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European Microscopy Congress 2016: Proceedings || Studying membrane proteins in intact cells using nanoparticle labels and liquid-phase electron microscopy
摘要： Cells have receptor proteins in their plasma membranes ‘listening’ to chemical signals from the outside world. These signals consist of ligands, small molecules that bind specifically to a receptor. But how those signals are interpreted and lead to decisions is incompletely understood mainly on account of limitations of present analytical methods. It is typically extremely difficult to directly see how endogenously expressed individual proteins respond to ligand binding in the intact cell, which can lead, for example, to the formation of protein complexes triggering signaling processes. Much knowledge about cellular function has been obtained via biochemical methods but these analyze pooled material from many thousands of cells and the knowledge is thus based on population averages. But we need to look at the individual cell in order to understand the fundamentals of how a cell interprets a signal. Studying membrane proteins at the nanoscale in intact eukaryotic cells is now possible using liquid-phase scanning transmission electron microscopy (STEM) [1, 2]. The key step is to specifically label the proteins of interest in a one-to-one ratio with small probes combined with nanoparticles, for example, gold nanoparticles or quantum dots. Cells in liquid are then placed in a microfluidic chamber enclosing the sample in the vacuum of the electron microscope, and are imaged with STEM. It is not always necessary to enclose the cells in the microfluidic chamber. For some studies, it is sufficient to obtain information from the thin outer regions of the cells, and those can be imaged with high resolution using environmental scanning electron microscopy (ESEM) with STEM detector [3]. Liquid-phase STEM was used to explore the formation of the epidermal growth factor HER2 at the single-molecule level in intact SKBR3 breast cancer cells in liquid state [4]. HER2 is a membrane protein and plays an important role in breast cancer aggressiveness and progression. Data analysis based on calculating the pair correlation function from individual HER2 positions revealed remarkable differences in functionality between different cellular regions, and between cells with possible relevance for studying cancer metastasis and drug response.
关键词： quantum dots,STEM,ESEM,whole cells,liquid-phase,EGFR,HER2
更新于2025-09-23 15:21:21
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Synthesis and biological evaluation of phthalocyanine-peptide conjugate for EGFR-targeted photodynamic therapy and bioimaging
摘要： To improve the biocompatibility and tumor selectivity, we employed an epidermal growth factor receptor (EGFR) binding peptide (namely GE11, with a sequence of Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile) as a tumor directing vector for the delivery of zinc(II) phthalocyanine for targeted photodynamic therapy and bioimaging. The photophysical properties, cellular uptake, in vitro cytotoxicity, and in vivo biodistribution of this phthalocyanine-peptide conjugate (namely Pc-GE11) have been evaluated. Pc-GE11 exhibited higher cellular uptake on EGFR-overexpressing epidermoid carcinoma A431 cells when compared with that on human breast adenocarcinoma MCF7 cells (low EGFR expression). Moreover, pretreatment of A431 cells with GE11 peptide inhibited the cellular uptake of Pc-GE11 significantly and it exhibited exclusive light-activated cytotoxicity toward A431 cells. Furthermore, Pc-GE11 showed much higher tumor accumulation than the non-targeted control compound containing a random peptide sequence (Tyr-Trp-Gly-Pro-Asn-Ile-His-Tyr-Tyr-Thr-Gln-Val) after intravenous administration in A431-tumor bearing mice, indicating the potential application of this GE11 peptide-conjugated photosensitizer for targeted photodynamic therapy and bioimaging.
关键词： EGFR-targeted,tumor selectivity,phthalocyanine-peptide conjugate,photodynamic therapy,bioimaging
更新于2025-09-23 15:21:21
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Synthesis and analysis of 64Cu-labeled GE11-modified polymeric micellar nanoparticles for EGFR-targeted molecular imaging in a colorectal cancer model
摘要： Polymeric micellar nanoparticles represent versatile and biocompatible platforms for targeted drug delivery. However, tracking their biodistribution, stability, and clearance profile in vivo is challenging. The goal of this study was to prepare surface-modified micelles with peptide GE11 for targeting the epidermal growth factor receptor (EGFR). In vitro fluorescence studies demonstrated significantly higher internalization of GE11-micelles into EGFR-expressing HCT116 colon cancer cells versus EGFR-negative SW620 cells. Azo coupling chemistry of tyrosine residues in the peptide backbone with aryl diazonium salts was used to label the micelles with radionuclide 64Cu for positron emission tomography (PET) imaging. In vivo analysis of 64Cu-labeled micelles showed prolonged blood circulation and predominant hepatobiliary clearance. The biodistribution profile of EGFR-targeting GE11-micelles was compared with non-targeting HW12-micelles in HCT116 tumor-bearing mice. PET revealed increasing tumor-to-muscle ratios for both micelles over 48 h. Accumulation of GE11-containing micelles in HCT116 tumors was higher compared to HW12-decorated micelles. Our data suggest that the efficacy of image-guided therapies with micellar nanoparticles could be enhanced by active targeting, as demonstrated with cancer biomarker EGFR.
关键词： polymeric micelles,epidermal growth factor receptor (EGFR),64Cu,GE11 peptide,Colorectal cancer,positron emission tomography (PET)
更新于2025-09-23 15:21:01
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Real-time molecular optical micro-imaging of EGFR mutations using a fluorescent erlotinib based tracer
摘要： Background: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM). Methods: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 μM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 μM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment. Results: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours. Conclusion: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.
关键词： Lung cancer,Erlotinib,Theranostic,EGFR,Epidermal growth factor,Fibred confocal fluorescence microscopy,Molecular imaging
更新于2025-09-19 17:15:36
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Fluorescein- and EGFR-Antibody Conjugated Silica Nanoparticles for Enhancement of Real-time Tumor Border Definition Using Confocal Laser Endomicroscopy in Squamous Cell Carcinoma of the Head and Neck
摘要： Intraoperative de?nition of tumor free resection margins in head and neck cancer is challenging. In the current proof-of-principle study we evaluated a novel silica nanoparticle-based agent for its potential use as contrast enhancer. We synthesized silica nanoparticles with an average size of 45 nm and modi?ed these particles with the ?uorescence stain ?uorescein isocyanate (FITC) for particle detection and with epidermal growth factor receptor (EGFR)-targeting antibodies for enhanced tumor speci?city. The nanoparticles exhibited good biocompatibility and could be detected in vitro and in vivo by confocal laser scanning microscopy. Additionally, we show in an ex vivo setting that these modi?ed nanoparticles speci?cally bind to tumor samples and could be detected using a handheld confocal ?uorescence endomicroscope. From a clinical point of view, we believe that this method could be used for tumor border contrast enhancement and for better intraoperative de?nition of R-0 tumor resection.
关键词： silica nanoparticles,EGFR,contrast agent
更新于2025-09-19 17:13:59
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Bacterial phytochrome as a scaffold for engineering of receptor tyrosine kinases controlled with near-infrared light
摘要： Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt and PLC-gamma signaling. Absence of spectral crosstalk between the opto-RTKs and GFP-based biosensors enable simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.
关键词： FGFR,BphP1,EGFR,DrBphP,bacteriophytochrome,Dr-RTK
更新于2025-09-19 17:13:59
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Bypassing pro-survival and resistance mechanisms of autophagy in EGFR-positive lung cancer cells by targeted delivery of 5FU using theranostic Ag <sub/>2</sub> S quantum dots
摘要： Targeted drug delivery systems that combine imaging and therapeutic functions in a single structure have become very popular in nanomedicine. Near-infrared (NIR) emitting Ag2S quantum dots (QDs) are excellent candidates for this task. Here, we have developed PEGylated Ag2S QDs functionalized with Cetuximab (Cet) antibody and loaded with an anticancer drug, 5-fluorouracil (5FU). These theranostic QDs were used for targeted NIR imaging and treatment of lung cancer using low (H1299) and high (A549) Epidermal Growth Factor Receptor (EGFR) overexpressing cell lines. The Cet conjugated QDs effectively and selectively delivered 5FU to A549 cells and provided significantly enhanced cell death associated with apoptosis. Interestingly, while treatment of cells with free 5FU activated autophagy, a cellular mechanism conferring resistance to cell death, these EGFR targeting multimodal QDs significantly overcame drug resistance compared to 5FU treatment alone. The improved therapeutic outcome of 5FU delivered to A549 cells by Cet conjugated Ag2S QDs is suggested as the synergistic outcome of enhanced receptor mediated uptake of nanoparticles, and hence the drug, coupled with suppressed autophagy even in the absence of addition of an autophagy suppressor.
关键词： lung cancer,EGFR,theranostic,Ag2S quantum dots,autophagy,targeted drug delivery,5-fluorouracil
更新于2025-09-16 10:30:52
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Cancer signaling by plasmonic quantum probes
摘要： Although the use of gold quantum dots for non-cellular sensing is growing rapidly, the use of quantum gold dots are still limited because of comparatively large overall size (biocompatible layer), overlapped Raman readout of lower concentrated metabolites inside the cells and dispersion of QDs inside the cancer cells. This is due to the labelling of QDs which results in specific binding and non-uniform distribution of QDs inside the cells. Besides this, QDs are toxic in nature due to the presence of chemical residues from the wet synthesis, hampering its rendition to clinical application. To increase biocompatibility and surge the cellular uptake, gold probes are usually layered with bio-films (PEG). However, layering impedes the interaction of metabolites and gold QDs and damp the surface plasmonic resonance, resulting in low Raman signal. The addition of biofilm increases the overall hydrodynamic size of gold QDs, limiting the dispersion and internalization of plasmonic QDs inside the cell as well as the nucleus. Therefore, the unique properties that arise from the quantum size of the plasmonic probe are lost. To retain the quantum size of QDs, there is a need to use a physical method of synthesis and use gold QDs layering free. In this research manuscript, we introduce the quantum sized layering free Plasmonic Quantum Probes (PQPs), a potential tool for cancer sensing which provides the opportunity to attain the sensing of surface oncoprotein, intracellular oncoprotein and nuclear metabolites simultaneously. In this work, we introduce the concept of Plasmonic Quantum Probes (PQPs) for intercellular sensing. To the best of our knowledge, it is the first time the probe close to the size of cellular metabolites was synthesized. By using physical synthesis (multiphoton laser ionization), biocompatible PQPs were synthesized without the chemical adulteration and eliminated the need of biocompatible layering. Since the PQPs are non-toxic in nature, no layering is required, enabling the study of true quantum effect in plasmonic cellular signaling. The naked PQPs demonstrated self-cellular uptake with even dispersion and non-specific attachment to all the cell components. As a result, the Raman spectra revealed rich information of multiple cell components, including surface (e.g., EGFR), intracellular (e.g., HPV E6/HPV E7) and nuclear metabolites (e.g., DNA/RNA), simultaneously in one single Raman profile. Due to the difference in the number of probes inside the cells, the signal strength of cancerous cell is significantly higher than that of fibroblast cells, implying that the differentiation of cancerous cells is possible through the analysis of the intensity of Raman readout. The probe size is found to be inversely related to the signal strength of the plasmonic readout. The PQP provides a holistic picture of cell states and may open up new possibilities for accurate diagnosis of cancer.
关键词： Plasmonic signaling,EGFR,Sensing,HPV E6/HPV E7,Quantum probes
更新于2025-09-16 10:30:52
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Evaluation of Light-Emitting Diodes’ Effects on the Expression Level of P53 and EGFR in the Gingival Tissues of Albino Rats
摘要： Background and objectives: The light-curing unit is considered an essential piece of equipment in every dental office. This study was conducted to evaluate the effect of Light-Emitting Diodes (LEDs) by the light cure (LC) device on gingival tissues of albino rats histologically and by regarding the expression of P53 and epidermal growth factor receptor (EGFR). Materials and methods: Gingival tissues of the rats were exposed to LEDs for 30 s with an interval of 30 s for periods of 2 and 5 min and were examined after two and four weeks of light exposure. After the set time, histological sections were studied and the P53 and EGFR expressions were evaluated immunohistochemically and by molecular methods. Results: Mild hyperplasia and mild inflammatory response were detected in higher rates after two weeks of exposure when compared to 4 weeks postexposure. Whereas fibrosis was found at a higher rate after four weeks than that found after two weeks postexposure, parakeratosis was seen only in the group that was exposed for 5 min to LC and when biopsies were taken after 2 weeks. We found that the immunohistochemical expression of P53 was not changed. Similarly, the alteration of EGFR expression was statistically nonsignificant (p > 0.05) when compared to the control group. The data obtained from the qRT-PCR reaction was analyzed using the comparative CT (2???CT) method. Statistically, there was no significant difference in the expression of EGER and P53 gene transcripts. Conclusions: LED causes no serious alteration in P53 and EGFR expression, and only trivial histopathological changes occurred, most of which recovered after a 4-week interval.
关键词： P53,qRT-PCR,EGFR,dental curing lights,blue light hazard
更新于2025-09-12 10:27:22
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Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore
摘要： Purpose: Imaging techniques for highly specific detection of cancer cells in vivo can have applications ranging from preclinical drug discovery studies to clinical cancer diagnosis and surgical therapy. Although fluorescence imaging using cancer-targeted antibodies has shown promise, nonspecific probe accumulation in tissue results in significant background fluorescence, reducing detection sensitivity using traditional intensity–based continuous-wave (CW) fluorescence imaging. Here we demonstrate that fluorescence lifetime (FLT) imaging can provide significant tumor contrast enhancement over CW intensity in preclinical models of human breast cancer. Experimental Design: Mice bearing MDA-MB-231 tumors were injected with anti-EGFR antibody conjugated to the fluorescent dye IRDye 800CW (anti-EGFR-800). Time domain fluorescence imaging was performed in vivo and in situ up to 48 hours after dye injection. Results: Mice injected with anti-EGFR-800 showed a significantly longer FLT (0.7 ± 0.03 ns) compared with the FLT of nonspecific probe uptake in liver (0.63 ± 0.05 ns), providing a dramatic improvement in sensitivity and specificity compared with CW intensity. IgG antibody–conjugated IRDye 800CW did not show an increased FLT compared with normal tissue, suggesting that the FLT increase of anti-EGFR-800 in tumors was associated with receptor expression. Using serial surgery, we show that FLT allows the detection of smaller residual tumors in the surgical bed than possible using CW intensity. Conclusions: Our data suggest that FLT can significantly enhance tumor contrast using fluorescently labeled antibodies, thereby accelerating the efficient clinical application of these probes for margin assessment in image-guided surgery and for highly specific detection of tumor receptors in vivo.
关键词： near-infrared fluorophore,EGFR,fluorescence lifetime imaging,image-guided surgery,tumor contrast
更新于2025-09-11 14:15:04