Fabrication and Characterization of Cinnamaldehyde-Loaded Mesoporous Bioactive Glass Nanoparticles/PHBV-Based Microspheres for Preventing Bacterial Infection and Promoting Bone Tissue Regeneration

Abstract

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is considered a suitable polymer for drug delivery systems and bone tissue engineering due to its biocompatibility and biodegradability. However, the lack of bioactivity and antibacterial activity hinders its biomedical applications. In this study, mesoporous bioactive glass nanoparticles (MBGN) were incorporated into PHBV to enhance its bioactivity, while cinnamaldehyde (CIN) was loaded in MBGN to introduce antimicrobial activity. The blank (PHBV/MBGN) and the CIN-loaded microspheres (PHBV/MBGN/CIN5, PHBV/MBGN/CIN10, and PHBV/MBGN/CIN20) were fabricated by emulsion solvent extraction/evaporation method. The average particle size and zeta potential of all samples were investigated, as well as the morphology of all samples evaluated by scanning electron microscopy. PHBV/MBGN/CIN5, PHBV/MBGN/CIN10, and PHBV/MBGN/CIN20 significantly exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli in the first 3 h, while CIN releasing behavior was observed up to 7 d. Human osteosarcoma cell (MG-63) proliferation and attachment were noticed after 24 h cell culture, demonstrating no adverse effects due to the presence of microspheres. Additionally, the rapid formation of hydroxyapatite on the composite microspheres after immersion in simulated body fluid (SBF) during 7 d revealed the bioactivity of the composite microspheres. Our findings indicate that this system represents an alternative model for an antibacterial biomaterial for potential applications in bone tissue engineering.

Keywords: antibacterial activity; bioactive glass nanoparticles; bone tissue engineering; cinnamon oil; drug delivery systems; emulsion solvent evaporation method; polyhydroxyalkanoates.

Figure 1

Scanning electron micrographs of (a) PHBV/MBGN; (b) PHBV/MBGN/CIN5; (c) PHBV/MBGN/CIN10, and (d) PHBV/MBGN/CIN20 microspheres. Arrows indicate mesoporous bioactive glass nanoparticles.

Figure 2

FTIR spectra of (a) PHBV/MBGN; (b) CIN; (c) PHBV/MBGN/CIN5; (d) PHBV/MBGN/CIN10, and (e) PHBV/MBGN/CIN20 in the range of 4000–400 cm⁻¹. The relevant peaks are discussed in the text.

Figure 3

In vitro CIN release behavior of PHBV/MBGN/CIN5, PHBV/MBGN/CIN10, and PHBV/MBGN/CIN20 microspheres in simulated body fluid: (a) CIN cumulative amount (µg mL⁻¹), and (b) CIN cumulative release (%). Experimental data are reported as mean ± standard deviation. n = 3. A sample of PHBV/MBGN/CIN20 showed the highest CIN cumulative amount and CIN cumulative release.

Figure 4

Relative viability of (a) Staphylococcus aureus and (b) Escherichia coli on different concentrations of CIN-loaded PHBV/MBGN microspheres (PHBV/MBGN/CIN5, PHBV/MBGN/CIN10, and PHBV/MBGN/CIN20) at 3, 6, and 24 h incubation. Bacterial viability in free microspheres’ medium was used as reference. Experimental data are reported as mean ± standard deviation. n = 3. Means followed by the different letters within columns indicate a significant difference at p < 0.05 using Duncan’s new multiple range test. Each different time point was analyzed separately.

Figure 5

Cytotoxicity test of osteosarcoma MG-63 cells on the different microsphere concentrations (1000, 100, 10, and 1 µg mL⁻¹) for (a) day 1 and (b) day 5. The relative cell viability in free microsphere medium was used as reference. Experimental data are reported as mean ± standard deviation (n = 3). Means followed by the different letters within columns indicate a significant difference at p < 0.05 using Duncan’s new multiple range test. Each different microsphere concentration was analyzed separately; (c) Scanning electron micrographs showing cell adhesion and attachment on the surface of PHBV/MBGN/CIN5, PHBV/MBGN/CIN10, and PHBV/MBGN/CIN20 microspheres at the highest concentration of microspheres, i.e., 1000 µg mL⁻¹, after 5 d of culture, the arrows indicate the presence of cells or filopodia.

Figure 6

The bioactivity analysis of PHBV/MBGN/CIN20 microspheres by simulated body fluid (SBF) immersion test: (a) FTIR spectra; (b) XRD patterns; (c) Scanning electron micrograph indicating hydroxyapatite formation on the surface of microspheres, as shown by arrows; (d) A representative EDS spectrum showing the presence of C, O, P, and Ca. The peak of Au is detected from the substrate as a result of the gold sputtering process.