Evaluation of the physical and antifungal effects of chlorhexidine diacetate incorporated into polymethyl methacrylate

Abstract This study aimed to evaluate the physical properties and antifungal activities of polymethyl methacrylate (PMMA) acrylic resins after the incorporation of chlorhexidine diacetate salt (CDA). Methodology: First, acrylic resin specimens were fabricated with Vipi Cor® and DuraLay® resins with and without the incorporation of 0.5%, 1.0% or 2.0% CDA. The residual monomer and CDA release were measured at intervals ranging from 2 hours to 28 days using ultraviolet spectrometry combined with high-performance liquid chromatography. The antifungal activity against C. albicans was evaluated with the agar diffusion method. Fourier transform infrared spectroscopy was used to analyze the degree of resin conversion. Finally, the water sorption values of the resins were also measured. Results: The incorporated CDA concentration significantly changed the rate of CDA release (p<0.0001); however, the brand of the material appeared to have no significant influence on drug release. Subsequently, the inhibition zones were compared between the tested groups and within the same brand, and only the comparisons between the CDA 2% and CDA 1% groups and between the CDA 1% and CDA 0.5% groups failed to yield significant differences. Regarding the degrees of conversion, the differences were not significant and were lower only in the CDA 2% groups. Water sorption was significantly increased at the 1.0% and 2.0% concentrations. Conclusions: We concluded that the incorporation of CDA into PMMA-based resins enabled the inhibition of C. albicans growth rate, did not alter the degrees of conversion of the tested resins and did not change the release of residual monomers.


Introduction
A large variety of microorganisms take advantage of the environments generated by prostheses and use them as a substrate for colonization. 1,2 Candida albicans, one of these microorganisms, is an opportunistic pathogen capable of generating inflammatory responses in tissues, especially in immunocompromised patients. [3][4][5][6] Furthermore, it is a fact that acrylic resins are porous and have low resistance to abrasion. 7,8 Over time, increased surface roughness commonly leads to biofilm accumulation, creating a conducive environment to the development of microorganisms such as the C. albicans. 2,9,10 The principal form to control and eliminate infections caused by C. albicans is the use of proper hygiene aids and drugs with antifungal activity. Among these drugs, chlorhexidine has proven antifungal action against C. albicans and the advantage of being effective even against strains that are resistant to other local antifungal agents available to treat this infection in the oral cavity. [3][4][5]11,12 The chlorhexidine has been successfully used in Dentistry and is available in different formulations, including digluconate, hydrochloride, and diacetate formulations. The first formulation is most commonly used in mouthwashes 13 because it is more soluble in water, while the last two formulations are more soluble in ethanol. The use of chlorhexidine is considered the gold standard antiseptic treatment for this agent has strong bactericidal and bacteriostatic capacities. 14 Although the results of chlorhexidine use have proven satisfactory, this drug efficacy in the form of mouthwashes or gels depends directly on the patient's cooperation; thus, the expected result is not always obtained. 15 For this reason, several examinations have investigated the potential use of drug delivery systems involving the incorporation of antifungal or antimicrobial agents into denture acrylic resins or soft liners. [16][17][18] Silver nanoparticles, fluconazole, 16 nystatin, 17 ketoconazole 18 and chlorhexidine 18 are some substances that have been incorporated into polymers.
Chlorhexidine is one of the principal antifungals that has been tested in drug release systems following its incorporation into acrylic resins. [18][19][20][21] Moreover, soft liners placed in dentures have been used as carriers for this drug in the treatment of denture stomatitis. 22,23 However, few studies have tested the chlorhexidine diacetate (CDA) incorporation into auto-polymerized acrylic resins. Therefore, this study aims to assess this drug influence following its incorporation into auto-polymerized polymethyl methacrylate (PMMA)based resins and the ability of such resins to release the drug by testing the following null hypotheses: a) chlorhexidine incorporation does not alter residual monomers leaching from the resins; b) chlorhexidine incorporated into rigid acrylic resins is not released from PMMA; c) chlorhexidine incorporation into acrylic resins does not enable the drug to inhibit C. albicans growth rate; d) chlorhexidine incorporation does not change the degrees of the conversion of the evaluated resins; and e) chlorhexidine incorporation does not alter the water sorption of PMMA-based acrylic resins.

Methodology Test specimen preparation
To calculate sample size, SigmaPlot 14.0 tool with the test power set in 0.80 was used, and the values were obtained from the previous study by Bertollini, et al. 21 (2014). According to the calculation, 3 samples were needed in each group for chlorhexidine release, residual monomer leaching, and antifungal activity; in addition, for the degree of conversion n=6 and water sorption n=10. In total, 200 samples were used.
The specimens were made with two selfpolymerizable PMMA-based acrylic resins, i.e., Vipi Cor ® and DuraLay ® , and comprised 8 groups involving the incorporation of 0.5%, 1.0% or 2.0% CDA salt into each resin and 2 control groups without the drug, as shown in Figure 1.
To make the test specimens, resins were manipulated according to the manufacturers' recommendations in a ratio of 2 g polymer to 1 mL monomer. The chlorhexidine diacetate salt was separately weighed on a precision scale with 4-digit accuracy (Gehaka BG200; São Paulo, SP, Brazil) to obtain 0.5%, 1.0%, and 2.0% samples by weight for each polymer material. The powder/monomer/CDA proportions for each mixing group were as follows: 0.5% CDA, 5 g/2.5 mL/0.025 g; 1% CDA, 5 g/2.5 mL/0.05 g; and 2% CDA, 5 g/2.5 mL/0.1 g.
Initially, the chlorhexidine diacetate was dissolved in the monomer until achieving a homogeneous mixture. Subsequently, the polymer was added and mixed for 30 seconds. High-performance liquid chromatography (HPLC) HPLC was used to measure the chlorhexidine release and residual monomer leaching from the acrylic resins, for all groups. For this reason, after manipulating the resins, they were placed in discshaped silicon molds measuring 10.0 mm in diameter and 3.0 mm in thickness. At the plate, three discs of each group were placed, and the samples were collected in triplicate. 21,[24][25][26] The specimens were polymerized for 5 min at ambient temperature. 24 The specimens were stored individually in 24well cell culture plates (TPP Techno Plastic Products; Trasadingen, Switzerland) containing 1 mL of sterile distilled water in each well. The volume of liquid used was sufficient to fully cover the surface of the resin disc. The storage liquid was maintained at 37°C and was changed every 48 hours to obtain a release curve.
Following, the assessment of chlorhexidine and residual monomer release, the solution was removed after 2 hours and measured via HPLC, associated with ultraviolet spectroscopy (UV). This procedure was repeated every 7 days to create a 28-day interval. The

Antifungal activity
In total, 24 DuraLay ® and Vipi Cor ® disc-shaped samples were examined. The samples were divided into 4 groups for each materials with 0%, 0.5%, 1.0%, or 2.0% chlorhexidine, a total of 8 groups with 3 discs each. 21 Candida albicans strain obtained from the Oswaldo surfaces. After solidification of the media, plates were capped and placed in an oven at 37°C to ensure their sterility and then placed in a refrigerator until use. The agar diffusion test was performed using a technique previously described by Radnai, et al. 23 (2010).
The surface received a prepared C. albicans suspension of 100 µl (10 8 -10 9 CFU/mL) that was pipetted and inoculated by rubbing the swab across the sterile surface. Each group had three discs placed at the plate, and the samples were collected in triplicate. The specimens were distributed uniformly on the surface of each plate. 21 Then, the plates with the samples were incubated at 37°C for 48 hours.
After this period, the inhibition zone diameters of the C. albicans growth generated around the resin discs were measured using a calibrated digital caliper (Mitutoyo; Tokyo, Japan) and reflected light.
In total, five measurements were performed for each disc, their diameter (10 mm) was then calculation was then performed using the following mathematical formula: 29 m 0 is the initial sample weight, and V 0 is the initial sample volume.
The mean value of each group was obtained, and the results were then subjected to two-way ANOVA analysis with subsequent Tukey tests (p<0.05).

High-performance liquid chromatography (HPLC)
The HPLC analysis results are presented in Shapiro-Wilk test revealed that two of the three reviewed factors (i.e., drug concentration and storage time) significantly changed chlorhexidine release rate (p<0.0001), but the material brand did not appear to significantly influence the release of the drug.
The residual monomer release results exhibited numerical changes; however, these differences were not statistically significant for any of the assessed drug concentrations (p>0.05).

C. albicans inhibition zone -agar diffusion method
Based on the results shown in Table 1, both chlorhexidine diacetate resins were able to inhibit C. albicans growth rate because they induced an inhibition zone formation.
Moreover, the statistical analyses revealed no significant differences between the two tested brands in terms of their abilities to inhibit C. albicans. Regarding the chlorhexidine concentration, all tested concentrations differed significantly from the control groups, which showed no inhibitory capacities. There were no significant differences between the 2% and 1% concentration groups or the 1% and 0.5% groups.

Degree of conversion evaluation
Analyses of the degrees of the conversion of the acrylic resins indicated that the presence of the chlorhexidine salt caused no significant changes in resin conversion processes assessed in this study, which confirmed the initial hypothesis (p>0.05), as shown in

Water sorption
The analyses of the sorption values of the resins (Table 3) revealed that the two resins behaved similarly when comparing samples with the same chlorhexidine concentrations. However, the 1% and 2% groups were significantly different from both the control and 0.5% group. Moreover, a significant difference was detected between the 1% and 2% groups.

Discussion
The results obtained, related to the drug release profiles, corroborated the kinetics previously described in the literature for the release of antifungal agents because the initial concentration of the drug generates a significantly higher diffusion gradient than it does during the remaining 28 days. 19,[30][31][32] The diffusion gradient changes the drug output due to the release mechanism of these compounds, Previous studies have demonstrated that the inhibition zone size tends to increase in proportion to the amount of antifungal agent incorporated into the material either due to direct contact of the specimen with the plate culture 16,34,35 or with the storage solution into which the agar-containing wells are placed. 19,21,31 However, in this study, although statistically different drug amounts were released by each group, no significant differences were found between the groups with concentrations of 2% vs. 1% or 1% vs. 0.5%.
It was postulated that chlorhexidine salt presence would not alter the kinetics of polymerization or the medium viscosity. These hypotheses were confirmed by the lack of significant alteration in either parameter.   the salt loss leaves micrometric pores in the material structure increasing the surface of contact area with the environment, which enhances the water intake process into the material. 32 The sorption rates of both resins showed similarity between the groups with the same concentrations of