1. INTRODUCTION:

In recent years, the field of ophthalmology have undergone the significant advancement, to overcome the shortfall of traditional ocular drug delivery system.

The recent development focus on the nanoparticle-based drug delivery systems to overcome the drawbacks of conventional eye drops. These advanced carriers are engineered specially to address the challenges like the lower bioavailability, less drug retention and absorption in the ocular surroundings.

They are frequently made of poly (lactic-co-glycolic acid) or PLGA because of its compatibility and biodegradability. As the mucus layer at the eye surface is negatively charged, cationic polymers might interact with it¹. Various polymers have been examined to prepare mucoadhesive nanoparticles, developed nanoparticles made of Eudiragit^® RL 100 was good ocular tolerance, no inflammation or discomfort in the rabbit’s eye.² Positively charged nanoparticles can also be prepared when Eudiragit^® RL100 is combined with PLGA^3,4.

Nano pharmacology applies nanotechnology to enhance drug precision, improving bioavailability and targeting specific cells at the molecular level to reduce systemic toxicity. Furthermore, these smart delivery systems enable therapeutic agents to overcome significant biological obstacles, including the blood-brain barrier^5.

Polymeric nanoparticles function as effective drug carriers, offering controlled release, enhanced solubility for poorly soluble drugs, and reduced systemic toxicity. These sub-micron systems utilize natural and synthetic polymers to bypass biological barriers, protecting drugs from premature degradation^6.

Solid Lipid Nanoparticles (SLNs) to deliver anti-inflammatory steroids. The mucoadhesive nature of the system ensures the drug "sticks" to the tear film, improving its absorption into the deeper tissues of the eye. It notes that such nanoparticle preparations are vital strategies to increase ocular bioavailability by overcoming physiological barriers⁷. The Nanoparticles based formulation, In vivo showed a significant increase in ocular residence time⁸.

The biodegradable nanoparticles were optimized to provide a sustained drug release of 87.14% over 12 hours, specifically targeting viral keratitis to reduce dosing frequency⁹. The effectiveness of polymeric nanoparticles depends entirely on selecting the right fabrication technique to match the drug's properties¹⁰.The transition from conventional to smart drug delivery systems, identifying nanotechnology as the solution to poor drug solubility and lack of targeting specificity. The paper outlines how nanocarriers enhance biological navigation to pre-selected sites while transforming hydrophobic drugs for improved compatibility¹¹ Polymer concentrations and stirring speeds affect the particles' size and their ability to release the drug effectively over time¹².

Design of Experiment (DoE) approach to optimize the formulation resulting in enhanced stability and controlled drug release¹³. A solvent evaporation method to overcome the drug's poor solubility and low oral bioavailability. The resulting formulation achieved sustained release and improved dissolution, suggesting enhanced therapeutic potential with reduced dosing frequency.^14.

Besifloxacin ophthalmic suspension 0.6%w/v was approved by US-FDA in 2009. The formulation uses Duracite^® technology to increase the residence time of drug on the ocular^11,15. Besifloxacin is a novel fluoroquinolone antibiotic for ocular pathogens that are currently resistant to present fluoroquinolone antibiotics ¹⁶. Therefore, an attempt was made to prepare and characterize besifloxacin loaded ocular nanoparticles for the treatment of ocular infection. Nanoparticles were prepared by nanoprecipitetion technique followed by solvent evaporation methods. Physiochemical characterization of the nanoparticles was performed by measuring particle size, zeta potential, drug entrapment efficiency, in vitro drug release and in vivo study. Solid state characterization of the freeze-dried nanoparticles was performed Fourier Transform Infrared spectroscopy (FTIR), Differential Scanning Calorimetry (DSC). However, the aim of the current study was to develop positively charged nanoparticles of besifloxacin with Eudiragit^® RL100 and PLGA that could interact with the anionic mucins present in the mucous layer of the tear film and evaluate its physiochemical properties.

2. MATERIALS AND METHODS:

The standard API of besifloxacin hydrochloride was obtained as a kind gift from Maithri Drugs Pvt. Ltd, Telangana. Polymer- Eudragit^® RL100, PLGA and surfactant- Poloxamer-188 were purchased from Sigma 68, Mumbai, India. Acetone was obtained from Remkem Ltd., India. Methanol of (HPLC) grade (Fisher Scientific, Qualigen, India) , Acetonitrile and phosphoric acid were obtained from (RFCL Limited, New Delhi, India), The filters with pore size of 0.22μm were arranged from Pall Life Sciences, Mumbai used for filtration of mobile phase and sample solutions, Hydrochloric acid, disodium hydrogen phosphate, and Potassium dihydrogen phosphate, Dialysis membrane: (Himedia, Mumbai, India) of 12000 Daltons cut off provided from University.

2.1. Chromatographic conditions:

The content of drug product evaluated by validated stability indicating HPLC method of Besifloxacin by reverse phase techniques Chromatographic separation was carried out at 30⁰C on an Inertsil^® ODS-3, C18 column (250mm × 6mm × 5µm particle sizes, GL Sciences, Inc. USA, www.glsciencesinc.com). The BSF was separated gradient with a mobile phase consisting of inorganic phase-water in 0.5% triethylamine (pH=3) and organic phase (mixture of methanol and acetonitrile in 70:30 v/v) at 80:20 v/v ratio. The pH of inorganic phase (water) was adjusted with diluted orthophosphoric acid and sodium hydroxide. The mobile phase was filtered and degassed for five minutes in a bath sonicator prior to use. To reach equilibrium the analysis was usually started after the passage of 60-70mL of mobile phase. The flow rate was 2.25mL/min. The injection volume was 50µL, and the eluted analytes for drug was traced by UV-detection at 289nm.

2.1.1 Optimization of Nanoparticles By 2³ Full Factorial Designs:

Method/ Procedure:

Quadratic model was applied for optimization of nanoparticles using factorial design. Polymer (A), surfactant (B) and organic phase (C), each at two level (low and high concentration) and three replicates were selected as independent variables while % yield and entrapment efficiency as response variable. Design layout was shown in table 1, optimized formulation was in shown in table 2 and in Variable parameters used in the preparation of nanoparticles.

Table 1: Design Layout

Std	Run	Factor 1	Factor 2	Factor 3	Response 1	Response 2
		A: polymer	B: surfectant	C: organic phase	Yield	entrapmment
		Mg	%	Ml	%	mg/ml
20	1	10	0.3	9	94.459	5.54097
17	2	100	0.01	9	91.9635	8.03648
2	3	10	0.01	3	68.9167	31.0833
10	4	100	0.3	3	91.4774	8.53262
9	5	10	0.3	3	85.4068	14.5932
24	6	100	0.3	9	63.3639	36.6361
18	7	100	0.01	9	92.0998	7.901
12	8	100	0.3	3	82.6666	12.3334
6	9	100	0.01	3	76.3641	23.6359
16	10	100	0.01	9	63.356	5.604
5	11	100	0.01	3	82.0764	21.9237
4	12	100	0.01	3	84.4166	15.5834
8	13	10	0.3	3	87.971	12.029
15	14	10	0.01	9	96.4383	13.5617
19	15	10	0.3	9	97.7804	8.2196
1	16	10	0.01	3	93.858	26.142
7	17	10	0.3	3	88.789	17.211
22	18	100	0.3	9	77.3094	22.6906
3	19	10	0.01	3	72.236	27.764
14	20	10	0.01	9	91.1607	8.83927
23	21	100	0.3	9	69.97	30.365
21	22	10	0.3	9	95.1372	14.8628
13	23	10	0.01	9	97.9522	2.0478
11	24	100	0.3	3	86.0418	13.9582

Table 2: Constant parameter used in the preparation of nanoparticles.

S. No.	Constant parameter	Values
1.	Diameter of needle nozzle	0.8mm
2	Height of needle tip from solution surface	5cm
3.	Dropping rate	2mL/min
4.	Distilled water	10mL
5.	Drug	10mg

Table 3: Variable parameters used in the preparation of nanoparticles

S. No	Variable parameter	Range investigated
1.	Amount of polymer	10-100 mg
2.	Amount of organic solvent	3-9 mL
3.	Amount of surfactant	10-30 mg

2.2 Preparation of besifloxacin – loaded Eudiragit^® RL100 and PLGA nanoparticles:

A Nanoprecipitetion technique was applied to prepare BSF ocular nanoparticles. Typically, different ratio of drug, polymer (RL100 and PLGA), dispersing agent (Poloxamer 188) and drug concentration keeping constant at 10mg were used to formulate nanoparticles. The drug 10mg was first dissolved in to a sufficient quantity of methanol (1-3 mL). Preparation of polymeric solution by dissolving PLGA and RL 100 polymer in the ratio 3:1 (Range 10-100mg) in to the acetone (range 2-6 mL).

After, that drug solution was dropped by niddle into polymer solution at 1500rpm. W/O emulsion was obtained, and this emulsion was then immersed drop wise in to solution of polaxmer-188 (dispersing agent) and result W/O/W double emulsion was obtained. Prepared double emulsion was evaporated on magnetic stirrer at 30-40^◦ C and the final volume of the aqueous suspension was collected. The nanosuspension was then centrifuged at 180000rpm, 4^◦C for 1hr (Remi, Mumbai, India). Nanoparticles collected and then lyophilized by using with and without 1% wt/vol mannitol as lyoprotectent.

Formulation designs were based on the software Design Expert, 24 formulations was formulated by varying the variable parameter. The final optimized formulation was selected by the software. The design layouts of formulation design were shown in table 1, constant parameter and variables parameter used in formulation as shown in table 2 and 3 respectively.

2.2.1 Selection of optimized formulation:

The selection of optimized formulation was based on statistical software. Table 4

Table 4 Optimized formulation

S. No

Polymer

(mg)

Surfactant (mg)

Organic

Phase (mL)

Water (mL)

4.240

2.2 Physiochemical characterization:

2.2.1 Particle size and zeta potential:

Nanoparticles size distribution was determined using photon correlation spectroscopy (PCS) with Zetasizer 3000 (Malvern instrument Ltd., Malvern, Worcestershire United Kingdom). The size distribution analysis was performed at a scattering angle of 90^◦ and at a temperature of 25^◦C using samples appropriately diluted with filtered water. The mean particle size Z_avg of each sample was determined three times and the average values were calculated.

Zeta potential values were determined by electrophoretic light scattering (ELS) using the same instrument. Nanoparticles were suspended in filtered water and diluted with water. For each preparation three samples were injected into the capillary cell of the Zetasizer 3000. Then the average values of three replicates were calculated.

2.2.2 Surface morphology:

Scanning electron microscopy (JSM – 5200, Tokyo Japan) was used to analyze particle size and surface topography. The instrument was operated at 15 KV acceleration voltages. The sample was shadowed in a cathodic evaporator with a gold layer 20nm thick. Photographs were elaborated by an image processing program and individual NP diameters were measured to obtain mean particle size.

2.2.3 Solid state characterization:

· Infrared spectrophotometry:

Fourier transform infrared analysis (FTIR) was conducted to verify the possibility of interaction. FTIR transmission spectra were obtained using a FTIR – 8300 spectrophotometers (Shimdazu, Tokyo, Japan). A total of 2% (W/W) of sample, with respect to the potassium bromide (KBr, S.D. Fine Chem. Ltd., Mumbai India) disc, was mixed with dry KBr. The mixture was ground into fine power using an agate mortar before compressing into KBr disc under a hydraulic press at 10,000 psi. Each KBr disc was scanned at 4mm/s at a resolution of 2 cm over a wave number in the region of 400 – 4000 cm^-1 using IR solution software (ver.1.10). The characteristic peaks were recorded for different samples.

· Differential scanning calorimetry (DSC):

Polymeric nanoparticles, polymer, drug, surfactant and physical mixture of sample were separately sealed in aluminum cells and set in Perkin – Elmer DSC6 apparatus (Uberlingen, Germany) between 40⁰C – 320⁰C. Thermal analysis was performed at a heating rate maintained at 10⁰C per minute in a nitrogen atmosphere. Alumina was used as the reference standard.

2.2 In vitro drug release:

In - vitro drug release study was performing using dialysis membrane (12000mol. wt. cut off) in which 10 mL of the nanosuspension, API solution and blank nanoparticles were taken in dialysis membrane and it was sealed both sides by dialysis membrane closure clips, that was attached with paddles of dissolution apparatus. Then it was dipped into simulated tears pH 7.4 the dissolution was performed at 50rpm. The samples were collected in different intervals up to 24hours. The sampling volume was kept 5mL and same quantity 5ml of simulated tears was added after each sample collection. The samples were collected analyzed by UV- spectrophotometer to understand the release pattern.

2.3 Statistical analysis:

The results of in-vitro data were analyzed by statistical software by (E Kinetic Software BIT Mishra Ranchi, India) to obtain the best fit kinetic model for in vitro drug release.

2.4 Release kinetics evaluation:

Release data were fitted to different mathematical models to reveal the release patterns at nanoparticles. Zero order, first order and Higuchi release models were used for this purpose. The zero order plots were constructed by plotting cumulative percentage release versus time, first order plot was constructed by plotting log cumulative percentage release versus time and Higuchi plot was constructed by plotting cumulative percentage release versus square root of time.

2.5 Determination of drug entrapment efficiency:

Take 15mg of freeze-dried nanoparticles in a volumetric flask filled with distilled water for extraction of drug and kept for 24hrs. The mixture was sonicated for 20min then filtered by using vacuums filter to obtain complete clear solution and sample will be assayed by UV spectrophotometer at λ_max 289nm. The percentage of drug entrapment efficiency can be calculated by using following equation:

Weight of drug in nanoparticles

% Drug Entrapment = --------------------------------×100

Efficiency Weight of drug use

2.6 Nanoparticles recovery:

The recovery of nanoparticles suspension was analyzed by centrifugation methods, where 10 mL suspension was centrifuged at 15000rpm at 4^◦C. the sediment nanoparticles were collected, freeze dried and calculated % yield.

Weight of recover particlex100

% Yield = -----------------------------------------

Weight of drug and polymer

2.7 Sterility test:

The sterility test was performed according to Indian Pharmacopeia. Direct inoculation method was used 2mL of liquid from test container was removed with a sterile pipette. The test liquid was aseptically transferred to the fluid thioglycollate medium. The liquid was mixed with the media. The inoculated media were incubated for 14 days at 30-35^◦C.

2.8 Stability studies:

Optimized formulation was selected for short term stability studies. The nanoparticles suspension was placed at accelerated and stress condition, at room temperature, freeze (2-8C). Stability studies were performed according to ICH guidelines [ICH Q1A (R2), ICH Q1B, ICH Q1 C]²⁰.

3.0 RESULTS AND DISCUSSION:

3.1 Statistically model optimization:

To optimize BSF loaded nanoparticles formulations, relationship among independent variables polymer (A), surfactant (B) and organic phase (C) and response variables %yield and entrapment efficiency at two level and three replicates were evaluated by Design expert^®(version 8.0.7.1, Stat-Ease Inc., Minneapolis; MN). The best model for all two-response variable was found to be quadratic. For estimation of the significant of the model, analysis of variance (ANOVA) was determined as per the provision of design expert software as shown in table 5.

Table 5 Analysis of variance (ANOVA) of two response variables

Source	Yield		Entrapment
Source	F value	P value	F value	P value
Model	3.13	0.0277	13.30	<0.0001
A – polymer	7.05	0.0173	1.46	0.2442
B – surfactant	0.054	0.8193	0.054	0.8197
C – organic phase	0.56	0.4645	8.35	0.0107
AB	1.39	0.2555	14.18	0.0017
AC	8.64	0.0096	18.91	0.0005
BC	3.74	0.07909	44.62	<0.0001
ABC	0.49	0.4935	5.51	0.0321

3.2.0 Physiochemical characterizations:

3.2.1 Particle size and zeta potential analysis:

Formulations showed a small mean size, well suited for ocular administration and provided a good drug diffusional release. Particle size for ophthalmic application should not exceed 10µm because with larger size a scratching feeling might occur, reduced particle size improves the patient comfort. The effect of drug: polymer ratio and organic phase: aqueous phase ratio has an immerse effects on particle size and distribution. The obtained size was 652nm and less than one polydispersity index proves less fluctuation in sizes, figure-1.

The zeta potential values for BSF containing nanoparticles were remained in the range of positive value +40mV. The positive surface charge of the nanoparticles was observed due to the presence of quaternary ammonium groups of Euidragit RL 100. A positive charge can facilitate an adhesion to the cornea surface and account for for a strong interaction with negatively charged mucosa of the conjunctiva and anionic mucin present in tear film, prolonging the residence time of the formulation figure-2.

Figure-1. Particle size distribution.

Figure-2. Zeta potential of formulation.

3.2.2 Surface morphology:

SEM micrographs of optimized PLGA and RL100 ocular nanoparticles formulation under 8.00 KX magnification was shown in figure 3. SEM micrographs of besifloxacin hydrochloride ocular nanoparticles formulation showed the smooth surfaced nanoparticles with spherical shape and uniformly distributed.

Figure-3. SEM micrographs of BSF ocular nanoparticles.

3.2.3 Fourier transform infared analysis (FTIR):

Fourier transform infared analysis was conducted to verify the possibility of interaction of chemical bonds between drug and polymer. In the present investigation, FTIR spectra of pure drug, polymer, dispersing agent, physical mixture and drug loaded formulation of PLGA and RL 100 nanoparticles were analyzed using FTIR spectrophotometer for characteristic absorption bands, indicative of their interaction. There was no interaction was found between drug and polymer.

3.2.4 Thermal analysis:

Thermal analysis is an important evaluation technique to find any possible interaction between the drug and the polymers used. Any such interaction may reduce the drug entrapment efficiency of the polymer and may also alter the efficacy of the drug. Such interaction can be identified by any change in thermogram. The DSC runs were performed over a temperature range of 40-320◦C at a heating rate of 10◦C per minute. The thermogram of BSF shows T_g- 55.99⁰C, BSF- NPs shows T_g- 57.54⁰C, PLGA T_g- 40.65⁰C, Eudiragit RL 100 found T_g- 68⁰C, polaxmer 188 T_g- 59.78⁰C as shown in figure 4-8.This result suggests that no interaction had taken place in NPs, and the properties of both the drug and the polymer were unchanged.

3.2.4 Drug entrapment efficiency:

The entrapment efficiency was affected by drug: polymer ratio and organic phase (solvent): aqueous phase (non solvent) ratio. The entrapment efficiency was found to vary with drug and polymer ratio. It was observed that increase in polymer concentration in organic phase increases drug entrapment due to increase in organic phase viscosity. This might have increased the diffusion resistance to drug molecules from organic phase to aqueous phase, thereby entrapping more drugs in the polymer NPs. It was also found that the drug entrapment depends on organic phase and aqueous phase volume ratio. The change in phase volume ratio changed the entrapment efficiency.

3.2.5 In-vitro drug release:

The release rate was found to be influenced by drug: polymer ratio. Increase of drug release was observed as function of drug: polymer ratio. Such a finding can be related to the progressivesaturation of the polymer ammonium group by drug molecules occurring at higher drug: polymer ratio, which increases the dissolutive nature of drug release. In general, NPs formulations showed a prolonged release, no burst effect was observed as shown in figure 9. The release of Drug loaded nanoparticles follow higuchi matrix (R²= 0.8782) and mechanism of release was Fickian diffusion.

3.2.6 Mechanism of drug release:

The mechanism of release of besifloxacin hydrochloride nanoparticles was studied by treating the release data to zero order, first order, Higuchi and Peppas. It was found that In-vitro drug release was best explained by Higuchi equation (r²= 0.8782), as the plots showed the highest linearity, followed by first order (r²= 0.080) and zero order (0.081). Log cumulative percentage of drug release versus log time curves shows high linearity, and it proves that the formulation follows the Higuchi model and drug release through fickian diffusion.

3.3.0 Sterility test:

No turbidity was observed indicating absence of microbial growth when the formulations were incubated for 14 days at 30-35^◦C in case of fluid thioglycollate medium. The preparations were examined and found to pass the test for sterility.

3.4.0 Stability studies:

The result of stability studies as shown in table 6 indicate that the most suitable storage condition for nanoparticles of Besifloxacin hydrochloride was 4◦C followed by room temperature (37◦C±2^◦C) and accelerated stress condition 65% RH at 40◦C as shown in figure 10-12.

4.0 CONCLUSION:

Although PLGA nanoparticles are biodegradable, they possess a negative zeta potential and probably have a low interaction with anionic mucus. Therefore, the possibility of producing positively charged nanoparticles by adding Eudiragit to these formulations was investigated. In our current work, prepared PLGA, Eudiragit^® RL100 nanoparticles of besifloxacin using a modified nanoprecipitation technique. The drug from the biodegradable particles generally releases through several mechanisms such as desorption of the surface bound/ adsorbed drug, diffusion through the particle matrix, diffusion through the polymer wall and in case drug is encapsulated in the core, surface and bulk degradation and a combined degradation/ diffusion process. Evaluated parameters like rheological studies, drug entrapment efficiency, in vitro drug release, microbial studies, sterility testing and in vivo studies. Nanoparticles were characterized by particle size, zeta potential and surface morphology. Formulation containing variables such as different drug polymer ratio and different solvents ratio were prepared and treated for different kinetic models of drug release. Optimized besifloxacin HCL nanoparticles possess: Good ocular retention property due to unique particle size and zeta potential values was found to be suitable for ocular administration. The drug entrapment efficiency increased as the polymer concentration increased. The results of stability studies indicate that the most suitable storage condition for nanoparticles of besifloxacin was at 4-8^◦C. In future this is one of the leading approaches to control bacterial conjunctivitis with besifloxacin through this type of novel drug delivery system.

5.0. ACKNOWLEDGEMENT:

The authors would like to grateful of the authorities of Monad University Hapur, India for funded and providing required facilities to carry out the proposed work and thankful to the Guide- Dr. Amit Singh and Co-Guide, Dr. Rupesh Dudhe for remarkable support during the experimental design and successful completion of designed work.

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16. International Council for Harmonization. ICH Q1A (R2) 2003, Stability testing of new drug substance and drug products., ICH Q2(R2) 2024. Validation of analytical procedure, ICH Q8 (R2) 2009 Pharmaceutical Development, ICH Q14 (2023) Analytical procedure development.

Received on 03.04.2026 Revised on 06.05.2026

Accepted on 08.06.2026 Published on 10.07.2026

Available online from July 25, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(3):171-177.

DOI: 10.52711/2231-5675.2026.00026

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Study model	observation	Zero order	First order	Higuchi model	Peppas model	Hixcrowl model
Freeze	_R2	0.74	0.68	1.0	1.0	0.74
Freeze	K	49.99	- 4.29	9.992	7.38	0.47
Room Temperature	_R2	0.75	0.89	1.0	1.0	0.78
Room Temperature	K	48.20	-4.12	9.996	7.32	0.47
Accelerated stability	_R2	0.74	0.50	1.0	1.0	0.67
Accelerated stability	K	49.65	-2.41	9.97	7.3	0.40