Study On Preparation And Performance Of Plasmid PUDK-HGF Freezing And Spray Drying Preparation

Plasmid pUDK-HGF generally requires storage at low temperature,which is not conducive to the storage,transportation and application of drugs;the active ingredient is unstable in solution,the concentration is low,and it cannot be used at high concentration.This article aims to use mannitol,sucrose and other protective agents added to pUDK-HGF solution to prepare a solid dosage form with good fluidity,low moisture absorption and good stability by vacuum freeze drying and spray drying methods,so that the integrity of pUDK-HGF it will not be destroyed.The integrity and stability of the solid dosage form pUDK-HGF are determined and analyzed through cell experiments,animal experiments,and accelerated experiments.The main research results of this article are as follows:(1)Preparation of recombinant plasmid pUDK-HGF,the production process includes:a series of processes such as fermentation,lysis,concentration,purification of bacteria,etc.,to obtain a recombinant plasmid pUDK-HGF with higher purity.(2)Freeze-drying Through the screening of the ratio of high supercoil and low hygroscopicity,the optimal formulation of mannitol+sucrose(16+4)mg/m L was determined.Accelerated experiments show that:under 4?and-20?,the super spiral ratio of the sample stored for 6 months will not be reduced.Under 40?and 25?,it can be stored for 2 months.The higher the temperature after two months,the super spiral ratio The greater the degree of fracture.(3)Spray drying through the measurement of the hygroscopicity of the samples,the results show that:the higher the plasmid content,the higher the hygroscopicity,the measurement results of the fluidity of the four samples show that they meet the production requirements,and the accelerated experiment shows:4?and-20?Under the conditions,the supercoiled ratio of the samples stored for 6 months is slightly reduced.At 40?and 25?,the samples stored for6 months(mannitol:sucrose=4:1):plasmid=50:1 The smallest.(4)Both liquid dosage forms and spray-dried pce-EGFP(carrying luciferase and having a similar structure to pUDK-HGF)can transfect 293T cells,and the expression levels of the two are basically the same under the same dosage conditions.pUDK-HGF promotes cell migration,and cells treated with 50 ng/m L pUDK-HGF migrated faster between cells.A model of mouse back trauma was established,and it was found that spray-dried pUDK-HGF can promote wound healing in mice,and the appropriate dosage promotes better expression.Too high a concentration may hinder expression for a certain period of time.The dose of 4 mg works best,about 400?g/cm~2.