RESUMEN

The objective of this study is to prepare and characterize solid dispersions of nifedipine (NP) using porous spherical silicate micro beads (MB) that were approximately 100 µm in diameter with vinylpyrrolidone/vinyl acetate copolymer (PVP/VA) and a Wurster-type fluidized bed granulator. Compared with previously reported solid dispersion using only MB, the supersaturation of NP dissolved from the proposed system of MB and PVP/VA was maintained during dissolution tests. The proposed system produced a solid dispersion product coated on MB, and morphology was maintained after the coating process to prepare solid dispersion; therefore, the powder characteristics, such as flowability of the proposed solid dispersion product, was tremendously preferable to that of the conventional spray-dried solid dispersions of NP with PVP/VA, expecting to make the consequent manufacturing processes easy for development. Another advantage in the terms of manufacturing is its simple process to prepare solid dispersion by spraying the drug and polymer that were dissolved in an organic solvent onto a MB in a Wurster-type fluidized bed granulator, thus, simplifying the optimization and scale-up with ease.

Asunto(s)

Microesferas , Nifedipino/administración & dosificación , Povidona/análogos & derivados , Silicatos/química , Tecnología Farmacéutica/métodos , Cristalización , Liberación de Fármacos , Tamaño de la Partícula , Povidona/química , Polvos/química , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Agua/química

RESUMEN

The purpose of this study is to establish a novel approach for preparing a solid dispersion drug product using spherical silicate by a Wurster-type fluidized bed granulator. The spherical silicate used in this study has porous structure and ideal particle size for loading by a Wurster-type fluidized bed granulator. As model drugs, ibuprofen (IBU), indomethacin (IMC), and phenytoin (PNT) were used and the proposed approach was applied to prepare amorphous drug. All drugs could be loaded on the spherical silicate in an amorphous state. On the other hand, spray drying of spherical silicate suspended in IBU solution was conducted to prepare amorphous product of IBU as a reference; however, complete amorphization was not achieved. Dissolution profiles of each drug after loading on spherical silicate by a Wurster-type fluidized bed granulator were evaluated, and dramatic improvement of dissolution was observed compared with those of crystalline drug. In the proposed approach, specific surface area and particle size of spherical silicate were determined as a key factors to contribute to high yield of amorphous product.

Asunto(s)

Ibuprofeno/química , Indometacina/química , Fenitoína/química , Silicatos/química , Cristalización , Tamaño de la Partícula , Porosidad , Solubilidad , Soluciones/química , Propiedades de Superficie , Tecnología Farmacéutica/métodos

RESUMEN

Genetic modification of stem cells could be applied to initiate/enhance their secretion of therapeutic molecules, alter their biological properties, or label them for in vivo tracking. We recently developed a negatively charged gene carrier ("anioplex") based on pullulan-spermine, a conjugate prepared from a natural polysaccharide and polyamine. In rat mesenchymal stem cells (MSCs), anioplex-derived reporter gene activity was comparable to or exceeded that obtained using a commercial cationic lipid reagent. Transfection in the growth medium with 15% serum and antibiotics was approximately sevenfold more effective than in serum-free conditions. Cytotoxicity was essentially indiscernible after 24 h of anioplex transfection with 20 µg/mL DNA, in contrast to cationic lipid transfection that resulted in 40%-60% death of target MSCs. Anioplex-derived reporter gene activity persisted throughout the entire 3-week study, with post-transfection MSCs appearing to maintain osteogenic, adipogenic, and chondrogenic multipotency. In particular, chondrogenic pellet formation of differentiating human MSCs was significantly inhibited after lipofection but not after aniofection, which further indicates the biological inertness of pullulan-spermine/DNA anioplexes. Collectively, these data introduce a straightforward technology for genetic engineering of adult stem/progenitor cells under physiological niche-like conditions. Moreover, reporter gene activity was observed in rat spinal cords after minimally invasive intrathecal implantation, suggesting effective engraftment of donor MSCs. It is therefore plausible that anioplex-transfected MSCs or other stem/progenitor cells with autologous potential could be applied to disorders such as neurotrauma or neuropathic pain that involve the spinal cord and brain.

Asunto(s)

ADN/metabolismo , Ingeniería Genética/métodos , Glucanos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Suero/metabolismo , Espermina/metabolismo , Tejido Adiposo/citología , Animales , Transporte Biológico , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Muerte Celular , Diferenciación Celular , Supervivencia Celular , Humanos , Espacio Intracelular/metabolismo , Células Madre Mesenquimatosas/citología , Ratas , Ratas Wistar , Médula Espinal/patología , Células del Estroma/citología , Células del Estroma/metabolismo , Células del Estroma/trasplante , Transfección

RESUMEN

The objective of this study is to prepare a non-viral carrier of gene transfection from various polysaccharides and evaluate the feasibility in gene expression for mesenchymal stem cells (MSCs). Various amounts of spermine were chemically introduced into pullulan, dextran and mannan with a molecular weight of around 40 000 or pullulan with different molecular weights to prepare cationized polysaccharides with different extents of spermine introduced (spermine-polysaccharide). Each cationized polysaccharide was complexed with a plasmid DNA at various ratios and in vitro gene transfection was investigated for rat bone marrow-derived MSCs. The level of gene expression depended on the type of cationized polysaccharide. The highest level was observed for the complex of spermine-pullulan and plasmid DNA. Additionally, the level also depended on the molecular weight of pullulan and the extent of spermine introduced to pullulan. Suppression of gene expression with chlorpromazine and methyl-beta-cyclodextrin of endocytosis inhibitors demonstrated that the cellular uptake of spermine-pullulan-plasmid DNA complexes was mediated by clathrin- and raft/caveolae-dependent endocytic pathways. The cationized pullulan is a promising non-viral carrier of plasmid DNA for MSCs.

Asunto(s)

Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Transfección/métodos , Animales , Supervivencia Celular/efectos de los fármacos , ADN/genética , ADN/metabolismo , Endocitosis/efectos de los fármacos , Glucanos/química , Glucanos/metabolismo , Glucanos/toxicidad , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Peso Molecular , Plásmidos/genética , Polisacáridos/toxicidad , Ratas , Ratas Wistar , Espermina/química

RESUMEN

The objective of this study is to prepare a non-viral carrier of gene expression from the polysaccharide dextran and evaluate the effect of amine compounds introduced to dextran on the level of gene expression. Dextran with a molecular weight of 74 x 10(3) was cationized by the chemical introduction of different amine compounds. The cationized dextran was complexed with a plasmid DNA and the vitro gene transfection was investigated for HeLa cells. The level of gene expression depended on the amine compound introduced to dextran. The highest level was observed for the complex of spermine-introduced dextran and plasmid DNA. The highest cellular internalization and the best buffering effect were observed among every cationized dextran. Every complex did not show any cytotoxicity. It is concluded that the superior properties of spermine-introduced dextran enabled the plasmid DNA to enhance the expression level to a great extent compared with other cationized dextrans. Cationized dextran is a promising non-viral carrier of plasmid DNA.

Asunto(s)

Aminas/química , ADN/genética , ADN/metabolismo , Dextranos/química , Dextranos/metabolismo , Plásmidos/genética , Transfección/métodos , Animales , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Dextranos/toxicidad , Etidio/metabolismo , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Sustancias Intercalantes/metabolismo

RESUMEN

The objective of this study is to investigate the possibility of small interfering RNA (siRNA) complexed with a cationized dextran of nonviral carrier to biologically modify the adipogenesis extent of bone marrow-derived mesenchymal stem cells (MSC). Spermine was chemically introduced to the hydroxyl groups of dextran to prepare the cationized dextran (spermine-dextran). The spermine-dextran could form a complex with siRNA, and the physicochemical properties were changed by the molecular weight of dextran, the molar percentage of spermine introduced to dextran, and the molar ratio of nitrogen molecule of spermine-dextran to the phosphorous ones of siRNA (N/P ratio). The gene expression level of luciferase or green fluorescence protein was significantly suppressed by transfection with the complex of spermine-dextran and siRNA. The gene expression level by the complex decreased with an increase in the extent of complex internalized. Biochemical experiments revealed that culture in an adipogenic differentiation medium allowed MSC to differentiate into adipogenic cells. However, upon culturing with siRNA of anti-transcription coactivator containing PDZ-binding motif (TAZ) for osteogenic differentiation complexed with the spermine-dextran, the adipogenesis of MSC was further promoted. It is concluded that the spemine-dextran was a promising nonviral carrier to suppress the expression level of differentiation gene, resulting in the modification of cell differentiation direction.

Asunto(s)

Adipogénesis/genética , Dextranos/farmacología , Células Madre Mesenquimatosas/metabolismo , ARN Interferente Pequeño/genética , Transfección/métodos , Adipogénesis/efectos de los fármacos , Secuencias de Aminoácidos/fisiología , Animales , Dextranos/química , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Células Madre Mesenquimatosas/citología , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Espermina/química , Espermina/farmacología

RESUMEN

Overactive bladder is a highly prevalent clinical condition that is often caused by bladder outlet obstruction (BOO). Increased coupling of bladder smooth muscle cells (BSMC) via gap junctions has been hypothesized as a mechanism for myogenic bladder overactivity in BOO, although little is known about the regulatory system underlying such changes. Here, we report the involvement of basic fibroblast growth factor (bFGF) and connexin 43, a bladder gap junction protein, in bladder overactivity. BOO created by urethral constriction in rats resulted in elevated bFGF and connexin 43 levels in the bladder urothelium and muscle layer, respectively, and muscle strips from these bladders were more sensitive than those from sham-operated controls to a cholinergic agonist. In vitro bFGF treatment increased connexin 43 expression in cultured rat BSMC via the ERK 1/2 pathway. This finding was supported by another in vivo model, where bFGF released from gelatin hydrogels fixed on rat bladder walls caused connexin 43 upregulation and gap junction formation in the muscle layer. Bladder muscle strips in this model showed increased sensitivity to a cholinergic agonist that was blocked by inhibition of gap junction function with alpha-glycyrrhetinic acid. Cystometric analyses of this model showed typical features of detrusor overactivity such as significantly increased micturition frequency and decreased bladder capacity. These findings suggest that bFGF from the urothelium could induce bladder hypersensitivity to acetylcholine via gap junction generation in the smooth muscle, thereby contributing to the myogenic overactivity of obstructed bladders.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/metabolismo , Uniones Comunicantes/metabolismo , Músculo Liso/metabolismo , Vejiga Urinaria Hiperactiva/metabolismo , Vejiga Urinaria/metabolismo , Animales , Células Cultivadas , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Vejiga Urinaria/citología , Vejiga Urinaria/efectos de los fármacos , Obstrucción del Cuello de la Vejiga Urinaria/complicaciones , Vejiga Urinaria Hiperactiva/etiología

RESUMEN

Introduction of various kinds of exogenous genes is an important step for control of differentiation in stem cell biology and regenerative medicine. However, some kinds of cells are vulnerable to manipulations such as gene delivery. In this context, a gene introduction method with higher efficiency and safety is required. Bone marrow stromal cells (BMSCs) offer possibilities for clinical application because of their potential for expandability and ability to be auto-transplanted. In this study, we established an efficient induction system of dopamine-producing neuronal cells from BMSCs in several species using the spermine-pullulan-mediated reverse transfection technique. In this system, introduced exogenous plasmid genes were successfully transcribed and expressed as proteins in the cytoplasm of BMSCs with the smallest number of cell death. Microtubule-associated protein 2 and anti-beta-tubulin class III+ neurons were successfully delivered from human, monkey, and mouse BMSCs, and further treatment with trophic factors promoted differentiation of induced neuronal cells into dopamine-producing cells that were positive for tyrosine hydroxylase and secreted dopamine after high K+ stimulation in high-performance liquid chromatography analysis. Our study indicates the availability of the reverse transfection method for the induction of dopamine-producing neuronal cells from BMSCs, which is expected to apply to cell-based therapy in Parkinson's disease.

Asunto(s)

Células de la Médula Ósea/citología , Dopamina/biosíntesis , Glucanos/farmacología , Neuronas/metabolismo , Espermina/farmacología , Células del Estroma/citología , Transfección/métodos , Animales , Células de la Médula Ósea/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/genética , Glucanos/toxicidad , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Luciferasas , Macaca , Ratones , Microscopía Confocal , Neuronas/citología , Neuronas/efectos de los fármacos , Plásmidos/genética , Especificidad de la Especie , Espermina/toxicidad , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Transgenes

RESUMEN

The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 x 10(3). Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion.

Asunto(s)

Expresión Génica , Vectores Genéticos/química , Glucanos/química , Plásmidos/química , Transfección/métodos , Asialoglicoproteínas/farmacología , Cationes/química , Línea Celular Tumoral , ADN/química , ADN/genética , Fetuínas , Vectores Genéticos/efectos de los fármacos , Vectores Genéticos/genética , Humanos , Hígado/metabolismo , Luciferasas/análisis , Luciferasas/genética , Peso Molecular , Plásmidos/efectos de los fármacos , Plásmidos/genética , Espermina/química , alfa-Fetoproteínas/farmacología