Development, Statistical Optimization and Characterization of Ganciclovir Laden Ethyl Cellulose Nanoparticles: A 3² Factorial Design Approach

Susanta Paul¹*, Subhabrota Majumdar², Mainak Chakraborty³, Soumyadip Ghosh²

¹Department of Pharmaceutical Technology, NSHM Knowledge Campus, Kolkata, West Bengal-700041, India.

²Calcutta Institute of Pharmaceutical Technology and Allied Health Sciences, Uluberia, Howrah-711316, India.

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

*Corresponding Author E-mail: susanta.paul@nshm.com

ABSTRACT:

Keratitis left untreated can lead to blindness. Various factors contribute to ocular keratitis, primarily viral infections such as VZV and HSV. Ganciclovir is effective in treating viral keratitis by inhibiting viral DNA polymerase replication, thereby suppressing viral reproduction and delaying the progression of the disease, which ultimately reduces the risk of corneal scarring and perforation. However, the efficacy of ganciclovir in treating ocular keratitis is limited by challenges related to medication delivery and absorption, particularly with conventional eye drops and ointments that quickly evaporate upon contact with the cornea, necessitating frequent administration that may impact patient compliance. To overcome these limitations, researchers have explored nanoparticle-based drug delivery systems. Nanoparticles, particularly those made from biocompatible and biodegradable materials like ethyl cellulose, offer advantages such as controlled drug release, enhanced bioavailability, and improved ocular permeability. Ethyl cellulose nanoparticles can effectively transport drugs to ocular tissues, protect them from degradation, and improve drug absorption, thereby reducing the frequency of dosing and minimizing side effects. In this study, ganciclovir was incorporated into ethyl cellulose nanoparticles using the emulsion-solvent evaporation method. The formulation parameters, including ethyl cellulose and polyvinyl alcohol concentrations, were optimized using a 3² full factorial design. The optimized formulation (B6) demonstrated the lowest errors and had particle size, zeta potential, and encapsulation efficiency values of 178.1nm, -21.53mV, and 53.51%, respectively. Furthermore, the optimized batch exhibited sustained drug release, with 87.14% release observed at 12 hours. Overall, the study highlights the potential of ethyl cellulose nanoparticles as a promising drug delivery system for improving the treatment outcomes of ocular keratitis, with the optimized formulation showing favorable characteristics for effective and sustained drug delivery.

KEYWORDS: Ganciclovir, Nanoparticle, Optimization, Ocular drug delivery, In-vitro study.

INTRODUCTION:

Ocular keratitis, an inflammation of the cornea, can cause blindness if untreated. It often results from viral infections like varicella-zoster virus(VZV) and herpes simplex virus(HSV). Ganciclovir, an antiviral drug, inhibits viral DNA replication, reducing disease progression and complications^1-5. However, traditional ganciclovir eye drops and ointments have poor corneal penetration and require frequent dosing, affecting patient compliance.

To improve delivery, researchers are developing nanoparticle-based formulations, particularly ethyl cellulose nanoparticles (ECNPs). ECNPs enhance corneal permeation, sustain drug release, and increase bioavailability. In this study, ganciclovir-loaded ECNPs were prepared via emulsion-solvent evaporation, examining the effects of ethyl cellulose and polyvinyl alcohol concentrations on drug release, particle size, and entrapment efficiency over 12 hours. Characterization included surface morphology, zeta potential, and in vitro dispersion. These findings could inform future in vivo studies and advance nanoparticle-based ocular drug delivery systems⁶.

MATERIALS AND METHODS:

Materials

Ganciclovir was procured via Yarrow Chem Products in Mumbai, India. Sigma-Aldrich (Bangalore, India) provided the EC, PVA with molecular weight of 31, 000–50, 000Da, and dichloromethane. Fisher Scientific Co. was the source of all buffer ingredients and solvents (Pittsburgh, PA, USA). All buffers and mobile phases were prepared with the addition of distilled deionized (DDI) water. Sigma-Aldrich provided the remaining necessary ingredients (Bangalore, India)⁷.

Methods:

Drug –polymer interaction study:

The KBr pellet method was employed with an FTIR spectrometer (Alpha – II, Bruker, Germany) in the 400-4000 cm⁻¹ range to analyze samples. Compatibility between the active ingredient and polymers was assessed by comparing their spectra. Thermograms were obtained using a differential scanning calorimeter (DSC) (Pyris 6 DSC, Perkin-Elmer Schweiz AG, Switzerland). Three milligram samples were sealed in aluminum pans and scanned at 10°C/min from 50-350°C. The experiments were repeated twice for accuracy^8-11.

Statistical design of experiments:

The research involved investigating two independent variables within their specified ranges, as outlined in Table 1, along with the dependent variables. The formulations obtained are described in Table 2. Optimization was conducted using a 3-level, 2-component combined response surface design, which incorporates three responses in the quadratic system. The experiments were conducted using the Design-Expert program (Version 13) developed by Stat-Ease Inc. (Minneapolis, MN)^12,13.

Table 1: Response - surface plan variables

Independent - variables	Used levels
	Low ( - 1 )		Medium ( 0 )		High ( + 1 )
Experimental strategy
X ₁: Quantity of E C (milligram)	150.0		200.0		250.0
X ₂: Quantity of P V A (% w/v)	0.50		1.00		1.50
Mathematical optimisation
Dependent-variables		Range of Constraints		Goal
Y ₁: % of Entrapment efficiency (%)		In-range		Maximum
Y ₂: Size of the particle (nanometre)		In-range		Minimum
Y ₃: % of Drug released at 12 hours (%)		In-range		Minimum

Preparation of ganciclovir nanoparticles:

The preparation of ganciclovir nanoparticles involved the use of the biocompatible polymer ethyl cellulose (EC-4) via the emulsion-solvent evaporation method¹⁰. Encapsulating polymers were dissolved in ethyl acetate (EtOAc) to create the organic phase, with amounts ranging from 150 milligrams to 250 milligrams¹⁴. Distilled deionized water was used to dissolve polyvinyl alcohol (PVA) and ganciclovir (GCV) to form the aqueous phases, with concentrations of PVA were ranging from 0.5% w/v to 1.50% w/v and the amount of ganciclovir was same for all batches. Subsequently, the organic phase was homogenized with the GCV and PVA emulsifier-containing aqueous phase using a sonicator probe (Labman, India) at 50% intensity for 30 seconds^{20, 21}. The resulting emulsions were allowed to evaporate overnight at room temperature using a 1000 rpm magnetic stirrer (Remi, Germany). GCV nanoparticles were obtained by centrifuging the resulting nanosuspensions at 18,000 revolutions per minute. The particle size, zeta potential, and % encapsulation efficiency of the nanoparticles were determined after freezing using a freeze dryer (Southern Scientific, India)¹⁵.

Table 2: Response surface design of experimental preparation (runs) and practical outcomes.

Batch code	Independent-variables		Dependent-variables
Batch code	X₁: Quantity of E C (milligram)	X₂: Quantity of P V A (% w / v)	Y₁: % Entrapment efficiency (%)	Y₂: Size of the particle (nm)	Y₃: % Drug released at 12 hours (%)
B ₁	200.0	1.00	52.241	534.42	81.110
B₂	200.0	0.50	48.372	468.51	74.971
B₃	150.0	1.00	55.491	450.51	81.293
B₄	250.0	0.50	50.651	168.81	76.470
B₅	250.0	1.50	52.563	153.31	86.011
B₆	250.0	1.00	53.515	178.12	87.141
B₇	150.0	1.50	62.321	371.0	77.230
B₈	200.0	1.50	57.224	475.61	77.670
B₉	150.0	0.50	49.332	574.31	78.221

Determination of particle size, zeta potential and size distribution:

Using the zetasizer (Malvern Instruments, Malvern, UK) and the dynamic light scattering (DLS) method, the particle size and size distributions of the freshly made nanosuspensions were ascertained. An operating temperature of 25°C was used for the measurements. For every sample, five size measures were taken at the same time^16-19.

Determination of encapsulation efficiency:

The same technique was employed to determine the drug loading and encapsulation efficiency in NP. By measuring the expected number of nanoparticles necessary to ideally contain 150 mg of the ganciclovir, the amount of ganciclovir (GCV) in the EC nanomaterials was determined. To fully decompose the 150 mg of freeze-dried NP, it was diluted in 2 milliliters of dichloromethane and vigorously agitated for one minute. The resulting supernatant was collected for analysis after centrifugation at 12,000 rotations per minute for 10 minutes followed by vortexing. A double beam spectroscopy (UV-spectrophotometer, Shimadzu 1800, Japan) operating at a wavelength of 256 nm was used to measure the concentration of GCV. The following equation was employed to calculate the encapsulation efficiency of several batches of nanoparticles. Three separate measurements were conducted²⁰.

Initial drug added – Amount of free unentrapped drug

% Entrapment = ---------------------------------------------- x 100

efficiency Initial drug added

In-vitro drug release study:

In the in-vitro drug release study, Ganciclovir (GCV) loaded nanoparticles were evaluated using a dialysis sack with a 12,000–14,000 kDa molecular weight cut-off. Samples (5 mL) were immersed in simulated tear fluid (STF) and agitated at 50 rpm. Sampling occurred at set intervals (1, 2, 3, 4, 6, 8, and 12 hours), replacing withdrawn samples with fresh medium. UV spectrophotometry (Shimadzu 1800) at 256 nm post-dilution measured drug concentration. Various kinetic models (zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson–Crowell) were applied to interpret release data, selecting the model with the highest coefficient of regression (R²) value to determine the releasing mechanism²¹.

Surface morphology of nanoparticle:

The freeze-dried nanoparticles were placed on one side of an adhesive for scanning electron microscopy examination. Subsequently, gold was applied to the dry nanoparticles. The surface morphology of the pellet was investigated using a scanning electron microscope (FEI Quanta-200 MK2) with electron microscopy capabilities²¹.

Optimization by factorial design:

Optimization was conducted using a complete factorial design with 2 components at three levels each to optimize the response of the variables. The two factors that were manipulated were the quantity of polyvinyl alcohol (PVA) (X₂) and ethyl cellulose (EC) (X₁). These factors were appropriately labeled as -1, 0, and +1, indicating low, medium, and high levels, respectively²¹. The response variables included the percentage of drug released at 12 hours, the size of particles (nm) (Y₂), and the encapsulation efficiency (EE) (%) (Y₁). Experimental trials were conducted in this design for each of the nine potential combinations. Throughout the investigation, all other preparation factors and processing variables remained constant. The polynomial response is represented by equation:

Y = b₀ + b₁A + b₂B + b₃AB + b₄A²+ b₅B²

In this equation, 'Y' represents the response variable. The coefficients of regression and the impact of each factor are denoted by b₀, b₁, b₂, b₃, b₄, and b₅. Parameters A and B correspond to the factors in the equation, and the interaction between the two variables is represented by the term AB. The validity of the model and the significance of each parameter can be assessed using an ANOVA One-Way model.

RESULT:

Statistical designs of experiment and formulation of ganciclovir (GCV) nanoparticles:

The statistical design of experiments and formulation of ganciclovir (GCV) nanoparticles involved allocating variable factors to the response surface as depicted in (Table 1). This formed the basis for the surface response design. Subsequently, nine formulations were arranged based on the response surface elements listed in (Table 2). A quadratic model with two distinct factors and three responses was utilized to construct the design.

Drug –polymer interaction study:

The FTIR spectra of pure GCV or the mixtures of GCV and EC showed no discernible interaction between EC and GCV (Fig. 1). According to the DSC analysis, no observable endotherm was detected, suggesting that the GCV is present in a molecularly dispersed state. The DSC thermograms of all three materials displayed in (Fig. 2) revealed the fusion endotherms of pure drug, ethyl cellulose, and a physical mixture of GCV and EC at 255.25°C and 57.50°C, respectively.

Fig. 1: FTIR spectra of (a) ganciclovir in pure form (b) ethyl cellulose in pure form (c) physical mixture of ganciclovir and ethyl cellulose.

Fig. 2: DSC thermograms of (a) ganciclovir in pure form (b) ethyl cellulose in pure form (c) physical mixture of ganciclovir and ethyl cellulose.

Zeta potential, particle size, and polydispersity index:

Table 3 summarizes the particle size, zeta potential, and polydispersity index (PDI) of various formulations containing GCV-loaded nanoparticles. The particle sizes ranged from 153.3 nm to 574.3 nm. Zeta potential, crucial for colloidal stability, ranged from -8.7 mV to -36.6 mV, indicating stable colloidal systems. The PDI, a measure of particle size distribution, ranged from 0.179 to 0.751, suggesting high particle homogeneity. Fig. 3 illustrates the zeta potential and particle size of the optimized batch.

Fig. 3: The optimised formulation batch (B₆)'s (a) zeta potential and (b) Size of the particle.

Table 3: The zeta potential, polydispersity index, and particle size of ganciclovir nanoparticles containing varying amounts of polyvinyl alcohol and ethyl cellulose.

Formulation code	Size of the particle (nm)	Zeta-potential (m V)	P D I
B ₁	534. 4	-31. 44	0. 751
B ₂	468. 5	-22. 33	0. 646
B ₃	450. 5	-29. 9	0. 598
B ₄	168. 8	-16. 6	0. 179
B ₅	153. 3	-8. 7	0. 195
B ₆	178. 1	-21. 53	0. 2044
B ₇	371. 0	-23. 26	0. 491
B ₈	475. 6	-24. 4	0. 592
B ₉	574. 3	-36. 6	0. 733

Entrapment efficiency:

Table 2 shows the encapsulation effectiveness (% EE) of nanoparticles for ophthalmic delivery, ranging from 48.37% to 62.32%. This variation reflects differences in drug encapsulation efficiency across preparations. The highest % EE of 62.32% indicates more effective encapsulation, potentially enhancing drug delivery and therapeutic effects, while the lowest % EE of 48.37% suggests room for improvement.

These % EE values highlight the need to optimize nanoparticle formulation for stable and effective drug encapsulation in ocular administration. Optimization can ensure consistent therapeutic delivery and minimize the wasteful use of active pharmaceutical ingredients. Future research could focus on identifying variables influencing encapsulation efficiency and refining manufacturing processes to enhance drug loading and delivery efficiency.

In-vitro drug release study:

The nanoparticles designed for ocular use underwent a 12-hour in vitro drug release study across nine batches (B1-B9). Results showed consistent drug release patterns, indicating potential for prolonged ocular administration. Initially, no drug release was observed, confirming successful encapsulation. Gradual, controlled release followed, with B5, B6, and B9 exhibiting higher release percentages, possibly due to faster kinetics or increased drug loading. B1, B2, and B3 showed lower release, indicating reduced loading or slower kinetics. A burst release phase occurred between three to six hours, followed by a plateau, indicating sustained release. This sustained release is advantageous for ocular applications, ensuring prolonged efficacy and reduced dosing frequency.

Kinetic analysis of the GCV release:

The release data for all developed formulations were analyzed using the Higuchi, Korsmeyer-Peppas, Zero-order, and First-order models. Table 4 presents and illustrates the 'R' values for the first-order, zero-order, Higuchi's kinetic model, and Korsmeyer-Peppas approach. The study indicates that the first-order model, which represents a non-Fickian diffusion approach, most accurately describes the release kinetics of the produced GCV nanoparticles.

Fig. 4: Cumulative percentage of ganciclovir released vs time curve for (a) batches B₁, B₂, and B₃ (b) batches B₅, B₆, and B₉; and (c) batches B₄, B₇, and B₈.

The surface structure of nanoparticles:

The surface morphology of the nanoparticles is depicted in Figure 5, showcasing the surface structure of the dried GCV nanoparticles as observed through scanning electron microscopy (FEI Quanta-200 MK2, Netherlands).

Fig. 5: SEM image of a prepared nanoparticle loaded with ganciclovir.

Formulation optimisation by statistical analysis and experimental design:

Formulation optimization was conducted using a 3² full-factorial design to optimize the incorporation of the active ingredient into nanoparticles, as outlined in Table 1. The variables dependent on this investigation were the quantities of polyvinyl alcohol (PVA) and ethyl cellulose polymer (EC-4). These quantities were adjusted based on the outcomes of multiple trial batches at three different levels: low (-1), medium (0), and high (+1). The responses observed were the entrapment efficiency percentage (%), particle size (nm), and the percentage of drug released (%) at 12 hours. The different responses investigated were represented using a quadratic equation, as follows:

Entrapment efficiency (%) = 52.83 + 1.73 A + 3.95 B + 2.77 A B + 1.36 A ² + 0.33B ²

Where, F-value = 28.19 ; R ² = 0.97920 ; P < 0.05

Particle size (nm) = 505.55 + 149.26 A + 35.28 B + 46.95A B + 176.83 A ² + 19.08 B ²

Where, F-value = 18.520 ; R ² = 0.96860 ; P < 0.05

% of drug released at 12 hrs. (%) = 81.070 + 2.14 A + 1.87 B + 2.63 A B + 3.14 A ² + 4.74B ²

Where, F-value = 31.800; R² = 0.98150; P < 0.05

Table 4: In-vitro release kinetic data of nanoparticles loaded with ganciclovir.

Batch code	Zero-order model		Higuchi kinetic model		Korsmeyer-peppa’s model		First-order model
Batch code	r ²	k ₀	r ²	k _h	r ²	n	r ²	k ₁
B ₁	0.9185	8.2634	0.9072	31.723	0.8672	1.7880	0.9817	-0.082
B ₂	0.9237	7.360	0.9113	28.281	0.8658	1.7960	0.9832	-0.062
B ₃	0.9728	8.1220	0.9198	30.511	0.9452	1.4850	0.9761	-0.079
B ₄	0.9486	7.7650	0.919	29.531	0.9133	1.5850	0.9843	-0.070
B ₅	0.9367	8.640	0.915	33.020	0.9171	1.5540	0.9835	-0.097
B ₆	0.9372	8.630	0.924	33.130	0.9362	1.2850	0.9764	-0.104
B ₇	0.9513	7.8340	0.918	29.752	0.9260	1.5600	0.9855	-0.071
B ₈	0.9443	7.90	0.908	29.962	0.8834	1.6490	0.9813	-0.071
B ₉	0.8968	7.740	0.912	30.192	0.8860	1.4020	0.9784	-0.073

Table 5: An ANOVA summary for quadratic response surface models.

Foundation	Sum of square	Degree of freedom	Mean square	‘F’- value	‘P’- value
Encapsulation efficiency (%)
Model	146.760	05	29.350	28.190	0.01000
A - Quantity of ethyl cellulose	18.100	01	18.100	17.380	0.02510
B - Quantity of polyvinyl alcohol	94.010	01	94.010	90.280	0.00250
A B	30.690	01	30.690	29.470	0.01230
A ²	3.740	01	3.740	3.590	0.15450
B ²	0.22890	01	0.22890	0.21990	0.67110
Size of the particle (nanometre)
Model	2.1320	05	42647.660	18.520	0.01830
A - Quantity of ethyl cellulose	1.3370	01	1.330	58.050	0.00470
B - Quantity of polyvinyl alcohol	7469.480	01	7469.480	3.2400	1.16950
A B	8817.210	01	8817.210	3.8300	0.14530
A ²	62540.03	01	62540.060	27.160	0.01370
B ²	728.350	01	728.350	0.31630	0.61310
% of drug released at 12 hrs (%)
Model	141.370	05	28.270	31.800	0.00840
A - Quantity of ethyl cellulose	27.580	01	27.580	31.010	0.01140
B - Quantity of polyvinyl alcohol	21.160	01	21.160	23.790	0.01650
A B	27.730	01	27.730	31.190	0 .01130
A ²	19.800	01	19.800	22.260	0 .01800
B ²	45.100	01	45.100	50.720	0 .00570

DISCUSSION:

Ganciclovir (GCV) and ethyl cellulose (EC) in nanoparticles show no significant chemical interaction, confirmed by FTIR and DSC. Both retain their thermal properties. Key parameters like particle size (153.3-574.3 nm), zeta potential (-8.7 to -36.6 mV), and encapsulation efficiency (48.37%-62.32%) were assessed. GCV-loaded nanoparticles demonstrated stable in-vitro drug release over 12 hours, with a first-order release model indicating non-Fickian diffusion. Using a 3² full-factorial design, varying polyvinyl alcohol (PVA) and EC optimized drug release, particle size, and entrapment efficiency. Statistical optimization identified formulation F6 as optimal. These findings enhance nanoparticle formulations for ocular drug delivery, improving therapeutic efficacy and patient outcomes.

CONCLUSION:

The study optimizes ganciclovir-laden ethyl cellulose nanoparticles for ocular drug delivery using a 3² factorial design. By varying polyvinyl alcohol and ethyl cellulose concentrations, researchers identified conditions for sustained release and high encapsulation efficiency. Characterization confirmed favorable physical properties and controlled release kinetics, indicating potential for effective ocular drug delivery. These findings underscore the value of statistical optimization and suggest promising applications in ocular treatments, pending further clinical validation.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This research was done at Department of Pharmaceutical technology, NSHM Knowledge Campus, Kolkata. We thank everyone for establishing a conducive setting for experiments.

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Received on 03.04.2024 Revised on 25.07.2024

Accepted on 10.10.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):537-544.

DOI: 10.52711/0974-360X.2025.00080

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