Preparation of Terpenoid-Invasomes with Selective Activity against S. aureus and Characterization by Cryo Transmission Electron Microscopy

Abstract

Terpenoids are natural plant-derived products that are applied to treat a broad range of human diseases, such as airway infections and inflammation. However, pharmaceutical applications of terpenoids against bacterial infection remain challenging due to their poor water solubility. Here, we produce invasomes encapsulating thymol, menthol, camphor and 1,8-cineol, characterize them via cryo transmission electron microscopy and assess their bactericidal properties. While control- and cineol-invasomes are similarly distributed between unilamellar and bilamellar vesicles, a shift towards unilamellar invasomes is observable after encapsulation of thymol, menthol or camphor. Thymol- and camphor-invasomes show a size reduction, whereas menthol-invasomes are enlarged and cineol-invasomes remain unchanged compared to control. While thymol-invasomes lead to the strongest growth inhibition of S. aureus, camphor- or cineol-invasomes mediate cell death and S. aureus growth is not affected by menthol-invasomes. Flow cytometric analysis validate that invasomes comprising thymol are highly bactericidal to S. aureus. Notably, treatment with thymol-invasomes does not affect survival of Gram-negative E. coli. In summary, we successfully produce terpenoid-invasomes and demonstrate that particularly thymol-invasomes show a strong selective activity against Gram-positive bacteria. Our findings provide a promising approach to increase the bioavailability of terpenoid-based drugs and may be directly applicable for treating severe bacterial infections such as methicillin-resistant S. aureus.

Keywords: E. coli; S. aureus; bactericidal; camphor; cineol; invasomes; menthol; terpenoids; thymol.

Figure 1

Structural formulas of the terpenoids thymol, menthol, camphor and cineol used for encapsulation into invasomes.

Figure 2

Successful production of invasomes encapsulating thymol, menthol, camphor and cineol by extrusion. (A) Cryo transmission electron microscopy (TEM) showing control invasomes without terpenoids (small representative section of original micrograph). (B) Schematic view of terpenoids encapsulated by invasomes. Localization of terpenoids within the aqueous phase of the invasome was chosen only for visualization reasons and does not represent their natural localization. (C–F) Small representative sections of original cryo electron micrographs revealed invasomes comprising thymol (C), menthol (D), camphor (E) or cineol (F) after extrusion. TEM: Cryo transmission electron microscopy.

Figure 3

Invasomes without tepenoids (A) and encapsulating the terpenoids thymol (B) menthol (C) camphor (D) or cineol (E) reveal neutral Zeta potentials.

Figure 4

Characterization of lamellarity of the produced terpenoid-comprising invasomes. (A) Schematic view of individual types of lamellar phase lipid bilayers. (B) Without the addition of a terpenoid (control, B), mostly unilamellar and bilamellar vesicles were formed in equal proportions. (C–E) Invasomes comprising of thymol or camphor showed mostly unilamellar vesicles and a significantly smaller amount of bilamellar vesicles, while menthol-invasomes revealed no changes between MLV1 and MVL2. (D) Cineol-invasomes revealed a similar distribution between unilamellar and bilamellar vesicles. Distribution of the individual vesicle types is depicted in relation to their lamellarity measured from respective cryo electron micrographs. * p < 0.05 was considered significant (Mann–Whitney test, one-tailed). MLV: Multilamellar vesicles. (n.s. means not significant)

Figure 5

Encapsulation of terpenoids directly affects invasomes size. (A) Example of a small representative section of an original cryo-electron micrograph used for measuring invasomes’ size. (B) Control invasomes without terpenoid showing a mean radius of 40 ± 15 nm. (C) Thymol-comprising invasomes revealed a smaller radius of 33 ± 18 nm compared to control. (D) Production of invasomes with menthol resulted in an increased invasome radius of 59 ± 22 nm. (E) Like thymol, camphor-invasomes also shower a smaller invasome size (30 ± 16 nm radius) compared to control. (F) With a mean radius of 43 ± 17 nm, the size of invasomes with cineol was similar to control. Frequency plots of the radius distribution of the invasomes. A fit with the Gaussian function is displayed as a red line.

Figure 6

Encapsulation of terpenoids does not affect the bilayer thickness of invasomes. (A) Example of a small representative section of an original cryo-electron micrograph used for measurement of the bilayer thickness. (B–F) A similar liposomal bilayer thickness was observable for control invasomes (4 ± 0.5 nm), thymol-invasomes (5 ± 1 nm) menthol-invasomes (4 ± 0.3 nm), camphor-invasomes (4 ± 0.5 nm) or cineol-invasomes (4 ± 0.5 nm). Frequency plots of the bilayer thickness distribution of invasomes. A fit with the Gaussian function is displayed as a red line.

Figure 7

Invasomes encapsulating thymol, camphor and cineol show bactericidal activity against S. aureus in a growth-inhibition zone assay. (A) Control invasomes without terpenoids did not affect bacterial growth. (B) Strong growth inhibition of S. aureus exposed to invasomes comprising thymol. (C) Invasomes encapsulating menthol did not affect growth of S. aureus. (D–E) Growth inhibition of S. aureus exposed to invasomes comprising camphor and cineol. (F) Statistical evaluation of the measured zones of inhibition validated the significant inhibition of S. aureus growth by thymol-containing invasomes compared to all other terpenoid-invasomes and control. * p < 0.05 was considered significant (Mann–Whitney test, one-tailed). (n.s. means not significant)

Figure 8

Flow cytometric analysis of cell death validate invasomes encapsulating thymol to be strongly bactericidal against Gram-positive bacteria like S. aureus. (A,B) Compared to untreated negative control, 0.5 mg/mL or 1 mg/mL thymol packaged in invasomes resulted in a profound cell death depicted by PI-staining. (C) 2 mg/mL thymol packaged in invasomes resulted in only 9% PI-stained S. aureus. (D) Assessment of cell flow revealed only around 1000 cells/second in the S. aureus population treated with 2 mg/mL thymol-comprising invasomes, suggesting a nearly complete cell death prior to following flow cytometric analysis. PI: Propidium iodide.

Figure 9

Invasomes encapsulating thymol are not bactericidal against Gram-negative bacteria such as E. coli. (A–C) Compared to untreated negative control, treatment of E. coli. with 0.5 mg/mL, 1 mg/mL or 2 mg/mL thymol packaged in invasomes did not result in elevated amounts of cell death. (D) Assessment of cell flow revealed no relevant effects of the different concentrations of invasome-encapsulated thymol on the growth of E. coli.

Figure 10

Invasomes encapsulating cineol reveal bactericidal activity against E. coli in a growth-inhibition zone assay. (A,B) Control invasomes without terpenoids or thymol invasomes did not affect bacterial growth. (C,D) While menthol-invasomes led to a slight inhibition of E. coli growth, camphor-invasomes did not affect growth of S. aureus. (E) Strong growth inhibition of E. coli exposed to invasomes comprising cineol. (F) Statistical evaluation of the measured zones of inhibition validated the significant inhibition of E. coli growth by cineol-containing invasomes compared to all other terpenoid-invasomes and control. * p < 0.05 was considered significant (Mann–Whitney test, one-tailed). (n.s. means not significant)

Figure 11

Schematic view on invasomal packaging of thymol and its selective bactericidal activity against Gram-positive S. aureus.