Design, Optimization, and Characterization of Hyaluronic Acid-Conjugated Nanoparticle loaded Gel of Imatinib Mesylate for Targeted Delivery in Dermatofibrosarcoma Protuberans

%EE=Amount of total drug - Amount of free drug in supernantAmount of total drug×100

High EE% ensures accurate dosing, maximized therapeutic efficacy, and minimized off-target effects.

Preparation of Nanogel by Incorporation of Nanoparticles of Imatinib Mesylate

On the basis of Box–Behnken design (BBD) approach, the optimized nanosuspension batch (NANOGEL) will be selected for further formulation of nanogel. The nanogel was prepared using Carbopol-934. Briefly, required amount of Carbopol-934 (2 % w/v) was added into nanosuspension (NANOGEL) and pH was adjusted using triethanolamine with gentle stirring. It allows to stand for 30 min to complete humectation of polymer chains. Similarly, the gel containing unprocessed IMT was also prepared. Prepared IMT-Gel and Nanogel were subjected for various quality control tests viz. Gel viscosity study, Drug content, pH, Spreadability, In-vitro drug diffusion studies, Ex- vivo drug diffusion studies (11) (12).

Evaluation of Gel

Gel Viscosity Study

The rheological property of prepared gel formulations was evaluated by determining the viscosity. Viscosity was measured using Brookfield viscometer equipped with Spindle 64 at 25 °C. (LV-DV III, Brookfield Engineering Laboratory, Incorporation, Middleboro) (13).

Drug Content

The prepared gel formulations were analyzed for drug content by transferring 1 gm of formulation in 100 mL volumetric flask. In this volumetric flask, 50 mL methanol was added, followed by continuous shaking until the gel was totally dispersed to give a clear solution. Final volume was adjusted to 100 ml with the help of methanol. Drug concentration of solution was determined spectrophotometrically at 315 nm using UV‑Visible spectrophotometer (Shimadzu 1800, Japan) (14).

pH Measurement

The pH of Nanogel was measured by using a digital pH meter which was calibrated before use with the standard buffer solutions of pH 4 and 7.1 g of gel was dissolved in 100ml of distilled water and stored for 2 h. The measurement of pH of IMT nanogel was done in triplicate and average values were calculated.(15)

Spreadability

The Spreadability of nanogel as determined by placing 0.5 g of respective gel within a circle of diameter 1 cm, pre-marked on a glass plate over which a second glass plate was placed. A weight of 500 g was allowed to rest on the upper glass plate for about 5 min.(15)

In-Vitro Drug Diffusion Studies

The In-vitro drug release study of Imatinib mesylate gel was determined using a Franz‑diffusion cell. The cellophane membrane was fixed on the receptor cell.(16) The donor cell was filled with 1 g of gel formulation. The receptor compartment was filled with 25mL of phosphate buffer pH 7.4 and constantly stirred with a small magnetic bar at a speed of 50 rpm during the experiments to confirm homogeneity. The 0.5 mL samples were withdrawn at scheduled time intervals (0, 15, 30, 45, 60, 90, 120, 180, 240, 300, 360 min) and were replaced with same volume of pH 7.4 phosphate buffer to maintain the sink condition. Samples were analyzed at 315 nm on UV‑visible spectrophotometer.(15)

Ex-Vivo Drug Diffusion Study

The ex-vivo drug diffusion study of gel was determined using a diffusion cell. In which dermatomed (500 μm thickness) pig’s ear skin (provided by a local slaughterhouse) was mounted between the donor and receptor compartments of vertical Franz-type diffusion cells with an effective permeation area of1.5 cm2. The The donor cell was filled with 1 g of gel formulation. The receptor compartment was filled with 25 mL of phosphate buffer pH 7.4 and constantly stirred with a small magnetic bar at a speed of 50 rpm during the experiments to confirm homogeneity, The 0.5 mL samples were withdrawn at scheduled time intervals (0, 15, 30, 45, 60, 90, 120, 180, 240, 300, 360 min) and were replaced with same volume of pH 7.4 phosphate buffer to maintain the sink condition. Samples were analyzed at 315 nm on UV‑visible spectrophotometer.(15, 17)

Stability Studies

The Imatinib mesylate loaded Nanoparticle incorporated gel optimized formulation (NANOGEL) was subjected for stability evaluation as per ICH guideline. It was evaluated at different temperatures and relative humidity such 25 ± 2 °C / 60 ± 5 % RH and 40 ± 2°C / 75 ± 5 % RH for 1 month. The evaluation parameters were pH, viscosity Spreadability and Assay.(18-20)

RESULT AND DISCUSSION

Formulation of IMT-nanoparticles using box behnken design

Based on the Preliminary Trails a Box-Behnken Design (BBD) was applied to optimize the formulation of an imatinib mesylate-loaded nanoparticles by evaluating the effects of three independent variables on the dependent variable.

Table 1 Results for BBD Design for Optimized Batch

Model fitting and regression analysis

The Imatinib-loaded PLGA nanoparticle was optimized using Box- Behnken design and response surface methodology. ANOVA and F-test analyses confirmed that the independent variables had a significant impact on all response outcomes, as indicated by low p-values (p < 0.05) and high F-values. These results demonstrated a strong correlation between experimental and predicted values. The high R², along with close alignment between adjusted and predicted R² values, further validated the model’s accuracy and reliability. To streamline the models, statistically non-significant terms were removed, resulting in simplified reduced regression equations. These reduced models maintained strong predictive power while improving clarity and efficiency. They will be used for further optimization and interpretation of formulation responses.

Table 2 ANOVA Summary and Model Reduction Analysis for Selected Responses

All models (full and reduced) are highly significant, with Fcalculated > Fcritical and p-values <0.0001 for the models. The models have high R² values (ranging from 0.9916 to 0.9995), showing they explain most of the variation in the responses. The lack of fit tests show that the models are a good fit for the data. The reduced models are simpler but still provide reliable predictions with only minor reductions in model fit compared to the full models. This analysis suggests that the factors involved in the formulation significantly influence the responses, and the models can be used confidently for optimization.

Nonetheless, since both interaction and quadratic terms are crucial, interpreting the equations in isolation could be misleading. Consequently, contour and 3D surface plots were created, which uncovered nonlinear relationships between the variables and responses. These graphical representations assisted in defining the optimal design space and offered a more comprehensive understanding of how variables affect formulation outcomes.

Figure 1 Contour Plots and 3D surface plots for Response Y1, Y2 & Y3

The predicted versus actual plots for particle size (Y₁), polydispersity index (Y₂), and entrapment efficiency (Y₃) collectively demonstrate the excellent predictive performance of the Box–Behnken Design model. In all three plots, the experimental observations closely align with the predicted values, indicating minimal residual error and the absence of systematic bias. The high degree of correlation between predicted and observed responses confirms the adequacy, accuracy, and robustness of the developed regression models. These results validate the suitability of the QbD-based optimization approach for the development of Imatinib Mesylate-loaded PLGA nanoparticles, ensuring reliable prediction of the critical quality attributes and supporting the selection of an optimized formulation for further characterization.

Figure 2 Predicted vs Actual Plot for response Y1, Y2 & Y3

Checkpoint analysis and formulation optimization

The Design of Experiments (DoE) approach was used to evaluate checkpoint of two batches to identify the one with the highest desirability score. Among them, Batch OBIN-2 demonstrated a desirability value of 1 and exhibited the lowest percentage error when compared to OBIN 1. Due to its superior predictive accuracy and performance, OBIN 2 was selected as the final optimized formulation. This batch was subsequently designated as OBIN-IMT-NP and considered the optimized batch for further formulation and evaluation.

Figure 3 Overlay Plot

Table 3 Checkpoint analysis and optimized batch

Characterization of Optimized Imatinib Mesylate Loaded Nanoparticles

Particle Size and Polydispersity Index (PDI)

An average particle size of 234.5 nm and a polydispersity index (PDI) of 0.204 were observed for the optimized formulation. These findings support the formulation results and affirm that OBIN-IMT-NP was successfully created with a suitable size distribution.

Figure 4 Particle size and PDI of optimized nanoparticle

Scanning Electron Microscopy (SEM) of IMT-Nanoparticles

SEM analysis was conducted to examine the size, shape, and surface characteristics of the optimized imatinib-loaded nanoparticles (IMT-NP). The nanoparticles appeared uniformly dispersed, spherical in shape, smooth-surfaced, and nonporous. These morphological features indicate effective formulation and structural integrity. Additionally, the particle size observed via SEM closely matched the values, confirming the accuracy and consistency of the particle size analysis.

Figure 5 SEM image of IMT loaded Nanoparticles

Evaluation of Optimized Nanogel

Viscosity of nanogel

 The average viscosity of IMT nanogel was observed 1904.07 ± 3.28 indicate good reproducibility and uniformity in the formulations compare to the IMT gel.

Drug Content of Optimized Nanogel

The Drug content of Optimized Nanogel is performed using UV-Visible spectrophotometer. IMT Gel compare to IMT nanogel showed an average drug content of 95.86% ± 0.71 and 99.26% ± 0.94.

pH measurement

The pH of the IMT-NPs loaded nanogel was measured using a calibrated digital pH meter at room temperature. The Gel showed a pH value of 7.19 ± 0.014, which is within the acceptable range for topical application and is considered non-irritant to the skin.

Spreadability Study

The IMT nanogel exhibited good average spreadability 28.0 ± 0.29, indicating ease of application and suitability for topical delivery.

In-Vitro drug Release study

The IMT gel showed a gradual increase in drug release from 2.59 ± 0.52% to 32.95 ± 3.56%, whereas the IMT nanogel showed significantly higher release, increasing from 7.58 ± 0.63% to 99.72 ± 6.20% over 360 min. The enhanced release from the nanogel may be attributed to its smaller particle size, increased surface area, and improved drug dispersion, which facilitate faster dissolution and diffusion of the drug.

Table 4 Results of in-vitro drug release study of optimized nanogel

Figure 6 Cumulative drug release study of IMT gel and IMT Nanogel

Release Kinetics of IMT Nanogel

The release kinetics study showed that the drug release from both IMT gel and IMT nanogel followed different mathematical models with varying degrees of correlation. For IMT gel, the highest correlation coefficient was observed for the Korsmeyer–Peppas model (R² = 0.976), with an n value of 0.672, indicating anomalous (non-Fickian) drug release involving both diffusion and polymer relaxation/erosion mechanisms. Similarly, the IMT nanogel showed the highest R² for the Korsmeyer–Peppas model (R² = 0.972), with an n value of 0.664, suggesting a comparable non-Fickian release mechanism. The results indicate that drug release from both formulations was predominantly governed by a combination of diffusion and polymer relaxation/erosion mechanisms.

Table 5 Result of Release kinetic study

Stability study of optimized IMT nanogel

The stability study indicated that the formulation remained stable under accelerated conditions of 40 ± 2°C/75 ± 5% RH, with no significant changes in its evaluated parameters. The pH was maintained at 7.14 ± 0.04, while viscosity and spreadability were 1852 ± 2.23 and 27.67 ± 0.02, respectively. The similarity factor (f₂) of 74.74 (>50) indicated a high degree of similarity between the freshly prepared and stability-tested formulation, suggesting that the formulation maintained its physicochemical characteristics during storage. The dissolution profiles of the reference and test formulations showed a comparable and progressive increase in drug release with time. The test formulation exhibited slightly higher drug release during most of the initial and intermediate time points, while both formulations approached nearly complete drug release at 360 min (≈99%). The close overlap between the two dissolution curves, along with the relatively small standard deviations, indicates good reproducibility and similarity in drug release behavior between the test and reference formulations.

Figure 7 Stability study of optimized nanogel