In healty condition, the cornea is a translucent, avascular connective tissue. However, a number of inflammatory, viral, and degenerative ocular conditions can lead to corneal neovascularization (CV) [1]. Furthermore, CV is a significant contributor to corneal transplant rejection and opacity, ultimately leading to blindness or other visual impairments. CV and other eye tissues has become a significant global public health issue [2]. The avascular state of the cornea relies on a delicate balance between proangiogenic and antiangiogenic forces. Among these, vascular endothelial growth factor (VEGF) plays a crucial role, being constitutively expressed in the endothelium, limbal vascular endothelial cells, and corneal epithelium. These cells typically have soluble VEGFR-1 and VEGFR-3 acting as a buffer to preserve avascularity and corneal clarity [3]. When pathological circumstances including infection, inflammation, and hypoxia arise, invading monocytes, neutrophils, and repair epithelium express VEGF [4]. Despite its role in tissue repair, VEGF becomes a key contributor to reduced vision and a major cause of allograft rejection in corneal transplant recipients. It is estimated that 1.4 million people in the USA alone are affected by CV [5,6].

Steroids, Cox 2 inhibitors, photodynamic therapy, fine needle diathermy, and anti-VEGF have been shown to be effective in treating CV among the various therapeutic approaches identified thus far [7]. Regardless of their clinical advantages, all are associated with significant side effects. Steroid use is restricted due to its association with cataract formation, ocular hypertension, and corneal thinning [8]. Topical NSAID use may result in corneal perforation and ulceration [9]. Inflammation is induced by the combination of photodynamic therapy and fine needle diathermy [10]. Anti-VEGF treatment can lead to decreased epithelial healing, corneal thinning [11], and epithelial erosion [12]. Therefore, it's imperative to look for safe and effective CV therapy.

One common drug used to treat type 2 diabetes is metformin (MT), which influences blood glucose uptake by activating adenosine 5′-monophosphate (AMP)-activated protein kinase. According to a recent discovery, MT may have the potential to prevent cancer and other chronic diseases [13]. Furthermore, MT has the ability to prevent neovascularization. According to Tan et al. (2009), MT inhibits angiogenesis by elevating the level of antiangiogenic thrombospondin-1 through the nuclear factor kappa-B (NF-ĸB) pathway [14]. In the conducted study, women were administered metformin treatment for 6 months. As a result of this treatment, approximately a 50% increase in Thrombospondin-1 (TSP-1) levels in volunteers was observed. Additionally, a decrease in the activity of NF-ĸB, Erk1/2, and Erk5 pathways was detected. When a TSP-1 neutralizing antibody was added, it was shown that the activity of NF-ĸB, Erk1/2, and Erk5 pathways returned to normal levels, confirming that this effect was due to increased TSP-1. In the scope of this study, the aim was to increase TSP-1 levels by administering metformin, thereby reducing NF-ĸB pathway activation and, consequently, suppressing angiogenesis to treat corneal neovascularization.

Drug bioavailability to the cornea remains a challenge as tear fluid continuously washes away eye drops. Additionally, the cornea's unique anatomy creates a strong physiological barrier that prevents drug penetration [2,15,16]. Consequently, for topical eye drops to achieve therapeutic effectiveness, frequent application throughout the day is required, leading to eye irritation and potential temporary blurriness after each application [17]. Our hypothesis posits that the delivery of silk fibroin (SF) microparticles can prolong the retention of metformin (MT) in the cornea, thereby enhancing its therapeutic efficacy against corneal neovascularization (CV).

In an effort to increase drug delivery effectiveness, mitigate side effects, increase drug bioavailability, and extend drug retention in the cornea, many innovative drug delivery systems have been developed. Nano-sized carriers, such as liposomes, lipid or polymeric micro-nanoparticles, and nanoemulsions, have shown great promise in ocular drug delivery [18]. Due to the structures of micro- and nano-based systems are composed of atoms and molecules with different sizes and morphologies, these systems have the unique ability to be tailored to specific requirements depending on the intended application. By encapsulating active pharmaceutical ingredients, these structures can be used in the field of pharmaceutics to enhance toxicity profile, distribution, sustainability, biocompatibility, and efficacy [19].

SF is a naturally occurring polymer extracted from silkworm cocoons, comprising heavy and light chains connected by a disulfide bond [20]. The heavy chain consists of eleven hydrophilic amorphous domains and twelve hydrophobic crystallizable domains [21]. Due to its excellent mechanical properties, high cellular uptake, tunable biodegradability, and excellent biocompatibility, SF has been used extensively in drug delivery. Moreover, the crystalline content and secondary structure of SF could be altered to regulate the drug release [22].

In this study, we formulated silk fibroin (SF)-based encapsulated metformin (MT) microparticles for the treatment of corneal neovascularization. Following particle size measurement, scanning electron microscopy (SEM) imaging, drug loading procedures, and in vivo release studies of MT-loaded microparticles, cytotoxicity and permeability tests were conducted in cell culture. Subsequently, MT-loaded SF microparticles were applied to rat eyes for a week to evaluate their efficacy against corneal neovascularization.