Self-nanoemulsifying drug delivery system of black soldier fly ( Hermetia illucens ) oil: Optimization, formulation, and characterization

The purpose of this study was to formulate a self-nanoemulsifying drug delivery system (SNEDDS) from black soldier fly oil (BSFO) as a possible antibiotic substitute in chicken. Using Design Expert Ver.13.0.1.0, an I-optimal combination was created by altering the concentrations of the oil components BSFO, polyethylene glycol 400 (PEG 400) ( v / v ), and Tween 80. This mixture was then used to analyze the optimal formula. The parameters for optimization were chosen to be the emulsification time and transmittance percentage. Droplet size, polydispersity index (PDI), zeta potential (ZP), viscosity, thermodynamic stability


INTRODUCTION
The use of antibiotics as growth promoters in poultry feed has been restricted in several countries [1].Using an antibiotic growth promoter inconsistent with the recommended dosage and continuous administration can cause antibacterial resistance, livestock product residues [2], weakening disease resistance, and environmental pollution [3].Feed antibiotics have been prohibited in the European Union since January 2006 (Regulation EC/1831/2003).Antibiotic bans can result in economic losses.Prohibition of the use of antibiotics has encouraged efforts to develop alternatives to antibiotics [4], namely traditional antibiotics as feed additives.Various feed additives have been utilized as antibacterial agents, including phytobiotics, probiotics, prebiotics, synbiotics, enzymes, and organic acids.Plant organic acids have been widely investigated, whereas those of animal origin have rarely been studied.Several additional organic acids also exhibit antibacterial effects.Insect-based biorefineries are a potential alternative for manufacturing feeds and foods because of the increasing demand for renewable and sustainable products [5].One potential source of animal organic acid is lauric acid obtained from black soldier fly oil (BSFO).BSFO contains lauric acid (C:12) as the main fatty acid, which is a mediumchain fatty acid, which has the potential as an antibacterial agent, and boosts immunity [6][7][8][9].BSFO contains 44.55% of lauric acid.Lauric acid can damage bacterial cell membranes and walls through physicochemical processes.250 rpm.The formulations were sonicated using an ultrasonic cleaner (Aczet model number CUB 10-liter Frequency 40 KHz power 120 W Germany) at 40ºC for 10 minutes and visual observation was carried out (Table 1).

Measurement of transmittance
Five milliliters of distilled water and one hundred microliters of BSFO SNEDDS were vigorously mixed using a vortex for 2 minutes.The self-nanoemulsification effectiveness of SNEDDS was shown by transmittance values of the formulations exceeding 90% [16].The transmittance percentage was measured using a UV spectrophotometer at 650 nm wavelength [17].

Measurement of emulsification time
To determine the emulsification time, separate glass beakers set at 37ºC were set, and 50 ml of AGF with a pH of 1.42 was added along with 100 μl of BSFO SNEDDS.The mixture was then gently stirred using a magnetic stirrer at 100 rpm until the solution became clear.The amount of time it took for the solution to become clear was considered selfemulsification [18].The SNEDDS with good emulsification and solubility characteristics along with BSFO, Tween 80, and PEG 400 in various ratios were selectively chosen.

Optimization of BSFO SNEDDS
In a further experimental study, a three-part system, comprising X1, the BSFO phase; X2, the surfactant (Tween 80); and X3, the cosurfactant (PEG 400), was used.The responses (Y1 = emulsification time; Y2 = transmittance) were interpreted using Design Expert software version 13.0.1.0.For emulsification time and transmittance, the results of the BSFO Broiler performance can be partially explained by the improvement in intestinal morphology and microbial balance associated with the modulation of intestinal microflora and pathogen inhibition [10].Toxic emissions from pathogenic microorganisms compromise the physical integrity of the small intestine cell wall, which is crucial for vitamin absorption.Traditional antibiotics can be easily administered through drinking water; however, BSFO, in the form of an oil phase, does not dissolve in water.One way to overcome this solubility problem is to use nanotechnology as nanoemulsions [in the self-nanoemulsifying drug delivery system (SNEDDS)].Selfemulsifying drug delivery systems are utilized to overcome poorly mixed and insoluble substances by improving their solubility composed of a mixture of oils, surfactants, and cosurfactans [11].SNEDDS are homogenous anhydrous liquid solutions of cosurfactant, surfactant, and oil.With its potential to speed up and expand the absorption of drugs with low water solubility properties, SNEDDS has been an exciting object of study [12].When these naturally occurring oil-in-water nanoemulsions were diluted with water while being gently stirred, they had a size of approximately 5-100 nm [11].The nanoparticles can dissolve in water [13].Nanoparticles are gaining attraction for functional applications in interdisciplinary fields.In general, the nanoparticles are 10 −9 cm in size.The size of nanoemulsions ranges from 1 to 100 nm [14].In sick animals, the beneficial effect of nanoparticles is that active substances are more quickly and effectively carried to the site of infection.SNEDDS has been proposed to improve the formulations' solubility [15].The BSFO has been used as a novel feedstock for preparing nanoemulsions [5].However, BSFO has limited oral applications owing to its low oral bioavailability.In this study, a new SNEDDS was created to improve the solubility and absorption of BSFO when administered to poultry via drinking water.

MATERIALS AND METHODS
The primary material, BSFO, was acquired from a black soldier fly farmer in Bekasi Regency, West Java, Indonesia, utilizing a mechanical pressing machine.Then, 14-day-old fresh black soldier fly larvae were dried at 60ºC for 24 hours.Black soldier fly larvae that had been dried were pulverized and pressed to produce oil.After letting the oil remain for 2 days, the sediment was separated.In order to create SNEDDS, the solution was then filtered in the laboratory using Whatman filter paper.The chemicals and other components used were: Tween 80 and polyethylene glycol 400 (PEG 400) are both available from PT. Brataco in Cikarang, Bekasi, Indonesia, and Sigma Aldrich in Germany, respectively.Distilled water (Jaya Santosa, Indonesia), hydrochloric acid (37%), and sodium chloride (MERCK, Germany) were used to make artificial gastric fluid (AGF).

BSFO SNEDDS preparation
BSFO, Tween 80, and PEG 400 were mixed in various proportions to generate SNEDDS with a ratio of 1:1:1 to 1:8:1.BSFO is first combined with PEG 400 by means of a magnetic stirrer for 10 minutes at 37ºC, 250 rpm.The Tween 80 was then added and thoroughly mixed for another 20 minutes at 37ºC,  Table 2.The components that were examined using an I-optimal mixture design ranged in value.

Independent variables (factors) Range
Low High O n l i n e F i r s t SNEDDS were replicated in triplicate.A t-test using a single sample was used to evaluate the data.

Measurement of droplet size
Using a Zetasizer Nano (Malvern, Germany), the polydispersity index (PDI) and nanoemulsion droplet size were calculated [19].The experiment was carried out in triplicate, with the samples diluted with distilled water at a ratio of 1: 1,000 (v/v) [20].

Measurement of zeta potential (ZP)
Using a particle size analyzer from Malvern Instrument Germany, dynamic light scattering (DLS) was utilized to estimate the ZPs of the most optimal formulations.Results were captured once the samples were put into clear disposable cuvettes [21].The samples were thoroughly mixed with distilled water at a ratio of 1:1,000 (v/v), and the experiment was conducted thrice [20].

Measurement of viscosity
Employing a small-sample adapter (Brookfield digital viscometer DV-I Prime, Brookfield Engineering Laboratories, INC, Middleboro, MA 02346), BSFO SNEDDS and nanoemulsion viscosities were evaluated.The viscosity of BSFO SNEDDS was evaluated using Spindle 03 at 100 rpm for 15 seconds at room temperature (25ºC) in triplicate.The Spindle 02 was used to measure the nanoemulsion's viscosity at 100 rpm at room temperature (25ºC) for 15 seconds in triplicate.

Measurement of morphology
Applying a Joel JEM-100 CX (Joel, Tokyo, Japan) transmission electron microscopy, The morphology of BSFO SNEDDS was carefully investigated.A sample drop taken from SNEDDS samples was diluted with water (1:1,000) and stained for 30 seconds with a 2% phosphotungstic acid solution, followed by being deposited on a copper grid [22].

Studies on the thermodynamic stability of BSFO SNEDDS
Additional research on the thermodynamic stability of the ideal formula was conducted.The first step was heating and cooling cycles.Six cycles were examined, each lasting between 4ºC and 45ºC for at least 48 hours.Centrifugation was applied on formulations that passed it through this temperature without exhibiting any indications of instability (cracking or creaming).Second was centrifugation.The mixtures were centrifuged at 3,500 rpm for 30 minutes.The freeze-thaw cycle was implemented for formulations that demonstrated no evidence O n l i n e F i r s t of instability (cracking or creaming).The last step was the cycle of freezing and thawing.The formulations were stored between −21ºC and 25ºC for no less than 48 hours at each temperature (using a deep freezer).As described by Parmar et al. [23], the formulations were centrifuged for 5 minutes at 3,000 rpm.

Formulation optimization of BSFO SNEDDS
Table 1 shows the effects of the BSFO, Tween 80, and PEG 400 ratios on visual observation.Because of the clarity of the observations, the ideal ratio was calculated using the formula 1:6:1.The BSFO: Tween 80: PEG 400 were 1: 5: 1 (lower limit) and 1:7:1 (upper limit) (v/v), in a respective manner.Lower and upper limits, which are shown in Table 2, were established using this data.With the aid of response surface methodology and considering three independent parameters (BSFO, PEG 400, and Tween 80) effect on two dependent variables (transmittance and emulsification time), this study seeks to identify the variables' levels at which the product is of the highest quality.The selectively chosen formulation relied on the "trading off" among multiple variable responses, i.e., increasing transmittance percentage while reducing emulsification time.In order to identify the ideal formula for an array of formulas, this study optimized using an I-optimal mixture design (Table 3).
Transmittance and emulsification values were determined using the 20 formulas in Table 3.The responses were transmittance and emulsification time.Table 4 displays the result from the analysis of variance and lack-of-fit tests.

Emulsification time
The hues that correspond to the light spectrum serve as an illustration of the emulsification time [24].The closer distance to light green indicates a high emulsification time.The residuals' normal probability plot for emulsification time appears in Figure 1(a).The obtained data revealed no evidence of whether the straight line contains any outliers; the residuals are normally distributed.Meanwhile, the data in Figure 1(b) are centered on the zero line, and no particular value sticks out.The model (Fig. 2) clearly illustrates the relationship between quantity (BSFO, Tween 80) and response (emulsification time).The projected value and the actual values were very closely matched, as shown by the lack of fit analysis (Table 4), which revealed no significance.For creating emulsions in gastric fluid with SNEDDS properties, emulsification time is a crucial component to consider [25].When in contact with biological fluids, the nonionic surfactant Tween 80 quickly disperses due to its low toxicity, strong solubility, and good emulsification ability [25].Figure 5 shows the amount of time needed for   O n l i n e F i r s t quicker emulsification with a higher Tween 80 content.In nanoemulsion formulations, reduced BSFO is frequently employed to enhance solubility and bioavailability.A singlesample t-test with Open-Stat was used to confirm the outcomes of the optimum formula.The emulsification time's p-value was 1,000 (Table 5), which was practically perfect and showed no difference between the expected and actual data.

Percentage of transmittance
The solution's transmittance percentage was utilized to well document the process of self-emulsification while the solution was dissolving and the process of selfemulsification was taking place [26].The colors that follow the light spectrum serve as an example of the transmittance value [24].A normal probability plot of the transmittance residuals is shown in Figure 3(a).There is no evidence of the potential of outliers, and the straight line displays residuals with a normally distributed distribution.The data are close to the zero line, as shown in Figure 3(b), and nobody sticks out.
In the unique cubic model (Fig. 4), the redder the shift, the greater the transmittance value.The transmittance increases as the BSFO% decreases.The fit analysis result in Table 4 did not provide any significant results, demonstrating that the predicted values and the observed data were closely matched.With desirability of one, the suggested ideal formulas for BSFO, Tween 80, and PEG 400 were 11.426%, 73.364%, and 11.177%, respectively (Fig. 5).Through the use of Open-Stat software and a single-sample t-test, the outcomes of the optimum formula were confirmed.Since the transmittance's p-value was 0.999 (Table 5), higher than 0.05, no statistically significant discrepancy existed between the projected value and the observed data.

Particle size and PDI
Assuming the presence of spherical particles, the mean particle size and PDI were derived using the volume, intensity, and bimodal distribution.According to the sample's   O n l i n e F i r s t size, the PDI calculates the sample's heterogeneity [27].This instrument allows the measurement of particle homogeneity with a size range from 0.0 to 1.0 [11].It was discovered that the preferred formulation's particle size distribution was 10.02 ± 0.12 nm (Fig. 6), indicating a small size distribution of the system.The PDI was (0.36% ± 0.0%), indicating the homogeneity distribution of the system.As it controls the pace and volume of drug release and absorption and increases bioavailability, droplet size is a crucial component in SNEDDS formulations [23].Fewer surfactants are needed since the shock wave that results from ultrasonication can efficiently mix the layers.Consequently, smaller droplet emulsions within the size distribution were produced [28].The interfacial coating was condensed and stabilized by the addition of Tween 80 to the BSFO nanoemulsion.

Zeta potential
The BSFO nanoemulsion droplet had a ZP of −19.4 mV (Fig. 7).The ZP measurements showed that the SNEDDS formulation was stable.According to Balakumar et al. [20], the formulation's inclusion of fatty acids was likely the reason for the significant negative charge.The use of ZP measurements with DLS measurements to determine particle size and surface charge has grown in popularity [29].

Morphology of BSFO SNEDDS
With a spherical shape and uniform droplet size, the BSFO nanoemulsion was visible as a bright spot against a dark background (Fig. 8).Numerous parameters, including emulsion composition, homogenization, and environment, must be tuned to produce stable nanoemulsions [31].The cosurfactant's surfactant ratio impacted the nanoemulsions morphology [5].Lauric acid-containing samples were more spherical and included remnants of the membrane [32].

Test for thermodynamic stability
The environmental conditions, including temperature, markedly affect the stability of nanoemulsion [33].This study used centrifugation and freeze-thaw cycle techniques in thermodynamic stability tests to find metastable formulations [34].The thermodynamic stability tests were successful for the ideal formulation (Table 6).The system's stability was demonstrated by the best formulation, which displayed no signs of instability.In order to create a nanoemulsion, BSFO  O n l i n e F i r s t it would be possible to employ it as an antibacterial agent and a possible antibiotic substitute in the production of chicken.SNEDDS should spontaneously emulsify in the digestive system.The BSFO SNEDDS system needs to be of high enough caliber to resist instability and prevent creaming, cracking, or precipitation.The prepared BSFO SNEDDS was verified to be stable for the shortest time using thermodynamic stability.The chosen formulation was centrifuged, exposed to a freeze-thaw cycle, and subjected to a heating-cooling cycle [35].

CONCLUSION
The ratios of 11.426% BSFO, 73.364% of Tween 80, and 11.177% PEG 400 were used to create the SNEDDS of BSFO.The formulation's properties included a high percent transmittance of 95.2 ± 0.72, a brief emulsification time of 47.89 ± 0.78 seconds, a small droplet size of 10.02 ± 0.12 nm with a PDI of 0.36% ± 0.0%, a ZP of −19.4 mV, and BSFO SNEDDS; and nanoemulsion had viscosities of 387.33 ± 22.81 mPa•s and 10.47 ± 0.42 mPa•s.BSFO SNEDDS passed the thermodynamic stability test and showed signs of having a sphere-like form.This investigation showed that this formulation might increase BSFO water solubility and stability.If this formula is improved,   O n l i n e F i r s t : indicates the separation of particles.Clearly: indicates homogeneity.

Figure 1 .
Figure 1.Graph of a residual parameter of emulsification time.

Figure 2 .
Figure 2. Special model cubic of emulsification time graphic values.

Figure 3 .
Figure 3. Graph of a residual parameter of transmittance.

Figure 4 .
Figure 4. Special model cubic of transmittance graphic values.

Figure 5 .
Figure 5. Superimposed image of particular model cubic emulsification time and transmittance.

Figure 8 .
Figure 8. Transmission electron micrograph of BSFO self-nanoemulsifying drug delivery system with spherical shape.

Table 1 .
The effect of surfactant ratio on visual observation.

Table 5 .
Single sample t-test verification of the optimal formula.

Table 6 .
Test for thermodynamic stability.