3,4-Ethylenedioxythiophene Hydrogels: Relating Structure and Charge Transport in Supramolecular Gels.

Ionic charge transport is a ubiquitous language of communication in biological systems. As such, bioengineering is in constant need of innovative, soft, and biocompatible materials that facilitate ionic conduction. Low molecular weight gelators (LMWGs) are complex self-assembled materials that have received increasing attention in recent years. Beyond their biocompatible, self-healing, and stimuli responsive facets, LMWGs can be viewed as a "solid" electrolyte solution. In this work, we investigate 3,4-ethylenedioxythiophene (EDOT) as a capping group for a small peptide library, which we use as a system to understand the relationship between modes of assembly and charge transport in supramolecular gels. Through a combination of techniques including small-angle neutron scattering (SANS), NMR-based Van't Hoff analysis, atomic force microscopy (AFM), rheology, four-point probe, and electrochemical impedance spectroscopy (EIS), we found that modifications to the peptide sequence result in distinct assembly pathways, thermodynamic parameters, mechanical properties, and ionic conductivities. Four-point probe conductivity measurements and electrochemical impedance spectroscopy suggest that ionic conductivity is approximately doubled by programmable gel assemblies with hollow cylinder morphologies relative to gels containing solid fibers or a control electrolyte. More broadly, it is hoped this work will serve as a platform for those working on charge transport of aqueous soft materials in general.

Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis.

The intrinsic heterogeneity of many nanoformulations is currently challenging to characterize on both the single particle and population level. Therefore, there is great opportunity to develop advanced techniques to describe and understand nanomedicine heterogeneity, which will aid translation to the clinic by informing manufacturing quality control, characterization for regulatory bodies, and connecting nanoformulation properties to clinical outcomes to enable rational design. Here, we present an analytical technique to provide such information, while measuring the nanocarrier and cargo simultaneously with label-free, nondestructive single particle automated Raman trapping analysis (SPARTA). We first synthesized a library of model compounds covering a range of hydrophilicities and providing distinct Raman signals. These compounds were then loaded into model nanovesicles (polymersomes) that can load both hydrophobic and hydrophilic cargo into the membrane or core regions, respectively. Using our analytical framework, we characterized the heterogeneity of the population by correlating the signal per particle from the membrane and cargo. We found that core and membrane loading can be distinguished, and we detected subpopulations of highly loaded particles in certain cases. We then confirmed the suitability of our technique in liposomes, another nanovesicle class, including the commercial formulation Doxil. Our label-free analytical technique precisely determines cargo location alongside loading and release heterogeneity in nanomedicines, which could be instrumental for future quality control, regulatory body protocols, and development of structure-function relationships to bring more nanomedicines to the clinic.

RegiSTORM: channel registration for multi-color stochastic optical reconstruction microscopy.

BACKGROUND: Stochastic optical reconstruction microscopy (STORM), a super-resolution microscopy technique based on single-molecule localizations, has become popular to characterize sub-diffraction limit targets. However, due to lengthy image acquisition, STORM recordings are prone to sample drift. Existing cross-correlation or fiducial marker-based algorithms allow correcting the drift within each channel, but misalignment between channels remains due to interchannel drift accumulating during sequential channel acquisition. This is a major drawback in multi-color STORM, a technique of utmost importance for the characterization of various biological interactions. RESULTS: We developed RegiSTORM, a software for reducing channel misalignment by accurately registering STORM channels utilizing fiducial markers in the sample. RegiSTORM identifies fiducials from the STORM localization data based on their non-blinking nature and uses them as landmarks for channel registration. We first demonstrated accurate registration on recordings of fiducials only, as evidenced by significantly reduced target registration error with all the tested channel combinations. Next, we validated the performance in a more practically relevant setup on cells multi-stained for tubulin. Finally, we showed that RegiSTORM successfully registers two-color STORM recordings of cargo-loaded lipid nanoparticles without fiducials, demonstrating the broader applicability of this software. CONCLUSIONS: The developed RegiSTORM software was demonstrated to be able to accurately register multiple STORM channels and is freely available as open-source (MIT license) at https://github.com/oystein676/RegiSTORM.git and https://doi.org/10.5281/zenodo.5509861 (archived), and runs as a standalone executable (Windows) or via Python (Mac OS, Linux).

High-throughput measurement of the content and properties of nano-sized bioparticles with single-particle profiler.

We introduce a method, single-particle profiler, that provides single-particle information on the content and biophysical properties of thousands of particles in the size range 5-200 nm. We use our single-particle profiler to measure the messenger RNA encapsulation efficiency of lipid nanoparticles, the viral binding efficiencies of different nanobodies, and the biophysical heterogeneity of liposomes, lipoproteins, exosomes and viruses.

Potent Virustatic Polymer-Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo.

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer-lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition.

Peptide-Folding Triggered Phase Separation and Lipid Membrane Destabilization in Cholesterol-Rich Lipid Vesicles.

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.

Excitability properties of mouse and human skeletal muscle fibres compared by muscle velocity recovery cycles.

Mouse models of skeletal muscle channelopathies are not phenocopies of human disease. In some cases (e.g. Myotonia Congenita) the phenotype is much more severe, whilst in others (e.g. Hypokalaemic periodic paralysis) rodent physiology is protective. This suggests a species' difference in muscle excitability properties. In humans these can be measured indirectly by the post-impulse changes in conduction velocity, using Muscle Velocity Recovery Cycles (MVRCs). We performed MVRCs in mice and compared their muscle excitability properties with humans. Mouse Tibialis Anterior MVRCs (n = 70) have only one phase of supernormality (increased conduction velocity), which is smaller in magnitude (p = 9 × 10-21), and shorter in duration (p = 3 × 10-24) than human (n = 26). This abbreviated supernormality is followed by a period of late subnormality (reduced velocity) in mice, which overlaps in time with the late supernormality seen in human MVRCs. The period of late subnormality suggests increased t-tubule Na+/K+-pump activity. The subnormal phase in mice was converted to supernormality by blocking ClC-1 chloride channels, suggesting relatively higher chloride conductance in skeletal muscle. Our findings help explain discrepancies in phenotype between mice and humans with skeletal muscle channelopathies and potentially other neuromuscular disorders. MVRCs are a valuable new tool to compare in vivo muscle membrane properties between species and will allow further dissection of the molecular mechanisms regulating muscle excitability.

Coupling Lipid Nanoparticle Structure and Automated Single-Particle Composition Analysis to Design Phospholipase-Responsive Nanocarriers.

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here Single Particle Automated Raman Trapping Analysis (SPARTA) is combined with small-angle X-ray and neutron scattering (SAXS/SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP-phospholipase D (PLD) interactions. This analysis demonstrates that PLD, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate is an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated.

Design of Lipid-Based Nanocarriers via Cation Modulation of Ethanol-Interdigitated Lipid Membranes.

Short-chain alcohols (i.e., ethanol) can induce membrane interdigitation in saturated-chain phosphatidylcholines (PCs). In this process, alcohol molecules intercalate between phosphate heads, increasing lateral separation and favoring hydrophobic interactions between opposing acyl chains, which interpenetrate forming an interdigitated phase. Unraveling mechanisms underlying the interactions between ethanol and model lipid membranes has implications for cell biology, biochemistry, and for the formulation of lipid-based nanocarriers. However, investigations of ethanol-lipid membrane systems have been carried out in deionized water, which limits their applicability. Here, using a combination of small- and wide-angle X-ray scattering, small-angle neutron scattering, and all-atom molecular dynamics simulations, we analyzed the effect of varying CaCl2 and NaCl concentrations on ethanol-induced interdigitation. We observed that while ethanol addition leads to the interdigitation of bulk phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers in the presence of CaCl2 and NaCl regardless of the salt concentration, the ethanol-induced interdigitation of vesicular DPPC depends on the choice of cation and its concentration. These findings unravel a key role for cations in the ethanol-induced interdigitation of lipid membranes in either bulk phase or vesicular form.

Clinical and genetic spectrum of a Chinese cohort with SCN4A gene mutations.

Skeletal muscle sodium channelopathies due to SCN4A gene mutations have a broad clinical spectrum. However, each phenotype has been reported in few cases of Chinese origin. We present detailed phenotype and genotype data from a cohort of 40 cases with SCN4A gene mutations seen in neuromuscular diagnostic service in Huashan hospital, Fudan University. Cases were referred from 6 independent provinces from 2010 to 2018. A questionnaire covering demographics, precipitating factors, episodes of paralysis and myotonia was designed to collect the clinical information. Electrodiagnostic studies and muscle MRI were retrospectively analyzed. The clinical spectrum of patients included: 6 Hyperkalemic periodic paralysis (15%), 18 Hypokalemic periodic paralysis (45%), 7 sodium channel myotonia (17.5%), 4 paramyotonia congenita (10%) and 5 heterozygous asymptomatic mutation carriers (12.5%). Review of clinical information highlights a significant delay to diagnosis (median 15 years), reports of pain and myalgia in the majority of patients, male predominance, circadian rhythm and common precipitating factors. Electrodiagnostic studies revealed subclinical myotonic discharges and a positive long exercise test in asymptomatic carriers. Muscle MRI identified edema and fatty infiltration in gastrocnemius and soleus. A total of 13 reported and 2 novel SCN4A mutations were identified with most variants distributed in the transmembrane helix S4 to S6, with a hotspot mutation p.Arg675Gln accounting for 32.5% (13/40) of the cohort. Our study revealed a higher proportion of periodic paralysis in SCN4A-mutated patients compared with cohorts from England and the Netherlands. It also highlights the importance of electrodiagnostic studies in diagnosis and segregation studies.

Engineering Swollen Cubosomes Using Cholesterol and Anionic Lipids.

Dispersions of nonlamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with porous internal structures) with water channel diameters of up to 171 Å, which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipid DOPA, DOPG, or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and that these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules and development of confined interfacial reaction environments.

Urogenital symptoms in mitochondrial disease: overlooked and undertreated.

BACKGROUND AND PURPOSE: Bowel symptoms are well documented in mitochondrial disease. However, data concerning other pelvic organs is limited. A large case-control study has therefore been undertaken to determine the presence of lower urinary tract symptoms (LUTS) and sexual dysfunction in adults with genetically confirmed mitochondrial disease. METHODS: Adults with genetically confirmed mitochondrial disease and control subjects were recruited from a specialist mitochondrial clinic. The presence and severity of LUTS and their impact on quality of life, in addition to sexual dysfunction and bowel symptoms, were captured using four validated questionnaires. Subgroup analysis was undertaken in patients harbouring the m.3243A>G MT-TL1 mitochondrial DNA mutation. A subset of patients underwent urodynamic studies to further characterize their LUTS. RESULTS: Data from 58 patients and 19 controls (gender and age matched) were collected. Adults with mitochondrial disease had significantly more overactive bladder (81.5% vs. 56.3%, P = 0.039) and low stream (34.5% vs. 5.3%, P = 0.013) urinary symptoms than controls. Urodynamic studies in 10 patients confirmed that bladder storage symptoms predominate. Despite high rates of LUTS, none of the patient group was receiving treatment. Female patients and those harbouring the m.3243A>G MT-TL1 mutation experienced significantly more sexual dysfunction than controls (53.1% vs. 11.1%, P = 0.026, and 66.7% vs. 26.3%, P = 0.011, respectively). CONCLUSIONS: Lower urinary tract symptoms are common but undertreated in adult mitochondrial disease, and female patients and those harbouring the m.3243A>G MT-TL1 mutation experience sexual dysfunction. Given their impact on quality of life, screening for and treating LUTS and sexual dysfunction in adults with mitochondrial disease are strongly recommended.

Cubosomes: The Next Generation of Smart Lipid Nanoparticles?

Cubosomes are highly stable nanoparticles formed from the lipid cubic phase and stabilized by a polymer based outer corona. Bicontinuous lipid cubic phases consist of a single lipid bilayer that forms a continuous periodic membrane lattice structure with pores formed by two interwoven water channels. Cubosome composition can be tuned to engineer pore sizes or include bioactive lipids, the polymer outer corona can be used for targeting and they are highly stable under physiological conditions. Compared to liposomes, the structure provides a significantly higher membrane surface area for loading of membrane proteins and small drug molecules. Owing to recent advances, they can be engineered in vitro in both bulk and nanoparticle formats with applications including drug delivery, membrane bioreactors, artificial cells, and biosensors. This review outlines recent advances in cubosome technology enabling their application and provides guidelines for the rational design of new systems for biomedical applications.

Fate of Liposomes in the Presence of Phospholipase C and D: From Atomic to Supramolecular Lipid Arrangement.

Understanding the origins of lipid membrane bilayer rearrangement in response to external stimuli is an essential component of cell biology and the bottom-up design of liposomes for biomedical applications. The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorus-oxygen bonds of phosphate esters in phosphatidylcholine (PC) lipids. The atomic position of this hydrolysis reaction has huge implications for the stability of PC-containing self-assembled structures, such as the cell wall and lipid-based vesicle drug delivery vectors. While PLC converts PC to diacylglycerol (DAG), the interaction of PC with PLD produces phosphatidic acid (PA). Here we present a combination of small-angle scattering data and all-atom molecular dynamics simulations, providing insights into the effects of atomic-scale reorganization on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated incorporation of DAG or PA. We observed that PC liposomes completely disintegrate in the presence of PLC, as conversion of PC to DAG progresses. At lower concentrations, DAG molecules within fluid PC bilayers form hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules and burrow into the hydrophobic region. This leads initially to membrane thinning followed by a swelling of the lamellar phase with increased DAG. At higher DAG concentrations, localized membrane tension causes a change in lipid phase from lamellar to the hexagonal and micellar cubic phases. Molecular dynamics simulations show that this destabilization is also caused in part by the decreased ability of DAG-containing PC membranes to coordinate sodium ions. Conversely, PLD-treated PC liposomes remain stable up to extremely high conversions to PA. Here, the negatively charged PA headgroup attracts significant amounts of sodium ions from the bulk solution to the membrane surface, leading to a swelling of the coordinated water layer. These findings are a vital step toward a fundamental understanding of the degradation behavior of PC lipid membranes in the presence of these clinically relevant enzymes, and toward the rational design of diagnostic and drug delivery technologies for phospholipase-dysregulation-based diseases.

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick's, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Spontaneous charged lipid transfer between lipid vesicles.

An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

How does ytterbium chloride interact with DMPC bilayers? A computational and experimental study.

Lanthanide salts have been studied for many years, primarily in Nuclear Magnetic Resonance (NMR) experiments of mixed lipid-protein systems and more recently to study lipid flip-flop in model membrane systems. It is well recognised that lanthanide salts can influence the behaviour of both lipid and protein systems, however a full molecular level description of lipid-lanthanide interactions is still outstanding. Here we present a study of lanthanide-bilayer interactions, using molecular dynamics computer simulations, fluorescence electrostatic potential experiments and nuclear magnetic resonance. Computer simulations reveal the microscopic structure of DMPC lipid bilayers in the presence of Yb3+, and a surprising ability of the membranes to adsorb significant concentrations of Yb3+ without disrupting the overall membrane structure. At concentrations commonly used in NMR experiments, Yb3+ ions bind strongly to 5 lipids, inducing a small decrease of the area per lipid and a slight increase of the ordering of the aliphatic chains and the bilayer thickness. The area compressibility modulus increases by a factor of two, with respect to the free-salt case, showing that Yb3+ ions make the bilayer more rigid. These modifications of the bilayer properties should be taken into account in the interpretation of NMR experiments.

A key to the backdoor into the castle: The clinical ramifications of immunoediting driven by antigenic competition.

Over the last decade the field of cancer biology has gained considerable data on genomic heterogeneity. This situation creates challenges and possibly opportunities for cancer treatment. The evolution of the tumor at all stages also requires the growing malignancy to confront and avoid the immune system. What we describe here is the interaction of two immune phenomena that work together to change the characteristics of the tumor, i.e., antigenic competition and immune editing. These two systems are mutually functional and their interaction is capable of altering the characteristics of the tumor for protection and survival in an immune competent host as well as restricting the diversity of the tumor clones. Therefore, the final outcome of these interactions can also become the key to the backdoor into the castle. Through an additional immune manipulation, autologous tumor cell immunization, we can achieve prevention of disease recurrence after surgical resection and by analyzing induced human monoclonal antibodies to the neoantigens, gain in site into the restriction of diversity of the mutant clones. These findings may also open the door for a pathway to immune prevention of cancer.

Cytosolic 5'-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis.

OBJECTIVES: Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. MATERIALS AND METHODS: Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. RESULTS: Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. INTERPRETATION: Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.