Formation Of Transient Pores Research Articles

Low-Intensity Focused Ultrasound-Responsive Phase-Transitional Liposomes Loaded with STING Agonist Enhances Immune Activation for Breast Cancer Immunotherapy.

Background: Pharmacologically targeting the STING pathway offers a novel approach to cancer immunotherapy. However, small-molecule STING agonists face challenges such as poor tumor accumulation, rapid clearance, and short-lived effects within the tumor microenvironment, thus limiting their therapeutic potential. To address the challenges of poor specificity and inadequate targeting of STING in breast cancer treatment, herein, we report the design and development of a targeted liposomal delivery system modified with the tumor-targeting peptide iRGD (iRGD-STING-PFP@liposomes). With LIFU irradiation, the liposomal system exploits acoustic cavitation, where gas nuclei form and collapse within the hydrophobic region of the liposome lipid bilayer (transient pore formation), which leads to significantly enhanced drug release. Methods: Transmission electron microscopy (TEM) was used to investigate the physicochemical properties of the targeted liposomes. Encapsulation efficiency and in vitro release were assessed using the dialysis bag method, while the effects of iRGD on liposome targeting were evaluated through laser confocal microscopy. The CCK-8 assay was used to investigate the toxicity and cell growth effects of this system on 4T1 breast cancer cells and HUVEC vascular endothelial cells. A subcutaneous breast cancer tumor model was established to evaluate the tumor-killing effects and therapeutic mechanism of the newly developed liposomes. Results: The liposome carrier exhibited a regular morphology, with a particle size of 232.16 ± 19.82 nm, as indicated by dynamic light scattering (DLS), and demonstrated low toxicity to both HUVEC and 4T1 cells. With an encapsulation efficiency of 41.82 ± 5.67%, the carrier exhibited a slow release pattern in vitro after STING loading. Targeting results indicated that iRGD modification enhanced the system's ability to target 4T1 cells. The iRGD-STING-PFP@liposomes group demonstrated significant tumor growth inhibition in the subcutaneous breast cancer mouse model with effective activation of the immune system, resulting in the highest populations of matured dendritic cells (71.2 ± 5.4%), increased presentation of tumor-related antigens, promoted CD8+ T cell infiltration at the tumor site, and enhanced NK cell activity. Conclusions: The iRGD-STING-PFP@liposomes targeted drug delivery system effectively targets breast cancer cells, providing a new strategy for breast cancer immunotherapy. These findings indicate that iRGD-STING-PFP@liposomes could successfully deliver STING agonists to tumor tissue, trigger the innate immune response, and may serve as a potential platform for targeted immunotherapy.

Curvature-Assisted Vesicle Explosion Under Light-Induced Asymmetric Oxidation.

Exposure of cell membranes to reactive oxygen species can cause oxidation of membrane lipids. Oxidized lipids undergo drastic conformational changes, compromising the mechanical integrity of the membrane and causing cell death. For giant unilamellar vesicles, a classic cell mimetic system, a range of mechanical responses under oxidative assault has been observed including formation of nanopores, transient micron-sized pores, and total sudden catastrophic collapse (i.e., explosion). However, the physical mechanism regarding how lipid oxidation causes vesicles to explode remains elusive. Here, with light-induced asymmetric oxidation experiments, the role of spontaneous curvature on vesicle instability and its link to the conformational changes of oxidized lipid products is systematically investigated. A comprehensive membrane model is proposed for pore-opening dynamics incorporating spontaneous curvature and membrane curling, which captures the experimental observations well. The kinetics of lipid oxidation are further characterized and how light-induced asymmetric oxidation generates spontaneous curvature in a non-monotonic temporal manner is rationalized. Using the framework, a phase diagram with an analytical criterion to predict transient pore formation or catastrophic vesicle collapse is provided. The work can shed light on understanding biomembrane stability under oxidative assault and strategizing release dynamics of vesicle-based drug deliverysystems.

Glucose and Oxygen Levels Modulate the Pore-Forming Effects of Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae.

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin's pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.

Effect of tryptophan position and lysine/arginine substitution in antimicrobial peptides on antifungal action

Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.

Surface Acoustic Wave (SAW)‐Based Sonoporation of Single‐Adherent‐Cell

Sonoporation refers to the formation of tiny transient pores in cell plasma membranes by using ultrasound to increase the permeability to bioactive materials. Recently, the Sonoporation technique has been widely researched in cell treatment applications, such as gene transfection. However, due to the random distribution of microbubbles, it is challenging to perform controllable and precise localized sonoporation of a target cell. In this work, a device based on the surface acoustic wave is developed to achieve a selective manipulation and cavitation of microbubbles. The device consists of a pair of microbubble positioning slant‐finger interdigital transducers (SFITs) for moving a selected microbubble in a two‐dimensional plane. Meanwhile, another narrow‐frequency‐band SFIT is integrated into the device for local cavitation control of the same microbubble. As a result, a microbubble can be transported orthogonal to and along the acoustic transmission path by continuously adjusting the input frequency and relative phase. Upon reaching the target cell, the selected microbubble can be cavitated without exciting other microbubbles, resulting in local sonoporation. The resolution of phase‐based positioning and frequency‐based transportation are 8.3 and 7.0 µm with 45° and 10 kHz settings, respectively.

Determining the Functional Oligomeric State of Membrane-Associated Protein Oligomers Forming Membrane Pores on Giant Lipid Vesicles.

Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

Experimental and computational study of membrane permeabilization and translocation of the dynorphin A neuropeptide.

Dynorphins are endogenous neuropeptides that function as ligands for the κ-opioid receptor. In addition to opioid activity, dynorphins can induce several pathological effects such as neurological dysfunctions and cell death. Previous studies have suggested that Dynorphin A (DynA) mediates some pathogenic actions through formation of transient pores in lipid domains or translocation across the plasma membrane. Here, we use planar bilayer electrophysiology to show that DynA induces pore formation in negatively charged membranes. We find a large variability in pore conformations showing equilibrium conductance fluctuations, what disregards electroporation as the dominant mechanism of pore formation. Ion selectivity measurements showing cationic selectivity indicate that positive protein charges of DynA are stabilized by phosphatidyl serine negative charges in the formation of combined structures. We complement this study with computational simulations that assess the stability of diverse peptide arrangements in the hydrophobic core of the bilayer. We show that DynA is capable of assembling in charged membranes to form water-filled pores that conduct ions. Furthermore, we find that DynA generates membrane permeabilization events compatible with a translocation of the protein across the planar bilayer when using a neutral lipid. In these conditions, DynA is still capable of forming water-filled pores similar to those in a charged membrane, but translocation seems to be favored.

Ion-Induced Transient Potential Fluctuations Facilitate Pore Formation and Cation Transport through Lipid Membranes.

Unassisted ion transport through lipid membranes plays a crucial role in many cell functions without which life would not be possible, yet the precise mechanism behind the process remains unknown due to its molecular complexity. Here, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. High-throughput wide-field non-resonant second harmonic (SH) microscopy of membrane water shows that membrane potential fluctuations are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.

Numerical study of opposed zero-net-mass-flow jet-induced erythrocyte mechanoporation

With the advantages of biosafety and efficiency, increasing attention has been paid to the devices for gene and macromolecular drug delivery based on mechanoporation. The transient pore formation on the cell membrane allows cargo transportation when the membrane areal strain is beyond the critical pore value and below the lysis tension threshold. Based on this principle, we propose a method to apply the proper fluid stress on cells moving in a microchannel under the action of zero-net-mass-flux (ZNMF) jets. In this study, an immersed finite element method (IFEM) is adopted to simulate the interaction between the cells and the fluid fields so as to investigate the cell movement and deformation in this mechanoporation system. To evaluate the efficiency of the cargo delivery, a pore integral is defined as the mean pore rate when the cell passes through the jet region. By analyzing the effects of the parameters, including the pressure gradient along the microchannel, the jet amplitude, and the jet frequency, on the pore integrals, a group of optimized parameters for cargo delivery efficiency are obtained. Additionally, the stability and safety of this system are analyzed in detail. These results are helpful in designing the mechanoporation devices and improving their efficiency of drug delivery.

Landscape of Cellular Bioeffects Triggered by Ultrasound-Induced Sonoporation.

Sonoporation is the process of transient pore formation in the cell membrane triggered by ultrasound (US). Numerous studies have provided us with firm evidence that sonoporation may assist cancer treatment through effective drug and gene delivery. However, there is a massive gap in the body of literature on the issue of understanding the complexity of biophysical and biochemical sonoporation-induced cellular effects. This study provides a detailed explanation of the US-triggered bioeffects, in particular, cell compartments and the internal environment of the cell, as well as the further consequences on cell reproduction and growth. Moreover, a detailed biophysical insight into US-provoked pore formation is presented. This study is expected to review the knowledge of cellular effects initiated by US-induced sonoporation and summarize the attempts at clinical implementation.

Calcium Electroporation Reduces Viability and Proliferation Capacity of Four Uveal Melanoma Cell Lines in 2D and 3D Cultures.

Simple SummaryCalcium electroporation (CaEP) is an innovative anti-tumor treatment modality that induces cell death by introducing supraphysiological concentrations of calcium into cells with a limited effect on normal cells. The objective of the present study is to assess the effect of CaEP in uveal melanoma (UM) cell lines in comparison to electrochemotherapy (ECT) with bleomycin using 2D monolayer cell cultures as well as 3D tumor spheroid models in four different UM cell lines. The morphological changes of the spheroids, the cell viability, growth rate as well as the cytotoxic effect of electroporation (EP) with calcium chloride and bleomycin were evaluated with various drug concentrations. The results of CaEP and ECT both suggest a comparable dose-dependent reduction in cell viability and proliferation rate in all tested 2D cell lines and 3D tumor spheroids. These data point out that CaEP is an established anticancer treatment causing cell death by ATP depletion in in vitro and in vivo, representing an efficient alternative therapy with a lower cytotoxic potency for the local UM tumor control.Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non-cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye.

Influence of Electroporation Medium on Delivery of Cell-Impermeable Small Molecules by Electrical Short-Circuiting via an Aqueous Droplet in Dielectric Oil: A Comparison of Different Fluorescent Tracers.

Membrane permeabilization stimulated by high-voltage electric pulses has been used to deliver cell-impermeable exogenous molecules. The electric field effect on the cells depends on various experimental parameters, such as electric field strength, the number of electric pulses, and the electroporation medium. In this study, we show the influence of the electroporation medium on membrane permeabilization stimulated by electrical short-circuiting via an aqueous droplet in dielectric oil, a novel methodology developed by our previous investigations. We investigated the membrane permeabilization by three methods, influx of calcium ions, uptake of nucleic acid-binding fluorophores (YO-PRO-1), and calcein leakage. We demonstrated that the external medium conductivity had a significant impact on the cells in all described experiments. The short-circuiting using a low-conductivity electroporation medium enhanced the formation of both transient and irreversible membrane pores. We also found that clathrin-mediated endocytosis contributed to YO-PRO-1 uptake when a cell culture medium was used as an electroporation medium.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1–like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Dynorphin A induces membrane permeabilization by formation of proteolipidic pores. Insights from electrophysiology and computational simulations

Dynorphins are endogenous neuropeptides that function as ligands for the κ-opioid receptor. In addition to opioid activity, dynorphins can induce several pathological effects such as neurological dysfunctions and cell death. Previous studies have suggested that Dynorphin A (DynA) mediates some pathogenic actions through formation of transient pores in lipid domains of the plasma membrane. Here, we use planar bilayer electrophysiology to show that DynA induces pore formation in negatively charged membranes.

Dynorphin A induces membrane permeabilization by formation of proteolipidic pores. Insights from electrophysiology and computational simulations

Dynorphins are endogenous neuropeptides that function as ligands for the κ-opioid receptor. In addition to opioid activity, dynorphins can induce several pathological effects such as neurological dysfunctions and cell death. Previous studies have suggested that Dynorphin A (DynA) mediates some pathogenic actions through formation of transient pores in lipid domains of the plasma membrane. Here, we use planar bilayer electrophysiology to show that DynA induces pore formation in negatively charged membranes. We find a large variability in pore conformations showing equilibrium conductance fluctuations, what disregards electroporation as the dominant mechanism of pore formation. Ion selectivity measurements showing cationic selectivity indicate that positive protein charges of DynA are stabilized by phosphatidyl serine negative charges in the formation of combined structures. We complement our study with computational simulations that assess the stability of diverse peptide arrangements in the hydrophobic core of the bilayer. We show that DynA is capable of assembling in charged membranes to form water-filled pores that conduct ions.

Light-Triggered Release of Large Biomacromolecules from Porphyrin-Phospholipid Liposomes

Liposomes containing small amounts of porphyrin-phospholipid (PoP) have been shown to encapsulate small molecular weight cargos and then release them upon exposure to red light. A putative mechanism involves transient pore formation in the bilayer induced by PoP-mediated photo-oxidation of unsaturated lipids. However, little is known about the properties of such pores. This study assesses whether large carbohydrate and protein molecules could be released from PoP liposomes upon red light exposure. A small fluorophore with ∼0.5 kDa in molecular weight, fluorescently labeled dextrans of ∼5 and ∼500 kDa, and a ∼240 kDa fluorescent protein were passively entrapped in PoP liposomes. When exposed to 665 nm irradiation, liposomes containing PoP, but not liposomes lacking it, released all these cargos in a size-dependent manner that occurred with oxidation of unsaturated lipids included in the bilayer. Thus, this study demonstrates the feasibility of light-triggered release of large biomacromolecules from liposomes.

Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles.

A minimal synthetic cell should contain a substrate for information storage and have the capability to divide. Notable efforts were made to assemble functional synthetic cells from the bottom up, however often lacking the capability to reproduce. Here, we develop a mechanism to fully control reversible cargo loading and division of DNA-containing giant unilamellar vesicles (GUVs) with light. We make use of the photosensitizer Chlorin e6 (Ce6) which self-assembles into lipid bilayers and leads to local lipid peroxidation upon illumination. On the time scale of minutes, illumination induces the formation of transient pores, which we exploit for cargo encapsulation or controlled release. In combination with osmosis, complete division of two daughter GUVs can be triggered within seconds of illumination due to a spontaneous curvature increase. We ultimately demonstrate the division of a selected DNA-containing GUV with full spatiotemporal control—proving the relevance of the division mechanism for bottom-up synthetic biology.

Molecular Mechanism of Ultrasound-Induced Structural Defects in Liposomes: A Nonequilibrium Molecular Dynamics Simulation Study

The use of ultrasound in combination with liposomes is a promising approach to improve drug delivery. To achieve an optimal drug release rate, it is important to understand how ultrasound induces pathways on the liposome surface where drugs can be released from the liposome. To this end, we carry out large-scale ultrasound-induced molecular dynamics simulations for three single lipid component liposomes formed from the commonly used phospholipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), or phosphatidylcholine (POPC). The results show that ultrasound induces the detachment of two leaflets of the DOPC surface, suggesting that the drug release pathway may be through the low lipid packing areas on the stretched surface. In contrast, ultrasound induces pore formation on the surface of DPPC and DOPC, where drugs could escape from the liposomes. While the leaflet detachment and transient pore formation are the mechanisms of DOPC and DPPC, respectively, in both liquid-ordered and liquid-disordered phases, the leaflet detachment mechanism is switched to the transient pore formation mechanism on going from the liquid-ordered phase to the liquid-disordered phase in the POPC liposome. By adding 30% mol cholesterol, the leaflet detachment mechanism is observed in all liposomes. We found that the molecular origin that determines a mechanism is the competition between the intraleaflet and interleaflet interacting energy of lipids. The connection to experimental and theoretical modeling is discussed in some detail.

Molecular dynamics simulations of the effects of lipid oxidation on the permeability of cell membranes

The formation of transient pores in their membranes is a well-known mechanism of permeabilization of cells exposed to high-intensity electric pulses. However, the formation of such pores is not able to explain all aspects of the so-called electroporation phenomenon. In particular, the reasons for sustained permeability of cell membranes, persisting long after the pulses’ application, remain elusive. The complete resealing of cell membranes takes indeed orders of magnitude longer than the time for electropore closure as reported from molecular dynamics (MD) investigations. Lipid peroxidation has been suggested as a possible mechanism to explain the sustainable permeability of cell membranes. However, theoretical investigations of membrane lesions containing excess amounts of hydroperoxides have shown that the conductivities of such lesions were not high enough to account for the experimental measurements. Here, expanding on these studies, we investigate quantitatively the permeability of cell membrane lesions that underwent secondary oxidation. MD simulations and free energy calculations of lipid bilayers show that such lesions provide a better model of post-pulse permeable and conductive electropermeabilized cells. These results are further discussed in the context of sonoporation and ferroptosis, respectively a procedure and a phenomenon, among others, in which, alike electroporation, substantial lipid oxidation might be triggered.

Dynorphin a Induces Membrane Permeabilization by Formation of Proteolipidic Pores

Dynorphins are endogenous opioid neuropeptides that function as ligands for the κ-opioid receptor. In addition to opioid actions, dynorphins can induce several pathological effects that are not mediated through the opioid receptors, such as neurological dysfunctions and cell death, probably related to direct membrane interactions. In particular, previous studies have suggested that Dynorphin a (DynA) - one of the dynorphin actin peptides - mediates some pathogenic actions through formation of transient pores in lipid domains of the plasma membrane.