Construction And Evaluation Of Protein Drug Sustained-release Preparations Via Inner Ear Administration

Inner ear disorders are common clinical diseases that seriously influence human health. Currently, clinical treatments of these two diseases rely heavily on systemic drug delivery routes. However, as the existence of blood labyrinth barrier(BLB), the clinical usefulness of many drugs is limited and the undesirable side effects are easy to happen. The inner ear administration through tympanic, which could transport drugs into inner ear through round window membrane(RWM), indicated that the local drug delivery can be used in the treatment of inner ear diseases. As an important cytokine used to treat a variety of diseases clinically, IFN was proved to have good curative effect in the treatment of sudden deafness. For that reason, we chose IFN as a model of protein drugs and investigated the in vivo pharmacokinetics after inner ear administration. According to its characteristics of short half-life, poor stability in vivo, the IFN was prepared to nanoparticles and further loaded into thermosensitive hydrogel. The in vitro and in vivo studies of IFN formulations were performed, which can provide a reference for the research of protein drugs via tympanic administration.The IFNα-2b PLGA nanoparticles were prepared using double emulsion-solvent evaporation method. The three factors in the preparation process of which have great influence on the entrapment efficiency were obtained: content of PLGA, concentration of PVA and the volume of external water phase. The formulation was optimized using these three factors as variables and encapsulation efficiency, particle size as target. The optimal prescription that obtained through Box-Behnken experimental design and further chose by response surface method were: 114 mg of PLGA, 1% PVA and 9 m L of external water phase. The prepared nanoparticles had high encapsulation efficiency, smaller particle size and good stability. Compared to solutions, the NPs had certain sustained-release effect, which could reach for 36 h in vitro.The thermosensitive in situ gel was prepared using Pluronic®F127(P407) and Pluronic®F68(P188). The phase transition temperature of poloxamer gel was determined by tube inversion method. The connection between the concentration of P407, P188 and phase transition temperature were investigated. The proper three formula were chosen to do the in vitro dissolution test, the optimal prescription was obtained: 20% P407 combined with 5% P188, which dissolve longest. Rheological measurements showed that the gelation temperature was consistent with the tube inversion method. In vitro release tests indicated that the BSA and BSA NPs loaded poloxamer gel have a good slow release effect which could sustain to release for 17 h and 48 h in PBS medium, respectively.The injectable thermosensitive hydrogels were prepared using chitosan and β-glycerophosphate(β-GP) as material and the gelation process relied on the adjustment of p H. The gelation time was determined by the tube inversion method and turbidimetric assay. The effects of the degree of deacetylation of chitosan, the concentration of chitosan and β-GP on the gelation time of gels were discussed. The optimal prescription was 2%(w/v) chitosan with 96.2% degree of deacetylation, adding 400 μL of 56%(w/v) β-GP, which could gelatinize at 3 minutes. The water retention and swelling property of CS/β-GP hydrogels were good enough and could sustain to release through RWM after tympanic administration. In vitro degradation experiments showed the CS/β-GP hydrogels could slowly degrade in the PBS solution within lysozyme. The results of infrared test indicated that the formation of CS/β-GP hydrogels was not only the simple physical mixture, but also the force of hydrogen bonding. The scanning electron microscope pictures showed that the NPs were adhered at the skeletons and the drugs could release through the huge mesh structure of hydrogels. The BSA and BSA NPs loaded CS/β-GP hydrogels emerged great slow release effect. The BSA/CS gel could continuely release for 36 h in vitro and the BSA NPs/CS gel was 72 h.The pharmacokinetic results showed that IFN in various dosage forms could enter into the inner ear perilymph(PL), after unilateral tympanic administration. Compared to solution, the NPs can increase the amount of IFN into the inner ear and the residence time in the administration side to some extent, the relative bioavailability of which in PL was 122.4%. To load IFN into thermosensitive hydrogels can significantly enhance the drug distribution in the inner ear PL. In comparison with IFN solution, the relative bioavailability of IFN/P, IFN/CS gel in PL were 216.9% and 277.0%, and the average residence time in the PL were also extended from 9.1 h to 10.8 h, 14.7 h, respectively. Compared to IFN gels, NPs loaded composite hydrogel preparation can further enhance the retention time of the drug in vivo. In comparison with IFN/P and NPs/P gel, the MRT in PL increased from 10.9 h to 12.9 h. In comparison with IFN/CS and NPs/CS gel, the MRT in PL increased from 14.6 h to 23.1 h. Both thermosensitive hydrogels could be served as carriers of IFN solution or NPs, but compared to poloxamer gel, the CS gel had much longer retention time of IFN in the PL after tympanum injection.In summary, our results show that to prepare protein drugs into NPs or thermosensitive hydrogels formulation can prolong the drug release time in vitro. Both NPs and gels could effectively deliver IFN into PL through the round window membrane and significantly improve the local bioavailability of the drug in the inner ear. Compared to a single NPs or gel formulation, the NPs/gel composite formulations can better enhance the residence time of IFN in vivo, which is more suitable as a drug delivery system for the inner ear. Compared to the poloxamer gel, the sustain-released effect of CS/β-GP gel is much better both in vitro and in vivo.