Preparation And Cellabsorption Of Folic Acid Targeted Nattokinase Microcapsules

Nattokinase is a kind of alkaline serine protease with strong fibrinolytic activity,which has a good role in preventing thrombosis and dissolving thrombus.The molecular weight of nattokinase is 27.2 kDa,which can be easily hydrolyzed by proteases and difficult to be absorbed in human gastrointestinal when taken orally.As a result,the oral bioavailability of nattokinase is low and the mechanism of in vivo absorption and thrombolysis of nattokinase is still unclear.To improve the oral bioavailability of nattokinase is very important for its basic research and application.Current studies mainly focus on preparation of enteric-coated microcapsules with natural wall materials to prevent nattokinase from being digested in vivo.Few studies on absorption in cells were conducted,and the absorption pathway is still unknown.In order to solve these problems,our research mainly focused on three aspects: the in vitro inhibition effect on thrombin and plasminogen activator inhibitor-1(PAI-1)of nattokinase and its hydrolysate;the preparation of nattokinase microcapsules and the evaluation of their absorption in Caco-2 cells.(1)Digestion of nattokinase in vitro was simulated by using pepsin,trypsin and chymotrypsin.It was found that the intact nattokinase had strong inhibition on thrombin and PAI-1,but nattokinase hydrolysate had no inhibitory effect,which suggests that only the intact nattokinase can inhibit thrombin and PAI-1and has anti-thrombotic effect.(2)The optimum technological parameters were obtained by double emulsion evaporation method using single factor and orthogonal experiment,with poly(ethylene glycol-b-(DL-lactic acid-co-glycolic acid)-b-ethylene glycol)(PEG-PLGA)as the wall material.Results were as follows,wall material concentration of 5 mg/mL,PVA(polyvinyl alcohol)concentration of 1.0%,homogenization time of 7.5 min,and homogenization speed of 17500 r/min.Two kinds of nattokinase microcapsules were respectively prepared using the optimum parameters with PEG-PLGA and folic acid poly(ethyleneglycol-b-(DL-lactic acid-co-glycolic acid)-b-ethylene glycol)(FA-PEG-PLGA)as wall materials.For the two microcapsules,the average particle sizes was271.3±7.1 nm and 255.8±3.3 nm,the polydispersity coefficient(PDI)was0.263±0.023 and 0.231±0.014,the Zeta potential was-20.17±1.42 mV and-24.73 ±2.36 mV,the encapsulation efficiency was 61.5±2.4% and 58.8±2.5%,respectively.Results of simulated release in vitro showed that more than 60% of nattokinase in the two microcapsules were sustained within 2 hours in gastric environment,and the microcapsules had well sustained-release ability in intestinal environment.All the results showed that the nanocapsules prepared in our study were nano-scale,and have desirable particle size distribution,dispersion and sustained-release properties.Meanwhile,tracer microcapsules with good characteristics were prepared by using coumarin-6 as fluorescent probes.(3)The monolayer cell model of Caco-2 was established to detect the cytotoxicity of the two microcapsules,and then the absorption of two nattokinase microcapsules in cells was evaluated by apparent permeability coefficient detection and laser scanning confocal microscopy observation.Results showed that the two kinds of nattokinase microcapsules had no obvious cytotoxicity in cells and both had good absorption in Caco-2 cells,and the FA-PEG-PLGA nattokinase microcapsules comparatively had better absorption in cells.