Preparation and evaluation of nattokinaseloaded self-double-emulsifying drug delivery system

Xiaona Wang,Sifan Jiang,Xinyue Wang,Jie Liao,Zongning Yin*

Key Laboratory of Drug Targeting and Drug Delivery Systems,West China School of Pharmacy,Sichuan University,No.17,Block 3,Southern Renmin Road,Chengdu 610041,China

Preparation and evaluation of nattokinaseloaded self-double-emulsifying drug delivery system

Xiaona Wang,Sifan Jiang,Xinyue Wang,Jie Liao,Zongning Yin*

Key Laboratory of Drug Targeting and Drug Delivery Systems,West China School of Pharmacy,Sichuan University,No.17,Block 3,Southern Renmin Road,Chengdu 610041,China

ARTICLEINFO

Article history:

Received 20 January 2015

Received in revised form 23 April 2015

Accepted 27 April 2015

Available online 12 May 2015

Self-double-emulsifying drug delivery system

Nattokinase

Yield

Release in vitro

Pharmacodynamics study

In the present study,we prepared nattokinase-loaded self-double-emulsifying drug delivery system(SDEDDS)and investigated its preliminary pharmacodynamics.The type and concentration of oil phase,inner aqueous phase and emulsifier were screened to prepare optimum nattokinase-loaded SDEDDS.Next,the optimum formulations were characterized based on microstructure,volume-weighted mean droplet size,self-emulsifying rate, yield,storage stability,in vitro release and in vivo pharmacodynamics studies.The water/ oil/water multiple emulsions exhibited typical multiple structure,with relatively small volumeweighted mean droplet size 6.0±0.7μm and high self-emulsifying ability(self-emulsifying time<2 min).Encapsulation of nattokinase was up to 86.8±8.2%.The cumulative release of nattokinase within 8 h was about 30%,exhibiting a sustained release effect.The pharmacodynamics study indicated that nattokinase-loaded SDEDDS could significantly prolong the whole blood clotting time in mouse and effectively improve the carrageenan-induced tail thrombosis compared with nattokinase solution.Moreover,we showed that SDEDDS could successfully self-emulsify into water/oil/water multiple emulsions upon dilution in dispersion medium with gentle stirring and effectively protect nattokinase activity in gastric environment.Our findings suggested that SDEDDS could be a promising strategy for peptide and protein drugs by oral administration.

?2015 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical University.This is an open access article under the CC BY-NC-ND license(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

With the development of biotechnology,such as genetic engineering,molecular biology and so on,the stability of peptide and protein drugs has been prominently increased[1]. Nattokinase was firstly discovered by Sumi H et al.[2]in 1987. Compared with other thrombolytic drugs(urokinase, lumbrukinase and so on),nattokinase has many unique advantages,such as abundant sources,low cost,long half-life,low side effects and high fibrinolytic activity.Moreover, nattokinase is also resistant to trypsin hydrolysis in intestine,and it can be administered either intravenously or orally [3,4],making it ideal for the prevention and treatment of embolism.Studies have shown that nattokinase is orally effective, and it can be absorbed in intestine[5-7].The optimum pH for fibrinolytic activity of Nattokinase was around 9 and the enzyme activity decreased rapidly at level below pH 5[2,8].Enteric coating is essential to protect the enzyme component of nattokinase from gastric acid degradation,there are nattokinase enteric-coated capsules[2],enteric-coated tablets[9]and other health care products on the market.Recently,lipid-based new drug delivery systems have shown great potential to further improve the absorption of peptide and protein drugs[10,11]. Especially,the water/oil/water multiple emulsions have received a great deal of attention[12-16].So far,there have no literature reports related to the nattokinase incorporated emulsions,included self-double-emulsifying drug delivery system (SDEDDS).

SDEDDS is the mixture of water-in-oil emulsions and hydrophilic emulsifiers,which can self-emulsify into water/oil/ water multiple emulsions followed by dilution with aqueous media under gastrointestinal motility or mild agitation at the ambient temperature[17].This type of drug delivery system has significant advantages as follows.On one hand,SDEDDS can avoid the inactivation and enzymatic degradation of peptide and protein drugs in gastrointestinal tract.Meanwhile,the absorption and pharmacological activity of the drugs can be significantly increased compare with other formulations[18-20]. On the one hand,SDEDDS can realize the spontaneous emulsification due to the gastrointestinal motility in vivo instead of artificial emulsification in vitro[17].Therefore,SDEDDS is more stable compared with conventional thermodynamically unstable multiple emulsions[12],and it can effectively avoid the poor stability of multiple emulsions during preparation and storage in vitro.Besides,SDEDDS greatly facilitates the patients by reducing the dose volume.

In the present study,we prepared nattokinase-loaded SDEDDS.Firstly,the optimum SDEDDS formulations were obtained by screening the type and concentration of the oil phase, inner aqueous phase and emulsifier.Then,the microstructure,volume-weighted mean droplet size,self-emulsifying rate, yield,storage stability,and nattokinase release from SDEDDS were evaluated in vitro.Finally,KM mice were utilized as an animal model for pharmacodynamics study in vivo.

2. Material and methods

2.1. Materials

Medium chain triglycerides(MCT)was purchased from Aviation Pharmaceutical Co.,Ltd.(Liaoning,China).Liquid paraffin was purchased fromTianjin Kermel Chemical Reagent Co.,Ltd. (Tianjin,China).Sorbitane monooleate(Span80)was purchased from Chengdu Kelong Chemical Reagent Factory (Chengdu,China).Polyethylene glycol(30)dimer hydroxy stearate(Arlacel P135)was purchased from Guangdong Peng Chemical Co.,Ltd.(Guangdong,China).Cetyl PEG/PPG-10/1 dimethicone(Abil?EM90)was purchased from Guangzhou Wei Xi Chemical Co.,Ltd.(Guangdong,China).Caprylocaproyl Macrogolglycerides EP(Labrasol?)was a gift sample from Gattefosse,France.Enhanced BCA protein Assay was purchased from Beyotime(Jiangsu,China).Nattokinase was purchased from National Enzyme Company(Beijing,China). Carrageenan was purchased from Sigma(Beijing,China).Labmade ultra-pure water.All other chemicals were of analytical grade.

Kunming mice,clean degree,weighting(20±2)g,half males and half females,were purchased from Experimental Animal Center of Sichuan University(Chengdu,China).All animal experiments were approved by the Animal Experimental Ethical Committee ofWest China School of Pharmacy,Chengdu,China, Animal production license numbers was SCXK(chuan)2013-026.

2.2. Screening the composition and preparation condition of SDEDDS formulations

Oil phase and lipophilic emulsifier were weighed at certain ratio and then well mixed.The coarse water/oil emulsions were prepared by slowly dropping the inner aqueous phase(water or 20 mg/mL nattokinase solution)into oil phase with magnetic stirring(1000 rpm)at room temperature.Fine and homogeneous water/oil emulsions were obtained under different homogenization condition(10000 rpm 3 min/6 min/9 min, 13000 rpm 3 min/6 min/9 min)(FJ-200 high-speed dispersion homogenizer,Jiangsujintan Jincheng Instruments Co.,Jiangsu, China).Finally,nattokinase-loaded SDEDDS was obtained by proportionally mixing the water/oil emulsions with Labrasol under magnetic stirring.

The type of commonly used self-emulsifying oil phase(MCT and liquid paraffin)and lipophilic emulsifiers(Abil EM90,Arlacel P135 and Span80)was investigated through microscopic morphology,volume-weighted mean droplet size and selfemulsifying area.Concentrations of lipophilic emulsifier varying from 10%to 25%(w/w),hydrophilic emulsifier varying from 10% to 20%(w/w)and inner aqueous phase varying from 30%to 50%(w/w)were also screened to optimize SDEDDS formulations.

The particle size and microscopic morphology of SDEDDS formulations after self-emulsifying into water/oil/water multiple emulsions were the index used for preparation optimization.The microscopic morphology was studied under inverted fluorescence microscope(×400,Axiovert 40CFL,Carl Zeiss,Germany)and the particle size distribution was determined by dynamic light scattering(Mastersizer 2000,Malvern Instruments Ltd.,Worcestershire,UK)equipped with a He-Ne laser(k=623 nm),respectively.The results were recorded as the typical volume-weighted mean droplet size D[3,4].Each formulation was carried out three times in parallel,and data were presented as Mean±SD.Pseudo-ternary phase diagrams were constructed with emulsifier titration method[18]. A series of mixtures consisting of water/oil emulsions and water phase were accurately weighed into vials at certain ratios(0:10, 1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,9:1,10:0),and then they were uniformly mixed under magnetic stirring at room temperature.Labrasol was then added into each mixture drop by drop. Meanwhile,samples were stirred and observed through microscopy.The total mass of samples at which water/oil/water multiple emulsions formed and disappeared under microscopic observation was recorded as m1and m2,respectively.These values were used to determine the boundaries of the multiple emulsions regions,through which optimum ratios of combination vehicles were selected to develop nattokinaseloaded SDEDDS and calculate self-emulsifying area.

2.3. Characterization of SDEDDS formulations

2.3.1. Microstructure observation

The morphology of SDEDDS formulations after transformation into water/oil/water multiple emulsions was observed respectively under inverted fluorescence microscope(×400, Axiovert 40CFL,Carl Zeiss,Germany),upright fluorescence microscope with an optical immersion microscope(×1,000,Leica DM1000,Germany),confocal laser scanning laser microscope (×2,000,CSLM,Leica SP5,Germany,both inner and outer aqueous phase containing 10μg/ml sodium fluorescein)using an Ar/ Kr laser at an excitation of 491 nm,and transmission electron microscope(×20,000,H-6001V,HITACHI,Japan).

2.3.2. Particle size analysis

The particle size distribution of SDEDDS formulations after transformation into water/oil/water multiple emulsions was determined by dynamic light scattering(Mastersizer 2000, Malvern Instruments Ltd.,Worcestershire,UK)equipped with a He-Ne laser(k=623 nm).The results were recorded as the typical volume-weighted mean droplet size D[3,4].Each formulation was carried out three times in parallel,and data were presented as Mean±SD.

2.3.4. Determination of the self-emulsifying rate of SDEDDS formulations

The turbidity method was used to determine the selfemulsifying rate of SDEDDS formulations.Briefly,appropriate amount of SDEDDS formulations was added into dissolution vessels containing 200 ml outer aqueous phase at 37±0.5°C, and the speed of the paddle was adjusted to 100 rpm.Samples were withdrawn at different time intervals,and the absorbance at a wavelength of 330 nm was measured until equilibrium was established.Absorbance A represented turbidity,and the relative turbidity A′was calculated according to the formula(1).Curves showing changes of turbidity and relative turbidity with time were drawn.T90(the time when turbidity reached 90%of the maximum turbidity)represented complete self-emulsifying time of SDEDDS formulations.

where A′is the relative turbidity,Aminis the minimum turbidity value throughout the whole process,Amaxis the maximum turbidity value at equilibrium,and Atis the turbidity value at a given time t.

2.3.5. Investigation of yield and storage stability

Yield,also called encapsulation efficiency,is the most important parameter characterizing the success of a multiple emulsion preparation procedure[21],which can be defined as the percentage of the aqueous phase marker added to the inner aqueous phase which remains entrapped in the inner aqueous phase on manufacture of the W/O/W multiple emulsions[22].

According to the above-mentioned method in section 2.1, optimum blank and nattokinase-loaded SDEDDS formulations(nattokinase was 20 mg/ml in inner aqueous phase) were constructed.They could self-emulsify into water/oil/ water multiple emulsions under mild agitation followed by dilution with outer aqueous phase.Briefly,1 ml freshly prepared water/oil/water multiple emulsions was diluted with 14 ml purified water and centrifuged at 10,000 rpm for 15 min at 4°C in a refrigerated centrifuge.The resulting lower outer aqueous phase was collected into a 1 ml syringe and filtered through a membrane filter(0.22-μm pore size)to eliminate the oil droplets.Subsequently,it was analyzed by Chemiluminescence Apparatus(Varioskan Flash,Thermo scientific,USA)with the BCA kit at a wavelength of 562 nm,and its absorbance was recorded as A1.The blank multiple emulsions without nattokinase were treated similarly and regarded as a control,and its absorbance was recorded as A0,then the absorbance of nattokinase which leaked into the outer aqueous phase was recorded as A2=A1-A0,the concentration C2of nattokinase in the outer aqueous phase was calculated according to the standard curve equation y=1.1541×+0.0968, R2=0.9992.The yield of nattokinase-loaded SDEDDS was determined according to the formula(2).Analyses for each formulation were carried out in triplicate,and data were presented as Mean±SD.

where W1is the original amount of nattokinase incorporated into the internal aqueous phase,W2is the amount nattokinase leaked into the outer aqueous phase at a given time t,it was calculated according to the formula W2=C2V2,where V2is the volume of outer aqueous phase.

The stability of freshly prepared SDEDDS formulations and after 30 d storage at room temperature was compared by monitoring the time-dependent changes in terms of microscopic morphology,oil droplet size,yield and so on.The leakage rates of the emulsions after storage may reflect the encapsulation stability,which can be defined as the percentage of the watersoluble drug added to the inner aqueous phase which remains entrapped in the inner aqueous phase following storage[22].

2.3.6. In vitro drug release study

The release of nattokinase from SDEDDS formulations in vitro was investigated on a dissolution apparatus ZRS-8G,Tian Da Tian Fa Technology Company(Tianjin,China).Briefly,appropriate amounts of blank and nattokinase-loaded SDEDDS formulations were separately added into the dissolution vessels containing 100 ml of different release media,such as water, phosphate buffer solution(pH 6.80),hydrochloric acid solution(pH 1.21)and acetic acid-sodium acetate buffer(pH 4.50). Taking into account the thermal instability of nattokinase,the temperature of the release medium was maintained at 25±0.5°C,and the speed of the paddle was adjusted to 100 rpm. Subsequently,2 ml sample was withdrawn and immediately replaced by fresh medium at fixed time intervals(5,15,30,45, 60,90,120,180,240,360 and 480 min).The cumulative release of nattokinase from SDEDDS formulations was determined,and then the release profiles were drawn.Analyses for each for-mulation were carried out in triplicate,and data were presented as Mean±SD.

Table 1-Results of self-emulsifying area of different SDEDDS formulations.

2.3.7. Pharmacodynamics study of nattokinase-loaded SDEDDS formulations

2.3.7.1. Influence of different prescription groups on whole blood clotting time of mouse.A total of 70 KM mice were randomly divided into seven groups as follows:normal saline control group;low-(8630 FU/kg.bw),medium-(12,945 FU/kg.bw)and high-dose(17,260FU/kg.bw)nattokinase solution groups;low-(8630 FU/kg.bw),medium-(12,945 FU/kg.bw)and high-dose (17,260FU/kg.bw)nattokinase-loaded SDEDDS formulation groups.Mice were daily fed by gavage for 14 days(n=10)and anesthetized with ether at 1 h after last intragastric administration.A drop of blood was collected from the eye ground vein onto the glass slide by a 0.5-mm capillary glass tube.The blood drop was immediately and gently provoked from the edge to the center with clean needles at regular intervals until the bloodshot was observed.This period was considered as the whole blood clotting time of mouse in vitro.

2.3.7.2. Influence of different prescription groups on tail thrombosis of mouse.Animals were grouped and administrated as same as in section 2.3.7.1.Briefly,2 h after last intragastric administration,mice were administered with 0.5%carrageenan (w/v)at a dose of 0.1 ml/10 g by peritoneal injection to induce tail thrombosis.The length of tail thrombus was determined at 8 h,24 h and 48 h,and then the relative length of tail thrombus was calculated according to the formula(3).

2.3.7.3. Statistical analysis.Data were presented as Mean±SD (n=10).Statistical significance of differences among groups was performed by one-way ANOVA and Kruskal-Walis H test, and then the statistical significance of the means was determined by SNK or Dunnett-t test.A P value of less than 0.05 was considered as statistically significant.

3. Results and discussions

3.1. Screening the composition and preparation condition of SDEDDS formulations

Fig.1 shows pseudo-ternary phase diagrams of four prescriptions shown in Table 1.The shaded area indicates that SDEDDS formulations could self-emulsify into water/oil/water multiple emulsions.By comparing the self-emulsifying area,we could conclude that MCT had a better emulsifying capacity than liquid paraffin;and EM90 had a better emulsifying capacity than Arlacel P135 and Span 80.The maximum self-emulsifying area of the prescription C was 41.8%.Then we optimized the concentrations of emulsifier and inner aqueous phase using MCT andAbil EM90 as oil phase and lipophilic emulsifier,respectively.

Fig.2A shows that the oil globule sizes of water/oil/water multiple emulsions were decreased when the concentration of hydrophilic emulsifier was increased from 10%to 15%(w/ w)with a constant concentration of EM90.However,when the concentration of the hydrophilic emulsifier reached 20%(data not shown),most droplets of water/oil/water emulsions occurred in phase inversion.It seemed that excess of hydrophilic emulsifier destabilized the multiple emulsions.This could be caused by that the excess of hydrophilic emulsifier formed mixed micelles,which could dissolve the oil phase and lipophilic emulsifier,leading to film rupture and accelerated release of drugs in inner aqueous phase.Therefore,the yield of formulations was also reduced[23,24].Moreover,the oil globule sizes of water/oil/water emulsions were increased with the increase of inner aqueous phase.This could be explained by that increased inner aqueous phase resulted in the high viscosity of the water/oil emulsions,leading to hard dispersion of the SDEDDS formulations into the outer aqueous phase to form water/oil/water multiple emulsions.Therefore,droplets of water/ oil/water emulsions with larger size finally formed[25].In addition,the oil globule sizes of water/oil/water emulsions were decreased when the lipophilic emulsifier concentration was increased from 10%to 20%with a constant concentration of Labrasol.However,the oil globule sizes were increased when the lipophilic emulsifier concentration was greater than 25% (Fig.2B).This could be explained by that the water-oil interfacial tension was reduced with the increase of the lipophilic emulsifier concentration from 10%to 20%,resulting in the reduced sizes of oil droplets.However,the lipophilic emulsifier was saturated in the water-oil interface at a concentration above 25%[26],and the interfacial tension was no longer reduced.On the contrary,the viscosity of water/oil emulsions was too large,leading to hard dispersion of SDEDDS[23].

Comprehensively considering the morphology,particle size and self-emulsifying area of water/oil/water multiple emulsions transformed from SDEDDS formulations,we found that MCT was the oil phase,Abil EM90 was the lipophilic emulsifier and Labrasol was the hydrophilic emulsifier in the optimum SDEDDS formulations.In addition,the optimum concentration of Abil EM90,Labrasol and inner aqueous phase was 20% (w/w),15%(w/w)and 40%(w/w),respectively.

Table 2 shows that the oil globule sizes of water/oil/water multiple emulsions were slightly increased with the homogenization time increasing at a constant 10,000 rpm or13,000 rpm.In the end,we determined the best w/o preparation condition was 10,000 rpm 3 min,at the moment the water/ oil/water multiple emulsions owned relatively small volumeweighted mean droplet size and good morphology.

Table 2-Results of oil globule sizes of water/oil/water multiple emulsions under different homogenization condition for preparing the w/o emulsions.

3.2. Characterization of SDEDDS formulations

3.2.1. Microscopic structure observation

Fig.3A-C show that the freshly prepared optimum SDEDDS formulations could self-emulsify into fine water/oil/water multiple emulsions after dilution with water(37±0.5°C)under gentle stirring.The structure analysis revealed that the dispersed oil droplets contained smaller dispersed inner water droplets, which was in accordance with the characteristics of multiple emulsions[25,27,28].

3.2.2. Particle size analysis

Fig.3D shows the microstructure of inner water droplets of multiple emulsions,the average particle size of water/oil emulsions was about 580±20 nm,and the particle size of water/oil emulsions was distributed in a range of 250-800 nm.

Fig.4 reveals that the oil droplet size of water/oil/water multiple emulsions transformed from SDEDDS formulations was 6.0±0.7μm,representing a typical bimodal distribution of multiple emulsions.This could be explained by that the small size internal water droplets promoted the formation of larger oil droplets,thereby forming the second globule peak which belonged to the bimodal oil droplet distribution[29-31].The polydispersity of the oil droplet size distribution was expressed as span[23],which was defined as follows:

where D(0,5)is the size in microns at which 50%of the sample is larger and 50%is smaller;D(0,1)and D(0,9)are the sizes of the droplets below 10%and 90%,respectively,of the sample lies.The span of the oil droplet size of water/oil/water emulsions transformed from SDEDDS formulations was 1.7±0.2.

3.2.3. Determination of the self-emulsifying rate of SDEDDS formulations

The self-emulsifying rate of SDEDDS formulations reflects the self-emulsifying ability[32].The faster self-emulsifying rate, the better self-emulsifying ability of SDEDDS formulations, and vice versa.Experimental results indicated that as thehydrophilic emulsifier concentration was 10%,15%and 20%, the T90was 2 min,1.6 min and 1.5 min,respectively.T90was all less than 2 min,indicating that SDEDDS formulations had high self-emulsifying ability.Fig.5 exhibits the curves of turbidity and relative turbidity of SDEDDS formulations versus time when the hydrophilic emulsifier concentration was 15%.

3.2.4. Investigation of yield and storage stability

The initial yield of nattokinase-loaded SDEDDS formulations was 86.8±8.2%,whereas it was changed to 82.6±9.1%after stored at room temperature for 30 d.The morphology of nattokinase-loaded SDEDDS formulations was barely changed. Fig.6 A-B show that the oil globule size was decreased from 5.3±0.4μm to 4.7±0.7μm for nattokinase-loaded SDEDDS formulations and from 6.9±0.4μm to 3.4±0.5μm for blank formulations.Data revealed that the nattokinase-loaded SDEDDS formulations had smaller change in particle size, showing better stability.This could be explained by that partial proteins could be adsorbed at the oil/water interface,then interact with the surfactants at the oil/water interface and enhance the flexibility of the interfacial film.This process prevented globule breaking,leading to the improved stability of the emulsions[25].

3.2.5. In vitro drug release study

Fig.7 shows the nattokinase release profiles from the optimum SDEDDS formulations.The cumulative release of nattokinase was about 30%after 8 h,providing a sustained release in four different release media.We found that the SDEDDS formulations were stable without an initial burst release due to the release of poorly entrapped nattokinase[33].In this study,the cumulative release of nattokinase from SDEDDS formulations was almost balanced within 8 h,which was consistent with many studies.It has been reported that the cumulative release of drugs from water/oil/water multiple emulsions in vitro ranges from 20%to 40%within 10 h[34-36].Especially for the water/oil/water multiple emulsions containing Abil EM90 as lipophilic emulsifier,their release is slow through the liquid oil membrane(60%release after 30 days)[37].We could also control the drug release rate by adjusting the type and concentration of oil phase and emulsifier.

The rank order of nattokinase release rate in vitro was pH 1.21>H2O>pH 4.50>pH 6.80.In our previous study,we found the rank order of oil droplet size of multiple emulsions in four releasemediawaspH6.80(5.47±0.19μm)>pH 4.50(5.09±0.20μm)>H2O(4.83±0.28μm)>pH 1.21 (4.69±0.32μm).The reason may be that the smaller oil droplet size,the larger interfacial area available for drug transport across the oil phase barrier,then the faster drug release[30].

Table 3 shows that the release data fitted to Weibull equation since the highest correlation coefficients were obtained (R2from 0.97 to 0.99).

3.2.6. Pharmacodynamics study

Table 4 shows the results of whole blood clotting time in vitro. We found that the whole blood clotting time was significantly prolonged in low-,medium-,high-dose groups of nattokinase-loaded SDEDDS formulations and high-dose group of nattokinase solution compared with the normal saline control group(P<0.05).However,only a tendency of extended whole blood clotting time was found in other groups compared with the normal saline control group(P>0.05).Carrageenaninducedtailthrombosismodelisusedtoevaluate antithrombotic and thrombolytic agents.This model is simple and non-invasive to the experimental animals.Moreover,it allows observing and measuring progression of thrombosis continuously and accurately[38,39].Table 4 shows that nattokinaseloaded SDEDDS formulations could significantly improve mouse tail thrombosis by reducing the mouse tail thrombosis average relative length(P<0.05).However,only a tendency of reduced mouse tail thrombosis average relative length was detected in nattokinase solution groups compared with the normal saline control group(P>0.05).In addition,the differences between the high-dose group of nattokinase-loaded SDEDDS formulations and the high-dose group of nattokinase solution were statistically significant(P<0.05),and the photos of mouse tail thrombosis at 48 h were shown in Fig.8.Therefore,we concluded that the encapsulation of nattokinase in SDEDDS formulations would better protect nattokinase against inactivation in the stomach,leading to a better efficiency through oral administration.Moreover,the efficacy of SDEDDS formulations was also in a dose-dependent manner,and the best efficacy was found in the high-dose group of nattokinaseloaded SDEDDS formulations.

4. Conclution

In the present study,we successfully prepared the nattokinaseloaded SDEDDS formulations.The optimal formulations werestill stable over a period of 30 d at room temperature.They could self-emulsify into fine water/oil/water multiple emulsions upon dilution in dispersion medium under gentle stirring,with small volume-weighted mean droplet size,high yield and selfemulsifying ability,and a sustained release of nattokinase in vitro.Besides,in vivo pharmacodynamics results indicated that the encapsulation of nattokinase in SDEDDS formulations could better protect nattokinase against inactivation in the stomach,leading to a better efficiency through oral administration.Our findings suggested that SDEDDS could be a promising strategy for peptide and protein drugs by oral administration.

Table 3-Results of nattokinase release data simulated by Weibull equation.

Table 4-Influence of different prescription groups on whole blood clotting time and tails thrombosis of mouse.(Data shown as Mean±SD,n=10).

AcAcknowledgements

This work was financially supported by National Natural Science Foundation of China(No.81373338).

REFERENCES

[1]Yun Y,Cho YW,Park K..Nanoparticles for oral delivery: targeted nanoparticles with peptidic ligands for oral protein delivery.Adv Drug Deliv Rev 2013;65:822-832.

[2]Sumi H,Hamada H,Tsushima H,et al.A novel fibrinolytic enzyme(nattokinase)in the vegetable cheese Natto;a typical and popular soybean food in the Japanese diet.Cell Mol Life Sci 1987;43:1110-1111.

[3]Dabbagh F,Negahdaripour M,Berenjian A,et al. Nattokinase:production and application.Appl Microbiol Biotechnol 2014;98:9199-9206.

[4]Wang SL,Wu YY,Liang TW..Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007.New Biotechnol 2011;28:196-202.

[5]Fujita M,Hong K,Ito Y,et al.Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat.Biol Pharm Bull 1995;18:1387-1391.

[6]Duan ZB,Dong GX,Wan XQ,et al.Study on absorption and distribution characters of nattokinase in small intestine of rabbits.Acta Nutr Sin 2008;2:022.

[7]Fujita M,Ohnishi K,Takaoka S,et al.Antihypertensive effects of continuous oral administration of nattokinase and its fragments in spontaneously hypertensive rats.Biol Pharm Bull 2011;34:1696-1701.

[8]Dubey R,Kumar J,Agrawala D,et al.Isolation,production, purification,assay and characterization of fibrinolytic enzymes(Nattokinase,Streptokinase and Urokinase)from bacterial sources.Afr J Biotechnol 2013;10:1408-1420.

[9]Law D,Zhang Z..Stabilization and target delivery of Nattokinase using compression coating.Drug Dev Ind Pharm 2007;33:495-503.

[10]Martinho N,Damgé C,Reis CP..Recent advances in drug delivery systems.J Biomater Nanobi 2011;2:510-526.

[11]Li P,Nielsen HM,Müllertz A..Oral delivery of peptides and proteins using lipid-based drug delivery systems.Expert Opin Drug Del 2012;9:1289-1304.

[12]Zhang L,Zhang Q,Zhou P,et al.Enhanced absorption of Stachydrine using a self-double-emulsifying drug delivery systems(SDEDDS):in vitro and in vivo studies.Life Sci 2013;10:895-899.

[13]Qi X,Wang L,Zhu J..Water-in-oil-in-water double emulsions:an excellent delivery system for improving the oral bioavailability of pidotimod in rats.J Pharm Sci 2011;100:2203-2211.

[14]Shima M,Tanaka M,Fujii T,et al.Oral administration of insulin included in fine W/O/W emulsions to rats.Food Hydrocol 2006;20:523-531.

[15]Li X,Qi J,Xie Y,et al.Nanoemulsions coated with alginate/ chitosan as oral insulin delivery systems:preparation, characterization,and hypoglycemic effect in rats.Int J Nanomed 2013;8:23-32.

[16]Siddhartha T,Senthil V,Kishan I,et al.Design and development of oral Nanoparticulated insulin in multiple emulsion.Curr Drug Deliv 2014;11:472-485.

[17]Qi X,Wang L,Zhu J,et al.Self-double-emulsifying drug delivery system(SDEDDS):a new way for oral delivery of drugs with high solubility and low permeability.Int J Pharm 2011;409:245-251.

[18]Garti N,Bisperink C..Double emulsions:progress and applications.Curr Opin Colloid Interface Sci 1998;3:657-667.

[19]Akhtar M,Murray BS,Afeisume EI,et al.Encapsulation of flavonoid in multiple emulsion using spinning disc reactor technology.Food Hydrocoll 2014;34:62-67.

[20]Sapei L,Naqvi MA,Rousseau D..Stability and release properties of double emulsions for food applications.Food Hydrocoll 2012;27:316-323.

[21]Koberstein-Hajda A,Dickinson E..Stability of water-in-oilin-water emulsions containing faba bean proteins.Food Hydrocoll 1996;10:251-254.

[22]O’Regan J,Mulvihill DM..Sodium caseinate-maltodextrin conjugate stabilized double emulsions:encapsulation and stability.Food Res Int 2010;43:224-231.

[23]Matos M,Gutiérrez G,Coca J,et al.Preparation of water-inoil-in-water(W1/O/W2)double emulsions containing transresveratrol.Colloid Surf A 2014;442:69-79.

[24]Shima M,Kobayashi Y,Kimura Y,et al.Effect of the hydrophilic surfactants on the preparation and encapsulation efficiency in course and fine W/O/W type emulsions.Colloid Surf A 2004;238:83-90.

[25]Cournarie F,Savelli M-P,Rosilio V,et al.Insulin-loaded W/O/W multiple emulsions:comparison of the performances of systems prepared with medium-chaintriglycerides and fish oil.Eur J Pharm Biopharm 2004;58:477-482.

[26]Csóka I,Eros I..Stability of multiple emulsions:I. Determination of factors influencing multiple drop breakdown.Int J Pharm 1997;156:119-123.

[27]Pays K,Giermanska-Kahn J,Pouligny B,et al.Double emulsions:how does release occur?J Control Release 2002;79:193-205.

[28]González-Ochoa H,Ibarra-Bracamontes L,Arauz-Lara JL.. Two-stage coalescence in double emulsions.Langmuir 2003;19:7837-7840.

[29]Garti N,Benichou A..Recent developments in double emulsions for food applications.Food Emuls 2004;10:353-412.

[30]Weiss J,Muschiolik G..Factors affecting the droplet size of water-in-oil emulsions(W/O)and the oil globule size in water-in-oil-in-water emulsions(W/O/W).J Dispers Sci Technol 2007;28:703-716.

[31]Carrillo-Navas H,Cruz-Olivares J,Varela-Guerrero V,et al. Rheological properties of a double emulsion nutraceutical system incorporating chia essential oil and ascorbic acid stabilized by carbohydrate polymer-protein blends. Carbohydr Polym 2012;87:1231-1235.

[32]Balakumar K,Raghavan CV,Abdu S..Self nanoemulsifying drug delivery system(SNEDDS)of rosuvastatin calcium: design,formulation,bioavailability and pharmacokinetic evaluation.Colloid Surf B 2013;112:337-343.

[33]Khoee S,Yaghoobian M..An investigation into the role of surfactants in controlling particle size of polymeric nanocapsules containing penicillin-G in double emulsion. Eur J Med Chem 2009;44:2392-2399.

[34]Bonnet M,Cansell M,Placin F,et al.Influence of the oil globule fraction on the release rate profiles from multiple W/O/W emulsions.Colloid Surf B 2010;78:44-52.

[35]Geiger S,Tokgoz S,Fructus A,et al.Kinetics of swellingbreakdown of a W/O/W multiple emulsion:possible mechanisms for the lipophilic surfactant effect.J Control Release 1998;52:99-107.

[36]Herzi S,Essafi W,Bellagha S,et al.Influence of the inner droplet fraction on the release rate profiles from multiple W/O/W emulsions.Colloid Surf A 2014;441:489-495.

[37]Sela Y,Magdassi S,Garti N..Release of markers from the inner water phase of W/O/W emulsions stabilized by silicone based polymeric surfactants.J Control Release 1995;33:1-12.

[38]Arslan R,Bor Z,Bektas N,et al.Antithrombotic effects of ethanol extract of Crataegus orientalis in the carrageenaninduced mice tail thrombosis model.Thromb Res 2011;127:210-213.

[39]Simkhada JR,Cho SS,Mander P,et al.Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model.Thromb Res 2012;129:176-182.

*Corresponding author.Sichuan University,No.17,Block 3,Southern Renmin Road,Chengdu 610041,China.Tel./fax:+86 28 85502917. E-mail address:yzn@scu.edu.cn(Z.Yin). Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2015.04.005

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