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Therapeutic proteins are subjected to a variety of stresses during manufacturing, storage or administration, that often lead to undesired protein aggregation and particle formation. Ultrafiltration-diafiltration (UF-DF) processing of monoclonal antibodies (mAbs) is one such manufacturing step that has been shown to result in such physical degradation. In this work, we explore the use of different analytical techniques and lab-scale setups as methodologies to predict and rank-order the aggregation potential of four different mAbs during large-scale UF-DF processing. In the first part of the study, a suite of biophysical techniques was applied to assess differences in their inherent bulk protein properties including conformational and colloidal stability in a PBS buffer. Additionally, the inherent interfacial properties of these mAbs in PBS were measured using a Langmuir trough technique. In the next part of the study, several different scale-down lab models were evaluated including a lab bench-scale UF-DF setup, mechanical stress (shaking/stirring) studies in vials, and application of interfacial dilatational stress using a Langmuir trough to assess protein particle formation in different UF-DF processing buffers. Taken together, our results demonstrate the ability of a Langmuir-trough methodology to accurately predict the mAb instability profile observed during large scale UF-DF processing.

Cell therapies are hampered by a lack of available delivery systems, resulting in inconsistent outcomes in animal studies and human clinical trials. Hydrogel encapsulants offer a broad range of tunable characteristics in the design of cell delivery vehicles. The focus of the hydrogel field has been on durable encapsulants that provide long-term paracrine function of the cells. However, some cell therapies require cell-to-cell contact in order to elicit their effect. Controlled release microencapsulants would be beneficial in these situations, but appropriate polymers have not been adaptable to microsphere manufacturing because they harden too slowly. We developed and tested a novel microencapsulant formulation (acrylated hyaluronic acid: AHA) with degradation characteristics as a controlled release cell delivery vehicle. The properties of AHA microspheres were evaluated and compared to those of poly(ethylene glycol) diacrylate (PEGDA), a durable hydrogel. AHA microspheres possessed a higher swelling ratio, lower diffusion barrier, faster degradation rate, a lower storage modulus, and a larger average diameter than microspheres composed of PEGDA. Additionally, in vitro cell viability and release and short-term in vivo biocompatibility in immune competent Sprague-Dawley rats was assessed for each microsphere type. Compared to PEGDA, microspheres composed of AHA resulted in significantly less foreign body response in vivo as measured by a lack of cellularity or fibrotic ring in the surrounding tissue and no cellular infiltration into the microsphere. This study illustrates the potential of AHA microspheres as a degradable cell delivery system with superior encapsulated cell viability and biocompatibility with the surrounding tissue.

Formation of submicron and subvisible protein particles (0.1-100 μm) present a major obstacle during processing and storage of therapeutic proteins. While protein aggregation resulting in particle formation is well-understood in bulk solution, the mechanisms of aggregation due to interfacial stresses is less understood. Particularly, in this study, we focus on understanding the combined effect of temperature and application of interfacial dilatational stresses, on interface-induced protein particle formation, using two industrially relevant monoclonal antibodies (mAbs). The surface activity of Molecule C (MC) and Molecule B (MB) were measured at room temperature (RT) and 4 °C in the absence and presence of interfacial dilatation stress using a Langmuir trough. These results were correlated with Micro-flow imaging (MFI) to characterize formation of subvisible protein particles at the interface and in the bulk solution. Our results show that the surface activity for both proteins is temperature dependent. However, the extent of the impact of temperature on the mechanical properties of the monomolecular protein films when subjected to dilatational stresses is protein dependent. Protein particle analysis provided evidence that protein particles formed in bulk solution originate at the interface and are dependent on both application of thermal stresses and interfacial dilatational stresses. In the absence of any interfacial stresses, more and larger protein particles were formed at the interface at RT than at 4 °C. When mAb formulations are subjected to interfacial dilatational stresses, protein particle formation in bulk solution was found to be temperature dependent. Together our results validate that mAb solutions maintained at 4 °C can lower the surface activity of proteins and reduce their tendency to form interface-induced protein particles both in the absence and presence of interfacial dilatational stresses.

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their “real” biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.

Monoclonal antibodies (mAbs) have demonstrated tremendous potential as therapeutic products for various target diseases ranging from oncology to neurodegenerative diseases. However, aggregation of monomeric mAbs into higher-order structures is an undesired consequence of bioprocessing, transportation, and storage of these proteins. Interfacial stresses that arise from agitation, storage, interactions with the solid container walls, as well as interactions with the silicone oil that is used to lubricate the plunger in prefilled syringes have all been shown to contribute to aggregation of mAbs, although the exact mechanism of aggregation remains under investigation. In this chapter, we present the various interfaces encountered by monoclonal antibodies and discuss recent research efforts and analytical techniques that focus on how dilatation and shear stresses at the air-water interface can be related to formation of protein particles in bulk solutions. Newer techniques such as interfacial viscosity and rheology will also be discussed. Finally, we will review research efforts that focus on understanding the role of solid-liquid and liquid-liquid interfaces in this aggregation process.

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.

Successful gene therapy requires the development of vectors that enable efficient delivery of genetic materials (e.g., pDNA or siRNA) to targeted cells, without degradation of the genetic materials. We have shown that nanoparticles formed by combining cell-penetrating peptide and pDNA (CPP-pDNA) into complexes and condensing them with calcium chloride can provide gene nanoparticles with high transfection efficiency and low cytotoxicity. In this work, we compare in situ measurements of the membrane insertion potential of three arginine-based gene nanoparticles (RW9-NPs, R9-NPs, and RH9-NPs) using four lipid compositions and two types of model membrane (Langmuir monolayers vs. supported bilayers) with their transfection efficiency in two human cancer cell lines. Using a Langmuir trough, we measured the membrane insertion potential of our gene nanoparticles to model membrane monolayers. A Quartz Crystal Microbalance with Dissipation (QCM-D) technique was used to monitor the adsorption of these nanoparticles to lipid bilayers of various compositions. Finally, gene expression using these nanoparticles was measured in breast cancer and cervical cancer cell lines. Our cell culture studies indicate that although R9-NPs and RW9-NPs show a significant increase in transfection efficiency compared to free pDNA, RH9-NPs do not show any significant difference. Both the Langmuir monolayer and QCM-D bilayer studies show that these results are best reflected in the in situ measurement assays when lipid systems containing a mixture of phospholipids, cholesterol, and sphingolipids are used. It is important to note that the mechanism of penetration is expected to differ for RW9 vs. R9; however, gene nanoparticles containing either of these CPPs show similar transfection efficiency. Our results therefore demonstrate that the design of predictive assays for gene therapy using CPPs must involve carefully chosen model lipid membrane systems that accurately represent the varying compositions of cell membranes.

The material properties of tissues and their mechanical state is an important factor during development, disease, regenerative medicine and tissue engineering. Here we describe a microrheological measurement technique utilizing aggregates of microinjected ferromagnetic nickel particles to probe the viscoelastic properties of embryonic tissues. Quail embryos were cultured in a plastic incubator chamber located at the center of two pairs of crossed electromagnets. We estimate the Young’s modulus of the ECM-rich region separating the mesoderm and endoderm in Hamburger Hamilton stage 6-10 quail embryos as 300±100 Pa. We found a pronounced viscoelastic behavior consistent with a Zener (standard generalized solid) model. The viscoelastic response is about 45% of the total response, with a characteristic relaxation time of 1.3 sec.

The capacity for a soft material to combine remote sensing and remote actuation is highly desirable for many applications in soft robotics and wearable technologies. This work presents a silicone elastomer with a suspension of a small weight fraction of ferromagnetic nickel nanorods, which is capable of both sensing deformation and altering stiffness in the presence of an external magnetic field. Cylinders composed of silicone elastomer and 1% by weight nickel nanorods experience large increases in compressive modulus when exposed to an external magnetic field. Incremental compressions totaling 600 g of force applied to the same silicone-nanorod composites increase the magnetic field strength measured by a Hall effect sensor enabling the material to be used as a soft load cell capable of detecting the rate, duration, and magnitude of force applied. In addition, lattice structures are 3D printed using an ink composed of silicone elastomer and 1% by weight nickel nanorods, which possess the same sensing capacity.

Most vaccines contain aluminum adjuvants; however, their exact mechanism of action remains unclear. A novel mechanism by Shi and colleagues proposes aluminum adjuvants may enhance immune activation by binding and reorganizing lipids that are key components of lipid rafts. To better understand the specificity of interaction between aluminum adjuvants and the cell membrane lipids, we present a biophysical study of lipid domain clustering in simple model phospholipid monolayers containing dipalmitoyl-phosphatidylcholine (DPPC) and dioleoyl-phosphatidylcholine (DOPC) exposed to two aluminum adjuvants, Alhydrogel and Adju-Phos. Surface pressure measurements and fluorescence microscopy images verified aluminum adjuvant-induced increase in lipid domain size, even in the key lipid raft components. Additionally, adjuvant induced lipid clustering differed based on the physicochemical properties of the adjuvants. Alhydrogel appeared to reduce monolayer compressibility and insert into the monolayer, while Adju-Phos induced more significant changes in domain size, without compromising the integrity of the monolayer. The Alhydrogel and Adju-Phos-mediated reorganization of phospholipid domains reported here supports the new mechanistic paradigm proposed by Shi and co-workers, and further suggests that lipid clustering is induced even in simple phospholipid membranes. The results present the basis for future exploration into lipid-mediated mechanisms of action for adjuvants.

Understanding interactions between inhaled nanoparticles and lung surfactants (LS) present at the air-water interface in the lung, is critical to assessing the toxicity of these nanoparticles. Specifically, in this work, we assess the impact of engineered carbon nanoparticles (ECN) on the ability of healthy LS to undergo reversible collapse, which is essential for proper functioning of LS. Using a Langmuir trough, multiple compression-expansion cycles are performed to assess changes in the surface pressure vs. area isotherms with time and continuous cyclic compression-expansion. Further, theoretical analysis of the isotherms is used to calculate the ability of these lipid systems to retain material during monolayer collapse, due to interactions with ECNs. These results are complemented with fluorescence images of alterations in collapse mechanisms in these monolayer films. Four different model phospholipid systems, that mimic the major compositions of LS, are used in this study. Together, our results show that the ECN does not impact the mechanism of collapse. However, the ability to retain material at the interface during monolayer collapse, as well as re-incorporation of material after a compression-expansion cycle is altered to varying extent by ECNs and depends on the composition of the lipid mixtures.

The interactions among biomacromolecules within insect cuticle may offer new motifs for biomimetic material design. CPR27 is an abundant protein in the rigid cuticle of the elytron from Tribolium castaneum. CPR27 contains the Rebers-Riddiford (RR) motif, which is hypothesized to bind chitin. In this study, active magnetic microrheology coupled with microscopy and protein particle analysis techniques were used to correlate alterations in the viscosity of chitosan solutions with changes in solution microstructure. Addition of CPR27 to chitosan solutions led to a 3-fold drop in viscosity. This change was accompanied by the presence of micrometer-sized coacervate particles in solution. Coacervate formation had a strong dependence on chitosan concentration. Analysis showed the existence of a critical CPR27 concentration beyond which a significant increase in particle count was observed. These effects were not observed when a non-RR cuticular protein, CP30, was tested, providing evidence of a structure-function relationship related to the RR motif.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Summary CP30 is a major component protein in the rigid cuticle of the beetle elytron which we recently identified. It has a highly unusual amino acid sequence comprised largely of alternating blocks of 3–5 anionic and cationic amino acid residues (three charged residues, glutamic acid, arginine and histidine, make up 76% of the sequence) which suggest an unusual stuctural role. The secondary structure of CP30 was evaluated at a variety of pH, ionic strength and temperature conditions. Circular dichroism (CD), fluorescence spectroscopy and static and dynamic light scattering showed that the protein had an extended α-helical structure at pH 3, 5 and 7 which unfolded around 60°C and reformed upon cooling back to 10°C. The protein aggregated or adhered to the dialysis membrane at pH 5, low salt, which is close to the protein's isoelectric point (5.82), preventing analysis at that condition. Further studies aimed at solubilizing the protein near that condition may uncover evidence of assembly. While the structural function of CP30 is currently unknown, the sequence of CP30 may offer new design motifs for the development of novel polymer or gel biomaterials.

Lipid membranes, a major component of cells, are subjected to significant changes in pH depending on their location in the cell: the outer leaflet of the cell membrane is exposed to a pH of 7.4 whereas lipid membranes that make up late endosomes and lysosomes are exposed to a pH of as low as 4.4. The purpose of this study is to evaluate how changes in the environmental pH within cells alter the fluidity of phospholipid membranes. Specifically, we studied pH-induced alterations in the surface arrangement of monounsaturated lipids with zwitterionic headgroups (phosphoethanolamine (PE) and phosphocholine (PC)) that are abundant in plasma membranes as well as anionic lipids (phosphatidylserine (PS) and phosphatidylglycerol (PG)) that are abundant in inner membranes using a combination of techniques including surface tension vs area measurements, interfacial microrheology, and fluorescence/atomic force microscopy. Using an active interfacial microrheology technique, we find that phospholipids with zwitterionic headgroups show a significant increase in their surface viscosity at acidic pH. This increase in surface viscosity is also found to depend on the size of the lipid headgroup, with a smaller headgroup showing a greater increase in viscosity. The observed pH-induced increase in viscosity is also accompanied by an increase in the cohesion pressure between zwitterionic molecules at acidic pH and a decrease in the average molecular area of the lipids, as measured by fitting the surface pressure isotherms to well-established equations of state. Because fluorescent images show no change in the phase of the lipids, we attribute this change in surface viscosity to the pH-induced reorientation of the P

Cell-penetrating peptides (CPPs) are often used as transporter systems to deliver various therapeutic agents into the cell. We developed a novel machine learning application which can quantitatively screen the insertion/interaction potential of various CPPs into three model phospholipid monolayers. An artificial neural network (ANN) was designed, trained, and ultimately tested on an external dataset using Langmuir experimental data for 13 CPPs (hydrophilic and amphiphilic) together with various features related to the insertion/interaction efficiency of CPPs. The trained ANN provided accurate predictions of the maximum change in surface pressure of CPPs when injected below three membrane models at pH 7.4. The accuracy of predictions was high for the dataset which was used to construct the model (r2 = 0.986) as well as for the external “prospective” dataset (r2 = 0.969). In conclusion, this study demonstrates the promising potential of ANNs for screening the insertion potential of CPPs into membrane models for efficient intracellular delivery of therapeutic agents.

Cell penetrating peptides (CPPs) have been established as excellent candidates for mediating drug delivery into cells. When designing synthetic CPPs for drug delivery applications, it is important to understand their ability to penetrate the cell membrane. In this paper, anionic or zwitterionic phospholipid monolayers at the air-water interface are used as model cell membranes to monitor the membrane insertion potential of synthetic CPPs. The insertion potential of CPPs having different cationic and hydrophobic amino acids were recorded using a Langmuir monolayer approach that records peptide adsorption to model membranes. Fluorescence microscopy was used to visualize alterations in phospholipid packing due to peptide insertion. All CPPs had the highest penetration potential in the presence of anionic phospholipids. In addition, two of three amphiphilic CPPs inserted into zwitterionic phospholipids, but none of the hydrophilic CPPs did. All the CPPs studied induced disruptions in phospholipid packing and domain morphology, which were most pronounced for amphiphilic CPPs. Overall, small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Designers of synthetic CPPs for efficient intracellular drug delivery should consider small nuances in CPP electrostatic and hydrophobic properties.

Noncovalent complexation of plasmid DNA (pDNA) with cell-penetrating peptides (CPPs) forms relatively large complexes with poor gene expression. Yet, condensing these CPP-pDNA complexes via addition of calcium chloride produces small and stable nanoparticles with high levels of gene expression. This simple formulation offered high transfection efficiency and negligible cytotoxicity in HEK-293 (a virus-immortalized kidney cell) and A549 (a human lung cancer cell line). Small changes in CPP charge type, charge spacing, and hydrophobicity were studied by using five arginine-rich CPPs: the well-known hydrophilic polyarginine R9 peptide, a hydrophilic RH9 peptide, and three amphiphilic peptides (RA9, RL9, and RW9) with charge distributions that favor membrane penetration. R9 and RW9 nanoparticles were significantly more effective than the other CPPs under most formulation conditions. However, these CPPs exhibit large differences in membrane penetration potential. Maximum transfection resulted from an appropriate balance of complexing with pDNA, releasing DNA, and membrane penetration potential.

The overall goal of this work is to study the combined effects of Mini-B, a 34 residue synthetic analog of the lung surfactant protein SP-B, and cholesterol, a neutral lipid, on a model binary lipid mixture containing dipalmitolphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), that is often used to mimic the primary phospholipid composition of lung surfactants. Using surface pressure vs. mean molecular area isotherms, fluorescence imaging and analysis of lipid domain size distributions; we report on changes in the structure, function and stability of the model lipid-protein films in the presence and absence of varying composition of cholesterol. Our results indicate that at low cholesterol concentrations, Mini-B can prevent cholesterol's tendency to lower the line tension between lipid domain boundaries, while maintaining Mini-B's ability to cause reversible collapse resulting in the formation of surface associated reservoirs. Our results also show that lowering the line tension between domains can adversely impact monolayer folding mechanisms. We propose that small amounts of cholesterol and synthetic protein Mini-B can together achieve the seemingly opposing requirements of efficient LS: fluid enough to flow at the air-water interface, while being rigid enough to oppose irreversible collapse at ultra-low surface tensions.

The size distribution of domains in phase-separated lung surfactant monolayers influences monolayer viscoelasticity and compressibility which, in turn, influence monolayer collapse and set the compression at which the minimum surface tension is reached. The surfactant-specific protein SP-B decreases the mean domain size and polydispersity as shown by fluorescence microscopy. From the images, the line tension and dipole density difference are determined by comparing the measured size distributions with a theory derived by minimizing the free energy associated with the domain energy and mixing entropy. We find that SP-B increases the line tension, dipole density difference, and the compressibility modulus at surface pressures up to the squeeze-out pressure. The increase in line tension due to SP-B indicates the protein avoids domain boundaries due to its solubility in the more fluid regions of the film.

Tracking the motion of lipid domains on a vesicle is a rheological technique allowing the measurement of surface shear viscosities of vesicular lipid phases. The ratio of surface to bulk viscosity defines a viscous length scale. Hydrodynamic interactions split the motion of the domains into different modes of diffusion. The measurability of surface shear viscosities from any mode of diffusion is limited to viscous length scales between the radius of the domains and the radius of the vesicle. The measurability of the surface shear viscosity results from the sensitivity of the diffusion to surface shear viscosities and from sufficient spatial resolution to resolve the diffusive motion. Switching between the various modes of diffusion is a trade between sensitivity gained and resolution lost by the hydrodynamic interactions leaving the measurability unchanged. Measurability drops with the number of domains making single-domain rheology the best technique to measure surface shear viscosities. Ultimately confinement of the domains to small vesicles renders measurements of surface rheological properties with domain-tracking rheology impossible. Experiments on domains in vesicles of a mixture of dioleoylphosphatidylcholine (DOPC), dipalmytoylphosphatidylcholin (DPPC) and cholesterol (Chol) exhibit diffusion that is entirely controlled by dissipation into the water. The diffusion is suppressed compared to the diffusion of isolated domains in a flat membrane due to confinement to the curved vesicle and by hydrodynamic interactions between the domains. Effects of surface shear viscosity can be neglected.

Lung surfactant (LS) is a mixture of lipids and proteins that line the alveolar air–liquid interface, lowering the interfacial tension to levels that make breathing possible. In acute respiratory distress syndrome (ARDS), inactivation of LS is believed to play an important role in the development and severity of the disease. This review examines the competitive adsorption of LS and surface-active contaminants, such as serum proteins, present in the alveolar fluids of ARDS patients, and how this competitive adsorption can cause normal amounts of otherwise normal LS to be ineffective in lowering the interfacial tension. LS and serum proteins compete for the air–water interface when both are present in solution either in the alveolar fluids or in a Langmuir trough. Equilibrium favors LS as it has the lower equilibrium surface pressure, but the smaller proteins are kinetically favored over multi-micron LS bilayer aggregates by faster diffusion. If albumin reaches the interface, it creates an energy barrier to subsequent LS adsorption that slows or prevents the adsorption of the necessary amounts of LS required to lower surface tension. This process can be understood in terms of classic colloid stability theory in which an energy barrier to diffusion stabilizes colloidal suspensions against aggregation. This analogy provides qualitative and quantitative predictions regarding the origin of surfactant inactivation. An important corollary is that any additive that promotes colloid coagulation, such as increased electrolyte concentration, multivalent ions, hydrophilic non-adsorbing polymers such as PEG, dextran, etc. added to LS, or polyelectrolytes such as chitosan, also promotes LS adsorption in the presence of serum proteins and helps reverse surfactant inactivation. The theory provides quantitative tools to determine the optimal concentration of these additives and suggests that multiple additives may have a synergistic effect. A variety of physical and chemical techniques including isotherms, fluorescence microscopy, electron microscopy and X-ray diffraction show that LS adsorption is enhanced by this mechanism without substantially altering the structure or properties of the LS monolayer.

Mechanical agitation of monoclonal antibody (mAb) solutions often leads to protein particle formation. In this study, various formulations of an immunoglobulin G (IgG) 1 mAb were subjected to different controlled interfacial stresses using a Langmuir trough, and protein particles formed at the interface and measured in bulk solution were characterized using atomic force microscopy and flow digital imaging. Results were compared to mAb solutions agitated in glass vials and unstressed controls. At lower pH, mAb solutions exhibited larger hysteresis in their surface pressure versus area isotherms and increased number of particles in bulk solution, when subjected to interfacial stresses. mAb samples subjected to 750-1000 interfacial compression-expansion cycles in 6 h contained high particle numbers in bulk solution, and displayed similar particulation trends when agitated in vials. At compression rates of 50 cycles in 6 h, however, particle levels in mAb solutions were comparable to unstressed controls, despite protein aggregates being present at the air-solution interface. These results suggest that while the air-solution interface serves as a nucleation site for initiating protein aggregation, the number of protein particles measured in bulk mAb solutions depends on the total number of compression cycles that proteins at the air-solution interface are subjected to within a fixed time.

The focus of this work is to elucidate how phospholipid composition can modulate lipid nanoparticle interactions in phospholipid monolayer systems. We report on alterations in lipid domain formation induced by anionically engineered carbon nanodiamonds (ECNs) as a function of lipid headgroup charge and alkyl chain saturation. Using surface pressure vs area isotherms, monolayer compressibility, and fluorescence microscopy, we found that anionic ECNs induced domain shape alterations in zwitterionic phosphatidylcholine lipids, irrespective of the lipid alkyl chain saturation, even when the surface pressure vs area isotherms did not show any significant changes. Bean-shaped structures characteristic of dipalmitoylphosphatidylcholine (DPPC) were converted to multilobed, fractal, or spiral domains as a result of exposure to ECNs, indicating that ECNs lower the line tension between domains in the case of zwitterionic lipids. For membrane systems containing anionic phospholipids, ECN-induced changes in domain packing were related to the electrostatic interactions between the anionic ECNs and the anionic lipid headgroups, even when zwitterionic lipids are present in excess. By comparing the measured size distributions with our recently developed theory derived by minimizing the free energy associated with the domain energy and mixing entropy, we found that the change in line tension induced by anionic ECNs is dominated by the charge in the condensed lipid domains. Atomic force microscopy images of the transferred anionic films confirm that the location of the anionic ECNs in the lipid monolayers is also modulated by the charge on the condensed lipid domains. Because biological membranes such as lung surfactants contain both saturated and unsaturated phospholipids with different lipid headgroup charges, our results suggest that when studying potential adverse effects of nanoparticles on biological systems the role of lipid compositions cannot be neglected.

Noncovalently condensed complexes of genetic material, cell penetrating peptides (CPPs), and calcium chloride present a nonviral route to improve transfection efficiency of nucleic acids (e.g., pDNA and siRNA). However, the exact mechanisms of membrane insertion and delivery of macromolecule complexes to intracellular locations as well as their stability in the intracellular environment are not understood. We show that calcium condensed gene complexes containing different hydrophilic (i.e., dTAT, K9, R9, and RH9) and amphiphilic (i.e., RA9, RL9, and RW9) CPPs formed stable cationic complexes of hydrodynamic radii 100 nm at neutral pH. However, increasing the acidity caused the complexes to become neutral or anionic and increase in size. Using zwitterionic and anionic phospholipid monolayers as models that mimic the membrane composition of the outer leaflet of cell membranes and intracellular vesicles and pHs that mimic the intracellular environment, we study the membrane insertion potential of these seven gene complexes (CPP/pDNA/Ca(2+) complexes) into model membranes. At neutral pH, all gene complexes demonstrated the highest insertion potential into anionic phospholipid membranes, with complexes containing amphiphilic peptides showing the maximum insertion. However, at acidic pH, the gene complexes demonstrated maximum monolayer insertion into zwitterionic lipids, irrespective of the chemical composition of the CPP in the complexes. Our results suggest that in the neutral environment the complexes are unable to penetrate the zwitterionic lipid membranes but can penetrate through the anionic lipid membranes. However, the acidic pH mimicking the local environment in the late endosomes leads to a significant increase in adsorption of the complexes to zwitterionic lipid headgroups and decreases for anionic headgroups. These membrane-gene complex interactions may be responsible for the ability of the complexes to efficiently enter the intracellular environment through endocytosis and escape from the endosomes to effectively deliver their genetic payload.

This is the published version. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

This is the published version. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

We study the autonomous motion of catalytic nanorods in Gibbs monolayers. The catalytic activity of the rods on a hydrogen peroxide aqueous subphase gives rise to anomalous translational and rotational diffusion. The rods perform a Levy-walk superdiffusive motion that can be decomposed into thermal orientation fluctuations and an active motion of the rods with a constant velocity along their long axis. Since interfacial dissipation increases relative to bulk phase dissipation when miniaturizing the size of objects moving in the interface, the autonomous nanorods allow for precise measurements of surface shear viscosities as low as a few nN s/m. The cross over from active motion toward passive diffusion when increasing the surfactant concentration is explained by a loss of friction asymmetry of the rods.

Modern materials science requires efficient processing and characterization techniques for low dimensional systems. Raman spectroscopy is an important non-destructive tool, which provides enormous information on these materials. This understanding is not only interesting in its own right from a physicist's point of view, but can also be of considerable importance in optoelectronics and device applications of these materials in nanotechnology. The commercial Raman spectrometers are quite expensive. In this article, we have presented a relatively less expensive set-up with home-built collection optics attachment. The details of the instrumentation have been described. Studies on four classes of nanostructures - Ge nanoparticles, porous silicon (nanowire), carbon nanotubes and 2D InGaAs quantum layers, demonstrate that this unit can be of use in teaching and research on nanomaterials.

Lung surfactant is crucial for optimal pulmonary function throughout life. An absence or deficiency of surfactant can affect the surfactant pool leading to respiratory distress. Even if the coupling between surfactant dysfunction and the underlying disease is not always well understood, using exogenous surfactants as replacement is usually a standard therapeutic option in respiratory distress. Exogenous surfactants have been extensively studied in animal models and clinical trials. The present article provides an update on the evolution of surfactant therapy, types of surfactant treatment, and development of newer-generation surfactants. The differences in the performance between various surfactants are highlighted and advanced research that has been conducted so far in developing the optimal delivery of surfactant is discussed.

The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid-general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid-general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step.

We present a study of static and dynamic interfacial properties of self-assembled polyelectrolyte complex nanoparticles (size 110-120 nm) containing entrapped surfactant molecules at a fluid/fluid interface. Surface tension vs time measurements of an aqueous solution of these polyelectrolyte complex nanoparticles (PCNs) show a concentration-dependent biphasic adsorption to the air/water interface while interfacial microrheology data show a concentration-dependent initial increase in the surface viscosity (up to 10(-7) N·m/s), followed by a sharp decrease (10(-9) N·m/s). Direct visualization of the air/water interface shows disappearance of particles from the interface over time. On the basis of these observations, we propose that the PCNs at fluid/fluid interfaces exist in two states: initial accumulation of PCNs at the air/water interface as nanoparticles, followed by interface induced disassembly of the accumulated PCNs into their components. The lack of change in particle size, charge, and viscosity of the bulk aqueous solution of PCNs with time indicates that this disintegration of the self-assembled PCNs is an interfacial phenomenon. Changes in energy encountered by the PCNs at the interface lead to instability of the self-assembled system and dissociation into its components. Such systems can be used for applications requiring directed delivery and triggered release of entrapped surfactants or macromolecules at fluid/fluid interfaces.

We present alterations in the phase, morphology and mechanical properties of a native replacement surfactant film induced either by the presence or absence of surfactant specific proteins SP-B and SP-C. Using Langmuir isotherms and fluorescence microscopy, the individual lipid-protein interactions in a complicated native surfactant system are explored. The surface tension lowering property of SurvantaTM, a native surfactant, is significantly compromised in the absence of the proteins, as is the ability of the film to undergo reversible collapse. A lack of proteins also causes the characteristic shoulder, prevalent at ∼ 40 mN/m in most lung surfactant mixtures, to disappear. A lack of this characteristic shoulder illustrates the inability of the film to undergo reversible squeeze out by forming “surface associated surfactant reservoirs”. Addition of SP-B causes an increase in the amount of surfactant material adsorbed from the sub-phase. Further it increases the monolayer stability and the compressibility modulus of the protein deficient film. SP-B is therefore responsible for helping the film achieve a high enough surface pressure during compression, as well as quick re-absorption of material during expansion. SP-C plays a dominant role in the formation of bilayer patches containing unsaturated lipids. SP-C also changes the mechanisms of monolayer collapse, and the film collapses via the formation of reversible collapse cracks. However, it is only in the presence of both SP-B and SP-C that the monolayer films are able to perform all the biophysical functions necessary for the proper working of the lung surfactant. These observations provide conclusive evidence showing that both SP-B and SP-C have distinct biophysical functions in the lung surfactant system, making them equally necessary for the long term survival of air-breathing mammals.

The magnetically driven rotation of 300 nm diameter rods shows the surface viscosity of albumin at an air-water interface increases from 10−9 to 10−5 Ns/m over two hours while the surface pressure saturates in minutes. The increase in surface viscosity is not accompanied by a corresponding increase in elasticity, suggesting that the protein film anneals with time, resulting in a more densely packed film leading to increased resistance to shear. The nanometer dimensions of the rods provide the same sensitivity as passive microrheology with an improved ability to measure more viscous films.

Electrostatic interactions strongly affect the immersion depth of nanoparticles into an interface. We prove this statement by measuring the diffusion constant of charged nanoparticles at a sodium chloride solution/air interface. Interfacial diffusion of nanoparticles slows down with increasing ionic strength of the sodium chloride solution. Hydrodynamic calculations are used to estimate the immersion depth from the diffusion constant, suggesting that nanoparticles with a carboxylate surface are only slightly immersed into a bare air/water interface. With increasing molarities of sodium chloride, the immersion depth increases to complete immersion for a 10(-2) molar solution. Our experiments show that the location of nanoparticles at interfaces is determined by an intricate interplay between the electrostatic properties of the solution/air interface, the solution/solid interface, and the classical contact angle.

Magnetic fields varying on the colloidal length scale are used for the directed transport of magnetically labeled biological cells. The transport is achieved by using the ratchet effect which relies on an asymmetric, symmetry broken periodic potential where nonequilibrium fluctuations or oscillations generate a net cell current. Ferrofluid ingested mouse macrophages were placed on a magnetic garnet film with alternating stripe domain patterns, and a pulsating magnetic potential is provided by superposing an oscillating magnetic field normal to the film. The symmetry of the resulting periodic stripe potential is broken locally by the curvature of the stripes. We show, both experimentally and theoretically, the curvature of such stripes required for inducing directed transport of the macrophages in the ratchet. This may be useful for microfluidic devices such as a digital colloidal shift register for magnetically labeled biological cells.

Overdamped magnetic nanorod-gyroscopes driven by a rotating magnetic field undergo a series of reorientations when sedimenting on top of a surface in a viscous liquid. By changing the amplitude and the rotation frequency of the driving magnetic field, the nanorod-gyroscope either synchronizes or desynchronizes with the field and rotates either around its long or short axis. The different regimes of motion are explained theoretically by coupling the nanorod-gyroscopes motion to the creeping flow equations of the surrounding fluid. It is shown that friction anisotropy plays an important role for the orientation of the nanorod-gyroscopes.

Cell penetrating peptides (CPPs) have been established as excellent candidates for mediating drug delivery into cells. When designing synthetic CPPs for drug delivery applications, it is important to understand their ability to penetrate the cell membrane. In this paper, anionic or zwitterionic phospholipid monolayers at the air-water interface are used as model cell membranes to monitor the membrane insertion potential of synthetic CPPs. The insertion potential of CPPs having different cationic and hydrophobic amino acids were recorded using a Langmuir monolayer approach that records peptide adsorption to model membranes. Fluorescence microscopy was used to visualize alterations in phospholipid packing due to peptide insertion. All CPPs had the highest penetration potential in the presence of anionic phospholipids. In addition, two of three amphiphilic CPPs inserted into zwitterionic phospholipids, but none of the hydrophilic CPPs did. All the CPPs studied induced disruptions in phospholipid packing and domain morphology, which were most pronounced for amphiphilic CPPs. Overall, small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Designers of synthetic CPPs for efficient intracellular drug delivery should consider small nuances in CPP electrostatic and hydrophobic properties.

Biological processes offer several intriguing examples of directed self-assembly into higher order structures that influence human health and well-being. The aggregation of microtubule associating protein Tau, an intrinsically disordered soluble protein that forms neurofibrillar tangles in cells in Alzheimer's disease may be directed by the presence of hydrophobic templates. Using various lipid monolayers and lipid-free surfaces we monitor the adsorption and aggregation kinetics of Tau proteins to these model membranes. Monitoring the surface tension change shows a two-step adsorption process followed by these proteins. A novel microrheology and quartz crystal microbalance study shows that the first step is dominated by the N-terminus of peptide, while the second step is possibly due to an intermediate hair-pin structure. Our microrheology data also show that the viscoelastic properties of these proteins is dominated by the proteins propensity to aggregate.

Increased manufacturing of engineered nanoparticles (ENPs) with commercial and biomedical applications has led to an increase in the release of these particles into the environment. Since the pulmonary route is an obvious one for the intake of these nanoparticles, concerns regarding potential health risks of these NPs has also seen a rapid increase in recent years.In this work, we describe the interaction of an engineered carbon nanoparticle (nanodiamond ENDs) with a model lung surfactant system (DPPC) both as a function of time of exposure and concentration. Using surface pressure vs. area per molecule coupled with fluorescence microscopy, we study lipid/END interactions for several weeks. Our results indicate that over short times (0-2 days exposure) the ENDs do not significantly affect the biophysical properties of this model surfactant. However after longer exposure, the ENDs begin to alter the surface tension lowering ability and domain morphology of DPPC. Our results indicate that long term exposure to these ENDs cause a lowering of line tension of the film, in a manner similar to interactions between DPPC and small (

Abnormal aggregation of microtubule associating protein, tau, into neurofibrillar aggregates, due to protein mutations, is a defining hallmark of several neurological diseases. Recent research indicates that the polymerization of soluble tau proteins into paired helical filaments may be influenced by the hydrophobic properties of its monomers, the presence of inducers and the local environment. In this work, we will discuss our results from using five HTau 40 protein variations: wild type (WT), pseudophosphorylated (7-phos), mutations on the binding domain (P301L), assembly incompetent protein (I277/308P), and mutations on the N terminal (R5L), to study template induced adsorption and aggregation of Tau proteins at a model hydrophobic interface.Traditional biophysical techniques such as surface pressure vs. time (adsorption isotherms) are used to record adsorption kinetics. We find that even though tau is a soluble protein, it is highly surface active at nanomolar concentrations and demonstrate a two-step adsorption to the hydrophobic interface. Further, the adsorption kinetics is dependent both on the concentration and protein mutation. However, almost all the proteins studied here demonstrate a saturation concentration of a few hundred nanomoles, which is much lower than the bulk concentration where protein aggregation is recorded. Using an active microrheology technique unique to our lab, we also find that surface viscosity of the adsorbed protein films increase by orders of magnitude with time, indicating protein-protein interactions. However, the kinetics of this increase depends on the mutations on the protein. Further, TEM images of the protein solution obtained from the surface indicate the formation of protein oligomers. In summary, our results indicate that the soluble Tau proteins have interesting surfactant properties even at nanomolar concentrations that may play a role in their aggregation during Alzheimer's disease.