Diabetes mellitus is a chronic metabolic condition marked by hyperglycemia [[1], [2], [3]]. It's one of the most prominent risk factors for the onset and progression of atherosclerosis and a major contributor to deaths from cardiovascular disease [[4], [5], [6], [7]]. Atherosclerosis is a progressive lipid and inflammatory cell buildup within the arterial intima [8,9]. Hyperglycemia in diabetes mellitus exacerbates vascular disarray in atherosclerosis by damaging endothelial cells and vascular smooth muscles [1,2]. This means that anti-atherosclerotic drugs should be recommended for diabetic patients to reduce the risk of cardiovascular mortality. Multiple studies show that activating beta-adrenergic receptors has proven effective in treating metabolic and peripheral vascular diseases [[10], [11], [12]]. Isoxsuprine hydrochloride (IXS) is an orally administered compound that acts as a beta-adrenergic agonist [[10], [11], [12]]. Its primary mechanism of action involves the relaxation and dilation of vascular smooth muscle, leading to improved arterial blood flow [[10], [11], [12]]. This effect is achieved by the activation of numerous pathways, including the NO/cGMP and H2S/KATP pathways, as well as the blockage of L-type voltage-gated calcium channels [[10], [11], [12]]. IXS also lowers the viscosity of the blood, stops the platelet aggregation and boosts glucose absorption, insulin secretion, and fat breakdown [[13], [14], [15]].

Oral IXS has various limitations that compromise its therapeutic efficacy, including low bioavailability and hepatic first-pass metabolism [[16], [17], [18]]. Oral IXS shows patient incompliance due to its high dose frequency, short biological half-life and fast clearance [16,[18], [19], [20]]. Numerous investigations have been conducted to address these limitations through the development of controlled release formulations of IXS [16,19,20]. In their study, Sullad et al. successfully created an oral controlled release formulation for IXS [19]. This was achieved by utilizing a combination of chitosan and polyurethane mix microspheres, which were generated through the emulsion cross-linking process. The study conducted by Kagade et al. aimed to develop a microsponge drug delivery system for IXS using cellulose acetate phthalate to achieve targeted and controlled drug release in order to enhance the absorption of the IXS [20]. Oral matrix tablets containing hydroxypropylmethylcellulose and Gaur Gum polymers were designed by Jain et al. to provide a method of controlled administration of IXS [16].

The nasal route has been employed as a delivery system due to its vast surface area, porous endothelium membrane, high total blood flow, and lack of first-pass metabolism [21,22]. The epithelial barrier and fast mucociliary clearance represent the main disadvantage of nasal delivery [21,22]. Lipid-based nanoparticles have been identified as a possible approach to overcome these limitations and enhance the efficacy of medication delivery via the nasal route [[23], [24], [25]]. The low particle size of nanoparticles produced a high surface area, which increased the drugs' bioavailability [26,27]. Invasomes formulation is a liposomal nano-vesiclar delivery system made of phospholipids, cholesterol, and penetration enhancers such as ethanol and terpenes [23,24]. The presence of phospholipid can promote the residence time of drugs by increasing the system's viscosity and forming thick, cohesive layers around individual vesicles [28,29]. Cholesterol is incorporated to strengthen and stabilize the structure and produce stiffer invasomes [24]. The presence of terpenes and ethanol as penetration enhancers promotes drug absorption by increasing membrane fluidity and permeability [23,25]. Invasomes show therapeutic and pharmacological promise for enhancing drug's bioavailability, efficacy, and selectivity [[23], [24], [25]]. In addition, the controlled and sustained release of the drug from invasomes results in a longer half-life and clearance of the drug, which in turn reduces the dose frequency of the drug and improves patient compliance [[23], [24], [25]]. Based on literature reviews, no studies have explored the development of a nasal delivery system for IXS loaded invasomes.

It is important to understand the interaction behavior of drugs and plasma proteins to elucidate the pharmacodynamics and pharmacokinetics of drugs [26,30]. Human serum albumin (HSA) and human holo-transferrin (HTF) are the most thoroughly researched proteins due to their substantial role in drug transportation and their numerous vital physiological and pharmacological roles [26,30]. Utilizing molecular docking and molecular dynamic simulation analysis will enable us to gain a comprehensive understanding of the mechanisms by which drugs interact with plasma proteins and how these interactions influence cellular function [30]. Accordingly, we have explored the interaction behavior between IXS and plasma proteins. Cytotoxicity assays are used to evaluate the potential harmful effects of drugs using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [27,31]. RAW 264.7 cells are a murine macrophage-like cell line that is commonly used as a model for the study of the pathogenesis of atherosclerosis [32,33]. Accordingly, we have explored the cytotoxicity of IXS-loaded invasomes on Murine macrophage (RAW264.7) cells.

The goal of this research was to sustain IXS's release and improve its delivery, permeation, efficacy, and patient compliance as a potential diabetes-accelerated atherosclerosis treatment by developing a nasal formulation of IXS-loaded invasomes. Production of nasal IXS-loaded invasomes formulation can sustain the release of IXS in order to increase its biological half-life and decrease its elimination and dose frequency. Different IXS-loaded invasomes formulations were made using design expert software and characterized in vitro to select the optimum formulation. Additionally, the optimum formulation was characterized in vitro to study its cytotoxicity and its interaction behavior with serum proteins. After that, the optimum formulation was examined in vivo for experimental diabetes and atherosclerosis in rats.