RESUMEN
The aim of this study was to prepare recombinant human erythropoietin (rhEPO) loaded poly(lactic-co-glycolic acid) (PLGA) microspheres using human serum albumin (HSA) as a stabilizer. Prior to encapsulation, the rhEPO-HSA mixture microparticles were fabricated using a modified freezing-induced phase separation method. The microparticles were subsequently encapsulated into PLGA microspheres. Process optimization revealed that the polymer concentration in the organic phase and the sodium chloride (NaCl) concentration in the outer water phase of the s/o/w emulsion played critical roles in determining the properties of the resultant microspheres. An in vitro release test showed that rhEPO was released from PLGA microspheres in a sustained manner up to 30 days. A single injection of rhEPO-loaded PLGA microspheres in Sprague-Dawley rats resulted in elevated hemoglobin and red blood cell concentrations for about 33 days. The stability of the rhEPO within the PLGA microspheres was systematically investigated by size-exclusion high-performance liquid chromatography (SEC-HPLC), SDS-PAGE, western blot and in vivo biological activity assay. The stability of rhEPO released from rhEPO-loaded microspheres was also examined by western blot. The results suggested that the integrity of rhEPO was successfully protected during the encapsulation process and the release period from polymeric matrices.
Asunto(s)
Composición de Medicamentos/métodos , Estabilidad de Medicamentos , Eritropoyetina/química , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Albúmina Sérica/química , Animales , Emulsiones/administración & dosificación , Emulsiones/síntesis química , Emulsiones/química , Recuento de Eritrocitos/estadística & datos numéricos , Eritropoyetina/farmacología , Excipientes/química , Hemoglobinas/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas , Ratas Sprague-DawleyRESUMEN
Using tissue engineering, a complex of neural stem cells (NSCs) and collagen type I was transplanted for the therapy of cerebral ischemic injury. NSCs from E14 d rats were dissociated and cultured by neurosphere formation in serum-free medium in the presence of basic fibroblast growth factor (bFGF), then seeded onto collagen to measure cell adhesive ability. BrdU was added to the culture medium to label the NSCs. Wistar rats (n=100) were subjected to 2-hour middle cerebral artery occlusion. After 24 hours of reperfusion, rats were assigned randomly to five groups: NSCs-collagen repair group, NSCs repair group, unseeded collagen repair group, MCAO medium group, and sham group. Neurological, immunohistological and electronic microscope assessments were performed to examine the effects of these treatments. Scanning electronic microscopy showed that NSCs assemble in the pores of collagen. At 3, 7, 15, and 30 d after transplantation of the NSC-collagen complex, some of the engrafted NSCs survive, differentiate and form synapses in the brains of rats subjected to cerebral ischemia. Six d after transplantation of the NSC-collagen complex into the brains of ischemic rats, the collagen began to degrade; 30 d after transplantation, the collagen had degraded completely. The implantation of NSCs and type I collagen facilitated the structural and functional recovery of neural tissue following ischemic injury.