Fusion Of Liposomes Research Articles

Modulating the elasticity of milk exosome-based hybrid vesicles to optimize transepithelial transport and enhance oral peptide delivery.

To address challenges such as limited loading capacity, restricted targeting precision, and low yield of natural exosomes as drug carriers, the fusion of liposomes and exosomes to create hybrid vesicles has emerged as a viable solution approach. While current research mainly focuses on designing functionalized liposomes, less attention is given to how liposome membrane materials affect the elasticity of these hybrids and their delivery efficiency. This study utilized milk exosomes (mExos) as model exosomes, and generated hybrid vesicles with varying elasticity through the fusion of phospholipids with differing chain lengths, examining the disparities among various hybrid vesicles in their ability to overcoming the gastrointestinal barriers. It was observed that while hard hybrid vesicles exhibited reduced mucus penetration compared to soft hybrid vesicles, they demonstrated a notably higher efficacy in traversing the epithelial cell barrier. The enhanced transepithelial cell capability of hard vesicles can be attributed to their reduced tendency to aggregate in the lysosome through the down-regulated clathrin-mediated endocytosis pathway, as well as by the strengthening of the endoplasmic reticulum-Golgi exocytosis pathway due to their rigid characteristics. In comparison to soft hybrid vesicles, semaglutide (SET) loaded hard hybrid vesicles demonstrated improved in vivo epithelial permeability, enhanced oral bioavailability, and better therapeutic effectiveness. This study could provide valuable insights for determining the optimal elasticity of exosome-liposome hybrid vesicles in the development of oral nanocarriers.

Fusion of polymer-coated liposomes and centrifugally spun microfibers as hybrid materials to enhance sustained release.

Liposomes are employed for the delivery of molecular cargo in several classes of systems. For instance, the embedding of loaded liposomes in polymeric fibrous scaffolds has enabled the creation of hybrid materials that mimic biological membranes. Liposomes with unmodified surfaces have been predominantly integrated into fibers, which leads to instabilities due to interfacial incompatibility. In addition, electrospinning has been almost exclusively employed for fiber fabrication, which limits the potential for scale-up production. Here, we present the fabrication of hybrid biomimetic materials by fusing polymer-coated liposomes to force-spun microfibers to increase the stability of the hybrid materials and enhance the sustained release of the cargo. l-α-Phosphatidylcholine liposomes were coated with chitosan or polyethylene glycol (PEG). The nano-differential scanning calorimetry results confirm that polymer coating does not affect the phase transition temperature (T m) of the liposomes, where only the model drug, quercetin, reduced T m. Centrifugal spinning was employed to fabricate hydrophobic polycaprolactone (PCL) microfibers at various polymer concentrations and using various solvents and spinning parameters to increase the yield at the lowest fiber diameter. The highest microfiber production rate obtained occurred at a 20% (w/v) PCL concentration in 50 : 50 (v/v) chloroform and methanol solution with an average fiber diameter of 584.85 ± 26.30 nm. The non-chemical fusion of the polymer-coated liposomes and the fibrous scaffolds was promoted by immersion at T > T m, under ultrasonication. We hypothesize that the fusion is driven by hydrophobic interactions between the liposomes and the fibers, which merge the materials through the lipid bilayer. The fused hybrid material solved the burst release problem observed when adhering plain liposomes to nanofibers. Both PEG and chitosan yielded a sustained release, where the release rate with the former was faster. These results demonstrate that the fusion of polymer-coated liposomes and microfibers enables more effective blending of the loaded carriers into the polymer microfibers. Ultimately, the fused liposome/microfiber hybrids are stable matrices and enhance the sustained release of molecular cargo.

Efficient single-channel current measurements of the human BK channel using a liposome-immobilized gold probe.

The human BK channel (hBK) is an essential membrane protein that regulates various biological functions, and its dysfunction leads to serious diseases. Understanding the biophysical properties of hBK channels is crucial for drug development. Artificial lipid bilayer recording is used to measure biophysical properties at the single-channel level. However, this technique is time-consuming and complicated; thus, its measurement efficiency is very low. Previously, we developed a novel technique to improve the measurement efficiency by rapidly forming lipid bilayer membranes and incorporating ion channels into the membrane using a hydrophilically modified gold probe. To further improve our technique for application to the hBK channel, we combined it using the gold probe with a liposome fusion method. Using a probe on which liposomes containing hBK channels were immobilized, the channels were efficiently incorporated into the lipid bilayer membrane, and the measured channel currents showed the current characteristics of the hBK channel. This technique will be useful for the efficient measurements of the channel properties of hBK and other biologically important channels.

Liposomal fusion of plant-based extracellular vesicles to enhance skin anti-inflammation

This study reports a hybrid extracellular vesicle (EV)/liposome system developed through vesicular membrane fusion between anti-inflammatory Hydrangea macrophylla leaf EVs (HML-EVs) and terpinen-4-ol/azelamide monoethanolamine-loaded liposomes. Successful liposomal fusion between HML-EVs and terpinen-4-ol/azelamide monoethanolamine-loaded-liposome membranes, confirmed using the two-color fluorescence resonance energy transfer mechanism, enabled the scaled production of a stable hybrid HML-EV/liposome dispersion at a length scale of 100–200 nm. We show that the hybrid EV/liposome significantly inhibits apoptosis by nitric oxide and tumor necrosis factor-α production. Furthermore, HML-EV/liposomes can inhibit the synthesis of protease-activated receptor-2, interleukin-6, c-Jun, and thymic stromal lymphopoietin, thus exhibiting anti-inflammatory performance. These findings highlight that the hybrid EV/liposome system established in this study alleviates skin irritation and improves skin anti-inflammation.

Reversible Modulation of Cell-Cell Interactions Using Electrochemistry.

Cell-cell interactions play an important role in many biological processes, and various methods have been developed for controlling the cell-cell interactions. However, the effective and rapid control of intercellular interactions remains challenging. Herein, we report a novel, rapid, and effective electrochemical strategy without destroying the basic life processes for the dynamic control of intercellular interactions via liposome fusion. In the proposed system, bioorthogonal chemical groups and hydroquinone (HQ)- and aminooxy (AO)-tethered ligands were modified on the surface of living cells on the basis of the liposome fusion, enabling dynamical intercellular assemblies. Upon application of the corresponding oxidative potential, the "off-state" HQ could be oxidized to the "on-state" quinone (Q), which subsequently reacts with AO-tethered ligands to form stable oxime linkages under physiological conditions. This reaction effectively shortens the distance between cells, promoting the formation of cell clusters. When the corresponding reverse reductive potential is applied, the oxime linkage is cleaved, resulting in the release of the cells. Furthermore, we employed HQ- and AO-tethered ligands to modify mitochondria, inducing mitochondrial aggregation. This noninvasive and label-free strategy allows for the dynamic reversible regulation of intercellular interactions, enhancing our understanding of intercellular communication networks, and has the potential for improving the antitumor therapy efficacy.

Electrochemical Investigation of the Stability of Poly-Phosphocholinated Liposomes.

Poly[2-(methacryloyloxy)ethyl phosphorylcholine] liposomes (pMPC liposomes) gained attention during the last few years because of their potential use in treating osteoarthritis. pMPC liposomes that serve as boundary lubricants are intended to restore the natural lubrication properties of articular cartilage. For this purpose, it is important that the liposomes remain intact and do not fuse and spread as a lipid film on the cartilage surface. Here, we investigate the stability of the liposomes and their interaction with two types of solid surfaces, gold and carbon, by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). With the aid of a hydrophilic species used as an electroactive probe in the solution, the charge transfer characteristics of the electrode surfaces are obtained. Additionally, from EIS, the capacitance characteristics of the surfaces are derived. No decrease of the peak currents and no displacement of the peak potentials to greater overpotentials are observed in the CV experiments. No decrease in the apparent capacitance and increase in the charge transfer resistance is observed in the EIS experiments. On the contrary, all parameters in both CV and EIS do change in the opposite direction. The obtained results confirm that there is only physical adsorption without fusion and spreading of the pMPC liposomes and without the formation of lipid films on the surfaces of both gold and carbon electrodes.

Stability of Free and Liposomal Encapsulated RNA on a Mucoadhesive PVA Polymer for Esophageal RNA Drug Targeting Using the EsoCap System

AbstractThe development of RNA and oligonucleotide‐based therapeutics is a longstanding goal and is currently gaining significant attention. Several RNA‐based drugs are approved for clinical use. Others are under investigation or in preclinical trials. This study have initiated the development of RNA drugs for localized use in the esophagus, utilizing the EsoCap System. This system's core component is a mucoadhesive film that carries the RNA drug and is precisely applied to the esophagus. Research into the stability and properties of naked and liposomal‐encapsulated RNA on mucoadhesive polymer film reveals that RNAs remain stable in various conditions without degradation, RNA leakage, or liposome fusion observed. The liposome size also remains constant after application on the film, drying, and rehydration. These findings pave the way for RNA drug development for esophageal diseases and their administration via the EsoCap system.

Liposome fusion assisted delivery of silica nanoquenchers for rapid detection of exosomal MicroRNAs

Analysis of exosome information in bodily fluids is an important priority in liquid biopsy. Exosomal microRNAs (miRNAs) have been increasingly identified as credible objects due to their high stability and abundance. However, traditional detection techniques for exosomal miRNAs consume considerable time and require abundant professional equipment. Herein, we introduced a novel silica nanoquencher (qSiNP)-based nanoplatform with liposomal shell encapsulation. Liposomes were demonstrated to efficiently deliver qSiNPs into tumor exosomes via membrane fusion pathways. Subsequent loading of qSiNPs with fluorophore-labeled antisense oligonucleotides rapidly resulted in a specific fluorescent signal for “off-on” switchable in situ detection and imaging of targeted exosomal miRNAs without complicated RNA extraction or targeted amplification. With this strategy, the distinction of tumor-related miRNAs from normal exosomes was successfully achieved, which further indicated that our platform has the merits of easy preparation, stable encapsulation, efficient uptake, and an alterable molecular beacon, for improving the diagnostic value of liquid biopsy in various cancer diseases.

Strain-Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion.

Due to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide-alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide- and strained alkyne-functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

Strain‐Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion

AbstractDue to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide‐alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide‐ and strained alkyne‐functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

A practical guide for fast implementation of SNARE-mediated liposome fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAER) family proteins are the engines of most intra-cellular and exocytotic membrane fusion pathways (Jahn and Scheller 2006). Over the past two decades, in-vitro liposome fusion has been proven to be a powerful tool to reconstruct physiological SNARE-mediated membrane fusion processes (Liu et al. 2017). The reconstitution of the membrane fusion process not only provides direct evidence of the capability of the cognate SNARE complex in driving membrane fusion but also allows researchers to study the functional mechanisms of regulatory proteins in related pathways (Wickner and Rizo 2017). Heretofore, a variety of delicate methods for in-vitro SNARE-mediated liposome fusion have been established (Bao et al. 2018; Diao et al. 2012; Duzgunes 2003; Gong et al. 2015; Heo et al. 2021; Kiessling et al. 2015; Kreye et al. 2008; Kyoung et al. 2013; Liu et al. 2017; Scott et al. 2003). Although technological advances have made reconstitution more physiologically relevant, increasingly elaborate experimental procedures, instruments, and data processing algorithms nevertheless hinder the non-experts from setting up basic SNARE-mediated liposome fusion assays. Here, we describe a low-cost, timesaving, and easy-to-handle protocol to set up a foundational in-vitro SNARE-mediated liposome fusion assay based on our previous publications (Liu et al. 2023; Wang and Ma 2022). The protocol can be readily adapted to assess various types of SNARE-mediated membrane fusion and the actions of fusion regulators by using appropriate alternative additives (e.g., proteins, macromolecules, chemicals, etc.). The total time required for one round of the assay is typically two days and could be extremely compressed into one day.

Molecular Mechanisms of Cationic Fusogenic Liposome Interactions with Bacterial Envelopes.

Although fusogenic liposomes offer a promising approach for the delivery of antibiotic payloads across the cell envelope of Gram-negative bacteria, there is still a limited understanding of the individual nanocarrier interactions with the bacterial target. Using super-resolution microscopy, we characterize the interaction dynamics of positively charged fusogenic liposomes with Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria. The liposomes merge with the outer membrane (OM) of Gram-negative bacteria, while attachment or lipid internalization is observed in Gram-positive cells. Employing total internal reflection fluorescence microscopy, we demonstrated liposome fusion with model supported lipid bilayers. For whole E. coli cells, however, we observed heterogeneous membrane integrations, primarily involving liposome attachment and hemifusion events. With increasing lipopolysaccharide length, the likelihood of full-fusion events was reduced. The integration of artificial lipids into the OM of Gram-negative cells led to membrane destabilization, resulting in decreased bacterial vitality, membrane detachment, and improved codelivery of vancomycin─an effective antibiotic against Gram-positive cells. These findings provide significant insights into the interactions of individual nanocarriers with bacterial envelopes at the single-cell level, uncovering effects that would be missed in bulk measurements. This highlights the importance of conducting single-particle and single-cell investigations to assess the performance of next-generation drug delivery platforms.

Prevention of liposome cryoaggregation by a radish (Raphanus sativus L.) vacuolar calcium-binding protein (RVCaB)

Previously we found that the soluble fraction of radish (Raphanus sativus L.) taproots contained a large amount of an intrinsically disordered protein, which we identified as radish vacuolar calcium binding protein (RVCaB). Although RVCaB is known to inhibit cryo-damage to freeze-sensitive enzyme lactate dehydrogenase, there is no information on its cryoprotection of other types of macromolecules, such as biomembranes. In this study we found that RVCaB inhibited the cryoaggregation of phospholipid liposomes to a greater extent than common cryoprotectants, such as trehalose, glycine betaine, proline, bovine serum albumin (BSA), and polyethylene glycol (PEG). RVCaB inhibited the freeze/thaw-induced membrane fusion of liposomes. It became structurally disordered in solution, and this disorder was maintained in the presence of liposomes. Finally, the mechanism underlying RVCaB's inhibition of liposome cryoaggregation was discussed.

Programming assembly of biomimetic exosomes: An emerging theranostic nanomedicine platform.

Exosomes have emerged as a promising cell-free therapeutic approach. However, challenges in large-scale production, quality control, and heterogeneity must be overcome before they can be used clinically. Biomimetic exosomes containing key components of natural exosomes have been assembled through extrusion, artificial synthesis, and liposome fusion to address these limitations. These exosome-mimetics (EMs) possess similar morphology and function but provide higher yields, faster large-scale production, and similar size compared to conventional exosomes. This article provides an overview of the chemical and biological properties of various synthetic exosome systems, including nanovesicles (NVs), EMs, and hybrid exosomes. We highlight recent advances in the production and applications of nanobiotechnology and discuss the advantages, limitations, and potential clinical applications of programming assembly of exosome mimetics.

Beyond Extracellular Vesicles: Hybrid Membrane Nanovesicles as Emerging Advanced Tools for Biomedical Applications.

Extracellular vesicles (EVs), involved in essential physiological and pathological processes of the organism, have emerged as powerful tools for disease treatment owing to their unique natural biological characteristics and artificially acquired advantages. However, the limited targeting ability, insufficient production yield, and low drug-loading capability of natural simplex EVs have greatly hindered their development in clinical translation. Therefore, the establishment of multifunctional hybrid membrane nanovesicles (HMNVs) with favorable adaptability and flexibility has become the key to expanding the practical application of EVs. This timely review summarizes the current progress of HMNVs for biomedical applications. Different HMNVs preparation strategies including physical, chemical, and chimera approaches are first discussed. This review then individually describes the diverse types of HMNVs based on homologous or heterologous cell membrane substances, a fusion of cell membrane and liposome, as well as a fusion of cell membrane and bacterial membrane. Subsequently, a specific emphasis is placed on the highlight of biological applications of the HMNVs toward various diseases with representative examples. Finally, ongoing challenges and prospects of the currently developed HMNVs in clinical translational applications are briefly presented. This review will not only stimulate broad interest among researchers from diverse disciplines but also provide valuable insights for the development of promising nanoplatforms in precision medicine.

A Dual Protective Drug Delivery System Based on Lipid Coated Core-Shell Mesoporous Silica for Efficient Delivery of Cabazitaxel to Prostate Cancer Cells

Abstract Many advancements are happening in drug delivery to develop an excellent nanocarrier to deliver drugs to target sites bypassing clinical barriers, thereby improving cellular uptake. Lipid coating on mesoporous silica nanoparticles (MSNs) has significantly reduced the drawbacks of many MSNs and increased their compatibility. This study reports a dual protective acid stimuli-responsive lipid-coated core-shell mesoporous silica nanoparticle (CSMS) conjugated with cabazitaxel showing better drug release, cell uptake, and cytotoxicity, and suitability in the prostate cancer (PC-3) cell line. Initially, monodispersed CSMS were conjugated with cabazitaxel (CBZ) through a hydrazone linker (CBZ@Hy-CSMS), proving its appropriate use in designing a stimuli-responsive system. In the second part, CBZ-conjugated CSMS was coated with a lipid layer (L-CBZ@Hy-CSMS) by the liposome fusion method. The presented dual protective CSMS system showed a significant increase in drug delivery at pH 5.4 compared to 7.4, with a drastic decrease in premature drug release when exposed to pH 7.4. The lipid-coated CSMS showed excellent biocompatibility and better cellular uptake with enhanced cell cytotoxicity in PC-3 cancer cells as compared to the uncoated CSMS. CSMS with a lipid coating combined with a stimuli-responsive system could improve the therapeutic delivery and treatment difficulties in many other cell lines and diseases.

Dynamic Fusion of Nucleic Acid Functionalized Nano-/Micro-Cell-Like Containments: From Basic Concepts to Applications.

Membrane fusion processes play key roles in biological transformations, such as endocytosis/exocytosis, signal transduction, neurotransmission, or viral infections, and substantial research efforts have been directed to emulate these functions by artificial means. The recognition and dynamic reconfiguration properties of nucleic acids provide a versatile means to induce membrane fusion. Here we address recent advances in the functionalization of liposomes or membranes with structurally engineered lipidated nucleic acids guiding the fusion of cell-like containments, and the biophysical and chemical parameters controlling the fusion of the liposomes will be discussed. Intermembrane bridging by duplex or triplex nucleic acids and light-induced activation of membrane-associated nucleic acid constituents provide the means for spatiotemporal fusion of liposomes or nucleic acid modified liposome fusion with native cell membranes. The membrane fusion processes lead to exchange of loads in the fused containments and are a means to integrate functional assemblies. This is exemplified with the operation of biocatalytic cascades and dynamic DNA polymerization/nicking or transcription machineries in fused protocell systems. Membrane fusion processes of protocell assemblies are found to have important drug-delivery, therapeutic, sensing, and biocatalytic applications. The future challenges and perspectives of DNA-guided fused containments and membranes are addressed.

Development of Functional Chimeric Nanoparticles by Membrane Fusion of Small Extracellular Vesicles and Drug-Encapsulated Liposomes

Since small extracellular vesicle (sEVs) are involved in cell-to-cell communication via transfer of certain bioactive molecules and have the capability to overcome biological barriers against drug transport, their use as a drug delivery system (DDS) has been demonstrated in treatment of a diverse range of diseases. However, some issues in drug encapsulation have been pointed out, including low encapsulation efficiency and poor reproducibility. It was previously reported that liposomes containing phosphatidylserine (PS) can fuse together in the presence of calcium ion, which allows for drug encapsulation into the resultant liposomes (i.e., calcium fusion method). On the other hand, PS is reportedly present in lipid membrane of sEVs as a distinct lipid composition. We therefore hypothesized that PS-mediated membrane fusion of sEVs with PS-liposomes encapsulating therapeutic agents via the calcium fusion method can be applied to convenient drug encapsulation into sEVs. Membrane fusion of PS-liposomes and sEVs derived from murine melanoma B16F1 cells (B16-sEVs) was firstly confirmed. The obtained nanoparticles, termed chimeric nanoparticles (CM-NP), showed comparable cellular uptake to B16-sEVs into B16F1 cells. Moreover, CM-NP encapsulating an anticancer drug doxorubicin (DOX) (CM-NP-DOX) could be prepared by membrane fusion of PS-liposomes encapsulating DOX (PS-Lipo-DOX) and B16-sEVs. CM-NP-DOX exhibited a superior anticancer effect on B16F1 cells in vitro compared with PS-Lipo-DOX. These findings suggest that the calcium fusion method could be applied for membrane fusion of sEVs and PS-liposomes, and that this approach would likely be useful for efficient drug encapsulation into sEVs, as well as increasing liposome functionality.

Gas‐Liquid Flow‐Induced Characteristics of Dispersed Liposome‐Based Particles

AbstractLiposomes are closed bilayers of phospholipids formed in aqueous media. The hydrodynamic property of colloidal dispersions of liposomes is one of the factors which alter the structure of liposomes. The present review article discusses the mechanistic aspects of the deformation, breakage, and fusion of liposomes in different liquid shear flows. Then, characteristic features reported with respect to the particles consisting of liposomes and bubbles are discussed to explore the role of shear flows induced by the acoustically active bubbles. Furthermore, the gas‐liquid flow‐induced characteristics of liposomes are described for their application to kinetically controllable enzymatic reactions in bubble‐column bioreactors.

Three Compartment Liposome Fusion: Functional Protocells for Biocatalytic Cascades and Operation of Dynamic DNA Machineries

AbstractNucleic acid‐functionalized liposomes modified at their boundaries with o‐nitrobenzyl phosphate‐caged hairpin units and pH‐responsive C‐G·C+ triplex forming strands are used for the concomitant light and pH‐triggered fusion of three types of loaded liposomes. The fusion processes are followed by light‐scattering size enlargement measurements, optical methods, and biocatalytic cascades activated upon the mixing of the liposomes loaded with enzymes and their substrates and their fusion into the cell‐like containments. The fused liposomes act as functional protocells for the integration of biocatalytic machineries. This is exemplified by the operation of an autonomous polymerization/nickase machinery synthesizing a Mg2+‐ion‐dependent DNAzyme and of a transcription machinery yielding the Malachite Green‐RNA aptamer product.