| Insulin plays a vital role in the diabetes therapy.Nowadays,the main method of insulin administration is skin injection,but the patient can easily get pain by this method.So the study of non-skin injection is widely focused on,and pulmonary inhalation is regarded as the best non-skin injection for the best absorption.But inhalation insulin is prepared by vacuum freeze drying.Insulin is easy to lose activity,and reduce the effectiveness by this method;meanwhile,insulin is easy to lose its activity,owing to its low storage stability.So protectants should be explored to protect the activity of insulin during freeze-drying and enhance the storage stability of insulin.The research can be divided into three aspects:First,bioactive protection of LEA-motif(LEAM)and trehalose towards insulin was investigated respectively and synergistically.High performance liquid chromatography method was used to study the proficiency of insulin and differential scanning calorimetry was used to study glass transition temperature.According to the HPLC result,both LEAM and trehalose could protect the bioactivity of insulin.The protective effect of LEAM was better than that of trehalose.When insulin was under synergistic protection of LEAM and trehalose,adding a little trehalose could enhance the bioactive protection effect of LEAM towards insulin.According to the DSC result,lyophilized insulin under LEAM had better heat stability than that under trehalose.During lyophilization,both the glass transition temperature and the strength of the glassy state could be enhanced,while the heat stability of lyophilization became better.Second,classical constant temperature method was used to study the storage duration of lyophilized insulin and insulin within LEAM.The degradation of insulin followed the first-order kinetic reaction equation.At 25?,the 10% and 50% insulin degradation duration of lyophilized insulin within LEAM was 6.73 days and 44.22 days longer than that of lyophilized insulin,and when stored at 4?,the 10% and 50%insulin degradation duration of lyophilized insulin within LEAM was 228.11 days and1499.12 days longer than that of lyophilized insulin.As a result,no matter lyophilized drug is stored at 25 ? or 4 ?,lyophilized insulin within LEAM has longer storage duration,indicating that LEAM can effectively enhance the storage stability of lyophilized insulin.At last,sucrose was selected as the protectant to study the bioactivity protection towards insulin by molecular simulation method.The molecular simulation method was performed using replica exchange molecular simulation and Gromacs software with a Gromos96(53a6)force field.The results indicated that compared with the native structure of insulin,the alteration of the structures,RMSD values and secondary structures of insulin in the protection of sucrose were not very obvious,and the H-bond number of insulin and main chain within insulin could be protected within sucrose.So sucrose can be used as bioprotectant to preserve the bioactivity of insulin,and due to the H-bond interaction between insulin and sucrose,the bioactivity of insulin can be protected during lyophilization.In conclusion,according to the experiment result,LEAM can be used as protectant during lyophilization.Under the protection of LEAM,adding a little trehalose during lyophilization can synergistically enhance the protective effect towards insulin during lyophlization.Due to the good heat stability,LEAM can effectively protect the activity of lyophilized insulin during storage.According to the theoretical result,sucrose is a kind of good protectant,which can preserve the bioactivity of insulin.The above research can give a good guidance to the future development of insulin in new formulation,and support the following study on the selection of lyoprotectant by molecular simulation method. |
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