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Cytokine expression is an important biomarker in understanding hypoxia microenvironments in tumor growth and metastasis. In-droplet-based immunoassays performed above the target cell membrane were employed to track the cytokines of single cells with the aid of three types of immuno-nanoprobes (one capture nanoprobe and two reporter nanoprobes). Single cells and nanoprobes were co-packaged in water-in-oil microdroplets (about 100 µm in diameter) using a cross-shaped microfluidic chip. In each droplet, capture nanoprobes would be first fixed to the cell surface by linking to membrane proteins that have been streptavidinized. Then, the capture nanoprobes can collect cell-secreted cytokines (VEGF and IL-8) by the antibodies, followed by two reporter nanoprobes that emit distinguishable fluorescence. Fluorescence imaging was utilized to record the signal outputs of two reporter probes, which reflect cytokine expressions secreted by a single tumor cell. The cytokine levels at different degrees of hypoxia induction were assessed. Multiple chemometric methods were adopted to distinguish differences in the secretion of two cytokines and the results demonstrated a positive correlation. This study developed an in-droplet, dual-target, simultaneous biosensing strategy for a single cell, which is helpful for understanding the impacts of hypoxia microenvironments on cell cytokines that are vital for assessing early cancer diagnosis and prognosis.
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Análise de Célula Única , Imunoensaio/métodos , Humanos , Análise de Célula Única/métodos , Citocinas/metabolismo , Citocinas/análise , Interleucina-8/análise , Interleucina-8/metabolismo , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/metabolismo , Dispositivos Lab-On-A-Chip , Hipóxia Celular , Hipóxia/metabolismoRESUMO
With the vigorous development of biotechnology, genetically modified organisms (GMOs) have become more and more common. In order to effectively supervise and administrate them, the rapid and accurate detection of GMOs is urgently demanded. Here, GMO gene-specific sensing methods based on colorimetry and surface-enhanced Raman scattering (SERS) were proposed based on the lateral branch cleavage function of the CRISPR/Cas12a system. Two transgenes, pCaMV35S and M810 Cry1Ab, were chosen as targets for transgenic crops. By using these methods, we performed transgenic detection on five types of maize leaves and successfully distinguished transgenic from non-transgenic samples. The colorimetric method is rapid, economical and available for field detection. The SERS approach, giving a higher sensitivity to 100 fM, is more suitable for laboratory application scenarios. This study explores practical transgenic detection approaches and will be valuable for the supervision of GMOs.
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Sistemas CRISPR-Cas , Zea mays , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Sistemas CRISPR-Cas/genética , TransgenesRESUMO
The aberrant growth of cervical cells caused by the infection of human papillomavirus (HPV) may cause cervical cancer. In order to effectively prevent the occurrence of cervical cancer and for better follow-up treatment after surgery, a rapid and reliable detection method of HPV DNA is essential. Here, a surface-enhanced Raman scattering (SERS) detection method was developed based on the clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 technique and the enzyme catalysis amplification reaction, which achieved a simple and rapid detection of low-content HPV genes. The CRISPR/dCas9/sgRNA complex was anchored above a magnetic bead, which can precisely capture target DNA sequences, exhibiting high selectivity for HPV genes. When the biotinylated target DNAs exist, they can bridge a streptavidin-modified horse radish peroxidase (HRP) to the magnetic bead, producing an HRP-decorated conjugate. This conjugate allows an HRP-catalyzed reaction for its substrate (3,3',5,5'-tetramethylbenzidine, TMB). Gold nanostars with a silica shell exhibiting the lightning rod effect of SERS were employed to measure the SERS spectra of the oxidative product of TMB. Enzyme catalysis and SERS co-contribute to the SERS signal output, ensuring a high detection sensitivity. This method is a proof of concept for detecting HPV DNAs in a complex system. The current method can be applied to other target DNAs simply by changing the sgRNA sequence. Many superiors portend that the CRISPR/dCas9-based SERS method is promising for further clinical application.
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Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Análise Espectral Raman/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias do Colo do Útero/genética , DNA/química , Catálise , Ouro/químicaRESUMO
A microfluidic-based surface-enhanced Raman scattering (SERS) platform for analyzing cytokines secreted by single cells is reported based on the elaborate bioconjugation of the immuno-sandwich complex on the probed cell surface. This platform integrates the dual functions of microfluidic droplet separation of single cells and SERS measurement. Two immune nanoprobes (capture probe and SERS probe) are introduced into a microfluidic droplet along with a single cell. They were anchored to the cell membrane protein surface by capturing secreted cytokines to form an immune sandwich structure, realizing the enrichment effect of cytokines above the cell membrane surface and the amplification effect of SERS detection probes. This single-cell analytical platform was applied to track specific cell-secreted vascular endothelial growth factor (VEGF) of different cell lines (MCF-7, SGC, and T24), and highly sensitive detection of VEGF was achieved. Chemometric methods (principal component analysis and t-distributed stochastic neighbor embedding) were adopted for the SERS data analysis, and the support vector machine (SVM) discriminant model was established to test the data. These chemometric methods successfully identify significant differences in the secreting ability of cytokines among three kinds of cancer cell lines, revealing cell heterogeneity. In addition, the behavior of single cells secreting VEGF was monitored time-dependently and was shown to increase with time. This work demonstrates the importance of tracking specific cells secreting cytokines based on the cell surface bioconjugation strategy. Our developed platform provides guidelines for using the single-cell exocytosis factors as biomarkers to assess the early diagnosis of cancer and provide physiological cues for learning single-cell secretions.
Assuntos
Nanopartículas Metálicas , Técnicas Analíticas Microfluídicas , Membrana Celular , Citocinas , Nanopartículas Metálicas/química , Técnicas Analíticas Microfluídicas/métodos , Microfluídica , Análise Espectral Raman/métodos , Fator A de Crescimento do Endotélio VascularRESUMO
Despite recent advances in single-cell analysis techniques, the ability of single-cell analysis platforms to track specific cells that secreted cytokines remains limited. Here, we report a microfluidic droplet-based fluorescence imaging platform that can analyze single cell-secreted vascular endothelial growth factor (VEGF), an important regulator of physiological and pathological angiogenesis, to explore cellular physiological clues at the single-cell level. Two kinds of silica nanoparticle (NP)-based immunoprobes were developed, and they were bioconjugated to the membrane proteins of the probed cell surface via the bridging of secreted VEGF. Thus, an immunosandwich assay was built above the probed cell via fluorescence imaging analysis of each cell in isolated droplets. This analytical platform was used to compare the single-cell VEGF secretion ability of three cell lines (MCF-7, HeLa, and H8), which experimentally demonstrates the cellular heterogeneity of cells in secreting cytokines. The uniqueness of this method is that the single-cell assay is carried out above the cell of interest, and no additional carriers (beads or reporter cells) for capturing analytes are needed, which dramatically improves the availability of microdroplets. This single-cell analytical platform can be applied for determining other secreted cytokines at the single-cell level by changing other immune pairs, which will be an available tool for exploring single-cell metabonomics.
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Técnicas Analíticas Microfluídicas , Microfluídica , Citocinas , Técnicas Analíticas Microfluídicas/métodos , Imagem Óptica , Análise de Célula Única , Fator A de Crescimento do Endotélio Vascular/análise , Fatores de Crescimento do Endotélio VascularRESUMO
Although the chemical components of Panax notoginseng (PN) and Panax ginseng (PG) are similar, their bioactivities are different. In this study, the differential bioactivities of PN and PG were used as the research object. First, the different metabolites in the plasma after oral administration of PN and PG were analyzed using a UPLC-Q/TOF-MS-based metabolomics approach. Afterward, the metabolite-target- pathway network of PN and PG was constructed, and thus the pathways related to different bioactivities were analyzed. As a result, 7 different metabolites were identified in PN group, and 10 different metabolites were identified in the PG group. In the PN group, the metabolite N1 was related to hemostasis, N1 and N3 were related to inhibiting the nerve center, antihypertensive, and abirritation. The metabolites N1, N3, N4, N5, and N6 were related to liver protection. The results showed that the metabolites G1, G2, G3, G5, and G6 in PG group were related to heart protection, and G1, G2, G6, and G9 were related to increased blood pressure. There were 13 signaling pathways related to different biological activities of PN (8 pathways) and PG (5 pathways). These pathways further clarified the mechanism of action that caused the different bioactivities between PN and PG. In summary, metabolomics combined with network pharmacology could be helpful to clarify the material basis of different bioactivities between PN and PG, promoting the research on PN and PG.
Assuntos
Ginsenosídeos , Panax notoginseng , Panax , Ginsenosídeos/metabolismo , Ginsenosídeos/farmacologia , Metabolômica , Panax/metabolismo , PlasmaRESUMO
Traditional methods for single-nucleotide variants based on amplification and fluorescence signals require expensive reagents and cumbersome instruments, and they are time-consuming for each trial. Here, a porous anodised aluminium (PAA)-based sensing chip modified with deactivated Cas9 (dCas9) proteins and synthetic guide RNA (sgRNA) as the biorecognition receptor is developed, which can be used for the label-free sensing of the diffuse large B-cell lymphoma (DLBCL) MYD88L265P gene by integrating with electrochemical ionic current rectification (ICR) measurement. The sgRNA that can specifically identify and capture the MYD88L265P gene was screened, which has been proved to be workable to activate dCas9 for the target MYD88L265P. In the sensing process, the dCas9 proteins can capture the genome sequence, thus bringing negative charges over the PAA chip and correspondingly resulting in a variation in the ICR value due to the uneven transport of potassium anions through the ion channels of the PAA chip. The whole sensing can be finished within 40 min, and there is no need for gene amplification. The CRISPR/dCas9-based sensor demonstrates ultrasensitive detection performance in the concentration range of 50 to 200 ng µL-1 and it has been proved to be feasible for the genome sequence of patient tissues. This sensor shows the potential of targeting other mutations by designing the corresponding sgRNAs and expands the applications of CRISPR/dCas9 technology to the on-chip electrical detection of nucleic acids, which will be very valuable for rapid diagnosis of clinically mutated genes. This makes the hybrid CRISPR-PAA chip an ideal candidate for next-generation nucleic acid biosensors.
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Técnicas Biossensoriais , Sistemas CRISPR-Cas , Linfoma Difuso de Grandes Células B , Fator 88 de Diferenciação Mieloide , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/genética , Mutação , Fator 88 de Diferenciação Mieloide/genéticaRESUMO
Three marketed anti-PD-L1 antibodies almost have severe immune-mediated side effects. The therapeutic effects of anti-PD-L1 chemical inhibitors are not satisfied in the clinical trials. Here we constructed human-derived protein scaffolds library and screened scaffolds with a shape complementary to the PD-1 binding domain of PD-L1. The RNA binding domain of U1 snRNPA was selected as one of potential binders because it had the most favorable binding energies with PD-L1 and conformed to pre-established biological criteria for the screening of candidates. The recombinant U1 snRNPA (rU1 snRNPA) in Escherichia coli exhibits anti-cancer activity in melanoma and breast cancer by reactivating tumor-suppressed T cells in vitro and anti-melanoma activity in vivo. Considering hydrophobic and electrostatic interactions, three residues were mutated on the interface of U1 snRNPA and PD-L1 complex, and the ranked variants by PatchDock and A32D showed an increased active phenotype. The screening of human-derived protein scaffolds may become the potential development of therapeutic agents.
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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.
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Escherichia coli/química , Proteínas de Membrana/análise , Análise Espectral Raman , Ressonância de Plasmônio de Superfície , Propriedades de SuperfícieRESUMO
Autophagy plays a critical role in many vitally important physiological and pathological processes, such as the removal of damaged and aged organelles and redundant proteins. Although autophagy is mainly a protective process for cells, it can also cause cell death. In this study, we employed in situ and ex situ surface-enhanced Raman scattering (SERS) spectroscopies to obtain chemical information of lysosomes of HepG2 cells. Results reveal that the SERS profiles of the isolated lysosomes are different from the in situ spectra, indicating that lysosomes lie in different microenvironments in these two cases. We further investigated the molecular changes of isolated lysosomes according to the autophagy induced by starvation via ex situ SERS. During autophagy, the conformation of proteins and the structures of lipids have been affected, and autophagy-related molecular evidence is given for the first time in the living lysosomes. We expect that this study will provide a reference for understanding the cell autophagy mechanism.
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Lisossomos , Análise Espectral Raman , Autofagia , Células Hep G2 , Humanos , OrganelasRESUMO
Sorafenib is the first-line therapeutic option for advanced hepatocellular carcinoma (HCC). Many patients exhibit a primary resistance (PR) response after initial treatment. In previous studies, compared to acquired resistance, the mechanism of PR is unclear. The present study aimed to evaluate the response of patient samples to sorafenib by patient-derived xenograft (PDX) models, and the differences at the transcriptome level between the sorafenib PR group and the sorafenib sensitive group were analyzed by single-cell sequencing technology. A specific cell cluster may be differentiated by the liver bud hepatic cells, and the JUN transcription factors in this cell cluster were highly activated. The albumin is secreted by other cell clusters, and the cluster stimulates the FcRn complex receptor to activate the HIF pathway and cell proliferation, resulting in a poor response to sorafenib. These findings are validated by both cell communication analysis and experiments. Thus, the current studies provided a novel approach for the treatment of sorafenib-resistant HCC.
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Raman spectroscopy has emerged as a promising tool in biomedical analysis and clinical diagnosis. The development of surface-enhanced Raman scattering spectroscopy (SERS) improved the detection limit with ultrahigh sensitivity and simplicity. More and more Raman spectroscopy clinical trials (R-PCT) have been conducted recently. However, there is a lack of an up-to-date review summarizing the current status of Raman clinical trials performed until now. Hence, the clinical trials for Raman were retrieved from the International Clinical Trials Registration Platform. We summarized the clinical characteristics of 55 registered Raman spectroscopy clinical trials (R-RSCTs) and 44 published Raman spectroscopy clinical trials (P-RSCTs). This review could assist researchers and clinicians to understand the current status of Raman spectroscopy clinical research and perhaps could benefit the reasonable and accurate design of future SERS studies.
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A highly sensitive trypsin sensing system in serum was developed by using an anodic alumina oxide (AAO)-based, trypsin substrate-decorated hybrid ion permeation membrane. Owing to the trypsin-triggered peptide hydrolyzation reaction, the surface electrical feature of the peptide-decorated hybrid ion membrane changed. The electric double layer effect reduces the effective ion current diameter in the AAO nano unit, so that the ion current rectification ratio will be enhanced, realizing the quantitative detection of trypsin. The lowest detection concentration can be achieved as low as 0.1 pM. This method is no need for sample pre-preparation, easy to operate, highly sensitive, and also applicable to other enzyme evaluation systems by changing corresponding substrates. This study provides a new idea for selective measurements of proteases in complex biological samples.
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Óxido de Alumínio/química , Nanotecnologia/instrumentação , Peptídeos/química , Tripsina/análise , Tripsina/sangue , Técnicas Eletroquímicas , Humanos , Membranas Artificiais , Microscopia Eletrônica de VarreduraRESUMO
A nanotip sensitive to reactive oxygen species (ROS) and NAD+/NADH (oxidized/reduced forms of nicotinamide adenine dinucleotide) was designed and prepared to identify the redox events in a single living cell by surface-enhanced Raman scattering (SERS) spectroscopy. The nanotips were prepared by the one-step laser-induced Ag growth and deposition. A redox-reversible Raman reporter, 4-mercaptophenol (4-MP), was employed for the nanotip decoration along with the Ag deposition. 4-MP can be converted to SERS-inactive 4-mercaptocyclohexa-2,5-dienone (4-MC) by Fe3+ ions to complete signal rezeroing for multiple oxidative stress event loops. The SERS signal conversion from 4-MC to 4-MP provides a cue for the reduction process that is NADH-dependent. In contrast, by the conversion from 4-MP to 4-MC, the oxidative stress events and the signal transduction mechanism of cells stimulated by drugs (phorbol 12-myristate 13-acetate and H2O2) can be explored by SERS. This sensor is easy to fabricate and can be recycled. This tip-typed SERS nanosensor can be extendedly available for tracing other key markers in other NAD+/NADH-mediated respiratory chain and glycolysis, e.g., lactic acid, pyruvic acid, adenosine triphosphate, and antioxidants. It will be useful for investigating the diseases of abnormal oxidative stress and mitochondrial metabolism at the single-cell level.
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Peróxido de Hidrogênio , Estresse Oxidativo , NAD/metabolismo , Oxirredução , Análise Espectral RamanRESUMO
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.
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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
Ganoderma lucidum, also known as LINGZHI, has a long tradition of use in folk medicine of the Far East, which is documented in the oldest Chinese pharmacopoeia, declaring it a superior medicine. LINGZHI-8 (LZ-8) is an immunoregulatory fungal protein isolated from the fruiting body of Ganoderma lucidum. Neutropenia is a condition with an abnormally low levels of neutrophils in the blood, which is caused by numerous medical conditions or medications, such as chemotherapy. The current study demonstrated that recombinant LZ-8 (rLZ-8) from Pichia promoted the differentiation of bone marrow hematopoietic stem cells (HSCs) into granulocytes in a neutropenia mouse model induced by cyclophosphamide. Also, it regulated the CXCR4-SDF1 axis to promote the mobilization of HSCs and the release of neutrophils from the bone marrow to peripheral blood. Binding of rLZ-8 to the colony stimulating factor 1 receptor (CSF1R) promotes the differentiation of HSCs into primitive CFU colonies. These results suggested that rLZ-8 has a potential effect in the treatment of chemotherapy-induced neutropenia.
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A high-throughput single-cell analytical technique based on the microdroplet array integrated with the plasmon-enhanced-four-wave mixing (PE-FWM) imaging was developed, which is applicable for the highly sensitive and automatic assessment of the surface receptors of cells. The metal nanoprobes were prepared by simply decorating metal nanoparticles with capturing molecules (antibody or molecules with surface identification function). Owing to the multifrequency selection of lasers via resonating their plasmonic bands, these metal nanoprobes are highly recognizable under the FWM imaging and display high photostability above fluorescent dyes. This PE-FWM imaging technique shows superior to dark-field imaging due to almost no interference from off-resonant species and exhibits the antifade feature that is suitable for long-period cell monitoring. The automated processing of images is available for the analysis of cell heterogeneity according to the cell surface receptors. Emerging applications such as single-cell analysis, bioimaging, metabolite, and drug tracing offer many biological and medical possibilities with broad application prospects.
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Nanopartículas Metálicas/química , Imagem Óptica/métodos , Análise de Célula Única/métodos , Linhagem Celular , Fator de Crescimento Epidérmico/química , Humanos , Análise de Célula Única/instrumentação , Ressonância de Plasmônio de Superfície/métodosRESUMO
High-efficiency induction of bone marrow mesenchymal stem cells (BMSCs) to osteogenic differentiation in vitro can help solve a series of bone diseases such as bone injury, fracture repair, and osteoporosis. In order to explore the optimal conditions for different chemical inducers to promote BMSCs differentiation and the possible differentiation mechanisms, we developed a smart nanoprobe that can achieve in situ alkaline phosphatase (ALP) activity detection during osteogenic differentiation in cells. The smart nanoprobe (Au@BCIP) was designed as the surface decoration of gold nanoparticles (AuNPs) with 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The nanoprobe was co-cultured with differentiated BMSCs at different stages to monitor ALP activity based on an ALP-catalyzed hydrolysis reaction with BCIP as a substrate. The product can be quickly oxidized by dissolved oxygen to obtain a Raman-active species (5,5'-dibromo-4,4'-dichloro-1H,1H-[2,2'] biindolylidene-3,3'-dione). The SERS sensitivity was greatly improved by resonating the excitation wavelength of 632.8 nm. It is a new strategy for tracing bone disease-related ALP activity in an in vivo model with high sensitivity and selectivity and non-invasion. By using this nanoprobe, osteogenic differentiation of cells under osteogenic supplements was assessed and the p38 MAPK signaling pathway for osteogenic differentiation was experimentally evidenced, which are of significance for understanding BMSCs and regulating their osteogenic differentiation process.
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Nanopartículas Metálicas , Osteogênese , Fosfatase Alcalina , Diferenciação Celular , Ouro , Análise Espectral RamanRESUMO
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
The precise role of Epidermal Growth Factor Receptor (EGFR) in Hepatocellular carcinoma (HCC) cells is unknown and EGFR inhibitors have not achieved positive clinical results. The rapid and drastic internalization of EGFR has been proved to successfully treat EGFR inhibitor-resistant patients in recent clinical trials. Here, the anti-tumor efficacy of a protein (rLZ-8) from Ganoderma lucidum was evaluated, it was demonstrated that rLZ-8 could bind to EGFR specifically, drastically enter into Hepatoma cells, abrogate endosomal recycling and induce HCC cell death. Surprisingly, we screened a monoclonal antibody which possesses competitive binding site with rLZ-8, it also trigger catastrophic EGFR internalization. This result suggests that it is necessary to investigate the interface of EGFR and rLZ-8 complex. An internalization related epitope (S222/K269) was identified on the dimerization arm of EGFR extracellular domain (ECD). These results suggest vulnerability of HCC cells to catastrophic EGFR internalization that can be targeted by a novel epitope and point to the possible exploitation in the design of anti-EGFR therapeutic biologics for HCC therapy.