Poly(glutamic acid)-Based Viscosity Reducers for Concentrated Formulations of a Monoclonal IgG Antibody.

Above a concentration threshold, the viscosity of solutions of proteins increases abruptly, which hampers the injectability of therapeutic formulations. Concentrations above 200 g/L are an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (e.g., arginine) help decrease the viscosity, but they are used at concentrations as high as about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic monoclonal antibody (mAb) solution by poly(d,l-glutamic acid) and poly(l-glutamic acid) derivatives added at concentrations of <6.5 g/L (i.e., a mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethylene glycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be decreased to ∼20 mPa s as compared to values of ∼100 mPa s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1 g/L mAb) and by observation of phase separation at higher concentrations, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether, these results show that polyglutamate derivatives hold an untapped potential as an excipient for fluidification of concentrated protein solutions.

Quantitative Microscale Thermometry in Droplets Loaded with Gold Nanoparticles.

Gold nanoparticles (AuNPs) are increasingly used for their thermoplasmonic properties, i.e., their ability to convert light energy into heat through plasmon resonance. However, measuring temperature gradients generated at the microscale by assemblies of AuNPs remains challenging, especially for random 3D distributions of AuNPs. Here, we introduce a label-free thermometry approach, combining quantitative wavefront microscopy and numerical simulations, to infer the heating power dissipated by a 3D model system consisting of emulsion microdroplets loaded with AuNPs. This approach gives access to the temperature reached in the droplets under laser irradiation without the need for extrinsic calibration. This versatile thermometry method is promising for noninvasive temperature measurements in various 3D microsystems involving AuNPs as colloidal heat sources, including photothermal drug delivery systems.

Micelle kinetics of photoswitchable surfactants: Self-assembly pathways and relaxation mechanisms.

HYPOTHESIS: A key question in the kinetics of surfactant self-assembly is whether exchange of unimers or fusion/fission of entire micelles is the dominant pathway. In this study, an isomerizable surfactant is used to explore fundamental out-of-equilibrium kinetics and mechanisms for growth and dissolution of micelles. EXPERIMENTS: The kinetics of cationic surfactant 4-butyl-4'-(3-trimethylammoniumpropoxy)-phenylazobenzene was studied using molecular dynamics simulations. The fusion and exchange processes were investigated using umbrella sampling. Equilibrium states were validated by comparison with small-angle X-ray scattering data. The photo-isomerization event was simulated by modifying the torsion potential of the photo-responsive group to emulate the trans-to-cis transition. FINDINGS: Micelle growth is dominated by unimer exchange processes, whereas, depending on the conditions, dissolution can occur both through fission and unimer expulsion. Fusion barriers increase steeply with the aggregation number making this an unlikely pathway to equilibrium for micelles of sizes that fit with the experimental data. The barriers for unimer expulsion remain constant and are much lower for unimer insertion, making exchange more likely at high aggregation. When simulating photo-conversion events, both fission and a large degree of unimer expulsion can occur depending on the extent of the out-of-equilibrium stress that is put on the system.

Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids.

One of the biggest challenges in membrane protein (MP) research is to secure physiologically relevant structural and functional information after extracting MPs from their native membrane. Amphipathic polymers represent attractive alternatives to detergents for stabilizing MPs in aqueous solutions. The predominant polymers used in MP biochemistry and biophysics are amphipols (APols), one class of which, styrene maleic acid (SMA) copolymers and their derivatives, has proven particularly efficient at MP extraction. In order to examine the relationship between the chemical structure of the polymers and their ability to extract MPs from membranes, we have developed two novel classes of APols bearing either cycloalkane or aryl (aromatic) rings, named CyclAPols and ArylAPols, respectively. The effect on solubilization of such parameters as the density of hydrophobic groups, the number of carbon atoms and their arrangement in the hydrophobic moieties, as well as the charge density of the polymers was evaluated. The membrane-solubilizing efficiency of the SMAs, CyclAPols, and ArylAPols was compared using as models (i) two MPs, BmrA and a GFP-fused version of LacY, overexpressed in the inner membrane of Escherichia coli, and (ii) bacteriorhodopsin, naturally expressed in the purple membrane of Halobacterium salinarum. This analysis shows that, as compared to SMAs, the novel APols feature an improved efficiency at extracting MPs while preserving native protein-lipid interactions.

UCST-Type Polymer Capsules Formed by Interfacial Complexation.

Formation of aqueous-core polymer capsules exhibiting an upper critical solution temperature (UCST) was achieved using surfactant-polymer interfacial complexation in water-in-oil inverse emulsions. In fluorinated oil, Coulombic interactions between Krytox, an anionic oil-soluble surfactant, and a cationic poly(lysine) grafted with poly(acrylamide-co-acrylonitrile) enabled the formation of an adsorbed polymer shell at the surface of water droplets. The thermoresponsiveness of the polymer shell was assessed by fluorescence microscopy with and without the presence of nanoparticles, including gold particles. We show that, above the cloud point, polymers with a balanced fraction of UCST grafts form flat adlayers that (i) spontaneously entrap nanoparticles upon cooling and (ii) switch from fluid-like dynamics at high temperature to solid-like dynamics below the cloud point. This system offers a straightforward mean to prepare temperature-sensitive capsules in mild, biocompatible conditions and to concentrate nanoparticles (including nanoheaters) in their shell.

Inducible intracellular membranes: molecular aspects and emerging applications.

Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering an abnormal accumulation of membrane structures inside the cells (or membrane proliferation) share two major common features: (1) they promote the formation of highly curved membrane domains and (2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, and inspire new applications, either for academia (high-yield membrane protein production and nanovesicle production) or industry (biofuel production and vaccine preparation).

Solubilization and Stabilization of Membrane Proteins by Cycloalkane-Modified Amphiphilic Polymers.

Membrane proteins (MPs) need to be extracted from biological membranes and purified in their native state for most structural and functional in vitro investigations. Amphiphilic copolymers, such as amphipols (APols), have emerged as very useful alternatives to detergents for keeping MPs water-soluble under their native form. However, classical APols, such as poly(acrylic acid) (PAA) derivatives, seldom enable direct MP extraction. Poly(styrene maleic anhydride) copolymers (SMAs), which bear aromatic rings as hydrophobic side groups, have been reported to be more effective extracting agents. In order to test the hypothesis of the role of cyclic hydrophobic moieties in membrane solubilization by copolymers, we have prepared PAA derivatives comprising cyclic rather than linear aliphatic side groups (CyclAPols). As references, APol A8-35, SMAs, and diisobutylene maleic acid (DIBMA) were compared with CyclAPols. Using as models the plasma membrane of Escherichia coli and the extraction-resistant purple membrane from Halobacterium salinarum, we show that CyclAPols combine the extraction efficiency of SMAs with the stabilization afforded to MPs by classical APols such as A8-35.

Distinctive Low-Resolution Structural Features of Dimers of Antibody-Drug Conjugates and Parent Antibody Determined by Small-Angle X-ray Scattering.

Structural features of lysine-conjugated antibody-drug conjugate (ADC) from humanized IgG1 were studied by small-angle X-ray scattering (SAXS). As the physicochemical properties of the cytotoxic drug (payload) and linker may impact the conformational and colloidal stabilities of the conjugated monoclonal antibody (mAb), it is essential to characterize how the conjugation may affect the overall higher order structure and therefore the physical stability and integrity of the ADCs upon storage conditions. Here, the ADC monomer and aggregates generated upon thermal stress were analyzed by high performance liquid chromatography coupled to SAXS with a particular focus on the fraction of dimers (3-10% depending on the storage conditions at 25 and 40 °C). In addition to average parameters such as radius of gyration, molecular weight, and maximal end-to-end distance, the structural information obtained from SAXS patterns were visualized as a low-resolution average envelope of both monomers and dimers (implementation of two methods: ab initio reconstruction and modeling Fab and Fc as rigid bodies with a flexible hinge). We showed that the monomer envelope of the ADC was similar to the corresponding (nonconjugated) parent monoclonal antibody (mAb). ADC dimers appeared more compact and less polydisperse than the dimers of mAb, which was also confirmed by atomic force microscopy. The generated envelopes of the mAb dimers suggest elongated structures with one or few inter-mAb contacts at the outermost region of Fab or Fc domains. The structural features of ADC dimers are independent of the tested pH buffering system (pH 5.0/acetate and pH 6.0/histidine with or without NaCl) and characterized by multiple, tighter contacts between the Fab and Fc domains and distortion of the monomer native shape. Results from the SAXS structural study show in the present case that conjugation has favored innermost inter-ADC contacts in the dimer, which differ from the inter-mAb ones. In general, it is likely that many parameters affect inter-ADC association, including the chemical nature of linkers and drugs, degree of conjugation, conjugation sites, etc. Making a qualitative difference between mAb and ADC dimers as a function of these parameters can help point to the presence of tight associations that must be abolished in protein drug formulations.

Bacteria-Based Production of Thiol-Clickable, Genetically Encoded Lipid Nanovesicles.

Despite 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. Herein, a microbial platform for high-yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, 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, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria-based proteoliposomes are an attractive eco-friendly alternative that can outperform currently used liposomes.

Redox-Triggered Control of Cell Adhesion and Deadhesion on Poly(lysine)-g-poly(ethylene oxide) Adlayers.

Spontaneous adsorption of poly(lysine)-g-poly(ethylene glycol) comb-like copolymers (PLL-g-PEG) is a versatile mean to coat substrates with polymer layers that resist cell adhesion. We prepared redox cleavable PLL-g-PEG to switch adhesion on demand. Redox sensitivity was obtained by introducing disulfide linkers between the PLL backbone and PEG strands. This modification was done alone or in combination with an azide end on the PEG strands that enabled in situ conjugations of adhesion peptides or fluorescent labels (by a simple application of commercially available molecules for copper-free click chemistry compatible with cell survival). To balance the functional (adhesion-promoting) vs cell-repellent copolymers, mixed layers of adjusted compositions were obtained by coadsorption from mixed solutions of the cleavable copolymer with noncleavable and repellant PLL-g-PEG. The deposition of copolymers and quantitative cleavage as triggered by reductive conditions (application of solutions of tris(carboxyethyl)phosphine, dithiothreitol, or glutathione) were characterized by QCM-D, XPS, and fluorescence microscopy. In cell culture conditions, redox-triggered cleavage was obtained by a nontoxic application of TCEP for a few minutes, enabling either to release cell attachment points (i.e., cleavage of RGD-presenting areas) or to "open" nonspecific adherent areas (i.e., transition from PEG-presenting areas to adherent PLL-like coatings).

Tannin-controlled micelles and fibrils of κ-casein.

Effects of green tea tannin epigallocatechin-gallate (EGCG) on thermal-stress-induced amyloid fibril formation of reduced carboxymethylated bovine milk protein κ-casein were studied by dynamical light scattering and small angle X-ray 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 us to resolve micelles and fibrils so that the time dependence of the scattering profile revealed the structural evolution of the two populations. The low-Q scattering intensity prior to an expected increase in 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 conditions, 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 proteins participate in micelle-monomer (quasi)equilibrium. This release is reversible, indicating also that native-to-amyloid-prone monomer conversion is reversible as well. We show that EGCG does not bind to protein in fibrils, neither does it affect/prevent the proamyloid conversion of monomers. EGCG hinders the addition of monomers to growing fibrils. These facts allowed us to propose the 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.

Rolling and aging in temperature-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 poly(lysine)-grafted-poly(N-isopropylacrylamide) (PLL-g-PNIPAM) which is a temperature-sensitive polymer. We ramp the temperature from below to above T_{c}=32±1^{∘}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 immobilization. Experimental data are understood within a rate-equations-based model that includes aging effects and includes three populations: the untethered, the rolling, and the arrested colloids. We show that preadsorption dynamics casts a characteristic scaling function α(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 the untethered phase.

Refolding of Aggregation-Prone ScFv Antibody Fragments Assisted by Hydrophobically Modified Poly(sodium acrylate) Derivatives.

ScFv antibody fragments are a promising alternative to full-length antibodies for both therapeutic and diagnosis applications. They can be overexpressed in bacteria, which enables easy large scale production. Since scFv are artificial constructs, they are poorly soluble and prone to aggregation, which makes them difficult to manipulate and to refold. Here, stabilization and refolding of scFv fragments from urea-unfolded solutions are reported based on the use of micromolar amounts of polymers playing the role of artificial chaperons. Using fluorescence correlation spectroscopy, the size and aggregation number of complexes of scFv with unmodified or hydrophobically modified poly(sodium acrylate) are determined. The evolution of the secondary structure along the refolding procedure, in the presence or absence of 0.4 m l-arginine at scFv:polymer < 1:5 (w/w), is determined by high-sensitivity synchrotron-radiation circular dichroism. Measurements reveal that refolding in the presence of polymers yields native-like secondary structure, though a different folding pathway can be followed compared to refolding in the absence of polymer. This is the first report on the use of macromolecular additives to assist refolding of a multidomain protein of therapeutic interest.

Kinetics of Photocontrollable Micelles: Light-Induced Self-Assembly and Disassembly of Azobenzene-Based Surfactants Revealed by TR-SAXS.

The kinetics of micelles involving photosensitive surfactants is still not well understood. In this work, we unravel the mechanistic pathways involved in the micelle formation and dissolution of photocontrollable micelles. We focus on the fast self-assembly processes of photosensitive cationic azobenzene-containing surfactants (AzoTMA) that display a change in hydrophobicity induced by a reversible cis-trans conformational transition upon exposure to light. By combining both in situ time-resolved small-angle X-ray scattering (SAXS) and light scattering, we characterized the detailed structure and phase behavior of AzoTMA in mixtures of water and dimethylformamide (DMF). Time-resolved synchrotron SAXS with monochromatic light as a trigger enabled us to observe the nonequilibrium formation and dissolution process of micelles (demicellization) directly on the nanoscale with a time resolution starting from milliseconds. The structural results show that in pure water UV-light illumination leads to a 12% reduction of the aggregation number of the micelles and more than a 50% increase in the critical micelle concentration (CMC). Close to the CMC, adjusted by the addition of DMF, UV light illumination leads to a complete dissolution of the micelles, while shining blue light reverses the process and leads to the reformation of micelles. The UV-triggered dissolution follows a two-step mechanism; the first and rapid (second time scale) release of unimers is followed by a slower decomposition of the micelles (over tens of seconds) as a result of an increase in temperature due to optical absorption. Similarly, the reverse process, i.e., micelle formation, occurs rapidly upon photoconversion to trans conformers under blue light, and micelles are disrupted at long exposure time due to the optical absorption and corresponding increase in temperature. Interestingly, the coexistence of unimers with regular micelles is found at all times, and no other transient assemblies could be detected by SAXS.

Tailored stimuli-responsive interaction between particles adjusted by straightforward adsorption of mixed layers of Poly(lysine)-g-PEG and Poly(lysine)-g-PNIPAM on anionic beads.

We report a simple and versatile method to functionalize anionic colloid particles and control particle solubility. Poly(lysine)-based copolymers (PLL) grafted with polyethylene oxide (PLL-g-PEG) or poly(N-isopropylacrylamide) (PLL-g-PNIPAM) spontaneously adsorb on bare beads dispersed in aqueous solutions of the copolymers. The final composition of the mixed ad-layers formed (i.e. PEG/PNIPAM ratio) was adjusted by the polymer concentrations in solutions. While the (PLL-g-PEG)-coated particles were stable in a wide range of temperature, the presence of PLL-g-PNIPAM in the outer layer provided a reversible temperature-triggered aggregation at 32±1 °C. In the range of PNIPAM fraction going from 100% (beads fully covered by PLL-g-PNIPAM) down to a threshold 20% weight ratio (with 80% PLL-g-PEG), the particles aggregated rapidly to form micrometer size clusters. Below 20% weight fraction of PLL-g-PNIPAM, the kinetic was drastically lowered. Using PLL derivatives provides a straightforward route allowing to control the fraction of a functional chain (here PNIPAM) deposited on PEGylated particles, and in turn to adjust surface interaction and here the rate of particle-particle aggregation as a function of the density of functional chains. This approach can be generalized to many anionic surfaces onto which PLL is known to adhere tightly, such as glass or silica.

Prevention of aggregation and renaturation of carbonic anhydrase via weak association with octadecyl- or azobenzene-modified poly(acrylate) derivatives.

The prevention of aggregation during renaturation of urea-denatured carbonic anhydrase B (CAB) via hydrophobic and Coulomb association with anionic polymers was studied in mixed solutions of CAB and amphiphilic poly(acrylate) copolymers. The polymers were derivatives of a parent poly(acrylic acid) randomly grafted with hydrophobic side groups (either 3 mol % octadecyl group, or 1-5 mol % alkylamidoazobenzene photoresponsive groups). CAB:polymer complexes were characterized by light scattering and fluorescence correlation spectroscopy in aqueous buffers (pH 7.75 or 5.9). Circular dichroism and enzyme activity assays enabled us to study the kinetics of renaturation. All copolymers, including the hydrophilic PAA parent chain, provided a remarkable protective effect against CAB aggregation during renaturation, and most of them (but not the octadecyl-modified one) markedly enhanced the regain of activity as compared to CAB alone. The significant role of Coulomb binding in renaturation and comparatively the lack of efficacy of hydrophobic association was highlighted by measurements of activity regain before and after in situ dissociation of hydrophobic complexes (achieved by phototriggering the polarity of azobenzene-modified polymers under exposure to UV light). In the presence of polymers (CAB:polymer of 1:1 w/w ratio) at concentration ∼0.6 g L(-1), the radii of the largest complexes were similar to the radii of the copolymers alone, suggesting that the binding of CAB involves one or a few polymer chain(s). These complexes dissociated by dilution (0.01 g L(-1)). It is concluded that prevention of irreversible aggregation and activity recovery were achieved when marginally stable complexes are formed. Reaching a balanced stability of the complex plays the main role in CAB renaturation, irrespective of the nature of the binding (by Coulomb association, with or without contribution of hydrophobic association).

Quantitative characterization by asymmetrical flow field-flow fractionation of IgG thermal aggregation with and without polymer protective agents.

Complexes formed between poly(acrylates) and polyclonal immunoglobulin G (IgG) in its native conformation and after heat stress were characterized using asymmetric flow field-flow fractionation (AF4) coupled with on-line UV-Vis spectroscopy and multi-angle light-scattering detection (MALS). Mixtures of IgG and poly(acrylates) of increasing structural complexity, sodium poly(acrylate) (PAA), a sodium poly(acrylate) bearing at random 3 mol % n-octadecyl groups, and a random copolymer of sodium acrylate (35 mol%), N-n-octylacrylamide (25 mol%) and N-isopropylacrylamide (40 mol%), were fractionated in a sodium phosphate buffer (0.02 M, pH 6.8) in the presence, or not, of 0.1 M NaCl. The AF4 protocol developed allowed the fractionation of solutions containing free poly(acrylates), native IgG monomer and dimer, poly(acrylates)/IgG complexes made up of one IgG molecule and a few polymer chains, and/or larger poly(acrylates)/IgG aggregates. The molar mass and recovery of the soluble analytes were obtained for mixed solutions of poly(acrylates) and native IgG and for the same solutions incubated at 65 °C for 10 min. From the combined AF4 results, we concluded that in solutions of low ionic strength, the presence of PAA increased the recovery ratio of IgG after thermal stress because of the formation of electrostatically-driven PAA/IgG complexes, but PAA had no protective effect in the presence of 0.1 M NaCl. Poly(acrylates) bearing hydrophobic groups significantly increased IgG recovery after stress, independently of NaCl concentration, because of the synergistic effect of hydrophobic and electrostatic interactions. The AF4 results corroborate conclusions drawn from a previous study combining four analytical techniques. This study demonstrates that AF4 is an efficient tool for the analysis of protein formulations subjected to stress, an important achievement given the anticipated important role of proteins in near-future human therapies.

Prevention of thermally induced aggregation of IgG antibodies by noncovalent interaction with poly(acrylate) derivatives.

Prevention of thermal aggregation of antibodies in aqueous solutions was achieved by noncovalent association with hydrophobically modified poly(acrylate) copolymers. Using a polyclonal immunoglobin G (IgG) as a model system for antibodies, we have studied the mechanisms by which this multidomain protein interacts with polyanions when incubated at physiological pH and at temperatures below and above the protein unfolding/denaturation temperature, in salt-free solutions and in 0.1 M NaCl solutions. The polyanions selected were sodium poly(acrylates), random copolymers of sodium acrylate and N-n-octadecylacrylamide (3 mol %), and a random copolymer of sodium acrylate, N-n-octylacrylamide (25 mol %), and N-isopropylacrylamide (40 mol %). They were derived from two poly(acrylic acid) parent chains of Mw 5000 and 150000 g·mol(-1). The IgG/polyanion interactions were monitored by static and dynamic light scattering, fluorescence correlation spectroscopy, capillary zone electrophoresis, and high sensitivity differential scanning calorimetry. In salt-free solutions, the hydrophilic PAA chains form complexes with IgG upon thermal unfolding of the protein (1:1 w/w IgG/PAA), but they do not interact with native IgG. The complexes exhibit a remarkable protective effect against IgG aggregation and maintain low aggregation numbers (average degree of oligomerization <12 at a temperature up to 85 °C). These interactions are screened in 0.1 M NaCl and, consequently, PAAs lose their protective effect. Amphiphilic PAA derivatives (1:1 w/w IgG/polymer) are able to prevent thermal aggregation (preserving IgG monomers) or retard aggregation of IgG (formation of oligomers and slow growth), revealing the importance of both hydrophobic interactions and modulation of the Coulomb interactions with or without NaCl present. This study leads the way toward the design of new formulations of therapeutic proteins using noncovalent 1:1 polymer/protein association that are transient and require a markedly lower additive concentration compared to conventional osmolyte protecting agents. They do not modify IgG permanently, which is an asset for applications in therapeutic protein formulations since the in vivo efficacy of the protein should not be affected.

Triggering cell adhesion, migration or shape change with a dynamic surface coating.

There's an APP for that: cell-repellent APP (azido-[polylysine-g-PEG]) is used to create substrates for spatially controlled dynamic cell adhesion. The simple addition of a functional peptide to the culture medium rapidly triggers cell adhesion. This highly accessible yet powerful technique allows diverse applications, demonstrated through tissue motility assays, patterned coculturing and triggered cell shape change.

Unfolding of cytochrome C upon interaction with azobenzene-modified copolymers.

Hydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered polarity switches. The effect of interaction between polymers and proteins on the stability of the proteins is, however, rarely investigated. Here we studied the unfolding/folding equilibrium of cytochrome c (cyt c) under its oxidized or reduced forms, in the presence of various amphiphilic copolymers (by circular dichroism and intrinsic fluorescence measurements). As models of stimuli-responsive amphiphilic chains, we considered poly(acrylic acid) derivatives, modified to contain hydrophobic, light-responsive azobenzene moieties. These copolymers are, thus, capable to develop both ionic (under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt c (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt c underwent unfolding, at [urea] of 9-10 M, which was analyzed under the framework of the equilibrium between two states (native-unfolded). In the presence of polymers, the native folding of cyt c was preserved at low concentrations of urea (typically <4M). However, the presence of polymers facilitated unfolding, which occurred at urea concentrations lowered by 2-4 M as compared to unfolding in the absence of polymers (polymer/cyt c ratio of 1:1 g/g). The predominant contribution of coulombic interactions was shown by both the lack of significant impact of the amount of (neutral) azobenzene moieties in the copolymers and the disappearance of destabilization at ionic strength higher than 150 mM. In addition, stability was similar to that of an isolated cyt c, in the presence of a neutral chain bearing acryloyl(oligoethyleneoxide) units instead of the ionized sodium acrylate moieties. DSC measurements showed that in the presence of polymers, cyt c is thermally unfolded in aqueous buffer at temperatures lowered by >20 °C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt c was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose pK(a) can be photoshifted), unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we conclude that remote control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarity.