Nanoscale Advances

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.

Surface functionalization of biomaterials enables the immobilization of proteins and other molecules and can be utilized to direct the biological response to devices and implants. Fetuin-A is a blood plasma protein involved in numerous physiological processes, including the regulation of mineralization. Notably, many investigations of fetuin-A have explored its cellular interaction when in solution, but limited studies report the role of fetuin-A when used as a surface modifier. The present investigation explores the response elicited by fetuin-A on Saos-2 cells when it is immobilized on a model gold surface through the covalent reaction with dithiobis(succinimdyl propionate) (DSP). Comparative surface characterization using x-ray photoelectron spectroscopy (XPS), atomic force microscopy - infrared spectroscopy (AFM-IR) and surface plasmon resonance (SPR) confirmed the surface modifications but indicate partial inhomogeneity in the functionalizer surface coverage. The interaction of albumin and fetuin-A with the surface was quantified by radiolabeling, quartz crystal microbalance with dissipation (QCM-D) and SPR, demonstrating a higher mass of fetuin-A bound to the surface in comparison to serum albumin. Over 7 days, cells bound to the surfaces with immobilized fetuin-A showed significantly hindered proliferation of osteoblast-like cells compared to the positive control (fibronectin), presumably due to a decrease in cell metabolism. This study provides new insights into the role of fetuin-A in regulating Saos2 cell response and elucidates its potential use in combination with chemical functionalizers for biomedical applications requiring surface modification.

Silver has been used as a biocide for centuries, mostly in health-oriented applications. However, as a biocide, silver is toxic not only to its intended targets, mainly bacteria and fungi, but also to all living cells. Because of this toxicity, it is desirable to use forms of silver that maximize the required biocidal activity while minimizing the amount of silver that will be released in the environment at the end of life of the product. Silver nano objects are a good compromise for such requirements. The high surface to volume ratio allows for good reactivity and thus good biocidal activity, while the small amount of silver present in nano objects allows for a limited environmental release at the product end of life. In this work, we tested three types of silver nano objects. The first type, polyvinylpyrrolidone-coated silver nanoparticles (nAg-PVP) were used as a control nanoparticle, as this type of nanoparticle is now widespread. We also manufactured and tested maltodextrin-coated silver nanoparticles (nAg-MD) and micrometric (20 {micro}m in two dimensions and a few nanometers in the third one) silver flakes ({micro}AgSF). For these three silver nano objects, we investigated the biocidal activity by stringent tests using both Staphylococcus aureus and Escherichia coli as target bacteria. In addition, we investigated toxicity on mammalian macrophages or keratinocytes cell lines, as well as on an insect hemocyte cell line. Our results showed that the two innovative silver nano objects (nAg-MD and even more {micro}AgSF), showed both a better bactericidal activity and a lesser toxicity than the reference nAg-PVP nanoparticles. In addition, we also checked that beyond toxicity, the silver nano objects did not induce an inflammatory reaction, making them safer to use.

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.

Cancer cell membrane coated biomimetic nanoparticles have been shown to be highly efficient in cellular uptake, homotypic tumour targeting, and the ability to suppress tumour growth compared to uncoated nanoparticles. Long duration anti-cancer treatment regimens require highly stable cancer cell membrane coated biomimetic nanoparticle. To manufacture such highly stable cancer cell membrane coated biomimetic nanoparticle, we used "Click-chemistry" to encapsulate cancer cell membrane on nanoparticles. In situ characterization was done to confirm the functionality of the novel Click-chemistry based formulation to encapsulate cancer cell membrane on nanoparticles. Gold nanoparticles were encapsulated with the cell membranes of cell lines of lung adenocarcinoma, malignant melanoma, high-grade serous epithelial ovarian cancer, colorectal cancer, oral cancer, esophageal adenocarcinoma, adenoid cystic carcinoma of salivary gland, and breast cancer. Functional group analysis, size, morphology, and surface charge confirmed long-stability of the biomimetic nanoparticles after incubating in complete growth medium for 12-months.

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

Upon near-infrared (NIR) irradiation, combined treatment comprising of photothermal therapy (PTT) and chemotherapy (CHT) offers synergistic effects by inducing localized heat to intended tumor sites and simultaneously allowed delivering drugs thus to minimize undesired side-effects but enhance cytotoxic therapies. In this study we developed a novel platform that enables simultaneously to respond light stimuli with localized heat and released drugs using drug contained gold nanorods (GNRs). Methotrexate (MTX), a model anticancer drug is attached through hydrolytic ester bonding to targeting molecular hyaluronic acid (HA) that is coated onto GNRs. Based on the rationale, HA provides a good scaffold for high biocompatibility to shield risky GNRs, targeting for a CD44 receptor, and easy chemical binding of drugs. Upon a single light irradiation, MTX-HA functionalized GNRs (MTX-HA @GNRs) provide localized heat to cancer areas for PTT and the elevated temperature accelerates hydrolytic cleavage of the ester bond onto GNRs in physiological condition for CHT, ultimately releasing MTX to cells. In contrast to previous combination therapies that do not concurrently offer heat and drugs upon light stimuli, our NIR triggered CHT with PTT provides clinically effective options with combinatorial treatment that possesses high efficacy resulted in in vitro tests. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/719030v1_figsh1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@759596org.highwire.dtl.DTLVardef@1afcdf7org.highwire.dtl.DTLVardef@fb1505org.highwire.dtl.DTLVardef@2100e4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO Schematic illustration of our nanoplatform a) The light responsive combinational therapy using GNRs scaffold for photo thermal therapy and chemotherapy b) The formation of TGNRs@RHO.B-HA and their light responsive mechanism with active CD44 receptor binding affinity c) light triggered hydrolytic release of model drug Rhodamine.B from TGNRs@RHO.B-HA. 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.

Conventional fluorescent imaging probes, including organic dyes and semiconductor quantum dots, suffer from inherent limitations such as photobleaching, cytotoxicity, poor aqueous dispersibility, and complex synthetic routes, necessitating the development of next-generation nanoscale fluorophores suitable for biological imaging. Carbon dots (CDs) have emerged as a compelling alternative owing to their nanoscale dimensions, tunable photoluminescence, excellent biocompatibility, and amenability to green synthesis from biomass-derived precursors. Herein, we report a comparative synthesis and systematic physicochemical evaluation of nitrogen-doped and undoped carbon dots derived from chamomile (Matricaria chamomilla L.) extract, prepared via solvothermal and microwave-assisted routes. Among the four synthesized variants--CM ST-U, CM ST-N, CM MW-U, and CM MW-N--the solvothermally synthesized nitrogen-doped carbon dots (CM ST-N) exhibited markedly superior optical performance, characterized by a high fluorescence quantum yield of 57.2%, which is among the highest reported for biomass-derived nitrogen-doped carbon dots. Comprehensive characterization using UV-visible spectroscopy, photoluminescence (PL) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), zeta potential analysis, and atomic force microscopy (AFM) confirmed the nanoscale dimensions (~8.3 nm), surface-rich functional groups, successful nitrogen incorporation (10.86 %), and moderate colloidal stability (zeta potential: -17.3 mV). Photoluminescence stability studies across seven solvent systems including biologically relevant media--phosphate-buffered saline (PBS), Dulbeccos modified Eagles medium (DMEM), and serum-free medium (SFM) demonstrated sustained fluorescence emission over 72 hours. In vitro cytotoxicity assessment using the MTT assay on RPE-1 retinal pigment epithelial cells confirmed high cell viability (>70%) across a broad concentration range (10-500 {micro}g mL-1) over multiple exposure durations. Collectively, these results establish CM ST-N as a highly fluorescent, biocompatible, and colloidally stable nanoprobe with strong potential for fluorescence-based bioimaging applications.

Cancer immunotherapies show clinical promise but often rely on T-cell priming and are limited by tumor heterogeneity and the immunosuppressive tumor microenvironment (TME). Innate immune activation offers a complementary strategy, with specific aim in natural killer (NK) cell activation for antigen-independent response. Biomimetic nanoparticles combining virus-like morphology with cell membrane (CM) coating offer a strategy to engage this innate immune axis. This study investigates virus-like mesoporous silica nanoparticles (VLPSi) with tunable spikes, surface functionalization, and CM coating as innate immunity modulators. Optimization revealed that longer spikes, amine functionalization, and CM coating synergistically enhance NK cell activation within human PBMCs, as indicated by CD69/CD25 upregulation and IFN-{gamma} secretion. CD14+ monocyte depletion attenuated activation, identifying monocyte-dependent crosstalk as a key mechanism. In purified NK cells, engineered CM-coated VLPSi induced early activation and supported feeder-free expansion. These results define topology, surface chemistry, and CM coating as parameters for innate immune modulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/720074v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@db5cdorg.highwire.dtl.DTLVardef@1ab41eorg.highwire.dtl.DTLVardef@127428dorg.highwire.dtl.DTLVardef@82609a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sustainable synthesis of photoluminescent nanomaterials with tuneable surface chemistry and defined biological activity remains a central challenge in green nanoscience. Here we show that the energy-input route used to carbonise a single bearberry (Arctostaphylos uva-ursi) extract precursor system exerts a decisive and mechanistically coherent influence over the surface chemistry, optical performance, and bioactivity of the resulting carbon quantum dots (CQDs). Hydrothermal processing (160 {degrees}C, 6 h) yields particles of 7.13 nm hydrodynamic diameter enriched in surface hydroxyl and carbonyl groups, a higher graphitic sp{superscript 2} carbon fraction (43.06%), and potent DPPH radical scavenging activity. In contrast, microwave-assisted synthesis yields 9.65 nm particles with a higher surface carboxylate content (O-C=O: 19.06%), enhanced fluorescence quantum yield, and increased intracellular uptake. Uptake is statistically significant in retinal epithelial cells at 200 {micro}g/mL (p < 0.001) and shows concentration-dependent accumulation in zebrafish larvae from 100 {micro}g/mL (p < 0.05). Combined XPS C 1s deconvolution and FTIR difference spectroscopy indicate that incomplete decarboxylation under microwave conditions underlies these distinct properties. Both formulations maintained full cytocompatibility across 10-250 {micro}g/mL in both RPE-1 and HeLa cells, with no statistically significant reduction in viability at any tested concentration. These findings define a synthesis-route-encoded structure property relationship that enables rational selection between antioxidant-optimised and imaging-optimised CQD formulations from an identical green precursor system.

Tetrahedral DNA nanostructures (TDNs) are promising nanocarriers due to their structural precision, biocompatibility, and efficient cellular uptake. However, their stability under physiological conditions remains a key challenge. In this study, TDNs were synthesized via a one-pot thermal annealing method and characterized using native PAGE, dynamic light scattering (DLS), and zeta potential analysis, confirming uniform size ([~]13 nm) and negative surface charge. Their stability was systematically evaluated across different biological media (DMEM complete, serum-free DMEM, and E3), temperatures (4 {degrees}C, 25 {degrees}C, and 37 {degrees}C), and pH conditions (4.0, 7.0, and 8.5) over 24 h. Results revealed rapid degradation in serum-containing medium, increased instability at higher temperatures, and reduced stability under acidic conditions, while serum-free, lower-temperature, and neutral to mildly basic environments enhanced structural integrity. These findings highlight the strong environmental dependence of TDN stability and provide insights for optimizing their design for biomedical applications.

Red-emitting carbon quantum dots (HP-CQDs) were synthesised for the first time from aqueous leaf extracts of Hamelia patens through single-step, reagent-free microwave-assisted carbonisation (750 W). The resulting nanoparticles displayed a narrow hydrodynamic size distribution centred at 3.9 nm, consistent with atomic force microscopy measurements showing a maximum height of 2.81 nm. Under 400 nm excitation, the CQDs exhibited a characteristic red emission maximum at 675 nm, representing a rare example of long-wavelength-emitting green CQDs derived from plant biomass. UV-Vis absorption bands at 224 and 256 nm were assigned to {pi}-{pi}* transitions of aromatic carbon domains and n-{pi}* transitions associated with carbonyl-containing surface groups, respectively. X-ray photoelectron spectroscopy (XPS) indicated a carbon-rich composition (C: 67.24%, O: 31.25%, N: 1.52%) with prominent C-O (42.67%) and C-C/C=C (42.64%) contributions. ATR-FTIR further confirmed the retention of hydroxyl, ether, and aliphatic functionalities following carbonisation. The excitation-wavelength-independent emission peak position implicates discrete surface molecular states rather than a heterogeneous distribution of emitters. HP-CQDs exhibit potent DPPH radical scavenging activity (IC50 = 141.8 {micro}g mL-1), comparable to ascorbic acid (IC50 = 114.8 {micro}g mL-1), and maintain >95% cell viability in both HeLa and RPE-1 cells up to 250 {micro}g mL-1. Confocal microscopy demonstrates concentration-dependent cytoplasmic accumulation and selective perinuclear localization at 300 {micro}g mL-1. In vivo biodistribution in zebrafish larvae confirms systemic uptake with statistically significant fluorescence enhancement at 500 {micro}g mL-1 (p < 0.01), establishing HP-CQDs as biocompatible red-fluorescent probes with dual imaging-antioxidant functionality. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/724069v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1dbe864org.highwire.dtl.DTLVardef@763ed0org.highwire.dtl.DTLVardef@115e9b9org.highwire.dtl.DTLVardef@1a3941e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nowadays N95 facial mask has gain huge attention due to COVID19 pandemic situation and it serves as the prime PPE. Though the microbes can be restricted to get inside the human body due to the presence of mask temporarily, but over the time, bacteria and other microbes may get entrapped into the threads of the mask itself and thus acting as a storage chamber of microbes. It is necessary to eliminate them from the mask surface. To do so different floral structured Nano-ZnO with variable oriented arrangement of petals were fabricated on the surface of the N95 mask and further characterized through instrumentations including XRD, FTIR,UV-Vis, Fluorescence-Spectroscopy, SEM, DLS. The average crystallite size calculated for synthesized four different ZnO nanoflower were 25.19 nm, 23.46 nm, 27.27 nm and 31.78 nm (for glycerol, PEG, EDTA, Chitosan assisted) respectively. The antimicrobial activity was investigated by standard microbial broth dilution assay and Kirby-Bauer test which assured the inhibition of the bacterial growth. The MIC-MBC value of ZnO nanoflowers for E.coli and B. subtilis were found to be effective at dilution of 250 {micro}g/ml and 100 {micro}g/ml. Additionally a modified Kirby-Bauer assay has been designed to investigate the killing efficiency of the bacteria (E.coli). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/719592v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@a76030org.highwire.dtl.DTLVardef@9bf1b3org.highwire.dtl.DTLVardef@19232forg.highwire.dtl.DTLVardef@54fe68_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. - Graphical AbstractC_FLOATNO C_FIG

The optimization of mRNA-lipid nanoparticles (mRNA-LNPs) for therapeutic applications is limited in part by the inadequate characterization of mRNA payload heterogeneity. One current challenge is accurately measuring the number of mRNA copies within individual LNPs, where the standard method of intensity-based mRNA number determination is sensitive to fluorescent dye-dye interactions and heterogeneity of mRNA labeling. Here we present a single-particle microscopy method that combines direct counting of the mRNA copies per LNP with LNP size measurements. While confined in microwells, individual mRNA-LNPs are lysed to release their cargo and stained with a dye such that the number of mRNA molecules in each well can be directly counted using fluorescence microscopy. Since the method stains the mRNA cargo in situ, it enables characterization of LNPs formulated with therapeutic grade (e.g., unlabeled) mRNA. We applied this approach to two Onpattro(R)-based LNP formulations prepared using different formulation buffers, where the two formulations had different average mRNA copy number, particle size, and fraction of LNPs lacking mRNA. The ability to directly count the number of mRNA molecules in LNPs establishes a complimentary method to intensity-based mRNA number determination and supports the characterization and screening of clinically relevant LNP formulations.

Lipid nanoparticles (LNPs) have demonstrated strong potential in COVID-19 mRNA vaccines nevertheless they still face the challenges in low mRNA delivery efficacy. Virus-like porous silica (VLPSi) nanoparticles (NPs) represent a promising biomimetic delivery platform because their spiked morphology may enhance cellular internalization and promote endosomal membrane disruption. However, the application of VLPSi for mRNA has been rarely explored. In this study, hybrid lipid-VLPSi NPs were developed by combining VLPSi with either lipoplexes (LPs) or LNPs. The effects of lipid types, mass ratio of different compositions, and amine modifications of VLPSi on mRNA delivery were studied. The results demonstrated that both LP and LNP could be successfully integrated with VLPSi to form hybrid delivery systems for mRNA transfection. VLPSi could significantly enhance mRNA delivery of both LPs and LNPs due to improved cellular uptake, structural stabilization of the mRNA complex, and enhanced endosomal escape mediated by the rigid virus-like surface architecture. Among the tested lipid formulations, the ionizable lipid ALC-0315 and helper lipid DOPE with mass ratio of 5:3 was the most effective lipid composition to be integrated with VLPSi, showing the highest mRNA delivery performance. In addition, amino modification of VLPSi was found to be a critical factor for efficient mRNA delivery. Hybrid LNPs containing amino-modified VLPSi showed significantly higher transfection efficiency than those containing unmodified VLPSi. Notably, amino-modified LNP-VLPSi achieved up to fivefold higher gene expression than conventional LNPs. Overall, this study establishes VLPSi as an efficient platform for amplifying lipid-mediated mRNA delivery. Owing to its straightforward integration into widely used LNP systems, VLPSi offers an adaptable and effective strategy for advancing next-generation mRNA therapeutics.

Carbon monoxide (CO) poisoning is responsible for around 50,000 emergency department visits per year in the U.S. alone. With the present standard of care, persistent neurological sequelae occur in [~]30-40% of severe CO poisoning cases. Currently, there is no available targeted molecular antidote for CO poisoning. In previous work, we have developed an antidotal therapy for CO poisoning based on an engineered hemeprotein, human neuroglobin (Ngb-H64Q-CCC). Intravenous infusion of Ngb-H64Q-CCC removes CO from the circulating red blood cells and improves survival in a lethal CO-poisoning mouse model. However, the infusion of heme-containing proteins has inherent heme toxicity risks that may limit the dose that can be used safely without liver or kidney toxicity. In order to overcome these problems, we have investigated the development of immobilized Ngb in a solid matrix. This approach allows for the development of a CO removal system using an extracorporeal blood circulating system coupled with a stationary matrix with immobilized Ngb-H64Q-CCC. Such system avoids drug infusion and possible organ injury, allows for antidote recycling, and provides advantages for storage and handling of the antidote. By assessing the efficacy of Ngb-H64Q-CCC immobilized through different linkage strategies, we have identified N-hydroxysuccinimide agarose resin as a viable stationary phase. The immobilized protein shows preserved heme redox activity, can be chemically reduced/oxidized for activation/CO release purposes, and retains its CO removal capacity after successive regeneration cycles. We expect that this novel approach will advance the development of new scavenger-based therapies for CO poisoning.

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.

Surface functionalization of inorganic quantum dot nanoparticles is of great interest in the application of these materials toward a wide range of biological applications where membrane interactions are critical. The use of amphiphilic lipids to functionalize the surfaces of quantum dots represents a promising alternative to produce water-soluble and membrane-active materials with facile tuning of the quantum dots surface properties. Here, we demonstrate an experimental approach that yields lipid-coated quantum dots with highly tunable surface charge by controlling the concentration of cationic lipids during preparation. Through fluorescence-activated cell sorting assays, we show that these cationic lipid-coated quantum dots can enhance membrane interactions and increase membrane labeling density in live HEK293 cells. We further employed coarse-grained molecular dynamics simulations to model the lipid self-assembly process using an implicit solvent force field and subsequently model the adsorption of lipid-coated quantum dots to model membranes. Our simulations show that we can control the effective surface charge of lipid-coated quantum dots and influence the strength of adsorption to oppositely charged lipid membranes, a process that is mediated by the release of counterions at the quantum dot-membrane interface. This work supports the future development of biocompatible and water-soluble inorganic nanoparticles with highly tunable surfaces, and provides mechanistic insight into how different lipids can influence nanoparticle-membrane interactions at a molecular scale.