Cationically modified inhalable nintedanib niosomes: enhancing therapeutic activity against non-small-cell lung cancer

Abstract

Aim: This study was designed to develop and test nintedanib-loaded niosomes as inhalable carriers for enhancing its therapeutic efficacy via localized drug accumulation and addressing issues such as low bioavailability and severe toxicity. Methods: Niosomes were prepared by thin-film hydration method and were evaluated for in vitro therapeutic effectiveness in lung cancer cells. Results: The optimized niosomal formulation displayed optimized vesicle size, controlled and extended release of drug, and efficient aerodynamic properties indicating its suitability as an aerosolized formulation. In vitro studies revealed significantly superior cytotoxicity of nintedanib-loaded niosomes which was further validated by 3D spheroids. Conclusion: These findings establish the effectiveness of niosomes as inhalable delivery carriers which could serve as a promising strategy for delivery of nintedanib to treat several lung cancers.

Keywords: aerosol; cationically modified vesicles; inhalable delivery carrier; nintedanib; niosomes; non-small-cell lung cancer; tyrosine kinase.

Figure 1.. Physicochemical characterization of niosomes during development and optimization.

(A) Cryo-TEM micrographs of nint reg nio (F2) and nint DO nio (F4). Scale bar 200 nm. (B) Calorimetric study confirming the encapsulation of nint in niosomes. (C) Powder x-ray diffraction characterization of samples: (i) Nintedanib (nint), (ii) blank DO Nio, (iii) physical mixture of blank DO nio and nint, and (iv) nint DO nio. (D) In vitro release profile of optimized niosomes revealing biphasic release nature of the niosomal vesicles. Data represent mean ± standard deviation (n = 3). DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt); Nint DO nio: Nint-loaded niosomes containing DOTAP; Nint Reg nio: Nint-loaded niosomes prepared without DOTAP.

Figure 2.. Cellular uptake studies.

(A) Qualitative illustration by representative florescent images of A549 cells after treatment with control (coumarin-6) and coumarin-loaded niosomes for 1 h and 3 h. Scale bar = 100 μm. (B) Quantitative representation by measuring the fluorescence intensity of intracellular coumarin after lysing the cells. Data represent mean ± standard deviation (n = 3). *p < 0.05; ****p < 0.0001. DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt); Nint DO nio: Nint-loaded niosomes containing DOTAP; Nint Reg nio: Nint-loaded niosomes prepared without DOTAP.

Figure 3.. Cytotoxicity studies performed in five different non-small-cell lung cancer (NSCLC) cell lines.

Cell lines (A) A549, (B) H2122, (C) H1299, (D) H358 and (E) H460. Additionally, cytotoxicity of blank niosomes was performed in (F) A549 cancer cells and (G) HEK-293 normal cells HEK cell. Data represent mean ± standard deviation of three individual experiments with n = 6 for each experiment. HEK: Human embryonic kidney; Nint: Nintedanib; Nint DO nio: Nint-loaded niosomes containing DOTAP; Nint Reg nio: Nint-loaded niosomes prepared without DOTAP.

Figure 4.. In vitro aerosolization performance of nint DO nio.

(A) Aerodynamic distribution expressed as percentage deposition at each stage in Next Generation Impactor™. (B) Percentage of cumulative deposition of nint DO nio against effective aerodynamic cut-off diameter of respective stages of Next Generation Impactor™. (C) Aerosolized properties of niosomes; MMAD, percentage FPF and GSD for nint DO nio. Data are represented as mean ± standard deviation for n = 3 experiments. FPF: Fine particle fraction; GSD: Geometric standard deviation; MMAD: Mass median aerodynamic diameter; Nint DO nio: Nint-loaded niosomes containing DOTAP.

Figure 5.. Migration studies performed using scratch assay on A549 cancer cells.

(A) Representative microscopic images of scratch after treatment with nint and nint DO nio at 1.25 mM and 7.5 μM capture at 0 h (pretreatment), 24 h and 48 h post-treatment. (B) Quantitative representation of the studies measured as percentage scratch closure at different time intervals. Data represent mean ± standard deviation (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Nint: Nintedanib; Nint DO nio: Nint-loaded niosomes containing DOTAP.

Figure 6.. Clonogenic studies performed in A549 cells.

(A) Representative images of colonies of different treatment groups. (B) Quantitative representation of treatment measured as percentage reduction in colonies at 1.25 μM and 7.5 μM concentration of the treatments. Data represent mean ± standard deviation (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001. Nint: Nintedanib; Nint DO nio: Nint-loaded niosomes containing DOTAP.

Figure 7.. Tumor simulation studies performed in A549 cells.

(A) Single-dose treatment regimen: antitumor activity of treatment groups represented as spheroid volume versus time. (B) Multiple-dose treatment regimen: representative images of spheroids from various treatment groups at days 1, 6 and 12. (C) Multiple-dose treatment regimen: quantitative representation of treatment measured as spheroid volume versus time for different treatment groups. Data represent mean ± standard deviation (n = 6). Scale bar = 100 μm. *p < 0.05; ***p < 0.001. Nint: Nintedanib; Nint DO nio: Nint-loaded niosomes containing DOTAP.

Figure 8.. Evaluation of antitumor activity of treatment groups on spheroids after 15 days of incubation.

(A) Quantitative representation by measuring percentage cell viability obtained using CellTiter-Glo 3D calculated relative to untreated cells (control) considered as 100%. Data represent mean ± standard deviation (n = 3). (B) Representative florescent images of spheroid from live–dead cell assay, where green dye = viable cells and red dye = dead cells in spheroids. Scale bar = 100 μm. (C) Quantitative representation of live–dead cell assay measured as red fluorescent protein (relative to control) for different treatment groups at different treatment regimens. Data represent mean ± standard deviation (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Nint: Nintedanib; Nint DO nio: Nint-loaded niosomes containing DOTAP.