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Giant vesicles are widely used as a model membrane system, both for basic biological systems and for their promising applications in the development of smart materials and cell mimetics, as well as in driving new technologies in synthetic biology and for the cosmetics and pharmaceutical industry. The reader is guided to use giant vesicles, from the formation of simple membrane platforms to advanced membrane and cell system models. It also includes fundamentals for understanding lipid or polymer membrane structure, properties and behavior. Every chapter includes ideas for further applications and discussions on the implications of the observed phenomena towards understanding membrane-related processes. The Giant Vesicle Book is meant to be a road companion, a trusted guide for those making their first steps in this field as well as a source of information required by experts. Key Features • A complete summary of the field, covering fundamental concepts, practical methods, core theory, and the most promising applications • A start-up package of theoretical and experimental information for newcomers in the field • Extensive protocols for establishing the required preparations and assays • Tips and instructions for carefully performing and interpreting measurements with giant vesicles or for observing them, including pitfalls • Approaches developed for investigating giant vesicles as well as brief overviews of previous studies implementing the described techniques • Handy tables with data and structures for ready reference Part I: The making of Chapter 1 Preparation methods for giant unilamellar vesicles - Rumiana Dimova, Pasquale Stano, Carlos M. Marques and Peter Walde Chapter 2 Preparation and properties of giant plasma membrane vesicles and giant unilamellar vesicles from natural membranes - Joseph H. Lorent and Ilya Levental Chapter 3 Protein reconstitution in giant vesicles - Matthias Garten, Daniel Lévy and Patricia Bassereau　 Chapter 4 GUVs with cytoskeleton - Tobias Härtel and Petra Schwille Part II: Giant vesicles theoretically and in silico Chapter 5 Understanding giant vesicles – a theoretical perspective - Reinhard Lipowsky Chapter 6 Simulating membranes, vesicles, and cells - Thorsten Auth, Dmitry A. Fedosov and Gerhard Gompper Chapter 7 Theory of vesicle dynamics in flow and electric fields - Petia M. Vlahovska and Chaouqi Misbah Chapter 8 Particle-membrane interactions - Jaime Agudo-Canalejo, Reinhard Lipowsky Chapter 9 Theory of polymer-membrane interactions - Fabrice Thalmann and Carlos M. Marques Part III: GUV-based techniques and what one can learn from them Chapter 10 Application of optical microscopy techniques on giant unilamellar vesicles - Luis A. Bagatolli Chapter 11 Mechanics assays of synthetic lipid membranes based on micropipette aspiration - Elisa Parra and David Needham Chapter 12 Atomic force microscopy of giant unilamellar vesicles - Andreas Janshoff Chapter 13 Manipulation and biophysical characterization of GUVs with an optical stretcher - Gheorghe Cojoc, Antoine Girot, Ulysse Delabre and Jochen Guck Chapter 14 Vesicle fluctuation analysis - John Hjort Ipsen, Allan Grønhøj Hansen and Tripta Bhatia Chapter 15 Using electric fields to assess membrane material properties in GUVs - Rumiana Dimova and　　Karin A. Riske Chapter 16 Creating membrane nanotubes from GUVs - Coline Prévost, Mijo Simunovic and Patricia Bassereau Chapter 17 Measuring GUV adhesion - Kheya Sengupta and Ana Smith Chapter 18 Phase diagrams and tie lines in GUVs - Matthew C. Blosser, Caitlin Cornell, Scott P. Rayermann and Sarah L. Keller Chapter 19 Vesicle dynamics in flow: an experimental approach - Victor Steinberg and Michael Levant Chapter 20 Membrane permeability measurements - Begoña Ugarte-Uribe, Ana J. García-Sáez and Mireille M. A. E. Claessens Part IV: GUVs as membrane interaction platforms Chapter 21 - Lipid and protein mobility in GUVs - Begoña Ugarte-Uribe, Kushal Kumar Das and Ana J. García-Sáez Chapter 22 Shining light on membranes - Rosangela Itri, Carlos M. Marques and Mauricio S. Baptista Chapter 23 Protein-membrane interactions - Eva M Schmid and Daniel A Fletcher Chapter 24 Effects of antimicrobial peptides and detergents on GUVs - Karin A. Riske Chapter 25 Lipid-polymer interactions: effect on GUVs shapes and behavior - Brigitte Pépin-Donat, François Quemeneur and Clément Campillo Part V: GUVs as complex membrane containers Chapter 26 Polymersomes - Praful Nair, David Christian and Dennis E. Discher Chapter 27 Giant hybrid polymer/lipid vesicles - Thi Phuong Tuyen Dao, Khalid Ferji, Fabio Fernandes, Manuel Prieto, Sébastien Lecommandoux, Emmanuel Ibarboure, Olivier Sandre and Jean-François Le Meins Chapter 28 Giant unilamellar vesicles: from protocell models to the construction of minimal cells - Masayuki Imai and Peter Walde Chapter 29 Encapsulation of aqueous two-phase systems and gels within giant lipid vesicles - Allyson M. Marianelli and Christine D. Keating Chapter 30 Droplet-supported giant lipid vesicles as compartments for synthetic biology - Johannes P. Frohnmayer, Marian Weiss, Lucia T. Benk, Jan-Willi Janiesch, Barbara Haller, Rafael B. Lira, Rumiana Dimova, Ilia Plazman and Joachim P. Spatz Appendices Appendix 1 List of lipids and physical constants of lipid bilayers Appendix 2 List of membrane dyes and fluorescent groups conjugated to lipids Appendix 3 List of detergents Appendix 4 List of water-soluble dyes or their fluorescent groups and their structures Rumiana Dimova leads an experimental lab in biophysics at the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany. She has been working with giant vesicles already from the beginning of her scientific career. After being introduced into the magic of their preparation during her studies as a student in Bulgaria, she remained fascinated by their application and over the years pursued a variety of projects employing giant vesicles as a platform to develop new methods for the biophysical characterization of membranes and processes involving them. Until now, these studies have resulted in more than hundred peer-reviewed publications. Recently, she was also awarded the Emmy Noether distinction for women in physics of the European Physical Society. Carlos Marques , a CNRS senior scientist, founded the MCube group at the Charles Sadron Institute in Strasbourg, France, where he gears experimental and theoretical research towards the understanding of the physical properties of self-assembled lipid bilayers. Trained as a polymer theoretician, Carlos first got interested in membranes because they interact with polymers and published the first prediction for the membrane changes expected when polymers adsorb on lipid bilayers. He then expanded the scope of his group to include experiments and numerical simulations, and has now published many papers based on research with giant unilamellar vesicles, including the first study of lipid oxidation in GUVs and the discovery of the so-called PVA method for vesicle growth.

A method for the study of conjugated polyelectrolyte (CPE) photophysics in solution at the single-molecule level is described. Extended observation times of single polymer molecules are enabled by the encapsulation of the CPEs within 200-nm lipid vesicles, which are in turn immobilized on a surface. When combined with a molecular-level visualization of vesicles and CPE via cryo-transmission electron microscopy, these single-molecule spectroscopy studies on CPEs enable us to directly correlate the polymer conformation with its spectroscopic features. These studies are conducted with poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylene-vinylene] (MPS-PPV, a negatively charged CPE), when encapsulated in neutral and in negatively charged lipid vesicles. MPS-PPV exists as a freely diffusing polymer when confined in negatively charged vesicles. Individual MPS-PPV molecules adopt a collapsed-chain conformation leading to efficient energy migration over multiple chromophores. Both the presence of stepwise photobleaching in fluorescence intensity-time trajectories and emission from low-energy chromophores along the chain are observed. These results correlate with the amplified sensing potential reported for MPS-PPV in aqueous solution. When confined within neutral vesicles, single MPS-PPV molecules adopt an extended conformation upon insertion in the lipid bilayer. In this case emission arises from multiple chromophores within the isolated polymer chains, leading to an exponential decay of the intensity over time and a broad blue-shifted emission spectrum.

Lipid polymer hybrid nanoparticles (LPHNPs) for the controlled delivery of hydrophilic doxorubicin hydrochloride (DOX.HCl) and lipophilic DOX base have been fabricated by the single step modified nanoprecipitation method. Poly (D, L-lactide-co-glicolide) (PLGA), lecithin, and 1,2-distearoyl-Sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000 (DSPE-PEG 2000) were selected as structural components. The mean particle size was 173-208 nm, with an encapsulation efficiency of 17.8±1.9 to 43.8±4.4% and 40.3±0.6 to 59. 8±1.4% for DOX.HCl and DOX base, respectively. The drug release profile was in the range 33-57% in 24 hours and followed the Higuchi model (R =0.9867-0.9450) and Fickian diffusion (n<0.5). However, the release of DOX base was slower than DOX.HCl. The in vitro cytotoxicity studies and confocal imaging showed safety, good biocompatibility, and a higher degree of particle internalization. The higher internalization of DOX base was attributed to higher permeability of lipophilic component and better hydrophobic interaction of particles with cell membranes. Compared to the free DOX, the DOX.HCl and DOX base loaded LPHNPs showed higher antiproliferation effects in MDA-MB231 and PC3 cells. Therefore, LPHNPs have provided a potential drug delivery strategy for safe, controlled delivery of both hydrophilic and lipophilic form of DOX in cancer cells.

The purpose of this study was to develop a novel lipid-polymer hybrid drug carrier comprised of folate (FA) modified lipid-shell and polymer-core nanoparticles (FLPNPs) for sustained, controlled, and targeted delivery of paclitaxel (PTX). The core-shell NPs consist of 1) a poly(ε-caprolactone) hydrophobic core based on self-assembly of poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL) amphiphilic copolymers, 2) a lipid monolayer formed with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000), 3) a targeting ligand (FA) on the surface, and were prepared using a thin-film hydration and ultrasonic dispersion method. Transmission electron microscopy and dynamic light scattering analysis confirmed the coating of the lipid monolayer on the hydrophobic polymer core. Physicochemical characterizations of PTX-loaded FLPNPs, such as particle size and size distribution, zeta potential, morphology, drug loading content, encapsulation efficiency, and in vitro drug release, were also evaluated. Fluorescent microscopy proved the internalization efficiency and targeting ability of the folate conjugated on the lipid monolayer for the EMT6 cancer cells which overexpress folate receptor. In vitro cytotoxicity assay demonstrated that the cytotoxic effect of PTX-loaded FLPNPs was lower than that of Taxol(®), but higher than that of PTX-loaded LPNPs (without folate conjugation). In EMT6 breast tumor model, intratumoral administration of PTX-loaded FLPNPs showed similar antitumor efficacy but low toxicity compared to Taxol(®). More importantly, PTX-loaded FLPNPs showed greater tumor growth inhibition (65.78%) than the nontargeted PTX-loaded LPNPs (48.38%) (P<0.05). These findings indicated that the PTX loaded-FLPNPs with mixed lipid monolayer shell and biodegradable polymer core would be a promising nanosized drug formulation for tumor-targeted therapy.

An optimal design of nanocarriers is required to overcome the gap between synthetic and biological identity, improving the clinical translation of nanomedicine. A new generation of hybrid vehicles based on lipid–polymer coupling, obtained by Microfluidics, is proposed and validated for theranostics and multimodal imaging applications. A coupled Hydrodynamic Flow Focusing (cHFF) is exploited to control the time scales of solvent exchange and the coupling of the polymer nanoprecipitation with the lipid self-assembly simultaneously, guiding the formation of Lipid–Polymer NPs (LiPoNs). This hybrid lipid–polymeric tool is made up of core–shell structure, where a polymeric chitosan core is enveloped in a lipid bilayer, capable of co-encapsulating simultaneously Gd-DTPA and Irinotecan/Atto 633 compounds. As a result, a monodisperse population of hybrid NPs with an average size of 77 nm, with preserved structural integrity in different environmental conditions and high biocompatibility, can be used for MRI and Optical applications. Furthermore, preliminary results show the enhanced delivery and therapeutic efficacy of Irinotecan-loaded hybrid formulation against U87 MG cancers cells.