Nanoscale Advances

Copper chalcogenide nanocrystals (NCs) are promising candidates for biophotonic applications due to their tunable optical properties. Concrete methods to examine the relationship between their degradation and toxicity are necessary to enable development of nanoconstructs with reduced toxicity. This study compares the degradation and acute cytotoxicity of three compositions of micelle-coated copper chalcogenide NCs: the fluorescent semiconductor copper indium sulfide (CuInS2), and the plasmonic semiconductors copper sulfide (Cu2-xS) and chalcopyrite copper iron sulfide (CuFeS2). We developed a quantitative degradation assay to assess ion release from these ultra-small nanocrystals, revealing that while all three particles biodegrade, CuInS2 and CuFeS2 undergo rapid degradation in artificial lysosomal fluid, leading to a burst release of indium and iron ions. In cellular toxicity assays, CuInS2 exhibited significantly higher acute cytotoxicity than Cu2-xS and CuFeS2, primarily due to indium-induced necrosis. To mitigate this toxicity, an alternative surface-binding polymer coating was introduced, effectively reducing both the degradation rate and cytotoxicity of CuInS2. These findings highlight the influence of both nanocrystal composition and coating chemistry in moderating the acute cytotoxity of degradable nanocrystals, demonstrating that tuning of composition and degradation rate can be used to moderate nanoparticle toxicity.

Edible plant-derived nanovesicles (PDNVs) have emerged as promising nanotherapeutic strategies for various diseases, including cancer. The biochemical composition and functional properties of PDNVs vary considerably on the basis of their botanical source. Bacopa monnieri (L.) Wettst is a medicinal plant renowned for its rich phytochemical profile, yet the isolation and biological activities of B. monnieri -derived nanovesicles (BMNVs) remain unexplored. We report, for the first time, the isolation, molecular cargo profiling, and in vitro functional evaluation of BMNVs against neuroblastoma cells. The isolated BMNVs displayed a characteristic bilayer morphology with an average particle size of [~]112 nm. Mass spectrometry-based metabolite analysis revealed an enrichment of triterpenoids and triterpene saponins, whereas protein cargo analysis revealed superoxide dismutase, which is correlated with their intrinsic free radical scavenging activity. In vitro assays demonstrated that BMNVs significantly suppress neuroblastoma cell growth and induce morphological alterations. Confocal three-dimensional reconstruction confirmed the cellular internalization of the BMNVs, revealing a distinct perinuclear distribution. This study provides the first evidence of the use of BMNVs as bioactive carriers, highlighting their potential as novel nanotherapeutic agents and establishing B. monnieri as a valuable natural resource for the development of bioactive plant-derived nanovesicles for nanomedicine. HighlightsO_LIFirst isolation and biophysical characterization of Bacopa monnieri-derived nanovesicles (BMNVs). C_LIO_LIBMNVs have diverse metabolite profiles and are notably enriched in triterpenoids and triterpene saponins. C_LIO_LIThe superoxide dismutase (SOD) identified within BMNVs confers intrinsic free radical-scavenging activity. C_LIO_LIBMNVs exhibit therapeutic potential as anti-neuroblastoma agents. C_LIO_LIThese edible plant-derived nanovesicles offer a versatile, biogenic platform to explore for further development in diverse therapeutic and nutraceutical applications. C_LI

Cathodoluminescence (CL) microscopy has the potential to achieve a key goal in biological imaging: the simultaneous visualization of proteins and cellular ultrastructure. This goal can be attained by tagging proteins of interest with spectrally distinct cathodoluminescent probes for detection in electron microscopy. To this end, lanthanide nanoparticles (LNPs) are promising probe candidates due to their stability under the electron beam and their distinct ion-dependent emission spectra suitable for multiplexed detection. However, the hydrophobic surface chemistry of LNPs limits their use in biological samples and requires surface functionalization compatible with aqueous environments and EM sample preparation protocols. Here, we use a DNA-based ligand exchange strategy that renders cathodoluminescent LNPs hydrophilic and compatible with further functionalization for specific protein labeling. We characterize the CL emission of DNA-functionalized LNPs following aqueous transfer and common EM preparation steps, including osmium tetroxide staining and drying protocols based on hexamethyldisilazane and critical point drying, and show that LNPs retain their CL emission under all tested conditions. Finally, we demonstrate multicolor CL imaging of spectrally distinct, DNA-functionalized LNPs on the surface of mammalian cells, enabling simultaneous visualization of cellular ultrastructure via secondary electrons and LNPs via multiple CL color channels.

Inflammation is a fundamental immune response but, when dysregulated, contributes to the pathogenesis of numerous inflammatory disorders. Although there are several conventional anti-inflammatory drugs which are effective, their long term use is often associated with adverse side effects, which highlights the need for safer alternative therapeutic drugs. Probiotic derived membrane vesicles (MVs) have recently emerged as biologically active nanostructures capable of modulating host immune responses. In the present study, MVs isolated from Lactobacillus acidophilus MTCC 10307 were evaluated for their anti-inflammatory efficacy and safety profile using in vitro and in vivo models. In RAW 264.7 macrophages, L. acidophilus MVs significantly attenuated lipopolysaccharide induced expression of the pro-inflammatory mediators Il-1{beta}, Il-6, and iNOS, accompanied by reduced nitric oxide and reactive oxygen species production which was abolished in the proteinase K treated MVs. The protein levels of NF{kappa}B and IL1{beta} were also reduced in the treatment groups. Repeated dose oral toxicity studies revealed no adverse effects, as evidenced by body weight and histopathological evaluation of major organs. The anti-inflammatory properties of L. acidophilus MVs were further validated in an in vivo hind paw edema model, which shows inflammation resolution demonstrated by molecular and histological analysis. Proteomic analysis using LC-MS/MS identified the presence of surface-layer protein A (SlpA) which is a potential bioactive component which might contribute to the observed immunomodulatory effects. Collectively, these findings demonstrate that L. acidophilus MVs exert potent anti-inflammatory activity while maintaining an excellent safety profile using integrated in vitro and in vivo models.

Environmental degradation and accumulation of plastics results in micro- and nanoplastics (MNPLs) that are small enough to cross biological barriers, including the blood-brain barrier. Microglia, resident immune cells of brain, are critical regulators of neuroimmune homeostasis and represent a cellular target of nanoplastic exposure. In this study, we assessed the neurotoxic effects of two sizes of polystyrene nanoplastics (PS-NPs; 100 nm and 500 nm) using integrated in vivo and in vitro exposure and washout paradigms. In vivo exposure in mice (60 days; 0.15 or 1.5 mg/day) showed the accumulation of both PS-NP sizes in the cerebral cortex without histopathological damage. However, cortical microglia showed pronounced morphological remodeling, observed as increased expression of Iba1 and GFAP. Transcriptomic profiling of cortical tissue revealed a strong size-dependent response. The 100 nm PS-NP group revealed 18 DEGs (|log2FC| [&ge;] 2, padj < 0.05), whereas the 500 nm PS-NPs showed more than 4,000 DEGs, including upregulation of immune- and microglia-associated genes (CCL5, CXCL10, LCN2, LYZ2) and downregulation of synaptic and neuronal signaling genes (GRIN2B, SYN1, STX1B, MAP1B, ITPR1/2). In vitro assessment, using BV2 microglia cells, showed internalization of PS-NPs via the endolysosomal pathway, with strong co-localization to Rab7- and LAMP2-positive compartments and prolonged intracellular retention following exposure washout. Also, microglial activation markers (Iba1, CD68) exhibited a transient, size- and concentration-dependent increase, correlated with intracellular particle burden rather than cumulative exposure. Overall, these findings demonstrate that PS-NPs accumulate in brain, driving size-dependent microglia activation and transcriptomic reprogramming, even after cessation of exposure to PS-NPs. HighlightsO_LIPS-NPs (100 nm and 500 nm) reach mouse cerebral cortex following 60-day oral exposure. C_LIO_LIPS-NPs were internalized by microglia; accumulated in endolysosomal compartments. C_LIO_LIPS-NP exposure induced transient microglial activation without sustained cytotoxicity. C_LIO_LIMicroglial activation was correlated with intracellular PS-NPs burden. C_LIO_LITranscriptomics revealed disruption of neuroimmune and microglial regulatory pathways. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/712727v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aba3eaorg.highwire.dtl.DTLVardef@1967641org.highwire.dtl.DTLVardef@12da637org.highwire.dtl.DTLVardef@1fb8441_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Dimethyl sulfoxide (DMSO) is a polar aprotic organic solvent that is widely used in biological applications. It is routinely applied as a cryoprotectant for long-term cell freezing as well as to dissolve peptides or drugs for immune cell functional assays. We report on a remarkable impact of low concentrations of DMSO on in vitro expansion of the total CD3+ pool isolated from human PBMCs as well as on the purified CD8+ T cell fraction thereof in the presence of different cytokine combinations typically used for therapeutic T cell expansion. Characterizing survival, proliferation, activation, exhaustion and differentiation, we demonstrate that DMSO at low concentrations substantially skews the differentiation of T cells towards a memory phenotype in a dose-dependent way. This is a desirable outcome for the field of adoptive T cell therapies for cancer, where it has been established that T cells with a memory phenotype exert superior anti-cancer immune responses.

Correlative microscopy techniques are used for many different applications in the biological sciences because the comparison of different imaging methods allows researchers to gain more insight and data from samples. Correlative light and electron microscopy (CLEM) methods have been developed to preserve biological samples to withstand the harsh environments necessary for electron microscopy. After first being imaged using widefield (WF) and super-resolution structured illumination fluorescence microscopy (SIM), a NanoSuit chemical treatment was applied to a mammalian testis sample before imaging with scanning electron microscopy (SEM). This was done to compare the image quality and resolution of each technique. SEM yields higher resolution and offers validation of results from SIM.

The design of multifunctional nanomaterials that combine chemotherapy with photothermal therapy (PTT) has emerged as a promising strategy to overcome the limitations of conventional cancer treatments. Here, we report the fabrication of a novel therapeutic hydrogel system composed of Folic Acid-functionalized iron oxide nanoparticles (IO NPs) synthesized via an arc-discharge method, loaded with doxorubicin (DOX), and embedded within a bacterial cellulose/polyvinyl alcohol (BC/PVA) matrix. The arc-discharge technique produced crystalline FeNPs with high purity and narrow size distribution. Folic acid conjugation enabled tumor-targeted delivery, while DOX was efficiently incorporated via electrostatic and {pi}-{pi} stacking interactions. Embedding in the BC/PVA hydrogel facilitated sustained drug release and improved biocompatibility. Structural and functional characterization was conducted using X-ray diffraction (XRD), scanning electron microscopy (SEM), UV-Vis spectroscopy, magnetization studies, swelling and rheological analysis, and photothermal heating experiments. In vitro cancer cell studies demonstrated enhanced therapeutic efficacy of the hydrogel system under near-infrared (NIR) irradiation, where synergistic chemo-photothermal effects resulted in significant reduction in cell viability compared to single-mode treatments. This study highlights a multifunctional nanoplatform that integrates targeted delivery, controlled release, and dual therapeutic modalities for effective cancer treatment.

Cellular immunotherapy has revolutionized cancer treatment by enabling more targeted and personalized disease management. As the field progresses, there is an increasing need for high-throughput in vitro assays to efficiently assess the cytotoxicity of therapeutic cells. Conventional cytotoxicity assays pose various limitations in the workflow and scalability. Here, we present an mRNA lipid nanoparticle (mRNA-LNP) approach to efficiently and robustly deliver reporter genes to target cells for assessing immune effector cell-mediated cytotoxicity. This approach enables the rapid, homogenous reporter expression without compromising the viability of target cells. The cytotoxicity results obtained using mRNA-LNP-transfected cells are highly consistent and comparable to those obtained using cell lines with stable reporter gene expression. Finally, we highlight the mRNA-LNP approachs compatibility across a diverse range of tumor models, including primary tumor-derived models, enabling rapid and high-throughput assessment of the potency of various cytotoxic therapeutic cells.

Silica-encapsulated gold nanostars (AuNStar-SiO2) are a widely used plasmonic nanoparticle platform for surface-enhanced resonance Raman scattering (SERRS) bio-applications. In this paper, we demonstrate that coupled nanostar subpopulations can dominate the ensemble-average SERRS response of the suspension and that near-neutral standard cell culture conditions are sufficient to hydrolyze the silica nanoshell and introduce variability in signal intensity following in vitro endocytosis. Monomeric and oligomeric AuNStar-SiO2 fractions were isolated using continuous density-gradient centrifugation and monomeric populations were found to exhibit significantly weaker SERRS compared to their oligomeric counterparts. Using monomer-enriched AuNStar-SiO2, we investigated the stability of the silica nanoshell under conditions representative of sequential acidification during endocytosis and characterized the subsequent changes to nanoparticle optical properties. In acidic environments, reflecting lysosomal pH, the silica shell was stable, whereas near-neutral and alkaline conditions in cell culture medium induced silica-shell hydrolysis, nanostar release, and interparticle aggregation, leading to transient SERS amplification. When cells were treated with AuNStar-SiO2 under near-neutral and acidic conditions, we observed the opposite trend in SERS signal strength. At pH 7.4, the SERRS signal was suppressed even though transmission electron microscopy (TEM) images of intracellular nanoparticles showed progressive extents of silica hydrolysis, while at pH 6.4 SERS signal was strong and the silica shell of intracellular nanoparticles remained intact. Together, these findings show how SERRS output can differ between control conditions and biological applications, highlighting the role that local environmental factors play in nanoparticle stability and performance. Our results highlight the previously overlooked role of silica nanoshell instability on SERRS signal output in physiological environments and describe opportunities to harness silica nanoshell hydrolysis to improve the biomedical application of silica-coated plasmonic probes.

Both for diagnostic purposes and regenerative medicine, it is essential to develop advanced imaging platforms capable of tracking the biodistribution of small extracellular vesicles (sEVs), as current methods are limited by inadequate resolution and sensitivity. In this study, we introduce a novel labeling strategy utilizing the radioisotope zirconium-89 (89Zr), which boasts a half-life of 78.4 h and is cost-effective to produce. To achieve this, we designed a new chelator tailored for 89Zr4+ that offers enhanced stability compared to the conventional deferoxamine (DFO). This chelator forms a robust complex with 89Zr4+ at room temperature, suitable for sEV labeling for PET imaging applications. The radiolabeling process involved a two-step procedure: first, conjugation of the chelator to the sEVs, and second, radiolabeling with 89Zr4+. The resulting sEV-L1-Zr demonstrated a radiochemical yield of approximately 60% and maintained around 80% stability in plasma over seven days. Importantly, our modifications did not alter the morphology, surface protein composition, internal RNA content, or bioactivity of the sEVs. We successfully visualized sEVs at very low doses in the mouse heart following intravenous injection of sEV-L1-Zr. Additionally, ex vivo experiments using a Langendorff rat heart perfusion model confirmed targeted accumulation of the vesicles in cardiomyocytes as compared to other cells in the heart compartment. This approach provides a promising platform for sensitive and stable in vivo tracking of sEVs, advancing their application in both diagnostic imaging and regenerative therapies.

Silver (Ag+) ions are known to be toxic to bacteria, cells, organisms and living systems; yet its impacts on the locomotion of surface-crawling organisms remain poorly quantified. Here we investigated the short-term (0-6 hours) effects of Ag+ ions on the locomotion of Drosophila melanogaster larvae on flat agarose surfaces containing Ag+ ions at different concentrations (0, 1, 10, and 100 mM). By quantifying their locomotion, we found that Drosophila larvae showed shorter accumulated distances and reduced crawling speed. Additionally, we quantified the go/stop dynamics and peristalsis of the larvae and observed that Ag+ ions disrupted the normal, rhythmic, peristaltic contraction of the larvae and "trapped" them in the stop phase. Such toxic effects were dependent on Ag+ concentration and exposure duration.

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

Understanding lipid metabolism in peripheral glial cells is crucial for elucidating the molecular mechanisms underlying neurodegeneration, cancerogenesis and therapy resistance. Here, we introduce a spectrolipidomic sensing approach that integrates Raman, FT-IR, and AFM-IR spectroscopy to monitor nanoscale cholesterol remodeling in glial cells exposed to cannabidiol (CBD). Deuterated cholesterol (dChol) was employed as an intrinsic, spectroscopically active molecular probe, enabling selective tracking of cholesterol transformations through characteristic C-D vibrational signatures within the 2300-2000 cm-1 silent spectral region. Multimodal vibrational spectroscopy provided label-free, spatially resolved insight into lipid organization, redistribution, and metabolic reprogramming across micro- and nanoscales. The dChol probe enabled semi-quantitative evaluation of cholesterol uptake, esterification, and membrane integration, revealing that the sequence of CBD exposure, before or after probe addition, triggers distinct lipid metabolic pathways. Raman spectroscopy demonstrated superior sensitivity, with reliable detection of intracellular dChol at concentrations as low as 10 {micro}M, outperforming FT-IR imaging and confirming its suitability for cell lipid sensing. This analytical platform establishes deuterium-labeled lipids as powerful vibrational sensors for probing lipid metabolism and CBD-induced remodeling in situ. The presented spectrolipidomic framework paves the way for next-generation, spectroscopy-based biosensing systems capable of visualizing lipid dynamics, membrane restructuring, and drug- lipid interactions under pharmacological or environmental stress conditions. HighlightsO_LIDeuterated cholesterol (dChol) used as an intrinsic vibrational sensor C_LIO_LILower detection threshold of intracellular dChol for Raman than FT-IR C_LIO_LIAFM-IR reveals phases of lipid droplet formation in nanoscale C_LIO_LICBD alters cholesterol uptake, esterification, and lipid unsaturation profiles C_LI

The present study introduces a novel design and analysis of a sensor based on a terahertz metamaterial absorber (MMA) to identify Glioblastoma cell by employing microwave imaging techniques. Terahertz (THz) frequencies offer unique advantages for biomedical applications. Computer Simulation Technology (CST) employs a finite integration (FIT) approach to simulate the suggested structure in the resonant frequency (RF) range of 4.5 THz to 6 THz. The crystal structure displays three distinct absorption peaks at resonance frequencies. The MTA can absorb energy in three specific spectral bands: 4.782 THz, 5.30 THz, and 5.7319 THz. At these frequencies, the MTM achieves exceptionally high absorption, reaching 99.99%, 99.98%, and 99.68% peak absorption, respectively. The electric field (E), magnetic field (H), and surface current of the MTM are also examined. Finally, detecting Glioblastoma cells is also being investigated by analyzing the E-field H-field using microwave imaging. The suggested biosensor features a high-quality factor of 143.63, a frequency shifts per refractive index of 1.45 THz/RIU. In this study, the PCR value is measured at 0.95 at a frequency of 4.782 THz, indicating a high efficiency in polarization conversion. Polarization Conversion Ratio (PCR) quantifies the efficiency of a metamaterial in converting the polarization of an incident electromagnetic wave. Extensive simulation studies confirm the sensors capability to distinguish between healthy and cancerous cervical tissue. The suggested MMA-based sensor has numerous advantages and can be utilized for Glioblastoma cell detection.

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.

Nanoplastics generated from plastic waste in our ecosystems are becoming increasingly prevalent as bulk plastics exposed to natural factors like water and sunlight fragment to the nanoscale over time. These incidental nanoplastics span a wide range of physicochemical properties, which makes studying nanoplastic interactions in biological systems difficult. Here, we characterized the behavior of incidental nanoplastics generated through mechanical abrasion within coacervate droplets to probe the surface properties of the nanoplastics. We used elastin-like polypeptides (ELPs) to create hydrophobic or charged coacervate microenvironments. Using optical microscopy and fluorescence quantification, we observed that nanoplastics made from polyethylene terephthalate (nPET), nylon 6 (nPA), and polystyrene (nPS) exhibited distinct partitioning behavior with more favorable interactions with hydrophobic droplets. This indicated that the hydrophobic polymer backbone was the predominate surface feature despite exposed functional groups of the incidental nanoplastics, in contrast to findings with model carboxylated latex nanospheres (nPS-COOH). Furthermore, the selective partitioning of incidental nanoplastics into the hydrophobic droplets was able to capture over 80% of nPET in solution, and after recovery of the protein droplet, was able to cumulatively capture over 75% of the nPET feedstock across multiple cycles. This work explores the nuanced surface characteristics of incidental nanoplastics, expands the application of coacervates as chemical probes, and demonstrates a biopolymer approach for effective nanoplastic removal.

The presence of hydroxyapatite (HAp) in the Cuticle of laying hen eggshells was investigated through an extensive and detailed study combining scanning electron microscopy (SEM) coupled with energy-dispersive spectroscopy (EDS), as well as micro-Raman, micro-FTIR, X-ray diffraction (XRD), and thermogravimetric analysis (TG). Additionally, Raman and FTIR spectra of Cuticle HAp were compared with those obtained from the internal surface of chicken femur fragments and from a bovine HAp sample. Examination of the same region by SEM in SE (topographic) and BSE (subsurface compositional contrast) modes revealed unidirectional (nanofibrous) calcite growth within the Vertical Layer (VL) and Palisade Layer (PL), which together constitute nearly the entire eggshell thickness. Shell thickening in the VL and PL layers appears to proceed via an additive mechanism characterized by the successive deposition of nanodroplets containing, according to our hypothesis, the mineral phase, water, and organic components. This multiphasic system generates lamellae that progressively increase in thickness through the continuous incorporation of new nanodroplets onto the pre-existing surface. This additive nanodroplet-mediated growth contributes to understanding how micropores form in the PL and VL. Biomineralization via an additive mechanism is strongly supported by the presence of nano-hemispheres attached to growing lamellae in the VL and PL. Statistical analyses corroborate the relationship between the diameter of Cuticle nanospheres and that of nano-hemispheres in the Vertical Layer. Fractures observed in the VL indicate structural continuity between the Cuticle and the Vertical Layer, suggesting that additive growth involves a continuous supply of HAp -- possibly across the entire uterine surface -- which, through a yet undescribed mechanism, dissolves and/or transforms calcium phosphate nanospheres into calcium carbonate nanofibers.

Magic-angle spinning (MAS) solid-state NMR (SSNMR) has been widely used to determine amyloid fibril structures at atomic resolution. Such studies typically rely on homogeneous fibril preparations that produce narrow linewidths and high spectral resolution, enabling reliable resonance assignment and structural analysis. However, many biologically relevant amyloid aggregates are structurally heterogeneous, resulting in spectral broadening and reduced sensitivity that hinder atomic-resolution characterization. Lipids are known to modulate amyloid aggregation pathways and promote the formation of toxic species that are often less homogeneous, further complicating NMR-based investigations. Here, we evaluate the feasibility of utilizing the benefits associated with high-field (1.1 GHz) SSNMR for studying ganglioside GD3-catalyzed A{beta}42 aggregates. Uniformly-13C,15N-labeled A{beta}42 was incubated with GD3 to generate lipid-associated aggregates and analyzed under MAS conditions. 13C cross-polarization magic-angle spinning (CPMAS) spectra and 2D 13C-13C chemical shift correlation experiments using CORD (COmbined R2nv-Driven) mixing were acquired and compared with data collected at 600 MHz. Despite the heterogeneous nature of the GM1-associated assemblies, the 1.1 GHz spectra exhibit enhanced sensitivity and improved spectral resolution. Better resolved resonances corresponding to selectively structured regions of A{beta}42 are observed, indicating the presence of an ordered core within the lipid-associated aggregates. These results demonstrate that ultrahigh-field SSNMR significantly improves the characterization of heterogeneous amyloid assemblies and provides a promising approach for atomic-level investigation of biologically relevant, lipid-modulated A{beta} aggregates.