Rayya Ahmed AL BALUSHI ^1* AISWARYA CHAUDHURI ² Raghuram Kandimalla ³ Ashanul Haque ⁴ Khalaf M. Alenezi ⁴ Mohd Saeed ⁵ Mohammad Changez ⁶ Thuraya Salim Al Harthi ¹ Mohammed Al Hinaai ¹ Samra Siddiqui ⁷ Ashish Kumar Agrawal Agrawal ² Farrukh Aqil ^3,8

• ¹ Department of Basic and Applied Sciences, College of Applied and Health Sciences, A’Sharqiyah University, Ibra, Oman
• ² Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (BHU), Varanasi, Uttar Pradesh, India
• ³ Brown Cancer Center, University of Louisville, Louisville, Colorado, United States
• ⁴ Department of Chemistry, College of Science, University of Hail, Ha'il, Hail, Saudi Arabia
• ⁵ Department of Biology, College of Science, University of Hail, Ha'il, Hail, Saudi Arabia
• ⁶ College of Health Sciences, University of Buraimi, Buraimi, Oman
• ⁷ Public Health Management, College of Public Health and Informatics, University of Hail, Ha'il, Hail, Saudi Arabia
• ⁸ Department of Medicine, School of Medicine, University of Louisville, Louisville, Colorado, United States

Frankincense demonstrates in vitro anticancer activity, yet its conventional delivery faces significant challenges due to limited oral bioavailability. In this study, frankincense resins were extracted and characterized using gas chromatography-mass spectrometry (GC-MS) and liquid chromatographymass spectrometry (LC-MS). Among others, isopinocarveol, 1,5-dimethyl-8-(prop-1-en-2-yl) cyclodeca-1, 5-diene, 4,6,6-trimethylbicyclo [3.1.1]hept-3-en-2-ol, 4,6,6-trimethyl bicyclo[3.1.1]hept-3-en-2-one, methyl 14-methylpenta decanoate, methyl 16-methylheptadecanoate, methyl octadec-9enoate, methyl-octadeca-9,12-dienoate, α-thujene, p-cymene, carvone, α-cubebene, germacrene A were the principal metabolites. FO was used to prepare nanoemulsions (FO-NEs) and was optimized using a 3-factor, 3-level Box Behnken Design (BBD), comprising 10% FO (v/v), 40% surfactant (cremophor EL) and co-surfactant (Transcutol P). The optimized FO-NEs showed an average particle size of 65.1 ± 4.21 nm, a polydispersity index (PDI) of 0.258 ± 0.04, and a zeta potential of -22.3 ± 1.2 mV, respectively. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) confirmed the spherical shape of FO-NEs. In addition to the physicochemical evaluation, in vitro cytotoxicity studies, wound healing assay, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) generation assay against breast cancer (MDA-MB-231 and MDA-MB-231-TR) and lung cancer (A549, A549-TR, and H1299) cell lines were also carried out. We demonstrate that FO-NEs (13.2 μg/mL) exhibit higher activity as compared to FO alone (22.5 μg/mL) against resistant breast cancer MDA-MB-231-TR cell line. Over, the result presented herein clearly demonstrates that the FO-NEs significantly enhance cancer cell internalization and activity compared to free FO.