Nattokinase (NK) is a kind of biological enzyme separated from the traditional Japanese food natto [1]. It is a straight chain of amino acids without space folding, with a molecular weight of 27.7 kDa and 275 amino acid residues [2]. NK can dissolve thrombus characterized by low plasminogen levels [3]. And compared with other instant drugs, such as urokinase, streptokinase, and tissue plasminogen activator, NK can directly degrade the thrombus in the blood clot and cross-linked fibrin [3], [4]. Meanwhile, oral administration of natto or NK has been proven to enhance the release of an endogenous plasminogen activator [5], [6]. Due to its long half-life, safety, easy preparation, high activity and low cost, NK has become a promising new safe thrombolytic nutraceutical or supplement [7] .

Fibrinolytic activity in NK can be achieved by taking the drug orally. Studies have shown that NK can be absorbed in the intestine, but it is very sensitive to changes in temperature and pH value, especially in an acidic environment [8]. The optimum pH value of NK fibrinolytic activity is about 9. When the pH value is below 5, the enzyme activity decreases rapidly [9]. However, the pH value of gastric acid is far below 5, which greatly limits its effectiveness due to the inactivation of NK in the gastric environment [10]. There are currently many different approaches to achieving improved stability of NK so that it is protected from gastric acid degradation.

The embedding technique is an effective method to stabilize the activity of NK in the stomach and release it in the intestine. Therefore, it is of great significance to construct an NK vector, to embed NK and protect NK from the adverse effects of gastric acid [11]. At present, NK drug delivery systems include liposomes [12], emulsion [13], polysaccharide [14], proteins [7] and molecular complexes, etc. (such as polyethylene glycol-polylactic acid-glycolic acid, PEG-PLGA) [15]. Each drug delivery system has its unique characteristics. γ-Polyglutamic acid (γ-PGA) is a new type of natural molecular material with unique properties [16]. Due to its non-toxicity, γ-PGA is widely used in the fields of food, medicine and cosmetics and is an excellent wall material. Chitosan (CS) is a type of natural cation derived from chitin, which has good chemical and biological properties such as biocompatibility, biodegradability, pH response, adsorption hydrophilicity and aqueous [17]. In previous studies, CS was mucoadhesive, which can prolong the contact time between drug and biofilm, so it is suitable for increasing intestinal absorption time and is widely used as a carrier in drug delivery systems [18].

The layer-by-layer (LBL) self-assembly technique is a novel method for preparing polyelectrolyte nanoparticles [19], [20], [21]. It uses electrostatic interactions, hydrogen bonds and covalent bonds as the driving force of self-assembly, and alternately deposits positively charged and negatively charged polyelectrolytes on the template surface. After removing the template, multi-layer nanoparticles with high embedding rate can be obtained [21][22]. In recent years, melamine formaldehyde (MF) [20], [23], silica [19], polystyrene [24] and CaCO3 [25] etc. have been considered ideal sacrificial template candidates. CaCO3 can be selected as an ideal sacrificial template material because of its low synthesis cost and good biocompatibility [25].

In this study, CaCO3 was used as a sacrificial template, CS and γ-PGA were used as wall materials, and NK-CaCO3 was embedded layer by layer, and microcapsules were obtained by core removal. The microcapsules are then evaluated for their size, morphology, and other characteristics. The encapsulation efficiency of NK and the fibrinolytic activity and stability of microcapsules were also determined. Finally, NK-loaded microcapsules were subjected to simulated digestion in simulated gastrointestinal fluid and modeled by pharmacokinetics. This study intends to protect NK by embedding and to verify its environmental stability and digestive stability.