Simultaneous Protein Colorful Imaging via Raman Signal Classification.

Protein imaging aids diagnosis and drug development by revealing protein-drug interactions or protein levels. However, the challenges of imaging multiple proteins, reduced sensitivity, and high reliance on specific protein properties such as Raman peaks or refractive index hinder the understanding. Here, we introduce multiprotein colorful imaging through Raman signal classification. Our method utilized machine learning-assisted classification of Raman signals, which are the distinctive features of label-free proteins. As a result, three types of proteins could be imaged simultaneously. In addition, we could quantify individual proteins from a mixture of multiple proteins over a wide detection range (10 fg/mL-1 µg/mL). These results showed a 1000-fold improvement in sensitivity and a 30-fold increase in the upper limit of detection compared to existing methods. These advances will enhance our understanding of biology and facilitate the development of disease diagnoses and treatments.

Artificial Intelligence-Based Major Depressive Disorder (MDD) Diagnosis Using Raman Spectroscopic Features of Plasma Exosomes.

In vitro diagnosis using biomarkers for major depressive disorder (MDD) can offer considerable advantages in overcoming the lack of objective tests for depression and treating more patients. Plasma exosomes can be novel biomarkers for MDD based on their ability to pass through the blood-brain barrier and offer brain-related information. Here, we demonstrate a novel and precise MDD diagnosis using deep learning analysis and surface-enhanced Raman spectroscopy (SERS) of plasma exosomes. Our system is implemented based on 28,000 exosome SERS signals, providing sample-wise prediction results. Notably, this approach shows remarkable performance in predicting 70 test samples unused in the training step, with an area under the curve (AUC) of 0.939, a sensitivity of 91.4%, and a specificity of 88.6%. In addition, we confirm that the diagnostic scores were correlated with the degree of depression. These results show the utility of exosomes as novel biomarkers for MDD diagnosis and suggest a novel approach for prescreening techniques for psychiatric disorders.

Permanent Anticoagulation Blood-Vessel by Mezzo-Sized Double Re-Entrant Structure.

Having a permanent omniphobicity on the inner surface of the tube can bring enormous advantages, such as reducing resistance and avoiding precipitation during mass transfer. For example, such a tube can prevent blood clotting when delivering blood composed of complex hydrophilic and lipophilic compounds. However, it is very challenging to fabricate micro and nanostructures inside a tube. To overcome these, a wearability and deformation-free structural omniphobic surface is fabricated. The omniphobic surface can repel liquids by its "air-spring" under the structure, regardless of surface tension. Furthermore, it is not lost an omniphobicity under physical deformation like curved or twisted. By using these properties, omniphobic structures on the inner wall of the tube by the "roll-up" method are fabricated. Fabricated omniphobic tubes still repels liquids, even complex liquids like blood. According to the ex vivo blood tests for medical usage, the tube can reduce thrombus formation by 99%, like the heparin-coated tube. So, the surface will soon replace typical coating-based medical surfaces or anticoagulation blood vessels.

A Proteomic Approach to Understand the Clinical Significance of Acute Myeloid Leukemia-Derived Extracellular Vesicles Reflecting Essential Characteristics of Leukemia.

Extracellular vesicle (EV) proteins from acute myeloid leukemia (AML) cell lines were analyzed using mass spectrometry. The analyses identified 2450 proteins, including 461 differentially expressed proteins (290 upregulated and 171 downregulated). CD53 and CD47 were upregulated and were selected as candidate biomarkers. The association between survival of patients with AML and the expression levels of CD53 and CD47 at diagnosis was analyzed using mRNA expression data from The Cancer Genome Atlas database. Patients with higher expression levels showed significantly inferior survival than those with lower expression levels. ELISA results of the expression levels of CD53 and CD47 from EVs in the bone marrow of patients with AML at diagnosis and at the time of complete remission with induction chemotherapy revealed that patients with downregulated CD53 and CD47 expression appeared to relapse less frequently. Network model analysis of EV proteins revealed several upregulated kinases, including LYN, CSNK2A1, SYK, CSK, and PTK2B. The potential cytotoxicity of several clinically applicable drugs that inhibit these kinases was tested in AML cell lines. The drugs lowered the viability of AML cells. The collective data suggest that AML cell-derived EVs could reflect essential leukemia biology.

Levels of Extracellular Vesicles in Pulmonary and Peripheral Blood Correlate with Stages of Lung Cancer Patients.

BACKGROUND: The extracellular vesicle (EV) concentration is known to be higher in cancer patients than in healthy individuals. Herein, we report that EV levels differ in the tumor-draining pulmonary vein blood and the peripheral blood of animal models and human subjects at different pathological stages of lung cancer. METHODS: Ten rabbits and 40 humans formed the study cohorts. Blood was collected from the peripheral vein of members of all groups. Pulmonary blood was collected intraoperatively from all groups except for the healthy human controls. Quantitative analysis of EV levels was performed using a nanoparticle tracking assay, a CD63 enzyme-linked immunosorbent assay, and western blotting. RESULTS: The EV levels in the peripheral blood of animals and patients with lung cancer were higher than those in the peripheral blood of healthy controls (p < 0.01 and p < 0.001, respectively). Moreover, for both animals and patients with lung cancer, the EV levels in the pulmonary blood were significantly higher than those in the preoperative peripheral blood (p < 0.01 and p < 0.0001, respectively). In patients, the pathological stages of lung cancer showed a higher correlation with the pulmonary EV levels than the peripheral EV levels. CONCLUSIONS: EV levels increased with increasing lung cancer grade, and this trend was more prominent in the pulmonary blood than in the peripheral blood.

Extracellular Vesicle Identification Using Label-Free Surface-Enhanced Raman Spectroscopy: Detection and Signal Analysis Strategies.

Extracellular vesicles (EVs) have been widely investigated as promising biomarkers for the liquid biopsy of diseases, owing to their countless roles in biological systems. Furthermore, with the notable progress of exosome research, the use of label-free surface-enhanced Raman spectroscopy (SERS) to identify and distinguish disease-related EVs has emerged. Even in the absence of specific markers for disease-related EVs, label-free SERS enables the identification of unique patterns of disease-related EVs through their molecular fingerprints. In this review, we describe label-free SERS approaches for disease-related EV pattern identification in terms of substrate design and signal analysis strategies. We first describe the general characteristics of EVs and their SERS signals. We then present recent works on applied plasmonic nanostructures to sensitively detect EVs and notable methods to interpret complex spectral data. This review also discusses current challenges and future prospects of label-free SERS-based disease-related EV pattern identification.

Identification of Newly Emerging Influenza Viruses by Detecting the Virally Infected Cells Based on Surface Enhanced Raman Spectroscopy and Principal Component Analysis.

Rapid diagnosis and quarantine of influenza virus mutant-infected people is critical to contain the fatal viral infection spread because effective antiviral drugs are normally not available. Conventional methods, however, cannot be used for the diagnosis because these methods need predefined labels, likely also unavailable for just emerging viruses. Here, we propose label-free identification of cells infected with different influenza viruses based on surface-enhanced Raman spectroscopy (SERS) and principal component analysis (PCA). Viral envelope proteins that are displayed on the surface of cells after infection of influenza viruses were targeted for this identification. Cells that expressed the envelope proteins of A/WSN/33 H1N1 or A/California/04/2009 H1N1 influenza viruses produced distinct SERS signals. Cells that displayed combinations of the envelope proteins from these two viral variants, an indication of emergence of a new virus, also generated characteristic SERS patterns. However, the cell's own surface proteins often hindered the identification of virally infected cells by producing SERS peaks similar to viral ones. PCA of the obtained SERS patterns could effectively capture the virus-specific signal components from the jumbled SERS peaks. Our study demonstrates a potential of combination of SERS and PCA to identify newly emerging influenza viruses through sensing the cells infected with the viruses.

Aqueous synthesis of highly monodisperse sub-100 nm AgCl nanospheres/cubes and their plasmonic nanomesh replicas as visible-light photocatalysts and single SERS probes.

Despite the distinctive electrochemical and photocatalytic properties of nanostructured silver chloride (AgCl), the shape- and size-dependence of their properties have not been thoroughly investigated to date. The most substantial reason responsible for this incomplete study and the subsequent limited applications is the failure in controlling the structure of AgCl nanomaterials, mainly owing to the challenging synthetic conditions including organic phase and high reaction temperature. In this work, we reported a rapid one-pot room-temperature aqueous synthesis of highly monodisperse sub-100 nm AgCl nanomaterials with various shapes and sizes by controlling the precursor (Ag+ and AuCl4 -) ratios. The remaining unreacted metal precursors (Ag+ and AuCl4 -) used to produce AgClNC were subsequently reduced by ascorbic acid on the surface of the synthesized AgCl nanomaterials to form Ag/Au bimetallic nanomesh structures (AgClNC#AuAgCMs and SMs). After the removal of the AgCl nanotemplates, only nanomesh structures (AuAgCMs and SMs) were obtained. Importantly, we successfully decreased the size of the AgCl nanomaterials which were replicated into bimetallic spherical and cubic nanomesh structures that were small enough (∼100 nm) to show intense surface-plasmon-absorption bands. Based on these unique chemical and physical properties, we could take advantage of the plasmonic photocatalysis properties of the complex comprising semiconducting AgCl/metallic nanomesh replica for the complete removal of the environmentally harmful Cr6+ in the presence of sacrificial agents such as formic acid. Finally, the novel bimetallic nanomesh structures proved themselves to exhibit intense surface-enhanced Raman scattering properties in a single-particle enhancing the electromagnetic field.

Stem cell transplantation for Huntington's diseases.

Therapeutic approaches based on stem cells have received considerable attention as potential treatments for Huntington's disease (HD), which is a fatal, inherited neurodegenerative disorder, caused by progressive loss of GABAergic medium spiny neurons (MSNs) in the striatum of the forebrain. Transplantation of stem cells or their derivatives in animal models of HD, efficiently improved functions by replacing the damaged or lost neurons. In particular, neural stem cells (NSCs) for HD treatments have been developed from various sources, such as the brain itself, the pluripotent stem cells (PSCs), and the somatic cells of the HD patients. However, the brain-derived NSCs are difficult to obtain, and the PSCs have to be differentiated into a population of the desired neuronal cells that may cause a risk of tumor formation after transplantation. In contrast, induced NSCs, derived from somatic cells as a new stem cell source for transplantation, are less likely to form tumors. Given that the stem cell transplantation strategy for treatment of HD, as a genetic disease, is to replace the dysfunctional or lost neurons, the correction of mutant genes containing the expanded CAG repeats is essential. In this review, we will describe the methods for obtaining the optimal NSCs for transplantation-based HD treatment and the differentiation conditions for the functional GABAergic MSNs as therapeutic cells. Also, we will discuss the valuable gene correction of the disease stem cells by the CRISPR/Cas9 system for HD treatment.

Exosome Classification by Pattern Analysis of Surface-Enhanced Raman Spectroscopy Data for Lung Cancer Diagnosis.

Owing to the role of exosome as a cargo for intercellular communication, especially in cancer metastasis, the evidence has been consistently accumulated that exosomes can be used as a noninvasive indicator of cancer. Consequently, several studies applying exosome have been proposed for cancer diagnostic methods such as ELISA assay. However, it has been still challenging to get reliable results due to the requirement of a labeling process and high concentration of exosome. Here, we demonstrate a label-free and highly sensitive classification method of exosome by combining surface-enhanced Raman scattering (SERS) and statistical pattern analysis. Unlike the conventional method to read different peak positions and amplitudes of a spectrum, whole SERS spectra of exosomes were analyzed by principal component analysis (PCA). By employing this pattern analysis, lung cancer cell derived exosomes were clearly distinguished from normal cell derived exosomes by 95.3% sensitivity and 97.3% specificity. Moreover, by analyzing the PCA result, we could suggest that this difference was induced by 11 different points in SERS signals from lung cancer cell derived exosomes. This result paved the way for new real-time diagnosis and classification of lung cancer by using exosome as a cancer marker.

Autoenhanced Raman Spectroscopy via Plasmonic Trapping for Molecular Sensing.

As a label-free and sensitive biosensor, surface-enhanced Raman spectroscopy (SERS) is a rapidly emerging technique. However, because SERS spectra are obtained in the area of light excitation and the enhancement effect can be varied depending on the position of a substrate, it is important to match the enhanced area with an illuminated spot. Here, in order to overcome such difficulty, we demonstrated a new technique combining SERS with plasmonic trapping. By plasmonic trapping, we can collect gold nanoparticles (GNPs) in the middle of initially fabricated nanobowtie structures where a laser is excited. As a result of trapping GNPs, hot-spots are formed at that area. Because SERS is measured in the area irradiated by a laser, hot-spot can be simultaneously coincided with a detection site for SERS. By using this, we detected Rhodamine 6G to 100 pM. To further verify and improve the reproducibility of our technique, we also calculated the electric field distribution, trapping force and trapping potential.

Identification of Newly Emerging Influenza Viruses by Surface-Enhanced Raman Spectroscopy.

In this work, we demonstrate in situ virus identification based on surface-enhanced Raman scattering (SERS). We hypothesized that newly emerging influenza viruses possess surface proteins and lipids that can generate distinctive Raman signals. To test this hypothesis, SERS signals were measured from the surface of a noninfluenza virus, two different influenza viruses, and a genetically shuffled influenza virus. To ensure the safety for experimenters we constructed nonreplicating pseudotyped viruses that display main influenza virus surface components. Pseudotype with influenza virus components produced enhanced Raman peaks, on gold nanoparticles, that are easily distinguishable from those of pseudotype with a noninfluenza virus component, vesicular stomatitis virus G protein (VSVG). Furthermore, virus with the surface components of a newly emerging influenza strain, A/California/04/2009 (H1N1), generated Raman peaks different from those of viruses with components of the conventional laboratory-adapted influenza strain, A/WSN/33 (H1N1). Interestingly, the virus simultaneously displaying surface components of both influenza strains, a model mutant with genome reassortment, also produced a Raman signal pattern that is clearly distinguishable from those of each strain. This work highlights that SERS can provide a powerful label-free strategy to quickly identify newly emerging and potentially fatal influenza viruses.

Graphene nanopore with a self-integrated optical antenna.

We report graphene nanopores with integrated optical antennae. We demonstrate that a nanometer-sized heated spot created by photon-to-heat conversion of a gold nanorod resting on a graphene membrane forms a nanoscale pore with a self-integrated optical antenna in a single step. The distinct plasmonic traits of metal nanoparticles, which have a unique capability to concentrate light into nanoscale regions, yield the significant advantage of parallel nanopore fabrication compared to the conventional sequential process using an electron beam. Tunability of both the nanopore dimensions and the optical characteristics of plasmonic nanoantennae are further achieved. Finally, the key optical function of our self-integrated optical antenna on the vicinity of graphene nanopore is manifested by multifold fluorescent signal enhancement during DNA translocation.

Thoracoscopic color and fluorescence imaging system for sentinel lymph node mapping in porcine lung using indocyanine green-neomannosyl human serum albumin: intraoperative image-guided sentinel nodes navigation.

PURPOSE: This study was performed to validate a newly developed sentinel lymph node (SLN) targeting tracer, indocyanine green-neomannosyl human serum albumin (ICG:MSA), and a thoracoscopic version of the intraoperative color and fluorescence imaging system (ICFIS) for lung cancer SLN mapping. METHODS: ICG alone or ICG:MSA (5 µg/kg) was injected into the rat thigh, and the results were compared. The fluorescence signal-to-background ratios of SLNs were recorded and evaluated over a 2-h period by using ICFIS. Additionally, a SLN biopsy was performed via video-assisted thoracoscopic surgery with the use of ICG:MSA in porcine lung by using thoracoscopic ICFIS. RESULTS: The newly developed ICG:MSA showed a significantly improved signal-to-background ratio compared with ICG alone throughout the trials. All SLNs were identified in both rats (ten SLNs in ten rat thighs) and pigs (ten SLNs in ten porcine lungs) under in vivo conditions. All SLNs were dissected successfully by using video-assisted thoracoscopic surgery with the help of thoracoscopic ICFIS. DISCUSSION: ICG:MSA accumulates in the SLN by uptake and retention through the mannose-specific receptors on macrophages. Thoracoscopic ICFIS successfully assisted SLN mapping despite low near-infrared light transmission in the commercial thoracoscope. On the basis of the results of the thoracoscopic SLN mapping, we anticipate that ICG:MSA and thoracoscopic ICFIS can be translated to clinical trials in the near future.

Highly sensitive and selective anticancer effect by conjugated HA-cisplatin in non-small cell lung cancer overexpressed with CD44.

In spite of severe side effects, chemotherapy is widely used as a major anticancer treatment in non-small cell lung cancer (NSCLC). In order to enhance the therapeutic properties and reduce side effects, enormous efforts have been devoted to direct anticancer agents specifically to tumor tissues by the use of nanoparticles, or cancer cell marker attached drugs. However, cell-specific chemotherapy is still in its infancy and is not applicable to all types of cancers due to the complexity of the cancer occurrence and progress. In this study, we demonstrate that hyaluronan (HA)-conjugated cisplatin has highly selective and sensitive anticancer effects in NSCLC cells that overexpress the trans-membrane receptor, CD44, as HA is a specific ligand for CD44. In proliferation experiments, HA-conjugated cisplatin showed dramatic cell viability decreases (from 100% to 42.31%) in H1299 cells, which overexpress CD44, whereas no such change was observed in A549 and HFL1, which have little to no expression of CD44. More importantly, conjugation with HA decreased the dosage concentration of cisplatin by 50-fold for both cytotoxic and anti-metastatic effects. In addition, HA-cisplatin conjugate treatment selectively decreased migration (from 100% to 7.8%) and invasiveness (from 100% to 21.4%, respectively) of H1299. Based on our experimental results, we strongly believe that HA-cisplatin conjugate is a potential anticancer chemo-agent, which target CD44 overexpression in NSCLC, with minimal side effects and high therapeutic properties.

Dose Calculation Accuracy of Beam Models in RadCalc for a 1.5 T MR-Linac.

The purpose of this study is to evaluate RadCalc, an independent dose verification software, for patient-specific quality assurance (PSQA) in online adaptive planning with a magnetic resonance linear accelerator (MR-linac) of a 1.5 T. Version 7.1.4 of RadCalc to introduce the capability to establish a beam model that incorporates MR field characteristics. A total of six models were established, with one using manufacturer-provided data and the others differing in percentage depth dose (PDD) data sources. Overall, two models utilized PDD data from the treatment planning system (TPS), and three used commissioned PDD data from gantry angles of 0° and 270°. Simple tests on a virtual water phantom assessed dose-calculation accuracy, revealing percentage differences ranging from -0.5% to -20.6%. Excluding models with significant differences, clinical tests on 575 adaptive plans (prostate, liver, and breast) showed percentage differences of -0.51%, 1.12%, and 4.10%, respectively. The doses calculated using RadCalc demonstrated similar trends to those of the PSQA-based measurements. The newly released version of RadCalc enables beam modeling that considers the characteristics of the 1.5 T magnetic field. The accuracy of the software in calculating doses at 1.5 T magnetic fields has been verified, thereby making it a reliable and effective tool for PSQA in adaptive plans.

Charge-Based Isolation of Extracellular Vesicles from Human Plasma.

Extracellular vesicles (EVs) have garnered significant attention due to their potential applications in disease diagnostics and management. However, the process of isolating EVs, primarily from blood samples, is still suboptimal. This is mainly attributed to the abundant nature of soluble proteins and lipoproteins, which are often separated together with EVs in the end products of conventional isolation methods. As such, we devise a single-step charge-based EV isolation method by utilizing positively charged beads to selectively remove negatively charged major impurities from human plasma via electrostatic interaction. By carefully controlling the buffer pH, we successfully collected EVs from undesired plasma components with superior purity and yield compared to conventional EV collection methods. Moreover, the developed process is rapid, taking only about 20 min for overall EV isolation. The charge-based isolation can ultimately benefit the EV-based liquid biopsy field for the early diagnosis of various diseases.

Targeted delivery of anti-miRNA21 sensitizes PD-L1high tumor to immunotherapy by promoting immunogenic cell death.

Rationale: Growing evidence has demonstrated that miRNA-21 (miR-21) upregulation is closely associated with tumor pathogenesis. However, the mechanisms by which miR-21 inhibition modulates the immunosuppressive tumor microenvironment (TME) and improves tumor sensitivity to immune checkpoint blockade therapies remain largely unexplored. In this study, we demonstrate the precise delivery of anti-miR-21 using a PD-L1-targeting peptide conjugate (P21) to the PD-L1high TME. Methods: Investigating miR-21 inhibition mechanisms involved conducting quantitative real-time PCR, western blot, flow cytometry, and confocal microscopy analyses. The antitumor efficacy and immune profile of P21 monotherapy, or combined with anti-PD-L1 immune checkpoint inhibitors, were assessed in mouse models bearing CT26.CL25 tumors and 4T1 breast cancer. Results Inhibition of oncogenic miR-21 in cancer cells by P21 efficiently activates tumor suppressor genes, inducing autophagy and endoplasmic reticulum stress. Subsequent cell-death-associated immune activation (immunogenic cell death) is initiated via the release of damage-associated molecular patterns. The in vivo results also illustrated that the immunogenic cell death triggered by P21 could effectively sensitize the immunosuppressive TME. That is, P21 enhances CD8+ T cell infiltration in tumor tissues by conferring immunogenicity to dying cancer cells and promoting dendritic cell maturation. Meanwhile, combining P21 with an anti-PD-L1 immune checkpoint inhibitor elicits a highly potent antitumor effect in a CT26.CL25 tumor-bearing mouse model and 4T1 metastatic tumor model. Conclusions: Collectively, we have clarified a miR-21-related immunogenic cell death mechanism through the precise delivery of anti-miR-21 to the PD-L1high TME. These findings highlight the potential of miR-21 as a target for immunotherapeutic interventions.

In-situ nanospectroscopic pH monitoring by plasmon resonance energy transfer (PRET).

Real-time detection of pH value in a living cell is in central importance for research about cells and diseases. In spite of the great advances in science and technology, pH measurement in a living cell is still limited in spatial resolution, in-situ detection, and intracellular monitoring. Here, we designed a nanoscale pH meter by Plasmon Resonance Energy Transfer (PRET). In order to highly sensitively measure pH with nanoscale spatial resolution, we choose 80 nm spherical gold nanoparticle (GNP) and phenol red which is commonly used in cell media for pH determination. The resonance energy of GNP is transferred to phenol red because the scattering intensity of GNP is overlapped with the second absorption intensity of phenol red at near 560 nm. Meanwhile, the absorption intensity of phenol red molecules is changing with pH value of the solution. For that reason, the intensity of PRET from GNP to phenol red molecules also changes by the acidity of phenol red solution. Then we can detect pH values with nanoscale spatial resolution through the Rayleigh scattering intensity of GNP. As we changed pH value from 6.0 to 9.0, the scattering intensity of GNP is decreased because the absorbance of phenol red at 560 nm wavelength is increased with increasing pH value. The Gaussian peak of a difference in Rayleigh scattering spectra of GNP between pH 6.0 and pH 9.0 indicates exactly the same as UV-vis spectral difference between basic and acidic phenol red solution. We expect that this pH measuring technique has a significant impact on the pH detection of living cells with nanoscale, and it can make possibility to image the cell structure by pH variation.

Three-dimensional reduced-symmetry of colloidal plasmonic nanoparticles.

Owing to their novel optical properties, three-dimensional plasmonic nanostructures with reduced symmetry such as a nanocrescent and a nanocup have attracted considerable current interest in biophotonic imaging and sensing. However, their practical applications have been still limited since the colloidal synthesis of such structures that allows, in principle, for in vivo application and large-scale production has not been explored yet. To date, these structures have been fabricated only on two-dimensional substrates using micro/nanofabrication techniques. Here we demonstrate an innovative way of breaking symmetry of colloidal plasmonic nanoparticles. Our strategy exploits the direct overgrowth of Au on a hybrid colloidal dimer consisting of Au and polystyrene (PS) nanoparticles without the self-nucleation of Au in an aqueous solution. Upon the overgrowth reaction, the steric crowding of PS leads to morphological evolution of the Au part in the dimer ranging from half-shell, nanocrescent to nanoshell associated with the appearance of the second plasmon absorption band in near IR. Surface-enhanced Raman scattering signal is obtained directly from the symmetry-broken nanoparticles solution as an example showing the viability of the present approach. We believe our concept represents an important step toward a wide range of biophotonic applications for optical nanoplasmonics such as targeting, sensing/imaging, gene delivery, and optical gene regulations.