Formulation And Evaluation of Ezetimibe Nanoemulsion

Nanoemulsions are thermodynamically stable colloidal dispersions formed by mixing two immiscible liquids with the aid of surfactants and co-surfactants, which lower interfacial tension and yield a uniform phase. They are usually transparent or translucent and are also described as ultrafine, submicron, or mini-emulsions. Depending on the arrangement of dispersed and continuous phases, nanoemulsions are categorized as oil-in-water (O/W), where oil droplets are distributed in water; water-in-oil (W/O), where water droplets are distributed within oil; and bi-continuous systems, in which microdomains of both oil and water coexist throughout the formulation.[1]

COMPONENTS OF NANOEMULSION

Table 1: Lists of components

METHODS OF PREPARATION

There are many techniques used to create nanoemulsion drug delivery systems, and many of them overlap. We have grouped these methods based on how much energy they require, the type of phase inversion involved, and whether they use self-emulsification. High energy methods include high-pressure homogenization, microfluidization, and ultrasonication. Low energy methods include phase inversion emulsification, which can be transitional (such as phase inversion temperature or composition) or catastrophic (such as emulsion inversion point), as well as the self-nanoemulsification method.^[2]

1. High energy methods

1. High-Pressure Homogenization

This technique involves the application of high pressure to a system containing an oil phase, an aqueous phase & a surfactant or co-surfactant. The pressure is exerted using specialized equipment known as a homogenizer. However, there are several issues associated with homogenizers, including low productivity, component degradation due to challenges in mass production, and excessive heat generation. This method is limited to producing only oil-in-water (o/w) liquid nanoemulsions with an oil phase of less than 20%, and it is not capable of creating cream nanoemulsions with high viscosity or hardness that have a mean droplet diameter of less than 200nm.^[3]

2. Microfluidization

Microfluidization is a technique for mixing that employs a device known as a microfluidizer. This device operates using a high-pressure positive displacement pump, which can range from 500 to 20,000psi, to propel the product through an interaction chamber composed of small channels referred to as 'microchannels'. As the product traverses these microchannels, it reaches an impingement area, leading to the formation of extremely fine particles within the sub-micron range. The two phases, namely the aqueous & oily phases, are amalgamated & processed in an inline homogenizer to create a coarse emulsion. Subsequently, this coarse emulsion is introduced into a microfluidizer for further processing, aiming to achieve a stable nanoemulsion. The coarse emulsion is cycled through the interaction chamber of the microfluidizer multiple times until the target particle size is reached. Finally, the bulk emulsion is filtered through a filter under nitrogen to eliminate larger droplets, resulting in a consistent nanoemulsion.^[4]

3. Ultrasonication

Sonication, also known as ultrasonic homogenization, is employed to produce kinetically stable nanoemulsions. In this process, a sonicator probe is placed in contact with a dispersion containing liquids, surfactants, and cosurfactants. The probe generates mechanical vibrations and cavitation, supplying the energy required to form small droplets. Sonication is commonly utilized for small-scale nanoemulsion production. To avoid shear-induced coalescence, operational parameters must be carefully controlled. The particle size of the dispersed phase decreases as the duration of sonication, power input, and surfactant concentration increase. Achieving a droplet size of approximately 20 nm requires optimization of the ultrasonic reaction chamber design, operating conditions, and formulation variables such as surfactant concentration and oil phase composition. A primary limitation of sonication is its unsuitability for preparing large volumes of nanoemulsions.^[5]

2. Low energy methods

For the preparation of nanoemulsion systems, these methods require low energy.
Low-energy emulsification techniques are more energy efficient as they use internal chemical energy of the systems & only require gentle stirring for the creation of nanoemulsions.^[2]

1. Phase inversion temperature

In the phase inversion temperature (PIT) method, the temperature of the emulsion system is raised to alter the solubilizing behavior of the surfactant (from hydrophilic to lipophilic), resulting in the formation of bicontinuous microemulsions and subsequent emulsion inversion. This process consists of four steps:

(i) When the temperature is below the PIT, a macro-emulsion is present, characterized by nonionic surfactants, primarily hydrophilic.

(ii) As the temperature increases, the surfactants slowly transition to a lipophilic state and become solubilized by the oil phase.

(iii) At the PIT, bicontinuous microemulsions are formed.

(iv) When the temperature exceeds the PIT, the emulsion undergoes inversion, with water being dispersed into the mixture of oil and lipophilic surfactant. The system is then rapidly cooled through water dilution, which instantaneously renders the surfactant hydrophilic, thereby prompting spontaneous and swift migration to the aqueous phase. This turbulent displacement facilitates the generation of nanoemulsions.^[6]

2. Emulsion Inversion point (EIP)

The EIP method represents a low-energy & spontaneous approach to emulsification. Under constant temperature conditions, this method facilitates the alteration of the intrinsic characteristics of thermodynamically stable microemulsions or liquid crystals, transforming them into nano-structured forms through a gradual dilution with either water or oil. This process leads to the formation of thermodynamically unstable yet kinetically stable direct or inverse nanoemulsions. A minor adjustment in the ratio of water or oil within the established microemulsion system will modify the surfactant hydration pattern & its affinity for the aqueous phase. Consequently, this induces instabilities within the microemulsion system, ultimately resulting in its disintegration into nano-emulsion.^[6]

3. Spontaneous Emulsification Method

Spontaneous emulsions are generated in this method by combining water & oil with an emulsifier through gentle stirring at a specific temperature. The gentle magnetic stirring facilitates the mixing of phases, allowing the emulsifier to migrate into the aqueous phase, which increases the oil-water interfacial area & leads to the formation of oil droplets. Spontaneous emulsification consists of three primary steps, which are as follows:

• The creation of a homogeneous organic solution that includes oil & a lipophilic surfactant in a water-miscible solvent, along with a hydrophilic surfactant.
• The organic phase is then injected into the aqueous phase while magnetic stirring is applied, resulting in the formation of the oil-in-water emulsion.
• Finally, the water-miscible solvent is eliminated through evaporation under reduced pressure.^[7]

Applications of Nanoemulsions

The nanoemulsions are used in a wide range of fields like pharmaceuticals, biomedical fields, cosmetics, food, agriculture, and industry because of their unique physicochemical properties and ability to improve solubility, stability, and targeted delivery of active compounds.

Pharmaceutical Applications

In pharmaceutical industry, the nanoemulsions are used in various drug delivery systems as they can increase the solubility of poorly water soluble drugs, thus increasing their bioavailability and therapeutic efficacy. Nanoemulsions facilitate the controlled and targeted release of active ingredients, thereby decreasing the frequency of doses and minimizing side effects. Furthermore, they provide safeguarding for delicate compounds from enzymatic or oxidative breakdown, thereby guaranteeing stability throughout formulation and storage. Owing to their small droplet size and extensive surface area, nanoemulsions can be efficiently delivered via various routes including oral, parenteral, transdermal, ocular, and pulmonary, positioning them as a promising platform for contemporary pharmaceutical applications.

Cosmetic Applications

Nanoemulsion is extensively utilized in the cosmetic sector because its active components are readily absorbed, resulting in effective action due to its characteristic properties, including a small droplet size and the capacity to minimize water loss from the skin. Nanoemulsions are employed in moisturizers, lotions, and creams. As an appealing delivery system in cosmetics, the droplet size of nanoemulsions is extremely small, preventing creaming and flocculation, which contributes to a more refined product that enhances uptake by antigen-presenting cells in stable formulations. The incorporation of nanoemulsion in cosmetic products facilitates the swift penetration of active ingredients through the skin, attributed to the large surface area of the droplets. It is also observed that nanoemulsions can penetrate effectively through rough skin. This characteristic of nanoemulsion reduces the need for additional penetration enhancers, which can lead to formulation incompatibility.^[8]

Nanoemulsion in Food industry

Nanoemulsions present a diverse array of applications owing to their compositional versatility across various sectors, particularly in the food and beverage industries. In contrast to microemulsions, nanoemulsions have gained significant traction in food processing due to their diminutive size, thermodynamic stability, and the ability to continuously self-assemble with both hydrophilic and hydrophobic components. Their transparency and minimal light wave scattering capabilities ultimately facilitate their integration into optically clear products, such as enhanced soft drinks and bottled waters. Unlike traditional micro or other standard emulsions, nanoemulsions can be formulated to exhibit greater viscosity or a gel-like consistency, even at very low droplet concentrations, making them suitable for creating products with reduced fat content and innovative textures.^[9]

Advantages and Disadvantages of Nanoemulsions

ADVANTAGES

• Increased solubility of drugs that are poorly-water soluble.
• Bioavailability enhancement.
• Reduce dose frequency.
• Improve patient compliance.
• It has rapid onset of action.

DISADVANTAGES

• It requires high surfactant concentrations which leads to the risk of irritation or toxicity.
• The nanoemulsion stability is affected by the environmental factors like pH, temperature etc.
• High production costs.
• Thermodynamically unstable.

MATERIALS AND METHODS

MATERIALS

Table 3: Lists of Chemicals

Standard calibration curve of Ezetimibe in Acetonitrile

• Determination of λ_max using UV spectrophotometer

The λ_max of drug sample was determined by scanning 10ppm standard stock solution in the range from 200-400nm using Shimadzu-1800 UV spectrometer.

• Construction of calibration curve by UV spectroscopy

Ezetimibe (10mg) was dissolved in Acetonitrile (10ml).[1000µg/ml] 1ml was taken from the above solution and made upto 10ml with acetonitrile. [100µg/ml] Dilutions were made 5ppm, 10ppm, 15ppm, 20ppm, 25ppm and were subjected to UV spectrophotometer. Absorbance was measured at 231nm.

SOLUBILITY STUDIES

Solubility of Ezetimibe in various components (oils, surfactants & cosurfactants) was determined by adding an excess amount of drug in 500mg of selected vehicle in 5ml capacity stoppered vials, and mixed using a cyclomixer. These vials were then kept at 25±1⁰C in an shaking incubator for 72h to reach equilibrium. The balanced samples were taken out of the shaker and subjected to centrifugation at 3000rpm for a duration of 15 minutes. The supernatant was collected & the concentration of drug was determined using UV spectrophotometer.

FTIR (Fourier Transform Infrared Spectroscopy)

FTIR analysis was performed to evaluate potential interactions between the pure drug (Ezetimibe) & the excipients in the optimized nanoemulsion formulation. A small quantity of each sample (pure drug and nanoemulsion formulation) was taken and dissolved in a minimal amount of chloroform in a clean china dish using a mortar and pestle to aid dispersion. Using a capillary tube, a thin film of the solution was carefully applied onto clean, dry potassium bromide (KBr) or sodium chloride (NaCl) discs and allowed to form a uniform layer. The discs were subsequently positioned within the sample holder of the FTIR spectrophotometer for measurement. The spectra were in the range of 4000 to 400cm^-1. A background spectrum was collected prior to each sample scan. The recorded spectra were analyzed for the presence, shift, or disappearance of characteristic peaks, indicating potential interactions or encapsulation of the drug within the formulation matrix.

Construction of pseudo ternary phase diagrams

• According to solubility studies, capryol 90 was chosen as the oil phase for the development of the nanoemulsion.
• Tween 80 and Transcutol HP served as the surfactant and co surfactant, respectively. Distilled water constituted the aqueous phase.
• The surfactant and cosurfactant were mixed (S_mix) in volume ratios of 4:1, 3:1, 1:3, 2:1, and 1:1. For each phase diagram, the oil and S_mix ratios were thoroughly mixed in various volume ratios ranging from 1:9 to 9:1.
• Phase diagrams were constructed utilizing the aqueous titration method.
• The physical state of the nanoemulsion was indicated on a pseudo three component phase diagram, with one axis denoting the aqueous phase, another representing the oil, and the third illustrating the mixture of surfactant and cosurfactant at a fixed volume ratios (S_mix), using TernaryPlot.com.

FORMULATION OF NANOEMULSION

Prepare the homogeneous mixture of oil and S_mix (surfactant & cosurfactant). Ezetimibe was precisely measured and subsequently incorporated into the aforementioned mixture. Oil phase then added drop wise to the aqueous phase with continuous mixing using homogenizer leads to the formation of O/W nanoemulsion.

Optimized formulae of Nanoemulsions

THERMODYNAMIC STABILITY STUDIES

Selected formulations underwent a series of thermodynamic stability assessments to evaluate their physical stability.

Centrifugation

The prepared formulations were centrifuged at 5000rpm for 30min & observed for phase separation, creaming or cracking.

Freeze Thaw Cycle

A required quantity of the nanoemulsion was transferred into a vial & subjected to three freeze–thaw cycles by storing it at −25?°C for one hour followed by room temperature for one hour. Three freeze-thaw cycles between -21°C & +25°C with formulation storage at each temperature for not less than 48 h were performed.

Determination of particle size and polydispersity index (PDI)

The particle size & PDI of nanoemulsions are evaluated using photon correlation spectroscopy (PCS) with the Malvern Zetasizer, which observes the changes in light scattering due to the Brownian motion of particles over time. PCS operates on the principle that smaller particles move at a higher velocity compared to larger particles. To measure particle size, a specific amount of the formulation is dispersed in distilled water to create a homogeneous dispersion, which must be utilized immediately for the measurement of particle size and PDI.

Determination of zeta potential

The zeta potential serves as a technique for assessing the surface charge of particles when immersed in a liquid medium. This measurement is instrumental in forecasting the stability of dispersions, with its value being influenced by the physicochemical characteristics of the drug, polymer, vehicle, and the presence and adsorption of electrolytes. The Malvern Zetasizer instrument is utilized for this measurement. To determine the zeta potential, the nanoemulsion is diluted & its value is derived from the electrophoretic mobility of the oil droplets. A zeta potential of ±30mV is considered adequate for maintaining the physical stability of the nanoemulsion.

Drug content

A 1mL aliquot of the nanoemulsion formulation was transferred into a 10mL volumetric flask & diluted to volume with acetonitrile. The resulting solution was analyzed for drug content using a UV-visible spectrophotometer at a wavelength of 231nm.

pH

The measured apparent pH of the formulations was determined using a pH meterin triplicate at a temperature of 25°C.

Viscosity

The viscosity of the formulated nanoemulsions was measured undiluted using a Brookfield Viscometer with spindle #C50-1 at a temperature of 25±0.5?C. 1ml of the formulation was utilized for the purpose of determining viscosity. The speed of the spindle was adjusted to 50rpm & a single run was performed at a temperature of 25±0.5?C.

In vitro diffusion studies

A study on in vitro diffusion was performed using a Franz diffusion cell. The investigation was performed using an eggshell membrane. The membrane was prepared appropriately and preserved in a pH 7.4 phosphate buffer prior to its application. A phosphate buffer solution with a pH of 7.4 served as the diffusion medium, and the receptor compartment was filled with this solution. The membrane of the eggshell was placed between the donor & recipient sections of the diffusion cell. One milliliter of the test formulation was added to the donor compartment. The temperature of the diffusion medium was regulated at 37±1°C, while the buffer solution was continuously agitated with a magnetic bead at a rate of 500 to 700 rpm. Samples were extracted from the release medium at intervals of 5, 10, 15, 30, 45, 60, 90, 120, 180, and 240 minutes & replaced with an equivalent volume of fresh buffer solution to sustain sink conditions. The samples were analyzed spectrophotometrically at a wavelength of 231 nm against their corresponding blank.

Stability studies

Stability evaluation of the optimized nanoemulsion was performed at accelerated storage conditions (40 °C/75% RH).

RESULTS AND DISCUSSION

Calibration plot of Ezetimibe

Determination of λ_max

λ_max of Ezetimibe in acetonitrile was found to be 231nm. When examined in the range of 200-400nm, Ezetimibe showed an absorption maxima at 231nm in acetonitrile. Different concentrations were prepared using acetonitrile. A correlation coefficient of R²=0.9941 was shown in the calibration data.

Fig1: Spectrum of Ezetimibe

Calibration curve of Ezetimibe

Table 5: Calibration Curve of Ezetimibe

n=3

Fig2: Calibration Curve of Ezetimibe at λ_max 231nm(n=3)

SOLUBILITY STUDIES OF EZETIMIBE

Table 6: Solubility of Ezetimibe in different oils

n = 3

Table 7: Solubility of Ezetimibe in different Surfactants & Co-Surfactants

n = 3

Fig 3: Graph depicting solubility of Ezetimibe in different oils(n=3)

Fig 4: Graph depicting solubility of Ezetimibe in different surfactants & co-surfactants(n=3)

Using the FTIR spectrophotometer, the compatibility of pharmacological excipients was examined. FTIR data of the drug alone and the drug combined with the excipients suggested that there was no apparent interaction between the medication & the used excipients.

Table 8: Interpretation of pure drug (Ezetimibe) and optimized formulation (F2)

PSEUDO TERNARY PHASE DIAGRAM

Pseudo ternary phase diagram of S_mix ratio 4:1

Fig10: Different ratios of oil and S_mix (4:1)

Fig11: Different ratios of oil and S_mix (3:1)

Fig12: Different ratios of oil and S_mix (2:1)

Fig14: Different ratios of oil and S_mix (1:1)

EVALUATION OF NANOEMULSION

Table 15: Particle size, zeta potential and PDI of optimized aqueous titration solutions.

Diffusion Studies

Table 17: % cumulative drug release of optimized formulation F2

Fig 20: Graph depicting the %CDR graphs of optimized formulation F2(n=3)

Stability Studies

Stability studies for the F2 formulation were carried out at 40 °C and 75% relative humidity.