INTRODUCTION:

Corneal ulcers, painful open sores on the cornea's surface, can result from various causes, with bacterial infections being a prominent culprit. Bacterial corneal ulcers occur when microorganisms, such as Staphylococcus aureus or Pseudomonas aeruginosa, invade the cornea, often through a corneal abrasion or injury, take advantage of compromised corneal integrity, multiplying rapidly and triggering an inflammatory response.

Symptoms include intense eye pain, redness, blurred vision, and increased sensitivity to light¹.Among the antibiotics employed in managing bacterial corneal ulcers, levofloxacin has emerged as a key player, showcasing its efficacy in combating a spectrum of bacteria². Levofloxacin, a fluoroquinolone antibiotic, is known for its broad-spectrum activity against various bacteria, including the ones commonly implicated in corneal infections such as Staphylococcus aureus and Pseudomonas aeruginosa. Its ability to penetrate ocular tissues makes it a valuable choice for topical application in the form of eye drops^3,4. However, conventional eye drops often suffer from poor bioavailability due to rapid drainage and low retention on the ocular surface. The unique anatomy and physiology of the eye present inherent limitations that must be addressed to optimize drug delivery and enhance patient outcomes. One significant challenge lies in the complex barriers that protect the eye, including the corneal and conjunctival barriers, which can hinder the penetration of drugs into the ocular tissues. Additionally, the rapid clearance mechanisms, such as tear turnover and drainage, pose obstacles to prolonged drug contact with the ocular surface^5,6. The design of effective drug formulations is another hurdle. Previous studies had developed several delivery systems, such as nanoparticle^7,8, microparticle⁹, liposome¹⁰, self-nanoemulsifying drug delivery system (SNEDDS)¹¹ and in situ gel¹², to overcome these challenges and prolong drug release.

Thermosensitive in situ gels in ocular drug delivery present a breakthrough in optimizing therapeutic outcomes. Responsive to physiological temperature, these gels undergo a transition from liquid to gel upon contact with the ocular surface¹³. This unique property enhances drug bioavailability and extends retention time, overcoming challenges posed by rapid tear turnover. The gel viscosity promotes sustained drug release, minimizing the need for frequent administrations¹⁴. This innovative approach not only ensures prolonged contact with ocular tissues but also improves patient compliance. By addressing anatomical barriers and maximizing drug efficacy, thermosensitive in situ gels prove advantageous in revolutionizing ocular drug delivery for enhanced bioavailability and prolonged retention¹⁵.Poloxamer 407, a thermosensitive polymer, has gained prominence in pharmaceutical formulations, particularly as an in situ gel polymer. At lower temperatures, it exists as a free-flowing liquid, but upon contact with body heat, it undergoes a rapid and reversible sol-gel transition, forming a gel-like depot at the application site¹². This property makes it ideal for sustained drug release and enhanced bioavailability in ocular and other local drug delivery systems. Poloxamer 407's thermo-responsive nature offers advantages. When instilled as a liquid, it adheres well to the ocular surface, ensuring prolonged contact. As it transforms into a gel, it provides a sustained release of the incorporated drug, minimizing the need for frequent applications. Its biocompatibility and ease of formulation make it an attractive choice for controlled drug release systems^14,16. Chitosan, a natural biopolymer derived from chitin, has demonstrated remarkable anti-ulcerative effects, presenting a potential therapeutic avenue. Its unique properties, including mucoadhesion and bioadhesion, contribute to a protective barrier on the mucosa. Research suggests that chitosan's positive charge interacts with negatively charged mucosal surfaces, forming a shield against ulcerogenic factors^17,18.

In this study, a thermosensitive polymer base of poloxamer 407 was combined with chitosan to produce an in situ levofloxacin gel. Characterisation was carried out to see the effect of both polymers on the physicochemical properties of the resulting gel such as appearance, pH, gelation time and temperature, viscosity, and drug release. Subsequently, the antibacterial activity was comparatively evaluated with a market product.

MATERIALS AND METHODS:

Materials:

Levofloxacin Hemihydrate (LFX) was provided by PT Pharos Indonesia. Poloxamer 407 (PLX407) in the trade name of Kolliphor P407 and Chitosan (CHT) were procured from Sigma-Aldrich, Singapore. Other chemicals namely Sodium chloride (NaCl), benzalkonium chloride, glacial acetic acid, sodium hydroxide were of analytical grade and purchased from PT Brataco, Indonesia. Pseudomonas aeruginosa strain ATCC 27853 was acquired from the Centre for Health Laboratory and Calibration Yogyakarta, Indonesia.

Preparation of LFX in situ gel:

Levofloxacin (LFX) in situ gel was prepared using 5 different formulations with different ratios of PLX407 and CHT as shown in Table 1. Firstly, a solution of 1% CHT in 1% glacial acetic acid was prepared. Technical abbreviations will be defined upon first use. In a separate vessel, PLX407 was dissolved in 60 mL of deionised water. LFX was then added and the solution was stirred until a homogeneous solution was obtained. An appropriate amount of stock solution was added to achieve the desired final CHT concentration, after which deionised water was added up to a total volume of 100mL. All remaining ingredients were sequentially incorporated, beginning with NaCl and phosphate buffer components, and concluding with benzalkonium chloride as a preservative^16,19.

Table 1. LFX in situ gel formula with various concentrations of PLX407 and CHT

Formulas	PLX407 (%)	CHT (%)
F1	23	0.1
F2	23	0.25
F3	23	0.4
F4	24	0.25
F5	25	0.25

Determination of gelation temperatures:

A volume of 1 mL of the in situ gel solution was carefully measured and transferred into a test tube. The test tube then was placed in a shaking water bath set to room temperature. The temperature was incrementally raised by 1°C intervals. After each increment, the test tube was observed for signs of gelation. Gelation was considered to occur when the solution solidified, as indicated by the cessation of flow and the meniscus no longer moving when the test tube was tilted to 90°C²⁰.

Determination of gelling capacity:

Gelation time and gel viscosity were measured to determine gelling capacity. The gelation time was determined by placing a single drop of the formulated solution into a vial containing 2 ml of freshly prepared Simulated Tear Fluid (STF) at a temperature of 34°C. The initiation of gel formation was marked by the visual observation of the drop within the STF. The precise time at which gel formation was initiated and the subsequent time required for complete gelation were recorded as gelation time, after which the viscosity is measured using a rheosys viscometer²⁰.

In vitro drug release study and kinetic modelling of the LFX in situ gel:

The drug release study was carried out using the membrane dialysis method. An aliquot of 5.0 mL of the levofloxacin-loaded in situ gel was carefully introduced into a sealed dialysis bag, ensuring airtight closure, and submerged in 50.0 mL of Simulated Tear Fluid (STF) with a pH of 7.4. The entire experimental system was meticulously maintained at a constant temperature of 37°C ± 0.5°C, accompanied by continuous agitation at a rate of 20 rpm. Sampling was conducted at specific time intervals: 15, 30, 45, 60, 120, 240, and 360 minutes, extracting samples from the receptor compartment. To compensate for media loss during sampling, fresh STF solution at pH 7.4 was replenished after each withdrawal. The quantification of released drug content was determined by spectrophotometric UV²¹. A commercially available eye drop product was used for comparison purposes. The kinetic modelling was performed using DD Solver to conduct curve fitting.

Antibacterial activity test:

Antibacterial activity test was carried out using the agar diffusion method. Levofloxacin eye drops that are marketed were employed as a positive control while a placebo formula lacking any active ingredient served as the negative control. Each solution was subjected to testing, with three replications performed. Samples from all the groups were poured into sterilised nutrient agar plates that had previously been inoculated with Pseudomonas aeruginosa ATCC 27853. The agar plates were incubated for 24 hours at 37 ± 0.5ºC. With the exception of incubation, all treatments were conducted under laminar air flow (LAF). Subsequently, the zone of inhibition generated by levofloxacin gel in situ plates was compared with the control¹².

Statistical analysis:

All measurements were conducted in a minimum of three replications and presented as mean + standard deviation. Statistical analyses were executed at a 95% confidence level to determine significant distinctions between two or more data points.

RESULT:

Preparation and characterization of the LFX in situ gel:

a) Gelation time and gelation temperature of the LFX in situ gel:

LFX in situ gel formulations are anticipated to undergo sol-gel transition at body temperature, while maintaining a rapid retention time, to enhance user comfort. Figure 1 illustrates the effect of polymer quantities, particularly PLX 407 and CHT, on gelation temperature and time. Chitosan inclusion is known to expedite the gelation process by reducing both time and temperature²². The role of chitosan in the gelation process was evident in Figure X, which illustrates the impact of varying chitosan concentrations on gelation time and temperature. As anticipated, the addition of chitosan led to a notable acceleration in gelation, demonstrated by the reduction in both time and temperature when chitosan concentration increased from 0.1% to 0.25%. This finding aligns with previous studies highlighting the gelation-enhancing properties of chitosan²³. However, an intriguing reversal in the trend was observed when the chitosan concentration was further increased to 0.4%. Contrary to expectations, this higher concentration resulted in a significant increase in gelation time and temperature.

Drug release and kinetical model of the LFX in situ gel:

The comparative study between in situ gel and conventional eye drops revealed distinct drug release kinetics, emphasizing the prolonged release characteristics of the formulated in situ gel. Conventional eye drops exhibited a rapid drug release, with over 89% of the drug released from the dialysis membrane within the first 3 hours. In stark contrast, the in situ gel, during the same timeframe, demonstrated a significantly slower drug release, releasing only 27.56% of the drug. These findings underscore the efficacy of the formulated in situ gel in prolonging the drug release process.

Figure 3. In vitro drug release of the LFX in situ gel compared to marketed eye drop. The drug release study was carried out using the membrane dialysis method. An aliquot of 5.0 mL of sample was carefully introduced into a sealed dialysis bag, ensuring airtight closure, and submerged in 50.0 mL of Simulated Tear Fluid (STF) with a pH of 7.4. The entire experimental system was meticulously maintained at a constant temperature of 37°C ± 0.5°C, accompanied by continuous agitation at a rate of 20 rpm.

Model fitting analysis was employed to elucidate the drug release profile (Table 2), and the first order model emerged as the preferred model with a high coefficient of determination (r² = 0.99768). This finding suggests that the drug release process is predominantly controlled by the concentration gradient between the gel and the surrounding media. The adoption of a first-order release model aligns with the objectives of a prolonged release preparation, where the drug is released gradually and consistently over time. Further insight into the drug release mechanism was gained through the application of the Korsmeyer-Peppas model. The obtained value of n, 0.99089, indicates that the drug release process follows the super case II mechanism²⁵.

Table 2. Kinetic Modelling of the LFX in situ gel

Release Kinetic Model	R²
Higuchi	0.83588
Zero-order	0.99427
First-order	0.99768^*
Korsmeyer-Peppas	0.99089^**

^**- n Korsmeyer-Peppas: 0.9799; ^*-Best Model

Antibacterial activity of the LFX in situ gel:

The antibacterial testing of LFX in situ gel revealed notable inhibitory effects against Pseudomonas aeruginosa, a clinically significant pathogen. The inhibition zone observed for the LFX in situ gel was statistically comparable to that of the positive control (p>0.05). Both the LFX in situ gel and the positive control exhibited substantial inhibition zones, with average values of 28 mm and 29.13 mm, respectively. Conversely, the negative control exhibited no inhibition zone, indicating the absence of antibacterial activity in the absence of the formulated in situ gel. This underscores the specificity of the antibacterial effect attributed to the LFX in situ gel. Crucially, the antibacterial activity observed in the LFX in situ gel did not compromise or hinder the efficacy of the drug. The formulation successfully retained its antibacterial potency, as demonstrated by the comparable inhibition zone to the positive control. This result is pivotal in ensuring that the therapeutic benefits of the drug are not compromised during the incorporation into the gel matrix.

Figure 4. Commercially available levofloxacin eye drops were used as the positive control and a placebo with no active ingredient was used as the negative control. The assay was conducted against Pseudomonas aeruginosa ATCC 27853, Except for incubation, all treatments were conducted in a laminar air flow (LAF). n.s: not significant (p>0.05); n.a: not applicable due to the absence of any zone of inhibition in the negative control.

DISCUSSION:

The primary objective of this study was to develop Levofloxacin (LFX) in situ gel utilizing a combination of polymers, namely poloxamer 407 (PLX407) and chitosan (CHT). The investigation into gel characteristics revealed significant influences of both polymers on gelation temperature, gelation time, and viscosity. The gelation temperature observed in the range of 28°C to 35°C, and gelation times ranging from 18 to 39 seconds are crucial parameters, aligning with the physiological temperature of the eye mucosa, which is approximately 34°C²⁶. These findings suggest that all formulations in the study are well-suited for ocular applications, ensuring user comfort and acceptance. PLX 407, known for its thermosensitive properties, played a vital role in the gelation process²⁷. The increase in temperature initiated a decrease in the solubility of PLX 407, leading to hydrophobic interactions and micelle formation. Subsequent temperature elevation triggered the aggregation of micelles, facilitating gelation. The synergistic involvement of PLX 407 and CHT in the gelation process, as illustrated in Figure 1, highlighted that higher concentrations of PLX 407 correlated with lower gelation temperatures and shorter gelation times, consistent with previous findings in the literature²⁸. At low concentrations (<0.4%), an increase in CHT concentration was found to decrease gelation temperature and time. CHT ability to accelerate dehydration during micellization, form crosslinks with poloxamer 407, and attract water created a conducive environment for faster gel structure formation^23,29. The observed increase in temperature and gelation time, potentially due to the dissolution of CHT requiring acetic acid, which has an opposing effect on the gelation process, showcased the complex interplay of factors influencing the gelation process. Acetic acid competes with the interaction between the PLX 407-CHT compound and PLX 407 itself³⁰.

Moreover, the impact of polymers on gel viscosity was explored, revealing that CHT had a more dominant influence than PLX 407. CHT incorporation enhanced the mechanical strength of the gel by increasing interactions between components and increasing crosslink density. This result is consistent with previous studies²⁴ and emphasizes the importance of considering the specific contributions of each polymer in achieving the desired rheological properties. The drug release profile, modeled using the first order model, indicated a controlled release mechanism. However, the value of n in the Korsmeyer-Peppas model suggested a super case II mechanism, characterized by non-Fickian diffusion, polymer relaxation, and swelling as determining factors. The discrepancy between the models highlights the intricate nature of drug release mechanisms.In practice, blinking and the tear response cause shear forces and dilution, leading to dissolution and erosion of the gel. The use of a dialysis membrane in this study may have restricted the observation of dissolution and erosion, emphasizing the importance of practical relevance in experimental design³¹.

Figure 5. PLX407 and CHT as the thermosensitive base for the sol-gel transition process of LFX in situ gel. The incorporation of CHT improves the mechanical properties of the gel and synergistically affects the gelation process.

CONCLUSION:

In conclusion, the comprehensive examination of the LFX in situ gel formulation revealed the intricate interplay between PLX 407 and CHT in modulating gelation characteristics and viscosity. The formulation exhibited promising features for ocular applications, demonstrating controlled drug release and mechanical strength. The observed super case II mechanism further underscores the relevance of considering real-world conditions in drug release modeling. Future studies may explore in vivo applications and address the impact of external factors on the gel's performance, moving towards the practical implementation of this promising drug delivery system.

CONFLICT OF INTEREST:

The authors declared there is no conflicts of interest.

ACKNOWLEDGMENTS:

This research was funded by Final Project Recognition Grant Universitas Gadjah Mada Number 5075/UN1.P.II/Dit-Lit/PT.01.01/2023

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Received on 06.12.2023 Modified on 14.03.2024

Research J. Pharm. and Tech 2024; 17(11):5349-5355.

DOI: 10.52711/0974-360X.2024.00817