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Acute kidney injury (AKI) is the sudden decrease in renal function that can be attributed to dysregulated reactive oxygen species (ROS) production and impaired mitochondrial function. Irisin, a type I membrane protein secreted by skeletal muscles in response to physical activity, has been reported to alleviate kidney damage through regulation of mitochondrial biogenesis and oxidative metabolism. In this study, a macrophage membrane-coated metal-organic framework (MCM@MOF) is developed as a nanocarrier for encapsulating irisin to overcome the inherent characteristics of irisin, including a short circulation time, limited kidney-targeting ability, and low membrane permeability. The engineered irisin-mediated biomimetic nanotherapeutics have extended circulation time and enhanced targeting capability toward injured kidneys due to the preservation of macrophage membrane proteins. The irisin-encapsulated biomimetic nanotherapeutics effectively mitigate acute ischemia-reperfusion injury by protecting mitochondrial function and modulating SOD2 levels in renal tubular epithelial cells. The present study provides novel insights to advance the development of irisin as a potential therapeutic approach for AKI.
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Ischemia/reperfusion injury of the kidney is associated with high morbidity and mortality, and treatment of this injury remains a challenge. G protein-coupled receptor kinase 4 (GRK4) plays a vital role in essential hypertension and myocardial infarction, but its function in kidney ischemia/reperfusion injury remains undetermined. Among the GRK subtypes (GRK2-6) expressed in kidneys, the increase in GRK4 expression was much more apparent than that of the other four GRKs 24 hours after injury and was found to accumulate in the nuclei of injured mouse and human renal tubule cells. Gain- and loss-of-function experiments revealed that GRK4 overexpression exacerbated acute kidney ischemia/reperfusion injury, whereas kidney tubule-specific knockout of GRK4 decreased injury-induced kidney dysfunction. Necroptosis was the major type of tubule cell death mediated by GRK4, because GRK4 significantly increased receptor interacting kinase (RIPK)1 expression and phosphorylation, subsequently leading to RIPK3 and mixed lineage kinase domain-like protein (MLKL) phosphorylation after kidney ischemia/reperfusion injury, but was reversed by necrostatin-1 pretreatment (an RIPK1 inhibitor). Using co-immunoprecipitation, mass spectrometry, and siRNA screening studies, we identified signal transducer and activator of transcription (STAT)1 as a GRK4 binding protein, which co-localized with GRK4 in the nuclei of renal tubule cells. Additionally, GRK4 phosphorylated STAT1 at serine 727, whose inactive mutation effectively reversed GRK4-mediated RIPK1 activation and tubule cell death. Kidney-targeted GRK4 silencing with nanoparticle delivery considerably ameliorated kidney ischemia/reperfusion injury. Thus, our findings reveal that GRK4 triggers necroptosis and aggravates kidney ischemia/reperfusion injury, and its downregulation may provide a promising therapeutic strategy for kidney protection.
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Injúria Renal Aguda , Traumatismo por Reperfusão , Animais , Humanos , Camundongos , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/complicações , Morte Celular , Regulação para Baixo , Rim/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Acoplados a Proteínas G/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/prevenção & controleRESUMO
Background Oral NaCl produces a greater natriuresis and diuresis than the intravenous infusion of the same amount of NaCl, indicating the existence of a gastro-renal axis. As one of the major natriuretic hormones secreted by both the intestines and the kidney, we hypothesized that renal uroguanylin interacts with dopamine receptors to increase sodium excretion synergistically, an impaired interaction of which may be involved in the pathogenesis of hypertension. Methods and Results In Wistar-Kyoto rats, the infusion of uroguanylin or fenoldopam (a D1-like receptor agonist) induced natriuresis and diuresis. Although subthreshold dosages of uroguanylin or fenoldopam had no effect, the coinfusion of subthreshold dosages of those reagents significantly increased sodium excretion. The coinfusion of an antagonist against D1-like receptors, SCH23390, or an antagonist against uroguanylin, 2-methylthioadenosine triphosphate, prevented the fenoldopam- or uroguanylin-mediated natriuresis and diuresis in Wistar-Kyoto rats. However, the natriuretic effects of uroguanylin and fenoldopam were not observed in spontaneously hypertensive rats. The uroguanylin/D1-like receptor interaction was also confirmed in renal proximal tubule cells. In renal proximal tubule cells from Wistar-Kyoto rats but not spontaneously hypertensive rats, stimulation of either D1-like receptors or uroguanylin inhibited Na+-K+-ATPase activity, an effect that was blocked in the presence of SCH23390 or 2-methylthioadenosine triphosphate. In renal proximal tubule cells from Wistar-Kyoto rats, guanylyl cyclase C receptor (uroguanylin receptor) and D1 receptor coimmunoprecipitated, which was increased after stimulation by either uroguanylin or fenoldopam; stimulation of one receptor increased renal proximal tubule cell membrane expression of the other. Conclusions These data suggest that there is synergism between uroguanylin and D1-like receptors to increase sodium excretion. An aberrant interaction between the renal uroguanylin and D1-like receptors may play a role in the pathogenesis of hypertension.
Assuntos
Fenoldopam , Hipertensão , Peptídeos Natriuréticos , Receptores de Dopamina D1 , Animais , Fenoldopam/farmacologia , Hipertensão/metabolismo , Rim , Túbulos Renais Proximais/metabolismo , Natriurese , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de Dopamina D1/metabolismo , Sódio/metabolismo , Cloreto de SódioRESUMO
Challenges in studying the structures and functions of cell membrane proteins lie in their lipophilicity, which makes them hard to be stabilized, crystallized, and expressed by E. coli. Herein, we propose an evanescent field excited surface-enhanced Raman scattering (EF-SERS) strategy for label-free analysis of membrane proteins in situ. Extracted cell membranes tightly wrapped the metal nanoparticles by an extruder, which ensures the SERS signals of the membrane proteins precisely benefit from the localized surface plasmons (LSPs). The leaky mode of a waveguide was employed to improve the plasmon excitation coupling. Thus, the LSPs and waveguide modes together enable the achievement of high-quality SERS profiles of membrane proteins. By spectral analysis, the structural changes of membrane proteins can be deeply understood at the molecular level. This method has broader applicability in establishing the Ramanomics of membrane proteins and unraveling the exact changes of membrane proteins during physiological processes.
Assuntos
Escherichia coli/química , Proteínas de Membrana/análise , Análise Espectral Raman , Ressonância de Plasmônio de Superfície , Propriedades de SuperfícieRESUMO
The multi-drug resistance (MDR) is the leading reason resulting in the failure of cancer treatment. Decreasing the development chance of MDR and fighting against the MDR cancer are still facing severe challenges. In order to overcome MDR via disrupting the original metabolic pathway of cancer cells, we designed a multi-functionalized nano-conjugate based on the starvation therapy to make cancer cells availably sensitized to chemotherapy. The nano-conjugate constitutes of the nano-carrier (AuNP-PEG-RGD) and glucose oxidase (GOx, activity equivalent), which not only can specifically target cancer cells with the help of the cancer-targeting peptide (RGD) laid on the surface, but also can deplete glucose and O2 with the simultaneous generation of H2O2. Insufficient glucose, excess H2O2, and hypoxia microenvironments can suppress cell proliferation and induce cell apoptosis. With the hypothesis that the specific damage induced by the nano-conjugate can make cancer cells much vulnerable to chemotherapy, we further evaluated the therapeutic effect of an anti-cancer drug (doxorubicin, Dox) with the assistance of the low dose of nano-conjugate for the breast cancer cell. The results indicate that 0.2⯵g/mL of Dox in the combination of 22.5 pM of the nano-conjugate can kill 80% cancer cells, which effectively improves the treatment efficiency compared with the nano-conjugate or Dox alone based on the synergism effect (the combination index<1). More importantly, our developed strategy can be used for sensitizing the MDR cancer cells to the traditional ineffective drugs, which owns potential applications in decreasing the chance of MDR development and overcoming drug-resistant cancers.
Assuntos
Neoplasias da Mama , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Nanoconjugados , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Humanos , Peróxido de Hidrogênio , Microambiente TumoralRESUMO
Confronted with the rapid evolution and dissemination of antibiotic resistance, there is an urgent need to develop alternative treatment strategies for drug-resistant pathogens. Here, an unconventional approach is presented to restore the susceptibility of methicillin-resistant S. aureus (MRSA) to a broad spectrum of conventional antibiotics via photo-disassembly of functional membrane microdomains. The photo-disassembly of microdomains is based on effective photolysis of staphyloxanthin, the golden carotenoid pigment that gives its name. Upon pulsed laser treatment, cell membranes are found severely disorganized and malfunctioned to defense antibiotics, as unveiled by membrane permeabilization, membrane fluidification, and detachment of membrane protein, PBP2a. Consequently, the photolysis approach increases susceptibility and inhibits development of resistance to a broad spectrum of antibiotics including penicillins, quinolones, tetracyclines, aminoglycosides, lipopeptides, and oxazolidinones. The synergistic therapy, without phototoxicity to the host, is effective in combating MRSA both in vitro and in vivo in a mice skin infection model. Collectively, this endogenous chromophore-targeted phototherapy concept paves a novel platform to revive conventional antibiotics to combat drug-resistant S. aureus infections as well as to screen new lead compounds.
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Mitochondrion is one of the most important organelles and becomes a target in many cancer therapeutic strategies. Mitochondrial microenvironments in response to therapeutic methods are the key to understand therapeutic mechanisms. However, they are almost rarely studied. Herein, the mitochondrial microenvironments, including mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) after different photodynamic therapy (PDT) dosages, were monitored by fluorescent imaging and compared among three cell lines (HepG2, MCF-7, and LO2). Furthermore, the fluctuations of intramitochondrial pHs were revealed via a plasmonic mitochondrion-targeting surface-enhanced Raman scattering (SERS) pH nanosensor. Results indicate that the MMP decreases gradually with the ROS generation and the cancerous cells exhibit less response to excess ROS relative to normal cells. On the other hand, the pH value in the mitochondria decreases initially and then increases when the amount of ROS increases. The LO2 cell is preliminarily evidenced to have a higher self-adjustment ability due to its better tolerance to differential intra/extracellular pHs. This study may provide a basis for an in-depth understanding of the mechanisms of the mitochondrial targeting-based PDT therapeutic processes. It is also helpful for more accurate and useful diagnosis according to intramitochondrial microenvironments and improvement on therapy efficiency of cancers.
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Mitocôndrias/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Imagem Óptica , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismoRESUMO
Subcellular mitochondrion has become a target for improving the therapeutic efficiency and reducing side damage to normal cells via a combination of many therapeutic strategies. However, the underlying molecular mechanisms associated with cell death induced by subcellular dysfunction remain unknown or disputed. In this study, we investigated the dynamic molecular changes of living mitochondria upon phototherapy (photothermal therapy plus photodynamic therapy, PTT & PDT) by surface-enhanced Raman scattering spectroscopy (SERS) and intended to disclose the photo-induced cell death route in breast cancer cells (MCF-7) taking into account the mitochondrion. Indocyanine green (ICG), a Food and Drug Administration (FDA)-approved clinic blood-injection near-infrared angiographic contrast agent and a PTT & PDT drug, was used for the evaluation of the phototherapy effect. The results revealed that the content of phenylalanine (Phe) in mitochondria evidently increased during the phototherapy-induced cell death process. Moreover, the phototherapy-induced cell apoptosis was mainly regulated through the DNA structures. We expect that the understanding of mitochondrial molecular stress responses will be helpful for the diagnosis and therapy of cellular processes associated with mitochondria and provide valuable guidance for the further design and development of more effective therapeutic platforms and methods at the sub-cellular level.
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Mitocôndrias/metabolismo , Análise Espectral Raman/métodos , Dano ao DNA/efeitos dos fármacos , Ouro/química , Humanos , Hipertermia Induzida/métodos , Verde de Indocianina/farmacologia , Verde de Indocianina/efeitos da radiação , Verde de Indocianina/toxicidade , Raios Infravermelhos , Células MCF-7 , Nanotubos/química , Fenilalanina/metabolismo , Fotoquimioterapia/métodosRESUMO
Cytobiological methods for cell nucleus-related studies start with the extraction processes of intranuclear components with many cell lysis buffers, following with the structural characterizations and quantitative analysis of the extracted components. In this study, we tried to evaluate the availability and reliability of these extraction-based analytical methods from their spectral features. We implemented an in situ surface-enhanced Raman scattering spectroscopy (SERS) strategy with the help of the nucleus-targeting nanoprobes to investigate the molecular information of nucleus, in comparison with these ex situ methods. This study provides valuable references for choosing an appropriate detection method according to different detection purposes, and also points out the risks of many developing cell-related analytical methods that combine the traditional cytobiological techniques from exogenous interferences during sample preprocesses. Graphical abstract.
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Núcleo Celular/química , DNA/análise , Nanopartículas Metálicas/química , Proteínas Nucleares/análise , Análise Espectral Raman/métodos , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Ouro/química , Células Hep G2 , Humanos , Nanopartículas Metálicas/ultraestrutura , Peptídeos/química , Polietilenoglicóis/químicaRESUMO
Confronted with the severe situation that the pace of resistance acquisition is faster than the clinical introduction of new antibiotics, health organizations are calling for effective approaches to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, an approach to treat MRSA through photolysis of staphyloxanthin, an antioxidant residing in the microdomain of S. aureus membrane, is reported. This photochemistry process is uncovered through transient absorption imaging and quantitated by absorption spectroscopy, Raman spectroscopy, and mass spectrometry. Photolysis of staphyloxanthin transiently elevates the membrane permeability and renders MRSA highly susceptible to hydrogen peroxide attack. Consequently, staphyloxanthin photolysis by low-level 460 nm light eradicates MRSA synergistically with hydrogen peroxide and other reactive oxygen species. The effectiveness of this synergistic therapy is well validated in MRSA planktonic culture, MRSA-infected macrophage cells, stationary-phase MRSA, persisters, S. aureus biofilms, and two mice wound infection models. Collectively, the work demonstrates that staphyloxanthin photolysis is a new therapeutic platform to treat MRSA infections.
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A high-throughput, dual-channel single cell analytical method is described for the detection of sialic acid (SA) on single cell based on the use of microfluidic droplets integrated with plasmonic imaging and surface-enhanced Raman spectroscopy (SERS) with the assistance of a multifunctional metal nanoparticle-based probe. The multifunctional plasmonic nanoprobe was prepared by modifying silver nanoparticles (AgNPs) with 4-mercaptophenylboronic acid (MPBA) that both warrants SA recognition and acts as a Raman reporter. This nanoprobe is a high-contrast indicator under bright field imaging due to the strong energy loss feature of AgNPs, and also owns possesses a strong SERS enhancement capability toward MPBA. Cells incubated with the plasmonic nanoprobes were isolated in water-in-oil droplets and then were re-dispersed in a chamber array chip. High-precision profiles of SA on a single cell in one droplet were obtained by the bright field imaging and image processing. The SA expression levels on different cell lines (MCF-7, HepG2, SGC and BNL.CL2) traced by SERS spectroscopy were compared. The statistical data among different cell lines confirm that the SA expression levels on cancer cells are much higher than that on normal cells. Single cell analysis further revealed that the cell-to-cell variations are more obvious in cancer cell lines. This study provides a valuable tool for understanding glycan-related biochemical processes. Graphical abstract A high-throughput, dual-channel microfluidic droplet platform succeeded in distinguishing different cancer cell lines at single living cell level integrated with plasmonic imaging and surface-enhanced Raman spectroscopy with assistance of a multifunctional metal nanoparticle-based probe.
Assuntos
Biomarcadores Tumorais/análise , Técnicas Analíticas Microfluídicas/métodos , Ácido N-Acetilneuramínico/análise , Análise de Célula Única/métodos , Análise Espectral Raman/métodos , Animais , Ácidos Borônicos/química , Linhagem Celular Tumoral , Humanos , Dispositivos Lab-On-A-Chip , Nanopartículas Metálicas/química , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Neoplasias/diagnóstico , Prata/química , Compostos de Sulfidrila/químicaRESUMO
Identification of specific and reliable biomarkers or unique characteristics is significant for cancer molecular diagnosis and cancer therapeutic assessment. As a biomarker, sialic acid expression in human biofluid or on cell surface is one of interest to determine the tumor malignancy and metastasis since it involves in many crucial metabolic processes. In this work, we aimed to develop a molecular diagnosis method to make it possible to distinguish human breast cancer and normal tissues by capturing rich spectral features of phenyboronic acid-based nanoprobe (MPBA@AgNP) when it binds to sialic acid on cell surface. We analyzed and found that the marker bands at 1074 and 1570â¯cm-1 recorded by Surface-enhanced Raman spectroscopy (SERS) displayed discernible spectral differences in vitro cell lines. Density functional theory (DFT) was further used to explore and support the detailed changes of vibrational modes affected by sialic acid. This method is generally applicable by testing three types of in vitro cell lines (HepG2, Hela, BNL.CL2) and one pair of the tissue sections (cancer tissue and normal tissue) from the human breast regions. Besides, the area under receiver operating characteristic (ROC) curves for 1074 and 1572â¯cm-1 are 0.9419 and 0.9023, confirming determination of the specific molecular expression by the spectral features holds potential promise for improving cancer detection accuracy. Furthermore, sialic acid expression and distribution acquired of breast tissues by confocal SERS mapping further indicated our method is possible for cancer early diagnosis and toward to real-time in vivo study.
Assuntos
Neoplasias da Mama/diagnóstico , Membrana Celular/química , Ácido N-Acetilneuramínico/análise , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Células HeLa , Células Hep G2 , Humanos , Teoria Quântica , Análise Espectral RamanRESUMO
Subcellular organelles, for example, nucleus, mitochondria, and lysosome, are the vital organelles with responsibilities that maintain cell operation and metabolism. Owing to their roles in energy production and programmed cell death, these organelles have become prime therapeutic targets in different diseases and states. In this study, biocompatible, organelle-targeting nanoprobes were developed by modifying gold nanorods (AuNRs) with specific targeting peptides. These nanoprobes were employed to directly profile subcellular biomolecules and vital organelles by surface-enhanced Raman scattering (SERS) spectroscopy. Macromolecular spectral profiles of subcellular organelles were achieved and compared. Further, these organelle-targeting AuNRs were used for the photothermal treatment of cancer cells (HepG2, HeLa, and MCF-7 cell lines). The cell viability assays show that the nucleus- and mitochondria-targeting AuNRs provide higher photothermal efficiencies under an 808 nm laser relative to the lysosome-targeting ones. This study makes critical insights into the spectral profiles of subcellular organelles and also inspires people in the development of high-efficacy cancer therapeutic strategies by subcellular organelle-targeting drugs.
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Nanotubos , Sistemas de Liberação de Medicamentos , Ouro , Humanos , Células MCF-7 , Análise Espectral RamanRESUMO
The expression levels of glycans on the surfaces of cancer and normal cells show different, however, this difference is not noticeable enough to distinguish them directly. So, herein, based on the targeted molecular recognition of the glycans on cell surfaces by 4-mercaptophenyl boronic acid (MPBA), a novel surface-enhanced Raman scattering (SERS) nanoprobe (glucose-MPBA@AgNPs) was prepared by inducing controllable assembly of MPBA decorated Ag nanoparticles (MPBA@AgNPs) in a certain level via the bridge of glucose to amplify such a limited difference in SERS measurements. On the basis of the aggregation-induced 3D SERS hot spot effect, this multi-particle nanoprobe possesses over 10 times stronger SERS enhancement ability than the individual MPBA@AgNPs. As the different sialic acid (SA) expression on the surfaces of cancer and normal cells led to the different accumulation of glucose-MPBA@AgNPs, the results we obtained (mean intensities recorded from five cells) indicate the SA amounts on two kinds of cells can provide 5-7 times signal contrast grade in SERS band intensities (P < 0.001). Compared with the monodispersed nanoprobe, our developed nanoprobe amplifies the SA expression difference on cell surfaces and supports high sensitivity for cancer cell recognition, which might be useful in providing highly effective recognition of the edges of tumor tissues in clinic field.
Assuntos
Membrana Celular/química , Nanopartículas Metálicas/química , Sondas Moleculares/química , Polissacarídeos/análise , Ácidos Siálicos/análise , Prata/química , Ácidos Borônicos/química , Linhagem Celular , Membrana Celular/patologia , Glucose/química , Células Hep G2 , Hepatócitos/química , Hepatócitos/patologia , Humanos , Nanopartículas Metálicas/ultraestrutura , Sondas Moleculares/síntese química , Especificidade de Órgãos , Polissacarídeos/química , Ácidos Siálicos/química , Análise Espectral Raman/métodos , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
Photodynamic therapy (PDT) involves the uptake of photosensitizers by cancer cells and the irradiation of a light with a specific wavelength to trigger a series of photochemical reactions based on the generation of reactive oxygen, leading to cancer cell death. PDT has been widely used in various fields of biomedicine. However, the molecular events of the cancer cell nucleus during the PDT process are still unclear. In this work, a nuclear-targeted gold nanorod Raman nanoprobe combined with surface-enhanced Raman scattering spectroscopy (SERS) was exploited to investigate the dynamic intranuclear molecular changes of B16 cells (a murine melanoma cell line) treated with a photosensitizer (Chlorin e6) and the specific light (650 nm). The SERS spectra of the cell nucleus during the PDT treatment were recorded in situ and the spectroscopic analysis of the dynamics of the nucleus uncovered two main events in the therapeutic process: the protein degradation and the DNA fragmentation. We expect that these findings are of vital significance in having a better understanding of the PDT mechanism acting on the cancer cell nucleus and can further help us to design and develop more effective therapeutic platforms and methods.
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A surface-enhanced Raman scattering (SERS) method for in situ detection and analysis of the intranuclear biomolecular information of a cell has been developed based on a small, biocompatible, nuclear-targeting alkyne-tagged deoxyribonucleic acid (DNA) probe (5-ethynyl-2'-deoxyuridine, EDU) that can specially accumulate in the cell nucleus during DNA replications to precisely locate the nuclear region without disturbance in cell biological activities and functions. Since the specific alkyne group shows a Raman peak in the Raman-silent region of cells, it is an interior label to visualize the nuclear location synchronously in real time when measuring the SERS spectra of a cell. Because no fluorescent-labeled dyes were used for locating cell nuclei, this method is simple, nondestructive, non- photobleaching, and valuable for the in situ exploration of vital physiological processes with DNA participation in cell organelles. Graphical abstract A universal strategy was developed to accurately locate the nuclear region and obtain precise molecular information of cell nuclei by SERS.
Assuntos
Alcinos/análise , Núcleo Celular/patologia , Desoxiuridina/análogos & derivados , Neoplasias/patologia , Núcleo Celular/química , Desoxiuridina/análise , Humanos , Células MCF-7 , Microscopia de Fluorescência/métodos , Neoplasias/química , Imagem Óptica/métodos , Análise Espectral Raman/métodosRESUMO
The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.
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Concentração de Íons de Hidrogênio , Nanotubos , Organelas/química , Análise Espectral Raman , Núcleo Celular/química , Sistemas de Liberação de Medicamentos , Ouro , Células Hep G2 , Humanos , Lisossomos/química , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/química , PiridinasRESUMO
Sialoglycan expression is critical for assessing various diseases progression. Especially, its abnormal levels are commonly believed to be associated with tumor and metastatic cancer types. While, complicated structures, multiple types and dynamic distributions make it challenging for in situ investigating sialoglycans at the physiological status. Herein, we developed a 4-mercaptophenylboronic acid (MPBA)-based surface-enhanced Raman scattering (SERS) nanosensor to in situ study sialoglycan levels and dynamic expression processes of different cell types based on molecular recognition between phenylboronic acid and sialoglycans at physiological condition. This nanosensor is designed by the MPBA decorated silver nanoparticle (AgNP), which is unique and multifunctional because of its three-in-one role involving the Raman signal enhancer (AgNP), the sensing reporter of MPBA and the target receptor based on the recognition of phenylboronic acid and sialoglycans. When this nanosensor binds to sialoglycans, the molecular vibrational modes of MPBA will change, which can be traced by ultrasensitive SERS technique. The superiority of this study is that we built the relation between the spectral changes of MPBA (relative intensities) in molecular recognition with the sialoglycan dynamic expression of cells. We believe that our SERS strategy could be further extended to explore crucial physiological processes and significant biological system that glycans are involved in.
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Técnicas Biossensoriais/métodos , Ácidos Borônicos/química , Glicolipídeos/isolamento & purificação , Nanopartículas Metálicas/química , Compostos de Sulfidrila/química , Glicolipídeos/química , Humanos , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
Targeted delivery of chemotherapeutic agents to pathology areas can improve drug efficiency and reduce serious side effects on normal regions. However, their treatment mechanism on cells or cell nuclei is still mysterious due to the lack of in situ characterization methods. In this paper, the specific diagnosis and treatment processes of a targeted antitumor agent (doxorubicin, Dox) functionalized aptamer complex (TLS11a-GC-Dox) toward HepG2 cells, a human hepatocellular carcinoma cell line, were tracked in real time by the surface-enhanced Raman scattering (SERS) spectroscopic technique and dark-field imaging with the assistance of gold nanorod-based nuclear targeted probes, which possess remarkable SERS enhancement ability, specific targeting, and excellent biological compatibility. This is the first time to explore the acting mechanism of an aptamer-based targeted drug on cell nucleus based on the spectral information on components inside the cell nucleus. The results demonstrate that this aptamer/drug conjugate has targeting and sustained-release actions and its therapeutic effect is achieved by the gradual damage of relevant proteins and DNA in nuclei. Better understanding of the mechanism of aptamer-drug conjugates acting on cancer cells is conductive to increasing cancer therapy efficiency and is also helpful for the design of highly effective drug delivery methods.
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Aptâmeros de Nucleotídeos/química , Doxorrubicina/química , Análise Espectral Raman/métodos , Aptâmeros de Nucleotídeos/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Ouro/química , Células Hep G2 , Humanos , Microscopia Confocal , Nanotubos/químicaRESUMO
Investigating the molecular changes of cancer cell nucleus with drugs treatment is crucial for the design of new anticancer drugs, the development of novel diagnostic strategies, and the advancement of cancer therapy efficiency. In order to better understand the action effects of drugs, accurate location and in situ acquisition of the molecular information of the cell nuclei are necessary. In this work, we report a microspectroscopic technique called dark-field and fluorescence coimaging assisted surface-enhanced Raman scattering (SERS) spectroscopy, combined with nuclear targeting nanoprobes, to in situ study Soma Gastric Cancer (SGC-7901) cell nuclei treated with two model drugs, e.g., DNA binder (Hoechst33342) and anticancer drug (doxorubicin, Dox) via spectral analysis at the molecular level. Nuclear targeting nanoprobes with an assembly structure of thiol-modified polyethylene glycol polymers (PEG) and nuclear localizing signal peptides (NLS) around gold nanorods (AuNRs) were prepared to achieve the amplified SERS signals of biomolecules in the cell nuclei. With the assistance of dark field/fluorescence imaging with simultaneous location, in situ SERS spectra in one cell nucleus were measured and analyzed to disclose the effects of Hoechst33342 and Dox on main biomolecules in the cell nuclei. The experimental results show that this method possesses great potential to investigate the targets of new anticancer drugs and the real-time monitoring of the dynamic changes of cells caused by exogenous molecules.