Acyclovir Nanoparticle-Loaded Cream: A Novel Topical System for Enhanced Antiviral Delivery

Nanotechnology has significantly advanced since the 19th century, producing materials with improved strength, stability, and unique nanoscale properties that have revolutionized healthcare, catalysis, electronics, and diagnostics1. In drug delivery, nanoparticles offer advantages such as targeted delivery, controlled release, and reduced systemic side effects compared to conventional formulations2,3. Acyclovir, an antiviral drug used against herpes simplex and varicella zoster, suffers from poor skin penetration and low systemic bioavailability (10–26.7%). Thus, there is a pressing need to improve its delivery through innovative nanocarrier-based topical formulations.

Nanoparticles (1–1000 nm) are advanced drug carriers with unique physicochemical properties such as small size, large surface area, and tunable surface chemistry1. They can encapsulate, adsorb, or bind therapeutic agents, enhancing stability, bioavailability, targeted delivery, and controlled release while reducing systemic side effects2,3.Common types include polymeric nanoparticles, liposomes, solid lipid nanoparticles, metallic nanoparticles, and nanocrystals, often prepared using methods like nanoprecipitation, ionic gelation, solvent evaporation, or interfacial polymerization4. Biodegradable polymers such as chitosan, CMC, polylactide, and PEG are widely used for safe formulations5.Applications such as cancer therapy, infectious disease treatment, vaccine and gene delivery, and biomedical imaging6. In topical drug delivery, nanoparticles in semisolid bases (creams, gels, ointments) improve skin penetration, sustained release, local targeting, and patient compliance, making them a promising strategy to enhance therapeutic efficacy and safety7.

1.1 Review of Literature

1. May Bin-Jumah et al;2020, (International Journal of Nanomedicine) developed clarithromycin-loaded chitosan nanoparticles using ionotropic gelation. The optimized formulation (152 nm, EE 70.05%, zeta potential +35.2 mV) showed sustained drug release (82.98% in 12 h), 2.7-fold higher corneal permeation, and improved antibacterial activity, suggesting potential for bacterial conjunctivitis treatment.
2. Karol Yesenia Hernandez et al;2020, (RSC Advances) investigated formulation parameters in the preparation of PLGA nanoparticles via emulsification-solvent evaporation and nanoprecipitation. Particle size and uniformity were significantly influenced by polymer and PVA concentrations, agitation speed, and cryoprotectants. Optimized conditions yielded stable, reproducible nanoparticles suitable for therapeutic use.
3. Ramatu Omenesa Bello et al;2020 (Nanomaterials) formulated acyclovir-loaded solid lipid nanoparticles (SLNs) with Biogapress Vegetal 297 ATO and Tween 80 to improve oral bioavailability. The optimized SLNs (134 nm) achieved a four-fold higher plasma acyclovir concentration compared to commercial suspension, indicating a promising alternative for oral delivery.
4. Haniza Hassan et al;2021, (Molecules) developed acyclovir-loaded SLNs using Compritol 888 ATO to enhance oral bioavailability and reduce dosing frequency. The optimized nanoparticles (108.67 nm, EE 91.05%) demonstrated stability for 3 months and a five-fold increase in oral bioavailability in rats compared to commercial suspension.

The objective of this study is to overcome the limitation of poor skin penetration commonly observed with conventional topical acyclovir formulations. To achieve this, a novel nanoparticle-based topical formulation will be developed with the aim of enhancing drug permeation, improving localized retention, and ultimately increasing the therapeutic efficacy of acyclovir.

2. MATERIALS AND METHODS

Acyclovir (Yarrow Chem, Mumbai), chitosan (Sisco Research Laboratories, Maharashtra), carboxymethyl cellulose (LOBA Chemie, Mumbai), and sodium tripolyphosphate (Nice Chemicals, Kochi) were used. Materials were weighed using an electronic balance (Prince Scale Industries, Ahmedabad). Characterization was performed using FT-IR (Jasco FT/IR-4700), UV–Visible spectrophotometer (Systronics, Ahmedabad), SEM (Hitachi SU 3500), and zeta potential analyzer (Microtrac Nano Trac, USA). A magnetic stirrer (Rotek Equipments, Kerala) was used for mixing.

2.1 Determination of UV λmax

10mg of acyclovir were accurately weighed and dissolved in 10ml of acetate buffer (pH 5.5) in a volumetric flask to obtain a 1000 µg/ml stock solution. From this, 1 ml was diluted to 10 ml with the same buffer to prepare a 100 µg/ml working solution. Acetate buffer was used as the blank, and the UV absorbance of the solutions was scanned from 200–400 nm to determine the λmax8.

2.2 Preparation of standard calibration curve of Acyclovir in Acetate buffer (pH 5.5)

10 mg of acyclovir was dissolved in  10 ml acetate buffer (pH 5.5) to prepare a 1000 µg/ml stock solution. From this, 1 ml was diluted to 10 ml to obtain a 100 µg/ml working solution. Serial dilutions (0, 2.5, 5,...15) were prepared, and absorbance was measured at 252 nm using acetate buffer as the blank. A standard curve was then plotted between absorbance and concentration9.

2.3 Solubility studies

To determine the solubility of Acyclovir in various solvents, saturation solubility studies were conducted.The pure drug’s solubility was tested in distilled water, ethanol, acetate buffer (pH 5.5), and acetic acid. The solubility was found from the experimental data9.

2.4 Determination of melting point

The melting point of Acyclovir was found out by using the capillary tube method.A small amount of the drug was placed in a capillary tube sealed at one end and then inserted into a melting point apparatus. The temperature at the point when the drug melted was recorded. The average of triplicate measurements was used as the final melting point value10.

2.5 Drug and excipient compatibility study

FT-IR spectroscopic method was used to conduct drug-excipient compatibility study. FT-IR spectra of pure drug, chitosan, CMC, STPP and their mixture, were taken by KBr pellet technique between 400-4000cm-1 . Once the spectra were recorded, the peaks were compared for incompatibility.

2.6 Preparation of Acyclovir  Nanoparticle By Ionic Gelation Method.

• The ionic gelation method was utilized to produce Acyclovir Nanoparticle
• Dissolve 100 mg of chitosan in 10 ml of 1% (v/v) acetic acid. Stir the chitosan solution for 3–4 hours at room temperature until a clear solution is obtained.
• Add 120 mg of acyclovir into the chitosan solution.Stir the mixture thoroughly to form a homogeneous drug-polymer solution.
• Prepare 5 ml of 0.84% (w/v) sodium tripolyphosphate (STPP) solution in distilled water.
• Add the STPP solution dropwise into the chitosan-acyclovir mixture under magnetic stirring.
• Maintain stirring at 800–1000 rpm for 30–60 minutes.
• Ionic crosslinking occurs between positively charged chitosan and negatively charged STPP, forming nanoparticles.
• Centrifuge the suspension at 10,000–15,000 rpm for 20–30 minutes.Wash the resultant nanoparticle pellet with distilled water to remove excess STPP or unreacted components.
• Dry the washed nanoparticles in a hot air oven at an appropriate temperature until a dry powder is obtained.

Table No. 1 : Formulation table of Acyclovir Nanoparticle

2.7.1 FTIR-spectrophotometer:

In order to find out the possible interactions between drug and polymers used in formulation, Fourier Transform Infra-red Spectroscopy(FT-IR) analysis was carried out on pure substances and their physical mixtures. FT-IR spectra of pure drug,Chitosan, CMCand their physical mixtures were carried out by direct method between 400-4000 cm²¹. This is to ensure that there is no incompatibility between drug and polymers. Once spectra was recorded, the peaks of pure drug, polymers and physical mixtures of polymers, drug and penetration enhancers were compared for incompatibility

2.7.2 Determination of drug content and entrapment efficiency:

Nanoparticle formulations in acetate buffer (pH 5.5) were analyzed for drug content by dissolving and diluting samples to 10 µg/mL, then measuring absorbance with a UV–Visible spectrophotometer at 252nm wavelength; entrapment efficiency was determined by centrifuging the samples, removing the supernatant, and measuring the amount of free drug remaining11.

% DEE = (Practical drug loading/ Theoretical drug load) x 100

2.7.3 In-vitro drug release study by dialysis diffusion method.

Eggshell membranes were isolated, washed, hydrated in a buffer, and mounted in a diffusion cell. The donor contained the drug formulation, and the receptor contained an acetate buffer (pH 5.5) under constant stirring. Samples were withdrawn at 0, 1, 2, 4, 5, 6, and 7?h, replaced with fresh buffer, and drug concentration was measured by UV-Visible spectrometer to plot the cumulative release profile12.

2.7.4 Optimization By Design Expert Stat Ease Software Version-13

Statistical design of experiments using Design-Expert Software (Version 13) was applied to optimize formulation. Chitosan and CMC amounts were selected as factors, while entrapment efficiency and in vitro drug release were responses. Nine trials were conducted, contour plots generated, and the best formulation identified based on optimization criteria.

2.7.5 Scanning Electron Microscopy (SEM)

Nanoparticle morphology, size, and surface characteristics were examined using SEM. Samples were diluted (1:5) with ultra-pure water and a drop was placed on polished aluminum for imaging13.

2.7.6 Zeta potential

Vesicle size, polydispersity, and zeta potential were measured using a Zetasizer (Nano ZS 90) after diluting 100?µL of formulation in acetate buffer (pH 5.5) in a disposable capillary cell14.

2.7.7 Drug release kinetics

Drug release data of optimized nanoparticles were fitted to various kinetic models to reveal the drug release mechanism from the nanoparticle. Those consist of Zero order, first order, Higuchi model and Korsmeyer-Peppas plot and R2 values were determined.

1. Zero Order Kinetics Model – Cumulative %drug release versus Time T.
2. First Order Kinetics Model - Log cumulative percentage drug remaining versus Time T.
3. Higuchi’s model – cumulative percent drug release versus square root of Time T.
4. Korsmeyer- Peppas model –Log cumulative percent Drug released versus log time.

2.8 Formulation of Acyclovir loaded Nanoparticle cream:

To make an oil-in-water (O/W) cream base, first heat the oily ingredients (like stearic acid, cetostearyl alcohol, and mineral oil) together at 75°C. At the same time, heat the water-based ingredients (like water, glycerin, triethanolamine, and methyl paraben) to the same temperature. Gradually pour the melted oil phase into the water phase while stirring constantly to create a uniform blend. Continue stirring until the cream cools to room temperature15.

2.8.1 Incorporation of formulated nanoparticles in o/w cream base:

15g of cream base and prepared nanoparticles (equivalent to the dose) were mixed on tile using geometric mixing pattern part by part16.

2.9 Evaluation parameters of Fexofenadine HCL loaded Nanoparticle cream:

Spreadability:

The spreadability of the cream was measured by spreading 0.5g of the cream on a circle of 2cm diameter premarked on a glass plate and the second glass plate was employed. A weight of 500 grams was placed on the upper glass plate for 5 minutes, after which the spread diameter of the cream was measured. The spreadability (S) can be measured by the using the formula17.

S = m* l/t.

2.9.1 Viscosity measurements:

Viscosity of formulated cream of acyclovir nanoparticle was measured by Brookfield viscometer. Keeping the nanoparticulate cream under the Brookfield viscometer at room temperature, set the rpm at 100, set the spindle No-6 and obtained values are noted18.

2.9.2 pH:

The pH of the formulation was measured with a digital pH meter using a glass electrode. The glass electrode is dipped into the solution and the reading which is showing on the display is noted down19.

2.9.3 In vitro drug release:

Nanoparticle cream was applied on egg membrane in a Franz diffusion cell with acetate buffer (pH 5.5) at 37?±?1°C and stirred at 500?rpm. Samples (1?mL) were withdrawn at 0, 1, 2, 4, 5, 6, and 7?h replaced with fresh buffer, and drug concentration measured by UV spectrophotometry at 252?nm20.

3. RESULT AND DISCUSSION

3.1 Determination of UV λmax

Fig no. 1: UV spectrum of Acyclovir in Acetate buffer (pH 5.5)

The pure drug of Acyclovir was scanned by UV spectroscopy and λmax was found to be 252nm which complies with the official standards.

3.2 Standard calibration curve of Acyclovir in acetate buffer (pH5.5)

Acyclovir solution in acetate buffer (pH 5.5) showed a λmax at 252?nm, which was used to construct the standard calibration curve.

Absorbance of Acyclovir standards (0–15?µg/mL) in acetate buffer was measured. The curve was linear, obeying Beer-Lambert’s law.

Table no 2: concentration vs absorption

Fig no. 2: Standard calibration curve of Acyclovir in acetate buffer (pH 5.5)

Acyclovir was analyzed using UV spectroscopy in acetate buffer (pH 5.5), and the maximum absorbance (λmax) was found at 252 nm, which matched the official standard value. A calibration curve was prepared at this wavelength for concentrations between 0–15 μg/ml. The curve was linear across the tested range and followed Beer–Lambert’s law.

3.3 Solubility studies

Table No 3.: Solubility profile of Acyclovir in different media

Solubility studies revealed that the drug was slightly soluble in distilled water and ethanol, whereas it was soluble in acetic acid and acetate buffer (pH 5.5).

3.4 Melting point determination

Melting point of Acyclovir was found to be 256 ± 0.01oC (n=3)

3.5 Drug – excipient compatibility

Both the excipients and pure drugs infrared spectra are examined. It has been found in this investigation that there is no significant shifting of the peaks, indicating that there was no physical interaction due to bond formation between the drug and excipients.

Fig no. 3: FTIR spectrum of Drug + chitosan + CMC + STPP

The FT-IR spectrum of Acyclovir exhibited characteristic peaks at 786 cm?¹ (Ar–CH stretching), 1386 cm?¹ (C–N stretching), 1509 cm?¹ (C=N stretching), and 1729 cm?¹ (C=O stretching), confirming the presence of its functional groups. A comparison of the spectra of pure Acyclovir and its physical mixture with polymers revealed no additional or shifted peaks, indicating the absence of any significant drug–excipient interactions.

3.6 EVALUATION PARAMETERS OF NANOPARTICLE:

3.6.1 Drug Content

The Drug Content of the prepared nanoparticle were conducted and the results are illustrated in the table below.

Table No 4. Drug content of Acyclovir loaded Nanoparticle

All values are expressed as mean ± SD, n=3.

Fig no. 4: Drug Content of Nanoparticle

The drug content of the prepared Acyclovir-loaded nanoparticles ranged from 60.2% to 82.3% across the formulations. Among them, F7 showed the highest drug content, while F8 exhibited the lowest. These results indicate that increase in polymer concentration affected the amount of drug incorporated into the nanoparticles

3.6.2 Entrapment efficiency (%)

Table no 5: Entrapment efficiency of Acyclovir loaded nanoparticles

All values are expressed as mean ±SD , n=3

Fig No 5. Entrapment Efficiency of Nanoparticle

The entrapment efficiency of the nanoparticles varied between 61.7% and 89.3%. F7 showed the highest entrapment efficiency, whereas F5 had the lowest. This suggests that an increase in polymer concentration favors better drug retention within the nanoparticles.

3.6.3 In vitro diffusion study

Table 6: Percentage in-vitro diffusion study of formulations

Fig no. 6 in vitro drug release of fomulation (F1-F9)

The in vitro diffusion study of Acyclovir-loaded nanoparticles was performed using a Franz diffusion cell with acetate buffer (pH 5.5) as the diffusion medium. Among the formulations, F7 showed the lowest release 59.74% at 420min. F8 exhibited the highest cumulative drug diffusion, These results indicate that an increase in polymer concentration contributes to a more sustained drug release from the nanoparticles.

3.6.4. Optimization by design expert stat ease software

The formulation is optimized by Design expert Software version 13.0.7.0. Central composite design was used to find the optimized formulation. Two input factors chitosan and CMC was selected and concentration of these factors for preparing the formulations were suggested by the software.9 formulations were prepared and the values of responses, ie., drug entrapment efficiency and in vitro drug release were given to the software for optimization. After optimization of the analyzed data, one was selected by considering the drug entrapment efficiency and in vitro drug release. The batch with chitosan-200mg and CMC-200mg with desirability 1 was found to be optimum.

Table no 7: Numerical test results of model adequacy checking for influence of independent variables on response variables

Fig no 7: 3-D response surface plot showing the effect of Chitosan and CMC for

Fig no 8: 3-D response surface plot showing the effect of amount of Chitosan and CMC for in vitro drug release (%)

Fig no. 9: Overlay plot of optimized formulation

Table 8: Desirability table

Table 9: Response values of predicted, experimental and percentage error obtained at optimal levels of the factors

Fig no.10: Optimized formulation of Acyclovit loaded nanoparticles

3.6.5 Surface morphology

The surface morphology and shape features of the microspheres were studied with the help of scanning electron microscopy (SEM). High-resolution images were captured at appropriate magnifications. The photograph is shown below:

Fig no.11: SEM of Acyclovit loaded nanoparticles

The surface morphology of the nanoparticles was studied with the help of scanning electron microscopy (SEM) to assess their shape and size. The resulting image revealed that the prepared nanoparticles were spherical in shape and in nano range

3.6.6 Zeta Potential

Fig no.12: Zeta Potential of Acyclovit loaded nanoparticles

Zeta potential of the formulation F7 was observed, the value was – 8.4 (mv) which indicates the prepared formulation was stable.

3.6.7 Particle size distribution

Fig no.13:Particle size distribution of Acyclovit loaded nanoparticles

Particle size of the formulation F7 was observed, the value was  211.3 (nm) which indicates the prepared formulation was in the nano range.

3.6.8 Drug release kinetics

The release kinetics of the optimized Acyclovir-loaded nanoparticle formulation (F7) followed the Higuchi model, indicating a diffusion-controlled mechanism. The Korsmeyer–Peppas release exponent (n = 0.714) further confirmed anomalous (non-Fickian) diffusion, suggesting that drug release occurred through a combination of diffusion and erosion-controlled processes

Table 10:  Kinetic studies of formulation of Nanoparticle

Fig no 14: zero order release kinetics of optimized formulation

fig no.15: first order release kinetics of optimized formulation

Fig no.16: higuchi plot of optimized formulation

Fig no.17: korsmeyer-peppas plot of optimized formulation

The release kinetics of the optimized Acyclovir-loaded nanoparticle formulation F7 were analyzed using zero-order, first-order, Higuchi, and Korsmeyer–Peppas models. The correlation coefficients (R²) were 0.900 for zero-order, 0.944 for first-order, 0.984 for Higuchi, and 0.947 for Korsmeyer–Peppas. The drug release data was best described by the Higuchi model, indicating a diffusion-controlled release mechanism. The release exponent (n) from the Korsmeyer–Peppas model was 0.714, suggesting anomalous (non-Fickian) diffusion, where drug release is governed by a combination of diffusion and erosion-controlled mechanisms.

3.6.9 STABILITY STUDIES

Accelerated stability studies were carried out for optimized formulation as per ICH guidelines. From the stability studies data which was carried out for a period of 3 months showed that the optimized formulation passes stability studies with no significant changes in drug entrapment efficiency and invitro drug release. The results of stability data were shown in Table

Table 11: Stability studies of optimized formulation

3.7 Evaluation of Nanoparticle loaded Cream

3.7.1 Spreadability

Table 12: Spreadability of Nanoparticle loaded cream

Mean (X ± S.D) (n = 3)

3.7.2 Viscosity measurements

Table 13: Viscosity of Nanoparticle loaded cream

Mean (X ± S.D) (n = 3)

3.7.3 Determination of pH

Table 14: pH of Nanoparticle loaded cream.

Mean (X ± S.D) (n = 3)

3.7.4 In-vitro drug release study

Table 15: In-vitro drug release of Nanoparticle loaded cream

Fig no.18: In-vitro drug release of Nanoparticle loaded cream

3.7.5 Kinetic evaluation of Nanoparticle loaded cream

Table 16:  Kinetic studies of formulation of Nanoparticle loaded cream (r2 )

Fig no.19: zero order release kinetics of Nanoparticle loaded cream

fig no.20: first order release kinetics of Nanoparticle loaded cream

Fig no.21: Higuchi plot of nanoparticle loaded cream

Fig no.22: Korsmeyer-peppas plot of Nanoparticle loaded cream