(T1030-01-01) Nifuroxazide-Loaded Lipid Nanocarriers – An Effective Treatment against Lung Cancer

Purpose: Lung Cancer is a highly invasive and most commonly diagnosed cancer worldwide with high incidence rates. According to statistics, it is the leading cause of cancer-related deaths worldwide. In 2020, approximately 2.2 million people were diagnosed with an estimated 1.8 million deaths making it a huge burden on global mortality. Despite progress in surgical, chemotherapy, and targeted therapy, the 5-year survival rate remains low, with only 23% of patients surviving across all stages of lung cancer. Moreover, the current treatment strategies are severely limited by systemic adverse effects, low concentration of drug at the primary tumor site, and development of resistance emphasizing the need for new treatment strategies. The development of new drug candidates is a costly and time-consuming process. Therefore, there is an increased interest in repurposing existing drugs for new uses. Nifuroxazide (NIF) is an oral nitrofuran antibiotic, indicated as an anti-diarrheal agent. Considering its anti-tumor activity on colorectal cancer we hypothesize that nifuroxazide might be effective against lung cancer as well. The anti-cancer activity of nifuroxazide on lung cancer has not yet been investigated. The aim of our research is to evaluate the efficacy of nifuroxazide on lung cancer cells and further encapsulate nifuroxazide into lipid nanocarriers for improved therapeutic response against lung cancer.

Methods: In vitro cell culture studies were initially conducted on A549, H1299 and H358 cell lines to evaluate the effectiveness of the NIF against lung cancer. To select the appropriate lipid, lipid solubility studies were performed on Compritol 888 ATO^®, Geleol^®, Precirol ATO^®. Excess drug was added to 100 mg of the melted lipid and allowed to solubilize. The drug solubility in the solid lipid was determined by visual inspection, a clear solution indicated solubility, while turbidity indicated insoluble drug crystals. SLNs were prepared using ultrasonication technique. Briefly, the NIF-loaded SLNs (NIF-SLNs) were prepared by heating Geleol^® with 0.1% tween and 2.5 mg of NIF at 65^◦C for 40 minutes. In parallel, the aqueous phase containing 1% PVA as the stabilizer was also heated to the same temperature. Subsequently, 5 mL of the heated aqueous phase was dispersed in the fused lipid and subjected to ultrasonication for 10 minutes at an amplitude of 40%. Finally, the resulting dispersion was cooled to 4^◦C. Further, the NIF-SLNs were passed through Sephadex G25 M column to remove unentrapped drug. Then, NIF-SLNs were freeze-dried and the solid-state characterization was performed. Further, the formulation was analyzed for in vitro release and in vitro aerosolization performance. In vitro three-dimensional tumor studies are being conducted on A549 cell line to evaluate the effectiveness of the formulation.

Results: Preliminary cell viability studies of NIF on various lung cancer cell lines namely A549, H1299 and H358 cell lines exhibited a concentration-dependent decline in cell viability with IC₅₀ value ranging in 12 to 14.4 µM. Therefore, cell viability studies confirm the anti-tumor activity of NIF. Once the anti-tumor efficacy was confirmed we developed a Nifuroxazide-loaded solid lipid nanoparticle for treatment of lung cancer. The preliminary lipid screening study revealed that among all three lipids, Geleol^® has allowed complete solubilization of 2.5 mg of NIF exhibiting a transparent clear solution (Figure 1A). NIF-SLNs prepared with Geleol^® as solid lipid exhibited a particle size of 179.30 ± 14.33 nm, PDI of 0.32 ± 0.06 and zeta-potential of -26.56 ± 1.68 mV. The developed NIF-SLNs demonstrated an entrapment efficiency of 72.73 ± 4.67 % w/w and drug loading of 12.63 ± 1.19 µg of NIF per mg of lipid (Figure 1B). The DSC study reveal a sharp exothermic peak representing the melting point of NIF at 295 °C (Figure 2A). This characteristic NIF peak is absent in thermograms of both blank nanoparticles and NIF-loaded nanoparticles indicating the successful encapsulation of NIF in solid lipid nanoparticles. The formulation showed an initial burst release of 41.45 % in 10 h followed by a prolonged release for 48 h (figure 2B). In vitro aerodynamic studies showed good aerosolization properties, NIF-SLNs exhibited fine particle fraction (FPF) of about 72.45 ± 2.51 % and mass median aerodynamic diameter (MMAD) of 5.5 ± 0.05 µm (figure 3B and 3C). The slightly higher MMAD could be due to the viscosity of the formulation. The IC₅₀ of the free drug A549 was around 14.45 ± 0.30 µM while the formulation displayed 2-fold reduction in IC₅₀ and is more effective in inhibiting tumor progression compared to the free drug.

Conclusion: This study is at the forefront of research, as it focuses on two crucial aspects: demonstrating the efficacy of NIF against lung cancer and developing an inhalable NIF-loaded formulation. NIF-SLNs exhibits favorable particle characteristics, efficient drug entrapment, and optimal aerodynamic diameter when formulated as lipid nanocarriers. Additionally, we are conducting further research to improve the MMAD of the formulation by reducing the viscosity of formulation. Further, we will evaluate its in vitro efficacy in a three-dimensional tumor model.

Figure 1. (A) Preliminary solubility studies on various solid lipids. (B) Particle characterization of NIF-SLN formulation after optimization.

Figure 2. (A) DSC thermograph of NIF shows nifuroxazide characteristic melting peak at 295˚C, that is absent in NIF-SLN representing complete drug encapsulation into nanoparticle. (B) In-vitro release profile of NIF from NIF-SLN indicating burst release of 41.45 % in 10 h.

Figure 3. (A) Drug deposition of NIF-SLN represented as percent drug deposited on each stage of the Next Generation Impactor (NGI). (B) In vitro aerosolization parameters of NIF-SLN formulation.