Langmuir 1995,11, 599-606
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Dilatational Properties of Poly(D,L-lacticacid) and Bovine Serum Albumin Monolayers Spread at the Airmater Interface F. Boury, Tz.Ivanova,: I. Pana'iotov,t and J. E. Proust" Pharmacie Galknique et Biophysique Pharmaceutique, Facultk de Pharmacie, 16 Boulevard Daviers, 49100 Angers, France Received June 11, 1994. I n Final Form: August 23, 1994@ Dynamic behavior of monolayers of a biodegradable polymer (poly(D,L-lacticacid),PLA50)and a protein (bovine serum albumin, BSA) spread at the aidwater interface are studied to clarify the organization of the films with regard to the composition and the state of compression. A rheological approach based on the Maxwell's model was found to be useful to discriminatethe influenceofeach constituentin the monolayer. Before the transition phase of PLABO,rheological properties of the mixed monolayer are governed by the PLA50 which constitutes probably the continuous phase of the system. During the transition phase of PLA50,condensation ofthe PIA50 segments allow the formation of a continuous phase mainly constituted from BSA. After the transition phase of PLABO,rheological properties ofthe mixed monolayer were shown to be identical to the properties of a pure BSA monolayer. This indicates that BSA is present between PIA50 aggregates and prevents the connection of these domains at least until the higher studied pressure (Le. 21 mN/m). The obtained results suggest that BSA could be an efficient stabilizer of the interfacial region during preparation of PLA50 particles by an emulsion technique.
Introduction Interactions of proteins with polymer substrates have been widely investigated to understand the events which take place when an artificial surface is brought in contact with biological It is in fact well-known that the adsorption of proteins is the initial stage governing the in vivo polymeric polydispersed system (micro or submicrometer ~ i z e ) .Another ~,~ point which motivates the study of polymer-protein interactions is the development of a drug carrier which could liberate a protein (vaccine for example) at a controlled time after administration of the carrier. In this way, a biodegradable polymer like poly(D,L-lacticacid) (PLA50)appears convenient for the design of a colloidal carrier in which the protein has been e n ~ a p s u l a t e d Formulation .~~~ of such particles is generally related to an oil in water emulsion process combined with a solvent evaporation m e t h ~ d The . ~ use of a surface active agent is necessary to stabilize the emulsion. For this purpose, poly(viny1alcohol)(PVA)is the most used surface active agent but presents some adverse effects which limit intravenous administration.1° An alternative which has been proposed recently to improve biocompatibility of the systems is the preparation of a drug carrier (nanoparticles) + Permanent Address: Biophysical Chemistry Laboratory, University of Sofia, J. Bourchier 1 str., 1126 Sofia, Bulgaria
Abstract published inAdvance ACSAbstracts, January 1,1995. (1)Amiji, A.; Park, K. Biomaterials 1992,13,682. (2) Brynda, E.; Cepalova, N. A.; Stol, M. J . Biomed. Mater. Res. 1984, 18,685. (3) Lee, S. H.; Ruckenstein, E. J . Colloid Interface Sci. 1988,125, 365. (4)Baszkin, A.; Deyme, M.; Perez, E.; Proust, J. E. Proteins at interface;Brash, J. L., Horbett, T. A,, Eds.; ACS symposium series 343; American Chemical Society: Washington, DC, 1987; p 451. ( 5 ) Makino, K.; Ohshima, H.; Kondo, T. J . Colloidlnterface Sci. 1987, 115, 1987. (6)Brash, J. Proteins at interface; Brash, J. L., Horbett, T. A,, Eds.; ACS symposium series 343; American Chemical Society: Washington, DC, 1987; p 490. (7) Cohen, S.; Yoshioka, T.; Lucarelli, M.; Hwang, L. H.; Langer, R. Phurm. Res. 1991,8,713. (8)Bodmer, D.; Kissel, T.; Traechslin, E. J . Controlled Release 1992, 21, 129. (9) Vernier Julienne, M. C.; Alonso, M. J.;Gomez Amoza, G. L.; Benoit, J. P. Drug Dew. Ind. Pharm. 1992,18,1063. (10) Burgener, F. A.; Gutierrez, 0.H., Logsdon, G. A. Radiology 1982, 143,379. @
0743-7463/95/2411-0599$09.00/0
by using bovine serum albumin (BSA) as a stabilizing agent.ll This could probably have some consequences in the in vivo behavior of the particle; on the other hand, in the schema of vaccination, the presence of a protein at the surface of the particles could be turned to account for liberation of the protein after a short delay.12 The use of a rheological approach based on the Maxwell model applied to a viscoelastic bidimensionnal body was found to correlate with the dynamic behavior of PVA and PLABOPVA monolayers spread a t the airlwater interface or adsorbed at a liquiuiquid interface.13 Results obtained by this approach have permitted a conclusion of strong interactions between the two polymers and have pointed out the probable irreversible anchoring of the PVA at the surface of the microparticles; these results justify the use of an alternative way of preparation of such particles. In order to clarify the interactions of PLABO with BSA which could be involved during preparation of the particles, we adopted a similar approach. The dynamic processes occurring during both compression and relaxation of BSA and BSAPLA50 monolayers spread a t the airlwater interface are analyzed with regard to the developed model.
Materials and Methods Polymers. PLA50, a poly(D,L-lacticacid) stereocopolymer, was obtained from CRBPA (URA CNRS 1465 Montpellier, France). According to the Vert clas~ificatiod~ it contains 50% L-repeating units. Its mean molecular weight (M,) determined by size exclusion chromatography is 41 600. Polymolecularity index was kept in the range 1.6-1.9. The bovine serum albumin was obtained from Sigma (Paris, France); its molecular weight was 69 000. Solvents. Dichloromethane (DCM) was supplied by Prolabo (Paris, France) and used without further purification. Water was ultrapure and obtained from a Millipore system (Milli-Q Plus, Millipore, France). (11)Verrechia, T.; Huve, P.; Bazile, D.; Veillard, M.; Spenlehauer, G.; Couvreur, P. J . Biomed. Mater. Res. 1993,27,1019. (12) Aguado, M. T., Lambert, Immunobiology 1992,184,113. (13) Boury, F.; Ivanova, Tz.; Panafotov, I.; Proust, J. E.; Bois, A.; Richou, J. J . Colloid Interface Sci., in press. (14)Vert, M.; Christel, P.; Chabot, F.; Leray, J. Macromolecular Biomaterials; Hasting, G. W., Ducheyne, P., Eds.; CRC Press: Boca Raton, FL, 1984; p 119.
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Spread Polymer Monolayers. BSA and PLA50 were spread by means of Exmire microsyringe on an aqueous subphase on the maximum available area (927cm2)of a Langmuir film balance (LAUDA FW2, Germany) after the water interface was cleaned by suction. Insoluble BSA monolayerswere formed by spreading the protein aqueous solution (0.5 mg/mL) containing 0.1% of propylic alcohol (Merck, Germany). The values of the surface pressure after spreading were less than 0.1 mN/m. Monolayers were left for about 15 min before measurement. Monolayers of pure PLA50 were formed by spreading a solution of PLA50 in dichloromethane (1mg/mL) (Prolabo, France) and were left for about 15 min. Mixed monolayers of BSA and PLA50 can be formed by using various modes of spreading. The simplest one is the successive spreading of the two constituent solutions. First, a dilute BSA monolayer was formed following the above described procedure. Then, PLA50 was spread by depositing droplets of the organic solution in many places on the surface. As a result of a spreading of the organic solution along the surface, a mixed BSALPLABO monolayer is expected to be formed. Because of the presence of a preformed dilute BSA film, a certain inhomogeneity has to be expected. An alternative way is also used by spreading firstly PLA50 and then BSA. Isotherm Measurements. Surface pressure (n)-surface area (A) isotherms were obtained by continuous compression of = 150 cm2/min. the spread monolayers at constant velocity RelaxationMeasurements. Surface pressure as a function of time was measured during small compressionor expansion of the monolayers and during the relaxation process which follows the perturbation. A Sartorius balance fitted with a Wilhelmy plate and connected to a computer was used to measure surface pressure variations (with an accuracy of 0.02 mN/m) versus the time. The quicker rate of acquisition was about 5 value&. Small compression or expansion of the monolayers was done from a given surface pressure with different velocities by using the mobile barrier of the film balance. The distance between the mobile and floating barrier was in all cases equal to about 7 cm.
is for characteristic times in the range of 1to lo3 s.24-28 In general, the viscoelastic properties of a surface film depend on the characteristic time ofthe perturbation, since various relaxation processes involving molecular relaxation mechanism with different characteristic times can take place. Compressed polymer layers are often far from equilibrium. Slow surface pressure relaxation after stopping the monolayer compression and surface pressure-area hysteresis has been o b ~ e r v e d . ~ These ~ , ~ effects ~ are related to conformational changes involving desorption of segments from the surface. Quantitative interpretation of the observed effects is difficult, because the relaxations are measured mainly after large compressions and the effects are nonlinear. In order to develop a theoretical approach, based on linear relationships of the thermodynamics of irreversible processes, the relaxations must be obtained after small, well-defined changes in surface pressure. Then, the observed time-dependent effects, during and after compression caused by the displacement of segments from interface to the adjacent phase(s), can be described phenomenologically using the equations of the two-dimensional rheology or of the formal chemical kinetic. In order to describe the surface pressure change, An = J&) - ni (Figure l),during the time T of the compression c with a constant velocity u b followed by a relaxation r, we will suppose that at any moment the total surface pressure change An = n(t)- ni can be represented as a sum of one equilibrium An, and one nonequilibrium Anne contribution.
Atomic Force Microscopy on Langmuir-Blodgett (LB) Film. The LB transfer was carried out on a freshly cleaved mica
An = Ane iAnne
plate. The mica plate was firstly immersed into the aqueous subphase; after the monolayer was spread, the film was compressed to the desired surface pressure and then deposited a t a constant surface pressure on the mica. The speed of the transfer was 5 m d m i n . We carried out AFM imaging in the noncontact mode with a commercial instrument (Topometrix, Santa Clara, CAI. (We thank A. Kerrien from Fondis Electronic for her technical assistance.)
Theoretical Approach RheologicalDilatational Properties. The dynamic response of a surface film to a dilatational (compressional) mechanical stress has been investigated on a large time < t < lo3s. Data for the viscoelastic dilational scale, properties of surface films have been obtained in several ways: (1)Analysis of thermally excited surface waves, probed by surface light scattering. This is for characto s;15-17(2) teristic times in the range of Mechanically generated surface waves by periodic compression-expansion of the surface film. This is for to 1 s18-23 (3) characteristic times in the range of Continuous compression (or expansion)of a surface. This (15) Earnshaw, J. C. InPolymersurfacesand interfaces II; Feast, W. J., Munro, H. S., Richards, R. W., Eds.; J. Wiley & Son: Chichester, New York, 1993; p 101. (16) Langevin, D.; Bouchiat, A. M. C.R.Acad. Sci., Ser. B 1971,272, 1422. (17) Hard, S.; Jofgren, H. J . Colloid Interface Sci. 1977,60, 529. (18) Lucassen-Reynders, E. H. In Anionic Surfactant; LucassenReynders, E. H., Eds.; Marcel1 Dekker: New York and Basel, 1983; p 173. (19) Lucassen-Reynders, E. H.; Lucassen, J. Adu. Colloid Znterface Sci. 1969,2,347. (20) Mann, J. A. In Techniques o f Surface Chemistry and Physics; Good, R. J., Stroberg, R. R., Patrick, R. L., Eds.; Marcel Dekker: New York, 1972; Vol. 1, p 77. (21) Lucassen, J.; Barnes, G. T. J . Chem. SOC.,Faraday Trans. 1 1972,68, 2129.
(1)
The equilibrium part, Ane,is related to the equilibrium surface dilatational elasticity E,. Ubt
An, = E, Ai where Ai is the initial surface area before the compression and W A i , the correspondingstrain E (see Figure la). This elastic behavior is represented by the upper branch of the mechanical model in Figure la. The nonequilibrium part of the total surface pressure change Anneis associated with the accumulationof elastic energy during the compression. Dissipation of this accumulated energy through formation of loops and tails of segments in the adjacent phase(s) occurs during compression as well as relaxation. This viscoelastic behavior can be described with Maxwell’s equation (22) Edwards, D.; Brenner, H.; Wasan, D. T. In Interfacial transport processes and rheology; Butterworth-Heinemann: Oxford, 1991. (23) Van der Tempel, M.; Van de Ried, R. P. J . Chem. Phys. 1966, 42,2769. (24) Van Voorst Vader, F.; Erkens, J. F.; Van den Tempel, M. Trans. Faraday SOC.1964,60, 1170. (25) Dimitrov, D. S.; Panaiotov, I.; Richmond, P.; Ter-MinassianSaraga, L. J . Colloid Znterfuce Sci. 1978,65, 483. (26) Panaiotov, I.; Dimitrov, D. S.;Ter-Minassian-Saraga,L. J . Colloid Interface Sei. 1979,72,49. (27) Panaiotov, I.; Dimitrov, D. S.; Ivanova, M. J . Colloid Interface Sci. 1979,69, 318. (28) Panaiotov, I.; Sanfeld,A,; B0is.A.; Baret, J. F. J.Colloid Interface Sci. 1983,96, 315. (29) Mac Ritchie, F. Chemistry at interfaces; Academic Press: San Diego, CA, 1990. (30) Kimura, M.; Kamaki, M.; Nakajima, T.; Nakahara, H.; Fukuda, K. J . A m . Oil Chem. SOC.1990,67, 698.
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Dilatational Properties of Polymer Monolayers
T
part, En,, of the surface dilatational elasticity, that is due to pushing out of segments from the surface. When the time of compression T is much smaller than t),the following useful the time of relaxation process t(T
