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Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.
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Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas Cullin/metabolismo , Células Madre Embrionarias/fisiología , Colorantes Fluorescentes/metabolismo , Proteínas Luminiscentes/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cullin/genética , Células Madre Embrionarias/citología , Genes Reporteros , Células HeLa , Humanos , Proteínas Luminiscentes/genética , RatonesRESUMEN
Nanomedicine is promising to improve conventional cancer medicine by making diagnosis and therapy more accurate and more effective in a more personalized manner. A key of the cancer nanomedicine is construction of medical nanodevices by programming various requisite functions to nanoparticles (NPs). As compared to that of soft NPs, including organic micelles and polymers, fabrication of an inorganic NP based nanodevice is still challenging; the approved nanoformulations have been confined to the limited number of superparamagnetic iron oxide NPs (SPIONs). The major challenges lie in how to program the requisite functions to inorganic NPs. In spite the much denser and less hydrophilic properties of inorganic NPs, most of the following functions have to be programmed for their in vivo applications: (A) high dispersibility in a physiological environment, (B) high stealth efficiency to slip through the trap by liver and spleen, (C) high targeting efficiency to cancer tissue, (D) clear visualization of cancer for diagnosis, and (E) high anticancer activity for treatment.In our approach, poly(glycerol) (PG), containing a hydroxy group at every monomer unit, was found as a better alternative to poly(ethylene glycol) (PEG), the most commonly used hydrophilic polymer, giving (A) high dispersibility to inorganic NPs. Although most of the inorganic NPs are not dense in functional groups, the hyperbranched structure with many hydroxy groups in PG turns the less functional surface into highly functional one, imparting not only good hydrophilicity but also (B) high stealth efficiency as we reported recently. In addition, a number of hydroxy groups in PG afford the structural or functional extensibility to introduce the additional layer or function. This enables us to design and construct a three-layer architecture consisting of a core inorganic NP, a hydrophilic and stealthy PG layer, and a functional molecule layer, where their interfaces are connected firmly by covalent bonds. The three-layered nanodevice is very flexible in its design for the following reasons: The PG coating can be applied to a wide variety of inorganic NPs with various functions, and various functional moieties can be introduced on the PG layer as a functional molecule layer. Owing to the versatility of the three-layer model, the rest of the above functions (C)-(E) can be programed in the NP core and/or the outmost layer in nanodevices.In this Account, the author described first the methodology for precise construction and quantitative characterization of various biomedical nanodevices. This fundamental aspect of this research has been achieved by "applying organic chemistry to nanomaterials" which is the concept of our research. That is, the rich chemistry in synthesis and characterization of organic compounds has been applied to the nanodevice fabrication and characterization. Second, evaluation of the functions programmed in the nanodevices is described in terms of stealth and targeting efficiencies, cancer diagnosis and therapy, and biomedical sensing. This stage in our research made us more interdisciplinary from chemistry and nanoscience to biology and medicine. The following research spiral has been established in our group to strongly promote the improvement of our biomedical nanodevices; nanodevice design â precise construction â quantitative characterization â functional evaluation.
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Nanopartículas , Neoplasias , Humanos , Nanomedicina , Glicerol , Nanopartículas/uso terapéutico , Nanopartículas/química , Polímeros/química , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológicoRESUMEN
We have been working with carbon nanotube separation through host-guest chemistry. Herein, a new macrocyclic host molecule, Cu-tethered square nanobrackets, is designed, synthesized and applied to single-walled carbon nanotubes (SWNTs) for their diameter-based separation. The complexation between copper ions and dipyrrin moieties of the nanobracket gives Cu-tethered square nanobrackets, which is confirmed by absorption, Raman and MALDI-TOF mass spectra. Upon extraction of SWNTs with the nanobracket and copper(II), in situ-formed square Cu-nanobrackets are found to interlock SWNTs to disperse them in 2-propanol. The interlocking is confirmed by Raman spectroscopy after thorough washing of the extracted SWNTs. Pristine SWNTs were recovered through demetalation of the interlocked ones along with the nanobracket. Raman and absorption spectroscopies of the extracted SWNTs reveals the diameter enrichment of only several kinds of SWNTs in the diameter range from 0.94 to 1.10 nm among ≈20 kinds of SWNTs from 0.76 to 1.20 nm in their diameter range. The diameter selectivity is supported by the theoretical calculations with the GFN2-xTB method, indicating that the most preferred SWNT diameter for the square Cu-nanobrackets is 1.04 nm.
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Boron neutron capture therapy (BNCT), advanced cancer treatment utilizing nuclear fission of 10 B atom in cancer cells, is attracting increasing attention. As 10 B delivery agent, sodium borocaptate (10 BSH, 10 B12 H11 SH â 2Na), has been used in clinical studies along with L-boronophenylalanine. Recently, this boron cluster has been conjugated with lipids, polymers or nanoparticles to increase selectivity to and retentivity in tumor. In this work, anticancer nanoformulations for BNCT are designed, consisting of poly(glycerol) functionalized detonation nanodiamonds (DND-PG) as a hydrophilic nanocarrier, the boron cluster moiety (10 B12 H11 2- ) as a dense boron-10 source, and phenylboronic acid or RGD peptide as an active targeting moiety. Some hydroxy groups in PG were oxidized to carboxy groups (DND-PG-COOH) to conjugate the active targeting moiety. Some hydroxy groups in DND-PG-COOH were then transformed to azide to conjugate 10 B12 H11 2- through click chemistry. The nanodrugs were evaluated inâ vitro using B16 murine melanoma cells in terms of cell viability, BNCT efficacy and cellular uptake. As a result, the 10 B12 H11 2- moiety is found to facilitate cellular uptake probably due to its negative charge. Upon thermal neutron irradiation, the nanodrugs with 10 B12 H11 2- moiety exhibited good anticancer efficacies with slight differences with and without targeting moiety.
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Terapia por Captura de Neutrón de Boro , Nanodiamantes , Neoplasias , Ratones , Animales , Boro , Glicerol , Compuestos de BoroRESUMEN
Boron neutron capture therapy (BNCT) is a non-invasive cancer treatment with little adverse effect utilizing nuclear fission of 10 B upon neutron irradiation. While neutron source has been developed from a nuclear reactor to a compact accelerator, only two kinds of drugs, boronophenylalanine and sodium borocaptate, have been clinically used for decades despite their low tumor specificity and/or retentivity. To overcome these challenges, various boron-containing nanomaterials, or "nanosensitizers", have been designed based on micelles, (bio)polymers and inorganic nanoparticles. Among them, inorganic nanoparticles such as boron carbide can include a much higher 10 B content, but successful in vivo applications are very limited. Additionally, recent reports on the photothermal effect of boron carbide are motivating for the addition of another modality of photothermal therapy. In this study, 10 B enriched boron carbide (10 B4 C) nanoparticle is functionalized with polyglycerol (PG), giving 10 B4 C-PG with enough dispersibility in a physiological environment. Pharmacokinetic experiments show that 10 B4 C-PG fulfills the following three requirements for BNCT; 1) low intrinsic toxicity, 2) 10 B in tumor/tumor tissue (wt/wt) ≥ 20 ppm, and 3) 10 B concentrations in tumor/blood ≥ 3. In vivo study reveals that neutron irradiation after intravenous administration of 10 B4 C-PG suppresses cancer growth significantly and eradicates cancer with the help of near-infrared light irradiation.
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Terapia por Captura de Neutrón de Boro , Nanopartículas , Neoplasias , Boro/farmacología , Compuestos de Boro/farmacología , Glicerol , Humanos , Neoplasias/tratamiento farmacológico , Neutrones , Terapia Fototérmica , PolímerosRESUMEN
Functionalization of nanoparticles (NPs) with targeting moieties has a high potential to advance precision nanomedicine. However, the targeting moieties on a NP surface are known to be masked by a protein corona in biofluids, lowering the targeting efficiency. Although it has been demonstrated at the cellular level, little is known about the influence of the protein corona on the subcellular targeting. Herein, we adopted triphenylphosphonium (TPP) as a mitochondrial targeting moiety and investigated the effects of protein coronas from fetal bovine serum and human plasma on its targeting ability and cytotoxicity. Specifically, we introduced TPP in low (l) and high (h) densities on the surface of nanodiamond (ND) functionalized with polyglycerol (PG). Despite the "corona-free" PG interface, we found that the TPP moiety attracted proteins to form a corona layer with clear linearity between the TPP density and the protein amount. By performing investigations on human cervix epithelium (HeLa) and human lung epithelial carcinoma (A549) cells, we further demonstrated that (1) the protein corona alleviated the cytotoxicity of both ND-PG-TPP-l and -h, (2) a smaller amount of proteins on the surface of ND-PG-TPP-l did not affect its mitochondrial targeting ability, and (3) a larger amount of proteins on the surface of ND-PG-TPP-h diminished its targeting specificity by restricting the NDs inside the endosome and lysosome compartments. Our findings will provide in-depth insights into the design of NPs with active targeting moiety for more precise and safer delivery at the subcellular level.
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Glicerol/química , Mitocondrias/efectos de los fármacos , Nanodiamantes/química , Neoplasias/tratamiento farmacológico , Compuestos Organofosforados/administración & dosificación , Polietilenglicoles/química , Polímeros/química , Corona de Proteínas/química , Células A549 , Proliferación Celular , Portadores de Fármacos/química , Células HeLa , Humanos , Mitocondrias/metabolismo , Neoplasias/patología , Compuestos Organofosforados/químicaRESUMEN
The electronic and magnetic structures of diamond nanoparticles with a hydrogenated surface are investigated as a function of annealing temperature under vacuum annealing up to 800-1000 °C. Near edge X-ray absorption fine structure (NEXAFS) spectra together with elemental analysis show successive creation of defect-induced nonbonding surface states at the expense of surface-hydrogen atoms as the annealing temperature is increased above 800 °C. Magnetization and ESR spectra confirm the increase in the concentration of localized spins assigned to the nonbonding surface states upon the increase of the annealing temperature. Around 800 °C, surface defects collectively created upon the annealing result in the formation of graphene nano-islands which possess magnetic nonbonding edge states of π-electron origin. Interestingly, extremely slow spin relaxation is observed in the magnetization of the edge state spins at low temperatures. The relaxation time is well explained in terms of a lognormal distribution of magnetic anisotropy energies instead of the classical Néel relaxation mechanism with a unique magnetic anisotropy energy, in addition to the contribution of the quantum mechanical tunnelling mechanism. The spin-orbit interaction enhanced by the electrostatic potential gradient created at the interface between the core diamond particle and surface graphene nano-islands is responsible for the slow spin relaxation.
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The extracellular signal-regulated kinase (ERK) plays a central role in the signaling cascades of cell growth. Here, we show that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner. A fluorescence resonance energy transfer (FRET) biosensor revealed that stochastic ERK activity pulses fired spontaneously or propagated from adjacent cells. Frequency, but not amplitude, of ERK activity pulses exhibited a bell-shaped response to the cell density and correlated with cell proliferation rates. Consistently, synthetic ERK activity pulses generated by a light-switchable CRaf protein accelerated cell proliferation. A mathematical model clarified that 80% and 20% of ERK activity pulses are generated by the noise and cell-to-cell propagation, respectively. Finally, RNA sequencing analysis of cells subjected to the synthetic ERK activity pulses suggested the involvement of serum responsive factor (SRF) transcription factors in the gene expression driven by the ERK activity pulses.
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Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Algoritmos , Animales , Benzamidas/farmacología , Comunicación Celular , Recuento de Células , Línea Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Regulación de la Expresión Génica , Humanos , Imidazoles/farmacología , Cinética , Modelos Biológicos , Regiones Promotoras Genéticas , Ratas , Análisis de Secuencia de ARN , Procesos Estocásticos , Imagen de Lapso de Tiempo , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismoRESUMEN
BACKGROUND: Tumor-associated macrophages (TAMs) are the most abundant stromal cells in the tumor microenvironment. Turning the TAMs against their host tumor cells is an intriguing therapeutic strategy particularly attractive for patients with immunologically "cold" tumors. This concept was mechanistically demonstrated on in vitro human and murine lung cancer cells and their corresponding TAM models through combinatorial use of nanodiamond-doxorubicin conjugates (Nano-DOX) and a PD-L1 blocking agent BMS-1. Nano-DOX are an agent previously proved to be able to stimulate tumor cells' immunogenicity and thereby reactivate the TAMs into the anti-tumor M1 phenotype. RESULTS: Nano-DOX were first shown to stimulate the tumor cells and the TAMs to release the cytokine HMGB1 which, regardless of its source, acted through the RAGE/NF-κB pathway to induce PD-L1 in the tumor cells and PD-L1/PD-1 in the TAMs. Interestingly, Nano-DOX also induced NF-κB-dependent RAGE expression in the tumor cells and thus reinforced HMGB1's action thereon. Then, BMS-1 was shown to enhance Nano-DOX-stimulated M1-type activation of TAMs both by blocking Nano-DOX-induced PD-L1 in the TAMs and by blocking tumor cell PD-L1 ligation with TAM PD-1. The TAMs with enhanced M1-type repolarization both killed the tumor cells and suppressed their growth. BMS-1 could also potentiate Nano-DOX's action to suppress tumor cell growth via blocking of Nano-DOX-induced PD-L1 therein. Finally, Nano-DOX and BMS-1 achieved synergistic therapeutic efficacy against in vivo tumor grafts in a TAM-dependent manner. CONCLUSIONS: PD-L1/PD-1 upregulation mediated by autocrine and paracrine activation of the HMGB1/RAGE/NF-κB signaling is a key response of lung cancer cells and their TAMs to stress, which can be induced by Nano-DOX. Blockade of Nano-DOX-induced PD-L1, both in the cancer cells and the TAMs, achieves enhanced activation of TAM-mediated anti-tumor response.
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Antígeno B7-H1/efectos de los fármacos , Doxorrubicina/farmacología , Inhibidores de Puntos de Control Inmunológico/farmacología , Nanodiamantes/química , Macrófagos Asociados a Tumores , Células A549 , Animales , Antígeno B7-H1/genética , Línea Celular Tumoral , Citocinas/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Unique properties and potential applications of 2D materials draw much attention for mass production of thin-layer 2D materials. Ball milling exfoliation of 2D materials has been rarely used, in spite of a promising dry phase production method, because of the superficial information in the mechanism and the effect of the operating parameters on the yield, size and thickness. Here, we investigate systematically the ball milling operating parameters in the exfoliation of bulk MoS2 in the presence of sodium cholate (SC) as an exfoliant. The yield and dimensions of the exfoliated MoS2 nanosheet were monitored by changing the parameters such as the weight ratio of bulk MoS2 and SC (SC/MoS2), the filling ratio in the volume of milling ball and container (φ), milling ball size (d B), milling revolution speed (n R ), and initial mass of bulk MoS2 ([Formula: see text]). The yield of exfoliation is found to be 95% at the optimum ball milling conditions (SC/MoS2 = 0.75, φ = 50%, [Formula: see text] = 0.20 g). In addition, yield and size of the exfoliated MoS2 were controlled by the conditions of the ball milling. As for the evaluation of the exfoliated MoS2, we developed a novel method by use of the XRD profile to determine the size and thickness of the ball-milled MoS2 powder with less than 30% difference from those determined by the well-known absorption method. Finally, the size and thickness of the MoS2 nanosheets prepared by ball milling exfoliation were correlated with their electrocatalytic and photoelectrocatalytic activities.
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Preferential accumulation of nanoparticles in a tumor is realized commonly by combined effects of active and passive targeting. However, passive targeting based on an enhanced permeation and retention (EPR) effect is not sufficient to observe clear tumor fluorescence images in most of the in vivo experiments using tumor-bearing mice. Herein, polyglycerol-functionalized nanodiamonds (ND-PG) conjugated with cyanine dye (Cy7) are synthesized and it is found that the resulting ND-PG-Cy7 is preferentially accumulated in the tumor, giving clear fluorescence in in vivo and ex vivo fluorescence images. One of the plausible reasons is the longer in vivo blood circulation time of ND-PG-Cy7 (half-life: 58 h determined by the pharmacokinetic analysis) than that of other nanoparticles (half-life: <20 h in most of the previous reports). In a typical example, the fluorescence intensity of tumors increases due to continuous tumor accumulation of ND-PG-Cy7, even more than one week postinjection. This may be owing to the stealth effect of PG that was reported previously, avoiding recognition and excretion by reticuloendothelial cells, which are abundant in liver and spleen. In fact, the fluorescence intensities from the liver and spleen is similar to those from other organs, while the tumor exhibits much stronger fluorescence in the ex vivo image.
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Benzotiazoles/química , Carbocianinas/química , Glicerol/química , Rayos Infrarrojos , Nanodiamantes/química , Neoplasias/diagnóstico por imagen , Polímeros/química , Animales , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Hidrodinámica , Ratones Endogámicos BALB C , Ratones Desnudos , Nanodiamantes/ultraestructura , Imagen Óptica , Electricidad Estática , Factores de TiempoRESUMEN
Metal-free magnetic mixed micelles (mean diameter: 16 nm) composed of biocompatible surfactant Tween 80 and hydrophobic pyrrolidine-N-oxyl radical were prepared by mixing them in phosphate-buffered saline. The magnetic mixed micelles were characterized by dynamic light scattering and small angle neutron scattering measurements. The stability of the micelles is found to depend on the length of alkyl side chain in the nitroxide compounds and degree of unsaturation in the hydrophobic chain in the surfactant. The size of the mixed micelle can be tuned by changing the molar ratio of Tween 80 and nitroxyl radical. In view of theranostic application of the micelle, the cytotoxicity and stability in a physiological environment was investigated; the mixed micelle exhibited no cytotoxicity, high colloidal stability and high resistance towards reduction by large excess ascorbic acid. The in vitro and in vivo magnetic resonance imaging (MRI) revealed sufficient contrast enhancement in the proton longitudinal relaxation time (T 1) weighted images. In addition, hydrophobic fluorophores and an anticancer drug are stably encapsulated in the mixed micelles and showed fluorescence (FL) upon reduction by ascorbic acid and cytotoxicity to cancer cells, respectively. For example, the paclitaxel-loaded mixed micelles efficiently suppressed cancer cell growth. Furthermore, they were found to give higher MRI contrast (higher r 1 value) in vitro than the micelles without paclitaxel. The magnetic mixed micelles presented here are promising theranostic agents in nanomedicine due to their high biocompatibility and high resistivity towards reduction as well as functioning as a drug carrier in therapy and MR or FL imaging probe in diagnosis.
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BACKGROUND: Triple negative breast cancer (TNBC) has the poorest prognosis of all breast cancer subtypes and is one of the most fatal diseases for women. Combining cytotoxic chemotherapy with immunotherapy has shown great promise for TNBC treatment. However, chemotherapy often leads to the development of chemoresistance and severe systemic toxicity compromising the immune functions that are crucial to anti-TNBC immune therapy. Tumor-induced immunosuppression also poses a great hindrance to efficacious anti-TNBC immunotherapy. Nanomedicine holds great promise to overcome these hurdles. RESULTS: Doxorubicin-polyglycerol-nanodiamond conjugate (Nano-DOX) was firstly found to be a cytostatic agent to the 4T1 cells and displayed a lower apparent therapeutic potency than DOX. However, the tumor-bearing animals, particularly some key immune cells thereof, showed good tolerance of Nano-DOX as opposed to the severe toxicity of DOX. Next, Nano-DOX did not induce significant upregulation of P-gp and IL-6, which were demonstrated to be key mediators of chemoresistance to DOX in the 4T1 cells. Then, Nano-DOX was shown to downregulate tumor-derived granulocyte-colony stimulating factor (G-CSF) and suppresses the induction and tissue filtration of myeloid-derived suppressor cells (MDSCs) that are the principal effectors of cancer-associated systemic immunosuppression. Nano-DOX also alleviated the phenotype of MDSCs induced by 4T1 cells. Finally, Nano-DOX induced the 4T1 cells to emit damage associated molecular patterns (DAMPs) that stimulated the tumor immune microenvironment through activating key immune effector cells involved in anti-tumor immunity, such as macrophages, dendritic cells and lymphocytes in the tumor tissue. CONCLUSIONS: Nano-DOX is a cytostatic agent with good host tolerance which is capable of evading chemoresistance and reversing cancer-induced immunosuppression both at the systemic level and in the tumor microenvironment in TNBC. Our work presents Nano-DOX as an interesting example that a chemotherapeutic agent in nano-form may possess distinct biochemical properties from its free form, which can be exploited to join chemotherapy with immunotherapy for better treatment of cancer.
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Antineoplásicos/administración & dosificación , Citostáticos/administración & dosificación , Doxorrubicina/administración & dosificación , Glicerol/química , Nanoconjugados/química , Polímeros/química , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Citostáticos/uso terapéutico , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Ratones Endogámicos BALB C , Nanodiamantes/química , Neoplasias de la Mama Triple Negativas/inmunología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Nanodiamond(ND)-based technologies are flourishing in a wide variety of fields spanning from electronics and optics to biomedicine. NDs are considered a family of nanomaterials with an sp3 carbon core and a variety of sizes, shapes, and surfaces. They show interesting physicochemical properties such as hardness, stiffness, and chemical stability. Additionally, they can undergo ad-hoc core and surface functionalization, which tailors them for the desired applications. Noteworthy, the properties of NDs and their surface chemistry are highly dependent on the synthetic method used to prepare them. In this Minireview, we describe the preparation of NDs from the materials-chemistry viewpoint. The different methodologies of synthesis, purification, and surface functionalization as well as biomedical applications are critically discussed. New synthetic approaches as well as limits and obstacles of NDs are presented and analyzed.
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Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.
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Técnicas Biosensibles , Quinasas Asociadas a rho/metabolismo , Secuencia de Aminoácidos , Western Blotting , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Microscopía Fluorescente , Fosforilación , Plásmidos/genética , Plásmidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especificidad por Sustrato , Imagen de Lapso de Tiempo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
With a view to developing a theranostic nanomedicine for targeted drug delivery systems visible by magnetic resonance (MR) imaging, robust metal-free magnetic nanoemulsions (mean particle size less than 20â nm) consisting of a biocompatible surfactant and hydrophobic, low molecular weight 2,2,5-trimethyl-5-(4-alkoxy)phenylpyrrolidine-N-oxyl radicals were prepared in pHâ 7.4 phosphate-buffered saline (PBS). The structure of the nanoemulsions was characterized by electron paramagnetic resonance spectroscopy, and dynamic light scattering and small-angle neutron-scattering measurements. The nanoemulsions showed high colloidal stability, low cytotoxicity, enough reduction resistance to excess ascorbic acid, and sufficient contrast enhancement in the proton longitudinal relaxation time (T1 ) weighted MR images in PBS in vitro (and preliminarily in vivo). Furthermore, the hydrophobic anticancer drug paclitaxel could be encapsulated inside the nanoparticles, and the resulting paclitaxel-loaded nanoemulsions were efficiently incorporated into HeLa cells to suppress cell growth.
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Antineoplásicos Fitogénicos/química , Portadores de Fármacos/química , Nanopartículas de Magnetita/química , Óxidos de Nitrógeno/química , Paclitaxel/química , Animales , Antineoplásicos Fitogénicos/toxicidad , Ácido Ascórbico/química , Encéfalo/diagnóstico por imagen , Proliferación Celular/efectos de los fármacos , Dispersión Dinámica de Luz , Espectroscopía de Resonancia por Spin del Electrón , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Imagen por Resonancia Magnética , Ratones , Peso Molecular , Difracción de Neutrones , Oxidación-Reducción , Paclitaxel/toxicidad , Tamaño de la Partícula , Dispersión del Ángulo PequeñoRESUMEN
Thin-layer 2D materials have been attracting enormous interest, and various processes have been investigated to obtain these materials efficiently. In view of their practical applications, the most desirable source for the preparation of these thin-layer materials is the pristine bulk materials with stacked layers, such as pristine graphite. There are many options in terms of conditions for the exfoliation of thin-layer materials, and these include wet and dry processes, with or without additives, and the kind of solvent. In this context, we found that the versatile exfoliant hexahydroxytriphenylene works efficiently for the exfoliation of typical 2D materials such as graphene, MoS2 , and hexagonal boron nitride (h-BN) by both wet and dry processes by using sonication and ball milling, respectively, in aqueous and organic solvents. As for graphene, stable dispersions with relatively high concentrations (up to 0.28â mg mL(-1) ) in water and tetrahydrofuran were obtained from graphite in the presence of hexahydroxytriphenylene by a wet process with the use of bath sonication and by a dry process involving ball milling. Especially, most of the graphite was exfoliated and dispersed as thin-layer graphene in both aqueous and organic solvents through ball milling, even on a large scale (47-86 % yield). In addition, the exfoliant was easily removed from the precipitated composite by heat treatment without disturbing the graphene structure. Bulk MoS2 and h-BN were also exfoliated by both wet and dry processes. Similar to graphene, dispersions of MoS2 and h-BN of high concentrations in water and DMF were produced in high yields through ball milling.
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A nanodiamond-polyglycerol-gadolinium(ll) conjugate has been designed and prepared as novel nanodiamond-based magnetic resonance (MR) contrast agent dispersible in physiological media. Detonation nanodiamond (dND) was first grafted with polyglycerol (PG) through ring-opening polymerization of glycidol to impart dispersibility to dND in physiological media. Since the hydroxyl group in PG serves as a scaffold for further surface functionalization, diethylenetriaminepentaacetic acid (DTPA) was immobilized on the surface of dND-PG through multistep organic transformations and Gd(III) ion was complexed in the last step. The resulting dND-PG-Gd(III) exhibited good dispersibility (> 4.5 mg/mL) and stability (> 3 months) in phosphate buffered saline (PBS). In vitro MR evaluation indicates that water proton T1 relaxivity or r1 of dND-PG-Gd(III) in aqueous solutions is larger than that of Magnevist® and the difference in the relaxivity becomes larger under weaker magnetic fields. The good dispersibility together with relatively high T1 relaxivity makes dND-PG-Gd(III) a promising contrast agent for in vivo MR imaging.
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Gadolinio/química , Glicerol/química , Imagen por Resonancia Magnética/métodos , Nanocápsulas/química , Nanoconjugados/química , Nanodiamantes/química , Polímeros/química , Difusión , Imagen por Resonancia Magnética/instrumentación , Ensayo de Materiales , Nanocápsulas/ultraestructura , Nanoconjugados/ultraestructura , Fantasmas de Imagen , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP) has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (-OH â -OTs (tosylate) â -N3) in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8) terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL) and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA) through electrostatic attraction, making them promising as nonviral vectors for gene delivery.