| For acute ischemic stroke, the first-line therapy strategy in clinical is thrombolysis and promoting the reperfusion in the ischemic tissue in time; however, which will lead to further damage to the ischemic tissue. Therefore, reducing the reperfusion injury is the key forthe treatment of ischemic stroke. At present, superoxide dismutase (SOD) and edaravone are often used in clinical cases for the treatment of cerebral ischemia-reperfusion (I/R) injury. The application of traditional Chinese medicine (TCM) in the treatment of cerebral ischemic stroke has a history of 1500 years.Paeoniflorin (PF), a characteristic monoterpene glucoside isolated from Paeoniae Radix, can ameliorate mortality, infarct size and deficits in neurological symptoms induced by t-MCAO. Its solubility is high, while the half-life time is short and the bioavailability is very low.The 4AC-PF, a derivative of the paeoniflorin (PF), increased lipophilicity and extended half-life. The study of physic-chemical property showed that 4AC-PF was a poorly water-soluble drug, and its solubility in aqueousis only about 2μg/mL.Recently, the polymeric micelles with core-shell structure have been widely studied in promoting the solubility of insoluble drugs. Pluronic P123 (EO20-PO70-EO20), amphiphilic block copolymers, has the outstanding advantage in this field. Its PO segments is long, which can form the hydrophobic core to solubilize the insoluble drugs significantly; while its EO segments can form the hydrophilic shell which making the long cycle in vivo possible. In order to achieve the purpose of solubilization and control releasing, we take Pluronic P123 as the carrier material to build the 4AC-PF loadedpolymeric micelles drug delivery system to explore its therapeutic effecton cerebral I\R injury.The contents of this thesis were including building the analysis method of 4AC-PF in vitro and in vivo; preparation and optimizing the prescription of 4AC-PF loaded Pluronic P123 polymeric micelles drug delivery system; investigation of the effect of reducing cerebral I/Rinjury ont-MCAO rats; and study of biodistribution of 4AC-PF loaded Pluronic P123 polymeric micelles delivery system on t-MCAO rats.The 4AC-PF-loaded polymeric micelles were prepared by thin-film hydration methods. Entrapment efficiency (EE) and drug loading coefficient (DL) were used as the evaluation index, the single factor experiments showed that with the increasing amount of 4AC-PF, the EE was decreased while the DL was increased; with the increasing volume of water, both the EE and DL were increased and then decreased as inverted bell-shaped. These two factors had significant influence to the EE and DL. Hence, based on the results of single factor experiments, micelle formulation was optimized employing the central composite design-response surface methodology with two factors and five levels.All dependent variables (EE% and DL%) can be well-fitted by the quadratic polynomial equations with a high correlation coefficiency (R>0.95, P<.05) as follows:EE%=1.16+1.04X-0.90Y-1.11X2-0.67Y2+ 0.97XY;DL%= 0.02+0.22X-0.11Y-0.15X2-0.01Y2+0.12XY(where X represents the amount of 4AC-PF, Y represents the volume of water).The common part of the contour plot between EE% and DL% was the favorable area for the feeding of 4AC-PF and volume of water/The final optimized formulation was 1.2mg of 4AC-PF and 1mL of water with 10mg carrier. Studies of the physico-chemical property of micelles showed that the mean diameters of 4AC-PF loaded Pluronic P123 polymeric micelles were close to 20 nm with an acceptable polydispersity index (PDI) between 0.23 and 0.25;The morphology of polymeric micelles was observed by transmission electron microscopyshowed that the polymeric micelles exhibited a spherical shape of homogeneous particle size, which was in good agreement with that measured by the laser scattering technique.The sobility of 4AC-PF was increased 585-fold by the polymeric micelles formulation. And in vitro release of 4AC-PF from the micelles was only 28.5% during 48hand the polymeric micelles were structurally stable in PBS containing l%oTween-80 (pH 7.4).The pharmacodynamics experiments of 4AC-PF-loaded Pluronic P123 polymeric micelles on the t-MCAO model rats had proved that,4AC-PF polymeric micelles group, compared with MCAOgroup, blank micelles group and PF solution group, can significantly reduceabout 35.30% of the brain infarct size (P<0.05) and improve the SOD activityof the ischemichemisphere about 2.8-fold (P<0.001). Furthermore, 4AC-PF loaded polymeric micelles can also ameliorate nucleoplasm shrinkage and mitochondrial swelling of neurouscells in the hippocampus CA1 region and striatal region.Afteriv.administration of 4AC-PF solution and 4AC-PF-loaded Pluronic PI23 micelles to MCAO rats, the biodistribution was assessed, to investigate the concentration of 4AC-PF in brain and other organs. The results indicated that compared with the non-ischemic hemisphere, the amout of 4AC-PF in micellar formulation is 5.00-fold (P<0.01),8.33-fold (P<0.01) and 5.25-fold (P<0.001) high than the solution formulation at 3h,12h and 24h, respectively; Compared with 4AC-PF solution, the amount of 4AC-PF in polymeric micelles formulation accumulated in the ischemic hemishphere is 4.23-fold high at 3h (P<0.01),2.08-fold high at 12h (P<0.05), and 10.50-fold high at 24h (P<0.01),respectively.Furthermore, the blood circulation time of 4AC-PF in MCAO rats was significantly prolonged, and the accumulation of 4AC-PF in kidney, spleen and lung was increased while the drug in liver was decreased.In summary,4AC-PF loaded Pluronic P123 polymeric micelles benefits 4AC-PF in increasing solubility in aqueous solution, sustained-releasing and targeting tothe ischemic hemisphere; The polymeric micelles can ameliorate the neurological impairment (P<0.01); approximately narrow 35.30% of the cerebral infarct size (P<0.05); improve the SOD activity about 2.8-fold (P<0.001), and ameliorate the cytoplasm shrinkage and the swelling of the cell lysis.Therefore,4AC-PF loaded Pluronic P123 polymeric micelles are promising drug delivery system in the treatment of cerebral I/R injury. |
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