WO2025114570A1

WO2025114570A1 - Remineralising chlorhexidine crystal forms containing fluoride and uses in dentistry and medicine

Info

Publication number: WO2025114570A1
Application number: PCT/EP2024/084165
Authority: WO
Inventors: Michael Cattell; Eleni TRIKOILI; Rui Sun; Ama JOHAL; Gleb Sukhorukov; Simon Charles FIELDING RAWLINSON; David James GOULD; Simon Holmes
Original assignee: Queen Mary University of London
Current assignee: Queen Mary University of London
Priority date: 2023-12-01
Filing date: 2024-11-29
Publication date: 2025-06-05
Anticipated expiration: 2026-06-01

Abstract

A crystalline salt of chlorhexidine (CHXD) fluoride having a spherical morphology under Scanning Electron Microscopy (SEM) and comprising a fluoride anion and a cation selected from the group consisting of sodium, ammonium, potassium, or tin, and a process for the preparation of monodisperse crystals of the same. There is provided a pharmaceutical 5 composition, and uses thereof in dentistry and medicine.

Description

REMINERALISING CHLORHEXIDINE CRYSTAL FORMS CONTAINING FLUORIDE AND USES IN DENTISTRY AND MEDICINE

FIELD OF THE INVENTION

The present invention relates to the synthesis and surface crystallisation of re-mineralising novel crystal forms of salts of chlorhexidine containing fluoride together with other ions and uses thereof in Dentistry and Medicine.

BACKGROUND OF THE INVENTION

Chlorhexidine (N,N""1,6-Hexanediylbis[N'-(4-chlorophenyl)(imidodicarbonimidic diamide)] or (1 E)-2-[6-[[amino-[(E)-[amino-(4-chloroanilino)methylidene]amino]methylidene]amino] hexyl]-1-[amino-(4-chloroanilino) methylidene]guanidine) is a bis-biguanide antiseptic and disinfectant with bactericidal and bacteriostatic action against gram-positive and gram-negative bacteria. It is used as a topical, antimicrobial mouth wash or coating to reduce tooth decay. Several patents refer to varnishes (US 4,496,322) containing antimicrobial agents, specifically chlorhexidine diacetate/acetate, a benzoin gum, and a solvent applied to teeth to provide sustained release of the antimicrobial agent, and US 4,883,534 providing a sealing composition, applied to the varnish, to extend the antimicrobial protection of the varnish. There are many reported benefits of using chlorhexidine products for the maintenance of oral hygiene reported in numerous studies in the form of dentifrices, mouth rinses, toothpaste, sprays, and gels etc., (Loe and Harald, eds., In; Supplement No. 16, Vol. 21 , 1986 to the Journal of Periodontal Research, presented articles entitled "Chlorhexidine in the Prevention and Treatment of Gingivitis"). US 4,569,837, "Pharmaceutical Preparation for Remedy of Periodontal Disease and Process for Production Thereof," describes films for insertion in the gingival sulcus which contain and release chlorhexidine gluconate. Although these antibacterial agents such as chlorhexidine digluconate mouth rinses are effective, there are problems with their substantivity, and when bacterial infections are inaccessible, they can be ineffective. For instance, when these antimicrobial agents are unable to penetrate, periodontal pockets produced because of periodontitis and peri-implantitis. The chlorhexidine concentrations used in dental applications can also cause tooth staining which is not desirable.

Patents EP3429990B1 and US10640463B2 provided solutions to these problems by the synthesis of a crystalline salt of chlorhexidine calcium chloride which provided a sustained, pH reactive release or controlled release via light, ultrasound, or magnetic fields, the disclosures of which are incorporated herein by reference. It was able to be dispersed in polymers and gels, encapsulated and spun into fibres and membranes and functionalised with iron and gold nanoparticles. This technology although anti-bacterially effective in maintaining oral hygiene, does not have any tooth re-mineralising properties, which are useful to treat caries and white spot lesions occurring in orthodontic treatment. These lesions develop adjacent to orthodontic brackets and are disfiguring, compromising the overall treatment. Plaque establishment and gingivitis also occur during orthodontic treatment posing problems (Kirschneck et al., 2016. Efficacy of fluoride varnish for preventing white spot lesions and gingivitis during orthodontic treatment with fixed appliances-a prospective randomized controlled trial. Clinical Oral Investigation, 20:2371- 2378). Single application of fluoride varnish in this study however gave no additional prevention advantage compared with sufficient dental hygiene with fluoride toothpaste use. WO 2016/131642 A1 describes a varnish containing chlorhexidine, fluoride cations and bleaching agents as well as numerous other components. This invention necessitates the use of a polymer carrier and time-consuming applications and did not claim tooth remineralisation.

Several publications discussed the use of combining the antibacterial effects of chlorhexidine and inhibitory effects of fluoride in one mouth rinse, and that they were useful in controlling plaque deposition and gingival bleeding (Joyston-Bechal and Hernaman 1993). The effect of a mouth rinse containing chlorhexidine and fluoride on plaque and gingival bleeding. Journal of Clinical Periodontology, Vol. 20,1 , 49 - 53). Fluoride-containing mouth rinses and dentifrices are also effective in caries lesion remineralisation (Parkinson et al., 2018. A randomised clinical evaluation of a fluoride mouth rinse and dentifrice in an in-situ caries model. Journal of Dentistry, 70:59-66). There are several mouth rinses, gels and dentifrices, which contain sodium fluoride and chlorhexidine; they however do not react to produce a crystalline form rapidly onto surfaces, and with a slow chlorhexidine and fluoride release and substantivity. They are readily washed away by the action of the saliva or require multiple time-consuming daily applications and a polymer carrier. WO 2022/189604 A1 describes a chlorhexidine containing toothpaste but without remineralising properties. Toothpastes are however available with high fluoride content (>1000 ppm; EP 3393426 B1) or containing bioactive glasses with high fluoride content and other ions (WO 1997/027148 A1), which induce remineralisation. There are however concerns with the cytotoxicity of fluoride and high fluoride containing toothpastes (Tabatabaei et al., Cytotoxicity of the ingredients of commonly used toothpastes and mouthwashes on human gingival fibroblasts. Front. Dent 2019 Nov-Dec; 16(6):450-457; Jeng et al., Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanisms. Cell Biology and Toxicology. 1998; 14: 383-389). This process also requires that fluoride/ bioglass containing toothpastes are left in-situ after brushing to produce remineralisation and it is often washed away by salivary flow or requiring multiple daily applications. The sites of bacterial infections including root caries, periodontal pockets, interdental contact areas and orthodontic brackets are also often inaccessible to antimicrobial agents/remineralising agents such as toothpastes, gels and varnishes used in the oral cavity or their substantivity limits their effectiveness.

The present invention provides rapid surface crystallisation of novel crystal forms of salts of chlorhexidine containing fluoride in their structure and the unique ability to rapidly bind to the tooth/substrates during synthesis, providing a sustained chlorhexidine and fluoride release and remineralisation of the tooth structure, together with the maintenance of oral hygiene. The fluidity of this novel rinse ensures it accesses and penetrates difficult sites in the mouth leaving a bed of crystallites, with no polymer carrier needed. These crystal formulations have a much lower fluoride content (17-136ppm, Table 5) than current fluoride containing adjuncts and are rapidly delivered and attached to the site of infection with no gross tooth staining.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a crystalline salt of chlorhexidine (CHXD) fluoride having a spherical morphology under Scanning Electron Microscopy (SEM) and comprising a fluoride anion and a cation selected from the group consisting of sodium, ammonium, potassium, or tin. There is also provided the synthesis of the crystalline salt of chlorhexidine (CHXD) fluoride.

The crystalline salt of chlorhexidine fluoride may preferably further comprise a chloride anion and a cation selected from the group consisting of sodium, zinc, magnesium, or strontium. Preferably, the cation may be sodium or zinc, more preferably sodium.

There may also be provided a crystalline salt of chlorhexidine ammonium, tin, potassium or sodium fluoride/sodium, zinc, magnesium, or strontium chloride having a spherical morphology under Scanning Electron Microscopy (SEM). In other words, the salt may comprise either fluoride or mixed fluoride/chloride anions, and a cation selected from the group consisting of sodium, ammonium, zinc, magnesium, potassium, strontium, or tin.

Chlorhexidine is a cationic polybisguanide and the formal IUPAC name is A/, /V""1, 6- Hexanediylbis[/V'-(4-chlorophenyl)(imidodicarbonimidic diamide)]. It is sometimes referred to as 1 , 6-bis(4-chloro-phenylbiguanido) hexane. Prior art uses as an antibacterial agent the dihydrochloride, diacetate or digluconate salt forms.

References to cations selected from the group consisting of calcium, sodium, potassium, ammonium, magnesium, zinc, tin, or strontium include references to Ca²⁺, Na⁺, K⁺, Mg²⁺ Zn²⁺, NH₄ ⁺, Sn²⁺, Sr²⁺, respectively.

Examples of a crystalline salt of chlorhexidine (CHXD) chloride/fluoride of the invention having a spherical morphology under SEM are shown in Figures 1-4 and Figures 11 , 12, 19, 23, 24. These figures show a morphology that could also be described as dendritic, i.e. , having crystalline dendrites of a branched form. Accordingly, the crystalline salt of chlorhexidine according to the invention may preferably have a spherical and dendritic morphology under SEM.

Crystals of the crystalline salt of chlorhexidine chloride/fluoride of the invention having a spherical morphology under SEM are preferably monodisperse in form.

Preferably, the cation may comprise sodium and the crystalline salt has an X-ray diffraction pattern comprising peaks, in terms of 2-theta, at about; 5.2, about 8.4, about 9.8, about 12.2, about 12.7, about 13.3, about 14.0, about 15.6, about 15.8, about 16.1 , about 18.1 , about 18.6, about 19.0, about 19.7, about 20.0, about 20.5, about 20.8, about 21.6, about 22.7, about 23.7, about 24.7, about 25.5, about 26.1 , about 26.5, about 28.8, about 28.9, about 29.8, about 31.2, about 31.7, about 34.2, about 34.3, about 35.4, about 38.8, about 38.9, about 44.3, about 45.5, about 56.1 degrees.

Preferably, the crystalline salt comprises a cation that is sodium and may have an X-ray diffraction pattern comprising main peaks, in terms of 2-theta, at about 7.48, about 12.77, about 14.99, about 16.96, about 20.43, about 20.84, about 22.25, about 22.65, about 22.90, about 23.25, about 27.08, about 27.79 and about 38.87 degrees. Preferably, the crystalline salt wherein the cation comprises ammonium may have an X-ray diffraction pattern comprising main peaks, in terms of 2-theta, at about; about 8.48, about 12.67, about 13.34, about 15.73, about 15.79, about 18.16, about 18.09, about 18.57, about 20.02, about 20,58, about 20.52, about 25.53, and about 26.51 degrees.

Preferably, the crystalline salt wherein the cation comprises sodium or ammonium may have X-ray diffraction (XRD) patterns shown in Figures 9 and 20 and 22. The 2 theta peaks are shown in Tables 7, 8 and 9.

Examples of a crystalline salt of chlorhexidine chloride/fluoride having a spherical morphology under SEM are shown in Figures 1 , 2, 3, 4, 11 , 12, 19, 23, 24.

Unless stated otherwise, X-ray diffraction analysis is performed from 5 to 70° 2-theta, with a step size of 0.0334° and a count time of 200 s, and Ni-filtered Cu-K_a radiation at wavelengths of 0.1540598 nm and 0.15444260 nm is used. A reflection mode, divergence slit, flat plate 0/0 geometry may also be used.

The XRD pattern for the chlorhexidine chloride/fluoride of the invention can be compared to that for chlorhexidine diacetate (Figures 9, 10 and 21). The XRD indicates crystal structural differences in the crystalline salt of chlorhexidine chloride/fluoride of the invention compared to chlorhexidine diacetate. The differences include the provision of new peaks and peak broadening in the XRD pattern in comparison to chlorhexidine diacetate.

The crystalline salt of chlorhexidine chloride/fluoride of the invention may have a particle diameter range of from about 3 to about 50pm, suitably from about 4pm to about 30 pm. Crystal synthesis and size are responsive to the dispersion of inclusions and surface roughness/flaw size.

Crystalline forms of chlorhexidine in accordance with the present invention provide controlled release or delayed (slow) release forms of chlorhexidine and fluoride. According to a second aspect of the invention, there is provided a process for the preparation of monodisperse crystals comprising; (a) chlorhexidine/sodium fluoride (b) chlorhexidine sodium chloride/sodium fluoride; (c) chlorhexidine zinc chloride/sodium fluoride; (d) chlorhexidine sodium chloride/potassium fluoride; (e) chlorhexidine sodium chloride/tin fluoride; or (f) chlorhexidine sodium chloride/ammonium fluoride salts of the invention, comprising; (i) mixing and dissolving a first aqueous solution of chlorhexidine acetate with a second aqueous solution of a sodium, ammonium, zinc, potassium or tin fluoride, the second aqueous solution at a concentration from about 0.1M to about 1.0M, and a third aqueous solution of sodium or zinc chloride at a concentration of 0.1 M to 1.0M;

(ii) allowing the CHXD-NaF, CHXD-NaCI/NaF, CHXD-ZnCh/NaF, CHXD-NaCI/KF, CHXD-SnF2/NaCI, or CHXD-NaCI/NF F salts to precipitate;

(iii) centrifuging the precipitate formed in (ii) to obtain a solid mass of precipitated salt crystals; and

(iv) washing the precipitated solid mass of (iii).

It will be appreciated that the three aqueous solutions in step (i) can be mixed and dissolved in any order.

The above process may use a metal chloride of the formula MCI_X, where x is equal to 1 or 2, and M is selected from the group consisting of Ca²⁺, Na⁺, K⁺, Mg²⁺, Zn²⁺, NH4⁺, Sn²⁺, and Sr²⁺. The preparation of crystalline salts of chlorhexidine chloride including the cations Ca²⁺, Na⁺, K⁺, Mg²⁺, Zn²⁺, and Sr²⁺ is described in EP3429990 B1 which is incorporated herein by reference.

The concentration of the chlorhexidine acetate in the first aqueous solution may be from about 0.3mg/ml, or from about 5mg/ml to about 50mg/ml, or preferably about 10mg/ml to about 40mg/ml, suitably about 15 mg/ml.

The concentration of the metal fluoride ion in the second aqueous solution may be from about 0.05M to about 1.00M, or from about 0.30M to about 0.80M, preferably from about 0.45M to about 0.70M.

The concentration of the metal chloride in the third aqueous solution may be from about 0.07M to about 1M, or preferably from about 0.5M to about 0.7M.

The concentration of the metal chloride may suitably be about 0.66M and the concentration of the chlorhexidine acetate may be about 15mg/ml.

Sodium Fluoride (NaF), Potassium Fluoride (KF), Ammonium Fluoride (NF F) or Tin Fluoride (SnF2) may be suitable metal fluoride salts for use in this process. The molar ratio (mol/l) of metal chloride to metal fluoride in the above process may be from about 2:0.215 to about 2:2, suitably from about 2:0.215 to about 2:1 , more suitably from about 2:0.25 to about 2:0.5. The metal chloride may be sodium chloride and the metal fluoride may be sodium fluoride, and the molar ratio of metal chloride to metal fluoride may be from about 2:0.75 to about 2:2.

The crystalline salt may comprise from about 5 wt.% to about 25 wt.%, suitably from about 7 wt.% to about 15 wt.% of fluoride according to EDS elemental analysis.

Suitably, the crystalline salt releases from about 17 ppm to about 140 ppm of fluoride (F-), measured by dissolving in distilled water and using an F- Ion Selective Electrode (ISE) (ELIT 9808, 8 Channel Ion-Analyser, NICO 2000 LD, 7.2.84sa, UK).

Crystalline chlorhexidine chloride prepared by this process may have a particle size distribution of a mean (SD) diameter of about 11.0 to about 16 pm, suitably of from about 11.0 to about 13 pm.

The present invention provides a crystalline chlorhexidine chloride/fluoride salt prepared by a process of the invention as defined above. In this process, controlled crystallisation (nucleation and crystal growth) of the chlorhexidine chloride/fluoride salts (particles) can be achieved by the introduction of nuclei in the form of; emulsions, colloids, micro and nanoscale inorganic/ metallic oxides, to change the size, number, and morphology of the synthesized particles. Synthesis of the particles onto materials with different surface roughness will also have this effect.

According to a third aspect of the invention, there is a rapid surface crystallisation of CHXD- NaF/NaCI or CHXD-CaCh crystals in Example 2 and crystal variants described in Examples 1 , 3, 4 and 5 when used as a mouth rinse delivered via the application/mixing of two solutions. These solutions can be delivered via a pipette, emersion techniques or using bottled sprays. The particle synthesis is a low energy pH driven process requiring no energy input and is rapid (0.1 -15 seconds (secs) depending on the reagents and molarities used).

CHXD-NaF/NaCI and CHXD-CaCh crystals and crystal variants identified herein can be deposited onto biological/biomaterial surfaces via the rapid growth of dendritic tendrils onto the tooth/bone or biomaterial substrates including; polymers, glass-ceramics, ceramics, composites, and metals (titanium, stainless steel, chrome cobalt). The crystalline salts of chlorhexidine identified herein may advantageously rapidly bond to tooth and biomaterial surfaces. These novel coatings provide a sustained CHXD/Fluoride localised release, with the potential to maintain oral hygiene/prevent tooth decay at the required site. Novel particles in the above invention may also be rapidly deposited onto Personal Protective Equipment such as surgical gloves and masks to extend their antibacterial effectiveness.

Surface crystallisation of the particles is effective for natural and synthetic fibres (cellulose, cotton, polyurethane and nylon) for filters, wound dressings, socks, catheters, contact lenses, blood bags, packaging, and polymer films for biomedical/commercial applications.

The surface crystallisation of the current invention allows CHXD-NaF/NaCI crystals and variants to be coated onto human bone, exposed dental implant surfaces, dentures and tooth cavities or root canals prior to filling. This is particularly useful in the treatment of periimplantitis, denture stomatitis, root canal treatment and dental carries treatment, prevention, and tooth remineralisation.

According to a fourth aspect of the invention, CHXD-NaF/NaCI and CHXD-CaCh mouth rinses and crystalline variants can be applied weekly to teeth without encountering gross tooth staining and encouraging maintenance of oral hygiene. The crystalline salts of chlorhexidine identified herein advantageously do not cause gross tooth staining, preferably when applied weekly to teeth. Thus, reducing treatment time due to stain removal needed in current chlorhexidine mouth rinse products.

According to a fifth aspect of the invention a process of precipitating fluorapatite via precipitation of a CHXD-NaF/NaCI crystals (or crystal variants in Examples 1-5) to the tooth surface (via a mouth rinse) and allowing a sustained fluoride release to allow tooth remineralisation and maintain oral hygiene. The crystalline salts of chlorhexidine identified herein may advantageously cause the precipitation of fluorapatite at a surface when applied to teeth. This aspect is beneficial in the treatment and reversal of dental carries.

According to a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a crystalline chlorhexidine fluoride/chloride salt of the invention. The pharmaceutical compositions of the invention may be administered in any effective, convenient manner effective for treating a patient’s disease including, for instance, administration by oral, topical, intranasal, or intradermal routes, among others. In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for Example as a sterile aqueous dispersion, preferably isotonic.

For administration to mammals, and particularly humans, it is expected that the daily dosage of the active agent will be from 0.01 mg/kg up to 10mg/kg body weight, typically around 1mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependent on factors including the age, weight, sex, and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention.

The pharmaceutical compositions of the invention may be employed in combination with pharmaceutically acceptable carrier or carriers. Such carriers may include but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, ethanol and combinations thereof.

According to a seventh aspect of the invention, there is provided crystalline chlorhexidine fluoride/chloride salts according to the invention as defined above, and the pharmaceutical compositions comprising the same, for use in medicine and dentistry. Medical uses in accordance with the present invention extend to and include use in a method of treatment of a disease or medical condition as defined herein. Such methods of treatment comprise the step of administering a composition of the invention to a subject in need thereof. The invention also includes uses in the manufacture of a medicament for use in the treatment of such diseases or conditions.

The crystalline chlorhexidine salts according to the invention as defined above, and the pharmaceutical compositions comprising the same, may be for use in treating bacterial infections including root caries, periodontal pockets, interdental contact areas and orthodontic brackets. In particular, the crystalline chlorhexidine salts according to the invention as defined above, and the pharmaceutical compositions comprising the same, may be for use in treating dental caries and periodontal disease.

According to an eighth aspect of the invention, the crystalline chlorhexidine fluoride/chloride salts may be dispersed into several carriers. These can include polymerising (via heat, UV light or chemical cure) into polymerizable methacrylate monomers (comprising initiators) including hydroxyethyl methacrylate (HEMA), urethane dimethacrylate (HEMA-UDMA), or polymethylmethacrylate (PMMA). They may also be processed into fibres using electrospinning to form a mat, web, or substrate suitable for use as a membrane or bandage. Novel particles may also be encapsulated in multiple layers of the polyelectrolyte and/or polymer containing a crystalline chlorhexidine fluoride/ chloride salt of the invention. The composition comprising a crystalline chlorhexidine salt according to the invention as defined above may be encapsulated or suspended in a polyelectrolyte, or in a polymerizable monomer.

Further, the composition comprising a crystalline chlorhexidine salt according to the invention as defined above may be in the form of a mouthwash, toothpaste, gel, or polymer. Preferably, the mouthwash, toothpaste, gel, or polymer is for use in a medical method and/or a cosmetic method as defined herein.

Where the composition is in the form of a mouthwash, toothpaste, gel, or polymer, the composition may comprise from 0.06 to 2 wt.% of the crystalline chlorhexidine salt relative to the total weight % of the composition.

Where the composition is in the form of a mouthwash, the composition may comprise from about 0.06 to about 0.2 wt.% of the crystalline chlorhexidine salt relative to the total weight % of the composition.

Where the composition is in the form of a toothpaste or a gel, the composition may comprise from about 0.6 to about 1.2 wt.% of the crystalline chlorhexidine salt relative to the total weight % of the composition.

The mouthwash, toothpaste, gel, or polymer may comprise additives, including but not limited to one or more of a sweetener, a stabilizer, and a colouring agent.

According to the ninth aspect of the invention, there is provided chlorhexidine fluoride salt or variants that can be freeze-dried to produce powders, which can be incorporated into polymerizable dental/medical polymers or gels. They can also be used as a tooth/ root canal filling material, to fabricate denture bases or polymerised to a solid state using various methods. The polymerized polymer provides a matrix for the controlled/sustained release of chlorhexidine and fluoride from the compounds. These powdered compounds can similarly be incorporated into dental cement (including; glass ionomers/resin modified versions, light-cured resins, zinc phosphate or phenolate cement), or prophy/prophylaxis pastes, toothpaste, light-activated fissure sealants and gels and membranes for Dental/Medical applications.

In other aspects of the invention, there is provided the surface crystallisation of chlorhexidine fluoride/chloride salts (CHXD-NaF/NaCI and CHXD-CaCh) as described herein for use in the form of an aqueous composition, e.g. as a mouth rinse, on teeth, bone and/or biomaterial substrates including; polymers, glass-ceramics, ceramics, composites, and metals (titanium, stainless steel, chrome cobalt) to provide sustained and localised CHXD/Fluoride drug release.

The present invention provides a process of precipitating fluorapatite via application of a CHXD-NaF/NaCI mouth rinse or variants as described herein to effect tooth remineralisation and maintain oral hygiene.

As described herein, CHXD-NaF/NaCI and CHXD-CaCh mouth rinses which can be applied weekly without gross tooth staining, reducing treatment time for stain removal with current products and encouraging maintenance or oral hygiene.

Preferred aspects for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be described by way of reference only with reference to the following Examples which are present for the purposes of reference only.

BRIEF DESCRIPTION OF THE FIGURES

In the Examples the following Figures are present in which:

Figure 1 shows SEM images of CHXD-NaCI-NaF crystals containing (a) OM NaCI/ 0.33M NaF; (b) 0.033M NaCI/ 0.297M NaF; (c) 0.165M NaCI/ 0.165M NaF; (d) 0.198M NaCI/ 0.132M NaF (e) 0.297M NaCI/ 0.033 M NaF; (f) 0.33M NaCI/ OM NaF.

Figure 2 shows SEM images of CHXD-NaCI-NaF crystals containing (a) 0.33M NaCI/ 0.165 M NaF; (b) 0.33M NaCI/ 0.25 M NaF; (c) 0.33M NaCI/ 0.33 M NaF.

Figure 3 shows (a-f), SEM images showing CHXD/NaCI-NaF crystals containing; (a) 0.66M NaCI/0.07M NaF; (b) 0.66M NaCI/0.0825M NaF; (c) 0.66M NaCI/0.165M NaF; (d) 0.66M NaCI/0.33M NaF; (e) 0.66M NaCI/ 0.5M NaF; (f) 0.66M NaCI/ 0.66M NaF.

Figure 4 shows EDS results of the CHXD/ NaCI-NaF (0.66M NaF); (a) spectrum position (b) the EDS Spectra. Figure 5 shows mean CHXD (SD) release for the CHXD/ NaCI-NaF particles.

Figure 6 shows cumulative CHXD release for the CHXD/ NaCI-NaF particles.

Figure 7 shows fluoride content for the CHXD/NaF-NaCI particles.

Figure 8 shows (a) ISE calibration curve for the F- standards; (b) Cumulative Fluoride release for CHXD/ NaCI-NaF particles.

Figure 9 shows XRD plots showing the; (a) CHXD; (b) 0.66M NaCI/0.07M NaF; (c) 0.66M NaCI/0.0825MNaF; (d) 0.66M NaCI/0.165M NaF; (e) 0.66M NaCI/0.33M NaF; (f) 0.66M NaCI/ 0.5M NaF; (g) 0.66M NaCI/ 0.66MNaF.

Figure 10 shows XRD plots showing overlapped patterns for CHXD and CHX/NaCI -NaF particle groups.

Figure 11 shows SEM images showing the surface crystallisation of CHXD/NaCI - NaF crystals attached to the surface of; (a) teeth; (b) a PMMA denture base; (c) an orthodontic bracket; (d) lithium disilicate crown; (e) YTZP crown and (f) Titanium Leonard button.

Figure 12 shows SEM images showing the surface crystallisation of CHXD-CaCh crystals attached to the surface of; (a, b) smooth and rough implant surfaces; (c, d) a PMMA denture base and attached dendritically; (e) an orthodontic bracket; (f) tooth and composite surfaces; (g) surgical gloves surfaces; (h) surgical mask surfaces.

Figure 13 (a) Digital photos showing instantaneous reaction of CHXD (15mg/ml) with 0.66M NaCI/0.0825M NaF; and (b) CHXD (15mg/ml) with CaCI₂ (0.33M).

Figure 14 shows CHXD released from a CHXD/0.33M NaCI/ 0.33M NaF coated teeth (n=3) in artificial saliva (PH=7) showing sustained drug release (MIC <2.5ppm after 11 days indicated by yellow triangle).

Figure 15 shows Fluoride released from a CHXD/0.33M NaCI/ 0.33M NaF coated teeth (n=3) in artificial saliva (PH=7) showing sustained release. Figure 16 shows CHXD released from a CHX-CaCh coated orthodontic bracket (n=3) in artificial saliva (PH=7) showing sustained drug release (MIC <2.5ppm after 11.5 days indicated by yellow triangle).

Figure 17 shows CHXD released from a CHXD/0.66M NaCI/ 0.0825M NaF coated teeth (n=3) in artificial saliva (PH=7), showing sustained drug release (MIC <2.5ppm after 7 days indicated by yellow triangle).

Figure 18 shows Fluoride released from a CHXD/0.66M NaCI/ 0.0825M NaF coated teeth (n=3) in artificial saliva (PH=7) showing sustained release.

Figure 19 shows SEM images of NaF reacted with CHXD solution at different reaction times; (a) 40 mins; (b) 60 mins; (c) 80 mins; (d) 100 mins; (e) 110 mins; (f) 120 mins.

Figure 20 shows XRD plot for the CHXD/NaF (0.96M) particles.

Figure 21 shows XRD plots showing overlapped patterns for CHXD and the CHX- NaF (0.96M NaF) particle.

Figure 22 shows XRD plot for the CHXD/NH4F/NaCI particles (0.33M NH4F/0.33M NaCI).

Figure 23 shows SEM image of CHXD/NH4F/NaCI crystals containing (0.33M NH4F/0.33M NaCI).

Figure 24 shows SEM images of CHX/ZnCh/NaF crystals containing (a) 0.33M ZnCI₂/0.125M-NaF; (b) (0.33M ZnCI₂/0.25M NaF); (c) CHXD/KF/NaCI crystal containing (0.33M KF/0.33M NaCI); (d) CHXD/SnF₂/NaCI crystal containing (0.33M SnF₂/0.33M NaCI).

Figure 25 shows a bar chart showing differences between tooth staining groups.

Figure 26 shows digital images of CHXD-CaCI₂ Group 1 ; (a) Day 1; (b) after 14 Days in artificial saliva after CHX-CaCI₂ and tea application. Figure 27 shows digital images of CHXD/NaCI/NaF Group 2; (a) Day 1 ; (b) after 14 Days artificial saliva after CHXD/NaCI/NaF and tea application.

Figure 28 shows digital images of Corsodyl Group 3; (a) Day 1 ; (b) after 14 Days in artificial saliva after Corsodyl and tea application.

Figure 29 shows digital images of Control Group 4; (a) Day 1 ; (b) after 14 Days of Distilled water and tea application.

Figure 30 shows 19F MAS-NMR spectra of the Hydroxyapatite disc after coating with CHXD/NaF/NaCI mouth rinse and emersion in Artificial Saliva for 2 weeks.

Figure 31 shows hydroxyapatite discs; before coating (a) and after coating with CHXD/NaF/NaCI mouth rinse and emersion in artificial saliva after; (b) 1 week, (c, d) 2 weeks.

Figure 32 shows XRD plots indicating the shift of the hydroxyapatite to fluorapatite phases for the HA/demineralised discs and the CHXD/NaF-NaCI coated HA discs.

Figure 33 shows effects of; a) NaF; b) CHXD and c) CHXD/NaF-NaCI solutions on the DNA proliferation of the L929 fibroblast cells at 24, 48 and 168 hours. All treated groups were normalised to the control groups. A single asterisk “*” indicates significant differences (p<0.05), whereas two asterisks “**” indicates highly significant differences (p<0.001) between tested and control groups.

Figure 34 shows antimicrobial MTT assays on S. Mutans at 24 hours.

Figure 35 shows antimicrobial MTT assays on P. Gingivalis at 24 hours. DETAILED DESCRIPTION OF THE INVENTION

Example 1

Novel CHXD/NaCI/NaF particles were synthesised by precipitation of chlorhexidine diacetate (CHXD) (Sigma, Lot: WXBD1046V) with NaF (Sigma, S7920, Lot: SLCB1955) and NaCI (Sigma, 746398, Lot: SLCC4856). The concentration of the CHXD was 15mg/ml. Solutions of NaCI and NaF with various ratios were synthesised according to Table 1. All (7) solutions were reacted with the CHXD solution in 2 ml tubes (Eppendorf, UK) at 1 : 1 by volume at room temperature. The mixtures were reacted for 1 min and then centrifuged at 2400 rpm for 1 min (Eppendorf centrifuge 5417C, Germany). Turbidity was evident in all samples on mixing at (0.01-0.5 seconds (secs), Figure 13a). The supernatants of each tube were removed, and the precipitates were washed once with 1 ml of deionised water. Following that, the tubes were centrifuged again at 2400 rpm for another 1 min. The deionised water excess was removed from each tube and subsequently, all tubes were placed in nitrogen liquid for 30 minutes. Thereupon, the lid of the tubes was removed, and the tubes were covered with parafilm (Sigma-Aldrich, UK) and placed in the Freeze-dryer (ScanVac 39336805, Labogene, Germany) at -110°C and 4*10-4 mbar for 24 hours. Powder specimens synthesised from Table 1 and other formulations were characterised using scanning electron microscopy (FEI Inspect F, Nano Port, Eindhoven, and The Netherlands) in the secondary electron imaging mode. The accelerating voltage used was 10 kV, spot size was set at 3.0.

Table 1. Ratios of the compounds for synthesis of novel CHXD/ NaCI-NaF particles.

Varying ratios from 0 to 0.33M for both reagents (NaCI and NaF) were also carried out before mixing with CHXD, which resulted in agglomerates (Figs. 1a-b), partially formed crystallites (Figs. 1c) and crystal spheres which increased with NaCI content (Fig 1 d-f). Keeping NaCI content constant at 0.33M and mixing with 0.165-0.33M NaF and reacting with CHXD resulted in partially or fully formed crystal spheres (Figure 2 a-c).

Increasing NaCI to 0.66M resulted in well-formed crystal sphere particles throughout the NaF compositional range (Figures 3a-f). Therefore, formulations in Table 1 of the invention gave the required spherical crystal morphology which may affect the solubility and drug release efficacy. Figures 3 a-f illustrates novel CHXD/NaCI-NaF particles showing a spherical morphology.

Table 2. Particle size of the novel CHXD/ NaCI-NaF particles

The CHXD/NaF-NaCI (0.66M NaF) was analysed using energy-dispersive X-ray spectroscopy (EDS). A powder sample was carbon coated in an automatic sputter coater (Agar Scientific Ltd, U.K.) under vacuum for 60 seconds. The sample was analysed in an SEM (FEI Inspect F, NanoPort, Eindhoven, The Netherlands) using 20 kV, spot size set at 5.0, and the working distance of 8-10 mm. Quantitative elemental analysis indicated 11.13 wt.% fluoride ions incorporated in the particle (Table 3, Fig. 4a, b). It is noteworthy that the CHX-NaF-NaCI particles were washed with deionised water to remove unreacted elements and any physically absorbed divalent metal ions prior to this analysis.

Table 3. EDS elemental analysis for the CHXD/NaF-NaCI (0.66M NaF) particle.

The CHX release assay of the CHXD/ NaCI/NaF particles (Table 1) was measured using 0.1g of particles in a 2 ml tube (Eppendorf, UK) containing 2 ml of artificial saliva. The CHXD concentration of each sample was measured using UV-Vis Spectroscopy (Lambda 265, PerkinElmer, USA) at a wavelength of 254 nm at specific time points (Table 4). At each time point, the artificial saliva was replaced with fresh saliva before storage in an incubator (Benchmark Scientific, Inc., New Jersey, USA) at 37°C. The absorption measurement was used to calculate the CHXD release using the calibration curve (r²= 0.999) and the equation: y = mx+ b; where m is the slope and b is the intercept. The CHXD release of the CHXD/NaCI/NaF particles shows the Mean (SD) and the Cumulative CHXD Release plots in Figures 5 and 6, demonstrating a sustained CHXD release. The F- concentration of the synthesised CHXD/NaCI/NaF (Table 1) and the supernatant was measured using an F- Ion Selective Electrode (ISE) (ELIT 9808, 8 Channel Ion-Analyser, NICO 2000 LD, 7.2.84sa, UK). The calibration procedure used F’ standards, with serial dilutions of 0.5, 1 , 10, 100 and 1000 ppm ion-standards. The ISE calibration curve demonstrated the F’ Molar concentration (mol/L) was highly correlated with estimated mV of the F’ standards (Fig. 8a, r²=0.99). Aliquots from the artificial saliva samples utilised in the previous Uv-vis testing were measured at specific time points collected. The F’ concentration (ppm) of selected CHXD/NaCI/NaF samples plotted versus time (days) is shown in Figure 8b, showing a sustained release of F'from 2-10 ppm release after 21 days. The F’ content in the residual supernatant is shown in Table 6, indicating it was possible to control the residual F’ content for cosmetic regulatory and other therapeutic applications.

CHXD/NaCI/NaF particle (0.03g) samples (0.07, 0.0825, 0.165, 0.33, 0.5, 0.66 mol/L NaF) were also dissolved in distilled water and ISE measurements indicated F’ available in the particle groups (Table 5, Figure 7).

In particular, the 0.33M NaF particle group demonstrated an effective and sustained release of both CHXD (11 days above the MIC) and F’ after 21 days (Figures 14, 15). The current invention therefore provides a particle with the sustained and synergistic antibacterial effect of F’ and CHXD, and the availability of F’ for tooth remineralisation as demonstrated in Example 7. The CHXD/NaF/NaCI particles afford a unique combination in a single vehicle which will provide unique clinical advantages. The particle synthesis is a low energy process which requires no energy input as the reaction is partially pH driven. Table 4. CHXD release schedule.

Table 5. Fluoride content of the novel CHXD/ NaCI/NaF particles.

Table 6. Fluoride content of the novel CHXD/NaCI/NaF particles after reaction.

Synthesised CHXD/NaCI/NaF particles (Table 1), were characterised using X-ray diffraction. Powder samples were analysed in an X-ray diffractometer (Xpert Pro X-ray Diffractometer, Malvern Panalytical Ltd., The Netherlands). A reflection mode, divergence slit, flat plate 0/0 geometry and Ni-filter Cu-Ka radiation were used (A1 = 0.1540598 nm and A2 = 0.15444260 nm). The powdered samples were scanned in a 20 range between 5° and 70°. A step size of 0.0334° was used, with a step time of 200 s. The CHXD/NaCI/NaF particles produced changes in 2 theta positions (missing peaks, peak shifts, peak broadening and new peaks) compared to the chlorhexidine diacetate powder (Figs. 9a-f). XRD peaks and intensities are listed in Table 7, with many similar peaks present across sample groups in particular about; 5.2, 8.4, 9.8,12.2, 12.7, 13.3, 14.0, 15.6, 15.8,16.1 , 18.1 , 18.6,19.0,19.7, 20.0, 20.5, 20.8, 21.6, 22.7, 23.7, 24.7, 25.5, 26.1 , 26.5, 28.8, 28.9, 29.8, 31.2, 31.7, 34.2, 34.3, 35.4, 38.8, 38.9, 44.3, 45.5, 56.1 degrees 2 theta. The data for the spherical CHXD/NaCI/NaF particles and chlorhexidine diacetate was overlapped to illustrate distinct differences in peaks/peak positions, intensities (Fig. 10, Table 7) indicating crystal structural differences for the CHXD/NaCI/NaF particles compared with chlorhexidine diacetate. Table 7. XRD data for the novel CHXD/NaCI/NaF particles.

Table 7 continued: XRD data for the novel CHXD/NaCI/NaF particles.

Example 2

This Example demonstrates the surface crystallisation of the novel CHXD particles onto teeth/bone and biomaterial substrates using a mouth rinse. Teeth/bone or substrates including polymers, titanium implants, stainless steel orthodontic brackets/wires, yttria stabilised zirconia polycrystalline (YTZP) ceramics, lithium disilicate glass-ceramics or composite prostheses can be copiously coated with CHXD-CaCh or CHXD/NaCI/NaF particles or crystalline variants containing Zinc, ammonium, Strontium, copper, Tin, potassium, gold, silver, or iron nanoparticles. Various methods can be used to deliver these crystalline coatings via rinses including pipetting, sprays, and immersion techniques.

In particular; for the coating teeth/orthodontic brackets or prostheses, 1 ml of the chlorhexidine diacetate (15mg/ml) (CHXD) (Sigma, Lot: WXBD1046V) is reacted with 1 ml of NaCI/NaF (0.33 or 0.66M NaCI/0.33M NaF) (NaF: Sigma, S7920, Lot: SLCB1955) (NaCI: Sigma, 746398, Lot: SLCC4856) for 10 seconds in a 2ml tube, followed by immersion in the pre-reacted solution for 1 minute which produces spherical CHX/NaCI/NaF crystals attached dendritically to the material surfaces (Fig. 11 a-f). For coating orthodontic brackets, a more directed method using a pipette may be used for a more precise rinse application. 1 ml of the 15mg/ml CHX is reacted with 1 ml of NaCI/NaF (0.33 or 0.66M NaCI/0.33M NaF) for 10 seconds in a 2ml tube (Eppendorf, UK). This formulation prepared using 0.66 NaCI reacted with 0.33 NaF (2:1 ratio-Table 1) increases the speed of reaction to 0.01-0.5 secs (Figure 13a). The pre-reacted solution is next applied to orthodontic bracket by applying 2 drops (0.18 pl) using a pipette and left for 1 minute (Fig. 11c). SEM images indicate copious crystallisation of CHXD/NaCI/NaF particles on tooth surfaces (Fig. 11a), polymethyl methacrylate (PMMA) denture bases (Fig. 11 b) lithium disilicate glass-ceramics (Fig. 11 d), yttria stabilised zirconia polycrystalline ceramics (Fig. 11e) and titanium Leonard button surfaces (Figs. 11 f). There is a surface crystallisation and crystallite size effect associated with and responsive to different material surfaces and flaws. The crystallites are attached to the surface via root like extended tendrils (Fig. 11a-f) which grow rapidly at room temperature.

Alternately emersion in chlorhexidine diacetate (15mg/ml) for 1 minute followed by immersion in CaCh (0.33M) for 1 minute produces spherical CHX-CaCh crystals rapidly attached to the tooth, prosthesis surfaces or surgical gloves and masks (Fig 12 a-h). The surface crystallisation and crystallite size precipitated are again related to the surface roughness of the prosthesis (Figs. 12 a, c, d). A more bimodal crystal size was evident for PMMA surfaces, (Fig. 12c) and with rapid particle growth (0.3-seconds, Fig. 13b) of tendrils onto the material surface (Fig. 12d). The invention allows the copious precipitation of spherical crystallites across the tooth, composite and their respective interfaces attached via radiating root-like tendrils (Fig. 12f).

These solutions can also be added via emersion techniques, via a pipette at the volume ratio of 1 :1 or using bottled sprays. These processes produce rapid turbidity of the solutions (0.3 secs for CHX- CaCh, Fig. 13b) or from 0.01-15 secs (CHX/NaCI/NaF) at room temperature, which requires limited energy input to synthesise spherical particles. The synthesis reaction for the CHX/NaCI-NaF crystallisation can be increased by increase of NaCI content (0.66) to 0.01-0.5 secs (Figure 13 b). pH measurements of the solutions (n=3 per solution) were carried out using a pH meter (MP 220, Mettler, Toledo) and pH electrode (pH-2011 , ELIT, UK) at 25.0°C. Calibration was using pH 7.00 (Orion 910107, Thermo Scientific, UK) and pH 4.01 buffer calibration solutions (Orion 910104, Thermo Scientific, UK). The Mean (SD) pH of the solutions was; 7.25 (0.04) for CHXD 15 mg/mL; 6.25 (0.07) for NaCI (0.66 M); 8.66 (0.06) for NaF (0.66M); 7.83 (0.02) for NaF (0.33 M); 6.83 (0.06) for NaCI (0.33M) and 5.53 (0.02) for CaCI₂ (0.33M). The pH solution differences are in part responsible for this reactive crystallisation and producing a lower solubility solute, with higher concentration and allowing copious surface crystallisation as demonstrated in Figs. 11 and 12. The pH differences are significant since the pH scale is logarithmic (1 pH unit increase leads to tenfold increases in H⁺ concentration).

The above invention allows the sustained release of CHXD and fluoride at the required site. In particular, the following describes either CHXD-CaCh or CHXD/NaCI/NaF solutions (0.33M for both NaCI and NaF) (1 : 1 ratio) reacted in a tube (5ml, Eppendorf, UK) with CHXD for 10 seconds. This synthesis can also be carried out for all formulations in Table 6 in order that the residual F’ content can be controlled after reaction (in particular, CHXD/0.66M NaCI- 0.0825M NaF). Human molars (n=3) were stabilised onto the tube’s lid and inserted into the tubes and their surfaces fully immersed into the mouthwash for 1 minute.

For the orthodontic samples human teeth (n=3) were air-dried (20 secs) and etched using an etching gel (super etch SDI, Australia) for 10 secs, then rinsed with water (20 secs) and lightly blow-dried for 10 secs with an airline. Bonding agent (Ortho Solo™, USA), was applied using a micro-brush, followed by 10 secs air-drying and light curing for 20 secs (3TECH led-1007 Curing light, UK) in the gradual mode. After curing, the orthodontic bracket (Victory Series, 3M Unitek, Monrovia, Calif. USA) was placed in each labial tooth surface, and the excess bonding cement was removed using a dental probe. The bracket was cured using a light gun in gradual mode on brackets top side and bottom side for 20 secs for each position. The wires (Nitinol Super-Elastic, USA) and elastics (Elast-0 loop Ligatures, DB03-0040, Dbortho, UK) were placed on the brackets. 15mg/ml of CHXD solution was syringed (Terumo, Japan) onto the surface of the brackets using a syringe (Terumo, Japan) and a 30ga bendy-tip (Schottlander, UK) and left for 60 seconds, followed by 20ul of 0.33M CaCh left for 60 seconds.

Coated teeth with and without brackets (n=3 per experiment) were placed in 2ml of artificial saliva and stored in an incubator (Benchmark Scientific, Inc., New Jersey, USA) at 37°C. Samples were measured twice daily using UV-Vis Spectroscopy (Lambda 265, PerkinElmer, USA) at a wavelength of 254 nm. At each time point, the artificial saliva (pH 7) was replaced with fresh saliva before storage. For UVA/is calibration, serial dilutions of CHXD (Chlorhexidine Diacetate) were run and used to plot the standards calibration curve.

Figures 14 and 16 demonstrate the sustained release of CHXD from the particle-coated teeth and orthodontic brackets for 11 days (above minimum inhibitory concentration (MIC) of 2.5ppm). This contrasts with a commercial mouth rinse (Corsodyl Daily GSK, UK) which was identically evaluated using the same methods and was below MIC (2.5ppm) on day 1. Figures 15 and 18, demonstrate the sustained release of fluoride from particle coated teeth using CHXD/NaCI/NaF particle formulations (with tailored NaF concentrations).

This process can also be demonstrated on a range of biomaterials to give a sustained CHXD for the novel particles described in Examples 1 , 4 and 5. This process, therefore, allows controlled and rapid deposition of CHXD-CaCh or CHXD/NaCI/NaF particles or variants at the localised tooth or biomaterial interface to provide sustained antibacterial and remineralisation effects (Example 7) at the site of decay or infection.

Example 3

Novel CHXD/NaF particles were synthesised for inclusion into biomedical prostheses or as antibacterial powders. Mixtures of 0.33M NaF (Sigma, S7920, Lot: SLCB1955) reacted with CHXD (15mg/ml) produced no reaction at feasible timescales and crystallisation of particles was not evident. Therefore, 15mg/ml CHXD was mixed with higher concentrations of 0.96M NaF solution at 1 : 1 by volume at room temperature. The mixtures were left for 20, 40, 60, 80, 100, 120, 140 mins and then centrifuged at 2400 rpm for 1 min (Eppendorf centrifuge 5417C, Germany). The precipitates were washed with 1ml deionised water (three-stage Millipore Milli-Q 185 water purification system, Millipore, USA), and the precipitates were placed into liquid nitrogen for 30 mins, and then transferred to a freeze dryer (ScanVac CoolSafe Freeze Drying, Denmark) at -107°C, 0.009 mBar for 24 hours. Particles were stored at 4°C and wrapped in aluminium foil paper to exclude the light.

The freeze-dried novel CHXD/NaF particles were characterized using scanning electron microscopy (SEM, FEI inspect-F, Hillsboro); Energy-Dispersive Spectroscopy (EDS) (FEI Inspect F, NanoPort, Eindhoven, The Netherlands) and analysed using X-ray diffraction (XRD) (Panalytical, Almelo, The Netherlands). CHXD/NaF particles were only partially formed at 40 mins and there was increased crystal growth at 60 and 80 mins and a dense dispersal of dendritic CHXD/NaF particles and partially formed sheath bundles at 100- and 120-min reaction time (Figure 19 a-f). Table 8 and the XRD plots in Figures 20 and 21 indicate the CHX-/NaF particles had numerous differing and missing peaks when compared to chlorhexidine diacetate, indicating crystal structural differences for the CHXD/NaF particles (Table 8). There were also signs of peak broadening for the CHXD/NaF particles (Figure 21).

The F’ concentration of the synthesised CHXD/NaF particles (0.96 Mol) was found by dissolving the particles in distilled water and F’ concentration was measured using an F’ Ion Selective Electrode (ISE) (ELIT 9808, 8 Channel Ion-Analyser, NICO 2000 LD, 7.2.84sa, UK). The calibration procedure used F’ standards, with serial dilutions of 0.5, 1 , 10, 100 and 1000 ppm ion-standards. The ISE calibration curve demonstrated the F’ Molar concentration (mol/L) was highly correlated with estimated mV of the F’ standards (Fig. 8a, r²=0.99). CHXD/NaF particles (0.96 Mol) produced a fluoride content of 86.1 ppm available for release from biomedical prosthesis or dental polymers, fibres, gels, membranes, toothpaste, or dentifrices.

Table 8. XRD data for the novel CHXD/NaF particles and Chlorhexidine Diacetate (CHXD).

Example 4

Novel CHXD/NaCI-NH4F particles were synthesised by precipitation of Chlorhexidine Diacetate (CHXD C6143-25G, Lot: WXBO1046V) with NH₄F (Sigma Aldrich, 338869-25G, Lot: STBJ3936) and NaCI (Sigma, 746398, Lot: SLCC4856). The concentration of the CHXD was 15mg/ml. Solutions of 0.33M NaCI (or 0.66) mixed with 0.33M NH₄F were reacted for 1 minute with the CHXD solution in 2ml tubes (Eppendorf, UK) at 1 :1 volume at room temperature. The mixtures were then centrifuged at 2400 rpm for 1 min (Eppendorf centrifuge 5417C, Germany). The supernatants of each tube were removed, and the precipitates were washed once with 1 ml of deionised water and centrifuged again at 2400 rpm for 1 min. The deionised water excess was removed from each tube and subsequently, all tubes were placed in liquid nitrogen for 30 minutes. Thereupon, the lid of the tubes was removed, and the tubes were covered with Parafilm (Sigma-Aldrich, UK) and placed in the Freeze-dryer (ScanVac 39336805, Labogene, Germany) at -110°C and 4^x10^-4 mbar for 24 hours. This method demonstrated an alternate method of producing fluoride containing antibacterial CHXD particles using NH₄F when viewed using scanning electron microscopy (FEI inspect-F, USA) (Figure 23). Table 9 and the XRD plot (Figure 22) indicate the CHXD/NaCI-NFLF particles with differing 2 theta positions compared to chlorhexidine diacetate, indicating crystal structural differences. There were also signs of peak broadening. Novel CHXD/NaCI-NH4F particles can be synthesised by mixing NaCI (0.33 or 0.66M) with a range of NH4F solutions in a preferred range from 0.07 to 0.66M and reacting with CHXD.

Table 9. XRD data for the novel CHXD/NaCI-NF F particles.

Example 5

0.33M ZnCh (Sigma, 229997, lot no; MKCC2307) and NaF solutions prepared in a range from 0.125, 0.25, and 0.33M (Sigma, S7920, Lot # 051 M0215V) were at mixed in equal quantities at room temperature. 0.33M NaCI (Sigma, 746398, Lot: SLCC4856) was similarly prepared and mixed with either 0.33M KF (Sigma, Lot # MKBV0191V) or 0.33M SnF2 (Sigma 334626, Lot # MKBH4297V) at room temperature. Chlorhexidine diacetate solution 15 mg/ml (Sigma-Aldrich, C-6143, Lot:4G013891) was next mixed using a pipette (Eppendorf, Germany) with the respective salt solutions at a ratio of 1 :1 by volume. The mixtures were left for 1 min producing rapid turbidity and then centrifuged at 2400 rpm for 1 min (Eppendorf centrifuge 5417C, Germany). The precipitate was washed with deionized water (three stage Millipore Milli-Q 185 water purification system, Millipore, USA) and centrifuged again. The precipitate was freeze-dried (ScanVac Cool Safe Freeze Drying, Denmark) at - 107°C, 0.009 mBar for 24 hours. The morphology of the precipitates was characterised using scanning electron microscopy (FEI inspect-F, USA) at an accelerating voltage of 10 kV (spot size of 3.0 and working distance of 10 mm). The novel synthesised CHXD/ZnCh/NaF, CHXD/KF/NaCI and CHXD/SnF₂/NaCI particles indicate a spherical morphology with a similar structure (Figures 24 a, b, c, d). Novel CHXD/ZnCh/NaF, CHXD/KF/NaCI and CHXD/SnF2/NaCI particles can be prepared by mixing NaCI (0.33 or 0.66M) with a range of ZnCI₂, KF or SnF₂ solutions in preferred ranges from 0.07 to 0.66M and reacting with CHXD.

These particles offer a sustained CHXD release and the smart pH-responsive release of Zn, which is advantageous in acidic environments that encourage dental caries. There is also sequentially the added benefit of sustained fluoride release from these alternate variants (Potassium fluoride-KF, Tin fluoride-SnF2) to induce tooth remineralisation demonstrated in Example 7. These formulations (and Example 4) can also be delivered rapidly to the surface of tooth/bone or biomaterials via the low energy techniques described in Example 2.

Example 6

The current Example describes the surface crystallisation of novel CHXD particles onto teeth without significant surface staining compared to current chlorhexidine containing commercial products. The sustained chlorhexidine release of the invention and its biomimetic application to the tooth surface reduces the milliard reaction with drinks such as tea, and the need for gross stain removal from the tooth following application of current chlorhexidine products used for maintenance or oral hygiene.

For this Example, medical ethics approval (no. QMREC 2014/17) was gained for the use of human teeth from the tissue bank in the Institute of Dentistry, Bart’s and London, Queen Mary. The buccal, palatal and lingual surfaces of the premolar teeth (n=3 per group) were cleaned and polished using water and fluoride-free pumice with a prophylaxis brush at slow speed, then rinsed with water and dried using an air syringe. The roots of each premolar were covered with lab putty (Delta-SP80, Intertrading Dental AG, Kaltbrunn). Teeth were emersed in either a novel CHXD-CaCh or CHX/NaCI/NaF mouth-rinse (Table 10). Additional teeth were also immersed in 0.2% CHX (Corsodyl; GSK, Oral Healthcare, Weybridge, UK) used as a commercial comparison and distilled water (negative control, Table 10).

Table 10. Test groups for the staining study.

Formulation of the novel mouth rinses was as follows; Chlorhexidine diacetate (CHXD) (15 mg/ml) (Lot. WXBC5197V, Sigma Aldrich, UK) was mixed with calcium chloride solution (0.33 M) (Lot. SLBW0813, Sigma Aldrich, UK) at a 1 : 1 ratio for 10 seconds in 10 ml tubes (Eppendorf, UK) and the teeth immersed in the solution for 1 min (Group 1). NaCI (0.33) was mixed with NaF (0.33) and reacted with CHXD (15 mg/ml) mixed at a 1 : 1 ratio for 10 seconds in 10 ml tubes (Eppendorf, UK) and the teeth were immersed in the solution for 1 min (Group 2).

Colour values of maxillary premolars were recorded at baseline (before mouth rinse application), and after 1 day, 1 and 2 weeks of mouth rinse application. All teeth were stored in artificial saliva (Table 11), removed, washed in distilled water for 30 seconds and placed into the respective solutions for 1 min (Table 10). The novel CHX-CaCh or CHX/ NaCI/NaF mouth rinses were applied once a week for 1 min, whereas the Corsodyl mouth rinse was twice daily for 1 min, according to the manufacturer’s instructions. The artificial saliva was changed daily, and teeth were stored in an incubator (Binder, Binder GmbH, Germany) at 37.7°C. Tooth specimens were then removed, washed with distilled water for 30 secs and placed into a standard tea solution for 60 mins per day. For the standard tea solution, 1 g of tea leaves (Marks and Spencer Extra Strong, Marks and Spencer, London, UK) was added to 100 ml of water for 3 minutes then decanted and cooled to room temperature. After tea solution application, the specimens were rinsed in distilled water (30 secs) and allowed to bench dry for 1min. For the distilled water control, the same cycling procedure was followed. The optical density (OD) was then recorded for each test group.

A spectrometer (CM-2600d, Konica Minolta, Tokyo, Japan) was used to interpret the colour measurements by measuring the cervical, middle, and incisal sections of each premolar, (4 groups, n=3 per group) according to the CIE L*a*b* coordinates. The colour change (AE) for each premolar was evaluated by the following equation: AE = [(AL*) ² + (Aa*) ² + (Ab*) ²] 1 where AL*, Aa*, and Ab* are the differences in the respective values at baseline and after 1 day, 1 and 2 weeks of mouth rinse and tea application. The values (L*, a*, b*) were recorded with a 10° observer angle at a D65 illumination and were collected through spectrophotometry software (Spectra Magic Nx, Konica Minolta, Tokyo, Japan), as suggested by the CIE LAB 1976 standards.

The accuracy of the colour of the tooth specimens was established by using a colour space reference scale (1976 CIE L*a*b, International Commission on Illumination, Copyright© 2020 CIE). To enhance the accuracy of the colour measurements of each specimen, the spectrometer was calibrated three times using a white calibration plate (White Calibration Plate CM-A145, Konica Minolta, Tokyo, Japan). The colour measurements of each specimen were set down using the spectrophotometer at baseline (TO), at day 1 (T1), at week 1 (W1) and week 2 (W2). Two values for each tooth sample were recorded between TO and W2, which are the specular component included (E*abSCI) and the specular component excluded (E*abSCE).

Means of (AE) of each tested group for both SCI and SCE colour systems were evaluated and were normally distributed. ANOVA (2-way analysis) and Tukey’s post hoc tests were applied to interpret the colour differences of each tested group. The commercial group (0.2% Corsodyl) demonstrated higher colour changes (p = 0.0339) compared with the other groups for E*abSCI (p <0.001). There were no significant differences (p>0.05) between the control and the CHX-CaCh groups for both E*abSCI and E*abSCE parameters (Figure 25). Digital Images of the buccal surfaces of the tooth test groups (Table 10), were obtained using a digital optical microscope (Keyence VHX-2000, Keyence International, Mechelen, Belgium). Images were taken at baseline (day 1) and after 2 weeks of mouth rinse and tea applications (Figures 26, 27, 28, 29). This Example illustrates that both novel CHXD-CaCh and CHXD/NaCI-NaF mouth rinses used weekly do not lead to gross staining of the teeth, which reduces treatment time for stain removal and encourages the maintenance of oral hygiene. Example 7

This Example describes a CHXD/NaF-NaCI mouth rinse which can coat the tooth surface and give a sustained release of CHXD and F’ giving antibacterial effects and deposition of fluorapatite at the surface and remineralisation. This is particularly beneficial in the treatment and reversal of carries.

To demonstrate remineralisation effects, fully dense hydroxyapatite (HA) disks (12mm x 2mm, n=16) (Plasma Biotal, Ltd, UK) were used. The HA disks were immersed in acidic acetate buffer solution to be demineralised. The acidic acetate buffer preparation required 0.5L of distilled water, 1.225g of sodium acetate ^HsNaCh) (Lot; SLBD428IV, Sigma Aldrich, UK), mixed with 3.69ml of glacial acetic acid (CH₃COOH) (Lot: 18C274126, VWR, UK). The pH of the buffer was 4.7. The HA disks (n=16) were placed in 5ml tubes (Eppendorf, UK) filled with 3ml of acetate buffer. The disks remained in the acidic acetate buffer for 48 hours.

The discs were coated using a mouth rinse consisting of 0.5 ml of NaF (0.33M)/ NaCI (0.33M) reacted in 5ml tubes (Eppendorf, UK) with 0.5ml of CHXD (15mg/ml) for 10 seconds and subsequently, the HA disks were immersed in the reacted CHXD/NaF/NaCI solution for 1 minute. This procedure was then repeated. The disks were next transferred in tubes containing 2ml artificial saliva (AS, Table 11 , pH=7) and stored in an incubator (37°C). The disks were analysed using XRD (X-ray diffraction), and NMR (nuclear magnetic resonance) analysis after 1 week and 2 weeks after the CHX/NaF/NaCI coating.

Table 11. Chemicals for Artificial Saliva.

The presence of fine spherical crystallites and larger coarsened fluorapatite platelets were seen on the surface of the discs after 1 and 2 weeks of emersion in AS, when compared with the uncoated disc (Figs. 30 a-c), when viewed using scanning electron microscopy (FEI inspect-F, USA) at an accelerating voltage of 10 kV. The two-week sample was also analysed using solid-state ¹⁹F magic angle spinning nuclear magnetic resonance (MAS NMR), using a 600 MHz Bruker Advance NMR spectrometer (14.1 Tesla), operating at 564.7 MHz and using a low fluorine background probe. 1 M NaF solution was used as a reference with a signal of -120ppm. A 2.5 mm zirconia rotor was used and ¹⁹F MAS-NMR spectra were recorded at 21 kHz (spinning frequency), using a 60 s recycle delay. The ¹⁹F MAS-NMR spectra (Figure 30) were identified by a resonance line at -105ppm corresponding to the presence of fluorapatite in the range reported (Gao et al., RSC Adv., 2016, 6, 103782).

X-ray diffraction was used to characterise the HA disks before and after the 48h demineralisation treatment and after 1 and 2 weeks of the CHX/NaF/NaCI coating. The disk specimens were analysed in an X-ray diffractometer (Xpert Pro X-ray Diffractometer, Malvern Panalytical Ltd., The Netherlands). A reflection mode, divergence slit, flat plate 0/0 geometry and Ni-filter Cu-Ka radiation were used (A1 = 0.1540598 nm and A2 = 0.15444260 nm). The disks were scanned in a 20 range between 5° and 70°. A step size of 0.0334° was used, with a step time of 200 s. ICCD patterns of fluorapatite (FA) (00-015-0876) were used to identify the FA peaks/ phases. There was a shift in peak positions for the 2-week CHXD/NaF-NaCI coated specimens which fitted the peak positions for a fluorapatite phase (Figure 32).

The CHXD/NaF/NaCI mouth rinse therefore has the potential to provide a sustained remineralisation effect at the tooth or bone surface surfaces making it particularly attractive in the prevention and treatment of white spot lesions in Orthodontics and prevention and reversal of carries in restorative and maxillo-facial surgery.

Example 8

The cytotoxicity of the CHXD/NaF-NaCI (Example 1 , 0.66M NaF) particles against controls and the CHXD and NaF reagents was carried out in the following example.

L929 cells (ECACC, 85011425) were cultured in DMEM, (Lot: RNBL1427, Sigma-Aldrich, UK) supplemented with 10% foetal bovine serum (FBS), 1% penicillin and 1 % streptomycin (2199830, Gibco, UK), and 1 % L-glutamine (Lot:8MB025, Lonza, UK). The cells maintained in 75 cm2 Nunc flasks (Thermo Scientific, UK) under controlled conditions of 5% carbon dioxide (CO2) in a humidified atmosphere at 37 °C in an incubator (Wolf Laboratories, UK). All procedures were carried out in a sterile safety cabinet (TriMAT2, UK). Culture medium was aspirated and replenished every 48 hours until the cells reached 70% confluence. To passage the cells, DMEM was aspirated and subsequently, the cells were washed twice with 10 mL of PBS to eliminate excess cell debris and dilute out any foetal calf serum (an inhibitor of trypsin). The excess PBS was further aspirated, followed by the addition of 1mL of 0.25% trypsin solution (Lot:2457717, Gibco, UK) into the flask and incubated at 37°C for a duration of 5 minutes to achieve cellular detachment. The cells were then examined under a microscope (Hund, Wetzlar, Germany) to ensure cellular detachment from the flask and from neighbouring cells resulting in a single cell suspension, and a change in morphology to a rounded shape. Once cell detachment was confirmed, cell counting was conducted using a glass haemocytometer counting chamber and coverslip (Hirschmann, Germany, REF: 8100103, Lot:8039406) to determine cell numbers. The cell count was assessed in each of the 16 grid squares of the glass haemocytometer. The mean count per mL was calculated using the following equation (STEMCELL Technologies, online): Total number of nucleated cells/mL = average cell counts per square x dilution factor x 10⁴. Known numbers of cells of trypsinised cell suspension were pipetted into new flasks containing fresh DM EM and incubated at 37°C.

For the cytotoxicity assay three experimental groups comprising of dilutions of CHXD, NaF solutions (Lots: WXBC5197V and SLCB1955, Sigma-Aldrich, UK) and CHXD/NaF-NaCI (Example 1 , 0.66M NaF) in DMEM were conducted and compared to a control of DMEM alone in 96 well plates. Three individual plates were prepared (one for each treatment) for three time points (24 hours, 48 hours, 7 days), (n=9). The assessment of CHXD, NaF and the CHXD/NaF-NaCI (0.66M) particle was determined by measuring the DNA content of each experimental well. Cells were seeded at an initial density 10,000 cells per well. Following an overnight incubation period in DMEM, the medium was aspirated, and cells were subjected to two washes with phosphate-buffered saline (PBS). The first and the last rows, and the top and bottom columns were filled solely with DMEM, while negative controls (columns 2 and 9) encompassed a cell-only group devoid of CHXD.

The remaining cells were exposed to serial dilutions of CHXD and NaF, which were made of 50ppm, 25ppm, 12.5ppm, 6.25ppm, 3.125ppm, 1.5625ppm along with the dilutions of CHX/NaF-NaCI particles (n=3 for each time point). Exposure durations were 24, 48 hours and 7 days and stored in a humidified incubator at 37°C. A sterile syringe filter 0.10-0.22pm (Appleton, UK) was used to sieve the CHXD/NaF-NaCI particles prior to dilutions. This measure was implemented to verify the absence of any residual crystals or impurities that might impact the cell studies. At each designated time point (24, 48 hours, and 7 days), three plates corresponding to each treatment were removed from the incubator. The medium from each plate was discarded, and each well of the plates was rinsed twice with 100pl of PBS (Figure 100). Subsequently, the plates were placed into the designated freezer at -20°C. Once all the plates had been collected, on the day of the DNA proliferation reading, all experimental plates were retrieved from the freezer. The cultures underwent a brief incubation in 50pl of distilled water, which was added to each well. Following this, the plates were placed in the incubator for a duration of 10 minutes. Subsequently, they were placed again in the designated freezer at -20°C for half an hour until frozen, after which they were transferred to a hot bath (Aqua 5 Plus, Grant, JB Nova) at 37°C until thawed. This gradual process facilitated cellular breakdown, releasing the DNA and making it more available to the dye and ensured a thorough and effective integration of the fluorochrome with the cellular DNA.

A stock solution of the DNA dye, Hoechst 33258, was prepared at 1 mg/mL concentration in distilled water and stored wrapped in foil at 4°C. The TNE buffer was formulated with 10 mM Tris, 1 mM EDTA, and 2 M NaCI, pH=7.4. A working solution of the fluorochrome dye was next prepared by diluting the stock solution 1 :50 to achieve a concentration of 20 pg/mL in TNE buffer. Subsequently, 100 pl of this working solution was added to each well, resulting in a final Hoechst 33258 concentration of 10 pg/mL in a total volume of 200 pl per well, with a final NaCI concentration of 1 M.

After the treatment, the solutions were removed, and fluorescence readings were taken using a luminescence microplate reader (Clariostar Plus, BMG, Labtech, Germany) with an excitation wavelength of 355-20nm and emission of 455-30nm. The evaluation of cell DNA proliferation for each sample (CHXD/NaF-NaCI, NaF, and CHXD) involved three independent experiments (n=6 each) and was assessed using the MARS data analysis software (BMG, Labtech, Germany).

The results of the DNA proliferation of the L929 fibroblast cells at different time points (Figure 33) indicated that the CHXD/NaF-NaCI particle (Example 1) may promote cell proliferation and pose no cytotoxic effects over the range of concentrations encountered during its sustained release. In Figure 33, a single asterisk “*” indicates significant differences (p<0.05), whereas two asterisks “**” indicates highly significant differences (p<0.001) between tested and control groups. Example 9

Antibacterial studies were carried out on the CHXD/NaF-NaCI (Example 1 , 0.66M NaF) particles against controls and the CHXD and NaF reagents in the following example. Serial dilutions of CHXD and NaF and the CHX/NaF-NaCI particles were made according to Example 8. This example used P. gingivalis (W50) and S. mutans (NCTC 10449). S. mutans were cultured on Tryptone Soy Agar (Lot 2426701 , Oxoid, Basingstoke, UK) on agar plates and incubated at 37°C for 2 days. Colonies were then inoculated in 10ml Tryptone soya broth (TSB, Oxoid) and incubated for 24 hours. P. gingivalis were inoculated in 10ml Brain Heart Infusion (BHI). The broth was supplemented with equal volume amounts of 100 L of Vitamin K and Haemin and incubated overnight. The optical density was achieved by running a sample of the culture broth in a Bio Photometer (Eppendorf AG, Hamburg, Germany) and diluting to 0.1 using fresh BHI broth at 600nm.

An MTT assay was conducted using 100 pL of inoculated bacterial suspension in TSB or BHI pipetted into 96 well plates and incubated for 24 hours to form a biofilm. The culture medium supernatant was next removed from the plates and 100 pL of the serial dilutions of either CHXD, NaF or the CHX/NaF-NaCI particles was added to each well. The controls were the bacterial suspensions (TSB or BHI, n=3) and further incubated for 15 mins. 50 pL of TSB or BHI and 50 pL of MTT reagent was next added then incubated at 37°C for 5 mins. 150 pL of MTT solvent solution was next pipetted into each well which was wrapped with tinfoil and placed on a vibrational plate for 15 mins. The 96 well plate was then placed in the CLARIOstar (BMG labtech, UK) reader for analysis at the absorbance of 590 nm. The results are shown in Figures 34 and 35.

The MTT assay results indicates a substantial decrease in the viability of S. mutans (Figure 34) and P. gingivalis (Figure 35) following 24-hour treatment at all concentrations illustrating their antibacterial efficacy against aerobic and anaerobic bacteria.

Claims

1. A crystalline salt of chlorhexidine (CHXD) fluoride having a spherical morphology under Scanning Electron Microscopy (SEM) and comprising a fluoride anion and a cation selected from the group consisting of sodium, ammonium, potassium, or tin.

2. A crystalline salt of chlorhexidine fluoride according to claim 1 , further comprising a chloride anion and a cation selected from the group consisting of sodium, zinc, magnesium, or strontium.

3. A crystalline salt of chlorhexidine fluoride according to claim 2, wherein the cation is sodium and the crystalline salt has an X-ray diffraction pattern comprising 2-theta peaks at about 5.2, about 8.4, about 9.8 about 12.2, about 12.7, about 13.3, about 14.0, about 15.6, about 15.8, about 16.1 , about 18.1 , about 18.6, about 19.0, about 19.7, about 20.0, about 20.5, about 20.8, about 21.6, about 22.7, about 23.7, about 24.7, about 25.5, about 26.1 , about 26.5, about 28.8, about 28.9, about 29.8, about 31.2, about 31 .7, about 34.2, about 34.3, about 35.4, about 38.8, about 38.9, about 44.3, about 45.5, and about 56.1 degrees.

4. A crystalline salt of chlorhexidine fluoride according to claim 1 , wherein the cation is sodium, and wherein and the crystalline salt has an X-ray diffraction pattern comprising 2- theta peaks at about 7.48, about 12.77, about 14.99, about 16.96, about 20.43, about 20.84, about 22.25, about 22.65, about 22.90, about 23.25, about 27.08, about 27.79 and about 38.87 degrees.

5. A crystalline salt of chlorhexidine fluoride according to claim 1 wherein the cation is ammonium, and wherein the crystalline salt has an X-ray diffraction pattern comprising 2- theta peaks at about 8.48, about 12.67, about 13.34, about 15.73, about 15.79, about 18.16, about 18.09, about 18.57, about 20.02, about 20,58, about 20.52, about 25.53, and about 26.51 degrees.

6. A process for the preparation of monodisperse crystals of CHXD/NaF, CHXD- NaCI/NaF, CHXD/ZnCh/NaF, CHXD-NaCI/KF, CHXD/SnF₂/NaCI or CHXD/NaCI/NH₄F salts of any one of claims 1-5 comprising;

(i) mixing and dissolving aqueous solutions of chlorhexidine acetate with an aqueous solution of a sodium, ammonium, zinc, or tin fluoride at a concentration of 0.1 M to 1 ,0M and a sodium or zinc chloride at a concentration of 0M to 1 ,0M; (ii) allowing the CHXD/NaF, CHXD/NaCI/NaF, CHXD/ZnCh/NaF, CHXD/NaCI/KF, CHXD/SnF₂/NaCI or CHXD/NaCI/NH₄F salts to precipitate;

(iv) washing the precipitated solid mass of (iii).

7. A pharmaceutical composition comprising a crystalline chlorhexidine salt according to any one of claims 1 to 5

8. A composition comprising a crystalline chlorhexidine salt according to any one of claims 1 to 5 encapsulated or suspended in a polyelectrolyte, or in a polymerizable monomer.

9. A composition according to claim 7 in the form of a mouthwash, toothpaste, gel, or polymer for cosmetic use.

10. A method of treating a disease or condition in a subject, comprising the step of administering a crystalline chlorhexidine salt according to any one of claims 1 to 5 or a composition of any one of claims 7 to 9 to the subject in need thereof.

11. A crystalline chlorhexidine chloride salt according to claims 1 to 5 or a composition of any one of claims 7 to 9 for use in a method of treating a disease or condition in a subject.