Coregulated assembly of actin-like FtsA polymers with FtsZ during Z-ring formation and division in Escherichia coli

In Escherichia coli, the actin homolog FtsA localizes the cell division machinery, beginning with the Z-ring, to the cytoplasmic membrane through direct interaction with FtsZ. FtsZ polymers are first to assemble at the Z-ring at midcell, where they direct constriction and septation. While FtsZ polymerization is critical for establishing a functional Z-ring that leads to constriction, the assembly state of FtsA and the role of FtsA ATP utilization during division in E. coli remain unclear. Here, we show that ATP hydrolysis, FtsZ interaction, and phospholipid vesicle remodeling by FtsA are impaired by a substitution mutation at the predicted active site for hydrolysis. This mutation, Glu 14 to Arg, also impairs Z-ring assembly and division in vivo. To further investigate the role of phospholipid engagement and ATP utilization in regulating FtsA function, we characterized a truncated E. coli FtsA variant, FtsA(ΔMTS), which lacks the region at the C-terminus important for engaging the membrane and is defective for ATP hydrolysis. We show that E. coli FtsA(ΔMTS) forms ATP-dependent actin-like filaments and assembly is antagonized by FtsZ. Polymerization of FtsZ with GTP, or a non-hydrolyzable analog, blocks inhibition of ATP-dependent FtsA assembly, and instead favors coassembly of stable FtsA/FtsZ polymers. In the cell, FtsA/FtsZ coassembly is favored at midcell, where FtsZ polymerizes, and inhibited at regions where FtsZ polymers are destabilized by regulators, such as MinC at the poles or SlmA at the nucleoid. We show that MinC prevents recruitment of FtsZ, via FtsA, to phospholipids, suggesting that local interactions of MinC with FtsZ block membrane tethering and uncouple the Z-ring from its major membrane contact. During Z-ring formation, the coassembly of FtsZ polymers with FtsA is coordinated and is a critical early step in division. This step also serves as a checkpoint by responding to the suite of FtsZ assembly regulators in the cell that modulate Z-ring position and dynamics prior to initiating cell wall synthesis.

The fabrication of phospholipid vesicle-based artificial cells and their functions

Phospholipid vesicles as artificial cells are used to simulate the cellular structure and function.

SELECTION OF THE COMPOSITION OF A LIPOSOMAL DOSAGE FORM OF A RUSSIAN SOMATOSTATIN ANALOGUE WITH ANTITUMOR ACTIVITY

Objective: Was to create the composition of the liposomal pharmaceutical form for injections of somatostatin analogue cyphetrylin using soybean phosphatidylcholine (SPC). Methods: The cyphetrylin, active pharmaceutical ingredient (API), developed in the Chemical Synthesis Laboratory, the N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation; SPC and polyethylene glycol-2000-distearoylphosphatidylethanolamine (PEG-DSPE, Lipoid, Germany); cholesterol ≥99% (Sigma-Aldrich, Japan). The lipid film hydration method with subsequent liposomal dispersion filtration/extrusion through nylon membrane filters was used for the phospholipid vesicle production. Based on API and lipid components in different molar ratios, we studied over 15 model liposomal compositions and assessed each lipid's impact in use on quality attributes of resulting dispersions. Derived model samples of liposomal dispersion were estimated in terms of quality and efficiency of cyphetrylin encapsulation into vesicles, their average size and the surface charge (zeta potential), polydispersity index (PDI) and dispersion viscosity. We used spectral photometry, dispersion laser spectroscopy, electrophoretic particle mobility assay, and viscometry to assess these features. Results: Pharmaceutical form components' desirable molar ratios determined: cyphetrylin/SPC at 1:60.0 and SPC/cholesterol/PEG-DSPE at 1:0.2:0.004, were determined. This composition allows cyphetrylin liposomal dispersion production with relatively stable vesicles of uniform size, 176 nm in diameter, and a 100% maximum rate of API encapsulation into the bilayer. Conclusion: Technological and chemical/pharmaceutical studies resulted in selecting a preferable composition of an injectable liposomal pharmaceutical form model of somatostatin analog-based on the SPC.

Phospholipid Vesicles for Dermal/Transdermal and Nasal Administration of Active Molecules: The Effect of Surfactants and Alcohols on the Fluidity of Their Lipid Bilayers and Penetration Enhancement Properties

This is a comprehensive review on the use of phospholipid nanovesicles for dermal/transdermal and nasal drug administration. Phospholipid-based vesicular carriers have been widely investigated for enhanced drug delivery via dermal/transdermal routes. Classic phospholipid vesicles, liposomes, do not penetrate the deep layers of the skin, but remain confined to the upper stratum corneum. The literature describes several approaches with the aim of altering the properties of these vesicles to improve their penetration properties. Transfersomes and ethosomes are the most investigated penetration-enhancing phospholipid nanovesicles, obtained by the incorporation of surfactant edge activators and high concentrations of ethanol, respectively. These two types of vesicles differ in terms of their structure, characteristics, mechanism of action and mode of application on the skin. Edge activators contribute to the deformability and elasticity of transfersomes, enabling them to penetrate through pores much smaller than their own size. The ethanol high concentration in ethosomes generates a soft vesicle by fluidizing the phospholipid bilayers, allowing the vesicle to penetrate deeper into the skin. Glycerosomes and transethosomes, phospholipid vesicles containing glycerol or a mixture of ethanol and edge activators, respectively, are also covered. This review discusses the effects of edge activators, ethanol and glycerol on the phospholipid vesicle, emphasizing the differences between a soft and an elastic nanovesicle, and presents their different preparation methods. To date, these differences have not been comparatively discussed. The review presents a large number of active molecules incorporated in these carriers and investigated in vitro, in vivo or in clinical human tests.

The Vaginal-PVPA: A Vaginal Mucosa-Mimicking In Vitro Permeation Tool for Evaluation of Mucoadhesive Formulations

Drug administration to the vaginal site has gained increasing attention in past decades, highlighting the need for reliable in vitro methods to assess the performance of novel formulations. To optimize formulations destined for the vaginal site, it is important to evaluate the drug retention within the vagina as well as its permeation across the mucosa, particularly in the presence of vaginal fluids. Herewith, the vaginal-PVPA (Phospholipid Vesicle-based Permeation Assay) in vitro permeability model was validated as a tool to evaluate the permeation of the anti-inflammatory drug ibuprofen from liposomal formulations (i.e., plain and chitosan-coated liposomes). Drug permeation was assessed in the presence and absence of mucus and simulated vaginal fluid (SVF) at pH conditions mimicking both the healthy vaginal premenopausal conditions and vaginal infection/pre-puberty/post-menopause state. The permeation of ibuprofen proved to depend on the type of formulation (i.e., chitosan-coated liposomes exhibited lower drug permeation), the mucoadhesive formulation properties and pH condition. This study highlights both the importance of mucus and SVF in the vaginal model to better understand and predict the in vivo performance of formulations destined for vaginal administration, and the suitability of the vaginal-PVPA model for such investigations.

Impact of Mucin on Drug Diffusion: Development of a Straightforward In Vitro Method for the Determination of Drug Diffusivity in the Presence of Mucin

Mucosal drug delivery accounts for various administration routes (i.e., oral, vaginal, ocular, pulmonary, etc.) and offers a vast surface for the permeation of drugs. However, the mucus layer which shields and lubricates all mucosal tissues can compromise drugs from reaching the epithelial site, thus affecting their absorption and therapeutic effect. Therefore, the effect of the mucus layer on drug absorption has to be evaluated early in the drug-development phase, prior to in vivo studies. For this reason, we developed a simple, cost-effective and reproducible method employing UV-visible localized spectroscopy for the assessment of the interaction between mucin and drugs with different physicochemical characteristics. The mucin–drug interaction was investigated by measuring the drug relative diffusivity (Drel) in the presence of mucin, and the method was validated by fitting experimental and mathematical data. In vitro permeability studies were also performed using the mucus-covered artificial permeation barrier (mucus–PVPA, Phospholipid Vesicle-based Permeation Assay) for comparison. The obtained results showed that the diffusion of drugs was hampered by the presence of mucin, especially at higher concentrations. This novel method proved to be suitable for the investigation on the extent of mucin–drug interaction and can be successfully used to assess the impact that the mucus layer has on drug absorption.