(T1130-10-57) Preparation of Nebulised Luteolin-Loaded Nano-formulations as a Potential Management for Pulmonary Atrial Hypertension

Purpose: Luteolin is a natural flavonoid abundant in several fruits, vegetables, and medicinal herbs [1]. It exhibits multiple pharmacological activities, including potentially treating pulmonary arterial hypertension [1, 2]. However, its low water solubility and bioavailability restrict its therapeutic efficacy [3]. Nano-formulations such as nano-liposomes and nano-emulsions are widely used in pharmaceutical research to enhance the compound’s solubility and absorption [4]. This research aims to develop inhalable luteolin-loaded nano-liposomes and nano-emulsions to seek a suitable nano-formulation to overcome luteolin drawbacks. In addition, the effectiveness of the developed luteolin nano-formulations as a potential treatment for pulmonary arterial hypertension will be examined.

Methods: The thin-film rehydration method was applied to formulate the inhalable luteolin nano-liposomes [5]. Luteolin and a total of 40 mg of two phospholipids with high-phase transition temperatures and cholesterol were dissolved in (1:1, v/v) methanol and chloroform. The organic solvents were evaporated to form a lipid-thin film using a vacuum rotary evaporator at 30 mbar. A hand-mixing technique was used to rehydrate the thin film. Luteolin nano-emulsion formulations were developed by dissolving an appropriate amount of luteolin in a pre-measured quantity of limonene oil (oil phase), Tween 80 (surfactant), and ethanol (co-surfactant). The aqueous phase was added after completely dissolving the compound in the oily phase. Ten minutes of sonication have been applied to the nano-emulsion to enable the mixing of all the ingredients. The nano-emulsion formulation with the lowest percentage of Tween 80 and ethanol and the highest luteolin loading has been chosen to be further tested and compared with the nano-liposome. The selected nano-emulsion has the following percentages for its components: limonene oil (0.2%), Tween 80 (0.3%), ethanol (0.3%), and deionized water (99.2%). All the percentages in this formulation were calculated using the (w/w) ratio. The mean particle sizes (Z-average) and polydispersity index (PDI) of the newly developed luteolin nano-liposome and nano-emulsion formulations were measured via Malvern Zetasizer NanoZs with dynamic light scattering (DLS, Malvern Instruments, Malvern, UK) at 25 °C and a scattering angle of 173°. Cell-based assays were executed according to previously published protocols, including cell culture and DNA transfection [6, 7]. The impact of newly developed nano-formulations on the TGF-β pathway was determined by a TGFβ-responsive reporter assay using HEK 293 cells. In addition, their anti-proliferative effect was assessed by an anti-proliferative test (MTT) using mutated (bmpr2R899X+/-) mouse pulmonary arterial smooth muscle cells (PASMCs). The aerodynamic behaviour of the developed luteolin nano-liposome was determined using the next-generation impactor (NGI). The luteolin was quantified using reverse-phase HPLC (ACQUITY Arc, Waters) with a diode array detector (λ = 352).

Results: Nanoscale luteolin-loaded nano-liposomes and nano-emulsions have been developed with average sizes equal to 117± 7.4 nm and 76± 5.8 nm, respectively. Both formulations have a narrow PDI ≤ 0.2 and high encapsulation efficiency. The TGF-β pathway is stimulated in pulmonary arterial hypertension lungs [8]. Inhibiting this pathway offers a promising treatment option for patients with pulmonary arterial hypertension [8]. Free luteolin and luteolin-loaded nano-liposomes significantly inhibit TGF-β signalling using a TGFβ-responsive reporter assay (p ≤ 0.001). MTT assay results revealed that the newly formulated luteolin nano-liposomes have a significantly higher anti-proliferative effect than free luteolin on mutated (bmpr2R899X+/-) PASMCs (p ≤ 0.001), as depicted in Figure 1. Interestingly, none of the nano-liposome vehicles or luteolin-loaded nano-liposomes showed cytotoxicity or cell morphology changes (see Figures 1 and 2). However, it was found in this study that luteolin-loaded nano-emulsion has a cytotoxic effect on both HEK293T cells and mutated (bmpr2R899X+/-) PASMCs (p ≤ 0.001). See Figure 2 and Figure 3. Consequently, only the developed luteolin-loaded nano-liposome has been chosen to assess its aerodynamic behaviour further using NGI. The results revealed that the newly developed luteolin-nano-liposome exhibited excellent aerodynamic properties, as follows: The mass median aerodynamic diameter was 3.91 ± 0.04 µm, the fine particle fraction ≤ 5 µm was 58.59 ± 0.34%, and the percentage of particles ≤ 3 µm was 38.84 ± 0.28%. These findings indicate that the newly developed luteolin liposome possesses all the necessary characteristics to be inhalable and is anticipated to be effectively delivered deeply into the lungs.

Conclusion: Luteolin-loaded nano-liposomes and nano-emulsions were developed, and their therapeutic potential as a treatment for pulmonary arterial hypertension has been assessed. The newly developed luteolin nano-liposome holds promise in treating pulmonary arterial hypertension. Moreover, it has all the characteristics to be inhalable and to deposit deeply in the lungs. Meanwhile, luteolin nano-emulsion has been shown to have a cytotoxic effect.

References: 1. Ganai, S.A., et al., Anticancer activity of the plant flavonoid luteolin against preclinical models of various cancers and insights on different signalling mechanisms modulated. Phytotherapy Research, 2021. 35(7): p. 3509-3532.
2. Zhang, J.-j., M.-m. Shao, and M.-c. Wang, Therapeutic potential of natural flavonoids in pulmonary arterial hypertension: A review. Phytomedicine, 2024: p. 155535.
3. Zhang, N., et al., Formulation and evaluation of luteolin supersaturatable self-nanoemulsifying drug delivery system (S-SNEDDS) for enhanced oral bioavailability. Journal of Drug Delivery Science and Technology, 2020. 58: p. 101783.
4. Reddy, T.S., R. Zomer, and N. Mantri, Nanoformulations as a strategy to overcome the delivery limitations of cannabinoids. Phytotherapy Research, 2023. 37(4): p. 1526-1538.
5. Bangham, A.D., M.M. Standish, and J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of molecular biology, 1965. 13(1): p. 238-IN27.
6. Chowdhury, H., et al., Aminoglycoside-mediated promotion of translation readthrough occurs through a non-stochastic mechanism that competes with translation termination. Human Molecular Genetics, 2018. 27(2): p. 373-384.
7. Adan, A., Y. Kiraz, and Y. Baran, Cell proliferation and cytotoxicity assays. Current pharmaceutical biotechnology, 2016. 17(14): p. 1213-1221.
8. Sharmin, N., C.C. Nganwuchu, and M.T. Nasim, Targeting the TGF-β signaling pathway for resolution of pulmonary arterial hypertension. Trends in Pharmacological Sciences, 2021. 42(7): p. 510-513.

Acknowledgements: This research was supported by Schlumberger Foundation faculty for the future in the form of PhD funding awarded to Fatma Haddad.
Conflicts of Interest: “The authors declare no conflict of interest.”

Figure. 1. Relative antiproliferative effects of free luteolin, luteolin-loaded nano-liposome (Nano-liposome-lut), and free liposome (FL) using MTT assay.

Figure. 2. Epifluorescence microscope images of (a) untreated mutated (bmpr2R899X+/-) mouse pulmonary arterial smooth muscle cells (PASMCs) after 24 hr; (b) untreated mutated (bmpr2R899X+/-) PASMCs after 72 hr; (c) mutated (bmpr2R899X+/-) PASMCs after 72 hr from the treatment with luteolin 20 µM; (d) mutated (bmpr2R899X+/-) PASMCs after 72 from the treatment with luteolin nano-liposome 20 µM; (e) mutated (bmpr2R899X+/-) PASMCs after 72 hr from the treatment with luteolin nano-emulsion five µM; (f) mutated (bmpr2R899X+/-) PASMCs after 72 hr from the treatment with luteolin nano-emulsion 20 µM.

Figure .3. Cytotoxic effects of luteolin-loaded nano-emulsion using MTT assay.