Polymer International 41 (1996) 369-375

Preparation, Characterization and Biodegradable Characteristics of

Poly( I ,3-trimethylene carbonate-co-glycolide)

Cai Jie, K. J. Zhu* & Yang Shilin

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 3 10027, People's Republic of China

(Received 19 February 1996; accepted 22 May 1996)

Abstract: Poly( 1,3-trimethylene carbonate-co-glycolide) (PGCA) has been syn- thesized by ring-opening polymerization of 1,3-trimethylene carbonate (CA) and glycolide (GA) with stannous octoate as catalyst. The copolymers were charac- terized by 'H nuclear magnetic resonance (NMR), 13C NMR and differential scanning calorimetry. Water content and static contact angle of distilled water on the polymer surface were used to evaluate the hydrophobicity of the copoly- mers. There was no apparent difference in hydrophobicities of copolymers con- taining up to 30mol% GA. The biodegradation of PGCA was conducted in phosphate buffer solution at 37°C and in rats. The results indicated that the degradation rates of PGCA were higher than that of PCA and depended on the GA fraction in the copolymers. Furthermore, degradation occurred in the bulk when the GA content exceeded 20mol%. With less GA units the degradation became a surface reaction both in uitro and in uiuo. These properties of PGCA may be useful in protein delivery systems.

K e y words: poly (1,3-trimethylene carbonate-co-glycolide), polymer surface bio- degradation, protein-controlled release matrix.

IN TR 0 D U CTI 0 N

There have been many papers concerned with polymers to be applied in drug controlled release Among them, biodegradable polymers have an impor- tant role, because they can be converted to soluble frag- ments in vivo that can be metabolized or With developing biotechnology, many peptides and proteins have been refined, which are very useful in livestock farming and human the rap^.^ However, these drugs are usually unstable in the body. How to main- tain their bioactivity during the release process has

* To whom all correspondence should be addressed.

Polymer International 0959-8103/96/$09.00 0 1996 SCI. Printed in Great Britain

become a key problem in protein controlled release systems." Surface biodegradable polymers have two characteristics: one is the highly hydrophobic nature of the matrix; the other is the existence of unstable chemi- cal bonds in the polymer main chains. Surface bio- degradable polymers may, therefore, be useful in protein delivery systems. When the polymer is mixed with protein and a device made, the matrix only degrades at the surface and the protein in the surface layer diffuses out simultaneously. Also, because of the low per- meability of water in the highly hydrophobic polymer, protein inside the matrix remains in a stable solid form. Thus the protein bioactivity may be maintained in the release process.

369

370 C. Jie, K . J . Zhu, Y . Shilin

In recent years, polyanhydrides have been studied extensively by Langer et al.' 1-14 These polymers show bioerodible characteristics, but many of them are not suitable as a matrix for protein delivery, since they have high melting points and insolubility in common sol- vents, such as methylene chloride and chloroform. Poly- orthoesters with surface biodegradability have been investigated by Heller et al.' 5-17 However, the synthesis procedure for these polymers is rather complicated and some acidic or basic substances must be added to the polymer matrix, with possible loss of bioactivity of the proteins. Zhu et al." reported that poly(173-tri- methylene carbonate) shows surface biodegradable characteristics in vim, but the degradation rate is very low (molecular weight (MW) decreased to 50% in vivo in 6 months). In this paper, we report a new surface biodegradable polymer, poly(173-trimethylene carbonate-co-glycolide) (PGCA), and some degradation properties which may make it advantageous to use in protein delivery systems.

EXPERIMENTAL

Monomers

1,3-Trimethylene carbonate (CA) was synthesized according to the 1iterat~re.l~ 173-Propanediol (60.8 g), diethyl carbonate (114g) and a small cube of sodium metal were warmed until the sodium had completely dissolved, and the mixture was refluxed at 130°C for 4 h. Ethanol and residual diethyl carbonate were distilled off and the oil residue was dissolved in benzene, washed with water and then dried over calcium chloride. After removal of the solvent, the crude product was obtained by vacuum distillation (160-165"C/6 mm Hg) and then recrystallized from absolute ether (m.p. 46-47°C).

Glycolide (GA) was prepared by a method similar to that described in Ref. 20. Glycolic acid (500g) was mixed with 2-5 wt% zinc oxide in a three-necked flask, and the temperature raised to 120°C. As the rate of water elimination fell, the temperature increased to 180°C and the pressure was reduced gradually from 760 to 20mmHg over a period of 4-6h. When no more water was evolved, the vacuum was increased to 0.5- 1 mm Hg. The crude product was distilled out and then recrystallized from ethyl acetate three times. White crys- talline GA with a melting point of 8243°C was obtained.

Copolymerization

CA and GA were copolymerized in bulk. Monomers and catalyst in petroleum ether solution were intro-

duced to a well cleaned and dried vessel. The solvent was removed under vacuum and the vessel was heated to 140°C and maintained there for 36h. The product was dissolved in dimethylsulphoxide (DMSO) and pre- cipitated from methanol, then extracted with benzene to remove any possible PCA. Intrinsic viscosities were measured in DMSO at 25°C using an Ubbelohde vis- cometer. Molecular weights of copolymers were deter- mined by vapour pressure osmometry (VPO) (QX-08, Knaux Scientific Technical Supplies, Germany, in DMSO at 90°C using sucrose octaacetate (MW = 678.58) as reference).

Nuclear magnetic resonance (NMR) measurements

A JEOL 90Q NMR instrument was employed for NMR measurements using CDCl, as solvent. 'H NMR measurements were conducted in 5 mm 0.d. sample tubes with internal Me4% as shift reference at 89*55MHz, and in lOmm 0.d. sample tubes at 22.49 MHz for 13C NMR measurements.

Differential scanning calorimetry (DSC) measurements

Samples of 10-15mg were heated to melting in alu- minium pans with inverted lids on a Perkin Elmer DSC-7 thermal analyser at a heating rate of 20"Cmin-l. The instrument was calibrated with cyclo- hexane and indium prior to use.

Polymer hydrophobicity evaluation

Water content and static contact angle of distilled water on the polymer surface were used to evaluate the hydro- phobicity. Water content was defined as the percentage of water in wet polymer. It was measured gravimet- rically after the polymer had been immersed in distilled water to equilibrium at room temperature. Static contact angle was measured by a contact angle meter (JY-82)." The polymer membrane was made on a sila- nized glass microscopy slide. The coating thickness was about 2pm. Static contact angles were measured at 25°C on profiles of sessile drops using a microscope fitted with a goniometer eyepiece, with magnification 20 x . Readings were taken within 10-15 s and average drop size was 0.05 ml. Angles were measured on six dif- ferent regions of each polymer surface and the result averaged.

Polymer degradation

Samples of cylindrical shape (2-5 mm diameter, 7 mm length) were made by compression in a mould at 40°C

POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996

Preparation and characteristics of PGCA

,,/

371

for 5 min under a pressure of 100 kgcrn-,. Hydrolysis of the polymers in vitro was measured by immersing samples in phosphate buffer solution (pH 7-4) at 37°C. The samples were recovered periodically and the weight loss was measured. The molecular weight changes were determined by VPO.

I n vivo degradation was tested in rats. Polymer samples were weighed and sterilized by dipping in 70% ethanol solution before implanting subdermally in adult Kung ming rats in the scapular area lateral to the dorsal midline. At suitable time intervals the animals were sacrificed and the polymers were recovered. The samples were freed from adhering tissues, then rinsed with distilled water and dried. Weight loss was deter- mined and MW changes of the inner bulk and outer surface layer were measured by VPO.

RESULTS AND DISCUSSION

Polymer synthesis

Stannous octoate, stannous oxalate, stannous chloride and trimethyl aluminium can all be used as catalyst for this copolymerization. However, stannous octoate was selected for use in this work because of its lower tox- icity. The partial polymerization data are given in Table 1. When the reaction temperature was higher than 140"C, the molecular weight of the polymers decreased and the products became brown. With increasing reac- tion time the yield and viscosity increased, but beyond 40 h the viscosity decreased. The catalyst concentration

TABLE 1. Partial copolymerization data of 1.3- trimethylene carbonate (CA) and glycolide (GA)"

Catalyst/conc. Temperature Time Yield [Q] (M/L moll ("C) (h) (%) (dl g-'1

250 140 36 82.2 1.78 500 140 36 78.3 1.75

1000 1 40 36 76.4 1.67 500 120 36 73.8 1.19 500 140 36 78.2 1.75 500 160 36 84-4 1-20 500 140 10 43.2 1.25 500 140 22 58.0 1.31 500 140 59 86.3 1.52

a CA: GA = 85: 15 by mole in feed, Sn(oct), as catalyst. Intrinsic viscosities were measured in DMSO at 25°C

in the range examined (M/I, mole ratio, from 100 to 2000) had no apparent effect on the yield and viscosity. The copolymers (PGCA15, PGCA20 and PGCA30, the numbers representing the approximate mole percentage of GA) were completely soluble in DMSO, which means that no homopolymer PGA was formed.

'H NMR and I3C NMR spectra of PGCA are shown in Figs 1 and 2, respectively. In comparison with the spectra of PCA and PGA, it was clear that the peaks at 2.04, 4.25 and 4-64ppm in the 'H NMR spectrum belonged to the methylene groups - 0 - CH, -m, - CH, - OC(=O)-, -O-CH,-CH,-CH,-OC(=O)- of PCA units and -O-m2-C(=O)- of PGA units, respec- tively. The peaks at 28.27 (a), 154.95 (f) and 167.24 (g)

of

POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996

372 C. Jie, K . J . Zhu, Y . Shilin

GA-o ~ H , ~ H , ~ H , o ;to)- d l a 26 27 pprn b 61 97 pprn c 63 40 ppm

d6446pprn e6519ppm f15495pprn g16724pprn

I c

a

I I I , 10 180 160 140 120 100 80 60 40 20

s f ppm

Fig. 2. I3C NMR spectrum and assignment for poly(l,3-trimethylene carbonate-co-glycolide) (PGCAIS) measured in CDCl, at 30°C.

ppm in the I3C NMR spectrum corresponded to -CHz-Bz-CHz- of PCA segments, and C=O of PCA and PGA segments, respectively. The remaining peaks were assigned by comparison with model com- pounds of esters and carbonates (Sadtler Standard Carbon-13 NMR spectra V.49-56) and are as follows. The peak b (61.97ppm) was attributed to -OCH,- of CA in a GA-CA diad, peak c (63-48ppm) to -OCHz- of CA in a CA-CA diad. The peak d (64.46 ppm) and peak e (65.19 ppm) were assigned to -OCHz- of GA in a GA-GA-GA triad and a CA-GA-GA triad, respectively.

The reactivity ratios of GA and CA (rg and r,) were calculated according to the following equation :

GIL = (r, 911 + OMr, 11s + O (1) where g and 1 are feed compositions of GA and CA, and G and L are the compositions of the copolymers deter- mined by 'H NMR. The values of rg and r, obtained were 0.93 and 0.13, respectively.

Polymer hydrophobicity

The water content and static contact angles of PCA, PGCA15, PGCA20 and PGCA30 are listed in Table 2. There is almost no difference for the different composi- tions of the copolymers. This result seems unreasonable, because GA segments are much more hydrophilic than CA segments.'8*z2 In order to understand this result we studied the copolymers by DSC. Figure 3 shows DSC thermograms for PGCA15, PGCA20 and PGCA30. It can be seen that all the copolymers have only one glass transition temperature (q) and that the Tg values increase with increasing GA content. All are lower than body temperature (37°C). The curves show an amorp- hous state for PGCA15, and a semicrystalline state for PGCA2O and PGCA30 (with melting point at 192 and 198"C, respectively). The crystallinity of PGCA30 is higher than that of PGCA20 (enthalpy of melting is 7Jg-I for PGCA20 and 20.7Jg-' for PGCA30). Although the hydrophobicity of the copolymers

TABLE 2. Evaluation of hydrophobicity for PGCA and PCA"

Polymer PGCAI 5 PG CA20 PGCA30 PCA Water content (wt%) 1.83 (-10.02) 1.83 (-10.02) 1.85 (-10.02) 1.80 (-10.02) Contact angle (deg.) 58 (*2) 55 (-12) 53 (-12) 60 (-12)

a Water contents were measured gravimetrically after samples (n = 2) had been immersed in distilled water to equilibrium at room temperature. Static contact angles were measured by a contact angle meter at 25"C, average drop size was 0.05 ml and the readings were taken within 10-1 5 s (n = 6) .

POLYMER INTERNATIONAL VOL. 41, NO. 4. 1996

Preparation and characteristics of PGCA 373

1

-50 0 50 100 150 200 2 5 0

Temperature (OC)

Fig. 3. DSC thermograms of poly( 1,3-trimethylene carbonate- co-glycolide): (a) for PGCA15, T, - 19°C; (b) for PGCA20, T, -14°C and T, 192°C; (c) for PGCA30, T, -6°C and T,

198°C.

decreases with increasing GA content, the crystallinity increases. This may hinder water diffusing into the copolymer^.^^ The overall consequence is that the hydrophobicity of PGCA15, PGCA20 and PGCA30 is apparently little changed compared with PCA.

Polymer degradation

The degradation of PGCA copolymers in uitro and in uiuo was measured by change in weight and in molecu-

lar weight of polymer bulk and outer surface layers. The low shape stability and the deformation of the samples recovered prevented precise sampling of the surface and bulk. Therefore, the recovered samples were extracted with chloroform for 5 min to selectively separate the surface fraction (about 3wt% of sample). Control experiments showed that chloroform, a poor solvent for PGCA, slowly dissolved the surface layer of the polymer and did not selectively leach oligomeric com- ponents. The results are shown in Tables 3-5. The MW differences in both bulk and outer layers increased with increasing GA content for both in uitro and in uiuo test.

A comparison of the results is made in Figs 4 and 5. There is a large discrepancy in molecular weight changes between the surface and the bulk for PGCA15, while they are almost identical for PGCA30. For PGCA15, the bulk MW is almost unchanged after 40 days in rats, but the MW of the surface layers has decreased linearly to 60% of the initial molecular weight (Mno). The weight loss of PGCA15 also grad- ually decreased. These characteristics of PGCA15 rep- resent typical surface degradation. For PGCA30, the MW of the bulk and surface both fall to 30% of M,, over the same period. This shows a bulk degradation character. Comparing degradation rates of PGCA in uitro and in uiuo (Fig. 5), it can be seen that more rapid degradation occurs in uiuo than in uitro for both PGCA15 and PGCA30. This may indicate enzyme attack on the polymer matrix in uiuo."

TABLE 3. Degradation of PGCA in phosphate buffer solution (pH = 7-4.37"C)"

PGCA PGCAI 5 PG CA20 PG CA30

M n Mn/Mno (%) M, Mn/Mno (%) Mn M n I M n o (%I

Surface 21 300 95.2 20 700 90.3 21 500 77.9

Bulk 22 100 98.9 27 100 99.4 22 300 80.9 Surface 17 520 78.8 16 600 70.6 1 6 400 59.5

Bulk 21 100 94-4 19 900 84.4 1 8 400 66.6

10 days

28 days

* M , was determined by VPO in DMSO at 90°C (n =2). M,, of PGCA15, PGCA20 and PGCA30 was 22 300,23 200 at 27 600, respectively.

TABLE 4. Degradation of PGCAI 5 in wiwo

Days Wt loss (%)# Outer surface Inner bulk

M n Mn/Mno (%) M, M n I M n o

0 0 22 300 100 22 300 100 10 1.16 20 1 00 90 22 200 99.6 28 2.5 f 1 .I 1 6 200 72.5 22 200 98-4 39 3.5 f 2.2 12 700 58.4 20 400 97.7

a Weight loss refers to total implant mass (bulk plus surface) (n = 2).

POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996

374 C. Jie, K . J . Zhu, Y . Shilin

TABLE 5. Degradation of PGCASO in vivo

Days Wt loss (%)" Outer surface Inner bulk

Mn Mn/Mno(%) Mn M J M n o

0 0 27 600 100 27 600 100 10 1.67 18 200 66.2 18 400 66.7 28 2.3 f 1 a 4 9128 33.1 10 500 38.1 39 5.3 f 3-5 8 736 30.9 8 480 30.7

@Weight loss refers to total implant mass (bulk plus surface) (n = 2).

Measurements of the rates of hydrolysis of mono- meric aromatic esters and carbonates in aqueous solu- tion have shown that the carbonate groups are intrinsically more reactive than the ester.24 The degra- dation rate of PCA is, however, much slower than that of PGA. The degradation of PCA and PGA is recog- nized as mainly a hydrolysis process in aqueous solu- t i ~ n . " . ~ ~ The hydrolysis mechanism may be as follows.

Polyester degradation process :

0- I I

0 0 II I: RIGOR, = [Rl-C-OR,] = Rl-C-OR2

:OH, + OHz H O * . . H * - - O H , H 0 : H30' Q I:

= CR1-7. * 'OR21 - -> RICOOH + RZOH + HZO

HO : H a . .OH2

Polycarbonate degradation process:

80 - 70 -

h

55 2 "1 3 5 50 -

45-

30 - 20-

10-

i

-A- C (inner)

* - A ;0 io 30

time (days)

Fig. 4. Molecular weight changes of surface layer and bulk for PGCA15 and PGCA30 in uiuo.

0- I

0

ci R li

RIOCOR, = [R,O-C-OR2] = RIO- -OR,

: OH^ + OH^ HO.. .H.. .OH HO : H ~ O +

? I: I *

= [R1OC...OR,] - - > RIOCOOH

HO : H.-.OHz

+ RzOH + HZO --• RlOH + COZ

Hydrolytic chain scission of polyesters can be auto- catalysed by carboxylic acid end-groups (pK, = 3-5),26 but the carbonic acid group formed by degradation of polycarbonate is a weaker acid (pK, = 6-5). The degra- dation rate of PGA is higher than that of PCA.'8,27 For PGCA15, PGCA20 and PGCA30, there was no appar- ent difference in hydrophobicity by virtue of the balance between crystallinity and hydrophilicity in the GA seg-

' O t 0 0 10 20 30

time (days) I

Fig. 5. Molecular weight changes of surface layer for PGCA15 and PGCA30 in vitro and in uivo.

POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996

Preparation and characteristics of PGCA 375

ments. Hence the different degradation mechanism of CA and GA polymer chains plays an important role in PGCA copolymers. With increasing GA content, the autocatalysed hydrolysis effect of GA chains is enhanced, the degradation rate increased and the degra- dation character changed from surface to bulk.

CONCLUSIONS

The copolymer PGCA was prepared by ring-opening polymerization of CA and GA with stannous octoate. No apparent differences in hydrophobicities of PCA, PGCA15, PGCA20 and PGCA30 were observed. In vitro and in vivo tests showed that when the GA content was less than 20mol%, the degradation of the polymers occurred on the surface, otherwise polymer bulk degra- dation occurred.

ACKNOWLEDGEMENT

We are indebted to the National Natural Science Foun- dation of China for their support of this work. We also thank Ms Qiu Liyan for her assistance in the animal tests.

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