MEDUSA: A cloud-based tool for the analysis of X-ray diffuse scattering to obtain the bending modulus from oriented membrane stacks.

An important mechanical property of cells is their membrane bending modulus, κ. Here, we introduce MEDUSA (MEmbrane DiffUse Scattering Analysis), a cloud-based analysis tool to determine the bending modulus, κ, from the analysis of X-ray diffuse scattering. MEDUSA uses GPU (graphics processing unit) accelerated hardware and a parallelized algorithm to run the calculations efficiently in a few seconds. MEDUSA's graphical user interface allows the user to upload 2-dimensional data collected from different sources, perform background subtraction and distortion corrections, select regions of interest, run the fitting procedure and output the fitted parameters, the membranes' bending modulus κ, and compressional modulus B.

Alkylated EDTA potentiates antibacterial photodynamic activity of protoporphyrin.

Antibiotic resistance has garnered significant attention due to the scarcity of new antibiotics in development. Protoporphyrin IX (PpIX)-mediated photodynamic therapy shows promise as a novel antibacterial strategy, serving as an alternative to antibiotics. However, the poor solubility of PpIX and its tendency to aggregate greatly hinder its photodynamic efficacy. In this study, we demonstrate that alkylated EDTA derivatives (aEDTA), particularly C14-EDTA, can enhance the solubility of PpIX by facilitating its dispersion in aqueous solutions. The combination of C14-EDTA and PpIX exhibits potent antibacterial activity against Staphylococcus aureus (S. aureus) when exposed to LED light irradiation. Furthermore, this combination effectively eradicates S. aureus biofilms, which are known to be strongly resistant to antibiotics, and demonstrates high therapeutic efficacy in an animal model of infected ulcers. Mechanistic studies reveal that C14-EDTA can disrupt PpIX crystallization, increase bacterial membrane permeability and sequester divalent cations, thereby improving the accumulation of PpIX in bacteria. This, in turn, enhances reactive oxygen species (ROS) production and the antibacterial photodynamic activity. Overall, this effective strategy holds great promise in combating antibiotic-resistant strains.

Stabilization of Lipid Membranes through Partitioning of the Blood Bag Plasticizer Di-2-ethylhexyl phthalate (DEHP).

The safe storage of blood is of fundamental importance to health care systems all over the world. Currently, plastic bags are used for the collection and storage of donated blood and are typically made of poly(vinyl chloride) (PVC) plasticized with di-2-ethylhexyl phthalate (DEHP). DEHP is known to migrate into packed red blood cells (RBC) and has been found to extend their shelf life. It has been speculated that DEHP incorporates itself into the RBC membrane and alters membrane properties, thereby reducing susceptibility to hemolysis and morphological deterioration. Here, we used high-resolution X-ray diffraction and molecular dynamics (MD) simulations to study the interaction between DEHP and model POPC lipid membranes at high (9 mol %) and low (1 mol %) concentrations of DEHP. At both concentrations, DEHP was found to spontaneously partition into the bilayer. At high concentrations, DEHP molecules were found to aggregate in the aqueous phase before inserting as clusters into the membrane. The presence of DEHP in the bilayers resulted in subtle, yet statistically significant, alterations in several membrane properties in both the X-ray diffraction experiments and MD simulations. DEHP led to (1) an increase of membrane width and (2) an increase in the area per lipid. It was also found to (3) increase the deuterium order parameter, however, (4) decrease membrane orientation, indicating the formation of thicker, stiffer membranes with increased local curvature. The observed effects of DEHP on lipid bilayers may help to better understand its effect on RBC membranes in increasing the longevity of stored blood by improving membrane stability.

Anesthetics significantly increase the amount of intramembrane water in lipid membranes.

The potency of anesthesia was directly linked to the partitioning of the drug molecules in cell membranes by Meyer and Overton. Many molecules interact with lipid bilayers and lead to structural and functional changes. It remains an open question which change in membrane properties is responsible for a potential anesthetic effect or if anesthetics act by binding to direct targets. We studied the effect of ethanol, diethyl ether and isoflurane on the water distribution in lipid bilayers by combining all-atom molecular dynamics simulations and neutron diffraction experiments. The simulations show strong membrane-drug interactions with partitioning coefficients of 38%, 92% and 100% for ethanol, diethyl ether and isoflurane, respectively, and provide evidence for an increased water partitioning in the membrane core. The amount of intramembrane water molecules was experimentally determined by selectively deuterium labeling lipids, anesthetic drug and water molecules in neutron diffraction experiments. Four additional water molecules per lipid were observed in the presence of ethanol. Diethyl ether and isoflurane were found to significantly increase the amount of intramembrane water by 25% (8 water molecules). This increase in intramembrane water may contribute to the non-specific interactions between anesthetics and lipid membranes.

Biomimetic Nanomaterials for the Immunomodulation of the Cardiosplenic Axis Postmyocardial Infarction.

The spleen is an important mediator of both adaptive and innate immunity. As such, attempts to modulate the immune response provided by the spleen may be conducive to improved outcomes for numerous diseases throughout the body. Here, biomimicry is used to rationally design nanomaterials capable of splenic retention and immunomodulation for the treatment of disease in a distant organ, the postinfarct heart. Engineered senescent erythrocyte-derived nanotheranostic (eSENTs) are generated, demonstrating significant uptake by the immune cells of the spleen including T and B cells, as well as monocytes and macrophages. When loaded with suberoylanilide hydroxamic acid (SAHA), the nanoagents exhibit a potent therapeutic effect, reducing infarct size by 14% at 72 h postmyocardial infarction when given as a single intravenous dose 2 h after injury. These results are supportive of the hypothesis that RBC-derived biomimicry may provide new approaches for the targeted modulation of the pathological processes involved in myocardial infarction, thus further experiments to decisively confirm the mechanisms of action are currently underway. This novel concept may have far-reaching applicability for the treatment of a number of both acute and chronic conditions where the immune responses are either stimulated or suppressed by the splenic (auto)immune milieu.

Structural and mechanical properties of the red blood cell's cytoplasmic membrane seen through the lens of biophysics.

Red blood cells (RBCs) are the most abundant cell type in the human body and critical suppliers of oxygen. The cells are characterized by a simple structure with no internal organelles. Their two-layered outer shell is composed of a cytoplasmic membrane (RBC cm ) tethered to a spectrin cytoskeleton allowing the cell to be both flexible yet resistant against shear stress. These mechanical properties are intrinsically linked to the molecular composition and organization of their shell. The cytoplasmic membrane is expected to dominate the elastic behavior on small, nanometer length scales, which are most relevant for cellular processes that take place between the fibrils of the cytoskeleton. Several pathologies have been linked to structural and compositional changes within the RBC cm and the cell's mechanical properties. We review current findings in terms of RBC lipidomics, lipid organization and elastic properties with a focus on biophysical techniques, such as X-ray and neutron scattering, and Molecular Dynamics simulations, and their biological relevance. In our current understanding, the RBC cm 's structure is patchy, with nanometer sized liquid ordered and disordered lipid, and peptide domains. At the same time, it is surprisingly soft, with bending rigidities κ of 2-4 kBT. This is in strong contrast to the current belief that a high concentration of cholesterol results in stiff membranes. This extreme softness is likely the result of an interaction between polyunsaturated lipids and cholesterol, which may also occur in other biological membranes. There is strong evidence in the literature that there is no length scale dependence of κ of whole RBCs.

The Formation of RNA Pre-Polymers in the Presence of Different Prebiotic Mineral Surfaces Studied by Molecular Dynamics Simulations.

We used all-atom Molecular Dynamics (MD) computer simulations to study the formation of pre-polymers between the four nucleotides in RNA (AMP, UMP, CMP, GMP) in the presence of different substrates that could have been present in a prebiotic environment. Pre-polymers are C3'-C5' hydrogen-bonded nucleotides that have been suggested to be the precursors of phosphodiester-bonded RNA polymers. We simulated wet-dry cycles by successively removing water molecules from the simulations, from ~60 to 3 water molecules per nucleotide. The nine substrates in this study include three clay minerals, one mica, one phosphate mineral, one silica, and two metal oxides. The substrates differ in their surface charge and ability to form hydrogen bonds with the nucleotides. From the MD simulations, we quantify the interactions between different nucleotides, and between nucleotides and substrates. For comparison, we included graphite as an inert substrate, which is not charged and cannot form hydrogen bonds. We also simulated the dehydration of a nucleotide-only system, which mimics the drying of small droplets. The number of hydrogen bonds between nucleotides and nucleotides and substrates was found to increase significantly when water molecules were removed from the systems. The largest number of C3'-C5' hydrogen bonds between nucleotides occurred in the graphite and nucleotide-only systems. While the surface of the substrates led to an organization and periodic arrangement of the nucleotides, none of the substrates was found to be a catalyst for pre-polymer formation, neither at full hydration, nor when dehydrated. While confinement and dehydration seem to be the main drivers for hydrogen bond formation, substrate interactions reduced the interactions between nucleotides in all cases. Our findings suggest that small supersaturated water droplets that could have been produced by geysers or springs on the primitive Earth may play an important role in non-enzymatic RNA polymerization.

Perspective on the Application of Erythrocyte Liposome-Based Drug Delivery for Infectious Diseases.

Nanoparticles are explored as drug carriers with the promise for the treatment of diseases to increase the efficacy and also reduce side effects sometimes seen with conventional drugs. To accomplish this goal, drugs are encapsulated in or conjugated to the nanocarriers and selectively delivered to their targets. Potential applications include immunization, the delivery of anti-cancer drugs to tumours, antibiotics to infections, targeting resistant bacteria, and delivery of therapeutic agents to the brain. Despite this great promise and potential, drug delivery systems have yet to be established, mainly due to their limitations in physical instability and rapid clearance by the host's immune response. Recent interest has been taken in using red blood cells (RBC) as drug carriers due to their naturally long circulation time, flexible structure, and direct access to many target sites. This includes coating of nanoparticles with the membrane of red blood cells, and the fabrication and manipulation of liposomes made of the red blood cells' cytoplasmic membrane. The properties of these erythrocyte liposomes, such as charge and elastic properties, can be tuned through the incorporation of synthetic lipids to optimize physical properties and the loading efficiency and retention of different drugs. Specificity can be established through the anchorage of antigens and antibodies in the liposomal membrane to achieve targeted delivery. Although still at an early stage, this erythrocyte-based platform shows first promising results in vitro and in animal studies. However, their full potential in terms of increased efficacy and side effect minimization still needs to be explored in vivo.

Erythro-PmBs: A Selective Polymyxin B Delivery System Using Antibody-Conjugated Hybrid Erythrocyte Liposomes.

As a result of the growing worldwide antibiotic resistance crisis, many currently existing antibiotics have become ineffective due to bacteria developing resistive mechanisms. There are a limited number of potent antibiotics that are successful at suppressing microbial growth, such as polymyxin B (PmB); however, these are often deemed as a last resort due to their toxicity. We present a novel PmB delivery system constructed by conjugating hybrid erythrocyte liposomes with antibacterial antibodies to combine a high loading efficiency with guided delivery. The retention of PmB is enhanced by incorporating negatively charged lipids into the red blood cells' cytoplasmic membrane (RBCcm). Anti-Escherichia coli antibodies are attached to these hybrid erythrocyte liposomes by the inclusion of DSPE-PEG maleimide linkers. We show that these erythro-PmBs have a loading efficiency of ∼90% and are effective in delivering PmB to E. coli, with values for the minimum inhibitory concentration (MIC) being comparable to those of free PmB. The MIC values for Klebsiella aerogenes, however, significantly increased well beyond the resistant breakpoint, indicating that the inclusion of the anti-E. coli antibodies enables the erythro-PmBs to selectively deliver antibiotics to specific targets.

Erythro-VLPs: Anchoring SARS-CoV-2 spike proteins in erythrocyte liposomes.

Novel therapeutic strategies are needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. Here, we present a protocol to anchor the SARS-CoV-2 spike (S-)protein in the cytoplasmic membranes of erythrocyte liposomes. A surfactant was used to stabilize the S-protein's structure in the aqueous environment before insertion and to facilitate reconstitution of the S-proteins in the erythrocyte membranes. The insertion process was studied using coarse grained Molecular Dynamics (MD) simulations. Liposome formation and S-protein anchoring was studied by dynamic light scattering (DLS), ELV-protein co-sedimentation assays, fluorescent microcopy and cryo-TEM. The Erythro-VLPs (erythrocyte based virus like particles) have a well defined size of ∼200 nm and an average protein density on the outer membrane of up to ∼300 proteins/µm2. The correct insertion and functional conformation of the S-proteins was verified by dose-dependent binding to ACE-2 (angiotensin converting enzyme 2) in biolayer interferometry (BLI) assays. Seroconversion was observed in a pilot mouse trial after 14 days when administered intravenously, based on enzyme-linked immunosorbent assays (ELISA). This red blood cell based platform can open novel possibilities for therapeutics for the coronavirus disease (COVID-19) including variants, and other viruses in the future.