(T1230-06-37) Development of Chitosan-Coated Indole-3-Acetic Acid Nanoparticles: A Novel Nano Formulation Approach

Purpose: Oxidative stress and chronic inflammation are key contributors to the pathogenesis of cardiovascular diseases and neurodegenerative diseases. Persistent oxidative damage leads to cellular dysfunction, tissue injury, elevated morbidity, and mortality rates.[1] Indole 3 acetic acid (IAA) is a naturally occurring plant hormone classified under the Auxin family, possesses a molecular weight of 175.18g/mol. Beyond its established role as a growth regulator in plants, IAA has also demonstrated significant anti-inflammatory and antioxidant properties.[2] It has been shown to upregulate hemo oxygenase-1 (HO-1), an enzyme for the mitigation of inflammatory responses. Furthermore, its antioxidant potential is evidenced by its ability to reduce the generation of reactive oxygen species (ROS) and nitric oxide, in addition to neutralizing free radicals.[3] These characteristics collectively underscore the therapeutic potential of IAA in preclinical models of cardiovascular diseases. However, its poor aqueous solubility and chemical instability have hindered the clinical translation. Moreover, targeted therapies against oxidative stress remain inadequate. This study elucidates how IAA, when formulated as nanoliposomes shows potential as a novel therapeutic agent overcoming these limitations.

Methods: To formulate IAA as nano liposomes- a Design of Experiments (DOE) approach was employed, utilizing a Box Behnken Design to optimize the critical formulation parameters, including zeta potential, sonication time, and lipid concentration. The BBD was executed using JMP Software, and all 13 experimental runs were formulated as given by design matrix. Nanoliposomes were prepared using the thin film hydration method incorporating DMPG, DSPE PEG 2K, Cholesterol as lipid components. The lipids, along with the drug, were dissolved in a methanol-chloroform mixture and were subjected to solvent evaporation using a rotary evaporator, resulting in the thin film formation. This film was subsequently coated with chitosan and rehydrated with Phosphate Buffered Saline. The resulting mixture was then subjected to probe sonication to obtaain unilamellar vesicles. Following the optimization through DOE, a final batch was formulated under the same conditions. Characterization for Particle size, PDI and Zeta potential were measured using Dynamic light scattering in a Zetasizer. Morphology of the particles was studied using Scanning electron microscopy (SEM), and the entrapment efficiency of the particles was quantified via high performance liquid chromatography.

Results: The box Behnken Design has generated 13 runs to evaluate the influence of the given parameters. After formulating all the 13 batches and performing the analysis in the design matrix, DOE has suggested the optimal formulation to have -27.6mV zeta potential and 198.07 nm particle size. Four experimental runs were performed with the given parameters and the actual results were compared with the predicted results in SPSS software. There was no statistical insignificance found between both experimental and actual batches proving that the model fits. The final formulation had a particle size of 199 nm with 0.4 PDI and -27.1nm zeta potential. SEM imaging has shown even distribution of the small spherical molecules and displayed no aggregation. Entrapment efficiency as determined by the HPLC was found to be 68.2  ± 2.4% suggesting effective entrapment of the drug within the lipid bilayers.

Conclusion: The successful formulation of Indole-3-acetic acid (IAA) as nanoliposomes presents promising results in the development on the antioxidant and anti-inflammatory therapy. Through the systemic optimization, the formulation has achieved desirable physicochemical characteristics. These preliminary findings lay the groundwork for future in-vitro and in-vivo investigation to establish IAA as a novel compound for oxidative stress related cardiovascular diseases.

References: [1] Donia T, Khamis A. Management of oxidative stress and inflammation in cardiovascular diseases: mechanisms and challenges. Environmental Science and Pollution Research 2021;28:34121–53.
[2] Tang J, Li Y, Zhang L, Mu J, Jiang Y, Fu H, et al. Biosynthetic Pathways and Functions of Indole-3-Acetic Acid in Microorganisms. Microorganisms 2023;11. https://doi.org/10.3390/microorganisms11082077.
[3] Ji Y, Yin W, Liang Y, Sun L, Yin Y, Zhang W. Anti-Inflammatory and Anti-Oxidative Activity of Indole-3-Acetic Acid Involves Induction of HO-1 and Neutralization of Free Radicals in RAW264.7 Cells. IJMS 2020;21:1579. https://doi.org/10.3390/ijms21051579.

Acknowledgements:

Funding: American Heart Association AIREA grant to Raquibul Hasan, PhD
This project was also funded through Mercer University Internal grant (# DG00310)


Figure 1: Schematic representation of IAA formulation


Table 01: Formulation specifications given by the design matrix with the experimental values obtained


Figure 2: For Zeta Potential, the model achieved an RMSE of 6.9549 and R² = 0.85, with a significant p-value of 0.0286, indicating a strong predictive capability. For Particle Size, the model yielded an RMSE of 55.189 and R² = 0.83 with a p-value of 0.0395, suggesting a good model fit.