Development and Evaluation of a Thermosensitive in Situ Ophthalmic Hydrogel of Moxifloxacin for Enhanced Ocular Retention and Sustained Drug Delivery

The eye is one of the most delicate and functionally complex organs of the human body, playing a critical role in visual perception and overall quality of life. Because of its constant exposure to the external environment, the eye is highly susceptible to a wide range of infections and inflammatory disorders that may impair vision if not treated effectively. Ocular diseases such as bacterial conjunctivitis, keratitis, and other microbial infections require prompt therapeutic intervention to prevent complications and preserve visual function. For this purpose, ophthalmic drug delivery systems have been extensively developed to deliver therapeutic agents directly to ocular tissues. Among the available routes, topical administration in the form of eye drops remains the most widely used and patient-preferred method due to its simplicity, non-invasive nature, and ease of self-administration.^1-2 Despite its widespread use, topical ocular drug delivery faces several physiological and anatomical barriers that limit drug absorption and therapeutic efficacy. The eye possesses efficient protective mechanisms such as blinking, tear production, and nasolacrimal drainage that rapidly eliminate instilled medications from the ocular surface. Additionally, the corneal epithelium acts as a strong barrier to drug permeation, further reducing drug penetration into deeper ocular tissues. As a result, only a small fraction of the administered drug is able to reach the target site, and the bioavailability of conventional ophthalmic formulations is often reported to be less than 10%. Due to this rapid elimination and poor drug retention, conventional eye drops must be administered frequently to maintain therapeutic drug levels, which can lead to poor patient compliance and inconsistent treatment outcomes.^3-4 Traditional ophthalmic dosage forms such as solutions, suspensions, ointments, and inserts have been widely used for the treatment of ocular diseases. However, these conventional formulations present several drawbacks, including short precorneal residence time, rapid drug loss due to tear dilution, and variable drug absorption. In some cases, ointments may cause blurred vision and patient discomfort, while suspensions may show poor dose uniformity. Although the incorporation of viscosity-enhancing agents or mucoadhesive polymers has improved ocular retention to some extent, these approaches still fail to adequately overcome the rapid precorneal elimination of drugs. Therefore, the development of advanced drug delivery systems that can prolong ocular residence time and provide sustained drug release has become an important focus in ophthalmic pharmaceutical research. In recent years, in situ gelling drug delivery systems have emerged as a promising strategy for improving ocular drug bioavailability. These systems are designed as low-viscosity liquid formulations that undergo a sol-to-gel transition when exposed to physiological conditions such as temperature, pH, or ionic strength present in the ocular environment. Upon instillation into the eye, the formulation rapidly converts into a gel, forming a viscoelastic matrix that prolongs the residence time of the drug on the ocular surface. This transition helps reduce drug drainage, enhances drug absorption, and enables sustained release of the therapeutic agent over an extended period. Consequently, in situ gelling systems can significantly reduce dosing frequency and improve patient adherence to therapy.^5-8 Among the different types of stimuli-responsive systems, thermosensitive in situ hydrogels have gained considerable attention due to their practical advantages and patient convenience. These formulations remain in a liquid state at room temperature, allowing easy instillation into the eye, but undergo gelation at physiological temperature upon contact with the ocular surface. Ther more sponsive polymers such as poloxamers are commonly used in these systems because they exhibit reversible phase transition properties and possess good biocompatibility. The resulting hydrogel forms a three-dimensional network structure that can encapsulate the drug and release it gradually, thereby maintaining therapeutic drug concentrations for a prolonged duration. Such systems offer a promising approach for enhancing ocular drug retention, improving bioavailability, and minimizing systemic absorption. Bacterial infections of the eye are among the most common ocular disorders and can lead to serious complications if not treated appropriately. Moxifloxacin, a fourth-generation fluoroquinolone antibiotic, is widely used in ophthalmology due to its broad-spectrum antibacterial activity against both Gram-positive and Gram-negative pathogens. It acts by inhibiting bacterial DNA gyrase and topoisomerase IV, thereby preventing bacterial replication and growth. Moxifloxacin is frequently prescribed for the treatment of bacterial conjunctivitis, keratitis, and other ocular infections. However, when administered in conventional eye drop formulations, the drug is rapidly washed away from the ocular surface, resulting in reduced therapeutic efficiency and the need for frequent dosing.^7-10 To overcome these limitations, the development of an advanced ophthalmic formulation capable of enhancing drug retention and providing sustained release is highly desirable. Thermosensitive in situ hydrogels represent a promising platform for delivering antibiotics such as moxifloxacin more effectively. By undergoing gelation at ocular temperature, these systems can prolong drug contact time with the corneal surface, reduce drug loss through tear drainage, and maintain therapeutic concentrations for extended periods. Such formulations may significantly enhance ocular bioavailability while improving patient compliance and treatment efficacy.

Figure 1: Structure of Moxifloxacin

Therefore, the present study focuses on the development and evaluation of a thermosensitive in situ ophthalmic hydrogel of moxifloxacin aimed at improving ocular retention and achieving sustained drug delivery. The formulated system is designed to remain in liquid form during administration and undergo gelation upon exposure to physiological temperature in the eye. The developed hydrogel formulation is further evaluated for its physicochemical properties, gelation behavior, drug release characteristics, and overall suitability as an effective ophthalmic drug delivery system. Through this approach, the study seeks to provide an improved therapeutic strategy for the management of bacterial ocular infections while enhancing patient comfort and treatment outcomes.

MATERIALS AND METHODS:

MATERIALS:

All chemicals and reagents used in the present investigation were of analytical grade and utilized as received without further purification. Moxifloxacin hydrochloride, the active pharmaceutical ingredient, was procured from a Ajantha pharmaceutical. Poloxamer 407, employed as the primary thermosensitive polymer for the preparation of the in-situ gelling system, was obtained from BASF Ltd., Mumbai, India. Hydroxypropyl methylcellulose (HPMC) was used as a viscosity-enhancing and mucoadhesive polymer to improve the stability and ocular residence time of the formulation. Other excipients used in the formulation included benzalkonium chloride as a preservative, sodium chloride for isotonicity adjustment, and sodium hydroxide for pH adjustment of the formulation. Reagents such as sodium bicarbonate, calcium chloride dihydrate, disodium hydrogen phosphate, and potassium dihydrogen phosphate were used in the preparation of simulated tear fluid for evaluation studies. All the excipients and analytical reagents were procured from M/s Loba Chemie Pvt. Ltd., Mumbai, India. Distilled water was used throughout the experimental work for the preparation of formulations and analytical studies. All materials used in the study complied with pharmaceutical grade standards and were considered suitable for the formulation and evaluation of the thermosensitive in situ ophthalmic hydrogel of moxifloxacin for enhanced ocular retention and sustained drug delivery.

METHODS:

Characterization of Moxifloxacin

The active pharmaceutical ingredient, moxifloxacin hydrochloride, was characterized using standard physicochemical and analytical techniques to confirm its identity and purity prior to formulation development. Initially, UV–visible spectrophotometric analysis was performed to determine the maximum absorbance wavelength (λmax) of the drug. An accurately weighed quantity of moxifloxacin was dissolved in distilled water to prepare a solution of appropriate concentration, and the spectrum was scanned over a wavelength range of 200–400 nm using a UV–visible spectrophotometer to identify the characteristic absorption peak.¹¹ Further characterization of the drug and its compatibility with formulation excipients was carried out using Fourier Transform Infrared (FTIR) spectroscopy. A small quantity of the pure drug and a physical mixture of the drug with selected polymers were analyzed using an FTIR spectrophotometer to record the infrared spectra and identify the characteristic functional groups. The obtained spectra were evaluated to detect any possible interactions between the drug and polymers used in the formulation. In addition, the melting point of moxifloxacin hydrochloride was determined using the capillary method to confirm the purity and identity of the drug. The observed melting point was compared with the reported literature values to ensure the authenticity of the drug used for the formulation study.

Construction of Calibration Curve of Moxifloxacin

Preparation of Phosphate Buffer (pH 7.4)

Phosphate buffer of pH 7.4 was prepared using potassium dihydrogen phosphate and sodium hydroxide. Initially, 27.22 g of potassium dihydrogen phosphate (KH₂PO₄) was dissolved in distilled water and the volume was adjusted to 1000 ml to obtain a stock solution. Subsequently, 50 ml of this solution was transferred into a 200 ml volumetric flask, followed by the addition of 39 ml of 0.2 M sodium hydroxide solution. The final volume was adjusted with distilled water to obtain phosphate buffer with a pH of 7.4.¹²

Calibration Curve in Phosphate Buffer

An accurately weighed 10 mg of moxifloxacin hydrochloride was dissolved in 100 ml of phosphate buffer (pH 7.4) to obtain a stock solution of 100 µg/ml. From this stock solution, aliquots of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 ml were withdrawn and diluted to 10 ml with phosphate buffer to obtain solutions with concentrations ranging from 2–14 µg/ml. The absorbance of each solution was measured using a UV–visible spectrophotometer at the λmax of moxifloxacin (approximately 293 nm). A calibration curve was constructed by plotting absorbance versus concentration.¹³

Preparation of Simulated Tear Fluid (STF)

Simulated tear fluid was prepared to mimic the physiological conditions of the ocular environment. 0.670 g of sodium chloride, 0.200 g of sodium bicarbonate, and 0.008 g of calcium chloride dihydrate were dissolved in 100 ml of purified water with continuous stirring until a clear solution was obtained.¹⁴

Calibration Curve of Moxifloxacin in Simulated Tear Fluid

For the calibration curve in simulated tear fluid, 10 mg of moxifloxacin hydrochloride was accurately weighed and dissolved in 100 ml of STF to obtain a stock solution of 100 µg/ml. Aliquots of 0.2–1.4 ml were withdrawn and diluted up to 10 ml with STF to obtain concentrations ranging from 2–14 µg/ml. The absorbance of the prepared solutions was measured using a UV–visible spectrophotometer at the λmax of moxifloxacin, and a standard calibration curve was constructed.

Formulation Development of Thermosensitive In Situ Ophthalmic Hydrogel of Moxifloxacin^15-18

Method of Preparation

Thermosensitive in situ ophthalmic hydrogels of moxifloxacin were prepared using the cold method. Different formulations were developed by varying the concentration of Poloxamer 407 (Pluronic F127) as the primary thermosensitive gelling polymer in combination with secondary viscosity-enhancing polymers such as Carbopol 940 or Hydroxypropyl Methylcellulose (HPMC). Initially, aqueous dispersions of the selected concentrations of Carbopol 940 and HPMC were prepared separately in phosphate buffer pH 7.4. Subsequently, the required amount of Poloxamer 407 was gradually added to the polymer solutions with continuous stirring. The resulting dispersions were refrigerated for approximately 24 hours to allow complete dissolution of the polymer and obtain a clear homogeneous solution. Separately, the required quantity of moxifloxacin hydrochloride was dissolved in phosphate buffer (pH 7.4). Benzalkonium chloride (0.01%) was then added as a preservative with continuous stirring until a uniform drug solution was obtained. The prepared drug solution was slowly incorporated into the polymeric dispersion with continuous stirring to ensure uniform distribution of the drug within the formulation. The prepared in situ gel formulations were transferred into amber-colored glass containers to protect them from light. The filled containers were then subjected to thermal sterilization by autoclaving at 121°C under 15 psi pressure for 20 minutes. After sterilization, the formulations were stored under refrigerated conditions until further evaluation and characterization studies were carried out.^16-18

Table 1: Composition of Thermosensitive in Situ Ophthalmic Hydrogel Formulations of Moxifloxacin

Ten different thermosensitive in situ ophthalmic hydrogel formulations of moxifloxacin hydrochloride were developed using Poloxamer 407 as the primary ther more versible gelling polymer. Carbopol 940 and HPMC K4M were incorporated as secondary viscosity-enhancing and mucoadhesive polymers in varying concentrations to optimize gelation characteristics and ocular retention. Benzalkonium chloride (0.01% w/v) was used as a preservative, and phosphate buffer (pH 7.4) was used as the vehicle to maintain physiological compatibility.¹⁹

Evaluation of Formulations^20-40

Determination of Visual Appearance and Clarity

Clarity is an important quality attribute of ophthalmic formulations because the presence of particulate matter may cause irritation or discomfort to the eye. Therefore, the prepared moxifloxacin in situ ophthalmic hydrogel formulations were visually inspected to ensure their suitability for ocular administration. Each formulation was examined against both white and black backgrounds under adequate lighting conditions to detect the presence of any visible particles, turbidity, or cloudiness. Formulations that appeared clear and free from particulate matter were considered acceptable for further evaluation.

pH Determination

The pH of ophthalmic formulations plays a significant role in maintaining drug stability and ocular compatibility. A formulation with an inappropriate pH may cause irritation, lacrimation, and discomfort upon instillation. Ideally, ophthalmic formulations should possess a pH close to that of tear fluid, typically ranging between 5.0 and 7.4. The pH of the developed thermosensitive in situ hydrogel formulations of moxifloxacin was determined using a calibrated digital pH meter at room temperature. The measurements were performed to ensure that the formulations were within the acceptable physiological range and suitable for ocular administration.

Determination of Gelation Temperature

The gelation temperature represents the temperature at which the thermosensitive liquid formulation transforms into a gel, which is a critical parameter for in situ gelling systems. To determine the gelation temperature, 10 ml of the formulation was transferred into a transparent vial containing a magnetic stir bar. The vial was placed in a temperature-controlled water bath, and the temperature was gradually increased at a rate of approximately 2°C per minute while stirring continuously at 500 rpm. The temperature at which the magnetic bar ceased to move due to gel formation was recorded as the gelation temperature. The experiment was conducted in triplicate to ensure accuracy and reproducibility.

Effect of Dilution on Phase Transition Temperature

In vivo, ophthalmic formulations become diluted upon contact with tear fluid. Therefore, it is important to evaluate the effect of dilution on the phase transition behavior of the thermosensitive gel. For this study, the formulation was diluted with simulated tear fluid (STF) in a 40:7 ratio to mimic physiological conditions. The diluted sample was placed in a transparent beaker containing a magnetic stir bar and heated gradually while stirring at 100 rpm. The temperature at which gelation occurred, indicated by the cessation of magnetic stirring, was recorded as the phase transition temperature after dilution. This test helps predict the behavior of the formulation upon instillation into the eye.

Drug Content Determination

Drug content analysis was performed to ensure the uniform distribution and accurate concentration of moxifloxacin within the in situ gel formulations. An accurately weighed quantity of the formulation equivalent to 4 mg of moxifloxacin hydrochloride was dissolved in simulated tear fluid (pH 7.4) and stirred using a magnetic stirrer until complete dissolution was achieved. The solution was then filtered to remove any undissolved particles. From the filtrate, 10 ml of the solution was further diluted to 100 ml using simulated tear fluid. The absorbance of the resulting solution was measured using a UV–visible spectrophotometer at the λmax of moxifloxacin (approximately 293 nm) against STF as a blank. The drug content was calculated using the standard calibration curve.

In-vitro Permeation Study

Franz Diffusion Cell Assembly

The in vitro drug permeation of moxifloxacin from the thermosensitive hydrogel formulations was evaluated using a Franz diffusion cell apparatus. A cellophane membrane was mounted between the donor and receptor compartments. The receptor compartment was filled with 20 ml of simulated tear fluid (STF) and maintained at 37 ± 1°C to simulate physiological conditions. The hydrogel formulation containing 0.5% moxifloxacin was applied to the donor compartment. At predetermined time intervals, samples were withdrawn from the receptor compartment and replaced with an equal volume of fresh STF to maintain sink conditions. The collected samples were analyzed using UV spectrophotometry to determine the amount of drug permeated.

Ex-vivo Corneal Permeation Study Using Goat Cornea

Ex-vivo permeation studies were performed using fresh goat corneas obtained from a local slaughterhouse. The excised corneas were carefully isolated and stored in cold simulated tear fluid until use. The cornea was mounted between the donor and receptor compartments of the Franz diffusion cell, with the epithelial side facing the donor compartment containing the formulation. The receptor compartment was filled with STF and maintained at 37 ± 1°C with continuous stirring. Samples were collected at predetermined intervals and analyzed using a UV–visible spectrophotometer to determine the amount of drug permeated through the corneal membrane.

Rheological Study

The rheological behavior of the prepared formulations was evaluated to assess their viscosity and flow properties. The measurements were carried out using a Brookfield rheometer equipped with an appropriate spindle. The viscosity of the formulations was measured at different shear rates to determine their flow characteristics. The analysis was conducted at 37°C, simulating physiological ocular temperature. Evaluation of rheological properties helps ensure that the formulation possesses appropriate viscosity for easy instillation in liquid form and sufficient gel strength after administration.

Stability Studies

The stability of the developed thermosensitive in situ hydrogel formulations was assessed according to standard stability testing conditions. The formulations were stored in sealed glass vials at different temperature conditions including refrigerated temperature (4°C), room temperature (25°C), and accelerated conditions (40°C) for a period of two months. Samples were withdrawn at 15-day intervals and evaluated for changes in appearance, pH, gelation temperature, drug content, and in vitro drug release profile. These studies were performed to ensure the physical and chemical stability of the formulation during storage.

RESULTS AND DISCUSSION:

Pre-formulation Studies: Characterization of Moxifloxacin

The pre-formulation studies were performed to confirm the identity and purity of the drug prior to formulation development. Moxifloxacin hydrochloride was observed as a pale-yellow crystalline powder, which is consistent with the reported physical characteristics of the drug. The melting point was determined by the capillary method and found to be in the range of 235–238°C, which corresponds well with the literature values, confirming the purity and authenticity of the drug used in the study.

Spectroscopic Studies

UV Spectroscopy (Determination of λmax)

The UV absorption spectrum of moxifloxacin hydrochloride was recorded in phosphate buffer pH 7.4 and simulated tear fluid (STF) over the wavelength range of 200–400 nm using a UV–Visible spectrophotometer.

The maximum absorbance (λmax) of moxifloxacin was observed at 293 nm in both phosphate buffer (pH 7.4) and simulated tear fluid. This wavelength was selected for further quantitative analysis and calibration curve preparation.

Figure 2: U.V. spectra of moxifloxacin in PB F7.4

l max of moxifloxacin was found to be 293 nm in simulated tear fluid -STF