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Lysosomes are integration hubs for several signaling pathways, such as autophagy and endocytosis, and also crucial stores of ions, including Zn2+. Lysosomal dysfunction caused by changes in their morphology by fusion and fission processes can result in several pathological disorders. However, the role of Zn2+ in modulating the morphology of lysosomes is unclear. The resolution of conventional epifluorescence microscopy restricts accurate observation of morphological changes of subcellular fluorescence punctum. In this study, we used a modified epifluorescence microscopy to identify the center of a punctum from a series of z-stack images and calculate the morphological changes. We stained primary cultured rat embryonic cortical neurons with FluoZin3, a Zn2+-sensitive fluorescent dye, and Lysotracker, a lysosome-specific marker, to visualize the distribution of Zn2+-enriched vesicles and lysosomes, respectively. Our results revealed that treating neurons with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, a cell-permeable Zn2+ chelator, shrank Zn2+-enriched vesicles and lysosomes by up to 25% in an hour. Pretreating the neurons with YM201636, a blocker of lysosome fission, could suppress this shrinkage. These results demonstrate the usefulness of the modified epifluorescence microscopy for investigating the homeostasis of intracellular organelles and related disorders.
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Lisossomos , Neurônios , Animais , Autofagia , Células Cultivadas , Ratos , ZincoRESUMO
We describe a sensitive electrochemical immunoassay for Salmonella enterica serovar Typhimurium, a common foodborne pathogen which can cause infection at extremely small doses. The assay is based on the recognition of DNA biobarcode labels by differential pulse anodic stripping voltammetry (DPASV), following Ag enhancement. The biobarcodes consist of latex spheres (mean diameter 506 nm ± 22 nm) modified by ferromagnetic Fe3O4 particles. Each biobarcode is loaded by adsorption with approx. 27 molecules of mouse monoclonal antibody against S. Typhimurium and 3.5 × 10(5) molecules of 12 mer ssDNA. The assay is performed by adding the biobarcode, S. Typhimurium cells, and biotin-conjugated rabbit polyclonal antibody against Salmonella into well plates. After antigen-antibody binding, magnetic collection enables the excess polyclonal antibody to be washed off. Exposure to avidin-coated screen printed electrodes, and formation of the avidin-biotin bond, then enables the excess biobarcode to be removed. The biobarcode remaining on the electrode is quantified by DPASV measurement of Ag(+) ions following catalytic Ag deposition. The assay showed a negligible response to 10(7) CFU mL(-1)E. coli and had a limit of detection of 12 CFU mL(-1) in buffer, and 13 to 26 CFU mL(-1) for heat-killed and whole cell S. Typhimurium in plain milk, green bean sprouts and raw eggs. To the best of our knowledge, this is the lowest reported limit of detection for Salmonella by an electrochemical immunoassay not requiring sample pre-enrichment.
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DNA/química , Imunoensaio/métodos , Fenômenos Magnéticos , Salmonella typhimurium/isolamento & purificação , Prata/química , Animais , Eletroquímica , CamundongosRESUMO
Integrating the versatility of synthetic nanoparticles to natural biomaterials, such as cells or cell membranes, has gained considerable attention as promising alternative cargo delivery platforms in recent years. Extracellular vesicles (EVs), natural nanomaterials composed of a protein-rich lipid bilayer secreted by cells, have also shown advantages and great potential as a nano delivery platform in combination with synthetic particles due to their specific natural properties in overcoming several biology hurdles possessed in the recipient cell. Therefore, the preservation of EV's origin properties is critical for their application as nanocarriers. This chapter will describe the encapsulation procedure of MSN encapsulated in EV membrane derived from mouse renal adenocarcinoma (Renca) cells through biogenesis. The FMSN-enclosed EVs produced through this approach still contain preserved EV's natural membrane properties.
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Carcinoma de Células Renais , Vesículas Extracelulares , Neoplasias Renais , Nanopartículas , Animais , Camundongos , Carcinoma de Células Renais/metabolismo , Dióxido de Silício/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias Renais/metabolismoRESUMO
In the development of bioinspired nanomaterials for therapeutic applications, it is very important to validate the design of nanomaterials in the disease models. Therefore, it is desirable to visualize the change of the cells in the diseased site at the nanoscale. Heart diseases often start with structural, morphological, and functional alterations of cardiomyocyte components at the subcellular level. Here, we developed straightforward technique for long-term real-time intravital imaging of contracting hearts without the need of cardiac pacing and complex post processing images to understand the subcellular structural and dynamic changes in the myocardial infarction model. A two-photon microscope synchronized with electrocardiogram signals was used for long-term in vivo imaging of a contracting heart with subcellular resolution. We found that the structural and dynamic behaviors of organelles in cardiomyocytes closely correlated with heart function. In the myocardial infarction model, sarcomere shortening decreased from â¼15% (healthy) to â¼8% (diseased) as a result of impaired cardiac function, whereas the distances between sarcomeres increased by 100 nm (from 2.11 to 2.21 µm) in the diastolic state. In addition, T-tubule system regularity analysis revealed that T-tubule structures that were initially highly organized underwent significant remodeling. Morphological remodeling and changes in dynamic activity at the subcellular level are essential to maintain heart function after infarction in a heart disease model.
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The effect of the intracellular pH of macrophages after taking up biodegradable polymer nanoparticles (NPs) on immunomodulating functions has not been explored so far. Previous studies have demonstrated that biodegradable polyurethane (PU) NPs exhibit immunosuppressive activity. Yet, the intracellular mechanism is not clearly understood. In this study, a uniquely designed pH nanosensor is employed for tracking the intracellular pH value of macrophages to reveal the intracellular journey of PU NPs and to clarify the intracellular pH effect on the corresponding inflammatory response. First, fluorescent mesoporous silica nanoparticles (FRMSNs) is used to detect the pH change in macrophages after endo/phagocytosis of PU NPs. Second, PU is coated on the external surface of FRMSNs to examine the intracellular trafficking process of PU in the macrophages. The results show that the majority of PU-coated FRMSNs remain to stay at the cytosol-early endosome/phagosome regions. The intracellular pH value and other supporting results show that the immune response of PU NPs may be correlated to their internalization journey. The retardation in the degradation process of the PU NPs may intervene with the lysosome activity and repress the immunostimulatory effect, which contributes to the low immune response of PU NPs.
Assuntos
Nanopartículas , Poliuretanos , Humanos , Concentração de Íons de Hidrogênio , Inflamação , Macrófagos , FagossomosRESUMO
Mesoporous silica nanoparticles (MSNs) have emerged as a prominent nanomedicine platform, especially for tumor-related nanocarrier systems. However, there is increasing concern about the ability of nanoparticles (NPs) to penetrate solid tumors, resulting in compromised antitumor efficacy. Because the physicochemical properties of NPs play a significant role in their penetration and accumulation in solid tumors, it is essential to systematically study their relationship in a model system. Here, we report a multihierarchical assessment of the accumulation and penetration of fluorescence-labeled MSNs with nine different physicochemical properties in tumor spheroids using two-photon microscopy. Our results indicated that individual physicochemical parameters separately could not define the MSNs' ability to accumulate in a deeper tumor region; their features are entangled. We observed that the MSNs' stability determined their success in reaching the hypoxia region. Moreover, the change in the MSNs' penetration behavior postprotein crowning was associated with both the original properties of NPs and proteins on their surfaces.
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Nanoparticle (NP)-based targeted drug delivery is intended to transport therapeutically active molecules to specific cells and particular intracellular compartments. However, there is limited knowledge regarding the complete route of NPs in this targeting scenario. In this study, simultaneously performing motion and dynamic pH sensing using single-particle tracking (SPT) leads to an alternative method of gaining insights into the mesoporous silica nanoparticle's (MSN) journey in targeting lysosome. Two different pH-sensitive dyes and a reference dye are incorporated into mesoporous silica nanoparticles (MSNs) via co-condensation to broaden the measurable pH range (pH 4-7.5) of the nanoprobe. The phosphonate, amine, and lysosomal sorting peptides (YQRLGC) are conjugated onto the MSN's surface to study intracellular nano-biointeractions of two oppositely charged and lysosome-targetable MSNs. The brightness and stability of these MSNs allow their movement and dynamic pH evolution during their journey to be simultaneously monitored in real time. Importantly, a multidimensional analysis of MSN's movement and local pH has revealed new model intracellular dynamic states and distributions of MSNs, previously inaccessible when using single parameters alone. A key result is that YQRLGC-conjugated MSNs took an alternative route to target lysosomes apart from the traditional one, which sped up to 4 h and enhanced their targeting efficiency (up to 32%). The findings enrich our understanding of the intracellular journey of MSNs. This study offers complementary information on correlating the surface design with the full pathway of nanoparticles to achieve targeted delivery of therapeutic payload.
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Lisossomos/química , Nanopartículas/química , Dióxido de Silício/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Porosidade , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
Rapid and recent progress in fluorescence microscopic techniques has allowed for routine discovery and viewing of biological structures and processes in unprecedented spatiotemporal resolution. In these imaging techniques, fluorescent nanoparticles (NPs) play important roles in the improvement of reporting systems. A short overview of recently developed fluorescent NPs used for advanced in vivo imaging will be discussed in this mini-review. The discussion begins with the contribution of fluorescence imaging in exploring the fate of NPs in biological systems. NP applications for in vivo imaging, including cell labeling, multimodal imaging and theranostic agents, are then discussed. Finally, despite all of the advancements in bioimaging, some unsolved challenges will be briefly discussed concerning future research directions.
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Corantes Fluorescentes/análise , Nanopartículas/análise , Animais , Humanos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Nanomedicina Teranóstica/métodosRESUMO
The unique features of Mesoporous Silica Nanoparticles (MSNs) provide a suitable platform to carry fluorescence dyes for various bioimaging applications. Several strategies have been developed to conjugate a variety of dyes either in the pores or on the surfaces of MSNs to form the fluorescence MSNs (FMSNs). In this chapter, we will discuss recent research progress and future development of FMSNs for living system imaging. We will first describe different strategies for the fabrications of FMSNs. Then, we will discuss the recent developments of cellular and intracellular imaging including self-probe for the interactions of FMSNs with the cells, receptor and organelle labeling, sensing and tracking of biological system, and monitoring the drug delivery and release processes. Moreover, we will include the applications of FMSNs as contrast agents for in vivo imaging. Finally, we will conclude and highlight the challenges and opportunities for MSNs in medical applications.
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Sistemas de Liberação de Medicamentos , Corantes Fluorescentes/administração & dosagem , Imagem Molecular/métodos , Nanopartículas/administração & dosagem , Dióxido de Silício/administração & dosagem , Meios de Contraste/administração & dosagem , Meios de Contraste/química , Corantes Fluorescentes/análise , Humanos , Nanopartículas/química , Porosidade , Dióxido de Silício/químicaRESUMO
The introduction of light sheet fluorescence microscopy (LSFM) has overcome the challenges in conventional optical microscopy. Among the recent breakthroughs in fluorescence microscopy, LSFM had been proven to provide a high three-dimensional spatial resolution, high signal-to-noise ratio, fast imaging acquisition rate, and minuscule levels of phototoxic and photodamage effects. The aforementioned auspicious properties are crucial in the biomedical and clinical research fields, covering a broad range of applications: from the super-resolution imaging of intracellular dynamics in a single cell to the high spatiotemporal resolution imaging of developmental dynamics in an entirely large organism. In this review, we provided a systematic outline of the historical development of LSFM, detailed discussion on the variants and improvements of LSFM, and delineation on the most recent technological advancements of LSFM and its potential applications in single molecule/particle detection, single-molecule super-resolution imaging, imaging intracellular dynamics of a single cell, multicellular imaging: cell-cell and cell-matrix interactions, plant developmental biology, and brain imaging and developmental biology.
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Técnicas Citológicas , Microscopia de Fluorescência , Animais , Células Cultivadas , Humanos , Camundongos , Imagem ÓpticaRESUMO
We have constructed biobarcode labels based on 468nm diameter latex spheres. Modification with polyallylamine and then glutaraldehyde was used to attach a high DNA loading, consisting of aminated probe DNA (approx. 1.01×102 molecules per sphere) and biobarcode DNA (approx. 1.66×104 molecules per sphere). Detection of the biobarcodes was performed by application of a Ag enhancer solution, causing association of the Ag+ ions with the phosphate groups of the DNA. The deposited Ag was detected by differential pulse voltammetry. A 30 mer sequence from the BL21 strain of E. coli was detected with an LOD of 2.6fM (calibration range 10 aM to 0.1pM, r2=0.91, n=45). The LOD was lowered to 0.56aM (calibration range 100zM to 0.1nM, r2=0.991, n=50) by utilizing a sandwich assay with PNA-modified screen printed electrodes, which lowered the Ag background current. The sandwich assay platform was used to calibrate E. coli strain BL2(DE3) with an LOD of 17.0 CFU mL-1 (calibration range 10 to 106 CFU mL-1, r2=0.99, n=33) with good discrimination against Salmonella.
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DNA Bacteriano/análise , DNA Bacteriano/química , Eletroquímica/instrumentação , Látex/química , Ácidos Nucleicos Peptídicos/química , DNA Bacteriano/genética , Eletrodos , Escherichia coli/genética , Hibridização de Ácido Nucleico , ImpressãoRESUMO
We present a novel ratiometric pH sensor design using water-soluble, dual-emission, Mn(2+)-doped quantum dots (Qdots) decorated with D-penicillamine (DPA-MnQdots). In contrast to more commonly used ratiometric pH sensors that rely on the coupling of two fluorophores, our design uses only a single emitter, which simplifies ratiometric sensing and broadens the applications of the sensor. Our single-emitter DPA-MnQdots exhibit two emission bands, at 510nm (green) and 610nm (red), which are, respectively, attributable to exciton recombination and emission of the Mn(2+) dopants. The emission intensity ratio (I510/I610) of the DPA-MnQdots depends linearly on surrounding pH values within physiological conditions (from pH 4.5 to 8.5). Moreover, the biocompatible DPA-MnQdots were used for long-term monitoring of local pH values in HeLa cells.