Chemical Modification of Dental Dimethacrylate Copolymer with Tetramethylxylylene Diisocyanate-Based Quaternary Ammonium Urethane-Dimethacrylates—Physicochemical, Mechanical, and Antibacterial Properties

In this study, two novel quaternary ammonium urethane-dimethacrylates (QAUDMAs) were designed for potential use as comonomers in antibacterial dental composite restorative materials. QAUDMAs were synthesized via the reaction of 1,3-bis(1-isocyanato-1-methylethyl)benzene with 2-(methacryloyloxy)ethyl-2-decylhydroxyethylmethylammonium bromide (QA10+TMXDI) and 2-(methacryloyloxy)ethyl-2-dodecylhydroxyethylmethylammonium bromide (QA12+TMXDI). Their compositions with common dental dimethacrylates comprising QAUDMA 20 wt.%, urethane-dimethacrylate monomer (UDMA) 20 wt.%, bisphenol A glycerolate dimethacrylate (Bis-GMA) 40 wt.%, and triethylene glycol dimethacrylate (TEGDMA) 20 wt.%, were photocured. The achieved copolymers were characterized for their physicochemical and mechanical properties, including their degree of conversion (DC), glass transition temperature (Tg), polymerization shrinkage (S), water contact angle (WCA), flexural modulus (E), flexural strength (FS), hardness (HB), water sorption (WS), and water leachability (WL). The antibacterial activity of the copolymers was characterized by the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) against Staphylococcus aureus and Escherichia coli. The achieved results were compared to the properties of a typical dental copolymer comprising UDMA 40 wt.%, Bis-GMA 40 wt.%, and TEGDMA 20 wt.%. The introduction of QAUDMAs did not deteriorate physicochemical and mechanical properties. The WS and WL increased; however, they were still satisfactory. The copolymer comprising QA10+TMXDI showed a higher antibacterial effect than that comprising QA12+TMXDI and that of the reference copolymer.

Another concept of providing DCRMs with antimicrobial properties is based on the covalent incorporation of a monomeric biocide into the polymer network structure, constituting the DCRM matrix.It involves the copolymerization of universal dental dimethacrylates (DMAs), such as bisphenol A glycerolate dimethacrylate (Bis-GMA), its ethoxylated derivative (Bis-EMA), urethane-dimethacrylate monomer (UDMA), and triethylene glycol dimethacrylate (TEGDMA), with dimethacrylates having bioactive groups, of which dimethacrylates with quaternary ammonium (QA) groups (QADMAs) are the best known [18][19][20][21][22].The QADMA content in a copolymer ranges from a few to several dozen percent.This alternative offers the stable, non-leaching, and long-term antimicrobial action of a DCRM [23,24].The huge advantages of monomeric antimicrobials include lower cytotoxic effects in comparison to physically dispersed antibacterial agents [16,25] and an alternative to antibiotics, whose application should be limited due to the increasing resistance of microorganisms [26][27][28].The aforementioned features of QADMAs explain the great interest among scientists in designing novel monomer structures containing QA groups and the development of related materials.
QAn+MDEAs showed high antibacterial activity against many bacteria strains, including S. aureus, Streptococcus mutans, Actinomyces viscosus, Lactobacillus acidophilus, Streptococcus sanguinis, Porphyromonas gingivalis, Prevotella melaninogenica, and Enterococcus faecalis.In addition, these monomers showed cytotoxicity lower than Bis-GMA [29].The copolymer of QAn+MDEA 10 wt.% with Bis-GMA/TEGDMA 90 wt.% showed antibacterial activity against S. mutans [30].QAn+DMAEMA 1 wt.% was used to modify a commercial adhesive resin (Tetric N-Bond) and the resulting material also showed antibacterial activity against S. mutans [31].The introduction of QAn+Bis-GMA 5 wt.% into the Bis-GMA/TEGDMA formulation resulted in a copolymer with antibacterial activity against E. coli, S. aureus, S. mutans, and Bacillus subtillis.The antifungal activity of the modified copolymer against Candida albicans was observed too.As the QAn+Bis-GMA concentration increased, the antimicrobial activity increased; however, it was accompanied by an increase in cytotoxicity and the deterioration of mechanical properties [32].The modification of the same Bis-GMA/TEGDMA formulation with QAn+MDEA+IEM 5 wt.% resulted in an antibacterial copolymer only for QA16+MDEA+IEM.It showed antibacterial activity against S. mutans.All other copolymers gained antibacterial activity as the QAn+MDEA+IEMs concentration increased to 10 wt.%, but this was accompanied by the deterioration in mechanical properties [33,34].The QAn+MDEA+IPDI 50 wt.%and TEGDMA 50 wt.%copolymers also showed high antibacterial activity against S. mutans.However, the disadvantages of these copolymers included a low flexural strength, low modulus of elasticity, and high water sorption [35,36].By reducing the QAn+MDEA+IPDI content to 30 wt.% and using a formulation of Bis-GMA/TEGDMA 70 wt.%,an improvement in mechanical properties was achieved [36].QAn+TMDIs and their copolymers were the most extensively studied [37][38][39][40][41][42].Copolymers of QAn+TMDI 60 wt.% and TEGDMA 40 wt.% were characterized by very high antibacterial activity against S. aureus and E. coli.However, they had insufficiently good mechanical parameters, excessively high water sorption, and water leachability [38,39].Copolymers of QAn+TMDI 40 wt.%,Bis-GMA 40 wt.%, and TEGDMA 20 wt.% also showed high antimicrobial activity against S. aureus and E. coli [40].In addition, antifungal activity against C. albicans was observed for these copolymers [41].Many of their physicochemical, mechanical, and biological parameters were good.However, the detailed analysis showed that an increase in the N-alkyl substituent length resulted in a decrease in the antibacterial activity, and the deterioration of mechanical properties [40][41][42].This analysis led to the conclusion that copolymers based on QA10+TMDI and QA12+TMDI contain the optimum combination of properties [40].
The promising results obtained for copolymers based on QA10+TMDI and QA12+TMDI motivated us to replace the TMDI core with a 1,3-bis(1-isocyanate-1-methylethyl)benzene (TMXDI) core to obtain new urethane-dimethacrylate monomers with two QA groups located in the wings and 1,3-phenylene ring located in the core.We formulated a hypothesis that the copolymerization of the achieved quaternary ammonium urethane-dimethacrylates (QAUDMAs) with UDMA, Bis-GMA, and TEGDMA results in an antibacterial copolymer with good physicochemical and mechanical properties.Therefore, the goal of this work was to synthesize two novel urethane-dimethacrylates with the TMXDI core and two methacrylate-ended wings comprising the QA group in the middle.The QA groups were substituted with an N-alkyl chain of 10 and 12 carbon atoms (QA10+TMXDI and QA12+TMXDI, respectively).The two novel monomers were compounded with common dental dimethacrylates in the following weight fractions: QAUDMA 20 wt.%, UDMA 20 wt.%, Bis-GMA 40 wt.%, and TEGDMA 20 wt.%.The two novel monomers were also photocured.The novel monomers, as well as their compositions with dental dimethacrylates, were characterized for molecular weight (MW), concentration of double bonds (x DB ), viscosity (η), refractive index (RI), and density (d m ).The degree of conversion (DC), density (d p ), polymerization shrinkage (S), glass transition temperature (T g ), water contact angle (WCA), water sorption (WS), water solubility (SL), hardness (HB), flexural strength (FS), flexural modulus (E), minimum bactericidal concentration (MBC), and minimum inhibitory concentration (MIC) were all measured for the resulting copolymers (the first comprising QA10+TMXDI (20(QA10+TMXDI) p ) and the second comprising QA12+TMXDI (20(QA12+TMXDI) p ).The results were subjected to a comparative analysis of the properties of the standard dental copolymer consisting of UDMA 40 wt.%,Bis-GMA 40 wt.%, and TEGDMA 20 wt.% (40(UDMA) p ).
The novelty of this work combines the development of two new urethane-dimethacrylate monomers with quaternary ammonium groups (QAUDMAs) and the complex physicochemical, mechanical, and antibacterial characteristics of their copolymers with common dimethacrylates used in dentistry.So far, QAUDMAs synthesized from 1,3-bis(1-isocyanato-1methylethyl)benzene have not been described in the literature.
The development of such materials is very important for dental science and may contribute to reducing the scale of dental carries in the future.

Chemical Syntheses
The transesterification reaction reagents-MMA 1.00 mol (100.12 g), and MDEA 0.67 mol (79.85 g), the catalyst-K 2 CO 3 8 wt.% (14.40 g), the polymerization inhibitor-PTZ 0.05% (0.09 g) and the solvent-toluene 400 cm 3 were introduced into a single-necked round-bottom flask equipped with a Vigreux column and distillation head.The reaction mixture was brought to a boil.The equilibrium of the reaction was moved in favor of HAMA by the continuous collection of a distillate consisting of an azeotropic mixture of methanol (condensation reaction by-product), MMA (used in excess), and toluene (solvent).The reaction was carried out until the temperature at the column head reached 100 • C, which was achieved after 2.5 h.The cooled reaction mixture was filtered and washed three times with distilled water in a 1:2 volume ratio (HAMA and MDEA were soluble in water).The aqueous fractions were combined and extracted three times with chloroform in a 1:3 volume ratio (only HAMA was soluble in chloroform; as MDEA was water-insoluble, it remained in a water fraction).The chloroform fractions, containing only HAMA, were also combined, dried with MgSO 4 overnight, and chloroform was evaporated on a rotary evaporator under reduced pressure (30 and then 3 mbar).The obtained raw product was subjected to vacuum distillation (3 mbar), collecting a boiling fraction from 110 to 130 • C. The process yielded HAMA 14%.The Menshutkin reaction reagents-HAMA 0.107 mol (20.00 g), and alkyl bromide 0.107 mol (DB 23.66 g and DDB 26.67 g), and the polymerization inhibitor-PTZ 0.05 wt.% (0.022 and 0.023 g, respectively, in the reaction with DB and DDB) were introduced into a three-necked round-bottom flask equipped with a mechanical stirrer, reflux condenser, and thermometer.The reaction was carried out at 80 • C for 90 h.The reaction yielded 100% of 2-(methacryloyloxy)ethyl-2-decylhydroxyethylmethylammonium bromide (QAHAMA-10) and 2-(methacryloyloxy)ethyl-2-dodecylhydroxyethylmethylammonium bromide (QAHAMA-12).

Quaternary Ammonium Urethane-Dimethacrylates (QAUDMA)
A 50% solution of an addition reaction reagent-QAHAMA-n 0.070 mol (QAHAMA-10 28.50 g and QAHAMA-12 28.6 g), a catalyst-DBTDL 0.035 wt.% (0.013 g) and a solventdichloromethane 21 cm 3 were introduced into a three-necked round-bottom flask equipped with a reflux condenser, thermometer, and dropping funnel.A 50% solution of the second reagent, composed of TMXDI 0.035 mol (8.55 g) and dichloromethane 6.5 cm 3 , was placed in a dropping funnel.The flask content was brought to a boil (approximately 42 • C), and then a TMXDI solution was added dropwise for 1.5 h.The reaction was carried out for 5 h, maintaining the reaction mixture at the boiling point.Dichloromethane was evaporated on a rotary evaporator under reduced pressure (30 and then 3 mbar).A light-yellow, viscous, liquid substance remained in the flask, constituting the product.The flask with the product was placed in the laboratory dryer (SLW 53 STD, POL-EKO, Wodzisław Śl ąski, Poland) and heated at 42 • C for 24 h.The reaction yielded 100% both QAUDMAs, i.e., the addition reaction of TMXDI and QAHAMA-10 yielded QA10+TMXDI, and that of QAHAMA-12 yielded QA12+TMXDI.

Curing Procedure and Sample Preparation
Two experimental monomer compositions were prepared using mechanical stirring at 50 • C.They consisted of QAUDMA (QA10+TMXDI and QA12+TMXDI) 20 wt.%, UDMA 20 wt.%, Bis-GMA 40 wt.%, and TEGDMA 20 wt.%.The reference composition consisted of UDMA 40 wt.%,Bis-GMA 40 wt.%, and TEGDMA 20 wt.%.The components of the initiating system, comprising CQ 0.4 wt.% and DMAEMA 1 wt.%, were introduced into homogeneous monomer mixtures and stirring was continued at the same temperature until CQ dissolved.The achieved compositions were poured into the molds, covered with the PET foil, and irradiated with the UV-VIS lamp (Ultra Vitalux 300, Osram, Munich, Germany) at room temperature for 1 h.The lamp emitted radiation in the range of 280 to 780 nm and radiation exitance of 2400 mW/cm 2 .The achieved casts were processed following the guidelines for specimen preparation for testing particular properties.Each specimen was polished with fine sandpaper before experiments.

Nuclear Magnetic Resonance Spectroscopy (NMR)
The NMR 300 MHz spectrometer (UNITY/INOVA, Varian, Palo Alto, CA, USA) was used to collect 256 scan 1 H spectra and 40,000 scan 13 C NMR spectra of products achieved.CD 2 Cl 2 and CDCl 3 were used as solvents and TMS was used as an internal standard.
Monomers were tested as a very thin layer between two KBr pellets.Copolymers were powdered, sieved to a diameter of less than 25 µm, and dispersed in KBr pellets.
The degree of conversion (DC) in copolymers was calculated from the following equation: The variables in the equation are defined as follows: A C=C -the absorbance of the stretching vibration band of the carbon-carbon double bond in the methacrylate group at 1636 cm −1 ; A Ar -the absorbance of the aromatic stretching skeletal vibrations band at 1608 cm −1 .

Viscosity
The rotating viscometer (Visco Star Plus L, Brookfield Fungilab Viscometer, Barcelona, Spain) was used to determine the viscosity (η).The procedure was carried out at 25 • C according to the ISO 2555-2018 standard [43].

Refractive Index
The digital refractometer (DR 6100T, Krüss Optronic, Hamburg, Germany) was used to determine the refractive index (RI).The procedure was carried out according to the ISO 489:2022 standard [44].

Density and Polymerization Shrinkage
The density of monomers (d m ) was determined with the 1 mL pycnometer.The density of copolymers (d p ) was determined with the analytical balance (XP Balance, Mettler Toledo, Greifensee, Switzerland) analytical balance equipped with the density determination kit.
The polymerization shrinkage (S) was calculated from the following equation: The variables in the equation are defined as follows: d m -the monomer density; d p -the polymer density.

Differential Scanning Calorimetry (DSC)
The differential scanning calorimeter (DSC 3, Mettler Toledo, Greifensee, Switzerland) was used in the measurements, utilizing powdered polymer samples and standard aluminum crucibles.The 10 K/min heating rate, 0 to 200 • C temperature range, and air atmosphere were used in all experiments.
The glass transition temperature (T g ) of copolymers was read off the DSC curve as the midpoint of the transition region, following the 11357-2:2020 standard [45].

Water Contact Angle
The goniometer (OCA 15EC, Data Physics, Filderstadt, Germany) was used to determine the water contact angle (WCA) of copolymer surfaces.The analysis was performed on disc-like specimens with dimensions of 15 mm × 1.5 mm (diameter × thickness) utilizing the sessile drop method and 4 µL of deionized water.The measurement was performed immediately after the drop was placed on the sample surface.

Water Sorption and Solubility
The water sorption (WS) and solubility (SL) of polymers were determined according to the guidelines of the ISO 4049:2019 standard [46] on disc-like specimens with dimensions of 15 mm × 1.5 mm (diameter × thickness).The analytical balance (XP Balance, Mettler Toledo, Greifensee, Switzerland) was used in experiments.
The specimens were dried to constant weight (m 0 ) in a laboratory dryer (SLW 53 STD, POL-EKO, Wodzisław Śl ąski, Poland) and immersed in deionized water at room temperature for 7 days.After that, samples were removed from the water, dried with blotting paper, weighed (m 1 ), and dried again to a constant weight (m 2 ).
WS and SL were calculated using the following equations: The variables in the equation are defined as follows: m 0 -the initial mass of the dried samples; m 1 -the mass of the swollen samples; m 2 -the mass of the dried samples after immersion in water; V-the initial volume of the dried samples.
HB was calculated using the following equation: The variables in the equation are defined as follows: F m -the test load; d-the ball intender diameter (d = 5 mm); h-the immersion depth; h r -the reduced immersion depth (h r = 0.25 mm).

Flexural Properties
The universal testing machine (Z020, Zwick, Ulm, Germany) was used to determine the flexural strength (FS) and flexural modulus (E).The procedure was carried out according to the ISO 178:2019 standard [48] on bar specimens with dimensions of 64 mm × 10 mm × 3.3 mm (length × width × thickness).
FS and E were calculated using the following equations: The variables in the equation are defined as follows: P 1 -the load at the selected point of the elastic region of the stress-strain plot; P-the maximum load; l-the support span; b-the sample width; d-the sample thickness; δ-the deflection of the sample at P 1 .

Antibacterial Properties
The antibacterial properties were characterized by the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) against S. aureus (ATCC 25923) and E. coli (ATCC 25922).The tryptic soy broth (TSB) nutritious medium was used for the bacteria cultivation, which was carried out at 37 • C for 18 h in an incubator (ILW 53, POL-EKO, Wodzisław Śl ąski, Poland).The experiments were carried out on polymer powders sieved to a grain diameter of less than 25 µm.
A series of six copolymer suspensions in TSB were prepared with concentrations of 50, 25, 12.5, 6.25, 3.125, and 1.563 mg/mL.Next, 20 µL of S. aureus and E. coli bacterial suspensions in TSB (~5 × 10 8 CFU/mL) were introduced into the copolymer suspensions, vortexed (10 s, 2000 rpm), and incubated at 37 • C for 18 h.Suspensions were then vortexed again (10 s, 2000 rpm), and 100 µL of each was applied on an agar plate (Müller-Hinton agar, Diag-Med, Raszyn, Poland).The agar plates were incubated at 37 • C for 18 h.The number of bacterial colonies was qualitatively assessed by visual comparison to the control.The lowest concentration at which no bacterial colonies were observed on agar plates was taken as the MBC.The lowest concentration at which the bacteria growth was reduced was taken as the MIC.

Statistical Analysis
The software (Statistica 13.3, TIBCO Software Inc., Palo Alto, CA, USA) was used to perform the non-parametric Wilcoxon test with a significance level (p) of 0.05.The statistical significance of the results was determined for five samples.The results were expressed as the average value (AV) and corresponding standard deviation (SD).

Monomer Synthesis and Characterization
As part of this work, two QAUDMA monomers, QA10+TMXDI and QA12+TMXDI, were obtained.To achieve this goal, a procedure developed for the QAn+TMDI monomer synthesis was adopted [37].
The synthesis route of QA10+TMXDI and QA12+TMXDI included three stages (Figure 1): • The transesterification of MMA with MDEA that led to HAMA formation.It was conducted with the MMA excess and K 2 CO 3 catalyst; • The Menshutkin reaction by the reaction of HAMA with DB and DDB resulted in a conversion of the HAMA tertiary amine group into a quaternary ammonium group in QAHAMA-n.QAHAMA-10 and QAHAMA-12 were achieved, respectively; • The addition reaction of QAHAMA-n and TMXDI was conducted with the DBTDL cat- alyst and resulted in the formation of QAUDMAs: QA10+TMXDI and QA12+TMXDI; • The chemical structure of the obtained monomers was confirmed using spectroscopic methods, including1 H NMR, 13 C NMR, and FTIR.
Figure 2 shows the 1 H NMR spectra of the QA10+TMXDI and QA12+TMXDI monomers.Table 1 lists the proton signals present in the spectra with their assignment to chemical groups.Figure 4 shows the FTIR spectra of the QA10+TMXDI and QA12+TMXDI monomers.Table 3 lists of absorption bands present in the spectra with their assignment to chemical groups.Figure 4 shows the FTIR spectra of the QA10+TMXDI and QA12+TMXDI monomers.Table 3 lists of absorption bands present in the spectra with their assignment to chemical groups.1. The intensity of the absorption bands in the FTIR spectra was referred to as strong (s), medium (m), and weak (w). 2. Refers to the isocyanate group absorption band present in the FTIR spectra of the QAUDMA monomers before their thermal treatment.
The QA10+TMXDI and QA12+TMXDI monomers were characterized by their molecular weight (MW), concentration of double bonds (xDB), refractive index (RI), and density (dm).Table 4 presents the results for the experimental monomers and three commercial  The QA10+TMXDI and QA12+TMXDI monomers were characterized by their molecular weight (MW), concentration of double bonds (x DB ), refractive index (RI), and density (d m ).Table 4 presents the results for the experimental monomers and three commercial dental dimethacrylate monomers (Bis-GMA, UDMA, and TEGDMA), for comparison.The SD for the RI values was 0.0001. 2 The SD for the d m values was 0.01. 3 Taken from [37].
The molecular weight (MW) of QA10+TMXDI was 1061 g/mol and QA12+TMXDI had a MW that was 5% higher.Compared to Bis-GMA, UDMA, and TEGDMA, QA10+TMXDI as well as QA12+TMXDI had a higher MW, on average by 53, 57, and 74%, respectively.The MW was converted to the concentration of double bonds (x DB ).The x DB of QA10+TMXDI was 1.89 mol/kg and QA12+TMXDI had a x DB that was 5% lower.Compared to Bis-GMA, UDMA, and TEGDMA, QA10+TMXDI as well as QA12+TMXDI had a lower x DB , on average by 53, 57, and 74%, respectively.
The refractive index (RI) of QA10+TMXDI was 1.5230 and QA12+TMXDI had a RI that was 0.6% lower.This difference did not have statistical significance.Compared to Bis-GMA, QA10+TMXDI as well as QA12+TMXDI had a lower RI, on average by 2%.Compared to UDMA and TEGDMA, QA10+TMXDI as well as QA12+TMXDI had a higher RI, on average by 4 and 3%, respectively.
The d m of 20(QA10+TMXDI) m was 1.13 g/cm 3 and 20(QA12+TMXDI) m had a d m that was 2% lower.This difference was statistically significant.Compared to 40(UDMA) m , 20(QA10+TMXDI) m had a d m that was 2% lower, and this difference was statistically significant, whereas 20(QA12+TMXDI) m had the same d m , and this result did not have statistical significance.

Characterization of Copolymers
20(QA10+TMXDI) m , 20(QA12+TMXDI) m, and 40(UDMA) m were subjected to photopolymerization, utilizing an initiation system consisting of CQ and DMAEMA, which is typical for the DCRM photocuring.The structure of the obtained copolymers was characterized by the degree of conversion (DC).The physical properties were characterized by determining the polymerization shrinkage (S), glass transition temperature (T g ), wa-ter sorption (WS), water solubility (SL), and water contact angle (WCA).The mechanical properties were characterized by determining the hardness (HB), flexural modulus (E), and flexural strength (FS).The copolymers were also tested for antibacterial properties by determining the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) against S. aureus (ATCC 25923) and E. coli (ATCC 25922).

Degree of Conversion (DC)
The DC was determined utilizing the FTIR spectroscopy and internal standard method.As the tested systems comprised the TMXDI moiety, it was possible to use the aromatic skeletal vibration band as a standard (Figure 5).This is the most widely used method in the DC characterization of poly(dimethacrylate)s because this band does not change its specificity due to polymerization [49].Table 7 shows the DC in the copolymers tested.The DC was determined utilizing the FTIR spectroscopy and internal standard method.As the tested systems comprised the TMXDI moiety, it was possible to use the aromatic skeletal vibration band as a standard (Figure 5).This is the most widely used method in the DC characterization of poly(dimethacrylate)s because this band does not change its specificity due to polymerization [49].Table 7 shows the DC in the copolymers tested.The DC in 20(QA10+TMXDI)p was 0.57 and 20(QA12+TMXDI)p had a DC that was 21% higher.This difference was statistically significant.Compared to 40(UDMA)p, 20(QA10+TMXDI)p as well as 20(QA12+TMXDI)p had a higher DC, by 6 and 28%,  The DC in 20(QA10+TMXDI) p was 0.57 and 20(QA12+TMXDI) p had a DC that was 21% higher.This difference was statistically significant.Compared to 40(UDMA) p , 20(QA10+TMXDI) p as well as 20(QA12+TMXDI) p had a higher DC, by 6 and 28%, respectively.The difference for 20(QA10+TMXDI) p was statistically insignificant, whereas that for 20(QA12+TMXDI) p was statistically significant.

Physical Properties
Table 8 shows the results obtained for the d p , S, and T g of the copolymers tested.The DSC thermograms of the copolymers are presented in Supplementary Materials in Figure S1.The d p of 20(QA10+TMXDI) p was 1.21 g/cm 3 and 20(QA12+TMXDI) p had a d p that was 1% lower.Compared to 40(UDMA) p , 20(QA10+TMXDI) p had a higher d p , by 1%, whereas 20(QA12+TMXDI) p had the same d p .All the results for the d p were statistically insignificant.
The T g of 20(QA10+TMXDI) p was 53.46 • C and 20(QA12+TMXDI) p had a T g that was 13% higher.This difference was statistically significant.Compared to 40(UDMA) p , 20(QA10+TMXDI) p as well as 20(QA12+TMXDI) p had a lower T g , by 15 and 4%, respectively.The difference for 20(QA10+TMXDI) p was statistically significant.The other copolymer did not have statistical significance.
Table 9 shows the results obtained for the WS, SL, and WCA of the tested copolymers.The goniometry camera images of the deionized water droplets on the surfaces of the copolymers are presented in Supplementary Materials in Figure S2.
Table 9.The physical properties characterizing copolymers' interaction with water: water sorption (WS), water leachability (SL), and water contact angle (WCA).The lowercase letters "a"-"c" in the superscripts indicate couples with statistically significant results (p ≤ 0.05).The remaining results did not have statistical significance (p > 0.05).

Mechanical Properties
Table 10 shows the results obtained for the HB, E, and FS of the tested copolymers.The HB of 20(QA10+TMXDI) p was 234.8 MPa and 20(QA12+TMXDI) p had a HB that was 17% lower.Compared to 40(UDMA) p , 20(QA10+TMXDI) p had a HB that was 4% higher, and 20(QA12+TMXDI) p had a HB that was 14% lower.All these differences were statistically significant.

Antibacterial Properties
Table 11 shows the results obtained for the MIC and MBC of the tested copolymers concerning the S. aureus (ATCC 25923) and E. coli (ATCC 25922) bacteria strains.The results of the experiments are presented in Supplementary Materials in Figures S3-S8.20(QA10+TMXDI) p had an MBC of 12.5 mg/mL, and an MIC of 6.25 mg/mL concerning both bacteria tested.20(QA12+TMXDI) p had an MBC as well as MIC greater than 50 mg/mL concerning S. aureus.The same copolymer showed a lower MBC and MIC concerning E. coli, respectively, 25 and 12.5 mg/mL.40(UDMA) p had an MBC as well as MIC greater than 50 mg/mL in each case, i.e., concerning both bacteria strains.

Discussion
As part of this study, two novel urethane-dimethacrylate monomers with two QA groups were obtained.For this purpose, the methodology that we developed to obtain an analogous series of urethane-dimethacrylate monomers with two QA groups and a core derived from 1,6-diisocyanato-2,2,4-trimethylhexane (TMDI) was adopted (QAn+TMDI, where n describes the number of carbon atoms in the N-alkyl substituent) [37].Qan+TMDIs were obtained in a three-step process in which HAMA, QAHAMA-n, and Qan+TMDI were successively synthesized.In this study, we used TMXDI instead of TMDI.TMXDI is classified as an aliphatic isocyanate, but it has a central 1,3-phenylene ring [50].The addition reaction of QAHAMA-n and TMXDI resulted in the formation of QA10+TMXDI and QA12+TMXDI (Figure 1).
The spectroscopic analysis confirmed the chemical structure of the obtained monomers.The 1 H NMR (Figure 2, Table 1), as well as 13C NMR (Figure 3, Table 2) spectra of QA10+TMXDI and QA12+TMXDI, showed the presence of signals coming from the urethane linkage, TMXDI core, methacrylate group, MDEA moiety, and N-alkyl substituent.The signal locations of the -CH 3 and -CH 2 -groups adjacent to the quaternary nitrogen were specific to the QA group neighborhood.Signals derived from the proton and carbon atoms of the -CH 3 group neighboring to N + were found from 3.30 to 3.77 ppm in the 1 H NMR spectra and at 53 ppm in the 13 C NMR spectra.In the 1 H NMR spectrum of HAMA, that -CH 3 group gives a singlet at a higher field, i.e., ca.2.4 ppm.In the 13 C NMR spectrum of HAMA, a signal derived from the -CH 3 group adjacent to N + can be also found at a higher field, i.e., ca.42 ppm [37]).The formation of the urethane linkage was confirmed by the presence of signals of the -NHCOO-proton, present in the range from 7.10 to 7.80 ppm in the 1 H NMR spectra, and the -NHCOO-carbon atom, present at 157 ppm in 13 C NMR spectra.The quaternization and formation of the urethane linkage were additionally confirmed from the FTIR analysis.In the FTIR spectra, a band of the C-N + bond vibrations was present at 943 cm −1 , whereas the C-N bond vibrations of the -NHCOO-linkage resulted in two bands at 3215 and 1529 cm −1 (Figure 4, Table 3).
Physicochemical properties QA10+TMXDI and QA12+TMXDI depended on the Nalkyl substituent length (Table 4).Its extension with two methylene groups resulted in a 5% increase in the MW and x DB .The RI decreased by 0.6% and the d m increased by 15%.
QA10+TMXDI and QA12+TMXDI had a MW of 1061 and 1117 g/mol and a x DB of 1.89 and 1.79 mol/kg, respectively (Table 4).This showed that the extension of the N-alkyl substituent with two methylene groups resulted in a 5% increase in the MW and the same decrease in the x DB .The Ris of QA10+TMXDI and QA12+TMXDI were 1.5230 and 1.5138, respectively.This corresponded to the statistically insignificant decrease in the RI of 0.6% when the N-alkyl substituent length increased.The d m increased from 1.16 to 1.33 g/cm 3  with the increase in the N-alkyl substituent length.This was a 15% change, and this had statistical significance.It can be seen that the physicochemical properties of experimental QAUDMAs depended on the N-alkyl substituent length.The more noticeable change was observed for density.The increase in density may suggest the tighter packing of the QA12+TMXDI molecule than that of QA10+TMXDI, regardless of whether it had longer N-alkyl substituents.
The light-bending ability of QA10+TMXDI and QA12+TMXDI was adequate for a DCRM matrix.Their Ris (respectively, 1.5230 and 1.5138) were very close to that of dental DMAs (1.5493, 1.4614, and 1.4852, respectively, for Bis-GMA, UDMA, and TEGDMA [37]) and were within the range of 1.46 to 1.55, which is recommended for dental systems [51,52].
The densities of QA10+TMXDI (1.16 g/cm 3 ) and QA12+TMXDI (1.33 g/cm 3 ) were higher than those of Bis-GMA, UDMA, and TEGDMA (1.15, 1.09, and 1.07 g/cm 3 , respectively [37]).If compared to fully aliphatic UDMA and TEGDMA, it is understandable that the synthesized QAUDMAs had a higher density, as the TMXDI benzene ring is heavy and capable of tight packing [53].The comparison of the d m of QA10+TMXDI and QA12+TMXDI to that of Bis-GMA showed that they had higher densities.Listing the important differences in the chemical structure of experimental QAUDMAs and Bis-GMA may be useful in explaining this observation:
It can be concluded that the asymmetrical substitution in the central benzene ring and the long N-alkyl substituents do not disturb the tight packing of QA10+TMXDI and QA12+TMXDI.
Only a negligible effect of the N-alkyl substituent on the 20(QA10+TMXDI) m and 20(QA12+TMXDI) m properties was observed (Table 6).The extension of the N-alkyl substituents by two methylene groups resulted in a 1% decrease in the x DB , a 15% decrease in the η, a 0.05% increase in the RI, and a 2% increase in the d m .These differences were statistically insignificant, except for the d m , which was statistically significant.The most notable change occurred in the viscosity.Its decrease can suggest the weakening of intermolecular interactions in 20(QA12+TMXDI) m caused by the N-dodecyl substituent [53].
Compared to 40(UDMA) m , the experimental compositions had about a 10% higher MW and 10% lower x DB .This suggests that the replacement of half of UDMA with one of QAUDMAs would have a minor impact on a decrease in the theoretical crosslink density of the resulting copolymer [54].
The comparison of 40(UDMA) m and the experimental compositions by the viscosity, refractive index, and density also led to satisfactory conclusions.40(UDMA) m had the η of 3.53 Pa•s, which was in the range limited by the η of 20(QA10+TMXDI) m and 20(QA12+ TMXDI) m , i.e., 3.79 and 3.22 Pa•s, respectively.In addition, differences in the η between 40(UDMA) m and 20(QA10+TMXDI) m or 20(QA12+TMXDI) m were only 7 and 9%, respectively, and they did not have statistical significance.This showed that the experimental formulations would have flowability similar to the reference formulation, which would be beneficial to the DCRM preparation.The transparency of 20(QA10+TMXDI) m and 20(QA12+TMXDI) m also was suitable for dental materials.Their Ris were of 1.5101 and 1.5094, respectively.This matches the RI range of 1.46 to 1.55, which is recommended for dental materials [51,52].In addition, the difference in the RI of the experimental compositions and reference composition was statistically insignificant.Finally, the d m of 20(QA12+TMXDI) m and 40(UDMA) m had the same value of 1.11 g/cm 3 .20(QA10+TMXDI) m had a d m that was 2% higher, and that difference was statistically significant.20(QA10+TMXDI) m and 20(QA12+TMXDI) m were subjected to photopolymerization towards two experimental copolymers 20(QA10+TMXDI) p and 20(QA12+TMXDI) p .They were characterized by the DC as the fundamental parameter used to characterize the polymer network structure (Table 7).An appropriately high DC is responsible for the adequate physical and mechanical properties of the DCRM matrices and the effective functioning of a dental filling [55,56].The DC of novel QAUDMAs increased statistically significantly with the extension of the N-alkyl substituent.20(QA10+TMXDI) p had a DC of 0.57 and 20(QA12+TMXDI) p had a DC of 0.69.Their DCs also were higher than that in 40(UDMA) p ., which was 0.54.In addition, The DCs of 20(QA10+TMXDI) p and 20(QA12+TMXDI) p were higher than 0.55, which is recommended for clinical applications [57].Thus, we can conclude that polymerization of 20(QA10+TMXDI) m and 20(QA12+TMXDI) m exhibited high efficiency and was not negatively influenced by a central benzene ring (which increases system stiffness [54]) and N-alkyl substituents (which increase the distance between polymerizing species [58,59]) present in their molecules.
A significant effect of the N-alkyl substituent on the density, volumetric contraction, glass temperature, water sorption, and leachability of 20(QA10+TMXDI) p and 20(QA12+ TMXDI) p was not observed (Tables 8 and 9).20(QA10+TMXDI) p had a d p of 1.21 g/cm 3 , S of 7.04%, T g of 53.46 • C, WS of 10.43 µg/mm 3 , and SL of 2.18 µg/mm 3 .20(QA12+TMXDI) p had a 1% lower d p , and this change was statistically insignificant.The S and T g statistically significantly increased, and these changes were 10% and 13%, respectively.The WS decreased statistically insignificantly by 1%, whereas the SL increased by 13% and this change was statistically significant.Although the decrease in the WS was insignificant, the chemical character of the copolymer surface can explain this change.20(QA12+TMXDI) p was characterized by a WCA of 91.30 • (Table 9), which corresponds to a hydrophobic surface [60,61].20(QA12+TMXDI) p had a statistically insignificantly lower WS and WCA.However, its WCA was 91.30 • , which corresponds to a hydrophobic surface [60,61].An effect of the N-alkyl substituent on the SL can be attributed to a higher MW of QA12+TMXDI compared to that of QA10+TMXDI.It is possible that a smaller number of unreacted monomer molecules leached out from 20(QA12+TMXDI) p because of a higher DC compared to 20(QA10+TMXDI) p .A similar effect was also observed in other work on the copolymer of Qan+TMDI [39].
The reference copolymer, 40(UDMA) p , had a 7.42% S, which was within the range limited by the S of 20(QA10+TMXDI) p and 20(QA12+TMXDI) p (Table 8).Compared to 40(UDMA) p , 20(QA10+TMXDI) m had a 5% lower S and 20(QA12+TMXDI) m had a 4% higher S, and both changes were statistically significant.The small scale of these differences suggests that the experimental and reference formulations had similar volumetric contractions caused by polymerization.Therefore, the potential DCRM matrices modified with QA10+TMXDI and QA12+TMXDI would probably have a marginal gap similar to common dental dimethacrylate copolymers [62].
The thermal behavior of 20(QA10+TMXDI) p and 20(QA12+TMXDI) p also was appropriate for their application in the potential DCRM matrix.On one hand, the experimental copolymers had a T g of 53.46 and 60.30 • C, respectively.These T g s were lower than the T g of 40(UDMA) p (62.97 • C).The difference in T g s between the reference copolymer and 20(QA10+TMXDI) p was 15% and was statistically significant, whereas that for 20(QA12+TMXDI) p was 4% and was statistically insignificant (Table 8).However, the T g s in both cases were higher than 50 • C.This is a highly satisfactory result because it means that the experimental copolymers occur in a glassy state up to a temperature of at least 50 • C. Therefore, we can assume that the experimental copolymers would maintain constant mechanical performance over the entire temperature range in the oral cavity [63].
Compared to 40(UDMA) p , which had a WS of 5.31 µg/mm 3 and SL of 0.17 µg/mm 3 , the experimental copolymers had WSs almost twice as high and SLs approximately fourteen times higher (Table 9).These were statistically significant differences, but the values of the WS and SL were noticeably lower than those defined by ISO 4049, i.e., 40 µg/mm 3 and 7.5 µg/mm 3 , respectively [46].A higher WS (10.43 µg/mm 3 ), which was determined for 20(QA10+TMXDI) p , was 26% of the maximum recommended by the ISO standard, and a higher SL, which was determined for 20(QA12+TMXDI) p (2.46 µg/mm 3 ), was 33% of the maximum recommended by the ISO 4049 standard.This is a highly satisfactory outcome, as the hydrophilic QA groups usually cause an increase in a polymer's WS [39].The increased WS and SL of the experimental copolymers showed that the QA groups promote interactions with water molecules, even though the N-alkyl substituent decreased the hydrophilicity of the copolymer surface.This was confirmed by the result achieved for 40(UDMA) p , which was characterized by the lowest WCA (74.07 • , Table 9), and thus its surface was the most hydrophilic [60].
The mechanical properties of 20(QA10+TMXDI) p and 20(QA12+TMXDI) p decreased as the N-alkyl substituent length increased.The E, FS, and HB for 20(QA10+TMXDI) p were 3244.9Mpa, 100.6 Mpa, and 234.8 Mpa, respectively.They decreased by 7%, 10%, and 17% for 20(QA12+TMXDI) p (Table 10).Only the result for the HB was statistically significant.This led to the conclusion that hardness was the most sensitive to the N-alkyl chain extension, as its decline was the most pronounced.40(UDMA)p exhibited the E, FS, and HB of 3597.7 Mpa, 125.4 Mpa, and 225.9 Mpa, respectively.Its modification by introducing QAUDMA resulted in statistically significant decreases of E, by 10 and 16%, and FS, by 20 and 28%, respectively, for 20(QA10+TMXDI) p and 20(QA12+TMXDI) p .The HB of 20(QA10+TMXDI) p increased by 4% and this difference was statistically significant.As the length of the N-alkyl substituent increased to twelve carbon atoms, the HB decreased statistically significantly by 14%.However, the overall mechanical performance of 20(QA10+TMXDI) p as well as 20(QA12+TMXDI) p was fully satisfactory, as the tested parameters were high.
Finally, the experimental copolymers revealed antibacterial activity against S. aureus and E. coli (Table 11).20(QA10+TMXDI) p had a lower MIC and MBC than 20(QA12+ TMXDI) p .This means that the extension of the N-alkyl substituent in the QAUDMA with two methylene groups decreased the copolymer's antibacterial activity.If compared to 40(UDMA) p , both copolymers had higher antibacterial activity.In addition, intraspecies differences were observed for 20(QA12+TMXDI) p .If compared to 40(UDMA) p , 20(QA12+TMXDI) p showed the same antibacterial activity concerning S. aureus, and greater antibacterial activity concerning E.coli.20(QA12+TMXDI) p had a MBC 50% lower and MIC 75% lower than 40(UDMA) p in tests against E.coli.20(QA10+TMXDI) p showed more significant antibacterial activity and differentiation according to bacterial strain was no longer observed.20(QA10+TMXDI) p had a 75% lower MBC and 87.5% lower MIC than 40(UDMA) p in tests against both bacteria strains.
We can additionally characterize the experimental copolymers using a common classification of antimicrobials as bactericidal or bacteriostatic.This classification assumes that if the MBC to MIC ratio is lower than four, a biocide can be recognized bactericidal, and if the MBC to MIC ratio is higher than six, a biocide can be recognized bacteriostatic [64].As this ratio for 20(QA10+TMXDI) p was two, one can assume this copolymer is bactericidal and it may be possible to administer the 20(QA10+TMXDI) p dosages to kill 99.9% of the bacteria.
The monomer compositions had adequate flowability and transparency required for dental materials.
A comparative analysis of the tested copolymers' properties showed that their polymerization shrinkage, glass transition temperature, and mechanical properties, were satisfactory and were usually similar to the properties of the reference copolymer.The water sorption and water solubility mostly deteriorated; however, they were satisfactory.Their values were far below the limits pointed at the international standard ISO 4049.
The antibacterial properties of the copolymers were characterized against S. aureus (ATCC 25923) and E. coli (ATCC 25922).The copolymer comprising QA10+TMXDI showed a significantly higher antibacterial effect than both the copolymer comprising QA12+TMXDI and the reference copolymer, as it had a lower minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC).
This study demonstrated that the introduction of a benzene ring into the QAUDMA structure resulted in copolymers with very good physicochemical and mechanical characteristics and antibacterial activity.However, it was observed that the shorter the N-alkyl chain, the higher the antibacterial activity.Therefore, it is worth conducting further research with monomers that have shorter N-alkyl substituents.A more in-depth investigation of antibacterial properties should also be carried out for the tested copolymers, as the aim of this work was only to determine whether they have antibacterial activity.
)ethyl-2-hydroxyethylmethylalkylammonium Bromides (QAHAMA-n, Where n Is the Number of Carbon Atoms in the N-Alkyl Substituent)

Figure 3 Figure 3 .
Figure3shows the13 C NMR spectra of the QA10+TMXDI and QA12+TMXDI monomers.Table2lists carbon atom signals present in the spectra with their assignment to chemical groups.

Table 4 .
Properties of QA10+TMXDI and QA12+TMXDI related to properties of Bis-GMA, UDMA, and TEGDMA.The lowercase letter "a" in the superscripts indicate couples with statistically significant results (p ≤ 0.05).The remaining results did not have statistical significance (p > 0.05).

Table 6 .
Properties of 20(QA10+TMXDI) m and 20(QA12+TMXDI) m experimental monomer compositions related to properties of the 40(UDMA) m reference monomer composition.The lowercase letters "a" and "b" in the superscripts indicate couples with statistically significant results (p ≤ 0.05).The remaining results did not have statistical significance (p > 0.05).

Table 7 .
The DC in copolymers.The lowercase le ers "a" and "b" in the superscripts indicate couples with statistically significant results (p ≤ 0.05).The remaining results did not have statistical significance (p > 0.05).

Table 7 .
The DC in copolymers.The lowercase letters "a" and "b" in the superscripts indicate couples with statistically significant results (p ≤ 0.05).The remaining results did not have statistical significance (p > 0.05).

Table 8 .
The physical properties of copolymers: density (d p ), polymerization shrinkage (S), and glass transition temperature (T g ).The lowercase letters "a"-"c" in the superscripts indicate couples with statistically significant results (p ≤ 0.05).The remaining results did not have statistical significance (p > 0.05).