| OBJECTIVESParaquat (PQ) poisoning is becoming increasingly popular and has become the second common pesticide poisoning following acute organophosphate poisoning. The most serious damage of PQ poisoning are acute lung injury and subsequent pulmonary fibrosis. Currently there are no effective therapy for PQ poisoning, the mortality up to80%. Fluorofenidone (AKF) is a potential therapeutic compounds to PQ-induced acute lung injury. The distribution of AKF in body has no tropism. If AKF is accumulated in the lung tissue, that targeted to alleviate the PQ-induced lung damage, the therapeutic effect will be better. This project aims to prepare lung-targeting AKF-PLGA-MS and its lung targeting study in rats. And investigate the treatment of AKF on PQ-induced acute lung injury.METHODS AND RESULTS1. The prearation and evaluation of AKF-PLGA-MSAKF-PLGA-MS was prepared by the emulsion solvent evaporation method using PLGA as the carrier. The formulation was screened by single factor, such as the concentration of PVA, Drug/Copolymer ratio, the ratio of the water and organic phases, stirring speed, and evaluated bymorphology, encapsulation efficiency, loading efficiency, size and distribution. The optimal formulation of AKF-PLGA-MS was obtained by orthogonal design, and then appearance and morphology, size distribution, stability and vitro release behavior were evaluated.The obtained optimum experimental formulation were:the volume ratio of methylene chloride to PVA (2%) solution was1:10, the mass ratio of AKF to PLGA was1.5:5, stirring speed1500rpm. The average particle size of AKF-PLGA-MS was18.1μm with80%of AKF-PLGA-MS the range from7to30μm, and the encapsulation efficiency was80±2.53%, drug loading was8.2±1.90%. The AKF-PLGA-MS was stable at4℃and25℃for15days. No significant change was observed in its particle size, encapsulation efficiency, which indicated the stability was good. Prepared in the same manner the microspheres, the average particle diameter is3.9μm with a span of of1.59, for comparison.In vitro release tests showed that the cumulative release rate of AKF-PLGA-MS was about20%within6h, about67%within96h. The drug release profile of AKF-PLGA-MS in PBS was in lined with Korsmeyer-Peppas model, Q=11.141·t0.292(R2=0.9797), speculated that the release mechanism of AKF-PLGA-MS in PBS was Fick’s diffusion.2. In vivo distribution and lung targeting of AKF-PLGA-MS in PQ-induced acute lung injury ratsIn vivo experiment of AKF injection, AKF-PLGA-MS (3.9μm)、 AKF-PLGA-MS (18.1μm), the high performance liquid chromatography with UV detector was utilized for the determination of AKF contention in rats tissues.The lung targeting effect was evaluated by relative drug exposure value (Re)，targeting efficiency (Te) and peak concentration ratio (Ce).The results shows that the AKF concentrations in lung of the AKF-PLGA-MS (18.1μm)group is significantly higher than AKF injection group and AKF-PLGA-MS (3.9μm) group,at point12、24、48h, the difference was statistically significant (P<0.05). And AKF concentration in lung is significantly higher than plasma, heart, liver, spleen and kidney from AKF-PLGA-MS (18.1μm) group. The AKF concentration of injection group was not detected at24、48h point, as the concentration below the detection limit. However, the AKF concentration in lung of AKF-PLGA-MS (18.1μm) group were2.74±1.23,1.47±0.77μg·mL-1. It indicated that AKF-PLGA-MS (18.1μm) showed a certain slow release.Compared with AKF injection, Rel of AKF-PLGA-MS (18.1μm) in lung was19.92; Compared with AKF-PLGA-MS (3.9μm), Re2of AKF-PLGA-MS (18.1μm) in lung was8.74. Te of lung compared to blood and other tissues all were more than1(compared with heart, liver, spleen, kidney, the Te value of lung is83.91、21.49.27.08.29.43.44.77). The TCe of AKF-PLGA-MS (18.1μm), AKF-PLGA-MS (3.9μm) and AKF injection were86.83%.18.91%.13.55%, respectively. The Ce of AKF-PLGA-MS (3.9μm) and AKF-PLGA-MS (18.1μm) were5.74,1.15. The lung targeting effect of AKF-PLGA-MS (18.1μm) was proved remarkable.3. The therapeutic research of AKF on rats acute lung injury induced by paraquatTo evaluate the treatment of AKF on PQ-induced acute lung injury,93male SD rats were divided into five groups:blank group, negative controls group, AKF injection treatment group, AKF-PLGA-MS (3.9μm) treatment group and AKF-PLGA-MS (18.1μm) treatment group. To establish a model of paraquat induced acute lung injure, negative controls group and treatment groups were treated with intraperitoneal injection of parquet at the dosage of30mg/kg.0.5h after PQ injection, treatments groups were were intravenously administered (equivalent to30mg/kg AKF). Rats were killed at0.5,6,12,24,48h after exposure to PQ, TNF-α、IL-1β and NF-κB were assessed by ELISA, and lung pathological changes were observed.Compare with the blank group, TNF-α, IL-1β and NF-κB of negative controls group were greatly increased at each point (except for0.5h, TNF-α P>0.05, each of the time points P<0.05), the TNF-α, IL-1β and NF-κB of the treatments groups were increased (P<0.05), but lower than negative controls group. Compare with the negative controls group, At the0.5and24h point IL-1β of AKF-PLGA-MS (3.9μm) and AKF-PLGA-MS (18.1μm) group were significant decreased (P<0.05). At the48h point, TNF-α、IL-1β、NF-κB of AKF-PLGA-MS (18.1μm) were significant decreased (P<0.05), IL-1β、NF-κB of AKF-PLGA-MS(3.9μm) were significant decreased (P<0.05), While TNF-a expression was no signifieant difference (P>0.05). IL-1β of AKF injection were significant decreased (P<0.05), While TNF-a and NF-κB expression were no signifieant difference (P>0.05). And IL-1β、NF-κB of AKF-PLGA-MS (18.1μm) and AKF injection treatment group were significant decreased (P<0.05).Pathology results showed that the structure of lung of the control group is clear, alveolar walls is thin and no significant inflammatory cell infiltration. At the6h point, negative controls group shows a wide range of red blood cells and pulmonary interstitial inflammatory cell infiltration, At the24,48h point, shows alveolar septum fracture and thickening, but less vascular congestion and serous leakage reduction in the treatments groups.CONCLUSIONSAKF-PLGA-MS was prepared by the emulsion solvent evaporation method using PLGA as the carrier. The drug-loaded microspheres have small size, high encapsulation efficiency, good stability and a slow release. Compared with AKF injection group and AKF-PLGA-MS (3.9μm), AKF-PLGA-MS (18.1μm) group has certain lung-targeting property, and better therapeutic effect on acute lung injure induced by PQ. The therapeutic effect of AKF may relate to inhibit the expression of TNF-a, IL-1β and NF-κB, then reduce the inflammation in lung tissue. |
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