Langmuir 1996,11, 2131-2136
2131
Dilatational Properties of POly(D,L-laCtiCacid) and Bovine Serum Albumin Monolayers Formed from Spreading an Oil-in-Water Emulsion at the AirMTater Interface F. Boury, Tz. Ivanova,? I. Panaiotov,t and J. E. Proust* Pharmacie Galdnique et Biophysique Pharmaceutique, Facultd de Pharmacie, 16 Boulevard Daviers, 49100 Angers, France Received August 26, 1994. In Final Form: March 6, 1995@ The surface-pressure, surface-area isotherms of pure bovine serum albumin (BSA) and mixed poly@&-lactide)(PLABO)/BSAmonolayers spread from a water-in-oilemulsion at the airlwater interface were obtained and compared with those of monolayers of the same constituents obtained by usual methods. In order to obtain additional information about the film organization, a rheologicalapproach based on Maxwell's model and AFM imaging were used. The results show that the two constituents are better mixed in the monolayers formed by spreading an oil-in-water emulsion, while a greater segregation exists in the monolayers formed by successivespreading. The presence ofBSA aggregates was observed in the monolayers formed from emulsion. This could have some consequences on the preparation and the properties of the PLA50 particles obtained by an emulsion technique.
Introduction There has been a growinginterest to improve the efficacy of available vaccines by using novel antigen delivery systems.lS2 One possible way is to reduce the number of repeated administrations required for long-term protection. In this way, the design of biodegradable controlleddelivery systems has been to encapsulate various peptides and p r ~ t e i n . The ~ , ~use of a suitable poly(a-hydroxy acid) matrix permits, one by controlling the degradability of microspheres to liberate the vaccine a t different times after a single i n j e ~ t i o n .The ~ liberation is then dependent on the polymer weight, its crystallinity, and the ratio of lactic acidfglycolic a ~ i d . ~Another ,~ factor, much less studied than the preceding ones, which influences the properties of the final product, is the surface state of the material. These surface properties are mainly dependent on the process that leads to obtaining solid carriers. We showed in two previous paper^^,^ that the use of PVA (polyvinyl alcohol) or BSA as colloidal stabilizer of the DCM (dichloromethane) in water emulsion (the organic phase containing the PLABO), led probably to the irreversible anchoring of the stabilizer in the interfacial region. In the case of the protein,1° this could be account for liberation of the protein a t short delay. On the other hand, it seems to be adequate5J1 to formulate particles in which the water-soluble protein or
' Permanent address: Biophysical Chemistry Laboratory, University of Soda, J. Bourchier lstr., 1126 Sofia, Bulgaria. Abstract published in Advance A C S Abstracts, June 1, 1995. (1)Aguado, M. T.; Lambert, P. H. Immunobiology 1992,184, 125. (2)Bodmer, D.; Kissel, T.; Traechslin, E. J.ControlledRelease 1992, 21.129 - -, __ - . (3)Cohen, S.;Yoshioka, T.; Lucarelli, M.; Hwang, L. H.; Langer, R. Pharm. Res. 1991,8,713. (4)Wane, H. T.: Schmidt, E.: Flanaean, - D. R.: Linhardt. R. J. J. ControlledXeZease 1991,17, 23. (5) Jeffery, H.; Davis, S. S.; O'Hagan, D. T. J.Pharm. Res. 1993,10, @
362. (6) Spenlehauer, G.;Vert, M.; Benoit, J. P.;Boddaert, A. Biomaterials 1989,10, 557. (7)Vert, M.; Li, S.; Garreau, H. J. Controlled Release 1991,16, 15. (8) Boury, F.; Olivier, E.; Proust, J. E.; Benoit, J. P. J.ColZoidInteface Sci. 1994,163, 37. (9)Boury,F.;Ivanova,Tz.;Panai'otov,I.;Proust, J.E.;Bois,A.;Richou, J. J . Colloid Interface Sci. 1996,169, 380. (10)Boury, F.; Ivanova, Tz.; Panaiotov, I.; Proust, J. E. Langmuir 1996,11, 599. (11)Hora, M. S.; Rana, R. K.; Nunberg, J. H.; Tice, T. R.; Gilley, R. M.;Hudson, M. E. Pharm. Res. 1990,7,1190.
peptide is entrapped from a (water in oil)-in-water (wlol w) solvent evaporation technique. The aqueous solution ofthe protein is emulsified into a n organic solution (DCM) of the hydrophobiccoating polymer (PLA50). This primary water-in-oil emulsion (wlo)is then dispersed in water with formation of the wlolw emulsion. The organic solvent is finally removed with formation of solid microparticles. It is thus probable that the encapsulation of the protein is strongly dependent on the localization of both protein and polymer in bulk and interfacial regions of the primary emulsion. A general idea of the organization of the polymer matrix could be obtained by spreading the primary emulsion on a plane airlwater interface followed by evaporation of the DCM solvent. A study of the properties of the BSA and PLA50 monolayers obtained in this way could be developed and compared with monolayers spread by using the classical procedure. In fact, it is known that the state of protein films spread a t the airlwater interface depends on the mode of spreading. At least two effective methods for the formation of protein films spread from aqueous solution has been proposed by G ~ a s t a l l a ' ~and J ~ Trurnit,14 both leading to spreading yields near 100% and slightly different r e ~ u 1 t s . l ~ The purpose of this paper is to report the properties oi pure BSA and BSA/PLA50 monolayers obtained by spreading the emulsion a t the airlwater interface. We compare the effectiveness and the state of protein film obtained by emulsion spreading with those obtained by the usual methods. In order to obtain additional information about the film organization, we have analyzed the dynamic properties of the surface film by using a rheological approach previously developed to study the interaction of PLA50 and PVA at the airlwater and DCM/ water i n t e r f a ~ e .Morever, ~ a structural analysis of the films sampled by the Langmuir-Blodgett technique was performed by atomic force microscopy.
Materials and Methods Polymers. The PLA50, a poly(D,L-lacticacid) stereocopolymer, was obtained from CRBPA (URACNRS 1465,Montpellier, France). According to the Vert classification,16it contains 50% (12)Guastalla, J. C. R. Acad. Sei., Paris 1939,208, 1078. (13)Guastalla, J. Thesis, Universit6 de Montpellier, 1948. (14)Trurnit, H. J. J. Colloid Interface Sci. 1960,15, 1. (15)Ivanova, M.;Verger, R.; Bois, A.; Panai'otov, I. Colloids Surf. 1991,54,279.
0743-746319512411-2131$09.00/0 0 1995 American Chemical Society
Boury et al.
2132 Langmuir, Vol. 11,No.6, 1995
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Figure 1. Size distribution of the aqueousdroplets in the waterin-oil emulsion.
L-repeatingunits. Its mean molecular weight (M,)determined by size exclusion chromatography was 41 600. The 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) and 2-propanol were supplied by Prolabo (Paris, France) and used without further purification. Water was ultrapure and obtained from a Millipore system (Milli-Q plus, Millipore, France). Preparation of the Emulsion. The water-in-oil emulsion was prepared by dispersing the BSA aqueous solutions (25 mg/ mL) in DCM containing or not the dissolved PLA50 (1mg/mL). Thiswas achievedby sonicatingduring30 s the systemcontaining 200 pL of aqueous phase and 5 mL of organic phase by use of a Bronson sonifier cell disruptor B30. The resulting emulsionwas controlled in a Coulter apparatus (submicron particle analyzer Coulter model (N4MD;Coulter Electronics, Hialeah, FL). The detection angle was 90"; measurements duration was 5 min. According to Figure 1, 95% of the microdroplets of the system containing PLA50 had diameters between 520 and 561nm with an estimated mean diameter of 540 nm. For the systems emulsioned without PLA50, the emulsion was too unstable to permit the measurement of the droplet size. Isotherm Measurements. The studied monolayers were spread on an aqueous subphase on the maximum available area (927 cm2)of a Langmuir film balance (LAUDAFW2, Germany) after the water interface was cleaned by suction. An Exmire microsyringe was used to spread the monolayers at the water surface. Thevalues of the surface pressure after spreading were less than 0.1 d i m . Monolayers were left for about 10min before compression of the film at a velocity of 150 cm2min-l. Accordingto Guastalla's usual p r o ~ e d u r e ,BSA ~ ~ Jmonolayers ~ were formed by spreading the protein aqueous solution (0.5mg/ mL)containing0.1%of 2-propanolin order to facilitate spreading. An alternative way of spreading the BSA was used by directly spreading the emulsion at the aidwater interface. Monolayers of pure PLA50 were formed by spreading a solution of PLA50 in dichloromethane (1 mg/mL). Mixed monolayers of BSA and PLA50 were formed by successive spreading of the two constituents or by spreading the emulsion of the two constituents.
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I Time Figure2. (a)Rheologicalmodel ofthemonolayer. Deformation UbtlAi and change of surface pressure A n during the time T of compressionc with constant velocity ub,followed by a relaxation r. (b)Determination of the characteristictime z with Maxwell's
model (T
