Layered double hydroxides for controlled fluoride release

: This study aims to develop a nano-sized fluoridated layered double hydroxide (LDH)-based release system via hydrothermal treatment for the controlled delivery of fluoride (F - ) ions in the oral environment. The synthesis of conventional LDH-type (C-LDH) precursor nanomaterials was conducted using a co-precipitation method at constant pH, and the nanoparticulate-LDH (N-LDH) was synthesized by a hydrothermal procedure. Fluoride LDH (F-LDH) products were obtained through indirect synthesis using the precursor ion-exchange technique by varying the agitation time (2 and 24 h) and temperature (25 and 40 °C) to produce 12 material samples. The materials were characterized by energy dispersive x-ray, hexamethyldisilazane, digital radiography x-ray, Fourier-transform infrared, thermogravimetric analysis, and scanning electron microscopy. Additionally, the F-release kinetic profile was evaluated for 21 d in neutral and acid media with mathematical model analysis. Products with varying F-quantities were obtained, revealing specific release profiles. In general, there was a higher F-release in the acid medium, with emphasis on F-LDH-8. Fluoride-LDH and controlled fluoride delivery was successfully obtained, proving the potential of these nanomaterials as alternative anti-caries agents.


Introduction
Although a worldwide decline in caries has been observed, tooth decay remains a prevalent disease.Despite improvements in dental technology, dental caries affects children and adults regardless of their socioeconomic status. 1 Insufficient fluoride use contributes to this issue.Fluoride presence can reduce demineralization (mineral loss) and increase dental remineralization. 1,2Therefore, it is essential to discuss efficient fluoride use and improve fluoride-releasing methods for better action in the oral environment. 3ultifunctional nanosystems have offered unique benefits related to nano-sized particles, including large surface area, dental biofilm penetration ability, and controlled drug delivery. 4Layered double hydroxides (LDHs), also known as anionic clays or hydrotalcite-like compounds, are nano-sized compounds.They are octahedron-shaped comprising hydroxyls forming lamellae and an ion-exchange switchable anion. 5,6claration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
These materials exhibit crystalline conformation and their composition depends on the cation type, molar ratio, and interlamellar anion type. 5ecently, LDH has been utilized as an adsorbent to remove excess fluoride ions from aqueous solutions via adsorption or ion exchange. 7,8,9,10,11,12n dentistry, LDHs are used for calcium release to target hydroxyapatite creation or zinc ion release to reduce demineralization. 13,14Additionally, LDHs have a non-soluble structure, which remains unchanged in dental material like toothpaste and mouthwash. 15Studies evaluating fluoridated LDH (F-LDH) for controlled fluoride ion release and dental applications have not been sufficiently explored.Thus, this study aims to develop a nano-F-LDHbased release system synthesized via hydrothermal treatment for the controlled delivery of fluoride (F -) ions in the oral environment.

Methodology
This study comprises F-LDH synthesis, its characterization, and fluoride release kinetic evaluation from synthesized materials.For this, 12 fluoridated products were obtained from the synthesis of conventional (C-LDH) and nanoparticulate (N-LDH) precursors.Each product was obtained by varying the agitation conditions and temperature, as shown in Table 1.

Precursors synthesis: C-LDH and N-LDH
LDHs were synthesized using the direct co-precipitation method at constant pH and hydrothermal method in an autoclave/reactor for C-LDH and N-LDH, respectively.Both methods are described by the formula [M +2 1 , where M +2 and M +3 are bi and trivalent metal cations, respectively. 5MgCl 2 and AlCl 3 were mixed in a 3:1 ratio. 16,17The solutions were stirred vigorously at room temperature.Following this, the products were centrifuged (4500 rpm; 30 min, Sigma 6-15, Rio de Janeiro, Brazil) and washed 4 to 7 times with Milli-Q water.The C-LDH was dried in an oven (NOVA instruments, São Paulo, Brazil) for 12 h at 60 °C.N-LDH was resuspended with Milli-Q water, and the obtained colloidal suspension was transferred to a reactor/autoclave (MAITEC -Fornos INTI, São Paulo, Brazil) to undergo hydrothermal treatment for 16 h at 100 °C in an oven with air-forced circulation (NOVA instruments, São Paulo, Brazil) to obtain a final volume of 50 mL.Half the suspension was reserved, and the other half was frozen at −20 °C for 48 h and subsequently freeze-dried (TERRONI ® -

Fluoridated material synthesis
F-LDHs were indirectly synthesized by the ion exchange technique. 18A 500 mg/L NaF solution was mixed with two synthesized precursors types. 15ccording to Table 1, the stirring conditions (2 and 24 h) and the temperatures (25 and 40 °C) were varied to obtain 12 products.The materials were centrifuged, washed thrice with deionized water, dried in an oven (NOVA instruments, São Paulo, Brazil) for 3 h at 60°C, and reserved for characterization.Potentiometry analysis was conducted to confirm the synthesis.This utilized a specific fluoride iron electrode (Orion 9409) by the hexamethyldisiloxane (HMDS)-facilitated microdiffusion technique, following a modified Taves method (1968). 19,20he HMDS analysis consisted of placing the analite (powder of the 12 materials, 0.025 g) in individual polystyrene Petri dishes (60 × 15 mm) that serve as an alkaline trap for fluoride.All procedures were duplicated.First, a small hole (± 2 mm) was burned into the lid of polystyrene Petri dishes (60 × 15 mm).Then, the internal lid periphery was ringed with petroleum jelly for the posterior seal.The alkaline trap consisted of five NaOH drops (0.05 M) carefully placed in the inner part of the lid.The lid was fixed on a Petri dish by sealing the system.Finally, 2 mL of 3 M HMDS-saturated sulfuric acid was added through the hole in the lid, which was sealed with petroleum jelly.
After overnight agitation (Orbital Gyratory Shaker TE-141, Tecnal, São Paulo, Brazil), the lids were carefully removed and the NaOH fluoride trap was buffered with 25 µL of 0.2 M acetic acid.The total volume was adjusted to 75 µL by adding deionized water and the same pipette was used to mix the solution.
The calibration curve was obtained using five standards with known fluoride contents prepared by serial dilution from a 100 mg F/L stock solution (Orion Research, # 940907, Cambridge, USA): 0.01, 0.02, 0.04, 0.08, and 0.16 µgF.These standards were diffused in triplicate like for the samples.The millivoltage potentials were converted to µgF using Microsoft Office Excel and an r > 0.99 correlation coefficient was accepted.The mean repeatability of the duplicate sample readings was above 90%.

Characterization
Dispersive energy x-ray fluorescence spectrometry (EDX) was performed by a Shimadzu Spectrometer EDX 7000 under vacuum with a 10 mm collimator for Mg, Al, and Cl quantification.As EDX does not quantify elements with low atomic numbers, the fluoride ion quantification (total fluoride) was assessed according to the Taves method (1968) 19 by indirectly facilitated hexamethyldisiloxane (HMDS) microdiffusion through an Orion-specific ion electrode 9409 coupled to a potentiometer (Orion, 720 A).X-ray diffraction (XRD) was performed using a Shimadzu X-ray diffractometer, using Cu Kα radiation at 30 mA and 30 kV, in a 0.02° (2θ) range from 3° to 90°.The basal spacing d (003) was calculated using the Bragg equation (n•λ = 2•d •sinθ).Fourier-transform infrared spectrometry (FTIR) was conducted using a spectrometer (Thermo Scientific Nicolet TM iS TM 10) in the 4000-500 cm -1 range, using potassium bromide tablets (KBr); 2 mg sample and 200 mg dry KBr.Thermogravimetric analysis (TGA) was performed using a Shimadzu TGA-50 thermoanalyzer (Kyoto, Japan), at a 10 °C/min heating rate in a 50 mL/min nitrogen gas flow from 25 to 800 °C.Micrographs were obtained using scanning electron microscopy (SEM) from the TESCAN-VEGA3.The samples were previously coated with gold in a QUORUM-Q150R ES metallizer at a 10 mA current for 10 min.

Fluoride release kinetics
Fluoride release kinetics analysis was conducted for 21 d.The specimens (4.3 cm 2 ) were individually distributed in plastic vials, then immersed in deionized water (pH 7) and a sulfuric acid (pH = 5.5)-H 2 SO 4 solution. 21Sulfuric acid was used to acidify the solution medium to maintain the HMDS acidic conditions.
T he f luor ide a mou nt wa s me a su r e d at predetermined time intervals (1, 2, 3, 5, 7, 14, and 21 days).For F-released determination, 1 mL of the sample (F-LDH) was used with 1 mL total ionic strength adjustment buffer II (TISAB II).The standard solutions were prepared in triplicate under conditions similar to that of the samples (0.4-6.4 mg F/mL).The calibration and concentrations were determined by testing a linear regression curve using the Excel software.The electrode potential was related to the logarithm of the measured ion concentration by the Nernst equation.This program transformed the mV values provided by the electrode to ppm (µg F/mL).For each fluoride material, two fluoride release experimental repetitions were conducted.24 samples were obtained for neutral and acid media, each.Finally, the 48 F-contained solutions released were analyzed in duplicate and the analyses were validated using internal standards.A coefficient variation under 5% was considered acceptable.
The kinetics data were converted into percentages from total fluoride (HMDS) quantification (ppm).To investigate the release mechanism, the obtained data were analyzed using the following mathematical models: zero-order kinetics (Equation 1), first-order kinetics (Equation 2), Higuchi (Equation 3), and Korsmeyer-Peppas (Equation 4).Statistical calculations were performed using the non-linear regression module Statistica 13.3 software by the Quasi-Newton method with lamellar LDH geometry. 22t = k Z * t (1) where q t is the amount of drug dissolved in time (t), q e is the initial amount of drug in the solution, k Z is the zero-order release kinetic constant, k F is the firstorder release kinetic constant, k H is the Higuchi release kinetic constant, M t is the cumulative amount of drug released in time (t), and M ∞ is the initial drug loading.

Results
The EDX analysis results are presented in Table 2, which confirm the C-LDH and N-LDH formation.In addition to the presence of Cl in the precursors, the Mg/Al ratio of approximately 3:1 was ratified.
T he HMDS te c h n ique a l lowed f luor ide quantification in all analyzed samples (Table 2).The total fluoride quantification values, referring to 100% F present in fluoridated products, are presented in Table 2.For F-LDH-1 to -4 materials, chloride (Cl -) was quantified in smaller proportions when compared to the precursor (C-LDH).For materials synthesized with N-LDH nanoparticulate precursor using the colloidal suspension, the chloride ion was quantified at ≈1%.Only the F-LDH-8 sample reproduced the results of F-LDH-9 to -12, which utilized the chlorinated precursor in lyophilized powder form.The chloride ions were not quantified for these products.
The diffractograms of the precursors C-LDH and N-LDH are shown in Figures 1(A) and 1(B),   Figure 3 depicts the thermal degradation, where the different fluoridated sample behaviors of the fluoride-free samples can be observed.
The SEM micrographs in Figures 4(A -E) reveal particles with irregular and rough surfaces.

Fluoride release kinetics
Figure 5 shows the fluoride release kinetic curves for neutral and acidic media.The fluoride release pattern was similar for all products; however, minor differences in the amount of fluoride released for each material, comprising log release profiles, implied higher quantifiable anion values in the acidic medium.Among all products, F-LDH-9 presented 6.0% release values (64 µg/mL F) in both media and F-LDH-11 exhibited the highest F -amount as the experiment ended: 45.7% (56.32 µg/mL F, pH= 7.0) and 46.6% (57.40 µg/mL F, pH= 5.5) (Figures 5(A -B)).Moreover, Tables 3 and 4 summarize all estimated kinetic parameters with the determination coefficient (R2) based on the experimental data from Equations 1-4 for the neutral and acid media, respectively.The adjusted kinetic model results showed that F-LDH-8 (24 h synthesis at 40°C) exhibited a better F release in the acidic medium following a Fick model, whereas, in a neutral medium, a non-Fick behavior was observed.) synthesis from N-LDH powder (E).Absorptions associated with asymmetric axial and angular deformation of the hydroxyl group (ʋOH-3445 cm -1 ; δH 2 O-1638 cm -1 ) bonds correspond to the adsorbed and interlamellar water molecules, respectively.At approximately 2056 cm -1 , there is a band corresponding to a CO 2 medium.1355 cm -1 and 1097 cm -1 correspond to asymmetric axial stretching and deformation mode of the carbonate anion [ʋ(CO 3 2-)].The band at 600 cm -1 is related to the metal presence.Note, for (C), (D), and (E), the corresponding fluoride products showed a higher band frequency relative to CO 3 2-(1385 cm -1 ).

Discussion
This is a novel study conducted with fluorideloaded LDHs for cont rol led release, usi ng hydrothermal-treated LDH nanoparticulates under high pressure and temperature.LDHs are capable of anionic exchange; thus, they have attracted interest as promising materials for several applications.LDH is biocompatible and has been studied for numerous biological purposes, including controlled release systems.In dentistry, calcium release in hydroxyapatite or zinc ion formation is beneficial.Additionally, small crystals may be used to control dental hypersensitivity, occluding the dentinal tubules.LDH can adsorb and release fluoride that controls the carious process, which was the subject of this study. 15he EDX analysis provided the material elemental composition without revealing specific organic composition results of the respective elements.The EDX results suggest a partial anion exchange of Cl -by F -. A total anion exchange of Cl -by F -for F-LDH-9 to -12 was observed and confirmed by potentiometry analysis.
Figures 1(A) and 1(B) display diffractograms similar to that of the crystallographic pattern of clay seen in previous literature. 23The basal spacing d ( 003) value for all LDH precursors was 7.9 Å, which is similar to the values reported in studies for chloride presence. 22,24All F-LDH products presented the same reduction behavior of the basal spacing relative to the precursor (d (003) = 7.9 Å to 7.8/7.6Å), regardless of synthesis procedures, corroborating with the literature. 9,10,13,18,25An increase in the F-LDH crystallinity was observed as compared to that of C-LDH, which indicates the ordered accommodation of F -within the interlamellar region. 18F-LDH-5 (Figure 1(D)) synthesized from colloidal suspension showed greater crystallinity as compared to that of N-LDH (colloidal suspension and powder).This suggested freeze-drying process interference with the property, as previously reported. 26he FTIR analysis revealed bands centered at 3445 cm -1 and 1638 cm -1 , which are characterized by an asymmetric axial (ʋOH) and angular (δH 2 O) hydroxyl group deformation corresponding to the adsorbed or interlamellar water molecules.The 2056 cm -1 band corresponds to the CO 2 medium. 24,27Additionally, there were weak bands at 1355 cm -1 and 1097 cm -1 , which is attributed to the asymmetric axial stretching or deformation mode of the carbonate anion [ʋ(CO 3

2-
)], formed due to atmospheric CO 2 absorption during washing. 28,29At 600 cm -1 , the bands related to metal, Fluoride Release (%) Mg and Al, presence were observed bound to hydroxyl groups. 2,3,30When fluoride ion intercalation occurs, the band relative to the CO 3 2-(1355 cm -1 ) shifts to a higher frequency, comprising values centered on 1385 cm -1 (Figure 2(A)-(E)). 3,18n general, TGA revealed an initial mass loss between 25 and 200°C, corresponding to the phases: volatilization of adsorbed water (up to 100°C) and interlamellar water (up to 200°C).The third and fourth decomposition stages occurred at approximately 200-430°C and 450-800°C, respectively.This was related to structural dehydroxylation and CO 2 loss, with the consequent loss of the lamellar structure. 23,24n the last two steps, fluoride presence is clear as higher temperatures are necessary for material degradation.This is explained by the formation of strong hydrogen bonds, which increases the interaction between molecules and restricts structural loss, showing that the fluoridated samples are more stable (Figures 3(A)-(E)). 3,25,27According to SEM micrographs, the particles have irregular and rough surfaces, as described in previous literature (Figures 4(A)-(E)). 3,4or the F --release kinetic profiles of the fluoridated samples (Figures 5(A)-(B)), the analysis revealed the potential use of this material in dentistry.Slow and fast fluoride releases from synthesized materials were observed, thus proving the applicability of the material.According to the literature, the main anti-caries effect occurs when the material is present in the oral medium in its free (ionic) soluble form.Its high continuous concentration in the buccal environment is an excellent advantage for demineralization and dental remineralization. 31,32ost products exhibited a high amount of fluoride release in acidic medium, except for two: F-LDH-9 with a 6.0% release value (64 µg/mL F) in both media and F-LDH-11 with 45.7% (56.32 µg / µg/mL F) and 46.6% (57.40 µg / µg/mL F) release values in neutral and acidic media, respectively.These products, however, did not differ in pH change.Despite their crystalline structure, these characteristics corroborate with their diffractograms.They are less intense when compared to products from the precursor in suspension and configure a lower fluoride accommodation in the interlamellar space. 19his suggests greater anion availability by setting similar release values.Moreover, such products were obtained at room temperature and, as seen in the thermal analysis, fluoridated products need higher temperatures for degradation. 25,33These products were more unstable, less crystalline, and independent of the pH value, at room temperature.
The highest R 2 was obtained from the Korsmeyer-Peppas model (Tables and 3 and 4).In both media, the priority kinetic mechanism of fluoride products 1-4, originating from the same precursor and synthesis conditions, comprised non-Fick behavior.However, F-LDH-2 and -4 presented a release of Case II and Fick in neutral and acid media, respectively.Products -5 to -8 all followed Super Case II, excluding a neutral medium F-LDH-8, which presented Fickian behavior.
In the acid medium, two products, F-LDH-7 and -8, switched the release kinetics to non-Fick.Among products -9 to -12, only F-LDH-12 in the neutral medium showed non-Fick behavior.All other products followed the Fick behavior in the two media.
Regarding the fluoride release kinetic mechanism, Fick-like formulations characterization of slow fluoride diffusion through the F-LDH outer layer was observed. 34The non-Fick mechanism comprises F-LDH swelling and relaxation, and fluoride diffusion.For Case II or Super Case II, fluoride release is related to matrix swelling, diffusion, and erosion.A more marked release occurs in these phases due to matrix internal chain breakage, resulting in an easier F -release. 35ased on the results, the medium type and synthesis conditions affected the F-release kinetics.Although the F-LDH-11 and F-LDH-7 products presented a higher F -release %, F-LDH-8, synthesized at 40°C for 24 h, showed better fluoride release performance in the acid medium according to Fick's kinetic adjustment, in neutral medium to non-Fick in acid medium.Dental demineralization and remineralization processes are characterized by the critical pH of the medium.Although the expensive process develops in acidic pH, remineralization is activated when the pH is higher than 4.5. 36To obtain a product with dental application potential, F-LDH, selective for the acid medium, is suggested as dental caries development occurs in an acidic medium (critical pH = 5.5).
Thus, focusing on the clinical application of fluoridated LDHs, these formulations have good prospects as specific agents for patients at risk of developing caries.They can be used as a powdered product isolated or embedded in dental materials, such as dental varnishes, orthodontic cement, composite resins, toothpaste, and mouthwash. 15As caries is a biofilm and sugar-dependent disease affecting people of varying socioeconomic levels, easy-to-obtain and low-cost fluoride products are promising for good prophylactic therapy. 37urther research is needed to assess the behavior of these new materials in artificial saliva (in vitro experiments), which is a limitation of this study.Future research should address the material behavior in oral environments by applying in situ and in vivo models.This in vitro study shows the

Conclusions
F-LDHs were successfully synthesized and characterized in this study, thus providing a new approach to nanometric materials with potential application in dentistry for the prevention and control of dental caries.The F-LDH-8 product synthesized at 40°C for 24 h demonstrated the best fluoride release performance in the acidic medium.F-LDHs are a promising material for the prevention and control of dental caries, owing to the continuous fluoride ion release in the oral environment.
LD1500, São Paulo, Brazil) following under 1 mm/h with a 250 mm Hg vacuum to obtain the powder.The colloidal suspensions and powders were reserved for characterization and the materials were used for fluoridated product synthesis.

Table 2 .
Constitution of elements present in the studied samples.
Mg/Al: molar ratio of magnesium and aluminum; Mg, Al, and Cl were obtained using EDX; the F -ion was determined by HMDS.

Table 3 .
Estimated kinetic parameters of drug release (Neutral medium).

Table 4 .
Estimated kinetic parameters of drug release (Acid medium).