Biology subjects

Pazzi, J.

Publications and source records attributed to Pazzi, J..

4 recordsLinked to original sources

bioRxiv2025

Polymer-assisted assembly of giant vesicles in physiological solutions

Thin film hydration methods are widely used to assemble giant unilamellar vesicles (GUVs), but their efficiency drops sharply in solutions of physiological ionic strength due to enhanced adhesion between stacked lipid bilayers, which suppresses bud and foam-like mesophase formation. Here, we introduce polymer-coated nanocellulose paper (PCP) as a nanostructured substrate that overcomes this limitation. Hydration of lipid films on PCP--termed PCP-assisted hydration-- achieves high GUV yields across a broad temperature range (4-45 {degrees}C) using diverse soluble polymers, including ultralow-gelling agarose, hyaluronic acid, dextran, carrageenan, and polylysine. The nanoscale curvature of the cellulose fibers and the osmotic pressure generated by polymer dissolution act synergistically to promote membrane budding even under physiological salt conditions. The approach is scalable--supporting GUV production from millimeter-sized pieces to large-format sheets--and biocompatible, enabling encapsulation of complex biochemical systems such as cell-free expression mixtures and actin-fascin assemblies. PCP-assisted hydration thus provides a robust, versatile, and high-yielding platform for generating functional GUVs under physiological conditions.

biophysics↗

bioRxiv2024

Synthesis and Evaluation of Carmofur Analogs as Antiproliferative Agents, Inhibitors to the Main Protease (Mpro) of SARS-CoV-2, and Membrane Rupture-Inducing Agents

Initially developed as a derivatized analog of 5-fluorouracil for the treatment of colorectal cancer, carmofur has more recently demonstrated potent covalent inhibition of the main protease (Mpro) of SARS-CoV-2. Harnessing our previously described workflow for the optimized preparation of carmofur using benchtop 19F NMR spectroscopy, here, we prepared and evaluated a synthetic library of nine carmofur analogs with a selection of side chain motifs or single-atom substitution to explore the diversifiability of these compounds as Mpro inhibitors, where we discovered that a hexyl carbamate analog outperformed carmofur, and as antiproliferative agents in model human cell lines to identify differences in potency when the carbonyl electrophilicity and/or alkyl side chains are modified. Finally, we describe a novel workflow for the evaluation of membrane-rupturing small molecules through imaging of fluorescently labeled giant unilamellar vesicles (GUVs), and through this, we identified two lipophilic urethane analogs of carmofur bearing dodecyl urethane and octadecyl urethane side chains that have potent membrane-rupturing capability in the nanomolar range, providing insight into a potential mechanism for the in vitro activities of lipidated 5-fluorouracil analogs.

biochemistry↗

bioRxiv2023

Dynamics of giant vesicle assembly from thin lipid films

Giant unilamellar vesicles (GUVs) are micrometer-scale lipid assemblies that emulate key characteristics of biological cell membranes. GUVs can be obtained when solid-supported thin films of lipids are hydrated in aqueous solutions. However, a comprehensive understanding of their assembly dynamics has been lacking, impeding mechanistic insights. Here, we report the time dependence of the distribution of sizes and molar yield of GUVs obtained through a novel stopped-time technique. We compare three commonly used techniques, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions) gentle hydration, and electroformation. We demonstrate that all three techniques show sigmoidal yield curves. Yields increase monotonically before reaching a plateau, with surprisingly high yields 60 seconds after hydration. Gentle hydration shows limited time evolution in contrast to PAPYRUS and electroformation. Exploration of bud dynamics on the surfaces uncovers bud emergence, diameter growth, and merging phenomena. To provide a comprehensive explanation of our observations, we employ the thermodynamic budding and merging model. This work expands our understanding of GUV assembly dynamics and offers fundamental insights into the underlying thermodynamic principles governing this process.

biophysics↗

bioRxiv2020

Nanoscale curvature promotes high yield spontaneous formation of cell-mimetic giant vesicles

To date, surface-assisted assembly of cell-like giant vesicles use planar surfaces and require the application of electric fields or dissolved molecules to obtain adequate yields. Here, we present the use of nanoscale surface curvature and hydrophilic surface chemistry to promote the high yield assembly of GUVs. We show that assembly on surfaces composed of entangled hydrophilic nanocellulose fibers results in an unprecedented 100,000-fold reduction in costs while increasing yields compared to extant techniques. Quantitative measurements of yields provide mechanistic insight on the effect of nanoscale curvature and the effect of surface chemistry. We present a thermodynamic budding and merging, BNM, model that unifies observations of assembly. The BNM model considers the change in free energy by balancing elastic, adhesion, and membrane edge energies in the formation of surface-attached spherical buds. Due to curvature and the hydrophilicity of cellulose, energetically unfavorable formation of buds on planar and spherical surfaces becomes favorable (spontaneous) on surfaces composed of cylindrical cellulose nanofibers. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/227686v2_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@3452a4org.highwire.dtl.DTLVardef@11da2a1org.highwire.dtl.DTLVardef@a021a1org.highwire.dtl.DTLVardef@1000acb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗