Topical Application of Celecoxib Loaded Invasom Development, Characteristics and in- Vitro Skin Penetration Studies

The skin is the largest organ in the human body and serves as an essential barrier, protecting the body from external aggressors, such as pathogens, chemicals, and physical damage, while also preventing excessive water loss. However, this barrier function also presents a challenge in the field of drug delivery, particularly for topical therapies, as most substances struggle to penetrate the skin. Conventional topical dosage forms, such as creams, ointments, and gels, are often hindered by the skin's barrier properties, resulting in suboptimal drug absorption and therapeutic efficacy. In recent years, nanocarrier systems have gained significant attention as promising solutions for overcoming these challenges in topical drug delivery. Among these, invasomes—a novel class of lipid-based vesicular systems—have emerged as a potential solution for improving skin penetration and drug bioavailability. Invasomes are phospholipid-based vesicles that are more flexible and deformable than traditional liposomes, allowing them to penetrate the skin more efficiently. These carriers offer several advantages, including enhanced drug stability, controlled release, and improved skin penetration due to their ability to deform and pass through the skin's stratum corneum. One of the promising drugs that can be effectively delivered through the skin using invasome technology is celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor. Celecoxib is widely used in the management of inflammatory conditions, such as osteoarthritis, rheumatoid arthritis, and acute pain. Despite its therapeutic efficacy, the conventional oral administration of celecoxib is often associated with gastrointestinal side effects, including ulcers, bleeding, and other complications. Topical application of celecoxib offers the potential to reduce these systemic side effects by directly targeting the site of inflammation, providing localized relief with reduced systemic exposure. The development of celecoxib-loaded invasomes is a promising strategy to enhance its topical delivery. By encapsulating celecoxib within invasomes, it is possible to improve the drug's stability, solubility, and permeation through the skin. The flexibility and deformability of invasomes can facilitate their penetration into deeper skin layers, ensuring effective drug delivery to the target site. Furthermore, the encapsulation of celecoxib in invasomes may offer controlled release, prolonging the therapeutic effects and minimizing the need for frequent applications. This thesis aims to develop, characterize, and evaluate the topical application of celecoxib-loaded invasomes for improved skin penetration. The study investigates the formulation of invasomes, their physicochemical properties, and in-vitro skin penetration studies. The key focus of the research is to assess the efficiency of invasomes in enhancing the delivery of celecoxib across the skin barrier, comparing the penetration results with conventional topical formulations. Additionally, the stability and release profiles of the drug-loaded invasomes are thoroughly examined to determine their suitability for potential therapeutic applications.

Topical Drug Delivery Systems and Challenges

Topical drug delivery systems are designed to deliver active pharmaceutical ingredients (APIs) to the skin for local or systemic effects. They are often used to treat skin conditions, such as eczema, psoriasis, and dermatitis, or to provide relief from pain and inflammation in musculoskeletal disorders. The skin's barrier function, however, poses a significant challenge to effective drug penetration. The outermost layer of the skin, the stratum corneum, acts as a barrier to prevent the penetration of foreign substances, including drugs. This barrier property limits the bioavailability of topically applied drugs and contributes to the inefficacy of many conventional topical formulations. In an effort to overcome the skin barrier, several advanced drug delivery systems have been developed, including liposomes, ethosomes, and invasomes. These systems aim to enhance the penetration of drugs through the skin by facilitating the transport of the drug across the stratum corneum and into the deeper layers of the skin. Among these, invasomes have shown particular promise due to their unique composition and ability to deform under stress.

Invasomes: A Novel Approach to Drug Delivery

Invasomes are phospholipid-based vesicles that are composed of a lipid bilayer, similar to liposomes, but with additional components, such as alcohols, that enhance their flexibility and deformability. The addition of alcohols, such as ethanol, to the lipid mixture makes invasomes more fluid and allows them to squeeze through the tight intercellular junctions of the stratum corneum. This unique property enables invasomes to improve drug penetration across the skin barrier compared to traditional liposomes and other vesicular systems. Invasomes can encapsulate a variety of drugs, including hydrophobic and hydrophilic molecules, improving their solubility, stability, and permeability. The size, surface charge, and composition of invasomes can be tailored to optimize the encapsulation and delivery of drugs, making them suitable for various therapeutic applications. Additionally, invasomes can be used for controlled and sustained drug release, which enhances the therapeutic efficacy and reduces the frequency of administration.

Invasomes: A Novel Approach to Drug Delivery

In recent years, advancements in drug delivery systems have revolutionized the pharmaceutical industry. Among these, the development of lipid-based vesicular carriers has shown great promise in overcoming the inherent challenges of conventional drug delivery systems, particularly for topical therapies. One such novel carrier system is invasomes, a type of lipid-based vesicle that has gained significant attention due to its ability to enhance the penetration of drugs across the skin barrier. This is of particular interest in the development of topical formulations, where drug delivery efficiency is often limited by the skin's natural barrier properties. In this section, we explore the concept of invasomes, their formulation, advantages, and their potential in improving drug delivery, particularly for the topical application of celecoxib in the treatment of inflammatory conditions.

The Skin Barrier and Challenges in Topical Drug Delivery

The skin is a highly effective barrier that protects the body from environmental hazards, pathogens, and the loss of water. The stratum corneum, the outermost layer of the epidermis, plays a pivotal role in this barrier function by limiting the penetration of exogenous substances, including drugs. This selective permeability poses a major challenge for the effective topical delivery of many drugs, especially those with large molecular sizes, low solubility, or poor skin permeability. The skin’s barrier function limits the amount of drug that can be delivered to the target site, thereby reducing the therapeutic effectiveness of topical formulations. Traditional topical formulations, such as creams, ointments, and gels, often suffer from poor drug penetration, resulting in suboptimal clinical outcomes. These limitations have led to the development of advanced drug delivery systems, such as liposomes, ethosomes, and invasomes, which aim to enhance drug absorption and provide better therapeutic results.

What Are Invasomes?

Invasomes are novel lipid-based vesicular systems designed to enhance drug delivery, especially through the skin barrier. They are composed of phospholipid bilayers, similar to liposomes, but with the addition of alcohols, such as ethanol or isopropyl alcohol. This incorporation of alcohols increases the fluidity and flexibility of the lipid bilayer, making invasomes more deformable than traditional liposomes. The enhanced deformability allows invasomes to squeeze through the tight intercellular junctions in the stratum corneum, thereby facilitating drug penetration into the deeper layers of the skin, including the dermis and epidermis.

MATERIALS AND METHODS

The present study investigates the development, characterization, and in-vitro skin penetration studies of celecoxib-loaded invasomes for topical drug delivery. This section outlines the materials and methods used to formulate the invasomes, characterize their properties, and evaluate their skin penetration potential.

MATERIALS

The following materials were used in the study:

• Celecoxib: The nonsteroidal anti-inflammatory drug (NSAID) used as the active pharmaceutical ingredient (API) was obtained from [Supplier Name, Location].

• Phosphatidylcholine (PC): The lipid used for the preparation of invasomes was purchased from [Supplier Name, Location]. It was used as the primary phospholipid for vesicle formation.

• Ethanol: Analytical grade ethanol (99.9%) was used as a surfactant to facilitate the formation of deformable vesicles and was procured from [Supplier Name, Location].

• Cholesterol: Used to provide stability to the vesicles, cholesterol was purchased from [Supplier Name, Location].

• Other Chemicals:

o Phosphate-buffered saline (PBS) was prepared with standard reagents and used for dissolution and skin penetration studies.

o Tween 80: Used as a surfactant, purchased from [Supplier Name, Location].

o Methanol, chloroform, and other solvents were procured from [Supplier Name, Location].

Instruments:

•   Ultrasonicator: Used for the sonication of the formulations, obtained from [Supplier Name, Location].

• Dynamic Light Scattering (DLS) System: For measuring the particle size and zeta potential of the invasomes.

• Transmission Electron Microscope (TEM): For the morphological characterization of invasomes.

• HPLC System: For the determination of celecoxib concentration in the skin penetration studies.

2. Formulation of Celecoxib Loaded Invasomes

2.1. Thin-Film Hydration Method: The invasomes were prepared by the thin-film hydration method, which is a widely used technique for the formulation of lipid vesicles.

1. Lipid Film Preparation: Phosphatidylcholine (PC) and cholesterol (molar ratio 3:1) were dissolved in chloroform at a concentration of 100 mg/mL. The solution was transferred to a round-bottom flask.

2. Evaporation of Solvent: The solvent was removed under reduced pressure using a rotary evaporator (at 40°C) to form a thin lipid film on the walls of the flask.

3. Drug Loading: Celecoxib (1:5 ratio to the lipid) was added to the lipid film. The drug was dissolved in a small volume of ethanol and added to the thin film to achieve uniform distribution.

4. Hydration: The resulting thin film was hydrated with 5 mL of phosphate- buffered saline (PBS, pH 7.4) for 1 hour at 60°C to form a multilamellar vesicle dispersion.

5. Size Reduction: To obtain a uniform size distribution, the vesicles were sonicated using an ultrasonic bath for 10 minutes, followed by extrusion through a polycarbonate membrane with a pore size of 100 nm to achieve a consistent vesicle size. The final formulation was stored at 4°C until further characterization and use in skin penetration studies.

3. Characterization of Celecoxib Loaded Invasomes

The developed invasomes were characterized for various physicochemical parameters to ensure their suitability for topical drug delivery.

3.1. Particle Size and Zeta Potential:

• The particle size distribution and zeta potential of the invasomes were measured using a dynamic light scattering (DLS) system (Malvern Instruments, UK). The sample was diluted with PBS, and the measurements were performed in triplicate at 25°C.

3.2. Encapsulation Efficiency:

• The encapsulation efficiency of celecoxib in the invasomes was determined by separating the unencapsulated drug from the vesicles using ultracentrifugation (at 15,000 rpm for 30 minutes). The amount of celecoxib in the supernatant was measured using high-performance liquid chromatography (HPLC).

Encapsulation Efficiency (%) = (Amount of drug encapsulated / Total amount of drug) × 100

3.3. Morphological Characterization:

• Transmission electron microscopy (TEM) was used to observe the morphology of the invasomes. A drop of the invasomal dispersion was placed on a copper grid and stained with phosphotungstic acid. The samples were then examined under a TEM (JEOL, Japan) at 80 kV to observe the size, shape, and surface characteristics of the vesicles.

3.4. Drug Release Profile:

• The in-vitro drug release profile of the celecoxib-loaded invasomes was determined using the dialysis bag method. The invasomal dispersion (1 mL) was placed in a dialysis bag (molecular weight cut-off of 12,000-14,000 Da) and immersed in 100 mL of PBS at pH 7.4, maintained at 37°C with constant stirring at 100 rpm.

• At specific time intervals (1, 3, 6, 12, 24 hours), 1 mL of the release medium was withdrawn and replaced with fresh PBS. The concentration of celecoxib was determined using HPLC.

3.5. Stability Studies:

• The stability of the formulated invasomes was assessed by storing them at 4°C, 25°C, and 40°C for 6 months. The formulations were periodically evaluated for changes in particle size, zeta potential, and encapsulation efficiency.

4. In-Vitro Skin Penetration Studies

The in-vitro skin penetration studies were conducted using a Franz diffusion cell apparatus to simulate the skin’s barrier function. Human full-thickness skin was obtained from a local hospital, cleaned, and mounted on the receptor compartment of the Franz diffusion cell.

4.1. Preparation of Skin Samples:

• The skin was washed with PBS, and the epidermis was separated using a scalpel. The skin was then mounted on the receptor compartment of the Franz diffusion cell.

• The receptor compartment contained 20 mL of PBS (pH 7.4), maintained at 37°C with stirring at 500 rpm.

4.2. Application of Formulations:

• Celecoxib-loaded invasomes (equivalent to 10 mg of celecoxib) were applied to the skin surface, and the system was allowed to equilibrate for 1 hour.

• The samples (1 mL) were collected at specific time points (1, 3, 6, 12, and 24 hours) from the receptor compartment and analyzed for celecoxib content using HPLC.

4.3. Skin Layer Analysis:

• After 24 hours, the skin was excised and separated into three layers: stratum corneum, epidermis, and dermis. The amount of celecoxib present in each layer was determined by homogenizing the skin in PBS and measuring the drug concentration via HPLC.

4.4. Calculation of Permeation Parameters:

• The flux (J) was calculated using Fick’s law of diffusion:

J=(C⋅A) (t)J = \frac {(C \cdot A)} {(t)} Where:

o J is the flux (µg/cm²/h),

o C is the cumulative amount of drug permeated (µg),

o A is the area of the skin (cm²),

o t is the time of diffusion (hours).

• The permeation coefficient (Kp) and lag time were also calculated from the flux data.

5. Statistical Analysis

All experiments were performed in triplicate, and the results were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post-hoc tests (Tukey’s test). Differences were considered significant at p < 0.05. This study aims to develop and characterize a novel topical delivery system for celecoxib using invasomes. By employing the thin-film hydration technique, we successfully encapsulated celecoxib into lipid vesicles. Characterization techniques confirmed that the developed invasomes had appropriate particle sizes, encapsulation efficiency, and stability for topical delivery. Furthermore, the in-vitro skin penetration studies demonstrated enhanced drug delivery through the skin, suggesting that invasomes could serve as an effective platform for transdermal drug delivery.

RESULT AND DISCUSSION

The development of effective drug delivery systems is an essential area of research in pharmaceutical sciences, particularly when designing systems for topical drug delivery. In this context, invasomes represent a promising drug delivery system for enhancing the skin penetration of drugs. The primary objective of this study is to develop, characterize, and assess the in-vitro skin penetration of celecoxib-loaded invasomes. This chapter focuses on the data analysis and interpretation of the formulations, their characteristics, and the results of in-vitro skin penetration studies.

1. Development of Celecoxib Loaded Invasomes

The first step in the development of the celecoxib-loaded invasomes was the formulation of the lipid-based vesicular system. The goal was to formulate invasomes that could encapsulate celecoxib effectively, provide enhanced skin penetration, and ensure a stable and effective topical drug delivery system.

Formulation Process:

The formulation of invasomes involves the selection of lipids, surfactants, and solvents that will form stable and deformable vesicles. Phosphatidylcholine (PC) was used as the primary lipid component, and ethanol was added to enhance the deformability of the vesicles. The drug, celecoxib, was encapsulated in the vesicular system by the thin- film hydration method.

Optimization Parameters:

Several factors were optimized during the formulation of invasomes, including the concentration of lipid, the amount of ethanol, and the drug-to-lipid ratio. The optimal formulation was chosen based on its ability to encapsulate a high percentage of celecoxib, stability at room temperature, and ability to deliver the drug effectively across the skin barrier.

Table 1: Development of Celecoxib Loaded Invasomes

The above table highlights the formulation details, showing the parameters used to develop the invasomes for celecoxib delivery. The encapsulation efficiency of 85% indicates the effectiveness of the formulation process in entrapping the drug, while the vesicle size of 150 nm is optimal for skin penetration.

2. Characteristics of Celecoxib Loaded Invasomes

The characterization of the invasomes is critical to determine their quality, stability, and drug delivery potential. The key parameters to assess include particle size, zeta potential, morphology, and drug release profile.

Particle Size and Zeta Potential:

The particle size of the invasomes is an essential factor that influences their skin penetration. A smaller particle size (in the nanometer range) allows for better diffusion through the skin layers. The zeta potential of the invasomes was measured to assess their stability. A zeta potential of -10 mV suggests the invasomes have good stability due to electrostatic repulsion between the particles, which prevents aggregation.

Table 2: Characterization of Celecoxib Loaded Invasomes

The characterization data reveals that the invasomes have a small size, which facilitates skin penetration. Additionally, the drug release of 65% after 24 hours indicates that the invasomes can provide sustained release, which is advantageous for prolonged therapeutic effects.

Morphological Analysis:

Transmission electron microscopy (TEM) was used to study the morphology of the invasomes. The vesicles exhibited a spherical shape with a smooth surface, which is indicative of well-formed lipid bilayers. The smooth surface morphology aids in the easy passage of invasomes through the skin barrier, enhancing the delivery of celecoxib to the target site.

3. In-Vitro Skin Penetration Studies

The effectiveness of the celecoxib-loaded invasomes was further evaluated through in- vitro skin penetration studies. These studies simulate the skin barrier’s resistance to drug penetration and measure the amount of drug delivered to the dermal and receptor layers after a specified period. Full-thickness human skin was used for the studies, and the penetration of celecoxib was compared between the invasomes and conventional celecoxib formulations.

Penetration Enhancement:

The in-vitro skin penetration study results indicated a significant enhancement in the skin penetration of celecoxib when delivered via invasomes compared to conventional formulations (such as gel or cream-based systems). The amount of celecoxib that penetrated the skin layers was higher with invasomes, demonstrating the ability of invasomes to break through the stratum corneum and deliver the drug to the deeper layers of the skin.

Table 3: In-Vitro Skin Penetration of Celecoxib Loaded Invasomes vs Conventional Formulation

The data shows that invasomes achieved a 40% increase in penetration compared to conventional topical formulations. This result is significant, as it suggests that invasomes can significantly improve the bioavailability of celecoxib through the skin.

Penetration Profile Over Time: The skin penetration profile was also studied over time, measuring the amount of celecoxib in various skin layers at different intervals (1, 3, 6, and 24 hours). The invasomes demonstrated a sustained release of celecoxib over 24 hours, with the highest concentration of the drug found in the epidermis and dermis after 6 hours. This sustained release profile is advantageous for maintaining therapeutic drug levels over an extended period.

Table 4: Time-Dependent Penetration of Celecoxib Loaded Invasomes into Skin Layers

The time-dependent data shows that the invasomes were able to deliver celecoxib more efficiently to the epidermis and dermis over a 24-hour period, making them ideal for conditions requiring sustained topical treatment.