Formulation and In Vitro Evaluation of Polymer-Based Fluvoxamine Nanoemulsion

INTRODUCTION TO NANOEMULSIONS

Nanoemulsion drug delivery systems are a promising tool for delivering and improving the bioavailability of hydrophobic drugs and bioactive food components in the blood. The majority of drugs are hydrophobic (lipophilic) in nature, thus leads to low solubility and bioavailability problems [1]. The bioactive food components also show low bio availabilities in conventional doses. Such drug and food component formulations have low oral bioavailability, uncertain absorption profiles, dose variations, wide intra and inter-subject variability’s, and increased the chance of food effect [2]. Lipid-based formulations are a good choice for delivering drug and bioactive food components, which have low oral bioavailability and other formulation problems [3]. Nanoemulsion drug delivery systems are lipid-based formulation system which improves the solubility and bioavailability of hydrophobic drugs and bioactive food components [4].

Flavonoids (flavanols, flavones, flavanones, and isoflavones), nonflavonoids (hydroxybenzoic acids, stilbenes, and curcuminoids), and carotenoids (carotenes and xanthophylls) are food bioactive compounds that have been encapsulated successfully in nanoemulsions formulations [5, 6].

Nanoemulsion is defined as a colloidal dispersion of two immiscible liquids that is thermodynamically unstable. In nanoemulsion, one of the liquids forms the dispersed phase and other liquid forms the dispersing medium [7]Thus, design of well-controlled and versatile delivery systems is the main emphasis of present research initiatives. A promising method for delivering a variety of molecules to specific sites in the body is the association of therapeutic medicines with nanoparticles displaying special physicochemical and biological characteristics and structuring their paths for appropriate targeting [8, 10]. Targeted therapy in disease treatment involves administering precise quantities of therapeutic agents over an extended duration to the specific area of the body that is affected. The development of safer and more effective therapeutic nanoparticles is essential and represents one of the primary objectives of nanomedicine [11]. Surface modification can also enhance the recognition capability for targeted delivery. Therefore, the cleanability and biodistribution of therapeutic nanoparticles must be. carefully considered during the design process[12,13]

1.2. INTRODUCTION TO COMPONENT OF NANOEMULSION:

Components of nanoemulsion systems include oils, lipids, surfactants, water-soluble co-solvents, and water. In the formulation of nanoemulsions, the oil phase may include triglycerides like tri-, di-, or mono-acylglycerols, vegetable oils, mineral oils, free fatty acids etc[14]. Oil selection is generally based on the drug solubility. Oil phases which have high drug loading is generally used for development of nanoemulsion[15] Common surfactants used in the nanoemulsion systems for drug delivery and food ingredients are spans (sorbitan fatty acid esters), tweens [polyoxyethylene (POE) derivatives of sorbitan fatty acid ester], Cremophor^® EL (polyoxyl-35 castor oil), lauroyl macro-golglycerides (Gelucire^® 44/14), polysaccharides (gum and starch derivatives), phospholipids (egg, soy, or dairy lecithin), and amphiphilic proteins (whey protein isolate and caseinate) [16]

1.3. ADVANTAGES OF NANOEMULSION DRUG DELIVERY SYSTEM:

Nanoemulsions drug delivery systems are effective in solubilizing active lipophilic compounds, and therefore have for several applications [17,18]. he very small particle sizes of nanoemulsion drug delivery systems is a promising advantage over conventional emulsions; these systems therefore have optically transparent appearances, and show greater stability against droplet flocculation and coalescence [17,18,19]. Nanoemulsion drug delivery system has shown potential for effective systemic delivery of active components, such as food ingredients and lipophilic drugs, via oral, parenteral, ocular, and topical routes [20,21]

1.4. INTRODUCTION TO FLUVOXAMINE LOADED POLYMER BASED NANOEMULSIONS 

Depressive disorders are some of the most prevalent, impairing and costly illnesses, having recently been estimated to affect more than 246 million people worldwide [22]. Although they can be divided according to subtype and level of severity, these disorders are generally characterized by a depressed mood or general loss of pleasure or interest, usually accompanied by symptoms such as feelings of guilt, worthlessness or hopelessness; low self-esteem; indecisiveness or difficulty in concentrating or thinking; fatigue; psychomotor agitation or retardation; change in appetite; insomnia or hypersomnia; mood swings; and, in most severe cases, recurrent thoughts of death or suicidal ideation[23] The depressed person usually has a loss in work productivity and difficulty in managing social situations, with a decrease in general quality of life, and increased risk of suicide. Moreover, coexisting with other diseases can exacerbate their symptoms, being associated, for example, with a higher risk of severe complications in diabetic patients (amputation, blindness, dementia), and increasing the relative risk of developing heart disease by 200%[24]. Pharmacological treatment of depressive disorders can be quite variable, but selective serotonin reuptake inhibitors (fluoxetine, paroxetine, sertraline) are usually considered as first-line options. Other alternatives include serotonin and norepinephrine reuptake inhibitors (duloxetine, venlafaxine), tricyclic antidepressants (amitriptyline, imipramine, nortriptyline), tetracyclic antidepressants (mirtazapine), and atypical antidepressants (trazodone, nefazodone, agomelatine). Adjuvant hormonal and psychological treatments are also recommended in some cases, as well as lifestyle changes [25]. With so many available treatments to choose from, the choice should be made carefully, in a case-by-case trial-and-error approach, being individualized according to the patient’s characteristics (ethnicity, gender, age, socioeconomic status, comorbidities, etc.) and symptomatology[23].

Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), is widely used for the treatment of depression, obsessive-compulsive disorder (OCD), and anxiety disorders [24]. Despite its therapeutic efficacy, fluvoxamine exhibits limitations such as poor aqueous solubility, extensive first-pass metabolism, and variable bioavailability, leading to inconsistent therapeutic outcomes [25]. To address these challenges, advanced drug delivery systems, such as polymeric nanoemulsions, have emerged as promising carriers for fluvoxamine.

Nanoemulsions are biphasic dispersions of oil and water stabilized by surfactants and co-surfactants, possessing droplet sizes in the nanometer range [26]. These formulations enhance drug solubility, improve absorption, and facilitate targeted delivery, making them an effective approach for improving the bioavailability of poorly water-soluble drugs [27]. Furthermore, the incorporation of polymeric excipients such as hydroxypropyl methylcellulose (HPMC) and ethyl cellulose in nanoemulsions enhances stability, prolongs drug release, and minimizes systemic side effects [28].The formulation of fluvoxamine-loaded polymeric nanoemulsions typically involves the solvent evaporation method, employing stabilizers like polyvinyl alcohol (PVA) and surfactants such as Tween 80 to achieve optimal particle size and drug loading [29]. Optimization techniques, such as the Box-Behnken design, aid in fine-tuning formulation parameters to ensure desirable physicochemical properties and therapeutic efficacy [30,31].

Recent studies have demonstrated that fluvoxamine nanoemulsions significantly enhance drug permeation across biological membranes, leading to improved antidepressant effects in preclinical and clinical settings [32,33].

2. Aim

To Formulation and In-Vitro Evaluation of Polymers Based Fluvoxamine Nanoemulsions

3. Objectives:

• Development of Polymers Based Fluvoxamine Nanoemulsions

• In-vitro evaluation of developed Polymers Based Fluvoxamine Nanoemulsions.

4. MATERIALS AND METHODS

4.1. List of Chemicals and Equipment Used

Fluvoxamine was obtained from Hetero Healthcare Ltd. Changsari Guwahati as gift sample .Ethyl Cellulose was purchased from Loba Chemie Pvt. Ltd, Mumbai, and Maharashtra. Tween 80 and organic solvents such as Acetone and Ethanol were purchased from Himedia Laboratories Pvt. Ltd, Thane, and Maharashtra.

Table No 1: List of chemicals

Table No 2: List of Equipment

Beakers (100, 250, 500, 1000 ml), Volumetric flask (10, 100 ml), Measuring cylinder (10, 50 ml), Glass rod, Spatula, Conical flask (100, 500 ml), Funnel, Round bottom flask (500 ml), Pipette, Test tube etc.

4.2. Drug Profile

4.2.1. Drug Name: Fluvoxamine

Fig. 1: 2D Structure of Fluvoxamine

Fig. 2: 3D Structure of Fluvoxamine

IUPAC Name:

2-[(E)-[5-methoxy-1-[4-(trifluoromethyl) phenyl] pentylidene] amino] oxyethanamine

Molecular weight: 318.33 g/mol

Molecular formula: C₁₅H₂₁F₃N₂O₂

BCS Class: It falls under Class I of the Bio pharmaceutics Classification System (BCS).

Melting Range: 120-140.5 °C

Solubility: 5.4 mg/mL in water.

Storage: Room temperature (20°Cto25°C).

Fig. 3: 2D Structure of Ethyl Cellulose

4.2.2. Ethyl Cellulose

IUPAC Name: Cellulose ethyl ether

Molecular Formula: C₂₀H₃₈O₁₁

?Molecular Weight: Varies (polymeric compound)

Physical Description: White to light tan powder or granules

BCS: It falls under Class IV of the Biopharmaceutics Classification System (BCS).

Melting Range: Softens and decomposes starting around 135°C (275°F)

Solubility: Insoluble in water, soluble in various organic solvents

Storage: Store in a tightly closed container, in a cool, dry place, away from moisture and heat.

Fig. 4: 2D Structure of HPMC

4.2.3. Hydroxypropyl Methylcellulose (HPMC)

IUPAC Name: 2-Hydroxypropyl methyl ether of cellulose

Molecular Formula: C₅₆H₁₀₈O₃₀

Molecular Weight: Varies (typically 10,000 to 1,500,000 Daltons)

Physical Description: White to off-white, fibrous or granular powder

BCS: It falls under Class II of the Bio pharmaceutics Classification System (BCS).

Melting Range: Decomposes above 200°C (392°F)

Solubility: Soluble in cold water, forming a colloidal solution; insoluble in most organic solvents

Storage: Store in a tightly closed container, in a cool, dry place, away from moisture and heat.

4.3. Methodology

4.3.1. Physio-Chemical Properties

Physical and chemical properties of the Fluvoxamine were identified by using different methods, such as colour, nature, form, melting point, pH and odour of the selected drug and the details are recorded.

4.3.2. Solubility Studies

The solubility of the drug was carried out in various solvents. A small excess quantity (about 25mg) was taken and put into 10ml of each investigated solvent in a 50ml volumetric flask, and the volume made up to the mark. The solubility study was done at room temperature (25°C). The selected drug of a specific amount was added to each conical flask until undissolved particles were observed even after equilibrium for 6 hours with continuous shaking. The supernatant liquid was analyzed using a UV spectrophotometer for the drug dissolved until two successful readings of analysis were constant. The solubility study of Fluvoxamine in various solvents is recorded as well as documented [34].

4.3.3. Preparation of Standard Calibration Curve

The standard curve calibration method for a drug involves preparing a series of standard solutions with known concentrations. These solutions are analyzed using an appropriate analytical technique, such as UV spectrophotometry, to measure the corresponding signal response (e.g., absorbance or peak area). The drug was dissolved in a minimal amount of methanol and then diluted with phosphate buffer (pH 6.8) to obtain final concentrations of 5, 10, 15, 20, 25, and 30µg/mL Absorbance for each concentration was recorded spectrophotometrically at 261 nm. The obtained data points were plotted on a graph with concentration on the x-axis and signal response on the y-axis, followed by linear regression analysis to generate a calibration curve [35].

Fig. 5:  Stock Solution of API mixed with PBS Solution

4.3.4. Drug-Excipients Interactions by Fourier Transform Infra-Red (FT-IR) Spectroscopy

The compatibility between the drugs and excipients was compared using FT-IR spectra within a frequency range of 4000-400 cm-1.The position of the peak in the FT-IR spectra of pure Fluvoxamine was compared with the FT-IR spectra of Fluvoxamine with excipients. It was observed that there was no disappearance or shift in the band position of functional groups in the spectrum of Fluvoxamine alone as well as with the excipients. This compatibility study proved that the physical state of Fluvoxamine and Fluvoxamine with excipients were compatible. Hence, it can be concluded that the drug can be used with the selected polymers without causing instability in the formulation [36].

Fig. 6: Image of FT-IR Instrument

4.5. Preparation of Phosphate Buffer

To prepare a phosphate buffer with a pH of 6.8, begin by dissolving approximately 27.21 grams of Potassium Dehydrogenate Orthophosphate (KH?PO?) in 1000 milliliters of distilled water. Next, add around 8 grams of Sodium Hydroxide (NaOH). Take 250 milliliters of this solution and pour it into a beaker. Separately, dissolve about 112 milliliters of NaOH in distilled water and add this to the beaker, followed by 250 milliliters of the potassium dehydrogenate orthophosphate solution. Add distilled water to bring the total volume to 1000 milliliters. Mix thoroughly. Check the pH of the mixture: if the pH is below 6.8, add more NaOH; if the pH is above 6.8, add HCl until the pH reaches 6.8. The phosphate buffer is ready once the pH is stabilized at 6.8 [37].

4.6. Formulation design

Different batches of Fluvoxamine loaded polymeric nanoemulsions were prepared based on the 22Box-Behnken factorial designs. The independent variables were Polymer conjugates concentration in terms of mg (X1) and HPH pressure in terms of bar (X2) with the drug concentration of 20 mg for all formulation batches.

Table No 3: List of Composition of Formulations

4.7. Preparation of Fluvoxamine Loaded Polymeric Based Nanoparticles

Fluvoxamine-loaded polymeric nanoemulsions were formulated using the solvent evaporation method, incorporating Hydroxypropyl methylcellulose (HPMC), ethyl cellulose (EC), and Tween 80, with ethanol as the organic solvent and distilled water as the aqueous phase. Ethyl cellulose was first dissolved in ethanol, followed by the addition of HPMC and fluvoxamine to form the organic phase, while Tween 80 was dissolved in distilled water to act as a stabilizer, enhancing nanoemulsion stability. The organic phase was then gradually introduced into the aqueous phase under continuous magnetic stirring for 3 hours, facilitating emulsification and ensuring the homogeneous distribution of EC and HPMC. The resulting emulsion was subjected to high-speed homogenization at 15,000 rpm for 1 hour, reducing droplet size and increasing uniformity. To further break down larger particles, the mixture underwent sonication for 30 minutes, ensuring fine dispersion of polymeric nanoparticles (PNPs). The nanoemulsion was then transferred to a rotary evaporator, where ethanol was gradually eliminated under controlled vacuum conditions, initially set at 250 mbar and gradually reduced to 140 mbar over 15 minutes, leading to nanoparticle sedimentation. Following this, the suspension was centrifuged at 15,000 rpm for 45 minutes, separating the nanoparticles, which were then filtered using microporous filter paper to remove any residual solvent and unencapsulated material. Finally, the filtrate was dried using a spray dryer for 20–30 minutes, yielding dry, stable, and well-dispersed fluvoxamine-loaded polymeric nanoemulsions suitable for pharmaceutical applications. This optimized formulation method enhances drug solubility, stability, and bioavailability, making it a promising approach for fluvoxamine delivery [38,39,40,41].

4.7.1. Preparation of Fluvoxamine loaded Polymeric Nanoemulsions (Steps)

4.8.1. Determination of Entrapment Efficiency (EE) of polymeric nanoemulsions

The PNP formulation was centrifuged at 5,000 rpm for 20 minutes. The supernatant solution underwent filtration and separation. After diluting 1 ml of the filtrate with water, the absorbance at 261 nm was measured using a UV spectrophotometer with Phosphate buffer (pH 6.8) as the blank. Then, the amount of free drug in the formulations was assessed to determine the entrapment efficiency. The entrapment efficiency of PNPs can be calculated using the given equation [42].

4.8.2. Determination of Percentage Yield of Nanoemulsions

Nanoemulsions smaller than 1 micron successfully pass through the filter paper, while larger aggregates consisting of drug, polymer, and stabilizer are effectively removed. The filtrate obtained from this process was further ultracentrifuged at 10,000 rpm for 20 minutes using a Remi RM-12C microcentrifuge for each batch to ensure the separation of nanoparticles from unencapsulated materials. Following centrifugation, the supernatant and pellet were separated using a Millipore filter with a pore size of 0.45 µm, ensuring efficient purification. The residue and pellet weight were determined by combining all collected residues and weighing them. The total weight of the nanoemulsions was then calculated by subtracting the residual weight from the initial weight of the solid materials introduced into the formulation process. The percentage yield of polymeric nanoparticles (PNPs), also known as nanoparticle recovery, was determined using the following equation: [43].

4.8.3. pH Measurement

The pH of fluvoxamine-loaded polymeric nanoemulsions was measured using a calibrated digital pH meter to ensure formulation stability and compatibility. A 10 mL sample of the nanoemulsion was taken and allowed to equilibrate at room temperature (25 ± 2°C). The pH meter was calibrated with standard buffer solutions (pH 4.0, 7.0, and 9.2) before measurement. The electrode was rinsed with distilled water, wiped dry, and then immersed in the nanoemulsion sample, ensuring complete contact. The pH value was recorded once a stable reading was displayed, and measurements were performed in triplicate for accuracy [44].

4.8.4.Viscosity & Rheology

The viscosity and rheology of fluvoxamine-loaded polymeric nanoemulsions were measured using a Brookfield viscometer to assess their flow behavior and stability. A 10 mL sample was equilibrated at 25 ± 2°C before measurement. The viscometer was calibrated, and an appropriate spindle (No. 61 or 62) was selected based on the expected viscosity range. Viscosity readings were recorded at different shear rates (10, 20, and 50 rpm) to analyze flow behavior. The results were used to determine whether the nanoemulsion exhibited Newtonian or non-Newtonian flow properties, ensuring formulation stability and pharmaceutical suitability [45].

4.8.5. Density Measurement

The density of fluvoxamine-loaded polymeric nanoemulsions was measured using a pycnometer to ensure consistency and formulation stability. A clean, dry pycnometer was weighed (W1), then filled with the nanoemulsion and reweighed (W2) after ensuring no air bubbles were present. The filled pycnometer was maintained at 25 ± 2°C to ensure accuracy.[46] The density was calculated using the formula:                   

4.8.4. In-vitro Drug Release Studies

The in-vitro drug release of Fluvoxamine -loaded nanoemulsions was investigated in Phosphate Buffer Saline (PBS) pH 6.8 solutions utilizing a dissolution test apparatus. 50 mg of nanoemulsions were weighed and placed into the capsules. The water level for the environment is maintained, and the temperature is set at 37?C and 80 rpm. The jar was filled with 900ml of PBS, and the sample was removed at regular intervals (5, 10, 15,20,25,30 minutes) and replenished with 5 ml of PBS. The extracted samples were stored in test tubes. After 30 minutes, the samples were checked using UV spectroscopy for the absorbance at 292 nm [47].

4.8.8. Kinetic modelling of drug release

The release data were fitted into different mathematical models (Zero-order, First-order, Higuchi, and Korsmeyer-Peppas models) using DD Solver (Excel Add-in) to determine the mechanism of drug release (8,9). The correlation coefficient (R²) values were used to identify the best-fit kinetic model, while the release exponent (n) from the Korsmeyer-Peppas equation was analyzed to determine whether the release followed Fickian or non-Fickian diffusion [48].

4.8.5. Particles Size Analysis:

The Zetasizer (HSA3000) was used to conduct particle size analysis research on the nanoparticulate formulation. The freeze-dried NPs were reconstituted using double-distilled demineralised water. Samples were placed in square glass curettes, and particle size distribution was measured for optimal formulations. The improved formulation was diluted with excess water, and particle size was calculated [49].

4.8.6. Morphology Analysis Scanning Electron Microscope (SEM)

SEM (ESEM EDAX XL-30) was used to characterize polymeric nanoemulsions. Freeze-dried samples were stored in a gold-coated graphite sample container. The chamber was maintained at 0.6 mm Hg pressure and 20 kV voltages. Scanning electron photomicrographs were taken at various magnifications [50].

4.8.7. Optimization Strategies Data Analysis and Model Validation

ANOVA was used to establish the statistical validation of the polynomial equations generated by Design Expert® Software. Fitting a multiple linear regression model to a 3 2 Factorial design give a predictor equation incorporating interactive and polynomial term to evaluate the responses: ???? = ????0+ ????1????1 +????2????2 + ????12????1????2+ ????11????12 +????22????22 ----Where Y is the measured response associated with each factor level combination; b0 is an intercept representing the arithmetic average of all quantitative outcomes of nine runs; bi (b1, b2, b11, b12 and b22) are regression coefficients computed from the observed experimental values of Y and X1 and X2 are the coded levels of independent variables. The terms X1 X2 represent the interaction terms. Three-dimensional response surface plots resulting from equations were obtained by the Design Expert® software [51].

5. RESULT AND DISCUSSION:

5.1. Physical and Chemical Properties of Drug: Fluvoxamine

5.4. Standard Curve of Drug:

Fig.8: Graph Plot of Standard Curve of Drug

Table No: 7. Statistical Parameters

5.5.1. FT-IR of Drug:

Fig.9: FT-IR of Drug:

5.5.2. FT-IR of Drug-Excipients Interaction:

Fig.10: FT-IR of Drug with Excipients

Table No.8:  FT-IR Peak of Functional Groups of drug and excipients:

FTIR analysis was performed to assess the compatibility of the drug with excipients in the Pharmaceutical formulation. The characteristic peaks of the pure drug were compared with those of the drug-excipients mixture to detect any significant shifts. The results confirm that no major chemical interactions or degradation occurred, demonstrating the suitability of the excipients for nanoparticle formulation.

5.8. Percentage Entrapment Efficiency of Prepared Polymeric nanoemulsions:

Table No.9: List of Percentage Entrapment efficiency of Prepared Polymeric nanoemulsions:

All the batches showed entrapment efficiency in between 66-90%. The resultant entrapment efficiency is an indication that the method can be appropriate for technology transfer that is production on large scale. The optimized batch showed entrapment efficiency was found to be 89.23%.

5.6. Percentage Yield of the Prepared Polymeric nanoemulsions:

Table No.10: List of Percentage Yield of the Prepared Polymeric nanoemulsions:

All the batches showed production yield in between 66 to 85%. The resultant yield is an indication that the method can be appropriate for technology transfer that is production on large scale. The F7 batch showed production yield was found to be 84.33%.

5.7. pH value of formulated nanoemulsion:

Table No. 11: List of pH value:

All the batches showed pH value in between 3.9 to 8.9 .The resultant pH value is an indication that the method can be appropriate for technology transfer that is production on large scale. The Formulation F7 batch showed pH value was found to be 6.1 which is best for oral administration 

5.8. Viscosity of the Prepared Polymeric nanoemulsions:

Table No.12: List of Viscosity of the Prepared Polymeric nanoemulsions:

All the batches showed viscosity in between 107.79 to 96.88cP The resultant yield is an indication that the method can be appropriate for technology transfer that is production on large scale. The F7 batch showed production yield was found to be 145.7 cP.

5.9. In-vitro Drug Released Studies of Prepared PNPs

Table No.13: List for % Drug Release of F1, F2, F3, F4, F5 and F6 Formulation:

Fig.11: Graph Plot of In-vitro Drug Release of Formulation F1, F2 and F3

Fig.12: Graph Plot of In-vitro Drug Release of Formulation F4, F5 and F6

Table No. 14: List of In-vitro % Drug Release of F7, F8, F9, F10, F11 and F12 Formulation:

Fig.13: Graph Plot of In-vitro Drug Release of F7, F8and F9 Formulation

Fig.14: Graph Plot of In-vitro Drug Release of F10, F11 and F12 Formulation

In-vitro drug release profiles of Fluvoxamine loaded NPs were obtained by dissolution test apparatus Electro lab in phosphate buffer solution (pH 6.8). NPs filled capsules were placed in a capsule shaker and sealed. This is immersed into 900ml phosphate buffer solutions and the system was maintained 37°C under agitation of 85rpm/min. Aliquots were collected for every 30 mints up to 210 mints and the same was replaced with fresh buffer. The samples were further analyzed using UV-Spectrophotometer and absorbance was measured 292nm. Fluvoxamine loaded NPs batch F7 showed 91.53% The expected characteristics of NPs of sustained released was verified.

5.10. In-vitro Drug Release Kinetics:

The in-vitro drug release kinetics of the optimized formulations was analyzed using the software named “DD Solver”. The results were highlighted in bold values and indicate that the Korsmeyer-Peppas model provided the best fit for the formulations F7. This suggests that the release mechanism follows a combination of diffusion and erosion processes, consistent with the characteristics of the Korsmeyer-Peppas model.

Table No.15:List of the in-vitro Kinetics Models:

Figure 22. Graph Plot of In-vitro Kinetics Model using DD Solver for F7

Fig.15: Graph Plot of In-vitro Kinetics Model using DD Solver for F7:

Fig.16: Graph Plot of In-vitro Kinetics Model using DD Solver for F7:

Fig.17: Graph Plot of In-vitro Kinetics Model using DD Solver for F7:

Fig.18: Graph Plot of In-vitro Kinetics Model using DD Solver for F7:

 5.11. Particles Size Analysis:

Fig.19: Images of Particles Size of F7:

In addition to its ability to control the pace and degree of drug release and absorption, the particle size of the nanoemulsion is a crucial issue. The bioavailability of the medicine is enhanced by the smaller particle size, which provides a greater interfacial surface area. Among all the twelve prepared formulation, the particles size (PS), poly dispensability Index (PDI) of the F7 was considered as the best formulation with particles size to be found 77.90nm and PDI values was found to be 0.585. The average particles size of the formulation F7 was considered to be 383.33, as considered the best formulation.

5.12. Scanning electron microscope (SEM):

Based on formulation optimization, including % yield, drug loading, entrapment efficiency, and in-vitro drug release experiments, the F7 formulation was shown to be consistent and optimal. As a result, the F7 formulation was studied using Scanning Electron Microscopy (SEM) to determine the surface shape of the polymeric nanoemulsions. The SEM image indicated that the nanoemulsions of the Formulation F7 were smooth and spherical in structure. This nanoscale size confirms the successful formation of polymeric nanoemulsions, which is a critical prerequisite of achieving enhanced drug bioavailability and therapeutic efficacy.

Fig.20:  Images of SEM of Polymeric nanoemulsions of F7

Independent variables demonstrate that the model was significant for all the response variables. It was observed that independent variables X1 (polymer concentration) and X2 (HPH pressure) had a positive effect on the entrapment efficiency and drug loading of the nano-formulation that was nanoemulsions was achieved. The statistical evaluation was performed by using ANOVA. The results were showed in below table. The coefficients in the regression equation that contain more than one factor term are called interaction terms. This demonstrates that the link between variables and responses is not necessarily linear. When more than one element is changed at the same time and at various levels in a formulation, the reactions might vary.

5.13.1. Fit Summary

Response 2: Entrapment Efficiency

Table No. 17: Model Comparison for Drug Loading Prediction

Table No.18:Sequential Model Sum of Squares [Type I]

Table No.19:Model Summary Statistics

Table No.20:ANOVA for Linear model

Table No.21:Fit Statistics

The Predicted R² of 0.5830 is in reasonable agreement with the Adjusted R² of 0.7451; i.e. the difference is less than 0.2.

Adeq Precision measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 8.807 indicates an adequate signal. This model can be used to navigate the design space.

The coefficient estimate represents the expected change in response per unit change in factor value when all remaining factors are held constant. The intercept in an orthogonal design is the overall average response of all the runs. The coefficients are adjustments around that average based on the factor settings. When the factors are orthogonal the VIFs are 1; VIFs greater than 1 indicate multi-colinearity, the higher the VIF the more severe the correlation of factors. As a rough rule, VIFs less than 10 are tolerable.

ANOVA for Linear model Diagnostic

Three dimensional surface plots were generated by the Design Expert® software are presented in (Figure no. Fig.32 and Figure no. Fig.33 for the studied responses, i.e. Mean Drug loading (Y1) and Entrapment Efficiency (Y2). Fig.32 depicts response surface plot of Polymer Conjugate Concentration (X1) and HPH Pressure (X2) on Mean of Drug loading. nanoemulsions being nanoparticulate structures, formulation batch amongst all the design batches giving least particle size will be preferred more and selected as an optimized batch. Where F7 Design Batch, with a polymer concentration of about 150mg and 200mg, show the least particles size i.e. 383.33nm. Fig.33 depicts response surface plot of Polymer Conjugate Concentration (X1) and HPH Pressure (X2) on entrapment efficiency. The 3-D surface image shows a linear response, which indicates with the increase in the polymer concentration the entrapment efficiency increases, as more the polymer available more will be the entrapment efficiency. Here, two design batches i.e. F2 and F7 showed maximum entrapment efficiency i.e. 81.73% and 89.23% respectively. But as seen in first response Surface graph being a nanoparticle formulation considering the least particle size is also a crucial factor. So, the Design Batch with least particle size and maximum entrapment efficiency is selected. Therefore, F7 is considered as an optimized Batch.

Fig. 22: 3D surface Plot for X1 and X2 on drug Entrapment Efficiency, where X1= Polymer conjugates concentration and X2 = HPH pressure