Puerarin-loaded Nasal Thermosensitive In Situ Hydrogels For Prevention Of Hypobaric Hypoxia-induced Brain Damage Of Rats

Objective:The puerarin loaded thermosensitive in situ hydrogels（TISGs）were prepared to improve the relative bioavailability and brain targeting effect by intranasal administration,and prevent hypobaric hypoxic-induced brain damage in rats.Methods:The in situ nasal circulatory perfusion technique was applied to examine the nasal absorption of puerarin and its derivatives by rats.Poloxamer 407 and 188 served as the stroma of in situ hydrogels,and propylene glycol was used to dissolve puerarin.Phase transition temperature and the gelation time were used as the evaluation indicators.Orthogonal method was designed to optimize the formulations.The drug release behavior was investigated by in vitro dialysis and the release mechanism was evaluated.The content of puerarin that passed through the nasal mucosa of sheeps were investigated by the Franz diffusion cell.Rheological properties of TISGs were verified by rheometer.The cilia toxicity test was defined by toad palate mucosa.Acute decompression hypoxia model was establisehd to investigate the oxygen-carrying capacity and the mortality rate of mice.Brain injury model of rats induced by hypobaric hypoxia（HH）was used to estimate the preventive effect of TISGs,which was evaluated by the behavior,the pathological changes,oxidative stress indexes and immunohistochemistry.The pharmacokinetics and tissue distribution of oral and intranasal administration were compared by liquid chromatography-mass spectrometry（LC-MS/MS）.Results:The absorption rate constant indicated that puerarin was absorbed well by nasal mucosa of rats.The optimized formulation of puerarin TISGs was 23%Poloxamer 407,4%Poloxamer 188,and 10%propylene glycol.Phase transition temperature was 34.3℃ and the gelation time was 2.9 min,which were satisfied the property of nasal administration.The pattern of puerarin release from TISGs and absorbed by nasal mucosa of sheep were conformed to the first order kinetics equation,which depended on the drug concentration.The rheology experiment showed that the modulus of TISGs increased with the increased temperature.When the temperature was higher than 30℃,the storage modulus were greater than the loss modulus,which indicated gels formed.The viscosity decreased with the increased angular frequency at 34℃,which indicated that TISGs could be retained in the nasal cavity.Acute decompression hypoxia test certified that the mortality of mice decreased with the preventive nasal administration of puerarin TISGs.TISGs of puerarin could improve the oxygen carrying capacity of blood by increasing hemoglobin（HGB）content and the red blood count（RBC）.The HH brain injury model was successfully established with the hypobaric simulation chamber.The degree of hippocampus injury of rats was observed by H.E.pathological section.Morris maze test showed that the puerarin TISGs of15,25 mg·kg^-1could significantly decrease the escape latency of rats which were exposed in HH circumstances and increase the crossing times of brain injury rats.The TISGs of puerarin improved learning and memory ability of rats.Open filed test certified that the puerarin TISGs of 15,25 mg·kg^-1could increased average speed and straight times of rats which were suffering from HH brain damage.The puerarin TISGs of 25 mg·kg^-1could improve anti-oxidation ability of rats by decreasing the malondialdehyde（MDA）level and increasing the glutathione（GSH）level.The rats with HH brain injury of 25 mg·kg^-1can improve the damage of hippocampal neurons significantly,and the expression of hypoxia-inducible factor-1αof 25 mg·kg^-1 group was lower than that of the model group.Pharmacokinetics indicated that the peak time of the intranasal administration(T_max=10.0±5.7 min)was earlier than that of the oral administration(T_max=36±13.4 min).The relative bioavailability of the intranasal administration was 300%in comparison with that of the oral route.AUC of intranasal group was 954.5±335.1 h*ng/m L,which indicated that the intranasal administration of puerarin had good brain-targeting effect.Conclusion:This study successfully prepared puerarin TISGs,which could improve the bioavailability and brain targeting effect,and effectively prevent hypobaric hypoxia-induced brain damage in rats.This medication provides a new promising method for preventing hypobaric hypoxic-induced brain damage.