Methotrexate (MTX), an analogue of folic acid, is a powerful chemotherapeutic agent and widely used for the treatment of human malignancies and autoimmune diseases [1, 2]. Its mechanism of action involves disrupting cellular folate metabolism by inhibiting dihydrofolate reductase, an essential enzyme for cell division. Despite its clinical efficacy, MTX faces challenges, including low specificity, dose-dependent undesirable side effects, the development of resistances in cancer cells and a potential risk of developing cancer [1, 3–5]. Furthermore, MTX is a weak dicarboxylic acid [molecular weight, MW: 54.44 g/mol (C20H22N8O5); pKa values of 3.8, 4.8 and 5.6] that exhibit a poor, pH-dependent solubility (aqueous solubility ≈ 0.01 mg/mL at 20°C) and low permeability
(C logP ≈ 0.53). As a result, it has a poor oral bioavailability of less than 40%, which impairs its therapeutic effect, and therefore it is classified in class IV of the Biopharmaceutical Classification System [3, 6, 7]. In addition, MTX is chemically unstable and, thus, undergoes rapid decomposition when exposed to light or subjected to extremes of pH or temperature. Consequently, it is often administered intravenously at high doses (≥ 500 mg/m2) [8]. To overcome these challenges, nanoparticles (NPs) have emerged as promising carriers for tailored MTX delivery [9].
Nanotechnology has revolutionized cancer therapy by enabling targeted drug delivery, minimizing side effects and enhancing treatment efficacy [10–17]. Nanocarriers act as sophisticated vehicles, circulating through biological barriers to reach the specific cellular environments where their cargo can exert the most significant therapeutic effect [11, 14, 18–23]. Numerous research endeavors have delved into the encapsulation of MTX within NPs, with the primary objective of overcoming drug resistances in cancer cells and enhancing therapeutic outcomes. The intricately tailored encapsulation within NPs aims to provide sustained release profiles, ensuring a prolonged and controlled delivery of MTX. This sustained release approach plays a pivotal role in circumventing the development of resistances. Furthermore, nanoformulations contribute to improved bioavailability, enhancing the amount of MTX reaching its target within the body, thereby maximizing its therapeutic impact [8].
Numerous polymers have recently been suggested to formulate MTX-loaded NPs for cancer therapy [4, 24–29]. Among these polymers, chitosan (CS), a naturally derived polysaccharide from chitin, offers a wealth of advantages that render it an ideal candidate for use in the design of nanoparticulate drug delivery systems. Known for its hydrophilicity, biocompatibility, low immunogenicity, biodegradability and mucoadhesive properties, CS has found widespread application in diverse medical formulations [30–32]. CS’s appeal in the medical field extends beyond its biological properties to include its ease of modification and versatility. The presence of reactive amino and hydroxyl groups allows for various chemical modifications, facilitating the tailoring of CS-based formulations to meet specific medical requirements [33–35]. Furthermore, the cationic nature of chitosan enables interactions with negatively charged biological surfaces, a feature strategically employed in NP development [36–38]. When employed as a coating polymer for NPs, chitosan not only imparts stability and controlled release but also introduces an additional layer of biocompatibility, offering a hydrophilic stealth coating that prevent opsonization and evade phagocytosis in mononuclear phagocyte system, which is crucial for navigating the blood circulation effectively [39–45].
Several MTX-loaded CS-based NPs have been designed to enhance controlled drug delivery to tumors [46–49]. These include actively targeted NPs, such as those using covalently conjugated to folate [48], as well as stimuli-responsive particles. Stimuli-responsive nanosystems encompass pH-responsive NPs, for instance, by incorporating the surfactant 77 KL (Nα,Nε-dioctanoyl lysine with a lithium counterion) [47], redox-responsive NPs, like those based on L-cysteine covalently linked to CS [47], and light-responsive NPs, functionalized with photocatalytic titanium dioxide NPs [48]. These innovative formulations, often utilizing modified forms of CS, aim to enhance the targeting capability of MTX-loaded CS NPs and exert increased control over the release of the chemotherapeutic agent. While many studies showcase the improved efficiency of MTX-loaded CS-based NPs, a notable limitation is the lack of comprehensive preclinical data in several of these investigations. Future research should aim to bridge this gap, providing a more robust foundation for the translation of these findings into clinical applications.
Furthermore, magnetically driven NPs can be advantageous in achieving targeted delivery to tumor cells and other disorders [50–52]. Magnetite (Fe3O4) stands out as a distinctive category of magnetic nanoparticles that has garnered considerable attention the medical field. Due to their biocompatibility, extensive chemical affinity and distinctive magnetic properties, Fe3O4 NPs have found widespread use in biomedical applications [53]. A primary application within the medical domain is evident in magnetic resonance imaging, where the superparamagnetic characteristics of Fe3O4 contribute to enhanced imaging contrast, facilitating clearer and more detailed visualization of tissues and organs. In addition, Fe3O4 particles play a crucial role in targeted drug delivery systems, enabling the precise attachment or encapsulation of drugs within these NPs for specific delivery to targeted cells or tissues [54]. This targeted strategy not only enhances the therapeutic effectiveness of drugs but also minimizes potential side effects. Despite their immense potential, the use of Fe3O4 NPs in medical applications presents certain challenges. One notable hurdle involves concerns about potential toxicity, as the body’s response to iron-based NPs needs careful consideration. Another obstacle pertains to the susceptibility of Fe3O4 to air oxidation, which can lead to the loss of magnetic properties. Maintaining the stability of Fe3O4 particles in physiological environments and preventing aggregation are critical for successful medical applications. In addressing these challenges, researchers have directed their efforts toward surface modifications of Fe3O4 NPs. Coating these particles with various materials, including natural substances such as CS or synthetic polymers like polyethylene glycol and polyvinylpyrrolidone, enhances their biocompatibility and stability. Furthermore, endeavors have been undertaken to optimize the size, shape, surface charge and functionalization of Fe3O4 NPs, aiming to enhance their overall performance in Biomedicine [50, 53].
When Fe3O4 is incorporated within the CS matrix the resulting core/shell nanocomposite combines the magnetic responsiveness of Fe3O4 NPs with the inherent benefits of CS [54–57]. These magnetically driven NPs have shown promise in targeted drug delivery, hyperthermia, and imaging applications. The magnetic responsiveness enables the NPs to be guided to specific sites within the body using external magnetic fields. This targeted approach enhances the precision of drug delivery, minimizing off-target effects and improving therapeutic outcomes. Furthermore, the combination of CS and Fe3O4 in NPs offers opportunities for multifunctionality. Beyond drug delivery, these particles can be explored for applications such as magnetic hyperthermia, where the magnetic properties are harnessed to generate localized heat for therapeutic purposes [58, 59].
In this study, we explore magnetically targeted and pH-responsive Fe3O4/CS (core/shell) nanocomposites as a drug delivery platform for MTX. The research delves into the formulation and characterization of these NPs, emphasizing their stability, MTX loading capacity and magnetic responsiveness. In addition, the potential pH-responsive MTX release behavior is investigated under the acidic microenvironment typical of tumors (pH ≈ 5.5). Furthermore, the hemocompatibility and cytotoxicity of these MTX-loaded magnetic nanocomposites against human breast cancer cells are thoroughly examined, providing crucial insights into their safety and efficacy profiles. To the best of our knowledge, this is the first time that these Fe3O4/CS (core/shell) NPs with dual targeted features, magnetic and pH responsiveness, have been developed as a platform for the delivery of MTX molecules to malignant cells. By combining the advantageous features of the developed NPs, this research aims to significantly contribute to the advancement of targeted MTX-based cancer therapy.