Red Blood Cell Membrane Research Articles

Red blood cell derived nanocarrier drug delivery system: A promising strategy for tumor therapy

AbstractNano drug delivery systems have been widely used in tumor therapy. Researchers have been devoted to exploring novel nano‐carriers to prolong circulation time biocompatibility and tumor targeting efficiency in vivo. Erythrocyte membranes as bionic nano‐drug delivery systems have attracted more attention recently. There are abundant red blood cells (RBCs) in circulation, which are natural carriers for nanomaterial delivery. There are mainly two strategies to use RBCs as nanomaterial carriers: RBC‐hitchhiking and bionic nanomaterial coated with erythrocyte membrane loading with drugs. Although nano‐carrier based on RBCs have yielded some progress, the mechanism of mutual effect between nanoparticles and RBCs is still unclear. Here, we review the effects of nanoparticles on RBCs (morphology and function, etc) and application of RBCs membrane as nano‐drug delivery carriers in tumor therapy.

In vitro anti-inflammatory and anticoagulant activities of alkaloïds extracted from nopals of inermis Algerian Opuntia ficus indica (L).

The dearth of information surrounding the utilization of Opuntia cladode alkaloids underscores a critical gap in understanding their pharmacological properties and therapeutic potential, emphasizing the need for further comprehensive investigations. The present study aims to investigate the in vitro anti-inflammatory, anticoagulant, and antimicrobial activities of alkaloïds belonging to young cladodes (nopals) of inermis Algerian Opuntia ficus indica. The assessed alkaloïds showed a moderate anti-inflammatory effect regarding the BSA protein protection with a maximum of 51.04±1.84 % compared to 84.22±2.38 % for Diclofenac sodium as a positive standard. However, their stabilization of red blood cells membrane against induced hemolysis was greater than Diclofenac (52.38±2.01 % vs 48.97±2.73 %). The two assays had a significant correlation (< 0.05) of 0,968. Nopal alkaloïds extended the coagulation time (1.24 fold) by significantly affecting the exogenous pathway PT only, whilst they were ineffective against the endogenous pathway APTT. Conversely, Algerian nopal alkaloïds had neither bacteriostatic nor bactericide influence at 2 mg ml-1 on Candida albicans yeast or the six tested pathogenic bacterial strains. In perspective, the purification of these Opuntia nopal alkaloïds and understanding their accurate mechanism of action are clearly the strategic steps to illustrate their overall curative potential.

Testosterone supplementation increases red blood cell susceptibility to oxidative stress, decreases membrane deformability, and decreases survival after cold storage and transfusion.

Blood collection from donors on testosterone therapy (TT) is restricted to red blood cell (RBC) concentrates to avoid patient exposure to supraphysiological testosterone (T). The objective of this study was to identify TT-related changes in RBC characteristics relevant to transfusion effectiveness in patients. This was a two-part study with cohorts of patients and blood donors on TT. In part 1, we conducted longitudinal evaluation of RBCs collected before and at three time points after initiation of T. RBC assays included storage and oxidative hemolysis, membrane deformability (elongation index), and oximetry. In part 2, we evaluated the fate of transfused RBCs from TT donors in immunodeficient mice and by retrospective analyses of NIH's vein-to-vein databases. TT increased oxidative hemolysis (1.45-fold change) and decreased RBC membrane deformability. Plasma free testosterone was positively correlated with oxidative hemolysis (r = .552) and negatively correlated with the elongation index (r = -.472). Stored and gamma-irradiated RBCs from TT donors had lower posttransfusion recovery in mice compared to controls (41.6 ± 12 vs. 55.3 ± 20.5%). Recipients of RBCs from male donors taking T had 25% lower hemoglobin increments compared to recipients of RBCs from non-TT male donors, and had increased incidence (OR, 1.80) of requiring additional RBC transfusions within 48 h of the index transfusion event. TT is associated with altered RBC characteristics and transfusion effectiveness. These results suggest that clinical utilization of TT RBCs may be less effective in recipients who benefit from longer RBC survival, such as chronically transfused patients.

Зміна форми еритроцитів при механічному перемішуванні та заміщенні в середовищі сульфату на хлорид

At low pH, red blood cells (RBCs) are unable to maintain their disc shape and become stomatocytes. At the same time, at pH 5.0 of a sulphate medium, under conditions of mechanical mixing of the cell suspension, RBCs are partially retransformed into discoid forms, whereas in a chloride medium (pH 5.0) this transformation is observed to a high degree.This indicates that the chloride-binding sites of RBC membranes may be an additional link in the regulation of the shape of RBC under conditions of mechanical action on the cell suspension. The work investigated the change in the RBC shape at a normal pH value (7,4) due to the replacement of Na2SO 4 in the medium with NaCl and recording an increase in the intensity of fluctuations in optical density (OD), as an indicator of an increase in the level of discoid cells (normocytes) in a stirred cell suspension. It has been 3 established that in a medium containing Na2SO4 (110 mmol/l), RBCs are transformed into small stomatocytes within ~20 s. With an increase in NaCl concentration in the range of 15-105 mmol/l, an increase in the level of discoid cells is noted. Increasing the NaCl concentration from 105 to 150 mmol/l, on the contrary, causes a decrease in the level of discoid cells. With the exception of mechanical mixing in a medium with NaCl concentrations of 30-90 mmol/l, RBCs are morphologically stomatocytes. Whereas in a medium containing 150 mmol/l NaCl, RBC are represented by disc-echinocytes and echinocytes. The results obtained show that mechanical mixing of the cell suspension promotes the retransformation of stomatocytic RBCs into discoid forms. The weakening of the retransforming efficiency of mixing with an increase in the NaCl concentration in the medium from 105 to 150 mmol/l and the development of echinocytosis at 150 mmol/l NaCl indicates the existence of 2 types of sites for chloride. Сhloride binding to type 1, at a NaCl concentration in the medium of 15-105 mmol/l, leads to the establishment of discoid forms of RBC. Chloride binding to type 2 sites, at a NaCl concentration in the medium of 105-150 mmol/l, leads to the development of echinocytosis. The retransforming property of mechanical stirring may be associated with a change in the degree of chloride binding to these types of sites.

Diagnosis and Vulnerability Risk Assessment of Atherosclerotic Plaques Using an Amino Acid-Assembled Near-Infrared Ratiometric Nanoprobe.

To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.

Comparison of cell delivery and cell membrane camouflaged PLGA nanoparticles in the delivery of shikonin for colorectal cancer treatment

Inspired by the "natural camouflage" strategy, cell-based biomimetic drug delivery systems (BDDS) have shown great potential in cancer therapy. Red blood cell (RBC) delivery vehicles and red blood cell membrane (RBCm)-camouflaged vehicles were commonly used strategies for drug delivery. We prepared shikonin-encapsulated PLGA nanoparticles (PLGA/SK) with different surface charges to obtain both RBC delivery and RBCm-camouflaged PLGA NPs. The physicochemical properties, in vivo circulation and antitumor effects of these biomimetic preparations were studied. Since the positive PLGA NPs may affect the morphology and function of RBCs, the biomimetic preparations prepared by the negative PLGA NPs showed better in vitro stability. However, positive PLGA NP-based biomimetic preparations exhibited longer circulation time and higher tumor region accumulation, leading to stronger anti-tumor effects. Meanwhile, the RBC delivery PLGA(+) NPs possessed better in vitro cytotoxicity, longer circulation time and higher tumor accumulation than RBCm-camouflaged PLGA(+) NPs. Collectively, RBC delivery vehicles possessed more potential than RBCm-camouflaged vehicles on drug delivery for tumor treatment, especially with positive NPs-loaded.

Red Blood Cell Membrane-Coated Nanoparticles Enable Incompatible Blood Transfusions.

Blood transfusions save lives and improve health every day. Despite the matching of blood types being stricter than it ever has been, emergency transfusions among incompatible blood types are still inevitable in the clinic when there is a lack of acceptable blood types for recipients. Here to overcome this, a counter measure nanoplatform consisting of a polymeric core coated by a red blood cell (RBC) membrane is developed. With A-type or B-type RBC membrane camouflaging, the nanoplatform is capable of specifically capturing anti-A or anti-B IgM antibodies within B-type or A-type whole blood, thereby decreasing the corresponding IgM antibody levels and then allowing the incompatible blood transfusions. In addition to IgM, the anti-RBC IgG antibody in a passive immunization murine model can likewise be neutralized by this nanoplatform, leading to prolonged circulation time of incompatible donor RBCs. Noteworthily, nanoplatform made by expired RBCs (>42 days stored hypothermically) and then subjected to lyophilization does not impair their effect on antibody neutralization. Most importantly, antibody-captured RBC-NP do not exacerbate the risk of inflammation, complement activation, and coagulopathy in an acute hemorrhagic shock murine model. Overall, this biomimetic nanoplatform can safely neutralize the antibody to enable incompatible blood transfusion.

Erythrocyte Membrane Coating Alleviate Immune Response and Promoted Adipogenesis in Adipose Matrix.

Xenotransplantation of acellular adipose matrix (AAM) has come to prominence as an intriguing option for soft tissue reconstruction. However, the presence of immunogenic antigens within AAM can trigger unfavorable immune reactions, leading to inadequate invivo regeneration outcomes. Therefore, the development of advanced technology capable of modulating immune responses is crucial for the therapeutic implementation of AAM xenografts. In this work, an innovative technique is created to bypass the immune system by covering the surface of both AAM and Arg-Gly-Asp (RGD) peptide-modified AAM xenografts with autologous red blood cell (RBC) membrane. The RBC membrane coating remained persistent and exhibited no significant decline even after 21 days. Moreover, it effectively reduced the expression of antigen major histocompatibility complex class 1 (MHC1) on the AAM surface. Following xenogeneic transplantation, the RBC-coated xenografts demonstrated increased expression of the adipogenic factor PPAR-γ, Adipoq, Fabp4, Fasn, and Plin1 and higher numbers of adipocytes. In addition, they exhibited decreased expression of immunological factors, including IL-6, IL-2, IFN-γ, and TNF-α, and fewer inflammatory cells. These findings indicate that RBC membrane coating successfully suppressed immune responses and promoted increased adipogenesis in AAM xenografts. Therefore, AAM camouflage coating with RBC has a lot of potential as a biomaterial for soft tissue reconstruction in clinical settings.

Yet more evidence that non-aqueous myelin lipids can be directly imaged with ultrashort echo time (UTE) MRI on a clinical 3T scanner: a lyophilized red blood cell membrane lipid study

Direct imaging of semi-solid lipids, such as myelin, is of great interest as a noninvasive biomarker of neurodegenerative diseases. Yet, the short T2 relaxation times of semi-solid lipid protons hamper direct detection through conventional magnetic resonance imaging (MRI) pulse sequences. In this study, we examined whether a three-dimensional ultrashort echo time (3D UTE) sequence can directly acquire signals from membrane lipids. Membrane lipids from red blood cells (RBC) were collected from commercially available blood as a general model of the myelin lipid bilayer and subjected to D2O exchange and freeze-drying for complete water removal. Sufficiently high MR signals were detected with the 3D UTE sequence, which showed an ultrashort T2* of ∼77–271 µs and a short T1 of ∼189 ms for semi-solid RBC membrane lipids. These measurements can guide designing UTE-based sequences for direct in vivo imaging of membrane lipids.

Hemoglobin Binding to the Red Blood Cell (RBC) Membrane Is Associated with Decreased Cell Deformability.

The deformability of red blood cells (RBCs), expressing their ability to change their shape as a function of flow-induced shear stress, allows them to optimize oxygen delivery to the tissues and minimize their resistance to flow, especially in microcirculation. During physiological aging and blood storage, or under external stimulations, RBCs undergo metabolic and structural alterations, one of which is hemoglobin (Hb) redistribution between the cytosol and the membrane. Consequently, part of the Hb may attach to the cell membrane, and although this process is reversible, the increase in membrane-bound Hb (MBHb) can affect the cell's mechanical properties and deformability in particular. In the present study, we examined the correlation between the MBHb levels, determined by mass spectroscopy, and the cell deformability, determined by image analysis. Six hemoglobin subunits were found attached to the RBC membranes. The cell deformability was negatively correlated with the level of four subunits, with a highly significant inter-correlation between them. These data suggest that the decrease in RBC deformability results from Hb redistribution between the cytosol and the cell membrane and the respective Hb interaction with the cell membrane.

Synthetic Biomimetic Liposomes Harness Efferocytosis Machinery for Highly Efficient Macrophages-Targeted Drug Delivery to Alleviate Inflammation.

Macrophages play pivotal roles in the regulation of inflammatory responses and tissue repair, making them a prime target for inflammation alleviation. However, the accurate and efficient macrophages targeting is still a challenging task. Motivated by the efficient and specific removal of apoptotic cells by macrophages efferocytosis, a novel biomimetic liposomal system called Effero-RLP (Efferocytosis-mediated Red blood cell hybrid Liposomes) is developed which incorporates the membrane of apoptotic red blood cells (RBCs) with liposomes for the purpose of highly efficient macrophages targeting. Rosiglitazone (ROSI), a PPARγ agonist known to attenuate macrophage inflammatory responses, is encapsulated into Effero-RLP as model drug to regulate macrophage functions in DSS-induced colitis mouse model. Intriguingly, the Effero-RLP exhibits selective and efficient uptake by macrophages, which is significantly inhibited by the efferocytosis blocker Annexin V. In animal models, the Effero-RLP demonstrates rapid recognition by macrophages, leading to enhanced accumulation at inflammatory sites. Furthermore, ROSI-loaded Effero-RLP effectively alleviates inflammation and protects colon tissue from injury in the colitis mouse model, which is abolished by deletion of macrophages from mice model. In conclusion, the study highlights the potential of macrophage targeting using efferocytosis biomimetic liposomes. The development of Effero-RLP presents novel and promising strategies for alleviating inflammation.

#2505 PTEN ablation in osteocytes exacerbates anemia in mice with subtotal nephrectomy

Abstract Background and Aims The identification of novel roles for bone as a principal endocrine organ has grown in recent decades, being now recognized for its contribution to the regulation of numerous systemic functions. In previous studies, we observed that 5/6Nx mice with osteocytic deletion of phosphatase and tensin homolog (PTEN), exhibited higher bone volume in 5/6Nx mice, consistent with studies under normal renal conditions. However, we also noted a decrease in blood hemoglobin levels, prompting further exploration. This study aims to elucidate the causative actions by which specific PTEN deletion in osteocytes influences red blood cell (RBC) homeostasis and to identify its possible involvement in anemia development. Method To achieve this goal, we used mice with targeted PTEN deletion in osteocytes (PTEN Ocy-cKO) by crossing DMP1-Cre ± and PTENflox/flox mice, subjected to subtotal nephrectomy (5/6Nx) at 2 months of age. Two weeks post-surgery, bone and blood samples were collected for plasma biochemistry, blood count, RBC membrane integrity, and preliminary bone+bone marrow proteomic analysis. Cre-negative mice and animals with normal renal function were included as controls. Results Our findings revealed that PTEN Ocy-cKO mice, under both normal and reduced renal function conditions, showed higher bone volume than corresponding controls. In terms of blood homeostasis, PTEN Ocy-cKO mice with normal renal function showed splenomegaly and a lower number of circulating RBCs. Notably, PTEN Ocy-cKO mice undergoing 5/6Nx experienced a profound decrease in RBC number in only two weeks, independent of renal erythropoietin expression. Further exploration ruled out bone marrow failure as a cause, as the erythroid progenitor (CD45-CD71+Ter119+) population remained similar in both groups. Extramedullary hematopoiesis was observed in the spleen, in addition to increased circulating reticulocyte number in the PTEN Ocy-cKO mice. Finally, altered RBC membrane protein patterns in PTEN Ocy-cKO mice suggested reduced mechanical/osmotic resistance. Preliminary proteomic analysis identified downregulation of Slc4a1 (Band3) and Phosphofructokinase (Pfkm), whose deficiencies have been previously linked to anemia. Conclusion In conclusion, our results propose that osteocytes contribute to RBC homeostasis through a pathway downstream of PTEN, and open a new avenue for understanding anemia in chronic kidney disease and a potential target for future treatments.

Hybrid cell membranes camouflaged copper-loaded nano-prodrug for tumor angiogenesis inhibition and cell cuproptosis

The establishment of hybrid cell membrane-camouflaged nanotherapeutics is crucial to preserve and impart their biological functionality inherited from the source cells. In this study, one side relies on the intrinsic affinity between the intracellular domain of Band 3 in the red blood cells membrane (Rm) and P4.2 peptide-modified nanoparticles (Rm-Que/Cu), promoting the self-assembly coupling-driven encapsulation of Rm on the surface of nanodrugs. On the other side, it involves integrating the bioengineered cell membrane with high Del-1 expression (Dm) with Rm-Que/Cu to form RDm-Que/Cu, targeting the role of integrin αvβ3 on tumor neovascularization and endothelial cells (ECs). This approach simultaneously integrates the multi-functions, including self-assembly encapsulation, targeted drug delivery, and on-demand drug release. Both in vitro and in vivo experimental results confirmed that RDm-Que/Cu were able to disrupt the cellular copper homeostasis, inhibit tumor angiogenesis, and induce cuproptosis. This discovery provides a feasible platform for the biomedical application of hybrid cell membrane-camouflaged biomimetic nanotechnology.

Catalase, Glutathione Peroxidase, and Peroxiredoxin 2 in Erythrocyte Cytosol and Membrane in Hereditary Spherocytosis, Sickle Cell Disease, and β-Thalassemia.

Catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2) can counteract the deleterious effects of oxidative stress (OS). Their binding to the red blood cell (RBC) membrane has been reported in non-immune hemolytic anemias (NIHAs). Our aim was to evaluate the relationships between CAT, GPx, and Prx2, focusing on their role at the RBC membrane, in hereditary spherocytosis (HS), sickle cell disease (SCD), β-thalassemia (β-thal), and healthy individuals. The studies were performed in plasma and in the RBC cytosol and membrane, evaluating OS biomarkers and the enzymatic activities and/or the amounts of CAT, GPx, and Prx2. The binding of the enzymes to the membrane appears to be the primary protective mechanism against oxidative membrane injuries in healthy RBCs. In HS (unsplenectomized) and β-thal, translocation from the cytosol to the membrane of CAT and Prx2, respectively, was observed, probably to counteract lipid peroxidation. RBCs from splenectomized HS patients showed the highest membrane-bound hemoglobin, CAT, and GPx amounts in the membrane. SCD patients presented the lowest amount of enzyme linkage, possibly due to structural changes induced by sickle hemoglobin. The OS-induced changes and antioxidant response were different between the studied NIHAs and may contribute to the different clinical patterns in these patients.

Impact of fucosyltransferase 1-mediated epidermal blood group antigen H on anti-inflammatory response in atopic dermatitis.

The presence of the blood group H2 antigen on the membrane of red blood cells determines blood type O in individuals and this H2 antigen serves as a precursor to the A and B antigens expressed in blood types A and B, respectively. However, the specific involvement of ABH antigens in skin diseases is unknown. Therefore, we aim to investigate the expression of ABH antigens in skin tissue of patients with atopic dermatitis (AD) and MC903-induced AD-like mice. We demonstrated that the expression of ABH antigen is primarily located in the granular and horny layers of the skin in healthy control individuals. However, in patients with AD, the expression of the ABH antigen was absent or diminished in these layers, while the H2 antigen expression increased in the spinous layers of the affected skin lesions. Then, we investigated the biological function of blood group H antigen mediated by fucosyltransferase 1 (Fut1) in the skin, utilizing an AD mouse model induced by MC903 in wild-type (WT) and Fut1-knockout mice. After the application of MC903, Fut1-deficient mice, with no H2 antigen expression on their skin, exhibited more severe clinical signs, increased ear swelling, and elevated serum IgE levels compared with those of WT mice. Additionally, the MC903-induced thickening of both the epidermis and dermis was more pronounced in Fut1-deficient mice than that in WT mice. Furthermore, Fut1-deficient mice showed a significantly higher production of interleukin-4 (IL-4) and IL-6 in skin lesions compared with that of their WT counterparts. The expression of chemokines, particularly Ccl2 and Ccl8, was notably higher in Fut1-deficient mice compared with those of WT mice. The infiltration of CD4+ T cells, eosinophils, and mast cells into the lesional skin was significantly elevated in Fut1-deficient mice compared with that in WT mice. These findings demonstrate the protective role of H2 antigen expression against AD-like inflammation and highlight its potential therapeutic impact on AD through the regulation of blood group antigens.

Black Phosphorus Quantum Dot Loaded Bioinspired Nanoplatform Synergized with aPD-L1 for Multimode Cancer Immunotherapy.

Efforts to prolong the blood circulation time and bypass immune clearance play vital roles in improving the therapeutic efficacy of nanoparticles (NPs). Herein, a multifunctional nanoplatform (BPP@RTL) that precisely targets tumor cells is fabricated by encapsulating ultrasmall phototherapeutic agent black phosphorus quantum dot (BPQD), chemotherapeutic drug paclitaxel (PTX), and immunomodulator PolyMetformin (PM) in hybrid membrane-camouflaged liposomes. Specifically, the hybrid cell membrane coating derived from the fusion of cancer cell membrane and red blood cell membrane displays excellent tumor targeting efficiency and long blood circulation property due to the innate features of both membranes. After collaboration with aPD-L1-based immune checkpoint blockade therapy, a boosted immunotherapeutic effect is obtained due to elevated dendritic cell maturation and T cell activation. Significantly, laser-irradiated BPP@RTL combined with aPD-L1 effectively eliminates primary tumors and inhibits lung metastasis in 4T1 breast tumor model, offering a promising treatment plan to develop personalized antitumor strategy.

Large-Scale Surface Modification of Decellularized Matrix with Erythrocyte Membrane for Promoting In Situ Regeneration of Heart Valve

In situ regeneration is a promising strategy for constructing tissue engineering heart valves (TEHVs). Currently, the decellularized heart valve (DHV) is extensively employed as a TEHV scaffold. Nevertheless, DHV exhibits limited blood compatibility and notable difficulties in endothelialization, resulting in thrombosis and graft failure. The red blood cell membrane (RBCM) exhibits excellent biocompatibility and prolonged circulation stability and is extensively applied in the camouflage of nanoparticles for drug delivery; however, there is no report on its application for large-scale modification of decellularized extracellular matrix (ECM). For the first time, we utilized a layer-by-layer assembling strategy to immobilize RBCM on the surface of DHV and construct an innovative TEHV scaffold. Our findings demonstrated that the scaffold significantly improved the hemocompatibility of DHV by effectively preventing plasma protein adsorption, activated platelet adhesion, and erythrocyte aggregation, and induced macrophage polarization toward the M2 phenotype in vitro. Moreover, RBCM modification significantly enhanced the mechanical properties and enzymatic stability of DHV. The rat models of subcutaneous embedding and abdominal aorta implantation showed that the scaffold regulated the polarization of macrophages into the anti-inflammatory and pro-modeling M2 phenotype and promoted endothelialization and ECM remodeling in the early stage without thrombosis and calcification. The novel TEHV exhibits excellent performance and can overcome the limitations of commonly used clinical prostheses.

ANTIOXIDANT, ANTI-INFLAMMATORY, ANTIDIABETIC, ANTHELMINTHIC, AND THROMBOLYTIC ACTIVITY OF THE METHANOL EXTRACT OF CITRULLUS LANATUS PEEL

Background: The present study aimed for in vitro evaluation of Citrullus lanatus Peel (CLP) methanol extract for antioxidative, anti-inflammatory, antidiabetic, anthelmintic, and thrombolytic activity. Method: CLP was extracted with methanol and tested for in vitro antioxidative (DPPH and H2O2 scavenging method), thrombolytic, anthelmintic, antidiabetic, and anti-inflammatory properties (human red blood cell membrane stabilization method, α-amylase inhibitory effect by using Iodine-Starch method. Results: The percentage yield was 7% and phytochemical profile showed the presence of steroid, tannins, saponins, flavonoids, glycoside and reducing sugar. In DPPH scavenging assay, 88.7% was at 1000 g/ml, whereas 34% to 48.9% (IC50 27.65 µg/ml) in H2O2 and from 61.8% to 95.2% (IC50 value: 07 µg/ml) in ASCOR. In in vitro anti-inflammatory activity, the maximum protection percentage was observed 58% and 44%. Consecutively, in anti-diabetic activity, IC50 values of 1137.64 μg/ml whereas reference (Acarbose) exhibited IC50 at 671.71 μg/ml. On the other hand, the inhibitory effect of Acarbose showed 57% and 98% respectively at 2000 μg/ml. In earthworms, it considered as very potential for the helminthic infection and helminthiasis. A significant clot lysis activity of 44% was noticed when the clots were treated with 100 µl of CLP extract (10 mg/ml) in in-vitro thrombolytic activity assay. Conclusions: The findings of this study revealed that the peel possessed the antioxidant, anti-inflammatory, antidiabetic, anthelminthic and thrombolytic activities. These findings will be motivated to do further research on molecular level to finds into more depth which is considered as the limitation of this study. Peer Review History: Received 3 February 2024; Revised 12 March 2024; Accepted 20 April; Available online 15 May 2024 Academic Editor: Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, amaka_mgbahurike@yahoo.com Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.5/10 Reviewers: Dr. Gehan Fawzy Abdel Raoof Kandeel, Pharmacognosy Department, National Research Centre, Dokki, 12622, Giza, Egypt, gehankandeel9@yahoo.com Dr. Francis Adou Yapo, Felix Houphouet Boigny, University of Abidjan, Ivory Coast, fyapo@yahoo.fr

RHD Genotyping to Resolve Weak and Discrepant RHD Phenotypes: The “Serenissima” Experience

Background: A considerable number of RHD alleles responsible for weak and partial D phenotypes have been identified. Serological determination of these phenotypes is often doubtful and makes genetic analysis of RHD gene highly desirable in transfusion recipients and pregnant women. Aim: We report the experience of Mestre Blood Bank in analysis of the RHD gene in six years from 2018 to 2023. Methods: Subjects for RHD gene analysis were selected for presence of a serological weak D phenotype, defined as reactivity of RBCs with an anti-D reagent giving no or weak (≤2+) score in initial testing but agglutinating moderately or strongly with anti human globulin (AHG). These samples were selected for genotyping using the microarray-based method Bead-Chip supplied by Werfen. Results: From 2018 to 2023, we selected, for RHD gene analysis, 555 subject with D weak phenotype; 86 subjects (15.5%) were D positive and 56 (10.1%) were D negative, without variant, in 413 subjects a D weak or a D variant was observed. Discussion: Many serological weak D phenotypes are associated to RHD gene mutations leading to one or more amino acids substitutions in the RhD protein predicted to be within or below the RBC membrane, causing decreased antigen expression on the red cell surface. Prevalence of serological weak D phenotypes varies by race and ethnicity. Serological weak D phenotypes are the most common D variants detected in Caucasians (0.2%-1.0%). The majority, as in our series, are associated with weak D type 1, 2 or 3. Our data confirmed a high prevalence of weak D type 1 and type 2, but we observed a high prevalence of type 11 and 15 and of the uncommon type 18 too. The most common partial D phenotypes in Europe are DNB, DVI, and DVII. Our data confirmed a high prevalence of D partial type VI. Studies indicate that D partial transfusion recipients are at risk of forming alloanti-D when exposed to conventional RhD-positive blood units.

Biomimetic “nano-spears” for CAFs-targeting: splintered three “shields” with enhanced cisplatin anti-TNBC efficiency

The treatment dilemma of triple-negative breast cancer (TNBC) revolves around drug resistance and metastasis. Cancer-associated fibroblasts (CAFs) contribute to cisplatin (Cis) resistance and further metastasis in TNBC, making TNBC a difficult-to-treat disease. The dense stromal barrier which restricts drug delivery, invasive phenotype of tumor cells, and immunosuppressive tumor microenvironment (TME) induced by CAFs serve as three “shields” for TNBC against Cis therapy. Here, we designed a silybin-loaded biomimetic nanoparticle coated with anisamide-modified red blood cell membrane (ARm@SNP) as a “nanospear” for CAFs-targeting, which could shatter the “shields” and significantly exhibit inhibitory effect on 4T1 cells in combination with Cis both in vitro and in vivo. The ARm@SNP/Cis elicited 4T1 tumor growth arrest and destroyed three “shields” as follows: disintegrating the stromal barrier by inhibiting blood vessels growth and the expression of fibronectin; decreasing 4T1 cell invasion and metastasis by affecting the TGF-β/Twist/EMT pathway which impeded EMT activation; reversing the immunosuppressive microenvironment by increasing the activity and infiltration of immunocompetent cells. Based on CAFs-targeting, ARm@SNP reversed the resistance of Cis, remodeled the TME and inhibited invasion and metastasis while significantly improving the therapeutic effect of Cis on 4T1 tumor-bearing mice, providing a promising approach for treating intractable TNBC.