| Objectives: The purpose of this study was to investigate the factors which affect the physical and chemical stability of proanthocyanidins(PC). The characteristics of PC microspheres were studied which prepared by spray drying method. In the sustained release drug delivery system, PC was used as model drug, while chitosan(CTS), lecithin(LC), and bletilla striata polysaccharide(BSP) were used as carrier materials. The therapeutical effects of PC microspheres on radiation-induced lung injury in rats were investigated, and the tissue distribution of PC in vivo was evaluated.Methods: The precision, method reproducibility and recovery rate were evaluated by Ultraviolet Spectrophotometry(UV) method, meanwhile, the effect of heating temperature, heating time and light on the stability of PC were studied by UV. The physicochemical properties of microspheres were characterized by UV, Scanning Electron Microscopy(SEM), Fourier Transform Infrared Spectroscopy(FTIR), Differential Scanning Calorimetry(DSC), dynamic dialysis method, in vitro hydrolysis and enzymatic hydrolysis method, et al. One hundred and five healthy adult male wistar rats were randomly divided into seven groups(n = 15): Normal group(A), Model group(B), Dexamethasone(DEX) group(C), PC group(D), PC/LC/CTS microspheres group(E), BSP group(F), PC/BSP/CTS microspheres group(G). The models of radiation induced lung injury in rats were established by whole lung irradiation 15 Gy using Switzerland ELEKTA two-photon high-energy linear accelerator. The rats were oral gavage administration daily, the doses were as follows: Physiological saline, 1.25 m L/100 g(A); Physiological saline, 1.25 m L/100 g(B); DEX, 50 mg/L, 1.25 m L/100 g(C); PC, 20 mg/Kg, 1.25 m L/100 g(D); PC/LC/CTS microspheres, 20 mg/Kg, 1.25 m L/100 g(E); BSP, 30 mg/Kg, 1.25 m L/100 g(F); PC/BSP/CTS microspheres 20 mg/Kg, 1.25 m L/100 g(G). The blood, bronchoalveolar lavage fluid(BALF) and lung tissue were obtained after irradiation in 2, 4, 8 weeks. The number of white blood cells(WBC) and red blood cells(RBC) in blood and BALF were analyzed by sysmex KX-21 blood cell analyzer. WBC classification and counting were using blood counting chamber. Pathological changes of lung tissue were observed by hematoxylin-eosin staining(HE). The drug distribution in various tissues and organs were detected by HPLC.Results: The standard curve regression equation of PC was A= 0.1438C-0.1068(r=0.9999, n=5), which has a good linear relation in the range of 0.988~4.940 g/m L. PC has good stability below 60 oC. The precision study of PC within day and between days was 4.19% and1.34%, respectively. The stability of PC solution is better within 8 h. In the low, medium and high concentrations, the average recovery was 99.68 ± 6.00%, 99.86 ± 5.91% and 99.94 ± 2.01%, respectively.The results of SEM demonstrated that the PC/LC/CTS microspheres were predominantly spherical in shape with much wrinkled surfaces. The blank microspheres were spherical in shape with smooth surfaces. The FT-IR spectrum of PC/LC/CTS microspheres showed that the carbonyl group of LC formed hydrogen bonds with the amide group of CTS and the hydroxyl group of PC at 1068 cm-1. DSC study displayed that the stability of microspheres was improved because of the temperature of endothermic peaks increased from 250.56 oC to 349.89 oC when comparing with PC. The yield, drug load, encapsulation efficiency, water content and solid density were 43.36~61.68%, 7.10~17.29%, 35.78~68.19%, 8.00~13.00% and 0.4 g/cm3, respectively. The swelling rate of microspheres was elevated with increasing the concentration of CTS in the formulations. The moisture uptake of microspheres was saturated at 40 oC/RH 75% within 12 h. The results of in vitro release study showed that the release of proanthocyanidins from PC/LC/CTS microspheres was prolonged in phosphate buffered solution(PBS, p H 6.8), and a sustained release of 48.92~76.92% within 48 h was observed. The results of in vitro degradation indicated that degradation in lysozyme solution is faster than that in PBS solution. PC/LC/CTS microspheres possess preferable degradation. The morphology of microspheres with lysozyme and PBS developed into rough surfaces, coliform, laminar structure, and microspheres aggregated within 3 weeks. The results of adhesiveness demonstrated that the adhesiveness of microspheres on mucous membrane of small intestine is better than that of gastric mucosa. The retention rate of microspheres was elevated with increasing the concentration of CTS.The results of SEM demonstrated that the PC/BSP/CTS microspheres were predominantly spherical in shape with much wrinkled surfaces. The blank microspheres were spherical in shape with smooth surfaces. The yield, drug load, encapsulation efficiency, water content and solid density were 47.86~53.87%, 18.12~21.51%, 54.48~86.47%, 14.00~25.00% and 0.4 g/cm3, respectively. The swelling rate of microspheres was related to the ratio of drug/carrier. The moisture uptake of microspheres was saturated at 40 oC/RH 75% within 12 h. The results of in vitro release study showed that the release of PC from PC/BSP/CTS microspheres was prolonged in PBS( p H 6.8), and a sustained release of 28.89%~72.86% within 48 h was observed. The results of in vitro degradation indicated that degradation in lysozyme solution is faster than that in PBS. PC/BSP/CTS microspheres possess preferable degradation. The morphology of microspheres with lysozyme and phosphate buffered solution developed into rough surfaces, ramified, laminar structure, and microspheres aggregated within 3 weeks. The results of adhesiveness demonstrated that the adhesiveness of microspheres on mucous membrane of small intestine is better than that of gastric mucosa. The retention rate of microspheres was elevated with increasing the concentration of CTS, which indicated that CTS is related to the adhesiveness of microspheres.The model of radiation-induced lung injury in rats were established, which were used to investigate the treatment of proanthocyanidins oral sustained release microspheres. In the model group, the numbers of white blood cells were significantly decreased compared with that of rats in the normal group. After administration the numbers of white blood cells and red blood cells in blood of rats were rised. The number of white cells in BALF was positively correlated with the degree of inflammation. Model group(B) shows the highest number of white blood cells with severe inflammation, after administration the number of white blood cells was decreased with varying degrees.The number of red cells in BALF was positively correlated with the degree of congestion. Model group(B) shows the highest number of red blood cells with serious congestion, after administration the number of white blood cells was decreased with varying degrees. Lung tissue HE staining results showed that the alveolar wall ruptured, vascular and interstitial lung congestion, pulmonary interstitial hyperplasia, a large number of inflammatory cell infiltrations in alveolar and interstitial lung. After administration(C, D, E, F, G group), the lung tissue structure was clear, inflammatory cell infiltration and congestion was significantly decrease when compared with model group. The treatment effectiveness was better in C, E, G group. The symptoms of rats in D, F group were improved. Otherwise, with the duration of treatment, lung inflammatory cell infiltration, hyperemia, alveolar wall rupture were gradually improved. The concentration of PC reached to the peak after oral administration in 0.5 h, while PC/ LC/CTS microspheres and PC/BSP/CTS microspheres reached the peak after oral administration in 5 h and 3 h, respectively. The results indicating that the microspheres prepared by spray drying method have sustained release action.Conclusion:(1) We have successfully established the UV, which is used to study PC.(2) PC/LC/CTS microspheres(microspheres E) could serve as a carrier of oral sustained release drug delivery system for better physical and chemical properties, which have thermal stability improved, wrinkled surfaces, and the characteristics of sustained release, good biodegradability and mucoadhesive ability.(3) PC/BSP/CTS microspheres(microspheres H) could serve as a carrier of oral sustained release drug delivery system for better physical and chemical properties, which have wrinkled surfaces, and the characteristics of sustained release, good biodegradability and mucoadhesive ability.(4) PC/LC/CTS microspheres(microspheres E) and PC/BSP/CTS microspheres(microspheres H) have therapeutic effect on irradiation induced lung injury in rats, and the therapeutic effects of PC/LC/CTS microspheres were better than PC/BSP/CTS microspheres. The results of in vitro release and in vivo distribution suggest that PC/LC/CTS microspheres and PC/BSP/CTS microspheres both had sustained release effect, which promising provide the theoretical basis for the prophylaxis and clinical treatment of irradiation induced lung injury. |
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