Nebulized Bacterioruberin/Astaxanthin-Loaded Nanovesicles: Antitumoral Activity and Beyond

Abstract

The membranes of halophilic archaea are a source of novel biomaterials, mainly of isoprenoid nature, with therapeutic properties practically unraveled. Here, we explored the antitumoral activity of neutral archaeolipids (NAs, such as bacterioruberin, astaxanthin, and dihydrosqualene) present in the total archaeolipids (TAs) (a fraction from the first step of lipid extraction by the modified Blight and Dyer technique) extracted from halophilic archaea Halorubrum tebenquichense, and formulated as TA-nanoarchaeosomes (TA: polar archaeolipids (PAs): Tween 80, 5:5:4 w:w:w, TA-nanoARC). The structure of 300.3 ± 84.2 nm TA-nanoARC of 0.59 ± 0.12 polydispersity index and -20 ± 3.7 mV ζ potential as determined by SAXS modelling, revealed that NA reduced the hydrophobic core and enlarged its hydrophilic section in comparison to TA-lacking bilayers (nanoARC), while preserving the width (~50 Å) and unilamellarity. Stable to storage and nebulization, TA-nanoARC was cytotoxic on A549 cells after 48 h, with an IC50 expressed as [bacterioruberin] of 0.15 μg/mL (~0.20 µM), comparable to or lower than the IC50 of docetaxel or cisplatin. Such cytotoxicity was exerted at a concentration harmless to macrophages (mTHP-1 cells). Besides, the conditioned medium from TA-nanoARC nebulized on A549 cells reduced the expression of the CD204/SRA-1, an M2 phenotype marker, and induced pro-inflammatory activity, comparable to or to a greater extent than that induced by lipopolysaccharide, including IL-6 and TNF-α, in mTHP-1 as a model of tumor-associated macrophages. The endocytosis of TA-nanoARC by A549 cells induced Lysotracker red fluorescence to fade and blur. This suggested the internalization of the highly viscous and ordered TA-nanoARC rich in NAs and subsequent lysosomal dysfunction (and not its antioxidant activity), as responsible for the selective damage on A549 cells. These are the first results showing that nebulized TA-nanoARC, lethal to A549 cells and modulating mTHP-1 cell phenotype, may act as antitumorals in the absence of cytotoxic drugs.

Keywords: inhalation; lungs; xanthophylls.

Figure 1

Characterization of total archaeolipids (TAs) from H. tebenquichense. (A) UV-vis spectra; (B) Electrospray ionization–mass spectrometry (ESI-MS) analysis of polar archaeolipids (PAs) in negative ion mode and (C) neutral archaeolipids (NAs) in positive ion mode.

Figure 2

SAXS data of nanoARC and TA-nanoARC. (A) Log–log profiles with solid lines corresponding to model fits. (B) Electron density profiles derived from data fitting. n = 3.

Figure 3

RAMAN spectra of TA-nanoARC and TA of H. tebenquichense.

Figure 4

Attenuated total reflectance—Fourier transform infrared spectroscopy (ATR-FTIR) spectra of TA-nanoARC, nanoARC, Tween 80 micelles (m-T80), TA, and neutral archaeolipids (NAs) of H. tebenquichense.

Figure 5

Antioxidant activity of TA-nanoARC, nanoARC, total archaeolipids (TAs), and Trolox. Scavenging of DPPH•⁺ (A,B) ABTS•⁺ radicals as a function of phospholipid (PL) or Trolox concentrations. Insets display Trolox as a standard antioxidant control. n = 3.

Figure 6

Colloidal stability under storage of TA-nanoARC and nanoARC. The statistical analysis indicating no significant difference was determined using Wilcoxon signed-rank test. n = 3.

Figure 7

Cytotoxicity of A549 cells (A) and mTHP-1 (B) upon 48 h incubation with TA-nanoARC, nanoARC, or m-T80 after nebulization, expressed as function of phospholipid (PL) and bacterioruberin (BR) or Tween 80 (T80) concentration, n = 3. The control group consisted of cells cultured in medium only. Statistical significance compared to control was determined using a one-way ANOVA followed by Dunnett’s test, * p < 0.05; ** p < 0.01,*** p < 0.001, **** p < 0.0001.

Figure 7

Cytotoxicity of A549 cells (A) and mTHP-1 (B) upon 48 h incubation with TA-nanoARC, nanoARC, or m-T80 after nebulization, expressed as function of phospholipid (PL) and bacterioruberin (BR) or Tween 80 (T80) concentration, n = 3. The control group consisted of cells cultured in medium only. Statistical significance compared to control was determined using a one-way ANOVA followed by Dunnett’s test, * p < 0.05; ** p < 0.01,*** p < 0.001, **** p < 0.0001.

Figure 8

Antioxidant activity on A549 cells (A) and mTHP-1 (B) upon 24 h incubation with TA-nanoARC or nanoARC. Statistical significance compared to control was determined using a one-way ANOVA followed by Dunnett’s test, * p < 0.05; ** p < 0.01, *** p < 0.001. n = 3.

Figure 9

Mitochondrial membrane potential of A549 cells upon 24 h incubation with TA-nanoARC or nanoARC. Statistical significance compared to control was determined using a one-way ANOVA followed by Dunnett’s test, ** p < 0.01, **** p < 0.0001.

Figure 10

(A) Confocal fluorescence images of A549 stained with Lysotracker (red, LR) and Hoechst (green) after incubation with medium, TA-nanoARC, or nanoARC. Magnification 63× (B) Lysotracker Red (LR) fluorescence intensity inside the cells per cell was quantified using Image J Significant differences between formulations were determined using a one-way ANOVA followed by Dunnett’s test, * p < 0.05; ** p < 0.01.

Figure 10

(A) Confocal fluorescence images of A549 stained with Lysotracker (red, LR) and Hoechst (green) after incubation with medium, TA-nanoARC, or nanoARC. Magnification 63× (B) Lysotracker Red (LR) fluorescence intensity inside the cells per cell was quantified using Image J Significant differences between formulations were determined using a one-way ANOVA followed by Dunnett’s test, * p < 0.05; ** p < 0.01.

Figure 11

CD-204 fluorescence intensity on mTHP-1 cells incubated with A549 cell-conditioned media at 6.3 µg/mL phospholipids (PLs) of TA-nanoARC or nanoARC. Significance considered at ** p < 0.01.

Figure 12

Proinflammatory cytokine production (IL-6, IL-8, and TNF-α) by mTHP-1 in response to conditioned media from A549 cells treated with TA-nanoARC, nanoARC, or culture medium as a negative control. LPS was used as a positive control. Treatment concentrations (µg/mL PL—µg/mL BR): Control: 0.00–0.00; TA-nanoARC: 6.30–0.04; nanoARC: 6.30–0.00; LPS (1 µg/mL). Statistical significance was assessed against LPS and between TA-nanoARC and nanoARC using a one-way ANOVA followed by Dunnett’s test, significance considered at * p < 0.05; ** p < 0.01 *** p < 0.001, **** p < 0.0001.

Scheme 1

Electron density modeling of the bilayer.