Tuberculosis (TB) is an infectious disease caused by Mycobacterium Tuberculosis (MTB), which can result in mortality [1]. Infection can manifest in multiple regions of the body, with the lungs being the most commonly affected organ [2]. The distinction between pulmonary tuberculosis and extrapulmonary tuberculosis is critical [3]. Symptoms such as chronic cough, chest pain, breathlessness, fatigue, weight loss, and fever are considered as pulmonary tuberculosis, which may develop progressively over time [4]. In 1940s, governments have collaborated to eradicate tuberculosis worldwide, due to the significant mortality rate linked to the disease without treatment [5].

Advancements in first- and second-line medications are impeded by physicochemical limitations and mycobacterial resistance from MTB, which complicate their efficacy against pathogens within alveolar macrophages [6]. However, current therapy with antimicrobial agents is ineffective due to some therapeutic factors such as low patient adherence, prolonged treatment, interactions with comorbidities, multidrug resistance, serious adverse effects, and drug-related issues like drug degradation and poor solubility [7,8]. Therefore, the identification and development of novel drug classes derived from plant species, marine organisms, and fungi represent an inexhaustible source of bioactive compounds essential for overcoming the limitations of existing tuberculosis treatments [9]. Phytomedicinal components, such as alkaloids, flavonoids, and terpenoids, exhibited anti-TB activity [10]. Flavonoids disrupt the TB enzymatic pathways and cell-wall integrity [11]. Their increased hydrophobicity significantly influences their antitubercular activity, attributed to their lipid-rich and highly impermeable molecular structure compared to hydrophilic compounds [12]. Hydroxyl substitutions at positions 5, 6, 7 (trihydroxy) or 3′, 4′ (dihydroxy) represent promising antitubercular pharmacophores [13].

Naringenin (NAR) is a natural flavonoid compound with antioxidant, anticancer, anti-inflammatory, and antidiabetic properties [[14], [15], [16]]. Recent studies have confirmed NAR's activity, against various Mycobacterium strains, including M. bovis, M. smegmatis, and M. tuberculosis H37Rv [11,[17], [18], [19], [20], [21], [22]]. In the context of M. tuberculosis, NAR inhibits growth and replication in vitro by disrupting cell wall synthesis, DNA replication, and protein synthesis, while also inducing caspase-mediated apoptosis [23]. Despite the efficiency of NAR as an anti-TB phytomedicinal agent, its medical application remains limited due to poor solubility, significant first-pass metabolism, and diminished intestinal permeability, resulting in low oral bioavailability [17,[24], [25], [26]].

Lipid-drug conjugation represents a novel approach to enhance the physical and chemical properties of pharmaceuticals, facilitating effective loading into nanocarriers with improved permeation [[27], [28], [29], [30]]. Conjugation with fatty acids, including linoleic, palmitic, caprylic, or stearic acid, significantly enhances oral bioavailability of pharmaceuticals [31,32], applicable to both hydrophilic and hydrophobic drug molecules [33]. This approach reduces drug toxicity by altering aggregation patterns, improving solubility, reducing aggregation, and controlling their release [32]. Currently, many fatty acid-based prodrugs are under development and investigations [34]. These fatty acids bind to pharmaceuticals by forming carboxylate linkages [23]. Lipid drug conjugates (LDCs) are formed when drugs with alcohol or amino functional groups couple with fatty acids, leading to the formation of esters or amides [35,36]. The resulting prodrug produces a different pharmacokinetic profile, such as high lipophilicity with long-lasting effect [36], redirecting poorly soluble drugs to the lymphatic pathway to bypass first-pass metabolism [37].

Encapsulating LDCs into NPs enhanced drug gastric stability, and increased permeation across Caco-2 monolayers, leading to improved bioavailability [38,39]. Recent studies have demonstrated the ability of LDCs to achieve high concentrations in lymph nodes for extended periods while minimizing systemic exposure, unlike free-drug-loaded nanocarriers that undergo rapid systemic absorption [40]. Furthermore, LDCs enhance the lipophilicity of conjugated drugs, thereby improving drug-loading efficiency into the formulated NPs [41]. Additionally, LDC-NPs demonstrated their effectiveness by enhancing drug potency, reducing IC50, and improving t1/2 compared to the free drug [28].

This study aims to assess NAR-LDC-NPs as a novel method for tuberculosis treatment. We hypothesized that conjugating NAR with a lipid and subsequently encapsulating it into nanoparticles would leverage the lymphatic absorption pathway, thereby circumventing extensive first-pass hepatic metabolism and enhancing NAR's oral bioavailability. Furthermore, we anticipated that this approach would improve NAR's targeting to Mycobacterium Tuberculosis storage sites, ultimately leading to superior antimycobacterial activity. Herein, six formulations were developed utilizing three fatty acids of different chain lengths: caprylic acid (C8), palmitic acid (C16), and stearic acid (C18). Labrasol and Tween 80 were employed as surfactants to stabilize LDC-NPs. The characterization and optimization of the synthesized NAR-LDC-NPs were performed in vitro by evaluating entrapment efficiency percentage (EE%), PS, and ZP. The optimized NAR-LDC-NP formula was further characterized via its morphology, thermograms, ex vivo permeation, stability, in vitro anti-TB activity, and molecular docking.