MEMO: dataset and methods for robust multimodal retinal image registration with large or small vessel density differences.

The measurement of retinal blood flow (RBF) in capillaries can provide a powerful biomarker for the early diagnosis and treatment of ocular diseases. However, no single modality can determine capillary flowrates with high precision. Combining erythrocyte-mediated angiography (EMA) with optical coherence tomography angiography (OCTA) has the potential to achieve this goal, as EMA can measure the absolute RBF of retinal microvasculature and OCTA can provide the structural images of capillaries. However, multimodal retinal image registration between these two modalities remains largely unexplored. To fill this gap, we establish MEMO, the first public multimodal EMA and OCTA retinal image dataset. A unique challenge in multimodal retinal image registration between these modalities is the relatively large difference in vessel density (VD). To address this challenge, we propose a segmentation-based deep-learning framework (VDD-Reg), which provides robust results despite differences in vessel density. VDD-Reg consists of a vessel segmentation module and a registration module. To train the vessel segmentation module, we further designed a two-stage semi-supervised learning framework (LVD-Seg) combining supervised and unsupervised losses. We demonstrate that VDD-Reg outperforms existing methods quantitatively and qualitatively for cases of both small VD differences (using the CF-FA dataset) and large VD differences (using our MEMO dataset). Moreover, VDD-Reg requires as few as three annotated vessel segmentation masks to maintain its accuracy, demonstrating its feasibility.

Comprehensive analysis of common polymers using hyphenated TGA-FTIR-GC/MS and Raman spectroscopy towards a database for micro- and nanoplastics identification, characterization, and quantitation.

Environmental contamination by micro- and nanoplastics (MNPs) is well documented with potential for their increased accumulation globally. Growing public concern over environmental, ecological, and human exposure to MNPs has led to exponential increase in publications, news articles, and reports (Casillas et al., 2023). Significant knowledge gap exists in standardized analytical methods for the identification and quantification of MNPs from real world environmental samples. Here, we report comprehensive datasets utilizing thermogravimetric analyzer (TGA) coupled to a Fourier transformed infrared spectrometer (FTIR) and a gas chromatography/mass spectrometer (GC/MS) with corresponding Raman spectral data for the most common polymers documented to be present in the environment (35 plastics of 12 polymer types), to serve as a base line reference for the identification and quantitation of MNPs. Various parameters for TGA-FTIR-GC/MS data acquisition were optimized. Commercial consumer plastic product compositions were identified using this analytical database. Case studies to showcase the utility of the method for polymer mixtures analysis is included. This dataset would serve towards the development of a collaborative, global, comprehensive, and curated public database for the identification of various MNPs and mixtures.

Interfacial charge transfer with exfoliated graphene inhibits fibril formation in lysozyme amyloid.

Amyloid fibrillation is known to contribute in a variety of diseases including neurodegenerative disorders (e.g., Alzheimer's and Parkinson's disease) and type II diabetes. The inhibition of fibrillation has been suggested as a possible therapeutic strategy to prevent neuronal and pancreatic ß-cell death associated with amyloid diseases. To this end, strong hydrophobic and π-π interactions between proteins and nanomaterials at the nanobio interface could be used to mitigate the stacking of amyloid structures associated with fibrillation. In this study, the authors show that exfoliated graphene effectively inhibits the formation of amyloid fibrils using a model amyloid-forming protein, viz., hen egg white lysozyme (HEWL). While previous theoretical models posit that hydrophobic and π-π stacking interactions result in strong interactions between graphene and proteins, the authors experimentally identified the presence of additional interfacial charge transfer interactions between HEWL and graphene using micro-Raman spectroscopy and Kelvin probe force microscopy. Their photoluminescence spectroscopy and transmission electron microscopy studies evince that the interfacial charge transfer combined with hydrophobic and π-π stacking interactions, specifically between the nanomaterial and the amino acid tryptophan, increase HEWL adsorption on graphene and thereby inhibit amyloid fibrillation.

Defect-induced electronic states amplify the cellular toxicity of ZnO nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) are used in numerous applications, including sunscreens, cosmetics, textiles, and electrical devices. Increased consumer and occupational exposure to ZnO NPs potentially poses a risk for toxicity. While many studies have examined the toxicity of ZnO NPs, little is known regarding the toxicological impact of inherent defects arising from batch-to-batch variations. It was hypothesized that the presence of varying chemical defects in ZnO NPs will contribute to cellular toxicity in rat aortic endothelial cells (RAECs). Pristine and defected ZnO NPs (oxidized, reduced, and annealed) were prepared and assessed three major cellular outcomes; cytotoxicity/apoptosis, reactive oxygen species production and oxidative stress, and endoplasmic reticulum (ER) stress. ZnO NPs chemical defects were confirmed by X-ray photoelectron spectroscopy and photoluminescence. Increased toxicity was observed in defected ZnO NPs compared to the pristine NPs as measured by cell viability, ER stress, and glutathione redox potential. It was determined that ZnO NPs induced ER stress through the PERK pathway. Taken together, these results demonstrate a previously unrecognized contribution of chemical defects to the toxicity of ZnO NPs, which should be considered in the risk assessment of engineered nanomaterials.

Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress.

Prior research has demonstrated cells exposed to silver nanoparticles (AgNPs) undergo endoplasmic reticulum (ER) stress leading to cellular apoptosis and toxicity, however, the fundamental mechanism underlying AgNP-induced ER stress is unknown. We hypothesize the biophysical interactions between AgNPs and adsorbed proteins lead to misfolded proteins to elicit an ER stress response. Our investigation examined rat aortic endothelial cells (RAEC) exposed to 20 or 100 nm AgNPs with or without a biocorona (BC) consisting of bovine serum albumin (BSA), high density lipoprotein (HDL) or fetal bovine serum (FBS) to form a complex BC. The presence of a BC consisting of BSA or FBS proteins significantly reduced uptake of 20 nm and 100 nm AgNPs in RAEC. Western blot analysis indicated robust activation of the IREα and PERK pathways in RAEC exposed to 20 nm despite the reduction in uptake by the presence of a BC. This was not observed for the 100 nm AgNPs. Hyperspectral darkfield microscopy qualitatively confirmed that the preformed BC was maintained following uptake by RAEC. Transmission electron microscopy demonstrated a size dependent effect on the sub-cellular localization of AgNPs. Overall, these results suggest that AgNP size, surface area and BC formation governs the induction of ER stress and alterations in intracellular trafficking.

Chemiplasmonics for high-throughput biosensors.

BACKGROUND: The sensitivity of ELISA for biomarker detection can be significantly increased by integrating fluorescence with plasmonics. In surface-plasmon-coupled emission, the fluorophore emission is generally enhanced through the so-called physical mechanism due to an increase in the local electric field. Despite its fairly high enhancement factors, the use of surface-plasmon-coupled emission for high-throughput and point-of-care applications is still hampered due to the need for expensive focusing optics and spectrometers. METHODS: Here, we describe a new chemiplasmonic-sensing paradigm for enhanced emission through the molecular interactions between aromatic dyes and C60 films on Ag substrates. RESULTS: A 20-fold enhancement in the emission from rhodamine B-labeled biomolecules can be readily elicited without quenching its red color emission. As a proof of concept, we demonstrate two model bioassays using: 1) the RhB-streptavidin and biotin complexes in which the dye was excited using an inexpensive laser pointer and the ensuing enhanced emission was recorded by a smartphone camera without the need for focusing optics and 2) high-throughput 96-well plate assay for a model antigen (rabbit immunoglobulin) that showed detection sensitivity as low as 6.6 pM. CONCLUSION: Our results show clear evidence that chemiplasmonic sensors can be extended to detect biomarkers in a point-of-care setting through a smartphone in simple normal incidence geometry without the need for focusing optics. Furthermore, chemiplasmonic sensors also facilitate high-throughput screening of biomarkers in the conventional 96-well plate format with 10-20 times higher sensitivity.

Three-photon imaging using defect-induced photoluminescence in biocompatible ZnO nanoparticles.

BACKGROUND: Although optical spectroscopy promises improved lateral resolution for cancer imaging, its clinical use is seriously impeded by background fluorescence and photon attenuation even in the so-called two-photon absorption (2PA) imaging modality. An efficient strategy to meet the clinical cancer imaging needs, beyond what two-photon absorption (2PA) offers, is to use longer excitation wavelengths through three-photon absorption (3PA). A variety of fluorescent dyes and nanoparticles (NPs) have been used in 3PA imaging. However, their nonlinear 3PA coefficient is often low necessitating high excitation powers, which cause overheating, photodamage, and photo-induced toxicity. Doped wide band gap semiconductors such as Mn:ZnS NPs have previously been used for 3PA but suffer from poor 3PA coefficients. METHODS: Here, we prepared ZnO NPs with intrinsic defects with high 3PA coefficients using a polyol method. We functionalized them with peptides for selective uptake by glioblastoma U87MG cells and used breast cancer MCF-7 cells as control for 3PA studies. Uptake was measured using inductively coupled plasma-mass spectrometry. Biocompatibility studies were performed using reactive oxygen species and cell viability assays. RESULTS: We demonstrate that ZnO NPs, which have a band gap of 3.37 eV with an order of magnitude higher 3PA coefficients, can facilitate the use of longer excitation wavelengths 950-1,100 nm for bioimaging. We used the presence intrinsic defects (such as O interstitials and Zn vacancies) in ZnO NPs to induce electronic states within the band gap that can support strong visible luminescence 550-620 nm without the need for extrinsic doping. The peptide functionalization of ZnO NPs showed selective uptake by U87MG cells unlike MCF-7 cells without the integrin receptors. Furthermore, all ZnO NPs were found to be biocompatible for 3PA imaging. CONCLUSION: We show that defect-induced luminescence 550-620 nm in ZnO NPs (20 nm) due to 3PA at longer excitation (975 nm) can be used for 3PA imaging of U87MG glioblastoma cells with lower background noise.

Variations in biocorona formation related to defects in the structure of single walled carbon nanotubes and the hyperlipidemic disease state.

Ball-milling utilizes mechanical stress to modify properties of carbon nanotubes (CNTs) including size, capping, and functionalization. Ball-milling, however, may introduce structural defects resulting in altered CNT-biomolecule interactions. Nanomaterial-biomolecule interactions result in the formation of the biocorona (BC), which alters nanomaterial properties, function, and biological responses. The formation of the BC is governed by the nanomaterial physicochemical properties and the physiological environment. Underlying disease states such as cardiovascular disease can alter the biological milieu possibly leading to unique BC identities. In this ex vivo study, we evaluated variations in the formation of the BC on single-walled CNTs (SWCNTs) due to physicochemical alterations in structure resulting from ball-milling and variations in the environment due to the high-cholesterol disease state. Increased ball-milling time of SWCNTs resulted in enhanced structural defects. Following incubation in normal mouse serum, label-free quantitative proteomics identified differences in the biomolecular content of the BC due to the ball-milling process. Further, incubation in cholesterol-rich mouse serum resulted in the formation of unique BCs compared to SWCNTs incubated in normal serum. Our study demonstrates that the BC is modified due to physicochemical modifications such as defects induced by ball-milling and physiological disease conditions, which may result in variable biological responses.

Charge-transfer interactions induce surface dependent conformational changes in apolipoprotein biocorona.

Upon introduction into a biological system, engineered nanomaterials (ENMs) rapidly associate with a variety of biomolecules such as proteins and lipids to form a biocorona. The presence of "biocorona" influences nano-bio interactions considerably, and could ultimately result in altered biological responses. Apolipoprotein A-I (ApoA-I), the major constituent of high-density lipoprotein (HDL), is one of the most prevalent proteins found in ENM-biocorona irrespective of ENM nature, size, and shape. Given the importance of ApoA-I in HDL and cholesterol transport, it is necessary to understand the mechanisms of ApoA-I adsorption and the associated structural changes for assessing consequences of ENM exposure. Here, the authors used a comprehensive array of microscopic and spectroscopic tools to elucidate the interactions between ApoA-I and 100 nm Ag nanoparticles (AgNPs) with four different surface functional groups. The authors found that the protein adsorption and secondary structural changes are highly dependent on the surface functionality. Our electrochemical studies provided new evidence for charge transfer interactions that influence ApoA-I unfolding. While the unfolding of ApoA-I on AgNPs did not significantly change their uptake and short-term cytotoxicity, the authors observed that it strongly altered the ability of only some AgNPs to generate of reactive oxygen species. Our results shed new light on the importance of surface functionality and charge transfer interactions in biocorona formation.

Contribution of engineered nanomaterials physicochemical properties to mast cell degranulation.

The rapid development of engineered nanomaterials (ENMs) has grown dramatically in the last decade, with increased use in consumer products, industrial materials, and nanomedicines. However, due to increased manufacturing, there is concern that human and environmental exposures may lead to adverse immune outcomes. Mast cells, central to the innate immune response, are one of the earliest sensors of environmental insult and have been shown to play a role in ENM-mediated immune responses. Our laboratory previously determined that mast cells are activated via a non-FcεRI mediated response following silver nanoparticle (Ag NP) exposure, which was dependent upon key physicochemical properties. Using bone marrow-derived mast cells (BMMCs), we tested the hypothesis that ENM physicochemical properties influence mast cell degranulation. Exposure to 13 physicochemically distinct ENMs caused a range of mast degranulation responses, with smaller sized Ag NPs (5 nm and 20 nm) causing the most dramatic response. Mast cell responses were dependent on ENMs physicochemical properties such as size, apparent surface area, and zeta potential. Surprisingly, minimal ENM cellular association by mast cells was not correlated with mast cell degranulation. This study suggests that a subset of ENMs may elicit an allergic response and contribute to the exacerbation of allergic diseases.

Defect density in multiwalled carbon nanotubes influences ovalbumin adsorption and promotes macrophage activation and CD4(+) T-cell proliferation.

Carbon nanotubes (CNTs) are of great interest for the development of drugs and vaccines due to their unique physicochemical properties. The high surface area to volume ratio and delocalized pi-electron cloud of CNTs promote binding of proteins to the surface forming a protein corona. This unique feature of CNTs has been recognized for potential delivery of antigens for strong and long-lasting antigen-specific immune responses. Based on an earlier study that demonstrated increased protein binding, we propose that carboxylated multiwalled CNTs (MWCNTs) can function as an improved carrier to deliver antigens such as ovalbumin (OVA). To test this hypothesis, we coated carboxylated MWCNTs with OVA and measured uptake and activation of antigen-presenting cells (macrophages) and their ability to stimulate CD4(+) T-cell proliferation. We employed two types of carboxylated MWCNTs with different surface areas and defects (MWCNT-2 and MWCNT-30). MWCNT-2 and MWCNT-30 have surface areas of ~215 m(2)/g and 94 m(2)/g, respectively. The ratios of D- to G-band areas (I D/I G) were 0.97 and 1.37 for MWCNT-2 and MWCNT-30, respectively, samples showing that MWCNT-30 contained more defects. The increase in defects in MWCNT-30 led to increased binding of OVA as compared to MWCNT-2 (1,066±182 µg/mL vs 582±41 µg/mL, respectively). Both types of MWCNTs, along with MWCNT-OVA complexes, showed no observable toxicity to bone-marrow-derived macrophages up to 5 days. Surprisingly, we found that MWCNT-OVA complex significantly increased the expression of major histocompatibility complex class II on macrophages and production of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin 6), while MWCNTs without OVA protein corona did not. The coculture of MWCNT-OVA-complex-treated macrophages and OVA-specific CD4(+) T-cells isolated from OT-II mice demonstrated robust proliferation of CD4(+) T-cells. This study provides strong evidence for a role for defects in carboxylated MWCNTs and their use in the efficient delivery of antigens for the development of next-generation vaccines.

From the Cover: Disease-Induced Disparities in Formation of the Nanoparticle-Biocorona and the Toxicological Consequences.

Nanoparticle (NP) association with macromolecules in a physiological environment forms a biocorona (BC), which alters NP distribution, activity, and toxicity. While BC formation is dependent on NP physicochemical properties, little information exists on the influence of the physiological environment. Obese individuals and those with cardiovascular disease exist with altered serum chemistry, which is expected to influence BC formation and NP toxicity. We hypothesize that a BC formed on NPs following incubation in hyperlipidemic serum will result in altered NP-BC protein content, cellular association, and toxicity compared to normal serum conditions. We utilized Fe3O4 NPs, which are being developed as MRI contrast and tumor targeting agents to test our hypothesis. We used rat aortic endothelial cells (RAECs) within a dynamic flow in vitro exposure system to more accurately depict the in vivo environment. A BC was formed on 20nm PVP-suspended Fe3O4 NPs following incubation in water, 10% normal or hyperlipidemic rat serum. Addition of BCs resulted in increased hydrodynamic size and decreased surface charge. More cholesterol associated with Fe3O4 NPs after incubation in hyperlipidemic as compared with normal serum. Using quantitative proteomics, we identified unique differences in BC protein components between the 2 serum types. Under flow conditions, formation of a BC from both serum types reduced RAECs association of Fe3O4 NPs. Addition of BCs was found to exacerbate RAECs inflammatory gene responses to Fe3O4 NPs (Fe3O4-hyperlipidemic > Fe3O4-normal > Fe3O4) including increased expression of IL-6, TNF-α, Cxcl-2, VCAM-1, and ICAM-1. Overall, these findings demonstrate that disease-induced variations in physiological environments have a significant impact NP-BC formation, cellular association, and cell response.

Biomolecular Interactions and Biological Responses of Emerging Two-Dimensional Materials and Aromatic Amino Acid Complexes.

The present work experimentally investigates the interaction of aromatic amino acids viz., tyrosine, tryptophan, and phenylalnine with novel two-dimensional (2D) materials including graphene, graphene oxide (GO), and boron nitride (BN). Photoluminescence, micro-Raman spectroscopy, and cyclic voltammetry were employed to investigate the nature of interactions and possible charge transfer between 2D materials and amino acids. Graphene and GO were found to interact strongly with aromatic amino acids through π-π stacking, charge transfer, and H-bonding. Particularly, it was observed that both physi and chemisorption are prominent in the interactions of GO/graphene with phenylalanine and tryptophan while tyrosine exhibited strong chemisorption on graphene and GO. In contrast, BN exhibited little or no interactions, which could be attributed to localized π-electron clouds around N atoms in BN lattice. Lastly, the adsorption of amino acids on 2D materials was observed to considerably change their biological response in terms of reactive oxygen species generation. More importantly, these changes in the biological response followed the same trends observed in the physi and chemisorption measurements.