Monocyclic β-lactams loaded on hydroxyapatite: new biomaterials with enhanced antibacterial activity against resistant strains

Abstract

The development of biomaterials able to act against a wide range of bacteria, including antibiotic resistant bacteria, is of great importance since bacterial colonization is one of the main causes of implant failure. In this work, we explored the possibility to functionalize hydroxyapatite (HA) nanocrystals with some monocyclic N-thio-substituted β-lactams. To this aim, a series of non-polar azetidinones have been synthesized and characterized. The amount of azetidinones loaded on HA could be properly controlled on changing the polarity of the loading solution and it can reach values up to 17 wt%. Data on cumulative release in aqueous solution show different trends which can be related to the lipophilicity of the molecules and can be modulated by suitable groups on the azetidinone. The examined β-lactams-HA composites display good antibacterial activity against reference Gram-positive and Gram-negative bacteria. However, the results of citotoxicity and antibacterial tests indicate that HA loaded with 4-acetoxy-1-(methylthio)-azetidin-2-one displays the best performance. In fact, this material strongly inhibited the bacterial growth of both methicillin resistant and methicillin susceptible clinical isolates of S. aureus from surgical bone biopsies, showing to be a very good candidate as a new functional biomaterial with enhanced antibacterial activity.

Figure 1

Sketch of the possible applications of the biomaterials developed in this paper. R and R’ are defined in Figure 3.

Figure 2

β-lactams evaluated in this study. Calculated logP (CLogP) values were obtained with ChemDraw 15.0 program (specific algorithms for calculating logP from fragment based methods were developed by the Medicinal Chemistry Project of CambridgeSoft and BioByte).

Figure 3

Synthesis of N-thiosubstituted β-lactams, reaction yields in parenthesis.

Figure 4

Medium effect on loading of azetidinones 1, 1a, 2, and 2a on HA. Loading efficiency % (grey), azetidinone residue in DCM (white) and in the aqueous layer (blue).

Figure 5

Left: ATR-FTIR spectra for samples 1-HA, 2-HA, 1a-HA, 1b-HA, 2a-HA, 2b-HA. Right: comparison between spectra of 2a-HA, HA, and 2a pure compound; assignments of the main bands are indicated. An enlarged view for all samples is reported in Fig. SI-4.

Figure 6

TEM images of HA, 1a-HA, and 2a-HA nanocrystals. Scale bar = 200 nm. All images have the same magnification.

Figure 7

Release of azetidinones 1a (♦ blue), 1b (▪ red), 2a (▴ green), and 2b (X violet) from 1a-HA, 1b-HA, 2a-HA, and 2b-HA, in aqueous solution(left), buffer solution at pH 7.4 (center), buffer solution at pH 5.0 (right) media. The cumulative release is reported as mol %.

Figure 8

WST1 assay (a), and LDH release (b) of MG63 after 48 and 72 hours of culture on HA, 1a-HA, 1b-HA, 2a-HA, 2b-HA samples and CTRs. Values are reported as mean ± SD (*p < 0.05; **p < 0.005; ***p < 0.0005). (a) ***CTR + , 1b-HA, 2a-HA vs HA, 1a-HA, 2b-HA, CTR– (48 and 72 h); *1a-HA vs HA, CTR- (48 h); *2b-HA vs HA, CTR- (48 and 72 h), *HA vs CRT– (48 h). (b) ***CTR + , 1b-HA, 2a-HA vs HA, 1a-HA, CTR– (48 and 72 h); *1a-HA vs HA, CTR- (48 h); **2b-HA vs HA, CTR- (48 h); *2b-HA vs CTR- (72 h).