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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.

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.

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 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.

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) 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.

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 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.

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.

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.

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.

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.

Although the presence of cholesterol, the major neutral lipid component, is well known in native surfactants (upto 10 % mass), its role in the surfactant remains uncertain. The most recently FDA approved clinical surfactant contain cholesterol, while two that have been used for 20 years have cholesterol carefully removed. However, they are all successful in treating neonatal respiratory distress syndrome (NRDS) resulting from a lack of surfactant. As a result the optimal concentration of cholesterol, if any at all, remains debated. Here we present indications for an optimal cholesterol concentration by presenting alterations to the phase and morphology of Survanta, a clinically used bovine lung surfactant extract, induced by both physiological and elevated concentrations of cholesterol when the monolayer is in contact with surfactant in the subphase. We find that low cholesterol concentrations (1-2 wt %) help to achieve a lower surface tension by enhancing surfactant material adsorption to the interface. However, increasing the cholesterol concentrations to higher values (≈ 20 wt %) significantly alters the normal surfactant isotherm. Alterations in a typical signature plateau for Survanta at ∼ 40 mN/m are noted suggesting a change in the solid phase fraction of the film. Fluorescence microscopic imaging reveals the coexistence of discrete monolayer along with “multilayer reservoir” adjacent to the air/water interface. Differences in the collapse structures of the monolayer are also noted indicating an alteration in the mechanical properties of the monolayer film. Alterations in these properties in the absence of surface active lung surfactant proteins were also observed indicating that interactions between the monolayer at the interface, the “multilayer reservoir” and the subphase are essential for the proper functioning of the lung surfactant.