Encapsulation of erythromycin and bacitracin antibiotics into natural sporopollenin microcapsules: antibacterial, cytotoxicity, in vitro and in vivo release studies for enhanced bioavailability

Abstract

Nature produces large quantities of superbly complex and highly reliable microcapsules. The micrometre-sized Lycopodium clavatum spores are one example of these robust capsules. The encapsulation of erythromycin (EM) and bacitracin (BAC) antibiotics into the Lycopodium clavatum sporopollenin (LCS) extracted from these spore species is explored for the first time. The LCS microparticles are extensively characterised before and after loading using SEM, CLSM, TGA and FTIR techniques. The loading capacity and entrapping efficiency of EM were 16.2 and 32.4%, respectively. The antibacterial activities of pure antibiotics, empty LCS and the antibiotic-loaded LCS were evaluated against Staphylococcus aureus (Gram-positive), Pseudomonas aeruginosa (Gram-negative), and Klebsiella pneumoniae (Gram-negative) human pathogenic bacterial strains. A remarkable increase in the antibacterial fold activity of both EM- and BAC-loaded LCS compared to that of the pure antibiotics is observed. Crucial for drug delivery applications, empty LCS, EM- and BAC-loaded LCS were found to be nontoxic against human epithelial colorectal adenocarcinoma cells Caco-2 as revealed by the cytotoxicity evaluation. The in vitro release mechanism of EM in pH 7.4 showed a deviation from Fick's law. In vivo release of EM from EM-loaded LCS (an oral dose of 50 mg kg^-1) revealed high values of the area under the plasma concentration-time curve (AUC_{0-6 h} and AUC_0-∞ were 1620 and 2147 μg h L^-1, respectively) indicative of the enhanced EM bioavailability. The successful loading of antibiotics into the nontoxic LCS and the enhanced bioavailability can open up intriguing applications in oral and topical drug delivery strategies.

Fig. 1. SEM images of LCS microcapsules showing the surface morphological characterisations. (A) Empty LCS before loading and (B) after passive-vacuum EM loading.

Scheme 1. Proposed mechanism of passive-vacuum antibiotic encapsulation into empty LCS microcapsules.

Fig. 2. CLSM images of single empty LCS microparticle before the loading process. The microparticle was scanned with three laser excitation wavelengths represented by the blue, green and red channels. An image for the overlay of the three channels and the improved contrast DIC image are shown. Large empty black cavity ready for loading is obvious. Images were captured on a slice of a depth (z-direction) of around 12.5 μm.

Fig. 3. Overlay CLSM images of (A) multiple empty LCS microcapsules. (B and C) LCS microparticle loaded with EM–eosin Y binary complex via passive-vacuum technique showing the successful loading of the EM antibiotic inside the cavity of the sporopollenin microparticles.

Fig. 4. TGA analysis for EM, empty and EM-loaded LCS.

Fig. 5. FTIR spectra of pure erythromycin, empty LCS and EM-loaded LCS.

Fig. 6. Digital images showing antibacterial activity obtained using the disc diffusion method for different bioagents against different bacteria strains (given). Numbers given above the different inhibition zones represent: (1) empty LCS; (2) erythromycin; (3) bacitracin; (4) EM-loaded LCS and (5) BAC-loaded LCS.

Fig. 7. Antibacterial and synergistic activity of empty LCS, EM-loaded LCS and BAC-loaded LCS.

Fig. 8. Cytotoxicity evaluation of empty LCS, EM-loaded LCS and BAC-loaded LCS.

Fig. 9. In vitro release profile of EM from EM-loaded LCS into phosphate buffered saline (PBS), pH = 7.4, at 37 °C. The inset is the initial release profile within 30 minutes.

Fig. 10. Plasma concentration–time profile of EM-loaded LCS after oral administration of 50 mg kg⁻¹ to male albino rats.