COLLOIDS AND SURFACES ELSEVIER
B
Colloids and Surfaces B: Biointerfaces 6 (1996) 373 378
Changes in charge density and softness of a poly(c-lactide) microcapsule surface in the hydrolytic degradation process Kimiko Makino *, Hiroyuki Ohshima Faculty qf Pharmaceutical Sciences and Institute of Colloid and Interface Science, Science University of Tokyo, Shinjuku-ku, Tokyo 162, Japan Received 29 December 1995; accepted 29 January 1996
Abstract
The changes in surface properties of poly(L-lactide) microcapsules caused by hydrolytic degradation have been studied with electrophoretic mobility measurements. An electrokinetic model has been applied to examine the electrophoretic mobility data, which were previously analyzed with a model that does not take into account the liquid flow inside the microcapsule membrane [K. Makino, H. Ohshima and T. Kondo, J. Microencapsulation, 4 (1987) 47]. The present new model involves two parameters, the charge density in the microcapsule membrane and a softness parameter, the latter of which characterizes the reciprocal of the frictional coefficient of the polymer exerted on the liquid flow. Information about the changes in charge density and in the softness of the poly(IAactide) microcapsule surface have been newly obtained. The surface charge density increases by the cleavage of ester bonds in the polymer chain in the initial stage of the degradation process. It then gradually decreases down to the value for intact poly(Llactide) microcapsules as a result of the release of degraded polymer segments from the microcapsule surface. Also, as the degradation proceeds, the softness parameter value increases, suggesting that the surface of the microcapsules becomes softer, probably because the surface becomes porous. The above change in the softness and the decrease in charge density at the later stage of the degradation both imply liberation of charged polymer segments. The degradation of poly(c-lactide) microcapsules proceeds by alternate repetition of cleavage of the ester bonds in the polymer chains and liberation of the degraded polymer segments from the surface.
Keywords: Degradation; Electrophoretic mobility; Poly(L-lactide) microcapsules; Soft particle
1. Introduction
Poly(IAactide) is a well-known bio-degradable polymer. Its degradation mechanism has been studied with a particular emphasis on the application to drug delivery systems in the last decade by a number of research groups (see e.g. Refs. [1,2]). In previous papers [ 3 - 6 ] we have reported that degradation of poly(c-lactide) microcapsules was * Corresponding author. Tel.: (+81)-3-3260-6725: Fax: (+ 81 )-3-3268-3045. 0927-7765/96/$15.00 © 1996 ElsevierScience B.V. All rights reserved PH S0927-7765 (96)01271-4
accelerated in strongly alkaline or acidic solutions with the lowest degradation rate at neutral pH. Also, we have discussed the change in charge distribution in the microcapsule membrane during the hydrolytic degradation process by the analysis of electrophoretic mobility data of microcapsules at various stages of the degradation process. In a previous paper [-6], the electrophoretic mobility of poly(c-lactide) microcapsules was analyzed with a membrane model derived by Ohshima and Ohki [-7], in which electrolyte ions can penetrate the microcapsule surface but liquid cannot flow into
374
K. Makino, H. Ohshima/ColloidsSurJaces B: Biointerfaces6 (1996) 373 378
the microcapsule membrane (we term this model a model for "semi-soft" particles). Theoretical curves obtained from this model did not agree well with the experimental data of poly(L-lactide) microcapsules during the whole degradation process, although the model could explain the charge distribution in the polymer layer before the degradation starts. In this article, we will discuss the changes in "softness" and the charge density of the poly(L-lactide) microcapsule surface caused by the hydrolytic degradation by reanalyzing the electrophoretic mobility data with Ohshima's model for electrophoretic mobility of "soft" particles [8]. This model involves a "softness" parameter which characterizes the reciprocal of the frictional coefficient of the polymer exerted on the liquid flow. Here, a "soft particle" means a particle where the electrolyte ions can penetrate the surface and liquid can flow inside. This analysis will give us information on how the degradation proceeds in the microcapsule membrane and how degraded polymer chains are liberated from its polymer matrix.
2. Materials and methods
All experiments have been done as previously reported [6]. 2.1. Degradation of poly(L-lactide) microcapsules
In the degradation experiments, poly(L-lactide) microcapsules were dispersed in solutions of various pH values (1.2, 3.2, 5.4, 7.6, and 9.8) at final concentrations of 0.15% (v/v) and the suspensions were shaken at 37°C. The ionic strength of the solution was 0.154 with the addition of NaC1. Aliquots of these suspensions were taken at suitable time intervals, followed by centrifugation at 3000 rev min 1 for 10 rain. The precipitated microcapsules were used to prepare samples to measure the electrophoretic mobility. 2.2. Measurement of the electrophoretic mobility
Poly(L-lactide) microcapsules in different stages of degradation prepared as described above were
redispersed in pH 7.6 phosphate buffer solutions of various ionic strengths: 0.01, 0.03, 0.075 and 0.154. The electrophoretic mobility of the microcapsules was measured in each solution.
3. Results and discussion
The weight-averaged molecular weight of poly(Llactide) decreases as the hydrolytic degradation proceeds, indicating that a number of ester bonds in poly(L-lactide) are cleaved to produce poly(Llactide) molecules of lower degrees of polymerization, as has been reported before [3-6]. In the degradation process, lactic acid and poly(L-lactide) with a lower degree of polymerization are produced and liberated from the matrix, as suggested from changes in the molecular weight distribution of poly(L-lactide). To find where in a microcapsule membrane the cleavage of poly(L-lactide) occurs and how it proceeds to destroy the membrane, we have studied the change in the electrophoretic mobility of the microcapsules during the degradation process at various pH values. On degradation, dissociable carboxyl groups are produced at the end of polymer chains, and the density of polymer chains in the microcapsule membrane decreases with the liberation of polymer chains with a lower degree of polymerization. These phenomena are expected to cause changes in charge density and "softness" of the surface layer of poly(L-lactide) microcapsules. In our previous paper [6], the zeta potential of microcapsules at various stages of degradation was measured at pH 7.6 as a function of the ionic strength of the medium, which has been analyzed by a model for an ion-penetrable surface [6,7]. In this model, it is assumed that ion molecules in a bulk solution can penetrate into the surface layer of microcapsules and that liquid flow is not possible in the membrane phase. In the present article, we have reanalyzed the electrophoretic mobility of poly(L-lactide) microcapsules at various stages of the degradation process with the electrophoretic model for soft particles [8], in which ions can penetrate into the microcapsule surface and the water can flow inside the microcapsule membrane.
375
K. Makino, H. Ohshima/Colloids Surfaces B. Biointerfaces 6 (1996) 373-378
O~ [
g
_1 (
-r -2 >
'7
>
E
E
.~, "r,
~-4 0 E
O
E o ...-~ O l-
g g
-3 O e-.
(
o -6
-4
-50
k~
I
I
I
I
20
40
60
80
-8 II
100
Time (day) Fig. 1. Change in the electrophoretic mobility of degraded poly(L-lactide) microcapsules with elapsed time at pH 9.8. Redispersion media are buffer solutions (pH 7.6) at ionic strengths: 0.01 (©), 0.03 (A), 0.075 (O), and 0.154 (12).
The experimental data points in Figs. 1 and 2 were all taken from our previous work [6]. The values of the electrophoretic mobility of poly(L-lactide) microcapsules degraded in a buffer solution at pH 9.8 were plotted as a function of the degradation time period as shown in Fig. 1. The samples were redispersed in buffer solutions at pH 7.6 with ionic strengths of 0.01, 0.03, 0.075 and 0.154 for the electrophoretic measurement. The obtained values were negative at all ionic strengths in the solution, implying that the surfaces of this type of microcapsule have a net negative charge in the degradation period. As is clearly seen in this Figure, the changes of the electrophoretic mobility values are most obvious in the first 7 days and the values at 7 days are the most negative during the experimental period (100 days) at all ionic strengths, although the absolute values decrease by gradual recovery up to the original ones with time after 7 days. To see the differences in the surface properties of poly(L-lactide) microcapsules among the intact
0
,
0.04
i
0.08 0.12 Ionic strength
0.16
Fig. 2. Changes in electrophoretic mobility of poly(L-lactide) microcapsules on degradation at pH 9.8. Symbols are experimental data measured as a function of the ionic strength in the suspending medium at pH7.6: (©) intact poly(L-lactide) microcapsules (t=0); (A) degraded poly(L-lactide) microcapsules (t = 7 days); 10 ) degraded poly(L-lactide)microcapsules (t = 100 days). Solid curves are theoretical ones calculated with zN= -0.02 M and 1/2 = 1.l nm (curve l), zN = -0.06 M and 1/2= 1.8 nm (curve 2), and z N = - 0 . 0 1 6 M and l/2=3.0nm (curve 31. ones, those stored for 7 days and those for 100 days, the electrophoretic mobility values of the microcapsules at t i m e = 0 , 7 and 100 days were replotted against the ionic strengths of the suspending medium as shown in Fig. 2. The electrophoretic mobility of intact poly(L-lactide) microcapsules changes from - 6 . 7 to - 0 . 3 3 (~tm s -~ V -1 cm) in solutions with ionic strengths between 0.001 and 0.154. The electrophoretic mobility of the microcapsules degraded for 7 days and 100 days changes from - 4.5 to - 2.2 (gm s - 1 V - 1 cm) and from - 2 . 5 to - 1 . 6 (gm s -1 V - l c m ) respectively, in solutions with ionic strengths between 0.01 and 0.154. In solutions with lower ionic strengths, the electrophoretic mobility is found to be more negative. The electrophoretic mobility tends to a non-
376
K. Mak&o, H. Ohshima/Colloids SurJdces B: Bio&terJdces 6 (1996) 373 378
zero value even in the solution with an ionic strength of 0.154, which is clearly seen for the sample degraded for 7 days. As the ionic strength increases, however, the electrophoretic mobility becomes less negative. This phenomenon cannot be predicted from the models for a rigid particle or a semi-soft particle, suggesting that the surface of the present microcapsules is "soft" and the obtained data can be discussed with Ohshima's electrokinetic theory for "soft particles". We give below a mobility expression for this model. In this model, ionized groups of valency z are uniformly distributed at a number density of N (m -3) in the microcapsule membrane. Suppose that the particle moves in a liquid containing a symmetrical electrolyte of valency v in the applied field, n (m 3) is the bulk concentration of symmetrical electrolytes in the dispersing medium. The electrophoretic mobility g is then expressed as Eq. (1) [8]
zeN +--~22
CrY0 IPO/Km -~ If/DON/2 /'/- r/
1/Km+l/2
(1)
with
kTln[Z N ~(zN~2+ }1/2] ¢ooN- ve ~ + (\~vn) 1 0o =
kT(lnIZN v~
~(zN)Z+l}U2 ]
2vn + ( \ 2 v n )
2vn[l_~(zN~2 + 1}1/21) -I-~(~2V./ 2 (7/q)u2 =
Km = K
[
(2)
I-'~-\~)
x = \ereokr)
(3) (4) (5)
(6)
Here, t/is the viscosity, • is the frictional coefficient of the microcapsule membrane, G is the relative permittivity of the solution, eo is the permittivity of a vacuum, ¢DOS is the Donnan potential of the membrane interior, 0o is the potential at the membrane surface, ~c is the Debye-H0ckel parameter, and ~m can be interpreted as the Debye-Ht~ckel parameter of the layer. The parameter 2 character-
izes the degree of friction exerted on the liquid flow in the microcapsule membrane and zeN represents the density of the fixed charges in the membrane. The reciprocal of 2, i.e. 1/2, has the dimension of length and can be considered to be a "softness" parameter since, in the limit 1/2--,0, the surface layer becomes rigid. Although Eq. (1) assumes symmetrical electrolytes, the electrolytes used are not symmetrical (cations are univalent but anions are not); the value of v is set approximately equal to unity (v = 1), since anions are less important for negative charged particles. Eq.(1) involves two unknown parameters, N(m -3) and 1/2 (nm), which represent the fixed charge density in the surface and its softness respectively. By a curve-fitting procedure reported previously [-9 11], zN and 1/2 were determined. The values of p calculated via Eq.(1) are shown in Fig. 2 as a function of the electrolyte concentration (solid lines 1 3). We found it is possible to draw a curve with a pair of single values of each of zN and 1/2 that is in good agreement with the experimental data over a wide range of ionic strengths (0.01 0.154). This means that the poly(L-lactide) microcapsule surface at each stage of degradation can be considered as a "soft surface" described by Eq. (1) at ionic strengths between 0.01 and 0.154. Thus this agreement enables us to estimate the values of the unknown parameters zN and 1/2 by a curve-fitting procedure. The best-fit curve is obtained with zN = -0.02 M and 1/2= 1.1 nm for the microcapsule surface before the degradation starts. In the calculation, we used the value of the relative permittivity er of distilled water. The bestfit values of the charge density (zN) and the softness parameter (1/2) of the microcapsule surface are shown in Fig. 3. In the first 7 days, the charge density increases and then gradually decreases back to its original value. In contrast, 1/2 increases with time, illustrating that the microcapsule surface becomes softer as the degradation proceeds. This change in the softness parameter is caused by a decrease in the frictional coefficient of water flow exerted in the polymer layer by the degradation. Degradation of poly(L-lactide) is considered to make the microcapsule membrane porous by producing void spaces in it, which decrease the softness parameter of the microcapsule surface.
377
K. Makino, H. Ohshima/Colloids Surfaces B: BiointerJaces 6 (1996) 373 378
3.5
0
3
-0.01 2.5 -0.02
2.5
-0.03
2
-0.05 2
.~
Z 1.5
-O.04
1.5 -0.1
-0.05 Ii
-0.07
1
0.5
-0.06 i 0
i 20
I 40
i 60
. 80
i 10o
0
Time [days] Fig. 3. Changes in zN and 1/2 on degradation at pH 9.8. Symbols indicate negativecharge density, zN (© 1, and softness parameter, 1/2 (A).
In the surface layer of poly(L-lactide) microcapsules degraded at other pH values, the negative charge density and the softness parameter exhibit oscillatory changes with time, since the hydrolytic degradation rates are slower than at pH 9.8 [3]. Fig. 4 shows the changes in z N and 1/2 of poly(Llactide) microcapsules degraded at pH 5.4. At this pH the degradation rate is lowest, i.e. the frequencies of both the liberation of degraded polymer segments and the cleavage of ester bonds are considered to be smaller than those at other pH values. In the first 7 days, the cleavage of ester bonds in the microcapsule membrane occurs, which increases both the negative charge density and the polymer chain density in the surface region of the microcapsule membrane, because polymer segments produced by the degradation are not small enough to be liberated from the matrix. Between 7 and 75 days, the softness parameter increases with time. This is probably because the polymer chains become shorter by the degradation process and are released from the matrix. At the same time, the cleavage of the polymer chains
-0.15
i 0
i 20
i i 40 60 Time [days]
i 80
i 100
0.5
Fig. 4. Changes in zN and 1/2 on degradation at pH 5.4. Symbols indicate negative charge density, zN (©) and softness parameter, 1/)~(•).
should occur. Therefore, the negative charge density does not change during this stage. After 75 days, the cleavage of the ester bonds and the liberation of degraded polymer segments continuously proceed repeatedly with a period of about 75 days. This period of repetition will become shorter with time, because the surface area of the microcapsules decreases on degradation. If the degradation rates were small, oscillatory changes in z N and 1/)~ would be observed. Actually, at pH 9.8 (see Fig. 3), they are not seen except for a change in the initial stage, implying that the degradation rate is quite high at this pH. These findings suggest that the hydrolytic degradation of poly(Llactide) microcapsules starts at the microcapsule surface and proceeds to the membrane interior, like an erosion seen in some hydrogel layers [ 12]. The possible degradation processes are shown schematically in Fig. 5. Once the hydrolytic degradation starts from the particle surface, either of the following two types of process may follow. One is the case that the degradation proceeds toward the particle core in random directions, while the original surface region remains partially intact even in
K. Makino, H. Ohshima/Colloids Surfaces B: Biointerfaces 6 (1996) 373-378
378
membrane has a uniform structure (Type 2). The observed changes in z N and 1/2 suggest that poly(L-lactide) microcapsules degrade by the process described as Type 2. The period of repetition (75 days at pH 5.4) observed in Fig. 4 is considered to be the period in which one polymer layer unit is degraded and liberated from the microcapsule surface.
O
Intact poly(L-lactide) microcapsule
Degradation
~
~ Degradation
~eration
Libera
Degradation ~,
Type 1
Degradation
Type 2
Fig. 5. Schematic representation of possible hydrolytic degradation of poly(L-lactide) microscapsule membranes.
the final stage of the degradation until destruction of the microcapsule membrane (Type 1). Another is the case that the degradation proceeds in the direction perpendicular to the surface, over a unit of polymer layer, whose thickness is unknown, and this process repeats with a period corresponding to the polymer unit layer. This type of degradation may occur as if the microcapsule membrane has an onion-like structure in the degradation process, in spite of the fact that the intact microcapsule
References [ 1] M. Donbrow (Ed.), Microcapsules and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, FL, 1992. I-2] P.B. Deasy (Ed.), Microencapsulation and Related Drug Processes, Drugs and The Pharmaceutical Sciences, Vol. 20, M. Dekker, New York, 1994. I-3] K. Makino, M. Arakawa and T. Kondo, Chem. Pharm. Bull., 33 (1985) 1195. [4] K. Makino, H. Ohshima and T. Kondo, J. Microencapsulation, 3 (1986) 195. [5] K. Makino, H. Ohshima and T. Kondo, J. Microencapsulation, 3 (1986) 203. [6] K. Makino, H. Ohshima and T. Kondo, J. Microencapsulation, 4 (1987) 47. 1-7] H. Ohshima and S. Ohki, Biophys. J., 47 (1985) 673. [8] H. Ohshima, J. Colloid Interface Sci., 163 (1994) 474. 1-9] K. Makino, M. Ikekita, T. Kondo, S. Tanuma and H. Ohshima, Colloid Polym. Sci., 272 (1994) 487. [10] K. Makino, S. Yamamoto, K. Fujimoto, H. Kawaguchi and H. Ohshima, J. Colloid Interface Sci., 166 (1994) 251. [11] K. Makino, F. Fukai, T. Kawaguchi and H. Ohshima, Colloids Surfaces B: Biointerfaces, 5 (1995) 221. [12] K. Park, W.S.W. Shalaby and H. Park (Eds.), Biodegradable Hydrogels for Drug Delivery, Technomic Publications, Lancaster, UK, 1993.