Comparison of Protein Particle Formation in IgG1 mAbs Formulated with PS20 Vs. PS80 When Subjected to Interfacial Dilatational Stress.

Polysorbates (PS) are nonionic surfactants that are commonly included in protein formulations to mitigate the formation of interfacial stress-induced protein particles and thus increase their long-term storage stability. Nonetheless, factors that dictate the efficiency of different polysorbates in mitigating protein particle formation, especially during the application of interfacial stresses, are often ill defined. Here, we used a Langmuir trough to determine the surface activity of two IgG1 monoclonal antibodies formulated with two different polysorbates (PS20 and PS80) when subjected to interfacial dilatational stress. Interfacial properties of these formulations were then correlated with characterization of subvisible protein particles measured by micro-flow imaging (MFI). Both mAbs, when formulated in PS20, demonstrate faster adsorption kinetics and higher surface activity compared to PS80 or surfactant-free formulations. Compression/expansion results suggest that when exposed to interfacial dilatational stresses, both mAb/PS20 formulations display interfacial properties of PS20 alone. In contrast, interfacial properties of both mAb/PS80 formulations suggest mAbs and PS80 are co-adsorbed to the air-water interface. Further, MFI analysis of the interface and the bulk solution confirms that PS20 is more effective than PS80 at mitigating the formation of larger particles in the bulk solution in both mAbs. Concomitantly, the efficiency of PS to prevent interface-induced protein particle formation also depended on the protein's inherent tendency to aggregate at a surfactant-free interface. Together, the studies presented here highlight the importance of determining the interfacial properties of mAbs, surfactants, and their combinations to make informed formulation decisions about the choice of surfactant.

Liposome-Tethered Gold Nanoparticles Triggered by Pulsed NIR Light for Rapid Liposome Contents Release and Endosome Escape.

Remote triggering of contents release with micron spatial and sub-second temporal resolution has been a long-time goal of medical and technical applications of liposomes. Liposomes can sequester a variety of bioactive water-soluble ions, ligands and enzymes, and oligonucleotides. The bilayer that separates the liposome interior from the exterior solution provides a physical barrier to contents release and degradation. Tethering plasmon-resonant, hollow gold nanoshells to the liposomes, or growing gold nanoparticles directly on the liposome exterior, allows liposome contents to be released by nanosecond or shorter pulses of near-infrared light (NIR). Gold nanoshells or nanoparticles strongly adsorb NIR light; cells, tissues, and physiological media are transparent to NIR, allowing penetration depths of millimeters to centimeters. Nano to picosecond pulses of NIR light rapidly heat the gold nanoshells, inducing the formation of vapor nanobubbles, similar to cavitation bubbles. The collapse of the nanobubbles generates mechanical forces that rupture bilayer membranes to rapidly release liposome contents at the preferred location and time. Here, we review the syntheses, characterization, and applications of liposomes coupled to plasmon-resonant gold nanostructures for delivering a variety of biologically important contents in vitro and in vivo with sub-micron spatial control and sub-second temporal control.

Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies.

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality.

Near-Infrared Light Triggered-Release in Deep Brain Regions Using Ultra-photosensitive Nanovesicles.

Remote and minimally-invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold-coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad-PC-Rad as a tool for the delivery of bioactive molecules into brain tissue. Near-infrared picosecond laser pulses activated the gold-coating on the surface of nanovesicles, creating nanomechanical stress and leading to near-complete vesicle cargo release in sub-seconds. Compared to natural phospholipid liposomes, the photo-release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.

Near Infrared-Triggered Liposome Cages for Rapid, Localized Small Molecule Delivery.

Photolabile chelating cages or protecting groups need complex chemical syntheses and require UV, visible, or two-photon NIR light to trigger release. Different cages have different solubilities, reaction rates,  and energies required for triggering. Here we show that liposomes containing calcium, adenosine triphosphate, or carboxyfluorescein are tethered to plasmon-resonant hollow gold nanoshells (HGN) tuned to absorb light from 650-950 nm. Picosecond pulses of near infrared (NIR) light provided by a two-photon microscope, or by a stand-alone laser during flow through microfluidic channels, trigger contents release with spatial and temporal control. NIR light adsorption heats the HGN, inducing vapor nanobubbles that rupture the liposome, releasing cargo within milliseconds. Any water-soluble molecule can be released at essentially the same rate from the liposome-HGN. By using liposomes of different composition, or HGN of different sizes or shapes with different nanobubble threshold fluences, or irradiating on or off resonance, two different cargoes can be released simultaneously, one before the other, or in a desired ratio. Calcium release from liposome-HGN can be spatially patterned to crosslink alginate gels and trap living cells. Liposome-HGN provide stable, biocompatible isolation of the bioactive compound from its surroundings with minimal interactions with the local environment.

Small-Scale Tools to Assess the Impact of Interfacial and Shear Stress on Biologic Drug Products.

Proper risk analysis needs to be in place to understand the susceptibility of protein to unfold and aggregate in the presence of interfacial and/or shear stress. Certain techniques, such as agitation/shaking studies, have been traditionally used to understand the impact of these stresses on the protein physical stability. However, the stresses applied in these systems are convoluted, making it difficult to define the control strategy (i.e., adjustment in process parameters to reduce foaming/bubble formation, change pump type). We have developed two small-scale tools that allow for the isolation of interfacial and shear stress, respectively. These systems, in combination with computational fluid dynamics and numerical approximations, help simulate the normal operating ranges as well as the proven acceptable ranges for different unit operations such as tangential flow filtration (TFF), mixing, and filling.

Perfluoroheptane-Loaded Hollow Gold Nanoshells Reduce Nanobubble Threshold Flux.

The threshold flux for nanobubble formation and liposome rupture is reduced by 50-60% by adding a liquid mixture of tetradecanol and perfluoroheptane to the interior cavity of 40 nm diameter hollow gold nanoshells (HGN), and allowing the tetradecanol to solidify to hold the perfluoroheptane in place. On absorption of picosecond pulses of near-infrared light, the perfluoroheptane vaporizes to initiate cavitation-like nanobubbles as the HGN temperature increases. The lower spinodal temperature and heat capacity of perfluoroheptane relative to water causes the threshold flux for nanobubble formation to decrease. The perfluoroheptane-containing HGN can be linked via thiol-PEG-lipid tethers to carboxyfluorescein-containing liposomes and shows a similar decreased flux necessary for liposome contents release.

Scaling relationships for translational diffusion constants applied to membrane domain dissolution and growth.

We compare the way that relationships for diffusion constants scale with the size of diffusing membrane domains and the geometry of their environments. Then, we review our experimental work on the dynamics of dissolution/growth of membrane domains in crowding induced mixing, phase separation, and Ostwald ripening in a highly confined environment. Overall, the scaling relationships applied to diffusion constants obtained by fits to our dynamic data indicate that dissolution and growth is influenced by the diffusion of clusters or small domains of lipids, in addition to kinetic processes and geometrical constraints.

Optimizing the NIR Fluence Threshold for Nanobubble Generation by Controlled Synthesis of 10 - 40 nm Hollow Gold Nanoshells.

The laser fluence to trigger nanobubbles around hollow gold nanoshells (HGN) with near infrared light was examined through systematic modification of HGN size, localized surface plasmon resonance (LSPR), HGN concentration, and surface coverage. Improved temperature control during silver template synthesis provided monodisperse, silver templates as small as 9 nm. 10 nm HGN with < 2 nm shell thickness were prepared from these templates with a range of surface plasmon resonances from 600 - 900 nm. The fluence of picosecond near infrared (NIR) pulses to induce transient vapor nanobubbles decreased with HGN size at a fixed LSPR wavelength, unlike solid gold nanoparticles of similar dimensions that require an increased fluence with decreasing size. Nanobubble generation causes the HGN to melt with a blue shift of the LSPR. The nanobubble threshold fluence increases as the irradiation wavelength moves off the nanoshell LSPR. Surface treatment did not influence the threshold fluence. The threshold fluence increased with decreasing HGN concentration, suggesting that light localization through multiple scattering plays a role. The nanobubble threshold to rupture liposomes is 4 times smaller for 10 nm than for 40 nm HGN at a given LSPR, allowing us to use HGN size, LSPR, laser wavelength and fluence to control nanobubble generation.

Modular plasmonic nanocarriers for efficient and targeted delivery of cancer-therapeutic siRNA.

We have combined a versatile and powerful route to deliver nucleic acids with peptide-based cell-specific targeting. siRNA targeting the polo-like kinase gene is in clinical trials for cancer treatment, and here we deliver this RNA selectively to cancer cells displaying the neuropilin-1 epitope using gold nanoshells. Release of the siRNA from the nanoparticles results from irradiation with a pulsed near-infrared laser, which also provides efficient endosomal escape within the cell. As a result, our approach requires 10-fold less material than standard nucleic acid transduction materials and is significantly more efficient than other particle-based methods. We also describe a particle-nucleic acid design that does not rely on modified RNA, thereby making the preparation of these materials more efficient and much less expensive. These improvements, when combined with control over when and where the siRNA is released, could provide the basis for diverse cell biological studies.

Nanoscale patterning of membrane-bound proteins formed through curvature-induced partitioning of phase-specific receptor lipids.

This work describes a technique for forming high-density arrays and patterns of membrane-bound proteins through binding to a curvature-organized compositional pattern of metal-chelating lipids (Cu(2+)-DOIDA or Cu(2+)-DSIDA). In this bottom-up approach, the underlying support is an e-beam formed, square lattice pattern of hemispheres. This curvature pattern sorts Cu(2+)-DOIDA to the 200 nm hemispherical lattice sites of a 600 nm × 600 nm unit cell in Ld - Lo phase separated lipid multibilayers. Binding of histidine-tagged green fluorescent protein (His-GFP) creates a high density array of His-GFP-bound pixels localized to the square lattice sites. In comparison, the negative pixel pattern is created by sorting Cu(2+)-DSIDA in Ld - Lß' phase separated lipid multibilayers to the flat grid between the lattice sites followed by binding to His-GFP. Lattice defects in the His-GFP pattern lead to interesting features such as pattern circularity. We also observe defect-free arrays of His-GFP that demonstrate perfect arrays can be formed by this method suggesting the possibility of using this approach for the localization of various active molecules to form protein, DNA, or optically active molecular arrays.

Compositional sorting dynamics in coexisting lipid bilayer phases with variations in underlying e-beam formed curvature pattern.

Nanometer-scale curvature patterns of an underlying substrate are imposed on lipid multibilayers with each pattern imparting distinctly different sorting dynamics to a metastable pixelation pattern of coexisting liquid ordered (Lo)-liquid disordered (Ld) lipid phases. Therefore, this work provides pathways toward mechanical energy-based separations for analysis of biomembrane-associate species. The central design concept of the patterned sections of the silica substrate is a square lattice pattern of 100 nm projected radius poly(methyl methacrylate) (PMMA) hemispherical features formed by electron beam lithography which pixelates the coexisting phases in order to balance membrane bending and line energy. In one variation, we surround this pattern with three PMMA walls/fences 100 nm in height which substantially slows the loss of the high line energy pixelated Lo phase by altering the balance of two competing mechanism (Ostwald ripening vs. vesiculation). In another walled variation, we form a gradient of the spacing of the 100 nm features which forces partitioning of the Lo phase toward the end of the gradient with the most open (400 nm spacing) lattice pattern where a single vesicle could grow from the Lo phase. We show that two other variations distinctly impact the dynamics, demonstrating locally slowed loss of the high line energy pixelated Lo phase and spontaneous switching of the pixel location on the unit cell, respectively. Moreover, we show that the pixelation patterns can be regenerated and sharpened by a heating and cooling cycle. We argue that localized variations in the underlying curvature pattern have rather complex consequences because of the coupling and/or competition of dynamic processes to optimize mechanical energy such as lipid diffusion, vesiculation and growth, and phase/compositional partitioning.

Metastability in pixelation patterns of coexisting fluid lipid bilayer phases imposed by e-beam patterned substrates.

We study the dynamic evolution of pixilation patterns of the liquid-ordered (Lo) phase in coexistence with the liquid-disordered phase in lipid multibilayers. The pixilation patterns were formed by imposing lattice patterns of localized high curvature on phase-separating multibilayers using curvature-patterned regions of an underlying support. The projected radius of underlying hemisphere-like features, that provided the local curvature, was varied from 60 nm to 100 nm and the square lattice spacing between the features was varied between 200 nm and 400 nm using standard electron (e) -beam lithography. Over time, the area fraction of the Lo phase on the patterned regions of the substrate decreased toward zero at room temperature. This apparent metastability of the pattern derives from the high line energy of a pixelation pattern where a Boltzmann distribution shows near zero equilibrium partitioning of the Lo phase in the patterned regions. Kinetic rate analysis identifies two pattern-dependent mechanisms that dominate the transition to zero Lo area fraction; diffusion limited dissolution of the Lo phase driven by an Ostwald ripening-type process or the cooperative formation of vesicles containing Lo phase lipids. Interestingly, we observed the spontaneous formation of tubules in the corners of the array due to the high local curvature applied to the membrane. Furthermore we show that it is possible to regenerate pixilation patterns on the curvature-patterned regions by cooling below room temperature. Regenerated area fractions are in agreement with a room-temperature composition of primarily Ld phase and the high degree of overlap with the original patterns is suggestive of fixed nucleation sites.

Lipid domain pixelation patterns imposed by e-beam fabricated substrates.

This work describes a technique for forming nanometer-scale pixilated lipid domains that are self-organized into geometric patterns residing on a square lattice. In this process, a lipid multibilayer stack is deposited onto a silica substrate patterned with a square lattice array of bumps, hemispherical on their sides, formed by electron beam lithography. Domain patterns are shown to be confined to the flat grid between the bumps and composed of connected and individual domain pixels. Analysis of lattices of varying sizes shows that domain pattern formation is driven by mechanical energy minimization and packing constraints. We demonstrate single lattice sizes and a gradient in lattice size varying from the micrometer to the 100 nm scale applicable to precise arraying, patterning, and transport of biomolecules that partition to lipid domains.