Preparation Of Nattokinase Transfersome Gel

Nattokinase（NK）,as a microbial enzyme,has good efficacy in the prevention and treatment of cardiovascular and cerebrovascular diseases such as thrombosis,hypertension,hyperlipidemia and atherosclerosis,and is commonly used in the form of oral administration.However,nattokinase is easily degraded or deactivated by chemical oxidation during oral administration,which is difficult to be absorbed directly through the gastrointestinal tract.In view of the above shortcomings,this paper developed a transdermal drug delivery preparation of nattokinase-transfersome gel,which aims to protect nattokinase from pH,enzymes and intestinal bacteria through non-invasive transdermal drug delivery.It can overcome the shortcomings of poor stability of nattokinase and difficult absorption of macromolecules,and improve bioavailability.The specific contents of study are as follows:Part Ⅰ: Preparation of Nattokinase Transfersome Gel（1）Technology research of nattokinase transfersome.Lecithin and Tween 80 were selected as capsule materials,and nattokinase transfersomes were prepared by film dispersion-ultrasonic method.Taking the encapsulation rate as an index,the optimal process and formulation of nattokinase transfersomes were investigated by response surface methodology.The results were as follows: The mass ratio of capsule material was 3.85:1,the mass ratio of lipid to drug was 10:0.7,and the ultrasonic time was 6.5min.Confirmatory experiments showed that the average encapsulation efficiency of nattokinase transfersomes was（72.30±1.32）%.（2）Study on freeze-drying process of nattokinase transfersome suspension.Taking particle size and appearance as indicators,the optimal freeze-drying conditions of nattokinase transfersome were determined by single factor and orthogonal experiments:mannitol as freeze-drying protective agent,pre-freezing temperature of-80 ℃,prefreezing time of 14 h,and mass ratio of mannitol to lipids of 1:1.（3）Structural characterization of nattokinase transfersomes.The results showed that the prepared nattokinase transfersome was spherical,with a particle size of（172.30±3.06）nm,a potential of（-39.07±1.20）mV.DSC and FTIR results both showed that the characteristic peaks of nattokinase disappeared because it was encapsulated in transfersome vesicles,indicating the formation of nattokinase transfersomes.（4）Preparation of nattokinase transfersome gel.A molding method of "postadding" the transfersome lyophilized sample to the blank gel was used.The drug content uniformity and viscosity were comprehensively scored,and the optimal formulation conditions of the gel were investigated by quadratic regression orthogonal rotation combination design test: glycerol concentration 16%,carbomer concentration 1%,pH value 6.3.Part Ⅱ: Investigating the in vitro drug release and transdermal penetration behavior of nattokinase transfersome gel（1）The dialysis method was used to investigate the drug release in vitro.The results showed that the nattokinase transfersome gel had a certain sustained release effect,the drug release conformed to the Higuchi model,and the Ritger-peppas model fitting results n=0.389<0.45,the drug diffuses in the Fick form.（2）Franz diffusion cell method was used to conduct the transdermal permeability experiment.The results showed that the transdermal penetration effect of nattokinase transfersome gel was better,which was about 4 times that of its liposome gel.The feasibility of molecular nattokinase entering the blood circulation through the skin barrier;the optimal penetration enhancer was 3% azone,and the cumulative penetration percentage of nattokinase transfersome gel was（55.26±3.02）%.Part Ⅲ: Establishment and stability of the quality standard of nattokinase transfersome gelA quality inspection system for nattokinase transfersome gels that meets the requirements of the 2020 edition of the Chinese Pharmacopoeia was established,and its stability was investigated.The results showed that the prepared nattokinase transfersome gel had a uniform and fine gel-like appearance,the pH was（6.35±0.07）,and the viscosity was（265.47±2.93）Pa.s;the preparation had good stability and the best storage temperature is 4 °C.Part Ⅳ: Research on the safety and in vitro thrombolytic properties of nattokinase transfersome gelThe skin irritation experiment was carried out on the intact skin of rabbits.The results showed that that there were no redness,swelling and plaques on the skin of the rabbits,indicating that it was safe.The in vitro thrombolysis experiment showed that the nattokinase transfersome gel has a good thrombolytic effect and a certain sustained release effect.In conclusion,this subject has successfully developed a preparation form for the transdermal administration of macromolecular nattokinase-nattokinase transfersome gel.The transfersome gel preparation process is simple,stable and safe.It has good properties and good transdermal penetration effect,providing a new preparation form for nattokinase to prevent and treat cardiovascular and cerebrovascular diseases,and laying a certain theoretical foundation for the subsequent research on macromolecular biological enzyme topical preparations.