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1.
Biology (Basel) ; 12(10)2023 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-37887007

RESUMO

Bisphosphonates (BPs) are successfully used to cure a number of diseases characterized by a metabolic reduction in bone density, such as Osteoporosis, or a neoplastic destruction of bone tissue, such as multiple myeloma and bone metastases. These drugs exert their therapeutic effect by causing a systemic osteoclast depletion that, in turn, is responsible for reduced bone resorption. Unfortunately, in addition to their beneficial activity, BPs can also determine a frightening side effect known as osteonecrosis of the jaw (ONJ). It is generally believed that the inability of osteoclasts to dispose of inflamed/necrotic bone represents the main physiopathological aspect of ONJ. In principle, a therapeutic strategy able to elicit a local re-activation of osteoclast production could counteract ONJ and promote the healing of its lesions. Using an experimental model of Vitamin D3-dependent osteoclastogenesis, we have previously demonstrated that Magnesium is a powerful inducer of osteoclast differentiation. Here we show that, surprisingly, this effect is greatly enhanced by the presence of Zoledronate, chosen for our study because it is the most effective and dangerous of the BPs. This finding allows us to hypothesize that Magnesium might play an important role in the topical therapy of ONJ.

2.
Biology (Basel) ; 12(7)2023 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-37508422

RESUMO

After amputation, granular hemocytes infiltrate the blastema of regenerating cephalic tentacles of the freshwater snail Pomacea canaliculata. Here, the circulating phagocytic hemocytes were chemically depleted by injecting the snails with clodronate liposomes, and the effects on the cephalic tentacle regeneration onset and on Pc-Hemocyanin, Pc-transglutaminase (Pc-TG) and Pc-Allograft Inflammatory Factor-1 (Pc-AIF-1) gene expressions were investigated. Flow cytometry analysis demonstrated that clodronate liposomes targeted large circulating hemocytes, resulting in a transient decrease in their number. Corresponding with the phagocyte depletion, tentacle regeneration onset was halted, and it resumed at the expected pace when clodronate liposome effects were no longer visible. In addition to the regeneration progress, the expressions of Pc-Hemocyanin, Pc-TG, and Pc-AIF-1, which are markers of hemocyte-mediated functions like oxygen transport and immunity, clotting, and inflammation, were modified. After the injection of clodronate liposomes, a specific computer-assisted image analysis protocol still evidenced the presence of granular hemocytes in the tentacle blastema. This is consistent with reports indicating the large and agranular hemocyte population as the most represented among the professional phagocytes of P. canaliculata and with the hypothesis that different hemocyte morphologies could exert diverse biological functions, as it has been observed in other invertebrates.

3.
Magnes Res ; 34(3): 114-129, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34859787

RESUMO

The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized.


Assuntos
Colecalciferol , Monócitos , Diferenciação Celular , Colecalciferol/farmacologia , Humanos , Magnésio/farmacologia , Células U937
4.
Mol Neurobiol ; 57(2): 589-599, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31401765

RESUMO

The majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO.


Assuntos
Apoptose , Calpaína/metabolismo , Mutação/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Dobramento de Proteína , Rodopsina/genética , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Dobramento de Proteína/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Degeneração Retiniana/patologia , Rodopsina/química , Rodopsina/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo
5.
Cell Death Dis ; 9(5): 560, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29752430

RESUMO

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Serpinas/metabolismo , Animais , Linhagem Celular , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/farmacologia , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/farmacologia , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/patologia , Serpinas/genética , Serpinas/farmacologia
6.
Molecules ; 22(6)2017 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-28598368

RESUMO

Recently, solid lipid nanoparticles (SLNs) have attracted increasing attention owing to their potential as an oral delivery system, promoting intestinal absorption in the lymphatic circulation which plays a role in disseminating metastatic cancer cells and infectious agents throughout the body. SLN features can be exploited for the oral delivery of theranostics. Therefore, the aim of this work was to design and characterise self-assembled lipid nanoparticles (SALNs) to encapsulate and stabilise iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) as a model of a theranostic tool. SALNs were characterised for physico-chemical properties (particle size, surface charge, encapsulation efficiency, in vitro stability, and heparin leakage), as well as in vitro cytotoxicity by methyl thiazole tetrazolium (MTT) assay and cell internalisation in CaCo-2, a cell line model used as an indirect indication of intestinal lymphatic absorption. SALNs of about 180 nm, which are stable in suspension and have a high encapsulation efficiency (>90%) were obtained. SALNs were able to stabilise the heparin coating of Fe@hepa, which are typically unstable in physiological environments. Moreover, SALNs-Fe@hepa showed no cytotoxicity, although their ability to be internalised into CaCo-2 cells was highlighted by confocal microscopy analysis. Therefore, the results indicated that SALNs can be considered as a promising tool to orally deliver theranostic Fe@hepa into the lymphatic circulation, although further in vivo studies are needed to comprehend further potential applications.


Assuntos
Portadores de Fármacos/química , Compostos Férricos/química , Heparina/química , Nanopartículas/química , Nanomedicina Teranóstica/métodos , Transporte Biológico , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/metabolismo , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Gorduras/química , Compostos Férricos/farmacologia , Glicerídeos/química , Heparina/metabolismo , Humanos , Absorção Intestinal , Modelos Biológicos , Nanopartículas/ultraestrutura , Óleos/química , Tamanho da Partícula , Eletricidade Estática , Propriedades de Superfície
7.
Hum Mol Genet ; 25(13): 2801-2812, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27149983

RESUMO

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.


Assuntos
Calpaína/metabolismo , Rodopsina/genética , Animais , Apoptose/genética , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio , Calpaína/genética , Modelos Animais de Doenças , Camundongos , Mutação , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/genética , Rodopsina/metabolismo
8.
Cell Cycle ; 14(16): 2578-89, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26102293

RESUMO

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.


Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Amantadina , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Expressão Gênica , Células HL-60 , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Células K562 , Leucemia Mieloide Aguda/patologia , Macrófagos/fisiologia , Piperidinas/farmacologia , Mapas de Interação de Proteínas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Tamoxifeno/farmacologia , Tretinoína/farmacologia
9.
Exp Cell Res ; 319(20): 3201-13, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-23973664

RESUMO

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.


Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Orosomucoide/metabolismo , Vitamina D/farmacologia , Perfilação da Expressão Gênica , Células HL-60 , Humanos , Macrófagos/metabolismo , Orosomucoide/genética , Orosomucoide/isolamento & purificação , Células U937
10.
Int J Pharm ; 440(2): 161-9, 2013 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22982257

RESUMO

Several advanced in vitro and in vivo studies have proved the broad potential of cationic solid lipid nanoparticles (SLN) as nonviral vectors. However, a few data are available about the correlation between lipid component of the SLN structure and in vitro performance in terms of cell tolerance and transfection efficiency on different cell lines. In this paper SLN were prepared using stearic acid as main lipid component, stearylamine as cationic agent and protamine as transfection promoter and adding phosphatidylcholine (PC), cholesterol (Chol) or both to obtain three different multicomponent SLN (SLN-PC, SLN-Chol and SLN-PC-Chol, respectively). Cytotoxicity and transfection efficiency of the obtained SLN:pDNA complexes were evaluated on three different immortalized cell lines: COS-I (African green monkey kidney cell line), HepG2 (human hepatocellular liver carcinoma cell line) and Na1300 (murine neuroblastoma cell line). Samples were characterized for the exact quantitative composition, particle size, morphology, zeta potential and pDNA binding ability. All the three SLN samples were about 250-300 nm in size with a positive zeta potential, whereas SLN:pDNA complexes were about 300-400 nm in size with a less positive zeta potential, depending on the SLN composition. Concerning the cell tolerance, the three samples showed a level of cytotoxicity lower than that of the positive control polyethylenimine (PEI), regardless of the cell lines. The best transfection performance was observed for SLN-PC-Chol on COS-I cells while a transfection level lower than PEI was observed on HepG2 cells, regardless the SLN composition. On Na1300 cells, SLN-Chol showed a double efficiency with respect to PEI. Comparing these results to those obtained with the same kind of SLN without PC and/or Chol, it is possible to conclude that the addition of Chol and/or PC to the composition of cationic SLN modify the cell tolerance and the transfection efficiency of the gene vector in a manner strictly dependent on the cell type and the internalization pathways.


Assuntos
Cátions/química , Vetores Genéticos/química , Lipídeos/química , Nanopartículas/química , Transfecção/métodos , Animais , Células COS , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA/química , Vetores Genéticos/toxicidade , Células Hep G2 , Humanos , Lipídeos/toxicidade , Camundongos , Nanopartículas/toxicidade , Tamanho da Partícula , Propriedades de Superfície
11.
Drug Deliv ; 19(1): 1-10, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22070724

RESUMO

Cationic solid lipid nanoparticles (SLN) have been recently proposed as non-viral vectors in systemic gene therapy. The aim of this study was to evaluate the effect of the protamine amount used as the transfection promoter in SLN-mediated gene delivery. Three protamine-SLN samples (Pro25, Pro100, and Pro200) prepared by adding increasing amounts of protamine were characterized for their size, zeta potential, and protamine loading level. The samples were evaluated for pDNA complexation ability by gel-electrophoresis analysis and for cytotoxicity and transfection efficiency by using different cell lines (COS-I, HepG2, and Na1300). The size of SLN was ~230 nm and only Pro200 showed few particle aggregates. Unlike the Pro25 sample with the lowest protamine loading level, the others SLN samples (Pro100 and Pro200) exhibited a good ability in complexing pDNA. A cell-line dependent cytotoxicity lower than that of the positive control PEI (polyethilenimmine) was observed for all the SLN. Among these, only Pro100, having an intermediate amount of protamine, appeared able to promote pDNA cell transfer, especially in a neuronal cell line (Na1300). In conclusion, the amount of protamine as the transfection promoter in SLN affects not only the gene delivery ability of SLN but also their capacity to transfer genes efficiently to specific cell types.


Assuntos
Desenho de Fármacos , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Nanopartículas/administração & dosagem , Protaminas/administração & dosagem , Transfecção/métodos , Animais , Células COS , Cátions , Chlorocebus aethiops , DNA/administração & dosagem , Vetores Genéticos/genética , Células Hep G2 , Humanos , Lipossomos , Protaminas/genética
12.
Invest New Drugs ; 29(1): 73-86, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19834646

RESUMO

The cellular effects of a novel DNA-intercalating agent, the bipyridyl complex of platinum(II) with diphenyl thiourea, [Pt(bipy)(Ph(2)-tu)(2)]Cl(2), has been analyzed in the cisplatin (cDDP)-sensitive human ovarian carcinoma cell line, 2008, and its -resistant variant, C13* cells, in which the highest accumulation and cytotoxicity was found among six related bipyridyl thiourea complexes. We also show here that this complex causes reactive oxygen species to form and inhibits topoisomerase II activity to a greater extent in the sensitive than in the resistant line. The impairment of this enzyme led to DNA damage, as shown by the comet assay. As a consequence, cell cycle distribution has also been greatly perturbed in both lines. Morphological analysis revealed deep cellular derangement with the presence of cellular masses, together with increased membrane permeability and depolarization of the mitochondrial membrane. Some of these effects, sometimes differentially evident between the two cell lines, might also be related to the decrease of total cell magnesium content caused by this thiourea complex both in sensitive and resistant cells, though the basal content of this ion was higher in the cDDP-resistant line. Altogether these results suggest that this compound exerts its cytotoxicity by mechanisms partly mediated by the resistance phenotype. In particular, cDDP-sensitive cells were affected mostly by impairing topoisomerase II activity and by increasing membrane permeability and the formation of reactive oxygen species; conversely, mitochondrial impairment appeared to play the most important role in the action of complex F in resistant cells.


Assuntos
2,2'-Dipiridil/farmacologia , Cisplatino/farmacologia , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Compostos Organoplatínicos/farmacologia , Neoplasias Ovarianas/patologia , 2,2'-Dipiridil/química , Ciclo Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Substâncias Intercalantes/química , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Magnésio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos Organoplatínicos/química , Neoplasias Ovarianas/enzimologia , Espécies Reativas de Oxigênio/metabolismo
13.
Ther Deliv ; 2(11): 1419-35, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22826874

RESUMO

BACKGROUND: The ability to efficiently cross cellular barriers and accomplish high-level transgene expression is a critical challenge to broad application of nonviral vectors, such as cationic solid lipid nanoparticles (SLN). AIMS: This study aims to design and characterize in vitro multicomposite SLN as a novel platform for pDNA delivery. RESULTS/DISCUSSION: The distribution of each component (stearic acid, stearylamine, phosphatidylcholine, cholesterol, protamine and Pluronic F68) in the SLN matrix was studied by electron spectroscopy for chemical analysis and NMR in order to establish its influence on SLN cytotoxicity and transfection efficiency. Multicomposite SLN mediated the expression of enhanced green fluorescent protein in a way comparable with the positive control, but inducing a lower cytotoxicity. Moreover, the carrier exhibited the ability to enter the nucleoli, probably as a result of the synergic action of the nuclear localization signal of protamine and the flexibility of the lipid matrix owing to the phosphatidylcholine. CONCLUSION: The multicomposite SLN showed good transfection efficiency and negligible cytotoxicity, both crucial factors for an efficient gene-delivery system. Considering the fact that nucleoli have emerged in recent years as important targets in many fields, this novel carrier could have significant future therapy involvements whenever there is a requirement to overcome subcellular barriers. However, further work needs to be carried out in order to fully characterize the formulation, to elucidate where alternative colloidal structures might exist and play a role in obtaining the results presented.


Assuntos
DNA/administração & dosagem , Lipídeos/química , Nanopartículas , Transfecção , Animais , Células COS , Cátions , Nucléolo Celular/metabolismo , Chlorocebus aethiops , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia Fotoeletrônica/métodos , Plasmídeos
14.
Drug Deliv ; 18(1): 26-37, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20726809

RESUMO

Polysaccharide microparticles for the oral administration of gentamicin were designed in order to obtain an increased drug absorption by means of microparticle transport across the intestinal epithelia. Alginate/chitosan microparticles with a size of ~2 µm were developed by spray-drying a water solution containing the drug complexed with the polyanionic alginate and subsequent alginate cross-linking process by calcium ions and chitosan. The pre-formulation study, performed by changing the concentration of both cross-linkers, led to the selection of the most suitable formulation which was assayed for its capacity to be translocated across intestinal epithelia, via both M cells contained in Follicle Associated Epithelium (FAE) of Peyer's patches and enterocytes of the mucosal epithelium. An ex vivo perfusion technique of rabbit and rat intestinal tissues containing Peyer's patches combined with an in vitro method by using Caco-2 cell monolayers demonstrated the microparticulate carrier ability to be taken up by both M cells and enterocytes. However, only the endocytosis by M cells appeared to provide the microparticle transport from the epithelium toward deeper sub-epithelial regions.


Assuntos
Alginatos/química , Cloreto de Cálcio/química , Quitosana/química , Gentamicinas/química , Gentamicinas/farmacocinética , Mucosa Intestinal/metabolismo , Administração Oral , Alginatos/administração & dosagem , Animais , Transporte Biológico , Células CACO-2 , Química Farmacêutica/métodos , Quitosana/administração & dosagem , Endocitose , Gentamicinas/administração & dosagem , Ácido Glucurônico/administração & dosagem , Ácido Glucurônico/química , Ácidos Hexurônicos/administração & dosagem , Ácidos Hexurônicos/química , Humanos , Absorção Intestinal , Intestino Delgado/metabolismo , Microesferas , Tamanho da Partícula , Nódulos Linfáticos Agregados/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley
15.
Eur J Pharm Biopharm ; 76(3): 384-93, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20691262

RESUMO

Protamine has attracted much attention as DNA condenser and nuclear transfer enhancer although the excess of hydrophilicity and the strong DNA pack restrain its potentialities. In order to overcome this limitation, we added Protamine in the composition of solid lipid nanoparticles (SLN-Protamine) and we compared this carrier with the same kind of SLN containing Esterquat 1 instead of Protamine (SLN-EQ1). Carriers cytotoxicity was assessed on COS-I cells evaluating the cell cycle by propidium iodide test, while the transfection efficiency was studied using pEGFP as plasmid model. The cell penetrating activity of Protamine inside the lipid vectors was evaluated studying cell internalization by confocal microscopy using Red Nile-labeled carriers. SLN-Protamine:pDNA showed a mean diameter five-times smaller than the size of SLN-EQ1:pDNA and a remarkably lesser cytotoxicity. Transfection by SLN-Protamine:pDNA was seven-times more effective compared with the Protamine:pDNA polyplexes while no transfection capacity was observed for SLN-EQ1:pDNA complexes due to their inability to be internalized owing to their larger dimension. Red Nile-SLN-Protamine were localized in endocytic-like vesicles into the nuclear membrane suggesting the inclusion of Protamine in nano-lipophilic systems may enhance the reduction in the complex dimensions, the nuclear pDNA translocation and the pDNA release in the cells.


Assuntos
Núcleo Celular/metabolismo , DNA/metabolismo , Lipídeos/química , Nanopartículas , Protaminas , Compostos de Amônio Quaternário/metabolismo , Transfecção , Animais , Células COS , Cátions , Chlorocebus aethiops , DNA/genética , Vetores Genéticos , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas/química , Tamanho da Partícula , Plasmídeos , Protaminas/química , Protaminas/metabolismo , Protaminas/toxicidade , Compostos de Amônio Quaternário/química
16.
Int J Pharm ; 389(1-2): 254-61, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20100555

RESUMO

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical-chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.


Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Nanopartículas , Neuroblastoma/metabolismo , Animais , Cátions , Linhagem Celular Tumoral , Liofilização , Vetores Genéticos/química , Lipídeos/química , Camundongos , Tamanho da Partícula , Plasmídeos , Protaminas/química , Compostos de Amônio Quaternário/química , Propriedades de Superfície , Transfecção
17.
J Liposome Res ; 19(3): 241-51, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19694606

RESUMO

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.


Assuntos
Linhagem Celular Tumoral/metabolismo , Citometria de Fluxo/métodos , Fluoresceína , Microscopia Confocal/métodos , Oligodesoxirribonucleotídeos , Animais , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Fluoresceína/química , Fluoresceína/metabolismo , Humanos , Lipossomos/química , Lipossomos/metabolismo , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo
18.
J Immunol ; 181(8): 5660-72, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18832725

RESUMO

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34(+) hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D(3) monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D(3) receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D(3)/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.


Assuntos
Antígenos CD34 , Diferenciação Celular/imunologia , Colecalciferol/farmacologia , Proteínas de Homeodomínio/imunologia , Fator de Transcrição MafB/imunologia , Monócitos/imunologia , Células Progenitoras Mieloides/imunologia , Vitaminas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células HL-60 , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Hematopoese/imunologia , Proteínas Homeobox A10 , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Fator de Transcrição MafB/biossíntese , Fator de Transcrição MafB/genética , Monócitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Elementos de Resposta/genética , Elementos de Resposta/imunologia , Retroviridae , Transdução Genética , Células U937 , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologia
19.
Nanomedicine ; 3(1): 1-13, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17379164

RESUMO

The relatively hydrophilic bile acid, ursodeoxycholic acid (UDCA), was used as an additive to DOTAP cationic liposomes to evaluate the effect on the cellular uptake of an oligonucleotide. Nuclear magnetic resonance studies were applied to estimate the relative amount of incorporated UDCA into the lipidic bilayers. DOTAP or DOTAP-UDCA vesicles (MixVes; DOTAP/UDCA molar ratios 1:0.25, 1:0.5, 1:1, and 1:2) formed complexes with 5'-fluorescein conjugated 29-mer phosphorothioate oligonucleotides (PS-ODNs) and studied using gel electrophoresis. In addition, the complexes were tested after transfection to assess the cellular uptake and the localization of the oligo in a HaCaT cell line by the use of cytofluorimetric and confocal microscopic analysis. DOTAP lipid formulated in the presence of a defined amount of UDCA forms more stable, flexible, and active MixVes. In particular, the MixVes at 1:0.25 and 1:0.5 molar ratios increase and modify the cellular uptake of PS-ODNs if compared with DOTAP liposomes 3 hours after the transfection studies. Moreover, the in vitro data suggest that these new formulations are not toxic.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Ácidos Graxos Monoinsaturados/farmacologia , Lipossomos/farmacologia , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/farmacologia , Compostos de Amônio Quaternário/farmacologia , Ácido Ursodesoxicólico/farmacologia , Morte Celular/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Microscopia Confocal , Transfecção
20.
Eur J Pharm Biopharm ; 67(2): 320-8, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17368876

RESUMO

Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.


Assuntos
Liofilização , Lipídeos/química , Nanopartículas/química , Varredura Diferencial de Calorimetria , Cátions , Crioprotetores/farmacologia , Congelamento , Proteínas de Fluorescência Verde/química , Microscopia de Força Atômica , Fótons , Plasmídeos/metabolismo , Ácidos Esteáricos/química , Fatores de Tempo
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