Chitosan-Based Intelligent Microneedles for Delivery of Amphotericin B Loaded Oleosomes: Antifungal Ocular Patch Targeting for Effective Against Fungal Keratitis Using Rabbit Model via TLR4/NLRP3 Pathway

Sammar Fathy Elhabal,1 Saeed Abdul-Kareem Saeed Al-Zuhairy,2 Mohamed Fathi Mohamed Elrefai,3,4 Mohamed A El-Nabarawi,5 Sandra Hababeh,6 Kristina Zarif Attalla,7 Mai S Shoela,8 Jakline Nelson,9 Marwa Fady,10,11 Nahla A Elzohairy,11,12 Mariam E Amin,13 Heba Sabry Ahmed,14 Tassneim M Ewedah,15 Ibrahim Mousa,16 Ahmed Mohsen Elsaid Hamdan17 1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Modern University for Tech-Nology and Information (MTI) Mokattam, Cairo, Egypt; 2Department of Pharmacy, Kut University College Kut, Wasit, 52001, Iraq; 3Department of Anatomy and Embryology, Faculty of Medicine, Ain Shams University, Cairo, 11591 Egypt; 4Department of Anatomy, Physiology and Biochemistry, Faculty of Medicine, The Hashemite Uni-Versity, Zarqa, 13133 Jordan; 5Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt; 6Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; 7Department of Pharmaceutics, College of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology, Giza, Egypt; 8Department of Clinical Pharmacology, Faculty of Medicine, Alexandria University, Alexandria, Egypt; 9Department of Microbiology and Immunology, Faculty of Pharmacy, Nahda University, Beni-Suef (NUB), Beni-Suef, 62511, Egypt; 10Zagazig University Hospitals, Infection Control Unit, Zagazig, 44519, Egypt; 11Department of Microbiology and Immunology, Faculty of Pharmacy, Modern University for Technology and Information (MTI) Mokattam, Cairo, 11571, Egypt; 12Air Force Specialized Hospital, Cairo, 19448, Egypt; 13Microbiology and Immunology Department, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt; 14Department of clinical pharmacology, faculty of medicine, Zagazig university, Zagazig, 44519, Egypt; 15Pharmaceutics and Pharmaceutical Technology Department, Faculty of Pharmacy, Egyptian Russian University, Cairo, Egypt; 16Pharmaceutics Department, Faculty of Pharmacy, Sinai University, Al-Arish, Egypt; 17Department of Pharmacy Practice, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi ArabiaCorrespondence: Sammar Fathy Elhabal, Email [email protected]; [email protected] Ahmed Mohsen Elsaid Hamdan, Email [email protected]: Fungal keratitis (FK), a major cause of blindness, remains challenging to treat due to poor drug penetration and antifungal resistance. Amphotericin-B (AmB), a water-insoluble and low-permeability, necessitates innovative delivery systems to improve its therapeutic efficacy.Methods: AmB was encapsulated within oleosomes (Ole) prepared using the ethanol injection method, using phosphatidylcholine (Lipoid S100) and sodium oleate, resulting in nanosized spherical globules. The optimized Ole were characterized, then the selected Ole were incorporated into sodium polyacrylate/PEG/chitosan-based microneedles (AmB-Ole/MNs) to improve ocular delivery by creating transient microchannels on the eye surface.Results: The optimized Ole showed a droplet size of (175 ± 0.78 nm), polydispersity index of (0.33 ± 0.04), zeta potential of (31 ± 0.43 mV), high entrapment efficiency (91± 0.63%), and improved stability, bioavailability, and controlled drug release. The AmB-Ole/MNs system increases corneal penetration and contact time via transient microchannels in the eye surface, achieving sustained drug delivery over 72 hours with 70% ex vivo permeation over 80 hours compared to AmB. In vitro antifungal activity and histopathological examination showed that the AmB-Ole/MNs system has potent biofilm disruption (> 90%) and 27 mm and 32 mm zones of inhibition against Candida albicans and Aspergillus niger, respectively. The Cytotoxicity test showed reduced AmB toxicity with biocompatibility and in vivo rabbit model, ocular tolerance by targeting TLR4/NLRP3 pathways and histopathological studies.Conclusion: The AmB-Ole/MNs system as an innovative ocular delivery platform for fungal keratitis offers sustained drug release, enhanced permeation, potent antifungal activity, and reduced toxicity. AmB-Ole/MNs showed promise for ocular AmB delivery for FK.Keywords: microneedles, fungal keratitis, amphotericin-B, antifungal, chitosan, ocular, candida albicans, aspergillus niger