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Synthesis of copoly(d,l -lactic acid) with relatively low molecular weight and in vitro degradation
Eur. Poh'm. J. Vol. 25. No. 10, pp. 1019 1026, 1989 Printed in Great Britain. All rights reserved
0014-3057/89 $3.00 + 0.00 Copyright c 1989 Pergamon Press pie
SYNTHESIS OF COPOLY(D,L-LACTIC ACID) WITH RELATIVELY LOW MOLECULAR WEIGHT A N D IN VITRO D E G R A D A T I O N HIRONOBU FUKUZAKI,* MASARU YOSHIDA, MASAHARUASANO and MINORU KUMAKURA Department of Development, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma 370-12, Japan (Received 30 November 1988; & ret'isedform 25 January 1989)
Copoly(o-lactic acid/L-lactic acid; o-LA/L-LA) with relatively low molecular weight (/14nll of 300-3800, was synthesized by direct copolycondensation without catalyst in an atmosphere of nitrogen at 200 C, to evaluate the difference in degradation between crystalline and amorphous copolymers. The non-enzymatic degradation of crystalline copolymer, e.g. a copoly(o-LA/L-LA, 2/98 mol%), showed a parabolic-type degradation pattern in the h3. 900 to 2700 range; degradation preferentially occurs in the amorphous regions because the matrix remains highly crystalline in the initial stages. In contrast, the non-enzymatic degradation of amorphous copolymer, e.g. a copoly(o-LA/L-LA, 50/50 mol%), can be divided into three types according to ~Stn, viz. parabolic-type for /Qn = 1500, linear-type for :Qn = 2200, S-type for M~ = 3500. For comparison, the copolymers were degraded in the presence of enzymes; the process was markedly accelerated by the action of esterase-type enzymes, of which Rhizopus delemer lipase gave the highest degradation activity. Abstract
INTRODUCTION Lactic acid, which has two optical isomers, L-lactic acid (L-LA) and D-lactic acid (D-LA), is present in nature either as an intermediate or as an end-product in carbohydrate metabolism. It is widely distributed in all living things, animals, micro-organisms and plants. Its polymer can be expected to degrade enzymatically or non-enzymatically [1-7] because of the ester bonds in the main chains, and so it is one of the most promising biodegradable materials for drug delivery systems [8 19]. For this purpose, we have studied the synthesis of ester bond-containing polymers with relatively low molecular weight by direct polycondensation in the absence of catalysts, e.g. poly(D_L-LA) with a number-average molecular weight of M, = 1800 2200 [20], copoly(L-LA/glycolic acid) with AIn = 1600-3300 [21] and copoly(L-LA/fvalerolactone) with Mn = 1500-2600 [22]. In this case, the use of catalysts for polycondensation is undesirable because catalyst residues remain in the polymer as terminal groups. On the other hand, for relatively high molecular weight polymers, the shaping treatment requires fusion at high temperature or the use of a suitable organic solvent, in which the polymer materials or drugs are liable to undergo degradation during treatment. To remove this defect, we have found a new technique for the shaping treatment, viz. a melt-pressing technique [23]. An important characteristic of this technique is that relatively low molecular weight polymers fuse easily under the mild heat-pressure conditions because of the low softing point, without the use of organic solvents; it is possible to shape into suitable forms such as needles, *Present address: Taki Chemical Co. Ltd, Midori-machi 2, Befu-cho, Kakogawa-shi, Hyogo 675-01. Japan.
particles, membranes and tablets on solidifications of the melt. In this report, we describe the synthesis of copoly(D-LA/I,-LA) with relatively low molecular weight of ~Q'n= 300-3800 by means of direct polycondensation without catalyst at 200°C under an atmosphere of N,, in order to obtain crystalline and amorphous copolymers. We also report the difference between the in vitro degradations of the crystalline and amorphous copolymers, in cylindrical form produced by the melt-pressing technique. EXPERIMENTAL PROCEDURES
Materials Aqueous solutions (90%) of lactic acid monomers, viz. o-LA with optical purity of 96% and L-LA with optical purity of 99%, measured by enzymatic methods [24], were purchased from C. V. Chemie Combinatie, Amsterdam. Other chemicals were special grades, Synthesis q/" copolv(D-LA /L-LA )
The synthesis of a relatively low molecular weight copoly(o-LA/t,-LA) was performed by direct polycondensation in the absence of catalyst. A suitable mixture of D-LA and L-LA was charged into a glass ampoule and then N~ was bubbled into the mixture at a rate of 200 ml/min. The ampoule was immersed in an oil bath at 200C, The copolymers thus obtained were used without further purification. Measurement of molecular weight
The molecular weight of copoly(D-LA/t,-LA) was measured by both terminal carboxyl group analysis and gel permeation chromatography (GPC). In the end-group analysis, the number-average molecular weight (~¢,) of copoly(D-LA/L-LA), dissolved in benzyl alcohol, was determined by titration using 0.025 N KOH in
1019
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benzyl alcohol with phenolphthalein as indicator; M, was calculated from: 1000 W (1) 0.025f(V- V0) where W is the weight of polymer (g), fis the titre of 0.025 N KOH, V is the volume of titrated solution and V0 is the blank volume of titrated solution, respectively. In GPC, the measurements were performed with a Waters high performance liquid chromatography, Model ALC-244, at 2 5 C at a flow rate of I ml/min through 102, l 0 3 and 104/~ Waters ultrastyragel columes, in tetrahydrofuran. In this measurement, the molecular weight of copolymer was calibrated by use of standard polystyrene. ]~n --
Instrumental analyses The melting point (m.p.) and glass transition temperature (Tg) of the copolymer were determined with a Seiko differential scanning calorimeter (DSC), Model DSC-10, at a heating rate of 5°C/rain. The crystallinity of the copolymer was obtained with a Rigaku X-ray diffractometer, using Ni-filtered CuKa radiation at 35 KV and 20mA. The optical rotation of the copolymer, [~]~, was measured by a Horiba polarimeter, Model SEPA-200, using D line of sodium at 20C. For this purpose, the copolymer was dissolved in CHCI 3 (1 wt% concentration).
Preparation of fine cylindrical copoly(o-LA /L-LA ) A fine cylindrical copoly(D-LA/L-LA) matrix was prepared by melt-pressing technique as reported previously [21]; the schematic diagram for its preparations is shown in Fig. 1. Crushed copolymer (50mg) was charged into the commercially available poly(tetrafluoroethylene) tube (2 mm i.d.) and then the piston rods (stainless steel) were inserted from both ends of the tube under a pressure of 100kg/cm 2. Under this condition, the copolymer was momentarily altered from the solid to the molten state when warmed at 40-5OC; the resulting solid matrix in a fine cylindrical form (2mm in dia, 10mm long) was finally obtained after removal of the heating treatment. This tube containing 50 mg of copolymer was sterilized by irradiating up to 30kGy at - 7 8 C with 7-rays from a 6°Co source.
t4vdrolysis (degradation) of copoly(D-LA / L-LA ) The degree of degradation of copoly(D-LA/L-LA) moulded in a fine cylindrical form was evaluated by noting change of weight of the copolymer on treatment in M/15 phosphate buffer solution (pH 7.2) containing either no enzyme or l mg of enzyme, e.g. carboxylic esterase (EC 3.1.1.1) from Porcine liver and lipases (EC 3.1.1.3) from Polymer powder
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Wheat germ, Hog pancreas and Rhizopus delemer (Sigma Chemical Co.). A fine cylindrical copolymer pushed out from the tube was immersed into the vial containing 10 ml of buffer solution with or without enzymes (one sample per vial, 5 vials per group). The buffer solutions with or without enzymes were freshly prepared every 24 hr. The hydrolysis was carried out at 37°C. At intervals, the specimen was collected from the vial, washed with distilled water, lyophilized and weighed. The degree of degradation was estimated from: Degree of degradation ( % ) -
100(D 0 - D)
(2) Do where D Ois the weight of copolymer before hydrolysis and D is the weight of copolymer after hydrolysis for a certain period.
Water absorption of copoly(o-LA /L-LA ) The water absorption of copoly(D-LA/L-LA) was measured in order to evaluate the degree of swelling which indicates the hydrophilicity of the copolymer. A fine cylindrical copolymer (5 samples per group) was immersed in 20 ml of M/15 phosphate buffer solution (pH 7.2) at 37"C. At intervals, the sample was removed from the flask, lyophilized and then weighed. The resulting water absorption was estimated from: Water absorption ( % ) -
lO0(Ww- Wo)
WD
(3)
where Ww is the weight of copolymer containing water after a certain time and WD is the weight of dried copolymer. RESULTS AND DISCUSSION
Reaction mechanism for direct copolycondensation of D-LA and L-LA without catalysts The synthesis of copoly(D-LA/L-LA) was performed by direct copolycondensation in the absence of catalyst at 200°C in a N 2 atmosphere, in order to obtain crystalline and a m o r p h o u s copolymers with relatively low molecular weights. The changes in molecular weight distribution of copoly(D-LA/L-LA) with compositions of 92/8, 50/50 and 2/98 r e a l % are shown in Fig. 2a as a function of reaction time. The distributions, a l t h o u g h showing plural peaks corresponding to the oligomers in the initial stages o f reaction, became single peaks after 3 hr reaction. In this case, the peak showed a right-side shift with the passage of time because of the f o r m a t i o n of higher molecular weight copolymer. The effect of m o n o m e r composition on M n of copoly(o-LA/L-LA) as a function of reaction time is shown in Fig. 3, which shows that the rate of copolycondensation is not affected by the difference in the two isomeric lactic acids. However, the value of ~ro as a function of m o n o m e r composition was markedly increased with increasing reaction time, e.g. 700 after 2 hr reaction, 1500 after 5 hr, 2200 after 10 hr a n d 3800 after 20 hr. 20 o f copoly(D-LA/e-LA) The specific r o t a t i o n ([~]D) is shown in Fig. 4. A copolymer c o m p o s e d o f equim o l a r p r o p o r t i o n s of o - L A and L-LA, copoly(o-LA/ L-LA, 50/50 m o l % ) , is optically inactive in the ~Qo 300-3800 range. On the contrary, the 92/8 and 2/98 m o l % D-LA/e-LA copolymers have optical activities but the direction of r o t a t i o n of the sodium D line is reversed, so that D-LA/L-LA with a m o n o m e r composition of 92/8 m o l % gives D ( + ) - p o l y ( L A ) a n d D-LA/L-LA with a m o n o m e r composition of 2/98 m o l % gives L ( - ) - p o l y ( L A ) . In this case, the [~]~
Synthesis of copoly(D,L-lactic acid) (o)
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Fig. 2. Molecular weight distributions of (a) copoly(o-LA/L-LA) as functions of monomer composition and reaction time, (b) crystalline copoly(D-LA/L-LA. 2/98 real%) as functions of M. and hydrolysis time (without enzyme), and (c) amorphous copoly(D-LA/L-LA, 50/50 mol%) as functions of h?o and hydrolysis time (without enzyme). value of the copolymer with an optically active configuration remained constant with increase in M, after reaching a maximum or a minimum at /Q, near 1500. This result means that no racemization occurs at .~, of 1500 or above. Using a linear relationship between [~]~ of copoly(D-LA/L-LA) with Mn = 2200 and monomer composition (Fig. 4b), the value of
[.~]~ of pure homopoly (LA) such as D(+) and L(--) was estimated: it was found to be + 1 5 0 ' for poly[D(+)-LA] and - 150 ~ for poly[L(--)-LA]. The crystallinity of copoly(D-LA/L-LA) with M, = 2200 was examined by the X-ray method. The X-ray diffraction patterns as a function of monomer composition are shown in Fig. 5. The copolymers
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Fig. 3. Effect of monomer composition on ~ , of copoly(o-LA/L-LA) as a function of reaction time. were annealed for 15 hr at 9 5 C in vacuo (10-3 mmHg) before examination. The D-LA and L-LA rich copolymers, e.g. copoly(D-LA/L-LA, 92/8 mol%) or copoly(D-LA/L-LA, 2/98 mol%), are crystalline; they show crystal lattice reflections of 20 = 16.7, 19.1 and 22.3 ~ for a 92 mol% D-LA-containing copolymer (Fig. 5a) and 20 = 14.7, 16.6 and 22.3 ° for a 98 mol% L-LA-containing copolymer (Fig. 5e). These reflections were completely missing for a wide range of L-LA compositions from 16 to 80mo1% (b, c and d in Fig. 5) because there is formation of amorphous copolymer. The DSC curves of copoly(D-LA/L-LA) with ~Q, = 2200 as a function of monomer composition are shown in Fig. 6a. The crystalline copoly(D-LA/L-LA) with compositions 92/8 and 2/98 tool% showed Tg and melting endotherm, in contrast to only Tg for amorphous copoly(D-LA/L-LA, 50/50 mol%). This finding agreed very closely with that obtained by X-ray measurement (Fig. 5),
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Fig. 5. X-ray diffraction patterns of copoly(D-LA/L-LA) (Mn = 2200) with monomer compositions of (a) 92/8 tool%, (b) 80/20mo1%, (c) 50/50mo1%, (d) 16/84mo1% and (e) 2/98 tool%. The effect of monomer composition on T~ of copoly(D-LA/L-LA) with hl, = 1500, 2200 and 3500 is shown in Fig. 7. The Tg of amorphous copoly(D-LA/L-LA), which forms over a wide range of L-LA compositions from 16 to 80 tool%, is generally lower than that of the crystalline copolymer. This difference may be due to difference in mobility of copolymer chains.
Hydrolysis mechanism o f copoly (D-LA /L-LA ) in the absence of enzymes The cylindrical sample of copoly(D-LA/L-LA) was treated in M/15 phosphate buffer solution (pH 7.2), in order to check the degradation of copolymer in the absence of enzyme at 37°C. The effect of hydrolysis
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Fig. 6. DSC curves of (a) copoly(D-LA,,'L-LA) with M~ = 2200 as a function of monomer composition and (b) crystalline copoly(D-LAL-LA, 2/98 mol%) as functions of .~n and hydrolysis time (without enzyme). time on the degradation of copoly(o-LA/L-LA) with M., =900, 1500, 2200, 2700 and 3500 is shown in Fig. 8 as a function of m o n o m e r composition. In the crystalline copoly(D-LA/L-LA) systems with compositions 92/8 and 2/98 mol%, the degree of degradation showed a gradual increase with time after rapid increase in the initial stage, i.e. a parabola-type degradation pattern (a and c in Fig. 8); as a result, it is strongly dependent on Mn of the copolymer, the rate of degradation increasing with decrease in ~Tfn. In contrast, the degradation pattern of an amorphous copoly(o-LA/L-LA, 50/50 tool%) can be divided into three types according to &co, parabola-type (Mo= 1500), linear-type (M'n=2200) and S-type (~3n = 3500), as seen clearly in Fig. 8b. The parabolatype is characterized by rapid degradation in the initial stage; a linear-type is characterized by steady degradation (zero-order), and a S-type is characterized by the existence of a lag time (induction period) after which the degradation suddenly accelerates after being greatly depressed during the first two weeks. On the basis of these phenomena, the difference in degradation pattern between crystalline and amor-
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was treated in M/15 phosphate buffer solution (pH 7.2) containing no enzyme at 37C. Mn: (O) 900, (V]) 1500, (•) 2200, (O) 2700, (11) 3500.
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HIRONOBU FUKUZAKIel al.
2/98 mol%) with Mn = 900 and 2200 are shown in Fig. 2b as a function of hydrolysis time. A crystalline copolymer with A4o=900 before hydrolysis has plural peaks corresponding to the oligomers and a single peak corresponding to the copolymer. The plural peaks corresponding to the oligomers completely disappeared during the first week of the test, because of dissolution. For this reason, a main single peak is gradually shifted to the left with time and it changes from broad to sharp. This means that the molecular weight of the copolymer decreases by scission of the main chain. Such a tendency was similarly observed in a crystalline copolymer with ~o = 2200. On the other hand, the changes in molecular weight distribution of amorphous copoly(o-LA/L-LA, 50/50 tool%) with •Scn= 1500, 2200 and 3500 are shown in Fig. 2c as a function of hydrolysis time. These copolymers have the three characteristic patterns of degradation according to 33~ (Fig. 8b). In these systems, the plural peaks corresponding to oligomers were observed for the copolymers with Mn = 1500 and 2200 before hydrolysis, but the peak areas decreased with increase in "Mn and so it was not observed for a copolymer with IQ, = 3500. The oligomer peaks disappeared completely with time. This disappearance may be attributed to the dissolution rather than degradation by the action of water. For this reason, the main single peak showed a slight shift to the left with increase of hydrolysis time and the shape of the distribution changed from broad to sharp. This tendency is independent of JQn" These findings mean that the cause of difference in degradation pattern, as seen in Fig. 8b, cannot be fully explained only from the information obtained by GPC. It should be pointed out that only the molecular weight distribution of copolymer with IQ~ = 3500 before hydrolysis shows a great variation during the second week of the test when the degradation of copolymer occurs rapidly; in other words, the peak is remarkably shifted to the left because of rapid formation of low molecular weight polymer. DSC measurements were performed to clarify the relationship between the changes in crystallinity of the copolymer and the degree of degradation of the copolymer by water (without enzyme). The DSC curves of crystalline copoly(D-LA/L-LA, 2/98 mol%) with M n = 900 and 2200, subject to treatment in M/15 phosphate buffer solution (pH 7.2) containing no enzyme at 3 7 C are shown in Fig. 6b. In this case, 10 mg of copolymer was used for the measurement of DSC in order to evaluate the quantitative heat of fusion. The DSC pattern of a copolymer with h4', = 900 before hydrolysis showed a small melting endotherm at about 110C owing to low crystallinity. The peak area corresponding to the melting endotherm was increased markedly accompanying a decrease in melting point of the copolymer with the passage of hydrolysis time. This fact means that the molecular weight of crystalline copolymer decreases by scission of the main chain and also that the hydrolysis by water occurs preferentially in the amorphous regions of the copolymer. This tendency can be similarly observed in a crystalline copolymer with ~¢. = 2200. Reed et al. reported that the in vitro degradation (without enzyme) of a poly(glycolic acid) Dexon"
suture occurs in 0.2 M citrate-phosphate buffer of pH 7.0 at 37°C; they proved that the material remains highly crystalline over the initial period of six weeks when degradation is taking place in the amorphous regions [6]. This conclusion agrees closely with our experimental data. From these findings, the degradation mechanism of crystalline copoly(D-LA/L-LA) with a parabolic-type degradation patter can be explained by the following three processes (1) dissolution of oligomer, (2) preferential hydrolysis of a relatively low molecular weight copolymer in the amorphous regions leading to a narrow molecular weight distribution to the left of the higher molecular weight and crystalline copolymers, and (3) rate-limiting degradation of the copolymer in the crystalline regions. The resulting degradation of crystalline copolymer mainly proceeds by the processes (1) and (2) in the initial stage and followed by (3), giving a parabolic-type degradation pattern. In a copoly(D-LA/L-LA, 50/50 mol%), the degradation mechanism cannot be explained by the difference in crystallinity because the copolymer is amorphous; it shows three degradation patterns according to /Qn, viz. parabolic (~¢n = 1500), linear (M n = 2200) and S-type (.~', = 3500). These matrices showed remarkably different shapes (morphologies) according to hS¢n when treated in M/15 phosphate buffer solution (pH 7.2) containing no enzyme at 37°C. The morphologies of amorphous copoly(D-LA/n-LA, 50/50 tool%)with M'n = 1500, 2200 and 3500, treated in M/15 phosphate buffer (pH7.2) containing no enzyme at 3 7 C for a period of 2 weeks, is shown in Fig. 9. The morphology of a copolymer with h4, = 1500 changes from cylindrical solid to soft and spongy, because of strong swelling and degradation (Fig. 9a); however, an amorphous copolymer with ~Sr = 2200 swells but retains the cylindrical form, because of moderate swelling and rate-limiting degradation (Fig. 9b). In contrast, in an amorphous copolymer with Mn = 3500, there are no apparent changes in morphology (Fig. 9c). Possibly this is due to low swelling and degradation. Therefore, it is reasonable to assume that the degradation of amorphous copoly(D-LA/L-LA) by the action of water is closely related to the degree of swelling as a function of ~r n. The changes in water absorption of samples of amorphous copoly(o-LA/L-LA, 50/50 mol%) with h4,= 1500, 2200 and 3500, treated in M/15 phosphate buffer solution containing no enzyme at 37~C is shown in Fig. 10a. The hydrophilicity of the copolymer is markedly dependent on .Q,, e.g. the water absorption of copolymers with ~¢n = 1500, 2200 and 3500 were 1030, 450 and 26% after seven days respectively. In this case, the water absorption-time relationships for the amorphous copolymers with h~, = 1500 and 2200 were similar except for the difference in degree of swelling, which reached saturation in the early stages (up to the seventh day) and then kept constant. On the contrary, the water absorption of a copolymer with ~Q'n= 3500 increased linearly with time. On the basis of data such as molecular weight distribution (Fig. 2c) and water absorption (Fig. 10a), an attempt was made to explain the degradation mechanism of amorphous samples of copoly(D-LA/L-LA, 50/50 mol%) with parabolic, linear- and S-type degradation patterns.
1025
Synthesis of copoly(D,L-lacticacid)
In an amorphous copolymer with a parabolic-type degradation pattern (Mn = 1500), the degradation proceeds by the two processes, (1) dissolution of oligomers and (2) preferential and marked degradations of low molecular weight copolymers accompanying the strongest degree of swelling. The degradation process of an amorphous copolymer with a4n = 3500 displays a S-type pattern because of the weakest degree of swelling in the initial stage. With the passage of time, a small amount of water gradually penetrates into the matrix. In this case, the cylindrical copolymer is much more subject to hydrolysis in the interior rather than the surface and the resulting acidity inside the matrix increases with time in which the changes in shape (cylindrical form) could not be observed apparently, This fact was confirmed by our finding that only copolymer inside the matrix changes from solid to pasty, owing to the degradation after 14 days and this is also suggested from the results of GPC (Fig. 2c). After a lag time of two weeks, weight loss of the copolymer occurs rapidly by bursting the barrier (copolymer membrane) keeping the cylindrical shape and followed by a parabolic-type degradation process, as for a copolymer with Mn = 1500. A linear-type degradation pattern of a copolymer with Mn = 2200 is intermediate between those of a parabolic- and S-types. This means that a linear-type degradation process can result from balance of a7¢. and water absorption in an amorphous copoly(o-LA/L-LA), for example ,~, is 2200 and water absorption is 400-500%.
Hydrolysis mechanism of copoly(D-LA /L-LA ) in the presence of enzymes Fig. 9. Changes in sh~,peof amorphous copoly(D-LA/L-LA, 50/50mo1%) with {a) 33"n=1500, (b) a4n=2200 and (c) M,~= 3500, which were treated in M/15 phosphate buffer solution (pH 7,2) containing no enzyme at 37C for a period of 2 weeks. The shape of the copolymer before the treatment of hydrolysis is a fine cylindrical matrix (2 mm in dia, 10 mm long),
The hydrolysis of copoly(D-LA/L-LA) may be expected to be accelerated by esterase-type enzymes, such as carboxylic esterase and lipase, because it has an ester bond in the main chain. The effect of monomer composition on the enzymatic degradation of copoly(D-LA/L-LA) with a~n = 2200 after 10 days is shown in Fig. 1la as a function of the kind of enzyme. The degradation was (b)
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Fig. 10. Changes in water absorption of (a) amorphous copoly(D-LA/L-LA, 50/50mo1%) with ([Z]) M,~= 1500, (~) 2200 and ( I ) 3500, and (b) copoly(D-LA/L-LA)with a4, = 2200 as functions of time and monomer composition. EPJ
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HIRONOBU FUKUZAKI et al.
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is suggested by the data on water absorption of copoly(D-LA/L-LA) as shown clearly in Fig. 10b. It is reasonable to conclude that the degradation of copoly(D-LA/L-LA) with relatively low molecular weight, obtained by direct copolycondensation without catalyst, is accelerated by esterase-type enzymes which cleave the ester bonds of the copolymer, especially in the amorphous regions.
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REFERENCES
20 ~
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Composition (mot-%) Fig. I I. Enzymatic degradations o f c o p o ] y ( D - L A / L - L A ) with M~ = 2200 treated at 37;C, (a) for 10 days as functions
of kind of enzyme and monomer composition, and (b) by Rhizopus delemer lipase as functions of hydrolysis time and monomer composition. Kind of enzyme: (11) Rhizopus delemer lipase; (A) Hog pancreas lipase; (D) Carboxylic esterase from Porcine liver; (Q) Wheat germ lipase; (O) control (without enzyme).
markedly accelerated by the addition of enzyme; the amorphous copolymers ranging in L-LA m o n o m e r composition from 16 to 80 m o l % are much more subject to hydrolysis by the action of enzyme than crystalline copolymers and the resulting Rhizopus delemer lipase gives the highest degradation ability. The effect of monomer composition on the enzymatic degradation of copoly(D-LA/L-LA) with hZFn = 2200 by Rhizopus delemer lipase is shown in Fig. l lb as a function of time. The rate of degradation of the copolymer tended to increase with time; the increase is especially marked in the amorphous regions, where there is complete degradation after 21 days. In contrast, in the crystalline regions, the degradation is very slow, e.g. the degree of degradation was found to be 38% for a crystalline copoly(D-LA/L-LA, 92/8 mol%) and 24% for a crystalline copoly(o-LA/L-LA, 2/98 tool,) even after 21 days. This is because the hydrophilicity of the crystalline regions is lower than that of amorphous regions and so it is difficult for the enzymes to penetrate into the crystalline regions. This
I . I . Tabushi, H. Yamada, H. Matsuzaki and J. Furukawa. J. Polym. Sci.; Polym. Lett. Edn 13, 447 (1975). 2. D. F. Williams. J. Bioengng 1, 279 (1977). 3. K. Makino, M. Arakawa and T. Kondo. Chem. Pharm. Bull. 33, 1195 (1985). 4. M. Asano, M. Yoshida, I. Kaetsu, R. Katakai, T. Mashimo, H. Yuasa, K. Imai and H. Yamanaka. Polym. J. 20, 281 (1988). 5. C. G. Pitt, M. M. Gratzl, G. L. Kimmel, J. Surles and A. Schindler. Biomaterials 2, 215 (1981). 6. A. M. Reed and D. K. Gilding. Polymer 22, 494 (1981). 7. R. A. Kenley, M. O. Lee, T. R. Mahoney 1I and L. M. Sanders. Macromolecules 20, 2398 (1987). 8. S. Yolles, T. D. Leafe anti F. J. Meyer. J. Pharm. Sci. 64, 115 (1975). 9. C. G. Pitt, A. R. Jeffcoat, R. A. Zweidinger and A. Schindler. J. Biomed. Mater. Res. 13, 497 (1979). 10. G. Benagiano and H. L. Gabelnick. J. Steroid Biochem. 11,449 (1979). 11. L. R. Beck, R. A. Ramos, C. E. Flowers Jr, G. Z. Lopez, D. H. Lewis and D. R. Cowsar. Am. J. Obstet. Gynecol. 140, 799 (1981). 12. N. Wakiyama, K. Juni and M. Nakano. Chem. Pharm. Bull. 30, 2621 (1982). 13. N. Wakiyama, K. Juni and M. Nakano. Chem. Pharm. Bull. 30, 3719 (1982). 14. M. Hirano, M. Sakatoku, R. Yamashita and T. Iwa. Japan. J. Art!£ Organs 13, 1176 (1984). 15. D. R. Cowsar, T. R. Tice, R. M. Gilley and J. P. English. Meth. Encym. 112, 101 (1985). 16. J. P. Kitchell and D. L. Wise. Meth. Enzym. 112, 436 (1985). 17. L. M. Sanders, B. A. Kell, G. I. McRae and G. W. Whitehead. J. Pharm. Sci. 75, 356 (1986). 18. J. M. Schakenraad, J. A. Oosterbaan, P. Nieuwenhuis, I. Molenaar, J.Olijslager, W. Potman, M. J. D. Eenink and J. Feijen. Biomaterials 9, 116 (1988). 19. J. Kost, K. Leong and R. Langer. Makromolek. Chem., Macromolec. Symp. 19, 275 (1988). 20. M. Asano, M. Yoshida, I. Kaetsu, K. Imai, T. Mashimo, H. Yuasa, H. Yamanaka, K. Suzuki and 1. Yamazaki. Makromolek. Chem., Rapid Commun. 6, 509 (1985). 21. M. Asano, H. Fukuzaki, M. Yoshida, T. Mashimo, H. Yuasa, K. Imai, H. Yamanaka and K. Suzuki. J. Control. Release accepted for publication. 22. H. Fukuzaki, M. Yoshida, M. Asano, Y. Aiba and 1. Kaetsu. Eur. Polym. J. 24, 1029 (1988). 23. M. Asano, M. Yoshida, I. Kaetsu, H. Yamanaka, K. Nakai, H. Yuasa, K. Shida and M. Oya. J. Macromolec. Sci.-Chem. A21, 561 (1984). 24. F. Holl. In Methods of Enzymatic Analysis (Edited by H. U. Bergmeyer), pp. 1475-1479. Verlag Chemie Weinheim and Academic Press, New York (1974).
0014-3057/89 $3.00 + 0.00 Copyright c 1989 Pergamon Press pie
SYNTHESIS OF COPOLY(D,L-LACTIC ACID) WITH RELATIVELY LOW MOLECULAR WEIGHT A N D IN VITRO D E G R A D A T I O N HIRONOBU FUKUZAKI,* MASARU YOSHIDA, MASAHARUASANO and MINORU KUMAKURA Department of Development, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma 370-12, Japan (Received 30 November 1988; & ret'isedform 25 January 1989)
Copoly(o-lactic acid/L-lactic acid; o-LA/L-LA) with relatively low molecular weight (/14nll of 300-3800, was synthesized by direct copolycondensation without catalyst in an atmosphere of nitrogen at 200 C, to evaluate the difference in degradation between crystalline and amorphous copolymers. The non-enzymatic degradation of crystalline copolymer, e.g. a copoly(o-LA/L-LA, 2/98 mol%), showed a parabolic-type degradation pattern in the h3. 900 to 2700 range; degradation preferentially occurs in the amorphous regions because the matrix remains highly crystalline in the initial stages. In contrast, the non-enzymatic degradation of amorphous copolymer, e.g. a copoly(o-LA/L-LA, 50/50 mol%), can be divided into three types according to ~Stn, viz. parabolic-type for /Qn = 1500, linear-type for :Qn = 2200, S-type for M~ = 3500. For comparison, the copolymers were degraded in the presence of enzymes; the process was markedly accelerated by the action of esterase-type enzymes, of which Rhizopus delemer lipase gave the highest degradation activity. Abstract
INTRODUCTION Lactic acid, which has two optical isomers, L-lactic acid (L-LA) and D-lactic acid (D-LA), is present in nature either as an intermediate or as an end-product in carbohydrate metabolism. It is widely distributed in all living things, animals, micro-organisms and plants. Its polymer can be expected to degrade enzymatically or non-enzymatically [1-7] because of the ester bonds in the main chains, and so it is one of the most promising biodegradable materials for drug delivery systems [8 19]. For this purpose, we have studied the synthesis of ester bond-containing polymers with relatively low molecular weight by direct polycondensation in the absence of catalysts, e.g. poly(D_L-LA) with a number-average molecular weight of M, = 1800 2200 [20], copoly(L-LA/glycolic acid) with AIn = 1600-3300 [21] and copoly(L-LA/fvalerolactone) with Mn = 1500-2600 [22]. In this case, the use of catalysts for polycondensation is undesirable because catalyst residues remain in the polymer as terminal groups. On the other hand, for relatively high molecular weight polymers, the shaping treatment requires fusion at high temperature or the use of a suitable organic solvent, in which the polymer materials or drugs are liable to undergo degradation during treatment. To remove this defect, we have found a new technique for the shaping treatment, viz. a melt-pressing technique [23]. An important characteristic of this technique is that relatively low molecular weight polymers fuse easily under the mild heat-pressure conditions because of the low softing point, without the use of organic solvents; it is possible to shape into suitable forms such as needles, *Present address: Taki Chemical Co. Ltd, Midori-machi 2, Befu-cho, Kakogawa-shi, Hyogo 675-01. Japan.
particles, membranes and tablets on solidifications of the melt. In this report, we describe the synthesis of copoly(D-LA/I,-LA) with relatively low molecular weight of ~Q'n= 300-3800 by means of direct polycondensation without catalyst at 200°C under an atmosphere of N,, in order to obtain crystalline and amorphous copolymers. We also report the difference between the in vitro degradations of the crystalline and amorphous copolymers, in cylindrical form produced by the melt-pressing technique. EXPERIMENTAL PROCEDURES
Materials Aqueous solutions (90%) of lactic acid monomers, viz. o-LA with optical purity of 96% and L-LA with optical purity of 99%, measured by enzymatic methods [24], were purchased from C. V. Chemie Combinatie, Amsterdam. Other chemicals were special grades, Synthesis q/" copolv(D-LA /L-LA )
The synthesis of a relatively low molecular weight copoly(o-LA/t,-LA) was performed by direct polycondensation in the absence of catalyst. A suitable mixture of D-LA and L-LA was charged into a glass ampoule and then N~ was bubbled into the mixture at a rate of 200 ml/min. The ampoule was immersed in an oil bath at 200C, The copolymers thus obtained were used without further purification. Measurement of molecular weight
The molecular weight of copoly(D-LA/t,-LA) was measured by both terminal carboxyl group analysis and gel permeation chromatography (GPC). In the end-group analysis, the number-average molecular weight (~¢,) of copoly(D-LA/L-LA), dissolved in benzyl alcohol, was determined by titration using 0.025 N KOH in
1019
HIRONOBU FUKUZAKIel a[.
1020
benzyl alcohol with phenolphthalein as indicator; M, was calculated from: 1000 W (1) 0.025f(V- V0) where W is the weight of polymer (g), fis the titre of 0.025 N KOH, V is the volume of titrated solution and V0 is the blank volume of titrated solution, respectively. In GPC, the measurements were performed with a Waters high performance liquid chromatography, Model ALC-244, at 2 5 C at a flow rate of I ml/min through 102, l 0 3 and 104/~ Waters ultrastyragel columes, in tetrahydrofuran. In this measurement, the molecular weight of copolymer was calibrated by use of standard polystyrene. ]~n --
Instrumental analyses The melting point (m.p.) and glass transition temperature (Tg) of the copolymer were determined with a Seiko differential scanning calorimeter (DSC), Model DSC-10, at a heating rate of 5°C/rain. The crystallinity of the copolymer was obtained with a Rigaku X-ray diffractometer, using Ni-filtered CuKa radiation at 35 KV and 20mA. The optical rotation of the copolymer, [~]~, was measured by a Horiba polarimeter, Model SEPA-200, using D line of sodium at 20C. For this purpose, the copolymer was dissolved in CHCI 3 (1 wt% concentration).
Preparation of fine cylindrical copoly(o-LA /L-LA ) A fine cylindrical copoly(D-LA/L-LA) matrix was prepared by melt-pressing technique as reported previously [21]; the schematic diagram for its preparations is shown in Fig. 1. Crushed copolymer (50mg) was charged into the commercially available poly(tetrafluoroethylene) tube (2 mm i.d.) and then the piston rods (stainless steel) were inserted from both ends of the tube under a pressure of 100kg/cm 2. Under this condition, the copolymer was momentarily altered from the solid to the molten state when warmed at 40-5OC; the resulting solid matrix in a fine cylindrical form (2mm in dia, 10mm long) was finally obtained after removal of the heating treatment. This tube containing 50 mg of copolymer was sterilized by irradiating up to 30kGy at - 7 8 C with 7-rays from a 6°Co source.
t4vdrolysis (degradation) of copoly(D-LA / L-LA ) The degree of degradation of copoly(D-LA/L-LA) moulded in a fine cylindrical form was evaluated by noting change of weight of the copolymer on treatment in M/15 phosphate buffer solution (pH 7.2) containing either no enzyme or l mg of enzyme, e.g. carboxylic esterase (EC 3.1.1.1) from Porcine liver and lipases (EC 3.1.1.3) from Polymer powder
(o)
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(T-rays, -78"C) Cylindricat potymer matrix (2ram in diameter, lOmm long) Fig. 1. Schematic diagram for the preparation of a fine cylindrical copolymer matrix by melt-pressing.
Wheat germ, Hog pancreas and Rhizopus delemer (Sigma Chemical Co.). A fine cylindrical copolymer pushed out from the tube was immersed into the vial containing 10 ml of buffer solution with or without enzymes (one sample per vial, 5 vials per group). The buffer solutions with or without enzymes were freshly prepared every 24 hr. The hydrolysis was carried out at 37°C. At intervals, the specimen was collected from the vial, washed with distilled water, lyophilized and weighed. The degree of degradation was estimated from: Degree of degradation ( % ) -
100(D 0 - D)
(2) Do where D Ois the weight of copolymer before hydrolysis and D is the weight of copolymer after hydrolysis for a certain period.
Water absorption of copoly(o-LA /L-LA ) The water absorption of copoly(D-LA/L-LA) was measured in order to evaluate the degree of swelling which indicates the hydrophilicity of the copolymer. A fine cylindrical copolymer (5 samples per group) was immersed in 20 ml of M/15 phosphate buffer solution (pH 7.2) at 37"C. At intervals, the sample was removed from the flask, lyophilized and then weighed. The resulting water absorption was estimated from: Water absorption ( % ) -
lO0(Ww- Wo)
WD
(3)
where Ww is the weight of copolymer containing water after a certain time and WD is the weight of dried copolymer. RESULTS AND DISCUSSION
Reaction mechanism for direct copolycondensation of D-LA and L-LA without catalysts The synthesis of copoly(D-LA/L-LA) was performed by direct copolycondensation in the absence of catalyst at 200°C in a N 2 atmosphere, in order to obtain crystalline and a m o r p h o u s copolymers with relatively low molecular weights. The changes in molecular weight distribution of copoly(D-LA/L-LA) with compositions of 92/8, 50/50 and 2/98 r e a l % are shown in Fig. 2a as a function of reaction time. The distributions, a l t h o u g h showing plural peaks corresponding to the oligomers in the initial stages o f reaction, became single peaks after 3 hr reaction. In this case, the peak showed a right-side shift with the passage of time because of the f o r m a t i o n of higher molecular weight copolymer. The effect of m o n o m e r composition on M n of copoly(o-LA/L-LA) as a function of reaction time is shown in Fig. 3, which shows that the rate of copolycondensation is not affected by the difference in the two isomeric lactic acids. However, the value of ~ro as a function of m o n o m e r composition was markedly increased with increasing reaction time, e.g. 700 after 2 hr reaction, 1500 after 5 hr, 2200 after 10 hr a n d 3800 after 20 hr. 20 o f copoly(D-LA/e-LA) The specific r o t a t i o n ([~]D) is shown in Fig. 4. A copolymer c o m p o s e d o f equim o l a r p r o p o r t i o n s of o - L A and L-LA, copoly(o-LA/ L-LA, 50/50 m o l % ) , is optically inactive in the ~Qo 300-3800 range. On the contrary, the 92/8 and 2/98 m o l % D-LA/e-LA copolymers have optical activities but the direction of r o t a t i o n of the sodium D line is reversed, so that D-LA/L-LA with a m o n o m e r composition of 92/8 m o l % gives D ( + ) - p o l y ( L A ) a n d D-LA/L-LA with a m o n o m e r composition of 2/98 m o l % gives L ( - ) - p o l y ( L A ) . In this case, the [~]~
Synthesis of copoly(D,L-lactic acid) (o)
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Fig. 2. Molecular weight distributions of (a) copoly(o-LA/L-LA) as functions of monomer composition and reaction time, (b) crystalline copoly(D-LA/L-LA. 2/98 real%) as functions of M. and hydrolysis time (without enzyme), and (c) amorphous copoly(D-LA/L-LA, 50/50 mol%) as functions of h?o and hydrolysis time (without enzyme). value of the copolymer with an optically active configuration remained constant with increase in M, after reaching a maximum or a minimum at /Q, near 1500. This result means that no racemization occurs at .~, of 1500 or above. Using a linear relationship between [~]~ of copoly(D-LA/L-LA) with Mn = 2200 and monomer composition (Fig. 4b), the value of
[.~]~ of pure homopoly (LA) such as D(+) and L(--) was estimated: it was found to be + 1 5 0 ' for poly[D(+)-LA] and - 150 ~ for poly[L(--)-LA]. The crystallinity of copoly(D-LA/L-LA) with M, = 2200 was examined by the X-ray method. The X-ray diffraction patterns as a function of monomer composition are shown in Fig. 5. The copolymers
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Fig. 5. X-ray diffraction patterns of copoly(D-LA/L-LA) (Mn = 2200) with monomer compositions of (a) 92/8 tool%, (b) 80/20mo1%, (c) 50/50mo1%, (d) 16/84mo1% and (e) 2/98 tool%. The effect of monomer composition on T~ of copoly(D-LA/L-LA) with hl, = 1500, 2200 and 3500 is shown in Fig. 7. The Tg of amorphous copoly(D-LA/L-LA), which forms over a wide range of L-LA compositions from 16 to 80 tool%, is generally lower than that of the crystalline copolymer. This difference may be due to difference in mobility of copolymer chains.
Hydrolysis mechanism o f copoly (D-LA /L-LA ) in the absence of enzymes The cylindrical sample of copoly(D-LA/L-LA) was treated in M/15 phosphate buffer solution (pH 7.2), in order to check the degradation of copolymer in the absence of enzyme at 37°C. The effect of hydrolysis
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Fig. 6. DSC curves of (a) copoly(D-LA,,'L-LA) with M~ = 2200 as a function of monomer composition and (b) crystalline copoly(D-LAL-LA, 2/98 mol%) as functions of .~n and hydrolysis time (without enzyme). time on the degradation of copoly(o-LA/L-LA) with M., =900, 1500, 2200, 2700 and 3500 is shown in Fig. 8 as a function of m o n o m e r composition. In the crystalline copoly(D-LA/L-LA) systems with compositions 92/8 and 2/98 mol%, the degree of degradation showed a gradual increase with time after rapid increase in the initial stage, i.e. a parabola-type degradation pattern (a and c in Fig. 8); as a result, it is strongly dependent on Mn of the copolymer, the rate of degradation increasing with decrease in ~Tfn. In contrast, the degradation pattern of an amorphous copoly(o-LA/L-LA, 50/50 tool%) can be divided into three types according to &co, parabola-type (Mo= 1500), linear-type (M'n=2200) and S-type (~3n = 3500), as seen clearly in Fig. 8b. The parabolatype is characterized by rapid degradation in the initial stage; a linear-type is characterized by steady degradation (zero-order), and a S-type is characterized by the existence of a lag time (induction period) after which the degradation suddenly accelerates after being greatly depressed during the first two weeks. On the basis of these phenomena, the difference in degradation pattern between crystalline and amor-
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was treated in M/15 phosphate buffer solution (pH 7.2) containing no enzyme at 37C. Mn: (O) 900, (V]) 1500, (•) 2200, (O) 2700, (11) 3500.
1024
HIRONOBU FUKUZAKIel al.
2/98 mol%) with Mn = 900 and 2200 are shown in Fig. 2b as a function of hydrolysis time. A crystalline copolymer with A4o=900 before hydrolysis has plural peaks corresponding to the oligomers and a single peak corresponding to the copolymer. The plural peaks corresponding to the oligomers completely disappeared during the first week of the test, because of dissolution. For this reason, a main single peak is gradually shifted to the left with time and it changes from broad to sharp. This means that the molecular weight of the copolymer decreases by scission of the main chain. Such a tendency was similarly observed in a crystalline copolymer with ~o = 2200. On the other hand, the changes in molecular weight distribution of amorphous copoly(o-LA/L-LA, 50/50 tool%) with •Scn= 1500, 2200 and 3500 are shown in Fig. 2c as a function of hydrolysis time. These copolymers have the three characteristic patterns of degradation according to 33~ (Fig. 8b). In these systems, the plural peaks corresponding to oligomers were observed for the copolymers with Mn = 1500 and 2200 before hydrolysis, but the peak areas decreased with increase in "Mn and so it was not observed for a copolymer with IQ, = 3500. The oligomer peaks disappeared completely with time. This disappearance may be attributed to the dissolution rather than degradation by the action of water. For this reason, the main single peak showed a slight shift to the left with increase of hydrolysis time and the shape of the distribution changed from broad to sharp. This tendency is independent of JQn" These findings mean that the cause of difference in degradation pattern, as seen in Fig. 8b, cannot be fully explained only from the information obtained by GPC. It should be pointed out that only the molecular weight distribution of copolymer with IQ~ = 3500 before hydrolysis shows a great variation during the second week of the test when the degradation of copolymer occurs rapidly; in other words, the peak is remarkably shifted to the left because of rapid formation of low molecular weight polymer. DSC measurements were performed to clarify the relationship between the changes in crystallinity of the copolymer and the degree of degradation of the copolymer by water (without enzyme). The DSC curves of crystalline copoly(D-LA/L-LA, 2/98 mol%) with M n = 900 and 2200, subject to treatment in M/15 phosphate buffer solution (pH 7.2) containing no enzyme at 3 7 C are shown in Fig. 6b. In this case, 10 mg of copolymer was used for the measurement of DSC in order to evaluate the quantitative heat of fusion. The DSC pattern of a copolymer with h4', = 900 before hydrolysis showed a small melting endotherm at about 110C owing to low crystallinity. The peak area corresponding to the melting endotherm was increased markedly accompanying a decrease in melting point of the copolymer with the passage of hydrolysis time. This fact means that the molecular weight of crystalline copolymer decreases by scission of the main chain and also that the hydrolysis by water occurs preferentially in the amorphous regions of the copolymer. This tendency can be similarly observed in a crystalline copolymer with ~¢. = 2200. Reed et al. reported that the in vitro degradation (without enzyme) of a poly(glycolic acid) Dexon"
suture occurs in 0.2 M citrate-phosphate buffer of pH 7.0 at 37°C; they proved that the material remains highly crystalline over the initial period of six weeks when degradation is taking place in the amorphous regions [6]. This conclusion agrees closely with our experimental data. From these findings, the degradation mechanism of crystalline copoly(D-LA/L-LA) with a parabolic-type degradation patter can be explained by the following three processes (1) dissolution of oligomer, (2) preferential hydrolysis of a relatively low molecular weight copolymer in the amorphous regions leading to a narrow molecular weight distribution to the left of the higher molecular weight and crystalline copolymers, and (3) rate-limiting degradation of the copolymer in the crystalline regions. The resulting degradation of crystalline copolymer mainly proceeds by the processes (1) and (2) in the initial stage and followed by (3), giving a parabolic-type degradation pattern. In a copoly(D-LA/L-LA, 50/50 mol%), the degradation mechanism cannot be explained by the difference in crystallinity because the copolymer is amorphous; it shows three degradation patterns according to /Qn, viz. parabolic (~¢n = 1500), linear (M n = 2200) and S-type (.~', = 3500). These matrices showed remarkably different shapes (morphologies) according to hS¢n when treated in M/15 phosphate buffer solution (pH 7.2) containing no enzyme at 37°C. The morphologies of amorphous copoly(D-LA/n-LA, 50/50 tool%)with M'n = 1500, 2200 and 3500, treated in M/15 phosphate buffer (pH7.2) containing no enzyme at 3 7 C for a period of 2 weeks, is shown in Fig. 9. The morphology of a copolymer with h4, = 1500 changes from cylindrical solid to soft and spongy, because of strong swelling and degradation (Fig. 9a); however, an amorphous copolymer with ~Sr = 2200 swells but retains the cylindrical form, because of moderate swelling and rate-limiting degradation (Fig. 9b). In contrast, in an amorphous copolymer with Mn = 3500, there are no apparent changes in morphology (Fig. 9c). Possibly this is due to low swelling and degradation. Therefore, it is reasonable to assume that the degradation of amorphous copoly(D-LA/L-LA) by the action of water is closely related to the degree of swelling as a function of ~r n. The changes in water absorption of samples of amorphous copoly(o-LA/L-LA, 50/50 mol%) with h4,= 1500, 2200 and 3500, treated in M/15 phosphate buffer solution containing no enzyme at 37~C is shown in Fig. 10a. The hydrophilicity of the copolymer is markedly dependent on .Q,, e.g. the water absorption of copolymers with ~¢n = 1500, 2200 and 3500 were 1030, 450 and 26% after seven days respectively. In this case, the water absorption-time relationships for the amorphous copolymers with h~, = 1500 and 2200 were similar except for the difference in degree of swelling, which reached saturation in the early stages (up to the seventh day) and then kept constant. On the contrary, the water absorption of a copolymer with ~Q'n= 3500 increased linearly with time. On the basis of data such as molecular weight distribution (Fig. 2c) and water absorption (Fig. 10a), an attempt was made to explain the degradation mechanism of amorphous samples of copoly(D-LA/L-LA, 50/50 mol%) with parabolic, linear- and S-type degradation patterns.
1025
Synthesis of copoly(D,L-lacticacid)
In an amorphous copolymer with a parabolic-type degradation pattern (Mn = 1500), the degradation proceeds by the two processes, (1) dissolution of oligomers and (2) preferential and marked degradations of low molecular weight copolymers accompanying the strongest degree of swelling. The degradation process of an amorphous copolymer with a4n = 3500 displays a S-type pattern because of the weakest degree of swelling in the initial stage. With the passage of time, a small amount of water gradually penetrates into the matrix. In this case, the cylindrical copolymer is much more subject to hydrolysis in the interior rather than the surface and the resulting acidity inside the matrix increases with time in which the changes in shape (cylindrical form) could not be observed apparently, This fact was confirmed by our finding that only copolymer inside the matrix changes from solid to pasty, owing to the degradation after 14 days and this is also suggested from the results of GPC (Fig. 2c). After a lag time of two weeks, weight loss of the copolymer occurs rapidly by bursting the barrier (copolymer membrane) keeping the cylindrical shape and followed by a parabolic-type degradation process, as for a copolymer with Mn = 1500. A linear-type degradation pattern of a copolymer with Mn = 2200 is intermediate between those of a parabolic- and S-types. This means that a linear-type degradation process can result from balance of a7¢. and water absorption in an amorphous copoly(o-LA/L-LA), for example ,~, is 2200 and water absorption is 400-500%.
Hydrolysis mechanism of copoly(D-LA /L-LA ) in the presence of enzymes Fig. 9. Changes in sh~,peof amorphous copoly(D-LA/L-LA, 50/50mo1%) with {a) 33"n=1500, (b) a4n=2200 and (c) M,~= 3500, which were treated in M/15 phosphate buffer solution (pH 7,2) containing no enzyme at 37C for a period of 2 weeks. The shape of the copolymer before the treatment of hydrolysis is a fine cylindrical matrix (2 mm in dia, 10 mm long),
The hydrolysis of copoly(D-LA/L-LA) may be expected to be accelerated by esterase-type enzymes, such as carboxylic esterase and lipase, because it has an ester bond in the main chain. The effect of monomer composition on the enzymatic degradation of copoly(D-LA/L-LA) with a~n = 2200 after 10 days is shown in Fig. 1la as a function of the kind of enzyme. The degradation was (b)
2000 1000
1000
500 2OO
500 200
tO0
100
50 2O
30 20
I0
10
+-
ff
g g
3
5 -tel 0
I
I
I
I
I
5
10
15
20
25
Time
(days)
" D-LA
I 50 Composition
L-LA (moL-%)
Fig. 10. Changes in water absorption of (a) amorphous copoly(D-LA/L-LA, 50/50mo1%) with ([Z]) M,~= 1500, (~) 2200 and ( I ) 3500, and (b) copoly(D-LA/L-LA)with a4, = 2200 as functions of time and monomer composition. EPJ
251{b ('
1026
HIRONOBU FUKUZAKI et al.
100 ,
is suggested by the data on water absorption of copoly(D-LA/L-LA) as shown clearly in Fig. 10b. It is reasonable to conclude that the degradation of copoly(D-LA/L-LA) with relatively low molecular weight, obtained by direct copolycondensation without catalyst, is accelerated by esterase-type enzymes which cleave the ester bonds of the copolymer, especially in the amorphous regions.
60 40
REFERENCES
20 ~
I
o
loo 1~/.-21d ~ o
14
8o iii
60 40 20 0 D-LA
50
L-LA
Composition (mot-%) Fig. I I. Enzymatic degradations o f c o p o ] y ( D - L A / L - L A ) with M~ = 2200 treated at 37;C, (a) for 10 days as functions
of kind of enzyme and monomer composition, and (b) by Rhizopus delemer lipase as functions of hydrolysis time and monomer composition. Kind of enzyme: (11) Rhizopus delemer lipase; (A) Hog pancreas lipase; (D) Carboxylic esterase from Porcine liver; (Q) Wheat germ lipase; (O) control (without enzyme).
markedly accelerated by the addition of enzyme; the amorphous copolymers ranging in L-LA m o n o m e r composition from 16 to 80 m o l % are much more subject to hydrolysis by the action of enzyme than crystalline copolymers and the resulting Rhizopus delemer lipase gives the highest degradation ability. The effect of monomer composition on the enzymatic degradation of copoly(D-LA/L-LA) with hZFn = 2200 by Rhizopus delemer lipase is shown in Fig. l lb as a function of time. The rate of degradation of the copolymer tended to increase with time; the increase is especially marked in the amorphous regions, where there is complete degradation after 21 days. In contrast, in the crystalline regions, the degradation is very slow, e.g. the degree of degradation was found to be 38% for a crystalline copoly(D-LA/L-LA, 92/8 mol%) and 24% for a crystalline copoly(o-LA/L-LA, 2/98 tool,) even after 21 days. This is because the hydrophilicity of the crystalline regions is lower than that of amorphous regions and so it is difficult for the enzymes to penetrate into the crystalline regions. This
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