PENETRATION OF THERAPEUTIC AGENTS..

PENETRATION OF THERAPEUTIC AGENTS THROUGH NATURAL PLAQUE BIOFILMS In BIOFILMS: Persistence and ubiquity Edited by: Andrew McBain, David Allison, Johnathan Pratten, David Spratt, Mathew Upton, and Joanna Verran. 7th meeting of the BIOFILM Club, Gregynog Hall, Powys, 7-9th September 2005 ISBN 0-9551030-0-2 C. Robinson and P. S. Watson. Division of Oral Biology, Leeds Dental Institute, Clarendon Way, Leeds, LS2 9LU Abstract. Natural plaque biofilms were generated in vivo on natural enamel surfaces using the device described by Robinson et al 1997. After 7 days these were recovered from the mouth and immersed in solutions of the therapeutic to be tested. After designated time intervals, devices were removed and immediately snap frozen using liquid nitrogen. After lyophilisation the accumulated biofilm was embedded in methacrylate and sectioned at 5-micron intervals from saliva interface to the enamel surface. Groups of 10 sections were pooled for determination of therapeutic content and between the 5th and 6th section a pair of 2μm sections was collected for image analysis from which biomass volume and distribution were determined. Penetration for all compounds examined was limited in 30 seconds to the outer third or so of the plaque layer. At 2 minutes concentrations in the outer regions increased but little more penetrated to the enamel surface. For fluoride, only after 30 minutes was there a semblance of equilibration with the supernatant solution. The distribution profiles obtained were compared with biomass distribution and were closely related to the surface area to mass curves suggesting that uptake may be dictated by available surface area. This may also limit penetration. From preliminary investigations, where radioactive materials were used in vitro, amine fluoride was often seen to reside at the surfaces of biomass. The much more hydrophobic riclosan, however, seemed to be located within the biomass, possibly in the region of bacterial cells. Key words: Penetration, human, intact-biofilms, fluoride, triclosan, amine –fluoride, architecture Introduction. Penetration and uptake of materials by all pathological biofilms and especially oral plaque biofilms is a key feature in the administration of therapeutics (Stewart 2003). This is of particular importance when these have to penetrate difficult to reach stagnation sites (Addy and Adriaens, 1998) or through plaque to the enamel itself (ten Cate, 1999, Robinson et al 2000). In the case of dental plaque, periods of exposure to therapeutics during tooth brushing and mouth rinsing can be very short. While 2 minutes is recommended this is more usually about 30 seconds (MacGregor and Rugg-Gunn 1985, van der Ouderra 1991). Despite a great deal of interest in this area there has been little definitive information concerning penetration through natural intact biofilms formed on natural surfaces in vivo. Many studies recovered plaque by mechanical scraping which disrupts plaque architecture and would not therefore permit accurate determination of location of, in this case fluoride within the plaque (Dibdin, 1993, McNee et al., 1982, Tatevossian, 1985). Efforts made in vivo achieved some success but control over period of exposure and amount administered is very difficult (Kato et al 1997). A model was therefore devised to generate plaque in vivo on natural enamel surfaces whereby penetration studies could be conducted both in vivo and in vitro on intact undisturbed human plaque biofilms. This revealed gradients of endogenous fluoride from surface to interior suggesting that there was some natural restriction to full penetration (Robinson et al 1997). The model also showed, using confocal microscopy, that natural plaque contained voids and channels often extending through the biomass to the underlying enamel (Wood et al 2000). In other biofilms, such architecture has been highlighted as possibly having considerable influence on mass transfer through biofilms (Zhang and Bishop 1994, de Beer and Stoodley , 1995). Specifically channels were considered to provide better penetration compared with biomass with both biofilm thickness and interaction of components with the biofilm playing important roles, (Stewart 1998, Stewart 2003). Such new information on plaque biofilm architecture raised a number of questions concerning mass transfer through the biofilm. Most importantly, to what extent does penetration occur via the channels and how easy is it to penetrate the biomass? These are crucial questions when assessing delivery of, for example, fluoride to the enamel or antibacterials to bacterial cells deep in the biomass. Studies were therefore undertaken to investigate the uptake and penetration of fluoride, triclosan and amine fluoride(s) into natural undisturbed plaque biofilms. Plaque biofilms were generated in vivo over 7 days; they were then removed from the mouth and immersed in solutions containing fluoride or radioactively labeled triclosan or amine fluoride for prescribed time intervals. The distribution of these materials throughout the plaque was then determined and distribution profiles were compared with biofilm architecture. Materials and Methods. Plaque generation devices. Devices were constructed and placed intra-orally as described in Robinson et al 1997. Briefly, nylon rings ~5mm diameter were attached to discs of surface human enamel using a cyanoacrylate adhesive which were then trimmed to be slightly larger than the rings themselves. The devices were then bonded to selected tooth surfaces using Scotchbond Multipurpose Dental Adhesive. After the required period in vivo, devices were removed using orthodontic debonding pliers. Exposure to and determination of therapeutics of interest. After 7 days plaque generation in vivo, devices were removed from the mouth and immersed in relevant solutions for specified time periods as follows. Devices were totally immersed in fluoride solutions while radioactive solutions were applied in 3μl drops to the device surface. All concentrations were similar to those currently used in toothpastes. 1. Fluoride: 1000 ppm fluoride ion was used as sodium fluoride at neutral pH. 2. 14 C labelled triclosan: Unlabeled triclosan carrier was supplied by Irgacare, 14 C- labelled triclosan (GS-14703) was obtained from Syngenta, as a crystalline solid, specific activity 5.43MBq/ mg (both obtained through Unilever R&D). 0.054MBq per ml of 14 C labelled triclosan was used for incubation. Final concentrations in the test preparation were 10ppm for 14 CTriclosan, with 3000ppm total triclosan present (almost all of this remained in suspension). 3. 14C labelled amine fluoride(s): These were supplied by GABA international. 0.185MBq per ml of an equal mixture of N-Oleyldiethanolamine hydrofluoride (oleyl1-1-14C) and NOleyldiethanolamine hydrofluoride (hydroxyethyl-14C) was used for incubation. Concentrations of cold amine fluoride i.e. non-radioactive carrier were added to a final concentration of 1400ppm fluoride. Fluoride distribution After the prescribed time, devices were removed from incubation solutions, placed on absorbent filter paper and excess solution wicked from their surfaces. For fluoride and amine fluoride investigations they were then lyophilized, embedded in a methacrylate mixture and then sectioned at 5 micron intervals using an ultramicrotome from the saliva plaque interface to the enamel surface. Groups of 10 contiguous sections were pooled for analysis. .A pair of 2μm sections was collected for each group and retained for toluidine blue staining and image analysis for determination of biomass. Chloroform was used to disperse the methacrylate and after this was removed by evaporation, the residue was dissolved in acetate buffer pH 5 and fluoride was determined using a fluoride- specific electrode (Hallsworth et al 1976). For more detailed methodology see Watson et al 2005a. Triclosan distribution To minimise the possibility of some redistribution of hydrophobic triclosan during embedding in methacrylate, devices were snap frozen immediately after removal from the mouth. After freezing (-20oC) and removal of the nylon ring, the frozen plaque sample was supported in a minimum of OCT compound. Frozen sections (25 μM) were then taken using a cryomicrotome (Bright Cryostat). Pooled groups of four sections were subsequently placed in scintillation vials for counting. To each scintillation vial containing plaque material was added 10ml SigmaFluor scintillation cocktail (Sigma-Aldrich, Poole, UK). Sample vials, and blank controls, were transferred to racks and dark adapted overnight in the scintillation counter (Tri-Carb Liquid Scintillation Analyzer, Packard). Scintillation counting was then performed, using a 14 C specific counting protocol. Each sample was counted twice, for consecutive twenty minute periods and the average count calculated. Counts per minute (CPM) were automatically converted into disintegrations per minute (DPM), using an established quench curve. Counting efficiency was validated daily using 14 C and background standards prior to measurement of experimental samples. For more detailed methodology see Watson et al 2005b. Amine fluoride distribution Plaque biofilms were lyophilised embedded and sectioned as described above for fluoride. Pooled sections were then placed in scintillation vials, scintillant added and counting performed as described above for triclosan. Determination of TCN distribution by microautoradiography In a preliminary parallel investigation, a biofilm-containing device was exposed to an aqueous solution containing 10ppm 14 C-TCN, frozen, then lyophilised, embedded in methacrylate and serially sectioned (as described above for fluoride-exposed samples). Pairs of 2µm thick sections were dried down onto APES-coated glass slides and used for microautoradiography and image analysis. Slides were prepared for microautoradiography under dark-room conditions. Glass slides, on to which sections of 14C-TCN exposed plaque biofilms had been dried, were immersed for five seconds in LM-1 Hypercoat Emulsion (Amersham Biosciences, Little Chalfont, UK) which had been pre-melted in a water bath at 43°C. Slides were carefully recovered, drained of excess emulsion, and incubated at room temperature (15-25°C) in a light-tight box for one hour to allow the emulsion to set completely. Anhydrous silica gel was added to the box, which was re-sealed and incubated for six weeks at 4°C. After this period, the box was allowed to equilibrate to room temperature (15-25°C) for one hour before being opened. The slides were then immersed in developer (Kodak D-19) for five minutes, transferred to stop solution (0.5% v/ v acetic acid) for 30 seconds, incubated in fix (30% w/ v sodium thiosulphate) for 10 minutes, rinsed for 15 min in running tap water and then allowed to dry. Developed slides were stained with toluidine blue as described previously and viewed using an Olympus BX50 light microscope (Olympus UK, Southall, UK) fitted with a Hitachi VK-C150ED colour video camera (Hitachi Europe Ltd, Maidenhead, UK). Images were saved and analysed using SPOT Advanced software (Diagnostic Instruments Inc., Sterling Heights, MI, USA). The same procedure was used for plaque samples exposed to radiolabelled amine fluoride. Determination of biomass volume 2µm-thick plaque sections, taken between the pooled groups used for analysis, were dried on to APES-coated microscope slides. These were flooded with 0.1% aqueous toluidine blue, gently heated over a flame (until beginning to steam) and incubated at room temperature (15-25°C) for 15 min. Slides were then gently rinsed in tap water and allowed to dry. Images of stained plaque sections were obtained using a video camera (JVC 3-CCD) and analyzed using Zeiss KS300 Imaging Software (Zeiss, Jena, Germany). For each pair of sections, the mean area and the mean total perimeter of the biomass (stained material) was calculated. Knowing the section thickness, this permitted calculation of the volume and approximate surface area of plaque biomass within each section. Results. Uptake and distribution of fluoride ion. Figure 1 illustrates mean fluoride distributions for plaque biofilms exposed to fluoride for 30seconds, 2 minutes and 30 minutes (n=6 for each exposure time). Data has been calculated assuming that fluoride is associated with the biomass. In the case of both 30 seconds and two minutes, fluoride concentrations decreased from the saliva plaque interface towards the enamel surface. Two minutes exposure showed a greater increase in the outer plaque layer compared with a much lower increase in the plaque interior. Within 200 microns of the enamel surface there was no significant difference between 30 second and 2 minute exposures. After 30 minutes exposure, however, fluoride had clearly penetrated throughout the biofilm almost equilibrating with the immersion solution (1000ppm F). Uptake and distribution of amine fluoride(s). Figure 2A illustrates the distribution of radioactively labeled amine fluoride in natural plaque biofilms after exposure to 14C labeled material for 30 seconds. Most uptake was seen in the outer third or half of the plaque layer. Unlike fluoride, a rise was noted in the extreme outer edge of the plaque prior to the fall towards the enamel surface. Insufficient labeled material precluded further time periods. Preliminary results from microautoradiography (Figure 2B) indicated that the amine fluorides were often associated with the surfaces of biomass with perhaps less penetrating into the biomass itself. Uptake and distribution of radioactively labeled triclosan. The solubility of triclosan in aqueous media at pH 8.5 (the pH of many toothpastes) was determined to be a maximum of 10ppm. This concentration of 14C labeled material was therefore used in all penetration studies. The total unlabelled material present was 3000ppm which is consistent with the concentration in many toothpastes, although almost all of this would be insoluble. The results were more variable than for fluoride or amine fluoride, so that 30 second (Figure 3A) and 2 minute exposure times (Figure 3B) are shown separately. Both showed a rise in the outer edge of the plaque layer followed by a fall towards the enamel surface. There was no obvious difference between 30 seconds and two minute exposure times. Preliminary results from microautoradiography (Figure 3C) indicated that triclosan was often located in clusters within the biomass possibly associated with bacterial cells. Architecture of Plaque biofilms. Measurement of the proportion of biomass throughout the plaque layer revealed an increase from the saliva plaque interface which plateaued in the mid region of plaque and decreased somewhat near to the enamel surface. This indicated that biomass free areas i.e. channels and voids decreased steeply towards the interior, with a small increase near to the enamel. (Watson et al 2005a, and Watson and Robinson 2005 this meeting). While surface area of biomass showed little change throughout biofilm depth, surface area to biomass ratios decreased dramatically towards the interior to rise again somewhat towards the enamel (Figure 4). Discussion. It is clear from the data that penetration of the therapeutic materials into intact undisturbed dental plaque for the short periods associated with tooth brushing or mouth rinsing is restricted. Only after 30 minutes did fluoride approach equilibration with the externally applied solution. The profiles obtained for each of the species examined indicated that in 30 seconds or so, penetration only occurred within the outer third or half of the plaque biofilm layer. In the case of fluoride and triclosan where 2 minute intervals were also employed some small improvement in penetration was observed but the profiles remained similar with no significant differences in the inner third or so of the biofilm. With fluoride it was possible to use 30 minute exposure periods, after which time the concentrations throughout the plaque approached equilibration with the topically applied solution. Considering fluoride, for which most information is available, this raises interesting questions concerning normal uptake kinetics. Earlier data using this device which had equilibrated in the mouth for 7 days (Robinson et al 1997), showed very similar profiles of endogenous fluoride to those obtained after 30 seconds exposure in the current study. Since normal salivary fluoride contains about 0.02ppm fluoride had clearly accumulated in the plaque. However, even 7days accumulation had not generated more penetration than that seen after 30 seconds in vitro. This suggests that topically applied fluoride is adsorbed/absorbed most effectively in the outer regions of the plaque. However, further inward progress appears to be restricted possibly by adsorption near the surface and also by increasing difficulty of deeper penetration. The fact that exposure in vitro to 1000ppm fluoride for 30 minutes achieved almost full penetration of the plaque indicates that diffusion is extremely slow and/or that binding sites for fluoride in the outer plaque must be saturated before further penetration occurs. Amine fluoride used in many dental preparations for antibacterial activity and enamel repair also showed similar restricted uptake after 30 seconds i.e. to the outer third or half of the plaque layer. Like triclosan but unlike fluoride, a rise at the extreme outer edge of the plaque layer preceded the fall towards the enamel surface. Triclosan profiles, while more variable than fluoride, were less steep suggesting perhaps less binding at the plaque outer surface. Triclosan was still restricted to the outer regions of the plaque, however, indicating that like fluoride, inward diffusion was restricted. The very small amounts actually measured may be due the extremely low solubility of triclosan in water. It is clear from these studies that all of the therapeutics investigated showed restricted penetration into intact plaque biofilms during the short exposures associated with toothbrushing or mouthrinsing. The reasons for the restricted penetration of the materials studied are not clear. All have very different chemical characteristics. Fluoride is anionic, the amine fluorides are neutral or cationic and triclosan is extremely hydrophobic. In view of this, the answer would appear to reside more with the plaque biofilm than with the nature of the materials applied. A comparison was therefore made with the architecture of the intact plaque layers. Confocal laser scanning microscopy had suggested that the outer plaque comprised frond like structures projecting from a more compact base (Wood et al. 2000). Quantitative measurements (Watson et al 2005b Robinson and Watson 2005 this meeting) confirmed this but, in addition, were also able to demonstrate that while biomass surface area remained relatively unchanged throughout depth, surface area to mass ratios decreased dramatically in this direction. These curves in fact were rather similar to the penetration profiles reported here (figure 4). This would suggest that uptake near the surface may be governed by high plaque surface area perhaps by adsorption to the biomass surface. Decrease in surface area to mass ratio combined with an increasing compactness of biomass might restrict penetration. The presence of amine fluoride at biomass surfaces, while not definitive proof, illustrates that charged molecules can be selectively taken up at biomass surfaces. This may mean such molecules may be more effective in biofilms with a high surface area to mass ratio. . Although concentrations of triclosan measured were small the apparent greater uptake of triclosan and possible location in or near bacterial cells may be due to its entry into the biomass and location in hydrophobic environments such as bacterial cell membranes, In general terms, selective concentration of orally applied therapeutics in the outer regions of plaque biofilms may mean that this is where most of their activity resides. Future improvement may lie in strategies to improve delivery to deeper layers as well as deeper into the biomass. Acknowledgements. The authors would like to acknowledge the support of Unilever Research and Development. for the studies on fluoride and triclosan and GABA International for the studies using amine fluorides References: Addy M, Adriaens PA (1998). Consensus Report of Group A: Epidemiology and Etiology of Periodontal Disease and The Role of Plaque Control in Dental Disease. Proceedings of The European Workshop on Mechanical Plaque Control - Status of the Art and Science of Dental Plaque Control, Berne, Switzerland: Quintessence. Bradshaw DJ, Marsh PD (2003). Novel microscopic methods to study the structure and metabolism of oral biofilms. In: Medical Implications of Biofilms. M Wilson and D Devine editors. Cambridge: Cambridge University Press. Balzar Ekenback S, Linder LE, Sund ML, Lonnies H (2001). Effect of fluoride on glucose incorporation and metabolism in biofilm cells of Streptococcus mutans. European Journal of Oral Sciences 109 (182-186). de Beer D, Stoodley P (1995). Relation between the structure of an aerobic biofilm and transport phenomena. Water Science and Technology 32 (11-18). Dibdin GH (1993). Effect of bathing fluid on measurements of diffusion in dental plaque. Archives of Oral Biology 38 (251-254). Hallsworth AS, Weatherell JA, Deutsch D (1976). Determination of sub-nanogram amounts of fluoride with the fluoride electrode. Analytical Chemistry 48 (1660-1664). McNee SG, Geddes DA, Weetman DA (1982). Diffusion of sugars and acids in human dental plaque in vitro. Archives of Oral Biology 27 (975-979). Tatevossian A (1985). The effects of heat inactivation, tortuosity, extracellular polyglucan and ion-exchange sites on the diffusion of 14C-sucrose in human dental plaque residue in vitro. Archives of Oral Biology 30 (365-371). Kato K, Nakagaki H, Arai K, Pearce EIF (2002). The influence of salivary variables on fluoride retention in dental plaque exposed to a mineral-enriching solution. Caries Research 36 (58-63). Kato K, Nakagaki H, Takami Y, Tsuge S, Ando S, Robinson C (1997). A method for determining the distribution of fluoride, calcium and phosphate in human dental plaque and the effect of a single in vivo fluoride rinse. Archives of Oral Biology 42 (521-525). (Kato et al., 1997) MacGregor ID, Rugg-Gunn AJ (1985). Toothbrushing duration in 60 uninstructed young adults. Community Dentistry and Oral Epidemiology 13 (121-122). Marquis RE (1995). Antimicrobial actions of fluoride for oral bacteria. Canadian Journal of Microbiology 41 (955-964). McNee SG, Geddes DA, Weetman DA (1982). Diffusion of sugars and acids in human dental plaque in vitro. Archives of Oral Biology 27 (975-979). Robinson, C., Shore, R.C., Brookes, S.J., Strafford, S., Wood, S.R. & Kirkham, J. (2000). The chemistry of enamel caries. Critical Reviews in Oral Biology and Medicine 11, 481-495. Robinson, C., Kirkham, J., Percival, R., Shore, R.C., Bonass, W.A., Brookes, S.J., Kusa, L., Nakagaki, H., Kato, K. & Nattress, B. (1997). A method for the quantitative sitespecific study of the biochemistry within dental plaque biofilms formed in vivo. Caries Research 31, 194-200. Stewart PS (1998). A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms. Biotechnology and Bioengineering 59 (261-272). Stewart PS (2003). Diffusion in biofilms. Journal of Bacteriology 185 (1485-1491). (Stewart, 2003 Tatevossian A (1985). The effects of heat inactivation, tortuosity, extracellular polyglucan and ion-exchange sites on the diffusion of 14C-sucrose in human dental plaque residue in vitro. Archives of Oral Biology 30 (365-371). ten Cate JM (1999). Current concepts on the theories of the mechanism of action of fluoride. Acta Odontologica Scandinavica 57 (325-329). van der Ouderaa FJG (1991). Anti-plaque agents - rationale and prospects for prevention of gingivitis and periodontal disease. Journal of Clinical Periodontology 17 (447-454). Watson P, Pontefract H, Devine D, Shore R, Nattress B, Kirkham J, Robinson C. (2005a). Penetration of fluoride into natural plaque biofilms. Journal of Dental Research. 84(5) 451-455 Watson P.S. (2005b). The uptake and distribution of fluoride and triclosan in plaque biofilms formed in vivo using the Leeds in situ device. PhD Thesis University of Leeds. Wood SR, Kirkham J, Marsh PD, Shore RC, Nattress B, Robinson C (2000). Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy. Journal of Dental Research 79 (21-27). Watson P and Robinson C, (2005c) The architecture and microbial composition of natural plaque biofilms. Biofilm Club, Gregynog. Zhang TC, Bishop PL (1994). Evaluation of Tortuosity Factors and Effective Diffusivities in Biofilms. Water Research 28 (2279-2287). 1500 Unexposed controls 30s NaF, no wash 2 min NaF, no wash 30 min NaF, no wash 1250 Fluoride (ppm) 1000 750 500 250 0 0 100 200 300 400 500 600 700 800 Distance ( µ m) of section from salivary interface Figure 1. Distribution of fluoride in intact human plaques biofilms after exposure to 1000 ppm fluoride for 30 seconds. 2 minutes and 30 minutes (n= 6 for each time period). Depth of Plaque (µ m) Related to DPM Amine Fluoride 120 DPM Amine Fluoride 100 80 GA-10 - Aqueous AmF GA-12 - Aqueous AmF 60 GA-16 - Aqueous AmF GA-20 - Aqueous AmF 40 GA-18 - Aqueous AmF GA-28 (Aqueous UNWASHED?) 20 0 0 200 400 600 D e p t h 800 1000 1200 o f µ Pm l Figure 2A. Distribution of 14C radio-labeled amine fluoride in intact human plaque biofilms after 30 second exposure to aqueous solution containing… BIOMASS RADIOLABELLED ANTIMICROBIAL CHANNEL / VOID BIOMASS BIOMASS Figure 2B. Autoradiographic localization of amine fluoride at the surfaces of biomass. 5 5 4 4 R e p lic a te R e p lic a te 1 1 14 C-TCN 3 4 5 2 3 2 (ppm) (ppm) 2 14 C-TCN 3 3 4 5 2 6 6 1 1 0 0 900 750 600 450 300 150 0 D is t a n c eµ m( ) o f s e c t io n fro m e n a m e l 900 750 600 450 300 150 0 D is t a n c µem( ) o f s e c t io n fro m e n a m e l 2 m in s a q u e o u s A B Figure 3. Distribution of 14C radio-labeled Triclosan in intact human plaque biofilms after (A) 30 seconds and (B) 2 minute exposure to aqueous solutions containing 10ppm 14 C labeled triclosan. Total (triclosan = 3000ppm) STAINED BIOMASS NON-STAINED VOID RADIO-LABELLED TCN Figure 3C. Autoradiographic localization of triclosan within dense stained biomass, possibly associated with clusters of bacterial cells. Ratio of biomass surface area (mm2): biomass volume (mm3) 65 55 45 35 25 0 100 200 300 400 500 600 700 800 Distance ( µ m) of plaque section from salivary interface Figure . 4. Surface area to mass ratio of intact human plaque biofilms derived from image analysis and section thickness.