synthesis and swelling characterizations of a poly(gamma-glutamic acid) hydrogel

Synthesis and Swelling Characterizations of a poly(gamma-glutamic acid) Hydrogel

DENIS GONZALES, KESUO FAN, and MARTIN SEVOIAN*

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01002

SYNOPSIS

Natural, biosynthesized poly(gumrna-glutamic acid) (7-PGA) was crosslinked using dihalogenoalkanes yielding hydrogels with various features. Crosslinking reactions of the polymer and swelling of the hydrogels were studied. Various reaction parameters, like temperature and catalyst content, were adjusted to give highest yields in the network production. Swelling of the hydrogels showed dramatic changes when varying experimental conditions such as the molecular weight of y-PGA, the nature and concentration of the crosslinker, and the solution used for the swelling (ionic strength, pH). 0 1996 John Wiley & Sons, Inc. Keywords: poly(garnrna-glutamic acid) hydrogel synthesis swelling

I N T R O D U CTlO N

Slow release systems currently are being used widely in many fields of application, such as for controlled release of pesticides for crops or, even more popular, in pharmaceutical applications for chemicals, drugs, etc., in controlled release systems.'-5

A controlled release device must posses some ba- sic requirements related to the targeted application. The design of such a system in medical fields should present, for most of the cases, a zero order kinetic of the active particle deliverance without any sig- nificant burst effect and be biodegradable and bio- compatible.

Among the numerous systems developed, matrix or monolithic devices commonly are used for this purpose. In these cases the active ingredient to be delivered is merely and conveniently diluted in the carrier, whereas the releasing rate (the diffusion throughout the matrix) is the result of the chemical and physical features of the matrix.

Swellable hydrogels, for example, crosslinked polymers, have been extensively studied. The diffusion coefficient through these devices is basically dependent on both the degradation and the swelling

* To whom all correspondence should be adressed. Journal of Polymer Science: Part A Polymer Chemistry, Vol. 34,2019-2027 (1996) 0 1996 John Wiley & Sons, Inc. CCC 0887-624X/96/l02019-09

phenomena of the gel, which are related, to a large extent, to the polymer used.

Poly(gamma-glutamic acid) (-y-PGA) is a natural, bacterium biosynthesized polymer (Bacillus licheformis ATCC9945a) formally studied by Thorne et a1.6 Its availability and features (such as a large range of high molecular weight, different structural con- formations, biodegradability, and biocompatibility) make this material suitable for our project of the development of a slow delivery system.

The work reported herein is about the synthesis of a y-PGA crosslinked hydrogel and the swelling studies upon various parameters.

EXPERIMENTAL

Analytical

'H NMR spectra were performed on Bruker spec- trometers using Larmor frequencies of 200 Mhz in a 5 mm diameter probe in either D 2 0 or D6 DMSO as solvents.

Size Exclusion Chromatograms (SEC) were obtained using a Water ELC chromatograph equipped with a Asahipak GSM-700 column (7.5 mm i.d., 500 mm length), coupled with a UV detector set at 220 nm. Eluent was phosphate buffer (0.05M, pH = 7.2, NaCl 0.05M).

2019

2020 GONZALES, FAN, AND SEVOAIN

Production and Purification of y-PGA

High purity y-PGAs having different structures were synthesized following a process adapted from Thorne et a1.6 and Birrer et aL7 experiments. The bacteria (Bacillus licheformis ATCC9945a) were grown on agar plates from which the most mucoid colony was selected to inoculate Trypticase Soy Broth, and grown for 24 h a t 37°C. The temperature of the bacterial culture was then elevated to 65OC for 30 min, allowing the formation of spores. Fer- mentation of the obtained spores in the medium E (Table I) produced the crude polymer. After fer- mentation, the medium was centrifuged (10,000) and membrane filtered (cellulose membrane 0.22 pm) to remove the bacteria. Low molecular weight contam- inants were removed from the medium by ultrafil- tration with distilled water using hollow fibers (Mo- lecular weight cutoff: MWCO : 10,000). The polymer, then in its salt form, was acidified with phosphoric acid at 4°C at pKa conditions (pKa = 2.27).1° The polymer was purified by precipitation in an ether/ isopropanol mixture and freeze drying of a new water solution. NMR and SEC data for structural confir- mations and atomic absorption data for salt content were in agreement with the expected structure of the y-PGA.

Synthesis of the y-PGA Hydrogels

Linkage of the polymer through the carboxylic residue is complicated by its strong acidity and its closeness to the backbone giving rise to an important steric hindrance. Thus, alteration of the linear chain through the peptide bond hydrolysis often occurs during the reactions that are still being investigated."-16 However, crosslinking of the polymer to form a network has been successfully achieved with little or no hydrolysis of the main chain by using

Table I. Medium E

mild esterification condition^.^^ Dihalogenoalkanes were used as crosslinkers forming ester bonds with the y-PGA carboxylic groups. Mostly, a DMSOIy- PGA mixture (80/20) added with sodium bicarbon- ate (NaHC03) as a catalyst was allowed to stand 4 h a t 80".

r P G A

-(CH,-CH2-CH-NH-CO),- I

+ Temp., NaHC03, DMSO

X-CH,-(CH2),-CH,-X Crosslinker

-CH2-CH2-CH-NH-CO-CH2-CH2-CH-NH-CO- I I

C o/ \o I

I 0

I

C 0' 'O-Na'

o\C/o-Na+ I

Different hydrogels using different experimental conditions were synthesized. Special care was used for the hydrogel washing (several times first with phosphate buffer 0.05M, pH = 7.2 then with deion- ized water). Hydrogels were ground in liquid nitrogen and freeze dried. For all the gels, average size of the particles was estimated by microscope to be in a 10- 30 pm range. The crosslinked fraction was verified by 'H NMR after complete hydrolysis of the ester functions in a carbonate buffer (pH 10, 40°C) and comparative integration of the CH proton of the y- PGA and the CH2 protons of the dialcohol produced after hydrolysis (respectively peaks a' and c in Figs. 2-5). These calculated crosslinker fractions will be

Products Weight in g/L Experimental

L. Glutamic acid 20.0 Citric acid 12.0 Glycerin 80.0 NH4Cl 7.0 K2HP04 0.5 MgS04 * 7Hz0 0.5 FeC1, + 6H20 0.04 CaCl,. 2Hz0 0.15 MnSO, 0.0008"

a MnSO, concentration controls the D and L ratio.* Fitted formula is [%D] = 5.18 Ln[MnS04] + 119.1 (Range accuracy 40% < D% < 80%).

Incubation time controls the molecular weight of the synthesized polymer according to a previous work done in our laboratory? High molecular weight polymers were produced (up to Mu: 2.106) with a yield of polymer from 5 to 15 g per liter of medium.

Total volume: 1 liter, pH = 7 (with NaOH), Steril ize 2 0 m i n at 120°C.

SWELLING CHARACTERIZATIONS OF A POLY(GAA4MA-GLUTAMIC ACID) HYDROGEL 202 1

used further in this work as such, knowing that these data overestimate the effective crosslinker fraction (see esterification studies).

RESULTS AND DISCUSSION

Esterification Reactions and Synthesis of the y P C A Hydrogel

Control of the density of crosslinking and of the condensation reaction must be achieved in the preparation of a network presenting predictable and reproducible features. Examples in the literature concerning the esterification of the y-PGA using either bromoalkyl compounds or diazo compounds have been described using large excess of reactants with various yields as a result."-16 Studies were first conducted using bromobutane as a model compound, by varying reaction parameters such as the temperature and the catalyst ratio (NaHC03). Temperature effects were studied in a range typically from 65 to 95°C. Results presented in Figure 1(A) indicated little or no effect concerning the catalyst ratio from 0.5 to 2 mol NaHC03 per mol of repeated unit in the polymer, although this parameter seemed to dramatically affect the hydrogel weight yield (Table 11). Noticeable differences were obtained when the reaction was carried out a t several temperatures [Fig.l(B-D)]. Complete disappearance of the bromobutane, either by direct esterification (reaction

a) or hydrolysis (reaction b), occurred rapidly at 80°C and beyond. For reactions operated at 65 and 80"C, esterification is achieved through both reactions a and c in Figure 1, while at higher temperatures, for instance, 95"C, hydrolysis of the ester functions was observed.

Crosslinking reactions to yield a hydrogel have been followed by 'H NMR (Fig. 2). Varying the molecular size of the crosslinker (from 2 to 10 carbons) had little effect on the kinetics of the dibromoalkane disappearance. From this prospective, the reactions were completed after 4 h at 80°C using bromoalkyl compounds. On the other hand, chloroalkyl compounds exhibited lower reactivities as expected.

As gel formation occurs by the crosslinking reaction, only qualitative information comes out from the liquid state NMR study. However, according to the chemical shifts given in the literat~re,".'~ it is assumed that the y-PGA secondary structure goes from an helix to a coil conformation. Ester functions were detected furtively during the process when be- longing to non elastic chains or noncrosslinked y- PGA. CP/MASS 13C NMR spectra allowed us to detect the ester functions of the network (not shown here), while a more precise quantification of the total crosslinker fraction was achieved by 'H NMR anal- ysis of the hydrolyzed gel in alkaline conditions (Fig. 2). Some experimental data in terms of weight of gel yield upon varying reaction parameters are presented in Table 11. Yields of gel were calculated on

Table 11. Effect of Various Parameters on the Hydrogel Synthesis

Reaction Processing Effects

-y-PGA Concentration

Hydrogel weight yield (f5) 85 % 94 % 38% NaHCO, concentration

Hydrogel weight yield (+5) 0% 3% 16% 94% 92%

(weight fraction in DMSO) 0.30 0.20 0.10

(mol of NaHC03/mol of repeated unit) 0 0.15 0.50 1.00 2.00

Crosslinker Effects

Crosslinker concentration

Hydrogel weight yield (f5) Crosslinker length

Hydrogel weight yield (k5) Grafting yield (+lo)

(Mol C5H10 BrJrnol of repeated unit)

(CnH,nBr,)

(checked by 'H NMR after hydrolysis)

0.10 93%

n = 2 91%

100%

0.15 94 %

n = 5 94 %

100%

0.20 85%

n = 10 84 %

40%

Experimental conditions were (0.2 g of y-PGA/0.8 g of DMSO, 1 mol of NaHCO,/mol of repeated unit, 0.15 Mol C5HloBr2/mol of repeated unit, 8OoC, 4 h) unless otherwise specified.


- COOC4H9 5 a -COOH + BrC4H9

r-PGA 5 / \ /+ r-PGA

+ HOC4Hg ) -CWH

r-PGA

loo T 1, Bromobutane Disappearance

Q) C m c $ 60 E ' 40 4 r

20

Effect of catalyst ratio

650c Mol. NaHC03 (per mol of repeat unit) 1

0,5 1 2

I 1 Error

0 50 100 150 Minutes

0 , I 1 1

1, Butanol Formation loo 1 Effect of temperature

Q) so (I m c z 60 E g- 40

20

r * --Et

1 1 Error

60 80 O c

95

I I I

0 50 1 00 1 50 Minutes

loo 7 1, Bromobutane Disappearance

a, so C m c $ 60 E

40 9

s r

20

I1 Effect of temperature

+- 65 + 80 + 95

=-w

"C

I I E r r c l r L m m

0 , - 1 Y 1 Y 1

0 50 100 150 Minutes

loo 7 Ester functions Formation Effect of temperature I D

-8- 65 -8- 80 * 95 "C

0 50 100 150 Minutes

Figure 1. Kinetics of esterification of y-PGA with 1-bromobutane as a model compound. Experimental conditions were (0.1 g of y-PGAIO.9 g of DMSO, 1 mol of NaHCO,/mol of repeated unit, 0.3 Mol C,H,Br/mol of repeated unit, 80°C) unless otherwise specified.

the base of the gel weights measured after washing and freeze drying.

Concentration of -/-PGA in the solvent was an important factor for the hydrogel synthesis. The yield of the crosslinked polymer dropped down dramatically between 20 and 10% in weight of -/-PGA in DMSO solution. Catalyst content, up to 1 mol of NaHC03 per mol of carboxylic residue, also affected the network yield. The NaHC03 is assumed to pre-

vent the peptide bond hydrolysis by both the y-PGA carboxylic acid groups and the hydrogen bromide generated with the crosslinking reaction.

No marked effect was observed in the studied range concerning the crosslinker ratio or its carbon content. However, quantification of the crosslinker after hydrolysis of the hydrogel showed a low yied for the esterification reaction when using the dibromodecane. No difference was observed in the kinet-

SWELLING CHARACTERIZATIONS OF A POLY(GAMMA-GLUTAMIC ACID) HYDROGEL 2023

a'

4 .O 3.0 PPn

Figure 2. 'H NMR spectra during the crosslinking reaction (0.2 g of y-PGAI0.8 g of DMSO, 1 mol of NaHCO,/ mol of repeated unit, 0.15 Mol C,H,,Br,/mol of repeated unit, 80OC); 5 to 3 ppm area (a: CH of helix y-PGA, a': CH of random coil 7-PGA, b: of C,H,,Br,, c: alcohol de- rivatives, e: ester functions, *: solvent residues, w: water). (1)-(4) in DMSO D6 spectra ( t = 0 min, 20 min, 60 min, and 100 min), (5) in D 2 0 spectrum of the gel mixture after purification and hydrolysis in alkaline conditions (pH 10, 40°C).

ics of disappearance of the dibromoalkyl compounds, so it is believed that the condensation of the crosslinker involves to some extent an esterification through the alcohol as a product of degradation with different kinetics depending on the alcohol size.

Swelling Studies of the y-PGA Hydrogels

y-PGA hydrogels crosslinked with dihalogenoalkanes belong to the "biological chemically cross-

linked anionic" hydrogel type?,21 After crosslinking, a residual negative charge results from a difference in the mobility of the carboxylate groups linked to the network and the counterion. Thus, the swelling and deswelling can be explained mostly in terms of electrostatic repulsion of the carboxylate groups of the 7-PGA. According to the Flory the0ry,4,~,'~ other parameters effect to the total swelling pressure, which individual contributions offset each other a t the equilibrium. Those parameters influencing the swelling are (1) the solvation of the polymer, (2) the network structure, (3) the ionization of the side residues and the electrostatic effects.

However, networks were found to have an helix- to-coil transition, which is possible after crosslinking, as described by Matsuoka et al." Changes in the hydrodynamic volume of the polymer caused by this helix-to-coil transition may also influence the swelling.

Some of those parameters were varied for the swelling study of this new biological hydrogel.

Swelling Measurements

Swelling degrees (Q) were calculated as the volume of the swollen gel (in mL) per mg of dry hydrogel. Volumes of the gel swollen 1 h in the solution (30 mg of gel in 2 mL of solution) were measured in a calibrated tube after spinning under different Rel- ative Centrifugal Forces (RCF). Degrees leveled off after 30 min for most of the RCF [Fig. 3(a)]. Degrees of swelling Q depended on the RCF as shown in Figure 3(b). Different profiles were observed of various gel parameters and solvatation conditions ex- pressing changes in the gel compressibility. Exper- iments were conducted a t 27°C.

Polymer-Solvent Interaction Effects on the Swelling

Changes of the solvent in swelling experiments resulted as a modification of the Flory-Huggins interaction parameter x between the solvent and the hydrogel, thus inducing changes in the osmotic pressure. Swelling experiments were conducted using different water/alcohol fractions using NaCl O.O5M, ethanol, and propanol,2 (Fig. 4). Alcohols were poor solvents for the hydrogel (high values of X ) , and the degree Q was observed to decrease as expected, while the alcohol fraction increased. On the other hand, alcohols as helix stabilizers2' may have also contributed in the decreasing of the swelling. Q profiles were in agreement with those theo- retically predicted in the literature with the inter-


action parameter x variation? Little or no effect was observed on the degree Q when increasing the ethanol fraction up to 30% while little addition of propano1,Z immediately promoted a swelling drop.

Network Structure Effects on the Swelling

The network structure was found to have dramatic effects on the swelling ratio. Parameters that can effect are the molecular weight of the y-PGA, the molecular size and concentration of the crosslinker, and the network inhomogeneities such as loops, en- tanglements, nonelastic chains etc.,21s22 generated mostly by the synthesis process. These parameters, except the inhomogeneities, were investigated (Fig. 5).

Uses of higher M , of the polymer exhibited higher degrees of swelling [Fig. 5(a)]. Similar observations were made when decreasing the crosslinker content [Fig. 5(c)]. Comparable results, in the study of synthetic hydrogels, were found by Kuiicke et aL2' who related this phenomena to a larger mesh width. However, the increasing of the carbon number in the crosslinker (typically from 2 to 10) led to a striking drop of the degree Q [Fig. 5(b)] as sometime describedF2 It is assumed that the explanation of it to be a decreasing of the network inhomogeneity as the linear chain of the crosslinker is shortened. A striking decrease of the degree Q was observed, up to 10% of the crosslinker, before it leveled off. On the other hand, the swelling is linearly dependent on the crosslinker carbon number. The use of di- bromoethane, instead of the dibromodecane as crosslinkers, doubled the swelling of the resulted hydrogel.

+ Ethanol ft 2, propanol

0 0.3 0.6 0.9 Alcohol fraction

Figure 4. Effect of alcohol fraction (in a NaCl solution, 0.1M) on the swelling of an hydrogel (Mwy.P~A = 5.3 X lo5, 0.019 mol C5HIoBr2 per mol of repeated unit, RCF = 200 G) .

Ionization and Salt Effects on the Swelling

These parameters are also important for the swelling of a based y-PGA network as an anionic hydrogel, because a positive osmotic pressure is the response to a residual electrostatic charge in the gel. The fraction of ionized carboxylic groups in the hydrogel dramatically modified the swelling, as shown in Fig- ure 6. Metallic salt nature and concentration acting as a electrostatic shield also contributed to reduce the expansion of the network (Figs. 7 and 8).

Carboxylic residues ionization was controlled by the pH using citric/phosphate buffers. Experiments were conducted from pH = 2.1 (pKa of y-PGA or about 50% of ionization) to pH = 8.1 (Fig. 7). The maximum degree of swelling was found around pH = 5.1, which corresponded to 100% of ionization of the carboxylate groups (checked with the pH of a Na y-PGA solution). Beyond that point, the degree

I 00.195 J -- I

0 500 loo0 1500 m RCF (G)

Figure 3. (A) Effect of the spinning time on the swelling in water of an hydrogel (Mu = 5.3 X lo5, 0.081 mol C2H,Br2 per mol of repeated unit) upon different Relative Centrifugal Forces (RCF). (B) RCF effect on the swelling in phosphate buffer (pH = 7.2, 0.05M) of different hydrogel with various crosslinker content (Mu T-PGA = 5.3 X lo5, X mol C6HloBr2 per mol of repeated unit).

SWELLING CHARACTERIZATIONS OF A POLY(GAMMA-GLUTAMIC ACID) HYDROGEL 2025

Q decreased slightly, probably because of changes in the buffer equilibrium or possibly to some helix stabilization in the salt form as it was observed for in a y-PGA study.,' No intermediary ionization degree2, or delayed pH" phenomena for maximum Q were shown as it was observed elsewhere, probably due to the strong acidity of the carboxylic groups in our case.

The nature and concentration of metallic counterions were varied for both monovalent (Na', K+, Cs+ as well as the anion nature: C1-, Br-, I-) and divalent (M2+, Ca2+, Ba2' counterions (Fig. 7). Very slight effects were observed by varying the monovalent cation nature in the order of increasing Q: Na' < K+ < Cs'. The explanation of it is believed to be the result of an enhanced electrostatic shielding through a better contact ion/carboxylate as the size of the couterion decrease in agreement with other results on the study on the a-PGA helix stabilization.,' Change of the anion also effected slightly (in the order of increasing Q: Cl- < Br- < I-), although its concentration was said to have no effect on the swelling.22

Strong effects were marked by varying the divalent counterion nature. Use of CaClz solutions exhibited a more expanded hydrogel when compared to those exposed in MgC1, or BaC1, solutions, which in this case, eroded the only explanation based on the ions sizes. Although monovalent and divalent counterion combinations (the case here because sodium phosphate buffer was used) sometimes have been found to give crossed phenomena,,' it is believed the low degree Q for M 2 + counterion was the

% 0.025 5 loooh coo- = U 8 .- +j 0.02 c 0

cn 0.015 s b 0.01

; 0.005

a, cn - E

\ I F d

0- 0 2 4 6 8 1 0

PH

Figure 6. Effect of the pH on the swelling degree (in citric/phosphate buffer, O.lM, RCF = 200 G). Hydrogel studied was: Mw). .pGA = 5.3 X lo5, 0.019 mol C5HI0Br2 per mol of repeated unit.

effect of an helix stabilization when compared with Ca2+. The Ba2+ case was more likely a chelation of the hydrogel because precipitation of a y-PGA with BaCl, occurred, whereas no precipitation was observed with MgC1, or CaCl,, using concentrations up to 10 times the BaC1, solution.

Figure 8 indicates the salt concentration effect for monovalent (Na+) and divalent ( Ca2+) couter- cation. The increasing of the Ca2+ content gave rise to a gel-solid transition between and lo-' M, while the effect of a Na' concentration led to a log- arithmic dependence for the swelling degree Q in the range studied. Gels with different crosslinker

Figure 5. Network parameters effects on the swelling degree Q in phosphate buffer (pH = 7.2, O.O5M, RCF = 200 G). (A) Effect of the y P G A molecular weight M , (gels were crosslinked with 0.04 t 0.005 mol C5HI0Br2 per mol of repeated unit). (B) Effect of the crosslinker molecular size (M,) . -PG~ = 5.3 X lo5, 0.08 2 0.005 mol CnHPnBr2 per mol of repeated unit). (C) Effect of the crosslinker content (Mwt.PGA = 5.3 X lo5, X f 0.005 mol C5H,J3r2 per mol of repeated unit).


Monovalent Divalent 6 0.02 Monovalent t Countercation Counteranion Countercation c 0 0, I E 5 a,

0

F

,” 0.01 - A - U - 0 9:

NaCl KCI Cscl KCI KBr KI MgC12 CaC12 BaC12 Ions (0.1 M)

Figure 7. Effect of the ion type used in the swelling solution (phosphate buffer pH =

7.2,0.0125M, RCF = 200 G). Hydrogel studied was: M w 7 . p G A = 5.3 X lo5, 0.019 mol C5H,,Br, per mol of repeated unit.

ratio gave same profile types with differences in the degree Q (unpublished data).

CONCLUSION

Synthesis of a y-PGA hydrogel was made possible by the use of dialogenoalkanes as crosslinkers. Dif- ferent reaction parameters were studied, allowing us to understand and improve the production of the hydrogel.

Different parameters influencing the swelling degree were investigated. The effects were strikingly marked for the crosslinker content and molecular weight, the -y-PGA molecular weight, the pH and salt concentration of the swelling solution. Agree- ments with theoretical or experimental published results on other hydrogels swelling were obtained.

0.001 0.01 0.1 1

Ion concentration (M)

Figure 8. Effect of ion concentration on the swelling degree (gel used: Mu7.pGA = 5.3 X lo5, 0.019 mol C5HI0Br2 per mol of repeated unit, RCF = 200 G). Swelling conditions: phosphate buffer (pH = 7.2,0.0125M) for NaCl experiments, phosphate buffer (pH = 7.2, 0.0125M, NaCl 0.05M) for CaCl, experiments.

According to published data on the swelling of classical hydrogels, the y-PGA hydrogels rank their swelling features in a good position between biological ( i.e., starch-crosslinked-POCL3) and synthetic ( i.e., polyDimEthylAminoEthy1- Acrylate) hydrogel.*’

The use of this based y-PGA hydrogel as a slow delivery system is promising because the swelling degree Q, which happened to be related to the diffusion coefficient in drug releases, *‘ can be easily controlled by modifying many parameters.

This work was supported in part by Grant No. 5-20876 from Roussel-Uclaf France. We would like to Thank Dr. R. W. Lenz for his suggestions for this work.

REFERENCES AND NOTES

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2. C. G. Pitt, Proc. Int. Symp. Control. Rel. Bioact. Mater., vol. 20, Controlled Release Society Inc., Deerfield, IL, 1993, p. 146.

3. P. L. Lee and W. R. Good, Control. Rel. Technol. Pharmaceut. Appl., 3, 56 (1987).

4. D. Severian, Polymeric Biomaterials, Marcel Dekker Inc., New York, 1994.

5. M. A. El-Nokaly, D. M. Piatt, and B. A. Charpentier, Polymeric Delivery Systems-Properties and Appli- cations, ACS Symposium series, Washington, DC, 1993.

6. C. B. Thorne, C. G. Gomez, H. E. Noyes, and R. D. Housewright, Bacteriology, 68, 307 ( 1954).

7. G . A. Birrer, A. Cromwick, and R. A. Gross, Znt. J. Biol. Macromol., 16-5, 265 (1994).

8. C. Gomez-Leonard, R. D. Housewright, and C. B. Thorne, J. Bacteriol., 76-5, 499 ( 1958).

SWELLING CHARACTERIZATIONS OF A POLY(GAA4MA-GLUTAMIC ACID) HYDROGEL 2027

9. K. Fan, D. Gonzales, and M. Sevoian, to appear. 10. H. Kubota, Y. Nambu, and T. Endo, Chem. SOC. Jpn.,

11. M. Borbely, Y. Nagasaki, J. Borbely, K. Fan, A. Bho-

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18. P. J. Flory, Principles of Polymer Chemistry, Cornell

19. Y. Matsuoka, K. Ryoichi, and M. Sisido, Polym. J.,

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23. The hydrolysis of the peptide bond in the y-PGA chain was found to be a minor phenomena during the esterification reaction (or crosslinking reaction) upon the experimental conditions described in the text. In- deed, a degradation study on both the gel and the y- PGA showed that the gel degradation occurred at alkaline pH (i.e., pH 10) leading to the hydrolysis of the crosslinker bonds, and to a much lesser extent to the linear chain a l t e r a t i ~ n . ~ This allowed us to check the molecular weight of the T-PGA after the crosslinking reaction. For instance, starting with a y-PGA with a M,: 5.3 X lo5 led after the reaction to a linear chain with a M,: 4.1 X lo5.

Received August 17, 1995 Accepted December 26, 1995

synthesis and swelling characterizations of a poly(gamma-glutamic acid) hydrogel

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