine differences in C3+RBCs among a C3-DAT range of reactivities (0+ to 3+) in diluted CCC samples. P values less than 0.05 were statistically significant. Results In healthy, DAT-negative individuals, the mean positivity for PS+RBCs was 0.28% and for C3+RBCs was 0.34%, indicating that healthy individuals have very low percentages of RBCs with PS and C3 on their cell membrane surface. In CCC diluted samples, the mean C3+RBC percentage was 1.4% in negative C3-DAT samples, 5.9% in weak+ C3-DAT samples, 28.1% in 1+ C3-DAT samples, 67.2% in 2+ C3-DAT samples, and 99.0% in 3+ C3-DAT samples. One-way ANOVA revealed a statistically significant difference in C3+RBCs amongst all C3-DAT reactivities in diluted CCC samples (P<0.001), and Tukey’s HSD test found a mean difference in C3+RBCs percentage of 26.7% between 0+ and 1+ samples (P<0.001), 39.2% between 1+ and 2+ samples (P<0.001), and 31.8% between 2+ and 3+ samples (P<0.01). Undiluted CCC samples had a mean PS+RBC percentage of 7.0% and was statistically different from the PS+RBC percentage in NL-RBCs (P<0.01). Conclusion We describe a feasible assay to measure both PS+RBCs and C3+RBCs using flow cytometry in which CCC can be used as a positive control for PS+RBCs. This assay could serve to evaluate the quality of transfused RBC units and provide insights into the role of phosphatidylserine expression and complement deposition for RBC clearance.

Intracerebral hemorrhage (ICH) occurs upon blood vessel rupture, allowing blood into brain tissue. Red blood cells (RBCs) make up 99%+ of blood cells. Brain-trapped RBCs undergo phagocytosis; however, many RBCs lyse prior to being phagocytosed due to their abundance. RBC lysis releases hemoglobin and toxic heme, driving oxidative stress especially in lipid-rich plasma membranes (PM), causing tissue damage and ferroptosis. Few studies have explored RBC fate in ICH, but unraveling the factors governing RBC lysis may yield ICH therapies.In in vitro human and murine RBC cultures, we observed RBCs are exquisitely sensitive to heme-induced death with increased oxidized fatty acids (FAs), a ferroptosis marker (Fig 1A-B). The ferroptosis inducer Erastin validated RBC susceptibility to ferroptosis (Fig 1C). Heme-induced cell death was countered by β-mercaptoethanol (β-ME), N-acetylcysteine (NAC), ferrostatin-1 (Fer-1) and Vitamin E (Vit. E), ferroptosis inhibitors, confirming RBC death was due to ferroptosis (Fig 1D-G). Heme and Erastin induced RBC ferroptosis differs from eryptosis, RBC specific programmed cell death, as it isn't Ca++ dependent (Fig1H&I). Unlike other cells, RBCs solely rely on the Pentose Phosphate Pathway (PPP) for NADPH production for oxidation defense. PPP inhibition with Dehydroisoandrosterone (DHEA) and treatment with ThioNa, an NADP+ synthesis inhibitor, both heightened heme-induced RBC ferroptosis (Fig 1J-L). These data support the PPP's role against heme-driven ferroptosis. Heme induced RBC ferroptosis may trigger a chain reaction of RBC lysis and ongoing heme release. Notably, 400 million people face G6PD deficiency, the rate-limiting PPP enzyme, leading to NADPH depletion in RBCs with enhanced susceptibility to oxidation. Hence, in addition to identifying the PPP in RBCs resistance to heme toxicity, our findings could aid in identifying a population that is particularly vulnerable to the devastating effects of ICH.

Interspecies variations in mammalian red blood cells (RBCs) are observed in circulating RBC lifespan, cell size, fluidity, aggregation, water permeability, metabolism, lipid composition, and the overall proteome. Bovine RBC cell membrane is deficient in phosphatidylcholine and exhibits anomalies in the arrangement of phosphatidylethanolamine within the lipid bilayer. However, like human RBCs, virtually all the aminophospholipid phosphatidylserine (PS) is found within the cytoplasmic side of the cell membrane of intact circulating bovine RBCs. During apoptotic cell death of human and murine RBCs, PS translocates to the outer leaflet of the cell membrane via Ca2+-dependent and -independent signaling mechanisms. However, little is known about this process in bovine RBCs. Using cytofluorometry analyses, we characterized and compared the cell death responses in bovine and human RBCs in vitro exposed to various pathophysiologic cell stressors. Ionic stress, by ionophore treatment, and oxidative stress enhanced cytoplasmic Ca2+ levels and cell membrane PS expression in both bovine and human RBCs. Fever-grade hyperthermia and energy starvation promoted Ca2+ influx and elevated reactive oxygen species levels in both human and bovine RBCs. However, bovine RBCs displayed minimal increases in PS expression elicited by hyperthermia, energy starvation, and extracellular hypertonicity as compared to human RBCs. In response to decreased extracellular osmolality, bovine RBCs exhibited significantly enhanced fragility as compared to human RBCs. Bovine RBCs display differential cell death patterns as compared to human RBCs, only partly explained by increased Ca2+ influx and oxidative stress. Premature removal of circulating RBCs could potentially contribute to the pathogenesis of anemia in cattle caused by a wide range of factors such as systemic diseases, parasitic infections, and nutritional deficiencies.

Cryptantigens: time to uncover the real significance of T-activation.

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Oral Administration of FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Has Potent Anti-Sickling Effects in a Sickle Cell Anemia (SCA) Mouse Model, Resulting in Improved RBC Survival and Hemoglobin Levels

Introduction: Red blood cell(RBC), which passes through capillaries and exchanges O2 and CO2 with surrounding tissues, plays a very important in the blood circulation in the human body. Imbalance in blood circulation could lead to various cardiovascular related diseases such as high blood pressure, arteriosclerosis, diabetes, and stroke.[1] This is closely related to RBC aggregation. Negatively charged RBC membrane arranges a compact layer of positive ions around the membrane by electrical attraction with cations in plasma.The rearranged ions layer in plasma forms zeta potential with compact layer. When the electrostatic interaction between RBCs, i.e., zeta potential increases, RBC aggregation is improved. It is known that zeta potential depends on electronegativity of the RBC membrane, the dielectric constant of the medium and ionic strength [2]. Since pulsed magnetic field (PMF) influences human physiology, many clinical studies demonstrated the effect of PMF for wound healing, reducing pain, increasing angiogenesis, as well as improving blood circulation.[3-5] In this study, we have tried to elucidate the effect of PMF on RBC aggregation by indirectly observing zeta potential using negatively-charged magnetic beads(MB). Our hypothesis is that the increase of negative charges on RBC membrane results in the increase of cations in the compact layer, thus the increase of the zeta potential. As a result, the number of MB attached to the RBC increases, as the binding force between MB and compact layer increases.Experimental Method: Blood needed for experiment was obtained from Korean Red Cross after reviewing IRB exemption in Sangji University Bioethics Committee. Whole blood of 6 ml was centrifuged at 3000 rpm for 5 min in 4 oC for separating RBCs and plasma. MB used this experiment has diameter 1 μm with OH- groups (SiMAG-Silanol, Chemicell). To clearly see the MB attached to RBCs and, in addition, to avoid blood clots caused by proteins in plasma, RBC suspension of hematocrit (Ht) of 10% was prepared using PBS(phosphate buffer saline). The amount of MB added to RBC suspension was 0.5 mg. RBCs were also oxidative stressed with tBHP(Tert-Butyl hydroperoxide) in 0.4 mM at 37 oCincubator for 30 min, in order to confirm the change of RBCs membrane charge due to oxidative stress under PMF stimulus. In oxidative stressed RBCs, hemoglobin(Fe2+) is altered to methemoglobin(Fe3+) because of loss an electron, thereby, electronegativity of RBC membrane will be changed.[6] Each sample was exposed to PMF for 3 min. Our PMF stimulator system consisted of single layered coil of 10 turns with an elliptical shape of 12.0 x 4.5 cm. The maximum intensity is 0.27 T at a transition time of 0.102 ms, with pulse intervals of 1 Hz. The numbers of RBC attached MB were compared with control group and PMF treatment group. Only RBCs with a distance of about 1 μm between the MB and the surface of RBC were counted using hemocytometer.Results and Discussion: Fig.1. shows the numbers of RBCs according to the number of attached MB in the control and PMF treatment group. The numbers of RBCs attached MB increased after PMF treatment. The more the number of MBs attached to RBCs, the thicker the compact layer surrounding RBC membranes. This phenomenon might be inferred due to an increase of zeta potential, thereby, increase of negative charges on the RBC membrane. Thus, it can be interpreted that MB having hydroxide OH- group are more attached to RBCs via binding force between MB and compact layer. Also the numbers of MB in the group treated tBHP and the group exposed to PMF after tBHP treatment (tBHP+PMF) were compared in Fig.2. Although the number of MB attached to RBCs was 1~2 in tBHP group, after PMF stimulus, i.e. in tBHP+PMF group it appears the number of MB attached to RBCs increased up to 3~4. This might be explained that PMF affects the increase of RBC membrane charge on not only normal RBCs but also oxidative stressed RBCs. Therefore, it can be deduced that high instantaneous flux change of PMF improves the aggregation of RBCs by increasing the zeta potential, due to the increase of the membrane charge. **

Activating Pyruvate Kinase Improves Red Blood Cell Integrity By Reducing Band3 Tyrosine Phosphorylation

Leukocyte-reduced blood transfusions: perioperative indications, adverse effects, and cost analysis.

Nonspecific absorption and clearance of nanomaterials during circulation is the major cause for treatment failure in nanomedicine‐based cancer therapy. Therefore, herein bioinspired red blood cell (RBC) membrane is employed to camouflage 2D MoSe2 nanosheets with high photothermal conversion efficiency to achieve enhanced hemocompatibility and circulation time by preventing macrophage phagocytosis. RBC–MoSe2‐potentiated photothermal therapy (PTT) demonstrates potent in vivo antitumor efficacy, which triggers the release of tumor‐associated antigens to activate cytotoxic T lymphocytes and inactivate the PD‐1/PD‐L1 pathway to avoid immunologic escape. Furthermore, in the ablated tumor microenvironment, the tumor‐associated macrophages are effectively reprogrammed to tumoricidal M1 phenotype to potentiate the antitumor action. Taken together, this biomimetic functionalization thus provides a substantial advance in personalized PTT‐triggered immunotherapy for clinical translation.

Background: The development of abnormalities in red blood cell (RBC) deformability in patients undergoing hemodialysis remains a major problem, because it is related to peripheral microcirculation, oxygen supply, and various complications in such patients. γ-Linolenic acid (GLA; 18:3n-6), one of the polyunsaturated fatty acids and a precursor of prostaglandin E₁, is reported to have a favorable effect on the deformability of circulating blood cells in diabetic patients. Methods: In order to clarify the efficacy of GLA on RBC deformability in 7 patients undergoing maintenance hemodialysis, we examined in a pilot study the changes in the deformability of RBC and the changes in the phospholipid fatty acid composition in both plasma and RBC membrane before and after high-dose oral supplementation with GLA derived from Mucor circinelloides for 12 weeks. Results: Before supplementation, the micropore passage time of RBC suspension, which is an indicator of RBC deformability, in these patients was markedly longer than that in healthy control subjects. After administering GLA, the prolonged passage time of the patients both rapidly and steadily decreased and nearly reached control levels. Light microscopic observations of RBCs using Giemsa stain revealed a decreased number of poikilocytes after supplementation. An analysis of the fatty acid composition before treatment and 8 weeks after starting the treatment showed the dihomo-γ-linolenic acid (DGLA; 20:3n-6) level in the plasma to have increased (p < 0.05), while the arachidonic acid (AA; 20:4n-6) concentration in the RBC membrane decreased (p < 0.05). The level of DGLA in the RBC membrane, the level of GLA, and the ratio of GLA + DGLA/AA in plasma and RBC membrane did not change significantly; however, these all tended to increase. Conclusion: The results of this pilot study indicate that the oral supplementation of GLA extracted from M. circinelloides improves the poor RBC deformability in hemodialysis patients, partly by inducing changes in the composition of fatty acids in plasma and RBC membrane.

Spatial analysis of erythrocyte membrane fluctuations by digital holographic microscopy

Comparison of Ixazomib, Lenalidomide Plus Dexamethasone (IRd) and Bortezomib, Lenalidomide Plus Dexamethasone (VRd) in Newly Diagnosed Multiple Myeloma in China

Red Blood Cells (RBCs)-derived particles are an emerging group of novel drug delivery systems. The natural attributes of RBCs make them potential candidates for use as a drug carrier or nanoparticle camouflaging material as they are innately biocompatible. RBCs have been studied for multiple decades in drug delivery applications but their evolution in the clinical arena are considerably slower. They have been garnering attention for the unique capability of conserving their membrane proteins post fabrication that help them to stay non-immunogenic in the biological environment prolonging their circulation time and improving therapeutic efficiency. In this review, we discuss about the synthesis, significance, and various biomedical applications of the above-mentioned classes of engineered RBCs. This article is focused on the current state of clinical translation and the analysis of the hindrances associated with the transition from lab to clinic applications.