Naringenin (NRG, C15H12O5, 4′,5,7- trihydroxyflavanone, molecular weight 272.25 g/mol, Scheme 1) is a naturally occurring flavonoid classified as flavonones present in citrus fruits, grapes, beans, cherries, cocoa, oregano and tomatoes [[1], [2], [3]]. NRG presents important properties: antioxidant [4,5], anticancer [6,7], anti-inflammatory [[8], [9], [10]], antiviral [11], antidiabetic [12], antimutagenic [13], antibacterial [14,15], neuroprotective [16], and anti-apoptotic [17], among others.

NRG is characterized by poor aqueous solubility in water (46 μg mL−1) [18], which seems to contribute to its low oral bioavailability (5.81 %) [18,19] requiring therefore high doses for pharmaceutical uses of this compound. New strategies allowing to increase the water solubility of NRG are needed in order to enhance its therapeutic activity. The general strategy utilized for limited solubility drugs is the generation of novel solid phases, such as amorphous, polymorphs, hydrates, solvates, salts, and cocrystals [[20], [21], [22], [23], [24]]. Recently, cocrystallization has received increasing attention because of its significant ability to regulate physicochemical properties like dissolution rates, bioavailability, stability, manufacturability (flow, compactness, and processability), and the hygroscopicity of substances that suffer from poor solubility [20,[25], [26], [27], [28], [29], [30], [31]].

Cocrystallization is based on the interaction between two or more chemically different molecules, generally in stoichiometric ratio [[32], [33], [34]]. Pharmaceutical cocrystal, can be obtained by a target molecule or API (Active Pharmaceutical Ingredient) and a coformer or guest molecule, normally included in the list of GRAS substances (Generally Recognized as Safe) [28,31].

The use of conventional methods of cocrystallization, such as solvent evaporation, solid grinding, liquid-assisted grinding, slurring, and extrusion (twin screw extrusion, hot melt crystallization) in the cocrystal preparation presents several drawbacks, such as scaling-up difficulties or the presence of homocrystals in the final product and often require post purification steps to eliminate solvents [35,36]. That said, the use of supercritical fluid (FSC) presents a new and interesting pathway for the formation of cocrystals, since it avoids most of the drawbacks of traditional methods while having as main advantages: high purity of products, versatile approach to produce cocrystals with different morphologies and particle sizes, the possibility of thermolabile molecule processing, single step process, as well as being an environmentally acceptable technology reducing the use of organic solvent and minimizing the residual amount of solvent in the cocrystal when compared to some conventional techniques [37,38]. In this work, we applied GAS (gas antisolvent) technique, which involves adding compressed CO2 to a solution containing the target molecule and coformer solutes until the desired pressure is reached. After that, CO2 is incorporated into the liquid solvent, expanding it and decreasing the solvent power, leading to the precipitation of solutes [37].

There are published studies involving the cocrystallization of NRG which involves the use of conventional techniques [[39], [40], [41], [42], [43], [44]]. Recently, some works made use of betaine (BTN, Scheme 1) as coformer to cocrystallize NRG. To date, the fabrication of cocrystals consisting of NRG: BTN via the GAS technique or using FSC has not been found.

The formation of the NRG: BTN cocrystal is justified by the fact that NRG has two competitive hydrogen bonding sites that can act as donors and acceptors (4′,5,7-hydroxyl, and a carbonyl group), which makes it a good candidate for cocrystal formation. Also, NRG is a neutral compound with pKa = 7.05, so it is unsuitable for salt formation [45,46]. BTN, also known as trimethylglycine (C5H11NO2, molecular weight: 117.148 g/mol), is a natural sweetener found in microorganisms, plants, and animals [47]. It has gained significant attention in pharmaceutical formulations as a coformer due to its ability to form hydrogen bonds with molecule targets [[47], [48], [49]].

Recent work indicates that cocrystallization can change the permeability properties of drugs [50,51]. Some studies investigating the permeability of cocrystals, physical mixtures, and pure drugs through intestinal monolayer cells evidenced that cocrystals can induce significant differences in influencing the integrity of intestinal cell monolayers compared to the pure drugs and the physical mixtures [[52], [53], [54]]. Other studies did not have evidence effects on permeability by coformers or cocrystals on cell monolayers [55].

The efficacy of most drugs depends critically on their ability to cross cellular barriers and this aspect appears crucial for drugs targeting the central nervous system (CNS). Ideally, drugs intended for the blood-brain barrier (BBB) crossing should be reasonably lipid soluble, easily permeable, and metabolically stable to reach their target [56]. Flavonoids such as NRG are known to be able to cross the BBB [57], even if this property requires to be improved. Very recently it has been demonstrated that neuroactive drugs, such as ketoprofen-gabapentin can synergistically and mutually increase BBB crossing, and that their cocrystal further enhances these effects, possibly leading to higher CNS permeation and central effects of these compounds [58].

This study aims to perform the cocrystalization between NRG and BTN using the GAS technique and the characterization. Also, in vitro assays investigated the permeability of NRG as raw drug, physical mixture with betaine (NRG-BTN Mix), and cocrystal naringenin: betaine (1:1) (NRG:BTN Coc) across cell monolayers of IEC-6 and a BBB model, which was established by ECV 304.