Nanoscale

It is an essential element of mechanobiology to measure the forces of biological cells. In microparticle traction force microscopy, they are inferred from the deformation of elastic microparticles. Two complementary variants have been introduced before: the volume method, which reconstructs surface stresses from the displacements of fiducial markers embedded inside the particles, and the surface method, which infers stresses directly from the deformation of the particle surface. However, a systematic comparison of the two methods has been lacking. Here, we quantitatively compare both approaches using simulated traction fields representing biologically relevant loading scenarios. We find that the surface method consistently reconstructs traction profiles with substantially lower errors than the volume method, which suffers from displacement tracking and stress calculation at the surface. At high noise levels, however, the performance gap becomes smaller. To compare the performance of the two methods in a realistic experimental setting, we developed DNA-based hydrogel microparticles equipped with both fluorescent surface labels and embedded fluorescent nanoparticles, enabling the direct comparison of the two methods within the same system. Compression experiments produced traction profiles consistent with Hertzian contact mechanics and confirmed the trends observed in the simulations. While our computational workflow establishes a framework to apply both methods, our experimental workflow establishes DNA microparticles as versatile and biocompatible probes for measuring cellular forces.

Lipid nanoparticles (LNPs) are the most successful drug delivery carrier to date, but optimizing lipid formulations to improve membrane fusion capabilities for effective drug release has been challenging due to lack of a quantitative measure for fusogenicity. Here we introduce a new framework based on small angle X-ray scattering to experimentally measure [Formula] for lipids used in LNP formulations such as glycerol monooleate (GMO) and ionizable lipids (SM-102 and ALC-0315). Q intrinsically captures spontaneous curvature (J0), which is traditionally used to assess fusogenicity. The change of cubic lattice parameters with temperature was measured for GMO-containing lipid mixtures, and the Q extracted quantitatively correlated with LNP fusogenicity power validated by fluorescence-based fusion assays and cryogenic electron microscopy. Fusogenicity of SM-102 and ALC-0315 was quantified by adding them to host membranes and assessing change in Q. This framework provides researchers with the ability to optimize the fusogenicity of LNP formulations for potent drug release and enhances understanding of parameters governing fusion in all biomembranes.

Dragonfly and cicada wing-inspired titanium nanopillar surfaces show promising bactericidal properties for antibacterial medical implant applications, but the precise mechanisms of bacteria-nanopillar interactions under hydrated conditions remain unclear. Cryo-electron tomography (cryo-ET) enables the visualisation of cellular organelles within their native hydrated cellular environment at molecular resolution. Visualising the bacteria-material interface on nanostructured surfaces by cryo transmission electron microscopy (cryo-TEM) requires the preparation of thin lamellae. Obtaining lamellae of bacteria directly on metal substrates while in a non-fixed and hydrated state requires cryo-focused ion beam (cryo-FIB) milling to isolate the targeted bacteria from the bulk sample. This approach faces additional challenges compared to tissues or cells on TEM grids, as titanium samples require a simultaneous cross-section of soft and hard materials at the same position and require vitrification, which embeds the sample in a thick layer of ice. Nonetheless, we demonstrate how to target a specific bacterium interacting with a titanium nanopillar surface using correlative cryo-fluorescence imaging, and how lamellae can still be prepared from vitrified samples by extracting the targeted bacterium and its surrounding as a small volume and transferring it to a receptor grid for thin lamella preparation, called targeted cryo-lift-out. Here, we outline the workflows and discuss their advantages and limitations for producing lamellae through lift-out techniques under cryogenic conditions, using methods that do not involve gas injection systems (GIS) for the lift-out transfer. These advances enhance cryo-ET applications, enabling in situ investigations of the interface between bacteria and nanopillars to effectively study the bactericidal mechanisms of biomimetic nature-inspired nanotopographies in a hydrated environment.

Efficient drug delivery using nanoparticles (NPs) critically depends on their ability to diffuse through biological tissues to reach target cells at therapeutic concentrations. The extracellular matrix (ECM) poses a key barrier to such transport, which directly influences bio-distribution, cellular uptake, and overall therapeutic efficacy. A key regulator of this transport is hyaluronic acid/hyaluronan (HA), a major ECM polysaccharide that forms a hydrated, viscoelastic network. Increased/reduced hyaluronan concentration can elevate/decrease ECM bulk and effective viscosity. Increase in effective viscosity at the nanometer/micrometer length scales can hinder NP mobility through steric obstruction and hydrodynamic drag. There is a large variability in the HA molecular weights and concentrations, especially across age, tissue/organ, and pathological conditions. This work aims to study the diffusion of different NP types in the mixtures of HA polymers with variable molecular weights using the dynamic light scattering technique (DLS). Furthermore, we perform coarse-grained molecular dynamics (CG-MD) simulations for a model system to complement our findings from the dynamic light scattering experiments. We observe NP undergo anomalous diffusion, which is strongly dependent on the ratio of particle size/HA network mesh size, especially for higher molecular weight mixtures. This is strongly influenced by the effective viscosity, which is defined at the local environment experienced by the NPs. Our work highlights developing a simplified predictive framework coupled with simulations for a target-specific extracellular matrix environment.

Preparing electron-transparent cryo-lamellae is inherently a serial and low-throughput process. Once the lamellae are milled, these thin structures endure both mechanical and thermal stress, and as a result many valuable lamellae crack or even disintegrate entirely. This loss is often regarded as a "lamella tax", i.e. an unavoidable cost of working with such fragile specimens. In this work, we introduce two modifications to the standard lamella-preparation workflow aimed at improving lamella mechanical resistance to crack formation and external stress. The first modification involves milling arrays of perforations directly within the lamella body. These perforations are designed to function as crack-arrest holes, intercepting cracks as they appear and preventing, or at least delaying their further propagation. By slowing crack growth, these features increase the likelihood that the lamella remains intact long enough to complete cryo-TEM imaging. The second modification replaces the conventional rigid attachment of the lamella to the surrounding cellular bulk material with a softer suspension using ring-shaped springs formed by ion beam milling. Mounting the lamella on smooth annular springs provides mechanical compliance both across and along the lamella axis, as well as at intermediate angles and in the out-of-plane direction. This flexibility allows the lamella to accommodate larger stresses and deformations without reaching its mechanical failure threshold. We fabricated a series of test lamellae incorporating different crack-arrest hole geometries, as well as lamellae suspended on soft annular springs. We performed high-resolution cryo-TEM imaging to characterise the perforations themselves and characterised the captured crack geometry within the lamellae at the highest level of detail achieved to date. TEM imaging shows crack interception and guided, non-catastrophic failure paths, while simulations confirm lowered stress in suspended lamellae.

The acoustoelectric (AE) effect, in which acoustic waves modulate the electrical properties of a conductive medium, holds significant potential for biomedical imaging. While classic models describe the phenomenon through conductivity modulation, a detailed understanding of its microscopic origins, particularly the role of ion behaviours, remains lacking. This study introduces a novel electrokinetic perspective by investigating how ultrasound modulates ion-solvent interactions, thereby bridging macroscopic AE signals with underlying ion dynamics. Through finite element simulations of a dilute NaCl solution, we demonstrate that acoustic pressure waves induce local variations in ion mobility and diffusion by altering ion hydration shells and solvent viscosity. These changes disrupt the balance among Coulombic, diffusive, and frictional forces on individual ions, leading to the local conductivity modulation. Furthermore, simulations reveal that acoustic perturbation of the electrode-electrolyte interface (EEI) significantly enhances AE signal generation, highlighting the EEIs critical role in AE-related applications. By linking acoustic modulation to fundamental ion-solvent interactions, this work not only provides a foundation for more accurate, microscopically grounded models of the AE effect but also connects AE effect modelling to the active research of solvation dynamics in physical chemistry.

Nanoscale bilayer mimetics such as protein or polymer-based nanodiscs are versatile tools to study the physical chemistry of lipid bilayers or the structures and functions of membrane proteins. Here, we introduce DNA-Lipid Nanodiscs (DLNs) in which the interface between hydrophobic lipids and the charged DNA is mediated through amphiphilic poly(ethylene)glycol (PEG). For this, we modified oligonucleotides with PEG and hybridized them to a single-stranded ring to form functionalized minicircles with a well-defined diameter. The center of these minicircles can be filled with a lipid bilayer through addition of detergent-solubilized lipids followed by detergent removal. Simulations reveal that the methylene groups in PEG form dynamic interactions with the acyl chains of lipids, effectively shielding the hydrophobic mismatch. As proof of concept towards incorporation of complex membrane proteins, we inserted the biotinylated transmembrane domain of synaptobrevin into these nanodiscs and bound them to streptavidin-modified quantum dots as a marker for successful incorporation. We envision these atomically precise, modular DNA scaffolds to be widely applicable in future studies of membrane proteins and nanoscale lipid membranes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/705827v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@17cfcb5org.highwire.dtl.DTLVardef@b2dd2corg.highwire.dtl.DTLVardef@d6899aorg.highwire.dtl.DTLVardef@e400c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

To achieve a therapeutic effect, nanoparticles delivering nucleic acids must facilitate endosomal escape (EE) of their cargo. Despite extensive research, the mechanisms that lead to an effective EE are not sufficiently understood. Herein, we utilized Molecular Dynamics (MD) simulations in All Atom (AA) and Coarse Grained (CG) resolutions to differentiate the interaction of four polymeric formulations (polyplexes) and one lipid nanoparticle (LNP) with endosomal membranes. On the one hand, the results emphasize the benefit of hydrophobic residues in the nanoparticles. On the other hand, the role of anionic lipids in the biological membranes is demonstrated. Furthermore, the identified interaction patterns were successfully correlated to the in vitro performance of the formulations. For the first time, different EE mechanisms of polyplex formulations are visualized in simulation and therefore distinguishable from one another. Hence, this work highlights the power of MD simulations for taking a big step towards better understanding EE efficiency. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/711661v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@abba74org.highwire.dtl.DTLVardef@5e2b8eorg.highwire.dtl.DTLVardef@7db144org.highwire.dtl.DTLVardef@1034e_HPS_FORMAT_FIGEXP M_FIG C_FIG

It was recently postulated that neural microtubules (neuro-MTs), which are densely packed inside axons and dendrites, are vacuum cylindrical nanotubes that can mediate neuroelectrical transmission with a unique form of quasi-superconductivity. In this work, the behaviors of free electrons inside a theoretical neuro-MT are modeled using computational analysis and calculations. We reveal that neuro-MTs can function as nanosized physiological devices that mediate neuroelectrical transmission with a super-high energy efficiency. Under physiological conditions, the binding of cytosolic cations (e.g., K+ and Na+) to the surface residues of a neuro-MT triggers its transition from the resting state to an active state, and the rapid dissociation of these cations triggers the opposite. The dipole ring structures of a neuro-MT will help terminate the free electron conduction inside with high efficiency. The proposed neuro-MT-mediated electrical transmission offers a novel mechanistic explanation for the saltatory conduction of the action potentials along an axon. This study also provides insights into the design of novel biomimicking room-temperature superconducting materials, such as the quasi-superconducting carbon or silicone nanotubes.

Backscattered electron scanning electron microscopy (BSE-SEM) provides compositional image contrast but has found limited application to biological samples due to the low atomic number difference between constituent elements, the thickness of the surrounding environment, and the need for complex sample preparation. Here, we demonstrate the use of room temperature liquid phase BSE-SEM (LPBSEM) for imaging Bacillus subtilis spores encapsulated in graphene liquid cells, preserving native hydration and reducing the thickness of the sample environment. This approach eliminates the need for staining and enables high-contrast visualisation of subcellular structures. Distinct structural layers within B. subtilis spores have been observed with a contrast similar to conventional thin-section transmission electron microscopy but without the need for sample preparation that potentially compromises sample integrity. We further investigate the influence of beam energy on the interaction volume depth and image contrast and propose optimal conditions for subsurface visualisation. Monte Carlo simulations have been used to validate our experimental observations and provide a quantitative framework for understanding BSE generation from hydrated, low atomic number specimens.

Efficient integration of proteins into amphiphilic polymer membranes offers new opportunities in synthetic biology and nanotechnology. Long-term protein reconstitution into artificial membranes remains challenging due to a lack of stabilising protein-membrane interactions found in native lipid bilayers. Here, we redesigned the transmembrane region of a CytK-4D {beta}-barrel nanopore for stable insertion into 3.5-6.6 nm thick PBD-PEO (poly(1,2-butadiene)-b-poly(ethylene oxide)) bilayers. PBD-PEO membranes offer high mechanical and chemical stability and low electrical noise, but the thick membrane hinders anchoring of biological nanopores. By systematically investigating the elongation of the {beta}-barrel, we engineered nanopore constructs suitable for PBD11PEO8 and PBD22PEO14 membranes. Efficient insertions were observed by adding amino acids that stabilised the transmembrane {beta}-barrel structure and enhanced anchoring of the nanopore into the membrane. Molecular dynamics simulations and single-molecule assays revealed that nanopores folded naturally into PBD-PEO bilayers, enabling successful detection of cyclodextrins and translocation of polypeptides and full-length proteins. Our study offers important lessons for the reconstitution of membrane proteins into artificial membranes. Moreover, these highly robust nanopore-membrane interfaces can be readily integrated into biosensing devices, enabling peptide and protein analysis directly from complex solutions.

Despite a few clinical successes, the efficacy of cancer nanomedicines remains limited by rapid clearance by the mononuclear phagocytic system and poor permeation across the abnormal tumor vasculature. We previously showed that methyl palmitate nanoparticles (MPN) can safely and reversibly inhibit the phagocytic activity of immune cells for several hours, thereby improving tumor accumulation and the efficacy of systemically administered nanomedicines. Here, we demonstrate that, on a shorter time scale, MPN can induce vasodilation, introducing an additional mechanism to enhance the accumulation of therapeutic agents within the malignant tissue. Upon internalization by macrophages and endothelial cells, MPN trigger the release of endogenous nitric oxide (NO), a key mediator of vasodilation, in a concentration-, and time-dependent manner. Following MPN administration, raster-scanning optoacoustic mesoscopy (RSOM) revealed vasodilation across multiple tissues, with the strongest effect observed in tumors. To assess enhanced tumor accumulation, we injected 70 kDa fluorescent dextran and demonstrated via histology a markedly increased fluorescence signal exclusively in MPN-treated tumors compared to controls 24 hours later. In addition, positron emission tomography (PET) imaging of 89Zr-labeled clinical iron oxide nanoparticles (Feraheme) showed significantly greater tumor accumulation after a 15-minute MPN pretreatment. Finally, general serum biochemistry panels and histological analyses of major organs in healthy mice revealed no toxicity following either single or repeated MPN dosing. Overall, this study demonstrates that MPN-induced vasodilation occurring within minutes enhances intra-tumoral deposition of macromolecules and small nanoparticles. Together with their longer-term effects on phagocytosis inhibition, these findings indicate that MPN can improve therapeutic delivery through complementary, time-dependent mechanisms that increase tumor perfusion and vascular permeability.

Nanotechnology is rapidly transforming medicine by enabling versatile platforms for targeted delivery, controlled release, and intracellular transport of therapeutic payloads. Polymeric mesoscale nanoparticles (MNPs) are 300 to 500 nm in diameter with a PEGylated surface that exhibit unique renal tropism, specifically toward renal tubular epithelial cells. Despite their well-described therapeutic applications and route of localization to the tubules, we do not yet understand their physicochemical stability and cellular internalization mechanisms. In this study, we investigated the stability of MNPs under stress conditions by subjecting them to repeated freeze-thaw cycles and varying storage conditions to evaluate the effects on particle size and polydispersity index. MNPs demonstrated negligible changes in size and PDI up to 4 freeze-thaw cycles. We found that both empty and dye-loaded MNPs demonstrated negligible change in size under standard -20{degrees}C storage conditions. While empty MNPs were only stable at room temperature for one day, and not at 37{degrees}C, dye-loaded nanoparticles were stable for at least eight days under both storage conditions. We then performed in vitro studies to evaluate MNP cellular uptake mechanisms using the human renal cell carcinoma cell line 786-O treated with pharmacological inhibitors of uptake pathways. We found that MNP internalization is almost entirely prevented by dynamin inhibitors, while macropinocytosis inhibition also reduced uptake, suggesting that such standard nanoparticle uptake pathways are robust to the mesoscale size range. These findings provide key insights into the stability profile and endocytosis mechanisms of MNPs, which are critical for materials scale-up and translation of novel kidney-targeted drug and gene therapies.

Ion channels are pore-forming transmembrane proteins that allow ions to move down an electrochemical gradient and across the channel pore and regulate many cell functions. Among them, are the G-protein-gated inwardly-rectifying K+ channels 1 (GIRK1) that are ubiquitously expressed with major functions in the brain and heart. Interestingly, significantly higher GIRK1 expression has been found in estrogen receptor positive (ER+) breast cancer patients compared to patients with HER2+ tumors or normal patients, and that was statistically correlated with shorter survival times and metastatic potential. Herein, we report the preparation of [~]4 nm GAT1508-coated poly(ethylene glycol) gold nanoparticle (PEGylated AuNP) biomarker for ER+ breast cancer cell screening through an optical microscope. A urea-based small molecule, GAT1508, with an N-methylpyrazole benzyl group on one side and a bromo-thiophene tail on the other side, has been shown to predominantly bind GIRK1 subunits and specifically activate GIRK1/2 channels. Two derivatives of GAT1508were synthesized and characterized: an ethylamine derivative (GAT1508-EA) with a chain extension from the benzyl ring, and a propylamine derivative (GAT1508-PA) with a chain extension from the pyrazole ring. Electrophysiology (TEVC and whole-cell patch-clump) experiments as well as fluorescence studies (Thallium assay) showed that only GAT1508-PA inhibited GIRK1/2-mediated K+ currents in transfected HEK293GIRK1 cells. Docking studies showed strong binding for the propylamine GAT1508 derivative, both in the amine form (GAT1508-PA) as well as in the amide form (GAT1508-PA-EG2; coupled with PEG as in the AuNPs). GAT1508-PEG-AuNPs (GAT1508-NPs) were synthesized subsequently with [~]65 wt% metal loading. UV-Vis studies revealed the presence of the conjugated ligand at 260 nm. Flow cytometry studies showed binding of Alexa 594-labeled GAT1508-NPs in ER+ MCF-7 breast cancer cells with a strong interaction, while incubation of fixed MCF-7 cells with a GAT1508-NP solution led to optical detection of ER+ breast cancer cells, without the need of fluorescent dyes and additional amplification steps. Detection was not feasible in MDA-MB-231 cells, a triple (-) breast cell line that does not express GIRK1. This is the first study, to our knowledge, that couples nanotechnology with small molecule drug design and electrophysiology to develop ion channel-tracing molecular probes for the detection/screening of ER+ breast cancer.

Protein cages are versatile platforms capable of encapsulating a wide range of nanoparticle cargo within biocompatible protein shells while providing tunable functionalities. Here, we investigated a self-assembly system that forms vesicle-like protein cages while simultaneously encapsulating nanoparticles at high density, yielding pomegranate-like protein- nanoparticle hybrid materials. Amphiphilic recombinant fusion protein building blocks based on elastin-like polypeptides, leucin zippers, and fluorescent proteins were employed to assemble vesicle-like protein cages via temperature-triggered liquid-liquid phase separation in the presence of fluorescent polystyrene nanoparticles. Analysis of nanoparticle encapsulation density and protein cage size indicates cooperative interactions between protein building blocks and nanoparticles that mediate the formation of protein-nanoparticle coacervate intermediates, which subsequently convert into core-shell hybrid protein cages, as further supported by kinetics studies. We demonstrate the self-assembly hybrid protein cages incorporating a fluorescent calcium sensor protein and titanium oxide nanoparticles, which exhibit a drastic enhancement in their calcium-sensing capability as a result of nanoparticle encapsulation. This platform offers a broadly applicable strategy that integrates protein biofunctionality with diverse nanoparticle properties for development of advanced hybrid materials.

The global rise of antimicrobial resistance has significantly reduced the effectiveness of conventional antibiotics, highlighting the urgent need for alternative and complementary therapeutic strategies. Nanotechnology-based drug delivery systems, particularly lipid nanoparticles, have emerged as promising tools to enhance antibiotic efficacy while limiting toxicity and resistance development. In this study, we evaluated the antimicrobial activity and drug carrier potential of Ohmline, a novel alkyl-ether glycolipid capable of self-assembling into nanotubes and lipid nanoparticles. First, a wide range of Gram-positive and Gram-negative bacteria were used to test Ohmline nanotubes antibacterial activity. All examined strains were partially inhibited, with a more noticeable effect on Gram-positive bacteria. Then, the synergistic potential of Ohmline combined with commercially available antibiotics (ampicillin, ceftriaxone, and ciprofloxacin) was evaluated using two different approaches: binary mixtures of Ohmline nanotubes and antibiotics and microfluidically produced Ohmline:DMPC (75:25) nanoparticles with the antibiotics encapsulated. Binary formulations demonstrated strong, strain-dependent synergistic effects at sub-MIC antibiotic concentrations, particularly against Enterococcus faecalis and Citrobacter braakii. Notably, antibiotic encapsulation within Ohmline nanoparticles further enhanced antimicrobial efficacy compared to non-encapsulated combinations, achieving near-complete growth inhibition in E. faecalis and significant inhibition in Klebsiella pneumoniae and C. braakii. Overall, our findings demonstrate that Ohmline possesses intrinsic antibacterial activity and acts as an effective lipid nanocarrier that potentiates antibiotic action. The dual functionality of Ohmline supports its potential as a versatile building block for next-generation antimicrobial formulations.

The structural and functional characteristics of membrane proteins can be influenced by the composition of the membrane. Consequently, native membranes are most relevant for the study of receptors and other membrane proteins. In this study, we investigated two types of cell-derived vesicles: natively shed extracellular vesicles (EVs) and mechanically derived vesicles (MVs). To this end, we utilized the human breast cancer cell line SKBR3, which strongly overexpresses the receptor HER2. We designed a protocol based on designed ankyrin repeat proteins (DARPins) to purify EVs and MVs enriched in HER2, and to ensure the native orientation of the HER2 receptors within the vesicle. The isolated HER2-containing EVs and MVs were characterized by cryo-EM, cryo-electron tomography (cryo-ET) and mass spectrometry (MS), which revealed fundamental differences between the different vesicle types. Our study highlights the greater structural diversity of EVs over MVs. A single particle cryo-EM analysis and classification of all visible receptors on the vesicle surface yielded electron density consistent with HER2 at modest resolution. Taken together, our results suggest that MVs can serve better than EVs as a suitable platform for the structure determination of membrane proteins within their native membrane environments.

Accurate characterization of the nanoparticle (NP) protein corona is essential for predicting biological fate, safety, and therapeutic efficacy, and for enabling robust biomarker discovery. Standard isolation techniques, most commonly centrifugation and magnetic separation, are widely used, yet they rarely account for co-isolating endogenous biological NPs such as extracellular vesicles (EVs). This oversight can distort the apparent "biological identity" of the NP. Here, we quantitatively demonstrate the magnitude and impact of EVs on the perceived protein corona composition. We incubated highly monodisperse polystyrene NPs (50-1000 nm) and superparamagnetic beads in either standard human plasma or plasma depleted of EVs by immunoaffinity capture targeting 37 EV surface epitopes. Mass spectrometry revealed that EV depletion reduced the number of proteins identified on polystyrene NPs by 60-75% and on magnetic beads by 45-50%. Importantly, EV depletion also altered the apparent abundance hierarchy; it restored the expected relative abundance and rank of major plasma proteins such as albumin and shifted the top-ranked proteins from intracellular cytoskeletal component, consistent with EV carryover, to genuine soluble plasma adsorbates (e.g., apolipoproteins, complement factors). These results highlight that standard corona workflows can inadvertently co-isolate a vast array of EV-associated proteins, yielding inaccurate proteomic profiles. Discriminating genuine corona proteins and EV-associated contaminants is critical for advancing nanomedicine, ensuring predictive safety and efficacy profiles, and enhancing the precision of NP-based biomarker discovery.

mRNA-lipid nanoparticles (LNP) have proven their potential as a rapidly adaptable vaccine platform and promise to revolutionize numerous therapeutic areas. A major hurdle towards the widespread adoption of mRNA-LNP vaccines and therapeutics is their limited liquid shelf-life compared to more established modalities currently necessitating an ultralow temperature cold-chain to enable their distribution and storage. While ongoing efforts aim to improve liquid stability through chemical modification of mRNA and lipid components, complementary strategies that are broadly applicable across chemistries may further accelerate translation. Here, we present an approach to improve the liquid shelf-life of mRNA-LNPs that does not rely on modifications to the mRNA or LNP chemistry. In particular, we show that bleb formation induced by high ionic strength acidic citrate buffers during LNP formation reduces mRNA degradation and retains in vitro activity during extended liquid storage. We observed an increase in the in vitro activity storage half-life from 2.8 to 18.9 days at 25{degrees}C when prepared using high ionic strength buffers translating into a [~]7-fold improvement in the liquid shelf-life of MC3-LNPs. This enhanced stability of LNPs with large amount of bleb formation was mainly attributed to reduced rates of lipid-mRNA adduct formation and mRNA fragmentation. Furthermore, the acidic buffer dependent stabilization was observed across different ionizable lipids with the extent dependent on the ionizable lipid head group. We envision that the induction of bleb formation via selection of appropriate acidic mixing buffers may represent a universal approach to enhance mRNA-LNPs stability and enable extended long-term refrigerated storage.

X-ray fluorescence microscopy (XFM) continues to develop as a powerful quantitative technique for high resolution, label-free, elemental mapping of biological, environmental, and material samples. Methods for rigorously fitting spectra, increasing throughput, accounting for background signals, and deconvoluting overlapping emission lines continue to evolve. We show here that quantitative fits of XFM data obtained after removing a baseline, calculated by connecting peak edges, can be unexpectedly dependent upon acquisition dwell-time and spectral aggregation leading to differences in apparent elemental content. Using mouse preimplantation embryos and ovarian follicles as model samples, we demonstrate how these variables influence quantitative comparisons between samples. We find that subtracting an empirically measured blank spectrum instead of a baseline provides quantitative XFM elemental mapping results that are independent of dwell time and spectral aggregation dependencies.