β‐Cyclodextrin Inclusion Complex of d ‐Limonene: A Promising Biocompatible Formula for Enhanced Wound Healing—In Vitro and In Vivo Studies

Wound healing, a complex multifactorial process, continues to pose a challenge, justifying the search for innovative therapeutic approaches to accelerate recovery. In this study, β-cyclodextrin-encapsulated d-limonene (βCD-d-limonene) was formulated and investigated as a promising strategy for wound management. The inclusion complex was successfully synthesized via a simple approach and thoroughly characterized, confirming enhanced physicochemical stability, encapsulation efficiency, and thermal stability. The βCD-d-limonene nanoparticles exhibited a high encapsulation efficiency of 91.97% ± 3.42%. Antimicrobial assessments demonstrated significantly higher antibacterial efficacy of βCD-d-limonene compared to free d-limonene against both Gram-positive and Gram-negative bacteria. A significant 90-fold decrease in the minimum inhibitory concentration (MIC) was recorded for the most susceptible bacterium, Pseudomonas aeruginosa (P. aeruginosa), with values decreasing from 111.2 mg/mL for free d-limonene to 1.24 mg/mL for the βCD-d-limonene complex. In vivo, wound healing studies revealed faster wound closure, reduced inflammation, and improved tissue regeneration. Gene expression analysis demonstrated modulation of key markers such as IL-6, MMP3, BAX, VEGF, and TGF-β1, supporting the inclusion complex's (βCD-d-limonene) role in regulating inflammation, apoptosis, and angiogenesis. Histological and immunohistochemical evaluations confirmed enhanced tissue architecture and cellular response in βCD-d-limonene-treated mice. These results underscore the potential of βCD-d-limonene as a stable, biocompatible, and highly effective therapy, offering a natural platform for wound care and related biomedical applications.