Mixtures of Mucin and Oppositely Charged Surfactant Aggregates with

Jun 28, 2005 - molecular weight polymer called mucin.1 Mucin is pro- ... phase behavior of mixtures of these surfactants and mucin was studied, and th...
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Langmuir 2005, 21, 7097-7104

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Mixtures of Mucin and Oppositely Charged Surfactant Aggregates with Varying Charge Density. Phase Behavior, Association, and Dynamics Ge´raldine Lafitte,*,† Krister Thuresson,†,‡ and Olle So¨derman† Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, and Camurus AB, Ideon Science Park, SE-223 70 Lund, Sweden Received November 5, 2004. In Final Form: May 24, 2005 The nonionic surfactant Tween80 is a commonly used excipient in drug formulations containing an active substance with low aqueous solubility. Model drug vehicles with varying charge density were obtained by mixing Tween80 (PS-80) with the cationic surfactant Tetradecyltrimethylammonium chloride (TTAC), thus forming mixed micelles. The micelles were mixed with the negatively charged polyelectrolyte mucin, which is a component in the protective mucus layer covering epithelial cell linings. Depending on the composition of the mixture, complex-formation could be followed by precipitation. Using X-ray diffraction, it was found that the precipitate contained a lamellar phase with properties sensitive to the proportion of PS-80. Higher amounts of PS-80 were found to oppose phase separation. Further analysis in the onephase region, or alternatively of the supernatant of two-phase samples, by 1H NMR, HPLC, and diffusion measurements with PGSE-NMR led to the conclusions that at low proportion of PS-80 aggregates composed of mixed (PS-80 and TTAC) micelles and mucin were formed, whereas increased concentrations of PS-80 favored the dissolution of the precipitate and limited the interactions between mixed micelles and the polymer.

Introduction For reasons of patient compliance, oral administration is the preferred route of administration for many drug molecules. It is, however, a challenging route since many chemical compounds are affected by the degrading environment of the gastrointestinal tract (GIT). Labile active pharmaceutical ingredients (API) need protection from harsh conditions such as exposure to enzymatic activity and a varying pH. API with less favorable solubility properties, like hydrophobic drug molecules, need to be solubilized in order to increase their chances to be presented to the gastrointestinal epithelial cell surface. This is done by incorporating the API in drug carriers, also called vehicles. A correctly designed vehicle can both protect a labile API as well as increase the solubilization of drug molecules. Another challenge to tackle is barriers against absorption. In the GIT, a protective layer called mucus covers the surfaces of the epithelial cells. This layer is a highly hydrated gel, which derives its gel properties from a high molecular weight polymer called mucin.1 Mucin is produced continuously by specialized epithelial goblet cells. Several investigations have focused on characterizing these complex polymers.2-4 An important observation is that, although mucin molecules may differ from one part in the body to another, they do have several features in * To whom correspondence should be addressed. Fax: +46 46 222 4413. E-mail: [email protected]. † Physical Chemistry 1. ‡ Camurus AB. (1) Roussel, P.; Lamblin, G.; Lhermitte, M.; Houdret, N.; Lafitte, J.; Perini, J.; Klein, A.; Scharfman, A. Biochimie 1988, 70, 1471-1482. (2) Nordman, H.; Davies, J. R.; Herrmann, A.; Karlsson, N. G.; Hansson, G. C.; Carlstedt, I. Biochem. J. 1997, 326, 903-910. (3) Bromberg, L. E.; Barr, D. P. Biomacromolecules 2000, 1, 325334. (4) Waigh, T. A.; Papagiannopoulos, A.; Voice, A.; Bansil, R.; Unwin, A. P.; Dewhurst, C. D.; Turner, B.; Afdhal, N. Langmuir 2002, 18, 71887195.

common. Indeed, they are constituted of two main parts, namely, a glycoprotein backbone and carbohydrate chains, which are found as oligomeric side chains covalently bound to the glycoprotein backbone. Different mucin molecules may be linked by disulfide bonds to larger super-molecular aggregates. This has been illustrated as a “windmill” with four mucin monomers joined to one central protein by disulfide bonds.5,6 The resulting complex has a very high molecular weight, as illustrated by pig gastric mucin reported to have molecular weights between (10-50) × 106 Da.7 The formation of super-molecular aggregates is one of the main reasons why differences are found between mucin molecules from different sources. For example, Strous and Dekker reported a difference in molecular organization of human cervical mucin and rat gastric mucin.6 In this investigation, we chose to work with pig gastric mucin, which is linear and has a molecular weight polydispersity.1 Electron microscopy studies have indicated that the structure of this mucin in solution is characterized by flexible threads.1,6,8,9 The oligo-carbohydrate side chains are composed of five different monosaccharides and are frequently terminated with sialic acid which, together with sulfate residues, give a negative charge to the mucin molecules. In addition to their strong influence on the gel properties of the mucus layer, the negatively charged polyelectrolytes are important for interactions with (oppositely charged) API and drug vehicles and will affect their transport properties. (5) Allen, A. In Physiology of the gastrointestinal tract; Press, R., Ed.; Johnson, L. R.: New York, 1981; p 617. (6) Strous, G. J.; Dekker: J. Crit. Rev. Biochem. Mol. 1992, 27, 5792. (7) Bastardo, L.; Claesson, P.; Brown, W. Langmuir 2002, 18, 38483853. (8) Stokke, B. T.; Elgsaeter, A.; Skjakbraek, G.; Smidsrod, O. Carbohydr. Res. 1987, 160, 13-28. (9) Slayter, H. S.; Wold, J. K.; Midtvedt, T. Carbohydr. Res. 1991, 222, 1-9.

10.1021/la0472807 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/28/2005

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As a model of drug vehicles with varying charge density, we used micelles formed by various ratios of one cationic and one nonionic surface-active compound. Nonionic surfactants are one of the most important excipients for poorly water-soluble drugs, and positively charged drug vehicles may be considered when gastro retentive properties are desired. We focused our study on the interactions between these model vehicles and pig gastric mucin. The phase behavior of mixtures of these surfactants and mucin was studied, and the dynamic properties of the surfactant aggregates in the mixtures were followed. The results are important in order to be able to predict the behavior of an oral drug formulation when it reaches the mucus layer in the GIT and to further develop the formulation in order to improve drug delivery. Materials and Methods Materials. Mucin type III from porcine stomach was purchased from Sigma-Aldrich (batch 061K7041). The proportion of sialic acid was reported to be about 1.3 wt %. Polyoxyethylene sorbitane mono-oleate or Tween80 (PS-80) of pharmaceutical quality was obtained from Uniqema (Evebreg, Belgium). Disodium hydrogen phosphate dehydrate, potassium dihydrogen phosphate, and sodium azide were obtained from Merck (Darmstadt, Germany). Tetradecyltrimethylammonium chloride (TTAC) was obtained from Tokyo Chemical Industries Europe (Zwijndrecht, Belgium), whereas deuterium oxide (99.8 atom-% D) and sodium chloride were obtained from Dr. Glaser Lab chemicals, Basel, Switzerland and Riedel-DeHa¨en AG (Seelze, Germany), respectively. All chemicals were used as received. The water was deionized and passed through a Millipore Mill-Q purification unit before use. Sample Preparation. Stock solutions of PS-80, TTAC, and mucin were prepared with a phosphate buffer saline (PBS), pH ) 7.1 and an osmolarity of about 320 mOsm (13.4 mM Na2HPO4, H2O; 1.40 mM KH2PO4; 137 mM NaCl and 6.15 mM NaN3). This buffer is close to a relevant physiological fluid with respect to ionic strength. Final samples were prepared by measuring the weight of each stock solution added. A mucin concentration of 0.25 wt % was used for all samples. Samples used for NMR measurements were prepared with D2O and PBS at a weight ratio of 20/80. Each sample was mixed overnight at a controlled temperature (4 °C or 25 °C), and if necessary, centrifuged (4000 rpm for 30 min at 25 °C) to obtain a clear supernatant in phase separated samples. Determination of Phase Behavior. Partial phase diagrams (one- or two-phase behavior) were determined by visual inspection. Samples were prepared and observed at two temperatures (4 and 25 °C). The visual inspection of phase behavior was normally performed after equilibration overnight at the relevant temperature. It can be noted that the Krafft temperature of TTAC (in the range of 12-15 °C) is intermediate to the two temperatures chosen for phases studies. This temperature has been estimated by comparison with that of related surfactants with longer and shorter hydrocarbon chains.10 PGSE-NMR. All experiments were performed at 25 °C on a Bruker DMX-200 spectrometer, equipped with a Bruker diffusion probe having a maximum gradient strength of 9.6 T/m. A stimulated echo pulse sequence was used and echo decays were analyzed with the Stejskal-Tanner equation

[

(

I ) I0 exp -(γgδ)2 ∆ -

)]

δ D ) I0 exp[-kD] 3

where γ is the magnetogyric ratio of the proton, g is the gradient strength, δ is the length of the gradient pulse, ∆ is the diffusion time, and D is the diffusion coefficient. Quantitative NMR. 1H NMR was used to determine the concentrations of the two surfactants in the supernatant of some samples. Those experiments were done with a (deuterium locked) (10) Holmberg, K.; Jo¨nsson, B.; Kronberg, B.; Lindman, B. In Surfactants and polymers in aqueous solution, 2nd ed.; John Wiley and Sons: New York, 2003; pp 67-96.

Lafitte et al. Bruker spectrometer operating at 500.20 MHz. By comparing with reference solutions, integral values of characteristic peaks were used to obtain the concentration of each surfactant. Aqueous mixtures of both surfactants without mucin were used as reference. SAXD. Small angle X-ray diffraction experiments were performed on a Kratky compact small angle system equipped with a position sensitive wire detector containing 1024 channels having a width of 53.6 µm. A Cu KR radiation with a wavelength of 1.542 Å was provided by a Seifert ID 3000 X-ray generator operating at 50 kV and 40 mA. To have an appropriate signalto-noise ratio the samples were analyzed for a period of 4 to 12 h. Diffraction patterns were obtained from precipitates that were first collected and freeze-dried, followed by reswelling and equilibration at a relative humidity close to 100%. Repeated weighing before and after swelling gave the water content in the precipitates. HPLC. By using the method developed by Irache et al.,11 the mucin concentration in the supernatants could be calculated from high-pressure liquid chromatography (HPLC) analysis. These measurements also gave information about size distribution of mucin molecules. The HPLC setup used was a Waters Ultrahydrogel linear column in series with a Waters Ultrahydrogel 250 column connected to a UV-detector operating at 280 nm. The mobile phase, with a flow rate of 1 mL/min, was a solution of Tris-HCl (15 mM) and NaCl (300 mM) with a pH ) 7.5. A sample volume of 80 µL was used and the concentration of mucin of each sample was obtained from a mucin calibration curve (0.01, 0.102, 0.247, and 0.501 wt %). The measurements were performed at 25 ( 1 °C. Optical Microscopy. A microscope (Zeiss Axiopan) equipped with a Hamamatsu camera (model L2400-08) and crossed polarizers were used to determine anisotropic properties of some samples. The samples were investigated at room temperature after placing them between a glass slide and a cover slip. Cryo-TEM. A droplet of each sample was placed on the surface of a holey carbon film supported on a copper grid. The films were vitrified by plunging the grid into liquid ethane (-183 °C). The vitrified samples were stored and transferred in liquid nitrogen (-196 °C) before examining them by using a Philips CM 120 microscope operating at 120 kV.

Results and Discussion Aqueous Mixtures of PS-80 and TTAC. The aqueous system that was investigated contains apart from mucin, a cationic and a nonionic surfactant (TTAC and PS-80, respectively). Since this is a rather complex system, it was important to first understand the properties of the surfactant mixture before introducing it into the polymer matrix. One convenient tool to study the structure and the behavior of a mixed surfactant system is PGSENMR.12-14 By studying the diffusion coefficients, one can deduce information with regards to the nature of the aggregate structures present in the system. No sign of phase separation was observed in the investigated aqueous mixture containing 0.2 wt % TTAC and a varying amount of PS-80. Thus, all PGSE-NMR measurements were performed on homogeneous samples. The echo decay for TTAC was evaluated by studying the peak arising from the N-methyl protons. In all cases, the echo decay for TTAC was found to be monoexponential. The study of the diffusion of PS-80 could be done either through the EO-methylene protons (δ ) 3.6 ppm) or through the aliphatic protons (δ ) 1.2 ppm). We chose to follow the peak at 3.6 ppm since that at 1.2 ppm overlapped with the peak belonging to the protons of the aliphatic chain of TTAC. In the case of PS-80, a biexponential fit (11) Irache, J. M.; Durrer, C.; Duchene, D.; Ponchel, G. J. Controlled Release 1994, 31, 181-188. (12) Price, W. S. Concept Magn. Res. 1997, 9, 299-336. (13) Price, W. S. Concept Magn. Res. 1998, 10, 197-237. (14) Soderman, O.; Stilbs, P. Prog. NMR Spectrosc. 1994, 26, 445482.

Mixtures of Mucin and Surfactant Aggregates

Figure 1. Diffusion of PS-80 and TTAC in a set of samples with fixed weight percentage of TTAC (0.2 wt %) and various amount of PS-80. Diffusion of PS-80 in a pure solution is plotted as a reference. Samples were prepared in phosphate buffer at pH 7.1.

Figure 2. Chemical structure of Tween80.

offered a better description of the echo decay. The presence of two diffusing fractions is likely due to impurities with different surface activities.15-17 The less surface active impurities are responsible for a faster diffusion, which was observed at the low gradient strengths. To avoid the influence of the impurities, the intensities from the lowest gradient strengths were discarded. Figure 1 presents the diffusion coefficients of PS-80 and TTAC plotted versus the concentration of PS-80 in the mixed system with the concentration of TTAC held constant at 0.2 wt %. Diffusion data for PS-80 in an aqueous solution without TTAC are also included. The diffusion coefficient for the pure PS-80 micelles in the infinite dilution limit (D ) 4.9 × 10-11 m2/s) gave a hydrodynamic radius of about Rh ) 44 Å using the StokesEinstein relation. In addition, the ratio between diffusion coefficients presented in Figure 1 at 0.2 and 2.5 wt % of PS-80 was equal to 1.15. The measured decrease is significant and is due to obstruction effect and/or aggregate growth.18 It is important to point out that, in any event, the aggregate growth is small and we estimated to be less than 20%. One can mention that PS-80 micelles have a “hairy” micelle corona on account of the large hydrophilic headgroup formed by the EO chains (see Figure 2). In the mixture of both surfactants, the diffusion coefficient of PS-80 increased whereas that of TTAC decreased compared to that of their pure surfactants aggregates as observed in Figure 1. This proved that the (15) Hakansson, B.; Hansson, P.; Regev, O.; Soderman, O. Langmuir 1998, 14, 5730-5739. (16) Momot, K. I., Kuchel, Philip W. Concept Magn. Res. 2003, 19A, 51-64. (17) Lafitte, G.; Jarwoll, P.; Nyden, M.; Thuresson, K., manuscript in preparation, 2005. (18) Lekkerkerker, H. N. W.; Dhont, J. K. G. J. Chem. Phys. 1984, 80, 5790-5792.

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mixed micelles were intermediate in size to the pure PS80 and TTAC micelles. The formation of mixed micelles was favored by the large difference in CMC between the two surfactants. PS-80 (average Mw ) 1310 g/mol) and TTAC (Mw ) 292 g/mol) have been reported to have CMC values of 0.01-0.02 mM19-22 (i.e., 0.0013-0.0026 wt %) and 5.4 mM (i.e., 0.16 wt %) in a salt free solution, respectively, and aggregation numbers of 124 for PS-80 and 6523 for TTAC. In the case of ideal mixing, the CMC is expected to be inversely proportional to the sum of 1/CMC of each surfactant weighted by their respective fraction.24 In the present study, this means that aggregation is strongly influenced by the low CMC of PS-80 and that the concentration of unimers of TTAC is reduced. This effect is expected to reduce the measured diffusion coefficient of TTAC as indeed it was found to be in Figure 1. Even if mixed micelles are formed, the diffusion coefficients of the two surfactants are not expected to be identical, since the diffusion coefficient of each surfactant measured by PGSE-NMR is the sum of the diffusion coefficient of the molecules included in the micelles and that of the unimers in solution according to25

D ) xunimer Dunimer + (1 - xunimer) Dmicelles

(1)

where D is the self-diffusion coefficient, xunimer is the fraction of surfactants as unimer in solution, Dunimer is the diffusion coefficient of the unimers, and Dmicelles is the diffusion coefficient of surfactant molecules included in mixed micelles. Because of the low CMC of PS-80 (mentioned above), xunimer is negligible for the nonionic surfactant. Then, one can see that the diffusion coefficient measured for PS-80 was equal to that of the mixed micelles. Furthermore, since the diffusion coefficient of PS-80 in the mixture increases rather substantially compared to the pure PS80 system, this shows that the mixed micelles were smaller in size than the pure PS-80 micelles. On the other hand, the diffusion coefficients of TTAC are higher than that of mixed micelles. This is due to a comparably high concentration of small unimers (expected from a high CMC) with fast transport properties. So, the contribution of the unimers must be considered in eq 1. It results in a higher diffusion coefficient of TTAC than that of the mixed micelles. In addition, Figure 1 shows that by increasing the concentration of PS-80 (keeping the concentration of TTAC fixed at 0.2 wt %) the diffusion coefficient of the nonionic surfactant approaches that in the pure PS-80 system; that is, the influence of TTAC decreases. Not unexpectedly, it appears that at concentrations of PS-80 higher than 1 wt %, the nonionic surfactant is the dominating component. Aqueous Mixtures of PS-80, TTAC, and 0.25 wt % Mucin. Before entering into the discussion regarding the microstructure and the dynamics of the surfactantspolymer aggregates, the phase behavior is discussed and (19) Patist, A.; Bhagwat, S. S.; Penfield, K. W.; Aikens, P.; Shah, D. O. J. Surfactants Deterg. 2000, 3, 53-58. (20) Prak, D. J. L.; Pritchard, P. H. Water Res. 2002, 36, 3463-3472. (21) Haque, M. E.; Das, A. R.; Moulik, S. P. J. Colloid Interface Sci. 1999, 217, 1-7. (22) Huttenrauch, R.; Fricke, S.; Kohler, M. Pharm. Res. 1988, 5, 726-728. (23) Cang, H.; Brace, D. D.; Fayer, M. D. J. Phys. Chem. B 2001, 105, 10007-10015. (24) Holmberg, K.; Jo¨nsson, B.; Kronberg, B.; Lindman, B. In Surfactants and polymers in aqueous solution, 2nd ed.; John Wiley and Sons: New York, 2003; pp 119-138. (25) Evans, D. F.; Wennerstro¨m, H. The colloidal domain: Where physics, chemistry, biology and technology meet; 2nd ed.; Wiley-VCH: New York, 1999.

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Figure 3. Partial phase diagram of Mucin/TTAC/PS-80 at fixed concentration of mucin of 0.25 wt % at 25 °C (×, phase separation; b, no phase separation). Insert is the partial phase diagram for the range of concentrations of TTAC of 0.003-0.03 wt %.

an overview of the system is presented. Figure 3 shows one- and two-phase regions in a system containing 0.25 wt % mucin and various concentrations of the surfactants. The two-phase samples appeared as liquid supernatant in equilibrium with a dense precipitate at 4 °C or a gel like opaque phase at 25 °C. The difference in the appearance of the precipitate at the two temperatures was most likely due to the Krafft temperature of TTAC. Although the phase behavior was investigated both well below and above the Krafft temperature, no difference of the location of the phase border between the one- and the two-phase regions could be observed. 1. Aqueous Mixtures of TTAC and Mucin. We first focus on the set of samples containing only TTAC and mucin, i.e., the x axis of the phase diagram in Figure 3. At a concentration of TTAC below 0.003 wt %, the system was totally miscible, whereas at higher concentrations phase separation occurred resulting in formation of a precipitate. As mentioned earlier, the proportion of sialic acid in pig gastric mucin has been estimated to be about 1.3 wt %. Sialic acid is a collective name for a family of more than 40 acids with different functional groups having a common backbone, N-acetylneuraminic acid (Mw ≈ 309 Da). Thus, in a solution of 0.25 wt % mucin, the overall charge neutrality of the system was calculated at a TTAC concentration of about 0.003 wt %. This corresponded well to the concentration where phase separation was first observed. Dedinaite et al. showed that mucin molecules have hydrophobic patches along the backbone and that the negatively charged surfactant SDS most likely adsorbs on these patches.7,26 As a result, it appears reasonable to describe mucin as a hydrophobically modified polymer. Typically in a solution of a hydrophobically modified polyelectrolyte and oppositely charged surfactant, the adsorption of the surfactant occurs along the polymer backbone until overall charge neutrality is reached.27,28 (26) Dedinaite, A.; Bastardo, L. Langmuir 2002, 18, 9383-9392. (27) Bai, G. Y.; Santos, L. M. N. B. F.; Nichifor, M.; Lopes, A.; Bastos, M. J. Phys. Chem. B 2004, 108, 405-413. (28) Linse, P.; Piculell, L.; Hansson, P. In Polymer-surfactant systems; Kwak, J. C. T., Ed.; Marcel Dekker: Basel, 1998; pp 193-238.

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Linse et al. showed also that in a solution of globular proteins, which in some respects are similar to a hydrophobically modified polymer, oppositely charged surfactants bind in a noncooperative way until charge neutrality is reached and then bind in an “anti-cooperative” way.28 In the case of globular proteins, the formation of a precipitate has been observed when the “anti-cooperative” binding region is entered. Along the same lines the interactions, in the initial stage, between TTAC and mucin could be viewed as a noncooperative binding of the surfactant along the polymer backbone. This binding occurs on the account of hydrophobic and electrostatic interactions originating from hydrophobic patches and the opposite charge of the mucin molecules. It resulted the formation of a precipitate at overall charge neutrality. Generally, in the case of hydrophobically modified polymers, a third stage is reached at a surfactant activity close to the CMC of the surfactant system, where formation of micellar like aggregates along the polymer backbone is normally observed, resulting in a net-charged complex having the same charge as the surfactant (i.e., opposite in sign to the polymer) and dissolution of the complex follows. This does not happen in the TTAC/mucin system since no redissolution of the precipitate was observed. On the other hand, when increasing the TTAC concentration, the amount of precipitate gradually increased. The formation of a lamellar phase in the precipitate is the reason for this observation. We will return to this fact below. 2. Aqueous Mixtures of Mucin/TTAC and PS-80. For a system containing Mucin, TTAC and PS-80, three different regions could be distinguished in the phase diagram (see Figure 3). (i) In the one-phase region from 0 to 0.003 wt % of TTAC, TTAC binds in a noncooperative way to the mucin molecules, as discussed above. Furthermore, in analogy with the formation of mixed micelles between TTAC and PS-80, it is plausible that the addition of PS-80 resulted in the formation of complexes between all three components. However, it cannot be ruled out that the formation of mixed micelles may decrease the amount of mucinbound TTAC or that pure PS-80 micelles may form and leave the TTAC/mucin complexes unaffected. (ii) In the range between 0.003 and 0.03 wt % TTAC, the data from Figure 3 imply that the phase border is either a horizontal line or a line with a modest slope (concentration of PS-80 in the range of 0.01-0.03 wt %). We note that the (total) TTAC concentration is always quite low as compared to the CMC in a salt free solution while PS-80 concentration is well above CMC and aggregation is expected to occur. The appearance of the phase diagram would mean that, in this range, approximately the same amount of nonionic surfactant dissolved the precipitate independently of the amount of TTAC present. Two mechanisms to explain such behavior are readily available. First, a comparison with a binding isotherm of oppositely charged surfactant to a hydrophobically modified polyelectrolyte28 suggests that in the anti-cooperative region, the amount of surfactant adsorbed to the polymer is more or less constant, since the TTAC concentration is rather low it is reasonable to assume that in this region the samples indeed are in the anti-cooperative region. This would imply that the same amount of TTAC was precipitated with the mucin molecules and the excess was in solution. The addition of PS-80 is expected to decrease the amount of free TTAC by formation of mixed micelles. Then, at some point, the activity of TTAC decrease to a level such that the region of noncooperative binding of TTAC to mucin was reached again. This would mean that

Mixtures of Mucin and Surfactant Aggregates

dissolution of the precipitate was due to a transition from the anti-cooperative binding to the noncooperative binding; that is, the polymer/TTAC complexes would again attain a net negative charge. This mechanism can be illustrated by a slope of the phase border as suggested in insert in Figure 3, since the amount of TTAC that PS-80 micelles have to dissolve increase. Hence, the precipitate is expected to suddenly disappear on increasing the PS-80 concentration in analogy with how it first appeared (see above). On the other hand, in this range, the amount of precipitate obtained is such that a swelling of the precipitate before the dissolution would be difficult to observe. A swelling would be a sign of the other possible mechanism, in which PS-80 molecules bind to the hydrophobic patches of mucin and soluble aggregates of TTAC/PS-80/mucin are formed. In that case, swelling of the precipitate could be expected before dissolution. As it will be discussed below, SAXD data indicate that some PS-80 molecules may actually be incorporated in the bottom phase. The modest slope of this region in Figure 3 shows an easy dissolution of the precipitate by PS-80. In this region, the precipitate is dominated by mucin with TTAC molecules bound by a combination of hydrophobic and electrostatic interactions and distributed along the polymer chains to form a charge neutral complex. PS-80, with a strong tendency to curve toward oil and form spherical micelles (high HLB-value), binds to hydrophobic patches in the precipitate. Thus, incorporation of PS-80 makes the complex more soluble by formation of mixed micelles and the precipitate eventually dissolves. (iii) The third part of the phase diagram was found above 0.03 wt % TTAC. It is clear that the slope of the phase border is at least five times steeper than in the previous region. The phase border is reached for a molar ratio between PS-80 and TTAC of 1:1. The change on slope suggests a transition from one mechanism to another, and we will discuss this in more detail below. We chose to consider one concentration of TTAC (0.2 wt %) in more detail. Most interestingly, we observed that the amount of precipitate decreased with increasing amount of PS80. This was characterized by a continuous dissolution of the precipitate, whereas at the same time no swelling of the precipitate was observed. As will be shown below, the dissolution process occurred by simultaneous dissolution of mucin and TTAC. To be able to understand this process, one needs to consider the physical properties of the precipitate and those of PS-80. As we will see below, SAXD data show that the bottom phase is a lamellar phase in which mucin is dissolved. Since PS-80 with its high HLBvalue is not expected to dissolve to a large extent in the lamellar geometry, the dissolution of the complex probably occurs with a lamellar to a micellar transition of the surfactant aggregates. In this scenario, mixed micelles between TTAC and PS80 form in the supernatant, whereas the incorporation of PS-80 in the precipitate occurs only to a certain extent. The formation of mixed micelles renders the precipitate soluble and continuously displaces TTAC and mucin to the supernatant. This mechanism implies formation of aggregates between mixed micelles and mucin molecules in the supernatant. Actually, despite of the fact that the bottom phase has TTAC in excess, mucin is continuously dissolved. It can be mentioned that further increase of PS-80 is expected to results in hairy mixed micelles opposing complexation with mucin due to steric repulsion. We will return to these issues below in connection with discussing results from other methods focusing on properties of the precipitate and on properties of the supernatant.

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Figure 4. SAXD pattern of sample with 0.50 wt % of PS-80, 0.2 wt % TTAC, and 0.25 wt % mucin.

Microstructure and Chemical Composition of PS80, 0.2 wt % TTAC and 0.25 wt % Mucin. 1. Microstructure of the Precipitate. SAXD was used for studying the structure of the precipitate of a set of samples where the concentration of mucin and TTAC was held constant at 0.25 and 0.2 wt % respectively, and the concentration of PS-80 was varied. A representative SAXD pattern with the first two Bragg peaks is shown in Figure 4. Despite the low intensity of the second Bragg peak, it is reproducible. From Bragg’s law, the repeat distance d of a lamellar phase can be calculated from29

2π q)n d where q is the scattering vector of the peak position, d is the repeat distance of the lamella, and n is a factor equal to 1: 2: 3: 4.... In Figure 4, peaks corresponding to n equal to 1 and 2 have q values of 0.139 and 0.279 Å-1. In addition, by studying the samples between crossed polarizers in a light microscope, a uniaxial marbled birefringence, typical of a lamellar phase,30,31 was observed. Thus, both optical microscopy and SAXD suggest the presence of a lamellar liquid crystalline phase in the precipitate. Normally, TTAC tends to form aggregates that curve toward oil, i.e., spherical micelles. The formation of a lamellar structure in the precipitate can be explained by the fact that the surfactant headgroup repulsion is effectively decreased by complexation with the oppositely charged mucin. In addition, the ionic strength of the buffer system was also rather high. Both effects would favor the formation of less curved aggregates. Formation of a lamellar structure is also in line with a report by SwansonVethamuthu et al. where formation of planar aggregated structures (disks) were promoted in alkyltrimethylammonium halide systems by addition of NaBr.32 The lamellar liquid crystalline phase is characterized by a repeat distance, which slightly increases with the concentration of PS-80; see Table 1. This indicates that (29) Vonk, C. G. In Small-Angle X-ray Scattering; Kratky, O., Ed.; Academic Press Inc.: New York, 1982; Vol. 1, pp 433-466. (30) Porte, G. In Neutrons, X.-Rays and Light: Scattering Methods Applied to Soft Condensed Matter 2nd ed.; Zemb, T., Ed.; Elsevier: Amsterdam, 2002; Vol. 1, pp 299-315. (31) Geraghty, P. B., Attwood, D.; Collett, J. H.; Dandiker, Y. Pharm. Res. 1996, 13, 1265-1271. (32) Swanson-Vethamuthu, M.; Feitosa, E.; Brown, W. Langmuir 1998, 14, 1590-1596.

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Table 1. Lamellar Distances Calculated from SAXS Measurements and from Water Content of the Precipitate

wt % PS-80

volume fraction of water in precipitate (%)

0(0 0.249 ( 0.002 0.50 ( 0.002 0.75 ( 0.003

40.2 ( 0.1 36.6 ( 0.1 39.4 ( 0.1 42.3 ( 0.1

D (Å)

d water: dw (Å)

d aliphatic chain: dhc (Å)

40.9 ( 0.5 43.5 ( 0.5 45.5 ( 0.5 48.3 ( 0.5

16.4 ( 0.5 16.0 ( 0.5 18.0 ( 0.5 20.5 ( 0.5

24.5 ( 0.5 27.7 ( 0.5 27.6 ( 0.5 27.9 ( 0.5

PS-80 dissolves in the lamellar phase to some extent. At the low PS-80 concentrations (0 and 0.249 wt %), the diffraction data indicated the coexistence of two lamellar structures. This is most probably a consequence of difficulties in attaining complete thermodynamic equilibrium after reswelling the freeze-dried precipitate in humid air. In a lamellar liquid crystalline phase, d is the sum of the thickness of the surfactant bilayer (dhc) and the water layer (dw). Since the water content was calculated from repeated weighing after drying and reswelling, dhc and dw could be calculated via the volume fraction of water in each sample (see Table 1)

Φw )

mwaterinprecipitate and from the relation mwetprecipitate dw ) Φwd and dhc ) d - dw

By comparing dhc with the all-trans length of the hydrocarbon chain, l, of TTAC calculated according to l ) 0.15 + 0.127nc (nc being the number of carbon atoms in the hydrocarbon chain),25 dhc was about 0.7l. This suggested that the bilayer was composed mainly of cationic surfactant molecules. Indeed, surfactant bilayers normally have a hydrocarbon core with a thickness of about 80% of the length of two extended alkyl chains.25,33 Since the hydrocarbon tail of PS-80 is composed of oleyl chains (C18), the all-trans length of this surfactant is expected to be significantly longer. From Table 1, we note that the increase in the value of d with an increasing amount of PS-80 mainly is due to an increase of dw, suggesting that the precipitate became more hydrophilic with the addition of PS-80. This is due to incorporation of the hydrophilic oligo ethyleneoxide headgroup in the water domains of the lamellar phase, although the incorporation of PS-80 in the lamellar phase is rather modest since the value of dhc suggests that the bilayers are mainly composed of TTAC. The dissolution of the precipitate by PS-80 is then due to a phase transition from lamellar to micellar of the surfactant phase and PS80 is mainly in the supernatant in micellar aggregates. It deserves to be repeated that redissolution of a precipitate, formed by a hydrophobically modified polyelectrolyte and an oppositely charged surfactant, is expected at excess surfactant if the added surfactant tends to form small polymer bound spherical micellar aggregates.27,28 If the surfactant forms a lamellar liquid crystalline phase instead of spherical micellar aggregates, redissolution of the complex cannot occur. 2. Chemical Compositions of Precipitate and Supernatant. The chemical composition of the precipitate was investigated with focus on TTAC and mucin since quantitative analysis based on proton spectra for three (33) Holmberg, K.; Jo¨nsson, B.; Kronberg, B.; Lindman, B. In Surfactants and polymers in aqueous solution, 2nd ed.; John Wiley and Sons: New York, 2003; pp 39-66.

sets of samples showed that virtually all PS-80 was in the supernatant, again suggesting that the amount of PS-80 in the lamellar phase must be small. The amount of TTAC incorporated in the precipitate was calculated by subtracting from the initial amount of TTAC the amount in the supernatant obtained by 1H NMR. In Table 2, one can see that the higher the global concentration of PS-80, the lower the amount of TTAC in the precipitate as expected from the mechanism for redissolution suggested above. Due to the low concentration of mucin, the same method to determine the amount of mucin in supernatant and in the precipitate could not be used. Instead, the amount of mucin in the supernatant was determined by HPLC, and the results are plotted in Figure 5. Two regions could be identified; first the amount of mucin increased with nonionic surfactant until the one phase region was reached, after which point the concentration of mucin was constant. These results showed that the amount of mucin in the precipitate decreased continuously as the amount of PS-80 increased. In addition, Table 2 shows that the precipitate always had a substantial excess of cationic charge in the investigated range, even if the charge decreased slightly with an increasing concentration of PS80. As we suggested above, these results imply complexation between mixed micelles and mucin in the supernatant since mucin is continuously dissolved despite an excess of TTAC in the precipitate It is interesting to note from Figure 5 that, even without any addition of PS-80, about half of the initial amount of mucin was in the supernatant. This shows that the mucin sample is chemically heterogeneous. As a consequence, different mucin molecules have different charge densities and/or different chemical composition. It is conceivable that the fraction that did not form a precipitate with the oppositely charged TTAC had no (or only minute) negative charge or that complexes with cationic TTAC are soluble due to others reasons than electrostatics. A fractionation due to charges during precipitation could be invoked to partly explain the high ratio between charges of TTAC and those of mucin (see Table 2), since this calculation assumes a random distribution of charges among the mucin molecules. It could however not explain the 50-fold excess of positive charges as suggested by the ratio between the amount of TTAC molecules and the estimated amount of sialic acid in the precipitate. Instead, according to the above suggested, a surfactant dominated liquid crystalline phase is formed which incorporated a certain fraction of the mucin molecules. Additional data for a more in-depth investigation of the supernatant is now presented. HPLC chromatograms can be used to investigate changes in the size distribution of the mucin fraction in the supernatant. Results of a set of measurements are summarized in Figure 6. A reference solution of pure mucin is composed of two rather polydispersed fractions with retention times of about 12 and 17 min, where the latter dominated strongly. The addition of TTAC (0.2 wt %) and precipitation of a part of the polymer material lead to changes in the chromatogram such that the area of the second peak became smaller and the maximum of that peak was shifted toward longer retention time. This suggests that mucin molecules in the supernatant after precipitation are smaller than the average size and that mucin molecules contributing to the precipitate were larger than the average size of the mucin sample. Subsequent addition of PS-80 was responsible for two changes: first a peak, whose intensity increased with increasing amount of PS-80, appeared at a retention time

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Table 2. Amount of TTAC and Mucin in Supernatant and in Precipitate for Samples at 0.2 wt % TTAC and 0.25 wt % Mucin

wt % PS-80

wt % TTAC initial

wt % mucin initial

NTTAC in supernatant (mol/100 g of solution)

NTTAC in precipitate (mol/100 g of solution)

Nsialic acid in supernatant (mol/100 g of solution)

Nsialic acid in precipitate (mol/100 g of solution)

0.26 ( 0.02

0.2 ( 0.02

0.50 ( 0.02

0.2 ( 0.02

0.80 ( 0.02

0.2 ( 0.02

0.250 ( 0.001 0.250 (0.001 0.250 (0.001

5.65 × 10-4 ( 3.2 × 10-6 6.18 × 10-4 (3.9 × 10-6 6.44 × 10-4 (5.2 × 10-6

15.7 × 10-5 (7 × 10-6 6.6 × 10-5 (7 × 10-6 4.5 × 10-5 (9 × 10-6

7.2 × 10-6 (3 × 10-7 9.0 × 10-6 (4 × 10-7 9.3 × 10-6 (4 × 10-7

3.2 × 10-6 (2 × 10-7 1.55 × 10-6 (7 × 10-8 1.17 × 10-6 (7 × 10-8

Figure 5. Evolution of percentage of mucin in solution when concentration of PS-80 is increased. [, amount of mucin in solution; 2, initial amount of mucin in each sample.

Figure 6. HPLC chromatograph of Mucin (0.25 wt %), TTAC (0.2 wt %) and various weight percentage of PS-80: 0 wt % (0); 0.259 wt % (O); 0.5 wt % (4); 1.2 wt % (]); and pure mucin at 0.25 wt % (-). Insert shows the chromatographs in the range of retention time of 10-14 min.

of about 13 min (see Figure 6), and the peak located at about 17 min became larger and shifted until it looked like the pure mucin sample. This illustrates the continuous dissolution of mucin upon addition of PS-80. We have also investigated the aggregates formed in the supernatant during the dissolution with two additional methods, namely Cryo-TEM and PGSE-NMR. Cryo-TEM pictures obtained from the supernatant of three samples (i) 0.25 wt % mucin solution, (ii) 0.25 wt %

NTTAC/Nsialic acid in precipitate 49.1 42.6 39.5

mucin/0.2 wt % TTAC/0.75 wt % PS-80, and (iii) 0.25 wt % mucin/0.2 wt % TTAC/1.2 wt %PS-80 (i.e., a one-phase sample) are presented in Figure 7. Mucin molecules appeared to form large randomly shaped “fluffy” aggregates having a size of about 500 nm, see Figure 7A. In the supernatant of the two-phase sample (ii), more organized elongated structures, “fibers”, were observed, see Figure 7B. It thus appears that TTAC actually influences mucin also in the supernatant and the “fibers” structure in Figure 7B correspond to aggregates of mucin and surfactants. These structures vanished at higher concentration of PS-80 (iii) where the observed structures were similar to those observed in the pure mucin system, see Figure 7C. This corresponds well to what was observed in the HPLC measurements. In Figure 8, diffusion coefficients of both surfactants in the supernatant of samples containing 0.2 wt % TTAC, 0.25 wt % mucin and various concentrations of PS-80 are plotted. In the two-phase region, diffusion coefficients of TTAC are on average higher than those of the same surfactant in mixed micelles (without mucin). However, a strong decrease was observed with increasing concentration of PS-80. At concentrations of PS-80 below 0.9 wt %, TTAC and mucin precipitated as shown above. This results in a reduced amount of TTAC in the supernatant, which will increase the measured diffusion coefficient due to a larger fraction of unimers (eq 1). For PS-80 in the same region, the measured diffusion coefficients were similar to those in the case of a pure PS-80 solution, i.e., lower than those in Figure 1. Again, because of the depletion of TTAC from the supernatant, the amount of TTAC involved in mixed micelles was lower and the mixed micelles had a size closer to the case in pure PS-80 micelles. Finally, if one focuses on the one-phase region, the diffusion coefficients of both the cationic surfactant and PS-80 are similar to those in the PS-80/TTAC samples (i.e., without mucin). Following the interpretation in that system, it appears that mixed micelles form, but the data do not give any additional information about possible interactions or obstructions effect due to the presence of the mucin molecules. In summary, results of HPLC, Cryo-TEM, and PGSENMR illustrate that the dissolution of the precipitate proceeded by the formation of mixed micelles between TTAC and PS-80 and a simultaneous dissolution of mucin. Interactions between mucin molecules and mixed micelles are suggested to exist in the two-phase region. Moreover, these interactions are further weakened by the steric repulsion from the EO-chains in micelles dominated by PS-80. Conclusions The phase behavior in the present system is intriguing. Analogies with mixtures of surfactant with oppositely charged hydrophobically modified polymers are used to

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Figure 7. Cryo-Tem picture of a solution of 0.25 wt % of mucin (A); 0.25 wt % mucin/0.2 wt % TTAC/0.75 wt % PS-80 (B); 0.25 wt % mucin/0.2 wt % TTAC/1.2 wt % PS-80 (C).

Figure 8. Diffusion of PS-80 and TTAC in samples with constant concentrations of TTAC (0.2 wt %) and mucin (0.25 wt %) at 25 °C.

understand features at low TTAC concentration, whereas we have found that normally used models to rationalize data have shortcomings in explaining the behavior at higher surfactant concentrations. The phase diagram has to be interpreted in the light of the structural and dynamic investigations that have been performed. Quantitative NMR and SAXD show that only

a minute fraction of PS-80 dissolves in the precipitate, whereas HPLC, Cryo-TEM, and PGSE-NMR show that complexes of both surfactants and mucin are found in the supernatant. These complexes seem to disintegrate to TTAC/PS-80 mixed micelles and mucin molecules at high PS-80 concentration. The latter may be due to an increased steric repulsion from a “hairy” micellar corona. The reason the precipitate continuously dissolves on addition of PS-80 is that TTAC is dissolved continuously from a lamellar phase into a micellar phase. Hence, the mixed aggregates of TTAC and PS-80 have a substantial positive curvature. This is only possible if new aggregates form. The latter have a high aqueous solubility and are therefore found in the supernatant. Apart from being a challenging system from an academic perspective a deeper understanding is very useful from a pharmaceutical point of view. For instance, the present data show that to have retention of a drug carrier to the negatively charged mucus layer it is important to have a correct charge density. Such knowledge may be used in design of new gastroretentive systems. Acknowledgment. The authors thank C. Nistor and G. Karlsson for the help in the measurements with HPLC and Cryo-TEM, respectively. O.S. acknowledges support from the Swedish Research Council (VR). LA0472807