Oral delivery of hydroxyapetite particles

Abstract

An oral care product has starch particles containing HAP, FHAP or FA particles. The product can be used, for example, as a treatment for dentinal hypersensitivity or to promote remineralization of teeth. The starch nanoparticles may be made in an emulsion process with a phosphorous compound such as STMP used as a crosslinking agent. The starch may be cationized. A calcium salt added to the emulsion provides calcium ions in the starch nanoparticles. HAP particles added to the emulsion become embedded in the starch particles. The starch particles may be cationic. The starch particles penetrate subsurface lesions or dentinal tubules where the starch particles dissolve and deposit the HAP.

Description

ORAL DELIVERY OF HYDROXYAPETITE PARTICLESRELATED APPLICATIONS

[0001] This application claims the benefit of, and priority from, US Provisional Application No. 63 / 759,844, filed on February 18, 2025, which is incorporated by reference.FIELD

[0002] This specification relates to oral care products containing hydroxyapatite (HAP).BACKGROUND

[0003] Biomimetic hydroxyapatite and caries prevention: a systematic review and meta-analysis, Limeback et al., Can J Dent Hyg. 2021 Oct 1;53(3):148-159, provides a review of the use of hydroxyapatite (HAP) as an ingredient on oral care products to combat caries.SUMMARY OF THE INVENTION

[0004] This specification describes an oral care product having starch particles containing HAP particles. The product can be used, for example, as a treatment for dentinal hypersensitivity or to promote remineralization of teeth. The starch nanoparticles may be made generally as described in US Patent number 11,666,515, Phosphate Crosslinked Starch Nanoparticle and Dental Treatments, which is incorporated herein by reference. As described therein, a phosphorous compound such as STMP is used as a crosslinking agent while making a starch particle in an emulsion process. The starch may be cationized, optionally while formulating the starch particles. A calcium salt can be added to the process to provide calcium ions in the starch nanoparticles. HAP particles added to the emulsion while making the starch nanoparticles are also driven into a water phase of the emulsion and become embedded in the starch particles. In some embodiments, the starch particles with HAP have a size, e.g. less than 500 nm or less than 300 nm, that allows them to penetrate into sub-surface lesions or dentinal tubules. In some embodiments the starch particles are cationic (i.e. have a positive zeta potential), which assists in depositing them deep into sub-surface lesions or dentinal tubules rather than having the particles aggregate near the enamel surface of the lesion of tubule. The starch particles dissolved leaving the HAP, and optionally phosphorous and calcium, in the lesion or tubes. The product may be delivered, for example, in a mouthwash, toothpaste or soluble pullulan strip.

[0005] The following section is intended to introduce the reader to the invention and the detailed description to follow but not to limit or define any claimed invention.

[0006] In a process described herein, starch submicron and nanoparticles are prepared with phase-inverse emulsion reactive phosphorous cross-linking, with addition of calcium with or without fluoride, for example as described in WO2019 / 191456A1, which is incorporated herein by reference. Nanoscale HAP, FHAP, or FA crystallites are dispersed and their aggregates are broken apart with their inclusion in this manufacturing process, resulting in the incorporation of these materials within the mineral-loaded starch particles. Similar tuning of particle properties can be achieved by adjusting manufacturing steps to control particle size, charge, and composition. These particles are readily compounded into dental product formulations, including dissolvable dental strips, toothpaste tablets, varnishes, and other formats. The particles improve the efficacy of HAP remineralization by disrupting aggregation of apatite crystallites and providing a targeting mechanism for biofilms and carious lesions due to the small size and cationic net charge. Further, the combined release of HAP with calcium, phosphate, with or without fluoride ions provides a novel mechanism for remineralization akin to providing tiny bricks and mortar as a superior mechanism of filling tooth porosities. Previously reported mineral-loaded starch particles show efficacy at delivering calcium, phosphate, and fluoride to the subsurface of caries lesions, improving the mineralization. The novel incorporation of HAP-type materials increases the mineral content of these materials, thereby increasing the remineralization efficacy. In addition to remineralization, various nanoparticles described herein may be used for the treatment of dentinal hypersensitivity, to help prevent or treat caries, or in professional restorative dental applications.

[0007] This specification describes methods of making starch-based nanoparticles made with a phosphate crosslinker according to an emulsion process, and the resulting starch nanoparticles with one or more phosphates which are loaded with HAP, FHAP or FA crystalline materials. Optionally, the nanoparticles have an anionic active agent, which maybe added while making the nanoparticles. Optionally, the nanoparticles have a cation, which may be an active agent, and / or cationic moieties on the starch. The cation and / or cationic moieties may be added while making the nanoparticles. The nanoparticles may be used in one or more methods of therapeutic treatment such as tooth remineralization, treatment of carious lesions, or treatment of dentinal sensitivity.

[0008] In various processes described herein, starch-based nanoparticles loaded with HAP, FHAP or FA crystals are made using an emulsion process such as a phase inversion emulsion process. The biopolymer is crosslinked with a phosphate crosslinker, for example STMP. Optionally, one or more of (i) a multivalent cation, such as calcium, (ii) an ionic active agent, such as fluorine (i.e. fluoride) and / or fluorescein, and (iii) one or more starch cationizing agents, are present in the water phase of a water-in-oil emulsion.Compounds may be added to the water phase while the water phase is emulsified (i.e. after phase inversion), during the phase inversion, or before the water phase is emulsified (i.e. before phase inversion). The water phase also contains the starch and phosphate crosslinker. In some examples, a fluoride salt and / or a fluorescein salt is added to the water phase before phase inversion. In some examples, a calcium salt and / or one or more starch cationizing agents are added to the water phase during or after phase inversion. HAP-type and preferably nano-HAP crystals are incorporated into the water phase during or after phase inversion, which facilitates the breakup of crystallite aggregates and their subsequent incorporation into the prepared starch particle material.

[0009] Various nanoparticles described herein comprises starch, phosphorous, HAP-type crystals (HAP, FA, and / or FHAP) and optionally one or more active agents such as calcium, and fluorine (i.e. fluoride).

[0010] This specification also describes a method of providing one or more elements to a tooth, and the use of a nanoparticle to provide one or more elements to a tooth.Optionally, the elements are delivered to the tooth in the form of one or more minerals which may be insoluble in saliva at least at ordinary pH, for example 6.2 to 7.6. Optionally, the elements are delivered to a tooth as ions or in salts or other compounds that are soluble in saliva at ordinary pH. Optionally, phosphorous is delivered to a tooth as part of one or more phosphorous-starch compounds. The nanoparticles may be delivered to the tooth by way of attachment to plaque on the tooth or by way of attachment to and / or entry into a cariouslesion. The HAP-type crystallite components are insoluble in non-acidic (>5.0) pH and are delivered to the tooth in addition to calcium, phosphate, and optionally fluoride mineral ions.

[0011] This specification also describes the use of nanoparticles to treat a tooth, or a method of treating a tooth. The method includes applying nanoparticles to a tooth. The treatment may provide, for example, one or more of remineralization, treatment of a carious lesion and treatment of dentinal hypersensitivity.

[0012] Nanoparticles as described herein can be used to carry phosphorous, HAP-type crystals, and optionally calcium and / or fluorine (i.e. fluoride) to a demineralized region of a tooth. The nanoparticles can be applied, for example, by rinsing the mouth with an aqueous dispersion of the nanoparticles. Alternatively, a gel or paste or strip having dispersed nanoparticles therein can be applied to a tooth. Optionally, some or all of the plaque and / or pellicle can be removed from a tooth before or during the application of the nanoparticles, which may help the nanoparticles attach to a carious surface of the tooth or to enter into pores of the tooth.

[0013] However, the removal of pellicle is optional even when targeting carious lesions. Alternatively, plaque may be left in place and the nanoparticles may be targeted to the plaque.

[0014] Without intending to be limited by theory, it is expected that the nanoparticles will help deliver one or more elements and HAP-type crystals to a demineralized area of a tooth by one or more of: increasing the concentration of an element and HAP-type crystal in a rinse, gel or paste relative to a solution or non-nanoparticle dispersion containing the element; targeting the elements and HAP-type crystals to a demineralized area or associated plaque with or without entry of the nanoparticle into a pre-carious lesion; carrying minerals and HAP-type crystals that can act as seed crystals, bio-active compounds or filling material; providing one or more elements for reaction with elements released from a tooth or present in saliva; or, providing a delayed or sustained release of one or more elements. The combination of delivery of insoluble HAP-type crystals and soluble mineral ions is expected to provide more rapid and effective filling of demineralized tooth porosities by a concept akin to using tiny bricks and mortar, where the HAP-type crystals fill large voids and gaps between the deposited HAP crystals are thereby filled with and secured by templated precipitation of released mineral ions.BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1 is a schematic process flow diagram of biopolymer nanoparticle formation by way of phase inversion emulsion.

[0016] FIG. 2 shows X-Ray Diffraction images of nano Hydroxyapetite (nHAP) and starch nanoparticles loaded with nHAP.

[0017] FIG. 3 shows TEM data of starch (CrystICareTM by GreenMark Biomedical) particles, starch particles with nHAP commercial nHAP and agreement of reference HAP with commercial nHAP.

[0018] FIG. 4 shows micrographs of nHAP and starch particles with nHAP.

[0019] FIG. 5 shows additional micrographs of starch particles with nHAP.DETAILED DESCRIPTION

[0020] Carious lesions initially form when bacteria in the dental biofilm ferment sugars and produce organic acids, which demineralize enamel. As minerals leach from enamel rods, the area becomes more porous and weakens. The early lesion is comprised of a surface layer (surface zone) which appears relatively unaffected by the carious attack compared to the subsurface (lesion body). The surface zone results from mineral precipitation and is explained by solubility gradients, dissolution / precipitation mechanisms and protection by adsorbed agents present in saliva. Reports indicate that the carious lesion body has a pore volume (measure of porosity) of 5-30% and the surface zone <5%, compared to sound enamel which has a pore volume of 0.1%. These areas of subsurface porosity present clinically as a milky white opacity known as a “white spot lesion” and identify early-stage caries to the clinician. If the process is not reversed, tiny open microchannels in the enamel surface allow acid to continue entering the subsurface. The lesion thus becomes more and more porous, until it eventually cavitates, requiring invasive surgical restoration (dental filling). However, the caries process is dynamic and early-stage caries is sometimes reversible with better hygiene and remineralization agents such as high fluoride treatments or toothpastes. Some caries become inactive or “arrested” naturally and do not require any treatment, because the porosity particularly on the surface has been reduced by mineral and / or protein deposition. A better understanding of the caries process is leading to aparadigm shift in caries management, which emphasizes enamel preservation and minimally invasive dentistry, leading to better oral health outcomes. However, there is a need for alternative and / or improved remineralization agents.

[0021] Dental caries and other remineralized areas of a tooth are negatively charged and often found near plaque, which may also be negatively charged. Phosphorous and calcium are lost when a tooth demineralizes and phosphorous-calcium minerals are useful when remineralizing a tooth. Fluoride is also useful for remineralizing teeth and in the prevention or treatment of caries. Hydroxyapatite (HAP) is the crystal form of calcium phosphate which makes up enamel and can be produced with crystallite sizes ranging from 10’s of nanometers to microns in size. HAP has gained popularity in dentistry as a “fluoride alternative”, and has been incorporated into toothpastes, varnishes, and other dental products, with a focus on nanoHAP (nHAP) as being more effective due to a smaller size. Fluorohydroxyapatite (FHAP) or fluorapatite (FA) are fluoridated variations of HAP with similar tunable properties and additional acid resistance. All of these materials are negatively charged and tend to aggregate into much larger particle agglomerates, which limits their ability to effectively target teeth or biofilm, including the negatively charged subsurface of non-cavitated carious lesions.

[0022] International Publication Number WO 2017 / 070578, Detection and Treatment of Caries and Microcavities with Nanoparticles, describes nanoparticles for detecting and / or treating active carious lesions or microcavities in teeth. The nanoparticle comprises starch bearing at least one cationic region and / or having a net positive charge and thereby capable of associating with carious lesions on a tooth. In some examples the nanoparticles comprise an anticaries agent or a remineralizing agent. In some examples, the nanoparticles are formed from starch by a reactive extrusion process as described in U.S. Pub. No.2011 / 0042821. After the nanoparticles are formed, they are cationized and fortified with a fluoride-containing component, a calcium-containing component or a calcium and phosphate-containing component by lyophilization. The nanoparticles have a positive zeta potential at the pH of saliva. In one example, an anionic fluoride salt was loaded into cationic starch nanoparticles. A 30-minute delay in the release of fluoride in artificial saliva through a dialysis membrane was observed for the nanoparticles relative to a reference solution of free fluoride salt. WO2019 / 191456A1 describes a manufacturing process for starch nanoparticles whichare prepared by reactive emulsion with sodium trimetaphosphate, which are further modified to incorporate calcium and fluoride ions for use in dental treatment applications. The particle charge can be tuned by varying amounts of phosphate, calcium, and cationic chemical modification of the starch polymer. These publications are incorporated by reference herein.

[0023] Dental cavities begin with one or more areas of demineralization that present as a white spot. The white spot can alternatively be called a pre-cavity, a pre-carious lesion or a white spot lesion. The enamel of the white spot has greater porosity than natural enamel due to erosion typically caused by oral bacteria that release acids. The enamel surface may remain intact, but in an active lesion open micropores allow acid to penetrate into the tooth. The area of the white spot thus becomes more and more porous and demineralized over time. Eventually the white spot area may become so weak that the enamel surface collapses and a cavity is formed. In this specification, references to demineralized areas of the tooth include white spots and cavities unless a particular type of demineralized area is specified. However, it is expected that the nanoparticles described herein will be particularly useful in treating white spots where it is difficult or damaging to access the porous structure by conventional means. For example, conventional topically applied fluoride solutions may merely seal the surface of the white spot, thus making the white spot inactive but not restore the dominant porous area underneath the surface. Hydroxyapatite treatments (toothpastes, rinses, varnishes, etc.) contain large, negatively charged aggregates of HAP crystals which are repelled from negatively charged lesions and their large size and aggregation reduces their ability to penetrate into carious lesions. Drilling the tooth to provide fillings or bio-active materials removes natural enamel which is then lost forever, as it cannot reform at this stage. The nanoparticles described herein may be targeted to the white spots themselves or to plaque that may be near the white spots. The nanoparticles described herein may also be useful in occluding or sealing pores in dentin, as in the treatment of dentinal hypersensitivity. It is expected that treatment with the nanoparticles will be most beneficial when the white spot is in a pre-cavitated stage and active with open micropores and subsurface porosity. However, treatment with the nanoparticles may also be done after a cavity (hole) is formed in more advanced stages of caries.

[0024] Without intending to be limited by theory, the inventors believe that as calcium leaches away from a demineralized area of a tooth, it leaves behind negative polyphosphatecharges in the interior of the tooth. Nanoparticles can enter the demineralized area through micropores in an active lesion. The concentration of useful elements loaded into a nanoparticle dispersion might exceed the concentration of a comparable solution of the same elements. If the nanoparticles are positively charged, they may also associate with the surface (including surfaces below the outer enamel surface) of a tooth or plaque as a result of electrostatic attraction, thereby increasing either the delivery or retention, or both, of elements and materials in the nanoparticle. Once on or inside the tooth, saliva degrades the biopolymers and one or more elements and / or minerals released by the nanoparticle can help restore the pores and / or subsurface area. Materials delivered by the nanoparticles may, for example, react with ions or compounds of the tooth or saliva, provide seed particles for mineral formation, act as a bioactive compound that can be incorporated into naturally produced minerals, or simply fill the demineralized subsurface area. Deposited HAP-type minerals may provide additional templating of mineral deposition along the lines of a “brick and mortar” filling concept.

[0025] In a method described herein, biopolymer, i.e. starch, nanoparticles are made using an emulsion process. In brief, one or more biopolymers are dispersed or dissolved in water, the water is then dispersed (i.e. emulsified) in another phase, for example an oil phase, and the biopolymer is crosslinked while in dispersed droplets of the water phase in the dispersion or emulsion. The use of an oil as a second phase is optional but helps to load water-soluble reactants into the droplets of the water phase. However, another non-solvent of starch, for example ethanol or hexane, or a multi-phase aqueous system, may be used. The crosslinker may be a phosphate or polyphosphate crosslinker such as sodium tri metaphosphate (STMP) or sodium tripolyphosphate (STTP).

[0026] Optionally, the process may be a phase inversion emulsion (PIE) process. A schematic of a phase inversion process is shown in FIG. 1. In the example illustrated, a starch-based nanoparticle is made with a sodium trimetaphosphate (STMP) crosslinker. Initially an oil-in-water emulsion is formed which, after an increase in temperature, becomes a water-in-oil emulsion. A surfactant may be used to assist in the oil-in-water to water-in-oil transition and to select the temperature at which this transition occurs. The STMP is added so that the crosslinking reaction occurs within water droplets of the water-in-oil emulsion. Additional elements may be added to the water phase by adding them at any of the threestages shown in FIG. 1 (separate water and oil phases, oil in water emulsion, water in oil emulsion).

[0027] Referring to FIG. 1, an oil phase is homogenized with a water phase containing dissolved starch or dispersed starch nanoparticles. The oil may be, for example, paraffin oil or a food grade mineral oil. Alternatively, other food grade oils such as sunflower oil or olive oil may be used. After forming an oil in water emulsion, the temperature is increased to more than the phase inversion temperature (PIT) for the reaction conditions. The PIT may vary depending on the ratio of water to oil, the presence and type of any surfactants (for example Tween 85), the presence and type of any catalysts (for example NaCI) and the type of oil. In some cases, the PIT may be in the range of 25-60 °C.Optionally, heating can be provided by the high shear mixer itself, for example by increasing the mixer speed to heat the mixture. As the water in oil emulsion is being heated or after the phase inversion is complete, the crosslinker is added. The reaction may then continue, for example for about an hour.

[0028] The biopolymer is crosslinked using a phosphate crosslinker such as STMP, typically under alkaline conditions. While other crosslinkers might be used, STMP is advantageously available in food grade preparations. The crosslinker provides a source of phosphorus, an element useful for restoring a demineralized tooth. Part of the crosslinker (the inventors believe the part to be about 10-50% or 10-30%) reacts to form internal non-reversible (i.e. covalently bonded) crosslinks within the nanoparticles by way of monophosphate linkage. However, in addition to distarch monophosphate, side reactions may form other compounds such as monostarch triphosphate, monostarch monophosphate. The reaction is somewhat inefficient but dangling phosphate groups in either inorganic or organic compounds produced in the reaction or side reactions are available to form a strong associative complex with calcium and / or fluoride either within the particle or later when deployed in the oral cavity.

[0029] In some examples, NaCI salt is used to provide high ionic strength in the water phase, which favors the STMP reaction to occur in a subsequent step. However, in other examples described herein, NaF is used in place of, or in combination with, NaCI and to also provide fluoride in the nanoparticle. The NaF can be added, for example, in the water phase produced prior to homogenizing to form the O / W emulsion in FIG. 1. Alternatively, the NaFcan also be added while homogenizing to form the O / W emulsion or after formation of the O / W emulsion or, though with a possible decrease in fluoride release time, after nanoparticle formation. In some examples, hydroxyapatite, fluorohydroxyapatite, or fluorapatite (or any combination, therein) can be added prior to the addition of STMP, for the dissociation of aggregates due to shear mixing, stabilization in high ionic strength W / O emulsion micro- / nano-droplets, and subsequent incorporation in the prepared nanoparticles. In some examples, a calcium salt such as calcium chloride is added in the water phase. Optionally, the calcium salt, dry or in aqueous solution, is added into the W / O emulsion of FIG. 1 after the STMP is added or, possibly with some decrease in calcium and / or fluoride release time, after nanoparticle formation. Water soluble components are driven into the water droplets and at least partially react or otherwise associate with the nanoparticles. In another option, a calcium salt such as calcium chloride can be added in the water phase produced prior to homogenizing to form the O / W emulsion in FIG. 1 either in place of NaF and NaCI or in addition to NaF and / or NaCI. STMP produces negative charges in the resulting nanoparticle. The addition of calcium can block some of these charges. However, an additional step such as cationization of the starch is typically required to produce nanoparticles that are positively charged at neutral pH or even an acidic pH (i.e 5.5 or less) that may be found within or near a carious lesion.

[0030] The fluoride and / or calcium are present with the phosphorous and biopolymer in a dispersion of small water droplets in an emulsion, for example a water-in-oil emulsion, optionally stabilized by surfactant. Each droplet containing biopolymer produces a crosslinked particle. Optionally, an emulsion of water in another phase may be used.

[0031] The emulsion or emulsions are preferably produced using an ultra-high shear mixer, for example a Silverson dissolver agitator. This mixer advantageously produces minimal air encapsulation and provides sufficient shear to produce nanoparticles averaging under 700 nm or under 500 nm in diameter most of which, considering their hydrogel nature and distributions in the sizes of the nanoparticles and pores, are able to enter the pores of carious lesions (which average 750-800 nm in size). Further, the high shear helps to disassociate HAP-type crystal aggregates, reducing their measurable particle size and allowing their incorporation into prepared particles. The starch may be cooked, chemically degraded and / or thermo-mechanically processed to help produce a solution or dispersion ofstarch in the water phase. Alternatively, smaller starch nanoparticles (20-200 nm) such as those produced by EcoSynthetix Inc. under the trademark EcoSphere™ can be used as the starch feed source. The resultant nanoparticles may have a mean or average size, measured for example by the peak in a dynamic light scattering (DLS) plot, the Z-average size (or harmonic intensity averaged particle diameter as described in ISO 13321 or ISO 22412) of a DLS measurement, or the mean or D50 value in a nanoparticle tracking analysis (NTA) measurement, of less than 1000 nm, for example 100-700 nm, 100-500 nm, 200-500 nm or 200-400 nm. After breaking the emulsion, the water phase can optionally be centrifuged, for example at 4000 rpm for 1 minute, to separate the nanoparticles in the supernatant from unassociated precipitates in the pellet. The nanoparticles are optionally washed to remove traces of oil although if a suitable, i.e. food-grade, oil is used it is not necessary to completely remove all traces of oil. The supernatant can be freeze dried to obtain dry nanoparticles. The nanoparticles can be stored dry or, for a more limited time, in an aqueous dispersion, gel or paste. An aqueous dispersion gel or paste can optionally be sterilized or stabilized with a biocide, antimicrobial preservative or biostatic additive.

[0032] The amount of crosslinker used may be 1% to 50 mol % of STMP based on anhydrous glucose repeating units (AGU). Preferred samples were produced with 3% to 50% STMP, or from 10% to 50% STMP, for example about 30% STMP. Particle size does not appear to be clearly related to the amount of STMP except that, in some examples, very low amounts of STMP (i.e. 1%) produced very small nanoparticles (about 100 nm), low amounts of STMP (i.e. 1-5%) produced large nanoparticles (average size of about 300-500 nm) while larger amounts of STMP (5% to 50%) produced intermediate nanoparticles (about 100-300 nm). Without intending to be limited by theory, it is possible that samples made with very low STMP (i.e. 1%) do not incorporate substantially all of the available starch into nanoparticles although 3% STMP seems to be sufficient. Once sufficient crosslinker is available, the smaller size with larger amounts of STMP may be due to higher crosslinking and less swelling (as predicted by the Stokes-Einstein equation related to volume swell ratio) since the particles are hydrogels and their size is measured in a swollen state. It is also possible that particle size is influenced more by the amount of shear energy applied or other factors that could affect droplet size of the water in oil emulsion. In some cases, nanoparticles made with added calcium had zeta potentials near neutral, for example in a range from -5 to +5mV at a pH of 7.0. Optionally, precipitates produced in the water phase that are not associated with the nanoparticles can be separated by centrifugation. The nanoparticles tend to remain in the supernatant of the centrifuged sample. The nanoparticles exhibit swelling behavior and appear to be hydrogels. For example, the nanoparticles retain water, but the amount of water retained by the nanoparticles decreases with increasing ion concentration.

[0033] The nanoparticles become more negatively charged (as measured by zeta potential) with increasing pH and STMP content. In some examples, the zeta potential of nanoparticles with 1-50 mol % AGU of STMP, without calcium salt added and without starch cationization, ranged from 0 to -65 mV across a range of pH and STMP content, or -10 to -22 mV at neutral pH. For example, samples made with 30% STMP, without calcium salt added and without starch cationization, were measured as having a zeta potential of -15 mV at a pH of 3, -45 mV at pH of 8, and further decreasing to -70 mV at pH of 12.

[0034] Adding calcium, for examples as CaCh, but still without starch cationization (as described in more detail below) reduces the negative zeta potential of the nanoparticles. At near neutral pH and a 5% STMP content, the charge of the nanoparticles with calcium added can be in the range of -5 mV to 0 mV. Nanoparticles made with calcium and 30% STMP have a zeta potential in the range of -30 mV to -25 mV near neutral pH and without starch cationization. Without intending to be limited by theory, the added calcium may be capping the phosphate groups provided by the STMP. Optionally, the starch may be cationized to produce a further decrease in negative zeta potential, or to produce a positive zeta potential over a desired range of pH.

[0035] As an alternative to STMP, sodium tripolyphosphate (STTP) may be used as the crosslinker.

[0036] Optionally, the nanoparticles can be cationized, for example by the method described in International Publication Number WO 2017 / 070578, Detection and Treatment of Caries and Microcavities with Nanoparticles. Optionally, the starch may be cationized while in the water in oil emulsion. For example, glycidyl trimethyl ammonium chloride (GTAC), optionally with or pre-mixed with water and isopropyl alcohol or 2-proponol, may be added to the water phase before or after forming the water in oil emulsion. Alternatively, the starch may be cationized after the nanoparticles are formed. Alternatively, the starch may be cationized before the nanoparticles are formed, although in this case the starch is preferablyfirst cooked or regenerated so that the cationization is not limited to the surface of the starch granules.

[0037] Optionally, a fluorophore could be added to the nanoparticles, for example by the method described in International Publication Number WO 2017 / 070578, Detection and Treatment of Caries and Microcavities with Nanoparticles. Alternatively, a fluorophore can be added during the formation of the nanoparticles, for example by adding the fluorophore to the water phase. Optionally, the nanoparticles could be co-dispersed with fluorescent cationic nanoparticles, for example nanoparticles described in International Publication Number WO 2017 / 070578, Detection and Treatment of Caries and Microcavities with Nanoparticles.

[0038] The incorporation of PO43", Ca2+and F" ions (as appropriate) into various samples containing phosphate only; phosphate and calcium only; and, phosphate, calcium and fluoride, was confirmed by energy-dispersive X-ray spectroscopy (EDX / EDS) used for elemental analysis of areas on SEM images. The incorporation of HAP nanocrystals was confirmed by EDX / EDS, TEM imaging, and X-Ray Diffraction (XRD) studies.

[0039] The ion content of various freeze-dried samples (moisture content less than 0.1%) was also measured by inductively coupled plasma (ICP) analysis. The phosphorous content of the nanoparticles increases with the amount of STMP used. The increase in phosphorous content increases generally linearly with STMP mol %. In one example, nanoparticles produced with from 1-50 mol % AGU of STMP had phosphorous contents ranging from about 1000 to about 21,000 ppm. In this example, phosphorous content was determined by ICP-MS after dialyzing the samples for 6 days and treating the samples with HCI and HNO3. The phosphorous content values relate to total phosphorous, which is believed to be phosphorous bound to the nanoparticles, but the type of phosphate species present was not determined. Optionally, the range of STMP may be 3-50 mol % AGU or IQ-50 mol % AGU, which gives 4,000-21,000 ppm phosphorous in the nanoparticle. Optionally, the range of STMP may be 20-40%, 25-35%, or about 30 mol % AGU.

[0040] For example, nanoparticles produced with 30 mol % AGU of STMP but without being cationized contained about 15,000 ppm of phosphorous. With 67 mol % dry CaCh) based on starch (anhydro-glucose repeating units) added, the nanoparticles also contained about 1,320,000 ppm calcium. With 70 mol % NaF and 67 mol % CaCh) (based on starch) also added, the nanoparticles contained 19,000 ppm fluoride and calcium contentincreased to 2,160,000 ppm. With 70 mol % NaF (based on starch) added without CaCy (based on starch), the nanoparticles contained about 700 ppm fluoride.

[0041] The results above indicate that fluoride retention increases when calcium is also added and that calcium retention increases when fluoride is added. Calcium is believed to interact with phosphate through ionic interactions. Without intending to be limited by theory, it is possible that fluoride can co-precipitate with phosphate and calcium to form fluorapatite (Cas(PO4)3F) or other minerals in the nanoparticle. It is not clear if any of the calcium or fluoride is reacted with the starch or phosphates attached to the starch or not, or how the calcium or fluoride are combined with the starch. However, the phosphorous, calcium and fluoride contents described herein are measured in nanoparticles extracted from the supernatant of a centrifuged sample, wherein precipitates not bound to the nanoparticles were collected in the pellet of the centrifuge and not part of the measured calcium or fluoride contents. While no separation is perfect (and so some unbound precipitates might still be in the nanoparticles), it is expected that the combination of phosphorous, calcium and fluoride with the nanoparticle is sufficiently durable for at least a material portion of them to be delivered by the nanoparticle to a carious lesion. It is also expected that the addition of a different multivalent cation, for example a different divalent or trivalent cation or alkaline earth metal ion, could similarly increase the retention of anionic active agents such as fluoride, though without the potential benefit of adding calcium.

[0042] The nanoparticle might enter the carious lesion through pores in the lesion or stay on the outside of the lesion. Optionally, the teeth may be cleaned to remove plaque and / or the dental pellicle, for example by patient brushing or cleaning by a dental hygienist, before or while applying the nanoparticles to improve access to a carious lesion. As the nanoparticles break down in saliva while near or inside of the lesion, the phosphorous and calcium and / or fluoride may diffuse into the tooth and can deposit or optionally react, with or without additional elements from the tooth or saliva, to form minerals in the tooth. It is also possible that already formed minerals, for example calcium phosphate, calcium hydroxyapatite or fluorapatite, present within the nanoparticles can act as seed crystal or bioactive agents that fill parts of the lesion or are incorporated into additional minerals produced in the tooth.

[0043] Optionally the nanoparticle has a zeta potential of at least +2.0 mV, for example between +2.0 mV and +10.0 mV at all pH values of about 7.0 or less or at all pH values of about 5.5 or less. In some examples, the nanoparticles have a positive zeta potential in acidic solutions but a negative zeta potential under neutral or basic conditions, i.e. pH of 7.0 or more. For example, the zeta potential of the nanoparticles may be at least +2.0 mV at all pH values of about 5.5 or less but negative at pH values of 7.0 or more. Since the pH of saliva in the mouth is roughly neutral (typically about 7.4), but the pH in or near an active carious lesion is typically acidic (typically about 4-5), nanoparticles that have a positive zeta potential only in acidic conditions may be even more selectively targeted to active carious lesions than nanoparticles that have a positive zeta potential even under neutral or mildly basic (i.e. pH of 7.0 or more) conditions. The nanoparticles are administered to teeth, for example as a dispersion used in a rinse or mouthwash, which optionally may include diagnostic fluorescent nanoparticles, or in a gel or paste applied to the teeth, or in toothpaste.

[0044] The nanoparticles may have a size of up to 2500 nm but preferably have a size of 1000 nm or less. The term “nanoparticles” as used herein is not limited to particles having a size of 100 nm or less as in the IUPAC definition but also includes larger particles, for example particles up to 2500 nm, or up to 1000 nm, for example in their largest dimension or in the diameter of a sphere of equivalent volume. Optionally, the nanoparticles may have a mean or average size as determined by peak intensity of a DLS plot, the z-average of a DLS measurement or the mean or D50 of an NTA measurement, in the range of about 100 nm to about 700 nm, about 100 nm to about 600 nm, or in the range of about 100 nm to about 500 nm, or in the range of about 200 nm to about 500 nm, or in the range of about 100 nm to about 400 nm. As mentioned above, particles in these size ranges will be called nanoparticles, which is consistent with common usage of that word in North America particularly for particles less than 1000 nm in size. However in other parts of the world, and according to IUPAC definition, particles larger than 100 nm in size may alternatively be called microparticles.

[0045] Biopolymers, for example polysaccharides and proteins, and in principle any other biopolymer, and mixtures thereof, may be the biopolymer used in these processes. Any starch, for example waxy or dent corn starch, potato starch, tapioca starch, dextrin, dextran,starch ester, starch ether, carboxymethyl starch (CMS), and in principle any other starch or starch derivative, including cationic or anionic starch, and mixtures thereof, may be the biopolymer used in these processes. Any polysaccharide, cellulosic polymer or cellulose derivative, for example microcrystalline cellulose, carboxymethyl cellulose (CMC), any nanofibrillar cellulose (CNF), nanocrystalline cellulose (CNC), or cellulose ester, cellulose ether, and in principle any other polysaccharide, cellulose or cellulose derivative, and mixtures thereof, may be the biopolymer used in these processes. Proteins, for example zein (corn protein), casein (milk) or soy protein, and in principle any other protein or modified protein, and mixtures thereof, may be the biopolymer used in these processes.

[0046] Optionally, the nanoparticles may be prepared by a phase inversion emulsion process as described in U.S. Pat. No. 6,755,915, Method for the Preparation of Starch Particles. In this method starch particles are prepared in a two-phase system comprising steps of a) preparation of a first phase comprising a dispersion of starch in water, b) preparation of a dispersion or emulsion of the first phase in a second liquid phase, c) crosslinking of the starch present in the first phase, d) separating the starch particles thus formed. In some examples the second phase consists of a hydrophobic liquid and step b) consists in forming an oil-in-water emulsion. In some examples the second phase consists of a water-miscible non-solvent for starch.

[0047] The nanoparticles are stable in dry form. If stored wet, in a closed container, a sterile 5% w / w aqueous dispersion of cationic-mineral containing starch nanoparticles, or non-sterile aqueous dispersion stabilized with a citric acid / potassium sorbate or other foodgrade biocide, may be prepared. However, the biocide might not be required. There are some indications of stability without biocide, but it is not yet known whether the nanoparticles have bacteriostatic or bactericidal properties. Nanoparticles containing fluoride appear to have bacteriostatic or bactericidal properties.

[0048] The nanoparticles can be combined with one or more supplemental carriers (i.e. water, excipients or extenders etc.) that are toxicologically and functionally acceptable to create a composition that can be administered to the mount of an animal or person. The composition may be, for example, a mouth rinse, dentrifice, gel, varnish, paint, toothpaste, tooth powder or mouthwash. The carriers can be selected from the usual components of one or more of these compounds. For example, the carriers may be one or more of water,alcohols, surfactants, emulsifiers, foaming agents, abrasives, humectants, viscosity modifiers, tackifiers, film-formers, plasticizers, diluents, pH modifiers, sweeteners, flavors, coloring agents and preservatives. For example, the nanoparticles can be used in a rinse or mouthwash that is swished in the mouth and then suctioned and / or rinsed, at home or in a dental clinic. Alternatively, the nanoparticles can be in a toothpaste administered by brushing the enamel surface at home, or as a paste or gel applied by a hygienist. After being introduced into a mouth, the nanoparticles adhere to plaque or the surface of caries and may travel inside of active carious lesions by passing through the porous surface of the lesion.

[0049] The nanoparticles can be used, for example, without, or at least without an effective amount of, any additional oral care active ingredient such as an added anticaries agent or remineralizing agent. In particular, the nanoparticles can be used without adding effective amounts of any of the oral care active ingredients described in United States Patent Application Publication Pub. No. US 2017 / 0112949 A1 (also published as International Publication Number WO 2017 / 070578, Detection and Treatment of Caries and Microcavities with Nanoparticles). For example, the nanoparticles can be used without adding, during or after formation of the nanoparticle, one or more of a) a fluoride-containing active ingredient, b) a calcium-containing component, c) a calcium and phosphate containing compound and d) amine fluoride, casein phosphopeptide, phosphoprotein and equivalents and combinations thereof. In this case, the nanoparticle is believed to be effective due to the presence of one or more phosphate-containing, or starch and phosphate-containing, compounds that are introduced by the addition of crosslinker or created in the reaction of the crosslinker and starch. Without intending to be limited by theory, although phosphate alone does not create mineral compounds, the phosphate in the nanoparticle may react with calcium in saliva or other elements, ions or compounds in the mouth to create mineral compounds on or in a tooth or to help nucleate crystal formation within the white spot or active pre-cavity lesion.

[0050] Other two-phase emulsions, for example water and alcohol or hexane, might be used. However, the oil phase helps achieve a high loading of non-oil soluble active agents in the nanoparticle. The oil may be a food grade mineral oil, or other, preferably food grade, oils such as sunflower oil or olive oil. A surfactant, for example Tween 85, is also used. The transition temperature may vary depending on the water to oil ratio, the type of oil, and the type and amount of surfactant.

[0051] The following examples are provided to illustrate various embodiments and to provide further enabling disclosure but are not intended to limit any claimed invention.Example 1 - Procedure Used for the Preparation of Starch particles with Cationization in the Emulsion and incorporation of commercial nano-HAP

[0052] Combine water, starch, sodium hydroxide, surfactant (Tween 85), and oil (mineral oil or paraffin oil) to mixing vessel. Start mixing with high shear mixer (20,000 RPM dissolver agitator) and mix until emulsion is formed "10 minutes. Measure batch pH and ensure pH > 12.

[0053] Add sodium chloride and allow to mix until temperature is >65 °C.

[0054] Add commercial nano-HAP and mix at the 20,000 RPM for minimum 20 mins.

[0055] To cationize starch, add a mixture of glycidyl trimethyl ammonium chloride (GTAC), Dl-water, and 2-propanol and allow to react for about 1.5 hour. Monitor temperature and maintain between 65-80 °C. by adjusting high speed mixer mixing rate.

[0056] Add STMP to mixture and allow to react / cross-link for about 1.5 hours. Monitor temperature and adjust mixing speed to maintain temperature less than 80 °C. but greater than 65 °C. Measure batch pH and ensure still basic (about pH 10).

[0057] Add calcium chloride (dihydrate), optionally pre-dissolved in water, and mix for an additional 30 minutes.

[0058] Neutralize batch (to pH 7-8) by addition of HCI or NaOH if necessary.

[0059] Allow sample to cool to room temperature and / or refrigerate to facilitate phase separation of emulsion. If phase separation happened during the dwell time, discard the supernatant before moving to next step.

[0060] Dilute sample about 50:50 with 2-propanol and mix. Centrifuge at 9,000 RPM for 10 minutes to induce multi-phase separation of particles, aqueous / hydrophilic phase, and oil / hydrophobic phase.

[0061] Redisperse the particle phase in Dl-water with high shear mixer and repeat last step for 2 more times or until there is no trace of mineral oil in the supernatant. After the last washing, resuspend particle phase in Dl-water and move to next step.

[0062] Freeze the sample and lyophilize to obtain a dry powder sample of isolated mineral-loaded nanoparticles.Example 2 - Production of Nanoparticles, Optionally with Fluoride and nanoHAP, with Cationization in the Emulsion

[0063] Combine water, starch, sodium hydroxide, surfactant (Tween 85), and oil (mineral oil or paraffin oil) to mixing vessel. Start mixing with high shear mixer and mix until emulsion is formed "10 minutes. Measure batch pH and ensure pH > 12.

[0064] Add sodium fluoride and allow to mix until temperature is >65 °C.

[0065] Add commercial nano-HAP.

[0066] To cationize starch, add a mixture of glycidyl trimethyl ammonium chloride (GTAC), Dl-water, and 2-propanol and allow to react for about 1.5 hour. Monitor temperature and maintain between 65-80 °C. by adjusting high speed mixer mixing rate.

[0067] Add STMP to mixture and allow to react / cross-link for about 1.5 hours. Monitor temperature and adjust mixing speed to maintain temperature less than 80 °C. but greater than 65 °C. Measure batch pH and ensure still basic (about pH 10).

[0068] Add calcium chloride (dihydrate), optionally pre-dissolved in water, and mix for an additional 30 minutes.

[0069] Neutralize batch (to pH 7-8) by addition of HCI or NaOH if necessary.

[0070] Allow sample to cool to room temperature and / or refrigerate to facilitate phase separation of emulsion. If phase separation happened during the dwell time, discard the supernatant before moving to next step.

[0071] Dilute sample about 50:50 with 2-propanol and mix. Centrifuge at 9,000 RPM for 10 minutes to induce multi-phase separation of particles, aqueous / hydrophilic phase, and oil / hydrophobic phase.

[0072] Redisperse the particle phase in Dl-water with high shear mixer and repeat last step for 2 more times or until there is no trace of mineral oil in the supernatant. After the last washing, resuspend particle phase in Dl-water and move to next step.

[0073] Freeze the sample and lyophilize to obtain a dry powder sample of isolated mineral-loaded nanoparticles.Example 3 - Particle Size and Charge Measurements of Nanoparticle Samples

[0074] Various samples of cationized nanoparticles were made according to the method in Example 1 and 2. Controls (nHAP) included commercial nano-hydroxyapatite and starch particles prepared without addition of nano-hydroxyapatite. Samples were diluted and measured for particle size and charge as a function of solution pH using a ZetaView instrument, with the results shown in Table 1. GMB-HAP refers to particles made according to Example 1.TABLE 1

[0075] Though commercial nHAP has a reported particle size of 20 nm, size measurements with the ZetaView show significantly larger aggregates. This is an expected result, as the high surface energy of the material favors aggregation. The data for sheared nHAP highlights a reduction in average particle size, which still is significantly larger than the reported size of individual nHAP crystallites. GMB-HAP particles show larger particle size that is consistent with GMB mineral-loaded starch particles. Zeta potential measurements show negative zeta potential of nHAP at neutral to basic pH. Interesting to note is that as the surface area increased for smaller agglomerates, the negative charge increased, presumably due to desorption of calcium cations from the surface , which is similar to what has been shown for the porous subsurface of caries lesions in tooth enamel. GMB particles prepared with nHAP meanwhile show positive zeta potential at pH 7.Example 4 - SEM of prepared Nanoparticles

[0076] Scanning Electron Microscopy (SEM) micrographs were taken of gold-coated powder samples. Samples were prepared by freeze-drying to a powder, then sprinklingpowder onto a sample holder, which was then sputter-coated with gold, prior to SEM imaging. Collected images are shown in Figure 1. Commercial nHAP showed large aggregated clusters of small and spiny nHAP crystals. These fine sharp details are indicative of the tiny high-aspect ratio nHAP crystallite structure. Mineral-loaded starch particles prepared with loaded nHAP show a very different image, with aggregates of small, more spherical structures that are approximately 100-200 nm in size. Given the swelling of hydrated starch particles, these sizes are consistent with the sizes measured by the ZetaView experiments. The smoothness of the individual particles on the SEM imaging suggests that any nHAP, if present, has been encapsulated inside the starch particle and the loaded nHAP therefore is not affecting the surface properties of the starch particles. Images are shown in Fig. 2.Example 5 - X-Ray Diffraction Testing of nHAP samples

[0077] Collected washed and lyophilized powder samples were evaluated by X-Ray Diffraction (XRD) testing. Mineral-loaded starch particles showed a broad peak, also referred to as the “amorphous halo” observed for amorphous polymers, which is typical of starch that has been ‘cooked’ in caustic, as opposed to sharp peaks observed in XRD for crystalline compounds (such as HAP, KCI, NaCI). The halo is at a diffraction angle of approximately 18°. Other detectable peaks indicated presence of residual sodium chloride. All loaded calcium and phosphate within the particles are therefore amorphous. Commercial nHAP samples did not have the starch peak, and showed distinctive crystal patterns for hydroxyapatite and potassium chloride, the latter being included presumably to stabilize the material in solution. Prepared nHAP-loaded mineral-starch particles show evidence of the broad starch region at 18°, in addition to the crystallite peaks for HAP. Peaks for sodium chloride and potassium chloride were not detectable on this sample, which had undergone additional washing. These plots show that prepared nHAP-loaded mineral-starch particles contain HAP and starch, thereby proving presence of the crystalline material and that the manufacturing process does not disrupt the crystallinity of the HAP material. Evidence of the successful washing with the loss of the KCI peak is expected. The plots are shown in Fig. 3.Example 6 -Transmission Electron Microscopy (TEM) of Prepared Particles

[0078] Dried lyophilized powder samples were scattered on a carbon-coated sample mesh grid and imaged with TEM, including plasma cleaning as a pre-imaging step.Micrographs of nHAP showed high aspect ratio crystallites with an apparent 20 nm diameter and lengths ranging from 40-100 nm. Aggregates of the crystallites were visible. The nHAP-loaded starch particles showed bright collections of nHAP crystals surrounded by a faint haze, resulting from less electro-opaque starch. An example of two such particles are shown in close proximity in the next set of images. Each particle is approximately 100-300 nm in dimension and contains many nHAP crystals. Elemental mapping experiments show the presence of calcium, phophorus, oxygen, and carbon. A carbon and oxygen layer surrounds the mineral materials in the particle core. In this first image, the carbon mesh grid is also visible, which provides significant carbon signal to the background. The images are shown in Fig. 4.

[0079] Additional images were taken of samples that were in a different focal plane to the carbon grid. These images show similar composition, particle size, and a more clearly delineated overlap of carbon (from starch) with the calcium and phosphorus (from nHAP and loaded calcium and phosphate mineral ions). These images validate the loading of nHAP within the starch particles at relatively high loading concentrations. The encapsulation of these crystals may allow the starch surface properties to prevent aggregation and for biospecific targeting of caries lesions by the tuning of the starch particle size and zeta potential. The images are shown in Fig. 5.Example 7 - Thermogravimetric Analysis of Prepared Nanoparticles

[0080] Lyophilized samples were analyzed by thermogravimetric (TGA) analysis under nitrogen atmosphere. Commercial nHAP showed a simple profile with minimal loss (~4-8%) from surface moisture. TGA curves of mineral-loaded starch particles show a more complex profile, with initial loss due to water evaporation (-150-250 °C), and starch degradation at higher temperatures (-250-350 °C) to a plateau indicative of the starch residuals and the highly heat-stable minerals. The main finding from this analysis is that the residue is significantly higher for particles loaded with HAP, which confirms mineral loading -residual content (normalizing for initial hydration) is presented in Table 2 - results show 61%and 70% residual content for nHAP loaded particles without and with fluoride, respectively, compared to loadings of 47% and 49% for comparative particles without nHAP.TABLE 2

[0081] This written description uses examples to disclose the invention and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art.

Claims

CLAIMS:We claim:

1. An oral care product comprising,starch particles; and,hydroxyapatite (HAP), FHAP or FA particles in the starch particles.

2. The product of claim 1 having a size of 1000 nm or less, 500 nm or less or 300 nm or less.

3. The product of claim 1 or 2 having a positive zeta potential at a pH of 7.

4. The product of any of claims 1 to 3 comprising calcium and / or phosphorous in addition to the HAP particles.

5. The product of any of clams 1 to 4 wherein the starch is cationic.

6. The product of any of claims 1 to 5 wherein the starch particles are made according to an emulsion process, for example as described in US Patent number 11,666,515, Phosphate Crosslinked Starch Nanoparticle and Dental Treatments.

7. The product of any of claims 1 to 6 in a mouthwash, toothpaste or soluble pullulan strip or other oral delivery carrier.

8. The product of any of claims 1 to 7 comprising fluoride.

9. A method of treating dentinal hypersensitivity or caries or remineralizing teeth comprising applying the product of any of claims 1 to 8 to a tooth.

10. A method of making an oral care product comprising,preparing a first phase comprising a solution or dispersion of starch in water; preparing a dispersion or emulsion of the first phase in a second liquid phase such as an oil phase;adding multi-valent cations and / or a starch cationizing agent to the first phase; adding HAP, FHAP or FA particles to the first phase; and,crosslinking the starch in the first phase with a phosphate crosslinker.

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