Synthesis of a chitosan nanoparticle suspension and its protective effects against enamel demineralization after an in vitro cariogenic challenge

Abstract Objective Our study aims to synthesize, characterize, and determine the effects of a ChNPs suspension on human enamel after cariogenic challenge via pH-cycling. Methodology ChNPs were synthesized by ion gelation and characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. Forty enamel blocks were divided into four groups (n=10/group): (i) ChNPs suspension; (ii) chitosan solution; (iii) 0.05% sodium fluoride (NaF) solution; and (iv) distilled water. Specimens were exposed to cariogenic challenge by cycling in demineralization solution (3 h) and then remineralized (21h) for 7 days. Before each demineralization cycle, the corresponding solutions were passively applied for 90 s. After 7 days, specimens were examined for surface roughness (Ra) and Knoop hardness (KHN) before and after the cariogenic challenge; % KHN change (variation between initial and final hardness), and surface topography by an optical profilometer. The data were analyzed by repeated-measures ANOVA, One-way ANOVA, and Tukey tests (α=0.05). Results TEM images showed small spherical particles with diameter and zeta potential values of 79.3 nm and +47.9 mV, respectively. After the challenge, all groups showed an increase in Ra and a decrease in KHN values. Optical profilometry indicated that ChNPs- and NaF-treated specimens showed uneven roughness interspersed with smooth areas and the lowest %KHN values. Conclusion The ChNPs suspension was successfully synthesized and minimized human enamel demineralization after a cariogenic challenge, showing an interesting potential for use as an oral formulation for caries prevention.


Introduction
Dental caries is caused by an imbalance between the presence of undisrupted cariogenic biofilms and the intake of fermentable carbohydrates over time. 1,2 Frequent toothbrushing with a fluoridated toothpaste is considered the main control mechanism for caries prevention. 2-4 However, children experiencing poor caries management due to a high frequency of exposure to fermentable carbohydrates, ineffective biofilm removal, or a decrease in salivary flow should use fluoridated mouthwashes as an auxiliary control method. 4,5 Fluoride prevents caries development through physicochemical mechanisms that minimize tooth demineralization and promote remineralization. 6 The constant contact of fluoride molecules with the mineral structure of the teeth reduces enamel solubility during the carious process. 5 Yet, the literature have shown that children, especially those under 6 years of age, may swallow fluoridated toothpaste or mouthwash, which could lead to the onset of dental fluorosis. 5 This has encouraged the development of alternative natural agents with antimicrobial activity and remineralization effects for the prevention and control of dental caries in susceptible patients. Natural polymers, such as chitosan, have been tested in oral applications due to their antimicrobial properties 7-9 and role in enamel remineralization. [10][11][12] Chitosan is obtained from chitin, which is a linear biopolymer with ß-(1-4) -linked 2-acetamido-2deoxy-d-glucopyranose and 2-amino-2-deoxy-dglucopyranose units extracted from the exoskeleton of crustaceans. 9 A cation derived from the amine group (NH 2 ) is amine-protonated at low pH (NH 3 + ), interacting with negatively charged components, such as proteins, anionic polysaccharides, and phospholipids, 7,8 fungal and bacterial membranes, 7-9 as well as with the negatively charged demineralized enamel surface. 12 After the dissolution of chitosan, the acidic pH can open gaps among the surface layer crystals. 11 Thus, chitosan may interact with the enamel surface and form a physical barrier and the mineral content loss is minimized by organic acids. 10-12 However, the actual mechanisms by which chitosan can interact with the demineralized enamel remain unclear.
Currently, chitosan is one of the biopolymers most widely used for the synthesis of nanoparticles due to its bioavailability, biocompatibility, biodegradability, and of the chitosan solution was 10 mL. The solution was filtered with 5.0 μm and 0.8 μm membrane filters.
The ChNPs suspension was synthesized through the ionic gelation method with the addition of sodium tripolyphosphate (TPP). TPP aqueous solution (Sigma-Aldrich, St. Louis, USA) at 2.4 mg/mL was prepared separately and 3 mL was added to the 10 mL chitosan solution under agitation at 6000 rpm at room temperature using a continuous infusion pump at the rate of 60 mL/h. The final concentration of the ChNPs suspension was 4.4 mg/mL using the chemical formula of mixing solutions. The pH of the suspension was adjusted to 5.5 with the addition of NaOH to match the pH value of some commercial mouthwashes. 18,19 The pH of the suspension was monitored for 7 days, and the presence of precipitation was also evaluated by visual analysis during the same period.

Specimen preparation, application of agents, and cariogenic challenge
The sample size was estimated on a pilot study.
Considering a standard deviation of 27.0 and a minimal intergroup difference of 50.0 to detect the hardness values of the enamel, a sample of 4 enamel slabs was required to provide 95% statistical power with an α error of 0.05 in a two-sided paired t-test. Since the present study tested an experimental agent (ChNPs) that was not tested following a cariogenic challenge, a sample of 10 slabs was used for each group.
Twenty-three healthy third molars were obtained with the approval of the Juiz de Fora Research Ethics Committee (protocol no. 64959517.3.0000.5147).
The teeth were stored in 0.1% thymol at 4°C and used within 1 month after extraction. In the proximal surfaces of each tooth, a window of 4 mm × 4 mm was designed. Two slabs (one from each proximal surface) were obtained using a low-speed cutting machine with a water-cooled diamond disc (  Within this period, no formation of precipitates was found by visual analysis in the suspension.

Discussion
The null hypothesis tested in our study was partially rejected, since ChNPs-treated specimens had their enamel topography and hardness significantly altered after a cariogenic challenge; however, their roughness was not affected.
Chitosan is one of the biopolymers most commonly used for the synthesis of nanoparticles. Several methods can be used in nanoparticle engineering, such as electrospray, emulsification, solvent diffusion, microemulsion, and ionic gelation. 9,13 In our study, ChNPs were synthesized by ionic gelation using TPP, which is a rapid and easy method for nanoparticle production. The suspension occurs spontaneously to act as a protective agent on the enamel during the demineralization process. This is the reason why the TPP solution was not tested in our study as a control group. formation was successful and that there was an interaction between chitosan and TPP molecules. ChNPs had a mean particle size of 79.3±12.5 nm.

The TEM and DLS analyses indicated that ChNPs
Several factors can influence the size of nanoparticles, such as the synthesis method, pH of the media, and the concentration and molecular weight of chitosan. 7,23 These parameters have explained most of the interstudy differences concerning the size of nanoparticles.
In our study, we observed the presence of small areas with nanoparticle aggregation (Figure 1) The pH of the media can also affect relevant physicochemical characteristics of ChNPs suspensions and their stability by changing parameters such as particle hydrophobicity, wettability, and surface homogeneity. 24 In our study, the pH of the media remained unaltered and, during seven days, no formation of precipitates was found in the suspension, suggesting that the ChNPs suspension was stable.
Surface roughness alterations were detected using profilometer. Their importance on the aggravation of carious lesions was previously discussed by Ando, et al. 25 The authors showed that, as the enamel undergoes continued demineralization, its surface roughness is progressively increased. 25 Thus, the authors suggested that the increase in the enamel In our study, treatment with different agents increased Ra values in all groups, with no significant difference among them (Table 2). No agent was able to prevent the increase in surface roughness, since progressive enamel demineralization was observed.
Ra values were obtained from the entire enamel area.
In contrast, profilometer images showed that ChNPs and NaF groups had an uneven surface roughness interspersed with smooth enamel areas (Figures 2a   and 2c) compared to the uniform roughness observed along the entire chitosan surface and control-treated specimens (Figures 2b and 2d). We hypothesize the application of 0.05% NaF and ChNPs suspension may have protected the enamel surface and diminished demineralization in smooth enamel areas, despite the increased Ra values found. This hypothesis is supported by the microhardness data that will be further discussed herein.
The microhardness test is one of the most used approaches to determine alterations of the enamel surface during the early stages of caries development. 11,21,26 Although this test cannot point out differences in the mineral content, the surface microhardness assessment is appropriate to investigate the strength of a hard tissue (e.g., enamel) that has a non-homogenous fine microstructure prone to cracking. 21 In our study, we found no significant difference in KHN initial values among the groups ( Table   1), which suggests that all specimens were matched for hardness. After the cariogenic challenge, all groups showed a reduction in KHN values (Table 1).
This indicates that no agent, not even the 0.05% NaF solution, was able to prevent enamel demineralization.
However, ChNPs and 0.05% NaF groups showed lower % change in KHN and higher KHN values compared to those of chitosan and distilled water groups (Table   1). Collectively, these findings indicate that ChNPs and 0.05% NaF successfully strengthened the enamel structure, minimizing the demineralization process and preventing further topographical alterations ( Figures   2a and 2c). According to the literature, only when a chitosanbioglass complex was used, subsurface mineral deposition was observed, resulting in remineralization of early carious lesions in artificial enamel. 12 In our study, the chitosan solution (5 mg/mL) could not prevent the reduction of enamel hardness after the cariogenic challenge. Only the ChNPs suspension could minimize the KHN reduction, with no significant difference when compared with the treatment with 0.05% NaF. To the best our knowledge, this is the first study that investigated the effects of ChNPs on enamel properties after pH-cycling. Liu, et al. 14 (2007) evaluated the adhesion of chitosan nanoparticles contained in a toothpaste but did not use the pHcycling method. These authors found that ChNPs have a better absorption and adhesion capacity than that of the chitosan solution. The mechanism of action of ChNPs on enamel lesions is still unclear; however, we hypothesize that chitosan nanoparticles are small enough to penetrate the surface layer into the remaining voids of the carious lesions and bond onto the walls of demineralized prisms in a coordinated bond reaction. 30 The bond can occur by the chemical reaction between hydroxyapatite (metal ions, as Ca 2+ ) and chitosan (amino groups). 30 Importantly, besides the beneficial effects of ChNPs on the physical properties of demineralized enamel, the ChNPs suspension can be incorporated into a mouthwash formulation to act as a controlled drug release system in the oral cavity. 16

Conclusions
Our study showed that chitosan nanoparticles were successfully synthesized by the ionic gelation method, and they minimized human enamel demineralization after a cariogenic challenge, showing an interesting potential for use as an oral formulation for caries prevention. Also, ChNPs suspension showed a protective effect on the tooth surface by strengthening the enamel structure and minimizing its demineralization after an in vitro cariogenic challenge.
Synthesis of a chitosan nanoparticle suspension and its protective effects against enamel demineralization after an in vitro cariogenic challenge