Silicone Oil-water Interface Research Articles

Experiment and simulation of the interface characteristics during the two-phase invasion between silicone oil and water

In this study, the interface characteristics during the two-phase invasion of silicon oil and water were investigated using high-precision optical experiments and molecular dynamics simulations. An experimental system composed of a CCD (charge coupled device) industrial camera and an optical microscope was designed to observe the interface state between the dispersed phase at the silicone oil-water interface. At the same time, the microscopic image and energy spectrum results of the silicone oil-water mixture were obtained using a cryo-scanning electron microscope (Cryo-SEM). The molecular dynamics method was also used to simulate the dynamic process of the mass transfer of the silicone oil-water mixture. Through experimental results and molecular dynamics simulations, the phenomenon of diffuse mass transfer during the phase invasion and the existence of silicon oil-water associations were confirmed. The results showed that the silicone oil and water molecules were associated with each other due to the hydrogen bonding and van der Waals interactions, but the interfacial association matter was loose and unstable. The C–H, Si–O, and H–O bonds were the main factor in the formation of the silicone oil-water dispersion association matter. The findings revealed the rationality of the dispersion associated substances at the silicon oil-water interface, and showed its microstructure characteristics. The study is expected to provide an insight for the study of silicon oil-water interface and enrich the emulsification and separation theories between silicon oil and water solutions.

Determining the Dependence of Interfacial Tension on Molecular Area for Phospholipid Monolayers Formed at Silicone Oil-Water and Tricaprylin-Water Interfaces by Vesicle Fusion.

Phospholipid monolayers formed at oil-water interfaces have been used to explore biological interface properties. Thus, monolayer systems need to be quantitatively understood. Previously, we investigated the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) monolayers at silicone oil-water interfaces to determine the dependence of interfacial tension, γ, on the area per lipid, a, compared to that of the closely packed monolayers, acp. This study aims to develop a method to obtain the γ-a relationship from the γ-a/acp data by investigating POPC monolayers at the silicone oil-water and tricaprylin-water interfaces. Pendant drop tensiometry was used to obtain the dependence of γ on a/acp. Furthermore, by calculating the surface pressure, Π, from γ and multiplying a/acp with an estimated acp value, the dependence of Π on a was obtained. When a value approximately equal to the a of POPC bilayers was assigned to acp, the resultant Π-a profile partially or approximately completely overlapped with the Π-a isotherms obtained for the monolayers at the air-water interface using a Langmuir trough. The overlap for the silicone oil-water interface occurred at a ≤ 77 Å2, while that for the tricaprylin-water interface occurred in approximately the entire a region. The results indicate that the Π of the condensed monolayers is little affected by bulk oil. Thus, the γ-a relationship for the oil-water interface can be determined by comparing the compression isotherm with the one obtained for the air-water interface.

Adsorption and Aggregation of Monoclonal Antibodies at Silicone Oil-Water Interfaces.

Monoclonal antibody (mAb) therapies are rapidly growing for the treatment of various diseases like cancer and autoimmune disorders. Many mAb drug products are sold as prefilled syringes and vials with liquid formulations. Typically, the walls of prefilled syringes are coated with silicone oil to lubricate the surfaces during use. MAbs are surface-active and adsorb to these silicone oil-solution interfaces, which is a potential source of aggregation. We studied formulations containing two different antibodies, mAb1 and mAb2, where mAb1 aggregated more when agitated in the presence of an oil-water interface. This directly correlated with differences in surface activity of the mAbs, studied with interfacial tension, surface mass adsorption, and interfacial rheology. The difference in interfacial properties between the mAbs was further reinforced in the coalescence behavior of oil droplets laden with mAbs. We also looked at the efficacy of surfactants, typically added to stabilize mAb formulations, in lowering adsorption and aggregation of mAbs at oil-water interfaces. We showed the differences between poloxamer-188 and polysorbate-20 in competing with mAbs for adsorption to interfaces and in lowering particulate and overall aggregation. Our results establish a direct correspondence between the adsorption of mAbs at oil-water interfaces and aggregation and the effect of surfactants in lowering aggregation by competitively adsorbing to these interfaces.

Determination of the Coverage of Phosphatidylcholine Monolayers Formed at Silicone Oil–Water Interfaces by Vesicle Fusion

Phospholipid monolayers at oil-water interfaces are used for various biological applications and often formed by vesicle adsorption. However, the adsorbed structures are not well characterized; therefore, fundamental investigation on vesicle adsorption behavior is necessary for correct understanding of the monolayer systems. Herein, we investigated the adsorption of phosphatidylcholine vesicles onto silicone oil-water interfaces using fluorescence microscopy and pendant drop tensiometry. The interfacial monolayer coverage, S, was determined by assuming S = 1 for tightly packed monolayers. Adsorption for 10 min with a lipid concentration of 0.2 mM resulted in S ≈ 0.4. An increase in lipid concentration (0.5-2 mM) and adsorption time (1 h) moderately increased monolayer coverage (S ≈ 0.6). However, extended adsorption for 24 h only slightly increased coverage (S ≈ 0.7). Monolayers with an S < 0.6 were homogeneous, while those with an S > 0.6 were associated with several vesicular structures. The surface density of these bound vesicles increased with increasing S. We conclude that vesicles readily fused with the interface to form monolayers at S < 0.6 and that their fusogenicity considerably decreased at S > 0.6. These results demonstrate that the ability of vesicles to form monolayers is determined by the interfacial coverage.

Interaction Mechanisms of Giant Unilamellar Vesicles with Hydrophobic Glass Surfaces and Silicone Oil-Water Interfaces: Adsorption, Deformation, Rupture, Dynamic Shape Changes, Internal Vesicle Formation, and Desorption.

Phospholipid monolayers at oil-water interfaces are often obtained via vesicle adsorption. However, the interaction mechanisms of vesicles with these oil-water interfaces remain unclear. Herein, we studied the adsorption of giant unilamellar vesicles (GUVs) of approximately 2-5 μm diameter onto silicone oil-water interfaces and glass surfaces modified with hexamethyldisilazane (HMDS) and octadecyltrimethoxysilane (ODTMS) using fluorescence microscopy. The GUVs exhibited various modes of interaction, adsorbing on the silanized glass surfaces without sizable deformation, whereas GUVs bound to the silicone oil-water interface exhibited large deformation. After adsorption, GUV rupture occurred within 350, 110, and 3 ms on HMDS-modified glass, ODTMS-modified glass, and silicone oil-water interface, respectively. On glass surfaces, GUV rupture was often initiated and proceeded with pore formation near the surface. The monolayer patches formed by GUV rupture on HMDS-modified glass remained for at least 1 h over an area approximately twice of that estimated from the original GUV. On the ODTMS-modified glass and silicone oil surfaces, the monolayer patch structures disappeared in milliseconds owing to lipid diffusion across the interface. When adsorbed on the oil-water interface, the GUVs spontaneously underwent dynamic shape changes, internal vesicle formation, and desorption without rupture. Thus, it can be concluded that these different pathways arose from different lipid-surface affinities.

Investigation on the dispersion mass transfer in the interface between silicone oil and water

In this work, the dispersion mass transfer of the silicone oil-water mixture, especially the material dispersion process in the silicone oil/water interface was studied. Adopt a combination of macro test methods and microscopic test methods, the Cryo-scanning electron Microscope (Cryo-SEM) and the Transmission Electron Microscope (TEM) were introduced into the work of this paper respectively. The interface region between the dispersed phase and the continuous phase in the silicone oil-water mixture was investigated by the Cryo-SEM, the boundary of pure silicone oil droplets was studied by the TEM. Moreover, a simple and practical silicone oil-water interface characteristic measuring instrument (ICMI) has been designed and made for the measurement of properties in the silicone oil-water mixture interface. The results show that the silicone oil boundary is continuous and free of other impurities, and the interface presents a three-layer structure of “silicone oil-associated matter-water”, the Cryo-SEM images show the associated matter formed a layer of shell structure at the interface. The ICMI measurements show that the formation of the associated matter will lead to a rapid increase in interfacial pressure, while the associated matter migration will lead to rapid relaxation of the interface, and the relaxation effect is greater than the diffusion effect. This study shows that the dispersion mass transfer between silicone oil and water are consists of two interrelated stages: in the initial stage of mixing, continuous liquid phase splitting produces a large number of droplets, the diameter of the droplet group has a centralized distribution law, and there exists a sole clockwise circulation in the droplet. In the second stage, a kind of associated matter is formed by molecular aggregation in the interface area between the dispersed phase and the continuous phase, the associated matter contains both the silicone oil component and the water component. The formation and migration of the associated matter will result in a compression and relaxation effect of the interface. This manuscript provided a useful study for observing the oil/water interface with an electron microscope.

Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra; even though in solution, PEG rhIL-1ra had a lower ΔGunf and exhibited a more perturbed tertiary structure at the interface. Net protein-protein interactions in solution for rhIL-1ra were attractive but increased steric repulsion because of PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were 5 times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.

Understanding Protein-Interface Interactions of a Fusion Protein at Silicone Oil-Water Interface Probed by Sum Frequency Generation Vibrational Spectroscopy

Protein adsorbed at the silicone oil-water interface can undergo a conformational change that has the potential to induce protein aggregation on storage. Characterization of the protein structures at interface is therefore critical for understanding the protein-interface interactions. In this article, we have applied sum frequency generation (SFG) spectroscopy for studying the secondary structures of a fusion protein at interface and the surfactant effect on protein adsorption to silicone oil-water interface. SFG and chiral SFG spectra from adsorbed protein in the amide I region were analyzed. The presence of beta-sheet vibrational band at 1635 cm−1 implies the protein secondary structure was likely perturbed when protein adsorbed at silicone oil interface. The time-dependent SFG study showed a significant reduction in the SFG signal of preadsorbed protein when polysorbate 20 was introduced, suggesting surfactant has stronger interaction with the interface leading to desorption of protein from the interface. In the preadsorbed surfactant and a mixture of protein/polysorbate 20, SFG analysis confirmed that surfactant can dramatically prevent the protein adsorption to silicone oil surface. This study has demonstrated the potential of SFG for providing the detailed molecular level understanding of protein conformation at interface and assessing the influence of surfactant on protein adsorption behavior.

Colloidal Instability Fosters Agglomeration of Subvisible Particles Created by Rupture of Gels of a Monoclonal Antibody Formed at Silicone Oil-Water Interfaces

In this study, we investigated the effect of ionic strength (1.25-231 mM) on viscoelastic interfacial gels formed by a monoclonal antibody at silicone oil-water interfaces, and the formation of subvisible particles due to rupture of these gels. Rates of gel formation and their elastic moduli did not vary significantly with ionic strength. Likewise, during gel rupture no significant effects of ionic strength were observed on particle formation and aggregation as detected by microflow imaging, resonance mass measurement, and size exclusion chromatography. Subvisible particles formed by mechanical rupturing of the gels agglomerated over time, even during quiescent incubation, due to the colloidal instability of the particles.

Surfactant Effects on Particle Generation in Antibody Formulations in Pre-filled Syringes

Protein aggregation and particle formation have been observed when protein solutions contact hydrophobic interfaces, and it has been suggested that this undesirable phenomenon may be initiated by interfacial adsorption and subsequent gelation of the protein. The addition of surfactants, such as polysorbate 20, to protein formulations has been proposed as a way to reduce protein adsorption at silicone oil-water interfaces and mitigate the production of aggregates and particles. In an accelerated stability study, monoclonal antibody formulations containing varying concentrations of polysorbate 20 were incubated and agitated in pre-filled glass syringes (PFS), exposing the protein to silicone oil-water interfaces at the siliconized syringe walls, air-water interfaces, and agitation stress. Following agitation in siliconized syringes that contained an air bubble, lower particle concentrations were measured in the surfactant-containing antibody formulations than in surfactant-free formulations. Polysorbate 20 reduced particle formation when added at concentrations above or below the critical micelle concentration (CMC). The ability of polysorbate 20 to decrease particle generation in PFS corresponded with its ability to inhibit gelation of the adsorbed protein layer, which was assessed by measuring the interfacial diffusion of individual antibody molecules at the silicone oil-water interface using total internal reflectance fluorescence (TIRF) microscopy with single-molecule tracking.

Molecular trajectories provide signatures of protein clustering and crowding at the oil/water interface.

Using high throughput single-molecule total internal reflection fluorescence microscopy (TIRFM), we have acquired molecular trajectories of bovine serum albumin (BSA) and hen egg white lysozyme during protein layer formation at the silicone oil-water interface. These trajectories were analyzed to determine the distribution of molecular diffusion coefficients, and for signatures of molecular crowding/caging, including subdiffusive motion and temporal anticorrelation of the instantaneous velocity vector. The evolution of these properties with aging time of the interface was compared with dynamic interfacial tension measurements. For both lysozyme and BSA, we observed an overall slowing of protein objects, the onset of both subdiffusive and anticorrelated motion (associated with crowding), and a decrease in the interfacial tension with aging time. For lysozyme, all of these phenomena occurred virtually simultaneously, consistent with a homogeneous model of layer formation that involves gradual crowding of weakly interacting proteins. For BSA, however, the slowing occurred first, followed by the signatures of crowding/caging, followed by a decrease in interfacial tension, consistent with a heterogeneous model of layer formation involving the formation of protein clusters. The application of microrheological methods to single molecule trajectories described here provides an unprecedented level of mechanistic interpretation of interfacial events that occurred over a wide range of interfacial protein coverage.

Gelation of a Monoclonal Antibody at the Silicone Oil–Water Interface and Subsequent Rupture of the Interfacial Gel Results in Aggregation and Particle Formation

The formation of viscoelastic gels by a monoclonal antibody (mAb) at the silicone oil-water interface was studied using interfacial shear rheology. At a concentration of 50 μg/mL, the mAb formed gels in less than 1 h, and the gelation time decreased with increasing protein concentration. To probe the effects of mechanical rupture of the interfacial gel layers, a layer of silicone oil was overlaid on the surface of aqueous solutions of mAb, and the interface was ruptured periodically with a needle. Rupture of the interfacial gel resulted in formation of subvisible particles and substantial losses of mAb monomer, which were detected by microflow imaging and quantified by size-exclusion chromatography, respectively. Resonance mass measurement showed that levels of both protein particles and silicone oil droplets increased as the gel was repeatedly ruptured with a needle. In contrast, in samples wherein the interfacial gels were not ruptured, much lower levels of aggregates and particles were detected. Addition of nonionic surfactants (polysorbate 20 or polysorbate 80) protected against aggregation and protein particle formation, with increased protection seen with increasing surfactant levels, and with the greatest inhibition observed in samples containing polysorbate 80.

IgG1 Aggregation and Particle Formation Induced by Silicone–water Interfaces on Siliconized Borosilicate Glass Beads: A Model for Siliconized Primary Containers

Understanding and mitigating particle formation in prefilled syringes are critical for ensuring stability of therapeutic proteins. In the current study, siliconized beads were used as a model for the silicone-water interface to evaluate subvisible particle formation and aggregation of a monoclonal antibody (IgG(1)). Agitation with siliconized beads greatly accelerated the formation of protein aggregates and particles, an effect that was enhanced at pH 7.4 relative to pH 5 and in the presence of 0.5 M sucrose or 150 mM NaCl. Aggregation and particle formation were minimal in samples agitated without siliconized beads or in quiescent samples with siliconized beads. At pH 5, 0.01% (w/v) polysorbate 20 substantially inhibited aggregation during agitation with siliconized beads, but had minimal protective effect at pH 7.4. Transient exposure of IgG(1) formulations to the silicone-water interface by flowing formulations through a column packed with siliconized beads led to the formation of subvisible particles, with increased levels observed at pH 7.4 compared to pH 5. Agitation of protein formulations in the presence of siliconized glass beads provides a model for baked-on silicone oil-water interface in prefilled syringes and a means by which to evaluate particle formation and aggregation during formulation screening.

Ionic Strength Affects Tertiary Structure and Aggregation Propensity of a Monoclonal Antibody Adsorbed to Silicone Oil–Water Interfaces

Therapeutic proteins formulated in prefilled syringes lubricated with silicone oil come in contact with silicone oil-water interfaces for their entire shelf lives. Thus, the interactions between protein and silicone oil were studied to determine the effect of silicone oil on a monoclonal antibody's stability, both at the interface and in the bulk solution. The influence of ionic strength on these interactions was also investigated through the addition of various monovalent and divalent salts to sample formulations. The tertiary structure of the antibody was perturbed when it adsorbed to the silicone oil-water interface in solutions at low ionic strength. However, the tertiary structure of the antibody at the interface was not perturbed when the ionic strength of the formulation was increased. Even at low ionic strength, the secondary structure of the antibody adsorbed to the silicone oil-water interface was retained, suggesting that at low ionic strength, the adsorbed antibody assumes a molten globule-like conformation. This partially unfolded species was aggregation-prone, especially during agitation. Silicone oil-induced aggregation of the antibody was inhibited at higher ionic strength.

Three-Dimensional Real-Time Tracking of Nanoparticles at an Oil–Water Interface

Single-particle tracking with real-time feedback control can be used to study three-dimensional nanoparticle transport dynamics. We apply the method to study the behavior of adsorbed nanoparticles at a silicone oil-water interface in a microemulsion system over a range of particles sizes from 24 nm to 2000 nm. The diffusion coefficient of large particles (>200 nm) scales inversely with particle size, while smaller particles exhibit an unexpected increase in drag force at the interface. The technique can be applied in the future to study three-dimensional dynamics in a range of systems, including complex fluids, gels, biological cells, and geological media.

Dynamics of protein aggregation at the oil–water interface characterized by single molecule TIRF microscopy

Single molecule total internal reflectance fluorescence microscopy was used to observe the dynamic mechanisms of bovine serum albumin layer formation at the silicone oil–water interface. After an initial induction period, the mean diffusion coefficient of protein objects was observed to decrease as a function of exposure time. Simultaneously, the total adsorption rate was observed to decrease; both observations are consistent with protein layer formation. Importantly, the distribution of diffusion coefficients broadened systematically with exposure time, suggesting the presence of surface objects with a wide range of hydrodynamic radii ranging from a few nm (consistent with monomers) to ∼180 nm. The spatial distribution of the adsorption rate, which was initially uniform, gradually became inhomogeneous, also consistent with the existence of large aggregates. The kinetics of protein layer formation was dependent on the bulk protein concentration in the aqueous phase, and a kinetic population balance model was used to explain the nonlinear concentration dependence.

The effect of alkane tail length of C iE 8 surfactants on transport to the silicone oil–water interface

Detailed surfactant transport studies have typically been restricted to the air–water interface. This is mainly due to the lack of experimental devices and techniques available to study liquid–liquid interfaces. As a result, there is a lack of relevant data and understanding of surfactant behavior in microfluidic studies and emulsion applications. Using a novel shape fitting algorithm for a pendant drop capable of handling fluids of similar densities, i.e. low Bond numbers, we measure the dynamic surface tension as a function of bulk surfactant concentration at the silicone oil–water interface for a homologous series of C i E 8 nonionic surfactants. We show that the isotherms governing equilibrium at the oil–water and air–water interfaces are very different. Using a scaling analysis comparing two governing mass transport timescales, we demonstrate that there exists a transition from diffusion-limited to kinetic-limited dynamics at the silicone oil–water interface. Adsorption rate constants are determined from a one parameter nonlinear fit to dynamic surface tension data. These results demonstrate that the dynamics of interfacial transport are highly dependent on the immiscible fluids that form the interface.

Single Molecule Observations of Multiple Protein Populations at the Oil−Water Interface

Single-molecule total internal reflectance fluorescence microscopy was used to observe the dynamic behavior of >4000 bovine serum albumin objects at the silicone oil-water interface. The surface residence time distribution indicated the presence of three populations at the interface. Each population had a characteristic fluorescence intensity and distinctive interfacial diffusion behavior. Larger fluorescence intensity correlated with longer residence times and slower diffusion. These combined observations of fluorescence intensity, surface residence time, and interfacial diffusion suggested that the three populations represent monomers, dimers, and trimers respectively.

Behavior of Immiscible Two Liquid Layers Contained in Cylindrical Vessel Suddenly Set in Rotation.

Immiscible silicone oil and water were contained in a cylindrical vessel. The ratio of their volumes, referred to as the volume ratio, was varied over a wide range. The vessel was suddenly set in rotation, and the flow velocities of the two liquids were measured with particle image velocimetry (PIV) and laser Doppler velocimetry (LDV). The flow establishment time was defined as the period from the start of rotation to the moment at which a steady state is established in the vessel. An empirical equation for the flow establishment time was proposed as a function of the volume ratio, the angular frequency of rotation, and the physical properties of the liquids. The deformation of the silicone oil-water interface was also observed to confirm the findings obtained from the velocity measurements.

Model study of turbulence structure in a bottom blown bath with top slag using conditional sampling

Water model experiments were carried out to study the effect of top slag on the turbulence structure in a molten steel bath agitated by bottom gas injection. Water and silicone oil were used as models of molten steel and slag, respectively. Air was injected through a single-hole bottom nozzle so that the reverse emulsification of the silicone oil occurred at the silicone oil-water interface. Silicone oil droplets thus generated were carried deeply into the lower water layer. Turbulence measurements were made using a two-channel laser Doppler velocimeter (LDV) in the presence and absence of the top oil layer. A conditional sampling method called the four-quadrant classification method was applied to detect large scale coherent motions in the vertical bubbling jet as well as in the recirculation region. The structure and intensity of turbulence inside the bubbling jet were strongly affected by bubbles. In the absence of the top oil layer, higher momentum fluid motions directed from the centerline of the bubbling jet to the sidewall of the vessel were mainly responsible for the turbulence production in the bubbling jet, while lower momentum fluid motions directed from the sidewall toward the centerline governed the turbulence production in the recirculation region. On the other hand, in the presence of the top oil layer, the coherent motion in the bubbling jet was also affected slightly by the top layer except for the center of the bubbling jet, whereas any distinguished coherent motion was not observed in the recirculation region, and hence, turbulence production was weak there.