Rolling and ageing in T-ramp soft adhesion

Immediately before adsorption to a horizontal substrate, sinking polymer-coated colloids can undergo a complex sequence of landing, jumping, crawling and rolling events. Using video tracking we studied the soft adhesion to a horizontal flat plate of micron-size colloids coated by a controlled molar fraction $f$ of the polymer PLL-g-PNIPAM which is temperature sensitive. We ramp the temperature from below to above $T_c=32\pm 1^{\circ}$C, at which the PNIPAM polymer undergoes a transition triggering attractive interaction between microparticles and surface. The adsorption rate, the effective in-plane ($x-y$) diffusion constant and the average residence time distribution over $z$ were extracted from the Brownian motion records during last seconds before immobilisation. Experimental data are understood within a rate-equations based model that includes ageing effects and includes three populations: the untethered, the rolling and the arrested colloids. We show that pre-adsorption dynamics casts analyze a characteristic scaling function $\alpha (f)$ proportional to the number of available PNIPAM patches met by soft contact during Brownian rolling. In particular, the increase of in-plane diffusivity with increasing $f$ is understood: the stickiest particles have the shortest rolling regime prior to arrest, so that their motion is dominated by untethered phase

Importance of the dynamics of adsorption and of a transient interfacial stress on the formation of aggregates of IgG antibodies

International audienceIt is common knowledge that aggregation of proteins may occur in aqueous solutions under mechanical stress (shaking or high shear), even in solutions that are stable at rest. Addition of surfactants is a practical generic means to prevent this stress-induced aggregation (e.g. in formulations of therapeutic proteins), which suggests that interfaces contribute to destabilization. We studied here the role of interfacial stress by applying brief mechanical impacts on the air-water interface, in the presence or absence of surfactants, in solutions of immunoglobulin G (IgG), a class of proteins of high importance to the developments of new therapeutics. A variety of surfactants was tested including the neutral ones Tween80, C10-C14 fos-cholines, alkylaminoxide, surfactin, and two ionic ones, TTAB and lauroylsarcosine sodium salt. We determined the presence of aggregates in solution by light scattering. Irrespective of the type of antibody, either human polyclonal or a monoclonal one, we show that the amount of aggregated IgG increases in proportion to the number of impacts on the interface. In the absence of stress, we recorded images of oblate aggregates of IgG (ca. 12 nm height and 200-1200 nm diameter) present at the air-water interface (fluorescence microscopy using anti-Fab or anti-Fc markers, and AFM scans after transfer on freshly cleaved mica). Our results evidence that aggregates are formed at the air-water interface, and are brought in solution by transient stresses applied on the water surface. Rupture of interfacial films is an important source of aggregates in solution. Finally, the role of surface dynamics in the protection brought by surfactants is discussed based on the comparison of protective efficiencies with dynamic surface tension properties (measured by the maximum bubble pressure method). Our work indicates that better protection is conferred by surfactants showing the faster interfacial dynamics, which corresponds also to conditions of faster lowering of the interfacial energy at a short time scale

Amphiphilic Macromolecules on Cell Membranes: From Protective Layers to Controlled Permeabilization.

International audienceAntimicrobial and cell-penetrating peptides have inspired developments of abiotic membrane-active polymers that can coat, penetrate, or break lipid bilayers in model systems. Application to cell cultures is more recent, but remarkable bioactivities are already reported. Synthetic polymer chains were tailored to achieve (i) high biocide efficiencies, and selectivity for bacteria (Gram-positive/Gram-negative or bacterial/mammalian membranes), (ii) stable and mild encapsulation of viable isolated cells to escape immune systems, (iii) pH-, temperature-, or light-triggered interaction with cells. This review illustrates these recent achievements highlighting the use of abiotic polymers, and compares the major structural determinants that control efficiency of polymers and peptides. Charge density, sp. of cationic and guanidinium side groups, and hydrophobicity (including polarity of stimuli-responsive moieties) guide the design of new copolymers for the handling of cell membranes. While polycationic chains are generally used as biocidal or hemolytic agents, anionic amphiphilic polymers, including Amphipols, are particularly prone to mild permeabilization and/or intracell delivery

Aggregation of antibody drug conjugates at room temperature: SAXS and light scattering evidence for colloidal instability of a specific subpopulation

Coupling an hydrophobic drug onto monoclonal antibodies via Lysine residues is a common route to prepare antibody-drug conjugates (ADC), a promising class of biotherapeutics. But a few chemical modifications on protein surface often increases aggregation propensity, without clear understanding of the aggregation mechanisms at stake (loss of colloidal stability, self- assemblies, denaturation...), and the statistical nature of conjugation introduces polydispersity in the ADC population, which raises questions on whether the whole ADC population becomes unstable. To characterize the average interactions between ADC, we monitored small angle X-ray scattering in solutions of monoclonal IgG1 human antibody drug conjugate, with average degree of conjugation of 0, 2, or 3 drug molecules per protein. To characterize stability, we studied kinetics of aggregation at room temperature. Intrinsic Fuchs stability ratio of the ADC was estimated from the variation over time of scattered light intensity and hydrodynamic radius, in buffers of varying pH, and at diverse sucrose (0% or 10%) and NaCl (0 or 100 mM) concentrations. We show that stable ADC stock solutions became unstable upon pH shift, well below the pH of maximum average attraction between IgGs. Data indicates that aggregation can be ascribed to a fraction of ADC population usually representing less than 30 mol% of the sample. In contrast to the case of (monodisperse) monoclonal antibodies, our results suggest that a poor correlation between stability and average interaction parameters should be expected as a corollary of dispersity of ADC conjugation. In practice, the most unstable fraction of the ADC population can be removed by filtrations, which affects remarkably the apparent stability of the samples. Finally, the lack of correlation between the kinetic stability and variations of the average inter-ADC interactions is tentatively attributed to the uneven nature of charge distributions and the presence of patches on the drug-modified antibodies.This work was supported by the French National Research Agency (program Blanc International, grant ANR 2010-INT 1501, and program Investissement d’Avenir ANR-11- LABX-0011-01, and by SANOFI research grant to BFP. Authors are grateful to Javier Perez and Aurélien Thureau for their help and advice in SAXS measurements at SOLEIL. We thank Sophie Norvez from MMC laboratory in ESPCI for her help with circular dichroism.This is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/acs.langmuir.6b0065

Colloidal stability of tannins: astringency, wine tasting and beyond

Tannin-tannin and tannin-protein interactions in water-ethanol solvent mixtures are studied in the context of red wine tasting. While tannin self-aggregation is relevant for visual aspect of wine tasting (limpidity and related colloidal phenomena), tannin affinities for salivary proline-rich proteins is fundamental for a wide spectrum of organoleptic properties related to astringency. Tannin-tannin interactions are analyzed in water-ethanol wine-like solvents and the precipitation map is constructed for a typical grape tannin. The interaction between tannins and human salivary proline-rich proteins (PRP) are investigated in the framework of the shell model for micellization, known for describing tannin-induced aggregation of beta-casein. Tannin-assisted micellization and compaction of proteins observed by SAXS are described quantitatively and discussed in the case of astringency

Propuesta de diseño de un sistema fotovoltaico para la óptima extracción y bombeo de agua para riego agrícola, en el C. P. Aguas Calientes, 2021

Se propuso un diseño de un sistema fotovoltaico para óptima extracción y bombeo de agua para riego agrícola en el Centro Poblado Aguas Calientes cuyo propósito es aprovechar el recurso hídrico y que los terrenos sean productivos. Para un área de terreno de 0.158m2; se requiere 75 puntos emisores de riego por goteo para las plantaciones de palta cuyo caudal por emisor será de 4 l/h, regadas 3 h/día, necesitando 2500 litros de agua al día. Para satisfacer la demanda de agua se diseñó la línea de impulsión con una altura real de 37.28m, con pérdidas de energía de 26.86m.c.a., según el Qmd= 0.083 l/s, y el Qb=0.202 l/s el agua será bombeada con una Electrobomba Superficie Horizontal Pedrollo 1HP – PKm90 a una altura máxima de 62.47 m. Como resultado final la potencia requerida de la bomba es 750 Watts/h y el diseño del sistema fotovoltaico propuesto beneficiará a la población y podrá aprovechar sus terrenos para el cultivo agrícola el cual consta de 3 panel solar de 270W de potencia con 1 controlador de voltaje de 29.51Amp. Se concluyó que el sistema si es factible económicamente con VAN= 13401.177, TIR= y B/c= 21.493; con un monto de inversión de S/.21509.34 nuevos soles

Aggregation of Antibody Drug Conjugates at Room Temperature: SAXS and Light Scattering Evidence for Colloidal Instability of a Specific Subpopulation

Coupling a hydrophobic drug onto monoclonal antibodies via lysine residues is a common route to prepare antibody–drug conjugates (ADC), a promising class of biotherapeutics. But a few chemical modifications on protein surface often increase aggregation propensity, without a clear understanding of the aggregation mechanisms at stake (loss of colloidal stability, self-assemblies, denaturation, etc.), and the statistical nature of conjugation introduces polydispersity in the ADC population, which raises questions on whether the whole ADC population becomes unstable. To characterize the average interactions between ADC, we monitored small-angle X-ray scattering in solutions of monoclonal IgG1 human antibody drug conjugate, with average degree of conjugation of 0, 2, or 3 drug molecules per protein. To characterize stability, we studied the kinetics of aggregation at room temperature. The intrinsic Fuchs stability ratio of the ADC was estimated from the variation over time of scattered light intensity and hydrodynamic radius, in buffers of varying pH, and at diverse sucrose (0% or 10%) and NaCl (0 or 100 mM) concentrations. We show that stable ADC stock solutions became unstable upon pH shift, well below the pH of maximum average attraction between IgGs. Data indicate that aggregation can be ascribed to a fraction of ADC population usually representing less than 30 mol % of the sample. In contrast to the case of (monodisperse) monoclonal antibodies, our results suggest that a poor correlation between stability and average interaction parameters should be expected as a corollary of dispersity of ADC conjugation. In practice, the most unstable fraction of the ADC population can be removed by filtration, which affects remarkably the apparent stability of the samples. Finally, the lack of correlation between the kinetic stability and variations of the average inter-ADC interactions is tentatively attributed to the uneven nature of charge distributions and the presence of patches on the drug-modified antibodies

Bacteria‐Based Production of Thiol‐Clickable, Genetically Encoded Lipid Nanovesicles

International audienceDespite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Here we present a microbial platform for high yield production of lipidic nanovesicles, with clickable thiol moieties in their outer corona. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented and customizable corona of transmembrane polypeptides. In addition, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients or detergents) and their straightforward surface functionalization taking advantage of the diversity of commercially-available maleimide derivatives, engineering bacterial-based proteoliposomes are an attractive eco-friendly alternative that can outperform current liposome preparation methods

Tannin-controlled micelles and fibrils of κ\kappa-casein

Effects of green tea tannin epigallocatechin-gallate (EGCG) on thermal-stress-induced amyloid fibril formation of reduced carboxymethylated bovine milk protein $\kappa$-casein (RCMK) were studied by dynamical light scattering (DLS) and small angle x-rays scattering (SAXS). Two populations of aggregates, micelles and fibrils, dominated the time evolution of light scattering intensity and of effective hydrodynamic diameter. SAXS experiments allowed to resolve micelles and fibrils so that the time dependence of scattering profile revealed structural evolution of the two populations. The low-Q scattering intensity prior to an expected increase with time due to fibril growth, shows an intriguing rapid decrease which is interpreted as the release of monomers from micelles. This phenomenon, observed both in the absence and in the presence of EGCG, indicates that under thermal stress free native monomers are converted to amyloid-prone monomers that do not form micelles. The consumption of free native monomers results in a release of native monomers from micelles, because only native protein participate in micelle-monomer (quasi-)equilibrium. This release is reversible, indicating also that native-to-amyloid-prone monomers conversion is reversible as well. We show that EGCG does not bind to protein in fibrils, neither does it affect/prevent the pro-amyloid conversion of monomers. EGCG hinders the addition of monomers to growing fibrils. These facts allowed us to propose kinetics model for EGCG-controlled amyloid aggregation of micellar proteins. Therein, we introduced the growth-rate inhibition function which quantitatively accounts for the effect of EGCG on the fibril growth at any degree of thermal stress

Cationic poly(amidoamine) promotes cytosolic delivery of bovine RNase A in melanoma cells, while maintaining its cellular toxicity

Ribonucleases are known to cleave ribonucleic acids, inducing cell death. RNase A, a member of the ribonuclease family, generally displayed poor in vitro activity. This has been attributed to factors such as low intracellular delivery. Poly(amidoamine)s have been used to promote the translocation of non-permeant proteins to the cytosol. Our objective was to demonstrate that poly(amidoamine)s could potentially promote the delivery of RNase A to selected cell line. Interactions of three cationic poly(amidoamine)s (P1, P2 and ISA1) with wild-type bovine RNase A were investigated using gel retardation assays, DLS and microcalorimetry. Although the polymers and the protein are essentially cationic at physiological pH, complexation between the PAAs and RNase A was observed. The high sensitivity differential scanning calorimetry (HSDSC) thermograms demonstrated that the thermal stability of the protein was reduced when complexed with ISA1 (Tmax decreased by 6.5 °C) but was not affected by P1 and P2. All the polymers displayed low cytotoxicity towards non-cancerous cells (IC50 > 3.5 mg mL?1). While RNase A alone was not toxic to mouse melanoma cells (B16F1), P1 was able to promote cytosolic delivery of biologically active RNase A, increasing cell death (IC50 = 0.09 mg mL?1)