Development of Novel Biocomposites with Antimicrobial-Activity-Based Magnesium-Doped Hydroxyapatite with Amoxicillin

Abstract

Background/Objectives: A biocomposite based on magnesium-doped hydroxyapatite and enriched with amoxicillin (MgHApOx) was synthesized using the coprecipitation method and is presented here for the first time. Methods: The stability of MgHAp and MgHApOx suspensions was evaluated by ultrasound measurements. The structure of the synthesized MgHAp and MgHApOx was examined with X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The crystalline structure was determined by X-ray diffraction. The FTIR data were collected in the range of 4000-400 cm^-1. The morphology of the nanoparticles was evaluated by scanning electron microscopy (SEM). Furthermore, the biocompatible properties of MgHAp, MgHApOx and amoxicillin (Ox) suspensions were assessed using human fetal osteoblastic cells (hFOB 1.19 cell line). The antimicrobial properties of the MgHAp, MgHApOx and Ox suspension nanoparticles were assessed using the standard reference microbial strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231. Results: X-ray studies have shown that the biocomposite retains the characteristics of HAp and amoxicillin. The SEM assessment exhibited that the apatite contains particles at nanometric scale with acicular flakes morphology. The XRD and SEM results exhibited crystalline nanoparticles. The average crystallite size calculated from XRD analysis increased from 15.31 nm for MgHAp to 17.79 nm in the case of the MgHApOx sample. The energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) analysis highlighted the presence of the constituent elements of MgHAp and amoxicillin. Moreover, XPS confirmed the substitution of Ca²⁺ ions with Mg²⁺ and the presence of amoxicillin constituents in the MgHAp lattice. The results of the in vitro antimicrobial assay demonstrated that MgHAp, MgHApOx and Ox suspensions exhibited good antimicrobial activity against the tested microbial strains. The results showed that the antimicrobial activity of the samples was influenced by the presence of the antibiotic and also by the incubation time. Conclusions: The findings from the biological assays indicate that MgHAp and MgHApOx are promising candidates for the development of new biocompatible and antimicrobial agents for biomedical applications.

Keywords: amoxicillin; antimicrobial activity; hydroxyapatite; magnesium; suspensions.

Figure 1

Time evolution of the recorded signals of MgHAp suspension from left to right over 5000 s (a); Recorded signals amplitudes during the experiment for MgHAp suspension (b).

Figure 2

Time evolution of the recorded signals of MgHApOx suspension from left to right over 4500 s (a); Recorded signals amplitudes during the experiment for MgHApOx suspension (b).

Figure 3

Spectral amplitudes of all recorded signals (a), time-averaged attenuation for the investigated frequency range (b) and relative spectral amplitudes vs. time (c) of MgHAp sample.

Figure 4

Spectral amplitudes of all recorded signals (a), time-averaged attenuation for the investigated frequency range (b) and relative spectral amplitudes vs. time (c) of MgHApOx sample.

Figure 5

XRD patterns of MgHAp (b), MgHApOx (c) and Ox (d) samples. The JCPDS # 09-0432 of HAp (a) and JCPDS # 39-1832 of Ox (e). The * indicates the maxima associated with amoxicillin structure.

Figure 6

XRD patterns of MgHAp (a), MgHApOx (b) relative shift on 2θ range of 25–35°. The * indicates the maxima associated with amoxicillin structure.

Figure 7

(a,b) SEM micrographs obtained at ×100,000 and at ×200,000 for MgHAp sample; (d,e) SEM micrographs obtained at ×100,000 and at ×200,000 for MgHApOx sample. (c,f) particle size distribution obtained for MgHAp and MgHApOx.

Figure 8

Energy-dispersive X-ray spectroscopy spectra of (a) MgHAp and (b) MgHApOx.

Figure 9

FTIR general spectra of MgHAp, MgHApOx and Ox.

Figure 10

FTIR spectra of MgHAp, MgHApOx and Ox obtained between 900 and 1200 cm⁻¹ and their second derivative curve.

Figure 11

Deconvoluted FTIR spectra of the MgHAp, Ox and MgHApOx obtained in the 900–1200 cm⁻¹ spectral domain.

Figure 12

General XPS spectra of MgHAp (a) and MgHApOx (b) samples.

Figure 13

High-resolution XPS spectra and curve-fitting results of C 1s for MgHAp (a) and MgHApOx (b); O1s for MgHAp (c) and MgHApOx (d); Ca2p for MgHAp (e) and MgHApOx (f); P2p for MgHAp (g) and MgHApOx (h).

Figure 14

High-resolution XPS spectra and curve-fitting results of Mg 2p for MgHAp (a) and MgHApOx (b).

Figure 15

High-resolution XPS spectra and curve-fitting results of N1s (a) and S 2p (b) for MgHApOx sample.

Figure 16

Percentage hemolysis of sheep red blood cells (RBCs) exposed to different concentrations of MgHAp, MgHApOx and Ox suspensions. The statistical analysis of the data was performed using one-way ANOVA. The calculated p-values were p < 0.002.

Figure 17

MTT assay of hFOB 1.19 cells incubated with MgHAp, MgHApOx and Ox suspensions for 24, 48 and 72 h. The results are represented as mean ± standard deviation (SD) and are expressed as percentages of control (100% viability). The statistical analysis of the data was performed using one-way ANOVA. The p-values indicated are * p ≤ 0.002, ** p ≤ 0.001, *** p ≤ 0.0001.

Figure 18

Lactate dehydrogenase (LDH) activity released in the culture medium of hFOB 1.19 cells after the treatment with MgHAp, MgHApOx and Ox suspensions for 24, 48 and 72 h. The results are represented as mean ± standard deviation (SD). The statistical analysis of the data was performed using one-way ANOVA. The p-values indicated are * p ≤ 0.002, ** p ≤ 0.001, *** p ≤ 0.0001.

Figure 19

Graphical representation of the logarithmic values of colony forming units (CFU)/mL of S. aureus ATCC 25923 (a), E. coli ATCC 25922 (b) and C. albicans ATCC 10231 (c) microbial strains after 24, 48 and 72 h of exposure to MgHAp, MgHApOx and Ox suspensions. #—depicts the bactericidal properties of the samples. The results are represented as mean ± standard error. Ordinary one-way ANOVA was used for the statistical analysis. The p-values indicated are * p ≤ 0.002, ** p ≤ 0.001, *** p ≤ 0.0001.

Figure 20

The schematic representation (a) and the image (b) of US experimental setup [95].