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1.
Nat Commun ; 11(1): 4964, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009394

RESUMO

Thrombosis leads to platelet activation and subsequent degradation; therefore, replenishment of platelets from hematopoietic stem/progenitor cells (HSPCs) is needed to maintain the physiological level of circulating platelets. Platelet-derived microparticles (PMPs) are protein- and RNA-containing vesicles released from activated platelets. We hypothesized that factors carried by PMPs might influence the production of platelets from HSPCs, in a positive feedback fashion. Here we show that, during mouse acute liver injury, the density of megakaryocyte in the bone marrow increases following an increase in circulating PMPs, but without thrombopoietin (TPO) upregulation. In vitro, PMPs are internalized by HSPCs and drive them toward a megakaryocytic fate. Mechanistically, miR-1915-3p, a miRNA highly enriched in PMPs, is transported to target cells and suppresses the expression levels of Rho GTPase family member B, thereby inducing megakaryopoiesis. In addition, direct injection of PMPs into irradiated mice increases the number of megakaryocytes and platelets without affecting TPO levels. In conclusion, our data reveal that PMPs have a role in promoting megakaryocytic differentiation and platelet production.


Assuntos
Plaquetas/metabolismo , Diferenciação Celular , Micropartículas Derivadas de Células/metabolismo , Megacariócitos/citologia , MicroRNAs/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Endocitose , Perfilação da Expressão Gênica , Humanos , Fígado/lesões , Fígado/patologia , Masculino , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Poliploidia , Proteína rhoB de Ligação ao GTP/metabolismo
2.
Nat Commun ; 11(1): 4765, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958780

RESUMO

Fatty acids (FAs) are essential nutrients, but how they are transported into cells remains unclear. Here, we show that FAs trigger caveolae-dependent CD36 internalization, which in turn delivers FAs into adipocytes. During the process, binding of FAs to CD36 activates its downstream kinase LYN, which phosphorylates DHHC5, the palmitoyl acyltransferase of CD36, at Tyr91 and inactivates it. CD36 then gets depalmitoylated by APT1 and recruits another tyrosine kinase SYK to phosphorylate JNK and VAVs to initiate endocytic uptake of FAs. Blocking CD36 internalization by inhibiting APT1, LYN or SYK abolishes CD36-dependent FA uptake. Restricting CD36 at either palmitoylated or depalmitoylated state eliminates its FA uptake activity, indicating an essential role of dynamic palmitoylation of CD36. Furthermore, blocking endocytosis by targeting LYN or SYK inhibits CD36-dependent lipid droplet growth in adipocytes and high-fat-diet induced weight gain in mice. Our study has uncovered a dynamic palmitoylation-regulated endocytic pathway to take up FAs.


Assuntos
Antígenos CD36/metabolismo , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Lipoilação , Células 3T3-L1 , Aciltransferases/metabolismo , Adipócitos/metabolismo , Animais , Antígenos CD36/deficiência , Antígenos CD36/genética , Cavéolas/metabolismo , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Obesidade/tratamento farmacológico , Fosforilação , Transdução de Sinais , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Ganho de Peso/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
3.
PLoS One ; 15(8): e0237883, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866169

RESUMO

Although whole-genome sequencing has provided novel insights into Neisseria meningitidis, many open reading frames have only been annotated as hypothetical proteins with unknown biological functions. Our previous genetic analyses revealed that the hypothetical protein, NMB1345, plays a crucial role in meningococcal infection in human brain microvascular endothelial cells; however, NMB1345 has no homology to any identified protein in databases and its physiological function could not be elucidated using pre-existing methods. Among the many biological technologies to examine transient protein-protein interaction in vivo, one of the developed methods is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina species: However, this method has never been applied to assign function-unknown proteins in pathogenic bacteria. In the present study, we developed a new method to genetically incorporate ncAAs-encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair and elucidated the biological function(s) of the NMB1345 protein. The results revealed that the NMB1345 protein directly interacts with PilE, a major component of meningococcal pili, and further physicochemical and genetic analyses showed that the interaction between the NMB1345 protein and PilE was important for both functional pilus formation and meningococcal infectious ability in N. meningitidis. The present study using this new methodology for N. meningitidis provides novel insights into meningococcal pathogenesis by assigning the function of a hypothetical protein.


Assuntos
Aminoácidos/genética , Reagentes para Ligações Cruzadas/metabolismo , Luz , Neisseria meningitidis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Encéfalo/irrigação sanguínea , Endocitose , Células Endoteliais/microbiologia , Fímbrias Bacterianas/metabolismo , Humanos , Microvasos/patologia , Mutação/genética , Plasmídeos/genética
4.
PLoS Negl Trop Dis ; 14(9): e0008365, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898130

RESUMO

Insecticide resistance poses a significant threat to the control of arthropods that transmit disease agents. Nanoparticle carriers offer exciting opportunities to expand the armamentarium of insecticides available for public health and other pests. Most chemical insecticides are delivered by contact or feeding, and from there must penetrate various biological membranes to reach target organs and kill the pest organism. Nanoparticles have been shown to improve bioactive compound navigation of such barriers in vertebrates, but have not been well-explored in arthropods. In this study, we explored the potential of polyanhydride micro- and nanoparticles (250 nm- 3 µm), labeled with rhodamine B to associate with and/or transit across insect biological barriers, including the cuticle, epithelium, midgut and ovaries, in female Ae. aeygpti mosquitoes. Mosquitoes were exposed using conditions to mimic surface contact with a residual spray or paint, topical exposure to mimic contact with aerosolized insecticide, or per os in a sugar meal. In surface contact experiments, microparticles were sometimes observed in association with the exterior of the insect cuticle. Nanoparticles were more uniformly distributed across exterior tissues and present at higher concentrations. Furthermore, by surface contact, topical exposure, or per os, particles were detected in internal organs. In every experiment, amphiphilic polyanhydride nanoparticles associated with internal tissues to a higher degree than hydrophobic nanoparticles. In vitro, nanoparticles associated with Aedes aegypti Aag2 cells within two hours of exposure, and particles were evident in the cytoplasm. Further studies demonstrated that particle uptake is dependent on caveolae-mediated endocytosis. The propensity of these nanoparticles to cross biological barriers including the cuticle, to localize in target tissue sites of interest, and to reach the cytoplasm of cells, provides great promise for targeted delivery of insecticidal candidates that cannot otherwise reach these cellular and subcellular locations.


Assuntos
Aedes/fisiologia , Nanopartículas , Polianidridos , Aedes/citologia , Animais , Linhagem Celular , Endocitose , Feminino , Controle de Mosquitos/métodos , Rodaminas/química , Distribuição Tecidual
5.
Int J Nanomedicine ; 15: 6167-6182, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922000

RESUMO

Background: Among the novel cancer treatment strategies, combination therapy is a cornerstone of cancer therapy. Materials and Methods: Here, combination therapy with targeted polymer, magnetic hyperthermia and chemotherapy was presented as an effective therapeutic technique. The DOX-loaded PLA-PEG-FA magnetic nanoparticles (nanocarrier) were prepared via a double emulsion method. The nanocarriers were characterized by particle size, zeta potential, morphology, saturation magnetizations and heat generation capacity, and the encapsulation efficiency, drug content and in-vitro drug release for various weight ratios of PLA:DOX. Then, cytotoxicity, cellular uptake and apoptosis level of nanocarrier-treated cells for HeLa and CT26 cells were investigated by MTT assay, flow cytometry, and apoptosis detection kit. Results and Conclusions: The synthesized nanoparticles were spherical in shape, had low aggregation and considerable magnetic properties. Meanwhile, the drug content and encapsulation efficiency of nanoparticles can be achieved by varying the weight ratios of PLA:DOX. The saturation magnetizations of nanocarriers in the maximum applied magnetic field were 59/447 emu/g and 28/224 emu/g, respectively. Heat generation capacity of MNPs and nanocarriers were evaluated in the external AC magnetic field by a hyperthermia device. The highest temperature, 44.2°C, was measured in the nanocarriers suspension at w/w ratio 10:1 (polymer:DOX weight ratio) after exposed to the magnetic field for 60 minutes. The encapsulation efficiency improved with increasing polymer concentration, since the highest DOX encapsulation efficiency was related to the nanocarriers' suspension at w/w ratio 50:1 (79.6 ± 6.4%). However, the highest DOX loading efficiency was measured in the nanocarriers' suspension at w/w ratio 10:1 (5.14 ± 0.6%). The uptake efficiency and apoptosis level of nanocarrier-treated cells were higher than those of nanocarriers (folic acid free) and free DOX-treated cells in both cell lines. Therefore, this targeted nanocarrier may offer a promising nanosystem for cancer-combined chemotherapy and hyperthermia.


Assuntos
Doxorrubicina/farmacologia , Ácido Fólico/farmacologia , Hipertermia Induzida , Nanopartículas de Magnetita/química , Neoplasias/terapia , Polietilenoglicóis/química , Animais , Apoptose/efeitos dos fármacos , Liberação Controlada de Fármacos , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Nanopartículas de Magnetita/ultraestrutura , Camundongos , Tamanho da Partícula , Polietilenoglicóis/síntese química , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
6.
Int J Nanomedicine ; 15: 6311-6324, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922003

RESUMO

Background: Hyaluronic acid (HA) is a major component of extracellular matrix (ECM) and its over expression in tumor tissues contributes to the increase of interstitial fluid pressure (IFP) and hinders the penetration of nanoparticles into solid tumors. Materials and Methods: We here reported a tumoral microenvironment responsive multistage drug delivery system (NPs-EPI/HAase) which was formed layer by layer via electrostatic interaction with epirubicin (EPI)-loaded PEG-b-poly(2-(diisopropylamino)ethyl methacrylate)-b-poly(2-guanidinoethylmethacrylate) (mPEG-PDPA-PG, PEDG) micelles (NPs-EPI) and hyaluronidase (HAase). In this paper, we focused on the hyaluronidase-combined nanoparticles (NPs-EPI/HAase) for tumor penetration in tumor spheroid and solid tumor models in vitro and in vivo. Results: Our results showed that NPs-EPI/HAase effectively degrade the HA in ECM and facilitate deep penetration of NPs-EPI into solid tumor. Moreover, NPs-EPI mainly employed clathrin-mediated and macropinocytosis-mediated endocytic pathways for cellular uptake and were subsequently directed to the lysosomes for further drug release triggered by proton sponge effect. Compared with NPs-EPI, the HAase coating group showed an enhanced tumoral drug delivery efficacy and inhibition of tumor growth. Conclusion: Overall, our studies demonstrated that coating nanoparticles with HAase can provide a simple but efficient strategy for nano-drug carriers to enhance solid tumor penetration and chemotherapeutic efficacy.


Assuntos
Antineoplásicos/uso terapêutico , Hialuronoglucosaminidase/metabolismo , Nanopartículas/química , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Portadores de Fármacos/química , Endocitose/efeitos dos fármacos , Epirubicina/farmacologia , Epirubicina/uso terapêutico , Humanos , Antígeno Ki-67/metabolismo , Masculino , Camundongos Nus , Nanopartículas/administração & dosagem , Neoplasias/patologia , Polímeros/química , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
7.
Int J Nanomedicine ; 15: 6385-6399, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922007

RESUMO

Purpose: The mononuclear phagocyte system (MPS) presents a formidable obstacle that hampers the delivery of various nanopreparations to tumors. Therefore, there is an urgent need to improve the off-MPS targeting ability of nanomedicines. In the present study, we present a novel preconditioning strategy to substantially increase the circulation times and tumor targeting of nanoparticles by regulating nanocarrier-MPS interactions. Methods: In vitro, the effect of different vacuolar H+-ATPase inhibitors on macrophage uptake of targeted or nontargeted lipid vesicles was evaluated. Specifically, the clinically approved proton-pump inhibitor esomeprazole (ESO) was selected as a preconditioning agent. Then, we further investigated the blocking effect of ESO on the macrophage endocytosis of nanocarriers. In vivo, ESO was first intravenously administered into A549-tumor-bearing nude mice, and 24 h later, the c(RGDm7)-modified vesicles co-loaded with doxorubicin and gefitinib were intravenously injected. Results: In vitro, ESO was found to reduce the interactions between macrophages and c(RGDm7)-modified vesicles by interfering with the latter's lysosomal trafficking. Studies conducted in vivo confirmed that ESO pretreatment greatly decreased the liver and spleen distribution of the targeted vesicles, enhanced their tumor accumulation, and improved the therapeutic outcome of the drug-loaded nanomedicines. Conclusion: Our findings indicate that ESO can regulate the nanoparticle-MPS interaction, which provides a feasible option for enhancing the off-MPS targeting of nanomedicines.


Assuntos
Portadores de Fármacos/química , Esomeprazol/farmacologia , Sistema Fagocitário Mononuclear/citologia , Nanopartículas/química , Células A549 , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transporte Biológico , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Endocitose , Esomeprazol/farmacocinética , Esomeprazol/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Células RAW 264.7 , Distribuição Tecidual/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo
8.
Int J Nanomedicine ; 15: 6451-6468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922011

RESUMO

Background: Non-small cell lung cancer (NSCLC) is one of the most lethal types of cancer with highly infiltrating. Chemotherapy is far from satisfactory, vasculogenic mimicry (VM) and angiogenesis results in invasion, migration and relapse. Purpose: The objective of this study was to construct a novel CPP (mmp) modified vinorelbine and dioscin liposomes by two new functional materials, DSPE-PEG2000-MAL and CPP-PVGLIG-PEG5000, to destroy VM channels, angiogenesis, EMT and inhibit invasion and migration. Methods and Results: The targeting liposomes could be enriched in tumor sites through passive targeting, and the positively charged CPP was exposed and enhanced active targeting via electrostatic adsorption after being hydrolyzed by MMP2 enzymes overexpressed in the tumor microenvironment. We found that CPP (mmp) modified vinorelbine and dioscin liposomes with the ideal physicochemical properties and exhibited enhanced cellular uptake. In vitro and in vivo results showed that CPP (mmp) modified vinorelbine and dioscin liposomes could inhibit migration and invasion of A549 cells, destroy VM channels formation and angiogenesis, and block the EMT process. Pharmacodynamic studies showed that the targeting liposomes had obvious accumulations in tumor sites and magnificent antitumor efficiency. Conclusion: CPP (mmp) modified vinorelbine plus dioscin liposomes could provide a new strategy for NSCLC.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Microambiente Tumoral , Células A549 , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Galinhas , Endocitose/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Hidrólise , Lipossomos , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Microambiente Tumoral/efeitos dos fármacos , Vinorelbina/farmacologia , Vinorelbina/uso terapêutico
9.
Methods Mol Biol ; 2203: 241-261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833217

RESUMO

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.


Assuntos
Coronavirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Microscopia Confocal/métodos , Linhagem Celular , Coronavirus/isolamento & purificação , Meios de Cultura/química , Endocitose , Humanos , Proteínas do Nucleocapsídeo/metabolismo , Ácidos Tri-Iodobenzoicos/química , Internalização do Vírus
10.
Biomolecules ; 10(9)2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32825713

RESUMO

Caveolae are flask-shaped invaginations of the plasma membrane found in numerous cell types and are particularly abundant in endothelial cells and adipocytes. The lipid composition of caveolae largely matches that of lipid rafts microdomains that are particularly enriched in cholesterol, sphingomyelin, glycosphingolipids, and saturated fatty acids. Unlike lipid rafts, whose existence remains quite elusive in living cells, caveolae can be clearly distinguished by electron microscope. Despite their similar composition and the sharing of some functions, lipid rafts appear more heterogeneous in terms of size and are more dynamic than caveolae. Following the discovery of caveolin-1, the first molecular marker as well as the unique scaffolding protein of caveolae, we have witnessed a remarkable increase in studies aimed at investigating the role of these organelles in cell functions and human disease. The goal of this review is to discuss the most recent studies related to the role of caveolae and caveolins in endothelial cells. We first recapitulate the major embryological processes leading to the formation of the vascular tree. We next discuss the contribution of caveolins and cavins to membrane biogenesis and cell response to extracellular stimuli. We also address how caveolae and caveolins control endothelial cell metabolism, a central mechanism involved in migration proliferation and angiogenesis. Finally, as regards the emergency caused by COVID-19, we propose to study the caveolar platform as a potential target to block virus entry into endothelial cells.


Assuntos
Betacoronavirus/fisiologia , Cavéolas/metabolismo , Endocitose , Endotélio Vascular/metabolismo , Internalização do Vírus , Animais , Betacoronavirus/patogenicidade , Caveolinas/metabolismo , Endotélio Vascular/virologia , Humanos
11.
Int J Nanomedicine ; 15: 4811-4824, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32753867

RESUMO

Purpose: Magnetic resonance imaging (MRI) contrast agents are pharmaceuticals that enable a better visualization of internal body structures. In this study, we present the synthesis, MRI signal enhancement capabilities, in vitro as well as in vivo cytotoxicity results of gold-coated iron oxide nanoparticles (Fe3O4@AuNPs) as potential contrast agents. Methods: Fe3O4@AuNPs were obtained by synthesizing iron oxide nanoparticles and gradually coating them with gold. The obtained Fe3O4@AuNPs were characterized by spectroscopies, transmission electron microscopy (TEM) and energy dispersive X-ray diffraction. The effect of the nanoparticles on the MRI signal was tested using a 7T Bruker PharmaScan system. Cytotoxicity tests were made in vitro on Fe3O4@AuNP-treated retinal pigment epithelium cells by WST-1 tests and in vivo by following histopathological changes in rats after injection of Fe3O4@AuNPs. Results: Stable Fe3O4@AuNPs were successfully prepared following a simple and fast protocol (<1h worktime) and identified using TEM. The cytotoxicity tests on cells have shown biocompatibility of Fe3O4@AuNPs at small concentrations of Fe (<1.95×10-8 mg/cell). Whereas, at higher Fe concentrations (eg 7.5×10-8 mg/cell), cell viability decreased to 80.88±5.03%, showing a mild cytotoxic effect. MRI tests on rats showed an optimal Fe3O4@AuNPs concentration of 6mg/100g body weight to obtain high-quality images. The histopathological studies revealed significant transient inflammatory responses in the time range from 2 hours to 14 days after injection and focal cellular alterations in several organs, with the lung being the most affected organ. These results were confirmed by hyperspectral microscopic imaging of the same, but unstained tissues. In most organs, the inflammatory responses and sublethal cellular damage appeared to be transitory, except for the kidneys, where the glomerular damage indicated progression towards glomerular sclerosis. Conclusion: The obtained stable, gold covered, iron oxide nanoparticles with reduced cytotoxicity, gave a negative T2 signal in the MRI, which makes them suitable for candidates as contrast agent in small animal MRI applications.


Assuntos
Meios de Contraste/química , Compostos Férricos/química , Ouro/química , Imagem por Ressonância Magnética , Nanopartículas Metálicas/química , Animais , Sobrevivência Celular , Endocitose , Inflamação/patologia , Masculino , Nanopartículas Metálicas/ultraestrutura , Ratos Wistar , Difração de Raios X
12.
Int J Nanomedicine ; 15: 4825-4845, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32753868

RESUMO

Background: Nanosized drug delivery systems (NDDSs) have shown excellent prospects in tumor therapy. However, insufficient penetration of NDDSs has significantly impeded their development due to physiological instability and low passive penetration efficiency. Methods: Herein, we prepared a core cross-linked pullulan-modified nanosized system, fabricated by visible-light-induced diselenide bond cross-linked method for transporting ß-Lapachone and doxorubicin prodrug (boronate-DOX, BDOX), to improve the physiological stability of the NDDSs for efficient passive accumulation in tumor blood vessels (ß-Lapachone/BDOX-CCS). Additionally, ultrasound (US) was utilized to transfer ß-Lapachone/BDOX-CCS around the tumor vessel in a relay style to penetrate the tumor interstitium. Subsequently, ß-Lapachone enhanced ROS levels by overexpressing NQO1, resulting in the transformation of BDOX into DOX. DOX, together with abundant levels of ROS, achieved synergistic tumor therapy. Results: In vivo experiments demonstrated that ultrasound (US) + cross-linked nanosized drug delivery systems (ß-Lapachone/BDOX-CCS) group showed ten times higher DOX accumulation in the tumor interstitium than the non-cross-linked (ß-Lapachone/BDOX-NCS) group. Conclusion: Thus, this strategy could be a promising method to achieve deep penetration of NDDSs into the tumor.


Assuntos
Doxorrubicina/uso terapêutico , Nanopartículas/química , Naftoquinonas/uso terapêutico , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Ultrassonografia , Animais , Ácidos Borônicos/química , Permeabilidade Capilar/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Feminino , Glucanos/química , Células Hep G2 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftoquinonas/farmacocinética , Tamanho da Partícula , Pró-Fármacos/farmacocinética , Espécies Reativas de Oxigênio/metabolismo , Distribuição Tecidual/efeitos dos fármacos
13.
Int J Nanomedicine ; 15: 4877-4898, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32753869

RESUMO

Background: Although dynamics and uses of modified nanoparticles (NPs) as orally administered macromolecular drugs have been researched for many years, measures of molecule stability and aspects related to important transport-related mechanisms which have been assessed in vivo remain as relatively under characterized. Thus, our aim was to develop a novel type of oral-based delivery system for insulin and to overcome barriers to studying the stability, transport mechanisms, and efficacy in vivo of the delivery system. Methods: NPs we developed and tested were composed of insulin (INS), dicyandiamide-modified chitosan (DCDA-CS), cell-penetrating octaarginine (r8), and hydrophilic hyaluronic acid (HA) and were physically constructed by electrostatic self-assembly techniques. Results: Compared to free-insulin, levels of HA-DCDA-CS-r8-INS NPs were retained at more desirable measures of biological activity in our study. Further, our assessments of the mechanisms for NPs suggested that there were high measures of cellular uptake that mainly achieved through active transport via lipid rafts and the macropinocytosis pathway. Furthermore, investigations of NPs indicated their involvement in caveolae-mediated transport and in the DCDA-CS-mediated paracellular pathway, which contributed to increasing the efficiency of sequential transportation from the apical to basolateral areas. Accordingly, high efficiency of absorption of NPs in situ for intestinal loop models was realized. Consequently, there was a strong induction of a hypoglycemic effect in diabetic rats of NPs via orally based administrations when compared with measures related to free insulin. Conclusion: Overall, the dynamics underlying and influenced by HA-DCDA-CS-r8-INS may hold great promise for stability of insulin and could help overcome interference by the epithelial barrier, and thus showing a great potential to improve the efficacy of orally related treatments.


Assuntos
Quitosana/química , Ácido Hialurônico/química , Insulina/administração & dosagem , Nanopartículas Multifuncionais/química , Nanopartículas/química , Administração Oral , Animais , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Morte Celular/efeitos dos fármacos , Quitosana/síntese química , Diabetes Mellitus Experimental/tratamento farmacológico , Impedância Elétrica , Endocitose/efeitos dos fármacos , Guanidinas/síntese química , Guanidinas/química , Humanos , Ácido Hialurônico/síntese química , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Insulina/uso terapêutico , Absorção Intestinal/efeitos dos fármacos , Masculino , Muco/metabolismo , Nanopartículas/ultraestrutura , Ratos , Solubilidade , Suínos
14.
Cell Physiol Biochem ; 54(4): 767-790, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32830930

RESUMO

The pandemic of the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 at the end of 2019 marked the third outbreak of a highly pathogenic coronavirus affecting the human population in the past twenty years. Cross-species zoonotic transmission of SARS-CoV-2 has caused severe pathogenicity and led to more than 655,000 fatalities worldwide until July 28, 2020. Outbursts of this virus underlined the importance of controlling infectious pathogens across international frontiers. Unfortunately, there is currently no clinically approved antiviral drug or vaccine against SARS-CoV-2, although several broad-spectrum antiviral drugs targeting multiple RNA viruses have shown a positive response and improved recovery in patients. In this review, we compile our current knowledge of the emergence, transmission, and pathogenesis of SARS-CoV-2 and explore several features of SARS-CoV-2. We emphasize the current therapeutic approaches used to treat infected patients. We also highlight the results of in vitro and in vivo data from several studies, which have broadened our knowledge of potential drug candidates for the successful treatment of patients infected with and discuss possible virus and host-based treatment options against SARS-CoV-2.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Betacoronavirus/fisiologia , Coronaviridae/patogenicidade , Infecções por Coronaviridae/epidemiologia , Infecções por Coronaviridae/virologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Infecções por Coronavirus/transmissão , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/prevenção & controle , Citocinas/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Previsões , Genoma Viral , Saúde Global , Humanos , Imunidade Coletiva , Imunização Passiva , Pandemias/prevenção & controle , Peptídeo Hidrolases/farmacologia , Peptídeo Hidrolases/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , RNA Viral/genética , Receptores Virais/antagonistas & inibidores , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Virais , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Zoonoses
15.
Nat Commun ; 11(1): 4261, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848136

RESUMO

Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis.


Assuntos
Neoplasias da Mama/metabolismo , Comunicação Celular/fisiologia , Vesículas Extracelulares/metabolismo , Integrina beta3/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Endocitose , Feminino , Quinase 1 de Adesão Focal/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos , Camundongos Nus , Modelos Biológicos , Metástase Neoplásica/patologia , Transplante de Neoplasias
16.
Virology ; 548: 136-151, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838935

RESUMO

Bovine herpesvirus envelope glycoprotein E (gE) and, in particular, the gE cytoplasmic tail (CT) is a virulence determinant in cattle. Also, the gE CT contributes to virus cell-to-cell spread and anterograde neuronal transport. In this study, our goal was to map the gE CT sub-domains that contribute to virus cell-to-cell spread property. A panel of gE-CT specific mutant viruses was constructed and characterized, in vitro, with respect to their plaque phenotypes, gE recycling and gE basolateral membrane targeting. The results revealed that disruption of the tyrosine-based motifs, 467YTSL470 and 563YTVV566, individually produced smaller plaque phenotypes than the wild type. However, they were slightly larger than the gE CT-null virus plaques. The Y467A mutation affected the gE endocytosis, gE trans-Golgi network (TGN) recycling, and gE virion incorporation properties. However, the Y563A mutation affected only the gE basolateral cell-surface redistribution function. Notably, the simultaneous Y467A/Y563A mutations produced gE CT-null virus-like plaque phenotypes.


Assuntos
Doenças dos Bovinos/virologia , Citoplasma/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Bovinos , Endocitose , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/genética , Proteínas Virais/genética , Rede trans-Golgi/virologia
17.
Int J Nanomedicine ; 15: 4899-4918, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764924

RESUMO

Purpose: The use of chemotherapeutic agents to combat cancer is accompanied by high toxicity due to their inability to discriminate between cancer and normal cells. Therefore, cancer therapy research has focused on the targeted delivery of drugs to cancer cells. Here, we report an in vitro study of folate-poly(ethylene glycol)-poly(propylene succinate) nanoparticles (FA-PPSu-PEG-NPs) as a vehicle for targeted delivery of the anticancer drug paclitaxel in breast and cervical cancer cell lines. Methods: Paclitaxel-loaded-FA-PPSu-PEG-NPs characterization was performed by in vitro drug release studies and cytotoxicity assays. The NPs cellular uptake and internalization mechanism were monitored by live-cell imaging in different cancer cell lines. Expression of folate receptor-α (FOLR1) was examined in these cell lines, and specific FOLR1-mediated entry of the FA-PPSu-PEG-NPs was investigated by free folic acid competition. Using inhibitors for other endocytic pathways, alternative, non-FOLR1 dependent routes for NPs uptake were also examined. Results: Drug release experiments of Paclitaxel-loaded PPSu-PEG-NPs indicated a prolonged release of Paclitaxel over several days. Cytotoxicity of Paclitaxel-loaded PPSu-PEG-NPs was similar to free drug, as monitored in cancer cell lines. Live imaging of cells treated with either free Paclitaxel or Paclitaxel-loaded PPSu-PEG-NPs demonstrated tubulin-specific cell cycle arrest, with similar kinetics. Folate-conjugated NPs (FA-PPSu-PEG-NPs) targeted the FOLR1 receptor, as shown by free folic acid competition of the FA-PPSu-PEG-NPs cellular uptake in some of the cell lines tested. However, due to the differential expression of FOLR1 in the cancer cell lines, as well as the intrinsic differences between the different endocytic pathways utilized by different cell types, other mechanisms of nanoparticle cellular entry were also used, revealing that dynamin-dependent endocytosis and macropinocytosis pathways mediate, at least partially, cellular entry of the FA-PPSu-PEG NPs. Conclusion: Our data provide evidence that Paclitaxel-loaded-FA-PPSu-PEG-NPs can be used for targeted delivery of the drug, FA-PPSu-PEG-NPs can be used as vehicles for other anticancer drugs and their cellular uptake is mediated through a combination of FOLR1 receptor-specific endocytosis, and macropinocytosis. The exploration of the different cellular uptake mechanisms could improve treatment efficacy or allow a decrease in dosage of anticancer drugs.


Assuntos
Antineoplásicos/química , Portadores de Fármacos/química , Ácido Fólico/química , Nanopartículas/química , Poliésteres/química , Polietilenoglicóis/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Endocitose/efeitos dos fármacos , Receptor 1 de Folato/metabolismo , Ácido Fólico/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Paclitaxel/química , Paclitaxel/farmacologia
18.
Cells ; 9(9)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854430

RESUMO

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.


Assuntos
Inibidores de Adenilil Ciclases/uso terapêutico , Adenilil Ciclases/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , AMP Cíclico/antagonistas & inibidores , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
19.
Arch Virol ; 165(10): 2165-2176, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32740830

RESUMO

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.


Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Infecções por Vírus de RNA/genética , Apoptose/genética , Autofagia/genética , Autofagia/imunologia , Citocinas/genética , Citocinas/imunologia , Endocitose/genética , Endocitose/imunologia , Filoviridae/genética , Filoviridae/metabolismo , Filoviridae/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Interferons/genética , Interferons/imunologia , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Orthomyxoviridae/patogenicidade , Paramyxoviridae/genética , Paramyxoviridae/metabolismo , Paramyxoviridae/patogenicidade , Fosfatidilinositol 3-Quinase/imunologia , Pneumovirinae/genética , Pneumovirinae/metabolismo , Pneumovirinae/patogenicidade , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Rhabdoviridae/genética , Rhabdoviridae/metabolismo , Rhabdoviridae/patogenicidade , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/imunologia , Internalização do Vírus , Replicação Viral
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