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1.
Pharm Res ; 32(2): 680-90, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25163981

RESUMEN

PURPOSE: To develop a bio-assay for measuring long-term bioactivity of released anti-inflammatory compounds and to test the bioactivity of celecoxib (CXB) and triamcinolone acetonide (TA) released from a new PLGA-based microsphere platform. METHODS: Human osteoarthritic chondrocytes were plated according to standardized procedures after batch-wise harvest and cultured for 3 days to prevent cell confluency and changes in cell behaviour. Prostaglandin E2 (PGE2) production stimulated by TNFα was used as a parameter of inflammation. A novel microsphere platform based on PTE-functionalised PLGA was used to incorporate CXB and TA. Loaded microspheres were added to transwells overlying the cells, with transfer of the wells to new cell cultures every 3 days. Inhibition of PGE2 production was determined over a period of 21 days. RESULTS: PLGA(75:25)-PTE microspheres were prepared and loaded with CXB and TA at 86 and 97% loading efficiency, respectively. In the bioactivity assay, PGE2 levels induced by TNFα were reduced to an average of 30% using microspheres loaded with 0.1 nmol CXB per transwell; with microspheres loaded with 0.1 nmol TA, PGE2 production was initially reduced to 3% and gradually recovered to 30% reduction. At 1 nmol loading, PGE2 was inhibited to 0-7% for CXB-loaded microspheres, and 0-28% for TA-loaded microspheres. CONCLUSIONS: We present a novel sustained release bioactivity assay which provides an essential link between in vitro buffer-based release kinetics and in vivo application. Novel PLGA-based microspheres loaded with TA and CXB showed efficient anti-inflammatory effects over time.


Asunto(s)
Antiinflamatorios/metabolismo , Portadores de Fármacos/metabolismo , Ácido Láctico/metabolismo , Microesferas , Ácido Poliglicólico/metabolismo , Pirazoles/metabolismo , Sulfonamidas/metabolismo , Triamcinolona Acetonida/metabolismo , Antiinflamatorios/química , Bioensayo/métodos , Celecoxib , Células Cultivadas , Condrocitos/metabolismo , Portadores de Fármacos/química , Humanos , Ácido Láctico/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Pirazoles/química , Sulfonamidas/química , Triamcinolona Acetonida/química
2.
Sci Rep ; 5: 13574, 2015 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-26338423

RESUMEN

Malignant pleural effusion (MPE) is a common clinical problem in non-small cell lung carcinoma (NSCLC) patients; however, the underlying mechanisms are still largely unknown. Recent studies indicate that the frequency of the L858R mutant form of the epidermal growth factor receptor (EGFR-L858R) is higher in lung adenocarcinoma with MPE than in surgically resected specimens, suggesting that lung adenocarcinoma cells harboring this mutation tend to invade the adjacent pleural cavity. The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to investigate the molecular mechanisms involved in the formation of MPE. We found that expression of EGFR-L858R in lung cancer cells resulted in up-regulation of the CXCR4 in association with increased cancer cell invasive ability and MPE formation. Ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We also indicated that Inhibition of CXCR4 with small interfering RNA, neutralizing antibody, or receptor antagonist significantly suppressed the EGFR-L858R-dependent cell invasion. These results suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring a specific type of EGFR mutation.


Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Receptores ErbB/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Derrame Pleural Maligno/metabolismo , Adenocarcinoma/genética , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Humanos , Neoplasias Pulmonares/genética , Mutación/genética , Invasividad Neoplásica , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patología , Receptores CXCR4/metabolismo , Transducción de Señal
3.
Acta Biomater ; 23: 214-228, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26022968

RESUMEN

Hybrid hydrogels composed of poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) are presented in this study as novel injectable and thermoresponsive materials for siRNA delivery, which could specifically target several negative regulators of tissue homeostasis in cartilaginous tissues. Effectiveness of siRNA transfection using pNIPAAM formulated with either MgAl-LDH or MgFe-LDH platelets was investigated using osteoarthritic chondrocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing. No significant adverse effects of pNIPAAM/LDH hydrogels on cell viability were noticed. Cellular uptake of fluorescently labeled siRNA was greatly enhanced (>75%) in pNIPAAM/LDH hydrogel constructs compared to alginate, hyaluronan and fibrin gels, and was absent in pNIPAAM hydrogel without LDH platelets. When using siRNA against GAPDH, 82-98% reduction of gene expression was found in both types of pNIPAAM/LDH hydrogel constructs after 6 days of culturing. In the pNIPAAM/MgAl-LDH hybrid hydrogel, 80-95% of GAPDH enzyme activity was reduced in parallel with gene. Our findings show that the combination of a cytocompatible hydrogel and therapeutic RNA oligonucleotides is feasible. Thus it might hold promise in treating degeneration of cartilaginous tissues by providing supporting scaffolds for cells and interference with locally produced degenerative factors.


Asunto(s)
Condrocitos/fisiología , Preparaciones de Acción Retardada/administración & dosificación , Microinyecciones/métodos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transfección/métodos , Resinas Acrílicas/química , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/química , Células Cultivadas , Condrocitos/química , Preparaciones de Acción Retardada/química , Geles/química , Calor , Humanos , Hidróxidos/química , ARN Interferente Pequeño/química
4.
Arthritis Res Ther ; 17: 214, 2015 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-26290179

RESUMEN

INTRODUCTION: Chronic low back pain due to intervertebral disc (IVD) degeneration is associated with increased levels of inflammatory mediators. Current medical treatment consists of oral anti-inflammatory drugs to alleviate pain. In this study, the efficacy and safety of a novel thermoreversible poly-N-isopropylacrylamide MgFe-layered double hydroxide (pNIPAAM MgFe-LDH) hydrogel was evaluated for intradiscal controlled delivery of the selective cyclooxygenase (COX) 2 inhibitor and anti-inflammatory drug celecoxib (CXB). METHODS: Degradation, release behavior, and the ability of a CXB-loaded pNIPAAM MgFe-LDH hydrogel to suppress prostaglandin E2 (PGE2) levels in a controlled manner in the presence of a proinflammatory stimulus (TNF-α) were evaluated in vitro. Biocompatibility was evaluated histologically after subcutaneous injection in mice. Safety of intradiscal application of the loaded and unloaded hydrogels was studied in a canine model of spontaneous mild IVD degeneration by histological, biomolecular, and biochemical evaluation. After the hydrogel was shown to be biocompatible and safe, an in vivo dose-response study was performed in order to determine safety and efficacy of the pNIPAAM MgFe-LDH hydrogel for intradiscal controlled delivery of CXB. RESULTS: CXB release correlated to hydrogel degradation in vitro. Furthermore, controlled release from CXB-loaded hydrogels was demonstrated to suppress PGE2 levels in the presence of TNF-α. The hydrogel was shown to exhibit a good biocompatibility upon subcutaneous injection in mice. Upon intradiscal injection in a canine model, the hydrogel exhibited excellent biocompatibility based on histological evaluation of the treated IVDs. Gene expression and biochemical analyses supported the finding that no substantial negative effects of the hydrogel were observed. Safety of application was further confirmed by the absence of clinical symptoms, IVD herniation or progression of degeneration. Controlled release of CXB resulted in a nonsignificant maximal inhibition (approximately 35 %) of PGE2 levels in the mildly degenerated canine IVDs. CONCLUSIONS: In conclusion, this study showed biocompatibility and safe intradiscal application of an MgFe LDH-pNIPAAM hydrogel. Controlled release of CXB resulted in only limited inhibition of PGE2 in this model with mild IVD degeneration, and further studies should concentrate on application of controlled release from this type of hydrogel in animal models with more severe IVD degeneration.


Asunto(s)
Celecoxib/farmacología , Preparaciones de Acción Retardada/farmacología , Degeneración del Disco Intervertebral/tratamiento farmacológico , Disco Intervertebral/efectos de los fármacos , Resinas Acrílicas/química , Animales , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/química , Celecoxib/administración & dosificación , Celecoxib/química , Ciclooxigenasa 1/genética , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/farmacología , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Perros , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Hidrogeles/administración & dosificación , Hidrogeles/química , Hidróxidos/química , Inmunohistoquímica , Inyecciones Subcutáneas , Disco Intervertebral/metabolismo , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/metabolismo , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Reología , Temperatura
5.
Eur J Pharm Sci ; 53: 35-44, 2014 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-24345796

RESUMEN

The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous cells. In conclusion, non-specific effects should not be overlooked in the application of RNAi for mesenchymal cell transfection and may need to be overcome for its effective therapeutic application.


Asunto(s)
Condrocitos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Agrecanos/genética , Cartílago Articular/citología , Ciclo Celular , Supervivencia Celular , Células Cultivadas , Quitosano/química , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo II/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ciclooxigenasa 2/genética , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Humanos , Ácido Hialurónico/química , Inflamación , Disco Intervertebral/citología , Articulación de la Rodilla , Lípidos/química , Vértebras Lumbares , Osteopontina/genética , Polietileneimina/química , Antígeno Nuclear de Célula en Proliferación/genética , ARN Interferente Pequeño/genética , Transfección
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