Interaction between Cyclosporin and Poly (isobutyl cyanoacrylate

Interaction between Cyclosporin and Poly(isobutyl cyanoacrylate) Nanoparticles in Monolayers. J. Minones, E. Yebra-Pimentel, O. Conde, E. Iribarnegara...
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Langmuir 1994,10, 1888-1893

1888

Interaction between Cyclosporin and Poly(isobuty1 cyanoacrylate) Nanoparticles in Monolayers J. Mifiones,. E. Yebra-Pimentel, 0. Conde, E. Iribarnegaray, and M. Casas Departamento de Fkico-QuEmica, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain

J. L. Vila Jato and B. Seijo Departamento de Tecnologfu Farmaceutica, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain Received October 29, 1993. I n Final Form: March 15, 1994" Mixed monolayers of cyclosporin with poly(isobuty1cyanoacrylate)nanoparticles containing Pluronic as the polymerizing agent exhibited marked deviation from ideal behavior. This deviation was increased by increasing the pH and decreased by raising the temperature, which suggests that it was largely due to ion-dipole and dipole-dipoleinteractions,though a contribution by hydrogen bonding between cyclosporin NH groups and poly(isobuty1cyanoacrylate) CO groups cannot be ruled out. The negative values of the molar excess free energy, entropy, and enthalpy of mixing, and the minima observed for monolayers with mole fractions of cyclosporin between 0.2 and 0.4, suggest the formation of a "complexnor well-defined surface structure of maximum stability favored by low enthalpy. The existence of such strong interactions between poly(isobuty1cyanoacrylate) nanoparticles and cyclosporin may have serious implicationsfor the bioavailability of cyclosporin administered in such nanoparticles.

Introduction

Chart 1. Molecular Structure of Cyclosporin. ca;

Cyclosporin A is a cyclic oligopeptide composed of 11 amino acid residues (Chart 1). Its potent immunosuppressive activity' has led to it being the drug most widely used to prevent rejection of transplanted organs, but because of its poor solubility in water and poor bioavailability2 when dissolved in oil, it must be administered at dosage levels that cause chronic kidney p~isoning.~ In view of the above, new cyclosporin formulations ensuring satisfactory bioavailability and sustained release are being sought.k7 One vehicle with great potential for sustained drug release is the nanoparticle or microsphere. Here the aim is for a drug loaded in a polymer matrix or capsule to diffuse slowly through the polymer to the external medium at a rate controlled in part by the slow hydrolytic or enzymatic erosion of the surface of the particle. The matrices of choice for nanoparticle formulations are currently poly(alky1cyanoacrylate)s, which apart from their satisfactory encapsulation propertiess have low toxicity (especially those with slower biodegradation rates due to longer alkyl chains) and satisfactory biocompatibilit~.~ ~~

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* To whom correspondence should be addressed.

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Key: Leu,leucine; Ala, alanine; Val, valine; Sar,sarcosine;

Abu, a-aminobutanoicacid; MeBmt, (2S)-(methylamino)-(3R)hydroxy-(rlR)-methyloct-6-en-l-oic acid.

The intracorporeal distribution and fate of nanoparticles depend to a large extent on physicochemical factors (polymer molecular weight, particle size, surface charge, hydrophobicity, etc.)loJ1 which in turn depend not only on the starting monomer but also on the polymerizing agent used and on the drug loaded in the nanoparticles.12J3 However, very little is known of the influence of the drug and polymerizingagent. Research on interactions between the drug and nanoparticle, and on the influence of polymerizing agents on such interactions, should therefore greatly assist the design of new nanoparticle formulations. In this work we investigated the existence of interactions

Phone: 981594629. FAX: 981-594912. 0 Abstract published in Advance ACS Abstracts, May 1, 1994. (1) Borel, J. F.; Feurer, C.; Gubler, H. V.; Stachelin, H. Agents Actions 1976, 6, 468. (2)Wood, A. J.; Maurer, G.; Niederberger, W.; Beveridge, T. Transplant. Proc. 1983, 15, 2409. (3)Strom, T.B.; Loertacher, R. N.Engl. J. Med. 1984, 311, 728. (4)Singh, M.; Ravin, L. J. J. Parenter. Sci. Technol. 1986, 40,34. (5)Ryman, B.E.;Tynel, D. A. In Essays in Biochemistry; Campbell, P. N., Marshall, R. D., Eds.; Academic Press: London, 1980;Vol. 16,pp (10)Douglas,S. J.;Illum,L.;Davis,S. S.;Kreuter, J.J. ColloidZnterface 49-98. (6)Smeesters, C.; Giroux, L.; Vinet, B.; Arnoux, R.; Chaland, P.; Sci. 1984, 101, 149. Corman, J.; St-Louis, G.; Daloze, P. Can. J. Surg. 1988,31 (l),34. (11)Grislain, L.; Couvreur, P.; Roland, M. S.T.P. Pharma 1985, 1, (7)D'Souza, M. J. Drug Deu. Znd. Pharm. 1988, 14(10),1351-1357. 1038. (8) Couvreur, P.; Kante, B.; Roland, M.; Guiot, P.; Baudin, P.; Speise, (12)Baszkm, A.; Couvreur, P.; Deyme, M.; HenryMichelland, S.; P. J. Pharm. Pharmacol. 1979,31, 331. Albrecht, G. J. Pharm. Pharmacol. 1987,39, 973. (9)Couvreur,P.;Vranckx,H.;Braseuer,F.;Roland,M.S.T.P.Pharma (13)Egea,M.A.;Alsina,M.A.;Garcia,M.L.;Valls,O.;Losa,C.;Alonso, 1989, 5,31. M. J.; Vila Jato, J. L. Znt. J.Pharm. 1991,67, 103. ~

0743-746319412410-l888$04.50/0 0 1994 American Chemical Society

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Cyclosporin-Poly(isobuty1 cyanoacrylate) Interaction

Langmuir, Vol. 10, No. 6,1994 1889

between cyclosporin and poly(isobuty1 cyanoacrylate) (PIBCA) nanoparticles by studying the behavior of monolayers of cyclosporin, PIBCA nanoparticles, and mixtures of the two.

pH=6

-

Experimental Section Cyclosporin was used as supplied by Sandoz, without further purification (HPLC of a sample showed a single peak). PIBCA nanoparticles were prepared by Couvreur et a1.V emulsion polymerization method in the Pharmaceutical Technology Laboratory of the University of Santiago de Compostela (briefly, by OH-initiated anionic polymerizationof a stirred, thermostated emulsion of isobutyl cyanoacrylate in 103N HCl containing 1% Pluronic F68 as stabilizer; Pluronic F68 is a nonionic surfactant of mean molecular weight 8400 formed by a hydrophobic poly(oxypropylene)core surrounded by hydrophilicpoly(oxyethy1ene) chains). The resulting nanoparticle suspension was filtered and brought to pH 7 with 0.1 N NaOH, and the nanoparticles were freeze dried. The mean polymer molecular weight was 2000. Spreading mixtures of cyclosporin and nanoparticles in tetrahydrofuran were deposited on Theorell-Stenhagen buffers made of sodium citrate, borate, and phosphate, the pH of which was adjusted with 2 N HC1. For these substrates, deionizedwater with a resistivity of 18 MQScm was obtained from a Milli-RO, Milli-Q reverse osmosis system (from Millipore). Surface pressures were measured with a KSV 5000 Wilhelmy plate balance with a sensitivity of 4 pN/m over an operational range of 0-100 mN/m. In the KSV apparatus, the trough containingthe substrate solutionis made of Teflon and is mounted on an aluminum base through which water from a thermostat circulates. The mobile barrier compressing the monolayer is of hydrophilic material (polyacetal) so as to prevent the monolayer from slipping under the barrier. The barrier speed can be varied from 0.01 to 400 mm/min; in this work, all film compressions were performed at a barrier speed of 75 mm/min (corresponding to a compression rate of 112.5 cm2/min),equivalent to 33.1-64.9 &.molecule-l.min-l depending on the mixture. Preliminary experiments showed the P A curves to be unaffected by changing the compression rate within this range.

Results Behavior at pH 6. Even before compression, PIBCA nanoparticle monolayers exhibited nonzero surface pressure because of their Pluronic content. However, in a preliminary experiment the P A curve of a 0.022:0.978 mixture of Pluronic and cyclosporin (the highest Pluronicl cyclosporin mole ratio in the cyclosporin/nanoparticle films) was identical to that of pure cyclosporin except for a uniform shift to higher surface pressures, showing that Pluronic does not interact with cyclosporin under our experimental conditions. The presence of Pluronic was therefore ignored thereafter, and its contribution to surface pressure was offset by zeroing the surface balance before compression after the film had been left to equilibrate for 10 min. The isotherm obtained during compression of the pure PIBCA nanoparticle monolayer (curve 1 in Figure 1) reflects a transition from an expanded, highly compressible phase at low surface pressures to a condensed phase at high surface pressures. The isotherm of the pure cyclosporin monolayer (curve 8 in Figure 1) reflects a condensed phase and, at higher surface pressures, a collapse region in which surface pressure increases gradually with decreasing film area (the behavior of the pure cyclosporin film has been described in greater detail elsewhere).l4 The isotherms of mixtures with mole fractions of cyclosporin (x,) of 0.1 or 0.2 resemble that of the pure nanoparticle film, except for being slightly shifted toward smaller molecular areas. The isotherms of the other (14) Mifiones, J.; Yebra-Pimentel, E.; Iribarnegaray, E.; Conde, 0.; Casas, M. Colloids Surf. 1993, 76,227.

50 I

0

100

200

300

400

500

A (X2/MoIec.) Figure 1. Surface pressure plotted against mean molecular area ( P A isotherms) for mixtures of cyclosporin and PIBCA nanoparticles at pH 6 and 20 "C ( x , = mole fraction of cyclosporin): (1)PIBCA, (2) x c = 0.1, (3) x , = 0.2, (4) zc= 0.4, ( 5 ) xc = 0.6,(6) z, = 0.8,(7)n, = 0.94, (8)cyclosporin.

mixtures used (0.4 Ix , I0.94) are globally similar to that of pure cyclosporin, but with molecular areas and collapse pressures that decrease progressively with decreasing xc. Plots of mean molecular area against x , for fixed surface pressure all lie below the line representing ideal (additive) mixing behavior (Figure 2, upper panel), showing surface condensation between the components of the mixture. The plot for a fixed surface pressure of 5 mN/m exhibits marked changes of slope a t x c = 0.2 (point B in Figure 2) and xc = 0.8 (point C). Extrapolation of the segment AB to x c = 1.0 affords a partial molecular area of 77 A2 for cyclosporin in mixtures in this composition range, a much smaller value than the 318 A2/moleculeof pure cyclosporin films a t this surface pressure. In the range 0.2 < x , < 0.8, the partial molecular area of cyclosporin is 290.5 A2 and that of the PIBCA 147.3 A2,both of which are smaller than the molecular areas of the corresponding pure film. In the range 0.8 < x c < 1.0, cyclosporin has the same partial molecular area as in pure cyclosporin films, but condenses the nanoparticle component, the partial molecular area of which falls to 49 A2. The plot for a fixed surface pressure of 20 mN/m shows similar negative deviation from ideal behavior, with maximum condensation at x, = 0.4, though it changes slope more smoothly than the plot for 5 mN/m. According to the Cadenhead and Phillips classificati~n,~~ this is type I11 behavior. The collapse pressure of the mixed nanoparticle/ cyclosporin films decreases with increasing cyclosporin content over the range 0 I xc I 0.4, and increases with x , over the range 0.4 I x , I 1.0 (Figure 2, lower panel). There thus exists a kind of "azeotrope" of minimum collapse pressure with a composition in the range 0.2 Ixc I 0 . 4 . Influence of pH. A t pH 2, the plot of mean molecular area against x c at low surface pressure (5 mN/m) changes (15)Cadenhead, D. A,; Phillips, M.C. Adu. Chem. Ser. 1968,84,131.

Miilones et al.

Langmuir, Vol. 10, No. 6, 1994

'

Table 1. A&. (Difference between Measured and Ideal-MixingValues of Mean Molecular Area) for Mixed Monolayers of Cyclosporin and PIBCA Nanoparticles at 20 OC, for Various Conditions of pH, Composition, and Surface Pressure (xo= Mole Fraction of Cyclosporin) Me.(AZ/molecule) pH 11 PH 2 PH 6 n = 5 n=20 n=5 n=2o n=5 n=20 xc mN/m mN/m mN/m mN/m mN/m mN/m

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pH=6 Figure 2. Behavior of mixed monolayers of cyclosporin and PIBCA nanoparticles at pH 6 and 20 OC: (upper panel) mean molecular area plotted against the mole fraction of cyclosporin (x3 at surfacepressuresof 5 and 20 mN/m;dotted lines correspond to ideal mixing behavior; (lower panel) film collapse pressure plotted against xc. o-oArea

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Influence of Temperature. Varying the temperature between 15 and 30 OC has hardly any effect on the isotherms of pure cyclosporin films at pH 6, but does slightly influence those of pure PIBCA nanoparticle films and those of nanoparticle/cyclosporin mixtures. In general, interaction between the two components, as reflected by AAe, (the difference between the measured mean molecular area and the mean molecular area corresponding to ideal mixing behavior), decreased with increasing temperature (Table 2). Excess Functions of Mixing. The excess free energy of mixing of the mixed films was calculated from the experimental a-A curves of the mixed and pure films, following Goodrich,l6 as AGe, = NS:,(Al2 - x1A1 - xzA,)da

where N is Avogadro's number, AI, Az, and A12 are the molecular areas of the pure components and of the mixture, and x1 and x2 are the mole fractions of the components in the mixture. Following other researchers,l7-lBthe lower limit of integration was taken as zero, and for the upper limit of integration values of 5,10,15, and 20 mN/m were used. Integration was performed by using a planimeter to measure the area under the A-?r curves. Figure 4 shows the results extracted from isotherms obtained at 20 "C and pH 2, 6, or 11. In all cases, AGe, is negative for all values of x,, and more so as surface pressure and pH increase, and its minimum occurs for xc between 0.2 and 0.4. AGe, becomes less negative with increasing temperature (Table 3). Values of ASex were calculated using the GibbsHelmholtz equation for two-dimensional systems:31

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Figure 3. As for the upper panel of Figure 2, but for pH 2 (upper panel) and pH 11 (lower panel). slope sharply at xc = 0.2 (as at pH 6) and xc = 0.6 (as against xc = 0.8 at pH 6); see Figure 3, upper panel. At pH 11 (Figure 3, lower panel), the changes in slope occur at xc = 0.2 and x, = 0.94. Figures 2 and 3 show that the

deviation of the mixed nanoparticle/cyclosporinfilms from ideal mixing behavior increases with pH and with surface pressure (Table 1).

where y is the surface tension of water. Over the range of temperatures used in this study, d r l d T has the practically constant small value of -0.154 mN.m-l-OC-l, but since Me,ranged from -5.7 to -94.7 %,2, the second term on the right-hand side of eq 2 is non-negligible and increases AS,,. Valuesof the first term, (dAG,Jd!O,, which were all positive, were obtained as the calculated slopes of plots of AG,, against temperature at constant surface pressure. The overall results (Figure 5 ) show that AS',, becomes increasingly negative with increasing surface pressure, and for all temperatures is a minimum at xc = 0.4. (16) Goodrich,F. C. Roceedings of the 2nd Znternationul Congress on Surface Activity; Butterworths: London; 1957; Vol. I, p 85. (17) Bacon, K. J.; Barnes, G. T. J. Colloid Interface Sci. 1978,67,70. (18) Guay, D.; Leblanc, R. M. Langmuir 1987,3, 575. (19) Grainger, D.W.; Sunamoto, J.; Akiyoshi, K.; Goto, M.; Knutaon, K. Langmuir 1992,8,2479.

Langmuir, Val. 10,NO.6, 1994 1891

Cyclosporin-Poly(isobuty1 cyanoacrylate) Interaction

Table 2. A&, (Difference between Measured and Ideal-Mixing Values of Mean Molecular Area) for Mixed Monolayers of Cyclosporin and PIBCA Nanoparticles at pH 6, for Various Conditions of Temperature, Composition, and Surface Pressure (IC = Mole Fraction of CyclosDorin)

60.0 51.1 42.2 26.7

46.7 46.7 33.3 44.4

46.7 46.6 40.0 26.7

77.8 80.0 80.0 60.0

44.4 40.0 44.4 40.0

73.3 73.3 73.3 60.0

26.7 33.1 22.2 4.5

26.7 28.9 28.8 20.0

Table 3. Excess Free Energy of Mixing (AG,, J/mol) of Mixed Monolayers of Cyclosporin and PIBCA Nanoparticles at pH 6 and a Surface Pressure of 10 mN/m, for Various Conditions of Temperature and Composition (G = Mole Fraction of Cyclosporin) AC, (J/mol) (II = 10 mN/m) xc 15 "C 20 "C 25 "C 30 "C

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negative with decreasing temperature (especially in the range 15-20 "C).

Discussion

-1t

The results obtained showed that the deviation of cyclosporin/nanoparticle mixtures from ideal surface behavior (a) increases with pH, (b) increases with surface pressure, (c) decreases with increasing temperature, and (d) varies with the composition of the mixture. It seems unlikely that cyclosporin-nanoparticle interaction can to any significant extent be due to van der Waals forces between the hydrophobic chains of cyclosporin and PIBCA, since these chains are very short. A significant hydrophobic contribution seems, moreover, to be ruled out by the slight increase in interaction with increasing pH: if the interaction is predominantly hydrophobic, it should decrease with increasing pH, which removes isobutyl chains from the interface due to hydrolysis of PIBCA.12

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Figure 4. Excess free energy of mixing of mixed monolayers of cyclosporin and PIBCA nanoparticles at 20 "Cand various surface pressures plotted against the mole fraction of cyclosporin (x,): (top) pH 2, (middle) pH 6,(bottom) pH 11. Excess molar enthalpies of mixing were calculated as usual: AHe, = AG,,

+ TAS,,

(3)

The minimum of A H e , lies at x c = 0.4 for high surface pressures of 15 or 20 mN/m, but elsewhere for lower pressures (Figure 6). The excess enthalpy and entropy of mixing are hardly affected by temperature at surface pressures of 15 or 20 mN/m, but do change considerably at lower surface pressures (Figures 5 and 61,a t which they become more

Since cyclosporin is nonionogenous, ionic interaction can also be ruled out. It therefore appears that the interaction between cyclosporin and PIBCA nanoparticles is largely due to ion-dipole and dipole-dipole attractions, and/or to hydrogen bonding between cyclosporin NH groups and PIBCA carbonyl groups. The significant contribution of ion-dipole attraction would explain the increase in interaction with pH, since raising the pH increases the degree of ionization of PIBCA, while the contribution of dipole-dipole attractions is in keeping with the structures of cyclosporin and PIBCA, which feature enough dipoles to allow significant interaction, and with the decrease in interaction with increasing temperature (since increasing temperature disorients dipoles). The decrease in interaction with increasing temperature is also interpretable in terms of cyclosporin-PIBCA hydrogen bonding: although a large contribution from such bonds seems unlikely (the four NH groups of the cyclosporinmoleculenormally being

Mifiones et al.

1892 Langmuir, Vol. 10, No. 6,1994

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Figure 5. Excess molar entropy of mixing of mixed monolayers of cyclosporin and PIBCA nanoparticlesat various temperatures and pressures plotted against the mole fraction of cyclosporin (ZJ. involved in intramolecular hydrogen bonds), Loosli et aLm reported that these intramolecular bonds are stronger a t higher than at lower temperatures, which makes hydrogen bonding to PIBCA easier at lower temperatures than higher. The breaking of the intramolecular hydrogen bonds of cyclosporin, and the formation of cyclosporinPIBCA hydrogen bonds, would presumably also be favored by forcing the PIBCA carbonyl groups into close proximity with the cyclosporin NH groups; this would explain why cyclosporin-PIBCA interaction increases with increasing surface pressure, but then so also does the existence of ion-dipole and dipole-dipole interactions, both of which increase significantly as the distance between the interacting units decreases. Contact and interaction between PIBCA and cyclosporin molecules depend not only on surface pressure but also on the relative proportions of the two components in the mixture. Maximum contact and interaction appear to be achieved with mole fractions of cyclosporin in the range 0.2-0.4 (depending on pH, temperature, and surface pressure), suggesting the formation of a “specific surface arrangement” or “well-defined complex” such as various other r e ~ e a r c h e r s ~have l - ~ ~ postulated to explain near discontinuities in the slopes of area-composition curves. Mixtures with the critical composition behave as “azeotropes” of minimum collapse pressure, a finding analogous to those recently reported by Heath and Arnett.z4 Mo(20) Loosi, H. R.; Keeeler, H.; Oschkinat, H.; Weber, H. p.; Petcher, T. J.; Widmer, A. Helu. Chim. Acta 1986,68, 682. (21) Dervichian, D. G. In Surface Phenomena in Chemistry and Biology; Danielli, J. F., Pankhurst, K. G. A., Riddiford, A. C., Eds.; Pergamon: New, 1958; p 70. (22) Shah.D. 0. J. Colloid Interface Sci. 1971.37. 744. (23) Albrecht, 0.; Gruler, H.; Sackmann, E. J.Colioid Interface Sci. 1981, 79, 319.

tomura et al. have described mixed films with negativez6 or positive26 azeotropes as those in which interaction between the components of the films is greater or less than intermolecular interactions in the corresponding pure films. The existence of a well-defined surface arrangement in cyclosporin/PIBCA films of suitable composition is supported by the values of the thermodynamic functions calculated from our experimental data. AGex is always negative (which confirms that the mixed films are more stable than those of the pure components) and has a minimum at a composition which is practically constant (varying only slightly-in the range 0.2 I x c I 0.4 mentioned above-under the influence of pH, temperature, and surface pressure). Similar results have been reported for a variety of mixtures, and though the AGex minima in question have generally been located near equimolar c o m p o ~ i t i o n ,other ~ ~ ? compositions ~~ have also been r e p ~ r t e d . ~In~ general, ?~ our AGex results are in keeping with published findings for simple molecules17~z7JO~31 if the values shown in Figure 4, which represent AGex per mole of “polymer” (polymer standing here for the mixture of cyclosporin and PIBCA nanoparticles), are divided by a factor of 11-13 to yield an estimate (24) Heath, J. G.;Amett, E. M. J. Am. Chem. SOC.1992,114,4600. (25) Hayami, Y.; Kawano, M.; Motomura, K. Colloid Polym. Sci. 1991, 269, 167. (26) Motomura, K.; Yano, T.; Ikematsu, M.; Matuo, H.; Matuura, R. J. Colloid Interface Sci. 1979, 69, 209. (27) Tancrede, P.; Munger, G.;Leblanc, R. Biochim. Biophys. Acta 1982, 689, 45. (28) Gabrielli, G.;Puggelli, M.; Faccioli, R. J. Colloid Interface Sci. 1972, 41, 63. (29) Puggelli, M.; Gabrielli, G.Colloid Polym. Sci. 1983,261,166. (30) Fukuda,K.;Kato,T.; Machida, S.;Shimizu,Y.J.Colloidlnterface Sci. 1979, 68, 82. (31) Mifiones, J.; Cid, L.; Conde, 0. An. Quim. 1989, 85, 60.

Langmuir, Vol. 10, No. 6, 1994 1893

Cyclosporin-Poly(isobuty1 cyanoacrylate) Interaction

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Figure 6. Excess molar enthalpy of mixing of mixed monolayers of cyclosporin and PIBCA nanoparticles at various temperatures and surface pressures plotted against the mole fraction of cyclosporin (~3.

of AGex per mole of monomer. For mixtures a t a surface pressure of 20 mN/m (at which interaction is greatest), the resulting estimates of AGESper mole of monomer for the composition of minimum AGex range from -400 to -700 J/mol (depending on pH and temperature); these values are alittle more negative than most published values for films at the same surface pressure, which attests to the great stability of the cyclosporin/nanoparticle complex formed under these conditions. The existence of a well-defined complex in cyclosporin/ PIBCA films of suitable composition is also supported by the values of ASexand AHe,, which are always negative and have a minimum at xc = 0.4 a t high surface pressure. That both these parameters are negative shows that the thermodynamic stability of the mixed films derives from their low enthalpy rather than from the entropy term, and like AGES,AHex is always minimum in the range 0.2 Ix c I0.4. The negative values of ASexdo not, of course, reflect an increase in order upon mixing, but rather that, due to the formation of well-defined surface structures mediated by intermolecular attraction, the mixed films are more ordered than ideal mixtures would be. Note

that at high surface pressures, i.e., when the close contact of the molecules in the film must make for maximum intermolecular interaction, raising the temperature from 15 to 30 OC hardly affects ASexand AHex (Figures 5 and 61, suggesting that at these surface pressures this rise in temperature is insufficientto disrupt surface packing. This behavior contrasts with the appreciable temperature dependence of ASex and AHex for films in an expanded state, which appear to be more susceptible to disruption of any well-defined structure. The existence of such strong interactions between cyclosporin and PIBCA nanoparticles may have serious implications for the bioavailability of cyclosporin administered in such particles. Further light might be thrown on this question by probing the nature of the cyclosporinPIBCA interactions using techniques such as NMR, FTIR, or ESR spectroscopy.

Acknowledgment. This work was financially supported by the Consellerfa de Educaci6n e Ordenaci6n Universitaria (Xunta de Galicia) under Project No. 20310b91.