Starch-fibers composites, a study of all-polysaccharide foams from microwave foaming to biodegradation.

Sustainable composite foams based on rice starch and cellulosic long fibers were successfully fabricated using microwave irradiation. They were presented as a promising method to recycle some of the textile industry waste. A deep study of the processability and functionality of the composites revealed the performance improvement of starch with the addition of long cellulosic fibers, especially with 6 wt% of Arbocel®, in terms of foamability, water, and mechanical resistance features. Moreover, sodium bicarbonate, which acted as a blowing and pulping agent, led to a lower density and better fiber distribution that resulted in an improvement of the foams' functionalities. The range of the study is new in the domain of long fiber foam composites in terms of the foaming capability, and mechanical, thermal, and water resistance properties. Furthermore, all foams showed excellent biodegradability properties against a fungus commonly found in the environment; for example, values around 60 % weight loss after 33 days. Finally, the assessment of the CO2 emission during the process underlines the environmental benefits of the method employed.

In Situ Monitoring of Block Copolymer Self-Assembly via Solvent Exchange through Controlled Dialysis with Light and Neutron Scattering Detection.

Solution self-assembly of amphiphilic block copolymers (BCs) is typically performed by a solvent-to-water exchange. However, BC assemblies are often trapped in metastable states depending on the mixing conditions such as the magnitude and rate of water addition. BC self-assembly can be performed under near thermodynamic control by dialysis, which accounts for a slow and gradual water addition. In this Letter we report the use of a specifically designed dialysis cell to continuously monitor by dynamic light scattering and small-angle neutron scattering the morphological changes of PDMS-b-PEG BCs self-assemblies during THF-to-water exchange. The complete phase diagrams of near-equilibrium structures can then be established. Spherical micelles first form before evolving to rod-like micelles and vesicles, decreasing the total developed interfacial area of self-assembled structures in response to increasing interfacial energy as the water content increases. The dialysis kinetics can be tailored to the time scale of BC self-assembly by modifying the membrane pore size, which is of interest to study the interplay between thermodynamics and kinetics in self-assembly pathways.

Switchable Lipids: From Conformational Switch to Macroscopic Changes in Lipid Vesicles.

It has been shown that the use of conformationally pH-switchable lipids can drastically enhance the cytosolic drug delivery of lipid vesicles. Understanding the process by which the pH-switchable lipids disturb the lipid assembly of nanoparticles and trigger the cargo release is crucial to optimize the rational design of pH-switchable lipids. Here, we gather morphological observations (FF-SEM, Cryo-TEM, AFM, confocal microscopy), physicochemical characterization (DLS, ELS), as well as phase behavior studies (DSC, 2H NMR, Langmuir isotherm, and MAS NMR) to propose a mechanism of pH-triggered membrane destabilization. We demonstrate that the switchable lipids are homogeneously incorporated with other co-lipids (DSPC, cholesterol, and DSPE-PEG2000) and promote a liquid-ordered phase insensitive to temperature variation. Upon acidification, the protonation of the switchable lipids triggers a conformational switch altering the self-assembly properties of lipid nanoparticles. These modifications do not lead to a phase separation of the lipid membrane; however, they cause fluctuations and local defects, which result in morphological changes of the lipid vesicles. These changes are proposed to affect the permeability of vesicle membrane, triggering the release of the cargo encapsulated in the lipid vesicles (LVs). Our results confirm that pH-triggered release does not require major morphological changes, but can result from small defects affecting the lipid membrane permeability.

Tear of lipid membranes by nanoparticles.

Health concerns associated with the advent of nanotechnologies have risen sharply when it was found that particles of nanoscopic dimensions reach the cell lumina. Plasma and organelle lipid membranes, which are exposed to both the incoming and the engulfed nanoparticles, are the primary targets of possible disruptions. However, reported adhesion, invagination and embedment of nanoparticles (NPs) do not compromise the membrane integrity, precluding direct bilayer damage as a mechanism for toxicity. Here it is shown that a lipid membrane can be torn by small enough nanoparticles, thus unveiling mechanisms for how lipid membrane can be compromised by tearing from nanoparticles. Surprisingly, visualization by cryo transmission electron microscopy (cryo-TEM) of liposomes exposed to nanoparticles revealed also that liposomal laceration is prevented by particle abundance. Membrane destruction results thus from a subtle particle-membrane interplay that is here elucidated. This brings into a firmer molecular basis the theorized mechanisms of nanoparticle effects on lipid bilayers and paves the way for a better assessment of nanoparticle toxicity.

Hybrid polymer/lipid vesicles: Influence of polymer architecture and molar mass on line tension.

Hybrid polymer/lipid vesicles are self-assembled structures that have been the subject of an increasing number of studies in recent years. They are particularly promising tools in the development of cell membrane models because they offer the possibility to fine-tune their membrane structure by adjusting the distribution of components (presence or absence of "raft-like" lipid domains), which is of prime importance to control their membrane properties. Line tension in multiphase membranes is known to be a key parameter on membrane structuration, but remains unexplored, either experimentally or by computer modeling for hybrid polymer/lipid vesicles. In this study, we were able to measure the line tension on different budded hybrid vesicles, using a micropipette aspiration technique, and show the influence of the molar mass and the architecture of block copolymers on line tension and its consequences for membrane structuration.

Large hybrid Polymer/Lipid Unilamellar vesicle (LHUV) at the nanoscale: An insight into the lipid distribution in the membrane and permeability control.

Membrane structuration of Large Hybrid Unilamellar Polymer/Lipid Vesicle (LHUV) is an important parameter on the optimization of their properties and thus their valuation in various fields. However, this kind of information is hardly accessible. In this work, we will focus on the development of LHUV obtained from the self-assembly of diblock poly(dimethylsiloxane)-b-poly(ethylene oxide) (PDMS-b-PEO) of different molar masses combined with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 15% and 25% w/w content. The hybrid character of the resulting vesicles as well as their membrane structure are characterized by the mean of different techniques such as small-angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM). We show that hybrid vesicles with homogeneous membrane structure are obtained whatever the molar mass of the block copolymer (from 2500 to 4000 g/mol), with of a small number of tubular structures observed with the higher molar mass. We also demonstrate that the permeability of the LHUV, evaluated through controlled release experiments of fluorescein loaded in LHUV, is essentially controlled by the lipid/polymer composition.

Membrane reinforcement in giant hybrid polymer lipid vesicles achieved by controlling the polymer architecture.

The physical properties of membranes of hybrid polymer lipid vesicles are so far relatively unknown. Since their discovery a decade ago, many studies have aimed to show their great potential in many fields of application, but so far, few systematic studies have been carried out to decipher the relationship between the molecular characteristics of the components (molar mass, chemical nature, and architecture of the copolymer), the membrane structure and its properties. In this work, we study the association of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and poly(dimethylsiloxane)-b-poly(ethylene oxide) (PDMS-b-PEO) diblock copolymers of different molar masses in giant hybrid vesicles and establish a complete phase diagram of the membrane structure. We also measured the mechanical properties of the giant hybrid unilamellar vesicle (GHUV) through micropipette aspiration at different lipid/polymer compositions. Thanks to a previous work using triblock PEO-b-PDMS-b-PEO copolymers, we were able to reveal the effect of the architecture of the block copolymer on membrane structure and properties. Besides, the association of diblock copolymers PDMS-b-PEO and POPC leads to the formation of hybrid vesicles with unprecedented membrane toughness.

Switchable Lipid Provides pH-Sensitive Properties to Lipid and Hybrid Polymer/Lipid Membranes.

Blending amphiphilic copolymers and lipids constitutes a novel approach to combine the advantages of polymersomes and liposomes into a new single hybrid membrane. Efforts have been made to design stimuli-responsive vesicles, in which the membrane's dynamic is modulated by specific triggers. In this investigation, we proposed the design of pH-responsive hybrid vesicles formulated with poly(dimethylsiloxane)-block-poly(ethylene oxide) backbone (PDMS36-b-PEO23) and cationic switchable lipid (CSL). The latter undergoes a pH-triggered conformational change and induces membrane destabilization. Using confocal imaging and DLS measurements, we interrogated the structural changes in CSL-doped lipid and hybrid polymer/lipid unilamellar vesicles at the micro- and nanometric scale, respectively. Both switchable giant unilamellar lipid vesicles (GUV) and hybrid polymer/lipid unilamellar vesicles (GHUV) presented dynamic morphological changes, including protrusions and fission upon acidification. At the submicron scale, scattered intensity decreased for both switchable large unilamellar vesicles (LUV) and hybrid vesicles (LHUV) under acidic pH. Finally, monitoring the fluorescence leakage of encapsulated calcein, we attested that CSL increased the permeability of GUV and GHUV in a pH-specific fashion. Altogether, these results show that switchable lipids provide a pH-sensitive behavior to hybrid polymer/lipid vesicles that could be exploited for the triggered release of drugs, cell biomimicry studies, or as bioinspired micro/nanoreactors.

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

Large and Giant Unilamellar Vesicle(s) Obtained by Self-Assembly of Poly(dimethylsiloxane)-b-poly(ethylene oxide) Diblock Copolymers, Membrane Properties and Preliminary Investigation of their Ability to Form Hybrid Polymer/Lipid Vesicles.

In the emerging field of hybrid polymer/lipid vesicles, relatively few copolymers have been evaluated regarding their ability to form these structures and the resulting membrane properties have been scarcely studied. Here, we present the synthesis and self-assembly in solution of poly(dimethylsiloxane)-block-poly(ethylene oxide) diblock copolymers (PDMS-b-PEO). A library of different PDMS-b-PEO diblock copolymers was synthesized using ring-opening polymerization of hexamethylcyclotrisiloxane (D3) and further coupling with PEO chains via click chemistry. Self-assembly of the copolymers in water was studied using Dynamic Light Scattering (DLS), Static Light Scattering (SLS), Small Angle Neutron Scattering (SANS), and Cryo-Transmission Electron Microscopy (Cryo-TEM). Giant polymersomes obtained by electroformation present high toughness compared to those obtained from triblock copolymer in previous studies, for similar membrane thickness. Interestingly, these copolymers can be associated to phospholipids to form Giant Hybrid Unilamellar Vesicles (GHUV); preliminary investigations of their mechanical properties show that tough hybrid vesicles can be obtained.

Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics.

Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) and outer monolayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 µm2 s-1 at 25 °C and D = 2.3 ± 0.7 µm2 s-1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

A new composite hydrogel combining the biological properties of collagen with the mechanical properties of a supramolecular scaffold for bone tissue engineering.

Tissue engineering is a promising alternative to autografts, allografts, or biomaterials to address the treatment of severe and large bone lesions. Classically, tissue engineering products associate a scaffold and cells and are implanted or injected into the lesion. These cells must be embedded in an appropriate biocompatible scaffold, which offers a favourable environment for their survival and differentiation. Here, we designed a composite hydrogel composed of collagen I, an extracellular matrix protein widely used in several therapeutic applications, which we associated with a physical hydrogel generated from a synthetic small amphiphilic molecule. This composite showed improved mechanical and biological characteristics as compared with gels obtained from each separate compound. Incorporation of the physical hydrogel prevented shrinkage of collagen and cell diffusion out of the gel and yielded a gel with a higher elastic modulus than those of gels obtained with each component alone. The composite hydrogel allowed cell adhesion and proliferation in vitro and long-term cell survival in vivo. Moreover, it promoted the differentiation of human adipose-derived stem cells in the absence of any osteogenic factors. In vivo, cells embedded in the composite gel and injected subcutaneously in immunodeficient mice produced lamellar osteoid tissue and differentiated into osteoblasts. This study points this new composite hydrogel as a promising scaffold for bone tissue engineering applications.

Modulation of phase separation at the micron scale and nanoscale in giant polymer/lipid hybrid unilamellar vesicles (GHUVs).

Phase separation in giant polymer/lipid hybrid unilamellar vesicles (GHUVs) has been described over the last few years. However there is still a lack of understanding on the physical and molecular factors governing the phase separation in such systems. Among these parameters it has been suggested that in analogy to multicomponent lipid vesicles hydrophobic mismatches as well as lipid fluidity play a role. In this work, we aim to map a global picture of phase separation and domain formation in the membrane of GHUVs by using various copolymers based on poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) (PEO) with different architectures (grafted, triblock) and molar masses, combined with phospholipids in the fluid (POPC) or gel state (DPPC) at room temperature. From confocal imaging and fluorescence lifetime imaging microscopy (FLIM) techniques, the phase separation into either micro- or nano-domains within GHUVs was studied. In particular, our systematic studies demonstrate that in addition to the lipid/polymer fraction or the lipid physical state, important factors such as line tension at lipid polymer/lipid boundaries can be finely modulated by the molar mass and the architecture of the copolymer and lead to the formation of stable lipid domains with different sizes and morphologies in such GHUVs.

Polymersome shape transformation at the nanoscale.

Polymer vesicles, also named polymersomes, are valuable candidates for drug delivery and micro- or nanoreactor applications. As far as drug delivery is concerned, the shape of the carrier is believed to have a strong influence on the biodistribution and cell internalization. Polymersomes can be submitted to an osmotic imbalance when injected in physiological media leading to morphological changes. To understand these osmotic stress-induced variations in membrane properties and shapes, several nanovesicles made of the graft polymer poly(dimethylsiloxane)-g-poly(ethylene oxide) (PDMS-g-PEO) or the triblock copolymer PEO-b-PDMS-b-PEO were osmotically stressed and observed by light scattering, neutron scattering (SANS), and cryo-transmission electron microscopy (cryo-TEM). Hypotonic shock leads to a swelling of the vesicles, comparable to optically observable giant polymersomes, and hypertonic shock leads to collapsed structures such as stomatocytes and original nested vesicles, the latter being only observed for bilayers classically formed by amphiphilic copolymers. Complementary SANS and cryo-TEM experiments are shown to be in quantitative agreement and highlight the importance of the membrane structure on the behavior of these nanopolymersomes under hypertonic conditions as the final morphology reached depends whether or not the copolymers assemble into a bilayer. The vesicle radius and membrane curvature are also shown to be critical parameters for such transformations: the shape evolution trajectory agrees with theoretical models only for large enough vesicle radii above a threshold value around 4 times the membrane thickness.

A thermosensitive low molecular weight hydrogel as scaffold for tissue engineering.

Hydrogels that are non-toxic, easy to use, cytocompatible, injectable and degradable are valuable biomaterials for tissue engineering and tissue repair. However, few compounds currently fulfil these requirements. In this study, we describe the biological properties of a new type of thermosensitive hydrogel based on low-molecular weight glycosyl-nucleosyl-fluorinated (GNF) compound. This gel forms within 25 min by self-assembly of monomers as temperature decreases. It degrades slowly in vitro and in vivo. It induces moderate chronic inflammation and is progressively invaded by host cells and vessels, suggesting good integration to the host environment. Although human adult mesenchymal stem cells derived from adipose tissue (ASC) cannot adhere on the gel surface or within a 3D gel scaffold, cell aggregates grow and differentiate normally when entrapped in the GNF-based gel. Moreover, this hydrogel stimulates osteoblast differentiation of ASC in the absence of osteogenic factors. When implanted in mice, gel-entrapped cell aggregates survive for several weeks in contrast with gel-free spheroids. They are maintained in their original site of implantation where they interact with the host tissue and adhere on the extracellular matrix. They can differentiate in situ into alkaline phosphatase positive osteoblasts, which deposit a calcium phosphate-rich matrix. When injected into subcutaneous sites, gel-encapsulated cells show similar biological properties as implanted gel-cells complexes. These data point GNF-based gels as a novel class of hydrogels with original properties, in particular osteogenic potential, susceptible of providing new therapeutic solutions especially for bone tissue engineering applications.

The in vivo behavior and antitumor activity of doxorubicin-loaded poly(γ-benzyl l-glutamate)-block-hyaluronan polymersomes in Ehrlich ascites tumor-bearing BalB/c mice.

The in vivo efficacy of doxorubicin (DOX)-loaded poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG(23)-b-HYA(10))-based polymersomes (PolyDOX) was evaluated. Samples were efficiently labeled with technetium-99m radionuclide with good stability for in vivo studies. PolyDOX enhanced circulation time compared to free DOX. Biodistribution studies revealed selective accumulation of PolyDOX in the Ehrlich ascites tumor (EAT) as a result of passive accumulation and active targeting (CD44-mediated endocytosis) in EAT-bearing mice. Toxicity studies demonstrated PolyDOX is a safe drug carrier, and no hemolysis was observed with PolyDOX equivalent to 200 µg/mL of free DOX. PolyDOX dominantly controlled tumor growth by delaying doubling time of EATs compared to free DOX over 30 days after treatment. PolyDOX also increased life span six times more than free DOX. Hence, it is reasonable to expect that higher DOX levels attributable to PolyDOX improve the therapeutic index and reduce side effects due to site-specific drug accumulation. FROM THE CLINICAL EDITOR: In this preclinical project, doxorubicin loaded polymersomes enhanced intracellular uptake of doxorubicin in a murine model of Ehrlich Ascites Tumor (EAT) through CD44 receptor mediated endocytosis, resulting in prolonged Tumor Doubling Time and increase in life span of mice.

Nanogels based on poly(vinyl acetate) for the preparation of patterned porous films.

The use of poly(vinyl acetate) (PVAc) nanogels for the fabrication of patterned porous surfaces is described. These nanogels were synthesized by controlled radical cross-linking copolymerization (CRCC) involving a xanthate-mediated reversible addition-fragmentation chain transfer (RAFT) mechanism. This synthesis methodology allowed for the preparation of nanogels based on PVAc with a controlled constitutive chain length and average numbers of chains and cross-links. Solutions of these branched polymers were prepared in THF with a fixed amount of water and spin coated onto a surface of graphite. The surface porosity of corresponding films was observed by atomic force microscopy (AFM). Compared with linear PVAc homologues with a degree of polymerization (DP) sufficiently high to favor the formation of porous structures (DP = 50), a sharper and better defined porosity was observed with nanogels, the constitutive chains of which had the same DP. For nanogels differing only in their cross-link density, the pores were smaller and better defined in the case of the higher cross-link density, suggesting an enhanced stabilization of the water droplets during film formation. To explain these observations, it is postulated that PVAc nanogels can behave as compact particles providing steric stabilization of water droplets, which is referred to as a Pickering effect. The coalescence of water droplets would be better prevented as the cross-link density of the nanogels increases, resulting in a smaller size pore.

Block copolymer vesicle permeability measured by osmotic swelling and shrinking.

Vesicle response to osmotic shock provides insight into membrane permeability, a highly relevant value for applications ranging from nanoreactor experimentation to drug delivery. The osmotic shock approach has been employed extensively to elucidate the properties of phospholipid vesicles (liposomes) and of varieties of polymer vesicles (polymersomes). This study seeks to compare the membrane response for two varieties of polymersomes, a comb-type siloxane surfactant, poly(dimethylsiloxane)-g-poly(ethylene oxide) (PDMS-g-PEO), and a diblock copolymer, polybutadiene-b-poly(ethylene oxide) (PBut-b-PEO). Despite similar molecular weights and the same hydrophilic block (PEO), the two copolymers possess different hydrophobic blocks (PBut and PDMS) and corresponding glass transition temperatures (-31 and -123 °C, respectively). Dramatic variations in membrane response are observed during exposure to osmotic pressure differences, and values for polymer membrane permeability to water are extracted. We propose an explanation for the observed phenomena based on the respective properties of the PBut-b-PEO and PDMS-g-PEO membranes in terms of cohesion, thickness, and fluidity.

Mastering a double emulsion in a simple co-flow microfluidic to generate complex polymersomes.

We show that the production and the geometrical shape of complex polymersomes can be predicted by varying the flow rates of a simple microdevice using an empirical law which predicts the droplet size. This device is constituted of fused silica capillaries associated with adjusted tubing sleeves and T-junctions. Studying the effect of several experimental parameters, double emulsions containing a controlled number of droplets were fabricated. First, this study examines the stability of a jet in a simple confined microfluidic system, probing the conditions required for droplets production. Then, multicompartmental polymersomes were formed, controlling flow velocities. In this work, poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO) and poly(butadiene)-block-poly(ethyleneoxide) (PBut-b-PEO) amphiphilic copolymers were used and dissolved in chloroform/cyclohexane mixture. The ratio of these two solvents was adjusted in order to stabilize the double emulsion formation. The aqueous suspension contained poly(vinyl alcohol) (PVA), limiting the coalescence of the droplets. This work constitutes major progress in the control of double emulsion formation in microfluidic devices and shows that complex structures can be obtained using such a process.

A simple method to achieve high doxorubicin loading in biodegradable polymersomes.

Doxorubicin (Dox), an anthracycline anticancer drug, was successfully incorporated into block copolymer vesicles of poly(trimethylene carbonate)-b-poly(L-glutamic acid) (PTMC-b-PGA) by a solvent-displacement (nanoprecipitation) method. pH conditions were shown to have a strong influence on loading capacity and release profiles. Substantial drug loading (47% w/w) was achieved at pH 10.5. After pH neutralization, aqueous dispersions of drug-loaded vesicles were found stable for a prolonged period of time (at least 6months) without vesicle disruption or drug precipitation. Dox-loaded vesicles exhibited in vitro pH and temperature-dependent drug release profiles: release kinetics fastened in acid conditions or by increasing temperature. These features strongly support the interest of developing PTMC-b-PGA polymersomes as carriers for the controlled delivery of Dox.