Synthesis and antibacterial activity of polymer-antibiotic conjugates incorporated into a resin-based dental adhesive

Abstract

This work reports on polymer-antibiotic conjugates (PACs) as additives to resin-based restorative dental materials as a new strategy to convey sustained antibacterial character to these materials. Such antibacterial performance is expected to improve their longevity in the oral cavity. Using the previously reported ciprofloxacin (Cip)-based PAC as a control, a penicillin V (PV)-based PAC was investigated. The monomer-antibiotic conjugate (MAC) containing a methacrylate monomer group and a PV moiety was prepared via nucleophilic substitution between 2-chloroethyl methacrylate (CEMA) and penicillin V potassium (PVK). The PV-based PAC was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of the MAC with hydroxyethyl methacrylate (HEMA), and further characterized by ¹H NMR and gel permeation chromatography (GPC) analysis. Antibiotic resistance was investigated by passaging bacteria in low concentrations of the antibiotic for 19 days, followed by a 48 h challenge at higher concentrations. Our results suggest that the development of antibiotic resistance is unlikely. Zone of inhibition (ZOI) assays revealed no clearing zones around PV-containing resins indicating minimal antibiotic leakage from the material. Similarly, MTT assay demonstrated that the antibiotic-containing specimens did not release cytotoxic byproducts that may inhibit human gingival fibroblast growth. Counting of colony-forming units in an S. mutans biofilm model was used to assess bacterial survival at baseline and after subjecting the antibiotic-containing resin specimens to an enzymatic challenge for 30 days. Significantly reduced bacterial counts were observed as the biofilm aged from 24 to 72 h, and salivary enzymatic exposure did not reduce the antibacterial efficacy of the discs, suggesting that PV-resin will be effective in reducing the re-incidence of dental caries.

Fig. 1.

Synthesis of A) HEMA-PV MAC (via nucleophilic substitution), B) p(HEMA-PV-co-HEMA) PAC (via RAFT polymerization), and C) PAC-incorporated dental resin by photocuring.

Fig. 2.

500 MHz ¹H NMR spectra of (a) HEMA-PV MAC and (b) p(HEMA-PV-co-HEMA) PAC in CDCl₃.

Fig. 3.

GPC curve of p(HEMA-PV-co-HEMA) PAC.

Fig. 4.

PV release from the PV-PAC film incubated in pH 7.4 PBS buffer at 37 °C, as monitored by UV-Vis analysis. Slow and sustained PV release was observed after day 1.

Fig. 5.

Minimum inhibitory concentration (MIC) of Cip (A) and PVK (B) for S. mutants. OD₆₀₀ represents bacterial growth. Graph displays standard error of the mean of triplicate wells from one of three similar experiments.

Fig. 6.

Antibiotic resistance development. Following passage in low concentrations of antibiotics, S. mutans did not develop resistance to Cip (A) or PVK (B). Graph points are the average OD₆₀₀ absorbance values of duplicate wells where OD₆₀₀ represents bacterial growth.

Fig. 7.

Zone of inhibition assessment with S. mutans. Antibiotic does not diffuse out from the resin matrix to inhibit bacterial growth. No ZOI were observed for control SB+ resin discs (A) or PV-PAC resin discs (B). Disc conditioned media on paper discs also produced no ZOI for SB+ resin (C) or PV-PAC resin (D). Paper disc alone (E) and paper disc with DMSO (F) do not inhibit bacterial growth. PV-PAC in DMSO on a paper disc (G) efficiently prevents bacterial growth, producing a large ZOI.

Fig. 8.

Cytotoxicity assessment. Resins did not release cytotoxic by-products that inhibit human gingival fibroblast growth. OD₅₄₀ represents metabolic activity as measured by MTT assay. Graph represents two replicate experiments showing the average of triplicate wells, error bars represent standard error of the mean. No statistical differences among groups were observed (t-test, p < 0.05).

Fig. 9.

Bacterial survival in biofilms on PAC modified resin discs represented as percent colony forming units at baseline (A) and after 30 days of incubation in simulated human salivary enzyme solution (B). Discs were incubated for 24, 48, and 72 h with S. mutans. Bars represent the normalized average of three experiments, error bars represent standard error of the mean. Significant differences within groups are identified by * (Tukey’s test, p<0.05).

Fig. 10.

Scanning electron microscopy (SEM) images of S. mutans biofilm formation (72-h) on resin discs following SHSE incubation. Control SB+ (A) and PV-PAC discs (B) were imaged to show the disc surface with no bacteria. Visibly more bacteria are present on the control resin disc (C) compared to the PV PAC-modified resin disc (D) at 10,000X. Classical coccus bacteria are present on the control resin observed at 25,000X, while the cell morphology is notably altered on the PV-PAC disc (F) as the cells appear long and extended (white arrows), suggesting the inability to divide correctly.