Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings.

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid-protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (K D ) and kinetics (kon and koff ). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached.

Scalable lipid droplet microarray fabrication, validation, and screening.

High throughput screening of small molecules and natural products is costly, requiring significant amounts of time, reagents, and operating space. Although microarrays have proven effective in the miniaturization of screening for certain biochemical assays, such as nucleic acid hybridization or antibody binding, they are not widely used for drug discovery in cell culture due to the need for cells to internalize lipophilic drug candidates. Lipid droplet microarrays are a promising solution to this problem as they are capable of delivering lipophilic drugs to cells at dosages comparable to solution delivery. However, the scalablility of the array fabrication, assay validation, and screening steps has limited the utility of this approach. Here we take several new steps to scale up the process for lipid droplet array fabrication, assay validation in cell culture, and drug screening. A nanointaglio printing process has been adapted for use with a printing press. The arrays are stabilized for immersion into aqueous solution using a vapor coating process. In addition to delivery of lipophilic compounds, we found that we are also able to encapsulate and deliver a water-soluble compound in this way. The arrays can be functionalized by extracellular matrix proteins such as collagen prior to cell culture as the mechanism for uptake is based on direct contact with the lipid delivery vehicles rather than diffusion of the drug out of the microarray spots. We demonstrate this method for delivery to 3 different cell types and the screening of 92 natural product extracts on a microarray covering an area of less than 0.1 cm2. The arrays are suitable for miniaturized screening, for instance in high biosafety level facilities where space is limited and for applications where cell numbers are limited, such as in functional precision medicine.

Odor Discrimination by Lipid Membranes.

Odor detection and discrimination in mammals is known to be initiated by membrane-bound G-protein-coupled receptors (GPCRs). The role that the lipid membrane may play in odor discrimination, however, is less well understood. Here, we used model membrane systems to test the hypothesis that phospholipid bilayer membranes may be capable of odor discrimination. The effect of S-carvone, R-carvone, and racemic lilial on the model membrane systems was investigated. The odorants were found to affect the fluidity of supported lipid bilayers as measured by fluorescence recovery after photobleaching (FRAP). The effect of odorants on surface-supported lipid multilayer microarrays of different dimensions was also investigated. The lipid multilayer micro- and nanostructure was highly sensitive to exposure to these odorants. Fluorescently-labeled lipid multilayer droplets of 5-micron diameter were more responsive to these odorants than ethanol controls. Arrays of lipid multilayer diffraction gratings distinguished S-carvone from R-carvone in an artificial nose assay. Our results suggest that lipid bilayer membranes may play a role in odorant discrimination and molecular recognition in general.

Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of varicella-zoster virus immunoglobulin G antibodies in fingerstick blood.

Varicella zoster virus (VZV) causes childhood chickenpox, becomes latent in sensory ganglia and reactivates years later to cause shingles (Zoster) and postherpetic neuralgia in the elderly and immunosuppressed individuals. Serologic IgG tests can be used to determine if a person has antibodies to VZV from past varicella infection or had received varicella or zoster (shingles) vaccination. Commercial enzyme-linked immunosorbent assays (ELISAs) are currently used for the detection of VZV IgG antibodies in patient serum samples. However, ELISA tests require collection and processing of blood samples in a CLIA laboratory to separate serum or plasma for further testing. In this paper, we describe the development and testing of an antibody based Lateral Flow Immunochromatographic assay (LFA) device for the detection of VZV IgG in fingerstick whole blood. Analytical and clinical analyses were performed to compare the performance characteristics of the Viro VZV IgG LFA (VZV LFA) and the Diamedix VZV IgG ELISA. Analytical studies demonstrated the higher sensitivity of the VZV LFA compared to the ELISA by testing dilutions of the WHO VZV IgG serum International Standard. Clinical performance characteristics of the VZV LFA fingerstick whole blood assay were assessed at three point of care (POC) facilities by untrained users testing samples from 300 prospectively enrolled study subjects. VZV LFA results were compared with results obtained by testing serum samples obtained from the same study participants by the Diamedix VZV IgG ELISA. Two specimens with invalid results by the LFA assay were not included in the LFA performance calculations and nine equivocal ELISA results were included as positive for IgG results. The results from all three POC clinical sites demonstrated the higher sensitivity/positive percent agreement (PPA) (99.26%, 95% CI: 97.34-99.80) of the VZV LFA compared to the Diamedix VZV IgG ELISA (94.08%, 95% CI: 90.72-96.27). The specificity/negative percent agreement (NPA) of the VZV LFA compared to the ELISA test was calculated initially to be 39.29% (95% CI: 23.57-57.59) with 19 discordant test results out of 298 test results between the two assays (17 LFA positive/ELISA negative and two LFA negative/ELISA positive). The PPA and true NPA of the VZV LFA were determined by testing all 298 samples, including the discordant (19) and all concordant negative and positive (279) study subject serum samples, before and after blocking VZV gE antibody sites in the samples by spiking with VZV LFA gE capture antigen. The NPA improved to 100% (95% CI: 74.12-100) after the procedure when compared to the ELISA test results. The comparator ELISA PPA based on the spiking/blocking study remained as 94.08%, (95% CI: 90.72-96.27), comparable to test results from untreated samples. The VZV LFA has been demonstrated to be simple and sufficiently robust for use in CLIA-waived POC facilities by untrained healthcare professionals and to detect VZV IgG in 20 min from fingerstick whole blood. The VZV LFA therefore provides a fast, reliable, and highly sensitive method of determining prior VZV viral infection or varicella and zoster vaccination status.

Conjugating Micropatches to Living Cells Through Membrane Intercalation.

Existing clinical cell therapies, which rely on the use of biological functionalities of living cells, can be further enhanced by conjugating functional particles to the cells to form cell-particle complexes. Disk-shaped microparticles produced by the top-down microfabrication approach possess unique advantages for this application. However, none of the current mechanisms for conjugating the microfabricated microparticles to the cells are principally applicable to all types of cells with therapeutic potentials. On the other hand, membrane intercalation is a well-established mechanism for attaching fluorescent molecules to living cells or for immobilizing cells on a solid surface. This paper reports a study on conjugating disk-shaped microparticles, referred to as micropatches, to living cells through membrane intercalation for the first time. The procedure for producing the cell-micropatch complexes features an unprecedented integration of microcontact printing of micropatches, end-grafting of linear molecules of octadecyl chain and poly(ethylene glycol) to the printed micropatches, and use of gelatin as a temperature-sensitive sacrificial layer to allow the formation and subsequent release of the cell-micropatch complexes. Complexes composed of mouse neuroblastoma cells were found to be stable in vitro, and the micropatch-bound cells were viable, proliferative, and differentiable. Moreover, complexes composed of four other types of cells were produced. The membrane-intercalation mechanism and the corresponding fabrication technique developed in this study are potentially applicable to a wide range of therapeutic cells and thus promise to be useful for developing new cell therapies enhanced by the disk-shaped microparticles.

Enhanced cellular uptake of size-separated lipophilic silicon nanoparticles.

Specific size, shape and surface chemistry influence the biological activity of nanoparticles. In the case of lipophilic nanoparticles, which are widely used in consumer products, there is evidence that particle size and formulation influences skin permeability and that lipophilic particles smaller than 6 nm can embed in lipid bilayers. Since most nanoparticle synthetic procedures result in mixtures of different particles, post-synthetic purification promises to provide insights into nanostructure-function relationships. Here we used size-selective precipitation to separate lipophilic allyl-benzyl-capped silicon nanoparticles into monodisperse fractions within the range of 1 nm to 5 nm. We measured liposomal encapsulation and cellular uptake of the monodisperse particles and found them to have generally low cytotoxicities in Hela cells. However, specific fractions showed reproducibly higher cytotoxicity than other fractions as well as the unseparated ensemble. Measurements indicate that the cytotoxicity mechanism involves oxidative stress and the differential cytotoxicity is due to enhanced cellular uptake by specific fractions. The results indicate that specific particles, with enhanced suitability for incorporation into lipophilic regions of liposomes and subsequent in vitro delivery to cells, are enriched in certain fractions.

Lipid multilayer microarrays for in vitro liposomal drug delivery and screening.

Screening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed. Small-molecule arrays and microfluidics are promising approaches. Here we introduce a simple method of surface-mediated delivery of drugs to cells from a microarray of phospholipid multilayers (layers thicker than a bilayer) encapsulating small molecules. The multilayer patterns are of sub-cellular dimensions and controllable thickness and were formed by dip-pen nanolithography. The patterns successfully delivered a rhodamine-tagged lipid and drugs only to the cells directly over them, indicating successful encapsulation and no cross-contamination to cells grown next to the patterns. We also demonstrated multilayer thickness-dependant uptake of the lipids from spots with sub-cellular lateral dimensions, and therefore the possibility of delivering different dosages from different areas of the array. The efficacies of two drugs were assayed on the same surface, and we were able to deliver dosages comparable to those of solution based delivery (up to the equivalent of 30 µg/mL). We expect our method to be a promising first step toward producing a single high-throughput liposome-based screening microarray plate that can be used in the same way as a standard well plate.