Langmuir 1992,8, 2781-2784
2781
Compression-Expansion Curves of Poly(DL-lactic acid-co-glycolic acid) Monolayers J. Miiiones,' E. Iribarnegaray, C. Varela, N. Vila, 0. Conde, L. Cid, and M. Casas Department of Physical Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain Received March 6, 1992. In Final Form: July 31, 1992 The copolymer poly(DL-lacticacid-co-glycolicacid), used in the design of nanoparticles as drug delivery systems, forms stable monolayers at the &/water interface when spread from solutions of chloroform or 4:l (v:v) chloroform-ethanol mixtures. The II-A curves obtained show a plateau at a surface pressure of about 9 mN.m-', corresponding to a change from high to low compressibility monolayer states. Since the surface pressure in the plateau region was hardly altered by increasing temperature from 10 to 35 OC, the plateau is interpreted as reflecting compression-inducedreorientation of the polar groups of the polymer. There was marked hysteresis when the film was compressed to the collapse surface pressure (60 mN.m-l) and then expanded to its initial area: the first compression-expansion cycle did not return the monolayer to its original state, probably due to irreversible lose of molecules during collapse. Below the collapse surface pressure, however, the f i i showed reversible behavior. Monolayers were spread from chloroform, benzene, ethanol, acetonitrile,or 41,3:2,23, and 1:4 (v/v)chloroform-ethanol and In recent years, certain biodegradable and biocompatible chloroform-benzene mixtures. All solutionscontained0.5 % amyl polymers, notably lactide/glycolide copolymers, have been alcohol as spreading agent. PLA/GA solutions in chloroformbenzene and chloroform-ethanolmixtures were prepared either successfully used in drug dosage forms for sustainedrelease by dissolving the polymer directly in the solvent mixture or by of drugs such as contraceptives,' antiparkinsonian agents? adding the cosolvent to PLAIGA-chloroform solution. antibiotics,3p4 and anti-inflammatories.6 Their future in Greatest spreading took place with chloroform or highthis role is very promising, though some limitations arising chloroform mixturesas solvent;limitingareas of only 5Az/residue from incompatibilities have been reported.6 There is were achieved with acetonitrile or high-benzeneor high-ethanol nevertheless insufficient information on the physicochemsolvents. Spreadingwas poorer when the polymer was dinsolved ical properties of the polymers and on the surface in the previously prepared mixture than when dissolved in pure interactions between the polymers and the additives used chloroform. in the polymerization p r o c e s ~ . Better ~ * ~ understanding of Since the isotherms obtained by spreading from pure chlothe molecular properties of polymers would help in roform practically coincided with those recorded in 4 1 chlorointerpreting the behavior of pharmaceutical c o a t i n g ~ . ~ J ~ form-ethanol, the latter was chosen as the spreadingsolvent due One approach to the study of polymer films is the use to the reduction in vapor pressure afforded by the presence of of techniques devised for investigation of insoluble monoalcohol, which avoids gradual concentration of the solution. Storing the solutionsin a refrigerator, in a desiccator containii molecular films at the aidwater interface. In this work the mixture of solvents used, also helped avoid concentration. we determined the surface behavior of a copolymer of lactic Reproducible results were obtained with solutionsstored for up acid and glycolic acid used in the manufacture of nanoto 1week in this way. particle dosage forms. It is hoped that our results will The substrate on which fiis were spread was a Theorellhelp future elucidation of the nature of possible interacStenhagen pH 7 buffer solution prepared with deionized water tions with other components of the pharmaceutical of resistivity 18Mfbcm from a Milli-RO,M a - Q (MilliporeCorp.) formulation, or with the components of cell membranes reverseosmosissystem. Thissystemconsistsof a w a r d prefiiter in the patient. and a cellulose acetate RO cartridge set in series with a carbon fiiter, two ion-exchangeresin cartridges,and a 0.22-~mmembrane Experimental Part filter for removal of any microscopic particles or microorganisms that may have escaped retention by the upstream elementa. pOly(DL-ladiC acid-co-glycolic acid) (5050 PWGA) was II-A curves were recorded using a Langmuir f i i balance supplied by Boehringer Ingelheimunder the name Resomer 506 (FW-1 Lauda, FRG), with Teflon-coated trough and mobile (intrinsic viscosity of 0.8; mean molecular weight 98 OOO) and barrier. The precision of surface pressure measurements was used without further purification. hO.1 mN.m-l, and films were spread over an initial area of 562 cm2. Varying the volume of a 0.218 mg1mL solution deposited (1) Beck,L. R.; Flowers, C. E., Jr.; Pope, V. Z.;Wilborn, W. H.; Tice, between 58.5and 219.5rL (equivalenttovaryinginitialspreading T. R. Am. J. Obstet. Gynecol. 1983,147, 815. area between 25 and 96 A2lresidue) had no significant effect on (2) Peters,F.; Del Pozo, E.; Cont,A.;Breakwaldt,M.Obstet. Gynecol. II-A curves; accordingly a spreading volume containing 1.18 X 1986, 67, 82. 1017 monomer units (corresponding to an initial area of 50 A*/ (3) Tice, T. R.; Rowe, C. E.; Stteretrom, J. A. h o c . Int.Symp. Cont. residue)was used throughout the remainder of the experiments. Rel. Bioact. Mater. 1984,11, 6. (4)Baker,R.W.;Krisko,E.A.;Kochinke,F.;Grassi,M.;Armitage,G.;Film compression was started 5 min after spreading,previous Robertson, P.A.Proc.Int. Symp. Cont. Rel. Bioact. Mater. 1988,15,238. experimentshaving shown that times ranging from 2.5 to 30 min (5)Tie, T. R.;Lewis, D. H.; C o w , D. R.;Beck,L. R. U.S.Patent led to identical results. Filmswere compressedand d e c o m p d 4,542,025, 1985. (6) Madding, H. V. J. Controlled Release 1987, 6, 167. at a rate of 6.5 cmsmin-l (97.5 cm*.min-l); the curves obtained (7)Baazkin, A.; Couvreur, P.; Deyme, M.;Henry-Michelland, S.; where independent of barrier speed over the range 1.7-6.5 Albrecht, G. J. Phurm. Pharmacol. 1987,39,973. cm.min-l(25.5-97.5 cma-min-l). In most experiments, compres(8) &ea, M. A.;Vab, 0.;Alsina, M.A.; Garcia, M.L.;Losa,C.; Vila sion WBB continued until the monolayer collapsed or the II-A Jato, J. L.; Aloneo, M.J. Int. J. Pharm. 1991,67, 103. curve reached a plateau; expansions were continued until the (9) Zatz, J. L.; Weiner, N.;Gibaldi, M.J. Pharm. Sci. 1968,57,1440. f i i occupied its original area. Curves were highly reproducible; (10) Zatz, J. L.; Knowles, B. J. Pharm. Sci. 1970,59,1750.
Introduction
0743-746319212408-2781$03.00/0 Q 1992 American Chemical Society
Miii~neeet al.
2782 Langmuir, Vol. 8, No. 11,1992 mN/m MI--
%-,
40-
1 30
20
A%.*
I 9.5
-
A?/rei
Figure 1. Compreeeion-expamion cycle for 5050 PLA/GA Substrate pH 7, temperature 20 "C. each experiment was repeated until three curves coinciding to within 0.5 A2/monomer were obtained. Allexperimentsexcept thoee carried out to evaluate the effects of temperature were performed at 20 f 1 O C . Substrate temperature was controlled by passing thermostated water through the hollow base upon which the balance trough was placed. Results Figure 1shows a PLA/GA compression-expansioncurve obtained at 20 "C and pH 7. Segment AB corresponds to a state of the monolayer characterized by high compressibility (6.5X m.mN-'), while segment CD reflecta a condensed stateof low compressibility(5.6X 1W3m.mN-'). Between these there is a plateau of almost constant surface pressure (segment BC). The film collapsed (DE) at a surface pressure of about 60 "am-' and an area of about 2.5 A2/residueand exhibited considerablehysteresis upon decompression. To study recovery from collapse, the monolayer was compressed to collapse and immediately expanded to ita initial area, and then subjectedto five cycles of compression to a pressure of 6 "em-' (chosen arbitrarily in the reversible region of the II-A isotherm1') followed be expansion to II = 0, with 5 min between successive cycles. The film showed some recovery in the course of cycles 2-6 (Figure 21, but not until more than 4 h after the fiit cycle did it recover characteristics approximating those of the first compression (data not shown). Figure 3 shows the more or less reversible response obtained when the monolayer was compressed to various pressures below ita point of collapse and then allowed to expand. Even for an endpoint pressure of 30 mN.m-', i.e. beyond the plateau, the compression-expansion curve (11) Nitsch, W.;Malreymiw, R. Colloid Polym. Sei. 1990,268,462.
Figure2. Behavior of PW GA monolayera during compreaaion-
expansion cycles 1, continoua compreeeion up to cohpea preseure; l', continoua expamion down to II = 0 "em-'; 24, compression-expansioncyclea running between n = 0 mN-m-1 and II = 6 "em-', with 5 min between suceeaaive cyclea.
shows little or no hysteresis. Hysteresiswas only apparent after collapse. The stability of the film was tested by inserting 10-min pauses into the compression-expansioncycle at the surface pressures indicated by the arrows in Figure 4. Below the plateau (at 3.5 and 5 mN-m-l) the film remained in equilibrium; pausing on the plateau (at 10 "em-') gave rise to a small drop in surfacepressure which was recovered when the barrier began to move again; while a pause in the low compressibility region, at 20 mN-m-l, caused surface pressure to drop to 15 most of this decrease occurred during the fiit 2 min. Halting compression during collapse of the film at 60 "am-' caused surface pressure to drop to about 23 "em-'. Stopping the barrier during expansion of the monolayer increased surface pressure (Figure4). When immediately following collapsethe surface preseure reached zero, halting the barrier increased surface pressure to 20 mN*m-l,most of the increase taking place during the first minute, while stopping at 4.8 mN0m-l brought about an increase of 3 mN-m-l. The above resulta show that the film was only in equilibrium when it occupied an area greater than 20 A2/ residue, when it only exerted a small surface pressure. At smaller areas equilibrium was only reached after a relatively long time (>1 min). An important finding was that the behavior of the filme was almost unaffected by temperature. The isotherma in Figure 5 almost coincide over the high compressibility and plateau regions. In the low compressibility region they differ from those of most other substances in that the area occupied decreases with increasing temperature, possibly because the solubility of the PLAIGA increases with temperature and surface pressure. Evidence in favor
Langmuir, Vol. 8, NO.11, 1992 2783
Compression-Expansion Curves mNhn
eo
40
10
10
0
--
Figure 3. Successive uninterrupted compreeeion-expaneion cycles of PLA/GA monolayers: -, f i i t cycle;. ,second cycle; ---,third cycle; 0 0 0,fourth cycle;X X X, f i i h cycle. Arrow indicate the point at which decompreseion was started in each cycle.
Figure 4. Compression-expansion curve of PLA/GA showing the effecta of stopping the barrier at various points during Compression and decompression of the monolayer. m
h
ea
of this hypothesis came from experiments at pH 10 in which the monolayer was unstable above 20 OC (data not shown), presumably because of greater solubility due to its total ionization at this pH.
Discussion It is reasonable to assume that under continuous compression of PLA/GA monolayers, the random orientation of the polymer chains (beyond point A in Figure 1) is replaced by a more ordered, densely packed arrangement (point B). Between these points, compression of the monolayer is totally reversible: decompression curves retraced the compression curve (Figure 31, and no accumulation of energywas reflected by any decrease in surface pressure when the barrier is halted (Figure 4). The area per residue at point B was 18A2,which is very close to the value of 17.9 A2 per monomer calculated from solid-state X-ray diffraction measurements of polymers under the assumption of hexagonal packing.12 The plateau of the II-A curve (segment BC in Figure 1)may be due to the formation of a bilayer, as suggested by Malcolm for polypeptides.l3J4 Malcolm's suggestion was confirmed by Takenaka et al.15and Takeda et al.,16917 who by means of polarized infrared and transmission ~
(12)Bamford, C.H.; Elliott, A.; Hanby, W. E. In Synthetic Polypeptidee; Academic Preee: New York, 1956;p 239. (13)Malcolm, B.R. Polymer 1966, 7,595. (14)Malcolm, B.R. h o c . R. SOC.London 1968,305,363. (15)Takenaka, T.; Harada, K.; Matsumoto, M.J. Colloid Interface Sci. ISSO, 73,569. (16)Takeda, F.; Mataumoto,M.;Takenaka,T.; Fujiyoehi,Y. J. Colloid Interface Sci. 1981,84,220. (17)Takeda, F.; Mataumoto, M.;Takenaka, T.; Fujiyoehi, Y.; Uyeda, N. J. Colloid Interface Sci. 1983,91, 267.
10
bo
30
10
10
a
Figure 6. Influence of temperature on II-A curves of PLA/GA: 1,LO OC;2,15 O C ; 3,20.5 O C ; 4,25 O C ; 5,30 "C;6,35 OC. Substrate pH 7.
techniques found that when films were built up on germanium plates by transfer of monolayers at surface
Miilone8 et al.
2784 Langmuir, Vol. 8, No.11, 1992
pressures above the plateau, they were almost twice as thick as when surface pressures below the plateau were used. In our system, the bilayer hypothesis is supported by the area per residue at the end of the transition (point C in Figure 1)being practically half the area per residue at the beginning of the transition (point B)and too small to accommodate any reasonable monolayer conformation. The bilayer hypothesis nevertheless seems to be contradicted by the fact that temperature hardly affected the isotherms in the plateau region, since zero slope in a plot of transition surface pressure against temperature (and hence zero values of enthalpy and entropy calculated from the Clausius-Clapeyron equation) suggests that no transition in fact takes place. If there is indeed no transition between one state and another, then the plateau in the II-A curve simply reflects compression-induced reorientation of the polar groups of the lactic and glycolic acid residues. Such reorientation can certainlytake place quite easily, because the side chains of PLAIGA, which are formed of randomly distributed lactic acid and glycolic acid residues, are so short that the corresponding change in internal energy and enthalpy is negligible.18 The polymer may, therefore, assume an “accordion” conformation in which its polar groups are immersed in the subphaseas completelyas possible. Under this hypothesis, surface pressure should not have changed at all on the plateau under ideal compression conditions, since the decrease in area should be completely accounted for by the immersion of polar groups in the subphase with no change in energy. The slight increase in surface pressure observed in practice is attributed to compression having been performed continuously, leaving no time for equilibrium to be attained by the establishment of attractive interactions between the hydrocarbon groups; this interpretation is supported by the reversible fall in surface pressure that occurred when the barrier was halted on the plateau (Figure 4). An alternative explanation for the plateau in the compression curve as being due to molecular distortion may be also be put forward. In this, compression would force out the molecular segments from the air-water ~
interface into a subsurface region, termed the “transition layer”by Nitsch and Maksymiw’l in their study of catalase monolayers. However, it is not easy to see how the C-C and C==O bonds of a more rigid molecule like PLA/GA could be sufficiently distorted for the formation of such a layer to occur. According to the “accordion” interpretation of the plateau region, further compression of the film (segment CD in Figure 1)is possible only by compressingthe apolar segments of polymer chain left at the interface, which would explain the extremely high collapse surfacepressure and the very small area per residue at collapse. Thie interpretation of the CD segment as reflecting an orderly compression process is also in keeping with the reversible behavior exhibited when decompression is initiated or the barrier is halted at points within this segment (Figures 3 and 4). The inelastic behavior reflected by the rapid fall in surfacepressure that occurs when decompressionis begun upon collapse (segmentEF in Figure 1) may be attributed to the disruption of the monolayer during collapse; it may be hypothesized that the disorganization involved in collapse not only caused the loss of polymer from the film, as appears to be shown by the observed hysteresis (irreversible collapse behavior;l9 see Figure 2), but also hindered decompression of polymer chains left in the film in a compressed state. As such hindrance disappeared upon withdrawal of the barrier, the forces exerted by increasing numbers of compressed polymer chaine will have been felt (segmentFG in Figure l),after which surface pressure fell off as the polymer expanded (segment GH). In conclusion, we point out that the above results may have significant implications for the preparation of pharmaceutical nanoparticles with PLAIGA, since the drug release characteristics of such particles may be expected to depend on the elasticity of the polymer, which, as we have seen, depends on its history.
Acknowledgment. The authors thank Professor Vila Jato of the Department of Pharmacy of the University of Santiago de Compostela for suggestingthis research topic and supplying polymer samples.
~~~
(18) Isemura, T.;Hamaguchi, K. Bull. Chem. SOC.1953,26,425.
(19) Gainee, G. L.,Jr. Langmuir 1991, 7,834.
