In this study, three dimethacrylate quaternary ammonium compounds N,N-bis[2-(3-(methacryloyloxy)propanamido)ethyl]-N-methyldodecyl ammonium iodide (QADMAI-12), N,N-bis[2-(3-(methacryloyloxy)propanamido)ethyl]-N-methylhexadecyl-ammonium iodide (QADMAI-16), and N,N-bis[2-(3-(methacryloyloxy)propanamido)ethyl]-N-methyloctadectyl ammonium iodide (QADMAI-18) were synthesized and proposed to be used as antibacterial and radio-opaque agents in dental resin.
All QADMAIs were synthesized through a 2-steps reaction route, and their structures were confirmed by FT-IR and 1 H NMR spectra. Antibacterial activities against Streptococcus mutans ( S. mutans ) of QADMAIs were measured by agar diffusion test. Each QADMAI was mixed with TEGDMA (50/50, w/w) and photoinitiation system (0.7 wt% of CQ and 0.7 wt% of DMAEMA) to form resin system. Degree of monomer conversion (DC) was determined by FT-IR analysis. The flexural strength (FS) and modulus (FM) of the polymer were measured using a three-point bending set up. Radiograph was taken to determine the radio-opacity of the polymer, and aluminum step-wedge (0.5–4 mm) was used as calibration standard. Surface charge density was measured using fluorescein binding. A single-species biofilm model with S. mutans as the tests organism was used to evaluate the antibacterial property of the polymer. Bis-GMA/TEGDMA resin system was used as control material in all of the tests.
FT-IR and 1 H NMR spectra showed that the structures of QADMAIs were the same as designed. ANOVA analysis revealed that antibacterial activity of QADMAI decreased with the increasing of alkyl chain length ( p < 0.05). QADMAI containing polymers had higher DC ( p < 0.05) but lower FS and FM ( p < 0.05) than control polymer. Alkyl chain length had no influence on DC ( p > 0.05), but FS and FM of QADMAI-12 containing polymer were better than those of QADMAI-16 and QADMAI-18 containing polymers ( p < 0.05). QADMAI containing polymers had much better radio-opacity than control polymer ( p < 0.05), and the radio-opacity of polymer decreased with the increasing of alkyl chain length ( p < 0.05). All of QADMAIs containing polymers had higher surface charge density than control polymer ( p < 0.05), and surface charge densities of QADMAI-12 and QADMAI-16 containing polymers were nearly the same ( p > 0.05) which were higher than that of QADMAI-18 containing polymer ( p < 0.05). All of QADMAI containing polymers had good inhibitory effect on biofilm formation.
QADMAIs had no miscibility problem with TEGDMA, and QADMAIs could endow dental resin with both antibacterial activity and radio-opacity. Formulation of QADMAI containing resin should be optimized in terms of mechanical stregth to satisfy the requirements of dental resin for clinical application.
Dental composites, which consist of methacrylate monomers and inorganic fillers, have been widely used in clinic because of their aesthetic and easy handling properties. However, the problem of dental composites materials is their limited longevity due to bulk fractures and secondary caries, and the failed restorations are commonly treated by total replacement, which always leads to a significant amount of tooth structure loss. . Secondary caries that often occurs at the interface between the restoration and the cavity preparation has been reported as the main reason for restoration failure of dental composites materials . The bacteria such as Streptococcus mutans present in the dental plaque most likely play a major role in the development of secondary caries . Plaque accumulation and development of secondary caries were studied to be influenced by the properties of dental restorations . Unfortunately, because they have no intrinsic antibacterial activity, dental composites materials have been reported as accumulating more plaque than the other restorative materials such as ceramics and metals in vitro or in vivo . Therefore, one approach to achieving long-lasting restorations is endowing dental composites materials with antibacterial activity.
Quaternary ammonium compounds are well known and effective antibacterial agents, which are used in many fields, such as water treatment, medicine and healthcare products, food applications, and textile products . Quaternary ammonium methacrylate monomers (QAM) which are able to copolymerize with other methacrylate monomers could immobilize the antibacterial quaternary ammonium group in the polymer backbone and give long-term antibacterial activity to acrylic polymer . 1,2-Methacryloyloxydodecylpridinium bromide (MDPB), synthesized by Imazato and his co-workers, is the first QAM used in dental materials. Studies showed that attachment of oral bacterial and plaque accumulation was decreased when MDPB was incorporated into dental composites materials . After that, several QAMs were synthesized with the aim of preparing long-lasting antibacterial dental composites materials .
Quaternary ammonium salts are usually consisted of quaternary ammonium cation and anions like halogen anions and some other acidic anions. In the halogen elements, iodine is quite radio-opaque because of its high electronic density . For this reason, QAMs with iodine anion cannot only endow methacrylate-based dental resin with antibacterial activity, but also with radio-opacity.
In our previous research, a QAM with iodine anion named 2-Dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) was synthesized and incorporated into 2,2-Bis[4-(2-hydroxy-3-methacryloyloxypropyl)-phenyl]propane (Bis-GMA)/tri-ethyleneglycol dimethacrylate (TEGDMA) dental resin system . The study revealed that DDMAI containing polymer had antibacterial activity and higher radio-opacity than polymer without DDMAI. However, just like some other mono-methacrylate quaternary ammonium compounds, DDMAI also has miscibility problem with hydrophobic dimethacrylate monomers those are commonly used in dental composites materials such as TEGDMA . Lower concentration of DDMAI made relevant polymer only had biofilm inhibitory effect on early S. mutans biofilm formation, and the radio-opacity of the polymer was merely around 30% of the radio-opacity of aluminum with the same thickness, which was still far away from the clinical requirement .
Studies of Huang et al. and Antonucci et al. showed that quaternary ammonium dimethacrylates could be miscible with normal dental resins and increase the concentration of quaternary ammonium structures. Therefore, in this work, three novel quaternary ammonium dimethacrylates with iodine anion (QADMAI) were synthesized and mixed with TEGDMA to form high quaternary ammonium containing dental resins. The hypotheses are (I): QADMAIs could be mixed well with TEGDMA; (II) QADMAIs could endow dental resin system with both antibacterial and radio-opaque functions. Double bond conversion, flexural strength and modulus, surface charge density, antibacterial activity, and radio-opacity of QADMAI containing dental resins were investigated and compared with the commonly used Bis-GMA/TEGDMA dental resin.
Materials and methods
N-Methyl diethanol amine (MDEA), 1-iodododecane, 1-iodohexadecane, 1-iodooctadecane, dibutyltin dilaurate (DBTDL) TEGDMA, camphoroquinone (CQ), N,N′-dimethyl aminoethylmethacrylate (DMAEMA), fluorescein sodium salt, cetyltrimethylammonium chloride (CTMAC), acetone, and diethyl ether were purchased from Sigma–Aldrich Co. (St Luois, MO, USA). 2-(methacryloyloxy)ethyl isocyanate (MEI) was purchased from Tokyo Chemical Industry Co., Ltd. (Zwijndrecht, Belgium). Bis-GMA was purchased from Esstech Inc. (Essington, PA, USA). All of the compounds were used without further purification.
Synthesis of quaternary ammonium dimethacrylates with iodine anion (QADMI)
As shown in Fig. 1 , QADMAI was synthesized through a 2-steps route. FT-IR (Spectrum One, PerkinElmer, Waltham, MA, USA) and 1 H NMR (AV 400 MHz, Bruker Co., Germany) spectra of intermediate products and final products were obtained to confirm their structures. The FTIR spectra were recorded with 16 scans at a resolution of 4 cm −1 . The chemical shifts of 1 H NMR spectra were reported in ppm on δ scale with tetramethylsilane as the internal reference and CDCl 3 as the solvent.
General procedure for the synthesis of intermediate products HQAI
A mixture of MDEA (0.052 mol), alky iodide (0.05 mol), and 20 ml acetone were stirred at reflux. After 24 h reaction, the acetone was removed by distillation under vacuum. The obtained raw product was washed with ethyl ether and filtered for several times. Then the white intermediate product HQA was dried under vacuum at 35 °C for 48 h.
N,N-bis(2-hydroxyethyl)-N-methyldodecyl ammonium iodide (HQAI-12) . 18.07 g, yield: 87%. FT-IR: v (cm −1 ) 3294, 2956, 2927, 2855, 1381, 1090, 720. 1 H NMR (CDCl 3 , 400 MHz): δ 4.22[4H, 2N + CH 2 CH 2 OH], 4.09[2H, 2N + CH 2 CH 2 OH], 3.77-3.89[4H, 2N + CH 2 CH 2 OH], 3.57–3.62[2H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ], 3.38[3H, N + CH 3 ], 1.80[2H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ], 1.31-1.42[18H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ], 0.94-0.95[3H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ].
N,N-bis(2-hydroxyethyl)-N-methylhexadecyl ammonium iodide (HQAI-16) . 21.22 g, yield: 90%. FT-IR: v (cm −1 ) 3294, 2953, 2921, 2852, 1378, 1087, 727. 1 H-NMR (CDCl 3 , 400 MHz): δ 4.23[4H, 2N + CH 2 CH 2 OH], 4.00–4.02[2H, 2N + CH 2 CH 2 OH], 3.77-3.70[4H, 2N + CH 2 CH 2 OH], 3.57–3.61[2H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ], 3.38[3H, N + CH 3 ], 1.79–1.81[2H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ], 1.31–1.44[26H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ], 0.91–0.95[3H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ].
N,N-bis(2-Hydroxyethyl)-N-methyloctadectyl ammonium iodide (HQAI-18) . 20.73 g, yield: 83%. FT-IR: v (cm −1 ) 3293, 2957, 2918, 2851, 1379, 1087, 727. 1 H NMR (CDCl 3 , 400 MHz): δ 4.19[4H, 2N + CH 2 CH 2 OH], 4.11–4.12[2H, 2N + CH 2 CH 2 OH], 3.76–3.83[4H, 2N + CH 2 CH 2 OH], 3.54–3.58[2H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ], 3.35[3H, N + CH 3 ], 1.76–1.78[2H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ], 1.28–1.38[30H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ], 0.89–0.92[3H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ].
General procedure for the synthesis of final product QADMAI
Acetone used here was dried over 4 Å molecular sieves for 2 weeks. A mixture of HQAI (0.04 mol), MEI (0.08 mol), 20 ml acetone, and 2 droplets of DBTDL were stirred at 45 °C. The reaction was continued until the infrared absorbance peak of the –NCO group (2270 cm −1 ) disappeared in the FTIR spectra of the samples taken from the reaction medium. After removing the acetone by distillation under vacuum, the product was washed with diethyl ether and centrifuged to remove DBTDL. Then the yellow high viscose liquid was dried under vacuum at 35 °C for 48 h to obtain QADMAI.
N,N-bis[2-(3-(methacryloyloxy)propanamido)ethyl]-N-methyldodecyl ammonium iodide (QADMAI-12) . 26.98 g, yield: 93%. FT-IR: v (cm −1 ) 3256, 2925, 2855, 1710, 1637, 1454, 1320, 1297, 1160. 1 H NMR (CDCl 3 , 400 MHz): δ 6.33 6.36[2H, 2-NH–], 6.14[2H, 2CH 2 C(CH 3 ) trans ], 5.60[2H, 2CH 2 C(CH 3 ) cis ], 4.60–4.68[4H, 2-NHCH 2 CH 2 O–], 4.23–4.26[4H, N + CH 2 CH 2 O–], 3.95[4H, N + CH 2 CH 2 O–], 3.57–3.61[2H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ], 3.45–3.48[7H, N + CH 3 and 2-NHCH 2 CH 2 O–], 1.94[6H, 2CH 2 C(CH 3 )], 1.76[2H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ], 1.26–1.37[18H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ], 0.87–0.90[3H, N + CH 2 CH 2 (CH 2 ) 9 CH 3 ].
N,N-bis[2-(3-(Methacryloyloxy)propanamido)ethyl]-N-methylhexadecyl ammonium iodide (QADMAI-16) . 28.46 g, yield: 91%. FT-IR: v (cm −1 ) 3256, 2924, 2854, 1712, 1638, 1455, 1320, 1297, 1160. 1 H NMR (CDCl 3 , 400 MHz): δ 6.32-6.35[2H, 2-NH–], 6.14[2H, 2CH 2 C(CH 3 ) trans ], 5.59[2H, 2CH 2 C(CH 3 ) cis ], 4.59[4H, 2-NHCH 2 CH 2 O–], 4.22–4.26[4H, N + CH 2 CH 2 O–], 3.94[4H, N + CH 2 CH 2 O–], 3.54–3.59[2H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ], 3.43-3.50[7H, N + CH 3 and 2-NHCH 2 CH 2 O–], 1.94[6H, 2CH 2 C(CH 3 )], 1.76[2H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ], 1.25–1.36[26H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ], 0.86–0.89[3H, N + CH 2 CH 2 (CH 2 ) 13 CH 3 ].
N,N-bis[2-(3-(Methacryloyloxy)propanamido)ethyl]-N-methyloctadectyl ammonium iodide (QADMAI-18) . 30.45 g, yield: 94%. FT-IR: v (cm −1 ) 3260, 2923, 2853, 1713, 1638, 1455, 1320, 1297, 1161. 1 H NMR (CDCl 3 , 400 MHz): δ 6.68–6.71[2H, 2-NH–], 6.15[2H, 2CH 2 C(CH 3 ) trans ], 5.60[2H, 2CH 2 C(CH 3 ) cis ], 4.60[4H, 2-NHCH 2 CH 2 O–], 4.24–4.26[4H, N + CH 2 CH 2 O–], 3.96[4H, N + CH 2 CH 2 O–], 3.54–3.58[2H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ], 3.44–3.50[7H, N + CH 3 and 2-NHCH 2 CH 2 O–], 1.95[6H, 2CH 2 C(CH 3 )], 1.74[2H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ], 1.26–1.36[30H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ], 0.88–0.91[3H, N + CH 2 CH 2 (CH 2 ) 15 CH 3 ].
Antibacterial activity against Streptococcus mutans of QADMAI
The agar diffusion method was used for the determination of the antibacterial activity of the QADMAIs on S. mutans 10449. First, overnight grown cells of S. mutans 10449 were suspended in phosphate-buffered-saline (PBS) to get a suspension of A 660 = 0.5. Then the diluted (1000×) bacterial suspensions were spread on Mitis Salivarius (Becton Dickinson and Company, Sparks, MD, USA) agar plates with a cotton stick.
All of QADMAIs monomers were dissolved in acetone to get solutions with concentration of 10 mg/ml, and pure acetone was used as a control group. The filter paper discs (diameter 5 mm) were dipped in 1.0 ml of the solutions and placed on the Mitis Salivarius plates. After incubation for three days in anaerobic atmosphere (80% N 2 , 10% CO 2 , 10% H 2 ) at +37 °C, the inhibition zones were measured. The inhibition zone was determined by measuring its diameter (mm) with a magnifying glass with a scale. The size of the inhibition zone included the diameter of the filter paper disc. The test was performed with quadruplicates of each monomer solution. The test was repeated at least once.
Preparation of experimental resin system
QADMAI (12, 16 or 18) was mixed with TEGDMA at the mass ratio of 50 wt/50 wt, 0.7 wt% of CQ and 0.7 wt% of DMAEMA were added as photoinitiatior system. Bis-GMA/TEGDMA (50 wt/50 wt) resin system with CQ (0.7 wt%) and DMAEMA (0.7 wt%) was used as control. All of the resins were well blended to obtain a homogenous mixture, and stored at darkness before used.
Monomer (double bond) conversion
The degree of monomer conversion (DC) was determined by using a FTIR spectrometer (Spectrum One, PerkinElmer, Waltham, MA, USA) with an attenuated total reflectance (ATR) accessory. The FTIR spectra were recorded with one scan at a resolution of 4 cm −1 . All the samples were analyzed in a mold that was 2 mm thick and 6 mm diameter. First, the spectrum of the unpolymerized sample was measured. Then, the sample was irradiated for 60 s with a visible light-curing unit (Curing Light 2500, λ = 400–520 nm, I ≈ 550 mW cm −2 , 3M Co., St Paul, MN, USA). The sample was scanned for its FTIR spectrum every 5 s until 15 min after the beginning of irradiation.
To determine the percentage of reacted double bonds, the absorbance intensities of the methacrylate C C absorbance peak at 1636 cm −1 , which were decreased after being irradiated, and an internal carbonyl group standard peak at 1720 cm −1 , were calculated using a baseline method. The ratios of absorbance intensities were calculated and compared before and after polymerization. The DC at every irradiation time was calculated according to the equation
DC ( t ) = 1 − ( A C = C / A C = O ) t ( A C = C / A C = O ) 0 × 100 %