Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size.

Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the single-nanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r2, being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of self-inserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.

Contactless Stimulation and Control of Biomimetic Nanotubes by Calcium Ion Gradients.

Membrane tubular structures are important communication pathways between cells and cellular compartments. Studying these structures in their native environment is challenging, due to the complexity of membranes and varying chemical conditions within and outside of the cells. This work demonstrates that a calcium ion gradient, applied to a synthetic lipid nanotube, triggers lipid flow directed toward the application site, resulting in the formation of a bulge aggregate. This bulge can be translated in a contactless manner by moving a calcium ion source along the lipid nanotube. Furthermore, entrapment of polystyrene nanobeads within the bulge does not tamper the bulge movement and allows transporting of the nanoparticle cargo along the lipid nanotube. In addition to the synthetic lipid nanotubes, the response of cell plasma membrane tethers to local calcium ion stimulation is investigated. The directed membrane transport in these tethers is observed, but with slower kinetics in comparison to the synthetic lipid nanotubes. The findings of this work demonstrate a novel and contactless mode of transport in lipid nanotubes, guided by local exposure to calcium ions. The observed lipid nanotube behavior can advance the current understanding of the cell membrane tubular structures, which are constantly reshaped during dynamic cellular processes.

Membrane Tubulation in Lipid Vesicles Triggered by the Local Application of Calcium Ions.

Experimental and theoretical studies on ion-lipid interactions predict that binding of calcium ions to cell membranes leads to macroscopic mechanical effects and membrane remodeling. Herein, we provide experimental evidence that a point source of Ca2+ acting upon a negatively charged membrane generates spontaneous curvature and triggers the formation of tubular protrusions that point away from the ion source. This behavior is rationalized by strong binding of the divalent cations to the surface of the charged bilayer, which effectively neutralizes the surface charge density of outer leaflet of the bilayer. The mismatch in the surface charge density of the two leaflets leads to nonzero spontaneous curvature. We probe this mismatch through the use of molecular dynamics simulations and validate that calcium ion binding to a lipid membrane is sufficient to generate inward spontaneous curvature, bending the membrane. Additionally, we demonstrate that the formed tubular protrusions can be translated along the vesicle surface in a controlled manner by repositioning the site of localized Ca2+ exposure. The findings demonstrate lipid membrane remodeling in response to local chemical gradients and offer potential insights into the cell membrane behavior under conditions of varying calcium ion concentrations.

A heating-superfusion platform technology for the investigation of protein function in single cells.

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.

A rapid microfluidic technique for integrated viability determination of adherent single cells.

Here, we report on a novel protocol for determining the viability of individual cells in an adherent cell culture, without adversely affecting the remaining cells in the sample. This is facilitated using a freestanding microfluidic perfusion device, the Multifunctional Pipette (MFP), which generates a virtual flow cell around selected single cells. We investigated the utility on four different cell lines, NG108-15, HEK 293, PC12, and CHO, and combined the assay with a cell poration experiment, in which we apply the pore-forming agent digitonin, followed by fluorescein diphosphate, a pre-fluorescent substrate for alkaline phosphatase, in order to monitor intracellular enzyme activity. The cell viability was instantly assessed through simultaneous perfusion with fluorescein diacetate (FDA) and propidium iodide (PI), both being dispensed through the same superfusion device used to porate and deliver the enzyme substrate. In this fluorescence assay, viable and non-viable cells were distinguished by their green and red emission, respectively, within 10 s. In addition, the enzyme activity was monitored over time as a secondary test for cellular activity. Our findings demonstrate that this microfluidic technology-assisted approach is a facile, rapid, and reliable means to determine the viability in single-cell experiments and that viability studies can be performed routinely alongside typical substrate delivery protocols. This approach would remove the need for global cell viability testing and would enable viability studies of only the cells under experimental analysis.

Probing enzymatic activity inside single cells.

We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 µM ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.

Radial sizing of lipid nanotubes using membrane displacement analysis.

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%.

Membrane protrusion coarsening and nanotubulation within giant unilamellar vesicles.

Hydrophobic side groups on a stimuli-responsive polymer, encapsulated within a single giant unilamellar vesicle, enable membrane attachment during compartment formation at elevated temperatures. We thermally modulated the vesicle through implementation of an IR laser via an optical fiber, enabling localized directed heating. Polymer-membrane interactions were monitored using confocal imaging techniques as subsequent membrane protrusions occurred and lipid nanotubes formed in response to the polymer hydrogel contraction. These nanotubes, bridging the vesicle membrane to the contracting hydrogel, were retained on the surface of the polymer compartment, where they were transformed into smaller vesicles in a process reminiscent of cellular endocytosis. This development of a synthetic vesicle system containing a stimuli-responsive polymer could lead to a new platform for studying inter/intramembrane transport through lipid nanotubes.

Rational antibody design for undruggable targets using kinetically controlled biomolecular probes.

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

Ultrasensitive and high-throughput fluorescence analysis of droplet contents with orthogonal line confocal excitation.

This paper describes a simple modification to traditional confocal fluorescence detection that greatly improves signal-to-noise (s/n) for the high-speed analysis of droplet streams. Rather than using the conventional epi geometry, illumination of the droplet was in the form of a line that is orthogonal to both the direction of flow and the light-collection objective. In contrast to the epi geometry where we observed high levels of scattering background from the droplets, we detected more than 10-fold less background (depending on the laser power used) when orthogonal-line-confocal illumination was used. We characterized this improvement using a standard microfluidic platform over a range of analyte concentrations and observed an improvement in limits of detection of greater than 10. Using this method, we were able to analyze picomolar concentrations of analytes contained within picoliter-volume droplets at a rate of greater than 350 droplets per second.

3D micro-organisation printing of mammalian cells to generate biological tissues.

Significant strides have been made in the development of in vitro systems for disease modelling. However, the requirement of microenvironment control has placed limitations on the generation of relevant models. Herein, we present a biological tissue printing approach that employs open-volume microfluidics to position individual cells in complex 2D and 3D patterns, as well as in single cell arrays. The variety of bioprinted cell types employed, including skin epithelial (HaCaT), skin cancer (A431), liver cancer (Hep G2), and fibroblast (3T3-J2) cells, all of which exhibited excellent viability and survivability, allowing printed structures to rapidly develop into confluent tissues. To demonstrate a simple 2D oncology model, A431 and HaCaT cells were printed and grown into tissues. Furthermore, a basic skin model was established to probe drug response. 3D tissue formation was demonstrated by co-printing Hep G2 and 3T3-J2 cells onto an established fibroblast layer, the functionality of which was probed by measuring albumin production, and was found to be higher in comparison to both 2D and monoculture approaches. Bioprinting of primary cells was tested using acutely isolated primary rat dorsal root ganglia neurons, which survived and established processes. The presented technique offers a novel open-volume microfluidics approach to bioprint cells for the generation of biological tissues.

A new USP Class VI-compliant substrate for manufacturing disposable microfluidic devices.

As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification. We report two production processes that are compatible with the existing methods of rapid prototyping and present characterizations of the resultant PUMA microfluidic devices.

Droplets for ultrasmall-volume analysis.

By using methods that permit the generation and manipulation of ultrasmall-volume droplets, researchers are pushing the boundaries of ultrasensitive chemical analyses. (To listen to a podcast about this feature, please go to the Analytical Chemistry Web site at pubs.acs.org/ancham.).

Optofluidic generation of Laguerre-Gaussian beams.

Laguerre-Gaussian (LG) beams have been extensively studied due to their unique structure, characterized by a phase singularity at the center of the beam. Common methods for generating such beams include the use of diffractive optical elements and spatial light modulators, which although offering excellent versatility, suffers from several drawbacks, including in many cases a low power damage threshold as well as complexity and expense. This paper presents a simple, low cost method for the generation of high-fidelity LG beams using rapid prototyping techniques. Our approach is based on a fluidic-hologram concept, whereby the properties of the LG beam can be finely controlled by varying the refractive-index of the fluid that flows through the hologram. This simple approach, while optimized here for LG beam generation, is also expected to find applications in the production of tunable fluidic optical trains.

Label-free spatio-temporal monitoring of cytosolic mass, osmolarity, and volume in living cells.

Microorganisms adapt their biophysical properties in response to changes in their local environment. However, quantifying these changes at the single-cell level has only recently become possible, largely relying on fluorescent labeling strategies. In this work, we utilize yeast (Saccharomyces cerevisiae) to demonstrate label-free quantification of changes in both intracellular osmolarity and macromolecular concentration in response to changes in the local environment. By combining a digital holographic microscope with a millifluidic chip, the temporal response of cellular water flux was successfully isolated from the rate of production of higher molecular weight compounds, in addition to identifying the produced compounds in terms of the product of their refractive index increment [Formula: see text] and molar mass. The ability to identify, quantify and temporally resolve multiple biophysical processes in living cells at the single cell level offers a crucial complement to label-based strategies, suggesting broad applicability in studies of a wide-range of cellular processes.

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

Fabrication improvements for thermoset polyester (TPE) microfluidic devices.

Thermoset polyester (TPE) microfluidic devices were previously developed as an alternative to poly(dimethylsiloxane) (PDMS) devices, fabricated similarly by replica molding, yet offering stable surface properties and good chemical compatibility with some organics that are incompatible with PDMS. This paper describes a number of improvements in the fabrication of TPE chips. Specifically, we describe methods to form TPE devices with a thin bottom layer for use with high numerical aperture (NA) objectives for sensitive fluorescence detection and optical manipulation. We also describe plasma-bonding of TPE to glass to create hybrid TPE-glass devices. We further present a simple master-pretreatment method to replace our original technique that required the use of specialized equipment.

Controlled shrinkage and re-expansion of a single aqueous droplet inside an optical vortex trap.

This paper describes the shrinkage and re-expansion of individual femtoliter-volume aqueous droplets that were suspended in an organic medium and held in an optical vortex trap. To elucidate the mechanism behind this phenomenon, we constructed a heat- and mass-transfer model and carried out experimental verifications of our model. From these studies, we conclude that an evaporation mechanism sufficiently describes the shrinkage of aqueous droplets held in a vortex trap, whereas a mechanism based on the supersaturation of the organic phase by water that surrounds the droplet adequately explains the re-expansion of the shrunk droplet. The proposed mechanisms correlated well with experimental observations using different organic media, when H2O was replaced with D2O and when an optical tweezer was used to induce droplet shrinkage rather than an optical vortex trap. For H2O droplets, the temperature rise within the droplet during shrinkage was on the order of 1 K or less, owing to the rapid thermal conduction of heat away from the droplet at the microscale and the sharp increase in solubility for water by the organic phase with slight elevations in temperature. Because most chemical species confined to droplets can be made impenetrable to the aqueous/organic interface, a change in the volume of aqueous droplets translates into a change in concentration of the dissolved species within the droplets. Therefore, this phenomenon should find use in the study of fundamental chemical processes that are sensitive to concentration, such as macromolecular crowding and protein nucleation and crystallization.

Endocytic uptake of monomeric amyloid-ß peptides is clathrin- and dynamin-independent and results in selective accumulation of Aß(1-42) compared to Aß(1-40).

Intraneuronal accumulation of amyloid-ß (Aß) peptides represent an early pathological feature in Alzheimer's disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of Aß(1-40) and Aß(1-42) monomers in cultured SH-SY5Y cells. We find that both variants are constitutively internalised via endocytosis and that their uptake is proportional to cellular endocytic rate. Moreover, SH-SY5Y cells internalise consistently twice the amount of Aß(1-42) compared to Aß(1-40); an imaging-based quantification showed that cells treated with 1 µM peptide for 8 h contained 800,000 peptides of Aß(1-42) and 400,000 of Aß(1-40). Both variants co-localised to >90% with lysosomes or other acidic compartments. Dynasore and chlorpromazine endocytosis inhibitors were both found to reduce uptake, particularly of Aß(1-42). Overexpression of the C-terminal of the clathrin-binding domain of AP180, dynamin2 K44A, or Arf6 Q67L did however not reduce uptake of the Aß variants. By contrast, perturbation of actin polymerisation and inhibition of macropinocytosis reduced Aß(1-40) and Aß(1-42) uptake considerably. This study clarifies mechanisms of Aß(1-40) and Aß(1-42) uptake, pinpoints differences between the two variants and highlights a common and putative role of macropinocytosis in the early accumulation of intraneuronal Aß in AD.

Spatial characterization of a multifunctional pipette for drug delivery in hippocampal brain slices.

BACKGROUND: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies. NEW METHOD: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues. RESULTS: We demonstrate herein the ability of the MFP to selectively perfuse one dendritic layer in the CA1 region of hippocampus with CNQX, an AMPA receptor antagonist, while not affecting the other layers in this region. Our experiments also illustrate the essential role of hydrodynamic confinement in sharpening the spatial selectivity in brain slice experiments. Concentration-response measurements revealed that the ability of the MFP to control local drug concentration is comparable with that of whole slice perfusion, while in comparison the required amounts of active compounds can be reduced by several orders of magnitude. COMPARISON WITH EXISTING METHOD: The multifunctional pipette is applied with an angle, which, compared to other hydrodynamically confined microfluidic devices, provides more accessible space for other probing and imaging techniques. CONCLUSIONS: Using the MFP it will be possible to study selected regions of brain slices, integrated with various imaging and probing techniques, without affecting the other parts of the slices.