Diabetic retinopathy (DR) is a severe diabetes-related complication which occurs due to abnormal blood vessel growth in the retina. It remains one of the significant causes of blindness worldwide [1,2]. In 2020, it was estimated that 103.12 million individuals were affected by DR, and the prevalence is expected to reach 160.50 million by the year 2045 [3]. Several pathogenic factors and signaling pathways contribute to the progression of DR. Among these, the excessive production of vascular endothelial growth factor-A (VEGF-A) plays a major role by increasing vascular leakage through the breakdown of tight junctions in endothelial cells [4].

Piperine (PIP), an alkaloid extracted from black pepper (Piper nigrum), has attracted attention for its wide range of biological activities [[5], [6], [7], [8]]. It has shown antidiabetic effects by lowering blood glucose levels and improving insulin levels in animal models with diabetes and is particularly useful in managing hyperglycemia [[9], [10], [11]]. A recent study by Zhang et al. (2021) demonstrated that PIP lowered the expression of HIF-1α and VEGF-A in the retina of diabetic mice, indicating its potential as a therapeutic agent for the treatment of DR [12]. However, the poor water solubility of PIP (0.04 mg/mL) hampers its absorption and bioavailability [13,14].

To overcome this limitation, drug complexation with cyclodextrins (CDs) is a promising strategy. CDs are cyclic oligosaccharides with a hydrophilic outer surface and a hydrophobic central cavity, which can form inclusion complexes with hydrophobic drugs and improve their solubility and stability [15,16]. Studies have reported that hydroxypropyl-β-cyclodextrin (HPβCD) can significantly increase the solubility of PIP through the formation of an inclusion complex [17,18]. Moreover, recent review articles have thoroughly summarized the application of CDs in ocular drug delivery systems, including CD-based nanoparticles for treatment of retinal disorders [[19], [20], [21], [22]].

Magnetic nanoparticles (MNPs), especially iron oxide nanoparticles (IONs), have gained attention in drug delivery due to their magnetic properties and biocompatibility. These particles can be guided to targeted sites in the body using an external magnetic field, which also allows for controlled drug release. IONs have been extensively evaluated and reported to exhibit good ocular biocompatibility [[23], [24], [25]]. Studies have reported the widespread application of iron-based nanomaterials in the treatment of DR and other retinal vasculopathies, demonstrating considerable therapeutic potential. Their rigid structural properties help mitigate the risk of off-target drug leakage, rendering them highly suitable as drug delivery platforms for anti-angiogenic agents [[26], [27], [28]]. Additionally, IONs have demonstrated the ability to improve drug permeation through ocular tissues without causing adverse effects [29,30]. Under a magnetic field, IONs can be directed through the sclera and into the retina, which is a potential tool for treating retinal diseases [[30], [31], [32]]. However, the use of MNPs in drug delivery presents certain limitations. One major drawback is the reduced ability to retain drug targeting once the external magnetic field is removed [33]. Additionally, magnetic drug delivery systems often require specialized instruments and technical expertise, which can increase both the cost and complexity of treatment. Prolonged exposure to a magnetic field may also cause unintended effects on surrounding tissues or organs.

Due to their inherent dipolar interactions, naked IONs are prone to aggregation, leading to limited dispersion stability in aqueous medium. To address this limitation, surface modification using water-soluble polymers and CDs has been proposed as an effective approach to enhance colloidal stability. CD-based nanoparticles take the benefits of both components, i.e., CDs improve drug solubility and loading, while nanoparticles help with controlled and targeted delivery [34,35]. Additionally, βCD and its derivatives (e.g., HPβCD, carboxymethyl-βCD, maleated-βCD) are often used to coat the surface of IONs, while polymer coatings can prevent aggregation, increase hydrophilicity, and provide functional groups to bind drug molecule [[36], [37], [38], [39]].

Delivering drugs to the eye remains a significant challenge various barriers and rapid clearance from the eye surface [40,41]. To address these challenges, in situ gelling systems have gained interest as effective approaches to improve ocular bioavailability by extending the drug residence on ocular surface. These systems are administered as liquid formulations that transform into gels upon exposure to environmental triggers such as changes in pH, temperature, or ionic strength [42]. Ion-activated in situ gels rapidly form a viscoelastic gel matrix upon interaction with monovalent and divalent cations (e.g., Na+, Ca2+) naturally present in tear fluid [43]. Several studies have demonstrated the efficacy of ion-activated in situ gels. For example, Mandal et al. (2012) developed an ion-activated in situ gel containing moxifloxacin, which showed rapid gelation after administration, sustained drug release, and enhanced precorneal retention for the treatment of bacterial conjunctivitis [44]. Similarly, Shajari et al. (2024) formulated a modified gellan gum-based in situ gel for timolol maleate delivery, exhibiting favorable physicochemical properties, good biocompatibility, and potential for use in glaucoma therapy [45]. The incorporation of IONs into the gel increased the porosity of the gel matrix, and their larger surface area contributed to enhanced drug adsorption, leading to approximately 20 % higher uptake of diclofenac sodium compared to the gel formulation without IONs [46]. To our knowledge, this study is the first to report a novel ocular delivery system for PIP that combines HPβCD complexation with polymer-coated IONs within an in situ gel. This integrated strategy is designed to overcome aqueous solubility of PIP and enable its effective delivery to the posterior segment of the eye.

In this study, we optimized the synthesis of hydrophilic polymer-coated IONs. The inclusion complex formation of PIP with CDs was investigated, and PIP/CD-loaded polymer-coated IONs were prepared and characterized. Their mucoadhesive properties, in vitro drug release, and eye irritation potential were evaluated. The optimized polymer-coated IONs were then incorporated into an ion-activated in situ gel. Toxicity was tested using ARPE-19 cell lines, and the anti-VEGF, anti-angiogenic, and anti-inflammatory activities were evaluated in both in vitro and in vivo models using diabetic rats.