A synthetic membrane shaper for controlled liposome deformation

Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a dumbbell shape, while organelles such as the autophagosome exhibit stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

Effects of Antibacterial Peptide F1 on Bacterial Liposome Membrane Integrity

Previous studies from our lab have shown that the antimicrobial peptide F1 obtained from the milk fermentation by Lactobacillus paracasei FX-6 derived from Tibetan kefir was different from common antimicrobial peptides; specifically, F1 simultaneously inhibited the growth of Gram-negative and Gram-positive bacteria. Here, we present follow-on work demonstrating that after the antimicrobial peptide F1 acts on either Escherichia coli ATCC 25922 (E. coli) or Staphylococcus aureus ATCC 63589 (S. aureus), their respective bacterial membranes were severely deformed. This deformation allowed leakage of potassium and magnesium ions from the bacterial membrane. The interaction between the antimicrobial peptide F1 and the bacterial membrane was further explored by artificially simulating the bacterial phospholipid membranes and then extracting them. The study results indicated that after the antimicrobial peptide F1 interacted with the bacterial membranes caused significant calcein leakage that had been simulated by different liposomes. Furthermore, transmission electron microscopy observations revealed that the phospholipid membrane structure was destroyed and the liposomes presented aggregation and precipitation. Quartz Crystal Microbalance with Dissipation (QCM-D) results showed that the antimicrobial peptide F1 significantly reduced the quality of liposome membrane and increased their viscoelasticity. Based on the study's findings, the phospholipid membrane particle size was significantly increased, indicating that the antimicrobial peptide F1 had a direct effect on the phospholipid membrane. Conclusively, the antimicrobial peptide F1 destroyed the membrane structure of both Gram-negative and Gram-positive bacteria by destroying the shared components of their respective phospholipid membranes which resulted in leakage of cell contents and subsequently cell death.

Versatile Encapsulation and Synthesis of Potent Therapeutic Liposomes by Thermal Equilibration

The wide-scale use of liposomal delivery systems is hampered by difficulties in obtaining potent liposomal suspensions. Passive and active loading strategies have been proposed to formulate drug encapsulated liposomes, but are limited by low efficiencies (passive) or high drug specificities (active). Here, we present an efficient and universal loading strategy for synthesizing therapeutic liposomes. Integrating a thermal equilibration technique with our unique liposome synthesis approach, co-loaded targeting liposomes can be engineered in an efficient and scalable manner with potencies 200-fold higher than typical passive encapsulation techniques. We demonstrate this capability through simultaneous co-loading of hydrophilic and hydrophobic small molecules and through targeted delivery of liposomal Doxorubicin to a metastatic breast cancer cell line MDA-MB-231. Molecular dynamic simulations are used to explain interactions between Doxorubicin and liposome membrane during thermal equilibration. By addressing the existing challenges, we have developed an unparalleled approach that will facilitate the formulation of novel theranostic and pharmaceutical strategies.

Vesicular Release Dynamics are Altered by Interaction between Chemical Cargo and Vesicle Membrane Lipids

We used liposomes loaded with different monoamines, dopamine (DA) and serotonin (5-HT), to simulate vesicular release and to monitor the dynamics of chemical release from isolated vesicles during vesicle impact electrochemical cytometry (VIEC). The release of DA from liposomes presents a longer release time compared to 5-HT. Modelling the release time showed that DA filled vesicles had a higher percentage of events where the time for the peak fall was better fit to a double exponential (DblExp) decay function, suggesting multiple kinetic steps in the release. By fitting to a desorption-release model, where the transmitters adsorbed to the vesicle membrane, the dissociation rates of DA and 5-HT from liposome membrane were estimated. DA has a lower desorption rate constant, which leads to slower DA release than that observed for 5-HT, whereas there is little difference in pore size. The alteration of vesicular release dynamics due to the interaction between chemical cargo and vesicle membrane lipids provides an important mechanism to regulate vesicular release in chemical and physiological processes. It is highly possible that this introduces a fundamental chemical regulation difference between transmitters during exocytosis.

Vesicular Release Dynamics are Altered by Interaction between Chemical Cargo and Vesicle Membrane Lipids

We used liposomes loaded with different monoamines, dopamine (DA) and serotonin (5-HT), to simulate vesicular release and to monitor the dynamics of chemical release from isolated vesicles during vesicle impact electrochemical cytometry (VIEC). The release of DA from liposomes presents a longer release time compared to 5-HT. Modelling the release time showed that DA filled vesicles had a higher percentage of events where the time for the peak fall was better fit to a double exponential (DblExp) decay function, suggesting multiple kinetic steps in the release. By fitting to a desorption-release model, where the transmitters adsorbed to the vesicle membrane, the dissociation rates of DA and 5-HT from liposome membrane were estimated. DA has a lower desorption rate constant, which leads to slower DA release than that observed for 5-HT, whereas there is little difference in pore size. The alteration of vesicular release dynamics due to the interaction between chemical cargo and vesicle membrane lipids provides an important mechanism to regulate vesicular release in chemical and physiological processes. It is highly possible that this introduces a fundamental chemical regulation difference between transmitters during exocytosis.

The Antimicrobial Peptide Gramicidin S Enhances Membrane Adsorption and Ion Pore Formation Potency of Chemotherapy Drugs in Lipid Bilayers

We recently published two novel findings where we found the chemotherapy drugs (CDs) thiocolchicoside (TCC) and taxol to induce toroidal type ion pores and the antimicrobial peptide gramicidin S (GS) to induce transient defects in model membranes. Both CD pores and GS defects were induced under the influence of an applied transmembrane potential (≈100 mV), which was inspected using the electrophysiology record of membrane currents (ERMCs). In this article, I address the regulation of the membrane adsorption and pore formation of CDs due to GS-induced possible alterations of lipid bilayer physical properties. In ERMCs, low micromolar (≥1 μM) GS concentrations in the aqueous phase were found to cause an induction of defects in lipid bilayers, but nanomolar (nM) concentration GS did nothing. For the binary presence of CDs and GS in the membrane-bathing aqueous phase, the TCC pore formation potency is found to increase considerably due to nM concentration GS in buffer. This novel result resembles our recently reported finding that due to the binary aqueous presence of two AMPs (gramicidin A or alamethicin and GS), the pore or defect-forming potency of either AMP increases considerably. To reveal the underlying molecular mechanisms, the influence of GS (0–400 nM) on the quantitative liposome (membrane) adsorption of CD molecules, colchicine and TCC, was tested. I used the recently patented direct detection method, which helps detect the membrane active agents directly at the membrane in the mole fraction relative to its concentrations in aqueous phase. We find that GS, at concentrations known to do nothing to the lipid bilayer electrical barrier properties in ERMCs, increases the membrane adsorption (membrane uptake) of CDs considerably. This phenomenological finding along with the GS effects on CD-induced membrane conductance increase helps predict an important conclusion. The binary presence of AMPs alongside CDs in the lipid membrane vicinity may work toward enhancing the physical adsorption and pore formation potency of CDs in lipid bilayers. This may help understand why CDs cause considerable cytotoxicity.

Chitosan-covered liposomes as a promising drug transporter: nanoscale investigations

In this paper, we studied the graft chitosan conformation and its influence on the liposome membrane structure and dynamics as a function of the grafting molar-fraction.

Use of phosphatidylcholine in Tris-based extender with or without egg yolk to freeze Bapedi ram semen

Traditionally, egg yolk is a protective agent that is used to freeze semen in various species. However, the addition of egg yolk in extender risks the introduction of disease. Therefore, an alternative cryoprotective agent should be found to preserve ram semen. The aim of this study was to evaluate the effect of phosphatidylcholine (PC) as a protective agent in extender with or without egg yolk on semen characteristics and acrosome integrity of frozen then thawed Bapedi ram semen. Semen was collected from four mature Bapedi rams, in the Agricultural Research Council (ARC) Germplasm Conservation Programme, using an artificial vagina. Following collection, semen samples were randomly diluted into Tris-based extender (1: 2), with and without egg yolk, and supplemented with four concentrations of PC liposome (0 mg/ml), 0.25 mg/ml, 0.5 mg/ml and 0.75 mg/ml). Supplementation of PC liposome in extender with or without egg yolk did not improve the semen total motility (TM), progressive motility (PM) and rapid motility (RM) rate. The sperm cell membrane integrity in extender with or without egg yolk was not influenced by the supplementation of PC liposome after thawing (P >0.05). The addition of PC liposome to Tris-based extender with egg yolk had a similar result to control (Tris-based extender with egg yolk) on sperm cell acrosome integrity. In conclusion, supplementation of PC liposome to Tris-based extender without egg yolk had lower sperm cell viability and motility rates compared with the extender with egg yolk, regardless of concentration.Keywords: acrosome, cryoprotectant, liposome, membrane, motility

A twofold strategy for the protection of therapeutic peptides by attachment to the protease-resistant and fusogenic protein, saposin C

ABSTRACTA great challenge of therapeutic peptides (biologics) is their short half-life. However, biologics can be protected by encapsulation in liposomes used as drug-delivery platforms. Liposomes are typically incorporated into cells by endocytic pathways, which eventually expose therapeutics to favorable proteolytic conditions. To enhance biologics protection, we report the design and characterization of a liposome-protein chimera combining the liposome fusogenic properties of peripheral-membrane protein saposin C, covalently linked to a proapoptotic peptide (the active domain of Bcl-2 protein PUMA). We show by NMR that the saposin C component of the chimera is capable of binding liposomes and that the peptide binds prosurvival Bcl-xL, thus following known PUMA’s mechanism to induce cell death. These results indicate that the function of the individual components is preserved in the chimera. Our results point to a promising twofold strategy for drug delivery to; 1) avoid endocytosis by promoting liposome-membrane fusion, 2) provide additional protection by attachment to a stable, protease-resistant protein, which is a well-known method commonly used to prolong biologics half-life.