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Mucoadhesion, hydration and rheological properties of non-aqueous delivery systems (NADS) for the oral cavity
journal of dentistry 36 (2008) 351–359
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Mucoadhesion, hydration and rheological properties of non-aqueous delivery systems (NADS) for the oral cavity Mustafa A. Zaman a, Gary P. Martin a,*, Gareth D. Rees b,** a b
King’s College London, Phamaceutical Science Division, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK GlaxoSmithKline R&D, St. George’s Avenue, Weybridge, Surrey KT13 0DE, UK
article info
abstract
Article history:
Objectives: Effective delivery of oral care actives from conventional hydrogel formulations is
Received 5 July 2007
often compromised by poor retention associated with shear forces present in the mouth,
Received in revised form
salivary washout and over-hydration of the gel which can lead to structural breakdown and
16 January 2008
adhesive failure. Non-aqueous gels offer the opportunity to formulate rheologically accep-
Accepted 17 January 2008
table vehicles with higher concentrations of bioadhesive polymer than is possible using water as the primary solvent. Accordingly, this study describes the formulation and characterisation of the rheology, hydration and bioadhesive properties of a range of non-
Keywords:
aqueous delivery systems (NADS).
Mucoadhesion
Methods: The formulations were composed principally of glycerol, with varying amounts of
Rheology
polyethylene glycol (PEG) 400 and Carbopol1 974P being incorporated in the ranges 0–31.34%
Hydration
and 0–4% (w/w). Work of adhesion (WOA) and maximum force of detachment (Fmax) were
Non-aqueous delivery systems
determined using a Dartec tensile tester after application of a normal force. Rheology was
(NADS)
assessed using a Bohlin CS CVO rheometer. Results: WOA and Fmax increased with increasing compression time and Carbopol concentration. Addition of 30% (w/w) PEG 400 to the formulation containing 2% Carbopol in glycerol improved bioadhesive function. Formulation rheology was largely controlled by the Carbopol concentration, and to a lesser extent by the concentration of PEG 400 and these, in turn, largely determined the bioadhesion parameters and rates of hydration. Conclusion: The results of this in vitro study suggest that bioadhesion, and consequently potential drug bioavailability, would be enhanced by use of a water miscible non-aqueous delivery vehicle such as glycerol containing a bioadhesive polymer such as Carbopol with the addition of controlled amounts of PEG as plasticiser. # 2008 Elsevier Ltd. All rights reserved.
1.
Introduction
Efficient local delivery of actives such as dental bleaches and antimicrobials to the oral cavity is compromised by a number of factors that dramatically reduce residence time, most notably the shear forces associated with speaking, swallowing and mastication, as well as dilution and washout caused
by continuous saliva production.1 Attempts to increase residence time and maintain therapeutically active concentrations have focused on the use of bioadhesive delivery systems.2 Formats include hydrogels and anhydrous buccal patches.3–8 However, over-hydration of hydrophilic polymers can lead to structural breakdown of the gel and adhesive failure.9,10
* Corresponding author. Tel.: +44 207 848 4791; fax: +44 207 848 4791. ** Corresponding author. Tel.: +44 1932 822179; fax: +44 1932 822120. E-mail addresses: [email protected] (G.P. Martin), [email protected] (G.D. Rees). 0300-5712/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2008.01.014
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journal of dentistry 36 (2008) 351–359
One possible approach for controlling the rate of gel hydration with a view to enhancing substantivity is to formulate bioadhesive hydrophilic polymers in a non-aqueous carrier. The non-aqueous vehicle can be either polar or apolar; polyols (e.g. glycerol) are a typical example of the former whilst paraffin or vegetable-based oils are typical examples of the latter. Co-formulation of hydrophilic polymers with hydrophobic carriers has been previously reported,11 however relatively few reports describe the formulation of hydrophilic polymers in a hydrophilic non-aqueous vehicle. A particular advantage associated with the use of non-aqueous vehicles is that higher concentrations of bioadhesive polymer can be incorporated whilst retaining acceptable flow properties. The bioadhesive properties of oral delivery formats are a major contributing factor in determining the in vivo substantivity of the formulation, and thus availability of the active. There is no universally agreed test method to determine bioadhesion, however the large majority of studies report the work of adhesion (WOA) and the maximum force of detachment (Fmax) as the preferred bioadhesion parameters. The majority of in vitro bioadhesion studies have employed animal mucosae as model substrates.9,11–14 The oral cavity is however unusual in having both soft tissues, such as the tongue, mucosa, gingiva and palate, as well as hard tissues such as dentine and enamel, all of which are potential targets for the delivery and retention of active agents. Carbopol1 is a cross-linked polyacrylic acidbased polymer that has excellent bioadhesive properties over the short term when formulated as an aqueous system. However rapid hydration can occur leading to breakdown of the gel structure and ultimately adhesive failure. In addition, previous workers have documented that an important contributor to favourable adhesion properties can be the presence of ‘adhesion promoters’, such as polyethylene glycol15,16. A possible strategy for prolonging adhesion might be to suspend the hydrophilic polymers in a non-aqueous solvent (such as glycerol) with a view to controlling the rate of hydration. This could allow a longer time for the interaction to develop between the gel and tissue, before hydration eventually disrupts gel structure. The aim of the present study was to develop a suitable nonaqueous polymer-based bioadhesive semi-solid formulation which might have potential for the localised prolonged delivery of active agents to the oral cavity. Bioadhesion parameters (WOA and Fmax) were determined following suitable modification of a tensile tester. The dynamic oscillatory rheology and hydration behaviour of these nonaqueous delivery systems (NADS) was also investigated.
2.
Materials and methods
2.1.
Materials
Polyethylene glycol 400 was supplied by BASF (Ludwigshafen, Germany), glycerol by Croda International (East Yorkshire, UK) and Carbopol1 974P by BF Goodrich (Ohio, USA). Calcium chloride, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen orthophosphate and AR methanol were purchased from BDH Chemicals Ltd., Poole. Potassium chloride was supplied by Fisher Scientific Ltd. (Leicestershire,
UK). Lab-lemco and yeast extract were sourced from Oxoid Ltd. (Hampshire, UK), and protease peptone from Becon, Dickinson & Co. (New Jersey, USA). Hog gastric mucin, type III, partially purified from porcine stomach, and 40% (w/v) urea were purchased from Sigma Chemical Ltd. (Poole, UK). Saliveze1 artificial saliva was purchased from Wyvern Medical Ltd. (Ledbury, UK). Saliveze1 had a measured pH of 6.9 and contained 1% (w/w) carboxymethylcellulose (CMC), calcium chloride, magnesium chloride, potassium chloride, sodium chloride and dibasic sodium phosphate. Phosphatebuffered saline (PBS) pH 6.8 contained 0.10% (w/w) potassium dihydrogen orthophosphate, 0.20% dipotassium hydrogen orthophosphate and 0.85% (w/w) sodium chloride.
2.2.
Preparation of NADS
Formulations shown in Table 1 comprising polyethylene glycol 400 (PEG), Carbopol1 974P and/or glycerol were prepared with the aid of a Silverson1 homogeniser (L4R Mixer). Carbopol was dispersed in PEG at room temperature and mixed for 2 min at 4000 rpm using an ‘emulsor screen’ homogenising head. The mixture was then added to glycerol (preheated to 70 8C) and mixed for a further 20 min at 4000 rpm using a general purpose ‘disintegrating’ head. Delivery system (DS) 12 was mixed for 5 min using a Heidolph1 propeller until homogeneous. DSs 1–6 contained different amounts of Carbopol but a fixed amount of PEG, whereas DSs 2 and 8– 11 contained different amounts of PEG but a fixed amount of Carbopol. DS 7 is based on a proprietary soft-solid formulation designed for oral use. DSs 12 and 13 served as control formulations, the latter being glycerol alone.
2.3.
Tissue preparation
Wistar rats, which had not been sacrificed for the purpose of the present experiments, were donated by King’s College London. The intestine was removed within 30 min of sacrifice and the first 4–5 cm discarded. The remaining section was gently washed with pH 6.0 isotonic phosphate buffer to remove any debris, laid flat and then frozen immediately at 20 8C in order to inhibit muscle contraction and preserve the tissue for future use. Prior to use, tissue samples were thawed
Table 1 – Composition of NADS Formulation
1 2 3 4 5 6 7 8 9 10 11 12 13
Component (% (w/w)) PEG 400
Carbopol1 974P
5.5 5.5 5.5 5.5 5.5 5.5 31.34 10 20 30 0 40 0
4 2 0.25 0.50 0.75 1 0.63 2 2 2 2 0 0
Glycerol 90.50 92.50 94.25 94.00 93.75 93.50 68.03 88 78 68 98 60 100
journal of dentistry 36 (2008) 351–359
Table 2 – Composition of mucin-containing artificial saliva Component Lab-lemco powder Yeast extract Protease peptone Mucin type III: partially purified from porcine stomach Sodium chloride Calcium chloride Potassium chloride
Weight (g dm3) 1.0 2.0 5.0 2.5 0.35 0.20 0.20
at room temperature and sectioned into 1 cm lengths, then opened longitudinally to expose the inner mucosal surface.
2.4.
Artificial and human saliva
Artificial saliva (AS) was prepared in deionised water from the components listed in Table 2. The AS was then autoclaved at ca. 120 8C for 15 min and allowed to cool before adding 1.25 ml of 40% filter-sterilised urea.17 Whole, unstimulated human saliva (WUHS) was obtained over 1 h from one healthy male volunteer, with collection being carried out at least 1.5 h after eating, drinking or brushing. Saliva samples were collected in 50 ml polypropylene tubes and centrifuged for 30 min at 4 8C (27,000 g); the resulting supernatant was kept on ice and used the same day.
2.5.
In vitro tensile testing
A Dartec (Dartec Ltd., M9500 control system, West Midlands, UK) tensile tester was modified in order to measure WOA and Fmax. The upper aluminium platform consisted of two parts: an outer cylinder, screwed onto the load cell and an inner cylinder (to which the substrate was attached) that was able to freely move up and down within the outer cylinder (Fig. 1). To reduce
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the risk of mucosal lifting during tensile testing, PVC sleeves were used to secure the tissue flat in place on the upper and lower supports. Samples (0.10 ml) of the various test formulations were syringed onto the mucosal substrate (0.75 cm diameter) secured to the lower platform. The upper platform with a second portion of mucosal substrate secured in place was then lowered and a compression force (CF) of either 0.63 N or 1.10 N was applied normally to the sample and tissues. The applied force was designed to mimic application of an oral gel using the finger, and is comparable to that used in brushing studies.18,19 CFs are more properly defined in terms of force per unit area and in the case of the present study were 14.26 kN m2 and 24.90 kN m2, respectively. It is relevant to note that in the case of mucosal tissues the total available surface area is larger than that of a uniform flat inert surface, and consequently these values should be considered estimates. Compression times (CTs) of 1 min, 5 min and 10 min were investigated. Although long in the context of the time involved in digitally applying an oral gel, the CTs are relevant because they provide insight into how the bioadhesive properties of the formulation are likely to change immediately following application to the mucosal surface. The CF was then removed and the upper platform raised at a rate of 0.10 mm s1, allowing calculation of the WOA and Fmax via Origin1 (version 5.0) analytical software.
2.6.
Hydration studies
Hydration studies employed a modified cell-insert design based on a previously published method.20 The rates of hydration of the NADS when placed in contact with deionised water, phosphate buffered saline (PBS) pH 6.8, Saliveze1 and AS were determined gravimetrically, according to Eq. (1):
weight gain ð%Þ ¼
Wt W0 100 W0
Fig. 1 – Photographic illustration of Dartec tensile tester used to determine bioadhesion parameters.
(1)
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where W0 and Wt are the weights of the dry (t = 0) and hydrated NADS at time, t, respectively. Approximately 2 g samples of the NADS were accurately weighed into Nunc Anopore1 (25 mm diameter 13 mm height) porous tissue culture cell inserts, then immediately transferred to individual multi-well dish chambers containing 2 ml of the relevant hydration fluid. Two membrane pore sizes (0.02 mm and 0.20 mm) were initially employed during model validation. Chambers were incubated under ambient conditions and the inserts removed at intervals, gently blotted on absorbent paper for a few seconds, and weighed. Inserts were then placed into fresh chambers containing new media and re-sealed. Inserts were weighed every 5 min for the first hour, and subsequently every 30 min for a further 3 h.
2.7.
Dynamic oscillatory rheology
Rheological investigations were conducted on NADS both 6 and 30 months after preparation, during which time they were stored in sealed glass containers at room temperature. A Bohlin CS (controlled stress) CVO rheometer (Bohlin Instruments, Cirencester, UK) fitted with a 40 mm stainless steel parallel plate and a gap setting of 1 mm was employed. The rheometer was thermostatted to 25 8C, and the linear viscoelastic region determined via a torque sweep for each formulation. A stress range of 0.05–10 Pa, at a frequency of 0.1 Hz was applied; 20 measurements were taken during each run. Frequency sweeps, enabling the elastic modulus (G0 ) and viscous modulus (G00 ) to be measured, were then carried out over a frequency range of 0.01–10 Hz. Rheological studies were conducted using a stress of 0.1 Pa, which ensured that all formulations were tested within their linear viscoelastic range.
2.8.
Statistical analyses
Statistical testing was carried out using a three-way ANOVA, in which the bio adhesion parameters (CT and CF) attained for all formulations were compared. Tukey’s and Dunnett’s (ANOVA) comparison methods were also used to display confidence intervals between means, and a confidence interval of 95% was employed in all cases. Pearson’s correlation coefficients (r2) were also determined where appropriate.
3.
Results
3.1.
Bioadhesion of NADS to rat mucosa
The WOA data obtained for the NADS using the two CFs were similar and those obtained employing a CF of 0.63 N are shown in Fig. 2. Formulations containing Carbopol at concentrations of 1% or less (i.e. DS 6, DS 7, DS 12 and DS 13) produced similar values for WOA at both CFs when CTs of 1 and 5 min were employed. The WOA tended to increase as CT increased but this was most clearly apparent for DS 6, where the WOA was greater than DS 12 and DS 13 ( p < 0.05) when a CT of 10 min was employed. There was no significant difference in WOA values obtained for DS 7 and the negative controls DS 12 and DS 13 under any of the
Fig. 2 – Comparison of work of WOA on rat mucosa determined as a function of CT (1 min, 5 min and 10 min, respectively) at a CF of 0.63 N (WS.D., n = 6).
selected experimental conditions. When the Carbopol concentration was increased to 2% or greater, statistically significant increases in WOA were observed at both CFs. At a CF of 0.63 N and CTs of 5 and 10 min, the WOA for DS 1 was found to be greater than that obtained for all other formulations ( p < 0.05). In contrast, after applying a CF of 1.10 N for 1 min only, the WOA value for DS 1 was at least comparable to all four formulations containing 2% Carbopol, but found to be lower ( p < 0.05) than the values obtained for DS 9 and DS 10 (data not shown). As the PEG concentration was increased from 5.5% (DS 2) to 30% (DS 10) in formulations containing 2% Carbopol the WOA was found to be at a minimum when 10% PEG was employed (DS 8) as shown in Fig. 2. The equivalent Fmax data set obtained at a CF of 0.63 N is summarised in Fig. 3, and was very similar to that obtained when the higher CF of 1.1 N was employed. The Fmax values were relatively insensitive to the CT, with only the Fmax associated with DS 1 being markedly different from the other values obtained. Addition of PEG to the formulations containing 2% Carbopol had no apparent effect on Fmax ( p 0.5), neither was there a consistent trend. The variation associated with the resultant Fmax values was markedly greater than that associated with WOA.
Fig. 3 – Comparison of Fmax on rat mucosa determined as a function of CT (1 min, 5 min and 10 min, respectively) at a CF of 0.63 N (WS.D., n = 6).
journal of dentistry 36 (2008) 351–359
Fig. 4 – Mean percentage increase in weight as a function of time for DS 2 and DS 6 hydrated with deionised water using a cell culture insert hydration model as a function of time with 0.02 mm and 0.20 mm pore sizes (mean W S.D., n = 6).
3.2.
Hydration studies
The hydration of formulations DS 6 and DS 2, containing 1% and 2% Carbopol, respectively, in a cell culture insert with deionised water showed that pore size influenced hydration rate (Fig. 4). Nevertheless, the hydration rates of the two formulations could be differentiated ( p < 0.05) using both 0.02 mm and 0.20 mm membranes, with DS 6 exhibiting the higher rate of water uptake. Based on these preliminary results, the membrane with the larger pore size (0.20 mm) was employed in subsequent hydration studies, since the larger pore size provided least resistance to the transport of water across the membrane, but still adequately contained the formulation throughout the study. This was considered to be particularly relevant when comparing the hydration rates using media of differing viscosities, in particular Saliveze1 which contains CMC as a viscosity modifier. The initial rates of water uptake by the NADS (Table 1) are shown in ranked order in Fig. 5. DS 1 was not tested since, as a very highly structured vehicle, the rheological properties prevented it from being loaded within the cell inserts. DS 13, used as a control, leaked from the cell insert irrespective of the choice of membrane, and was consequently not included in the analysis. DS 2, DS 9 and DS 11 exhibited the slowest initial hydration rates ( p < 0.05), whilst DS 3 was the most rapid. Water uptake was effectively linear for all formulations during the first 60 min of the experiment. Decreasing the Carbopol concentration significantly increased the hydration rate ( p < 0.05) with, for example, the initial hydration rate of DS 3 being twice that of DS 2 ( p < 0.05). Increasing the PEG concentration of NADS containing 2% (w/ w) Carbopol resulted in statistically significant increases in the hydration rate of the resultant formulation in comparison to DS 2.
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Fig. 5 – Initial water uptake rate for NADS hydrated using deionised water (mean W S.D., n = 6).
After hydration for 4 h, DS 5, DS 7 and DS 10 were found to have gained the greatest weight, and the weight increases were indistinguishable ( p > 0.05). The formulations that gained the least weight over this period were DS 3 and DS 2. When comparing the formulations containing 2% Carbopol, only when the PEG concentration was increased to 30% was the subsequent percentage weight gain after 4 h also increased ( p < 0.05). Hydration rates using PBS, AS and Saliveze1 were investigated for DS 2, DS 5 and DS 10. Selection of this limited group allowed comparison of the twin effects of PEG concentration and Carbopol concentration to be investigated and a typical data set showing the rate of hydration of DS 5 is shown in Fig. 6. When formulations were hydrated using Saliveze1, the percentage weight gain over the experimental
Fig. 6 – Mean percentage weight gain plotted as a function of hydration time (min) of DS 5 hydrated with deionised water, artificial saliva (AS), phosphate buffered saline (PBS) and SalivezeW (S) (mean W S.D., n = 6).
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journal of dentistry 36 (2008) 351–359
period was markedly decreased ( p < 0.05) in comparison to deionised water, PBS and AS. For the three formulations over the 4 h measurement period, the greatest weight gain was observed using deionised water as the hydration medium ( p < 0.05). Weight gain by hydrating the individual formulations with either AS or PBS was similar over this period. The formulation containing 30% PEG (DS 10) was found to increase in weight faster than the formulation containing 5.5% PEG (DS 2) when PBS, AS and deionised water were used as the hydrating solvents ( p < 0.05). Water uptake for all three NADS was effectively linear over the first 60 min of the experiment for three of the hydrating solvents, although Saliveze1 proved to be the exception. Beyond 60 min, weight gain profiles became non-linear, and in the case of Saliveze1 appeared to approach a plateau. For individual NADS, the initial rates of hydration using deionised water, AS and PBS were comparable, but in all cases were slowest from Saliveze1 ( p < 0.05). Formulation DS 5 consistently exhibited the fastest initial rate of water uptake, irrespective of the choice of hydration medium ( p < 0.05).
3.3.
Dynamic oscillatory rheology
The semi-solid formulations used in the present study were shown to be viscoelastic, exhibiting properties both of solids and liquids. The use of dynamic oscillatory testing allowed the solid-like (elastic) and liquid-like (viscous) properties of the formulations to be determined simultaneously. These properties are important in the context of the proposed use since the formulations should not be so elastic that they cannot be effectively applied, but nor should the formulations be so unstructured and liquid-like that they are immediately removed by saliva flow and speaking. These experiments determine the structure in the formulations in their rheologically ‘ground’ state, that is to say the method of measurement is not destroying the sample. The rheological parameters G0 and G00 of the various formulations were determined as a function of frequency (Figs. 7 and 8). As the frequency of oscillation increases then the formulations exhibit more solidlike properties (as shown by G0 increasing), this corresponds to the elastic characteristics exhibited by such systems over short-time intervals. At lower frequencies, the liquid-like properties are more dominant (since the material will flow over longer time intervals) and G00 becomes larger. The magnitude of G0 and G00 also exhibited a dependence on the Carbopol and PEG concentration, with G00 predominant for formulations containing 1% Carbopol or less ( p < 0.05). This is more clearly illustrated in Fig. 9, which shows the effect of varying the Carbopol concentration on the viscoelastic moduli at 1 Hz of formulations containing 5.5% PEG. At higher oscillation frequencies, 5 Hz and 10 Hz, the differences between the values of G0 and G00 were greater. The Carbopol concentration at which the plots of G0 and G00 intersect can be considered as defining the transition point between a highly structured liquid and a gel. Thus G0 was dominant for DS 2 and DS 11 across the frequency range employed, although DS 11 exhibited the greater elastic properties ( p < 0.05). Mean values of G00 for these two formulations were not statistically differentiable. Addition of up to 10% (w/w) PEG to the NADS formulations containing 2% (w/w) Carbopol, significantly lowered the G0
Fig. 7 – Mean elastic modulus (G0 ) of NADS plotted as a function of oscillation frequency (mean W S.D., n = 3).
Fig. 8 – Mean viscous modulus (G00 ) of NADS plotted as a function of oscillation frequency (mean W S.D., n = 3).
value to an apparent local minimum ( p < 0.05). The increase in the PEG concentration to 30% (w/w) resulted in a moderate increase in G0 . Mean G00 values obtained for DS 8, DS 9 and DS 10 were not statistically differentiable. No significant differences in either G0 or G00 were observed for NADS containing 1% Carbopol stored for either 6 or 30 months. For those formulations containing 2% Carbopol, values of G0 and G00 were marginally lower at 30 months, with the decrease in G0 of formulation DS 2 significantly lower, indicating a loss in structure (data not shown).
journal of dentistry 36 (2008) 351–359
Fig. 9 – The effect of Carbopol concentration (% (w/w)) on the elastic (G0 ) and viscous (G00 ) moduli, at a frequency of 1 Hz, in NADS containing 5.5% (w/w) polyethylene glycol 400 stored for 6 months (mean W S.D., n = 3). The dashed lines represent the trend lines for G0 and G00 .
4.
Discussion
The WOA reflects the total energy input required for adhesive failure to occur; Fmax is the maximum force the adhesive joint can withstand before fracture, and depends on the strength of the weakest component of the adhesive joint. In the case of rat mucosa, fracture could occur within the mucus layer itself or within the formulation if the cohesive forces are weaker than the bioadhesive forces, or at the interface if the converse is true. In the present study, contact of NADS with a hydrated mucosal surface results in water uptake by the formulation. At the microscopic level, local controlled hydration facilitates interpenetration of polymer chains at the interface, and promotes structure build. Among the variables affecting bioadhesion, formulation rheology would be expected to impact on WOA and Fmax as the flow behaviour of the formulations change under the influence of external forces. This affects the intimacy of contact and penetration of crenulations present on the mucosal surface, all of which impact on the effective contact area and consequently the magnitude of the adhesion force. In comparison to the formulations containing Carbopol, DS 13 and DS 12 were rheologically much less structured and this is reflected by the reduction in magnitude of the bioadhesion parameters compared to formulations containing Carbopol. Within the range used in this study, the effect of increasing CT was to increase WOA and to a lesser extent Fmax. Increased contact time under load promotes spreading, penetration and bond formation. The impact of changing CT on WOA and Fmax was markedly greater than that of changing CF. These observations are consistent with a number of previously reported studies.21,22 Increasing the PEG concentration to 30% (w/w) in formulations containing 2% Carbopol resulted in a net increase in WOA and Fmax. Similar behaviour has been
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previously reported for formulations enriched in PEG, where significant improvements in mucoadhesion were attributed to the penetration of PEG chains across the mucosa–polymer interface.15 In the present study, despite the Carbopol concentration of DS 7 being almost half that of DS 6 (0.63% vs. 1%, respectively), values of WOA and Fmax values were not statistically differentiable. This is consistent with the argument that the elevated concentration of PEG present in DS 7 (31.34%) contributes to its bioadhesive performance. Ideally, in vitro bioadhesion testing would be conducted under well-controlled conditions of temperature and humidity. Unfortunately, however, this presents significant logistical difficulties not least of which is maintaining equilibrium conditions of temperature and humidity when applying the formulation. In the present study, precise control of the temperature and humidity was not possible, however the laboratory was air-conditioned and a temperature record kept during the course of these studies gave an average working temperature of 20 2 8C. Since these formulations are designed for oral care, a humidified atmosphere with a relative humidity >90% would have been preferred. Practical limitations also prevent the determination of WOA and Fmax in the presence of shear forces that are clearly of importance in vivo. Whilst the rheological parameters provide information regarding how such formulation operate under shear, evaluation of their likely substantivity in vivo would be aided by in vitro testing under dynamic flow conditions,23 and this will form the focus of a forthcoming study. Water is intimately involved in the dynamic equilibrium process of bond making, bond breaking, and molecular reorganisation that accompanies and controls bioadhesion.24 As previously highlighted, however, excessive hydration can rapidly reduce bioadhesion, and this is particularly marked for formulations based on fast-hydrating, hydrophilic polymers such as polyacrylic acids.25–27 The preliminary hydration studies demonstrated that the cell culture insert model was able to differentiate between formulations containing different levels of Carbopol. Water uptake was shown to be dependent on the PEG and Carbopol concentration as well as the hydration medium itself. The choice of hydration model was driven, in part at least, by the concern that exposing semi-solid formulations to an environment where excess hydration medium was present from the outset would confound measurement of the hydration properties, due to the possibility of rapid dissolution and disintegration. A further reason for choosing this model stemmed from the fact that the oral cavity does not present an environment where an excess of hydration media would be present and it is the moist mucosal surface which provides the principal source of hydrating solvent. Generally, formulations containing Carbopol concentrations of 1% (w/w) hydrated at a significantly faster rate during the initial linear phase than formulations containing 2% (w/w) Carbopol. Increasing the PEG concentration in formulations containing 2% (w/w) Carbopol to 30% (w/w) also enhanced the initial hydration rate. A number of physicochemical factors can be expected to impact the results obtained. Hydration of the formulations is thermodynamically driven, and occurs as a consequence of the hydrophilic components of the formulations sorbing
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aqueous media from its environment. Aside from the presence of an obvious diffusion gradient, the capacity and availability of carboxyl groups in the Carbopol, as well as hydroxyl groups in the PEG and glycerol to hydrogen bond induces water uptake into the gels. The marked reduction in hydration capacity of DS 3 at longer incubation times was attributed to the low Carbopol concentration in the formulation, whereas the low apparent hydration capacity observed for DS 2 was attributed to the high viscosity inherent to the formulation. The rheological parameters G0 and G00 were dependent on both the Carbopol, and to a lesser extent, the PEG concentration of the formulations. Formulation rheology also impacted upon the hydration behaviour with the most highly structured systems, such as DS 2 and DS 11, exhibiting the slowest rates of water uptake. For formulations hydrated using PBS and AS the decrease in water uptake, although not dramatic, was attributed to their higher ionic strength of these media compared to water. The influence of ionic strength on the hydration of DS 5 was greater than when DS 2 and DS 10 were hydrated and this was possibly attributable to the lower viscosity of the DS 5, allowing the hydration medium to more readily penetrate the formulation. Workers investigating the hydration properties of polyethylene oxide, chitosan and xanthan gum gels have also shown a decrease in hydration rate when hydrated in 0.10 M PBS rather than water.18 The decrease in percentage weight gain observed when formulations were hydrated using Saliveze1 was considered to be due to the relatively high viscosity of this commercial saliva substitute, as well as the presence of ions in solution. When comparing the results obtained for the formulations hydrated in PBS and AS, the presence of mucin glycoproteins, which under physiological conditions are net negatively charged, might have been expected to slow the percentage weight gain. However, percentage weight gain by the three NADS was similar whether from PBS or AS and thus it was concluded that the addition of the freeze-dried mucin did not impact significantly on the ionic strength of the hydration medium. In conclusion, this initial in vitro study would suggest that the use of water miscible, non-aqueous vehicles containing bioadhesive polymers such as Carbopol show promise as delivery systems for the oral cavity. The bioadhesive properties were influenced by the initial rheological properties of the vehicle.
Acknowledgements The authors would like to thank GlaxoSmithKline R&D, St. George’s Avenue, Weybridge, Surrey for funding this work and supporting Dr. Zaman during his PhD studies.
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2. Chickering DE, Jacob JS, Desai TA, Harrison M, Harris WP, Morrell CN, et al. Bioadhesive microspheres: III. An in vivo transit and bioavailability study of drug-loaded alginate and poly(fumaric-co-sebacic anhydride) microspheres. Journal of Controlled Release 1997;48:35–46. 3. Martin L, Wilson CG, Koosha F, Uchegbu IF. Sustained buccal delivery of the hydrophobic drug denbufylline using physically cross-linked palmitoyl glycol chitosan hydrogels. European Journal of Pharmaceutics and Biopharmaceutics 2003;55:35–45. 4. Mallery SR, Stoner GD, Larsen PE, Fields HW, Rodrigo KA, Schwartz SJ, et al. Formulation and in vitro and in vivo evaluation of a mucoadhesive gel containing freeze dried black raspberries: Implications for oral cancer chemoprevention. Pharmaceutical Research 2007;24:728–37. 5. Abdel-Hamid SM, Abdel-Hady SE, El-Shamy AA, ElDessouky HF. Formulation of an antispasmodic drug as a topical local anaesthetic. International Journal of Pharmaceutics 2006;326:107–18. 6. Nair MK, Chein YW. Development of anticandidal delivery systems. (II) Mucoadhesive devices for prolonged drug delivery in the oral cavity. Drug Development and Industrial Pharmacy 1996;22:243–53. 7. Cosyn J, Sabzevar MM. A systematic review of subgingival chlorhexidine gel administration in the treatment of chronic periodontitis. Journal of Periodontology 2005;76:1805– 13. 8. Schiff T. Anticalculus effect of a cetylpyridinium chloride/ zinc gluconate mucoadhesive gel: results of a randomized, double blind, controlled clinical trial. Journal of Clinical Dentistry 2007;18:79–81. 9. Smart JD. Recent developments in the use of bioadhesive systems for delivery of drugs to the oral cavity. Critical Reviews in Therapeutic Drug Delivery Systems 2004;21:319–44. 10. Bromberg L, Temchenko M, Alakhov V, Hatton TA. Bioadhesive properties and rheology of polyether-modified poly(acrylic acid) hydrogels. International Journal of Pharmaceutics 2004;282:45–60. 11. Mortazavi SA, Smart JD. An investigation into the role of water movement and mucus gel dehydration in mucoadhesion. Journal of Controlled Release 1993;25:197–203. 12. Mortazavi SA, Smart JD. An investigation of some factors influencing the in vitro assessment of mucoadhesion. International Journal of Pharmaceutics 1995;116:223–30. 13. Wong CF, Yuen KH, Peh KK. Formulation and evaluation of controlled release Eudragit buccal devices. International Journal of Pharmaceutics 1999;178:11–22. 14. Smart JD. An in vitro assessment of some mucosa-adhesive dosage forms. International Journal of Pharmaceutics 1991;73:69–74. 15. Huang Y, Leobandung W, Foss A, Peppas NA. Molecular aspects of muco- and bioadhesion: tethered structures and site-specific surfaces. Journal of Controlled Release 2000;65:63– 71. 16. De Ascentis A, de Grazia JL, Bowman CN, Colombo P, Peppas NA. Mucoadhesion of poly(2-hydroxyethylmethacrylate) is improved when linear poly(ethylene oxide) chains are added to the polymer network. Journal of Controlled Release 1995;33:197–201. 17. Wilson M, Kpendema H, Noar JH, Hunt N, Mordan NJ. Corrosion of intra-oral magnets in the presence and absence of biofilms of Streptococcus sanguis. Biomaterials 1995;16:721–5. 18. Driesden GM, Warren PR, Ernst CP. A laboratory comparison of efficacy of two powered toothbrushes. Journal of Dental Research 2000;79:S1233. 19. Hefferren JJ, Coffman L, Berg ML. Functionality of toothbrush/toothpaste systems. Journal of Dental Research 2000;79:S582.
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20. Needleman IG, Martin GP, Smales FC. Characterisation of bioadhesives for periodontal and oral mucosal drug delivery. Journal of Clinical Periodontology 1998;25:74–82. 21. Wong CF, Yuen KH, Peh KK. An in vitro method for buccal adhesion studies: importance of instrument variables. International Journal of Pharmaceutics 1999;180:47–57. 22. Shojaei AH, Paulson J, Honary S. Evaluation of poly(acrylic acid-co-ethylhexyl acrylate) films for mucoadhesive transbuccal drug delivery: factors affecting the force of mucoadhesion. Journal of Controlled Release 2000;67: 223–32. 23. Kockisch S, Rees GD, Young SA, Tsibouklis J, Smart JD. In situ evaluation of drug-loaded microspheres on a mucosal surface under dynamic test conditions. International Journal of Pharmaceutics 2004;276:51–8. 24. Gu J-M, Robinson JR, Leung S-H. Binding of acrylic polymers to mucin/epithelial surfaces: structure–property
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relationships. CRC Critical Reviews in Therapeutic Drug Carrier Systems 1998;5:21–67. 25. Garcı´a-Gonza´lez N, Kellaway IW, Blanco Fuente H, Anguiano Igea S, Delgado Charro B, Otero Espinar FJ, et al. Influence of glycerol concentration and Carbopol molecular weight on swelling and drug release characteristics of metoclopramide hydrogels. International Journal of Pharmaceutics 1994;104:107–13. 26. Garcı´a-Gonza´lez N, Kellaway IW, Blanco Fuente H, Anguiano Igea S, DelgadoCharro B, Otero Espinar FJ, et al. Design and evaluation of buccoadhesive metoclopramide hydrogels composed of poly(acrylic acid) crosslinked with sucrose. International Journal of Pharmaceutics 1993;100:65–70. 27. Ponchel G, Touchard F, Duchene D, Peppas NA. Bioadhesive analysis of controlled release systems. I. Fracture and interpenetration analysis in poly (acrylic acid)-contained systems. Journal of Controlled Release 1987;5:129–41.
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Mucoadhesion, hydration and rheological properties of non-aqueous delivery systems (NADS) for the oral cavity Mustafa A. Zaman a, Gary P. Martin a,*, Gareth D. Rees b,** a b
King’s College London, Phamaceutical Science Division, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK GlaxoSmithKline R&D, St. George’s Avenue, Weybridge, Surrey KT13 0DE, UK
article info
abstract
Article history:
Objectives: Effective delivery of oral care actives from conventional hydrogel formulations is
Received 5 July 2007
often compromised by poor retention associated with shear forces present in the mouth,
Received in revised form
salivary washout and over-hydration of the gel which can lead to structural breakdown and
16 January 2008
adhesive failure. Non-aqueous gels offer the opportunity to formulate rheologically accep-
Accepted 17 January 2008
table vehicles with higher concentrations of bioadhesive polymer than is possible using water as the primary solvent. Accordingly, this study describes the formulation and characterisation of the rheology, hydration and bioadhesive properties of a range of non-
Keywords:
aqueous delivery systems (NADS).
Mucoadhesion
Methods: The formulations were composed principally of glycerol, with varying amounts of
Rheology
polyethylene glycol (PEG) 400 and Carbopol1 974P being incorporated in the ranges 0–31.34%
Hydration
and 0–4% (w/w). Work of adhesion (WOA) and maximum force of detachment (Fmax) were
Non-aqueous delivery systems
determined using a Dartec tensile tester after application of a normal force. Rheology was
(NADS)
assessed using a Bohlin CS CVO rheometer. Results: WOA and Fmax increased with increasing compression time and Carbopol concentration. Addition of 30% (w/w) PEG 400 to the formulation containing 2% Carbopol in glycerol improved bioadhesive function. Formulation rheology was largely controlled by the Carbopol concentration, and to a lesser extent by the concentration of PEG 400 and these, in turn, largely determined the bioadhesion parameters and rates of hydration. Conclusion: The results of this in vitro study suggest that bioadhesion, and consequently potential drug bioavailability, would be enhanced by use of a water miscible non-aqueous delivery vehicle such as glycerol containing a bioadhesive polymer such as Carbopol with the addition of controlled amounts of PEG as plasticiser. # 2008 Elsevier Ltd. All rights reserved.
1.
Introduction
Efficient local delivery of actives such as dental bleaches and antimicrobials to the oral cavity is compromised by a number of factors that dramatically reduce residence time, most notably the shear forces associated with speaking, swallowing and mastication, as well as dilution and washout caused
by continuous saliva production.1 Attempts to increase residence time and maintain therapeutically active concentrations have focused on the use of bioadhesive delivery systems.2 Formats include hydrogels and anhydrous buccal patches.3–8 However, over-hydration of hydrophilic polymers can lead to structural breakdown of the gel and adhesive failure.9,10
* Corresponding author. Tel.: +44 207 848 4791; fax: +44 207 848 4791. ** Corresponding author. Tel.: +44 1932 822179; fax: +44 1932 822120. E-mail addresses: [email protected] (G.P. Martin), [email protected] (G.D. Rees). 0300-5712/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2008.01.014
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journal of dentistry 36 (2008) 351–359
One possible approach for controlling the rate of gel hydration with a view to enhancing substantivity is to formulate bioadhesive hydrophilic polymers in a non-aqueous carrier. The non-aqueous vehicle can be either polar or apolar; polyols (e.g. glycerol) are a typical example of the former whilst paraffin or vegetable-based oils are typical examples of the latter. Co-formulation of hydrophilic polymers with hydrophobic carriers has been previously reported,11 however relatively few reports describe the formulation of hydrophilic polymers in a hydrophilic non-aqueous vehicle. A particular advantage associated with the use of non-aqueous vehicles is that higher concentrations of bioadhesive polymer can be incorporated whilst retaining acceptable flow properties. The bioadhesive properties of oral delivery formats are a major contributing factor in determining the in vivo substantivity of the formulation, and thus availability of the active. There is no universally agreed test method to determine bioadhesion, however the large majority of studies report the work of adhesion (WOA) and the maximum force of detachment (Fmax) as the preferred bioadhesion parameters. The majority of in vitro bioadhesion studies have employed animal mucosae as model substrates.9,11–14 The oral cavity is however unusual in having both soft tissues, such as the tongue, mucosa, gingiva and palate, as well as hard tissues such as dentine and enamel, all of which are potential targets for the delivery and retention of active agents. Carbopol1 is a cross-linked polyacrylic acidbased polymer that has excellent bioadhesive properties over the short term when formulated as an aqueous system. However rapid hydration can occur leading to breakdown of the gel structure and ultimately adhesive failure. In addition, previous workers have documented that an important contributor to favourable adhesion properties can be the presence of ‘adhesion promoters’, such as polyethylene glycol15,16. A possible strategy for prolonging adhesion might be to suspend the hydrophilic polymers in a non-aqueous solvent (such as glycerol) with a view to controlling the rate of hydration. This could allow a longer time for the interaction to develop between the gel and tissue, before hydration eventually disrupts gel structure. The aim of the present study was to develop a suitable nonaqueous polymer-based bioadhesive semi-solid formulation which might have potential for the localised prolonged delivery of active agents to the oral cavity. Bioadhesion parameters (WOA and Fmax) were determined following suitable modification of a tensile tester. The dynamic oscillatory rheology and hydration behaviour of these nonaqueous delivery systems (NADS) was also investigated.
2.
Materials and methods
2.1.
Materials
Polyethylene glycol 400 was supplied by BASF (Ludwigshafen, Germany), glycerol by Croda International (East Yorkshire, UK) and Carbopol1 974P by BF Goodrich (Ohio, USA). Calcium chloride, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen orthophosphate and AR methanol were purchased from BDH Chemicals Ltd., Poole. Potassium chloride was supplied by Fisher Scientific Ltd. (Leicestershire,
UK). Lab-lemco and yeast extract were sourced from Oxoid Ltd. (Hampshire, UK), and protease peptone from Becon, Dickinson & Co. (New Jersey, USA). Hog gastric mucin, type III, partially purified from porcine stomach, and 40% (w/v) urea were purchased from Sigma Chemical Ltd. (Poole, UK). Saliveze1 artificial saliva was purchased from Wyvern Medical Ltd. (Ledbury, UK). Saliveze1 had a measured pH of 6.9 and contained 1% (w/w) carboxymethylcellulose (CMC), calcium chloride, magnesium chloride, potassium chloride, sodium chloride and dibasic sodium phosphate. Phosphatebuffered saline (PBS) pH 6.8 contained 0.10% (w/w) potassium dihydrogen orthophosphate, 0.20% dipotassium hydrogen orthophosphate and 0.85% (w/w) sodium chloride.
2.2.
Preparation of NADS
Formulations shown in Table 1 comprising polyethylene glycol 400 (PEG), Carbopol1 974P and/or glycerol were prepared with the aid of a Silverson1 homogeniser (L4R Mixer). Carbopol was dispersed in PEG at room temperature and mixed for 2 min at 4000 rpm using an ‘emulsor screen’ homogenising head. The mixture was then added to glycerol (preheated to 70 8C) and mixed for a further 20 min at 4000 rpm using a general purpose ‘disintegrating’ head. Delivery system (DS) 12 was mixed for 5 min using a Heidolph1 propeller until homogeneous. DSs 1–6 contained different amounts of Carbopol but a fixed amount of PEG, whereas DSs 2 and 8– 11 contained different amounts of PEG but a fixed amount of Carbopol. DS 7 is based on a proprietary soft-solid formulation designed for oral use. DSs 12 and 13 served as control formulations, the latter being glycerol alone.
2.3.
Tissue preparation
Wistar rats, which had not been sacrificed for the purpose of the present experiments, were donated by King’s College London. The intestine was removed within 30 min of sacrifice and the first 4–5 cm discarded. The remaining section was gently washed with pH 6.0 isotonic phosphate buffer to remove any debris, laid flat and then frozen immediately at 20 8C in order to inhibit muscle contraction and preserve the tissue for future use. Prior to use, tissue samples were thawed
Table 1 – Composition of NADS Formulation
1 2 3 4 5 6 7 8 9 10 11 12 13
Component (% (w/w)) PEG 400
Carbopol1 974P
5.5 5.5 5.5 5.5 5.5 5.5 31.34 10 20 30 0 40 0
4 2 0.25 0.50 0.75 1 0.63 2 2 2 2 0 0
Glycerol 90.50 92.50 94.25 94.00 93.75 93.50 68.03 88 78 68 98 60 100
journal of dentistry 36 (2008) 351–359
Table 2 – Composition of mucin-containing artificial saliva Component Lab-lemco powder Yeast extract Protease peptone Mucin type III: partially purified from porcine stomach Sodium chloride Calcium chloride Potassium chloride
Weight (g dm3) 1.0 2.0 5.0 2.5 0.35 0.20 0.20
at room temperature and sectioned into 1 cm lengths, then opened longitudinally to expose the inner mucosal surface.
2.4.
Artificial and human saliva
Artificial saliva (AS) was prepared in deionised water from the components listed in Table 2. The AS was then autoclaved at ca. 120 8C for 15 min and allowed to cool before adding 1.25 ml of 40% filter-sterilised urea.17 Whole, unstimulated human saliva (WUHS) was obtained over 1 h from one healthy male volunteer, with collection being carried out at least 1.5 h after eating, drinking or brushing. Saliva samples were collected in 50 ml polypropylene tubes and centrifuged for 30 min at 4 8C (27,000 g); the resulting supernatant was kept on ice and used the same day.
2.5.
In vitro tensile testing
A Dartec (Dartec Ltd., M9500 control system, West Midlands, UK) tensile tester was modified in order to measure WOA and Fmax. The upper aluminium platform consisted of two parts: an outer cylinder, screwed onto the load cell and an inner cylinder (to which the substrate was attached) that was able to freely move up and down within the outer cylinder (Fig. 1). To reduce
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the risk of mucosal lifting during tensile testing, PVC sleeves were used to secure the tissue flat in place on the upper and lower supports. Samples (0.10 ml) of the various test formulations were syringed onto the mucosal substrate (0.75 cm diameter) secured to the lower platform. The upper platform with a second portion of mucosal substrate secured in place was then lowered and a compression force (CF) of either 0.63 N or 1.10 N was applied normally to the sample and tissues. The applied force was designed to mimic application of an oral gel using the finger, and is comparable to that used in brushing studies.18,19 CFs are more properly defined in terms of force per unit area and in the case of the present study were 14.26 kN m2 and 24.90 kN m2, respectively. It is relevant to note that in the case of mucosal tissues the total available surface area is larger than that of a uniform flat inert surface, and consequently these values should be considered estimates. Compression times (CTs) of 1 min, 5 min and 10 min were investigated. Although long in the context of the time involved in digitally applying an oral gel, the CTs are relevant because they provide insight into how the bioadhesive properties of the formulation are likely to change immediately following application to the mucosal surface. The CF was then removed and the upper platform raised at a rate of 0.10 mm s1, allowing calculation of the WOA and Fmax via Origin1 (version 5.0) analytical software.
2.6.
Hydration studies
Hydration studies employed a modified cell-insert design based on a previously published method.20 The rates of hydration of the NADS when placed in contact with deionised water, phosphate buffered saline (PBS) pH 6.8, Saliveze1 and AS were determined gravimetrically, according to Eq. (1):
weight gain ð%Þ ¼
Wt W0 100 W0
Fig. 1 – Photographic illustration of Dartec tensile tester used to determine bioadhesion parameters.
(1)
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journal of dentistry 36 (2008) 351–359
where W0 and Wt are the weights of the dry (t = 0) and hydrated NADS at time, t, respectively. Approximately 2 g samples of the NADS were accurately weighed into Nunc Anopore1 (25 mm diameter 13 mm height) porous tissue culture cell inserts, then immediately transferred to individual multi-well dish chambers containing 2 ml of the relevant hydration fluid. Two membrane pore sizes (0.02 mm and 0.20 mm) were initially employed during model validation. Chambers were incubated under ambient conditions and the inserts removed at intervals, gently blotted on absorbent paper for a few seconds, and weighed. Inserts were then placed into fresh chambers containing new media and re-sealed. Inserts were weighed every 5 min for the first hour, and subsequently every 30 min for a further 3 h.
2.7.
Dynamic oscillatory rheology
Rheological investigations were conducted on NADS both 6 and 30 months after preparation, during which time they were stored in sealed glass containers at room temperature. A Bohlin CS (controlled stress) CVO rheometer (Bohlin Instruments, Cirencester, UK) fitted with a 40 mm stainless steel parallel plate and a gap setting of 1 mm was employed. The rheometer was thermostatted to 25 8C, and the linear viscoelastic region determined via a torque sweep for each formulation. A stress range of 0.05–10 Pa, at a frequency of 0.1 Hz was applied; 20 measurements were taken during each run. Frequency sweeps, enabling the elastic modulus (G0 ) and viscous modulus (G00 ) to be measured, were then carried out over a frequency range of 0.01–10 Hz. Rheological studies were conducted using a stress of 0.1 Pa, which ensured that all formulations were tested within their linear viscoelastic range.
2.8.
Statistical analyses
Statistical testing was carried out using a three-way ANOVA, in which the bio adhesion parameters (CT and CF) attained for all formulations were compared. Tukey’s and Dunnett’s (ANOVA) comparison methods were also used to display confidence intervals between means, and a confidence interval of 95% was employed in all cases. Pearson’s correlation coefficients (r2) were also determined where appropriate.
3.
Results
3.1.
Bioadhesion of NADS to rat mucosa
The WOA data obtained for the NADS using the two CFs were similar and those obtained employing a CF of 0.63 N are shown in Fig. 2. Formulations containing Carbopol at concentrations of 1% or less (i.e. DS 6, DS 7, DS 12 and DS 13) produced similar values for WOA at both CFs when CTs of 1 and 5 min were employed. The WOA tended to increase as CT increased but this was most clearly apparent for DS 6, where the WOA was greater than DS 12 and DS 13 ( p < 0.05) when a CT of 10 min was employed. There was no significant difference in WOA values obtained for DS 7 and the negative controls DS 12 and DS 13 under any of the
Fig. 2 – Comparison of work of WOA on rat mucosa determined as a function of CT (1 min, 5 min and 10 min, respectively) at a CF of 0.63 N (WS.D., n = 6).
selected experimental conditions. When the Carbopol concentration was increased to 2% or greater, statistically significant increases in WOA were observed at both CFs. At a CF of 0.63 N and CTs of 5 and 10 min, the WOA for DS 1 was found to be greater than that obtained for all other formulations ( p < 0.05). In contrast, after applying a CF of 1.10 N for 1 min only, the WOA value for DS 1 was at least comparable to all four formulations containing 2% Carbopol, but found to be lower ( p < 0.05) than the values obtained for DS 9 and DS 10 (data not shown). As the PEG concentration was increased from 5.5% (DS 2) to 30% (DS 10) in formulations containing 2% Carbopol the WOA was found to be at a minimum when 10% PEG was employed (DS 8) as shown in Fig. 2. The equivalent Fmax data set obtained at a CF of 0.63 N is summarised in Fig. 3, and was very similar to that obtained when the higher CF of 1.1 N was employed. The Fmax values were relatively insensitive to the CT, with only the Fmax associated with DS 1 being markedly different from the other values obtained. Addition of PEG to the formulations containing 2% Carbopol had no apparent effect on Fmax ( p 0.5), neither was there a consistent trend. The variation associated with the resultant Fmax values was markedly greater than that associated with WOA.
Fig. 3 – Comparison of Fmax on rat mucosa determined as a function of CT (1 min, 5 min and 10 min, respectively) at a CF of 0.63 N (WS.D., n = 6).
journal of dentistry 36 (2008) 351–359
Fig. 4 – Mean percentage increase in weight as a function of time for DS 2 and DS 6 hydrated with deionised water using a cell culture insert hydration model as a function of time with 0.02 mm and 0.20 mm pore sizes (mean W S.D., n = 6).
3.2.
Hydration studies
The hydration of formulations DS 6 and DS 2, containing 1% and 2% Carbopol, respectively, in a cell culture insert with deionised water showed that pore size influenced hydration rate (Fig. 4). Nevertheless, the hydration rates of the two formulations could be differentiated ( p < 0.05) using both 0.02 mm and 0.20 mm membranes, with DS 6 exhibiting the higher rate of water uptake. Based on these preliminary results, the membrane with the larger pore size (0.20 mm) was employed in subsequent hydration studies, since the larger pore size provided least resistance to the transport of water across the membrane, but still adequately contained the formulation throughout the study. This was considered to be particularly relevant when comparing the hydration rates using media of differing viscosities, in particular Saliveze1 which contains CMC as a viscosity modifier. The initial rates of water uptake by the NADS (Table 1) are shown in ranked order in Fig. 5. DS 1 was not tested since, as a very highly structured vehicle, the rheological properties prevented it from being loaded within the cell inserts. DS 13, used as a control, leaked from the cell insert irrespective of the choice of membrane, and was consequently not included in the analysis. DS 2, DS 9 and DS 11 exhibited the slowest initial hydration rates ( p < 0.05), whilst DS 3 was the most rapid. Water uptake was effectively linear for all formulations during the first 60 min of the experiment. Decreasing the Carbopol concentration significantly increased the hydration rate ( p < 0.05) with, for example, the initial hydration rate of DS 3 being twice that of DS 2 ( p < 0.05). Increasing the PEG concentration of NADS containing 2% (w/ w) Carbopol resulted in statistically significant increases in the hydration rate of the resultant formulation in comparison to DS 2.
355
Fig. 5 – Initial water uptake rate for NADS hydrated using deionised water (mean W S.D., n = 6).
After hydration for 4 h, DS 5, DS 7 and DS 10 were found to have gained the greatest weight, and the weight increases were indistinguishable ( p > 0.05). The formulations that gained the least weight over this period were DS 3 and DS 2. When comparing the formulations containing 2% Carbopol, only when the PEG concentration was increased to 30% was the subsequent percentage weight gain after 4 h also increased ( p < 0.05). Hydration rates using PBS, AS and Saliveze1 were investigated for DS 2, DS 5 and DS 10. Selection of this limited group allowed comparison of the twin effects of PEG concentration and Carbopol concentration to be investigated and a typical data set showing the rate of hydration of DS 5 is shown in Fig. 6. When formulations were hydrated using Saliveze1, the percentage weight gain over the experimental
Fig. 6 – Mean percentage weight gain plotted as a function of hydration time (min) of DS 5 hydrated with deionised water, artificial saliva (AS), phosphate buffered saline (PBS) and SalivezeW (S) (mean W S.D., n = 6).
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journal of dentistry 36 (2008) 351–359
period was markedly decreased ( p < 0.05) in comparison to deionised water, PBS and AS. For the three formulations over the 4 h measurement period, the greatest weight gain was observed using deionised water as the hydration medium ( p < 0.05). Weight gain by hydrating the individual formulations with either AS or PBS was similar over this period. The formulation containing 30% PEG (DS 10) was found to increase in weight faster than the formulation containing 5.5% PEG (DS 2) when PBS, AS and deionised water were used as the hydrating solvents ( p < 0.05). Water uptake for all three NADS was effectively linear over the first 60 min of the experiment for three of the hydrating solvents, although Saliveze1 proved to be the exception. Beyond 60 min, weight gain profiles became non-linear, and in the case of Saliveze1 appeared to approach a plateau. For individual NADS, the initial rates of hydration using deionised water, AS and PBS were comparable, but in all cases were slowest from Saliveze1 ( p < 0.05). Formulation DS 5 consistently exhibited the fastest initial rate of water uptake, irrespective of the choice of hydration medium ( p < 0.05).
3.3.
Dynamic oscillatory rheology
The semi-solid formulations used in the present study were shown to be viscoelastic, exhibiting properties both of solids and liquids. The use of dynamic oscillatory testing allowed the solid-like (elastic) and liquid-like (viscous) properties of the formulations to be determined simultaneously. These properties are important in the context of the proposed use since the formulations should not be so elastic that they cannot be effectively applied, but nor should the formulations be so unstructured and liquid-like that they are immediately removed by saliva flow and speaking. These experiments determine the structure in the formulations in their rheologically ‘ground’ state, that is to say the method of measurement is not destroying the sample. The rheological parameters G0 and G00 of the various formulations were determined as a function of frequency (Figs. 7 and 8). As the frequency of oscillation increases then the formulations exhibit more solidlike properties (as shown by G0 increasing), this corresponds to the elastic characteristics exhibited by such systems over short-time intervals. At lower frequencies, the liquid-like properties are more dominant (since the material will flow over longer time intervals) and G00 becomes larger. The magnitude of G0 and G00 also exhibited a dependence on the Carbopol and PEG concentration, with G00 predominant for formulations containing 1% Carbopol or less ( p < 0.05). This is more clearly illustrated in Fig. 9, which shows the effect of varying the Carbopol concentration on the viscoelastic moduli at 1 Hz of formulations containing 5.5% PEG. At higher oscillation frequencies, 5 Hz and 10 Hz, the differences between the values of G0 and G00 were greater. The Carbopol concentration at which the plots of G0 and G00 intersect can be considered as defining the transition point between a highly structured liquid and a gel. Thus G0 was dominant for DS 2 and DS 11 across the frequency range employed, although DS 11 exhibited the greater elastic properties ( p < 0.05). Mean values of G00 for these two formulations were not statistically differentiable. Addition of up to 10% (w/w) PEG to the NADS formulations containing 2% (w/w) Carbopol, significantly lowered the G0
Fig. 7 – Mean elastic modulus (G0 ) of NADS plotted as a function of oscillation frequency (mean W S.D., n = 3).
Fig. 8 – Mean viscous modulus (G00 ) of NADS plotted as a function of oscillation frequency (mean W S.D., n = 3).
value to an apparent local minimum ( p < 0.05). The increase in the PEG concentration to 30% (w/w) resulted in a moderate increase in G0 . Mean G00 values obtained for DS 8, DS 9 and DS 10 were not statistically differentiable. No significant differences in either G0 or G00 were observed for NADS containing 1% Carbopol stored for either 6 or 30 months. For those formulations containing 2% Carbopol, values of G0 and G00 were marginally lower at 30 months, with the decrease in G0 of formulation DS 2 significantly lower, indicating a loss in structure (data not shown).
journal of dentistry 36 (2008) 351–359
Fig. 9 – The effect of Carbopol concentration (% (w/w)) on the elastic (G0 ) and viscous (G00 ) moduli, at a frequency of 1 Hz, in NADS containing 5.5% (w/w) polyethylene glycol 400 stored for 6 months (mean W S.D., n = 3). The dashed lines represent the trend lines for G0 and G00 .
4.
Discussion
The WOA reflects the total energy input required for adhesive failure to occur; Fmax is the maximum force the adhesive joint can withstand before fracture, and depends on the strength of the weakest component of the adhesive joint. In the case of rat mucosa, fracture could occur within the mucus layer itself or within the formulation if the cohesive forces are weaker than the bioadhesive forces, or at the interface if the converse is true. In the present study, contact of NADS with a hydrated mucosal surface results in water uptake by the formulation. At the microscopic level, local controlled hydration facilitates interpenetration of polymer chains at the interface, and promotes structure build. Among the variables affecting bioadhesion, formulation rheology would be expected to impact on WOA and Fmax as the flow behaviour of the formulations change under the influence of external forces. This affects the intimacy of contact and penetration of crenulations present on the mucosal surface, all of which impact on the effective contact area and consequently the magnitude of the adhesion force. In comparison to the formulations containing Carbopol, DS 13 and DS 12 were rheologically much less structured and this is reflected by the reduction in magnitude of the bioadhesion parameters compared to formulations containing Carbopol. Within the range used in this study, the effect of increasing CT was to increase WOA and to a lesser extent Fmax. Increased contact time under load promotes spreading, penetration and bond formation. The impact of changing CT on WOA and Fmax was markedly greater than that of changing CF. These observations are consistent with a number of previously reported studies.21,22 Increasing the PEG concentration to 30% (w/w) in formulations containing 2% Carbopol resulted in a net increase in WOA and Fmax. Similar behaviour has been
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previously reported for formulations enriched in PEG, where significant improvements in mucoadhesion were attributed to the penetration of PEG chains across the mucosa–polymer interface.15 In the present study, despite the Carbopol concentration of DS 7 being almost half that of DS 6 (0.63% vs. 1%, respectively), values of WOA and Fmax values were not statistically differentiable. This is consistent with the argument that the elevated concentration of PEG present in DS 7 (31.34%) contributes to its bioadhesive performance. Ideally, in vitro bioadhesion testing would be conducted under well-controlled conditions of temperature and humidity. Unfortunately, however, this presents significant logistical difficulties not least of which is maintaining equilibrium conditions of temperature and humidity when applying the formulation. In the present study, precise control of the temperature and humidity was not possible, however the laboratory was air-conditioned and a temperature record kept during the course of these studies gave an average working temperature of 20 2 8C. Since these formulations are designed for oral care, a humidified atmosphere with a relative humidity >90% would have been preferred. Practical limitations also prevent the determination of WOA and Fmax in the presence of shear forces that are clearly of importance in vivo. Whilst the rheological parameters provide information regarding how such formulation operate under shear, evaluation of their likely substantivity in vivo would be aided by in vitro testing under dynamic flow conditions,23 and this will form the focus of a forthcoming study. Water is intimately involved in the dynamic equilibrium process of bond making, bond breaking, and molecular reorganisation that accompanies and controls bioadhesion.24 As previously highlighted, however, excessive hydration can rapidly reduce bioadhesion, and this is particularly marked for formulations based on fast-hydrating, hydrophilic polymers such as polyacrylic acids.25–27 The preliminary hydration studies demonstrated that the cell culture insert model was able to differentiate between formulations containing different levels of Carbopol. Water uptake was shown to be dependent on the PEG and Carbopol concentration as well as the hydration medium itself. The choice of hydration model was driven, in part at least, by the concern that exposing semi-solid formulations to an environment where excess hydration medium was present from the outset would confound measurement of the hydration properties, due to the possibility of rapid dissolution and disintegration. A further reason for choosing this model stemmed from the fact that the oral cavity does not present an environment where an excess of hydration media would be present and it is the moist mucosal surface which provides the principal source of hydrating solvent. Generally, formulations containing Carbopol concentrations of 1% (w/w) hydrated at a significantly faster rate during the initial linear phase than formulations containing 2% (w/w) Carbopol. Increasing the PEG concentration in formulations containing 2% (w/w) Carbopol to 30% (w/w) also enhanced the initial hydration rate. A number of physicochemical factors can be expected to impact the results obtained. Hydration of the formulations is thermodynamically driven, and occurs as a consequence of the hydrophilic components of the formulations sorbing
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aqueous media from its environment. Aside from the presence of an obvious diffusion gradient, the capacity and availability of carboxyl groups in the Carbopol, as well as hydroxyl groups in the PEG and glycerol to hydrogen bond induces water uptake into the gels. The marked reduction in hydration capacity of DS 3 at longer incubation times was attributed to the low Carbopol concentration in the formulation, whereas the low apparent hydration capacity observed for DS 2 was attributed to the high viscosity inherent to the formulation. The rheological parameters G0 and G00 were dependent on both the Carbopol, and to a lesser extent, the PEG concentration of the formulations. Formulation rheology also impacted upon the hydration behaviour with the most highly structured systems, such as DS 2 and DS 11, exhibiting the slowest rates of water uptake. For formulations hydrated using PBS and AS the decrease in water uptake, although not dramatic, was attributed to their higher ionic strength of these media compared to water. The influence of ionic strength on the hydration of DS 5 was greater than when DS 2 and DS 10 were hydrated and this was possibly attributable to the lower viscosity of the DS 5, allowing the hydration medium to more readily penetrate the formulation. Workers investigating the hydration properties of polyethylene oxide, chitosan and xanthan gum gels have also shown a decrease in hydration rate when hydrated in 0.10 M PBS rather than water.18 The decrease in percentage weight gain observed when formulations were hydrated using Saliveze1 was considered to be due to the relatively high viscosity of this commercial saliva substitute, as well as the presence of ions in solution. When comparing the results obtained for the formulations hydrated in PBS and AS, the presence of mucin glycoproteins, which under physiological conditions are net negatively charged, might have been expected to slow the percentage weight gain. However, percentage weight gain by the three NADS was similar whether from PBS or AS and thus it was concluded that the addition of the freeze-dried mucin did not impact significantly on the ionic strength of the hydration medium. In conclusion, this initial in vitro study would suggest that the use of water miscible, non-aqueous vehicles containing bioadhesive polymers such as Carbopol show promise as delivery systems for the oral cavity. The bioadhesive properties were influenced by the initial rheological properties of the vehicle.
Acknowledgements The authors would like to thank GlaxoSmithKline R&D, St. George’s Avenue, Weybridge, Surrey for funding this work and supporting Dr. Zaman during his PhD studies.
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