Preparation, In-Vitro and Ex-Vivo Evaluation of Liposomal Gel Delivery system for Timolol Maleate

Anannya Bose¹*, Subhabrota Majumdar², Asim Halder³

¹APC College of Pharmacy, Daluigacha, Singur, West Bengal 712409, India.

²Calcutta Institute of Pharmaceutical Technology & A.H.S, Uluberia, Howrah, West Bengal 711316, India.

³Department of Pharmaceutical Technology, Adamas University, Barasat, West Bengal 700126, India.

*Corresponding Author E-mail: a.bose.midnapore@gmail.com

ABSTRACT:

This study focuses on the development and characterization of a novel liposomal gel formulation intended for ocular drug delivery. The liposomal gel was prepared using the thin-film hydration method, with Tween-80 employed as a stabilizing agent to prevent aggregation. The encapsulation efficiency, particle size, zeta potential, and polydispersity index (PDI) of the liposomal formulation containing the active ingredient, timolol maleate, were evaluated. The encapsulation efficiency was found to be 57.47%, with a particle size of 340.8 nm and a zeta potential of -29.0 mV. The polydispersity index was determined to be 0.576, indicating a uniform distribution of liposome sizes and good stability of the formulation. The developed liposomal gel aims to enhance drug permeation, optimize therapeutic response, and minimize potential side effects associated with conventional forms of the drug. The incorporation of multilamellar vesicles into the gel matrix is expected to prolong drug residence time at the ocular surface, thereby improving drug absorption and reducing dosing frequency. Additionally, the mucoadhesive properties of the carbopol 940 gel base further enhance ocular residence time, leading to improved patient compliance and comfort. Overall, the prepared liposomal gel formulation shows promise as an effective ocular drug delivery system. Further in vitro and in vivo studies are warranted to evaluate its pharmacokinetic and pharmacodynamic profiles, as well as its safety and efficacy for clinical use.

KEYWORDS: Liposome, Glaucoma, Ocular drug delivery, Thin-film hydration method, Draize Ocular irritancy test.

INTRODUCTION:

Drug delivery in ocular therapeutics poses significant challenges. Frequent drug administration is necessary for various ocular ailments such as glaucoma, conjunctivitis, and dry eye syndrome. One of the main challenges faced in delivering drugs to the eyes is ensuring that the drug concentration is optimal at the intended site of action. The limited effectiveness of traditional eye drops in delivering drugs is primarily caused by factors that result in the drugs being lost before they can be absorbed.

These factors include the rapid turnover of tears, inefficient absorption, short duration of time the drops stay in the eye, and the difficulty drugs have in passing through the corneal epithelial membrane¹.

Occasionally, the drug being absorbed systemically through the nasolachrymal duct may lead to certain unwanted side effects. Because of these biological limitations, merely a tiny portion of the given quantity (less than 1%) is absorbed by the eyes. The clinician is compelled to suggest a more frequent dosing regimen with a significantly higher concentration, which unfortunately leads to various adverse effects of ophthalmic formulations. Alternative ocular drug delivery systems provide certain advantages compared to traditional liquid dosage forms. However, issues such as indistinct vision (e.g., ointments) or lack of patient obedience (e.g., inserts) have hindered their widespread adoption. These problems may be overcome by the use of liposomal gel². Liposomes are highly regarded as promising and versatile drug vesicles. When it comes to drug delivery systems, liposomes have proven to be superior in many ways. They provide features such as targeted delivery to specific sites, extended or regulated release of therapeutic substances, safeguarding against degradation and rapid clearance, amplified therapeutic outcomes, and diminished toxic side effects. Certain liposomal drug formulations have gained acceptance and been utilized in clinical settings in recent years, showcasing their significant merits.

A low-viscosity liquid dispersion, liposome particles can aggregate. Liposomes' low viscosity limits medication contact time in the eye, hence the approach must be improved. It is common to disperse drug-loaded vesicular systems into gel bases to extend their precorneal residence duration. Such liposome dispersed in gel base is called as liposomal gel. These liposomal gel formulation shows dual approaches, where gel helps to extend the contact period of the formulations on the cul-de-sac region and liposome enhances the corneal penetrability of the drugs³.

In this investigation, timolol maleate (TM) was chosen as the representative drug. TM, a non-selective beta-blocker, stands as a cornerstone in the treatment of glaucoma, which ranks as the second most prevalent cause of blindness globally, affecting over 70 million individuals. TM was singled out due to its swifter onset, improved tolerance, and reduced side effects relative to other medications within its pharmacological class. By impeding sympathetic nerve endings in the ciliary epithelium, this drug diminishes intraocular pressure (IOP) primarily by curbing the production of aqueous humor. While conventional TM eye drops are economical, user-friendly, and efficacious in lowering IOP, the anatomical intricacies of the eye, particularly the corneal epithelial barrier, pose constraints on drug absorption from lacrimal fluid into the anterior segment post-administration of eye drops. Ineffective therapy may result from low bioavailability (<5%) of TM eye drops. Most topically administered TM eye drops leak through the nasolacrimal duct, causing hypotension, bradycardia, and bronchospasm due to poor corneal permeability. A mucoadhesive cubogel formulation of TM was developed and tested to reduce such issues⁴. This study sought a way to disseminate liposomes containing TM into carbopol 940 gel basis to improve mucoadhesion and sustained release.

MATERIALS AND METHODS:

Materials: Pure drug Timolol maleate (TM) was purchase from Yarrow Chem products, Mumbai, India. Carbopol 940, phosphatidylcholine (PC), cholesterol (CHOL) were acquired from Himedia, Mumbai. All other reagents used were analytical grade.

Preparation of liposome containing timolol maleate:

For its antiglaucoma properties, timolol is typically prescribed in concentrations ranging from 0.25% to 0.5% w/v solution. Therefore, a final drug strength of 0.25% was employed in the process. Timolol maleate liposomes were formulated utilizing the thin film hydration method employing a Rotary evaporator (Superfit®, India). The liposomal formulations were crafted by dissolving phosphatidylcholine and cholesterol in a 7:3 ratio in approximately 20ml of an organic phase composed of chloroform and methanol in a 2:1 ratio. This solution was then transferred into a 250 ml round bottom flask. The organic phase was vaporized under a vacuum of 100 mm/hg using a rotary evaporator (Supervac®, India) at 45 ºC, resulting in the formation of a thin film. Evaporation continued for 15 minutes until a dry residue developed. This process gradually eliminates the organic solvent, leaving a thin lipid coating on the flask interior. The films were vacuum-dried overnight to completely evaporate the organic solvent. After hydrating the film with phosphate buffer solution (pH 7.4) and adding the appropriate medication, it was rotated at 60ºC for 45 minutes. The liposomal suspension was left overnight at 4ºC to attain complete lipid hydration⁵.

Determination of % encapsulation efficiency:

After lysing the prepared liposomes with absolute alcohol and sonicating them for 10 minutes, the percentage of drug encapsulated was calculated. Using a UV-visible spectrophotometer (model UV-1800; Schimadzu, Kyoto, Japan), the concentration of timolol maleate in absolute alcohol was measured spectrophotometrically at 295nm. The following relationship was used to compute the encapsulation efficiency.

Total drug – free drug

% Encapsulation efficiency = ----------------------- x 100

Total drug

Determination of zeta potential:

Assessing the physical constancy of colloidal dispersions can greatly benefit from the use of zeta potential. The velocity of particle movement in an electric field can be used to measure the zeta potential. The range of Zeta limits extended from -200 mV to +200 mV. In this study, the dispersion of timolol maleate liposomal dispersion was diluted 10 times and analysed for zeta potential using the zetasizer apparatus (Malvern Instruments, UK). The samples were appropriately diluted with filtered distilled water (10 times) and then employed in a small disposable zeta cell. The zeta potential was determined in triplicate⁵.

Table 1: Modified Draize’s grading scale for assessing the ocular irritation potential.

Formulations	Rabbit 1 (preliminary test)				Rabbit 2 (a confirmation test)				Rabbit 3 (a confirmation test)
Formulations	Cornea	Iris	Conjunctiva	Edema	Cornea	Iris	Conjunctiva	Edema	Cornea	Iris	Conjunctiva	Edema
Blank group	0	0	0	0	0	0	0	0	0	0	0	0
Test group	0	0	0	0	0	0	0	0	0	0	0	0
Positive control group	2	2	1	1	2	2	1	1	2	1	1	0
Negative control group	0	0	0	0	0	0	0	0	0	0	0	0

Determination of particle size:

The timolol maleate liposomes were assessed for particle size and size distribution using laser light scattering with a particle size analyzer (Malvern Mastersizer Hydro-2000 SM, UK). To prevent multiscattering phenomena, the samples were diluted appropriately with filtered distilled water (at a ratio of 10:1). Subsequently, they were loaded into a small disposable zeta cell, and the particle size was determined in triplicate⁵.

In-vitro drug release study:

The in-vitro release of the drug from liposomes was assessed via dialysis bags. Prior to use, the dialysis bags underwent careful rinsing in distilled water at room temperature for 12 hours to eliminate any preservatives. The liposomal formulation was then placed inside a dialysis bag, and both ends of the bag were securely fastened with threads. Subsequently, the sealed dialysis bag was immersed into a 250ml Erlenmeyer flask containing 100ml of pH 7.4 phosphate buffer solution. The flask was positioned on a magnetic stirrer, and stirring was maintained at 100rpm at a temperature of 37°C with precise thermostatic control. Samples were withdrawn at one-hour intervals over a twelve-hours period and subjected to spectrophotometric analysis to determine the drug content at 295nm⁶.

Incorporation of prepared liposome into gel base:

Table 1 shows the new formulation's formula. TM liposomal suspension at 0.25% w/v was added to carbopol 940 gel basis. Preservative was 0.1% w/v methyl paraben. The created product was then dispensed into 20ml amber glass vials with caps and teats. The final product was autoclaved at 121°C and 15 pressure for 20 minutes 6. After sterilisation, the formulations were refrigerated (4–8°C) until usage. The formulation was tested for physicochemical parameters, as shown in Table 1.

Determination of pH and viscosity:

A liposomal gel sample weighing 10g was dispersed in 100mL of distilled water and mixed thoroughly. The pH of the solution was measured using a pH meter.

In order to assess the viscosity of the liposomal gel containing timolol maleate, a beaker was filled with 100mL of the gel and positioned on a viscometer equipped with spindle number 64. The spindle was carefully lowered onto the gel until it reached the specified limit. We analyzed the rheological parameters of gels at a temperature of 27.10⁰C. The measurement was conducted at 100rpm and utilized the viscosity value displayed on the instrument^7,8.

Determination of isotonicity:

Isotonicity is a crucial quality that the ophthalmic product must possess. Ensuring isotonicity is essential to avoid any possible harm or irritation to the eye. The augmented formulation was blended with a small quantity of blood, and the morphology of blood cells was examined under a microscope at × 45 magnification. The observed results were then associated with those of the control solution. Understanding the effects of different solutions on blood cells is crucial for maintaining their integrity. Isotonic solutions play a key role in preserving cell structure, while hypertonic solutions lead to cell shrinkage and hypotonic solutions effect in cell bulging⁹.

Ex-vivo Trans corneal permeation study:

The transcorneal penetration of timolol maleate was investigated using goat corneas as the membrane model. Goat eyeballs procured from a slaughterhouse were carefully elated to the laboratory in normal saline at a temperature of 4°C. Subsequently, the corneas were meticulously insulated along with a small portion of neighbouring tissue of sclera and gently rinsed with cold saline solution. Each cornea was then mounted as a diffusion membrane within a vertical Franz diffusion cell with a volume capacity of 50ml. Approximately 1 gram of the test formulation was placed into the donor chamber, while the receptor chamber was filled with 50 ml of simulated tear fluid (STF) and unceasingly stirred using a magnetic stirrer. The positioning of the donor chamber tube was adjusted to ensure direct contact between the excised corneal surface and the diffusion medium. The temperature of the diffusion structure was carefully attuned to achieve equilibrium at 37⁰C on the membrane surface. A fixed interval of 60 minutes was used to withdraw one milliliter of sample from the receptor compartment and replace it with fresh STF. The samples were tested using UV-spectrophotometer at a wavelength of 294nm¹⁰.

A calculation was made to determine the permeation parameter of TM. This involved intrigue the amounts of drug that infused through an excised cornea against the corresponding time. From analyzing the permeation graphs, we determined the steady-state flux (J) standards crossways the expunged cornea using a specific relationship.

J = dQ/ dtA [µg/ (cm²s)] ……….. (1)

Where, Q designates the amount of agent overpass cornea, A is the corneal part unprotected and t is the time of contact. The penetration coefficient P was calculated as

P = J/C₀ (cm/s) ………… (2)

The apparent permeability coefficient was calculated using following equation:

P_app.= dQ/dt × 1 / (A × C₀ × 60) ……….. (3)

Where, C₀ represents the initial drug concentration in the donor compartment.

Morphology of prepared liposomal gel:

Research was conducted utilizing scanning electron microscopy (Zeiss EVO 18 Special Edition, Cambridge, United Kingdom) to investigate the surface morphology of the liposomal gel^11,12.

Ocular irritancy test:

Draize was intended to evaluate ophthalmic products for ocular irritation before distribution. 100 microliters of the formulation was carefully applied to the lower cul-de-sac, and several criteria were assessed at 1, 3, 9, and 24 hours following administration. We got Institutional Animal Ethics Committee approval before starting the investigation. The Institutional Animal Ethical Committee, NSHM Knowledge Campus, Kolkata, approved us. Three 1.5–2 kilogram albino New Zealand rabbits were used in this study. All four recipes are tested on three rabbits, both preliminary and confirmation. The rabbits had 5 days to acclimate to the controlled surroundings before the experiment. They had clean housing and food at this time. Their eye health was then checked. One rabbit was used for a preliminary test. One rabbit eye was exposed to the preparation, while the other was a negative control. In the first experiment, researchers examined the rabbits' eyes at precise intervals after exposure to the test preparation. Two more rabbits were confirmed with tests. These two studies examined all formulations. Based on the regulation score, the test preparation's ocular response is assessed and recorded. Report eye injuries, including conjunctiva, cornea, iris, and systemic consequences. Assessing ocular irritation requires determining its degree and reversibility. Individual scores are not a definitive measure of test preparation irritability. Instead, use them as a reference. All test observations are included in the test preparation irritation score¹³.

RESULT:

Characterization of liposome:

The timolol maleate liposome was prepared using the thin-film hydration method, utilizing Tween-80 as a suitable agent to produce liposomes without any aggregation. The purpose of this study was to develop liposomal gel containing timolol maleate for ocular administration. The entire work was divided into two steps. First step was to develop multilamellar vesicle (MLV) in order to enhance drug permeation, optimize therapeutic response, and minimize potential side effects of the active drug. The second step was to incorporate the prepared liposome into carbopol 940 gel base to enhance the residential time at the ocular surface.

Percentage encapsulation efficiency, particle size and zeta potential:

The percentage of encapsulation efficiency, particle size and zetapotential of prepared liposome containing timolol maleate were found to be 57.47 %, -29.0 mV and 340.8 nm respectively. Polydispersity index of prepared liposomal suspension was found to be 0.576. Upon examining the liposomal formulations, it becomes apparent that the polydispersity index (PDI) is consistently below 1.0. The observation mentioned above suggests that the vesicles in the formulations show a consistent and uniform distribution of sizes, indicating monodispersity. Thus, from the existing evidence, it can be concluded that the liposomes remained well dispersed in the formulation, showing no signs of aggregation.

Figure 1: (a) Zeta potential distribution of liposome (b) Particle size distribution of liposome

In-vitro drug release study:

The fig. 3 presented in this study illustrates the in-vitro release profile of timolol maleate from the liposome formulation that has been optimized (referred to as B₁). The data presented in the release profile indicates that the release of timolol maleate (specific compound) after a period of 12 hours was found to be 66.69%. The investigation focused on determining the in-vitro release profile of timolol maleate (the specific drug under study) from the optimized liposome formulation. The results revealed that approximately 52.85% of the entrapped drug was released within a time frame of 10 hours. The initial rapid release of timolol maleate for a duration of three hours may be attributed to the presence of unentrapped drug molecules.

pH and viscosity: The viscosity and pH values of prepared liposomal gel was found to be 1432 Pa.s and 7.45. This pH value showed that the gel probably would not produce any irritation to the eye. Hence, the prepared liposomal gel is suitable for topical application.

Figure 3: Observation results of isotonicity test on preparations solution.

Determination of isotonicity:

Upon careful examination under a microscope, it was found that the four preparations yielded normal blood cells that exhibited similar characteristics to those in the isotonic control solution. This is evident in fig. 3, where observations were conducted using an isotonic control solution (NaCl 0.9%), a hypertonic control solution (NaCl 2%), and a hypotonic control solution (NaCl 0.2%). Thus, it can be concluded that the necessary arrangements have been made to ensure isotonicity.

Ex-vivo and In-vitro correlation:

The ex vivo penetration analysis demonstrated a prolonged release of TM from the prepared liposomal gel, as depicted in Figure 4. These ex vivo findings directly corresponded to the results found from the in vitro drug diffusion analysis (Table 2). A gradual and prolonged release profile was perceived for TM during its dissemination through the excised corneal membrane.

Figure 4. Ex vivo release profiles of prepared liposomal gel containing timolol maleate.

Morphology of prepared liposomal gel:

The scanning electron microscopy (SEM) images of prepared liposomal gel containing timolol maleate are shown in fig. 5 respectively.

Figure 5: Scanning electron photomicrograph of freeze dried liposomal gel.

Ocular irritancy test:

After carefully examining the data, it was found that there were no significant findings in any of the categories, including cornea, iris, conjunctiva, and edema. Each category has its own description for the purpose of this value of zero. The cornea shows no signs of ulceration or opacity, while the iris appears normal. The conjunctiva displays normal blood vessels and no redness, indicating a healthy condition. Additionally, a zero value in the edema category suggests that there is no swelling in the rabbit's eye.

Figure 6: Ocular irritancy test.

DISCUSSION:

Thin-film hydration-prepared timolol maleate liposomes with Tween-80 stabilizers are potential ocular medicine delivery methods. Drug penetration, therapeutic response, and adverse effects improve with this method. Multilayered multilamellar vesicles (MLVs) improve drug penetration for sustained release and therapeutic effects, making their development attractive. Timolol maleate encapsulated in MLVs may improve bioavailability and therapeutic results, reducing dose frequency. The mucoadhesive characteristics of carbopol 940 gel base prolong formulation residency on the ocular surface when liposomes are added. Drug absorption, administration frequency, patient compliance, and comfort improve with increased residence duration. Envelopment efficiency, particle size, zeta potential, and polydispersity index (PDI) indicate liposomal drug delivery formulation quality and stability. Encapsulation efficiency (57.47%) suggests drug encapsulation, enhancing therapeutic efficacy. Ocular administration is possible due to the particle size (340.8 nm), which balances tissue penetration and clearance. Negative zeta potential (-29.0 mV) indicates stability, preventing aggregation and guaranteeing uniform dispersion. Low PDI (0.576) suggests uniform liposome sizes, needed for consistent drug administration and minimized variability. Timolol maleate liposomes are stable and dispersed, making them ideal for ocular administration. Further in vitro and in vivo studies are needed to prove their efficacy. Timolol maleate released 66.69% and 52.85% after 12 and 10 hours, respectively, in vitro, indicating sustained release.

CONCLUSION:

The goal of the successfully prepared timolol maleate-entrapped multilamellar liposomes was to improve therapeutic response, enhance drug molecule retention in the ocular surface, and decrease potential side effects. The prepared liposomes were found to have good morphological properties, percentage of entrapment efficiency, and size distribution influenced by bilayer cholesterol content. The prepared liposomes were then incorporated into carbopol 940 gel base, resulting in greater drug retention time at the ocular surface. Ex-vivo and in-vitro studies demonstrated the extended drug release property. Based on the results of isotonicity and ocular irritancy.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors express their gratitude to the Department of Pharmaceutical Technology, NSHM Knowledge Campus, Kolkata, for their generous support during the ocular toxicity study and all other laboratory investigations.

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Received on 19.02.2024 Revised on 08.06.2024

Accepted on 11.08.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5761-5767.

DOI: 10.52711/0974-360X.2024.00876

Batch Code	Zero- order plot		First- order plot		Higuchi’s plot		Korsmeyer-Peppa’s plot
Batch Code	In-vitro	Ex-vivo	In-vitro	Ex-vivo	In-vitro	Ex-vivo	In-vitro	Ex-vivo
B₁	0.9856	0.943	0.9359	0.946	0.8774	0.964	0.98	0.955