How to prepare reduced glutathione nanoliposomes with high embedding rate?

Reduced glutathione (GSH) is composed of L-hemi, γ,-[and 1-2 into], containing sulfhydryl, γ-glutamyl, and other functional groups, and exhibits antioxidant, detoxification, scavenging of free radicals, anti-radiation, and maintenance of normal protein and immune system functions[3] . However, GSH has a short circulation cycle in the body, is easily oxidized, and cannot cross the cell membrane, which limits its application in health care and medical treatment[4- 5] . Studies have shown that the micro- and nanoembedding technology can effectively improve the stability of GSH and its ability to cross the cell membrane[6] . Du Ge [7] used gelatin and gum arabic as wall materials to prepare GSH-embedded microcapsules by composite coagulation method, which showed a high embedding rate and a certain degree of protection of GSH at high temperatures.Naji-tabasi et al. [8] utilized basil seed gum to encapsulate GSH, and the prepared GSH nanoparticles were still stable in the gastrointestinal environment. In addition, Joy et al.[9] found that GSH embedded in liposome nanoparticles enhanced drug delivery to the brain.

 

Liposomes, as a kind of micro vesicles formed by lipid bilayer wrapped in aqueous solution, are capable of embedding not only lipophilic compounds but also hydrophilic compounds[10] . When GSH is embedded in liposomes, the bilayer can reduce the influence of environmental factors such as oxygen and metal ions on GSH, and increase the stability of GSH; in addition, because the phospholipid bilayer has a high affinity for the cell membrane, it is easier to transport GSH into the cell, and it can avoid the decomposition and destruction of GSH, so that GSH can be released slowly and the bioavailability of GSH can be improved[11] . There are many methods for embedding GSH into liposomes, such as pH gradient, ethanol injection, reversed-phase evaporation, and thin-film-ultrasound [12] . In this paper, we use

 

Influencing factors (e.g., amount of lecithin added, amount of Tween-80 added,

The interactions of GSH addition, sonication time and lecithin to cholesterol ratio were investigated in order to obtain the optimal process parameters of high GSH nanoliposomes and to provide theoretical and experimental bases for their applications in the food and medical industries.

 

1 Materials and Methods

1.1 Materials and reagents

Reduced glutathione (GSH), Shandong Jincheng Bio-Pharmaceutical Co., Ltd; soy lecithin, Shanghai McLean Biochemical Science and Technology Co., Ltd; cholesterol, Soleilbao Bio-technology Co.

 

1.2 Instruments and equipment

JY99-Ⅱ D Ultrasonic Crusher, Beijing Jiayuan Xingye Technology Co.

Ltd; Multiskan FC, Thermo Fisher (Shanghai) Instruments Co., Ltd; 5039R centrifuge, Eppendorf, Germany;

FE20pH meter, METTLER TOLEDO INSTRUMENTS INC.

 

1.3 Test methods

1 .3 . 1 Plotting of GSH standard curves

The reaction produces 2-nitro-5-mercaptobenzoic acid and glutathione disulfide, and 2-nitro-5-mercaptobenzoic acid has a yellow color with a maximum absorption value at 412 nm[13] . Dissolve 1 mg of GSH standard in 1 mL of distilled water to make a GSH stock solution with a mass concentration of 1 mg/mL. Then, according to the instruction of the reduced glutathione test kit, add 20 μL of the standard solution, 140 μL of reagent 2 and 40 μL of reagent 3 into a centrifuge tube, mix well, and let it stand for 2 min before analyzing the GSH concentration at 412 nm. After standing for 2 min, the absorbance was measured at 412 nm. The mass concentration of GSH was taken as the horizontal coordinate, and the absorbance at 412 nm was taken as the vertical coordinate, and the standard curve of GSH was plotted.

 

1 .3 .2 Preparation of reduced glutathione nanoliposomes

In this paper, GSH-embedded nanoliposomes (GLip) were prepared according to the thin-film-sonication method of Lasicd[14] . A certain amount of lecithin, cholesterol and Tween-80 was dissolved in 15 mL of anhydrous ethanol, and the mixed solution was poured into a round-bottomed flask and rotary evaporated at 50 ℃ and - 0.1 MPa to remove the organic phase and form a uniform and transparent film on the wall of the flask. Then, add 20 mL, 0.05 mol/L, pH 6.0 of PBS solution at 50 ℃, under the conditions of atmospheric pressure to continue rotary hydration for 30 min to wash the film; after the film was washed down, the phosphate buffer solution (PBS) in an ice bath at 250 W power for ultrasonication, the processing of 1 s work 1 s pause 1 s mode, at 20 ℃ static for 2 h, to obtain the GLip, the GLip, the GLip, the GLip, the GLip, the GLip, the GLip, the GLip, the GLip, the GLip, the GLip, the GLip. The GLip sample was transferred to the inner tube of an ultrafiltration centrifuge tube (with a molecular weight cutoff of 10 kDa) and centrifuged at 3,000 r/min for 20 min to collect the filtrate from the outer tube of the ultrafiltration centrifuge tube. Add 2 mL of distilled water to the inner tube, re-distribute the GLip concentrated solution, centrifuge again at 3,000 r/min for 20 min, and collect the filtrate from the outer tube of the ultrafiltration centrifuge tube. This step is repeated three times in order to separate the non-GLip forming material from the GLip. Finally, the GLip concentrate in the inner tube of the ultrafiltration centrifuge tube is completely removed, and the collected filtrate is completely mixed and stored at 4 °C, pending determination.

 

1 .3 .3 Determination of GLip embedding rate

Pipette 100 μL of filtrate from the outer tube of the ultrafiltration centrifuge tube and dilute it appropriately. After appropriate dilution, the content of free GSH in the filtrate was determined according to the method described in 1.3.1 and the standard curve of GSH, and the embedding rate of GSH in GLip was calculated according to equation (1).

Embedding rate ×100% , (1) where :Wtotal is the total amount of GSH added during preparation; Wfree is the amount of free GSH measured.

 

1 .3 .4 Optimization of process parameters

In the process of GLip preparation, the amounts of lecithin (100, 150, 200, 250, 300, 350 mg), Tween-80 (40, 60, 80, 100, 120, 140 mg), GSH (30, 40, 50, 60, 70, 80 mg), sonication time (5, 10, 15, 20, 25, 30 min) and the mass ratio of lecithin to cholesterol (1:1, 2:1, 4:1, 6:1, 8:1, 10:1) were measured using the embedding rate as an indicator, Based on the results of the one-way experiments, the response surface analysis (RSA) experiments with 17 experimental points at three factors and three levels were designed with the addition of lecithin, GSH and Tween-80 as independent variables.

 

1 .3 .5 Determination of GLip Particle Size

1 mL of GLip sample was diluted 10 times with ultrapure water, and then the particle size and surface charge of the sample were determined by potentiometric-particle size analyzer at 20 ℃, and the refractive index of phospholipids and dispersive medium was 1 . 330 . Each sample was analyzed more than 3 times in parallel.

 

1 .3 .6 Data processing and analysis

Response surface analysis was performed using Design-Expert V8.0.6 software. 0.6 software to fit a second-order polynomial equation, and the coefficients of the linear regression equation were tested for significance (F-test) to summarize the effects of the selected single factors on the GSH incorporation rate in GLip. The obtained second-order polynomial equations were transformed into response surfaces using Design-Expert V8.0.6 software to further analyze the effects of the experimental factors and levels on the embedding rate of GLip[15] .

 

2 Results and Discussion

2.1 Measurement of GSH content

The standard curve of GSH according to 1 .3 . The standard curve of GSH according to 1.3.1 is shown in Fig. 1. As can be seen from Fig. 1, the mass concentration of GSH showed a good linear relationship with the absorbance in the range of 0~200 μg/mL, and the linear equation was y = 0.002 1x + 0.004 7 , R2 = 0.999 2 . The mass concentration of GSH in the sample was calculated from the standard curve, and then the mass of GSH was determined.

 

2.2 GLip average particle size

The average grain size of GLip was (77.70 ± 0.5 mm). 70 ± 0 . 87) nm , PDI was (0. 219 ± 0 .005) and zeta potential was (- 27 .20 ± 0 .031) V. The average particle size of GLip was (77 .70 ± 0 . 87) nm , PDI was (0. 219 ± 0 .005) and zeta potential was (- 27 .20 ± 0 .031) V. The particle size distribution of this nanosized GLip particles is relatively uniform and stable. 2.3 Effect of one-way experiment on GLip embedding rate

 

2.3 . 1 Effect of lecithin addition on embedding rate

According to the pre-test, the mass ratio of lecithin to cholesterol was fixed at 4:1, the amount of tween-80 was 100 mg, and the amount of GSH was 70 mg, so as to study the effect of lecithin addition of 100, 150, 200, 250, 300 and 350 mg on the embedding rate of GLip. As can be seen from Fig. 2, when the amount of lecithin added was less than 200 g, the embedding rate of GLip was proportional to the amount of lecithin added, which was mainly due to the fact that the increase of lecithin could form more phospholipid bilayers, which could encapsulate more GSH, thus increasing the embedding rate of GLip; when the amount of added lecithin was 200 g, the embedding rate of GLip reached the highest, which was 63%; when the amount of added lecithin was more than 200 g, the embedding rate of GLip reached the highest, which was 63%. When the added amount of lecithin is more than 200 g, the embedding rate of GLip is basically unchanged, which may be due to the fact that the phospholipid bilayer has a certain limit to embed GSH, and tends to be saturated after reaching the maximum amount of embedded GSH. In addition, the GLip was prepared by the film-ultrasonic method, if the amount of lecithin added in the preparation system was too high, it would cause uneven film formation after rotary evaporation, and it would be more difficult to wash the film with PBS solution.

 

2.3 .2 Effect of lecithin to cholesterol ratio on encapsulation rate

The mass ratio of lecithin to cholesterol (1:1, 2:1, 4:1, 6:1, 8:1, 10:1) was fixed at 200 mg of lecithin, 100 mg of tween-80, and 70 mg of GSH, and the effect of lecithin to cholesterol ratio (1:1, 2:1, 4:1, 6:1, 8:1, 10:1) on the embedding rate of GLip was investigated. As shown in Figure 3, with the increase of the ratio of lecithin to cholesterol, the embedding rate of GLip increased and then decreased, and when the ratio of lecithin to cholesterol was 4:1, the embedding rate of GLip reached a maximum of about 55%. This is mainly due to the fact that cholesterol is a kind of lipid, which can enter into the phospholipid bilayer and enhance the tightness of the phospholipid bilayer, which makes the membrane stable and improves the embedding rate of Glip. However, when the ratio of added lecithin to cholesterol was greater than 4:1, the phospholipid bilayer became unstable with the addition of cholesterol, resulting in increased membrane permeability and even membrane formation difficulties, which led to the exudation of GSH and a decrease in the embedding rate of GLip[16] .

 

2.3 .3 Effect of Tween-80 addition on embedding rate

The mass ratio of lecithin and cholesterol was fixed at 4:1, the addition of lecithin was 200 mg, the addition of GSH was 70 mg, and the effect of the addition of tween-80 (40, 60, 80, 100, 120, 140 mg) on the embedding rate of GLip was investigated. As shown in Fig. 4, with the trend of tween-80 addition, the embedding rate of GLip reached the maximum of about 60% when the addition of tween-80 was 100 mg. Tween-80 is a nonionic surfactant, which can be evenly distributed in the aqueous and lipid phases of liposomes and encapsulated into the lipid bilayer to form a thicker membrane, which makes the structure of phospholipid bilayer more stable. Therefore, when the addition of Tween-80 was less than 100 mg, the embedding rate of GLip increased with the increase of Tween-80. The effect of glutathione addition (30, 40, 50, 60, 70, 80 mg) on the embedding rate of GLip was investigated. As shown in Fig. 5, with the increase of GSH, the embedding rate of GLip increased and then decreased, and the embedding rate of GLip reached the maximum of about 60% when the addition of GSH was 70 mg. This is mainly because glutathione is a water-soluble drug.

 

 The ability to enter the aqueous phase inside the phospholipid bilayer increases the embedding rate of GSH liposomes. However, the volume of liposome vesicles is limited, and the amount of glutathione embedded is certain[18] . When the amount of glutathione added is more than 70 mg, the volume of the aqueous phase of liposome becomes saturated, and it is difficult to dissolve more glutathione, thus reducing the GLip embedding rate.

 

2.3 .5 Effect of ultrasound time on embedding rate

The mass ratio of lecithin and cholesterol was fixed at 4:1, the amount of lecithin added was 200 mg, the amount of tween-80 added was 100 mg, and the amount of GSH added was 70 mg, and the effect of the sonication time on the embedding rate of GLip was investigated. As shown in Fig. 6, with the increase of sonication time, the embedding rate of GLip showed an increasing and then decreasing trend. When the sonication time was 10 min, the embedding rate of GLip reached the maximum, which was about 56%. In the process of preparing liposomes, ultrasound is usually used to redispersed the particles in the system, so as to obtain the liposomes with small particle size and uniform distribution, in order to increase the stability of the system[19] . However, when the ultrasound time is too long, the effect of ultrasound is enhanced, the structure of liposome is destroyed, and GSH is leaked, which reduces the embedding rate of GLip.

 

2.4 Optimization of GLip preparation parameters

2.4 . 1 Box-Behnken design

Based on the results of the one-way experiment, it is clear that changing the lecithin addition

The maximum embedding rate of GLip with the amount of lecithin, GSH and Tween-80 was greater than that with the ratio of lecithin to cholesterol and the ultrasound time, so the amount of lecithin, GSH and Tween-80 were chosen to maximize the embedding rate.

The results are shown in Table 1. The results of the three-factor, three-level impact surface test were designed according to the Box-Beknhen Center Combined Experimental Design principle, with the addition amount and the amount of Tween-80 as the independent variables and the embedding rate of GLip as the dependent variable.

 

2.4 .2 Model equation development and significance test

The data in Table 2 were analyzed using GLip's embedding rate as the response value and fitted to a quadratic polynomial regression equation modeled as : Y = 63 . 92 + 3.20A + 3. 11B - 0.52C + 2.40AB - 0.75AC + 1 . 54BC - 3.74A2 - 2.28B2 - 1 .64C2 . The results obtained from the significance test of the coefficients of the regression equation are shown in Table 3 .

 

From the significance test of the coefficients of the regression equation and the analysis of variance (ANOVA), we can see that P < 0.000 1 , so the response surface regression model reaches a highly significant level; and because R2 = 0.983 5 , it shows that 98% of the data can be interpreted by the model equation, and the P-value of the out-of-fit term is 0.160 9 , which is insignificant, which means that the model chosen in this experiment is suitable for the preparation of GLip, and the model of the quadratic regression equation is meaningful. This indicates that the chosen model is suitable for the preparation of GLip and the quadratic regression model is meaningful, so the embedding rate of liposomes can be obtained and the liposome preparation process can be optimized. The analysis of variance (ANOVA) showed that A, B, AB, BC, A2, B2, and C2 had significant effects on the embedding rate of GLip, and the standardized regression coefficients of the factors were A > B > C. The model coefficient of variation (CV) of the model was CV = 1, and the model coefficient of variation (CV) was CV = 1. The coefficient of variation of the model, CV = 1.40%, indicated that the difference between the predicted and experimental values was small.

 

2.4 .3 Preparation process optimization results analysis

The graph of response surface methodology (RSM) is a three-dimensional contour plot on a two-dimensional plane of a specific response surface (Y) with corresponding factors A, B, and C. Each response surface is analyzed for two of the factors, and the other factor is fixed at the zero level. Since the graph can visualize the effect of the factors on the response values, their interactions in the preparation of GLip can be found on the response surface analysis plots obtained from the experiments.20 The effect of the interactions among the three factors, namely, lecithin addition, GSH addition, and Tween-80 addition, on the embedding rate of GLip is shown in Fig. 7, Fig. 9, and Fig. 9, Fig. 10. - The effects of the interactions between the three factors on the GLip embedding rate are shown in Fig. 7 and Fig. 9.

 

As shown in Fig. 7, the response surface shows a steeper slope, which indicates that the interaction between lecithin and GSH additions has a more significant effect on the embedding rate of GLip. From the contour lines in Fig. 7, the ellipses are more densely arranged in the region of lecithin addition, which indicates that the effect of lecithin addition on the embedding rate is greater than that of GSH addition. When the amount of GSH was constant, the embedding rate increased and then decreased with the increasing amount of lecithin, and the embedding rate reached the maximum when the amount of lecithin was 225-250 mg. When the added amount of lecithin was constant, with the increase of GSH, the embedding rate increased first and then decreased; when the added amount of GSH was 75-80 mg, the embedding rate reached the maximum.

 

The shell of the liposome is lecithin, and with a certain amount of lecithin, the volume of the liposome formed is certain, and with the addition of GSH, the volume of the liposome will be overloaded. As shown in Fig. 8, the steeper slope can be seen from the response surface plot, indicating that the interaction between the amount of lecithin added and the amount of Tween-80 added has a more obvious effect on the embedding rate. From the contour lines in Fig. 8, it can be seen that the ellipses are more densely arranged in the region of lecithin addition, which indicates that the effect of lecithin addition on the embedding rate is greater than that of tween-80 addition on the embedding rate. When the amount of lecithin was constant, the embedding rate with the increase in the amount of tween-80 increased and then decreased the wettability, so that the water is easy to penetrate and accelerate the disintegration rate; in addition, its strong solubility, anti-flocculation effect improves the dissolution of the drug, which reduces the embedding rate. When the added amount of Tween-80 was unchanged, with the increasing amount of lecithin, the embedding rate was the first to increase and then decrease, and the embedding rate was the highest when the added amount of lecithin was 225~250 mg.

 

2.4 .4 Validation of optimal preparation conditions for GLip

The optimal parameters for the preparation of GLip using Design-Expert V8.0.6 statistical software were 232.84 mg of lecithin, 75.91 mg of GSH, and 94.30 mg of threonine-80, and the predicted optimized embedding rate was 66.27%. In order to verify the validity of the model, combined with the actual experimental situation, the optimal preparation process was optimized to 232 mg of lecithin, 75 mg of GSH, 94 mg of Tween-80, and the embedding rate of GLip was (65.85±0.54)% after 3 repetitions. 85±0.54)%, and the absolute value of the error from the theoretical prediction was less than 5%. This indicates that the model parameters optimized by the response surface method are accurate, and the optimal preparation conditions are reliable and valuable.

 

3 Conclusion

In this paper, based on a one-way experiment using the embedding rate as an indicator, the

Response surface methodology was used to analyze the interactions among the influencing factors in the preparation of GLip by thin-film ultrasonication, and the optimal parameters for the preparation of GLip were obtained to improve the embedding rate of GLip. The optimal preparation conditions were as follows: 232 mg of lecithin, 75 mg of GSH, and 94 mg of tween-80, and the maximum embedding rate of (65.85±0.54)% was obtained. The results of this paper provide a reference for the development of GSH nanodelivery system, which can further promote the application of GSH in food and pharmaceutical fields.

 

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