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1.
J Vis Exp ; (160)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32568221

RESUMO

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.


Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Compostos de Boro/metabolismo , Sobrevivência Celular , Cor , Ácidos Graxos/metabolismo , Corantes Fluorescentes/metabolismo , Gotículas Lipídicas/metabolismo , Lisossomos/metabolismo , Leveduras/citologia
2.
Molecules ; 25(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033280

RESUMO

The proteasome is the central component of the main cellular protein degradation pathway. During the past four decades, the critical function of the proteasome in numerous physiological processes has been revealed, and proteasome activity has been linked to various human diseases. The proteasome prevents the accumulation of misfolded proteins, controls the cell cycle, and regulates the immune response, to name a few important roles for this macromolecular "machine." As a therapeutic target, proteasome inhibitors have been approved for the treatment of multiple myeloma and mantle cell lymphoma. However, inability to sufficiently inhibit proteasome activity at tolerated doses has hampered efforts to expand the scope of proteasome inhibitor-based therapies. With emerging new modalities in myeloma, it might seem challenging to develop additional proteasome-based therapies. However, the constant development of new applications for proteasome inhibitors and deeper insights into the intricacies of protein homeostasis suggest that proteasome inhibitors might have novel therapeutic applications. Herein, we summarize the latest advances in proteasome inhibitor development and discuss the future of proteasome inhibitors and other proteasome-based therapies in combating human diseases.


Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular/métodos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Proteostase/efeitos dos fármacos , Compostos de Boro/metabolismo , Compostos de Boro/uso terapêutico , Bortezomib/metabolismo , Bortezomib/uso terapêutico , Glicina/análogos & derivados , Glicina/metabolismo , Glicina/uso terapêutico , Humanos , Lactonas/metabolismo , Lactonas/uso terapêutico , Oligopeptídeos/metabolismo , Oligopeptídeos/uso terapêutico , Pirróis/metabolismo , Pirróis/uso terapêutico
3.
Bioelectrochemistry ; 132: 107433, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31891877

RESUMO

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) layer, which enabled electroporation with micro- and submicrosecond pulses. No oxidation was observed with the electric field directed towards the ITO (cathode), even at field strengths much higher than that needed for permeabilization. Oxidation was readily detectable with the opposite polarity of pulses, but with the threshold higher than the permeabilization threshold. Moreover, a decrease in the medium conductance had opposite effects on permeabilization and lipid oxidation (it enhanced the former and suppressed the latter). We conclude that lipid oxidation can indeed occur at the plasma membrane after electric pulses, but it is not the cause of lasting membrane permeabilization.


Assuntos
Membrana Celular/metabolismo , Eletroporação/métodos , Lipídeos de Membrana/metabolismo , Animais , Compostos de Boro/metabolismo , Células CHO , Cricetulus , Oxirredução
4.
Mol Pharm ; 17(1): 202-211, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31763850

RESUMO

Boron neutron capture therapy (BNCT) has received extensive attention as noninvasive cell-level oncotherapy for treating solid cancer tumors. However, boron-containing drugs such as l-boronophenylalanine (BPA) and sodium borocaptate have low boron content and/or poor tumor-targeting ability, limiting their application. In this study, we designed and synthesized a series of nontoxic, dual-target boron carriers (B139, B142, and B151) with the ability to accumulate specifically in tumor cells. We found that the B139 uptake into hypoxic tumor regions was high, with a 70-fold boron content compared to BPA. In addition, in vivo observation showed that B139 can be trapped in tumor cells for a prolonged period and maintains an effective therapeutic concentration, with a peak boron concentration of 50.7 µg/g and a high tumor: blood boron ratio of >3, achieving ideal BNCT conditions. Cytotoxicity evaluation in mice further proved that B139 is safe and reliable. Therefore, B139 has great potential for BNCT application as a dual-target, safe, and efficient boron carrier.


Assuntos
Terapia por Captura de Nêutron de Boro , Neoplasias/radioterapia , Animais , Boranos/farmacologia , Compostos de Boro/química , Compostos de Boro/metabolismo , Compostos de Boro/farmacocinética , Compostos de Boro/toxicidade , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Nanopartículas/química , Nanopartículas/efeitos da radiação , Nanopartículas/uso terapêutico , Neoplasias/sangue , Neoplasias/enzimologia , Neoplasias/metabolismo , Nitroimidazóis/química , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Pharm ; 576: 118847, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-31759994

RESUMO

Crisaborole ointment, 2%, is a non-steroidal phosphodiesterase 4 inhibitor for the treatment of mild-to-moderate atopic dermatitis. It contains 9% w/w propylene glycol (PG). Although PG is generally considered to be safe when used as a pharmaceutical excipient or food additive, the European Medicines Agency has recommended maximum daily limits for PG exposure. To determine the potential skin permeation of PG from crisaborole ointment, ex vivo human skin (normal abdominal skin from healthy volunteers without atopic dermatitis) and in vivo minipig experiments (dermal application on unabraded or abraded skin) were performed. Over a 24-h period, the extent of PG permeation in the ex vivo human skin experiment was 3.7% for crisaborole ointment. In the in vivo minipig study, the bioavailability of PG after dermally applied crisaborole ointment was 3.56% for unabraded skin and 3.65% for abraded skin. Experimental values from this study can serve to provide scientific justification for using a product's specific absorption value, as opposed to a maximum absorption of 100%, when attempting to estimate systemic exposure of PG from a topical product.


Assuntos
Compostos de Boro/administração & dosagem , Compostos de Boro/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Pomadas/metabolismo , Propilenoglicol/metabolismo , Pele/metabolismo , Administração Cutânea , Animais , Disponibilidade Biológica , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , Fármacos Dermatológicos/administração & dosagem , Fármacos Dermatológicos/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pomadas/administração & dosagem , Permeabilidade , Absorção Cutânea/fisiologia , Suínos , Porco Miniatura
6.
Ann Nucl Med ; 34(1): 58-64, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31650410

RESUMO

OBJECTIVE: The tumor-to-normal tissue (T/N) boron ratio is determined in a patient prior to boron neutron capture therapy (BNCT) using 4-borono-2-18F-fluoro-L-phenylalanine (18F-FBPA) positron emission tomography (PET). The T/N ratio is used as a reference parameter to calculate BNCT dose and to evaluate treatment effects. The boronophenylalanine (BPA) dosage for BNCT treatment is higher than the 18F-FBPA dosage for PET diagnosis. Therefore, we aimed to determine whether the T/N ratios between diagnosis and treatment were correlated. METHODS: In this study, SAS tongue cancer cells were used to develop an orthotopic nude mouse model. Micro-PET was performed after the mice were injected a dose of 3.7 ± 0.74 MBq of 18F-FBPA via the tail vein. The 18F radioactivity in the tumor, muscle, and heart blood pool was calculated using AMIND software. Organs and blood were collected for boron concentration analysis using inductively coupled plasma-atomic emission spectroscopy after the mice were injected with 400 mg/kg BPA at 15, 30, 45, and 60 min. RESULTS: Pharmacokinetics of the tumor and muscle from 45 to 60 min after 18F-FBPA and BPA injections were slightly increased, whereas that of blood was slightly decreased. Median T/N ratios at 60 min after 18F-FBPA and BPA injections were 3.5 and 3.43, respectively. Median value of the T/N ratio between them was 3.49 at 60 min. The T/N ratio at 60 min after 18F-FBPA injection was similar to that after BPA injection. However, median tumor-to-blood (T/B) boron ratios of 18F-FBPA and BPA at 60 min were 1.63 and 3.35, respectively. Median value between them was 1.83 at 60 min. CONCLUSIONS: In this study, the T/B ratios demonstrate the spread of a distribution between 18F-FBPA and BPA injections. At 60 min, the T/N ratio of the 18F-FBPA injection was similar to that of the BPA injection. Boron concentration in normal tissue was almost equal to that in blood. Therefore, the representative T/N ratio could be obtained at 60 min after 18F-FBPA injection, and it was used as a reference parameter for calculating accurate radiation dose.


Assuntos
Compostos de Boro/uso terapêutico , Terapia por Captura de Nêutron de Boro , Fenilalanina/análogos & derivados , Tomografia por Emissão de Pósitrons , Doses de Radiação , Neoplasias da Língua/diagnóstico por imagem , Neoplasias da Língua/radioterapia , Animais , Compostos de Boro/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fenilalanina/metabolismo , Fenilalanina/uso terapêutico , Dosagem Radioterapêutica , Distribuição Tecidual , Neoplasias da Língua/metabolismo
7.
Commun Biol ; 2: 442, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815197

RESUMO

Real-time monitoring of newly acidified organelles during autophagy in living cells is highly desirable for a better understanding of intracellular degradative processes. Herein, we describe a reaction-based boron dipyrromethene (BODIPY) dye containing strongly electron-withdrawing diethyl 2-cyanoacrylate groups at the α-positions. The probe exhibits intense red fluorescence in acidic organelles or the acidified cytosol while exhibiting negligible fluorescence in other regions of the cell. The underlying mechanism is a nucleophilic reaction at the central meso-carbon of the indacene core, resulting in the loss of π-conjugation entailed by dramatic spectroscopic changes of more than 200 nm between its colorless, non-fluorescent leuco-BODIPY form and its red and brightly emitting form. The reversible transformation between red fluorescent BODIPY and leuco-BODIPY along with negligible cytotoxicity qualifies such dyes for rapid and direct intracellular lysosome imaging and cytosolic acidosis detection simultaneously without any washing step, enabling the real-time monitoring of newly acidified organelles during autophagy.


Assuntos
Autofagia , Compostos de Boro/metabolismo , Corantes Fluorescentes/metabolismo , Imagem Molecular/métodos , Imagem Óptica/métodos , Organelas/metabolismo , Compostos de Boro/química , Corantes Fluorescentes/química , Lisossomos/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução , Processos Fotoquímicos , Espectrometria de Fluorescência
8.
Cell Chem Biol ; 26(12): 1643-1651.e4, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31604616

RESUMO

Degradable crosslinkers that respond to intracellular biological stimuli are a critical component of many drug delivery systems. With numerous stimuli-responsive drug delivery systems in development, it is important to quantitatively study their intracellular processing. Herein we report a framework for quantifying the rate of intracellular bond degradation in the endocytic pathway. Toward this end, we devised and synthesized a reduction-sensitive FRET-based crosslinker that can be readily conjugated to a variety of targeting ligands. This crosslinker was conjugated to trastuzumab, a humanized monoclonal antibody against the HER2 receptor. We developed a model based on mass-action kinetics to describe the intracellular processing of this conjugate. The kinetic model was developed in conjunction with live-cell experiments to extract the rate constant for intracellular disulfide bond degradation. This framework may be applied to other endocytosis pathways, bond types, and cell types to quantify this fundamental degradation rate parameter.


Assuntos
Imunoconjugados/metabolismo , Trastuzumab/metabolismo , Compostos de Boro/química , Compostos de Boro/metabolismo , Linhagem Celular Tumoral , Dissulfetos/química , Dissulfetos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Glutationa/química , Meia-Vida , Humanos , Imunoconjugados/imunologia , Cinética , Microscopia Confocal , Modelos Teóricos , Receptor ErbB-2/imunologia , Rodaminas/química , Rodaminas/metabolismo , Transglutaminases/metabolismo , Trastuzumab/imunologia
9.
J Mater Chem B ; 7(43): 6861-6867, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31613291

RESUMO

Hypochlorous acid (HClO) is a powerful microbicidal agent in the innate immue system; however, abnormal HClO levels can cause tissue damage and many diseases. Thus, it is vitally important to develop facile, rapid and accurate analytical methods for the detection of HClO/ClO-in vitro and in vivo. In this work, we have constructed three meso-substituted BODIPY selenides with different hydrocarbyl groups (ethyl for BSe-Et, benzyl for BSe-Bz and phenyl for BSe-Ph) as fluorescent probes for the detection of HClO/ClO-. All three non-fluorescent probes can sense HClO/ClO- to form fluorescent selenoxides by blocking the photo-induced electron transfer process. Their sensing properties display a clear relationship with the structure of the hydrocarbyl. The sensing reactivity is heavily dependent on the electron-donating ability of hydrocarbyls, with the order of the response time as BSe-Et (2 s) < BSe-Bz (5 s) ≪ BSe-Ph (>100 s). Both BSe-Et and BSe-Bz afford a large fluorescence response and very low detection limits (0.3 nM and 0.8 nM), and BSe-Bz displays a higher selectivity over BSe-Et. Finally, as a representative, BSe-Bz was successfully applied to the detection of exo- and endogenous HClO in living cells, and demonstrated to be a mitochondria-localized fluorescent probe.


Assuntos
Compostos de Boro/metabolismo , Ácidos Carboxílicos/química , Ácido Hipocloroso/química , Mitocôndrias/metabolismo , Compostos Organosselênicos/química , Fluorescência , Humanos
10.
Bioconjug Chem ; 30(11): 2727-2750, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31593454

RESUMO

Two-dimensional nanosheet-based materials such as graphene, hexagonal boron nitride, and MoS2 represent intriguing structures for a variety of biological applications ranging from biosensing to nanomedicine. Recent advances have demonstrated that peptides can be identified with affinity for these three materials, thus generating a highly unique bioconjugate interfacial system. This Review focuses on recent advances in the formation of bioconjugates of these types, paying particular attention to the structure/function relationship of the peptide overlayer. This is achieved through the amino acid composition of the nanosheet binding peptides, thus allowing for precise control over the properties of the final materials. Such bioconjugate systems offer rapid advances via direct property control that remain difficult to achieve for biological applications using nonbiological approaches.


Assuntos
Compostos de Boro/metabolismo , Dissulfetos/metabolismo , Grafite/metabolismo , Molibdênio/metabolismo , Nanoconjugados/química , Nanoestruturas/química , Fragmentos de Peptídeos/metabolismo , Compostos de Boro/química , Dissulfetos/química , Grafite/química , Molibdênio/química , Fragmentos de Peptídeos/química
11.
Eur J Pharm Biopharm ; 145: 76-84, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31639417

RESUMO

Oral drug delivery is a preferred administration route due to its low cost, high patient compliance and fewer adverse events compared to intravenous administration. However, many pharmaceuticals suffer from poor solubility and low oral bioavailability. One major factor that contributes to low bioavailability are efflux transporters which prevent drug absorption through intestinal epithelial cells. P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two important efflux transporters in the intestine functioning to prevent toxic materials from entering systemic circulation. However, due to its broad substrate specificity, P-gp limits the absorption of many therapeutics, including chemotherapeutics and antibacterial agents. Methods to inhibit P-gp with competitive inhibitors have not been clinically successful. Here, we show that micron scale devices (microdevices) made from a commonly used biomaterial, polyethylene glycol (PEG), inhibit P-gp through a biosimilar mucus in Caco-2 cells and that transporter function is restored when the microdevices are removed. Microdevices were shown to inhibit P-gp mediated transport of calcein AM, doxorubicin, and rhodamine 123 (R123) and BCRP mediated transport of BODIPY-FL-prazosin. When in contact with Caco-2 cells, microdevices decrease the cell surface amount of P-gp without affecting the passive transport. Moreover, there was an increase in mucosal to serosal transport of R123 with microdevices in an ex-vivo mouse model and increased absorption in vivo. This biomaterial-based approach to inhibit efflux transporters can be applied to a range of drug delivery systems and allows for a nonpharmacologic method to increase intestinal drug absorption while limiting toxic effects.


Assuntos
Transporte Biológico/efeitos dos fármacos , Hidrogéis/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Disponibilidade Biológica , Compostos de Boro/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polietilenoglicóis/química , Prazosina/análogos & derivados , Prazosina/metabolismo , Rodamina 123/metabolismo , Solubilidade/efeitos dos fármacos
12.
Analyst ; 144(21): 6214-6224, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31528921

RESUMO

The ability of secondary ion mass spectrometry (SIMS) to provide high sensitivity imaging of elements and small-medium mass molecules in biological tissues and cells, makes it a very powerful tool for drug distribution studies. Here we report on the application of a high-resolution dynamic SIMS instrument for the quantification and localisation of therapeutic levels of the BNCT agent l-para-(dihydroxyboryl)-phenylalanine (BPA) in primary cell cultures from human patients exhibiting glioblastoma multiform tumours. Boron uptake and distribution was determined quantitatively as a function of cell-sampling location and different treatment regimes. Importantly, BPA was found to accumulate in cancer cells invading the 'brain around tumour' tissue in addition to the main tumour site. Pre-treatment of samples with l-tyrosine was found not to increase the uptake of BPA, nor change the intracellular drug distribution. In cultured cells from the tumour core and brain around tumour, with and without l-tyrosine pre-treatment, normalised boron-related signals were higher from cell nuclei than from cytoplasm. An efflux treatment was found to reduce BPA levels, but at a rate slower than the original uptake, and did not affect the intracellular drug distribution. To the best of our knowledge, these data represent the first published study of BPA uptake and l-amino acid pre-treatment in cultured primary human cells using dynamic SIMS, and the most detailed, subcellular distribution study of a BNCT agent in any cellular system.


Assuntos
Compostos de Boro/metabolismo , Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Espectrometria de Massas , Imagem Molecular , Nanotecnologia , Fenilalanina/análogos & derivados , Compostos de Boro/uso terapêutico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Humanos , Espaço Intracelular/metabolismo , Fenilalanina/metabolismo , Fenilalanina/uso terapêutico
13.
Drug Discov Today ; 24(12): 2234-2246, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31494188

RESUMO

A soft drug (SD) displays a metabolically labile spot and, after having exerted its activity in the site of action, undergoes a fast metabolism, leading to inactive metabolites. The SD approach has recently found widespread application in the dermatological field because it provides a means of localising the therapeutic effect in skin, while minimising systemic exposure. The literature is rapidly growing of successful examples of compounds targeting sphingosine-1-phosphate receptor 1 (S1PR1), transient receptor potential vanilloid 1 (TRPV1), Janus kinase (JAK), caspase 1, and histone deacetylase (HDAC), for the treatment of skin inflammatory, autoimmune, and oncological diseases. As a demonstration of the potential of this strategy, the SD approach recently led to the approval of crisaborole, a soft phosphodiesterase 4 (PDE4) inhibitor, for atopic dermatitis, while other agents are in clinical development.


Assuntos
Fármacos Dermatológicos/administração & dosagem , Desenvolvimento de Medicamentos , Dermatopatias/tratamento farmacológico , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/metabolismo , Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dermatite Atópica/tratamento farmacológico , Fármacos Dermatológicos/metabolismo , Fármacos Dermatológicos/farmacologia , Humanos , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia
14.
Dokl Biochem Biophys ; 486(1): 220-223, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367826

RESUMO

Cardiolipin (CL) plays a central role in lipid peroxidation (LPO) of the mitochondrial inner membrane due to higher content of unsaturated fatty acids in CL in comparison with the other phospholipids. CL oxidation plays an important role in the regulation of various intracellular signaling pathways and its excessive oxidation contributes to the development of various pathologies and, possibly, participates in the aging process. Mitochondria-targeted antioxidants containing triphenylphosphonium (TPP+) effectively protect CL from oxidation. It is assumed that fluorescent probes on the basis of the C11-BODIPY fluorophore sensitive to LPO and containing TPP+ can selectively register CL oxidation. To test this possibility, we carried out a molecular dynamic simulation of such probes in a model mitochondrial membrane. It is shown that the probes are located in the membrane at the same depth as the unsaturated bonds in CL molecules sensitive to oxidation. Increasing the length of the linker that binds the fluorophore and TPP+ residue ha little effect on the position of the probe in the membrane. This indicates the possibility of modifying the linker to increase the selectivity of the probes to CL.


Assuntos
Corantes Fluorescentes/metabolismo , Peroxidação de Lipídeos , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Compostos de Boro/metabolismo
15.
Angew Chem Int Ed Engl ; 58(38): 13394-13399, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31344292

RESUMO

Organic building blocks are the centerpieces of "one-for-all" nanoparticle development. Herein, we report the synthesis of a novel aza-BODIPY-lipid building block and its self-assembly into a liposomal nanoparticle (BODIPYsome). We observed optically stable NIR J-aggregation within the BODIPYsome that is likely attributed to J-dimerization. BODIPYsomes with cholesterol showed enhanced colloidal stability while maintaining a high extinction coefficient (128 mm-1 cm-1 ) and high fluorescence quenching (99.70±0.09 %), which enables photoacoustic (PA) properties from its intact structure and recovered NIR fluorescence properties when it is disrupted in cancer cells. Finally, its capabilities for optical imaging (PA/fluorescence) were observed in an orthotopic prostate tumor mouse model 24 h after intravenous administration. Overall, the BODIPYsome opens the door for engineering new building blocks in the design of optically stable biophotonic imaging agents.


Assuntos
Compostos de Boro/metabolismo , Neoplasias Oculares/diagnóstico por imagem , Lipossomos/metabolismo , Imagem Óptica/métodos , Humanos
16.
J Nutr ; 149(10): 1732-1741, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204781

RESUMO

BACKGROUND: The intestine is the main organ for absorbing dietary fat. High dietary lipid intake leads to fat deposition in the intestine and adversely influences fat absorption and health, but the underlying mechanism is unknown. OBJECTIVES: We used yellow catfish and their isolated intestinal epithelial cells to test the hypothesis that endoplasmic reticulum (ER) stress, autophagy, and apoptosis mediate fat-induced changes in lipid metabolism. METHODS: Male and female yellow catfish (weight: 3.79 ± 0.16 g; age: 3 mo) were fed diets containing lipid at 6.98% (low-fat diet; LFD), 11.3% (middle-fat diet; MFD), or 15.4% (high-fat diet; HFD) (by weight) for 8 wk. Each dietary group had 3 replicates, 30 fish per replicate. Their intestinal epithelial cells were isolated and incubated for 24 h in control solution or various concentrations of fatty acids (FAs) with or without 2-h pretreatment with an inhibitor [3-methyladenine (3-MA), 4-phenyl butyric acid (4-PBA), or Ac-DVED-CHO (AC)]. Triglyceride (TG) contents, genes, and enzymes involved in lipid metabolism, ER stress, autophagy, and apoptosis were determined in intestinal tissue and cells; immunoblotting, BODIPY 493/503 staining, ultrastructural observation, and the detection of autophagic and apoptotic vesicles were performed on intestinal cells. RESULTS: Compared with the LFD and MFD, the HFD increased intestinal TG content by 120-226%, activities of lipogenic enzymes by 19.0-245%, expression of genes related to lipogenesis (0.77-8.4-fold), lipolysis (0.36-6.0-fold), FA transport proteins (0.79-1.7-fold), ER stress (0.55-7.5-fold), autophagy (0.56-4.2-fold), and apoptosis (0.80-5.2-fold). Using isolated intestinal epithelial cells and inhibitors (4-PBA, 3-MA, and AC), we found that ER stress mediated FA-induced activation of autophagy (11.0-50.1%) and apoptosis (10.4-32.0%), and lipophagy and apoptosis mediated FA-induced lipolysis (3.40-41.6%). CONCLUSIONS: An HFD upregulated lipogenesis, lipolysis, and FA transport, induced ER stress, and activated autophagy and apoptosis. ER stress, autophagy, and apoptosis play important regulatory roles in fat-induced changes in lipid metabolism in the intestine and intestinal epithelial cells of yellow catfish.


Assuntos
Peixes-Gato , Gorduras na Dieta/efeitos adversos , Estresse do Retículo Endoplasmático , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/citologia , Lipase/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Apoptose , Autofagia , Compostos de Boro/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Dieta/veterinária , Enzimas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos
17.
Mol Cells ; 42(6): 470-479, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31250620

RESUMO

Interstitial cells of Cajal (ICCs) are pacemaker cells that exhibit periodic spontaneous depolarization in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of ghrelin and motilin on the pacemaker potentials of ICCs isolated from the mouse small intestine. Using the whole-cell patch-clamp configuration, we demonstrated that ghrelin depolarized pacemaker potentials of cultured ICCs in a dose-dependent manner. The ghrelin receptor antagonist [D-Lys] GHRP-6 completely inhibited this ghrelin-induced depolarization. Intracellular guanosine 5'-diphosphate-ß-S and pre-treatment with Ca2+free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate (IP3), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the IP3 receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C, the Rho kinase inhibitor Y-27632, and the PKC inhibitors staurosporine, Go6976, and rottlerin. All inhibitors except rottlerin blocked the ghrelin-induced pacemaker potential depolarization of ICCs. In addition, motilin depolarized the pacemaker potentials of ICCs in a similar dose-dependent manner as ghrelin, and this was also completely inhibited by [D-Lys] GHRP-6. These results suggest that ghrelin induced the pacemaker potential depolarization through the ghrelin receptor in a G protein-, IP3-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular Ca2+ regulation. In addition, motilin was able to depolarize the pacemaker potentials of ICCs through the ghrelin receptor. Therefore, ghrelin and its receptor may modulate GI motility by acting on ICCs in the murine small intestine.


Assuntos
Grelina/farmacologia , Células Intersticiais de Cajal/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Motilina/farmacologia , Acetofenonas/farmacologia , Amidas/farmacologia , Animais , Benzopiranos/farmacologia , Compostos de Boro/metabolismo , Cálcio/metabolismo , Carbazóis/farmacologia , Motilidade Gastrointestinal/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Células Intersticiais de Cajal/fisiologia , Intestino Delgado/fisiologia , Compostos Macrocíclicos/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Oligopeptídeos/metabolismo , Oxazóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Piridinas/farmacologia , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/metabolismo , Transdução de Sinais , Estaurosporina/farmacologia , Tapsigargina/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
18.
Inorg Chem ; 58(13): 8587-8595, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31117633

RESUMO

A new N,O-based BODIPY ligand was synthesized and further utilized to develop highly fluorescent and photostable Ru(II), Rh(III), and Ir(III) metal complexes. The complexes were fully characterized by different analytical techniques including single-crystal XRD studies. The photostabilities and live cell imaging capabilities of the complexes were investigated via confocal microscopy. The complexes localized specifically in the mitochondria of live cells and showed negligible cytotoxicities at a concentration used for imaging purposes. They also exhibited high photostabilities, with fluorescence intensities remaining above 50% after 1800 scans.


Assuntos
Compostos de Boro/metabolismo , Complexos de Coordenação/metabolismo , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Transporte Biológico , Compostos de Boro/síntese química , Compostos de Boro/efeitos da radiação , Compostos de Boro/toxicidade , Complexos de Coordenação/síntese química , Complexos de Coordenação/efeitos da radiação , Complexos de Coordenação/toxicidade , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/efeitos da radiação , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Irídio/química , Ligantes , Microscopia Confocal , Fotodegradação , Ródio/química , Rutênio/química
19.
Radiat Environ Biophys ; 58(3): 455-467, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31123853

RESUMO

Boron neutron capture therapy (BNCT) is a promising cancer binary therapy modality that utilizes the nuclear capture reaction of thermal neutrons by boron-10 resulting in a localized release of high- and low-linear energy transfer (LET) radiation. Electrochemotherapy (ECT) is based on electroporation (EP) that induces opening of pores in cell membranes, allowing the entry of compounds. Because EP is applied locally to a tumor, the compound is incorporated preferentially by tumor cells. Based on the knowledge that the therapeutic success of BNCT depends centrally on the boron content in tumor and normal tissues and that EP has proven to be an excellent facilitator of tumor biodistribution of an anti-tumor agent, the aim of this study was to evaluate if EP can optimize the delivery of boronated compounds. We performed biodistribution studies and qualitative microdistribution analyses of boron employing the boron compound sodium decahydrodecaborate (GB-10) + EP in the hamster cheek pouch oral cancer model. Syrian hamsters with chemically induced exophytic squamous cell carcinomas were used. A typical EP treatment was applied to each tumor, varying the moment of application with respect to the administration of GB-10 (early or late). The results of this study showed a significant increase in the absolute and relative tumor boron concentration and optimization of the qualitative microdistribution of boron by the use of early EP + GB-10 versus GB-10 without EP. This strategy could be a tool to improve the therapeutic efficacy of BNCT/GB-10 in vivo.


Assuntos
Compostos de Boro/metabolismo , Terapia por Captura de Nêutron de Boro/métodos , Boro/metabolismo , Isótopos/metabolismo , Animais , Bochecha , Cricetinae , Modelos Animais de Doenças , Mesocricetus , Neoplasias Bucais , Distribuição Tecidual
20.
Biophys J ; 116(10): 1984-1993, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31053255

RESUMO

Gravity-sensitive cellular responses are regularly observed in both specialized and nonspecialized cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel, to our knowledge, viscosity-sensitive molecular rotor based on mesosubstituted boron-dipyrrin used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the large diameter centrifuge at the European Space Agency Technology Centre. Mouse osteoblastic (MC3T3-E1) and endothelial (human umbilical vein endothelial cell) cell lines were tested, and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first, to our knowledge, quantitative data for cellular viscosity changes under hypergravity, up to 15 × g.


Assuntos
Gravitação , Espaço Intracelular/metabolismo , Células 3T3 , Animais , Fenômenos Biomecânicos , Compostos de Boro/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Camundongos , Viscosidade
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