Pulmonary surfactant inhibition of nanoparticle uptake by alveolar epithelial cells.

Pulmonary surfactant forms a sub-micrometer thick fluid layer that covers the surface of alveolar lumen and inhaled nanoparticles therefore come in to contact with surfactant prior to any interaction with epithelial cells. We investigate the role of the surfactant as a protective physical barrier by modeling the interactions using silica-Curosurf-alveolar epithelial cell system in vitro. Electron microscopy displays that the vesicles are preserved in the presence of nanoparticles while nanoparticle-lipid interaction leads to formation of mixed aggregates. Fluorescence microscopy reveals that the surfactant decreases the uptake of nanoparticles by up to two orders of magnitude in two models of alveolar epithelial cells, A549 and NCI-H441, irrespective of immersed culture on glass or air-liquid interface culture on transwell. Confocal microscopy corroborates the results by showing nanoparticle-lipid colocalization interacting with the cells. Our work thus supports the idea that pulmonary surfactant plays a protective role against inhaled nanoparticles. The effect of surfactant should therefore be considered in predictive assessment of nanoparticle toxicity or drug nanocarrier uptake. Models based on the one presented in this work may be used for preclinical tests with engineered nanoparticles.

On the rheology of pulmonary surfactant: Effects of concentration and consequences for the surfactant replacement therapy.

The role of pulmonary surfactant is to reduce the surface tension in the lungs and to facilitate breathing. Surfactant replacement therapy (SRT) aims at bringing a substitute by instillation into the airways, a technique that has proven to be efficient and lifesaving for preterm infants. Adapting this therapy to adults requires to scale the administered dose to the patient body weight and to increase the lipid concentration, whilst maintaining its surface and flow properties similar. Here, we exploit a magnetic wire-based microrheology technique to measure the viscosity of the exogenous pulmonary surfactant Curosurf® in various experimental conditions. The Curosurf® viscosity is found to increase exponentially with lipid concentration following the Krieger-Dougherty law of colloids. The Krieger-Dougherty behavior also predicts a divergence of the viscosity at the liquid-to-gel transition. For Curosurf® the transition concentration is found close to the concentration at which it is formulated (117 g L-1versus 80 g L-1). This outcome suggests that for SRT the surfactant rheological properties need to be monitored and kept within a certain range. The results found here could help in producing suspensions for respiratory distress syndrome adapted to adults. The present work also demonstrates the potential of the magnetic wire microrheology technique as an accurate tool to explore biological soft matter dynamics.

The role of surface charge in the interaction of nanoparticles with model pulmonary surfactants.

Inhaled nanoparticles traveling through the airways are able to reach the respiratory zone of the lungs. In such an event, the incoming particles first come into contact with the liquid lining the alveolar epithelium, the pulmonary surfactant. The pulmonary surfactant is composed of lipids and proteins that are assembled into large vesicular structures. The question of the nature of the biophysicochemical interaction with the pulmonary surfactant is central to understand how the nanoparticles can cross the air-blood barrier. Here we explore the phase behavior of sub-100 nm particles and surfactant substitutes under controlled conditions. Three types of surfactant mimetics, including the exogenous substitute Curosurf®, a drug administered to infants with respiratory distress syndrome, are tested together with aluminum oxide (Al2O3), silicon dioxide (SiO2) and polymer (latex) nanoparticles. The main result here is the observation of spontaneous nanoparticle-vesicle aggregation induced by coulombic attraction. The role of the surface charges is clearly established. We also evaluate the supported lipid bilayer formation recently predicted and find that in the cases studied these structures do not occur. Pertaining to the aggregate internal structure, fluorescence microscopy shows that the vesicles and particles are intermixed at the nano- to microscale. With particles acting as stickers between vesicles, it is anticipated that the presence of inhaled nanomaterials in the alveolar spaces could significantly modify the interfacial and bulk properties of the pulmonary surfactant and interfere with lung physiology.

Design of eco-friendly fabric softeners: Structure, rheology and interaction with cellulose nanocrystals.

HYPOTHESIS: Concentrated fabric softeners are water-based formulations containing around 10-15 wt% of double tailed esterquat surfactants primarily synthesized from palm oil. In recent patents, it was shown that a significant part of the surfactant contained in today's formulations can be reduced by circa 50% and replaced by natural guar polymers without detrimental effects on the deposition and softening performances. We presently study the structure and rheology of these softener formulations and identify the mechanisms at the origin of these effects. EXPERIMENTS: The polymer additives used are guar gum polysaccharides, one cationic and one modified through addition of hydroxypropyl groups. Formulations with and without guar polymers are investigated using optical and cryo-transmission electron microscopy, small-angle light and X-ray scattering and finally rheology. Similar techniques are applied to study the phase behavior of softener and cellulose nanocrystals considered here as a model for cotton. FINDINGS: The esterquat surfactants are shown to assemble into micron-sized vesicles in the dilute and concentrated regimes. In the former, guar addition in small amounts does not impair the vesicular structure and stability. In the concentrated regime, cationic guars induce a local crowding associated to depletion interactions and leads to the formation of a local lamellar order. In rheology, adjusting the polymer concentration at 1/10th that of the surfactant is sufficient to offset the decrease of the elastic property associated with the surfactant reduction. In conclusion, we have shown that through an appropriate choice of natural additives it is possible to lower the concentration of surfactants in fabric conditioners by about half, a result that could represent a significant breakthrough in current home care formulations.

The enzyme-like catalytic activity of cerium oxide nanoparticles and its dependency on Ce3+ surface area concentration.

Cerium oxide nanoparticles are known to catalyze the decomposition of reactive oxygen species such as the superoxide radical and hydrogen peroxide. Herein, we examine the superoxide dismutase (SOD) and catalase (CAT) mimetic catalytic activities of nanoceria and demonstrate the existence of generic behaviors. For particles of sizes 4.5, 7.8, 23 and 28 nm, the SOD and CAT catalytic activities exhibit the characteristic shape of a Langmuir isotherm as a function of cerium concentration. The results show that the catalytic effects are enhanced for smaller particles and for the particles with the largest Ce3+ fraction. The SOD-like activity obtained from the different samples is found to superimpose on a single master curve using the Ce3+ surface area concentration as a new variable, indicating the existence of particle independent redox mechanisms. For the CAT assays, the adsorption of H2O2 molecules at the particle surface modulates the efficacy of the decomposition process and must be taken into account. We design an amperometry-based experiment to evaluate the H2O2 adsorption at nanoceria surfaces, leading to the renormalization of the particle specific area. Depending on the particle type the amount of adsorbed H2O2 molecules varies from 2 to 20 nm-2. The proposed scalings are predictive and allow the determination of the SOD and CAT catalytic properties of cerium oxide solely from physicochemical features.

Supported pulmonary surfactant bilayers on silica nanoparticles: formulation, stability and impact on lung epithelial cells.

Studies have shown that following exposure to particulate matter, ultrafine fractions (<100 nm) may deposit along the respiratory tract down to the alveolar region. To assess the effects of nanoparticles on the lungs, it is essential to address the question of their biophysicochemical interaction with the different pulmonary environments, including the lung lining fluids and the epithelia. Here we examine one of these interactive scenarios and study the role of supported lipid bilayers (SLB) in the effect of 40 nm fluorescent silica particles on living cells. We first study the particle phase behavior in the presence of Curosurf®, a pulmonary surfactant substitute used in replacement therapies. It is found that Curosurf® vesicles interact strongly with the nanoparticles, but do not spontaneously form SLBs. To achieve this goal, we use sonication to reshape the vesicular membranes and induce lipid fusion around the particles. Centrifugal sedimentation and electron microscopy are carried out to determine the optimum coating conditions and layer thickness. We then explore the impact of surfactant SLBs on the cytotoxic potential and interactions towards a malignant epithelial cell line. All in vitro assays indicate that SLBs mitigate the particle toxicity and internalization rates. In the cytoplasm, the particle localization is also strongly coating dependent. It is concluded that SLBs profoundly affect cellular interactions and functions in vitro and could represent an alternative strategy for particle coating. The current data also shed some light on the potential mechanisms pertaining to the particle or pathogen transport through the air-blood barrier.

Fabric Softener-Cellulose Nanocrystal Interaction: A Model for Assessing Surfactant Deposition on Cotton.

There is currently a renewed interest for improving household and personal-care formulations to provide more environment-friendly products. Fabric conditioners used as softeners have to fulfill a number of stability and biodegradability requirements. They should also display significant adsorption on cotton under the conditions of use. The quantification of surfactant adsorption remains however difficult because the fabric-woven structure is complex and deposited amounts are generally small. Here, we propose a method to evaluate cellulose-surfactant interactions with increased detection sensitivity. The method is based on the use of cellulose nanocrystals (CNCs) in lieu of micron-sized fibers or yarns, combined with different techniques, including light scattering, optical and electron microscopy, and electrophoretic mobility. CNCs are rod-shaped nanoparticles in the form of 200 nm laths that are negatively charged and can be dispersed in bulk solutions. In this work, we use a double-tailed cationic surfactant present in fabric softener. Results show that the surfactants self-assemble into unilamellar, multivesicular, and multilamellar vesicles, and the interaction with CNCs is driven by electrostatics. Mutual interactions are strong and lead to the formation of large-scale aggregates, where the vesicles remain intact at the cellulose surface. The technique developed here could be exploited to rapidly assess the fabric conditioner efficiency obtained by varying the nature and content of their chemical additives.

Polyelectrolyte assisted charge titration spectrometry: Applications to latex and oxide nanoparticles.

The electrostatic charge density of particles is of paramount importance for the control of the dispersion stability. Conventional methods use potentiometric, conductometric or turbidity titration but require large amount of samples. Here we report a simple and cost-effective method called polyelectrolyte assisted charge titration spectrometry or PACTS. The technique takes advantage of the propensity of oppositely charged polymers and particles to assemble upon mixing, leading to aggregation or phase separation. The mixed dispersions exhibit a maximum in light scattering as a function of the volumetric ratio X, and the peak position XMax is linked to the particle charge density according to σ∼D0XMax where D0 is the particle diameter. The PACTS is successfully applied to organic latex, aluminum and silicon oxide particles of positive or negative charge using poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate). The protocol is also optimized with respect to important parameters such as pH and concentration, and to the polyelectrolyte molecular weight. The advantages of the PACTS technique are that it requires minute amounts of sample and that it is suitable to a broad variety of charged nano-objects.

Local viscoelasticity of living cells measured by rotational magnetic spectroscopy.

When submitted to a magnetic field, micron-size wires with superparamagnetic properties behave as embedded rheometers and represent interesting sensors for microrheology. Here we use rotational magnetic spectroscopy to measure the shear viscosity of the cytoplasm of living cells. We address the question of whether the cytoplasm is a viscoelastic liquid or an elastic gel. The main result of the study is the observation of a rotational instability between a synchronous and an asynchronous regime of rotation, found for murine fibroblasts and human cancer cells. For wires of susceptibility 3.6, the transition occurs in the range 0.01-1 rad s(-1). The determination of the shear viscosity (10-100 Pa s) and elastic modulus (5-20 Pa) confirms the viscoelastic character of the cytoplasm. In contrast to earlier studies, it is concluded that the interior of living cells can be described as a viscoelastic liquid, and not as an elastic gel.

Biophysicochemical Interaction of a Clinical Pulmonary Surfactant with Nanoalumina.

We report on the interaction of pulmonary surfactant composed of phospholipids and proteins with nanometric alumina (Al2O3) in the context of lung exposure and nanotoxicity. We study the bulk properties of phospholipid/nanoparticle dispersions and determine the nature of their interactions. The clinical surfactant Curosurf, both native and extruded, and a protein-free surfactant are investigated. The phase behavior of mixed surfactant/particle dispersions was determined by optical and electron microscopy, light scattering, and zeta potential measurements. It exhibits broad similarities with that of strongly interacting nanosystems such as polymers, proteins or particles, and supports the hypothesis of electrostatic complexation. At a critical stoichiometry, micron-sized aggregates arising from the association between oppositely charged vesicles and nanoparticles are formed. Contrary to the models of lipoprotein corona or of particle wrapping, our work shows that vesicles maintain their structural integrity and trap the particles at their surfaces. The agglomeration of particles in surfactant phase is a phenomenon of importance that could change the interactions of the particles with lung cells.

Evidence of a two-step process and pathway dependency in the thermodynamics of poly(diallyldimethylammonium chloride)/poly(sodium acrylate) complexation.

Recent studies have pointed out the importance of polyelectrolyte assembly in the elaboration of innovative nanomaterials. Beyond their structures, many important questions on the thermodynamics of association remain unanswered. Here, we investigate the complexation between poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium acrylate) (PANa) chains using a combination of three techniques: isothermal titration calorimetry (ITC), static and dynamic light scattering and electrophoresis. Upon addition of PDADMAC to PANa or vice-versa, the results obtained by the different techniques agree well with each other, and reveal a two-step process. The primary process is the formation of highly charged polyelectrolyte complexes of size 100 nm. The secondary process is the transition towards a coacervate phase made of rich and poor polymer droplets. The binding isotherms measured are accounted for using a phenomenological model that provides the thermodynamic parameters for each reaction. Small positive enthalpies and large positive entropies consistent with a counterion release scenario are found throughout this study. Furthermore, this work stresses the importance of the underestimated formulation pathway or mixing order in polyelectrolyte complexation.

Preventing corona effects: multiphosphonic acid poly(ethylene glycol) copolymers for stable stealth iron oxide nanoparticles.

When dispersed in biological fluids, engineered nanoparticles are selectively coated with proteins, resulting in the formation of a protein corona. It is suggested that the protein corona is critical in regulating the conditions of entry into the cytoplasm of living cells. Recent reports describe this phenomenon as ubiquitous and independent of the nature of the particle. For nanomedicine applications, however, there is a need to design advanced and cost-effective coatings that are resistant to protein adsorption and that increase the biodistribution in vivo. In this study, phosphonic acid poly(ethylene glycol) copolymers were synthesized and used to coat iron oxide particles. The copolymer composition was optimized to provide simple and scalable protocols as well as long-term stability in culture media. It is shown that polymers with multiple phosphonic acid functionalities and PEG chains outperform other types of coating, including ligands, polyelectrolytes, and carboxylic acid functionalized PEG. PEGylated particles exhibit moreover exceptional low cellular uptake, of the order of 100 femtograms of iron per cell. The present approach demonstrates that the surface chemistry of engineered particles is a key parameter in the interactions with cells. It also opens up new avenues for the efficient functionalization of inorganic surfaces.

Surfactant-triggered disassembly of electrostatic complexes probed at optical and quartz crystal microbalance length scales.

A critical advantage of electrostatic assemblies over covalent and crystalline bound materials is that associated structures can be disassembled into their original constituents. Nanoscale devices designed for the controlled release of functional molecules already exploit this property. To bring some insight into the mechanisms of disassembly and release, we study the disruption of molecular electrostatics-based interactions via competitive binding with ionic surfactants. To this aim, free-standing micrometer-size wires were synthesized using oppositely charged poly(diallyldimethylammonium chloride) and poly(acrylic acid) coated iron oxide nanoparticles. The disassembly is induced by the addition of sodium dodecyl sulfates that complex preferentially the positive polymers. The process is investigated at two different length scales: the length scale of the particles (10 nm) through the quartz crystal microbalance technique and that of the wires (>1 µm) via optical microscopy. Upon surfactant addition, the disassembly is initiated at the surface of the wires by the release of nanoparticles and by the swelling of the structure. In a second step, erosion involving larger pieces takes over and culminates in the complete dissolution of the wires, confirming the hypothesis of a surface-type swelling and erosion process.

Poly(acrylic acid)-coated iron oxide nanoparticles: quantitative evaluation of the coating properties and applications for the removal of a pollutant dye.

In this work, 6-12 nm iron oxide nanoparticles were synthesized and coated with poly(acrylic acid) chains of molecular weight 2100 g mol(-1). Based on a quantitative evaluation of the dispersions, the bare and coated particles were thoroughly characterized. The number densities of polymers adsorbed at the particle surface and of available chargeable groups were found to be 1.9±0.3 nm(-2) and 26±4 nm(-2), respectively. Occurring via a multi-site binding mechanism, the electrostatic coupling leads to a solid and resilient anchoring of the chains. To assess the efficacy of the particles for pollutant remediation, the adsorption isotherm of methylene blue molecules, a model of pollutant, was determined. The excellent agreement between the predicted and the measured amounts of adsorbed dyes suggests that most carboxylates participate to the complexation and adsorption mechanisms. An adsorption of 830 mg g(-1) was obtained. This quantity compares well with the highest values available for this dye.

Magnetic wire-based sensors for the microrheology of complex fluids.

We propose a simple microrheology technique to evaluate the viscoelastic properties of complex fluids. The method is based on the use of magnetic wires of a few microns in length submitted to a rotational magnetic field. In this work, the method is implemented on a surfactant wormlike micellar solution that behaves as an ideal Maxwell fluid. With increasing frequency, the wires undergo a transition between a steady and a hindered rotation regime. The study shows that the average rotational velocity and the amplitudes of the oscillations obey scaling laws with well-defined exponents. From a comparison between model predictions and experiments, the rheological parameters of the fluid are determined.

Interfacial activity of phosphonated-PEG functionalized cerium oxide nanoparticles.

In a recent publication, we have highlighted the potential of phosphonic acid terminated PEG oligomers to functionalize strong UV absorption cerium oxide nanoparticles, which yield suspensions that are stable in aqueous or organic solvents and are redispersible in different solvents after freeze-drying. In the present work, we highlight the interfacial activity of the functional ceria nanoparticles and their potential to modify hydrophobic surfaces. We first investigated the phosphonated-PEG amphiphilic oligomers behavior as strong surface active species forming irreversibly adsorbed layers. We then show that the oligomers interfacial properties translate to the functional nanoparticles. In particular, the addition of a small fraction of phosphonated-PEG oligomers with an extra C16 aliphatic chain (stickers) into the formulation enabled the tuning of (i) the nanoparticles adsorption at the air/water, polystyrene/water, oil/water interfaces and (ii) the particle/particle interaction in aqueous solutions. We also found that dense and closely packed two-dimensional monolayers of nanoceria can be formed by spontaneous adsorption or surface compression using a Langmuir trough. A hexagonal organization controlled by reversible and repulsive interaction has been characterized by GISAXS. Mono- or multilayers can also be stably formed or transferred on solid surfaces. Our results are key features in the field of polymer surface modification, solid-stabilized emulsions (Pickering), or supracolloidal assemblies.

The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles.

Engineered inorganic nanoparticles are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. In the present paper, we show that by changing the coating of iron oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small carboxylated polymers (poly(acrylic acid)), the colloidal stability of the dispersion is improved and the adsorption/internalization of iron toward living mammalian cells is profoundly affected. Citrate-coated particles are shown to destabilize in all fetal-calf-serum based physiological conditions tested, whereas the polymer coated particles exhibit an outstanding dispersibility as well as a structure devoid of protein corona. The interactions between nanoparticles and human lymphoblastoid cells are investigated by transmission electron microscopy and flow cytometry. Two types of nanoparticle/cell interactions are underlined. Iron oxides are found either adsorbed on the cellular membranes, or internalized into membrane-bound endocytosis compartments. For the precipitating citrate-coated particles, the kinetics of interactions reveal a massive and rapid adsorption of iron oxide on the cell surfaces. The quantification of the partition between adsorbed and internalized iron was performed from the cytometry data. The results highlight the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.

Controlling electrostatic co-assembly using ion-containing copolymers: from surfactants to nanoparticles.

In this review, we address the issue of the electrostatic complexation between charged-neutral diblock copolymers and oppositely charged nanocolloids. We show that nanocolloids such as surfactant micelles and iron oxide magnetic nanoparticles share similar properties when mixed with charged-neutral diblocks. Above a critical charge ratio, core-shell hierarchical structures form spontaneously under direct mixing conditions. The core-shell structures are identified by a combination of small-angle scattering techniques and transmission electron microscopy. The formation of multi-level objects is driven by the electrostatic attraction between opposite charges and by the release of the condensed counterions. Alternative mixing processes inspired from molecular biology are also described. The protocols applied here consist in screening the electrostatic interactions of the mixed dispersions, and then removing the salt progressively as an example by dialysis. With these techniques, the oppositely charged species are intimately mixed before they can interact, and their association is monitored by the desalting kinetics. As a result, sphere- and wire-like aggregates with remarkable superparamagnetic and stability properties are obtained. These findings are discussed in the light of a new paradigm which deals with the possibility to use inorganic nanoparticles as building blocks for the design and fabrication of supracolloidal assemblies with enhanced functionalities.

Magnetic nanowires generated via the waterborne desalting transition pathway.

We report a simple and versatile waterborne synthesis of magnetic nanowires following the innovative concept of electrostatic "desalting transition". Highly persistent superparamagnetic nanowires are generated from the controlled assembly of oppositely charged nanoparticles and commercially available polyelectrolytes. The wires have diameters around 200 nm and lengths between 1 µm and 0.5 mm, with either positive or negative charges on their surface. Beyond, we show that this soft-chemistry assembly approach is a general phenomenon independent of the feature of the macromolecular building blocks, opening significant perspectives for the design of multifunctional materials.

Probing oppositely charged surfactant and copolymer interactions by isothermal titration microcalorimetry.

The complexation between charge-neutral block copolymers and oppositely charged surfactants was investigated by light scattering experiments and by isothermal titration calorimetry (ITC). The copolymer was poly(sodium acrylate)-b-poly(acrylamide) and the surfactant dodecyltrimethylammonium bromide (DTAB). In a previous report, we had shown that the copolymers and the surfactants coassembled spontaneously into colloidal complexes. Depending on the charge ratio, the complexes were either single surfactant micelles decorated by copolymers or core-shell hierarchical structures. ITC was performed in order to investigate the thermodynamics of the complex formation. Titrations of copolymers by surfactants and of surfactants by copolymers revealed that the electrostatic coassembly was an endothermic reaction, suggesting a process dominated by the entropy of the counterions. Here, we found that the thermodynamic quantities associated with the reaction depended on the mixing order. When surfactants were added stepwise to copolymers, the titration was associated with the formation of single micelles decorated by a unique polymer. Above a critical charge ratio, the micelles rearranged themselves into 100 nm colloidal complexes in a collective process which displayed the following features: (i) the process was very slow as compared to the time scale of Brownian diffusion, (ii) the thermodynamic signature was a endothermic peak, and (iii) the stoichiometry between the positive and negative charges was modified from n = 0.48 (single micelles) to 0.75 (core-shell complexes). When copolymers were added stepwise to surfactants, the titration resulted in the formation of the core-shell aggregates only. In both experiments, the amount of polyelectrolytes needed for complex formation exceeded the number required to compensate the net micellar charge, confirming the evidence of overcharging in the complex formation.