Nanoemulsions: A Recent Drug Delivery Tool

Abstract

The use of nano/sub-micron particles in food, cosmetic, and pharmaceutical technology is becoming more and more popular. In particular, this interest has been growing in tandem with improved stabilization and emulsification methods. High-energy and low-energy spontaneous emulsification techniques are the two primary categories of nanoemulsion preparation techniques. Stability ranging from a few hours to years is influenced by important characteristics related to preparation procedures and components used. Nanoemulsions do not worry about issues like creaming, coalescence sedimentation, and flocculation because of their small droplet size. Ostwald ripening for them is the primary destabilizing process, though. This chapter provides a thorough overview of nanoemulsions including an explanation of their preparation techniques and assessments.

1. Introduction

Recently, there has been a lot of interest in lipid-based formulations as a way to increase the permeability and bioavailability of poorly soluble drugs. As a result, many innovative drug delivery strategies have been used, and in each case, the nanoemulsion is essential to the active pharmaceutical ingredient’s transport to the target organ or location. Among many different technologies, nanoemulsions have proven to advance drug delivery systems more than others.

These are considered the best alternatives for raising the oral bioavailability of drugs listed in the Biopharmaceutical Drug Classification System (BCS) under classes II and IV. A nanoemulsion is a clear solution of two nonsoluble liquids, such as water and oil, that is thermodynamically stable and stabilized by an interfacial layer of surfactant molecules. Nanoemulsions are a novel approach to medicine administration that uses emulsified water and oil systems with mean droplet sizes ranging from 50 to 1000 nm.

The main distinctions between emulsions and nanoemulsions are the size and morphology of the particles dispersed in continuous phases. The particle size in typical emulsions is 1/20 μm, but in nanoemulsions, it is 10/200 nm. A nanoemulsion is a kinetically stable liquid that consists of an oil phase and a water phase that possesses the appropriate surfactant. The dispersed phase has a low oil/water interfacial tension and is primarily composed of tiny particles with sizes ranging from 5 to 200 nm. They are regarded as the greatest alternative colloidal dispersions with exact ratios of an oil phase, an aqueous phase, a surfactant, and a co-surfactant are called nanoemulsions. To create the nanoemulsions, both high energy and low-energy emulsification procedures were applied. While low-energy emulsification methods take advantage of the system’s physicochemical properties, which exploit phase transitions to produce nanoemulsion, high-energy emulsification methods utilize high shear mixing, high/pressure homogenization, or ultrasonification. Because oil, surfactant, and co-surfactant nanoemulsions are nontoxic and nonirritating, the Food and Drug Administration (FDA) has approved them for consumption by humans as “generally recognised as safe” [1, 2].

2. Composition of nanoemulsion

The following are the primary elements used in the creation of nanoemulsions:

1. Oil (for the solubilization of drugs or lipophilic molecules)

2. Surfactant

3. Water

4. Co-surfactant (amplify the surfactant’s effect).

The stability of the system is greatly dependent upon and impacted by the selection of the appropriate components for nanoemulsions. Thus, choosing them carefully is essential. Surfactants and/or co-surfactants/co-solvents, along with an oily phase and an aqueous phase, make up most nanoemulsions.

2.1 Oil

Thus, oil is the most crucial ingredient in nanoemulsions. This facilitates the completion of the intended tasks. As such, it may operate as a carrier for the lipophilic active pharmaceutical ingredients or act as an active agent in and of itself (the essential oil in our study, for example). The selected oil’s characteristics, including its polarity (low polarity oils are quickly disrupted by applied external force), viscosity (low viscosity oils are disrupted by applied external energy more quickly, resulting in the creation of tiny droplets more quickly), and interfacial tension (low interfacial tension in the oil facilitates size reduction and reduces the energy required to shrink the droplet size).

2.2 Surfactants

Without surfactants, the nanoemulsion system that stabilizes the thermodynamically unstable mixing of two immiscible liquids by lowering interfacial tension and altering dispersion entropy would be incomplete. The primary criteria for the surfactants used in the production of nanoemulsions are stability, high drug loading capacity, efficacious emulsification, and safety. Rapid adsorption of an appropriate surfactant used in the nanoemulsion formulation onto the interface between the two immiscible phases is necessary to prevent the coalescence of the nanodroplets and to dramatically lower interfacial tension. The four subgroups of surfactants are cationic, nonionic, zwitter-ionic, and anionic surfactants. Out of these four types, nonionic surfactants are used the most since they are known to have high biological absorption, are less sensitive to pH variations, and have a lower toxicity and irritating profile than ionic ones. The lipids in the stratum corneum can also be fluidized and solubilized by nonionic surfactants, which enhances the skin’s capacity to absorb the applied solution.

The scale used to classify nonionic surfactants is called the hydrophilic–lipophilic balance (HLB) which is a specific empirical expression of nonionic surfactants that characterizes the relationship between the hydrophilic and hydrophobic portions of the surfactant. In 1954, William C. Gryphon developed it. The values on the HLB scale range from 0 to 20. In order to form water/oil nanoemulsions, surfactants with HLB values between 3 and 6 are lipophilic (like SPANS) sorbitan monolaurate, whereas those with HLB values between 8 and 18 are hydrophilic (like TWEENS) polyoxyethylene sorbitan monolaurate. Co-surfactants are surfactants with an HLB value greater than 20. It is not a random addition of surfactants; rather, the surface becomes saturated with surfactants, and micelles begin to form when the critical micelle concentration is reached. It is claimed that a mixture of surfactants makes the nanoemulsion system more stable. The four subgroups of surfactants are cationic, nonionic, zwitter-ionic, and anionic surfactants. Nonionic surfactants are the most often used of these four types because they are less sensitive to pH changes and have a lower toxicity and irritating profile than ionic ones. They are also well known for having a high level of biological acceptability. The stratum corneum’s lipids can also be fluidized and dissolved by nonionic surfactants.

They thereby enhance the skin’s capacity to absorb and permeate the applied solution. William C. Gryphon developed a scale in 1954 that is used to group nonionic surfactants.

2.3 Co-surfactant

Co-surfactant helps the surfactant in the nanoemulsion system emulsify the oil in an aqueous phase. By combining with surfactants and penetrating the surfactant layer, co-surfactants in these systems dissolve the interfacial film, provide the necessary fluidity, reduce interfacial tension, and aid in the emulsification process. The interfacial film is made more flexible by the addition of a co-surfactant because surfactant alone frequently fails to produce temporary negative interfacial tension and fluid interfacial film.

Co-surfactants can also aid in the solubilization of the oil by altering the oil-water interface’s curvature. Because the interaction between the surfactant and co-surfactant impacts how therapeutic compounds or lipophilic drugs partition into the aqueous and oil phases, choosing the right co-surfactant is crucial. 1,2-Propylene glycol, Transcutol® HP (diethylene glycol monoethyl ether), polyethylene glycol-400, and absolute carbohol. Alcohols may increase the miscibility of the two phases because of the way they partition between the aqueous and oily phases. Thus, ethanol, isopropyl alcohol, 1-butanol, and propylene glycol were selected as co-surfactants. Additionally, because carbosol and polyethylene glycol 400 are generally well-tolerated and show increased permeability when added to formulations, they were selected. The selection of surfactants and co-surfactants in the preparation of nanoemulsions is based on their % transmittance. Transcutol P (diethylene glycol monoethyl ether) had a higher % transmittance than propylene glycol. It was also found that transcutol P (diethylene glycol monoethyl ether) worked well as a potent permeation enhancer. It has been found that the surfactant’s performance during the emulsification process is influenced by the co-surfactant concentration. Selecting a co-surfactant involves a number of factors, one of which is assessing the phase diagram’s nanoemulsion area. The capacity of the ratio of surfactant to co-surfactant to form nanoemulsions is significantly influenced by the size of the nanoemulsion area in the phase diagram [3, 4, 5].

3. Methods of preparation of nanoemulsion

The stratum corneum (SC), the top layer of the epidermis, acts as a powerful barrier to the applied components, as was previously discussed. This issue makes it difficult for the formulations to penetrate the skin. Consequently, numerous solutions including micro and nanosystems were explored and developed in order to get around this problem. Adding the active substance or ingredients to a nanoemulsion carrier was one of the better ways. An emulsion with droplets ranging in size from 20 to 200 nm is called a nanoemulsion. It is a colloidal system with two phases that are incompatible with one another: an aqueous phase and an oily phase. The system’s dispersion of the oily component into the aqueous phase is referred to as an oil-in-water (oil/water) nanoemulsion.

To maintain physical stability over the long term without any flocculation or coalescence in the system. A nanoemulsion is a transparent or translucent liquid-in-liquid dispersion system that is stable both thermodynamically and kinetically. Nanoemulsions offer an advantage over regular emulsions in that they are able to better encapsulate medications in the body. The ability of the nanometer-sized droplets to impede phase separation, coalescence, and flocculation improves the dispersibility of the system. Better long-term stability follows as a consequence [6, 7].

3.1 Phase inversion method

Chemical energy from phase changes that happen during the emulsification process produces fine dispersion. The required phase transitions are provided by changes in the polymer chain or composition at constant temperature. Predicated on the notion that temperature affects the solubility of polyoxyethylene/type surfactants. The surfactant monolayer exhibits a notable positive temperature change with constant composition at low temperatures. When temperatures rise, this surfactant becomes lipophilic and produces an oil-swollen micellar solution phase due to spontaneous curvature brought on by dehydration, as shown in Figure 1.

Figure 1.

Method of preparation of nanoemulsion.

3.2 Phase inversion temperature (PIT)

This method changes the composition without changing the temperature. It is important to note that nonionic surfactants, including polyethoxylated surfactants, have temperature-dependent solubility. Emulsification is the process of changing the affinities of surfactants for water and oil that are dependent on temperature. Heat causes polyethoxylated surfactants to become lipophilic due to the dehydration of polyoxyethylene groups. As a result, this circumstance validates the applicability of the PIT method for producing nanoemulsions. To make nanoemulsions with this method, the sample temperature must achieve the PIT (hydrophile–lipophile balance) or PIT level.

The PIT method produces the smallest droplet sizes and interfacial tensions that are feasible. This method improves emulsification by utilizing extraordinarily low interfacial tensions at the HLB temperature. It has been observed, therefore, that despite spontaneous emulsification at the HLB temperature, emulsions are incredibly delicate because of the incredibly fast coalescence rate. It has been reported that stable, fine emulsion droplets can be formed by rapidly cooling the emulsion to a temperature near PIT [8, 9, 10].

3.3 Phase inversion composition (PIC)

This method keeps the temperature constant while changing the composition. Nanoemulsions can be made by progressively adding water or oil to the water/surfactant or oil/surfactant mixture. The PIC technique is more appropriate for large-scale production than the PIT method since adding a single component to an emulsion is easier than producing a sudden change in temperature. When more water is supplied to the system, the water volume increases, and the system reaches a transition composition. As stated otherwise, the surfactant’s spontaneous curvature goes from negative to zero as its polyoxyethylene chains get more hydrated. The transition composition achieves a balance between the lipophilic and hydrophilic properties of the surfactant, much like the HLB temperature, as shown in Figure 1.

3.4 Sonication method

Sonication is the most efficient way to make nanoemulsions. This method reduced the droplet size of conventional emulsions or microemulsions by using a sonication process. This method can only be used to generate small batches of nanoemulsions; large batches cannot be prepared this way. Even though ultrasound can create emulsion directly, it is best to create coarse emulsion before applying acoustic power since breaking an interface requires a lot of energy. Due to its low product throughput, the ultrasonic emulsification process is mostly utilized in laboratories to create emulsion droplets as thin as 0.2 micrometers, as shown in Figure 1 [11].

3.5 Ultrasonic system

“Sonotrodes” (sonicator probes) are composed of piezoelectric quartz crystals that may expand and contract in response to alternating electrical voltage and provide the energy input for ultrasonic emulsification. Cavitations, which occur when the sonicator probe’s tip mechanically vibrates the liquid, are the main process that produces ultrasonically generated effects. Cavitation is the formation and collapse of vapor cavities in a moving liquid. This type of vapor cavity arises when fluctuations in local velocity cause the local pressure to decrease to the temperature of the flowing liquid. When these cavities collapse, strong shock waves are produced that break up the scattered droplets by radiating across the solution along the radiating face of the tip, as shown in Figure 1.

3.6 Microfluidizer

When making emulsion, much higher pressures can be reached—up to about 700 Mpa. This is accomplished at the nozzle of the microfludizer, in the interaction chamber, where two jets of crude emulsion from different channels collide with one another. The process stream is delivered by a pneumatically powered pump that can pressurize the on-site compressed air (150/650 Mpa) to a maximum of 150 Mpa. A high-pressure flow stream driven via microchannels and into an impingement spot results in an impressive shearing motion. This has the potential to create a very fine emulsion.

3.7 High/energy emulsification method

Systems known as nanoemulsions are incapable of self-formation or equilibrium. This is why mechanical or chemical energy must be added to them in order for them to form. Among the mechanical energy input tools utilized in the creation of nanoemulsions are high-pressure homogenizers, high-shear stirring, and ultrasonic generators. These mechanical devices generate strong forces that separate the oil and water phases to form nanoemulsions. In high-energy technologies, the input energy density is approximately 108/1010 W kg/1. In the shortest length of time, the system obtains the energy required to generate uniform, small-sized particles. High/pressure homogenizers are the most widely used instruments for producing nanoemulsions because of their ability to achieve this.

3.8 High-pressure homogenizer

It is the method for producing nanoemulsions that is most frequently utilized. Nanoemulsions with particle sizes as small as 1 nm can be produced using this technology by employing a piston homogenizer or a high-pressure homogenizer. During the process, a small aperture is used to force the macroemulsion through at a pressure of 500–5000 psi. When cavitation, severe turbulence, and hydraulic shear come together, the process creates incredibly small droplet-sized nanoemulsions.

This process can be continued until the final product reaches the desired polydispersity index (PDI) and droplet size. The uniformity of droplet size in nanoemulsions is specified by PDI. Higher PDI in nanoemulsions is connected with lower droplet size uniformity. A PDI of less than 0.08 indicates a monodisperse sample; a PDI of more than 0.3 indicates a narrow size distribution; and a PDI between 0.08 and 0.3 indicates a broad size distribution. However, the production of small droplets at the submicron level requires a large amount of energy. This energy combined with the increasing temperatures during the high-pressure homogenization process, could cause the components to degrade. Enzymes, proteins, and nucleic acids are a few examples of molecules that heat can damage.

3.9 High/shear stirring

This method uses high-energy mixers and rotor/stator systems to prepare nanoemulsions. The droplet sizes of the internal phase can be significantly decreased by increasing the mixing intensity of these devices. However, creating emulsions with an average droplet size of less than 200–300 nm could be difficult [12, 13, 14, 15, 16].

3.10 Low/energy emulsification method

Nanoemulsification can also be accomplished with low-energy methods, resulting in more homogeneous and smaller droplets. These methods such as phase inversion temperature and phase inversion component, produce smaller and more homogeneous droplets by making use of the physicochemical properties of the system. The types of oils and emulsifiers that low-energy techniques can employ are restricted, such as proteins and polysaccharides, even though they are frequently more effective at creating minute droplets than high-energy approaches. To solve this problem, large amounts of artificial surfactants are used in low-energy methods to produce nanoemulsions; nonetheless, this limits their range of uses, especially for many food processing as shown in Figure 1.

3.11 Spontaneous nanoemulsification

It benefits from the chemical energy replacement during the emulsification process, which is based on dilution with the continuous phase and usually proceeds at constant temperature without any phase transitions in the system. This method can produce nanoemulsions at room temperature, hence no specialized equipment is required. Essentially, the following variables influenced it: bulk and interfacial viscosity, phase transition region, surfactant concentration, surfactant structure, and interfacial tension. In the pharmaceutical industry, systems developed with this method are usually called self-emulsifying drug/delivery systems (SEDDS) or self-nano/emulsifying drug/delivery systems (SNEDDS). When water is mixed with an oil phase that has a water-soluble component, oil droplets naturally form. The process is driven by the movement of a chemical that dissolves in water from the oil phase to the water phase. This leads to interfacial turbulence, which helps oil droplets form naturally, as shown in Figure 1 [17, 18, 19].

4. Patents related to nanoemulsion

As the most effective type of intellectual property protection, patents are crucial to a nanotechnology company’s expansion. Patents will be crucial to the success of the global nanotechnology revolution, just as they were to the growth of the biotechnology and information technology industries. In fact, patents are already influencing the young, quickly developing field of nanoscience and small technologies. A company’s long-term survival will depend on its ability to get legitimate and defendable patent protection as it develops nanotechnology-related products and processes and starts looking for commercial uses for its ideas, as stated in Table 1 [20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49].

Patent application title	Patent App. No.	Date
Nanoparticles and nanoemulsions	14/893,123	2017/12/05
Nanoemulsion therapeutic compositions and methods of using the same	12/567571	2015/02/24
Antimicrobial nanoemulsion compositions and methods	8236335	2012/08/07
Antimicrobial nanoemulsion compositions and methods	8232320	2012/07/31
Topical compositions and methods of detection and treatment	20120039814	2012/02/16
Cancer vaccine compositions and methods of using the same	20110280911	2011/11/17
Methods of using nanoemulsion compositions having anti/inflammatory activity	20110200657	2011/08/18
Stable nanoemulsions for ultrasound/mediated of drug delivery and imaging	20110177005	2011/07/21
Method for the preparation of nanoparticles from nanoemulsion	20110135734	2011/06/09
Antimicrobial nanoemulsion compositions and methods	20110070306	2011/03/24
Nanoemulsion formulations for direct delivery	20110045050	2011/02/24
Lyophilized nanoemulsion	20110015266	2011/01/20
Nanoemulsion vaccines	20100316673	2010/12/16
Nanoemulsion of resveratrol/phospholipid complex and method for preparing the same and applications thereof	20100297199	2010/11/25
Per fluorocarbonnanoemulsion containing quantum dot nanoparticles and method for preparing the same	20100233094	2010/09/16
Compositions for treatment and prevention of acne, methods for making the compositions, and methods of use thereof	20100226983	2010/09/09
Nanoemulsion therapeutic compositions and methods of using the same	20100092526	2010/04/15
Stable mixed emulsions	20100069511	2010/03/18
Antimicrobial nanoemulsion compositions and methods	7655252	2010/02/02
Antimicrobial nanoemulsion compositions and methods	20100003330	2010/01/07
Nanoemulsion influenza vaccine	20090304799	2009/12/10
Nanoemulsion adjuvants	20090291095	2009/11/26
Oil/in/water nanoemulsion, a cosmetic composition, and a cosmetic product comprising it, a process for preparing said nanoemulsion	20090208541	2009/08/20
Nanoemulsion therapeutic compositions and methods of using the same	20080317799	2008/12/25
	PCT/AU2008/001714	2008/11/18
Nanoemulsion vaccines	20080254066	2008/10/16
Nanoemulsion vaccines	20080181905	2008/07/31
Nanoemulsion vaccines	7314624	2008/01/01
Nanoemulsion compositions having anti/inflammatory activity	20070036831	2007/02/15
Nanoemulsion formulations	20020155084	2002/10/24
Nanomulsion based on nonionic and cationic amphiphilic lipids and uses thereof	6039936	2000/03/21
Solid fat nanoemulsions as vaccine delivery vehicles	5716637	1998/02/10
Solid fat nanoemulsions as drug delivery vehicles	5576016	1996/11/19

Table 1.

Patents on nanoemulsion.

5. Evaluation of nanoemulsion

5.1 Determination of encapsulation efficiency

To determine the amount of drug entrapped in the formulation, a weighed quantity of the formulation is ultrasonically dispersed in an organic solvent to release the drug, which is then extracted into a suitable buffer. Once the right dilutions are made and compared to an adequate blank, the extraction is subjected to spectrophotometric analysis at the drug’s λ_max to determine the drug content. The drug’s loading efficiency (LE) and entrapment efficiency (EE) can be calculated using these formulas. Drug LE represents the amount of drug in the achieved product (mg)/total product weight (mg) × 100, while drug EE represents the amount of drug in the obtained product (mg)/total quantity of drug added (mg) × 100. To assess drug concentration, high-performance liquid chromatography (HPLC) in reverse phase can also be employed [50, 51, 52, 53, 54, 55, 56, 57].

5.2 Determination of particle size and polydispersity index (PDI)

Photon correlation spectroscopy (PCS) is used to measure the PDI and particle size of nanoemulsions using a Malvern Zetasizer. PCS calculates the variation in light scattering over time brought on by particle Brownian motion.

The fundamental tenet of PCS is that smaller particles accelerate larger ones. The laser beam is distorted by the submicron particles present in the solution. The rate at which particle diffusion causes the laser scattering intensity to vary around a mean value at a fixed angle depends on the particle size. The calculated photoelectron time correlation function yields a line width distribution histogram that is associated with particle size. A weighed quantity of formulation is combined with double or distilled water to generate a homogenous dispersion, which is then used to measure the particle size. The PDI and particle size must be measured using this mixture immediately. A 0 (zero) PDI represents a monodisperse system while a 1 PDI represents a polydisperse particle dispersion [20].

5.3 Determination of zeta potential

The zeta potential is a method for figuring out a particle’s surface charge in a liquid. Zeta potential is a helpful tool for predicting dispersion stability; the presence and adsorption of electrolytes, as well as the physicochemical properties of the drug, polymer, and medium, all affect its value. The Malvern Zetasizer apparatus is employed for its measurement. Zeta potential is determined by diluting the nanoemulsion and calculating its value using the electrophoretic mobility of the oil droplets. It is believed that a zeta potential of ±30 mV suffices to ensure the nanoemulsion’s physical stability.

5.4 Morphological study of nanoemulsion

The morphology of nanoemulsions is analyzed using transmission electron microscopy (TEM). In a transmission electron microscope (TEM), an electron beam is directed at a thin foil specimen. Upon interaction with the material, these incident electrons become unscattered, elastically scattered, or elastically scattered electrons. The distances between the objective lens and the specimen and between the objective lens and its image plane regulate the magnification.

The electromagnetic lenses focused the scattered or unscattered electrons and projected them onto a screen to produce a contrast/amplitude image, a phase/contrast image, an electron diffraction image, or a phantom image with distinctly darkened parts, depending on the density of unscattered electrons. At higher magnifications, diffraction modes can be used in conjunction with bright field imaging to reveal the size and structure of nanoemulsion droplets. To immobilize the sample for TEM investigation, a suspension of lyophilized nanoparticles or a few drops of nanoemulsion are prepared in double-distilled water and placed onto a holey film grid. The excess solution must be squeezed off the grid and dyed after immobilization [21, 22].

5.5 Atomic force microscope (AFM)

These days, the surface morphology of nanoemulsion formulations is studied using a relatively new technique called AFM. Nanoemulsions are diluted with water and then applied to a glass slide in order to conduct AFM. After that, the coated drops are dried in an oven and scanned at 100 mV/s.

5.6 In vitro drug release study

Performance of drug formulation drug release studies conducted in vitro can help estimate in vivo. The in vitro release rate of a medication is usually studied using a United state pharmacopoeia (USP) dissolving apparatus. Dried nanoparticles or nanoemulsion containing 10 mg of medication were applied to dialysis membrane pouches and placed in a flask containing buffer after being dissolved in buffer. This experiment is run at 37 ± 0.5° with a stirring speed of 50 rpm. Samples are removed on a regular basis and are always replaced with the same volume of fresh dissolving media.

Spectrophotometry is used to detect the absorbance of materials at a certain wavelength after they have been suitably diluted. The absorbance of the sample is used to compute the percentage of drug release at different time intervals using a calibration curve.

5.7 In vitro skin permeation studies

With the Keshary–Chien/diffusion cell, permeation investigations can be studied both in vitro and in vivo. For permeation studies, the abdominal skin of adult male rats weighing 250 ± 10 g is frequently utilized. The donor and recipient chambers of the diffusion cell are separated by the rat skin. Twenty percent ethanol-infused fresh water is added to receiver chambers that are kept at 37°C and rotated at 300 rpm all the time. The formulas are kept in the donor room.

At predetermined intervals, such as 2, 4, 6, and 8 hours, a predetermined volume (0.5 ml) of the receiver chamber’s solution was removed for gas chromatographic analysis. Each time, the sample was quickly replaced with an equivalent volume of brand-new solution. Three runs are made for each sample. The total amount of medication absorbed via rat skins at each time interval is plotted against a function of time following cumulative adjustments. In steady-state conditions, the plot slope is utilized to calculate medicine penetration rates [23].

5.8 Stability studies

The purpose of stability studies is to assess how stable a medicinal ingredient is in the presence of various environmental factors, including temperature, humidity, and light. The formulation is stored for 24 months in a dispersed and freeze/dried state before the stability studies of the nanoemulsion are carried out in accordance with the guidelines set forth by the International Conference on Harmonization. There are three storage conditions that are utilized: ambient (25 ± 2°/60 ± 5% RH), refrigerated (5 ± 3°), and freeze (−20 ± 5°). The necessary quantity of nanoemulsion is stored in glass vials with hermetically sealed closures. Samples are removed and analyzed for characteristics such as loading, particle size, EE, and the in vitro drug release profile at predetermined intervals [24].

5.9 Shelf life determination

Expedited stability studies are performed to determine the shelf life of a nanoemulsion. The formulations are stored at three distinct temperatures and relative humidity levels (30°, 40°, and 50 ± 0.5°) for almost 3 months. After a predetermined period of time (0, 30, 60, and 90 days), samples are taken out and analyzed using HPLC at λmax to find out how much medication remains. The samples that are removed at zero time are known as control samples. This determines the order of the reaction. Next, using the following equation, the reaction rate constant (K) for the deterioration is calculated at each elevated temperature based on the slope of the lines: An Arrhenius plot with slope = −K/2.303 is produced by plotting the logarithms of K at different elevated temperatures against the reciprocal of absolute temperature. This data yields the plot value of K at 25°, which is then used to calculate shelf life by plugging the value into the calculation t0.9 = 0.1052/K25. Here, t0.9—also referred to as shelf life—is the amount of time required for a 10% degradation of the drug [25].

5.10 Thermodynamic stability studies

The majority of thermodynamic stability studies are carried out in three steps. Initially, a cycle of heating and cooling is conducted to determine whether adjusting the temperature affects the stability of the nanoemulsion in any way. The nanoemulsion is subjected to six cycles of temperature between 4° (the refrigeration temperature) and 40° while the formulation is stored at each temperature for a minimum of 48 hours.

The formulations that show stability at these temperatures are used in centrifugation experiments. The second technique involves centrifuging the manufactured nanoemulsions for 30 minutes at 5000 rpm in order to check for phase separation, creaming, or cracking. Those who showed no signs of instability could experience a freeze-thaw cycle. The third technique is the freeze/thaw cycle, which entails freezing and thawing nanoemulsion formulations three times at temperatures between −21° and + 25°.

If a formulation shows no signs of instability, this test finds that it has good stability. These mixtures are then subjected to dispersibility tests to determine how well they self- or emulsify. This test determines that a formulation has good stability if it exhibits no indicators of instability. The next step is to put these mixtures through dispersibility tests to see how effectively they self- or emulsify.

5.11 Dispersibility studies

Dispersibility tests are performed utilizing conventional USP XXII dissolving equipment to assess the effectiveness of self-emulsification or nanoemulsion formation. Each formulation is added to 500 ml of distilled water that is kept at 37 ± 0.5°; 2.1 ml of each formulation is used. A typical dissolution paddle made of stainless steel spins at 50 revolutions per minute to gently agitate the mixture. The nanoemulsion formulations’ In vitro performance is assessed visually by the application of a grading system that is explained below. Level A nanoemulsions seem clear or bluish and form quickly—within 1 minute. Although they are somewhat less transparent, Grade B nanoemulsions develop quickly and have a bluish-white appearance. Within 2 minutes, grade C nanoemulsions, which are fine, milky emulsions form. Grade D emulsions are slower and more dull, with a grayish-white look and a hint of oiliness [26].

5.12 Determination of viscosity

Viscosity measurement is a crucial component of a nanoemulsion’s physicochemical assessment. Viscosity is measured using a number of tools, such as the Ostwald viscometer, Hoeppler falling ball viscometer, Stormer viscometer, Brookfield viscometer, and Ferranti/Shirley viscometer. Of all these devices, the Brookfield viscometer is the one that is suggested for figuring out the viscosity of a nanoemulsion.

The assessment of viscosity verifies whether the system is an O/W or W/O emulsion. Systems that exhibit low viscosity are classified as O/W types, whereas those that exhibit high viscosity are classified as water-in-oil types. Nonetheless, the survismeter has emerged as the most often utilized piece of apparatus since it assesses the particle size, hydrodynamic volumes, contact angle, dipole moment, interfacial tension, surface tension, and viscosity of the nanoemulsions.

5.13 Refractive index

The refractive index provides information on the transparency of the nanoemulsion and the transmission of light through the medium. The ratio of the wave’s phase speed (vp) in the medium to its speed (c) in the reference medium is the formula for determining a medium’s refractive index (n), and it is expressed as n = c/vp. The refractive index of the nanoemulsion can be determined by placing a drop on a slide and comparing it to the refractive index of water (1.333) using an Abbes-type refractometer set at 25 ± 0.5. In order for a nanoemulsion to be considered transparent, its refractive index must equal that of water.

5.14 pH and osmolarity measurements

A pH meter is used to determine a nanoemulsion’s pH, whereas a microosmometer uses the freezing point method to determine an emulsion’s osmolarity. Once 100 μl of nanoemulsion has been transferred into a microtube, measurements are taken.

5.15 Dye solubilization

A water soluble dye is dispersible in an O/W globule as opposed to soluble in the aqueous phase of the W/O globule. Similarly, an oil-soluble dye is dispersible in the W/O globule but dissolves in the oily phase of the O/W globule. An O/W nanoemulsion will absorb color evenly when water-soluble dye is applied; however, if the emulsion is W/O, the dye will only remain in the dispersed phase, and the color will not absorb evenly [27].

5.16 Dilutability test

The stability of a nanoemulsion can be preserved when a continuous phase is added in larger quantities which justifies the dilution test. Consequently, unlike O/W nanoemulsions, W/O nanoemulsions do not dilute with water and instead experience phase inversion into O/W nanoemulsions. W/O nanoemulsion can only be diluted with oil [28].

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

BCS	biopharmaceutical drug classification system
HLB	hydrophilic-lipophilic balance
SC	stratum corneum
PIT	phase inversion temperature
PIC	phase inversion composition
PDI	polydispersity index
SEDDS	self-emulsifying drug/delivery systems
SNEDDS	self-nano/emulsifying drug/delivery systems
LE	loading efficiency
EE	entrapment efficiency
HPLC	high-performance liquid chromatography
PCS	Photon correlation spectroscopy
TEM	transmission electron microscopy
AFM	atomic force microscope