| Pulmonary surfactant(PS)is a complicated mixture of lipids and proteins secreted by type II alveolar epithelial cell(AT II).The main function of PS lies in maintaining normal respiratory mechanics by reducing alveolar surface tension to prevent alveolar collapse.Besides,it also plays important roles in providing uniform lung inflation,improving efficiency of airway clearance,and alleviating lung inflammatory reaction.At present,exogenous surfactant of animal origin was wildly used for the treatment of lung injury diseases,nevertheless,safety issues like the spread risk of animal related diseases remain to be improved.Besides,the ratio of surface-active proteins to lipid was uncontrollable because the quantity of the anmial-derived agents are variable from batch to batch.Therefore,the synthetic PS can be a choice to avoid these problems.Surfaxin?,the only peptide-based synthetic pulmonary surfactant in clinical application,has been approved by Food and Drug Administration(FDA)in 2012 for the treatment of neonatal respiratory distress syndrome(NRDS).As a conventional suspension formulation,the dosage form and treatment method of Surfaxin still need further exploration for large single dose as well as simple treatment.In this thesis,we systematically studied the preparation process of Surfaxin? and quality standard of sinapultide.Furthermore,we constructed a novel drug carrier of microbubbles(MBs)for pulmonary delivery based on the formulation of Surfaxin?.The shell membrane of MBs is fabricated by the phospholipid and sinapultide,which was the main components and active ingredient of Surfaxin?.The structural characterization of the MBs was fully evaluated.Especially,the stability mechanism of the sinapultide polypeptides to MBs was studied by simulation methods.Finally,the therapeutic effect of MBs integrated with ultrasound to inflammatory alveolar epithelial cell function as well as lung injury animal models were also investigated.The following points are mainly involved in the dissertation:1.The dispersion method was used to prepare sinapultide suspension.By using sedimentation rate,redispersibility,appearance characters and quality stability of suspension as indicators,the optimal preparation process of sinapultide suspension was determined.The high performance chromotograph(HPLC)method was adopted to determine the content of sinapultide suspension,and the testing standard of sinapultide content was estabished.A Diamonsil C18 column(250×4.60 nm,5 ?m)with gradient elution system was used.The mobile phase was acetonitrile-water with 0.1% trifluoroacetic acid(TFA)was adoptedwith the initial ratio of acetonitrile to water of 40 to 60,and the ratio of 90 to 10 after 25 min.The flow rate was 1.0 m L min-1 with wavelength of 215 nm,and the column temperature was maintained at 30 ?.Sinapultide had a good linear relationship(r = 0.9994)with a concentration ranging from 0.06 mg/m L to 1.5 mg/m L.The average recovery as well as relative standard deviation(RSD)of preparation samples was 98.13% and 1.31%,respectively.Results showed that the establishment method to determine the content of sinapultide is reliable,sensitive and reproducible,which could be used for the quality control of sinapultide.2.Synthetic pulmonary surfactant microbubbles were prepared by film dispersion and ultrasonic method based on the prescription of Surfaxin?.Besides,MBs with different concentrations of sinapultide were prepared,and the optimized MBs exhibited an average diameter of 1.82 ± 0.15 ?m and zeta potential of-55.2 ± 3.9 m V.A fluorescent inverted microscope was used to observe FITC-labeled sinapultide MBs,and sinapultide was found mainly existed in the membrane shell of MBs.Transmission electron microscope(TEM)and scanning electron microscope(SEM)were used to characterize the sinapultide MBs,and the spherical structure of sinapultide MBs was confirmed.Furthermore,the stability experiment demonstrated that the mother liquor of sinapultide MBs could last for three months.Therefore,the prescription had good stability for further development of innovative dosage forms.3.The self-made agar phantom was used to investigate the contrast effect of sinapultide MBs with different concentrations in vitro.The results showed that the ultrasonic signal intensity of sinapultide MBs group was improved obviously compared with control group.When the sinapultide concentration was 0.1 mg/m L,the ultrasonicsignal intensity was the highest and could last for 30 min.Based on the experimental results,the mechanism of sinapultide stabilized MBs firstly was studied by molecular dynamics MARTINI force field simulation for the first time with,and results showed that the addition of proper proportion of sinapultide could decrease surface tension of MBs.Accordingly,the stability of the MBs has been greatly improved.However,the stability of membrane would decrease with the concentration increase of the sinapultide due to the steric effect.4.Finally,the sinapultide MBs on lipopolysaccharide(LPS)mediated model of alveolar epithelial cells(AT II)was studied.The results show that the sinapultide MBs could significantly improve the secretion of inflammatory cytokines and the expression of surfactant protein A(SPA)in AT II cells.Under the ultrasonic treatment,the secretion of inflammatory cytokines and SPA were further improved compared with the group without ultrasound treatment.Furthermore,the in vivo experiment based on the acute lung injury(ALI)mice model was carried.The pathological morphology of lung tissue,the ratio of lung wet/ dry weight,interleukin-6(IL-6),tumor necrosis factor-?(TNF-?)and SP-A expression level were used as indexes.Results showed that the treatment of MBs combined with ultrasound could significantly improve pulmonary edema.The secretion levels of inflammatory factors as well as SP-A expression level were improved compared with the control group.Therefor,it can be concluded that microbubble combined with ultrasonic therapy maybe provide with a new option for the treatment of lung injury diseases. |
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