Membrane Fragments Research Articles

Enhanced tumor self-targeting of lemon-derived extracellular vesicles by embedding homotypic cancer cell membranes for efficient drug delivery

Plant-derived nanovesicles (PDVs) as nanodrug delivery carriers have gained recognition due to their satisfactory biosafety. However, there remains a challenge to target tumor sites accurately due to the uncertain membrane protein components on the surface of vesicles. Herein, a composite nanodrug delivery system by encapsulating the chemotherapy drug DOX is establish to efficiently target breast cancer. The novel nanoplatform (LEVBD) is formed by embedding the membrane fragments from breast cancer cell with the lemon-derived nanovesicles (LEVs) as the foundational skeleton. LEVBD reveal wonderful homologous tumor targeting due to fusion of cancer cell membrane components with LEVs, and the encapsulation of hybrid vesicles facilitates the transcellular transport of drugs. After intravenous injection, LEVBD could efficiently and selectively home to homologous tumor sites even under competition from heterologous tumors and significantly inhibit tumor growth without any observable toxic side effects. The doping of homologous cancer cell membranes provides a paradigm for the precise delivery of drug delivery vehicles using plant-derived vesicles as the backbone.Graphical abstract

Characterization of cell membrane fragments containing muscle type nAChR from Tetronarce californica after preparation using high pressure homogenization.

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) commonly used as a model for receptors belonging to the Cys-loop superfamily. Members of pLGICs are standardly used in numerous toxicological investigations e.g., GABA and nAChR in the context of nerve agent poisoning. Organophosphorus compounds inhibit AChE, leading to accumulation of acetylcholine in the synaptic cleft and subsequently to a cholinergic crisis, in part through desensitization of nAChR. Due to the limitations of standard therapy, studies concerning functional ligand-receptor interactions of therapeutically active substances are of high importance. Therefore, we developed a novel method to obtain muscle type nAChR-containing membrane fragments from native tissue using high-pressure homogenization. The obtained microsomal fragments were characterized using Dynamic Light Scattering, laser Doppler electrophoresis and protein concentration. The microsomal membrane fragments were further purified, and the plasma membrane fraction was enriched using different density gradients. KD and BMax values were determined using a scintillation proximity assay (SPA) with [3H]epibatidine as reporter ligand. Measurement data showed that the ideal conditions to obtain microsomal membrane fragments with high pressure homogenization were four runs at 400bar. For density gradient centrifugation the under layering of the microsomal membrane fragments (bottom-up method) is to be preferred for further purification. Sucrose seems to be more efficient compared to xylitol or iodixanol density gradients. The nAChR-containing plasma membrane fractions resulting from the developed purification protocol achieve a high degree of quality and reproducibility, making them suitable to model physiological conditions. This system has the potential to be used in both bead- and filtration-based assays probing affinity parameters for ligand binding or functional experiments. The protocol can be easily modified for other LGICs or transmembrane proteins, allowing for further expansion of its use.

SELECTION OF POLYMER FOR STENT-GRAFT COATING IN TERMS OF BIOCOMPATIBILITY AND BIODEGRADATION CHARACTERISTICS

HighlightsCoated stents or stent-grafts are widely used in endovascular surgery to close arterial perforations, dissections and aneurysms, as well as in stenting of arteries with loose atherosclerotic plaques in order to reduce the risk of emboli and strokes. The material of stent coating is of great importance in the prevention of early thrombosis and restenosis of stent grafts. Biodegradable polymers have an advantage over non-biodegradable polymers because they do not remain in the patient's tissues for a long period of time and do not cause chronic inflammation. The study of the dynamics and biodegradation characteristics of polymer coating can provide information about its suitability and safety in the stent graft. Annotation Aim. To screen potentially suitable polymers for stent-graft coating with the following assessment of biocompatibility and biodegradation dynamics in an in vivo experiment.Methods. The coating was applied on the stent by electrospinning from a solution of polymers: Polycaprolactone (PCL); polydioxanone (PDO); polylactide-co-caprolactone (P(LA/CL)) with lactide: caprolactone ratio – 70:30; polylactide-co-glycolide (PLGA) with lactide: glycolide ratio – 50:50. Chloroform (CHCl3) and 1,1,1,1,3,3,3,3,3-hexafluoro-2-propanol (HFP) were used as a solvent. Gore-Tex polymer membrane made of polytetrafluoroethylene (ePTFE) was used as control. In order to evaluate biocompatibility and biodegradation dynamics in vivo, the studied polymeric samples were implanted subcutaneously in male Wistar rats for periods of 7 and 14 days, 1, 2, 3 and 6 months. After explantation all samples were studied histologically.Results. 14 days after implantation a moderate inflammatory reaction developed on all polymer specimens. The PTFE material was separated from the surrounding tissues by a thin ordered fibrous capsule, confirming its satisfactory biocompatible properties. The porous structure of PCL membranes was filled by fibroblasts and the specimens were tightly integrated into the surrounding tissues. P(LA/CL) degrades with the formation of large fragments. The composite polymer P(LA/CL)/PDO degraded to form small fragments that are tightly integrated with the fibrous capsule. PLGA membranes showed high rates of degradation - membrane fragments were detected 3 months after implantation, after 6 months the samples were completely degraded.Conclusion. The results of biocompatibility and biodegradation assessment in vivo indicate that the most promising polymers for stent-graft creation are PCL, PLGA and composite polymer P(LA/CL)/PDO. In order to finalize the choice of polymer, it is necessary to conduct further studies to assess the biocompatibility and degradation of polymer coating during implantation of stent-grafts into the arterial bed of large laboratory animals.

In situ self-reassembling nanosystem enhances PD-L1 blockade for cancer immunotherapy

Although immune checkpoint inhibitors (ICIs) have made great progress in cancer treatment, their off-tumor distribution, low affinity of traditional ICIs and insufficient T cells infiltration at tumor site limit immunotherapeutic efficacy. Herein, we engineer a highly specific and effective PD-L1 inhibitor (PEC) that modulates the level of binding sites with PD-L1. Specifically, PEC is a hybrid system composed of E. coli membrane expressing PD-L1 binding protein and cancer cell membrane. Notably, PEC can target the tumor site, produce oxygen in response to H2O2, rupture into membrane fragments, and reassemble to form vesicles retaining the PD-L1 binding protein. Through in situ fracture and reassembly, PEC transforms from a hybrid membrane to a single E. coli membrane, leading to the increased density of PD-L1 binding protein. Consequently, the reassembled vesicles can bind to more PD-L1 on tumor cells and induce its degradation in lysosomes. Furthermore, the cGAS-STING signaling activators HZD is encapsulated into PEC to promote T cells infiltration. We demonstrate that PEC@HZD achieves sequential T cells recruitment and functional enhancement, thus stimulating a powerful antitumor immune response. This work provides a new perspective on tailoring ICIs to improve cancer immunotherapy.

Quercetin preserves mitochondria-endoplasmic reticulum contact sites improving mitochondrial dynamics in aged myocardial cells.

Cardiomyocyte senescence plays a crucial role in the pathophysiology of age-related cardiovascular disease. Senescent cells with impaired contractility, mitochondrial dysfunction, and hypertrophic growth accumulate in the heart during aging, contributing to cardiac dysfunction and remodeling. Mitochondrial dynamics is altered in aging cells, leading to changes in their function and morphology. Such rearrangements can affect the spatially restricted region of the mitochondrial membrane that interacts with reticulum membrane fragments, termed mitochondria-endoplasmic reticulum (ER) contact sites (MERCs). Besides, oxidative stress associated with inefficient organelle turnover can drive cellular senescence. Therefore, in this study, we evaluated the possible association between the senolytic effect of the antioxidant quercetin (Q) and MERCs preservation in a D-galactose-induced cellular senescence model. We found that Q ameliorates the senescent phenotype of H9c2 cells in association with increased mitochondria-ER colocalization, reduced distance between both organelles, and lower ROS production. Moreover, regulation of fusion and fission processes was related with increased mitochondrial ATP production and enhanced transmembrane potential. Overall, our data provide evidence that the inhibitory effect of Q on cellular senescence is associated with preserved MERCs and improved mitochondrial function and morphology, which might contribute to the attenuation of cardiac dysfunction.

Evaluation of the effectiveness of ultrafast urease tests in the diagnosis of Helicobacter pylori-associated gastritis in children

Background: Helicobacter pylori (H. pylori) is one of the main causes of gastritis, peptic ulcer and stomach cancer. Ultrafast urease tests (UUT) make it possible to simplify patient management by notifying the result after 5 minutes and entering it into the protocol of endoscopic conclusion. There is no data on the accuracy of UUT in the pediatric population. Aim of the study: to identify the accuracy of UUT in comparison with histological examination in children. Methods: a single-center retrospective study of children (4-18 years old) who underwent esophagogastroduodenoscopy with biopsy of the gastric antrum mucosa for H.pylori UUT, followed by histological examination. The reaction time stated by the manufacturers was estimated to be no more than 5 minutes for a positive result. Histology was the “gold” standard. Results: 211 patients were examined (boys n=103, 48.8%; girls n=108, 51.2%) average age 11.7 years (±3.5, 4-18 years). H.pylori infection was detected in 31 out of 211 patients. There was a significantly higher number of positive reactions of both UUT’s in the age group of children from 11 to 18 years old compared with the group of children from 4 to 10 years old. The sensitivity of AMA RUT Pro UUT was 18.7%, specificity 93.8%, negative and positive predictive value 82% and 42%, respectively, accuracy 79%. The sensitivity of the BioHit UFT300 UFUT was 40%, specificity 96.5%, negative and positive predictive value 92% and 60%, respectively, accuracy 90%. Conclusion: our data demonstrate the low sensitivity of ultrafast urease tests in children when using a single fragment of tissue from the gastric antrum in the five-minute reaction time allotted by manufacturers to a negative result. Perhaps sensitivity indicators can be improved by placing several fragments of the mucous membrane of the body and the antrum of the stomach on one test, which is a direction for future research.

Fabrication of Biomimetic Hybrid Liposomes via Microfluidic Technology: Homotypic Targeting and Antitumor Efficacy Studies in Glioma Cells.

The treatment of glioblastoma is hindered by the blood-brain barrier (BBB) and rapid drug clearance by the immune system. To address these challenges, we propose a novel drug delivery system using liposomes modified with cell membrane fragments. These modified liposomes can evade the immune system, cross the BBB, and accumulate in tumor tissue through homotypic targeting, thereby delivering drugs like paclitaxel and carboplatin more effectively. In this work, the hybrid liposomes were synthesized using microfluidics and integrating 3D printing to produce the microfluidic devices. In vitro, we explored the homotypic targeting capability, BBB passing ability, and therapeutic efficacy of paclitaxel and carboplatin. The production of hybrid liposomes by microfluidics has been key to creating high-quality biomimetic nanoparticles, and the integration of 3D printing has simplified the production of microfluidic devices, making the process more efficient and economical. In vitro experiments have shown that these drug-loaded biomimetic hybrid liposomes are able to reach the homotypic target, cross the BBB, and maintain the efficacy of paclitaxel and carboplatin. The development of biomimetic hybrid liposomes represents a promising approach for the treatment of glioblastoma. By combining the advantages of liposomal drug delivery with the stealth properties and targeting capabilities of cell membrane fragments, these nanoparticles can potentially overcome the challenges associated with traditional therapies.

Vascular Basement Membrane Fragmentation in Keloids and the Expression of Key Basement Membrane Component Genes

Background: Keloids are growing scars that arise from injury to the reticular dermis and subsequent chronic local inflammation. The latter may be promoted by vascular hyperpermeability, which permits the ingress of chronic inflammatory cells/factors. Cutaneous capillaries consist of endothelial cells that generate, and are anchored by, a vascular basement membrane (VBM). Because VBM blocks immune cells/factors ingress, we investigated whether keloids are associated with altered VBM structure and/or VBM component expression by local endothelial cells. Methods: In total, 54 keloid (n = 27) and adjacent normal skin (n = 27) samples from 14 patients underwent transmission electron microscopy (TEM). Cross-sections of whole capillaries were identified. VBM thickness, continuity, and the number of layers in keloid and normal skin tissues were quantified. The differential expression of 222 previously reported VBM component genes in keloid and normal skin endothelial cells was analyzed using the GSE121618-microarray dataset. Results: TEM images showed that keloid VBMs were significantly thinner than adjacent skin VBMs (0.053 versus 0.078 nm; P < 0.001). They were also greatly fragmented (continuity was 46% versus 85% in normal skin; P < 0.001) and had fewer (1.2 versus 2.4) layers (P < 0.001). Keloidal endothelial cells demonstrated downregulation of 22 genes, including papilin, laminin-α5, and laminin-α2, and upregulation of 28 genes, including laminin-β1, laminin-β2, laminin-γ1, and laminin-γ2. Conclusions: VBMs are greatly fragmented in keloids. These changes support the notion that keloids are initiated/promoted, at least partly, by vascular hyperpermeability.

How NINJ1 mediates plasma membrane rupture and why NINJ2 cannot.

Ninjurin-1 (NINJ1) is an active executioner of plasma membrane rupture (PMR), a process previously thought to be a passive osmotic lysis event in lytic cell death. Ninjurin-2 (NINJ2) is a close paralog of NINJ1 but cannot mediate PMR. Using cryogenic electron microscopy (cryo-EM), we show that NINJ1 and NINJ2 both assemble into linear filaments that are hydrophobic on one side but hydrophilic on the other. This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. In contrast to the straight NINJ1 filament, the NINJ2 filament is curved toward the intracellular space, preventing its circularization or even assembly on a relatively flat membrane to mediate PMR. Mutagenesis studies further demonstrate that the NINJ2 filament curvature is induced by strong association with lipids, particularly a cholesterol molecule, at the cytoplasmic leaflet of the lipid bilayer.

Streptozotocin-induced morphological changes in rat lungs

ABSTRACT Streptozotocin (STZ) is a commonly used compound for the induction of type 2 diabetes (T2D) in animal models, but its effects on non-pancreatic tissues like the lungs are not well understood. This study aimed to examine the histopathological impact of STZ on the lungs using male Sprague-Dawley rats. The rats were divided into two groups: a control group on a normal diet and an STZ-treated group receiving a high-fat diet and 10% sucrose water for 8 weeks, followed by an STZ injection (30 mg/kg body weight). All rats were terminated 9 days after STZ administration, and lung samples were collected for light microscopy, transmission electron microscopy (TEM), and confocal laser scanning microscopy. Light microscopy revealed thickening of alveolar septa, narrowing of alveoli, and inflammatory infiltrates in the STZ group. TEM showed mitochondrial damage in type 2 pneumocytes, including membrane fragmentation, cristae loss, and formation of mitochondrial-derived vesicles. Confocal microscopy revealed significantly higher expression of myeloperoxidase, neutrophil elastase, and citrullinated histone 3 in the STZ group compared to controls. These findings suggest that STZ induces considerable lung damage, emphasizing the need to consider lung toxicity in studies involving STZ.

Proteomic analysis of the pyrenoid-traversing membranes of Chlamydomonas reinhardtii reveals novel components.

Pyrenoids are algal CO2-fixing organelles that mediate approximately one-third of global carbon fixation and hold the potential to enhance crop growth if engineered into land plants. Most pyrenoids are traversed by membranes that are thought to supply them with concentrated CO2. Despite the critical nature of these membranes for pyrenoid function, they are poorly understood, with few protein components known in any species.• Here we identify protein components of the pyrenoid-traversing membranes from the leading model alga Chlamydomonas reinhardtii by affinity purification and mass spectrometry of membrane fragments. Our proteome includes previously-known proteins as well as novel candidates.• We further characterize two of the novel pyrenoid-traversing membrane-resident proteins, Cre10.g452250, which we name Pyrenoid Membrane Enriched 1 (PME1), and LCI16. We confirm their localization, observe that they physically interact, and find that neither protein is required for normal membrane morphology.• Taken together, our study identifies the proteome of pyrenoid-traversing membranes and initiates the characterization of a novel pyrenoid-traversing membrane complex, building toward a mechanistic understanding of the pyrenoid.

Structural basis of antimicrobial membrane coat assembly by human GBP1

Guanylate-binding proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) mediating host defense against intracellular pathogens. Their antimicrobial activity hinges on their ability to self-associate and coat pathogen-associated compartments or cytosolic bacteria. Coat formation depends on GTPase activity but how nucleotide binding and hydrolysis prime coat formation remains unclear. Here, we report the cryo-electron microscopy structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and describe the molecular ultrastructure of the GBP1 coat on liposomes and bacterial lipopolysaccharide membranes. Conformational changes of the middle and GTPase effector domains expose the isoprenylated C terminus for membrane association. The α-helical middle domains form a parallel, crossover arrangement essential for coat formation and position the extended effector domain for intercalation into the lipopolysaccharide layer of gram-negative membranes. Nucleotide binding and hydrolysis create oligomeric scaffolds with contractile abilities that promote membrane extrusion and fragmentation. Our data offer a structural and mechanistic framework for understanding GBP1 effector functions in intracellular immunity.

Renal cancer cells acquire immune surface protein through trogocytosis and horizontal gene transfer.

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here, we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45, CD56, CD14, and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells, as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis, which also revealed colocalization of lymphoid markers with carbonic anhydrase 9 (CAIX), a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells in tumor cells following trogocytosis. Finally, we show evidence of chromosomal DNA being transferred from immune cells to tumor cells during trogocytosis. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell, resulting in a fusion phenotype that expressed both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally, further studies into trogocytosis will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other types of cancers.

Advancements in Engineering Planar Model Cell Membranes: Current Techniques, Applications, and Future Perspectives.

Cell membranes are crucial elements in living organisms, serving as protective barriers and providing structural support for cells. They regulate numerous exchange and communication processes between cells and their environment, including interactions with other cells, tissues, ions, xenobiotics, and drugs. However, the complexity and heterogeneity of cell membranes-comprising two asymmetric layers with varying compositions across different cell types and states (e.g., healthy vs. diseased)-along with the challenges of manipulating real cell membranes represent significant obstacles for in vivo studies. To address these challenges, researchers have developed various methodologies to create model cell membranes or membrane fragments, including mono- or bilayers organized in planar systems. These models facilitate fundamental studies on membrane component interactions as well as the interactions of membrane components with external agents, such as drugs, nanoparticles (NPs), or biomarkers. The applications of model cell membranes have extended beyond basic research, encompassing areas such as biosensing and nanoparticle camouflage to evade immune detection. In this review, we highlight advancements in the engineering of planar model cell membranes, focusing on the nanoarchitectonic tools used for their fabrication. We also discuss approaches for incorporating challenging materials, such as proteins and enzymes, into these models. Finally, we present our view on future perspectives in the field of planar model cell membranes.

An innovative method for fractionating the milk fat globule membrane and the major buttermilk proteins

Buttermilk (BM) is a by-product of butter manufacturing that shares a similar composition to skim milk but contains a higher concentration of phospholipids due to milk fat globule membrane (MFGM) fragments released during cream churning. Despite its high added-value components, MFGM is underutilized mainly due to the challenge in separating its components from the other constituents of buttermilk. Our work has focused on the use of calcium phosphate particles, called hydroxyapatite (HA), to extract and separate the different components from buttermilk. This study investigated the impact of various physicochemical parameters (temperature, ionic strength, and pH) on the adsorption of a mixture of micellar casein (CM), whey proteins, and MFGM from BM onto HA surfaces. A box-Behnken design was utilized to predict optimal physicochemical conditions for selectively separating components in buttermilk. Ionic strength (IS) played a crucial role, with low IS having a minimal positive influence and high IS negatively influencing adsorption. pH influenced adsorption by altering protein and HA surface charges. The optimal conditions for adsorption were an IS of 100 mM for a pH of 7, for 90 % of CM, 37 % of β-lactoglobulin, 11 % of α-lactalbumin and 7 % of MFGM. Desorption experiments using citrate and alkaline pH showed promising results for CM recovery, with citrate efficiently releasing the adsorbed CM. Additionally, MFGM were separated from whey proteins through selective precipitation at a pH of 4.0, allowing sedimentation of MFGM (100 %) while keeping whey proteins soluble. The presence of β-lactoglobulin in the protein fraction of MFGM (31 %) and CM (37 %) was attributed to pasteurization, which denatured a portion of β-lactoglobulin and led to its attachment to MFGM surfaces and interaction with CM. This protocol produces a nearly pure fraction of MFGM with a simple method which is suitable for industrial production, representing significant valorization for the dairy sector.

Preparation and characterization of BSA-loaded liraglutide and platelet fragment nanoparticle delivery system for the treatment of diabetic atherosclerosis

BackgroundDiabetic atherosclerosis is one of the main causes of morbidity and mortality worldwide, but its therapeutic options are limited. Liraglutide (LIR), a synthetic analog of GLP-1 approved as an anti-obesity drug by the FDA, has been reported as a promising drug for diabetic atherosclerosis. However, the main problem with LIR is its use that requires regular parenteral injections, which necessitates the improvement of drug delivery for increased efficiency and minimization of injection numbers.ResultsThe objective of our present study was to prepare and characterize nanoparticles (BSA@LIR-PMF) for targeted drug delivery using LIR-encapsulated platelet membrane fragments (PMF) coated bovine serum albumin (BSA). We used various methods to characterize the prepared nanoparticles and evaluated their efficiency on diabetes-induced atherosclerosis in vitro and in vivo. The results showed that the nanoparticles were spherical and had good stability and uniform size with intact membrane protein structure. The loading and encapsulation rates (LR and ER) of BSA@LIR-PMF were respectively 8.29% and 90.39%, while the cumulative release rate was around 77.06% after 24 h. Besides, we also examined the impact of BSA@LIR-PMF on the proliferation, migration, phagocytosis, reactive oxygen species (ROS) levels, oxidative phosphorylation, glycolysis, lactate and ATP levels, and lipid deposition in the aortas. The results indicated that BSA@LIR-PMF could effectively inhibit ox-LDL-stimulated abnormal cell proliferation and migration, reduce the level of ROS and lactate concentration, and enhance the level of ATP, thereby improving oxidative phosphorylation in ox-LDL-treated cells.ConclusionBSA@LIR-PMF significantly inhibited diabetes-induced atherosclerosis. It was anticipated that the BSA@LIR-PMF nanoparticles might be used for treating diabetes-associated cardiovascular complications.

Human glioblastoma-derived cell membrane nanovesicles: a novel, cell-specific strategy for boron neutron capture therapy of brain tumors

Glioblastoma (GBM), one of the deadliest brain tumors, accounts for approximately 50% of all primary malignant CNS tumors, therefore novel, highly effective remedies are urgently needed. Boron neutron capture therapy, which has recently repositioned as a promising strategy to treat high-grade gliomas, requires a conspicuous accumulation of boron atoms in the cancer cells. With the aim of selectively deliver sodium borocaptate (BSH, a 12 B atoms-including molecule already employed in the clinics) to GBM cells, we developed novel cell membrane-derived vesicles (CMVs), overcoming the limits of natural extracellular vesicles as drug carriers, while maintaining their inherent homing abilities that make them preferable to fully synthetic nanocarriers. Purified cell membrane fragments, isolated from patient-derived GBM stem-like cell cultures, were used to prepare nanosized CMVs, which retained some membrane proteins specific of the GBM parent cells and were devoid of potentially detrimental genetic material. In vitro tests evidenced the targeting ability of this novel nanosystem and ruled out any cytotoxicity. The CMVs were successfully loaded with BSH, by following two different procedures, i.e. sonication and electroporation, demonstrating their potential applicability in GBM therapy.

Tangential flow filtration-facilitated purification of human red blood cell membrane fragments and its preferential use in removing unencapsulated material from resealed red blood cell ghosts compared to centrifugation.

The biodistribution of many therapeutics is controlled by the immune system. In addition, some molecules are cytotoxic when not encapsulated inside of larger cellular structures, such as hemoglobin (Hb) encapsulation inside of red blood cells (RBCs). To counter immune system recognition and cytotoxicity, drug delivery systems based on red blood cell membrane fragments (RBCMFs) have been proposed as a strategy for creating immunoprivileged therapeutics. However, the use of RBCMFs for drug delivery applications requires purification of RBCMFs at large scale from lysed RBCs free of their intracellular components. In this study, we were able to successfully use tangential flow filtration (TFF) to remove >99% of cell-free Hb from lysed RBCs at high concentrations (30%-40% v/v), producing RBCMFs that were 2.68 ± 0.17 μm in diameter. We were also able to characterize the RBCMFs more thoroughly than prior work, including measurement of particle zeta potential, along with individual TFF diacycle data on the cell-free Hb concentration in solution and time per diacycle, as well as concentration and size of the RBCMFs. In addition to purifying RBCMFs from lysed RBCs, we utilized a hypertonic solution to reseal purified RBCMFs encapsulating a model protein (Hb) to yield resealed Hb-encapsulated RBC ghosts (Hb-RBCGs). TFF was then compared against centrifugation as an alternative method for removing unencapsulated Hb from Hb-RBCGs, and the effects that each washing method on the resulting Hb-RBCG biophysical properties was assessed.

SARS-CoV-2-related peptides induce endothelial-to-mesenchymal transition in endothelial capillary cells derived from different body districts: focus on membrane (M) protein.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19, may lead to multiple organ dysfunctions and long-term complications. The induction of microvascular dysfunction is regarded as a main player in these pathological processes. To investigate the possible impact of SARS-CoV-2-induced endothelial-to-mesenchymal transition (EndMT) on fibrosis in "long-COVID" syndrome, we used primary cultures of human microvascular cells derived from the lungs, as the main infection target, compared to cells derived from different organs (dermis, heart, kidney, liver, brain) and to the HUVEC cell line. To mimic the virus action, we used mixed SARS-CoV-2 peptide fragments (PepTivator®) of spike (S), nucleocapsid (N), and membrane (M) proteins. TGFβ2 and cytokine mix (IL-1β, IL-6, TNFα) were used as positive controls. The percentage of cells positive to mesenchymal and endothelial markers was quantified by high content screening. We demonstrated that S+N+M mix induces irreversible EndMT in all analyzed endothelial cells via the TGFβ pathway, as demonstrated by ApoA1 treatment. We then tested the contribution of single peptides in lung and brain cells, demonstrating that EndMT is triggered by M peptide. This was confirmed by transfection experiment, inducing the endogenous expression of the glycoprotein M in lung-derived cells. In conclusion, we demonstrated that SARS-CoV-2 peptides induce EndMT in microvascular endothelial cells from multiple body districts. The different peptides play different roles in the induction and maintenance of the virus-mediated effects, which are organ-specific. These results corroborate the hypothesis of the SARS-CoV-2-mediated microvascular damage underlying the multiple organ dysfunctions and the long-COVID syndrome.

Numerical model for electrogenic transport by the ATP-dependent potassium pump KdpFABC

In vitro assays of ion transport are an essential tool for understanding molecular mechanisms associated with ATP-dependent pumps. Because ion transport is generally electrogenic, principles of electrophysiology are applicable, but conventional tools like patch-clamp are ineffective due to relatively low turnover rates of the pumps. Instead, assays have been developed to measure either voltage or current generated by transport activity of a population of molecules either in cell-derived membrane fragments or after reconstituting purified protein into proteoliposomes. In order to understand the nuances of these assays and to characterize effects of various operational parameters, we have developed a numerical model to simulate data produced by two relevant assays: fluorescence from voltage-sensitive dyes and current recorded by capacitive coupling on solid supported membranes. Parameters of the model, which has been implemented in Python, are described along with underlying principles of the computational algorithm. Experimental data from KdpFABC, a K+ pump associated with P-type ATPases, are presented, and model parameters have been adjusted to mimic these data. In addition, effects of key parameters such as nonselective leak conductance and turnover rate are demonstrated. Finally, simulated data are used to illustrate the effects of capacitive coupling on measured current and to compare alternative methods for quantification of raw data.