Oxidation of Membrane Lipids Alters the Activity of the Human Serotonin 1A Receptor.

Lipid oxidation has significant effects on lipid bilayer properties; these effects can be expected to extend to interactions between the lipid bilayer and integral membrane proteins. Given that G protein-coupled receptor (GPCR) activity is known to depend on the properties of the surrounding lipid bilayer, these proteins represent an intriguing class of molecules in which the impact of lipid oxidation on protein behavior is studied. Here, we study the effects of lipid oxidation on the human serotonin 1A receptor (5-HT1AR). Giant unilamellar vesicles (GUVs) containing integral 5-HT1AR were fabricated by the hydrogel swelling method; these GUVs contained polyunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinPC) and its oxidation product 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) at various ratios. 5-HT1AR-integrated GUVs were also fabricated from lipid mixtures that had been oxidized by extended exposure to the atmosphere. Both types of vesicles were used to evaluate 5-HT1AR activity using an assay to quantify GDP-GTP exchange by the coupled G protein α subunit. Results indicated that 5-HT1AR activity increases significantly in bilayers containing oxidized lipids. This work is an important step in understanding how hyperbaric oxidation can change plasma membrane properties and lead to physiological dysfunction.

Liposome production and concurrent loading of drug simulants by microfluidic hydrodynamic focusing.

Liposomes are spherical vesicles enclosed by phospholipid bilayers. Nanoscale liposomes are widely employed for drug delivery in the pharmaceutical industry. In this study, nanoscale liposomes are fabricated using the microfluidic hydrodynamic focusing (MHF) approach, and the effects of flow rate ratio (FRR) on liposome size and drug loading efficiency are studied. Fluorescein isothiocyanate modified dextran is used as a hydrophilic drug simulant and Nile red is used as a hydrophobic drug simulant. The experiment results show that hydrophilic drug simulant loading efficiency increases as FRR increases and eventually plateaues at around 90% loading efficiency. The hydrophobic drug simulant loading efficiency and FRR have a positive linear correlation when FRR varies from 10 to 50. Concurrent loading of both hydrophilic and hydrophobic drug simulants maintains the same loading efficiencies as those of loading each drug simulant alone. A negative correlation between liposome size and FRR is also confirmed. Unloaded liposomes and hydrophilic drug-loaded liposomes are of the same sizes, and are smaller than the ones loaded with the hydrophobic drug simulants alone or combined. The results suggest tunable liposome size and drug loading efficiency with the MHF technique. This provides evidence to encourage further studies of microfluidic liposome fabrication in the pharmaceutical industry.

Discrete elements for 3D microfluidics.

Microfluidic systems are rapidly becoming commonplace tools for high-precision materials synthesis, biochemical sample preparation, and biophysical analysis. Typically, microfluidic systems are constructed in monolithic form by means of microfabrication and, increasingly, by additive techniques. These methods restrict the design and assembly of truly complex systems by placing unnecessary emphasis on complete functional integration of operational elements in a planar environment. Here, we present a solution based on discrete elements that liberates designers to build large-scale microfluidic systems in three dimensions that are modular, diverse, and predictable by simple network analysis techniques. We develop a sample library of standardized components and connectors manufactured using stereolithography. We predict and validate the flow characteristics of these individual components to design and construct a tunable concentration gradient generator with a scalable number of parallel outputs. We show that these systems are rapidly reconfigurable by constructing three variations of a device for generating monodisperse microdroplets in two distinct size regimes and in a high-throughput mode by simple replacement of emulsifier subcircuits. Finally, we demonstrate the capability for active process monitoring by constructing an optical sensing element for detecting water droplets in a fluorocarbon stream and quantifying their size and frequency. By moving away from large-scale integration toward standardized discrete elements, we demonstrate the potential to reduce the practice of designing and assembling complex 3D microfluidic circuits to a methodology comparable to that found in the electronics industry.

The Functional Activity of the Human Serotonin 5-HT1A Receptor Is Controlled by Lipid Bilayer Composition.

Although the properties of the cell plasma membrane lipid bilayer are broadly understood to affect integral membrane proteins, details of these interactions are poorly understood. This is particularly the case for the large family of G protein-coupled receptors (GPCRs). Here, we examine the lipid dependence of the human serotonin 5-HT1A receptor, a GPCR that is central to neuronal function. We incorporate the protein in synthetic bilayers of controlled composition together with a fluorescent reporting system that detects GPCR-catalyzed activation of G protein to measure receptor-catalyzed oligonucleotide exchange. Our results show that increased membrane order induced by sterols and sphingomyelin increases receptor-catalyzed oligonucleotide exchange. Increasing membrane elastic curvature stress also increases this exchange. These results reveal the broad dependence that the 5-HT1A receptor has on plasma membrane properties, demonstrating that membrane lipid composition is a biochemical control parameter and highlighting the possibility that compositional changes related to aging, diet, or disease could impact cell signaling functions.

G Protein-Coupled Receptors Incorporated into Rehydrated Diblock Copolymer Vesicles Retain Functionality.

G protein-coupled receptor (GPCR) is incorporated into polymeric vesicles made up of diblock copolymer bilayers. Successfully incorporated GPCRs exhibit correct biased physiological orientation and respond to various ligands. After extended dehydrated storage via lyophilization and subsequent rehydration, diblock copolymer polymersomes retain their shape and incorporated GPCR retains its function.

Dynamics of Hydrogel-Assisted Giant Unilamellar Vesicle Formation from Unsaturated Lipid Systems.

While current research is centered on observing biophysical properties and phenomena in giant unilamellar vesicles (GUVs), little is known about fabrication parameters that control GUV formation. Using different lipids and rehydration buffers, we directly observe varying dynamics of hydrogel-assisted GUV formation via fluorescence microscopy. We observe the effects of buffer ionic strength, osmolarity, agarose density, and pH on the formation of GUVs using neutral and charged lipids. We find that increasing rehydration buffer ionic strength correlates with increased vesicle size and rate of GUV formation. Increasing buffer acidity increased the rate of GUV formation, while more basic environments slowed the rate. For buffers containing 500 mM sucrose, GUV formation was overall inhibited and only tubules formed. Observations of GUV formation dynamics elucidate parametric effects of charge, ionic strength, pH, and osmolarity, demonstrating the versatility of this biomimetic platform.

Addition of Cleaved Tail Fragments during Lipid Oxidation Stabilizes Membrane Permeability Behavior.

Lipid oxidation has been linked to plasma membrane damage leading to cell death. In previous work, we examined the effect of oxidation on bilayer permeability by replacing defined amounts of an unsaturated lipid species with the corresponding phospholipid product that would result from oxidative tail scission of that species. This study adds the cleaved tail fragment, better mimicking the chemical results of oxidation. Permeability of PEG12-NBD, a small, uncharged molecule, was measured for vesicles with oxidation concentration corresponding to between 0 and 18 mol % of total lipid content. Permeability was measured using a microfluidic trap to capture the vesicles and spinning disk confocal microscopy (SDCM) to measure the transport of fluorescent PEG12-NBD at the equatorial plane. The thicknesses of lipid bilayers containing oxidized species were estimated by measuring capacitance of a black lipid membrane while simultaneously measuring bilayer area. We found that relative to chemically modeled oxidized bilayers without tail fragments, bilayers containing cleaved tail groups were less permeable for the same degree of oxidation. Curiously, membrane capacitance measurements indicated that the addition of tail fragments to chemically modeled oxidized bilayers also thinned these bilayers relative to samples with no tail fragments; in other words, the more permeable membranes were thicker. Above 12.5% chemically modeled oxidation, compositions both with and without the cleaved tail groups showed pore formation. This work highlights the complexity of the relationship between chemically modeled lipid bilayer oxidation and cell membrane properties.

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions.

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles.

Low levels of lipid oxidation radically increase the passive permeability of lipid bilayers.

Oxidation of unsaturated lipids in cellular membranes has been shown to cause severe membrane damage and potentially cell death. The presence of oxidized lipid species in the membrane is known to cause changes in membrane properties, such as decreased fluidity. This study uses giant unilamellar vesicles (GUVs) to measure passive transport across membranes containing defined concentrations of oxidized lipid species. GUVs consisting of a saturated phospholipid, an unsaturated phospholipid, and cholesterol were used as model membranes. By replacing defined amounts of the unsaturated lipid with a corresponding oxidized product, the oxidation process could be mimicked, yielding vesicles of varying oxidized lipid concentration. Oxidized lipid concentration was varied from 0 mol% to 18 mol% of the total lipid concentration. Passive transport of PEG12-NBD, an uncharged fluorescent molecule, was measured using a microfluidic trap to capture the GUVs and spinning disk confocal microscopy (SDCM) to track the transport of a fluorescent species in the equatorial plane of each GUV. Membrane permeability was determined by fitting the resulting concentration profiles to a finite difference model of diffusion and permeation around and through the membrane. Experiments showed three permeability regimes. Without oxidation, transport was slow, with a measured permeability on the order of 1.5 × 10(-6) cm s(-1). At 2.5-10% oxidized species permeation was fast (1.5 × 10(-5) cm s(-1)). Above 12.5% oxidized species, the bilayer was disrupted by the formation of pore defects. As passive transport is an important mechanism for drug delivery, understanding the relationship between oxidation and permeation could provide insight into the pharmaceutical characteristics of tissues with oxidative damage.

Viscoelastic deformation of lipid bilayer vesicles.

Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic.

Human serotonin receptor 5-HT(1A) preferentially segregates to the liquid disordered phase in synthetic lipid bilayers.

We demonstrate successful incorporation of the G protein coupled receptor 5-HT1A into giant unilamellar vesicles using an agarose rehydration method. With direct observation using fluorescence techniques, we report preferential segregation of 5-HT1A into the cholesterol-poor liquid disordered phase of the membrane, contradicting previous reports of lipid raft segregation. Furthermore, altering the concentration of cholesterol and sphingomyelin in ternary mixtures does not alter 5-HT1A segregation into the liquid disordered phase.

3D-printed microfluidic device for high-throughput production of lipid nanoparticles incorporating SARS-CoV-2 spike protein mRNA.

Lipid nanoparticles (LNPs) are drug carriers for protecting nucleic acids for cellular delivery. The first mRNA vaccines authorized by the United States Food and Drug Administration are the mRNA-1273 (Moderna) and BNT162b (BioNTech/Pfizer) vaccines against coronavirus disease 2019 (COVID-19). We designed a 3D printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) device for producing mRNA-loaded LNPs that closely resemble the Moderna vaccine: we used the same lipid formulations to encapsulate mRNA encoding SARS-CoV-2 spike protein. The OSEM device is made of durable methacrylate-based materials that can support flow rates in the mL min-1 range and was fabricated by stereolithography (SLA), incorporating readily adaptable interfaces using commercial fluidic connectors. Two key features of the OSEM device are: 1) a 4-way hydrodynamic flow focusing region and 2) a staggered herringbone mixer (SHM). Superior to conventional planar fluid junctions, the 4-way sheath flow channel generates an evenly focused, circular center flow that facilitates the formation of LNPs with low polydispersity. Downstream, fluid mixing in the SHM is intensified by incorporating a zig-zag fluidic pathway to deliver high mRNA encapsulation efficiency. We characterized the mRNA-loaded LNPs produced in the OSEM device and showed that the enhanced 3D microfluidic structures enable a 5-fold higher throughput production rate (60 mL min-1) of LNPs compared to commercial multi-thousand-dollar micromixers. The device produced LNPs of diameter less than 90 nm, with low polydispersity (2-8%) and high mRNA encapsulation efficiency (>90%). The 3D-printed device provides a cost-effective and easily prepared solution for high-throughput LNP production.

Liposomes with double-stranded DNA anchoring the bilayer to a hydrogel core.

Liposomes are important biomolecular nanostructures for handling membrane-associated molecules in the lab and delivering drugs in the clinic. In addition to their biomedical applications, they have been widely used as model cell membranes in biophysical studies. Here we present a liposome-based model membrane that mimics the attachment of membrane-resident molecules to the cytoskeleton. To facilitate this attachment, we have developed a lipid-based hybrid nanostructure in which the liposome bilayer membrane is covalently anchored to a biocompatible poly(ethylene) glycol (PEG) hydrogel core using short double-stranded DNA (dsDNA) linkers. The dsDNA linkers connect cholesterol groups that reside in the bilayer to vinyl groups that are incorporated in the cross-linked hydrogel backbone. Size exclusion chromatography (SEC) of intact and surfactant-treated nanoparticles confirms the formation of anchored hydrogel structures. Transmission electron microscopy (TEM) shows ~100 nm nanoparticles even after removal of unanchored phospholipids. The location of dsDNA groups at the hydrogel-bilayer interface is confirmed with a fluorescence assay. Using DNA as a linker between the bilayer and a hydrogel core allows for temperature-dependent release of the anchoring interaction, produces polymer nanogels with addressible hybridization sites on their surface, and provides a prototype structure for potential future oligonucleotide drug delivery applications.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs).

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Imaging molecular transport across lipid bilayers.

Low-molecular-weight carboxylic acids have many properties common to small molecule drugs. The transport of these acids across cell membranes has been widely studied, but these studies have produced wildly varying permeability values. Recent reports have even claimed that the transport behavior of these drugs is contrary to the rule of thumb called Overton's rule, which holds that more lipophilic molecules transport across lipid membranes more quickly. We used confocal microscopy to image the transport of carboxylic acids with different lipophilicities into a giant unilamellar lipid vesicle (GUV). Fluorescein-dextran, which acts as a pH-sensitive dye, was encapsulated in the GUV to trace the transport of acid. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short (<100 ms) exposure. Results show that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive permeabilities. The permeabilities change with the same trend as literature oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules.

Mechanisms of alveolar epithelial translocation of a defined population of nanoparticles.

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Confocal imaging to quantify passive transport across biomimetic lipid membranes.

The ability of a molecule to pass through the plasma membrane without the aid of any active cellular mechanisms is central to that molecule's pharmaceutical characteristics. Passive transport has been understood in the context of Overton's rule, which states that more lipophilic molecules cross membrane lipid bilayers more readily. Existing techniques for measuring passive transport lack reproducibility and are hampered by the presence of an unstirred layer (USL) that dominates transport across the bilayer. This report describes assays based on spinning-disk confocal microscopy (SDCM) of giant unilamellar vesicles (GUVs) that allow for the detailed investigation of passive transport processes and mechanisms. This approach allows the concentration field to be directly observed, allowing membrane permeability to be determined easily from the transient concentration profile data. A series of molecules of increasing hydrophilicity was constructed, and the transport of these molecules into GUVs was observed. The observed permeability trend is consistent with Overton's rule. However, the values measured depart from the simple partition-diffusion proportionality model of passive transport. This technique is easy to implement and has great promise as an approach to measure membrane transport. It is optimally suited to precise quantitative measurements of the dependence of passive transport on membrane properties.

Asymmetric giant lipid vesicle fabrication.

Synthetic lipid bilayers have long been used as models of cell membranes. The compositional asymmetry in the eukaryotic plasma membrane is a key chemical characteristic of this membrane that has traditionally been difficult to reproduce in synthetic systems. In this chapter, we describe recent technologies for fabricating compositionally asymmetric giant unilamellar lipid vesicles (GUVs) and provide detailed protocols for a microfluidic-based fabrication technique.

Glucose transport machinery reconstituted in cell models.

Here we demonstrate the production of a functioning cell model by formation of giant vesicles reconstituted with the GLUT1 glucose transporter and a glucose oxidase and hydrogen peroxidase linked fluorescent reporter internally. Hence, a simplified artificial cell is formed that is able to take up glucose and process it.

"Smart" mobile affinity matrix for microfluidic immunoassays.

There is a current need for simple methods for immobilizing biomolecules within microfluidic channels. Here, a technique is reported for reversibly immobilizing immunoassay components in a channel zone that can be simply controlled by integrated heating elements. Latex beads were modified with the temperature-responsive polymer poly(N-isopropylacrylamide)(PNIPAAm) and co-modified with biotinylated poly(ethylene glycol)(PEG). PNIPAAm undergoes a hydrophilic-to-hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST)( approximately 28 degrees C in the solutions used here). This reversible transition drives the aggregation and dis-aggregation of the modified beads in heated zones within poly(ethylene terephthalate)(PET) microchannels. Biotinylated monoclonal antibodies for the drug digoxin were bound via streptavidin to the biotin-PEG-coated beads. These antibody-functionalized beads were then reversibly immobilized by aggregation and hydrophobic adhesion to the surface of PET microfluidic channels in response to a thermal stimulus. The antibodies on the beads immobilized in the channel were shown to bind digoxin and a competitor fluorescent ligand from a flow stream in a quantitative competitive assay format that reported the digoxin concentration. The antibodies could be replenished for each immunoassay trial, using the reversible, temperature-controlled immobilization process. This technique allows reagent immobilization immediately prior to an analytical procedure, following the removal of previously utilized beads, guaranteeing fresh and active immobilized biomolecules. Furthermore, it provides a simple approach to multiplexing through the simultaneous or sequential injection of different antibody-coated bead species, potentially at multiple sites in the integrated device channels.