RESUMO
We report here a new class of biological reagents derived from luminescent rhenium(I) polypyridine complexes modified with a poly(ethylene glycol) (PEG) pendant. The PEG-amine complexes [Re(N(^)N)(CO)(3)(py-PEG-NH(2))](PF(6)) (py-PEG-NH(2) = 3-amino-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine, MW(PEG) = 5000 Da, PDI(PEG) < 1.08; N(^)N = 1,10-phenanthroline (phen) (1-PEG-NH(2)), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me(4)-phen) (2-PEG-NH(2)), 4,7-diphenyl-1,10-phenanthroline (Ph(2)-phen) (3-PEG-NH(2))) and [Re(bpy-PEG)(CO)(3)(py-NH(2))](PF(6)) (bpy-PEG = 4-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine; py-NH(2) = 3-aminopyridine) (4-PEG-NH(2)) have been synthesized and characterized. The photophysical properties, lipophilicity, water solubility, cytotoxic activity, and cellular uptake properties of these complexes have been compared to those of their PEG-free counterparts [Re(N(^)N)(CO)(3)(py-Et-NH(2))](PF(6)) (py-Et-NH(2) = 3-amino-5-(N-(ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-Et-NH(2)), Me(4)-phen (2-Et-NH(2)), Ph(2)-phen (3-Et-NH(2))) and [Re(bpy-Et)(CO)(3)(py-NH(2))](PF(6)) (bpy-Et = 4-(N-(ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine) (4-Et-NH(2)). The PEG complexes exhibited significantly higher water solubility and lower cytotoxicity (IC(50) = 6.6 to 1152 µM) than their PEG-free counterparts (IC(50) = 3.6 to 159 µM), indicating that the covalent attachment of a PEG pendant to rhenium(I) polypyridine complexes is an effective way to increase their biocompatibility. The amine complexes 1-PEG-NH(2)-4-PEG-NH(2) have been activated with thiophosgene to yield the isothiocyanate complexes [Re(N(^)N)(CO)(3)(py-PEG-NCS)](PF(6)) (py-PEG-NCS = 3-isothiocyanato-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-NCS), Me(4)-phen (2-PEG-NCS), Ph(2)-phen (3-PEG-NCS)), and [Re(bpy-PEG)(CO)(3)(py-NCS)](PF(6)) (py-NCS = 3-isothiocyanatopyridine) (4-PEG-NCS) as a new class of luminescent PEGylation reagents. To examine their PEGylation properties, these isothiocyanate complexes have been reacted with a model substrate n-butylamine, resulting in the formation of the thiourea complexes [Re(N(^)N)(CO)(3)(py-PEG-Bu)](PF(6)) (py-PEG-Bu = 3-n-butylthioureidyl-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-Bu), Me(4)-phen (2-PEG-Bu), Ph(2)-phen (3-PEG-Bu)), and [Re(bpy-PEG)(CO)(3)(py-Bu)](PF(6)) (py-Bu = 3-n-butylthioureidylpyridine) (4-PEG-Bu). Additionally, bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been PEGylated with the isothiocyanate complexes to yield bioconjugates 1-PEG-BSA-4-PEG-BSA and 1-PEG-PEI-4-PEG-PEI, respectively. Upon irradiation, all the PEGylated BSA and PEI conjugates exhibited intense and long-lived emission in aqueous buffer under ambient conditions. The DNA-binding and polyplex-formation properties of conjugate 3-PEG-PEI have been studied and compared with those of unmodified PEI. Furthermore, the in vivo toxicity of complex 3-PEG-NH(2) and its PEG-free counterpart 3-Et-NH(2) has been investigated using zebrafish embryos as an animal model. Embryos treated with the PEG complex at high concentrations revealed delayed hatching, which has been ascribed to hypoxia as a result of adhering of the complex to the external surface of the chorion.
Assuntos
Complexos de Coordenação/síntese química , Polietilenoglicóis/química , Rênio/química , Água/química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacocinética , Embrião não Mamífero/efeitos dos fármacos , Células HeLa , Humanos , Concentração Inibidora 50 , Luminescência , Microscopia Confocal , Modelos Animais , Estrutura Molecular , Fotoquímica , Solubilidade , Peixe-ZebraRESUMO
Multidrug resistance (MDR) is a major clinical obstacle to the success of cancer chemotherapy. Here we developed a gold-doxorubicin (DOX) nanoconjugates system to overcome MDR. Gold nanoparticles (AuNPs) were first PEGylated as Au-PEG-NH(2), and DOX was then grafted onto AuNPs via a cleavable disulfide linkage (Au-PEG-SS-DOX). Confocal images revealed that the extent of intracellular uptake of Au-PEG-SS-DOX was greater than that of free DOX in the MDR cells, and inductively coupled plasma mass spectroscopy analysis further confirmed that AuNPs significantly increased the level of drug accumulation in MDR cells at a nanoparticles dose greater than 15 µM. The cytotoxicity study demonstrated that the Au-PEG-SS-DOX nanoconjugates system efficiently released the anticancer drug DOX and enhanced its cytotoxicity against MDR cancer cells. This study highlights the potential of using AuNPs for overcoming of MDR in cancer chemotherapy. FROM THE CLINICAL EDITOR: This study demonstrates that gold nanoparticles can be successfully applied to overcome MDR in cancer chemotherapy.
Assuntos
Doxorrubicina/farmacologia , Portadores de Fármacos/química , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Ouro/química , Nanopartículas Metálicas/química , Doxorrubicina/química , Portadores de Fármacos/efeitos adversos , Ouro/efeitos adversos , Células Hep G2 , Humanos , Nanopartículas Metálicas/efeitos adversos , Microscopia Confocal , Estrutura Molecular , Polietilenoglicóis/químicaRESUMO
The acquisition of multidrug resistance poses a serious problem in chemotherapy, and new types of transporters have been actively sought to overcome it. In the present study, poly(ethylene glycol)-conjugated (PEGylated) multi-walled carbon nanotubes (MWCNTs) were prepared and explored as drug carrier to overcome multidrug resistance. The prepared PEGylated MWCNTs penetrated into mammalian cells without damage plasma membrane, and its accumulation did not affect cell proliferation and cell cycle distribution. More importantly, PEGylated MWCNTs accumulated in the multidrug-resistant cancer cells as efficient as in the sensitive cancer cells. Intracellular translocation of PEGylated MWCNTs was visualized in both multidrug-resistant HepG2-DR cells and sensitive HepG2 cells, as judged by both fluorescent and transmission electron microscopy. PEGylated MWCNTs targeted cancer cells efficiently and multidrug-resistant cells failed to remove the intracellular MWCNTs. However, if used in combination with drugs without conjugation, PEGylated MWCNTs prompted drug efflux in MDR cells by stimulating the ATPase activity of P-glycoprotein. This study suggests that PEGylated MWCNTs can be developed as an efficient drug carrier to conjugate drugs for overcoming multidrug resistance in cancer chemotherapy.
Assuntos
Portadores de Fármacos/química , Resistencia a Medicamentos Antineoplásicos , Nanotubos de Carbono/química , Polietilenoglicóis/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antibióticos Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Células Hep G2 , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de FluorescênciaRESUMO
The accurate delivery of precise amounts of drugs to a specific location can considerably affect various clinical applications. The precise control of drug amount and position is crucial to a successful drug delivery. This paper proposes the use of poly(lactide-co-glycolicacid) (PLGA)-based microspheres to contain precise amounts of drugs and an optical tweezer manipulator to transport these drug-containing microspheres to their targeted sites in vivo. The drugs were delivered by the PLGA-based microspheres to the yolk sac of zebrafish embryos, and a sustained drug release was observed to examine the anti-angiogenesis and angiogenesis activities. The PLGA-based microspheres degraded in zebrafish, thereby verifying that these microspheres can be used as drug carriers in vivo to ensure good biocompatibility and biodegradation. The proposed precise drug delivery approach can be used in protein tests and drug property characterization in vivo.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Microesferas , Pinças Ópticas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Indutores da Angiogênese/farmacocinética , Indutores da Angiogênese/farmacologia , Animais , Preparações de Ação Retardada , Embrião não Mamífero/metabolismo , Microscopia de Fluorescência , Neovascularização Fisiológica/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacocinética , Fator A de Crescimento do Endotélio Vascular/farmacocinética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Peixe-ZebraRESUMO
Tea is a popular beverage consumed at different temperatures. The effect of tea on teeth at different temperatures has not been studied previously. The present study used an in vitro green tea immersed tooth model at different tea temperatures (hot and cold) compared to an in vivo tea administration model allowing rats to drink tea over the course of a week. The elements present in tea leaves were identified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and compared to the elements in teeth (enamel surface) using Laser-Induced Breakdown Spectroscopy (LIBS). Here, LIBS demonstrated in vivo and in vitro green tea treatments resulted in a significant increase in the mineral elements found in enamel. For the in vitro assessment, elements in enamel varied based on cold-tea and hot-tea treatment; however, hot water reduced the elements in enamel. Atomic force microscopy found the in vivo tea group had a higher roughness average (RA) compared with the in vivo water group. Cold tea and hot tea in vitro groups demonstrated lower RA than in vitro water controls. Scanning electron microscopy found hot water induced cracks more than 1.3µm in enamel while cold tea and hot tea promoted the adhering of extrinsic matter to teeth. Overall, teeth treated to high temperature lost the mineral phase leading to demineralization. Our results indicate that green tea protects enamel, but its protective action in dental structures is enhanced at cold temperature.
Assuntos
Camellia sinensis/química , Extratos Vegetais/administração & dosagem , Chá/química , Dente/ultraestrutura , Animais , Temperatura Baixa , Temperatura Alta , Masculino , Espectrometria de Massas , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Modelos Animais , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Ratos , Propriedades de Superfície , Dente/efeitos dos fármacosRESUMO
Free gold nanoparticles easily aggregate when the environment conditions change. Here, gold nanoparticles (AuNPs) with average diameter of 3.7 nm were prepared and then modified with poly(ethylene glycol) (PEG) to improve stability. The gold nanoparticles were first surface-modified with 3-mercaptopropionic acid (MPA) to form a self-assembled monolayer and subsequently conjugated with NH(2)-PEG-NH(2) through amidation between the amine end groups on PEG and the carboxylic acid groups on the particles. The biocompatibility and intracellular fate of PEG-modified gold nanoparticles (AuNP@MPA-PEG) were then studied in human cervical cancer (HeLa) cells. Cell viability test showed that AuNP@MPA-PEG did not induce obvious cytotoxicity. Both confocal laser scanning microscopy and transmission electron microscopy demonstrated that AuNP@MPA-PEG entered into mammalian cells and the cellular uptake of AuNP@MPA-PEG was time-dependent. Inductively coupled plasma mass spectrometry and confocal microscopy imaging further demonstrated that AuNP@MPA-PEG penetrated into the nucleus of mammalian cells upon exposure for 24 h. These results suggest that surface modification can enhance the stability and improve the biocompatibility. This study also indicates that AuNP@MPA-PEG can be used as potential nuclear targeted drug delivery carrier.
Assuntos
Núcleo Celular/metabolismo , Ouro/toxicidade , Nanopartículas/toxicidade , Polietilenoglicóis/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Esquema de Medicação , Fluoresceína-5-Isotiocianato , Ouro/química , Células HeLa , Humanos , Estrutura Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Polietilenoglicóis/toxicidadeRESUMO
A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser-scanning confocal microscopy. The microscopy images indicated that complex 3a was localized in the perinuclear region upon interiorization. Temperature-dependence experiments suggested that the internalization of the complex was an energy-requiring process such as endocytosis. This has been confirmed by cellular-uptake experiments involving the luminescent conjugates Ir-BSA and Ir-TF (TF = holo-transferrin), which were prepared by conjugation of the proteins with the complex [Ir(pq)(2)(phen-NCS)](PF(6)) (phen-NCS = 5-isothiocyanato-1,10-phenanthroline).
Assuntos
Indóis/química , Irídio/química , Luminescência , Compostos Organometálicos/síntese química , Compostos Organometálicos/metabolismo , Polímeros/química , Piridinas/química , Absorção , Animais , Bovinos , Eletroquímica , Elétrons , Citometria de Fluxo , Células HeLa , Humanos , Microscopia Confocal , Compostos Organometálicos/química , Compostos Organometálicos/toxicidade , Ligação Proteica , Soroalbumina Bovina/metabolismo , TitulometriaRESUMO
The mechanism of coffee eliciting erosion on teeth is unclear as few studies have investigated the direct effect of coffee on enamel and dentin structures. The present study identified how coffee, the most popular beverage worldwide, induces staining and erosion on teeth. We show the grade of erosion of molars and incisors in Sprague Dawley rats from two different age groups, young (four weeks) and old (six months). We quantified the concentration of metals contained in coffee by mass spectrometry (ICP-MS). To determine elemental content in enamel (i.e. superficial) and dentin (i.e. substructure), we used Laser-induced Breakdown Spectroscopy (LIBS) and X-ray fluorescence (XRF) spectroscopy, respectively. For LIBS, a significant decrease of Ca, P, and Na was observed in the young coffee group relative to age-matched controls, whereas a significant increase in Mn, Fe, and K was observed. In the old coffee group, a significant increase of Mg, Fe, and K was observed along with a decrease of Mg, Ca, P, Na, Sr and Zn. For XRF, a significant decrease of the Ca/P ratio in the coffee group was observed. Spectroscopy results were correlated with scanning electron microscopy (SEM) and histological analysis. The SEM analysis showed pores and open spaces between young and old coffee groups, respectively. Thinning of enamel layers, loss of continuity in the enamel-dentin-junction, and wide spaces in dentin tubules with coffee use was found histologically. Coffee induces decalcification of teeth that corresponds to erosion, exposing the dentin structure by reducing enamel. Coffee immersion demonstrated an intrinsic staining in dentin by metal deposition.
Assuntos
Café/química , Esmalte Dentário/química , Dentina/química , Minerais/análise , Análise Espectral , Animais , Cafeína/farmacologia , Esmalte Dentário/efeitos dos fármacos , Dentina/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Gold nanoparticles have been widely explored as cancer therapeutics and diagnostic agents in recent years. With their unique subcellular size and good biocompatibility, gold nanoparticles are a promising drug delivery vehicle. In this study, folic acid-coated gold nanoparticles conjugated with fluorophore FITC through amine terminated poly(ethylene glycol) were prepared and confocal microscopy together with bright-field differential interference contrast imaging data showed that folic acid-coated gold nanoparticles accumulated mainly in cytoplasm of primary human fibroblasts, without causing any observable cytotoxicity upon exposure for 48 hours. Through the further development of a drug delivery system that conjugates doxorubicin onto the surface of gold nanoparticles with a poly(ethylene glycol) spacer via an SMCC linker, we demonstrated that multidrug resistance in cancer cells can be significantly overcome by a combination of highly efficient cellular entry and enhanced cytotoxicity of Au-SMCC-DOX nanoconjugates, as revealed both by confocal microscopy imaging and cytotoxicity assay. The prepared Au-SMCC-DOX nanoconjugates demonstrated enhanced drug accumulation and retention in multidrug resistant hepG2-R cancer cells when it was compared with free doxorubicin, with a cytoplasm accumulation profile. The results indicated that gold nanoparticles are a kind of promising drug delivery vehicle with good biocompatibility and suitable for further applications in drug delivery for improved chemotherapy, especially for overcoming multidrug resistance.
Assuntos
Materiais Revestidos Biocompatíveis , Sistemas de Liberação de Medicamentos , Ouro/química , Nanopartículas Metálicas/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/uso terapêutico , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/farmacocinética , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Hep G2 , Humanos , Teste de Materiais , Nanopartículas Metálicas/química , Modelos Biológicos , Nanoconjugados/administração & dosagem , Nanoconjugados/química , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Propriedades de SuperfícieRESUMO
We present a new class of phosphorescent cyclometalated iridium(III) polypyridine poly(ethylene glycol) (PEG) complexes [Ir(N(^)C)2(bpy-CONH-PEG)](PF6) (bpy-CONH-PEG = 4-(N-(2-(ω-methoxypoly-(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine, number average molecular weight (Mn) = 5272.23, weight average molecular weight (Mw) = 5317.38, polydispersity index (PDI) = 1.009; HN(^)C = 2-phenylpyridine, Hppy (1a), 2-((1,1'-biphenyl)-4-yl)pyridine, Hpppy (2a), 2-phenylquinoline, Hpq (3a), 2-phenylbenzothiazole, Hbt (4a), 2-(1-naphthyl)benzothiazole, Hbsn (5a)). The photophysical, photochemical, and biological properties of these complexes have been compared with those of their PEG-free counterparts [Ir(N(^)C)2(bpy-CONH-Et)](PF6) (bpy-CONH-Et = 4-(N-ethylaminocarbonyl)-4'-methyl-2,2'-bipyridine; HN(^)C = Hppy (1b), Hpppy (2b), Hpq (3b), Hbt (4b), Hbsn (5b)). Upon irradiation, all the complexes exhibited intense and long-lived green to orange-red emission under ambient conditions. The emission was phosphorescence in nature and can be quenched by O2 with the generation of singlet oxygen ((1)O2). The quantum yields for (1)O2 production of the complexes in aerated DMSO (0.24-0.83) were found to be dependent on the excited-state lifetimes of the complexes, which can be altered using different cyclometalating ligands (N(^)C). Cell-based assays indicated that the PEG complexes were noncytotoxic in the dark (IC50 > 300 µM); however, most of them became significantly cytotoxic upon irradiation (IC50 = 3.4 - 23.2 µM). Laser-scanning confocal microscopy images revealed localization of complex 3a in the mitochondrial region of HeLa cells and the induction of rapid necrotic cell death upon light activation. Additionally, the lack of dark toxicity and potential application of the PEG complexes as a visualizing reagent have been demonstrated using zebrafish (Danio rerio) as an animal model.
Assuntos
Irídio/farmacologia , Mitocôndrias/metabolismo , Fotoquimioterapia , Polietilenoglicóis/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Cisplatino/farmacologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Células HeLa , Humanos , Concentração Inibidora 50 , L-Lactato Desidrogenase/metabolismo , Lipídeos/química , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Espectrometria de Fluorescência , Distribuição Tecidual/efeitos dos fármacos , Peixe-ZebraRESUMO
In this paper, we describe a new biodegradable composite composed of polycaprolactone and magnesium. Incorporation of magnesium micro-particles into the polycaprolactone matrix yields mechanical properties close to those of human cancellous bone, and in vitro studies indicate that the silane-coated Mg/PCL composites have excellent cytocompatibility and osteoblastic differentiation properties. The bioactivity of the composites is manifested by the formation of calcium and phosphate after immersion in simulated body fluids. The bulk mechanical properties can be maintained for 2 months before obvious degradation takes place. The in vivo animal study reveals a larger amount of new bone formation on the silane-coated Mg/PCL composites compared to conventional PMMA and pure polycaprolactone and our results suggest potential clinical applications of the sliane-coated Mg/PCL composites.
Assuntos
Substitutos Ósseos/química , Materiais Revestidos Biocompatíveis/química , Magnésio/química , Fraturas por Osteoporose/cirurgia , Poliésteres/química , Implantes Absorvíveis , Animais , Substitutos Ósseos/metabolismo , Linhagem Celular , Materiais Revestidos Biocompatíveis/metabolismo , Feminino , Humanos , Magnésio/metabolismo , Teste de Materiais , Camundongos , Osteogênese , Poliésteres/metabolismo , Ratos , Silanos/química , Silanos/metabolismo , Estresse MecânicoRESUMO
The original hedgehog (hh) gene was found in Drosophila and named for the appearance of a mutant phenotype which causes an embryo to be covered with pointy denticles, thus resembling a hedgehog. The hedgehog family consists of sonic hedgehog (Shh), desert hedgehog (Dhh), and Indian hedgehog (Ihh). Shh is found in vertebrates and the best studied ligand of the hedgehog signaling pathway (Gilbert, 2000). It plays an important role in regulating vertebrate organogenesis, such as in the growth of digits on limbs and organization of the brain, and earlier studies also show that it is important during retinal development (for a review, see Amato et al., 2004). Hedgehog expression drives waves of neurogenesis in animal retina, although genetic programs that control its expression are poorly elucidated. Recently, a novel transcriptional cascade which involves the atonal and Iroquois gene family was proposed in the regulation of hedgehog waves during vertebrate retinal development (Choy et al., 2010). This chapter will focus on Shh by addressing its signaling mechanisms and roles in vertebrate eye development, as well as a novel finding in retinogenesis.
Assuntos
Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Neurogênese/genética , Retina/embriologia , Transdução de Sinais , Animais , Comunicação Celular , Drosophila/genética , Drosophila/metabolismo , Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Transdução de Sinais/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Although many studies have demonstrated that cell phenotype is affected by the surface properties of biomaterials, these materials often fail to mimic the complexity of the native tissue microenvironment (TME). In this study, we have developed a new experimental model that allows the characterisation and functional reconstruction of natural TME. We discovered that mesenchymal stem cells (MSC) cultured on cryostat sections of bovine Achilles tendon adopted an elongated and aligned morphology, and expressed tenocyte marker tenomodulin (TNMD). This suggests that tendon sections contain the signalling cues that guide MSCs to commit to the tenogenic lineage. To reconstruct this instructive niche, we prepared PDMS replica by using tendon sections as template. The resulting bioimprint faithfully copied the physical topography and elasticity of the section. This replica, when coated with collagen 1, supported tenogenesis of MSC without requiring exogenous growth factors. This study illustrates how extracellular biophysical and biochemical features intertwines to form a niche that influences the cell fate and demonstrated that such complex information could be conveniently reconstructed with synthetic materials and purified extracellular matrix proteins.
Assuntos
Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Impressão Molecular/métodos , Tendões/citologia , Tendões/efeitos dos fármacos , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Crioultramicrotomia , Dimetilpolisiloxanos/química , Cães , Imunofluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Microscopia Confocal , Modelos Biológicos , Água/químicaRESUMO
Single-walled carbon nanotubes (SWNTs) have been identified as an efficient drug carrier. Here a controlled drug-delivery system based on SWNTs coated with doxorubicin (DOX) through hydrazone bonds was developed, because the hydrazone bond is more sensitive to tumor microenvironments than other covalent linkers. The SWNTs were firstly stabilized with polyethylene glycol (H(2)N-PEG-NH(2)). Hydrazinobenzoic acid (HBA) was then covalently attached on SWNTs via carbodiimide-activated coupling reaction to form hydrazine-modified SWNTs. The anticancer drug DOX was conjugated to the HBA segments of SWNT using hydrazine as the linker. The resulting hydrazone bonds formed between the DOX molecules and the HBA segments of SWNTs are acid cleavable, thereby providing a strong pH-responsive drug release, which may facilitate effective DOX release near the acidic tumor microenvironment and thus reduce its overall systemic toxicity. The DOX-loaded SWNTs were efficiently taken up by HepG2 tumor cells, and DOX was released intracellularly, as revealed by MTT assay and confocal microscope observations. Compared with SWNT-DOX conjugate formed by supramolecular interaction, the SWNT-HBA-DOX featured high weight loading and prolonged release of DOX, and thus improved its cytotoxicity against cancer cells. This study suggests that while SWNTs have great potential as a drug carrier, the efficient formulation strategy requires further study.
Assuntos
Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Preparações de Ação Retardada/farmacocinética , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Nanotubos de Carbono/química , Antibióticos Antineoplásicos/farmacocinética , Benzoatos/química , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Doxorrubicina/farmacocinética , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Polietilenoglicóis/químicaRESUMO
Lanthanide-doped upconversion nanoparticles (UCNPs) are considered promising novel near-infrared (NIR) bioimaging agents with the characteristics of high contrast and high penetration depth. However, the interactions between charged UCNPs and mammalian cells have not been thoroughly studied, and the corresponding intracellular uptake pathways remain unclear. Herein, our research work involved the use of a hydrothermal method to synthesize polyvinylpyrrolidone-coated UCNPs (UCNP-PVP), and then a ligand exchange reaction was performed on UCNP-PVP, with the help of polyethylenimine (PEI) and poly(acrylic acid) (PAA), to generate UCNP-PEI and UCNP-PAA. These polymer-coated UCNPs demonstrated good dispersibility in aqueous medium, had the same elemental composition and crystal phase, shared similar TEM and dynamic light scattering (DLS) size distribution, and exhibited similar upconversion luminescence efficiency. However, the positively charged UCNP-PEI evinced greatly enhanced cellular uptake in comparison with its neutral or negative counterparts, as shown by multiphoton confocal microscopy and inductively coupled plasma mass spectrometry (ICP-MS) measurements. Meanwhile, we found that cationic UCNP-PEI can be effectively internalized mainly through the clathrin endocytic mechanism, as revealed by colocalization, chemical, and genetic inhibitor studies. This study elucidates the role of the surface polymer coatings in governing UCNP-cell interactions, and it is the first report on the endocytic mechanism of positively charged lanthanide-doped UCNPs. Furthermore, this study provides important guidance for the development of UCNPs as specific intracellular nanoprobes, allowing us to control the UCNP-cell interactions by tuning surface properties.
Assuntos
Érbio/química , Fluoretos/química , Imagem Molecular/métodos , Nanopartículas/química , Polímeros/química , Polímeros/metabolismo , Itérbio/química , Ítrio/química , Clorpromazina/farmacologia , Clatrina/antagonistas & inibidores , Clatrina/deficiência , Clatrina/genética , Vesículas Revestidas/efeitos dos fármacos , Vesículas Revestidas/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Ligantes , Medições Luminescentes , Nanopartículas/toxicidade , Polímeros/toxicidade , Propriedades de SuperfícieRESUMO
Four luminescent ruthenium(II) polypyridine estradiol complexes [Ru(NwedgeN)2(bpy-estradiol)](PF6)2 (NwedgeN = 2,2'-bipyridine (bpy), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen); bpy-estradiol = 5-(4-(17alpha-ethynylestradiolyl)phenyl)-2,2'-bipyridine (bpy-ph-est), 4-(N-(6-(4-(17alpha-ethynylestradiolyl)benzoylamino)hexyl)aminomethyl)-4'-methyl-2,2'-bipyridine (mbpy-C6-est)) have been designed as new luminescent biological probes. The lipophilicity and photophysical and electrochemical properties of these complexes have been investigated. Upon photoexcitation, all the complexes exhibited intense and long-lived triplet metal-to-ligand charge-transfer (3MLCT) (dpi(Ru) --> pi*(diimine)) emission in fluid solutions at 298 K and in low-temperature glass. The binding of the complexes to estrogen receptor-alpha (ERalpha) has been studied by emission titrations. The Ph2-phen complexes showed emission enhancement and increased lifetimes upon binding to the protein. Additionally, the cytotoxicity of the complexes toward the HeLa cell line has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay and the IC50 values ranged from 83.1 to 166.6 microM (cisplatin showed an IC50 value of 34.3 microM under the same experimental conditions). Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser-scanning confocal microscopy.
Assuntos
Estradiol/química , Sondas Moleculares/química , Compostos Organometálicos/química , Polímeros/química , Piridinas/química , Rutênio/química , Sobrevivência Celular/efeitos dos fármacos , Eletroquímica , Células HeLa , Humanos , Luminescência , Medições Luminescentes/métodos , Sondas Moleculares/síntese química , Sondas Moleculares/farmacologia , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Estereoisomerismo , Testes de ToxicidadeRESUMO
Carbon nanotubes (CNTs) have been shown to cross cell membranes and can mediate the internalization of macromolecules. These characteristics have constituted CNTs as an exciting new tool for drug delivery and biological sensing. While CNTs exhibit great potential in biomedical and pharmaceutical applications, neither the cell penetration mechanism of CNTs nor the intracellular fate of the internalized CNTs are fully understood. In this study, time-lapse fluorescence microscopy was used to investigate the intracellular distribution of FITC labeled PEGylated single-walled CNTs (FITC-PEG-SWCNTs) in living cells and shown that PEGylated SWCNTs entered the nucleus of several mammalian cell lines in an energy-dependent process. The presence of FITC-PEG-SWCNTs in the cell nucleus did not cause discernible changes in the nuclear organization and had no effect on the growth kinetics and cell cycle distribution for up to 5 days. Remarkably, upon removal of the FITC-PEG-SWCNTs from the culture medium, the internalized FITC-PEG-SWCNTs rapidly moved out of the nucleus and were released from the cells. Thus, the intracellular PEGylated SWCNTs were highly dynamic and the cell penetration of PEGylated SWCNTs appeared as bidirectional. These observations suggest SWCNTs may be used as an ideal nanovector in biomedical and pharmaceutical applications.