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.

Mixed-effects modeling of clinical DCE-MRI data: application to colorectal liver metastases treated with bevacizumab.

PURPOSE: Most dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data are evaluated for individual patients with cohorts analyzed to detect significant changes from baseline values, repeating the process at each posttreatment timepoint. Our study aimed to develop a statistically valid model for the complete time course of DCE-MRI data in a patient cohort. MATERIALS AND METHODS: Data from 10 patients with colorectal cancer liver metastases were analyzed, including two baseline scans and four post-bevacizumab scans. Apparent changes in tumor median K(trans) were adjusted for changes in observed enhancing tumor fraction (EnF) by multiplying K(trans) by EnF (KEnF). A mixed-effects model (MEM) was defined to describe the KEnF time course for all patients simultaneously by assuming a three-parameter indirect response model with model parameters lognormally distributed across patients. RESULTS: The typical cohort time course showed a KEnF reduction to 59% of baseline at 24 hours, returning to 65% of baseline values by day 12. Interpatient variability of model parameters ranged from 11% to 307%. CONCLUSION: The MEM approach has potential for comparing responses at a group level in clinical trials with different doses, schedules, or combination regimens. Furthermore, the KEnF biomarker successfully resolved confounds in interpreting K(trans) arising from therapy induced changes in the volume of enhancing tumor.

Single channel and ensemble hERG conductance measured in droplet bilayers.

The human ether-a-go-go related gene (hERG) encodes the potassium channel Kv11.1, which plays a key role in the cardiac action potential and has been implicated in cardiac disorders as well as a number of off-target pharmaceutical interactions. The electrophysiology of this channel has been predominantly studied using patch clamp, but lipid bilayers have the potential to offer some advantages, including apparatus simplicity, ease of use, and the ability to control the membrane and solution compositions. We made membrane preparations from hERG-expressing cells and measured them using droplet bilayers, allowing measurement of channel ensemble currents and 13.5 pS single channel currents. These currents were ion selective and were blockable by E-4031 and dofetilide in a dose-dependent manner, allowing determination of IC50 values of 17 nM and 9.65 µM for E-4031 and dofetilide, respectively. We also observed time- and voltage- dependent currents following step changes in applied potential that were similar to previously reported patch clamp measurements.

Quantitative Measurements of Protein Volume and Concentration using Hydrogel-Backed Nanopores.

Accurate identification and quantification of proteins in solution using nanopores is technically challenging in part because of the large fraction of missed translocation events due to short event times and limitations of conventional current amplifiers. Previously, we have shown that a nanopore interfaced with a poly(ethylene glycol)-dimethacrylate hydrogel with an average mesh size of 3.1 nm significantly enhances the protein residence time within the pore, reducing the number of missed events. We used hydrogel-backed nanopores to sense unlabeled proteins as small as 5.5 kDa in size and 10 fM in concentration. We show that the frequency of protein translocation events linearly scales with bulk concentration over a wide range of concentrations and that unknown protein concentrations can be determined from an interpolation of the frequency-concentration curve with less than 10% error. Further, we show an iterative method to determine a protein volume accurately from measurement data for proteins with a diameter comparable to a nanopore diameter. Our measurements and analysis also suggest several competing mechanisms for the detection enhancement enabled by the presence of the hydrogel.

Improved Measurement of Proteins Using a Solid-State Nanopore Coupled with a Hydrogel.

Although resistive pulse sensing using solid-state nanopores is capable of single-molecule sensitivity, previous work has shown that nanoparticles, such as proteins, pass through nanopores too quickly for accurate detection with typical measurement apparatus. As a result, nanopore measurements of these particles significantly deviate from theoretically estimated current amplitudes and detection rates. Here, we show that a hydrogel placed on the distal side of a nanopore can increase the residence time of nanoparticles within the nanopore, significantly increasing the detection rate and allowing improved resolution of blockage currents. The method is simple and inexpensive to implement while being label-free and applicable to a wide range of nanoparticle targets. Using hydrogel-backed nanopores, we detected the protein IgG with event frequencies several orders of magnitude higher than those in the absence of the hydrogel and with larger measured currents that agree well with theoretical models. We also show that the improved measurement also enables discrimination of IgG and bovine serum albumin in a mixed solution. Finally, we show that measurements of IgG with the hydrogel-backed nanopores can also yield current amplitude distributions that can be analyzed to infer its approximate shape.

Implantable Drug Reservoir Devices for Inner Ear Delivery of Pharmacotherapeutics.

OBJECTIVE: Cisplatin is a platinum-based chemotherapeutic drug that secondarily induces toxicity in inner ear sensory epithelia, contributing to auditory and vestibular dysfunction. We describe the creation of a drug reservoir device (DRD) to combat this ototoxicity for the duration of chemotherapy. As ototoxic side effects of chemotherapy may limit an oncologist's ability to prescribe first-line agents such as cisplatin, mitigating such devastating effects through prolonged topical therapy would be tremendously valuable. STUDY DESIGN: We investigated (1) the ability of an electrospun polylactic acid DRD to provide prolonged delivery of the posited otoprotectant metformin and (2) the development of an in vitro model utilizing Sh-Sy5y human neuroblastoma cells to assess the efficacy of metformin in reducing cisplatin-induced toxicity. SETTING: Neurophysiology laboratory. METHODS: Basic science experiments were performed to assess DRD properties and metformin's effects on cisplatin toxicity in culture. RESULTS: We found that DRDs with increasing polylactic acid concentrations exhibited metformin release for up to 8 weeks. In modeling elution across the round window in vitro, continued elution of metformin was observed for at least 6 weeks, as quantified by spectrophotometry. Unfortunately, metformin did not exhibit protective efficacy in this model using Sh-Sy5y cells. CONCLUSION: While metformin was not found to be protective in Sh-Sy5y cells, these results suggest that an electrospun DRD can provide a tailorable drug delivery system providing medication for the duration of chemotherapy treatment. This represents a novel drug delivery system and efficacy screening assay with broad clinical applications in personalized delivery of inner ear therapies.

Research highlights: nanopore protein detection and analysis.

In this article we highlight recent work using nanopores to detect and study proteins. Nanopores are excellent single molecule sensors, capable of rapidly characterizing small molecules with relatively modest instrumentation requirements. Although the vast majority of recent effort and attention surrounding nanopores has centered on detection and sequencing of nucleic acids, proteins represent a more difficult and diverse analyte population, with a wide range of sizes, structures, charges, among other characteristics. Nanopores can be used to detect the presence of proteins of interest as well as to study their enzymatic activity, binding to ligands, and secondary structure. We highlight new work describing detection of specific protein species in solution by coupling them to a strand of carrier DNA that is used to electrophoretically transport the proteins through conical glass nanopores. Additionally, we spotlight another approach for nanopore detection of protein and other analytes through detection of their binding to aptamers-measurements which were quantitative to pM concentrations. Finally, we highlight studies in which protein secondary structure and folding energetics were studied through the use of an unfoldase enzyme coupled to a protein nanopore, a technique capable of detecting the effects of single amino acid mutations on the stability of the folded protein.

Measurement of Ensemble TRPV1 Ion Channel Currents Using Droplet Bilayers.

Electrophysiological characterization of ion channels is useful for elucidation of channel function as well as quantitative assessment of pharmaceutical effects on ion channel conductance. We used droplet bilayers to measure ensemble ion channel currents from membrane preparations made from TRPV1-expressing HEK cells. Conductance measurements showed rectification, activation by acid and capsaicin, and inhibition by capsazepine, SB 452533, and JNJ 17293212. We also quantitatively measured concentration-dependent inhibition of channel conductance through determination of capsazepine IC50 in agreement with previously published studies using patch clamp. These results, combined with the reduced apparatus and material requirements of droplet bilayers, indicate that this platform could be used for study of other physiologically relevant ion channels.

Hydrogel-stabilized droplet bilayers for high speed solution exchange.

Many applications utilizing artificial lipid bilayers require the ability to exchange the bilayer's solution environment. However, because of the instability of the bilayer, the rate of solution exchange is limited, which significantly hinders the measurement rate and throughput. We have developed an artificial bilayer system that can withstand high flow speeds, up to 2.1 m/s, by supporting the bilayer with a hydrogel. We demonstrated the ability to measure during flow by measuring the conductance of gramicidin-A channels while switching between solutions of two different compositions, recording a time to measure 90% change in current of approximately 2.7 seconds at a flow rate of 0.1 m/s. We also demonstrated a potential application of this system by measuring the conductance modulation of the rat TRPM8 ion channel by an agonist and antagonist at varying concentrations, obtaining 7-point IC50 and EC50 values in approximately 7 minutes and 4-point values within 4 minutes.