Artificial Cell Membranes Research Articles

Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins

The development of artificial cells has led to fundamental insights into the functional processes of living cells while simultaneously paving the way for transformative applications in biotechnology and medicine. A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We modify the membrane translocating loop of α-hemolysin to translocate functional peptides up to 52 amino acids across lipid membranes. Full membrane translocation occurs in the absence of any translocase machinery and the translocated peptides are recognized by specific peptide-binding ligands on the opposing membrane side. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures.

Deletion within ameloblastin multitargeting domain reduces its interaction with artificial cell membrane

In human, mutations in the gene encoding the enamel matrix protein ameloblastin (Ambn) have been identified in cases of amelogenesis imperfecta. In mouse models, perturbations in the Ambn gene have caused loss of enamel and dramatic disruptions in enamel-making ameloblast cell function. Critical roles for Ambn in ameloblast cell signaling and polarization as well as adhesion to the nascent enamel matrix have been supported. Recentely, we have identified a multitargeting domain (MTD) in Ambn that interacts with cell membrane, with the majority enamel matrix protein amelogenin, and with itself. This domain includes an amphipathic helix (AH) motif that directly interacts with cell membrane. In this study, we analyzed the sequence of the MTD for evolutionary conservation and found high conservation among mammals within the MTD and particularly within the AH motif. We computationally predicted that the AH motif lost its hydrophobic moment upon deleting hydrophobic but not hydrophilic residues from the motif. Furthermore, we rationally designed peptides that encompassed the Ambn MTD and contained deletions of largely hydrophobic or hydrophilic stretches of residues. To assess their AH-forming and membrane-binding abilities, we combined those peptides with synthetic phospholipid membrane vesicles and performed circular dichroism, membrane leakage, and vesicle clearance measurements. Circular dichroism showed retention of α-helix formation in all peptides except the one with the largest deletion of eleven amino acids including seven that were hydrophobic. This same peptide variant failed to cause leakage or clearance of synthetic membranes, while smaller deletions yielded intermediate membrane interaction as measured by leakage and clearance assays. Our data revealed that deletion of key hydrophobic residues from the AH leads to the most dramatic loss of Ambn–membrane interaction. Pinpointing roles of residues within the MTD has important implications for the multifunctionality of Ambn.

Effect of Simulated Microgravity on Artificial Single Cell Membrane Mechanics

Effect of Simulated Microgravity on Artificial Single Cell Membrane Mechanics

Controlling transport across artificial cell membranes

Controlling transport across artificial cell membranes

Interfacial energy-mediated bulk transport across artificial cell membranes

Interfacial energy-mediated bulk transport across artificial cell membranes

Genetically Engineered Cellular Nanovesicles: Theories, Design and Perspective

AbstractThe use of cellular nanovesicles (CNVs) is a groundbreaking innovation in biomedical applications. Both natural extracellular vesicles (EVs) and artificial cell membrane nanovesicles (AVs) have emerged as innovative CNVs, but they have limitations in therapeutic effects and targeting abilities. Challenges such as stability, immunogenicity, and drug payload capacity hinder their widespread application. Genetic engineering has matured as a widely employed modification strategy, leading to the development of genetically engineered extracellular vesicles (gEVs) and genetically engineered artificial cell membrane nanovesicles (gAVs). This review meticulously examines the diverse types and inherent characteristics of CNVs, alongside various surface engineering strategies for CNVs, with a specific focus on elucidating the attributes and detailing the preparation methods relevant to gEVs and gAVs. Furthermore, this exploration delves into the expansive landscape of biomedical applications, developmental prospects, and current challenges associated with the utilization of gEVs and gAVs. With a comprehensive approach, the primary objective is to provide insights that not only illuminate the nuanced intricacies of these nanovesicles but also pave the way for their seamless integration into clinical research and eventual translation into practical applications.

Nanopore detection of sub-nanosized plastics in PE-coated paper cups and analysis of their inflammatory responses

In recent years, there has been growing global concern about environmental pollution, and the increasing use of plastic products has made microplastics a serious environmental pollutant worldwide. Accordingly, the development of microplastic detection methods and research on the harmful effects of microplastics on human health are being actively pursued. In this study, we detected particle leaching from PE-coated paper cups using label-free nanopore sensing to identify and quantify the presence of sub-nanosized plastic particles. Plastic particles leached from the paper cups were classified as < 1 nm and > 1 nm, and their interactions with artificial cell membranes and inflammatory responses in the cells were evaluated. The results demonstrated that in a commonly used paper cup, particles < 1.4 nm were present at a concentration of approximately 1.13 mM in hot water and 0.62 mM in room-temperature water. It was demonstrated that plastic particles < 1 nm could condense the artificial cell membrane, whereas particles > 1 nm could thicken the artificial cell membrane. Furthermore, both size ranges of particles leached from the paper cups triggered an inflammatory response in the cells, with the inflammatory response increasing in proportion to the concentration and treatment time. The detection and analysis revealed the presence of sub-nanosized plastic particles in PE-coated paper cups, which are commonly used in daily life. These particles pose health threats and contribute to environmental pollution.

A novel biomimetic nanoplasmonic sensor for rapid and accurate evaluation of checkpoint inhibitor immunotherapy.

Immune checkpoint inhibitors (ICIs) emerged as promising immunotherapies for cancer treatment, harnessing the patient's immune system to fight and eliminate tumor cells. However, despite their potential and proven efficacies, checkpoint inhibitors still face important challenges such as the tumor heterogeneity and resistance mechanisms, and the complex in vitro testing, which limits their widespread applicability and implementation to treat cancer. To address these challenges, we propose a novel analytical technique utilizing biomimetic label-free nanoplasmonic biosensors for rapid and reliable screening and evaluation of checkpoint inhibitors. We have designed and fabricated a low-density nanostructured plasmonic sensor based on gold nanodisks that enables the direct formation of a functional supported lipid bilayer, which acts as an artificial cell membrane for tumor ligand immobilization. With this biomimetic scaffold, our biosensing approach provides real-time, highly sensitive analysis of immune checkpoint pathways and direct assessment of the blocking effects of monoclonal antibodies in less than 20min/test. We demonstrate the accuracy of our biomimetic sensor for the study of the programmed cell death protein 1 (PD1) checkpoint pathway, achieving a limit of detection of 6.7ng/mL for direct PD1/PD-L1 interaction monitoring. Besides, we have performed dose-response inhibition curves for an anti-PD1 monoclonal antibody, obtaining a half maximal inhibitory concentration (IC50) of 0.43nM, within the same range than those obtained with conventional techniques. Our biomimetic sensor platform combines the potential of plasmonic technologies for rapid label-free analysis with the reliability of cell-based assay in terms of ligand mobility. The biosensor is integrated in a compact user-friendly device for the straightforward implementation in biomedical and pharmaceutical laboratories.

Investigating Early HIV Infection: Utilizing Artificial Cell Membranes for Capturing Unintegrated Viral RNA

The high mutation rate of HIV poses a significant challenge for research and treatment development. Obtaining unaltered viral genetic material before integration into the host cell's DNA would be a valuable tool for studying early infection stages and developing more effective therapies. This study explores the feasibility of using artificial cell membranes containing human immunodeficiency virus (HIV) receptors (CD4 and co-receptor) to capture unintegrated viral RNA. We discuss the potential advantages and limitations of this approach compared to existing methods for studying HIV infection.

Investigating Early HIV Infection: Utilizing Artificial Cell Membranes for Capturing Unintegrated Viral RNA

Investigating Early HIV Infection: Utilizing Artificial Cell Membranes for Capturing Unintegrated Viral RNA

Investigating Early HIV Infection: Utilizing Artificial Cell Membranes for Capturing Unintegrated Viral RNA

Investigating Early HIV Infection: Utilizing Artificial Cell Membranes for Capturing Unintegrated Viral RNA

Development of an Aptamer-Based QCM-D Biosensor for the Detection of Thrombin Using Supported Lipid Bilayers as Surface Functionalization.

Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.

Functional Evaluation of Bilayer Lipid for the Development of Artificial Cell-membrane Structures

Functional Evaluation of Bilayer Lipid for the Development of Artificial Cell-membrane Structures

Reconstruction of Protein/Liposome Complex.

Most ion channels and receptors are distributed in cell membranes and are known as membrane proteins. These membrane proteins are folded in the cell membrane and become functional proteins. Here, we demonstrate a method of reconstructing membrane proteins into liposome membranes, which are commonly used as artificial cell membranes.

Biomimetic Lipid Raft: Domain Stability and Interaction with Physiologically Active Molecules.

The cell membrane, also called the plasma membrane, is the membrane on the cytoplasmic surface that separates the extracellular from the intracellular. It is thin, about 10 nm thick when viewed with an electron microscope, and is composed of two monolayers of phospholipid membranes (lipid bilayers) containing many types of proteins. It is now known that this cell membrane not only separates the extracellular from the intracellular, but is also involved in sensory stimuli such as pain, itching, sedation, and excitement. Since the "Fluid mosaic model" was proposed for cell membranes, molecules have been thought to be homogeneously distributed on the membrane surface. Later, at the end of the twentieth century, the existence of "Phase-separated microdomain structures" consisting of ordered phases rich in saturated lipids and cholesterol was suggested, and these were termed "Lipid rafts." A model in which lipid rafts regulate cell signaling has been proposed and is the subject of active research.This chapter first outlines the physicochemical properties and thermodynamic models of membrane phase separation (lipid rafts), which play an important role in cell signaling. Next, how physiologically active molecules such as local anesthetics, cooling agents (menthol), and warming agents (capsaicin) interact with artificial cell membranes will be presented.It is undeniable that the plasma membrane contains many channels and receptors that are involved in the propagation of sensory stimuli. At the same time, however, it is important to understand that the membrane exerts a significant influence on the intensity and propagation of these stimuli.

Mimic the electric activity in a heat-sensitive membrane in circuit

The membrane potential of an isolated biological neuron results from the concentration difference between intracellular and extracellular ions. Any external disturbance and synaptic current from adjacent neurons will break the balance of electromagnetic field induced by gradient distribution of ions. Neurons develop different biophysical functions and then more neurons are clustered with controllable synapses to form different functional regions in the brain. In this Letter, two linear circuits (LCs) are coupled via a nonlinear resistor for mimicking the electric characteristic of cell membrane. Thermistors are included to approach a thermal-sensitive membrane. The neuron model developed from the coupled circuits with thermistors has two capacitive variables, which represent the external and inner electric field, and energy balance between inner and outer membranes are controlled by the coupling channel via a nonlinear resistor. The neuron excitability is relative to the temperature, two kinds of thermistors are used in the coupled LCs to investigate the consensus and difference between inner and outer media. Energy propagation is blocked when the thermal properties have distinct difference between two sides of the cell membrane, and potential diversity supports continuous firing patterns. The results provide possible clues to design artificial cell membrane which is sensitive to temperature.

Artificial Breakthrough of Cell Membrane Barrier for Transmembrane Substance Exchange: A Review of Recent Progress

AbstractCell membrane composed of lipid bilayer is a selectively permeable membrane that only allows specific molecules to pass through. While such selectivity is essential for the survival and function of cells, there exist instances when it is necessary to overcome the cell membrane barrier. Study on the artificial cell membrane barrier breakthrough strategies is of great significance for the development of drug delivery systems and the understanding of cellular behaviors. Herein, the advancements in the development of strategies for opening cell membrane barriers over the past decade are summarized. The main transmembrane mechanisms are elucidated and then divided into three categories, i.e., cell perforation via microinjection/external physical fields, cell endocytosis‐assisted construction of artificial transmembrane channels, and untethered micro/nanomachines. Next, the potential applications after opening the cell membrane are discussed, which mainly focus on the transmembrane cargo delivery into the cell and endogenous substance extraction out from the cell. Finally, this review outlines the current challenges that impede the realization of practical applications and presents an outlook of future opportunities to promote further development. Through overcoming the challenges, it is anticipated that artificial cell membrane breakthrough strategies will provide a revolutionized tool in the near future to advance the field of biomedicine and biotechnology.

Synthesis of methotrexate-betulonic acid hybrids and evaluation of their effect on artificial and Caco-2 cell membranes

An efficient protocol for the synthesis of novel methotrexate-betulonic acid hybrids with a (tert-butoxycarbonylamino)-3,6-dioxa-8-octanamine (Boc-DOOA) linkage has been developed. Reaction of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-betulonamide with methotrexate resulted in a mixture of isomeric conjugates which were separated by column chromatography. Their structures and composition have been fully established by 1H NMR, 13C spectra, FAB mass spectrometry and elemental analysis. The identity of conjugates was confirmed by LC-MS data. Membranotropic properties of the new hybrids were assessed on the basis of their interactions with artificial lipid membranes by differential scanning calorimetry (DSC) method. The ability of the conjugates to penetrate Caco-2 cells is inferior to methotrexate. Probably, this is due to the increasing lipophilicity, the affinity of these hybrid molecules for the lipid bilayer increases, which is confirmed by experiments with artificial membranes.

X-ray reflectivity study of the heat shock protein Hsp70 interaction with an artificial cell membrane model

Membrane-bound heat shock protein 70 (Hsp70) apart from its intracellular localization was shown to be specifically expressed on the plasma membrane surface of tumor but not normal cells. Although the association of Hsp70 with lipid membranes is well documented the exact mechanisms for chaperone membrane anchoring have not been fully elucidated. Herein, we addressed the question of how Hsp70 interacts with negatively charged phospholipids in artificial lipid compositions employing the X-ray reflectivity (XRR) studies. In a first step, the interactions between dioleoylphosphatidylcholine (DOPC) in the presence or absence of dioleoylphosphatidylserine (DOPS) and Hsp70 had been assessed using Quartz crystal microbalance measurements, suggesting that Hsp70 adsorbs to the surface of DOPC/DOPS bilayer. Atomic force microscopy (AFM) imaging demonstrated that the presence of DOPS is required for stabilization of the lipid bilayer. The interaction of Hsp70 with DOPC/DOPS lipid compositions was further quantitatively determined by high energy X-ray reflectivity. A systematic characterization of the chaperone-lipid membrane interactions by various techniques revealed that artificial membranes can be stabilized by the electrostatic interaction of anionic DOPS lipids with Hsp70.

Sulfated liposome-based artificial cell membrane glycocalyx nanodecoys for coronavirus inactivation by membrane fusion

As a broad-spectrum antiviral nanoparticle, the cell membrane nanodecoy is a promising strategy for preventing viral infections. However, most of the cell membrane nanodecoys can only catch virus and cannot induce inactivation, which may bring about a considerably high risk of re-infection owing to the possible viral escape from the nanodecoys. To tackle this challenge, sulfated liposomes are employed to mimic the cell membrane glycocalyx for constructing an artificial cell membrane glycocalyx nanodecoy that exhibits excellent anti-coronavirus activity against HCoV-OC43, wild-type SARS-CoV-2, Alpha and Delta variant SARS-CoV-2 pseudovirus. In addition, this nanodecoy, loaded with surface sulfate groups as SARS-CoV-2 receptor arrays, can enhance the antiviral capability to virus inactivation through destroying the virus membrane structure and transfer the spike protein to postfusion conformation. Integrating bio-inspired recognition and inactivation of viruses in a single supramolecular entity, the artificial cell membrane nanodecoy opens a new avenue for the development of theranostic antiviral nanosystems, whose mass production is favored due to the facile engineering of sulfated liposomes.