Calcium Carbonate-Stabilized Nano-Caffeine Emulsion Attenuates Diabetic Cardiomyopathy via Antioxidant, Anti-Inflammatory, and Anti-Fibrotic Pathways in Type 2 Diabetic Rats with HPLC-Quantified Cardiac Caffeine Levels

Rasha A Mansouri,1,2 Esam M Aboubakr,3 Huda F Alshaibi,2,4 Wafaa M Fouda,5 Abeer A Banjabi,2 Maha Mohammed Al-Bazi,2 Hamdoon A Mohammed,6 Adel M Ahmed7 1Chemistry Department, Faculty of Science and Humanities, Prince Sattam bin Abdulaziz University, Al-Kharj, 11942, Saudi Arabia; 2Biochemistry Department, Faculty of Sciences, King Abdulaziz University, Jeddah, 22254, Saudi Arabia; 3Department of Pharmacology and Toxicology, Faculty of Pharmacy, Qena University, Qena, 83523, Egypt; 4Embryonic Stem Cell Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 22254, Saudi Arabia; 5Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Sadat City, Sadat, Egypt; 6Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University, Buraidah, Saudi Arabia; 7Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Qena University, Qena, 83523, EgyptCorrespondence: Adel M Ahmed, Email adelpharma2004@svu.edu.eg Hamdoon A Mohammed, Email ham.mohammed@qu.edu.saIntroduction: Type 2 diabetes mellitus (T2DM) is one of the most commonly diagnosed metabolic diseases. Notably, two-thirds of diabetic patients may develop diabetic cardiomyopathy (DCM), a life-threatening condition for which no curative treatment currently exists.Methods: This study aimed to investigate the potential ameliorative effects of caffeine against DCM development, utilizing a novel oral sustained-release caffeine-loaded Pickering emulsion formula stabilized by calcium carbonate nanoparticles to enhance its pharmaceutical and pharmacological properties. Eighty-four rats were divided into seven groups: control, caffeine, nano-caffeine, diabetic, diabetic + rosuvastatin, diabetic + caffeine, and diabetic + nano-caffeine.Results: Our findings demonstrated that the newly developed nano-caffeine formulation significantly downregulated myocardial injury markers (CK-MB, cTnI, ALT, AST, and LDH) and markedly ameliorated myocardial tissue injury and fibrosis, as confirmed by histopathological examination and desmin/α-SMA expression analysis. Additionally, the nano-caffeine treatment reduced inflammatory cytokines (TNF-α and IL-1β), attenuated hyperlipidemia, decreased iNOS and NO myocardial concentrations, and upregulated protective antioxidants (Nrf2, GSH, GSH-Px, SOD, and catalase) compared to the control group. Importantly, the cardioprotective effects of nano-caffeine were more pronounced than those observed in caffeine-treated diabetic rats. Furthermore, a novel, simple, and validated HPLC method was employed to quantify caffeine levels in cardiac tissues in all groups. The analysis revealed significantly higher caffeine concentrations in the nano-caffeine group compared to other groups, indicating improved tissue delivery.Conclusion: The formulation significantly enhances the cardioprotective effects of caffeine against myocardial injury in T2DM rats by optimizing its pharmacodynamic and pharmacokinetic properties.Keywords: type 2 diabetes mellitus, HPLC-quantification, diabetic cardiomyopathy, nano-caffeine, Pickering emulsion, cardioprotection, nanotechnology