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Influence of Several Factors on the Response of Calcitonin Monolayers to Compression at the Air-Water Interface N. Vila Romeu, J. Min˜ones Trillo,* O. Conde, M. Casas, and E. Iribarnegaray Department of Physical-Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain Received April 1, 1996. In Final Form: September 30, 1996X Salmon calcitonin, a polypeptide hormone used in the treatment of osteopathies, formed stable monolayers when spread at the air-water interface from 4:1 (v/v) chloroform/methanol spreading solutions. Monolayers on subphases of pH 2 were more expanded and collapsed at lower surface pressures than those on subphases of pH 6 and 10. Areas and collapse surface pressures decreased with increasing temperature, most markedly at acid pH. Hysteresis was observed when monolayers were repeatedly compressed beyond collapse and decompressed, with compression isotherms shifting to smaller areas in successive cycles. Hysteresis was increased by reducing compression speed and by increasing ionic strength. The results are interpreted as showing that collapse was gradual and partially reversible, consisting in the progressive submersion of the amino acids with the most polar groups (which will have formed a “transition layer” just below the interface) without expulsion of the whole molecule from the film.
Introduction Calcitonins are polypeptide hormones with 32 amino acids and mole weights around 3500 g/mol. The primary structure of salmon calcitonin (the form most widely used clinically) is shown in Chart 1; other calcitonins that have been characterized (including those of man, sheep, pig, rat, and eel) differ mainly as regards the sequence and/or identity of the amino acids forming the central portion of the chain.1 Calcitonin is implicated in calcium homeostasis, reducing resorption of bone by inhibiting the release of bone calcium into serum. Salmon calcitonin administered clinically to osteopathy patients arrests bone loss2,3 (chiefly by inhibiting osteoclast activity4 ) and alleviates associated chronic skeletal pain. Osteopathies treated in this way include senile osteoporosis, Paget’s disease of bone (characterized by acute osteolysis),5 and osteoporoses in which bone turnover is intensified, such as postmenopausal osteoporosis.6,7 Currently, calcitonin is administered either intramuscularly, which is inconvenient for long-term treatment, or intranasally. The latter is the most usual route, but has the disadvantages of poor absorption of calcitonin and rapid onset of tolerance.8-10 With a view to circumventing these problems, oral dosage forms are being investigated.11-13 In particular, Lee et al.13 have reported that * To whom correspondence should be addressed. Fax: + 34-81594912. E-mail: qfnuria usc.es. X Abstract published in Advance ACS Abstracts, December 1, 1996. (1) Guttman, S. Calcitonin 1980. Proceedings of the International Symposium, Milan, 1980; Percile, E., Ed.; Excerpta Medica International Congress Series; Elsevier: Amsterdam, 1981, Vol. 540, p 11. (2) Copp, D. H. Endocrinology 1962, 70, 638. (3) Milhaud, G. C. R. Acad. Sci. 1965, 261, 813. (4) Chambers, T. J.; Dunn, C. G. Calcif. Tissue Int. 1983, 35, 566. (5) Singre, F. R. In Metabolic bone disease II, 19th ed.; Avioli, L. V., Krane, S. M., Eds.; Academic Press: New York, 1978, p 489. (6) Wallach, S. Curr. Ther. Res. 1977, 22, 556. (7) Gennari, C. Calcitonin 1980. Proceedings of the International Symposium, Milan, 1980; Percile, E., Ed.; Excerpta Medica International Congress Series; Elsevier: Amsterdam, 1981, Vol. 540, p 277. (8) Rojanasathit, S.; Rosenberg, E.; Haddad Jr. Lancet 1974, 2, 1412 (9) Gennari, C.; Passeri, M.; Chiericheti, S. M.; Piolini, M. Lancet 1980, 1, 594 (10) Eisenger, J.; Ouaniche, J. Abstracts for “Calcitonin in Osteoporosis”. International Symposium, Naxos, 1986; Sandoz SpA: Milan, 1986; pp 54 and 55. (11) Morimoto, K.; Akatsuchi, H.; Aikawa, R.; Morishita, M.; Morisaca, K. J. Pharm. Sci. 1984, 73, 1367.
S0743-7463(96)00309-5 CCC: $14.00
Chart 1. Primary Structure of Salmon Calcitonin
controlled release from microspheres of poly[DL-lactic acidco-glycolic acid] (PLA/GA) achieves a more stable, longerlasting hypocalcemic effect. The behavior of microsphere drug systems in the organism depends on the structures of their components and their interactions with cell membranes, which are determined largely by surface phenomena. Understanding of these interactions, and of those between the microsphere polymer and the drug, should aid the development of successful systems. In this paper, we report the behavior of salmon calcitonin monolayers under compression at the air/water interface, which as far as we know has not been studied previously. The behavior of mixed calcitonin/PLA/GA films will be the subject of a subsequent paper. Experimental Methods Salmon calcitonin was supplied by Pierre Fabre´ Laboratories (Barcelona, Spain). The monolayers were spread on TheorellStenhagen buffer subphase (54.88 g of sodium hydroxide, 28 g of citric acid, 14.16 g of boric acid, 13.56 g of phosphoric acid, and distilled water to 2 L). The buffer pH was adjusted by adding 2 M HCl. Its ionic strength ( pH 6; (2) the extent of the plateau region decreased in the order A(pH 2) . A(pH 6) > A(pH 10); and (3) plateau onset surface pressure increased in the order π(pH 2) , π(pH 6) < π(pH 10). Beyond the plateau, the isotherms for pH 2, 6, and 10 coincided.
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Figure 2. π-A isotherms of salmon calcitonin monolayers at temperatures of 10, 20, and 30 °C, on subphases of pH 2 and (inset) pH 10.
The major difference between the above pH effects and those observed at 30 °C (Figure 1, inset) was that increasing the temperature increased the difference between the plateau surface pressures at pH 6 and pH 10. Influence of Temperature. Figure 2 shows the π-A isotherms of calcitonin monolayers compressed on subphases of pH 2 and pH 10 (inset) at 10, 20, and 30 °C. As subphase temperature increased, the area occupied by the expanded monolayer decreased, as did the surface pressure at the onset of the plateau region, both these effects being more acute at acid than at alkaline pH. By contrast, the area at the onset of the plateau increased with temperature at pH 10 but varied very little or not at all with temperature at pH 2. Hysteresis. When films spread on a buffer of pH 7 at 20 °C were subjected to successive cycles of compression/ decompression at a barrier speed of 0.11 cm/s, with compression proceeding past the plateau region in each cycle, hysteresis loops were recorded, with the decompression curves at lower π than the compression curves; furthermore, the preplateau region of the isotherms shifted to smaller areas in successive compression cycles (Figure 3). Use of a slower barrier speed (0.044 cm/s) causes the hysteresis to be more marked as well as a shift between the first and subsequent compression cycles (Figure 3, inset). If compression was halted before the plateau region was reached, hysteresis was less marked and the first and successive compression/expansion cycles, performed toward decreasing area, coincided (Figure 4). Figure 5 shows the results of experiments in which the monolayer was compressed past the plateau (to π > 30 mN/m), decompressed to π ≈ 0 mN/m, and then subjected
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Figure 4. π-A isotherms of salmon calcitonin during successive total or partial compression/expression cycles (see text) on a subphase of pH 7 at 20 °C. Barrier speed: 0.11 cm/s. (1) first cycle; (6) last cycle.
Figure 3. π-A isotherms of salmon calcitonin during successive compression/expression cycles on a subphase of pH 7 at 20 °C. Barrier speed: 0.11 cm/s or (inset) 0.044 cm/s.
to several cycles, each consisting of a 5-min wait, of recompression to a preplateau surface pressure (9 mN/m) and decompression until recovery of the initial area, after which the monolayer was left a further 5 min before again being compressed to π > 30 mN/m and decompressed to its initial area. The figure suggests that the monolayer tended to regain its initial state in the course of the partial cycles but that recovery was not total, since the final full cycle did not coincide with the first one. The inset of Figure 5 shows the results of experiments carried out using a subphase prepared by adding 2.5 mol/L of NaCl to the buffer solution so that the ionic strength became approximately 2.6 M. The influence of the ionic strength of the subphase on monolayers has already been studied thoroughly.14 Therefore we have carried out one experiment only to show that on increasing ionic strength the area of the expanded monolayer increased as well as its stability (as evidenced by the higher surface pressures of the onset of the plateau region, 25-30 mN/m). On the other hand the hysteresis under cyclic compression/ decompression was markedly stronger in the presence of salt in the subphase than in its absence as was the shift toward smaller areas of the second compression isotherm relative to the first. In this situation the recovery of the original structure of the protein is very slow, as after ten partial compression/expression cycles the curve corresponding to the last compression does not coincide with the first one. Finally, Figure 6 shows the results of experiments carried out at pH 2, pH 7, and pH 9 in which, as soon as (14) Aston, M. S.; Herrington, T. M. J. Colloid Interface Sci. 1991, 141, 50.
a given surface pressure was reached during compression of the film, compression was halted and surface pressure at constant area was thereafter monitored as a function of time. When compression was halted before or just after the beginning of the plateau region, relaxation involved only a minimal drop in surface pressure, but halting compression in the postplateau region gave rise to much larger surface pressure drops. Discussion The usage of a relatively high compression velocity as well as a solvent which is miscible with water (methanol) suggests the possibility of losing calcitonin molecules from the monolayer by their solubility in the bulk water phase. However, our results presented in Figures 4 and 6 seem to confirm the film stability, at least before collapse, as there is no hysteresis when the expansion/compression cycles were performed before collapse (Figure 4), proving that there is no solubility of molecules from the monolayer under these conditions. On the other hand, the results presented in Figure 6 manifest that when the monolayer is being compressed to a pressure lower that that of collpase, the surface pressure does not decrease with time during relaxation experiments. In the case of partial or total solubility, variations of pressure with time might be expected. Definitely, our results seem to confirm that the calcitonin monolayers are stable in the expanded state under the conditions investigated. Although the morphology of the π-A curves obtained in this work might suggest that the calcitonin monolayer exists as an expanded phase at low surface pressures and a condensed phase at high surface pressures, the limiting areas estimated from the high-pressure regions of the isotherms, ≈ 8 Å2/residue, are much smaller than those generally found for polypeptides, which, depending on the experimental conditions under which the monolayer is
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Figure 5. π-A isotherms of salmon calcitonin during successive total or partial compression/expression cycles (see text) on a subphase of pH 7 and ionic strength 2.6 M (inset), at 20 °C. Barrier speed: 0.11 cm/s. (- - -) first cycle; several cycles to π ) 9 mN/m; (‚‚‚) last cycle.
spread, range from 14.4 to 23 Å2/residue15-17 (in keeping with electron diffraction data for crystalline polypeptides18 ). This suggests that the plateau of the isotherms in fact reflects collapse of the monolayer. This conclusion is further supported by the fact that the surface pressures at which the plateau began, 16-22 mN/m, are similar to the collapse pressures of monolayers of low molecular weight proteins19 and polypeptides with a β conformation,17 whose isotherms reflect collapse as a slightly upwardsloping plateau similar to those observed in this work. The above considerations suggest that, once preplateau compression has forced the calcitonin molecule to adopt a close-packed form with only solvated polar amino acids submerged in the subphase, collapse involves the progressive reconfiguration of each molecule as a chain of loops suspended from less polar units. These loops form a “transition layer”20 just below the interface. At the end of the plateau region of the π-A isotherms, the only moieties left at the surface are vertically oriented apolar groups. Further compression of these closely packed apolar groups gives rise to the observed sharp increase in surface pressure in the post-plateau region of the π-A isotherms. (15) Crisp, D. J. In Surface Chemistry Supplementary Research; Butterworths: London, 1949; Parts I and II, p 17-35. (16) Cumper, C. W. N.; Alexander, A. E. Trans. Faraday. Soc. 1959, 46, 235. (17) Isemura, T.; Yamashita, T. Bull. Chem. Soc. Jpn. 1959, 32, 1. (18) Birdi, K. S.; Fasman, G. D. J. Polym. Sci. 1972, A110, 2483. (19) Malcolm, B. R. Proc. R. Soc. London 1968, A305, 363. (20) Nitsch, W.; Maksymiw, R. Colloid Polym. Sci. 1990, 268, 452.
Figure 6. Relaxation of salmon calcitonin monolayers compressed on subphases of pH 2, 7, and 9 at 20 °C when compression was halted at the surface pressures indicated in the figures.
Support for the above thesis comes from the results of Figure 5, which show that the compressed monolayer was almost able to recover its initial state upon decompression and so rule out massive irreversible loss of polypeptide molecules to the subphase during collapse. However, the observed hysteresis and the shifting of successive full compression isotherms toward smaller areas (Figure 3) show that recovery is slow. Indeed, there are also two further indications of the relatively long time scale of molecular events in this system. Firstly, the fact that reducing the compression/decompression rate increases the drop in surface pressure upon decompression and the shift of the second isotherm relative to the first (Figure 3, inset) may be attributed to the slower barrier speed enhancing the probability of the transition layer being stabilized by interactions with water in the subphase. Secondly, the fact that the relaxation curves obtained when compression was halted at the end of the collapse region (from 12 to 19 mN/m), shown in Figure 6, in which the surface pressure decreased with time at constant area to an equilibrium value corresponding approximately to the begining of the plateau (where the monolayer is in its expanded state with all polar groups anchored in the subphase) confirm that when the monolayer is compressed
Response of Calcitonin Monolayers to Compression
at a pressure higher than that of collapse, the molecules are not in a stable state and it takes time to reach quilibrium. On the other hand when the monolayer is compressed after the collapse, the pressure decreases to a lower value that this of the beginning of the collapse due to the presence of the elastic forces in the investigated system. The proposed mechanism of collapse is only a reasonable hypothesis, but it is not supported by any experimental data. For better evidence, other physicochemical methods including for example ellipsometry would be necessary. The influence of pH on the behavior of the monolayer is explained by the isoelectric point of calcitonin21 being about 10.4. The monolayer will therefore have been much more positively charged at pH 2 than at the higher pH values, and the correspondingly greater electrostatic repulsions between the charged amino acids will have led to greater unfolding of the molecule and the observed increase in film area. The stronger electrostatic repulsion likewise explains why at acid pH the film began to collapse at a lower surface pressure (and greater area) than at higher pH. The influence of temperature on the monolayer is at first sight somehow surprising, since increasing temperature generally expands a monolayer due to an increase in molecular motion permitted by the increased flexibility of the hydrophobic chains.22 However, behavior like that shown by calcitonin (reduction in film area as temperature (21) Marier, R.; Brugger, M.; Bru¨ckner, H.; Kamber, B.; Riniker, B.; Rittel, W. Acta Endocrinol. 1977, 85, 102.
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rises) has also been reported for polypeptides of benzyl glutamate and aminolauric, aminocapric, and aminocaprilic acids.23 In the present case, this behavior may be attributed to the rise in temperature reducing interaction between hydrophobic groups and thus reducing the forces counterbalancing the tendency for polar groups to be pulled down into the subphase by solvation. It is therefore not surprising that the observed reduction in film area with rising temperature was greatest at acid pH, at which there are more charged groups for solvation. Finally, the influence of the ionic strength of the subphase may also be explained in terms of the solvation of calcitonin polar groups, which at high ionic strength will be reduced due to competition for water between these groups and the ions of the electrolyte (“salting-out”24 ). As a result of the reduction in polar group solvation, electrostatic repulsion between these groups increases, and hence film area also increases. This explanation can be applied to an expanded region only because, as has already been proven by Aston et al.,14 in the condensed state the presence of ions in the subphase does not influence the characteristics of monolayers. LA960309P (22) Gaines, G. L., Jr. In Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishing: New York, 1966. (23) Isemura, T.; Hamaguchi, K. Bull. Chem. Soc. Japan. 1953, 26, 425. (24) Phillips, M. C.; Jones, M. N.; Patrick, C. P.; Jones, N. B.; Rodgers, M. J. Colloid Interface Sci. 1979, 22, 98.
