INTRODUCTION:

Glaucoma is a complex eye disorder characterized by optic nerve and retinal ganglion cell impairment, posing a significant global health burden^1,2. This condition affects individuals across age groups, including young adults and children. Its primary hallmark symptom is elevated intraocular pressure (IOP), which, if left untreated, can lead to irreversible blindness. Elevated IOP remains the primary modifiable risk factor for glaucoma progression^3,4.

Glaucoma is classified based on the underlying cause of elevated IOP, with the two main categories being open-angle and angle-closure glaucoma. Regardless of the type, the progression of glaucoma is generally irreversible, but early detection and appropriate treatment can help slow or prevent the worsening of the condition. Glaucoma is a leading cause of permanent blindness worldwide, affecting an estimated 76 million individuals globally^5,6.

To address the limitations of traditional ocular drug delivery methods, such as increased precorneal elimination and variability, in-situ forming polymeric formulations have been explored. These formulations transition from a solution to a gel state after administration, allowing for controlled drug release and enhanced therapeutic efficacy. Furthermore, the integration of niosomes, lipid-based nano-vesicles, into in-situ gels can further improve drug delivery by ensuring sustained release and stability, as niosomes can encapsulate both hydrophilic and hydrophobic drugs^7,8.

Ripasudil, a novel Rho-kinase inhibitor, has shown promise in the treatment of glaucoma by targeting the trabecular meshwork and reducing IOP. Incorporating Ripasudil into a niosomal in-situ gel formulation may improve the drug's bioavailability and residence time at the site of action, thereby enhancing its therapeutic efficacy in the management of glaucoma^9,10.

MATERIALS AND METHODS:

Ripasudil was received as gift sample from Ajanta pharma, Guwahati. Cholesterol, span 60, poloxamer 407 and 188, NaCl, sodium benzoate was gifted by Vishal Chem, Mumbai, India.

Development of Niosomal Suspension:

Dissolve Ripasudil, span 60, cholesterol in adequate amount in 1:2 ratio of cholesterol and methanol. Allow it to rotate for a liquid period of time at adequate temperature with 100RPM rotation and allowed to produce thin film. Hydrate this thing film with phosphate buffer 7.4 pH. Sonicate for 10 minutes. Prepare the 13 batches based on the central composite design to optimize the niosomal formulation. The formulation was showed in table no 1 and 2^11-14.

Table no 1: Construction of central composite design^15,16

Central composite designed batches
Independent variable
Independent variable	levels
	lower	Middle	Higher
Concentration of drug(X1)	38	40	42
cholesterol and surfactant ratio(X2)	1:1	1:1.5	1:2
Dependant variable
Particle size (Y1)
% Entrapment efficiency (Y2)

Table no 2: Optimization batches ^17,18

Run	Formulation	Independent variable
		Coded value
		X1 (mg)	X2(mg)
1	F1	38	100
2	F2	40	150
3	F3	42	100
4	F4	40	150
5	F5	40	150
6	F6	40	220.711
7	F7	38	200
8	F8	40	150
9	F9	40	79.2893
10	F10	42.8284	150
11	F11	42	200
12	F12	40	150
13	F13	37.1716	150

Development of Niosomal in Situ Gel:

Take equivalent of 4mg of niosomal suspension and add it into the 10ml of gel prepared with the poloxamer 407(20% w/v) and poloxamer 188 (10% w/v) to make 10ml of in situ gel^19-21.

Characterization of ripasudil Niosomes:

Particle Size and Size Distribution (PDI):

The mean particle diameter and size distribution of the prepared nanosuspension was measured using Malvern particle size analyzer^22-24.

% Entrapment Efficiency:

Centrifuge niosomal dispersion at 20,000 rpm for 1 hour, filter supernatant, dilute, measure absorbance at 278 nm for % EE^25,26.

%EE= Total Drug -Free Drug/Total Drug *100

Zeta Potential:

The Malvern zeta sizer assesses Niosome zeta potential via the Helmholtz-Smoluchowski equation using laser-based particle highlighting and scattered light detection^27-29.

Scanning Electron Microscopy (SEM): Nanosuspension morphology examined by SEM (ESEM EDAX XL-30) at 15kV, with platinum/palladium coating, at 1000X and 2500X magnifications^30-32.

Characterization of Niosomal in Situ Gel:

Physical appearance and pH:

Evaluated in situ gel visually, tested clarity, measured pH thrice^33,34.

Determination of Gelation Time and Temperature:

Time for sol-gel determined via tube inversion method at 40°C±1^35,36.

Gelling capacity:

The gelling capacity of the formulations was determined by placing a drop in 2mL of newly made STF and visually recording the time it took to gel. Using active chemicals. Gelling capabilities were rated using the stated coding system.

Code for gelling capacity: + After a few minutes, the gel formed and quickly disintegrated, ++ The gel developed quickly and lasted for a few hours, +++The gel developed quickly and stayed for an extended period^37-40.

Isotonicity:

Isotonicity was assessed using local goat blood samples mixed with heparin. Blood slides were treated with hypertonic, hypotonic, isotonic solutions, and the test formulation. The samples were examined under a 45X microscope to evaluate their isotonicity and potential cellular damage. No special permissions are required for the use of goat blood^41-45.

Viscosity:

Viscosity measured at 60rpm, 37°C using Labman viscometer, triplicate readings, mean±SD calculated^46-49.

In vitro Drug Release Study:

In-vitro drug release study with Franz diffusion cell: place niosomal in-situ gel in donor, phosphate buffer in receptor, analyze UV spectrophotometer^50-52.

Ex Vivo Corneal Permeability Study:

Ex vivo corneal permeability was assessed using a Franz diffusion cell system. Freshly excised goat corneas were obtained from a local slaughterhouse without the need for higher authority approval. The niosomal formulation was applied to the epithelial side of the corneas, with the receptor chamber filled with an appropriate buffer solution maintained at 37°C ± 0.5°C to simulate physiological conditions. Samples were withdrawn at predetermined time intervals from the receptor chamber and replaced with fresh buffer to maintain sink conditions. Drug permeation was quantified via UV spectrophotometry at 278 nm, and the permeation profile was analyzed to determine the formulation's efficacy in enhancing corneal drug delivery. Relevant ethical considerations are addressed within the manuscript ⁵³.

P_app= J_ss/cd

Where, P_app=Trans corneal permeability co-efficient, J_ss= steady state flux, Cd = concentration of drug in doner compartment

RESULT:

Particle Size:

Avg. particle size of optimized batch was found to be 160 nm.

Fig 1 Particle size of factorial batches

% Entrapment Efficiency:

The %EE of the optimized batch was found to be 88.5 %

Fig 2 %EE of factorial batches

Zeta potential:

The zeta potential of the optimized batch was found to be -9.2 mv.

Fig 3 Zeta potential of factorial batches

Scanning Electron Microscopy (SEM):

Fig 4 SEM image of optimized batch

Physical appearance and pH:

The pH of the formulation is 7.4 and it was the clear solution.

Determination of Gelation Time and Temperature:

Fig 5 Gelation time and temperature

Gelling capacity:

Table no3: Gelling capacity

Gelling capacity

+++

Isotonicity:

Fig 6 Hypotonic Solution + RBC Fig no 7 Isotonic Solution + RBC

Fig 8 Hypertonic Solution + RBC Fig 9 Formulation + RBC

Viscosity:

Table no 4: Viscosity before and after gelling

Before	550 cps
After	2501 cps

In vitro drug release study:

Fig 10 Dissolution of Niosomes

Fig 11 Dissolution of Niosomal In situ gel

Ex Vivo Corneal Permeability Study:

Table no5: EX vivo permeation coefficient of niosomes

P_app(cm/hr)	*J_ss(Q/cm²hr)**	Cd(mg/ml)
0.0535	0.094789	1.77

Table no6: EX vivo permeation coefficient of niosomal in situ gel

P_app(cm/hr)	*J_ss(Q/cm²hr)**	Cd(mg/ml)
0.0703	0.124571	1.77

DISCUSSION:

Particle Size Optimization The small particle size (160nm) indicates improved drug delivery potential. Discuss implications for bioavailability and stability.

Entrapment Efficiency High %EE (88.5%) suggests effective drug encapsulation. Consider factors affecting entrapment (e.g., lipid composition).

Zeta Potential and Stability: Negative zeta potential (-9.2mV) may enhance stability. Address potential aggregation or flocculation.

SEM Findings: SEM confirms niosomes morphology. Discuss surface characteristics and uniformity.

Gelling Capacity and Formulation Behavior: Explore the impact of gelling capacity on drug release.

Isotonicity and Ocular Tolerance: Isotonic formulation is crucial for ocular comfort. Discuss implications for patient compliance.

Viscosity Changes: Increased viscosity after gelling may affect residence time. Consider implications for drug release kinetics.

Drug Release Profiles: Compare niosomes and in situ gel release. Discuss sustained release and potential therapeutic benefits.

Corneal Permeability: Ex vivo permeation coefficients (Papp) provide insights. Address implications for ocular bioavailability.

CONCLUSION:

A niosomal in situ gel for glaucoma treatment was developed, encapsulating Ripasudil in niosomes with 160 nm particle size, 0.185 PDI, 88.5% entrapment efficiency, and 99.25% drug content. The zeta potential was -9.2. Optimization via central composite design improved properties, and integration into a gel with poloxamer 407 and 188 increased stability and efficacy. Stability testing over 30 days showed no significant changes, validating the robustness and potential of this non-invasive therapeutic formulation.

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Received on 16.06.2025 Revised on 10.07.2025

Accepted on 29.07.2025 Published on 15.10.2025

Available online from October 30, 2025

Research J. Science and Tech. 2025; 17(4):257-264.

DOI: 10.52711/2349-2988.2025.00036

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