Formulation and Evaluation of Solid Lipid Nanoparticle of Valsartan Incorporated into Self Emulsifying Drug Delivery System

Hypertension, commonly known as high blood pressure, is a significant global health problem affecting over 20% of the adult population worldwide. It is especially concerning in developing countries, where rapid lifestyle changes have led to an increasing prevalence of chronic diseases, including hypertension. The World Health Organization (WHO) and the International Society of Hypertension (ISH), along with the 7th Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure (JNC7), define hypertension in adults (aged 18 years or older) as systolic blood pressure of 140 mmHg or higher and diastolic blood pressure of 90 mmHg or higher. Hypertension is a major risk factor for several life-threatening conditions, including coronary heart disease, stroke, congestive heart failure, end-stage renal disease, peripheral vascular diseases. It is associated with approximately 1.6 million deaths annually due to ischemic heart disease and stroke, out of a total of about 10 million deaths in the country^{1 2 3.4,5,6}.

Valsartan

Valsartan is an angiotensin II receptor blocker (ARB) used to treat hypertension, heart failure, and post-myocardial infarction. It works by selectively blocking the angiotensin II type 1 receptor (AT1), preventing vasoconstriction, aldosterone secretion and cardiac or vascular remodeling. These actions help reduce blood pressure, decrease fluid retention and protect the heart and kidneys from damage. Unlike ACE inhibitors, valsartan does not  interfere with bradykinin breakdown, which lowers the risk of side effects like cough and  angioedema. In hypertension, valsartan effectively reduces both systolic and diastolic pressures, especially at higher doses (160 mg and 320 mg). It is often combined with other antihypertensives such as hydrochlorothiazide (HCTZ) or amlodipine to enhance efficacy and minimize side effects. The combination with diuretics helps manage fluid overload, while its use with calcium channel blockers can reduce peripheral edema. Valsartan is a BCS Class II drug with low solubility and high permeability. It undergoes minimal liver metabolism and is mainly excreted unchanged in feaces and urine ^{7 8 9}.

Self-Emulsifying Drug Delivery System (SEDDS) as a Drug Delivery System

A significant number of pharmaceutical compounds, particularly Biopharmaceutics Classification System (BCS) Class II drugs, exhibit poor aqueous solubility and high hydrophobicity, resulting in low and variable oral bioavailability. To address these challenges, lipid-based drug delivery systems such as self- emulsifying drug delivery systems (SEDDS), self-micro emulsifying (SMEDDS) and self-nanoemulsifying systems (SNEDDS) have been developed. SEDDS are isotropic mixtures of oils, surfactants, cosurfactants and hydrophilic solvents that form fine oil-in-water (O/W) emulsions upon mild agitation and dilution in gastrointestinal (GI) fluids. This process enhances solubilization and absorption of lipophilic drugs by increasing surface area through small droplet formation (100–300nm). SEDDS are especially useful for poorly water-soluble drugs, improving both the rate and extent of drug absorption and ensuring more predictable plasma concentrations. These systems are also physically stable and easy to manufacture, making them an effective strategy for enhancing the bioavailability of lipophilic drugs ^{10, 11,12}.

Selection Of Drugs For SMEDDS

Selecting drugs for Self-Emulsifying Drug Delivery Systems (SEDDS) requires considering factors like lipophilicity, melting point, dose and Log P value. Drugs must dissolve well in lipid excipients to be incorporated effectively. Low-dose drugs with low melting points are ideal, while high-dose drugs need excellent solubility in the oil phase. A Log P value above 2, preferably exceeding 5, indicates strong suitability for lipid-based systems; Additionally, food effects enhance absorption of lipophilic drugs, especially when taken with high-fat meals, improving solubilization and bioavailability compared to fasting, where dissolution may be limited ¹³.

Selection Of Excipients for Lipid-Based Formulations

Oils

The oil phase is a crucial excipient in Self-Emulsifying Drug Delivery Systems (SEDDS) as it enhances the solubility of lipophilic drugs, supports self-emulsification and improves lymphatic transport, thereby increasing gastrointestinal absorption. Suitable oils include medium and long-chain triglycerides (MCTs and LCTs), fatty acids, fatty alcohols and mono/di-/triglycerides. Although unmodified edible oils are physiologically safe, their poor drug solubilization and weak emulsification limit their use. In contrast, modified or hydrolyzed vegetable oils offer better emulsification and solubility, closely mimicking natural digestive processes. Novel semi-synthetic medium-chain derivatives with surfactant properties are increasingly used for their superior emulsification capabilities. Vegetable oils such as soybean, sesame and peanut oils are common but require higher energy input and may form less stable emulsions. Other options include light liquid paraffin, squalene, ethyl oleate and isopropyl myristate. The choice of oil significantly influences formulation attributes like drug release, emulsion stability, particle size and encapsulation efficiency ^{14, 15}.

Surfactants

Surfactants are essential in SMEDDS for emulsification and drug solubilization. Non-ionic surfactants with high HLB values, like Tween 80 and ethoxylated glycerides are commonly used due to their safety and oral acceptability. They enhance drug absorption by improving dissolution, permeability and inhibiting P-glycoprotein efflux. Typically, 30– 60% w/w surfactant is used, but excess may cause GI irritation or alter intestinal permeability. Surfactant concentration also influences droplet size higher levels may reduce or increase it depending on interfacial stability. Natural surfactants are safer but less effective. Each formulation should be evaluated for surfactant-related mucosal effects. 

Cosurfactants

Self-Emulsifying Drug Delivery Systems (SEDDS), high surfactant concentrations are typically required, but incorporating co-surfactants can lower this need by reducing interfacial tension, promoting spontaneous emulsification into fine droplets. Cosurfactants such as ethanol, propylene glycol (PG) and polyethylene glycol (PEG) help dissolve both the drug and surfactant in the lipid base. Though some non-ionic surfactants work without co-surfactants, proper selection is critical for effective drug solubilization and microemulsion formation. Alcohol-free formulations may enhance capsule compatibility by preventing drug precipitation, though they may limit lipophilic drug dissolution. Therefore, selecting the right combination of components is key to SEDDS performance ¹⁶.

Mechanism Of Self-Emulsification¹⁷

Self-emulsification occurs when the entropy change driving the dispersion process is greater than the energy required to expand the surface area of the dispersed phase. In conventional emulsions, the free energy (ΔG) is directly proportional to the energy needed to create a new interface between the oil and water phases. Over time, phase separation occurs as the system seeks to minimize its interfacial energy by reducing the total interfacial area.  To stabilize an emulsion formed upon aqueous dilution, emulsifying agents create a protective monolayer around the droplets, reducing interfacial energy and preventing coalescence. The thermodynamic relationship governing free energy change in emulsification can be expressed as: 

???????? = ∑????????4????????????2????

Where: 

ΔG = Free energy associated with the process

r_i = Radius of the droplets 

N_i = Number of droplets 

???? = Interfacial energy 

The efficiency of self-emulsification depends on how easily water penetrates the lipophilic components or gel phases that form at the droplet surface. Upon mixing a binary system of oil and surfactant with water, an interface forms between the oil and the aqueous phase. Water then begins to solubilize within the oil phase, progressively penetrating the interface until it reaches the solubilization limit. Beyond this point, further water entry causes the formation of dispersed  lipid phases. Eventually, as gentle agitation is applied, water penetrates the inner aqueous regions, disrupting the interface and promoting the formation of fine droplets. The extent to which the lipophilic phase interacts at the interface depends on the concentration and type of surfactants used in the system. Studies using particle size analysis and low-frequency dielectric spectroscopy (LFDS) have been conducted to assess self-emulsification properties in lipid-based formulations ¹⁷.^18,19,20.

Advantages of SMEDDS ^{21, 22}:

• Enhanced Oral Bioavailability: Increases absorption of poorly water-soluble drugs by forming microemulsions with a large surface area. 
• Simplified Manufacturing: Requires minimal equipment, making scale-up cost effective and industry-friendly. 
• Reduced Variability: Ensures consistent absorption and plasma drug levels, regardless of food intake. 
• Protection of Sensitive Molecules: Shields peptides and macromolecules from enzymatic degradation in the GI tract. 
• Bypasses Lipid Digestion: Enables direct absorption without the need for bile salts or enzymes. 
• Higher Drug Loading: Accommodates more drug using surfactants, co-surfactants and cosolvents that improve solubility.

Solid Lipid Nanoparticle:   

Recent advances in nanoscale drug delivery systems have significantly improved the development of lipid-based nanocarriers. Among these lipid excipients and biocompatible polymers have gained attention due to their effectiveness in enhancing drug incorporation and delivery. Lipid excipients derived from physiological lipids are particularly valued for their low toxicity and high biocompatibility. Solid Lipid Nanoparticles (SLNs) have emerged as a promising delivery platform by combining the advantages of liposomes, emulsions and polymeric nanoparticles. These systems are made using safe, physiologically accepted excipients. The solid lipid matrix serves to protect the encapsulated drug from both chemical degradation and enzymatic breakdown. SLNs also allow for the customization of drug release profiles, offering flexibility for different therapeutic needs. SLNs are generally spherical in shape, with sizes ranging from 50 to 1,000 nm. Their composition typically includes solid lipids (solid at room and body temperature), stabilizing agents (emulsifiers) and suitable solvents.  Commonly used GRAS (Generally Recognized as Safe) lipids include triglycerides, cholesterol, fatty acids, esters and natural waxes like beeswax and carnauba wax. Emulsifiers such as Poloxamers, Pluronic F68/F127 and sodium cholate stabilize the formulation, while additional agents may be used to enhance targeting or surface properties ^23,24,25.

SLN Formulation ^{26, 27}:  

1. Lipid Components

Lipids serve as the core matrix material in SLN, playing a crucial role in defining their structural and functional properties. Approximately 70% of the lipids used in SLN formulations belong to the categories of free fatty acids, fatty alcohols and glycerol esters. Some commonly employed lipids include: Stearic acid, glyceryl behenate, tripalmitin, cetyl palmitate, tristearin, glyceryl monostearate. These lipids contribute to the stability and controlled drug release properties of SLN.  

2. Surfactants

Surfactants play a crucial role in stabilizing Solid Lipid Nanoparticles (SLNs) by reducing interfacial tension between lipid and aqueous phases, thereby preventing aggregation and enhancing dispersion stability. They form a stabilizing layer around nanoparticles, improving uniformity and shelf-life. Nonionic surfactants like Poloxamer 188 and lecithin are commonly used in oral, ocular and parenteral formulations due to their low toxicity and steric stabilization. Ionic surfactants such as sodium deoxycholate offer electrostatic stabilization, while cationic surfactants like quaternary ammonium compounds are used selectively. Additionally, stabilizers like soy lecithin, egg lecithin and polyvinyl alcohol (PVA) are frequently incorporated to improve formulation performance. 

3. Cryoprotectants

(e.g., glucose, fructose, sorbitol, mannitol) are used in lyophilized SLN to maintain stability during freeze-drying.

4. Coating agents

like chitosan can modify the surface properties of SLN for targeted delivery.

5. Preservatives

such as parabens help prevent microbial contamination.

6. Active Pharmaceutical Ingredients in SLN

Solid Lipid Nanoparticles (SLNs) can encapsulate a wide variety of active pharmaceutical ingredients (APIs), both individually and for co-delivery. SLNs are particularly effective for hydrophobic drugs with poor water solubility. They have been successfully used to deliver anesthetics, antipyretics, antibiotics, analgesics, antiretrovirals, anticancer agents and antihypertensive drugs. Beyond small molecules, SLNs have also shown promise in the delivery of nucleic acids and peptides, highlighting their versatility in advanced drug delivery systems. This capability makes SLNs a powerful tool in enhancing drug solubility, stability and controlled release for various therapeutic applications.

Principles Of Drug Release from Solid Lipid Nanoparticles (SLN): 

The drug release behavior of Solid Lipid Nanoparticles (SLNs) is largely influenced by particle size. Smaller nanoparticles with larger surface areas enable faster drug release due to a significant portion of the drug being near the surface. Larger particles, on the other hand, allow for greater drug entrapment in the core, resulting in a slower, more sustained release profile. Key factors in drug release include an inverse relationship between the drug's partition coefficient and its release rate and the high surface area of nanoparticles facilitating rapid release. A homogeneous drug dispersion within the lipid matrix leads to slower, controlled release. The SLN drug entrapment model, determined by formulation composition and production techniques, affects release rates, which can span over weeks. In the drug-enriched shell model, initial burst release occurs, but increasing particle size minimizes this, allowing prolonged release. Surfactant type and concentration, along with lipid content, significantly impact release profiles and drug solubility ²⁸.

Methods of Preparation of Solid Lipid Nanoparticles (SLN) ²⁹:

1. High Pressure Homogenization (HPH) is a powerful and widely used method for preparing solid lipid nanoparticles (SLNs). This technique involves forcing a liquid through a     narrow gap at high pressures (100–2000 bar), generating high velocities (over 1000 km/h) in a short distance. The intense shear stress and cavitation forces produced during this process break down particles to the submicron size range. Initially, HPH was used for formulating solid lipid nano-dispersions, but particle size limitations sometimes compromised the quality of the dispersion. HPH can be performed using two main approaches: hot high-pressure homogenization and cold high-pressure homogenization. Both approaches involve mixing the drug with melted solid lipid to achieve the desired formulation ³⁰.

A) Hot High-Pressure Homogenization (HPH) involves melting the lipid and dissolving the drug in the molten lipid. A preheated aqueous medium is then added and the mixture is stirred to form a rough pre-emulsion. The process is carried out at temperatures 5–10°C above the lipid’s melting point. The pre-emulsion is processed using high-pressure  homogenization typically at 500–800 bar for 2–3 cycles. After homogenization, the nanoemulsion solidifies upon cooling. This method is ideal for lipophilic, poorly soluble drugs as well as heat-sensitive drugs due to short heat exposure. However, it’s less effective for hydrophilic drugs, as they have lower entrapment efficiency. 

B) Cold High-Pressure Homogenization (HPH) follows a similar initial process to hot HPH, where the drug is dispersed in a mixture of solid lipid or liquid lipid and melted solid lipid. The mixture is cooled using dry ice or liquid nitrogen, then milled to 50–100 microns. The milled microparticles are added to a cool emulsifier solution to form a presuspension, which is then processed under high pressure at or below room temperature. This technique minimizes drug loss, particularly for hydrophilic drugs, by reducing lipid melting. Cold HPH requires 3–5 cycles at 500–1500 bar, with potential particle size increase if pressure or cycle number is excessive.

2. Microemulsion-based Solid Lipid Nanoparticles (SLNs) are prepared by diluting microemulsions. The process starts with creating an optically transparent mixture of a low melting-point fatty acid (e.g., stearic acid), emulsifier (e.g., polysorbate 20), co-emulsifier (e.g., butanol), and water. The mixture is stirred at 65–70°C, then rapidly mixed into cold water (2– 3°C) to induce lipid crystallization. This temperature shift helps prevent aggregation. Compared to High-Pressure Homogenization (HPH), microemulsions contain less lipid. After solid lipid is melted and mixed with the drug and surfactants at higher temperatures, a clear w/o microemulsion is formed, which is then added to a surfactant-cosurfactant solution to form lipid particle suspension. The particles are washed via ultrafiltration to form solid lipid nanoparticles ³¹.

3. Ultrasonication involves melting the lipid and dissolving the drug in it. A hot aqueous surfactant solution is then added to this mixture. The mixture is subjected to high shear mixing at 15,000 RPM, followed by ultrasonication using a probe sonicator until the desired particle size is achieved. Afterward, the mixture is cooled to room temperature, resulting in the formation of solid lipid nanoparticles ³².

4. Solvent evaporation, a hydrophobic drug and lipophilic material are dissolved in a nonpolar solvent (e.g., cyclohexane, chloroform). This solution is emulsified in an aqueous phase using high-speed homogenization. For better emulsification, the coarse emulsion is passed through a microfluidizer. The organic solvent is evaporated by stirring under reduced pressure at room temperature, leaving behind solid lipid nanoparticles (SLNs) ³³.

MATERIALS:

Valsartan (Dhamtec pharma), caproyl 90, labrasol, transcutanol (were gift sample from gatte fosse) glyceryl monostearate (Loba Chemic pvt ltd), Tween 80 (Himedia), polaxamer 188, Mannitol, Isopropyl myristate (thomas baker), Aerosil 200 (Srichem laboratories).

METHOD OF PREPARATION:   

This involves 2 steps, 

1. Formulation and evaluation of solid lipid nanoparticle by solvent evaporation method.
2. Formulation and evaluation of self-emulsifying drug delivery system by using pseudo ternary phase diagram.

STEP I- Formulation and Evaluation of Solid Lipid Nanoparticle

Drug–Excipient Compatibility Studies ⁶⁹:

Compatibility between the drug and excipients is essential to ensure stability, safety and therapeutic efficacy of the dosage form. Any potential interaction can lead to degradation,  altered drug release or reduced effectiveness, making pre-formulation studies a critical step  in dosage form design. Fourier Transform Infrared Spectroscopy (FT-IR) is widely used for this purpose, as it enables detection of functional group interactions through characteristic absorption bands. In this study, compatibility was evaluated using a Shimadzu FT-IR 8400 spectrophotometer. Samples of the pure drug and drug–excipient mixtures were prepared by the potassium bromide (KBr) pellet method. The blends were compressed into transparent discs and spectra were recorded in the 4000 cm?¹ –750 cm?¹ range.

Selection of formulation variables:  

Selection of Lipid⁷⁰: The lipid used in the formulation was chosen based on the results of solubility studies. To evaluate drug solubility, 100 mg of each lipid was accurately weighed into separate culture tubes and placed in a water bath shaker at 80 °C until complete melting. An excess amount of drug was then incorporated, and the mixtures were continuously shaken for 2 hours at 75 °C to facilitate solubilization. After cooling and standing for 1 hour, the solidified mass was carefully separated, and the drug-enriched portion of the lipid was remelted. Subsequently, 5 ml of methanol was added, and the solubilized drug was quantified using a UV–Visible spectrophotometer at λmax 248.2 nm. 

Selection of Surfactant^71,72: The choice of surfactant was guided by a literature with emphasis on the Hydrophilic– Lipophilic Balance (HLB) value, physiochemical nature functionality with an appropriate HLB range is known to promote stable SLN formulations. The laboratory experiments were conducted by formulating solid lipid nanoparticle using tween 80 and span 60 entrapment efficiency and particle size were estimated. 

Selection of Stabilizer⁷³: According to a reference the use of a stabilizer in combination with a surfactant further enhance stability of Solid lipid nanoparticle. A secondary surfactant acting as a stabilizer can lower surface and interfacial tension, reduce particle size and polydispersity index (PDI) which may improve entrapment efficiency. SLNs were prepared both with and without the addition of a stabilizer polaxamer 188 to evaluate its impact. 

Method of preparation of solid lipid nanoparticles:  

Solvent evaporation method ⁷⁴: Span 80 and polaxamer 188 is dissolved in distilled water and stirred at 69ºC using magnetic stirrer to form aqueous solution. Glyceryl monostearate and valsartan are melted at 65-70ºC. Ethanol is added to it and stirred continuously to form organic phase. The organic phase is slowly injected into aqueous phase with continuous stirring. The emulsion formed is further stabilized by continuing the stirring with homogenizer. The obtained nanoparticle is further centrifuged by Remi centrifuge at 10,000 RPM for 30 mins at 4? in refrigerator.

Percent drug entrapment study ⁷⁵: Supernatant containing unentrapped valsartan of 1 ml was withdrawn and was diluted with distilled water upto mark in 25ml(SS-I) from that 1 ml was pipetted and diluted in 10 ml with distilled water(SS-II). UV spectrophotometer studies at ?max 248.2nm the amount of valsartan entrapped in SLNs was determined using formula,

????????% = ???????????????????? ???????????????? ???????????????????????????? − ???????????????? ???????????????????????????? ???????? ???????????????????????????????????????????? X 100

???????????????????? ???????????????? ????????????????????????????

Particle size analysis:  The particle size and size distribution of VSLNs is characterized by laser light scattering using a particle size analyzer (Malvern Mastersizer Hydro-2000 SM UK), Each sample was suitably diluted with filtered distilled water (10 times) to minimize risk of multiple scattering. The diluted sample was transferred into zeta cell and particle size was recorded. 

Factorial design : Factorial design approach was applied to study the interaction effects. Three independent variables were selected and evaluated at two levels each. These variables were drug-tolipid ratio (D:L), surfactant concentration and homogenization speed. The responses are particle size (Y1) and percentage encapsulation efficiency (%EE, Y2) given in table 01. Analysis was performed using Design Expert software version 13 used to generate data of experiment.  The experiment was performed. A 2³ factorial design was employed to systematically study the effect of three independent formulation variables at different levels on the 1selected responses. For statistical testing, a significance level of p < 0.05 was considered. Data were expressed as mean ± standard deviation.

Table 01:Formulation trial run suggested by Design Expert software

Lyophilization⁷⁶,⁷⁷: Lyophilization was employed to enhance the stability of SLNs and prevent hydrolysis and Ostwald ripening. Mannitol was used as cryoprotectant was mixed with SLN suspension prior to freeze-drying in a pilot freeze dryer (EPSILON 2-6D, Martin Christ, Germany) under controlled conditions (freezing at –45 °C for 2 h, primary drying at – 30 °C for 12 h and secondary drying at 20 °C for 6 h). Mannitol at a 1:1 lipid ratio yielded free-flowing, stable SLN powder and was selected for the formulation.

Thermal Property Determination: The thermal behavior of the lyophilized solid lipid nanoparticle formulation was assessed to confirm drug incorporation within the lipid matrix. Differential Scanning Calorimetry (DSC) was employed for this evaluation. Both pure valsartan and drug-loaded lyophilized SLNs were subjected to DSC analysis and the thermograms were compared to identify possible interactions and confirm entrapment. For analysis, the lyophilized solid lipid nanoparticle was sealed in an aluminum pan. The sealed pan was placed in the DSC instrument, which was equipped with a refrigerated cooling system and operated under a nitrogen purge at a flow rate of 50 mL/min. Calibration was performed using standard reference materials. An empty thermetically sealed aluminum pan was used as a reference. The sample was equilibrated at 25 °C for 10 minutes prior to heating and thermal transitions were recorded over the range of 25 °C to 200 °C at a heating rate of 5 °C/min. 

Morphological Evaluation: Morphological assessment of solid lipid nanoparticles (SLNs) was carried out to study particle shape and surface characteristics, which are critical parameters influencing stability and performance. Scanning Electron Microscopy (SEM) was employed for this purpose using a JEOL JSM-7600F instrument. The lyophilized sample was then mounted on the SEM sample holder and examined under the microscope to visualize the surface morphology and structural attributes of the nanoparticles. 

In-vitro drug release ^78,79,80: The in-vitro release behavior of SLN was investigated using a modified Franz diffusion cell. A cellophane membrane (molecular weight cut-off 12,000–14,000; pore size ~2.4 nm) was employed as the diffusion barrier. Prior to the experiment, the membrane was soaked in double distilled water for 12 hours to ensure proper hydration.  For the study, 50mg drug equivalent of SLN dispersion was carefully placed in the donor compartment, while the receptor compartment was filled with phosphate buffer (pH 7.4) containing 0.75% sodium lauryl sulfate (SLS). The receptor phase was maintained at 37 ± 0.5°C and stirred continuously at 800 RPM using a Teflon-coated magnetic stir bar. At predetermined intervals, 1 mL aliquots were withdrawn from the receptor compartment via the sampling port and immediately analyzed by UV–Visible spectrophotometry at λmax 248.2nm. The withdrawn volume was replaced with fresh buffer to maintain sink conditions. The cumulative release data were further subjected to mathematical modeling using zero order, first-order, Higuchi and Korsmeyer–Peppas equations to elucidate the release kinetics and mechanism of drug release from SLN.

STEP II- Formulation and Evaluation of Self Emulsifying Drug Delivery System

Screening of ingredients by determination of solubility of valsartan in different systems at 25? ^71,72:

Screening of oils: The solubility of valsartan was determined in various oils including castor oil, sunflower oil, isopropyl myristate, sesame oil and olive oil. Accurately weighed 50 mg of valsartan was added to 10 ml of each oil in separate beakers. The mixtures were subjected to continuous stirring on a magnetic stirrer at 25 ± 1°C for 36 hours to attain equilibrium. Following equilibration, the dispersions were centrifuged at 3000RPM for 15 minutes to  separate undissolved drug. The supernatant was carefully collected and filtered through a 0.45μm membrane filter. From the filtrate, 0.05ml was withdrawn and suitably diluted 1000 times with methanol. The absorbance of the resulting solution was measured using a UV– Visible spectrophotometer at 248.2nm. 

Screening of surfactants and cosurfactants: Valsartan (50 mg) was accurately weighed and dispersed in 10 ml of different surfactants and surfactants (Tween 80, Tween 20, Span 20, Span 80, caproyl 90, transcutanol, labrasol, Polyethylene glycol 400) each taken separately in individual beakers. The mixtures were subjected to continuous stirring on a magnetic stirrer at 25 ± 1°C for 36 hours to allow the system to attain equilibrium. After equilibration, the samples were centrifuged at 3000 RPM for 15 minutes and the supernatants were carefully collected. These were then filtered through a 0.45µm membrane filter to remove any undissolved drug. An aliquot of 0.05ml from the filtrate was withdrawn, diluted 1000-fold with methanol and analyzed spectrophotometrically at 248.2nm using a UV–Visible spectrophotometer. 

Evaluation of the emulsification potential of isopropyl myristate using tween 80 and caproyl 90 ⁸¹: The emulsification potential of isopropyl myristate (IPM) was evaluated using Tween 80 as surfactant and Capryol® 90 as co-surfactant at different surfactant : co-surfactant ratios (1:1, 2:1, 3:1, 4:1, 5:1, 5.5:1, 5.5:1.25, 5.5:1.5 and 5.5:2). Smix was prepared by mixing the required volumes of Tween 80 and Capryol® 90 until homogeneous, and 1 ml of each Smix was further mixed with 1 ml of IPM to obtain the oil–Smix concentrate. The emulsification study was performed by adding 1 ml of this mixture dropwise into 100 ml of distilled water maintained at 37 °C under continuous stirring at 100RPM and the resulting dispersion was observed for spontaneity of emulsification, clarity, appearance and phase separation at different time intervals. Optical clarity was quantified by measuring percentage transmittance (%T) at 638 nm using a UV spectrophotometer. The optimal Smix ratio was determined on the basis of rapid emulsification, high clarity (%T ≥ 90%) and absence of phase separation after storage. 

Evaluation of the emulsification potential by titration method ^82,83,84: To investigate the microemulsion region, various oil: Smix ratios were prepared  systematically. The oil phase and Smix (surfactant : co-surfactant mixture) were combined in the proportions of 9:1, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 For each system, the Smix ratio was maintained at 5:1 (surfactant : co-surfactant), and the accurately measured to 3ml of the selected oil and Smix were transferred into a clean beaker. The mixtures were subjected to gentle stirring to ensure complete miscibility and the formation of a uniform homogeneous phase. Following this, distilled water was added to each mixture in a controlled manner using a burette, dropwise, under continuous stirring. The titration was continued until a visible change in phase behavior was observed, such as turbidity or phase separation. The exact volume of water required to induce this phase transition was recorded carefully. This method was employed to confirm the microemulsion existence region and to construct the pseudo-ternary phase diagram, which is crucial for identifying the composition range suitable for stable microemulsion formulation.  

Construction of the pseudo ternary phase diagram ^82,83,84: Pseudo-ternary phase diagrams were developed to identify the microemulsion region. The selection of oil, surfactant and co-surfactant was based on their solubilizing capacity and the diagrams were constructed using the aqueous titration method. At the point of phase change, the proportion of oil, Smix and water was recorded and these values were used to plot the pseudo-ternary phase diagrams with the aid of Chemixschool® software. 

Mixture design ^85,86: Mixture design approach was applied to study the interaction effects. Three independent variables were selected and evaluated at two levels each. These variables were oil (%) Smix (%) and water (%). The responses is  percentage transmittance (%T, Y1) was give in table 02. Analysis was performed using Design Expert software version 13 used to generate data of experiment. The experiment was performed. For statistical testing, a significance level of p < 0.05 was considered.

Table 02: Formulation trial run suggested by Design Expert software

SLN valsartan loaded SEDDS ⁸⁷:

SLN Valsartan loaded self-emulsifying drug delivery systems (SEDDS) were prepared following pervious mixture design. A series of formulations containing different proportions of oil, surfactant and co-surfactant were developed, with a fixed SLN Valsartan lyophilized product of 59.20 mg ( drug equivalent to 20mg) in each system. For preparation, the oil, surfactant and co-surfactant were blended in a glass vial maintained at 35 °C using a magnetic stirrer. To this mixture, SLN valsartan was added under continuous stirring until a clear and uniform dispersion was obtained. The final formulations were stored at room temperature until further use. 

Evaluation of SEDDS: 

Percentage transmittance measurement ⁸⁸: Microemulsion formulation was diluted 50 times with distilled water. The percent transmittance of various formulations was measured at 650nm using UV-VIS spectrophotometer against distilled water as a blank. Results were taken in triplicate and the average was taken in to consideration. 

Self-emulsifying time ^82,89,90: The self-emulsifying properties of the developed SEDDS formulations were examined by visual observation in order to determine the time required for spontaneous formation of microemulsion. This evaluation was carried out by dropwise addition of the liquid concentrate into aqueous media and the emulsification process was monitored with respect to the rate of emulsification, the appearance of droplets, and the clarity of the final dispersion.  An accurately measured quantity of LSEDDS (100 µL) was introduced into 20 ml of distilled water and 300 ml of 0.1 N hydrochloric acid solution, at room temperature. The dispersions were stirred continuously with a magnetic stirrer and the self-emulsification behavior was observed. A formulation was considered to possess good emulsification efficiency when the droplets spread readily and formed a fine, transparent microemulsion without evidence of precipitation or phase separation. 

Precipitation assessment ⁹¹: The prepared LSEDDS formulations, 100 μl of each formulation was introduced into a glass beaker containing 300 mL of double-distilled water and 0.1 N hydrochloric acid solution at room temperature. The resulting emulsions were stored for 24 hours and examined visually at predetermined intervals (2, 4, 6, 8, 12 and 24 hours) to detect any signs of phase separation or drug precipitation. The formulations that remained clear and free from precipitation were considered physically stable. 

Robustness to Dilution ⁹¹: For the dilution study, SEDDS pre-concentrate was diluted 50, 100 and 1000 times with different media, namely distilled water, 0.1 N hydrochloric acid solution and phosphate buffer (pH 6.8). The resulting emulsions were stored for 24 hours and observations were made for signs of phase separation or drug precipitation. Formulations that remained clear and homogeneous were considered robust to dilution. 

Drug content of microemulsion ^92.93: The drug content of the formulation was analysed by dissolving 1ml of the formulation in 100 ml methanol.(SS-I) from that 0.1 ml was diluted with 10 ml methanol. Absorbance was determined using the UV spectrophotometer (UV 1700, Shimadzu, Japan) keeping blank microemulsion as control at wavelength 262nm. 

Thermodynamic Stability Studies ⁹⁴: Thermodynamic stability studies were conducted to evaluate the physical stability of the prepared SEDDS formulations, The stability of SEDDS is critical for maintaining formulation integrity, as instability may result in precipitation of the active pharmaceutical ingredient (API) from the excipient matrix, thereby compromising bioavailability and therapeutic efficacy. The influence of temperature variations and mechanical stress was assessed in three sequential steps: 

Heating–Cooling Cycle: Formulations were subjected to six alternate cycles between refrigeration temperature (4°C) and elevated temperature (45°C), with storage at each condition for not less than 48 hours. Formulations that remained stable without signs of phase separation were carried forward to the next step. 

Centrifugation Test: Stable samples from the heating–cooling cycle were centrifuged at 3500 RPM for 30 minutes. Formulations showing no evidence of phase separation were selected for freeze–thaw studies. 

Freeze–Thaw Cycle: Selected formulations were exposed to three freeze–thaw cycles, each consisting of storage at −4°C for 48 hours followed by storage at 25°C for 48 hours. 

Preparation of solid SMEDDS ^64,96:

The optimized liquid SEDDS formulation of SLN Valsrtan was converted into a solid dosage form by the adsorption technique. The liquid SEDDS was added gradually onto the adsorbent Aerosil 200 at carrier to LSEDDS  ratio 1:1, 2:1, 1:2 seperately, and the mixture was homogenized using a glass rod to obtain uniform distribution. The resultant mass was then passed through sieve No. 80 to yield free-flowing granules and stored in a desiccator until further use. 

Evaluation of solid SMEDDS ^64,96:

Angle of Repose: 

The angle of repose of S-SEDDS was determined by the funnel method. An accurately weighed sample was placed in a funnel, with the funnel height adjusted so that its tip just touched the apex of the powder heap. The powder was allowed to flow freely to form a cone and the diameter was measured. The angle of repose (θ) was calculated using the formula

???? = ?????????????¹(?/????)

θ= angle of repose 

h= height                        

r= radius  

Bulk Density:

Bulk density (BD) and tapped bulk density (TBD) were measured. A 5 g sample of SSEDDS was introduced into a 10 mL graduated cylinder, and the initial volume was recorded. The cylinder was tapped until no further volume change was observed. BD and TBD were calculated using formula,

???????? = ????????????????????? ???????? ????????????????????????/???????????????????????? ???????? ???????????????????????????? 

???????????? = ????????????????????? ???????? ????????????????????????/???????????????????????? ???????????????????????? ???????? ???????????????????????????? 

Compressibility Index:

The compressibility of the granules was expressed as Carr's Compressibility Index, calculated using formula, 

????????????????’???? ???????????????????? (%) = [(???????????? − ????????) / ????????????] × 100  

Hausner Ratio:

The Hausner ratio, another index of flowability, was calculated as: 

???????????????????????????? ???????????????????? = ???????????? / ???????? 

The in-vitro drug release studies from SLN Valsartan incorporated solid SEDDS^97,98

The in-vitro dissolution behaviour of optimized formula of solid SEDDS formulations was investigated using a USP Type II (paddle) dissolution apparatus. Solid SEDDS equivalent to 20mg valsartan were accurately filled into size "0" hard gelatin capsules and placed in the dissolution vessels. The dissolution medium consisted of 900 ml of 0.1 N hydrochloric acid solution maintained at 37±0.5°C, with the paddle rotation speed fixed at 50RPM to ensure uniform mixing. 

At predetermined time intervals of 10, 20, 30, 40, 50, 60 and 120 minutes, 5 ml of the dissolution medium was withdrawn and immediately replaced with an equal volume of fresh preheated medium to maintain sink conditions and constant volume throughout the study. The withdrawn samples were filtered using Whatman filter paper to remove undissolved particles. The filtrates were then analyzed spectrophotometrically at a wavelength of λmax 248nm to determine the drug concentration and cumulative percentage release. 

Droplet size analysis: Solid SEDDS were diluted to 100ml with distilled water. The droplet size distribution and poly dispersibility index of the resultant microemulsions were determined using particle size analyzer (Malvern Zeta sizer nano ZSP, Malvern Instrument Limited, USA).

Zeta potential: potential of the diluted SEDDS formulation was measured using Malvern Zeta sizer nano ZSP, Malvern Instrument Limited, USA. The SEDDS were diluted in the ratio of 1:20 v/v with distilled water and mixed for 1 min using a magnetic stirrer. 

Scanning Electron Microscopy (SEM): The surface morphology of the SLN loaded solid SEDDS of valsartan was examined using an analytical electron microscope (JEOL-JSM-AS430, Japan). A small quantity of the sample was lightly sprinkled over double-sided adhesive tape fixed on an aluminium stub. The stubs were coated with a thin layer of gold–palladium (thickness >10 Å) under an argon atmosphere using a gold sputter module in a high-vacuum evaporator. The coated samples were subsequently placed in the SEM chamber for surface analysis.

Compatibility Study by FTIR ⁹⁹: To evaluate possible chemical interactions between the drug and formulation excipients, Fourier Transform Infrared Spectroscopy (FTIR) analysis was carried out. FTIR spectra of pure valsartan and the optimized SLN Valsartan incorporated solid SEDDS formulation were recorded using a Shimadzu FTIR spectrophotometer. Samples were placed in the sample holder, and scanning was carried out in the range of 4000–400 cm?¹. The obtained spectra were compared to detect any characteristic peak shifts, disappearance or appearance of new peaks, which would indicate potential drug–excipient interactions. 

RESULTS AND DISCUSSION:

STEP 1 RESULTS: Solid Lipid Nanoparticle  

Fourier Transform Infra-Red Spectroscopy:

The principal peaks of valsartan were compared when admixed with excipients. The obtained FTIR spectra reveals there is no significant shift in peak as shown in fig 01. This confirms the compatibility between drug and the excipients.

Fig 01: FTIR analysis of pure valsartan and valsartan + excipients

Selection of Lipid: Lipid was selected based on solubility.Valsartan shows maximum solubility of 20mg/g in glyceryl monostearate.

Fig 02: Solubility analysis of lipid

Selection of Surfactant and stabilizer:

Tween 80 was chosen as the surfactant; the laboratory experiments were conducted using span 60 x tween 80 entrapment efficiency and particle size were estimated. Analysis results fig 03, 04 revealed that tween 80 was choice of surfactant to enhance EE% and reduce particle size. Hence tween 80 was considered for further studies as in table 03, 04. Poloxamer 188 was selected as the stabilizer for the Valsrtan-SLN formulation. SLNs were prepared both with and without the addition of a stabilizer to evaluate its impact. Formulation without stabilizer resulted in particle aggregation with very high PDI as summarized in table 04, fig 04, 05. Formulations containing Poloxamer 188 exhibited better stability and improved characteristics compared to those prepared without any stabilizer, confirming its suitability for valsarta -SLN preparation.

Table 03: Particle size and %EE using Tween 80 and Span 60

Table 04: Particle size and PDI using Tween 80 with polaxamer 188  and without polaxamer 188

Fig 03: Particle size analysis using Span 60

Fig 04: Particle size analysis using tween 80 with polaxamer

Fig 05: Particle size and PDI analysis using tween 80 without polaxamer

Table 04: factorial design implementation

For the factorial design of SLNs, the half-normal Pareto chart of particle size indicated that the drug-to-lipid ratio (Factor A) and surfactant concentration (Factor B) exerted significant effects on particle size, with Factor A showing a positive correlation (higher lipid concentration increased particle size), whereas Factor B showed a negative correlation (higher surfactant levels reduced particle size due to improved stabilization of the emulsion droplets). Homogenization time (Factor C) showed a comparatively smaller but still noticeable effect, with longer homogenization times leading to reduced particle size through increased shear.

The residual analysis further validated the model. The normal probability plot of residuals revealed that most data points were aligned along the straight line, confirming that the errors were normally distributed and no major deviations were observed. This observation supports the adequacy of the selected model. The predicted vs. actual values plot demonstrated a strong correlation, with most points lying close to the central line, signifying good agreement between experimental and predicted values. Similarly, the residuals vs. predicted plot showed a symmetrical distribution of residuals around the zero line. This indicates balanced prediction errors, with no systemic bias in under or over-estimation of particle size.

As drug lipid ratio increases particle size increased. This may be attributed due to increased viscosity of dispersed phase at high lipid concentration which resists size reduction during homogenization. Due to high HLB value of Tween 80 which is more lipophilic particle size decreases by increase in surfactant concentration it reduces interfacial tension and polaxamer188 stabilizes the formed particles thus preventing aggregation and resulting in smaller size. Due to high shear force imparted to the system prolonged homogenization resulted in reduction of particle size. Above is shown using 2D, 3D and cubic plot and is confirmed by the polynomial equation,

???????????????????????????????? ???????????????? =  +176.29 + 18.59???? – 36.51???? – 41.49????  − 6.54???????? – 19.39????????

Where

1. = Drug : lipid
2. = concentration of Surfactant 
3. = homogenization time

Overall, the statistical analysis confirmed that the developed model was robust, reliable, and suitable for predicting particle size within the experimental range.

Table 05: Anova of particle size analysis

Analysis of Entrapment Efficiency (%EE): Entrapment efficiency was taken is one of the major response parameters. The experimental values of %EE across all formulations ranged between 77%and 99%, with an average of 88.66%. The Model F-value of 6.94 implies the model is significant. There is only a 1.95% chance that an F-value this large could occur due to noise. P-values of 0.0195 less than 0.05 indicate model terms are significant. The suitability of the statistical models was assessed using a lack-of-fit test, as summarized in Table 23. The Lack of Fit F-value of 130.02 implies the Lack of Fit is not significant relative to the pure error. Non-significant lack of fit is good as shown in table 06.

In the half-normal Pareto chart of entrapment efficiency the most influential variable was the drug-to-lipid ratio (Factor A), which had a strong positive effect, confirming that higher lipid levels increased drug entrapment. Surfactant concentration (Factor B) also  contributed positively but to a lesser extent, suggesting that surfactant not only stabilized the nanoparticles but also aided in better solubilization of valsartan within the lipid matrix. Homogenization time (Factor C) showed minimal effect, indicating that entrapment efficiency was largely governed by lipid and surfactant composition rather than processing time. The normal probability plot of residuals displayed an approximately linear pattern , confirming normal error distribution. The predicted vs. actual plot demonstrated close clustering of data points around the diagonal line, which indicates good predictive accuracy. Additionally, the residual vs. predicted plot revealed a random and symmetric distribution of residuals around zero, suggesting the absence of systematic error.

As drug-lipid ratio increased entrapment efficiency increased as more lipid matrix was available to encapsulate the drug. As moderate concentration of surfactant enhanced entrapment efficiency due to improved stabilization of the drug–lipid interface. However, very high surfactant levels caused a reduction in entrapment efficiency, likely due to solubilization of drug in the aqueous phase. Excessively long homogenization times reduced entrapment efficiency, possibly due to drug expulsion from the lipid matrix or destabilization of the system caused by excessive energy input. Above is shown using 2D, 3D and cubic plot and is confirmed by the polynomial equation,

%???????? =  +89.16 + 4.85???? − 4.14???? + 2.17????

Where,

1. = Drug : lipid
2. = concentration of Surfactant 
3. = homogenization time

Overall, the statistical evaluation confirmed that the selected model was significant, well-fitted and reliable for predicting the effect of formulation parameters on entrapment efficiency.

Table 06: Anova of Entrapment Efficiency analysis

Optimization of Formulation:

The optimization study suggested valsartan SLN formulation at a drug-to-lipid ratio of 1:2, surfactant concentration of 2%, and homogenization time of 30 minutes. The predicted values for this optimized formulation were a particle size of 192.446 nm, entrapment efficiency of 88.73%, and drug content of 98.92%. The experimental (observed) values were a particle size of 195.3nm entrapment efficiency of 90.8% and drug content of 99.16 summarized in table 08. The close agreement between predicted and experimental values, with percentage error less than 2%, confirmed the accuracy of the design model and validated the reliability of the factorial design approach for optimization of SLNs and zeta potential of 28.5mv and PDI of 0.247 indicating excellent stability and drug loading capacity. 

Table 07: Results of optimizatio

Table  08:Actual and predicted values of optimized formulation

Fig 06: Particle size  and zeta potential of optimized formulation:

The release profile exhibited the highest linearity in Zero-order and Korsmeyer–Peppas plots (fig 07,10) suggesting that the drug release from SLNs follows concentration-independent kinetics, ensuring a uniform and sustained release over 24 hours with %CDR 73.46%.

Fig 07: Data of in-vitro drug release of optimized SLN formulation

Fig 07: Zero order plot of in-vitro drug release of optimized SLN formulation

Fig 08: First order plot of in-vitro drug release of optimized SLN formulation

Fig 09: Higuchi plot of in-vitro drug release of optimized SLN formulation

Fig 10: Korsmeyer–Peppas plot of in-vitro drug release of optimized SLN formulation

Lyophilization:

Fig 11: Lyophilized SLN of valsartan

Thermal Property Determination:  The DSC thermogram of the pure valsartan exhibited broad endothermic transitions corresponding to the excipients, without showing any sharp or characteristic melting peaks fig 12, indicating its predominantly amorphous nature. In contrast, the valsartan-containing lyophilized SLN displayed notable differences. The characteristic melting endotherm of pure valsartan, observed at 98.8 °C, was absent and instead a shifted peak at 108.0 °C with reduced enthalpy was detected. Additionally, a new endothermic event appeared at 168.3 °C. These changes confirm that valsartan underwent partial loss of crystallinity upon lyophilization and established physical interactions with the excipients. Such modifications in the thermal behaviour of valsartan suggest successful incorporation of the drug into the lyophilized matrix, which may contribute to enhanced solubility and improved dissolution performance.

Fig 12:DSC report of valsartan and lyophilized sample

SEM image of Lyophilized SLN:

The SEM analysis of valsartan-loaded solid lipid nanoparticles (SLNs) before(A) and after lyophilization showed that the particles retained their spherical shape with smooth surface morphology. Prior to lyophilization, the SLNs were discrete, uniform and free from aggregation, confirming successful formulation. After lyophilization, the nanoparticles largely maintained their geometry with only slight variation in size and surface contrast, likely due to lipid matrix changes and cryoprotectant interaction. The absence of significant aggregation indicates that the cryoprotectant effectively preserved particle stability during freeze-drying. Overall, the results confirm that lyophilization enhances the long-term stability of valsartan-loaded SLNs without compromising their morphology, supporting their potential for controlled drug delivery.

Fig 13: SEM Analysis of SLN-valsartan before lyophilization(A) after lyophilization (B)

STEP 2 RESULTS : Self Emulsifying Drug Delivery System Containing Solid Lipid Nanoparticle of Valsartan.  

Solubility evaluation of valsartan in various ingredients at 25ºC

Oil is the most important excipient as it can facilitate self-emulsification and increase the fraction of lipophilic drug transported via the intestinal lymphatic system, thereby increasing absorption from the GI tract. Amongst the individual oil phases, the saturation solubility of valsartan in isopropyl myristate was far superior as compared to other oil twice as sunflower oil, 10 times than olive oil, twice than castor oil and five times more than sesame oil. IPM is lipophilic and valsartan is also lipophilic thereby increased solubility in IPM.

Surfactants assist the immediate formation of o/w droplets and/or rapid spreading of the formulation in the aqueous media. They form a layer around the emulsion droplets and reduce the interfacial energy as well as provide a mechanical barrier to coalescence. This can prevent precipitation of the drug within the GI lumen and permit prolonged existence of drug molecules. Mostly, non-ionic surfactants are used as they are known to be less toxic and less affected by pH and ionic strength as compared to ionic surface-active agents. The drug showed maximum solubility in Tween 80 (72.31 mg/mL), with other surfactants such as Cremophor EL (68.40 mg/mL), Labrasol (61.75 mg/mL), Tween 20 (54.20 mg/mL) and Span 80 (25.16 mg/mL) displaying comparatively lower solubilization capacity.

The cosurfactant along with the surfactant, lowers the interfacial tension to a very small and even transient negative value. They will be beneficial for formulation of microemulsions in the optimum concentration range. Among the co-surfactants, valsartan was most soluble in Capryol 90 (65.40 mg/mL), while Transcutol P (52.70 mg/mL), PEG 400 (41.55 mg/mL), propylene glycol (38.26 mg/mL) and ethanol (21.43 mg/mL) showed reduced solubility values.

Fig 14: Solubility evaluation of valsartan in different oils at 25ºC

Fig 15:  Solubility evaluation of valsartan in different surfactants at 25º

Fig 16: Solubility evaluation of valsartan in different cosurfactants at 25º

Evaluation of the emulsification potential of isopropyl myristate using tween 80 and caproyl 90.

The Smix ratio of 5:1 (Tween 80 : Caproyl 90) exhibited the highest % transmittance of 96.2% with a slightly bluish transparent emulsion, emulsification time under 1 minute and no phase separation, indicating excellent emulsification efficiency and physical stability. Hence, this Smix ratio is predicted to be optimal for constructing a pseudo-ternary phase diagram and this is further supported by aqueous titration results confirming the formation of a stable emulsion.

Table 08: Evaluation of the emulsification potential of isopropyl myristate using tween 80 and caproyl90.

Fig 17: Evaluation of the emulsification potential by titration method

To investigate the microemulsion region, various oil: Smix ratios were prepared systematically at 5:1 Smix and water consumed was noted down  at 4:6, 5:5, 6:4, 7:3 and 8:2 was clear and transparent.

Table 09: Evaluation of the emulsification potential by titration method

Table 10: Data of 5:1 Smix in (ml)

Fig 18: Evaluation of the emulsification potential by titration method

Construction of pseudo ternary phase diagram of varying concentration of oil and smix at 5:1 ratio of surfactant and cosurfactant

Selection of oil and surfactant and the mixing ratio of oil and other components play an important role in the formulation of SEDDS. Therefore, the phase behaviour of each SEDDS needs to be carefully studied using the phase diagram constructed a by using Chemixschool® software. Pseudo ternary phase-diagrams helps in determining concentration range for suitable ingredients in SEDDS formulations. These give an idea about composition of a selected system and the nature of the resultant dispersion, such as phase separation, coarse emulsion and microemulsion and hence, assist in selecting optimum formulation. In the present study, on the basis of titration method, various oil: Smix ratios were prepared systematically at 5:1 Smix.  4:6, 5:5, 6:4, 7:3, 8:2 nano-emulsion phase was recognized based on visual inspection where clear and transparent dispersions were obtained on titration method. At the endpoint, the percentage composition of oil, Smix and water consumed was noted down, which was used for constructing pseudo ternary phase diagrams by using Chemixschool® software.

Fig 19: Pseudo ternary phase diagram

Evaluation of microemulsion using Pseudo Ternary region:

The region (1) provided by pseudo ternary suggested oil of 39.6% and Smix of 37.9% (5:1) and water of 22.4%, the obtained nanoemulsion was clear and transparent.

Fig 20: Region for evaluation of microemulsion using Pseudo Ternary region

Mixture design implementation:

% Transmittance was the major response parameters recorded during each experimental run using a UV spectrophotometry . Across all the formulations, the % Transmittance ranged between 91.7% (minimum) and 99.5% (maximum), with an average % Transmittance of 96.89%.

Table 11: Mixture design implementation

Analysis of % Transmittance:  % Transmittance was the major response parameters recorded during each experimental run using a UV spectrophotometry . Across all the formulations, the % Transmittance ranged between 91.7% (minimum) and 99.5% (maximum), with an average % Transmittance of 96.89%.

The Model F-value of 52.02 implies the model is significant. There is only a 0.05% chance that an F-value this large could occur due to noise. P-values of 0.0005 less than 0.0500 indicate model terms are significant. The suitability of the statistical models was assessed using a lackof-fit test, as summarized in Table 12. The Lack of Fit F-value of 1.00 implies the Lack of Fit is not significant relative to the pure error. Non-significant lack of fit is good.

The residual analysis further validated the model. The normal probability plot of residuals

revealed that most data points were aligned along the straight line, confirming that  the errors were normally distributed and no major deviations were observed. This observation supports the adequacy of the selected model.

The predicted vs. actual values plot demonstrated a strong correlation, with most points lying close to the central line, signifying good agreement between experimental and predicted values. Similarly, the residuals vs. predicted plot showed a symmetrical distribution of residuals around the zero line. This indicates balanced prediction errors, with no systemic bias in under- or over-estimation of %transmittance.

Oil concentration is the primary determinant of % transmittance, with higher oil levels (50%) resulting in superior clarity (% transmittance >99%), In contrast, formulations with moderate oil content (40–45%) exhibited slightly lower transmittance (94–98%), and the lowest transmittance (91.7%) was recorded in a formulation with lower oil (40%) and higher water (30%) content.

Increasing the water fraction led to a noticeable decrease in % transmittance, likely due to dilution of the surfactant/co-surfactant (Smix), which reduced the stabilization of oil droplets and caused larger droplet sizes, thereby increasing light scattering. Variations in Smix (30– 40%) had a moderate effect; higher Smix ratios in balanced oil-water systems contributed to  improved clarity but were less influential than the oil-to-water ratio  this is confirmed by 2d, 3d and cubic plots.

Overall, the statistical analysis confirmed that the developed model was robust, reliable and suitable for predicting %transmittance within the experimental range.

Table 12: Anova of % Transmittance

Optimization of Formulation:

Table 13: Results of optimization

Actual and predicted results:

Table 14: Actual and predicted values of optimized formulation

%Transmittance analysis:

Table 15: Valsartan SLN incorporated SEEDS formulation %Transmittance results

Fig 22: Valsartan SLN incorporated SEEDS formulation

Robustness to dilution: Robustness to dilution was performed with distilled water, 0.1 N hydrochloric acid and  phosphate buffer (pH 6.4) in which the formulations were diluted up to 50X, 100X, 1000X and were stored for hours. The data were recorded. Precipitation and phase separation was seen only at RUN 2 and 5 Precipitation and phase separation absent on other indicates the stability of the formulations to dilution. 

Table 16:Valsartan SLN incorporated SEEDS formulation dilution results

Pass : stable formulation

Thermodynamically stability of valsartan SLN incorporated SEEDS formulation:

Thermodynamic stability study was attempted to verify and also to avoid the unstable SEDDS formulations. Kinetic instability leads to separation of phases. The optimized emulsions were tested for stability at different temperatures and RPM. The emulsions were stable during centrifugation at 3500 rpm and alternative temperature cycles of 45 °C and -4 °C. There was no phase separation and precipitation except RUN 5 which indicates that formulations were stable at different temperatures and speed of centrifugation. SEDDS formulations were found stable under the conditions of study and unstable formulations were avoided.

Table 17: Thermodynamically stability of valsartan SLN incorporated SEEDS formulation

Assessment of self-emulsification time, phase separation and precipitation:

The SEDDS formulations should emulsify soon after putting a drop of liquid concentrate into aqueous media under slow agitation. The assessment of emulsification showed that all selected 8 formulations could emulsify within 25 seconds, which suggests rapidity of the liquid concentrate to get convert into nano-emulsion. Periodic observation of phase separation and precipitation was attempted every 2 h upto 24 h. Phase separation and precipitation was for RUN 5 and other had no phase separation after 2, 4, 6, 8, 12, 24 h representing that the SEDDS formulations were resulting stable nano emulsions.

Table 18: Precipitation and self-emulsification  time assessment of valsartan SLN incorporated SEEDS formulation

Evaluation of optimized formulation: The optimized valsartan SLN formulation exhibited high clarity (% transmittance 96.8%) and efficient drug loading of 98% (19.6mg) (50mg SLN  equivalent to 20 mg drug), with no precipitation or phase separation with particle size of 197.9nm and zeta potential of -31.4 It remained stable upon dilution in various media and demonstrated excellent thermodynamic stability under stress conditions, indicating stable formulation. These results suggest the formulation is suitable for oral administration, with consistent drug release and resistance to destabilization. 

Table 19: Dilution results of valsartan SLN incorporated SEEDS of optimized formulation

Table 20: Thermodynamically stability of valsartan SLN incorporated SEEDS of optimized formulation

Fig 23: Analysis of particle size and zeta potential of optimized formulation:

Evaluation of solid SEDDS:

Flow properties of solid SEDDS: The LSEDDS : Aerosil powder blends show that increasing Aerosil content improves flow and compressibility. Formulation 1:2 exhibited the lowest Carr’s index (19.9%), moderate Hausner’s ratio (1.25), and lowest angle of repose (31.02°) indicating better flow compared to 1:1 and 2:1. This suggests that Aerosil acts as an effective glidant, reducing cohesiveness and enhancing powder handling for processing. 

Table 21: Flow properties of valsartan SLN incorporated solid SEDDS formulation.

The in-vitro release profile of valsartan from SLNs incorporated into SEDDS was best described by the Korsmeyer–Peppas model (R² = 0.988), indicating an anomalous (non-Fickian) release mechanism that involves a combination of drug diffusion through the lipid matrix and carrier erosion/relaxation fig 27. A high First-order fit (R² = 0.9312) indicates that release rate is concentration-dependent, fig 25 while the Higuchi fit (R² = 0.886) further supports diffusion as a major contributor fig 26. Poor fit to the Zero-order model (R² = 0.6412) rules out constant, time-independent release which is shown in table 24. Together, these results suggest the SLNs incorporated into  SEDDS formulation enhances valsartan solubilization and provides diffusion-dominated release beneficial for oral bioavailability.

Table 22: Data of in-vitro drug release of optimized valsartan SLN incorporated solid SEDDS formulation.

Fig 24: Zero order plot of in-vitro drug release of optimized valsartan SLN incorporated solid SEDDS formulation.

Fig 25: First order plot of in-vitro drug release of optimized valsartan SLN incorporated solid SEDDS formulation.

Fig 26: Higuchi plot of in-vitro drug release of optimized valsartan SLN incorporated solid SEDDS formulation.

Fig 27: Korsmeyer–Peppas plot of in-vitro drug release of optimized valsartan SLN incorporated solid SEDDS formulation.