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Back to Journal »International Journal of Nanomedicine» Volume 12

Authors: Su Ru, Yang Li, Wang Yan, Yu Sheng, Guo Yan, Deng Jie, Zhao Q, Jin Xin

Published on July 21, 2017, the 2017 volume: 12 pages 5203-5221

DOI https://doi.org/10.2147/IJN.S139975

Single anonymous peer review

Editor who approved for publication: Dr. Linlin Sun

Su Runping, 1 Li Yang, 2 Wang Yue, 1 Yu Shanshan, 1 Guo, 1 Deng Jiayu, 1 Zhao Qianqian, 1 Jin Xiangqun 1 1 School of Pharmacy, 2 Ministry of Education Key Laboratory of Zoonotic Diseases, Key Laboratory of Zoonotic Disease Research Zoonoses, School of Veterinary Medicine, Jilin University, Changchun, China Abstract: This research aims to develop and optimize a nanoemulsion-based preparation containing Ceramide IIIB, using a phase inversion composition for transdermal delivery. The effect of ethanol, propylene glycol (PG), octyldodecanol and glycerin in Tween 80 system on the size of the nanoemulsion area in the phase diagram was studied using the water titration method. Subsequently, the load of Ceramide IIIB was kept constant (0.05 wt%), and the proposed formula and conditions were optimized through preliminary screening and experimental design. In the preliminary screening experiment, factors such as the weight ratio of octyldodecanol/(Tween 80:glycerin), water content, temperature, addition rate and mixing rate were studied. Response surface method is used to study water content (30%–70%, w/w), mixing speed (400–720 rpm), temperature (20°C–60°C) and addition speed (0.3– 1.8 mL/min) The droplet size and polydispersity index. The mathematical model shows that the best formula and conditions for preparing Ceramide IIIB nanoemulsion are temperature 41.49℃, addition speed 1.74 mL/min, water content 55.08 wt%, and mixing speed 720 rpm. Under the optimal formula conditions, the corresponding predicted response values ​​of the droplet size and polydispersity index were 15.51 nm and 0.12, which were in good agreement with the actual values ​​(15.8 nm and 0.108, respectively) without significant difference (P>0.05). Keywords: response surface method, nanoemulsion, optimization, particle size, polydispersity index

Environmental changes and improper skin care can affect the condition of normal skin, and may cause various skin diseases, such as common dermatitis, and may reduce the barrier function of the skin. 1 This investigation mainly focuses on Ceramide IIIB. It is well known that Ceramide IIIB can increase moisture content and make human skin smooth after topical application. 2

Ceramide IIIB supports the renewal of the skin's natural protective layer and forms an effective barrier to prevent moisture loss. Therefore, this same molecule as human skin is particularly suitable for long-term protection and repair of sensitive and dry skin. 3 At present, to make Ceramide IIIB active, cosmetics should contain at least 0.05 wt% Ceramide. However, due to its low solubility, its application in cosmetics and medicines is difficult. Therefore, a carrier should be developed for incorporation into ceramide IIIB and used in cosmetics and medicines. It has the following characteristics: no irritation to the skin, good solubility to ceramide IIIB, good skin permeability, and bioavailability high.

Ceramide IIIB-containing nanoemulsion 3 has been prepared by a high-energy emulsification method. Compared with low-energy methods, these high-energy methods require complex equipment and high energy for industrial-scale production. The low-energy method is advantageous because the optimal establishment of the phase diagram will produce the smallest nanoemulsion, while the low-energy method makes the scale-up easy. 4 These methods include phase inversion temperature, phase inversion composition (PIC) and spontaneous emulsification. In most cases, it is generally not recommended to use organic solvents in the cosmetics field. 4 It should be pointed out that the use of phase inversion temperature is not suitable for heat-sensitive active compounds. Therefore, we prepared an oil-in-water (O/W) nanoemulsion using the water titration method. Because the PIC method is easy to form and has relatively low energy costs, it has great potential for expanding applications. 5 The phase change is produced by gradually adding water to the mixture of surfactant and oil to form an O/W nanoemulsion. 6

Emulsifiers are a common ingredient in most topical drugs and cosmetics because they can dissolve otherwise insoluble compounds. 7 TW80 is an odorless and tasteless nonionic surfactant with a hydrophilic-lipophilic balance of 15±1. Due to its surface activity and chemical structure, Polysorbate 80 can reduce the interfacial tension of the system. 8 It is commonly used as a solubilizer and wetting agent. 9 The skin-friendliness of the developed nanoemulsion system may be impaired with the increase of TW80. Therefore, we need to add a co-surfactant to reduce the concentration of TW80.

In order to prepare nanoemulsions, the following aspects should be considered. It is generally believed that nanoemulsions with a particle size of 1-100 nm have a larger interface area, 10,11 help to overcome the epidermal barrier that may facilitate skin penetration of active substances. The nanoemulsion has remarkable adhesion properties on the skin surface after administration, because the water in the preparation evaporates, leaving a film of oil droplets. Due to the capillary force of the nanopore, the fusion and formation of the film are promoted, and the pressure can enhance this effect. This leads to increased skin hydration, which may promote the absorption of topical drugs or cosmetics. 12 The very small droplet size greatly reduces gravity, and 13 Brownian motion prevents gravity-driven precipitation or cream to a large extent. 6,10,14 The properties of transparent O/W nanoemulsions, good fluidity and no thickeners give them a pleasant aesthetic and skin feel. 13

Nanoemulsions can improve the local efficacy of active ingredients due to their small particle size. When interactions between different variables occur, formulation and process variables will affect particle size and polydispersity index (PDI) in different ways. Response surface method (RSM) is a powerful and effective statistical tool that can be used for multivariate analysis of the relationship between independent variables and response variables. 15,16

In this article, an optimization procedure involving preliminary screening experiments and RSM was carried out. We aim to optimize the process conditions and formulation composition of the ceramide IIIB-loaded nanoemulsion with the smallest particle size and the lowest PDI value through RSM.

On the basis of a carefully formulated formula, a stable nanoemulsion was obtained. Tween 80 (polysorbate 80) is used as an emulsifier. In addition, short-chain alcohols (ethanol, glycerol, PG) were also tested as co-surfactants. The cosmetic oils Tegosoft G20 (octyldodecanol) and Tween 80 were purchased from Evonik (Essen, Germany), which also provides Ceramide IIIB. Ethanol was purchased from Merck (Darmstadt, Germany). PG (Propylene Glycol) was purchased from SKC (Seoul, South Korea). Glycerin was purchased from KLK Emmerich GmbH, (Emmerich am Rhein, Germany). The characteristics of all ingredients used in the formulation are shown in Table 1. All ingredients used are pharmaceutical grade, food grade or cosmetic grade. Distilled water was used throughout the study. The structures of Tween 80, Ceramide IIIB and Octyldodecanol are shown in Figure 1.

Table 1 Ingredient content screening Note: The numbers in bold indicate that the compound content remains unchanged, and other numbers have changed.

Figure 1 The chemical structures of Tween 80, Ceramide IIIB and Octyldodecanol used in this study (ChemDraw®, CambridgeSoft).

All components are weighed and mixed under magnetic stirring. The composition is expressed as the wt% ratio between the components and the w:w ratio. The phase diagram was constructed using the water titration method at ambient temperature. Figures S1 and S2 depict the phase diagram structure used to determine the largest area of ​​stable emulsion in formulation development.

Initially, Tween 80 was combined with three types of solubilizers as the co-surfactant tested in this study: PG, glycerin, and ethanol. In the case of a fixed surfactant: co-surfactant (Smix) ratio of 1:1, the oil and Smix ratios are mixed well with different mass ratios ranging from 1:9 to 9:1 under magnetic stirring. The nanoemulsion phase was identified as the area in the phase diagram where a clear, easy-flowing and transparent formulation was obtained based on visual observation. The co-surfactant that reaches the largest nanoemulsion area is selected for formulation development. 17

Use Tegosoft G20 as the oil phase, and Tween 80 and glycerin as the surfactant and co-surfactant to draw the phase diagram. Tween 80 and glycerin are mixed in weight ratios of 1:3, 1:2, 1:1, 1:0, 2:1, and 3:1. These Smix ratios are chosen to increase the concentration of surfactant relative to co-surfactant and to reduce the concentration of co-surfactant relative to surfactant in order to study the phase diagram carefully. With different oil and Smix weight ratios (1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 6:4, 7:3 and 9:1). Gradually add water to each mass ratio of oil and Smix, and visually observe the transparent and single-phase mixture. When an opaque fluid system is observed, record the amount of water added to complete the phase diagram. The three axes of the pseudo three-component phase diagram represent water, Tegosoft G20, and a mixture of surfactants and co-surfactants in a fixed weight ratio.

Preparation of Ceramide ⅢB Nanoemulsion

Use Tegosoft G20 as the dispersed oil phase, and use Tween 80 and glycerin as surfactants and co-surfactants to prepare O/W nanoemulsion. The results of pseudo-ternary phase analysis show that the combined use of surfactant-cosurfactant (Tween 80-glycerin, 1:1 w:w) expands the self-emulsification area. The oil is mixed with surfactants and co-surfactants in weight ratios of 1:9, 2:8, and 3:7. Dissolve Ceramide IIIB in Eutanol G above 100°C, and then cool to the specified temperature. TW80 and glycerin are dissolved in the oil phase. The nanoemulsion was prepared by continuously adding distilled water to the mixture of Eutanol G, Tween 80 and glycerin (Table 1). Each is prepared by heating the mixture in a constant temperature bath (Julabo F25-ME) under constant magnetic stirring (400–720 rpm) and an addition rate of 0.3–1.8 mL in different temperature ranges from 20°C to 60°C Recipe/minute. Then, each formulation was continuously stirred for 30 minutes. Nanoemulsions were prepared by changing the emulsification parameters (Table 2) to screen these parameters to obtain small emulsions with tight size distribution to obtain high physical stability. In the rapid screening study, the influence of different variables on the characteristics of the emulsion was examined. Three variables are considered: emulsification temperature, addition rate and mixing rate. The emulsion is sampled and evaluated based on particle size and PDI.

Table 2 Phase inversion composition used for preliminary nanoemulsion screening Note: Bold numbers indicate that the process parameters remain unchanged, while other parameters are different. A series of emulsions were prepared with similar overall composition (5 wt% Tegosoft G20, 22.5 wt% Tween 80, 22.5% glycerin, 50 wt% water).

After initially determining the ideal nanoemulsion area, Design Expert software (version 8.0.6; Stat-Ease, Minneapolis, MN, USA) was used to utilize a four-factor central composite design (CCD). The independent variables of CCD and its coding level and scheme matrix are shown in Table 3 and Table 4, respectively. CCD is used to determine temperature (30°C--70°C, X1), addition rate (0.9--2.1 mL/min, X2), water content (30--70 wt%, X3), and mixing rate (480--800 rpm, X4) The effect on two response variables: the average droplet size of the nanoemulsion (Y1) and PDI (Y2). Therefore, based on the CCD, a total of 30 experiments were run, involving 16 factor points, 8 axis points, and 6 repetitions of the center point. Experiments are performed in random order to minimize the impact of unexplained variability in the actual response due to external factors. Choose CCD as the experimental design to estimate factor effects, evaluate the interaction effects between factors, and allow optimization in the full factor space. 18

Table 3 Abbreviations of independent variables and dependent variables established based on CCD: CCD, central composite design; PDI, polydispersity index.

Table 4 CCD scheme: abbreviations of independent variables and response variables: CCD, central composite design; PDI, polydispersity index.

A response surface analysis was performed to obtain the required formulation of the sphingolipid-loaded nanoemulsion carrier related to temperature (X1), addition rate (X2), water content (X3) and mixing rate (X4). The main goal is to determine the best formula composition and conditions to achieve the smallest particle size (Y1) and the lowest PDI (Y2). Then jointly analyze the responses by assigning them the same importance or weight to optimize multiple responses at the same time. 19

The second-order polynomial equation effectively expresses the response to the selected variables. The generalized response surface model is expressed by the following equation:

Among them, Yi is the predicted response, Xi is the independent variable, a0 is a constant, and ai, aii, and aij are linear, quadratic, and interaction coefficients, respectively.

According to the statistical significance of the model (P<0.05) and the lack of fit of the model provided by Design-Expert software is not significant, select an appropriate polynomial model. 19 The goodness of fit of the model is evaluated by coefficient determination (R2) and analysis of variance (ANOVA). 20 Significant differences between independent variables are determined by ANOVA. Generate response surface and 3D contour plots of fitted polynomial regression equations to better visualize the interaction of independent variables on the response. twenty one

The average droplet size of the ceramide IIIB nanoemulsion prepared under the recommended conditions of CCD and the experimental data obtained by PDI are listed in Table 4. The final simplified model is verified by comparing the experimental value with the predicted value obtained from the response regression equation. 22

Emulsion droplet size and polydispersity index measurement

The average droplet size and size distribution were measured by dynamic light scattering with a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) by diluting 1 mL of nanoemulsion with 20 mL of water. Each nanoemulsion is diluted with distilled water to a faint milky white. 23 The measurement was performed at 25°C and the scattering angle was 90°. The size distribution is represented by the PDI value. PDI value <0.25 indicates that the particle size distribution is narrow and provides good stability for the nanoemulsion. twenty four

A transmission electron microscope (TEM) was used for morphological inspection and spherical size confirmation of the nanoemulsion. For TEM observation, first dilute the nanoemulsion containing Ceramide IIIB in water (1:10), then deposit a drop of the diluted emulsion directly on the porous copper grid, and use 2% (w: v) phosphotungstic acid and Leave it at room temperature for 30 seconds. Blot the excess liquid with a Whatman filter paper, and dry the sample at room temperature. A TEM (H-7650; Hitachi, Tokyo, Japan) was used to obtain a photo of the droplet under a high voltage of 80 kV.

The physical and chemical properties of nanoemulsion ingredients

The choice of octyldodecanol as the internal phase of the nanoemulsion is based on the following considerations. Regarding the solubility of sphingolipids, octyldodecanol is considered to be a very highly soluble excipient, suitable for use as a carrier for incorporation of ceramide IIIB. Table 5 shows the dissolution test results of 1 wt% Ceramide IIIB in different cosmetic oils. Based on these advantages, we chose octyldodecanol as the oil phase to improve the pharmacokinetic activity of the drug and enhance the skin permeability.

Table 5 Dissolution test results of 1 wt% Ceramide IIIB in different cosmetic oils Note: The information is provided by Essen, Germany.

Filter emulsions in the phase diagram structure

The most important criterion for the development of nanoemulsion systems is that all excipients depend on requirements and belong to the category "generally considered safe". 17 Pseudo-ternary phase diagrams can be used to illustrate the screening of the composition of the nanoemulsion formation area.

The influence of ethanol, PG and glycerol in Tegosoft G20 and TW80 systems

In order to select the co-surfactant, a pseudo ternary phase diagram was drawn with different co-surfactants, namely PG, glycerin and ethanol (Figure S1). A clear transition from a slightly viscous liquid to a clear liquid and finally to a turbid liquid was observed. For the same surfactant and oil phase, the size of the nanoemulsion area in the phase diagram was compared under a fixed Smix ratio (1:1). When the chain length was increased from ethanol (Figure 1A) to PG (Figure 1B), the incorporation of PG significantly increased the incorporation of water. The effect of PG on the area of ​​the nanoemulsion is slightly better than that of glycerin (Figure 1C).

The transformation from optically transparent liquid to slightly turbid liquid is affected by the influence of co-surfactant on the particle size and the influence of co-surfactant on the optical transparency of nanoemulsion. At the same time, the stability of the system is directly related to optical transparency. 25 The effect of PG, ethanol and glycerol on the properties of the system is expected to affect the tendency to spontaneously form small droplets. The emulsification method is as described elsewhere. 26,27 The difference in nanoemulsion size in the different systems studied is mainly due to the nature of the co-surfactant.

PG and ethanol have a similar skeleton structure, consisting of H3C-(CHOH)-; however, there is a –CH2OH group on the main chain of PG, and ethanol has only one –H attached. 27 PG and glycerol (one more hydroxyl group than PG) form hydrogen bonds like water, have a relatively high dielectric constant, and are immiscible with hydrocarbon solvents. 28 This difference in molecular properties will change their interaction with Tegosoft G20, water and TW80 molecules.

Co-surfactants can change the properties of surfactants in the emulsion, which may be due to different physical chemistry or molecular phenomena. 27 First, the presence of co-surfactants can adjust the solubility of surfactant monomers in aqueous solutions by changing the magnitude of the hydrophobic effect. Secondly, the co-surfactant molecule may change the stacking and optimal curvature of the surfactant monolayer through the ability to compete with water molecules, thereby dewatering the hydrophilic head group of the surfactant. 26,27 Third, some co-surfactants change their optimal curvature and reduce the elasticity of the surfactant film by penetrating the hydrophilic head group region of the surfactant monolayer. 29,30 Glycerin affects the aggregation and structure of surfactants due to dehydration. 29 Another part of the co-surfactant molecule resides in the water and reduces the polarity of the water by interference. 31,32 The mechanism of droplet formation is due in part to the solubility and optimal curvature of the surfactant. 33 According to Shiao et al., 34 the mutual miscibility between the hydrophobic part of the surfactant and the oil will affect the degree of oil penetration into the amphiphilic membrane (the boundary between the organic matter) c phase and the water phase). Affect the spontaneous curvature. 31

The dependence of droplet size on co-surfactants may be attributed to the differences in their ability to change the various mechanisms mentioned above, such as surfactant solubility, density, and aqueous solution viscosity, as well as optimal curvature, interfacial rheology , Thickness, interfacial tension, and 26,27 The nanoemulsion containing 20% ​​ethanol appears to be more opaque than the nanoemulsion containing 30% PG, although the former system contains slightly smaller droplets. 27 Saberi et al. showed that using higher concentrations of glycerol can produce nanoemulsions with smaller droplets. 26 According to reports, the refractive index has a significant effect on the optical properties of the emulsion. 35 Compared with larger droplets, smaller droplets have less intensity of scattered light. 36 An emulsion with a refractive index of 1.37 corresponds to a PG concentration of 30% and an ethanol aqueous solution concentration of 20%, and the corresponding refractive index is 1.34. It is reported that when the glycerin concentration increases from 0% to 50%, the glycerin aqueous solution The concentration increased from 1.33 to 1.40. 26 Although the oil phase in these experiments is different, compared with the trend of refractive index, there is a reference value in this regard. Therefore, it can be inferred that the light scattering of the nanoemulsion containing ethanol should be greater than that of the nanoemulsion containing ethanol. The light scattering of the nanoemulsion of PG, and the light scattering of the nanoemulsion containing PG is greater than the light scattering of the nanoemulsion containing glycerin, resulting in the size difference phase diagram area.

The skin moisturizing effect depends on the amount of moisturizer absorbed and the physical and chemical properties of the stratum corneum. 37 Through preliminary transparency studies 37-39, the important pharmacological properties of glycerin confirmed visually are as follows: it is hygroscopic, decomposes the stratum corneum through desmosome degradation, has a smoothing effect, and protects the emulsion system from irritation. In addition, treatment with glycerin in water can reverse dry skin and erythema. 40 In addition, recent research results prove that glycerin can stimulate barrier repair and improve stratum corneum hydration. 41 The survey showed that the effect of glycerin lasts for a very long time. Therefore, compared with PG, glycerin is considered to be a more ideal co-surfactant.

The interface balance is due to the intermolecular forces between Tegosoft G20, TW80 and the three types of co-surfactants. TW80 is a non-ionic surfactant, which is beneficial to van der Waals forces and hydrogen bonding. 42 It should be emphasized that as part of future efforts, these conjectures need to be proven by determining the precise mechanisms involved.

Surfactant: The effect of the mass ratio of co-surfactant on the formation of nanoemulsion

The relationship between the phase behavior of the nanoemulsion and the mass ratio of surfactant: co-surfactant can be captured with the aid of a pseudo-ternary phase diagram. 43 Through the rearrangement of the components in the nanoemulsion phase, the system undergoes a transition from transparent to translucent to opaque and the light scattering behavior of the system. Due to the different light scattering of different size droplets, nanoemulsions containing higher or lower levels of glycerol are optically transparent or opaque. 26 The area of ​​the nanoemulsion was used as an evaluation standard for evaluating the mass ratio of surfactant to co-surfactant. The larger the area of ​​the nanoemulsion area, the higher the nanoemulsification efficiency of the system.

The phase diagram of the Tegosoft G20–Tween 80–water three-component system is shown in Figure S2A. A limited microemulsion formation area was obtained. The changes in the phase diagram in the presence of glycerol are shown in Figure S2B-F. Glycerin was added to Tween 80 in a weight ratio of 1:1. The size of the phase diagram has changed significantly. The possible explanation for the increase in the area of ​​the phase diagram is the increase in the incorporation of glycerin into the surfactant film and the decrease in the polarity of water, which sufficiently reduces the interfacial tension and facilitates the formation of O/W nanoemulsions. 44 Further increase the TW80 concentration, that is, at Smix ratio 2:1 (Figure S2E), the size of the area increases compared with Smix ratio 1:0 and Smix ratio 1:1. Although the concentration of TW80 further increases when the Smix ratio is 3:1 (Figure S2F), the area is reduced compared with the Smix ratio 2:1. The phase diagram area shrinks, indicating that the lowest interfacial tension and the best curvature have been reached. Therefore, it is redundant to perform the test with a 4:1 Smix ratio. When the glycerol concentration relative to TW80 was increased to obtain a Smix ratio of 1:2 (Figure S2C), a decrease in area compared to the Smix ratio of 1:1 was observed. When the glycerol concentration is further increased to reach a Smix ratio of 1:3 (Figure S2D), the area is further reduced.

At relatively low glycerol concentrations, the average particle size increases with increasing surfactant concentration. The optimal concentration of glycerol provides an ideal balance between the dehydration rate of the Tween 80 polar head and the viscosity of the water phase, thereby providing a sufficient rate of surfactant molecules to insert into the interface. 45 TW80: The weight ratio of glycerin is a key factor affecting the appearance of nanoemulsion and the area of ​​nanoemulsion. High glycerol concentration may cause the polar heads of the polysorbate molecules to be dehydrated too fast and too strongly,29 leading to a significant reduction in the hydrophilic-lipophilic balance of surfactants and preventing their proper adjustment at the oil-water interface. 46 Glycerin contains three hydroxyl groups; therefore, the increase in glycerol concentration accompanied by the decrease in TW80 concentration makes it more difficult to spontaneously rupture the oil-water interface, but the interfacial tension does not decrease. It is known that the area of ​​the O/W nanoemulsion region depends on the content of the surfactant. 47 Considering that the increase in the amount of surfactant will irritate the skin and glycerin is beneficial to the skin, the mass ratio of TW80:glycerin is 1:1 (Figure 2)), which produces a wider nanoemulsion area in the phase diagram, and was selected to proceed further Research.

Figure 2 The proposed mechanism of the phase inversion composition for the production of ceramide IIIB nanoemulsion: the mass ratio of TW80 to glycerol is 1:1.

The influence of composition parameters was systematically studied, including the weight ratio of oil:surfactant and the fraction of water. The research is limited to Tegosoft G20: the weight ratio of Tween 80/glycerol is in the range of 1:9-3:7, and the weight ratio of water is in the range of 30-70. In addition, the formation of nanoemulsions also depends on the preparation conditions. 33,45 The purpose of the preliminary screening experiment is to screen appropriate factors for optimizing the experimental design. The factors considered in the preliminary screening experiment are emulsification temperature, addition rate and mixing rate.

In this series of experiments, we studied the effect of emulsification temperature on the particle size and PDI value of nanoemulsions produced by PIC. This information is important for the rational design of nanoemulsion-based delivery systems. The result is shown in Figure 3, which shows that the smallest PDI value is produced at 50°C. It was found that reducing the emulsification temperature from 50°C to 20°C had almost no effect on the average droplet size. Increasing the emulsification energy input by increasing the temperature from 50°C to 60°C resulted in a significant increase in droplet size from 14.74 nm to 30.19 nm. This result is consistent with a reported study,48 showing that when the temperature is increased to 60°C, a larger droplet size is produced. The observed change in particle size and PDI value with increasing temperature may have a variety of reasons.

Figure 3 The effect of temperature on the average droplet size and polydispersity index.

The original oil droplets quickly split into many small oil droplets at moderate high temperatures, resulting in rapid dispersion and high dispersion of the emulsion. 49 The nature of the oil phase also changes the phase inversion temperature. Glycerol can adjust the properties of TW80 and affect the size and size distribution of droplets.26 It may be because glycerol can dehydrate the polar head groups of nonionic surfactant molecules, thereby changing their optimal curvature and lowering their cloud point. 50,51 The same trend has also been observed in systems based on Tween 80.31. Previous studies have confirmed that the effect of Tween 80 is highly dependent on temperature. 24 An increase in temperature will enhance the solubility of TW80, make the surfactant more soluble in the oil phase and affect the spontaneous curvature of the surfactant film. As the temperature rises, the head of Tween 80 gradually becomes dehydrated. 52 In addition, the dehydration of the head means that the accumulation parameters of surfactant molecules tend to be uniform, resulting in ultra-low interfacial tension, which in turn promotes the formation of O/W nanoemulsions. 52 However, high temperature will pass through the extremes of nonionic surfactant molecules. Hydrophilic head group dehydration reduces the solubility of hydrophilic surfactants, which may cause TW80 to leak from the oil-water interface, 24 allowing droplets to aggregate. These factors may contribute to the formation of smaller droplets and tighter size distribution at 50°C, resulting in lower Ostwald maturity. In our study, 50°C was chosen as the optimal temperature because it will produce relatively uniform emulsion droplets.

The purpose of these experiments is to examine the effect of the addition rate on the properties of the nanoemulsion through PIC. The overall composition of different systems remains the same, but the addition rate is different. The droplet size and PDI value that may be produced by the low-energy method vary significantly, depending on the addition rate (Figure 4). We found that the smallest droplets were formed at an addition rate of 1.5 mL/min: the diameter was 11.93 nm, and the PDI value was 0.191. A nanoemulsion with a narrow size distribution was also produced at an addition rate of 0.6 mL/min (diameter 27.92 nm and PDI value 0.189) and 0.9 mL/min (diameter 23.65 nm and PDI value 0.187), but the droplets formed were significantly larger than those produced The droplet addition rate of 1.5 mL/min. The addition rate is an important factor and should be adjusted to ensure that it is slow enough to form the O/W phase. The low addition rate allows the system time to be balanced. If the addition time is long, these relatively unstable samples become unstable, resulting in an increase in droplet size. 53

Figure 4 The effect of the addition rate on the average droplet size and polydispersity index.

By preparing a series of emulsions with a fixed composition, the effect of mixing rate on the formation of small droplets and PDI value was studied (Figure 5). For the prepared emulsion, the smallest average droplet size was observed at a stirring speed of 640 rpm. After increasing the mixing rate from 320 rpm to 640 rpm, the droplet size is effectively reduced from 83.96 nm to 11.93 nm, which is consistent with the previous results. 54 The width of the particle size distribution is not strongly affected by the mixing rate, as described by Saberi et al. 55

Figure 5 The effect of mixing rate on average droplet size and polydispersity index.

Similar results were observed for the emulsion droplet size and mixing rate of nanoemulsion emulsification. 53,56 Previous studies55,57 showed that gentle mixing is required to form very fine droplets, which highlights the importance of controlling this parameter. The mixing must be more intense to obtain the phase change. As the mixing rate increases, the droplet size will decrease because the applied mechanical energy will break and ensure a uniform distribution of the surfactant-oil phase in the water phase. 58 If the mixing rate is too high, it will promote unstable mechanisms such as coalescence and precipitation, resulting in larger final droplets. 53

Tegosoft G20: The effect of Tween 80/glycerin weight ratio

In this series of experiments, we studied the effect of oil: (surfactant: co-surfactant) ratio (OSR) on the droplet size and particle size distribution of the nanoemulsion produced by PIC (Figure 6). After the OSR is reduced, the average particle size and PDI value both decrease. This change is consistent with the previous report on the nanoemulsion prepared by PIC.33,45.

Figure 6: The effect of oil:surfactant ratio on average droplet size and polydispersity index.

As the molecular volume of the surfactant and co-surfactant decreases, the interfacial pressure decreases rapidly. The presence of glycerin strongly affects the formation of PIC nanoemulsion. 46 After adding water, the normal tendency of Tween 80 molecules is to migrate to a water-rich environment and adapt to the oil-water interface. The presence of glycerin reduces the solubility of Tween 80 in the water phase. 59 Tween 80 The control of the migration rate of molecules from the oil phase to the water phase is also affected by the solubility of glycerol and by increasing the viscosity of the water phase containing a large amount of glycerol. 46

The formation of emulsions is affected by the equivalence between two main mechanisms: droplet rupture and droplet coalescence. Surfactants play a major role in the deformation and breaking of droplets. Surfactants allow an interfacial tension gradient, which is essential for stable droplet formation. 13 The speed of droplet coalescence depends on the ability of the emulsifier to quickly form an absorbing monolayer on the newly formed interface, and 22 is mainly controlled by the emulsifier. In fact, emulsifiers play a vital role in the formation of emulsions by reducing the interfacial tension. 22 The concentration of surfactant controls the total surface area of ​​the droplets, the speed of droplet dispersion and the speed of coalescence. 58 The increase in glycerin concentration affects the properties of the emulsion. TW80 also forces glycerol to penetrate the interface area, which is the reason for the observed droplet size and particle size distribution.

The influence of water content on the average droplet size and size distribution of the microemulsion is shown in Figure 7. There is a significant difference in the amount of water required to obtain the smallest droplets. Under constant OSR, reducing the water concentration from 70% to 30% has a tendency to produce smaller droplets and a narrower size distribution. The smallest droplet (diameter ~8.861 nm) and the smallest size distribution (PDI 0.088) are formed at a water concentration of 30%. The average particle size decreased slightly from 40% to 30% water, from 40% to 50% water remained relatively low, from 50% to 60% water increased slightly, and finally from 60% to 70% water increased sharply. The width of the PDI value increases with increasing water concentration (from 30% to 40% water), but the trend is similar to the size distribution obtained between 40% and 70% water. Our results are consistent with the research results, indicating that the particle size increases with increasing water content. 60 Increasing the water content will eliminate the rigid film, resulting in droplet coalescence and an increase in particle size. 58

Figure 7 The effect of water content on the average droplet size and polydispersity index.

According to the CCD matrix, Table 4 shows the experimental data of each response variable under different independent variables. The response surface model allows predicting the change in particle size and PDI value based on the composition of the nanoemulsion. The estimated regression coefficients, R2, adjusted R2, regression (P value and F value) and standard deviations related to the effects of the three independent variables are shown in Table 6. The ANOVA in Table 6 shows that the quadratic polynomial model is sufficient for prediction. The model shows no lack of fit, because the P value of particle size and PDI (0.1592 and 0.7885, respectively)> 0.05. The insignificant fit and high R2 and Ra2 values ​​indicate that the quadratic equation can represent the system in a given experimental domain.

Table 6 Model ANOVA Abbreviations: ANOVA, analysis of variance; PDI, polydispersity index.

Response surface analysis shows that the coefficient value of the particle size second-order polynomial response model (R2=0.9731) is higher than that of the PDI response surface model (R2=0.9901). The observation results show that the RSM model can fully explain more than 90% of the response changes of the independent variables (particle size and PDI). It should be mentioned that if the quadratic term or interaction term containing these variables is found to be significant (P<0.05), then the final simplified model will include insignificant (P<0.05) linear terms. 61 Determine the significance of the model and each coefficient using the F test value and the corresponding P value (Table 7). The larger F value and the smaller P value show more obvious effects on the respective response variables. 20,62,63 In this study, considering the F value (particle size and PDI are 38.80 and 23.37, respectively), the two models are significant (Table 7).

Table 7 Regression coefficients in the final simplified second-order polynomial model Note: aP<0.0001; bP<0.01; cP<0.05; dP>0.05. X1, X2, X3 and X4 are the linear, quadratic and interactive terms of the quadratic polynomial equation respectively; X1, temperature; X2, addition rate; X3, water content; X4, mixing speed. Abbreviation: PDI, polydispersity index.

After careful observation, the particle size model showed that all linear coefficients (X1, X2, X3, X4), quadratic coefficients (X12, X22) and interaction parameters (X1X3, X2X4, X3X4) were significant (P<0.05). Water content is the most significant single parameter that affects the size of the microemulsion, followed by mixing rate and temperature. The interaction between the amount of water and the mixing rate (X3X4) has a more significant impact on the size of the nanoemulsion droplets than other interactions.

For the PDI response, the results show that the linear and quadratic coefficients of temperature, addition rate and water content have significant effects, which can be seen from their respective F and P values ​​(Table 7). Based on the F values ​​obtained from ANOVA (Table 6), the interaction parameters (X2X3, X2X4, and X3X4) showed a high level of significance compared with other items. However, no significant difference (P>0.05) was observed in the interaction parameter (X1X4) or the quadratic coefficient of the mixing rate (X42). Obviously, the water content ratio is of overriding importance in evaluating the droplet size and PDI response changes within the scope of the study. For low-energy emulsification methods, the influence of composition variables is much greater than that of preparation variables. 64 Our results are consistent with other studies. 58,60 indicate that increasing water content leads to an increase in particle size.

Draw a 3D graph of ANOVA to understand the influence of independent variables on response variables. Response surface plots of particle size and particle size distribution are used to explain the significant interaction of the model (P<0.05).

Generally, the pharmaceutical and cosmetic industries are very keen to produce nanoemulsions with smaller droplet sizes because it provides the entire system with extremely low surface tension and O/W droplet interfacial tension. 22 Particle size is a key feature of nanoemulsions and affects the release rate and absorption rate of biological activity. 65

Figure 8 reveals the importance of effective parameters to the particle size of the nanoemulsion. The variables that have the greatest influence on the nanoemulsion droplet size are the linear terms of water content and the interaction terms of water content and mixing rate, followed by the linear terms of mixing rate, temperature and addition rate. Water content is one of the important factors that affect the size of droplets. The response surface plots (Figure 9A and C) show that the droplet size decreases significantly as the water content decreases. This trend is consistent with the trend observed in earlier studies, that is, the droplet size decreases as the emulsifier content increases. This may be due to the fact that a fixed amount of emulsifier molecules are not completely covered on the newly formed droplets, resulting in an increase in the particle size of the emulsion. 21,66,67 In addition, the increase in mixing rate (P<0.05) slightly decreases the droplet size (Figure 9B). These results are very consistent with the report, that is, the effect of mixing rate is more significant at high addition rate, and low addition rate and high mixing rate are conducive to the formation of small droplet size emulsions. 53 This mechanism may be partly attributable to the rapid diffusion of surfactant molecules from oil into water.

Figure 8 The importance of effective parameters to the particle size of the nanoemulsion.

Figure 9 The response surface plot shows a significant (P<0.05) interaction. Note: Droplet size (AC) and polydispersity index (DG) are functions of temperature, addition rate, water content, and mixing rate. Variables not shown in each figure remain unchanged at the center level.

As the temperature increases, the droplet size first decreases and then increases (Figure 9A). Temperature plays an important role in affecting the emulsification performance of non-ionic TW80. Thermal degradation of the emulsifier causes adjacent droplets to gather and increase the average droplet size. 68 The results are consistent with expectations. They are consistent with the results obtained by Yu et al. 45. They found that as the preparation temperature increases, the interfacial tension decreases due to the relatively low interfacial tension and low viscous resistance of the oil phase.

As shown in Table 7, in addition to the interaction between temperature and water content, the interaction between the addition rate and the water content, the addition rate and the mixing rate, and the interaction between the water content and the mixing rate have a significant impact on PDI (P<0.0001), the impact on PDI is minimal. The response surface plot (Figure 9D-G) shows that PDI is significantly affected by water content, temperature, mixing rate, and addition rate. Figure 10 shows the importance of effective parameters to the PDI value of the nanoemulsion. Figure 9D shows that PDI is almost unaffected by the combined effects of water content and temperature. Figures 9F and G show that the combination of increased mixing rate and water content and the combination of increased mixing rate and addition rate broadened the droplet size distribution of the final emulsion. These results are fully supported by previous reports on the high-pressure emulsification process18,24. In contrast, Figure 9D shows that the combination of increasing water content and addition rate narrows the droplet size distribution.

Figure 10 The importance of effective parameters to the polydispersity index of nanoemulsion.

If the applied optimization criteria results in the smallest average droplet size and narrow PDI value range in the presence of the lowest amount of emulsifier, then the nanoemulsion will be considered the best formulation. 22 The best processing conditions that lead to the desired response target are the 3D response surface and contour map determined by superposition. After analyzing various interactions from different angles and evaluating optimization constraints, the best formula with the most ideal performance was determined to be 41.49°C, addition rate 1.74 mL/min, water content 55.08%, and 720 rpm mixing rate. Under optimal conditions, the corresponding predicted response values ​​for the average droplet size and PDI value are estimated to be 15.51 nm and 0.12, respectively.

Table 8 shows the comparison between the experimental data and the predicted data obtained from the CCD final reduction model. However, through experiments under the recommended optimal conditions, the adequacy of the final simplified model in predicting the optimal response value was tested. 22 Three nanoemulsions were prepared according to the recommended optimal combination level, and the average emulsion droplet size and PDI were 15.8 nm and 0.108, respectively. Then compare the experimental value and the predicted value to verify the effectiveness of the response surface model. Interestingly, the response value of the experimental data obtained from freshly prepared emulsion samples was found to be slightly higher or slightly lower than the predicted value, within 10% of the prediction error. No significant difference was observed between the predicted value and the actual value, indicating the suitability and adequacy of the corresponding response regression equation for correlating the response with the independent variable.

Table 8 Abbreviations of experimental and predicted response variables obtained from the final simplified model of CCD: CCD, central composite design; PDI, polydispersity index.

TEM (Figure 11) shows that the nanoemulsion droplets (black dots on white background) are spherical and uniform in size, containing a large number of small droplets of 10-30 nm, similar to the droplet size measurement results, which shows the average emulsion droplet size and PDI The experimental values ​​of the values ​​are 15.80 nm and 0.108, respectively.

Figure 11 Transmission electron microscope image of a nanoemulsion containing 0.05 wt% Ceramide IIIB. Note: The magnification is 20,000 times.

When preparing ceramide IIIB-containing nanoemulsions, controlling the formulation and process parameters is essential to obtain the properties required for effective transdermal delivery. This study shows that RSM can be effectively used to explain and predict the response of the particle size and PDI value of the ceramide IIIB-loaded nanoemulsion. Due to the promising results of this study, we plan to carry out future studies to evaluate the main factors affecting the encapsulation efficiency and stability of ceramide IIIB-containing nanoemulsions, as well as the application of this formulation in the skin delivery of ceramide IIIB.

Thank you for the technical support of the life science core facilities of Jilin University.

The authors report no conflicts of interest in this work.

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Figure S1 Pseudo-ternary phase diagram of a nanoemulsion composed of Tegosoft G20, Tween 80, water and different co-surfactants. Note: (A) ethanol; (B) propylene glycol; (C) glycerol with a Smix ratio of 1:1. Abbreviation: Smix, surfactant: co-surfactant.

Figure S2 shows the pseudo ternary phase diagram of the oil-in-water nanoemulsion region. Note: Tegosoft G20 (oil), water, Tween 80 (surfactant) and glycerin (co-surfactant) in different mixing ratios: (A) 1:0; (B) 1:1; (C) 1:2; (D) 1:3; (E) 2:1; (F) 3:1. Abbreviation: Smix, surfactant: co-surfactant.

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