Langmuir 1996,11,1636- 1644
1636
Dilatational Properties of Adsorbed Poly(D,L-lactide)and Bovine Serum Albumin Monolayers at the DichloromethaneMaterInterface F. Boury, Tz.Ivanova; I. Panaiotov,t and J. E. Proust* Pharmacie Gal6nique et Biophysique Pharmaceutique, Facult6 de Pharmacie, 16 Boulevard Daviers, 49100 Angers, France
A. Bois and J. Richou Laboratoire d'OptoElectronique, Universit6 de Toulon et de Var, 83130, La Garde, France Received August 26, 1994. In Final Form: January 19, 1995@ The use of the dynamic pendant drop method has permitted the accumulation of information in real time on the kinetic and rheological processes that accompany the adsorption of bovine serum albumin (BSA)at the dichloromethane (DCM)/waterinterface in the presence and in the absence of poly(D,L-lactide) (PLA50). These results have been compared with those obtained at the aidwater interface. The study of the adsorption kinetics of BSA at the DCWwater and aidwater interfaces has shown that the adsorption process was diffusion controlled in both cases for relatively low concentrations of protein (0.005 g/L). In the presence of PLA50, the adsorption of BSA at the DCWwater interface led to a mixed interfacial layer. The mechanism of the formation of this mixed interface involves the penetration of BSA into the saturated layer of PLA50 with the displacement and condensation of the PLA50 segments. In all cases, rheological results indicated a pure elastic behavior for the mixed BSA/PLA50 interfacial layers. The absence of relaxation upon the application of dilatational stress was attributed to the instantaneous displacement of the PLA50 segments and the nonexpulsion of the BSA segments. These results are interpreted with respect to the preparation of biodegradable PLA50 microparticles related to an emulsion-based process.
Introduction The preparation of biodegradable colloidal carriers has been widely studied for the delivery of many different types of drugs.lY2 From these studies, poly(a-hydroxy acid)s appear to be the most useful biocompatible polym e r ~ .The ~ choice of the preparation mode of microparticles is generally influenced by the hydrophilidhydrophobic character of the active agent. Thus, the encapsulation of hydrophobic drugs involves the fabrication of an oil-in-water (o/w) emulsion: where both the polymer PLA50 and the drug, A, are dissolved in the organic phase (DCM) (Figure 1.Ia). This organic phase is emulsified in an aqueous phase containing a surface active agent (e.g. poly(viny1 alcohol) (PVA)5or BSA6). The emulsion step is followed by an evaporation step: DCM, which exhibits some miscibility in the nonsolvent (aqueous media), can diffuse in the nonsolvent media and can be evaporated at the aidwater interface. This step is generally performed under vacuum with stirring. Solid microparticles containing the encapsulated drug can then be obtained after washing and filtration (Figure 1.Ib). In the case of hydrophilic active agents (proteins or peptides for example), it is convenient' to formulate a primary water-in-oil (w/o) emulsion which allows the t Permanent address: Biophysical Chemistry Laboratory, University of Sofia, J. Bourchier lstr., 1126 Sofia, Bulgaria. Abstract published in Advance ACS Abstracts, April 15,1995. (1)Cohen, S.; Yoshioka, T.; Lucarelli, M.; Hwang, L. H.; Langer, R. @
Pharm. Res. 1991,8,713. (2)Bodmer, D.; Kissel, T.; Traechslin,E. J.Controlled Release 1992, 21, 129. (3)Vert, M.; Christel, P.; Chabot, F.; Leray, J. Macromolecular Biomaterials; Hasting, G. W., Ducheyne, P., Eds.; CRC Press: Boca Raton, FL, 1984. (4) Spenlehauer,G.; Vert, M.; Benoit,J. P.; Boddaert,A. Biomaterials 1989,10, 557. (5)Vernier Julienne,M. C.; Alonso, M. J.;GomezAmoza, G. L.; Benoit, J. P. Drug Dev. Ind. Pharm. 1992,18,1063. (6) Verrechia, T.; Huve, P.; Bazile, D.; Veillard,M.;Spenlehauer,G.; Couvreur, P. J. Biomed. Mater. Res. 1993,27,1019.
1
I1
Figure 1. Schematic view of the preparation of biodegradable microspheres. I. Single o/w emulsion process: (a) droplet of DCM containing dissolved PIA50 and hydrophobic drug (A) dispersed in the aqueous phase containing BSA, (b) solvent evaporation procedure leading to the preparation of solid PIA50 microspheres. 11. Double w/o/w emulsion process: (a)aqueous dropletscontaining the dissolved BSA dispersed in a DCM phase containing the dissolved PLA50 (Primary emulsion); (b) emulsification of the primary emulsion in an aqueous phase containing the dissolved BSA, (c)solvent evaporationprocedure leading to the preparation of solid PLA50 microspheres. dispersion of the hydrophilic drug (BSA is used here as a model) in the organic phase containing the biodegradable polymer (Figure 1.IIa). The second step involves the formation of a (water-in-oil)-in-water (w/o/w) emulsion by emulsifylng the primary emulsion in an aqueous phase containing a surface-active agent (Figure 1.IIb). Finally, (7) Ogawa, Y.; Yamamoto, M.; Takada, S.; Okada, H.; Shimamoto, T. Chem. Pharm. Bull. 1988,36,1502.
0743-7463/95/2411-1636$09.00/0 0 1995 American Chemical Society
Dynamic Properties of Polymer Monolayers as in the former case, the organic solvent is removed by evaporation leading to the formation of solid microspheres (Figure 1.11~). In a series of papers819we studied the behavior of PLA50 and BSAin the emulsion process. In the single-emulsionbased process, BSA was used a s the tensioactive agent (Figure 1.Ia). In the double-emulsion-based process, BSA was used both as a model of hydrophilic drug and as the tensioactive agent of the primary and final emulsion (Figure 1.IIb). In the latter case, the process led to the localization of the protein in the polymer matrix as well as a t the surface of the microparticles. In these two preparation modes, it is important to know the arrangements of the two components (polymer (PLA50) and tensioactive agent (BSA))composing the interfacial layer a t the DCWwater interface. With this goal in mind, we developed an initial approach whereby PLABO and BSA were successively spread a t the airlwater interface using classical techniquess Thereafter, we studied the dynamic properties of the resulting monolayers. Results showed that, below 15 mNlm, the continuous phase of the surface film was composed of PLA50. During the first-order phase transition of the PLASO (see ref 10 and 111, a phase inversion led to the formation of a continuous phase composed of BSA. Both rheological and AFM imaging showed the segregation of the two constituents in the surface film. However, due to the mode of spreading and the resulting segregation, this first attempt to obtain a model of the interfacial layers did not dkcurately reflect the arrangement of the constituents a t the DCWwater interface during the formulation of microparticles. A second approach was developed by spreading the primary wlo emulsion containing BSA and PLA50 a t the airlwater i n t e r f a ~ e .By ~ use of this spreading technique, better mixing of the two components was obtained in the monolayer. It was also noted that the presence of dispersed BSA aggregates could influence the properties of the final product. This approach could perhaps furnish a model of the polymer matrix (in a plane geometry). In order to obtain additional information on the organization of the interfacial layers under the experimental conditions employed in the preparation of the microparticles, it was necessary to study the properties of BSA and PLA50 a t the DCWwater interface. Several approaches have been developed to study oil/ water interfaces.12-14 Due to technical difficulties, however, these approaches do not permit convenient interpretations of the real-time adsorption kinetics and rheological properties of the interfacial film. To analyze the dynamic response of the interface to a dilatational mechanical stress, we used a method based on the pendant drop technique15 which gave several interesting results concerning the arrangements of poly(viny1 alcohol) and PLA50 a t the DCWwater interface.16 The recording of relaxation processes and compression responses in real time allowed us to use a rheological approach based on a Maxwell-type model. (8) Boury, F.; Ivanova, Tz.; Pana'iotov, I.; Proust, J. E., Langmuir 1995,11, 599. (9) Boury, F.; Ivanova, Tz.; Panai'otov,I.; Proust, J. E. submitted for publication in Langmuir. (10)Boury, F.; Gulik,A,;Dedieu, J. C.; Proust, J. E. Langmuir 1994, 10, 1654. (11)Boury, F.;Olivier, E.; Proust, J. E.; Benoit, J. P. J. Colloid Intprfnrp ,-- - Sri - ... -1993 - - -, -160 -. 1 (12)Merigow, R.; T&y; M. Rev. Inst. Fr. Pet. 1961,16, 150. (13)Tornberg, E.; Lundh, G. J. Colloid Interface Sci. 1981,79,76. (14)Lankveld,J. M. G.;Lyklema, J. J. Colloid Interface Sci. 1972,
41, 466. (15) Grimaldi,M.; Bois, A.; Nury, S.; Riviere, C.; Verger, R.; Richou, J. OPT0 91, Pans, (1991). (16) Boury, F.; Ivanova, Tz.; Panafotov, I.; Proust, J. E.; Bois, A,; Richou, J. J. Colloid Interface Sci. 1995,169, 380.
Langmuir, Vol. 11, No. 5, 1995 1637
Figure 2. (a) Profile of a pendant drop with the parameters used for the determination of the interfacial tension. (b)Scheme of the interfacial tensiometer used in this study.
In summary, the purpose of this work was to examine the dynamic properties of BSA and PLABO at the DCW water interface. Adsorption kinetics of BSA a t the DCW water interface in the presence and absence of PLA50 (dissolved in the DCM phase) were studied and compared to results obtained a t the airlwater interface. In order to obtain information on the organization of the two components a t the interface, the interfacial rheologies of the systems were studied.
Materials and Methods Materials. PLA50, a poly(D,L-lactic acid) stereocopolymer,
was obtained from CRBPA' (URA CNRS 1465 Montpellier, France). According to the Vert clas~ification,~ it contains 50% of repeating L-units. Its mean molecular weight (Mw,.,), determined by size exclusion chromatography was 41600. The polymolecularity index was kept in the range 1.6-1.9. Bovine serum albumin was obtained from Sigma (Paris, France); its molecular weight was 69 000. Dichloromethane (DCM)and 2-propanol were supplied by Prolabo (Paris, France) and used without further purification. Ultrapure water was obtained from a Millipore purification system (Milli-Q plus, Millipore, France). Measurements at the DCM/Water Interface. The interfacial tensiometer is based on the integrated form ofthe Laplace equation for the pendant drop interface, in which the volume is a function of two coordinates and the slope of the drop pr0fi1e.l~ For each point, Mi, of the drop profile (see Figure 2a), the coordinates xi, zi, the angular slope Oi, and the volume ui are related by the following equation15
where g is the gravity, A@ is the difference in densities of the liquid forming the drop and of the surrounding liquid, y is the
Boury et al.
1638 Langmuir, Vol. 11, No. 5, 1995 interfacial tension, and b is the radius of curvature at the apex of the drop (point 0 in Figure 2). The interfacial tension is calculated by a least-squares method from this equation. A schematic representation of the tensiometer is given in Figure 2b. The components of the system are as follows: an optical bench, A; parallel red light source, B; aplanetic objectives cf= 85 mm), C; a black and white CCD camera (RTC56470) with 512 x 512 pixels sensor, D; an Exmire 2.5-mL gas chromatography syringe, E; the pendant drop hanging on a stainless steel needle (internal diameter 2.04 mm) inside a HELLMA fluorometer cell (10 x 10 x 45 mm, ref 103) placed in a water circulating thermostated jacket, F; two-step motor for fast and slow motions of the syringe plunger (the slow motion is 0.84pm per step ofthe motor), G; an M:486DX33 PC computer, H; a digitizing board (Imagine TechnologyPC Vision +)connected to the CCD camera, I; a TV monitor, J; the computer display, K a 68 HC 11 micro controllerconnected to the computer by the RS 232 output driving the two-step motor, L; for ascending drop a J form needle and a (10 x 20 x 45 mm, ref 103) HELLMA fluorometer cell, N. The choice between pendant or ascending drops depends on the respective densities of the liquid drop and the surrounding liquid and on the needle,which must be wetted by the surrounding liquid. This tensiometric arrangement can allow up to five measurements of the interfacial tension per second with a precision of &0.5%,whereas interfacial tension variations may be determined with a precision greater than 0.1%. For each measurement, the interfacial tension, the area, and the volume of the drop are recorded. Unlike other methods, based on the Runge-Kutta integration of the Laplace equation,18J9 this method gives a real-time measurement ofthe interfacial tension and allows one to control and modify the drop volume during the experiment in order to (i) maintain a constant interfacial area during changes of interfacial tensions (whenthis tension decreases,the area slightly increases as the drop is deformed; this feature can be used to avoid drop detachment from the needle during the adsorption kinetics),(ii)slowly compress the adsorbed layer at the interface, and (iii) apply fast compressions to the adsorbed layer in a few tenths of a second and then follow the evolution ofthe interfacial tension with time. Measurements at the Airwater Interface. The surface pressure, as a function of time, was measured during the small compressions or expansions of monolayers and during the relaxation process which followsthese perturbations. A Sartorius balance, fitted with a Wilhelmy plate, connected to a computer was used to measure surface pressure variations (with an accuracy of 0.02 mN/m) against time. The faster rate of acquisition was 5 valuesh. Small compressions or expansions of the monolayers were conducted, for a given surface pressure, with different velocities using the mobile barrier of the film balance. The distance between the mobile and floating barrier was 7 cm in all cases. Adsorption Kinetics. In the generally accepted model, the adsorption rate is determined by two consecutive processes: diffusion of surface active molecules from the bulk phase to the subsurface and an exchange between the subsurface and the interface, where there is an energy barrier that controls adsorption-desorption. In the limiting case, correpsonding to a vanishing energy barrier and a steady adsorption equilibrium, the diffusion controlled kinetics can be described by the wellknown Ward and Tordai equationZo
When desorption is negligible, t
- 0, eq 2 reduces to (3)
Equation 2 requires knowledge of T(t),while changes in surface tension with time dt) or with concentration dc) are often experimentally accessible. Values of U t )are usually obtained from experimental values of dt)by comparing a(t)and d c ) data and using the Gibbs' adsorption isotherm equation. Thereafter, the values of the diffusion coefficient ijl can be calculated from (2). When the calculated value of G8 corresponds to the true value of the independently measured diffusion coefficient g o , the adsorption kinetics are truly diffusion controlled. When G4 is much less smaller than iflo, an adsorption energy barrier controls the exchange between the subsurface and the interface. In the case of the kinetics of adsorption of protein molecules, the process could be considered as an adsorption of a certain number ofresidues at the interface. Assumingthat adsorption of a protein molecule is irreversible, eq 2 must be used in the followingform
(4) where n(t)is the number of residues attached at the interface per unit area at time t and COis the residue concentration in the bulk. In order to use eq 4, we require a method to obtain the evolution of the number of residues attached at the interface n(t)from the experimental data dt). The followingapproach has alreadybeen used in the study of adsorption kinetics of polymers16 and proteins.21 The relationship between d t )and n(t)is determined experimentally by measuring the surface tension u and the surface elasticity
simultaneously during the adsorption process. It should be noted that dynamic measurements of surface elasticity can be made by a small number of rapid compressions followed by expansions during the adsorption process. Since JC = a0 - u(t)and n(t)are time dependent, it can be shown that E=----d n - & dt (5) d l n n dt d l n n After integration of eq 5 from t > 0 to t, and n(t)to ne, we obtain where t , is the time required to attain equilibrium and ne the respective equilibrium value. Rheological Measurements. The dynamic response of the protein monolayer to a dilatational (or compressional)mechanical stress was studied using a theoretical approach based on twodimensional rheo10gy.16~22In order to describe the surface t )nL(Figure 3), during the time T of pressure change, A n = ~ ( the compression c with a constant velocity ub followed by a relaxation r , we suppose that at any moment, the total surface pressure change, An = n(t)- n,, can be expressed as the sum of one equilibrium An, and one nonequilibrium Annecontribution. An = Ax, An,,, (7) The equilibrium part Axe is related to the equilibrium surface dilatational elasticity E,. Thus
+
Ane = E, Ai ubt
where Ai is the initial surface area before the compression and - hA Ai - Ai
Ubt
where r is the number of adsorbed molecules per unit area at timet, iDis the diffusioncoefficient,COis the bulk concentration, is the subsurface concentration. and (17)Hartland, S.;Hartley, R. W., hisymmetric Fluid-Liquid Interfaces; Elsevier: Amsterdam, 1976. (18)Girault, H. H.; Schiffrin, D. J.; Smith, B. D. V. J . Electroanal. Chem. 1982,137,207. (19)Rotenberg, Y.;Boluvka, L.; Newmann, W. J . Colloid Interface Sci. 1983,93,169. (20) Ward, A. F. H.; Tordai, L. J. Chem. Phys. 1946,14, 453.
the corresponding strain E (see Figure 3). This elastic behavior is represented by the upper branch of the mechanical model in Figure 3. (21) Ivanova, M.; Verger, R.; Bois, A.; Panai'atov, I. Colloids Surf. 1991,54,279. (22)Edwards, D.;Brenner, H.; Wasan, D. T. In Interfacial transport Drocesses and rheolom: Butterworth-Heinemann in Chemical En'gineering: Oxford, 1%1.
Langmuir, Vol. 11,No. 5, 1995 1639
Dynamic Properties of Polymer Monolayers
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