CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Patent Application Ser. No. 63170520, filed on April 4, 2021, entitled “NANOZYME BASED- TOOTH BLEACHING SYSTEM,” which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure generally relates to a teeth whitening kit, methods of preparation and methods of using the same to whiten one or more teeth, and more particularly, to a teeth whitening kit with a minimum tooth sensitivity comprising two compositions and methods of using the same which may be used in whitening procedure.

BACKGROUND

[0003] Tooth whitening has become very popular due to the increasing interests for having a beautiful smile and, in turn, experiencing a better social life. Most tooth whitening formulations contain peroxide as one of the major ingredients. To achieve a proper whitening effect, a predetermined amount of peroxide must be prepared and while being protected from decomposition before and during the procedure of teeth whitening. Peroxidase is a group of oxidoreductase enzyme or enzymatic peroxidase which can induce peroxide degradation into free radicals in whitening products. These free radicals have a whitening effect on teeth. In the process of teeth whitening, after applying peroxide on teeth, the peroxidase may be applied on teeth to activate peroxide to produce free radicals. Using the enzymatic peroxidase in the teeth whitening formulation may have some limitations such as unstable states of this enzyme in acidic conditions of hydrogen peroxide. With this context, there is need to develop a new formulation of the whitening kit based on a new generation of the catalyze with peroxidase properties such as nanozyme. SUMMARY

[0004] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0005] In one general aspect, the present disclosure relates to an exemplary teeth whitening kit which may comprise first composition comprising a peroxide and a second composition comprising cerium oxide nanoparticles.

[0006] In one or more exemplary embodiments, the peroxide in the first composition of the teeth whitening kit may be hydrogen peroxide.

[0007] In one or more exemplary embodiments, the first composition of the teeth whitening kit may further comprise at least one of curcumin, pearl powder, coconut oil and a combination thereof.

[0008] In one or more exemplary embodiments, the teeth whitening kit may further comprise a third composition comprising a calcium phosphate-based compound.

[0009] In one or more exemplary embodiments, the calcium phosphate-based compound in the third composition may be hydroxyapatite nanoparticles.

[00010] In one or more exemplary embodiments, the third composition of the teeth whitening kit may further comprise a fluoride- containing compound.

[00011] In one or more exemplary embodiments, the fluoride-containing compound comprises a fluoride salt.

[00012] In one or more exemplary embodiments, the fluoride salt may be sodium fluoride. [00013] This Summary may introduce a number of concepts in a simplified format; the concepts are further disclosed within the “Detailed Description” section. This Summary is not intended to configure essential/key features of the claimed subject matter, nor is intended to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[00014] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which a presently preferred embodiment of the present disclosure will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the present disclosure. Embodiments of the present disclosure will now be described by way of example in association with the accompanying drawings in which:

[00015] FIG. 1 illustrates a method of teeth whitening with the exemplary whitening kit, consistent with exemplary embodiments of the present disclosure;

[00016] FIG. 2 illustrates Field Emission Scanning Electron Microscopes (FE-SEM) images of hydroxyapatite nanoparticles (201) and the cerium oxide nanoparticles (202), consistent with exemplary embodiments of the present disclosure;

[00017] FIG. 3 illustrates Fourier-transform infrared spectroscopy (FT-IR) of the hydroxyapatite nanoparticles which was recognized through FT-IR analysis, consistent with exemplary embodiments of the present disclosure;

[00018] FIG. 4 illustrates FT-IR spectra of the cerium oxide nanoparticles which was recognized through FTIR analysis, consistent with exemplary embodiments of the present disclosure; [00019] FIG. 5A illustrates the viability and the treated Mouse Fetal Fibroblast (MFF) cell line with hydroxyapatite nanoparticles after 24 and 48 hours consistent with exemplary embodiments of the present disclosure;

[00020] FIG. 5B illustrates the viability and the treated MFF cell line with cerium oxide nanoparticles after 24 and 48 hours consistent with exemplary embodiments of the present disclosure.

DETAILED DESCRIPTION

[00021] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to the exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[00022] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. V arious modifications to the exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein. [00023] It must be noted that, the singular forms “a,” “an,” and “the,” as used in the present disclosure, may include plural referents unless the context clearly dictates otherwise.

[00024] As used herein, the terms “comprising,” “including,” “constituting,” “containing,” “consisting of,” and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unmentioned method/process steps or elements.

[00025] Reference herein to “one embodiment,” “an embodiment,” “some embodiments,” “one or more embodiments,” “one exemplary embodiment,” “an exemplary embodiment,” “some exemplary embodiments,” and “one or more exemplary embodiments” indicate that a particular feature, structure or characteristic described in connection or association with the embodiment may be included in at least one of such embodiments. However, the appearance of such phrases in various places in the present disclosure do not necessarily refer to a same embodiment or embodiments.

[00026] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments. Likewise, the term “embodiments of the invention” does not require that all embodiments of the invention include the discussed feature, advantage or mode of operation.

[00027] The term “about,” “substantially,” and “approximately” as used herein, may indicate that a value(s) may include an inherent variation of error for a method being employed, a device, or a variation that may exist among the subjects/factors of a study.

[00028] Provided here is an exemplary teeth whitening kit, methods of preparation, and routs of application. One or more exemplary embodiments of the present disclosure also directs to a formulation which may be prepared and used in the exemplary teeth whitening kit. The exemplary teeth whitening kit disclosed here, may be capable of whitening tooth in home or dental office. In one or more exemplary embodiments, the exemplary teeth whitening kit disclosed here may comprise two compositions; a peroxide and a nanozyme.

[00029] Nanozymes or nanostructured artificial enzymes are a group of nano particulate agents which are able to mimic the catalytic activations of enzyme. Cerium oxide is a nanozyme and oxide of metal used as an oxidant and catalyst in organic synthesis. Nanoparticles of “Cerium oxide”, “nano ceria”, “cerium dioxide”, “dioxo cerium”, “Ceria” and equivalents thereof are a nanozyme which have a peroxidase mimetic activity and attributed to their ability for reversibly switch from Ce3 ⁺ to Ce4 ⁺ and high oxygen mobility at its surface.

[00030] As stated above, the exemplary teeth whitening kit of the present disclosure may be more effective on teeth whitening procedure due to using nanozyme instead of peroxidases. Moreover, the exemplary teeth whitening kit may include less amount of peroxide contrast to commercial whitening gel in the market. Therefore, side effects of the exemplary teeth whitening kit may be decreased rather than commercial whitening gel in the market.

[00031] In one or more exemplary embodiments, the exemplary teeth whitening kit of the present disclosure may comprise at least two compositions, which may be stored separately. [00032] In one or more exemplary embodiments, the first composition in the exemplary teeth whitening kit of the present disclosure may use to whiten teeth. This composition may comprise a peroxide.

[00033] In one or more exemplary embodiments, the peroxide may be selected, from hydrogen peroxide, urea peroxide, carbamide peroxide, or a combination thereof. The peroxide may have a concentration between 10 weight percent (wt%) to 35 wt% of the total weight of the first composition.

[00034] In one or more exemplary embodiments, the peroxide may be hydrogen peroxide and have a concentration between 10 wt% to 35 wt% of the total weight of the first composition. In one exemplary embodiment, hydrogen peroxide may have a concentration of 15 wt% of the total weight of the first composition.

[00035] In one or more exemplary embodiments, the first composition in the exemplary teeth whitening kit of the present disclosure may use to whiten teeth. This composition may further comprise at least one of curcumin, coconut oil, and pearl powder.

[00036] In one or more exemplary embodiments, the curcumin in the first composition of the exemplary teeth whitening kit may have a concentration of 1 wt% of the total weight of the first composition.

[00037] In one or more exemplary embodiments, the pearl powder in the first composition of the exemplary teeth whitening kit may have a concentration of 2 wt% of the total weight of the first composition.

[00038] In one or more exemplary embodiments, the coconut oil in the first composition of the exemplary teeth whitening kit may have a concentration of 2 wt% of the total weight of the first composition.

[00039] In one or exemplary embodiments, the first composition of the exemplary teeth whitening kit may further comprise vitamin E, ascorbic acid, uric acid, a carotenoid, vitamin A, kojic acid, a flavonoid, a phenolic compound, a polyphenol, a coenzyme, an herbal antioxidant, melatonin, an amino indole, a lipoic acid, rosemary extract, tocopherol, tocotriene, a bioflavonoid, a plant extract, tetrahydrocurcumin, camphorol, quercetine, epigenine, and a combination thereof.

[00040] In one or exemplary embodiments, the first composition of the exemplary teeth whitening kit may further comprise marble powder, silicate compounds (such as silica), and a combination thereof.

[00041] In one or more exemplary embodiments, the first composition of the exemplary teeth whitening kit may further comprise a pH adjuster. In one or more exemplary embodiments, the pH adjuster in the first composition may be selected from triethanolamine, sodium hydroxide, sodium perborate, sodium acetate, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, calcium bicarbonate, sodium carbonate and calcium carbonate, TRIS, any other salt of an alkaline base which is safely used in the mouth, and a combination thereof.

[00042] In one or more exemplary embodiments, the pH adjuster in the first composition of the exemplary teeth whitening kit may be triethanolamine and have a concentration of 1 wt% of the total weight of the first composition.

[00043] In one or more exemplary embodiments, the second composition in the exemplary teeth whitening kit of the present disclosure may be used to activate peroxide. The second composition may comprise a nanozyme. In one exemplary embodiment, the nanozyme may be cerium oxide nanoparticles.

[00044] In one or more exemplary embodiments, the cerium oxide nanoparticles in the second composition of the exemplary teeth whitening kit may have a concentration between 0.02 wt% to 0.2 wt% of the total weight of the second composition. In one exemplary embodiment, cerium oxide nanoparticles in the second composition of the exemplary teeth whitening kit may have a concentration of 0.2 wt% of the total weight of the second composition.

[00045] In one or more exemplary embodiments, the exemplary teeth whitening kit of the present disclosure may further comprise a third composition which may comprise a calcium phosphate-based compound and stored separately from other compositions.

[00046] In one or more exemplary embodiments, the calcium phosphate -based compound may be selected from nanoparticles of hydroxyapatite Caio(P0 ₄ ) ₆ (OH) ₂ , fluoroapatite Caio(P0 ₄ ) ₆ F ₂ , chloroapatite Caio(P0 ₄ ) ₆ Ch, tricalcium phosphate Caio(P0 ₄ ) ₂ , and a combination thereof. [00047] In one or more exemplary embodiment, the third composition of the exemplary teeth whitening kit in the present disclosure may comprise hydroxyapatite nanoparticles. [00048] In one or more exemplary embodiment, hydroxyapatite nanoparticles in the third composition of the exemplary teeth whitening kit in the present disclosure may have a concentration between 0.01 wt% to 0.3 wt% of the total weight of the third composition. [00049] In one or more exemplary embodiments, the hydroxyapatite nanoparticles in the third composition of the exemplary teeth whitening kit in the present disclosure may have a concentration of 0.2 wt% of the total weight of the third composition.

[00050] In one or more exemplary embodiments, the third composition of the exemplary teeth whitening kit in the present disclosure may further comprise a fluoride-containing compound.

[00051] In one or more exemplary embodiments, the fluoride containing compound may be selected from sodium monofluorophosphate, sodium fluoride, strontium chloride and stannous fluoride, and a combination thereof.

[00052] In one or more exemplary embodiment, the fluoride-containing compound may have a concentration between 0.5 wt% to 10 wt% of the total weight of the third composition. [00053] In one or more exemplary embodiment, the fluoride-containing compound may be sodium fluoride and have a concentration between 0.1 wt% to 1 wt% of the total weight of the third composition. In one exemplary embodiment, the sodium fluoride in the third composition may have a concentration of 0.2 wt% of the total weight of the third composition. [00054] In one or more exemplary embodiments, the third composition in the exemplary teeth whitening kit of the present disclosure may reduce tooth sensitivity and protect enamel from negative effects of the peroxide.

[00055] In one or more exemplary embodiments, the first, second and third composition of the exemplary teeth whitening kit in the present disclosure may further comprise a gelling agent. In one or more exemplary embodiments, the gelling agent may be selected from tragacanth gum, starches, cellulosic gums, carbomers, poloxamer (Pluronic), gelatin, hydroxypropyl methylcellulose, carboxy methylcellulose, chitosan, alginate, and a combination thereof.

[00056] In one or more exemplary embodiments, the gelling agent may be tragacanth gum and have a concentration of 2 wt% of the total weight of the first, second or third composition.

[00057] In one or more exemplary embodiments, the first, second and third composition of the exemplary teeth whitening kit in the present disclosure may further comprise a sweetening agent. In one or more exemplary embodiments, the sweetening agent may be selected from sodium saccharin, aspartame, sucralose, mannitol, sorbitol, xylitol, dextrose, glucose, fructose, brown sugar, and a combination thereof.

[00058] In one or more exemplary embodiments, the sweetening agent may be sodium saccharin and have a concentration of 0.05 wt% of the total weight of the first, second or third composition.

[00059] In one or more exemplary embodiments, the first, second and third composition of the exemplary teeth whitening kit in the present disclosure may further comprise a cooling agent. In one or more exemplary embodiments, the cooling agent may be selected from menthol, thymol, eucalyptol, peppermint oil, and a combination thereof.

[00060] In one or more exemplary embodiments, the cooling agent may be menthol and have a concentration of lwt% of the total weight of the first, second or third composition. [00061] In one or more exemplary embodiments, the first, second and third composition of the exemplary teeth whitening kit in the present disclosure may further comprise a stabilizing agent. In one or more exemplary embodiments, the stabilizing agent may be selected from ethylenediaminetetraacetic acid, tin phosphates/phosphonates, phosphoric acid, tin sulphonates, sodium sulfite, metal chelants such as EDTA di sodium, amino phosponates chelants, stabilizers meant specifically for H202 (such as tin salts, phosphoric acid), and a combination thereof.

[00062] In one or more exemplary embodiments, the stabilizing agent may be ethylenediaminetetraacetic and have a concentration of 0.5 wt% of the total weight of first, second or third composition.

[00063] In one or more exemplary embodiments, the first, second and third composition may be provided in separate containers, for example, with each container containing the first composition, the second composition, and the third composition. For example, the first composition may be provided in a first container, the second composition may be provided in a second container, and the third composition may be provided in a third container. Before application, the first and the second composition may be mixed together and then applied on tooth by an applicator, then whitening procedure may be performed. Finally, after whitening procedure, the third composition may be applied on tooth.

[00064] In one or more exemplary embodiments, the first composition and the second composition, may be provided in a single container separated by a removable or reputable separator. Just prior to use the separator may be removed or ruptured to allow for mixing of the two compositions together to form the tooth whitening composition (comprising the first and the second composition) and whitening procedure may be performed. Finally, after whitening procedure, the third composition may be applied on tooth.

[00065] In one or more exemplary embodiments, the tooth whitening composition may be remained on tooth for 1 minute to 20 minutes and the third composition may be remained on tooth for 1 minute to 20 minutes.

[00066] In one or more exemplary embodiments, pH of the teeth whitening composition (comprising the first and second composition) after combination may be 8 to 9. [00067] The exemplary teeth whitening kit disclosed here, may advantageously prepare a stable peroxide-based composition which may be stored for extended periods of time. The first composition of the exemplary teeth whitening kit may be active immediately before use. Furthermore, the exemplary teeth whitening kit may allow whitening products to be prepared with different peroxide contents without need for complete reformulation of the entire whitening composition. For example, consumers may need different level of whitening, for example, for reduced levels of the peroxide due to various sensitivities to peroxide. Different whitening products, having different levels of whitening effects, may simply be prepared using the exemplary whitening kit by choosing the concentration of the peroxide in the first composition, and without need to reformulate the second composition. Therefore, the exemplary teeth whitening kit may also provide an economical means for preparing a whitening product line having a variety of whitening levels.

[00068] In one or more exemplary embodiments, FIG. 1 illustrated a method of teeth whitening with the exemplary teeth whitening kit, consistent with exemplary embodiments of the present disclosure. As shown in FIG. 1, the exemplary method of teeth whitening with the exemplary teeth whitening kit 100 may comprise 5 steps; preparing the whitening composition comprising the first and the second compositions 101, applying the whitening composition on teeth 102, eradiating laser light on teeth 103, removing the whitening composition from teeth 104, applying the third composition on teeth 105, and removing the third composition from teeth 106. At first 101 the first composition and the second composition may be combined to achieve the whitening composition. This step 101 may be performed immediately before whitening procedure. Then 102 the whitening composition may be applied on teeth and then allowing the tooth whitening composition to remain on teeth for 1 minute to 20 minutes. In dental office, one more step 103 may be performed to achieve the maximum efficacy of the whitening composition. At this step 103 laser light may be radiated on teeth covered by the whitening composition. Then 104 the whitening composition may be removed by washing. Finally, 105 the third composition may be applied on teeth and washed after 1 minute to 20 minutes 106.

EXAMPLES

[00069] Hereinafter, the present disclosure will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples may be for illustrative purposes only and are not to be interpreted to limit the scope of the present disclosure.

Example 1: Preparation of the exemplary teeth whitening kit

[00070] In this example, the preparation procedure of the first, second and third composition of the exemplary teeth whitening kit were described.

In one or more exemplary embodiments, in order to prepare the first composition, two solutions (A and B) were separately prepared. In order to prepare the solution A, the hydrogen peroxide, the curcumin, gum tragacanth and triethanolamine were combined. In order to prepare the solution B, the pearl powder was added to the coconut oil. Finally, the two solutions were combined in a room temperature to achieve a homogeneous mixture or gel. pH was adjusted in a range of 5 to 6 by a pH adjuster such as triethanolamine. Table 1 bellow shows the components of the first composition and their concentrations, consistent with exemplary embodiments of the present disclosure.

Table 1:

Components of the first composition and their concentrations, consistent with exemplary embodiments of the present disclosure.

[00071] In order to prepare the second composition of the exemplary teeth whitening kit of present disclosure, cerium oxide and gelling agent were used. Diluted ammonia (in concentration of about 1 molar) was added dropwise into cerium nitrate hexahydrate (in concentration of about 0.1 molar) until pH of the mixture reached to 10. Said mixture was mixed in room temperature on the Magnetic stirrer. After filtration, the produced precipitates were washed twice with distilled water and dried for 2 hours in an oven (110 °C). Finally, the resulted powder of the cerium oxide heat treated about 700 °C for 2 hours in a conventional furnace and the cerium oxide was achieved. Then, cerium oxide with diameters of approximately 4.5 nanometers (nm), gum tragacanth, and double distilled water were mixed together by stirrer in a room temperature in order to achieve a homogeneous mixture or gel. Table 2 bellow shows the components of the second composition and their concentrations, consistent with exemplary embodiments of the present disclosure. Table 2:

Components of the second composition and their concentrations, consistent with exemplary embodiments of the present disclosure.

[00072] Synthesis of the hydroxyapatite nanoparticles was conducted through wet perception approach with a calcium/phosphate molar ration of 1.67. Ortho phosphoric acid solution (100 ml, in concentration of about 0.6 molar) was added dropwise into calcium hydroxide solution (100 ml, in concentration of about 2 molar) to create a milky solution under vigorous stirring at ambient condition within 24 hours. Free ions were removed through washing of the collected precipitation with distilled water. Then the obtained cake, first was dried under 100 °C in an oven, overnight. The produced agglomerate of the hydroxyapatite was milled by ball mill. Then, 20ml of polyvinyl alcohol (in amount of about 1.5 wt/v %) was added to the above produced powder and a plastic mixture was achieved. Ammonium bicarbonate (20 ml in amount of about 1.5 wt/v %) was added to the produced mixture and the generated hydroxyapatite was sintered for 2 hours in the conventional furnace, at 1000 °C. Gum tragacanth, sodium fluoride, and double distilled water were added to the produced nano hydroxyapatite with diameters of approximately 29 nm. The final solution was mixed in a room temperature by stirrer in order to achieve a homogeneous mixture or gel at 2000 rounds per minute (rpm). Table 3 bellow shows the components of the third composition and their concentrations, consistent with exemplary embodiments of the present disclosure.

Table 3:

Components of the third composition and their concentrations, consistent with exemplary embodiments of the present disclosure.

[00073] Surface morphology evaluation of the synthesized nanoparticles was executed through a scanning electron microscopy (SEM), at 15 kV. Prior to examination, the nanoparticles were mounted on aluminum stubs and then coated with a thin layer of gold by a sputter apparatus. FIG.2 illustrates FE-SEM images of hydroxyapatite nanoparticles (201) and the cerium oxide nanoparticles (202), consistent with exemplary embodiments of the present disclosure. Morphology of the hydroxyapatite nanoparticles 201 and the cerium oxide nanoparticles 201 were presented in FIG. 2. As shown in FIG. 2, SEM image of the hydroxyapatite nanoparticles depicted the agglomerates of the flaky crystals and SEM image confirmed a nanoscale sized globular structure for the cerium oxide. The recognition of functional groups in the cerium oxide and the hydroxyapatite nanoparticles were conducted using an FT-IR spectrophotometer. The recorded spectra with resolution of 2 cm ¹ were in a range of 4000 to 400 cm. FIG. 3 illustrates Fourier-transform infrared spectroscopy (FT-IR) of the hydroxyapatite nanoparticles which was recognized through FT-IR analysis, consistent with exemplary embodiments of the present disclosure; and FIG. 4 illustrates FT-IR spectra of the cerium oxide nanoparticles which was recognized through FTIR analysis, consistent with exemplary embodiments of the present disclosure. The functional groups in the hydroxyapatite and the cerium oxide nanoparticles were recognized through FTIR analysis, as observed in FIG. 3 and FIG. 4, respectively. Characteristic absorption peaks were recorded for the hydroxyapatite at around 1043.61 cm ¹ (phosphate groups), 3642.97 cm ¹ (hydroxyl groups) and 1474.15 cm ¹ (carbonyl groups). In the cerium oxide spectrum, absorbance bands at 3410 and 500 cm ¹ observed, which the former clarified the presence of O-H group and the latter Ce- O group.

Example 6: Cytotoxicity assay

[00074] In this example, Mouse Fetal Fibroblast (MFF) was used to evaluate in vitro toxicity of hydroxyapatite nanoparticles and cerium oxide nanoparticles by using 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Studied cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose medium supplemented with 10% of fetal bovine serum (FBS) and 1% of penicillin- streptomycin solution in incubator with a humidified atmosphere of 5% CO2 and 37°C for 24 hours. Cells were seeded on 96-well culture plates at a density of 5 x 104 cells/well. After 24 hours and 48 hours of incubation, culture media from the wells were replaced with fresh aliquots of complete media containing the cerium oxide nanoparticles in the desired concentration range (0, 20, 40, 80, 160 and 200 pg/ml) of cerium oxide nanoparticles and a blank (empty wells, no cells). After 24 and 48 hours of exposure, media containing cerium oxide nanoparticles were removed, and the cells were washed two times in phosphate buffered saline (PBS) to remove freely suspended nanoparticles and detached dead cells. Fresh culture medium with 10 pi of MTT (5 mg/ml) was then added into each well and culture plates were incubated for 4 hours at 37 °C. Subsequently, culture solution was removed and 100 ml of Dimethyl sulfoxide solution (DMSO) was added to each well and plate kept at room temperature in dark for 1 to 2 hours until intracellular purple formazan crystals became visible under microscope. Fluorescent intensity was measured by using a micro-plate reader with excitation/emission wavelength of 570 nanometer. FIG. 5A illustrates the viability and the treated MFF cell line with hydroxyapatite nanoparticles after 24 and 48 hours, consistent with exemplary embodiments of the present disclosure; and FIG. 5B illustrates the viability and the treated MFF cell line with cerium oxide nanoparticles after 24 and 48 hours, consistent with exemplary embodiments of the present disclosure.

[00075] All readings were taken in triplicate. FIG. 5-A and FIG. 5-B depicts the viability of MFF cell line upon treatment with various concentrations of hydroxyapatite nanoparticles and the cerium oxide nanoparticles, respectively. Hydroxyapatite nanoparticles had a significant viability for the incubation times of 24 and 48 hours at lower concentrations (10- 100 pg g/ml). Although, with increasing the hydroxyapatite concentration (> 100 pg/ml), their cell viability was decreased that confirmed a dose-dependent cytotoxicity.

Example 7: Evaluation efficacy of the exemplary teeth whitening composition on color changes of teeth

[00076] In this example, 18 teeth samples were randomly divided into two groups of 9 teeth, which teeth of each group were separately stored into staining solutions (coffee and tea solution) for 72 hours. Then, prepared teeth were immersed into filtered solutions over 3 days- period at room temperature; coffee and tea solutions were renewed daily. After embedding period, teeth were washed and placed into distilled water. To evaluate staining, a standard Vita shade guide was used, which the shade tabs were arranged in a numeric value based on manufacturer suggestion (Bl, Al, B2, D2, A2, Cl, C2, D4, A3, D3, B3, A3.5, B4, C3, A4, C4). After artificial staining of teeth, only teeth with shade C4 were selected for whitening process (14 teeth). Selected teeth were brushed and washed with distilled water before applying of the whitening composition to avoid false-positive results. In the following, the dried teeth mounted separately in plaster, so that the root of each tooth was placed in a plaster cast and the crown of the tooth was visible at the plaster outside. Besides, pumice powder accompanied with brush was applied onto the teeth surface and area where around the teeth and on the plaster were covered by a special gingival protector. The crown thickness, for each tooth, was vertically sectioned in half; after cutting of each tooth into two equal halves of A and B, teeth were mounted in acrylic. Then, the teeth whitening composition comprising first and second composition of the exemplary whitening kit in this disclosure were separately applied to only one half of each tooth.

Two commercial whitening gel (Boost™ and FGM™) were used to evaluate whitening efficacy of the teeth whitening kit in this disclosure. The first group of seven teeth was divided randomly into two subgroups. In the first subgroup Boost™ and the exemplary teeth whitening composition comprising first and second composition in this disclosure were applied on 4 teeth (teeth with label of T01,T02, T03, T04) and in second subgroup, FGM™ and the exemplary whitening composition comprising first and second composition in this disclosure were applied on three teeth (teeth with label of T05, T06, and T07). The second group of seven teeth was divided randomly into two subgroups. In the first subgroup, Boost™ and the exemplary whitening composition comprising first and second composition in this disclosure were applied on 4 teeth (teeth with label of C01, C02, C03) and in second subgroup, FGM™ and the exemplary whitening composition comprising first and second composition in this disclosure were applied on three teeth (teeth with liable of C04, C05, C06, and C07). Then the erbium laser light was radiated twice on each tooth for twenty minutes. In this stage, color on each tooth was evaluated and color selection was performed based on Vita 3D Shade guide. After whitening process with the exemplary whitening composition comprising the first and second composition in this disclosure, this value altered to lighter type in Vita shade tabs. Color changes of the teeth whitened with selected commercial (BOOST™ and FGM™) and the exemplary whitening composition comprising the first and second composition in this disclosure were presented in Table 4.

Table 4:

Color changes of the teeth whitened with selected commercial (BOOST™ and FGM™) and the exemplary whitening composition comprising the first and second composition, consistent with exemplary embodiments of the present disclosure.

[00077] Out of 8 specimens, the whitening efficacy of the exemplary whitening composition comprising the first and second composition in this disclosure were similar to BOOST™ in 5 specimens; in 1 specimen was better than BOOST™ whilst in the 2 specimens showed weaker whitening effects compared to BOOST™. Besides, the whitening effect of the exemplary whitening composition comprising the first and second composition in this disclosure was better than FGM™ in all treated specimens.

Example 8: Evaluation efficacy of the exemplary whitening kit on enamel micro hardness [00078] Twelve healthy human teeth, were sectioned in a mesiodistal direction in equal halves (halves of A and B), embedded in chemically activated acrylic resin and surfaces were brushed and polished with soft discs. Four groups were prepared as per treatment modality (n = 3): group 1, whitened with the exemplary teeth whitening composition comprising the first and the second composition in this disclosure; group 2, whitened with the exemplary whitening kit comprising the first and the second composition in this disclosure and accompanied with the third composition; group 3, whitened with BOOST™ (comprising 35% hydrogen peroxide); group 4, whitened with FGM™ (comprising 35% hydrogen peroxide). After applying whitening composition on tooth, erbium laser was radiated on tooth. Finally whitening composition were washed from the teeth after twenty minutes and the samples were placed in distilled water to evaluate the surface hardness of the enamel. In the second group, after washing the whitening composition, the third composition was applied on tooth for 5 minutes. Fourteen days post- whitening, a micro hardness tester was employed to determine Knoop microhardness (KHN; kg/mm2), at a load of 200 g with the indentation time of 15 seconds. Vickers assay was employed to evaluate the enamel micro hardness of the treated teeth with the commercial and the exemplary whitening kit comprising the first and second composition in this disclosure. Table 5 below shows the effect of commercial gel and the exemplary teeth whitening kit or composition on the enamel micro hardness; consistent with exemplary embodiments of the present disclosure; and Table 6 below shows the Vickers result (enamel micro hardness); consistent with exemplary embodiments of the present disclosure. In agreement with the calculated data shown in Table 6, among of the selected whitening compositions, the exemplary teeth whitening kit including the first, second and third composition in this disclosure appeared significantly increase in the enamel micro hardness whereas BOOST™ displayed decrease in the micro hardness.

Table 5:

The effect of commercial gel and the exemplary whitening kit or composition on the enamel micro hardness; consistent with exemplary embodiments of the present disclosure. (A: tooth whitened with the exemplary whitening composition of the present disclosure comprising the first and second composition B : tooth whitened with the exemplary whitening kit of the present disclosure comprising the first, second and third composition. C: tooth whitened with BOOST™ whitening gel D: tooth whitened with FGM™ whitening gel)

Table 6 :

Vickers result (enamel micro hardness); consistent with exemplary embodiments of the present disclosure. (A: tooth whitened with the exemplary whitening composition of the present disclosure comprising the first and second composition B: tooth whitened with the exemplary whitening kit of the present disclosure comprising the first, second and third composition. C: tooth whitened with BOOST™ whitening gel D: tooth whitened with FGM™ whitening gel)

Example 9: Comparing whitening effect between nanozymes and enzymatic peroxidases

In this example, the efficacy of nanozymes and enzymatic peroxidases in the whitening process was evaluated. In this example, seventeen teeth samples were randomly divided into two groups of seven teeth, which teeth of first group (C20 - C26) were separately stored into coffee solution (12 g coffee in 200 mL boiling water) and teeth of second group (T20 - T26) were separately stored into tea solution (2 g tea in 100 mL boiling water) for one week. Then, the prepared teeth were immersed into filtered solutions over 3 days-period at room temperature; coffee and tea solutions were renewed daily. After the embedding period, the teeth were washed and placed into distilled water for two days.

[00079] At this stage, one tooth from each group was excluded from the study due to deep enamel cracks (T23, C23). In the following, the dried teeth mounted separately in plaster, so that the root of each tooth was placed in a plaster cast and the crown of the tooth was visible at the plaster outside. Besides, pumice powder accompanied with brush was applied onto the teeth surface. Then crown of the teeth was separated from the plaster and said crown was divided into two parts by the Gingival Protector. Teeth with appropriate color were selected and the whitening process was conducted with two prepared whitening gel Ml wherein peroxide activated by cerium oxide and M2 wherein peroxide was activated by hydrogen peroxidase. After 20 minutes, whitening gels were washed out from teeth and said process was performed again. Finally, the effect of whitening gel was compared between two groups of teeth and the whitening effect of Ml was more than M2.

[00080] While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings. [00081] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[00082] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed. [00083] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[00084] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element. [00085] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[00086] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study, except where specific meanings have otherwise been set forth herein. Relational terms such as “first” and “second” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[00087] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it may be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.