Furosemide (C12H11ClN2O5S, MW: 330.7 Da) is a loop diuretic used for treatment of edema associated with heart failure, cirrhosis, or kidney disease. Also it is used to treat hypertension [1]. The World Health Organization (WHO) has designated furosemide as an essential medicine. Food and Drug Administration (FDA) approved the use of furosemide, which was patented in 1959, in 1982 [2,3]. Furosemide is commercially available in tablet, injectable and oral solution formulations in several strengths (10, 20, 40, and 80 mg) [3]. Furosemide decreases the reabsorption of sodium, chloride, and potassium in the renal tubules. As a result, these ions remain in the tubule and are then transported to the distal nephron. This process leads to the production of dilute urine as water retention occurs within the tubule once the distal segment is reached [4]. According to Amidon et al. [5,6], furosemide is a Biopharmaceutics Classification System (BCS) Class IV compound with a low water solubility and low permeability. Class IV drugs typically exhibit high levels of intersubject and intrasubject variability due to their low solubility and low permeability; this also contributes to their extremely low oral bioavailability. Therefore, various experiments are being carried out to make these medications more soluble and permeable. Due to poor solubility and permeability properties of furosemide, its absorption and bioavailability from gastrointestinal (GI) tract (37–51 %) after oral administration is very variable [[7], [8], [9]]. Following an oral dose of furosemide, which is rather rapidly absorbed from the GI system, peak plasma concentrations are reached 1–2 h. Additionally, the volume of distribution of furosemide in adults is 0.13 L/kg [10]. In healthy individuals, over 95 % of furosemide is bound to plasma proteins, predominantly albumin. At therapeutic concentrations, only 2.3 %–4.1 % of furosemide is present in its unbound state. The primary biotransformation product of furosemide is furosemide glucuronide, which exhibits an active diuretic effect. Current findings suggest that the metabolic processing of furosemide in the liver is minimal [11]. Its half-life in healthy volunteers is 0.33–1.17 h. In uremic patients without liver disease, approximately 60–98 % of an intravenous dose of furosemide is excreted in bile within 24 h [12]. It is well known that furosemide can cause a wide range of side effects, including blood diseases, lymphatic system, immune system, metabolic, nutritional, nervous system, ear and inner ear, vascular, gastrointestinal, hepatobiliary, skin and subcutaneous tissue, kidney, and urinary tract and congenital and hereditary/genetic diseases. Studies are being carried out to enhance solubility and permeability of furosemide to reduce the dose and therefore its side effects.

Nanocrystal technology offers a versatile approach to increase the bioavailability of drugs affected by solubility challenges, achieved through particle size reduction. Nanocrystal technology offers significant advances in oral bioavailability by increasing saturation solubility and dissolution rates and facilitating interaction with cell membranes. This technology has also proven to be suitable for scale-up due to its efficient, fast, and reproducible manufacturing process. These systems have several advantages, including lower production costs compared to other formulations due to reduced dependence on excipients. Nanocrystal-based formulations allow for dose reduction due to higher drug concentration, potentially reducing systemic side effects. Particularly for lipophilic drugs with limited water solubility, nanocrystal technology is superior in increasing dose proportionality, decreasing differences in bioavailability, and reducing interindividual variability often observed in fasting and fed states. Furthermore, they can be administered via various routes including parenteral, pulmonary, topical, ophthalmic, and can be effectively sterilized by a variety of methods [[13], [14], [15], [16], [17], [18]].

The dispersion of drug nanocrystals in a liquid medium is called “nanosuspension”; wherein the formulation essentially comprises an active substance, a stabilizer, and a dispersion medium. Variety of surfactants (e.g. Tween 80, sodium lauryl sulfate, lecithin) and polymeric stabilizers (e.g. Pluronics F68 and F127, hydroxypropyl methylcellulose, polyvinyl alcohol) are used to ensure system stability with a various dispersion media including water, aqueous solutions, and non-aqueous solvents [14,19]. There are three main approaches to the preparation of nanocrystals: top-down methods (nanonization), bottom-up approaches (crystal growth or nucleation), and combination techniques. Nanonization, or top-down methods, involves the reduction of coarse drug particles to the nanometric (nm) size range using techniques such as media milling or high-pressure homogenization (HPH). The bottom-up method relies on the controlled principles of precipitation and evaporation. This approach is characterized using simple instrumentation, low energy requirements, minimal heat generation, and cost-effectiveness. It is applicable to both thermostable and thermolabile drugs. Examples of these methods include antisolvent precipitation and supercritical fluid technology [13,20,21]. Given the wide range of drug properties and device characteristics, it is often difficult to obtain desired nanocrystals with a single preparation technology. However, by selectively combining top-down and bottom-up technologies to create a hybrid approach, the drawbacks associated with individual preparation techniques can be mitigated, leading to increased efficiency in particle size reduction [22]. Combinatorial technologies include Nanoedge® technology, pioneered by Baxter [23], and SmartCrystal® technology (Abbott/Soliqs, Ludwigshafen/Germany), which integrates pre-treatment with a particle size reduction step, effectively addressing issues such as instrument clogging [24] and improving the stability of nanocrystals. Combinatorial technology has been proven to be adaptable to a wide range of insoluble drugs using a variety of technologies. However, it does present challenges in terms of economic considerations and process complexity [22,25].

Similar formulation strategies have been developed to address the challenges associated with the low solubility and permeability of furosemide that limit its oral bioavailability. For example, Barbosa et al. prepared furosemide nanocrystals using a rotation-revolution mixer and achieved improvements in solubility and dissolution rate. However, the method used in that study had limitations regarding particle size control and long-term stability [26]. In another study, the antisolvent:solvent ratio and the stabilizer:drug ratio had a significant effect on the particle size of furosemide nanosuspensions, particularly through strong interactions. This interaction introduced formulation sensitivity, which represents a potential limitation in terms of reproducibility and scalability [9]. Similarly, Sahu and Das obtained furosemide nanosuspensions using nanoprecipitation and sonication techniques, but these methods encountered difficulties in maintaining the stability of the amorphous structure. Sedimentation was observed under all storage conditions; although formulations can be redispersed by shaking, this can present practical difficulties and may indicate potential physical stability issues [27]. Although recent studies on furosemide nanosuspensions have mostly used a combination of single techniques, such as nanoprecipitation or ultrasonication and a single stabilizer, these methods have limitations such as particle size control, long-term stability, and inability to fully improve permeability. In contrast, the present study presents a novel approach combining ultrasonication and ball milling techniques to achieve more effective particle size reduction and greater homogeneity. Furthermore, the effects of two different stabilizers, polyvinylpyrrolidone K30 (PVPK30) and Tween 80, were examined individually and in combination to evaluate their roles in enhancing both solubility and permeability. The aim of this study is to increase the solubility and permeability of furosemide through the formulation of nanosuspensions using homogenization and milling techniques. Two stabilizers (PVPK30 and Tween 80) were used alone or in combination in the preparation of furosemide nanosuspensions. Particle size and zeta potential values were measured for the resulting nanosuspensions (both before and after lyophilization) and raw furosemide. The physicochemical properties (e.g. Differential Scanning Calorimetry Analysis (DSC), X-ray and Fourier Transform Infrared Spectrometer (FT-IR) analyses) of raw furosemide, PVPK30, Tween 80, the physical mixtures, and nanosuspension formulations were determined. Additionally, the permeability of raw furosemide, the physical mixtures, and the nanosuspension formulations was determined across Caco-2 cell monolayers.