| Objective:Plague,a zoonotic disease caused by Yersinia pestis,historically caused a devastating pandemic and killed an estimated 200 million people.It can be transmitted in the form of aerosol and act on lungs,and its onset is rapid and severe.Once the plague spreads on a large scale,it will cause panic in the whole society because of its contagious and deadly.What’s worse it will seriously threaten our national security and public health security if it is used by terrorists.Gentamicin sulfate is one of the commonly used drugs for plague treatment,but the shortcomings involving instability,large dosage and complicated operation of its intravenous/muscular injection inhibit its development.The advantages of strong lung targeting,fast onset,small dosage,and self-administration for the dry powder inhalation make it an ideal choice for the prevention and treatment of biological aerosol lung injury,and the treatment of emerging infectious diseases.Therefore,the topic of this paper focuses on the demand for the dry powder inhalation for biosafety prevention drugs,and prepare dry powder inhalation with good atomization performance and high stability using gentamicin sulfate as a model drug,which provides the research basis for the development of gentamicin sulfate and other biosafety prevention and cure drug dry powder inhalation.Methods:(1)A high performance liquid chromatographic(HPLC)method was established for the determination of gentamicin sulfate dry powder inhalation.(2)Two different types of gentamicin sulfate dry powder inhalation were prepared: L-leucine with gentamicin sulfate dry powder inhalation was prepared by spray drying method.The dosage of L-leucine,spray drying inlet temperature and liquid supply rate were used as factors,and the yield and particle size were used as indicators.Orthogonal experiments with three factors and three levels were carried out to optimize the prescription composition and preparation process parameters.Lipid polymer hybrid nanoparticles with gentamicin sulfate dry powder inhalation was prepared by double emulsion solvent evaporation method combined with spray drying method.The effects of lipid types,PLGA types,lipid to PLGA ratio,PVA concentration and water to oil phase volume ratio on the encapsulation efficiency and particle size of the nanoparticles were investigated.Different carriers were added for spray drying,and the best prescription was selected.(3)The quality evaluation and preliminary pharmacodynamics study of the prepared dry powder inhalation was carried out.The properties of dry powder inhalation such as morphology,particle size,fluidity and emptying rate were researched.The clinical symptoms,bacterial load and histopathology of mice were analyzed after subcutaneous injection or pulmonary delivery of different dosage forms(subcutaneous injection of gentamicin sulfate,inhalation atomization solution of gentamicin sulfate,L-leucine with gentamicin sulfate dry powder inhalation and lipid polymer hybrid nanoparticles with gentamicin sulfate dry powder inhalation).Results:(1)The HPLC analysis method has good specificity.The regression equation of sample concentration versus peak area was A=6.2509+13.557C(r=0.9999)in the concentration range of 73.8～516.5 μg/m L,with good linearity.The reproducibility,precision,stability and recovery of the method meet the requirements,indicating that it is suitable for the determination of gentamicin sulfate dry powder inhalation.(2)The optimal formulation and process for preparation of L-leucine with gentamicin sulfate dry powder inhalation were as follows: 15%(w/w)L-leucine content,120℃ inlet temperature and 4.5 m L/min(15%)inlet speed.The emitted dose(ED),mean mass aerodynamic diameters(MMAD),geometric standard deviation(GSD)and Fine particle fraction(FPF)of dry powder inhalation prepared by this method were 91.77 ±0.52%,4.94 ± 0.45 μm,3.62 ± 0.03 μm and 42.36 ± 0.42%.The optimal formulation for preparation of lipid polymer hybrid nanoparticles with gentamicin sulfate dry powder inhalation was 1:10 ratio of egg yolk lecithin to PLGA 50/50 Mw:7000-17000,1:10(V/V)ratio of water phase to oil phase,1.0%(w/v)PVA as external water phase,L-leucine as carrier.The ED,MMAD,GSD and FPF were 90.12 ± 1.45%,4.58 ± 0.63μm,3.98 ± 1.78 μm and 40.12 ± 0.16%.(3)Mice delivered with the L-leucine with gentamicin sulfate dry powder inhalation and the lipid polymer hybrid nanoparticles with gentamicin sulfate dry powder inhalation had significantly reduced mortality.No Yersinia pestis was found in the blood,liver,spleen and lung of the mice delivered with the L-leucine with gentamicin sulfate dry powder inhalation.Yersinia pestis was found after 24 h of challenge in the liver of the mice delivered with the lipid polymer hybrid nanoparticles with gentamicin sulfate dry powder inhalation,after 48 h in the spleen and 0 h in the lung,while no Yersinia pestis was detected in the organs after 14 d.Compared with subcutaneous injection and inhalation atomization routes,the mice delivered with the L-leucine with gentamicin sulfate dry powder inhalation showed fewer symptoms of lung edema,inflammation,and bleeding,while the mice delivered with the lipid polymer hybrid nanoparticles with gentamicin sulfate dry powder inhalation showed no significant reduction in symptoms such as enlargement and inflammation in all organs.Conclusion:The results showed that the two different types of gentamicin sulfate dry powder inhalation prepared by spray drying method had the characteristics of uniform particle size,good fluidity and excellent atomization,which met the requirements of pulmonary inhalation administration.Compared with subcutaneous injection and inhalation atomization solution,the above dry powder inhalation can effectively reduce mortality,inhibit the proliferation of Yersinia pestis in the blood,liver,spleen and lung,and reduce tissue damage.At the same time,it is the advantages that is being easily inhaled by patients,stable character and easy to carry.This provides a new research and development idea and route of drug administration for the treatment of plague and other emergent infectious diseases. |
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