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Limitations of the rheological mucoadhesion method: The effect of the choice of conditions and the rheological synergism parameter
European Journal of Pharmaceutical Sciences 18 (2003) 349–357 www.elsevier.com / locate / ejps
Limitations of the rheological mucoadhesion method: The effect of the choice of conditions and the rheological synergism parameter ¨ ¨ Katarina Edsman* Helene Hagerstrom, Department of Pharmacy, Uppsala University, Uppsala Biomedical Centre, Box 580, SE 751 23 Uppsala, Sweden Received 16 July 2002; received in revised form 20 December 2002; accepted 19 February 2003
Abstract This work demonstrates several limitations of the simple rheological method that is widely used to investigate mucoadhesion of polymer gels. We establish the importance of the choice of conditions and the synergism parameter for the results obtained in comparative studies. Dynamic rheological measurements were performed on gels based on four slightly different poly(acrylic acid) (Carbopol) polymers and their corresponding mixtures with porcine stomach mucin and bovine submaxillary gland mucin. The rationale for the comparison of the polymers had a large influence on the results obtained. The method does not give the same ranking order when two different comparison strategies are used. Moreover, we show that the results obtained are also sensitive to where in the ‘rheological range’ the comparison is made, e.g., at which value of G9. Positive values of the synergism parameters are, for example, only seen with weak gels. The choice of synergism parameter also has a bearing on the results obtained, and here we suggest a new refined relative parameter, 9 /G p9 )). We also investigated the adhesion of the gel preparations to porcine nasal mucosa, using tensile strength the log ratio (log(G mix measurements. Increased gel strength resulted in stronger adhesion, which is in contrast to the results from the rheological method, where the positive values of the synergism parameters were seen only with weak gels. On the basis of the limitations demonstrated and discussed, we recommend that the rheological method should not be used as a stand-alone method for the studying of mucoadhesive properties of polymer gels. 2003 Elsevier Science B.V. All rights reserved. Keywords: Mucoadhesion; Gel; Rheological synergism; Carbopol; Tensile strength
1. Introduction The mucoadhesive behaviour of polymer gels has been the subject of numerous studies during the last 10 years. Among the most common methods is a simple rheological approach that was first suggested in 1990 by Hassan and Gallo. Usually, dynamic oscillatory measurements are performed to investigate the potential rheological interaction between a polymer gel and mucin. For mucoadhesive polymers it is widely believed that the rheological response of a gel–mucin mixture should be larger than the sum of the contributions from the gel and the mucin, a phenomenon that is commonly described as ‘rheological synergism’. The rheological mucoadhesion method has been used extensively, presumably because of its simplicity, and *Corresponding author. Tel.: 146-18-471-4268; fax: 146-18-4714223. E-mail address: [email protected] (K. Edsman).
several attempts have been made to screen and rank a number of different polymers (e.g., Caramella et al., 1994; Mortazavi, 1995; Tamburic and Craig, 1995; Madsen et al., 1998). However, a wide variation in results is found in the literature, which has been attributed mainly to differences in the mucin type and concentrations used (Rossi et al., 1995; Madsen et al., 1996; Kocevar-Nared et al., 1997; ¨ ¨ et al., 2000) as well as to different polymer Hagerstrom concentrations (Mortazavi and Smart, 1994; Madsen et al., ¨ ¨ et al., 2000). Furthermore, in a previous 1998; Hagerstrom study of ours we found that the results obtained can also vary considerably depending on, for example, the ionsensitivity of the polymer, the quantity of ions present and ¨ ¨ et al., 2000). instrumental factors (Hagerstrom In this paper we study different factors that can affect the results obtained, especially factors encountered when performing comparative studies. One specific problem with comparative and ranking studies is that so far there has been no consensus regarding the conditions under which the comparison should be
0928-0987 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0928-0987(03)00037-X
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made. Should one, for example, compare the polymers at the same concentration, or maybe one should adjust the concentration to make a comparison between polymer preparations with the same rheological properties, i.e., at different concentrations? Does the method give the same results irrespective of the conditions chosen? In this study one objective was to establish how the choice of comparison strategy can influence the results obtained. The two different strategies mentioned above were therefore used to investigate the reliability of the method. The polymers used were a series of Carbopol polymers, which are wellknown commercially available poly(acrylic acid) polymers. The choice was based on the fact that they are well characterised and have been included frequently in rheological mucoadhesion studies (e.g., Mortazavi et al., 1993; Caramella et al., 1994; Mortazavi and Smart, 1994; Madsen et al., 1998; Riley et al., 2001), which makes them suitable for this kind of methodological evaluation. The other aim of this study was to establish how the calculated rheological synergism parameters are affected by the choice of comparison conditions. In the literature no consensus can be found regarding the synergism parameter that is used, which could provide another explanation for the wide variation of results that is found. It is not clear what kind of synergism parameter is the most suitable, an absolute or a relative parameter. In the work presented here this issue is elucidated. We show how the different synergism parameters are influenced by the comparison conditions and we also suggest a new, hopefully refined synergism parameter. We have also investigated the adhesion of the gel preparations to porcine nasal mucosa, using tensile strength measurements, with a view to comparing the results from the rheological approach to a more in vivolike method.
2. Materials and methods
2.1. Materials The poly(acrylic acid) polymers used in this study were the linear polymer Carbopol 907 (C907) and the crosslinked polymers Carbopol 981 (C981), Carbopol 934P (C934) and Carbopol 940 (C940). All polymers were the kind gifts of Noveon (Cleveland, OH). The mucins used, type III (PS, partially purified mucin from porcine stomach) and type I-S (BSMG, bovine submaxillary gland mucin) were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Sigma and were of analytical or ‘ultra’ quality. Ultra-pure water was used throughout the experiments.
2.2. Preparation of gels and mixtures for rheological measurements Gels were prepared by dispersing the required amount of
polymer in 0.9% NaCl, using a magnetic stirrer for about 1 h. The pH was then adjusted to approximately 6.5–7 using 4.5 M NaOH and the sample was equilibrated at 4 8C overnight. The next day the pH was finely adjusted to physiological pH (7.4). On the third day, the pH was checked again and adjusted where necessary. The sample weight was then adjusted with 0.9% NaCl to obtain exactly the concentration required and the sample was again allowed to equilibrate at 4 8C. Mixtures were prepared by dispersing polymer and mucin in 0.9% NaCl. The pH was adjusted and the mixtures equilibrated as for the pure gels. The final mucin concentration was 4% (w / w) in all mixtures. Three replicates were made of all samples and were stored at 4 8C until the measurements were performed, this being within 7 days.
2.3. Rheological measurements Dynamic measurements were carried out at 37 8C using a Bohlin VOR rheometer (Bohlin Reologi, Lund, Sweden), a controlled rate instrument of the couette type. The measuring system used was a concentric cylinder (C14). After being loaded in the measuring geometry, the samples were lightly centrifuged for 1 min at 2093g to remove entrapped air. The surface of the sample was covered with silicon oil to avoid dehydration during measurement, and the sample was allowed to equilibrate for 30 min. First, a strain sweep measurement was made at a constant frequency of 1 Hz to determine the linear viscoelastic region for each of the samples. Then a frequency sweep (0.01–5 Hz) was performed at a selected strain amplitude which was always within the linear region of the sample (i.e., significantly below the maximum strain amplitude). The elastic (storage) modulus (G9), the viscous (loss) modulus (G0), and the phase angle (d ), were determined from the frequency sweep. Values of the elastic modulus (G9) were used for the calculation of the rheological synergism parameters. The absolute synergism parameter (DG9), also called the interaction term, is the elastic component that is interpreted as the interaction between the polymer and mucin. It is usually calculated from
9 1 DG9 G 9mix 5 G p9 1 G m
(1)
9 is the elastic modulus of the mixture and G 9p where G mix 9 represent the elastic modulus of polymer and and G m mucin, respectively. The equation was simplified as previously described (Rossi et al., 1996; Tamburic and Craig, ¨ ¨ et al., 2000) because of the negligibly 1997; Hagerstrom small elastic modulus of the mucin solutions used in this study and instead DG9 was calculated from 9 5 G p9 1 DG9 G mix
(2)
Furthermore, the relative synergism parameter which has
¨ ¨ , K. Edsman / European Journal of Pharmaceutical Sciences 18 (2003) 349–357 H. Hagerstrom Table 2 Tensile strength parameters a for some of the polymer gels
been put forward as an alternative to the absolute synergism parameter (Rossi et al., 1995) was calculated from
9 2 G p9 DG9 G mix Relative DG9 5 ]] 5 ]]] G 9p G p9
(3)
Series
Polymer
Conc. (%)
Tensile work (mJ)
Fracture strength (mN cm 22 )
2%
C907 C981 C934 C940
2 2 2 2
0.0244 (0.0013) 0.071 (0.010) 0.065 (0.014) 0.088 (0.019)
4.85 (0.37) 11.7 (1.9) 22.0 (4.5) 37 (11)
10 Pa
C907 C981 C934 C940
7.4 0.5 0.75 0.45
0.0290 (0.0026) 0.0308 (0.0028) 0.0332 (0.0050) 0.0292 (0.0031)
4.86 5.51 5.53 5.02
2.4. Tensile strength measurements Mucoadhesion measurements were made using a texture analyser, TA.HDi (Stable Micro Systems, Haslemere, UK), and freshly excised porcine nasal mucosa, as previ¨ ¨ and Edsman, 2001). In brief, ously described (Hagerstrom the mucosa was attached to the upper moveable probe of the instrument and the gel was placed in a 70 ml container. The mucosa was then lowered against the gel surface and allowed to penetrate 1 mm into the gel. After 2 min in contact the mucosa was withdrawn at a speed of 0.1 mm / s until a failure occurred between the surfaces. During the entire measurement a force–distance curve was recorded from which the tensile work (i.e., the area under the force–distance curve during the withdrawal phase) and the fracture strength (peak force divided by contact area, i.e., 1.54 cm 2 ) were determined using the computer software Texture Expert Exceed (Stable Micro Systems, Haslemere, UK).
351
a
(0.41) (0.43) (0.54) (0.44)
Mean values (S.D.), n53.
maintain the overall type I error rate at the specified level (a 50.05). The tensile strength data presented in Table 2 were analysed by a one-way analysis of variance (ANOVA) and subsequently Bonferroni’s multiple comparison test, using the software Prism (GraphPad Software, San Diego, CA).
3. Results and discussion
3.1. The effect of the comparison strategy and the choice of frequency
2.5. Statistical analysis The rheological data presented in Table 1 were statistically analysed by an unpaired two-tailed t-test, to determine whether the calculated interaction terms were significantly different from zero. The significance level (a ) was set to 0.05. Subsequently to the t-test we used the sequentially rejective Bonferroni test (Holm, 1979) to
With a view to evaluating the reliability of the method, the first aim of the study was to establish how the rationale for comparison of the polymers can influence the results obtained. That is, to find out how the results and ranking order were affected when the comparison was made at the same polymer concentration and when it was made at the same elastic properties, i.e., where the gels have the same
Table 1 Rheological parameters a for the polymer gels and the polymer–mucin mixtures G p9 (Pa)
G mix 9 PS (Pa)
DG9 PS (Pa)
Rel. DG9 PS (2)
G mix 9 BSMG (Pa)
DG9 BSMG (Pa)
Rel. DG9 BSMG (2)
0.5 0.5 0.5
10.40 (0.40) 0.147 (0.065) 16.0 (3.7)
2.44 (0.80) 0.53 (0.21) 4.52 (0.39)
27.96 (0.89) s. 0.39 (0.22) n.s. 211.5 (3.7) s.
20.77 2.6 20.72
3.58 (0.22) 3.61 (0.42) 12.5 (1.6)
26.82 (0.46) s. 3.46 (0.42) s. 23.5 (4.0) n.s.
20.66 23.6 20.22
C907 C981 C934 C940
2 2 2 2
0.103 (0.014) 123.3 (7.2) 532 (17) 635.7 (4.6)
0.37 (0.11) 42.3 (4.2) 147.3 (3.5) 206 (10)
0.27 (0.11) s. 281.0 (8.3) s. 2385 (17) s. 2429 (11) s.
2.6 20.66 20.72 20.68
1.28 (0.21) 98.5 (25.1) 271.0 (9.9) 293.3 (5.1)
1.17 (0.21) s. 224.8 (26.1) n.s. 2261 (20) s. 2342.3 (6.9) s.
11.4 20.20 20.49 20.54
0.2 Pa
C907 C981 C934 C940
2.2 0.25 0.5 0.3
0.153 (0.026) 0.182 (0.076) 0.147 (0.065) 0.21 (0.11)
0.605 (0.042) 0.309 (0.069) 0.53 (0.21) 0.82 (0.42)
0.452 (0.049) s. 0.13 (0.10) n.s. 0.39 (0.22) n.s. 0.61 (0.84) n.s.
2.95 0.70 2.6 2.9
1.64 (0.47) 0.215 (0.025) 3.61 (0.42) 1.78 (0.38)
1.48 (0.47) s. 0.034 (0.228) n.s. 3.46 (0.42) s. 1.57 (0.44) s.
9.67 0.19 23.6 7.47
10 Pa
C907 C981 C934 C940
7.4 0.5 0.75 0.45
9.93 (0.32) 10.40 (0.40) 12.9 (1.2) 10.47 (0.058)
21.5 2.44 3.73 4.15
11.6 (1.1) s. 27.96 (0.89) s. 29.1 (1.3) s. 26.32 (0.52) s.
1.2 20.77 20.71 20.60
37.2 (2.4) 3.58 (0.22) 15.0 (2.4) 6.5 (1.8)
27.3 (2.4) s. 26.82 (0.46) s. 2.1 (2.6) n.s. 24.0 (1.8) s.
2.7 20.66 0.16 20.38
Series
Polymer
Conc.
0.5%
C907 C981 C934 C940
2%
b
(1.0) (0.80) (0.63) (0.51)
The notations s. and n.s. indicate whether DG9 is significantly (s.) or not significantly (n.s.) different from zero (a 50.05). a Mean values (S.D.), n53. Values shown correspond to the frequency 1 Hz. b Excluded.
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value of G9. For this methodological study we chose to use commercially available poly(acrylic acid) polymers having the same chemical structure, but exhibiting different rheological properties, because of differences in, e.g., cross-linking density and gel particle size. By choosing polymers that were chemically similar we expected to lower the risk of artefacts and errors which might occur if polymers were used that have very diverse chemical properties and thereby different abilities of chemical interactions with the mucins. With the intention of identifying four series on which to perform the mucoadhesion measurements, we first investigated the rheological properties of the polymers as a function of concentration. From Fig. 1 it can be seen that the cross-linked polymers showed a distinct increase in G9 at low concentrations, whilst at higher concentrations the preparations had typical gel properties, i.e., a frequencyindependent G9 which was considerably higher than G0 over a large frequency range (Ross-Murphy and McEvoy, 1986; Almdal et al., 1993). The linear polymer C907 had a flatter curve without a distinct increase in G9. From the curves, two series were chosen where all the polymers either had a concentration of 0.5 or 2%, but different values of G9. And in addition, two series were chosen where the preparations had approximately the same elastic properties, i.e., the same value of G9 (0.2 and 10 Pa), but different polymer concentrations. For these four series, mixtures were prepared with the two different mucins, the PS and the BSMG mucin. The results from the measure9 , DG9 and the ments, in terms of values of G p9 , G mix relative DG9 obtained at the frequency 1 Hz, are given in Table 1. The sign of DG9 is also indicated by the shading of the points in Fig. 1. The only linear polymer, C907, gave significantly positive synergism with both mucins at all concentrations tested. For the cross-linked polymers it was clear that the DG9 was positive only at the lowest concentrations, in this case, significant positive terms were obtained only in the 0.2 Pa series with BSMG mucin. At higher concentrations or at higher G p9 the synergism parameter was either not significantly different from zero or was significantly negative, with both the PS and the BSMG mucin. In the 0.2 and 10 Pa series there was no obvious trend with respect to the ranking order of the three cross-linked polymers. In the 2% series there seemed to be a trend of increasing negative values of DG9 (C981:C934:C940). However, this is probably a reflection of the increasing G p9 in the series, C940 having the highest G p9 . To sum up, it is evident that the method gives different results and ranking order depending on the rationale for the comparison. This implies that the method cannot give reliable information about the mucoadhesive properties of one specific polymer in relation to the other polymers. This cannot be a reflection of different capabilities of chemical interaction with the mucin because all the polymers used here are chemically similar. Instead it points to the fact that other properties of the gel, such as structural and physico-
Fig. 1. The elastic modulus (G9) as a function of polymer concentration. The thin lines indicate the four series chosen, 0.5 and 2%, and 0.2 and 10 Pa, for which polymer–mucin mixtures were prepared. Circles along the lines corresponding to the series indicate the sign of DG9 obtained with PS mucin (a) and BSMG mucin (b): positive (black), not significantly different from zero (P.0.05) (grey) or negative (white).
chemical factors, are important for the results. These factors could be, for example, the molecular weight, the cross-linking density and the molecular flexibility, which are features that are also related to the rheological properties of the gel (Gu et al., 1988; Leung and Robinson, 1990; Junginger, 1991). Moreover, these features are generally considered to be important for the formation of entanglements during the mucoadhesion process. Therefore, maybe one should not expect the comparison and ranking of different polymers to be a straightforward procedure.
¨ ¨ , K. Edsman / European Journal of Pharmaceutical Sciences 18 (2003) 349–357 H. Hagerstrom
Another factor that may affect the results is the choice of frequency at which the rheological data are obtained. This is especially true for linear polymers (entangled polymer solutions) where G9 and G0 are frequency dependent. In this study a clear frequency dependence was only observed for those preparations that contained the linear polymer C907, whereas the cross-linked polymers showed very little or no frequency dependence. In Fig. 2 we display the frequency sweeps for C907 and also for one of the cross-linked polymers, C940, at the concentrations corresponding to the 2% and the 10 Pa series. Furthermore, in Fig. 3 the calculated DG9 values for the entire 2% and 10 Pa series are plotted against the frequency. It can be seen that for the cross-linked gels DG9 is not affected by the choice of frequency. For the C907 preparations the size of DG9 increases as the frequency increases. However, the sign of DG9 is not affected by the choice of frequency. Thus, with respect to positive or negative values of the synergism parameter the observations made from Table 1 (data obtained at 1 Hz) would still have been the same even if another frequency had been chosen. It is evident from Fig. 1 and Table 1 that positive values of the synergism parameters are only seen at low concentrations and / or with weak gels (i.e., at low values of G p9 ). Conversely, the negative values are only seen at high concentrations and / or with strong gels (i.e., at high values
353
of G p9 ). The conventional interpretation of this would be that only the weak gels exhibit mucoadhesive properties and that the strong gels do not show any mucoadhesive properties. As it is evident that the rheological properties of the gel are important for the results obtained with this method, it seems logical to compare polymer preparations having approximately the same elastic properties, since the rheological properties can be very different even though the polymers have the same concentration. Within the group of cross-linked polymers, the results obtained in the 0.2 and 10 Pa series are more similar than the results in the 0.5 and 2% series where the synergism parameters even had different signs. Looking at these data, we feel, however, that it is still not obvious which synergism parameter is the most suitable, and neither is it clear how to interpret the size and the sign of the parameter. In the following sections we try to address these issues.
3.2. The effect of the choice of rheological synergism parameter In Fig. 4 the absolute (DG9) and the relative synergism parameters (DG9 /G p9 ) obtained with BSMG mucin are plotted against G p9 for all four polymers. The absolute parameter exhibits large negative values at high values of G p9 and moderate positive ones at low G p9 . The relative
Fig. 2. The frequency dependence of the elastic modulus (G9, filled symbols) and the viscous modulus (G0, open symbols), shown for a few polymer preparations (a,d) and their corresponding mixtures with PS (b,e) and BSMG mucin (c,f): (a–c) 2% C907 (d,s) and 7.4% C907 (j,h); (d–f) 0.45% C940 (♦,앳 ) and 2% C940 (m,n).
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Fig. 3. The frequency dependence of the absolute synergism parameter (DG9) obtained for the preparations in the 2% series (a) and the 10 Pa series (b).
parameter, on the other hand, shows large positive values at low G p9 but approaches its negative limit of 21 (see Eq. (3)) at high G p9 . Consequently, the results obtained are not only very sensitive to where in the ‘rheological range’ the study is performed, i.e., at what value of G 9p , but also to which synergism parameter is being considered. The ‘relative approach’ to synergism seems to be the most reasonable, that is, to consider how many times stronger (or weaker) the polymer–mucin mixture is compared to the gel itself. However, in our opinion the relative parameter in its present form (DG9 /G 9p ) is not very suitable because it has a negative limit of 21, whereas positive values approach infinity when G 9p approaches zero (see Eq. (3)). From this it follows that the magnitude of positive values cannot be compared to the magnitude of negative ones. We would therefore like to suggest a new ‘relative’ parameter, called the ‘log G9 ratio’, with which the magnitude of positive and negative values are fully comparable. It is calculated as
S D
G 9mix log G9 ratio 5 log ]] . G 9p
(4)
With this parameter a value of 1 would mean that the G9 of
Fig. 4. The absolute (DG9, a) and the relative synergism parameters (DG9 /G p9 , b) obtained with BSMG mucin, as functions of the elastic modulus of the polymer gels (G 9p ).
the polymer–mucin mixture is 10 times higher than that of the gel (i.e., the addition of mucin causes a 10 times increase in G9). Further, a value of 0 would mean that the addition of mucin brings about no change in G9, and a value of 21 represents a decrease in G9 by a factor of 10, etc. Of course the ‘log ratio’ can also be calculated on the basis of G0 or the complex modulus, G*. 9 /G p9 ) In Fig. 5a we have plotted the ‘log ratio’ log (G mix obtained with BSMG mucin against the G p9 . For the crosslinked gels it is evident that the addition of mucin causes an increase of G9 that is large at low G p9 , but becomes a decrease at higher G p9 . Corresponding plots for the mixtures with PS mucin give values that are generally lower than for the BSMG mixtures, which is in accordance with ¨ ¨ et al., previous results (Rossi et al., 1995; Hagerstrom
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355
of the phase angle would result in a relatively large error in G0. And conversely, if the phase angle is large, a small error in the phase angle would result in a relative error in G9 that is comparatively high. If one wants to compare ‘true’ gels (based on cross-linked polymers) with entangled polymer solutions (based on linear polymers), it may be more appropriate to use the complex modulus, G*, which incorporates both the elastic and the viscous properties of the preparation. In this study the log G* ratio plot (Fig. 5c) is very similar to the one of the log G9 ratio. The use of the log ratio instead of the conventional relative parameter (DG9 /G p9 ) would have the advantage that the magnitude of positive and negative values would be fully comparable, since the log ratio does not approach a fixed limit on the negative scale as does the conventional parameter. But, of course, it would not solve the problem that the method itself gives results that to a large extent are influenced by the rheology of the gel.
3.3. Positive or negative values of the synergism parameters?
Fig. 5. The calculated log ratios based on G9 (a), G0 (b) and G* (c) obtained with BSMG mucin, as functions of the corresponding modulus of the polymer gels.
2000). In Fig. 5 the log ratios based on G0 and G* are also shown. A decrease in the log G0 ratio is observed with increasing G p99 , but the values are positive over the entire range tested. It should be noted, however, that for gels with a very low phase angle, a small error in the determination
The phenomenon of negative synergism values observed with the cross-linked poly(acrylic acid) gels complicates the interpretation of the results. We have previously observed negative synergism values also with gellan gum ¨ ¨ et al., 2000). However, the concept of gels (Hagerstrom rheological synergism is not limited to polymer mixtures with mucin, but has been observed and discussed in previous studies with, e.g., polymer–polymer mixtures (Williams and Phillips, 1995; Nishinari et al., 1996; Rodriguez et al., 2001). From these studies, it is not obvious that an interaction automatically results in a positive rheological synergism term. An interaction might as well result in weaker properties of the mixture than the sum of the individual components. We are therefore not convinced of the soundness of the conventional interpretation model that is used with the rheological mucoadhesion method. In this work positive values of the synergism parameters are obtained at low concentrations (low G p9 ), which is in good agreement with other studies, for example, that of Madsen et al. (1998), in which several cross-linked poly(acrylic acids) gels were mixed with homogenised mucus. Negative values of the synergism parameters have also been previously observed with cross-linked poly(acrylic acids) and commercial mucins (Rossi et al., 1995; Tamburic and Craig, 1997). It has been suggested that ions remaining in the mucin samples could cause a weakening of the gels. This is probably not the only reason, since the BSMG mucin actually contains more ions than the PS ¨ ¨ et al., 2000), but gives larger positive values (Hagerstrom of DG9 than the PS at the low polymer concentrations and smaller negative values at higher concentrations. Moreover, in this study the samples were prepared in 0.9% NaCl to simulate physiological conditions, and at this salt
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concentration the relatively small number of ions present in the mucins is negligible. Instead the negative values of the synergism parameters may be a consequence of the particulate properties of the cross-linked gels. Besides giving molecular interactions with the polymer, the addition of mucin to a strong particulate gel (i.e., at high polymer concentration) could perhaps ‘dilute’ the gel particles by filling the interstitial space, resulting in weaker bulk properties. Correspondingly, for a weak gel in which the gel particles are not in contact, the addition of mucin may represent an increased ‘concentration’ of the preparation, making the bulk properties stronger because the interstitial space is filled with mucin solution instead of pure medium. The question can be raised whether negative values of the synergism parameter really would imply that the gel preparation would have a bad performance in an imaginable in vivo situation, and vice versa. With this method, the interpenetration layer is simulated by mixing the gel with mucin. The gel is considered to be advantageous if the interpenetration layer (i.e., the gel–mucin mixture) is stronger than the gel itself. Can this interpretation model be applied to the situation at a gel–mucosa interface? Consider the general situation where a gel dosage form is in contact with a mucous tissue. The mucoadhesive joint can be considered to consist of three regions—the gel, the mucus layer and the interface (the interpenetration layer). It has been put forward that the residence time of the gel on the mucous tissue would be associated with the failure of the weakest region and, therefore, the cohesive properties of each region are important (Smart, 1999). From this it can be deduced that the gel dosage form should have a sufficient level of cohesiveness to provide a long residence time, and that a strengthening of the mucus layer also would be advantageous, as well as a strong interpenetration layer. But it cannot be deduced that the interpenetration layer necessarily has to be stronger than the gel dosage form itself. In this respect, we believe that the basis of interpretation model that is used with the rheological method is questionable and not easily applied to a possible in vivo situation.
3.4. Comparison to tensile strength measurements With a view to comparing the results from the rheological approach to a more in vivo-like mucoadhesion method, we performed tensile strength measurements using a texture analyser. The adhesion to freshly excised porcine nasal mucosa was measured for the preparations in the 2% and the 10 Pa series. In Table 2 the tensile work and the fracture strength obtained are presented. In the 10 Pa series, where the preparations have approximately the same rheological properties, no significant difference is observed between these preparations with respect to the adhesion to the mucosa, neither in the tensile work nor in the fracture strength. In the 2% series on the other hand,
where the preparations have different rheological properties, the strong cross-linked gels give significantly higher tensile work and fracture strength than the much weaker C907 preparation. Moreover, if the 2% cross-linked gels are compared to the corresponding weaker gels in the 10 Pa series it is evident that the adhesion to the mucosa becomes stronger with increasing gel strength. This has been observed previously in other studies (see, for exam¨ ¨ and Edsman, 2001). ple, Jones et al., 1997; Hagerstrom From these results it can be deduced that with a more in vivo-like method such as the tensile strength approach, stronger rheological properties result in stronger adhesion to the mucosa. This is basically opposite to the results obtained using the rheological mucoadhesion method, where the positive values of the synergism parameters are seen only with weak gels. We believe that this lack of correlation gives rise to even more questions regarding the resemblance of the rheological approach to the situation at the gel–mucosa interface. We would therefore recommend that the rheological method should not be used as a stand-alone method for studying mucoadhesion of polymer gels.
4. Conclusions In the work presented here, we have shown that with the rheological method the results of a comparative mucoadhesion study are strongly influenced by the rationale for the comparison. The method does not give the same ranking order of the polymers when two different comparison strategies are used. A comparative study with different polymers might, however, be more accurate if the study is performed where the polymer preparations have approximately the same rheological properties, e.g., the same value of G9. Furthermore, we have shown that the results obtained in a comparative study are also sensitive to where in the ‘rheological range’ the comparison is made, e.g., at which value of G9. Positive values of the synergism parameters are, for example, only seen with weak gels. The choice of rheological synergism parameter also has a bearing on the results obtained. We have suggested a new 9 /G p9 )). refined relative parameter, the log ratio (log(G mix Despite the refinements of the method that are suggested here, it is still not obvious how to interpret the results, which makes the comparison and ranking of polymers difficult. From tensile strength measurements of the adhesion of the gel preparations to porcine nasal mucosa, we conclude that increased gel strength results in stronger adhesion. This is in contrast to the results from the rheological method, where the positive values of the synergism parameters are seen only with weak gels. On the basis of the limitations of the method that are demonstrated and discussed in this work, we recommend that the method should not be used as a stand-alone
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method for the studying of mucoadhesive properties of polymer gels.
Acknowledgements We are thankful to Ms Sofia Josefsson for excellent technical assistance. Financial support for this study was obtained from AstraZeneca, Sweden, and the Knut and Alice Wallenberg Foundation, and is gratefully acknowledged.
References Almdal, K., Dyre, J., Hvidt, S., Kramer, O., 1993. Towards a phenomenological definition of the term ‘gel’. Polymer Gels Networks 1, 5–17. Caramella, C., Bonferoni, M.C., Rossi, S., Ferrari, F., 1994. Rheological and tensile tests for the assessment of polymer–mucin interactions. Eur. J. Pharm. Biopharm. 40, 213–217. Gu, J.M., Robinson, J.R., Leung, S.H.S., 1988. Binding of acrylic polymers to mucin / epithelial surfaces: structure–property relationships. Crit. Rev. Ther. Drug Carrier Syst. 5, 21–67. Hassan, E.E., Gallo, J.M., 1990. A simple rheological method for the in vitro assessment of mucin-polymer bioadhesive bond strength. Pharm. Res. 7, 491–495. Holm, S., 1979. A simple sequentially rejective multiple test procedure. Scand. J. Statist. Theory Appl. 6, 65–70. ¨ ¨ Hagerstrom, H., Edsman, K., 2001. Interpretation of mucoadhesive properties of polymer gel preparations using a tensile strength method. J. Pharm. Pharmacol. 53, 1589–1599. ¨ ¨ Hagerstrom, H., Paulsson, M., Edsman, K., 2000. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. Eur. J. Pharm. Sci. 9, 301–309. Jones, D.S., Woolfson, A.D., Brown, A.F., 1997. Textural, viscoelastic and mucoadhesive properties of pharmaceutical gels composed of cellulose polymers. Int. J. Pharm. 151, 223–233. Junginger, H.E., 1991. Mucoadhesive hydrogels. Pharm. Ind. 53, 1056– 1065. Kocevar-Nared, J., Kristl, J., Smid-Korbar, J., 1997. Comparative rheological investigation of crude gastric mucin and natural gastric mucus. Biomaterials 18, 677–681. Leung, S.-H.S., Robinson, J.R., 1990. Polymer structure features contributing to mucoadhesion. II. J. Control. Release 12, 187–194.
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Madsen, F., Eberth, K., Smart, J.D., 1996. A rheological evaluation of various mucus gels for use in in-vitro mucoadhesion testing. Pharm. Sci. 2, 563–566. Madsen, F., Eberth, K., Smart, J.D., 1998. A rheological examination of the mucoadhesive / mucus interaction: the effect of mucoadhesive type and concentration. J. Control. Release 50, 167–178. Mortazavi, S.A., 1995. An in vitro assessment of mucus / mucoadhesive interactions. Int. J. Pharm. 124, 173–182. Mortazavi, S.A., Carpenter, B.G., Smart, J.D., 1993. A comparative study on the role played by mucus glycoproteins in the rheological behaviour of the mucoadhesive / mucosal interface. Int. J. Pharm. 94, 195–201. Mortazavi, S.A., Smart, J.D., 1994. Factors influencing gel-strengthening at the mucoadhesive-mucus interface. J. Pharm. Pharmacol. 46, 86–90. Nishinari, K., Miyoshi, E., Takaya, T., Williams, P.A., 1996. Rheological and DSC studies on the interaction between gellan gum and konjac glucomannan. Carbohydrate Polym. 30, 193–207. Riley, R.G., Smart, J.D., Tsibouklis, J., Dettmar, P.W., Hampson, F., Davis, J.A., Kelly, G., Wilber, W.R., 2001. An investigation of mucus / polymer rheological synergism using synthesised and characterised poly (acrylic acid)s. Int. J. Pharm. 217, 87–100. Rodriguez, R., Alvarez-Lorenzo, C., Concheiro, A., 2001. Rheological evaluation of the interactions between cationic celluloses and Carbopol 974P in water. Biomacromolecules 2, 886–893. Rossi, S., Bonferoni, M.C., Ferrari, F., Bertoni, M., Caramella, C., 1996. Characterization of mucin interaction with three viscosity grades of sodium carboxymethylcellulose. Comparison between rheological and tensile testing. Eur. J. Pharm. Sci. 4, 189–196. Rossi, S., Bonferoni, M.C., Lippoli, G., Bertoni, M., Ferrari, F., Caramella, C., Conte, U., 1995. Influence of mucin type on polymer–mucin rheological interactions. Biomaterials 16, 1073–1079. Ross-Murphy, S.B., McEvoy, H., 1986. Fundamentals of hydrogels and gelation. Br. Polym. J. 18, 2–7. Smart, J.D., 1999. The role of water movement and polymer hydration in mucoadhesion. In: Mathiowitz, E., Chickering, III D.E., Lehr, C.M. (Eds.), Bioadhesive Drug Delivery Systems: Fundamentals, Novel Approaches and Development. Marcel Dekker, New York, pp. 11–23. Tamburic, S., Craig, D.Q.M., 1995. An investigation into the rheological, dielectric and mucoadhesive properties of poly(acrylic acid) gel systems. J. Control. Release 37, 59–68. Tamburic, S., Craig, D.Q.M., 1997. A comparison of different in vitro methods for measuring mucoadhesive performance. Eur. J. Pharm. Biopharm. 44, 159–167. Williams, P.A., Phillips, G.O., 1995. Interactions in mixed polysaccharide systems. In: Stephen, A.M. (Ed.), Food Polysaccharides and Their Applications. Marcel Dekker, New York, pp. 463–500.
Limitations of the rheological mucoadhesion method: The effect of the choice of conditions and the rheological synergism parameter ¨ ¨ Katarina Edsman* Helene Hagerstrom, Department of Pharmacy, Uppsala University, Uppsala Biomedical Centre, Box 580, SE 751 23 Uppsala, Sweden Received 16 July 2002; received in revised form 20 December 2002; accepted 19 February 2003
Abstract This work demonstrates several limitations of the simple rheological method that is widely used to investigate mucoadhesion of polymer gels. We establish the importance of the choice of conditions and the synergism parameter for the results obtained in comparative studies. Dynamic rheological measurements were performed on gels based on four slightly different poly(acrylic acid) (Carbopol) polymers and their corresponding mixtures with porcine stomach mucin and bovine submaxillary gland mucin. The rationale for the comparison of the polymers had a large influence on the results obtained. The method does not give the same ranking order when two different comparison strategies are used. Moreover, we show that the results obtained are also sensitive to where in the ‘rheological range’ the comparison is made, e.g., at which value of G9. Positive values of the synergism parameters are, for example, only seen with weak gels. The choice of synergism parameter also has a bearing on the results obtained, and here we suggest a new refined relative parameter, 9 /G p9 )). We also investigated the adhesion of the gel preparations to porcine nasal mucosa, using tensile strength the log ratio (log(G mix measurements. Increased gel strength resulted in stronger adhesion, which is in contrast to the results from the rheological method, where the positive values of the synergism parameters were seen only with weak gels. On the basis of the limitations demonstrated and discussed, we recommend that the rheological method should not be used as a stand-alone method for the studying of mucoadhesive properties of polymer gels. 2003 Elsevier Science B.V. All rights reserved. Keywords: Mucoadhesion; Gel; Rheological synergism; Carbopol; Tensile strength
1. Introduction The mucoadhesive behaviour of polymer gels has been the subject of numerous studies during the last 10 years. Among the most common methods is a simple rheological approach that was first suggested in 1990 by Hassan and Gallo. Usually, dynamic oscillatory measurements are performed to investigate the potential rheological interaction between a polymer gel and mucin. For mucoadhesive polymers it is widely believed that the rheological response of a gel–mucin mixture should be larger than the sum of the contributions from the gel and the mucin, a phenomenon that is commonly described as ‘rheological synergism’. The rheological mucoadhesion method has been used extensively, presumably because of its simplicity, and *Corresponding author. Tel.: 146-18-471-4268; fax: 146-18-4714223. E-mail address: [email protected] (K. Edsman).
several attempts have been made to screen and rank a number of different polymers (e.g., Caramella et al., 1994; Mortazavi, 1995; Tamburic and Craig, 1995; Madsen et al., 1998). However, a wide variation in results is found in the literature, which has been attributed mainly to differences in the mucin type and concentrations used (Rossi et al., 1995; Madsen et al., 1996; Kocevar-Nared et al., 1997; ¨ ¨ et al., 2000) as well as to different polymer Hagerstrom concentrations (Mortazavi and Smart, 1994; Madsen et al., ¨ ¨ et al., 2000). Furthermore, in a previous 1998; Hagerstrom study of ours we found that the results obtained can also vary considerably depending on, for example, the ionsensitivity of the polymer, the quantity of ions present and ¨ ¨ et al., 2000). instrumental factors (Hagerstrom In this paper we study different factors that can affect the results obtained, especially factors encountered when performing comparative studies. One specific problem with comparative and ranking studies is that so far there has been no consensus regarding the conditions under which the comparison should be
0928-0987 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0928-0987(03)00037-X
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made. Should one, for example, compare the polymers at the same concentration, or maybe one should adjust the concentration to make a comparison between polymer preparations with the same rheological properties, i.e., at different concentrations? Does the method give the same results irrespective of the conditions chosen? In this study one objective was to establish how the choice of comparison strategy can influence the results obtained. The two different strategies mentioned above were therefore used to investigate the reliability of the method. The polymers used were a series of Carbopol polymers, which are wellknown commercially available poly(acrylic acid) polymers. The choice was based on the fact that they are well characterised and have been included frequently in rheological mucoadhesion studies (e.g., Mortazavi et al., 1993; Caramella et al., 1994; Mortazavi and Smart, 1994; Madsen et al., 1998; Riley et al., 2001), which makes them suitable for this kind of methodological evaluation. The other aim of this study was to establish how the calculated rheological synergism parameters are affected by the choice of comparison conditions. In the literature no consensus can be found regarding the synergism parameter that is used, which could provide another explanation for the wide variation of results that is found. It is not clear what kind of synergism parameter is the most suitable, an absolute or a relative parameter. In the work presented here this issue is elucidated. We show how the different synergism parameters are influenced by the comparison conditions and we also suggest a new, hopefully refined synergism parameter. We have also investigated the adhesion of the gel preparations to porcine nasal mucosa, using tensile strength measurements, with a view to comparing the results from the rheological approach to a more in vivolike method.
2. Materials and methods
2.1. Materials The poly(acrylic acid) polymers used in this study were the linear polymer Carbopol 907 (C907) and the crosslinked polymers Carbopol 981 (C981), Carbopol 934P (C934) and Carbopol 940 (C940). All polymers were the kind gifts of Noveon (Cleveland, OH). The mucins used, type III (PS, partially purified mucin from porcine stomach) and type I-S (BSMG, bovine submaxillary gland mucin) were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Sigma and were of analytical or ‘ultra’ quality. Ultra-pure water was used throughout the experiments.
2.2. Preparation of gels and mixtures for rheological measurements Gels were prepared by dispersing the required amount of
polymer in 0.9% NaCl, using a magnetic stirrer for about 1 h. The pH was then adjusted to approximately 6.5–7 using 4.5 M NaOH and the sample was equilibrated at 4 8C overnight. The next day the pH was finely adjusted to physiological pH (7.4). On the third day, the pH was checked again and adjusted where necessary. The sample weight was then adjusted with 0.9% NaCl to obtain exactly the concentration required and the sample was again allowed to equilibrate at 4 8C. Mixtures were prepared by dispersing polymer and mucin in 0.9% NaCl. The pH was adjusted and the mixtures equilibrated as for the pure gels. The final mucin concentration was 4% (w / w) in all mixtures. Three replicates were made of all samples and were stored at 4 8C until the measurements were performed, this being within 7 days.
2.3. Rheological measurements Dynamic measurements were carried out at 37 8C using a Bohlin VOR rheometer (Bohlin Reologi, Lund, Sweden), a controlled rate instrument of the couette type. The measuring system used was a concentric cylinder (C14). After being loaded in the measuring geometry, the samples were lightly centrifuged for 1 min at 2093g to remove entrapped air. The surface of the sample was covered with silicon oil to avoid dehydration during measurement, and the sample was allowed to equilibrate for 30 min. First, a strain sweep measurement was made at a constant frequency of 1 Hz to determine the linear viscoelastic region for each of the samples. Then a frequency sweep (0.01–5 Hz) was performed at a selected strain amplitude which was always within the linear region of the sample (i.e., significantly below the maximum strain amplitude). The elastic (storage) modulus (G9), the viscous (loss) modulus (G0), and the phase angle (d ), were determined from the frequency sweep. Values of the elastic modulus (G9) were used for the calculation of the rheological synergism parameters. The absolute synergism parameter (DG9), also called the interaction term, is the elastic component that is interpreted as the interaction between the polymer and mucin. It is usually calculated from
9 1 DG9 G 9mix 5 G p9 1 G m
(1)
9 is the elastic modulus of the mixture and G 9p where G mix 9 represent the elastic modulus of polymer and and G m mucin, respectively. The equation was simplified as previously described (Rossi et al., 1996; Tamburic and Craig, ¨ ¨ et al., 2000) because of the negligibly 1997; Hagerstrom small elastic modulus of the mucin solutions used in this study and instead DG9 was calculated from 9 5 G p9 1 DG9 G mix
(2)
Furthermore, the relative synergism parameter which has
¨ ¨ , K. Edsman / European Journal of Pharmaceutical Sciences 18 (2003) 349–357 H. Hagerstrom Table 2 Tensile strength parameters a for some of the polymer gels
been put forward as an alternative to the absolute synergism parameter (Rossi et al., 1995) was calculated from
9 2 G p9 DG9 G mix Relative DG9 5 ]] 5 ]]] G 9p G p9
(3)
Series
Polymer
Conc. (%)
Tensile work (mJ)
Fracture strength (mN cm 22 )
2%
C907 C981 C934 C940
2 2 2 2
0.0244 (0.0013) 0.071 (0.010) 0.065 (0.014) 0.088 (0.019)
4.85 (0.37) 11.7 (1.9) 22.0 (4.5) 37 (11)
10 Pa
C907 C981 C934 C940
7.4 0.5 0.75 0.45
0.0290 (0.0026) 0.0308 (0.0028) 0.0332 (0.0050) 0.0292 (0.0031)
4.86 5.51 5.53 5.02
2.4. Tensile strength measurements Mucoadhesion measurements were made using a texture analyser, TA.HDi (Stable Micro Systems, Haslemere, UK), and freshly excised porcine nasal mucosa, as previ¨ ¨ and Edsman, 2001). In brief, ously described (Hagerstrom the mucosa was attached to the upper moveable probe of the instrument and the gel was placed in a 70 ml container. The mucosa was then lowered against the gel surface and allowed to penetrate 1 mm into the gel. After 2 min in contact the mucosa was withdrawn at a speed of 0.1 mm / s until a failure occurred between the surfaces. During the entire measurement a force–distance curve was recorded from which the tensile work (i.e., the area under the force–distance curve during the withdrawal phase) and the fracture strength (peak force divided by contact area, i.e., 1.54 cm 2 ) were determined using the computer software Texture Expert Exceed (Stable Micro Systems, Haslemere, UK).
351
a
(0.41) (0.43) (0.54) (0.44)
Mean values (S.D.), n53.
maintain the overall type I error rate at the specified level (a 50.05). The tensile strength data presented in Table 2 were analysed by a one-way analysis of variance (ANOVA) and subsequently Bonferroni’s multiple comparison test, using the software Prism (GraphPad Software, San Diego, CA).
3. Results and discussion
3.1. The effect of the comparison strategy and the choice of frequency
2.5. Statistical analysis The rheological data presented in Table 1 were statistically analysed by an unpaired two-tailed t-test, to determine whether the calculated interaction terms were significantly different from zero. The significance level (a ) was set to 0.05. Subsequently to the t-test we used the sequentially rejective Bonferroni test (Holm, 1979) to
With a view to evaluating the reliability of the method, the first aim of the study was to establish how the rationale for comparison of the polymers can influence the results obtained. That is, to find out how the results and ranking order were affected when the comparison was made at the same polymer concentration and when it was made at the same elastic properties, i.e., where the gels have the same
Table 1 Rheological parameters a for the polymer gels and the polymer–mucin mixtures G p9 (Pa)
G mix 9 PS (Pa)
DG9 PS (Pa)
Rel. DG9 PS (2)
G mix 9 BSMG (Pa)
DG9 BSMG (Pa)
Rel. DG9 BSMG (2)
0.5 0.5 0.5
10.40 (0.40) 0.147 (0.065) 16.0 (3.7)
2.44 (0.80) 0.53 (0.21) 4.52 (0.39)
27.96 (0.89) s. 0.39 (0.22) n.s. 211.5 (3.7) s.
20.77 2.6 20.72
3.58 (0.22) 3.61 (0.42) 12.5 (1.6)
26.82 (0.46) s. 3.46 (0.42) s. 23.5 (4.0) n.s.
20.66 23.6 20.22
C907 C981 C934 C940
2 2 2 2
0.103 (0.014) 123.3 (7.2) 532 (17) 635.7 (4.6)
0.37 (0.11) 42.3 (4.2) 147.3 (3.5) 206 (10)
0.27 (0.11) s. 281.0 (8.3) s. 2385 (17) s. 2429 (11) s.
2.6 20.66 20.72 20.68
1.28 (0.21) 98.5 (25.1) 271.0 (9.9) 293.3 (5.1)
1.17 (0.21) s. 224.8 (26.1) n.s. 2261 (20) s. 2342.3 (6.9) s.
11.4 20.20 20.49 20.54
0.2 Pa
C907 C981 C934 C940
2.2 0.25 0.5 0.3
0.153 (0.026) 0.182 (0.076) 0.147 (0.065) 0.21 (0.11)
0.605 (0.042) 0.309 (0.069) 0.53 (0.21) 0.82 (0.42)
0.452 (0.049) s. 0.13 (0.10) n.s. 0.39 (0.22) n.s. 0.61 (0.84) n.s.
2.95 0.70 2.6 2.9
1.64 (0.47) 0.215 (0.025) 3.61 (0.42) 1.78 (0.38)
1.48 (0.47) s. 0.034 (0.228) n.s. 3.46 (0.42) s. 1.57 (0.44) s.
9.67 0.19 23.6 7.47
10 Pa
C907 C981 C934 C940
7.4 0.5 0.75 0.45
9.93 (0.32) 10.40 (0.40) 12.9 (1.2) 10.47 (0.058)
21.5 2.44 3.73 4.15
11.6 (1.1) s. 27.96 (0.89) s. 29.1 (1.3) s. 26.32 (0.52) s.
1.2 20.77 20.71 20.60
37.2 (2.4) 3.58 (0.22) 15.0 (2.4) 6.5 (1.8)
27.3 (2.4) s. 26.82 (0.46) s. 2.1 (2.6) n.s. 24.0 (1.8) s.
2.7 20.66 0.16 20.38
Series
Polymer
Conc.
0.5%
C907 C981 C934 C940
2%
b
(1.0) (0.80) (0.63) (0.51)
The notations s. and n.s. indicate whether DG9 is significantly (s.) or not significantly (n.s.) different from zero (a 50.05). a Mean values (S.D.), n53. Values shown correspond to the frequency 1 Hz. b Excluded.
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value of G9. For this methodological study we chose to use commercially available poly(acrylic acid) polymers having the same chemical structure, but exhibiting different rheological properties, because of differences in, e.g., cross-linking density and gel particle size. By choosing polymers that were chemically similar we expected to lower the risk of artefacts and errors which might occur if polymers were used that have very diverse chemical properties and thereby different abilities of chemical interactions with the mucins. With the intention of identifying four series on which to perform the mucoadhesion measurements, we first investigated the rheological properties of the polymers as a function of concentration. From Fig. 1 it can be seen that the cross-linked polymers showed a distinct increase in G9 at low concentrations, whilst at higher concentrations the preparations had typical gel properties, i.e., a frequencyindependent G9 which was considerably higher than G0 over a large frequency range (Ross-Murphy and McEvoy, 1986; Almdal et al., 1993). The linear polymer C907 had a flatter curve without a distinct increase in G9. From the curves, two series were chosen where all the polymers either had a concentration of 0.5 or 2%, but different values of G9. And in addition, two series were chosen where the preparations had approximately the same elastic properties, i.e., the same value of G9 (0.2 and 10 Pa), but different polymer concentrations. For these four series, mixtures were prepared with the two different mucins, the PS and the BSMG mucin. The results from the measure9 , DG9 and the ments, in terms of values of G p9 , G mix relative DG9 obtained at the frequency 1 Hz, are given in Table 1. The sign of DG9 is also indicated by the shading of the points in Fig. 1. The only linear polymer, C907, gave significantly positive synergism with both mucins at all concentrations tested. For the cross-linked polymers it was clear that the DG9 was positive only at the lowest concentrations, in this case, significant positive terms were obtained only in the 0.2 Pa series with BSMG mucin. At higher concentrations or at higher G p9 the synergism parameter was either not significantly different from zero or was significantly negative, with both the PS and the BSMG mucin. In the 0.2 and 10 Pa series there was no obvious trend with respect to the ranking order of the three cross-linked polymers. In the 2% series there seemed to be a trend of increasing negative values of DG9 (C981:C934:C940). However, this is probably a reflection of the increasing G p9 in the series, C940 having the highest G p9 . To sum up, it is evident that the method gives different results and ranking order depending on the rationale for the comparison. This implies that the method cannot give reliable information about the mucoadhesive properties of one specific polymer in relation to the other polymers. This cannot be a reflection of different capabilities of chemical interaction with the mucin because all the polymers used here are chemically similar. Instead it points to the fact that other properties of the gel, such as structural and physico-
Fig. 1. The elastic modulus (G9) as a function of polymer concentration. The thin lines indicate the four series chosen, 0.5 and 2%, and 0.2 and 10 Pa, for which polymer–mucin mixtures were prepared. Circles along the lines corresponding to the series indicate the sign of DG9 obtained with PS mucin (a) and BSMG mucin (b): positive (black), not significantly different from zero (P.0.05) (grey) or negative (white).
chemical factors, are important for the results. These factors could be, for example, the molecular weight, the cross-linking density and the molecular flexibility, which are features that are also related to the rheological properties of the gel (Gu et al., 1988; Leung and Robinson, 1990; Junginger, 1991). Moreover, these features are generally considered to be important for the formation of entanglements during the mucoadhesion process. Therefore, maybe one should not expect the comparison and ranking of different polymers to be a straightforward procedure.
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Another factor that may affect the results is the choice of frequency at which the rheological data are obtained. This is especially true for linear polymers (entangled polymer solutions) where G9 and G0 are frequency dependent. In this study a clear frequency dependence was only observed for those preparations that contained the linear polymer C907, whereas the cross-linked polymers showed very little or no frequency dependence. In Fig. 2 we display the frequency sweeps for C907 and also for one of the cross-linked polymers, C940, at the concentrations corresponding to the 2% and the 10 Pa series. Furthermore, in Fig. 3 the calculated DG9 values for the entire 2% and 10 Pa series are plotted against the frequency. It can be seen that for the cross-linked gels DG9 is not affected by the choice of frequency. For the C907 preparations the size of DG9 increases as the frequency increases. However, the sign of DG9 is not affected by the choice of frequency. Thus, with respect to positive or negative values of the synergism parameter the observations made from Table 1 (data obtained at 1 Hz) would still have been the same even if another frequency had been chosen. It is evident from Fig. 1 and Table 1 that positive values of the synergism parameters are only seen at low concentrations and / or with weak gels (i.e., at low values of G p9 ). Conversely, the negative values are only seen at high concentrations and / or with strong gels (i.e., at high values
353
of G p9 ). The conventional interpretation of this would be that only the weak gels exhibit mucoadhesive properties and that the strong gels do not show any mucoadhesive properties. As it is evident that the rheological properties of the gel are important for the results obtained with this method, it seems logical to compare polymer preparations having approximately the same elastic properties, since the rheological properties can be very different even though the polymers have the same concentration. Within the group of cross-linked polymers, the results obtained in the 0.2 and 10 Pa series are more similar than the results in the 0.5 and 2% series where the synergism parameters even had different signs. Looking at these data, we feel, however, that it is still not obvious which synergism parameter is the most suitable, and neither is it clear how to interpret the size and the sign of the parameter. In the following sections we try to address these issues.
3.2. The effect of the choice of rheological synergism parameter In Fig. 4 the absolute (DG9) and the relative synergism parameters (DG9 /G p9 ) obtained with BSMG mucin are plotted against G p9 for all four polymers. The absolute parameter exhibits large negative values at high values of G p9 and moderate positive ones at low G p9 . The relative
Fig. 2. The frequency dependence of the elastic modulus (G9, filled symbols) and the viscous modulus (G0, open symbols), shown for a few polymer preparations (a,d) and their corresponding mixtures with PS (b,e) and BSMG mucin (c,f): (a–c) 2% C907 (d,s) and 7.4% C907 (j,h); (d–f) 0.45% C940 (♦,앳 ) and 2% C940 (m,n).
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Fig. 3. The frequency dependence of the absolute synergism parameter (DG9) obtained for the preparations in the 2% series (a) and the 10 Pa series (b).
parameter, on the other hand, shows large positive values at low G p9 but approaches its negative limit of 21 (see Eq. (3)) at high G p9 . Consequently, the results obtained are not only very sensitive to where in the ‘rheological range’ the study is performed, i.e., at what value of G 9p , but also to which synergism parameter is being considered. The ‘relative approach’ to synergism seems to be the most reasonable, that is, to consider how many times stronger (or weaker) the polymer–mucin mixture is compared to the gel itself. However, in our opinion the relative parameter in its present form (DG9 /G 9p ) is not very suitable because it has a negative limit of 21, whereas positive values approach infinity when G 9p approaches zero (see Eq. (3)). From this it follows that the magnitude of positive values cannot be compared to the magnitude of negative ones. We would therefore like to suggest a new ‘relative’ parameter, called the ‘log G9 ratio’, with which the magnitude of positive and negative values are fully comparable. It is calculated as
S D
G 9mix log G9 ratio 5 log ]] . G 9p
(4)
With this parameter a value of 1 would mean that the G9 of
Fig. 4. The absolute (DG9, a) and the relative synergism parameters (DG9 /G p9 , b) obtained with BSMG mucin, as functions of the elastic modulus of the polymer gels (G 9p ).
the polymer–mucin mixture is 10 times higher than that of the gel (i.e., the addition of mucin causes a 10 times increase in G9). Further, a value of 0 would mean that the addition of mucin brings about no change in G9, and a value of 21 represents a decrease in G9 by a factor of 10, etc. Of course the ‘log ratio’ can also be calculated on the basis of G0 or the complex modulus, G*. 9 /G p9 ) In Fig. 5a we have plotted the ‘log ratio’ log (G mix obtained with BSMG mucin against the G p9 . For the crosslinked gels it is evident that the addition of mucin causes an increase of G9 that is large at low G p9 , but becomes a decrease at higher G p9 . Corresponding plots for the mixtures with PS mucin give values that are generally lower than for the BSMG mixtures, which is in accordance with ¨ ¨ et al., previous results (Rossi et al., 1995; Hagerstrom
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of the phase angle would result in a relatively large error in G0. And conversely, if the phase angle is large, a small error in the phase angle would result in a relative error in G9 that is comparatively high. If one wants to compare ‘true’ gels (based on cross-linked polymers) with entangled polymer solutions (based on linear polymers), it may be more appropriate to use the complex modulus, G*, which incorporates both the elastic and the viscous properties of the preparation. In this study the log G* ratio plot (Fig. 5c) is very similar to the one of the log G9 ratio. The use of the log ratio instead of the conventional relative parameter (DG9 /G p9 ) would have the advantage that the magnitude of positive and negative values would be fully comparable, since the log ratio does not approach a fixed limit on the negative scale as does the conventional parameter. But, of course, it would not solve the problem that the method itself gives results that to a large extent are influenced by the rheology of the gel.
3.3. Positive or negative values of the synergism parameters?
Fig. 5. The calculated log ratios based on G9 (a), G0 (b) and G* (c) obtained with BSMG mucin, as functions of the corresponding modulus of the polymer gels.
2000). In Fig. 5 the log ratios based on G0 and G* are also shown. A decrease in the log G0 ratio is observed with increasing G p99 , but the values are positive over the entire range tested. It should be noted, however, that for gels with a very low phase angle, a small error in the determination
The phenomenon of negative synergism values observed with the cross-linked poly(acrylic acid) gels complicates the interpretation of the results. We have previously observed negative synergism values also with gellan gum ¨ ¨ et al., 2000). However, the concept of gels (Hagerstrom rheological synergism is not limited to polymer mixtures with mucin, but has been observed and discussed in previous studies with, e.g., polymer–polymer mixtures (Williams and Phillips, 1995; Nishinari et al., 1996; Rodriguez et al., 2001). From these studies, it is not obvious that an interaction automatically results in a positive rheological synergism term. An interaction might as well result in weaker properties of the mixture than the sum of the individual components. We are therefore not convinced of the soundness of the conventional interpretation model that is used with the rheological mucoadhesion method. In this work positive values of the synergism parameters are obtained at low concentrations (low G p9 ), which is in good agreement with other studies, for example, that of Madsen et al. (1998), in which several cross-linked poly(acrylic acids) gels were mixed with homogenised mucus. Negative values of the synergism parameters have also been previously observed with cross-linked poly(acrylic acids) and commercial mucins (Rossi et al., 1995; Tamburic and Craig, 1997). It has been suggested that ions remaining in the mucin samples could cause a weakening of the gels. This is probably not the only reason, since the BSMG mucin actually contains more ions than the PS ¨ ¨ et al., 2000), but gives larger positive values (Hagerstrom of DG9 than the PS at the low polymer concentrations and smaller negative values at higher concentrations. Moreover, in this study the samples were prepared in 0.9% NaCl to simulate physiological conditions, and at this salt
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concentration the relatively small number of ions present in the mucins is negligible. Instead the negative values of the synergism parameters may be a consequence of the particulate properties of the cross-linked gels. Besides giving molecular interactions with the polymer, the addition of mucin to a strong particulate gel (i.e., at high polymer concentration) could perhaps ‘dilute’ the gel particles by filling the interstitial space, resulting in weaker bulk properties. Correspondingly, for a weak gel in which the gel particles are not in contact, the addition of mucin may represent an increased ‘concentration’ of the preparation, making the bulk properties stronger because the interstitial space is filled with mucin solution instead of pure medium. The question can be raised whether negative values of the synergism parameter really would imply that the gel preparation would have a bad performance in an imaginable in vivo situation, and vice versa. With this method, the interpenetration layer is simulated by mixing the gel with mucin. The gel is considered to be advantageous if the interpenetration layer (i.e., the gel–mucin mixture) is stronger than the gel itself. Can this interpretation model be applied to the situation at a gel–mucosa interface? Consider the general situation where a gel dosage form is in contact with a mucous tissue. The mucoadhesive joint can be considered to consist of three regions—the gel, the mucus layer and the interface (the interpenetration layer). It has been put forward that the residence time of the gel on the mucous tissue would be associated with the failure of the weakest region and, therefore, the cohesive properties of each region are important (Smart, 1999). From this it can be deduced that the gel dosage form should have a sufficient level of cohesiveness to provide a long residence time, and that a strengthening of the mucus layer also would be advantageous, as well as a strong interpenetration layer. But it cannot be deduced that the interpenetration layer necessarily has to be stronger than the gel dosage form itself. In this respect, we believe that the basis of interpretation model that is used with the rheological method is questionable and not easily applied to a possible in vivo situation.
3.4. Comparison to tensile strength measurements With a view to comparing the results from the rheological approach to a more in vivo-like mucoadhesion method, we performed tensile strength measurements using a texture analyser. The adhesion to freshly excised porcine nasal mucosa was measured for the preparations in the 2% and the 10 Pa series. In Table 2 the tensile work and the fracture strength obtained are presented. In the 10 Pa series, where the preparations have approximately the same rheological properties, no significant difference is observed between these preparations with respect to the adhesion to the mucosa, neither in the tensile work nor in the fracture strength. In the 2% series on the other hand,
where the preparations have different rheological properties, the strong cross-linked gels give significantly higher tensile work and fracture strength than the much weaker C907 preparation. Moreover, if the 2% cross-linked gels are compared to the corresponding weaker gels in the 10 Pa series it is evident that the adhesion to the mucosa becomes stronger with increasing gel strength. This has been observed previously in other studies (see, for exam¨ ¨ and Edsman, 2001). ple, Jones et al., 1997; Hagerstrom From these results it can be deduced that with a more in vivo-like method such as the tensile strength approach, stronger rheological properties result in stronger adhesion to the mucosa. This is basically opposite to the results obtained using the rheological mucoadhesion method, where the positive values of the synergism parameters are seen only with weak gels. We believe that this lack of correlation gives rise to even more questions regarding the resemblance of the rheological approach to the situation at the gel–mucosa interface. We would therefore recommend that the rheological method should not be used as a stand-alone method for studying mucoadhesion of polymer gels.
4. Conclusions In the work presented here, we have shown that with the rheological method the results of a comparative mucoadhesion study are strongly influenced by the rationale for the comparison. The method does not give the same ranking order of the polymers when two different comparison strategies are used. A comparative study with different polymers might, however, be more accurate if the study is performed where the polymer preparations have approximately the same rheological properties, e.g., the same value of G9. Furthermore, we have shown that the results obtained in a comparative study are also sensitive to where in the ‘rheological range’ the comparison is made, e.g., at which value of G9. Positive values of the synergism parameters are, for example, only seen with weak gels. The choice of rheological synergism parameter also has a bearing on the results obtained. We have suggested a new 9 /G p9 )). refined relative parameter, the log ratio (log(G mix Despite the refinements of the method that are suggested here, it is still not obvious how to interpret the results, which makes the comparison and ranking of polymers difficult. From tensile strength measurements of the adhesion of the gel preparations to porcine nasal mucosa, we conclude that increased gel strength results in stronger adhesion. This is in contrast to the results from the rheological method, where the positive values of the synergism parameters are seen only with weak gels. On the basis of the limitations of the method that are demonstrated and discussed in this work, we recommend that the method should not be used as a stand-alone
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method for the studying of mucoadhesive properties of polymer gels.
Acknowledgements We are thankful to Ms Sofia Josefsson for excellent technical assistance. Financial support for this study was obtained from AstraZeneca, Sweden, and the Knut and Alice Wallenberg Foundation, and is gratefully acknowledged.
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