Development and Evaluation of Sprayable Nanoemulsion For Skin Cancer Using 5- Fluorouracil and Tretinoin

5-Fluorouracil (5-FU) is a widely used topical chemotherapeutic agent that acts by inhibiting thymidylate synthase, thereby interfering with DNA synthesis and leading to apoptosis in rapidly dividing cancer cells. However, its clinical utility is often limited by poor skin penetration, instability, and irritation at the site of application. Tretinoin, a derivative of vitamin A, is known to promote skin turnover, enhance permeability of active agents, and has shown synergistic effects when used in combination with other anticancer agents, including 5-FU. The use of nano emulsion -based drug delivery systems has emerged as a promising approach to overcome the limitations of conventional topical formulations. Nano emulsions, with droplet sizes in the Nano meter range (typically 20–200 nm), offer advantages such as increased drug solubility, enhanced skin permeation, controlled drug release, physical stability, and better cosmetic acceptability. Furthermore, incorporating such systems into a sprayable dosage form offers ease of application, uniform dosing, and improved patient compliance.

Nano emulsion: -

A nano emulsion is a thermodynamically or kinetically stable colloidal dispersion consisting of two immiscible liquids (typically oil and water), stabilized by surfactants and/or co-surfactants, with droplet sizes ranging from 20 to 200 nanometers. Unlike conventional emulsions, nano emulsions have ultra-small droplet sizes, which impart them with unique physicochemical and biological advantages, especially in pharmaceutical and cosmetic formulations.

Key Characteristics of Nano emulsions:

1. Droplet Size: Typically, between 20–200 nm, leading to a transparent or slightly bluish appearance.
2. High Surface Area: The small droplet size offers a large surface area for drug absorption.
3. Thermodynamic/Kinetic Stability: Some nano emulsions are thermodynamically stable, while others are kinetically stable with high resistance to coalescence, creaming, or phase separation.
4. Enhanced Solubility: Nano emulsions can solubilize poorly water-soluble drugs, increasing their bioavailability.
5. Improved Skin Permeation: Their nanometric size and composition enhance drug permeation through the skin, making them ideal for topical and transdermal delivery.
6. Controlled Release: They offer the possibility of sustained or controlled drug release at the site of action.

Components of a Nano emulsion:

1. Oil Phase: Solubilizes lipophilic drugs (e.g., isopropyl myristate, castor oil).
2. Aqueous Phase: Usually purified water.
3. Surfactant: Stabilizes the emulsion by reducing interfacial tension (e.g., Tween 80).
4. Co-surfactant: Enhances the flexibility of the surfactant film (e.g., Transcutol P, PEG 400).             

Types of Nano emulsions: -

• Oil-in-Water (O/W): Oil droplets dispersed in water; used for hydrophobic drugs.
• Water-in-Oil (W/O): Water droplets dispersed in oil; used for hydrophilic drugs.
• Bi-continuous: Complex systems where both phases are continuous.

Advantages in Dermatology and Cancer Therapy:

• Improved drug deposition in skin layers
• Reduced systemic side effects
• Non-greasy, cosmetically acceptable formulations
• Patient-friendly (sprayable or gel-based forms)
• Potential for targeted therapy in skin cancer and other dermal disorders.

Rationale: -

Skin cancer is a growing global health concern, with rising cases linked to prolonged ultraviolet exposure and lifestyle changes. Current treatment methods, including surgical removal, radiation and topical therapies, often have limitations such as incomplete drug delivery, adverse side effects and risk of reccurenece. Nanoemulsions due to their small droplet size, enhanced penetration and targeted delivery offer a promising solution. By incorporating Fluorouracil and Tretinoin into a sprayable nanoemulsion can provide a non-invasive effective and patient friendly approach to treating skin cancer, addressing the limitations of existing treatments. Fluorouracil and Tretinoin these two well-known powerful agents with proven anti-cancer properties, into a nanoemulsions, possible to create a targeted and localized treatment for skin cancer. This system can deliver drug directly to the affected area, minimizing systemic exposure amd enhancing therapeutic efficacy. Moreover, a sprayable formulation increases ease of use, improves uniform application and enhances patient compliance, especially in cases of hard to reach area of the skin.It fulfil the need of non- invasive effective and safe treatment methods for skin cancer . Additionally, it has the potential to overcome the limitations of existing therapies, improves patient outcomes, and contribute to the advanced treatments.

OBJECTIVE: -

To design and optimize a stable nanoemulsion system containg Fluorouracil and Tretinoin for enhanced skin penetration and localized drug delivery. To evaluate the physiochemical properties for the nanoemulsions including droplet size zeta potential, viscosity, and stability. To study the cytotoxic effects of the nanoemulsions on skin cancer cell lines to determine its therapeutic potential.

Plan of work: -

1. Literature Review
   • Collection and review of existing literature on skin cancer, nanoemulsions, 5-Fluorouracil (5-FU), Tretinoin, and their topical applications.
   • Study of various formulation strategies, surfactant/co-surfactant systems, and sprayable dosage forms.
2. Preformulation Studies
   • Determination of solubility of 5-FU and Tretinoin in various oils, surfactants, and co-surfactants.
   • Compatibility studies between active drugs and excipients using techniques like FTIR or DSC.
3. Selection of Components
   • Selection of oil phase, surfactant, and co-surfactant based on drug solubility and emulsification efficiency.
   • Construction of pseudo-ternary phase diagram to determine the nanoemulsion region.
4. Formulation of Nanoemulsion
   • Preparation of nanoemulsion using selected components by high-energy emulsification or spontaneous emulsification method.
   • Optimization of formulation parameters (oil/surfactant/co-surfactant ratio, drug concentration).
5. Conversion to Sprayable Form
   • Incorporation of the nanoemulsion into a sprayable system.
   • Evaluation of sprayability (spray pattern, droplet spreadability, and dose per actuation).
6. Characterization of Nanoemulsion
   • Droplet size and Polydispersity Index (PDI) using Dynamic Light Scattering (DLS).
   • Zeta potential for stability.
   • pH, viscosity, and refractive index.
   • Thermodynamic stability (heating-cooling cycles, centrifugation, freeze-thaw testing).
7. Drug Content Determination
   • Quantification of 5-FU and Tretinoin in the formulation using UV-Vis or HPLC analysis.
8. In Vitro Drug Release Study
   • Study of drug release profile using dialysis membrane or Franz diffusion cell.
9. Ex Vivo Skin Permeation Study
   • Evaluation of drug permeation and retention in skin using excised rat or porcine skin.
10. In Vitro Cytotoxicity Studies

• Assessment of cytotoxic effect on melanoma cell lines (e.g., A375) using MTT assay.

11. Skin Irritation Study

• Evaluation of skin irritation potential using animal models or reconstructed human skin models.

12. Stability Studies

• Short-term stability studies as per ICH guidelines to evaluate formulation stability over time.

13. Data Analysis and Interpretation

• Statistical analysis of results to determine significance and effectiveness of the formulation.

14. Conclusion and Future Scope

• Final summary of the study and suggestions for clinical or commercial application.

LITERATURE SURVEY: -

Selection of the drug compounds

Procurement and authentication

Formulation and development

Stability testing

Ph adjustment

Conclusion

Result

MATERIALS AND METHODS: -

MATERIALS-

The following materials and reagents were used in the preparation of the sprayable nanoemulsion containing 5-fluorouracil and tretinoin:

• Active Pharmaceutical Ingredients (APIs):
• 5-Fluorouracil (extracted from commercially available cream)
• Tretinoin (extracted from commercially available cream)

Solvents and Excipients: -

Ethanol (used for extraction and solubilization)

Distilled water (used as aqueous phase)

PEG-400 (used as a co-solvent and part of aqueous phase)

Coconut oil (used as the oil phase)

Tween 80 (non-ionic surfactant to stabilize the emulsion)

Sodium benzoate or methylparaben (used as preservatives)

Glassware and Apparatus: -

Beakers (100 mL and 250 mL)

Graduated cylinder

Stirring rods

Funnels

Filter paper (Whatman No.1)

Muslin cloth

Mortar and pestle

Glass slides

Compound microscope

Dropper bottles or spray bottles for storage

Extraction of Active Ingredients: -

• Extraction of 5-Fluorouracil-

Approximately 10 grams of a commercially available 5-fluorouracil cream was weighed and transferred to a clean beaker. To this, 40 mL of warm distilled water was added gradually while continuously stirring the mixture using a glass rod. The mixture was left undisturbed for about 30 minutes to allow proper diffusion of the active drug into the aqueous medium. The resulting semi-liquid mixture was filtered through a double layer of muslin cloth followed by filtration using Whatman No.1 filter paper. The clear aqueous extract containing dissolved 5-fluorouracil was collected and stored in a clean, dry container at refrigerated temperature until further use.

• Extraction of Tretinoin-

In a similar procedure, 10 grams of tretinoin cream was weighed and mixed with 10–15 mL of ethanol in a beaker. The contents were stirred thoroughly using a glass rod until the cream base broke down and tretinoin dissolved. The mixture was filtered through muslin cloth and then through filter paper to obtain a clear ethanolic solution of tretinoin. The extract was stored in a cool, dark place away from sunlight due to tretinoin’s light-sensitive nature.

Formulation of Nano emulsion

The nano emulsion was prepared using the aqueous phase and oil phase components with a low-energy emulsification method.

Preparation of Aqueous Phase-

The aqueous phase consisted of the 5-fluorouracil extract, PEG-400, and the required amount of distilled water. All components were mixed in a beaker and stirred well until a uniform solution was formed. If a preservative was being added (e.g., sodium benzoate or methylparaben), it was dissolved in this phase.

Preparation of Oil Phase-

The oil phase included coconut oil and Tween 80. Both were measured accurately and mixed thoroughly. Tween 80 was added as a surfactant to help emulsify the oil and water phases, reducing surface tension.

Emulsification Process-

Once both the aqueous and oil phases were ready, the oil phase was added dropwise into the aqueous phase under continuous stirring. A magnetic stirrer or manual stirring was used depending on availability. Stirring continued for at least 15–20 minutes to ensure complete mixing and formation of a uniform emulsion. The final volume was adjusted with distilled water as needed. The resulting nanoemulsion was then transferred into a clean, sterilized spray bottle or dropper bottle and stored under appropriate conditions (either refrigerated or at room temperature depending on preservative use).

Evaluation Parameters-

The following evaluation tests were performed on the final formulation to assess its stability and physical characteristics: -

Physical Appearance: - The emulsion was checked for color, consistency, and any signs of phase separation or grittiness.

pH Measurement: - A digital pH meter was used to ensure skin compatibility.

Viscosity: - Observed manually by flow behavior during pouring and spraying.

Stability Testing: - Samples were stored at both room temperature and refrigeration for 7 days to observe any physical or microbial changes.

Droplet Size Observation: -A drop of nanoemulsion was placed on a clean glass slide and examined under a compound microscope to assess the distribution and size of droplets.