The Effects of Some Structural Variations on the ... - ACS Publications

17 The Effects of Some Structural Variations on the Biodegradability of Step-Growth Polymers S. J. H U A N G , M . BITRITTO, K. W . L E O N G , J. P A V L I S K O , M . ROBY, and J. R. K N O X

Downloaded by PURDUE UNIV on July 8, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/ba-1978-0169.ch017

Department of Chemistry, Biological Science Group, and Institute of Materials Science, University of Connecticut, Storrs, C T 06268

Many new step-growth polymers containing one or more types of linkages such as amide, ester, urea, and methanes were biodegradable by fungi and enzymes. Suitable substituents, combinations of hydrophilic and hydrophobic segments and long repeating units, contribute to the increase of biodegradability of the polymers. The presence of hydrolyzable linkages and conformational flexibility are some of the important requirements for biodegradability. A ctivity i n the study of biodegradable polymers has been increasing i n recent years (1-4). Since most of the currently available polymers have not been found to be biodegradable, efforts have been directed toward the syntheses of new polymers that are biodegradable. In this chapter, our recent results and those of related reports by other research ers are discussed i n terms of the effects of structural variations on the biodegradabilities of step-growth polymers. Concerns with how to prevent or retard attack on polymer products by bacteria, fungi, insects, rodents, and other animals provided the early incentive for the study of the biodégradations of synthetic polymers. In recent years, the disposal of the mostly bioresistant polymer products now i n use has become increasingly difficult. The disposal of biode gradable polymers, on the other hand, is less difficult. This provides the current incentive for the study of biodegradable polymers. Moreover, biodegradable polymers are useful for the preparation of surgical i m plants, sutures, controlled-release drugs, fertilizers, fungicides, and agri cultural mulch. Successful uses of biodegradable polymers i n these newly developing areas w i l l result i n more effective utilization of resources. 0-8412-0381-4/78/33-169-205$05.00/l © 1978 American Chemical Society Allara and Hawkins; Stabilization and Degradation of Polymers Advances in Chemistry; American Chemical Society: Washington, DC, 1978.


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STABILIZATION A N D DEGRADATION OF P O L Y M E R S

Attempts to prepare biodegradable addition polymers have not been very successful. Incorporation of photodegradable units into polymer chains and mixing photosensitizers into polymer composites to prepare photodegradable materials so the products of photolyses might become biodegradable were the alternative approaches for making addition polymers biodegradable (2). Most of the recently discovered biodegradable polymers contain hydrolyzable linkages such as amide, ester, urea, and urethane along the polymer chains. These polymers are prepared generally by step-growth polymerizations. Increasing the hydrophilicity of the polymer, intro ducing substituents for better interactions between the polymer chains and enzymes, and increasing the polymer chain flexibility by copolymerization of different monomers have been some of the approaches taken by researchers with various degrees of success. Testing

Methods

Although soil burial testing affords a way to test samples for break down close to actual conditions of waste disposal, it lacks reproducibility because of the difficulties i n controlling climatic factors and the popula tions of various biological systems that are involved. F o r more repro ducible results, degradations by cultured fungi and bacteria as well as degradations by purified enzymes are used. A large part of the biodégradations of polymers is studied by using the polymers as the carbon and/or the nitrogen sources for the growth of microorganisms. F u n g i are used more frequently than bacteria. The degrees of degradation of the polymer samples are determined by study ing (a) the evidence of colony growth (the A S T M method) ( 3 - 6 ) , (b) the production of carbon dioxide ( 7 , 8 ) , (c) oxygen consumption (9), (d) the increase i n cell count or cell mass (9), (e) product forma tion analysis (9), (/) the changes i n the polymers' physical properties such as molecular weight, solution viscosity, tensile strength, etc. (10), (g) the weight loss of solid samples (10, I I ) , and (h) visible destruction of the samples (3,4). Degradation by enzymes is carried out generally by incubation of the samples with buffered enzyme solutions with suitable blanks to correct for buffer degradation and contamination proteins and/or degra dation of the enzymes ( I I ) . The degrees of degradation of samples can be followed by methods (e-h) mentioned above. Since degradation by microorganisms might be the result of multistep reactions catalyzed by enzymes and reactions not involving en zymes, they are very complex and the interpretation of data i n a quantita tive manner is often very difficult. O n the other hand, microorganisms can

Allara and Hawkins; Stabilization and Degradation of Polymers Advances in Chemistry; American Chemical Society: Washington, DC, 1978.


17.

HUANG ET A L .

Step-Growth Polymers

207

often utilize wide ranges of nutrients of different structures so positive results for biodégradation by microorganisms might be obtained without too much difficulty. Degradations by enzymes are comparatively simpler and there is a better chance for quantitative treatment of data. However, the high degrees of substrate specificity associated generally with enzymes make the selection of the right enzyme for degradation rather difficult. W e used both methods i n our study (9) and we found that fungi growth was a good screening test, whereas enzymes are found to be more effec tive degradation agents since relatively high concentrations of enzyme can be used. The ultimate but very time consuming approach of carrying out degradation by microorganisms, isolating the enzyme that is responsible for the degradation, and then studying the details of degradation by purified enzyme has been reported by the research groups of Suzuki (12,13) a n d O k a d a (14). Materials Our step-growth polymers were synthesized and characterized by standard methods. Details can be found i n the references cited i n the following sections. Results and

Discussion

Polyesters. In 1968, Darby and Kaplan reported that polyethylene adipate, mol wt 2,390, poly(trimethylene adipate), mol wt 5,240, and poly(tetramethylene adipate), mol wt 1,950, supported intense growth of fungi when tested with seven microorganisms: Aspergillus niger, A. flavus, A. versicolor, Chaetomium globosum, Pénicillium funiculosum, Pulhria pullulans, and Trichoderma (15). Potts, et al. studied several polyesters and found that aliphatic polyesters derived from c-caprolactone, succinic acid, and adipic acid supported heavy growth of fungi when tested with A . niger, A . flavus ,P. funiculosum, and C . globosum according to the A S T M method (3,4). The unsaturated poly(hexamethylene fumarate) supported only light growth and the polyesters derived from aromatic diacids d i d not support any growth. In a study using a fungus of Pénicillium sp. strain 26-1 capable of utilizing high molecular weight polycaprolactone, Tokiwa, Ando, and Suzuki found that among a series of polyethylene esters prepared from ethylene glycol and alkane diacids, polyethylene sebacate has the highest biodegradability. They found also that the polyesters derived from aromatic diacids were inert (9). These results suggest that replacements of the relatively flexible alkylene chain with more rigid olefin or aromatic rings prevent or retard degradation.


208

STABILIZATION A N D DEGRADATION O F P O L Y M E R S

Fields, Rodriguez (10), and Potts et al. (34) reported that poly esters of low molecular weight supported better fungal than polymers of the same structures but having higher molecular weight. To study the effect of changing the hydrophilicity of the polymer on its biodegradability we studied the poly(alkene D-tartrates) prepared from C - C i 2 diols and D-tartaric acid (16). The relative abilities of these polyesters to support the growth of A. niger are shown i n Table I. The polyesters derived from medium size diols ( C and Ce) are more degradable than those derived from smaller and larger diols. It is interesting to note that although polyethylene tartrate is water-soluble and non crystalline, its ability to support A . niger s growth is less than that of the partially crystalline, more hydrophobic and water-insoluble polyesters derived from 1,6-hexanediol and 1,8-octanediol. A l l polyesters tested were low molecular weight materials with M between 1,200-1,500. A balance of hydrophilicity and hydrophobicity appears to give the best result. Changing the hydrophilicity of poly(dodecamethylene tartrate) by acetylation or phosphoration decreased its ability to support A. niger growth. 2


6

n

Table I.

Extent of Growth of A. Niger on Poly (alkylene D-tartrates) after 14 Days at 3 7 ° C — h OCCH—CHCOO(CH )»0 2

I

I

OH

OH

η 2 4 6 8 10 12 12 acetate* 12 phosphate

Extent of Growth*

b

1 1 4 4 3 3 1 1

A S T M rating: 4-60 to 100% surface covered; 3-30 to 60% surface covered; 2-10 to 30% surface covered ; 1 < 10% surface covered ; and 0 = no visible growth.50% of available hydroxy groups reacted. e

b

Studies on enzyme degradations of polyesters provided some insight into the mechanism of the biodégradation. F r o m the Pénicillium sp. strain fungus that degraded polycaprolactone and other polyesters, Tokiwa and Suzuki isolated the enzyme and found that it splits endo genous ester bonds of the polyesters (12,13). They found also that the enzyme degraded polycaprolactone and polypropiolactone but not polyD,L-hydroxybutyrate (the D-isomer of w h i c h is known as one of the bac terial and algal storage materials). W e found that the viscosity molecular



17.

HUANG E T A L .

209


weight of a sample of polycaprolactone was reduced from 13,000 to 10,000 by exposure to a buffered solution of acid protease from Rhizopus chinensis for 10 days (17). Copolymers derived from phenyllactic acid and lactic acid were hydrolyzed by chymotrypsin with the rate of hydroly sis changing w i t h the phenyllactic acid residue content i n the polymer (18). It seems that some of the fungal degradations of polyesters proceed by enzyme-catalyzed hydrolysis of the ester linkages with certain degrees of substrate specificity. Polyamides. Although high molecular weight polyamides such as nylon-6, nylon-6,6, and nylon-12 resisted microbial (3, 4,19) and enzyme attack (17), low molecular weight cyclic and linear oligomers of caminocaproic acid were utilized by certain bacteria isolated from the effluent water of a nylon-6 plant. These include Corynebacterium aurantiacum B-2 reported by Fukumura (20,21) and Achromobacter guttatus KI 72 reported by Okada et al. (22). Bailey and co-workers reported that an alternating copolymer of glycine and c-aminocaproic acid was degradable by soil microorganisms (7). The corresponding copolymer derived from serine was water solu ble and could be degraded more rapidly. Since many proteolytic enzymes are specific in cleaving peptide link ages adjacent to substituent groups, we decided to prepare substituted polyamides, anticipating that the introduction of the substituents would make the polyamides more degradable. Oligomeric benzylated nylon-6,3, prepared from the achiral benzylmalonic acid, was hydrolyzed by chymo trypsin (7% hydrolysis owing to enzyme i n addition to 40% hydrolysis owing to buffer i n 10 days) (11). In the case of nylons-n,6 derived from D,L-«-benzyladipic acid, very little degradation was observed. However, all of the enzymes tested (chymotrypsin, subtilisin, thermolysin, pepsin, and elastase) were absorbed on the polymer surface after a 10-day test ing period. It is possible that the D-isomers of the D,L-polymers acted as enzyme inhibitors. -f-

NH(CH ) NHCOCHCO 2

-}

6

CH Ph 2

benzylated nylon 6,3 m p l 4 ( M 4 5 C Μ 2,000 0

Λ

• N H (CHo) N H C O ( C H ) C H C O n

2

3

- f

I CH Ph 2

benzylated nylons n,6

η = 2,4, 6,8


210

STABILIZATION A N D DEGRADATION OF P O L Y M E R S

Table II.

Degradation of Methyl- and Hydroxy-Substituted Polyamides by Fungi and Enzymes

- h NHCHCH NHCO(CH )«CO -jhr • • N H C H C H C H N H C O ( C H ) C O -jy 2

CH

2

2

2

OH

3

Extent of Fungal Growth*


2

I

x/y'

η

mp, °C

i/o i/o i/o 1/1 0/1

4 7 8 8 8

240-245 180-190 223-228 200-210 225 dec.

% Hydrolysis by Enzymes*

ChymoA . niger A . flavus trypsin

M

a

13,400 10,400 9,470 13,000 20,000

2 2 1 4 2

2 3 2 4 2

16.0 9.3 0

Elastase

4.5 13.5 32.0

A S T M rating: 4-60 to 100% surface covered; 3-30 to 60% surface covered; 2-10 to 30% surface covered; 1 < 10% surface covered; and 0 = no visible growth. 14-day exposure at 35-37 °C. Calculated on the % of total amount of susceptible amide linkages. Detected by ninhydrin analysis of the amino groups produced after a 5-day exposure at 30°C. Chymotrypsin was buffered (imidazole) at pH 7.5 and elastase was buffered (boraxboric acid) at pH 8.8 ± 0.4%. Ratio of diamines in the polyamides. e

b

0

A series of polyamides containing methyl and/or hydroxy substituents were prepared from the polymerizations of 1,2-diaminopropane and/or l,3-diamino-2-propanol with diacid chlorides. A l l of the substi tuted polymers supported the growth of A . niger and A . flavus whereas the unsubstiuted nylons prepared from ethylenediamine and 1,3-diaminopropane were resistant to fungal attack (Table I I ) (23,24). It is inter esting to note that the copolymer containing both the methyl and the hydroxy groups was the most degradable. W h e n the polyamides were tested with elastase the polyamide degradability increased wtih increasTable III.

Extent of Fungal Growth on Polyureas after 14 Days at 3 7 ° C

CONH(CH ) NHCONH(CH ) CHNH 2

6

2

4

COOR

Extent of Growth*

1

Sample

A . niger

A . flavus

L-lysine methyl ester · H C 1 L-lysine ethyl ester · H C 1 Polyurea H = M e M n 5,900 Polyurea R = E t M n 17,000

3 2 2 1

0 3 0 3

Same as Table I, note a.



17.

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E T

211


A L .

ing hydroxy content i n the polymer. The reverse pattern was found for chymotrypsin (25). Polyureas. Substituted polyureas were prepared from L-lysine methyl and ethyl esters (23). The methyl polyurea ester and the ethyl polyurea ester supported the growth of A . niger and only the ethyl poly urea ester supported the growth of A . flavus (Table III). After exposure to buffered chymotrypsin and subtilisin for seven days, the methyl polyurea became completely water-soluble. Ninhydrin analysis of the amino groups produced revealed 5 % and 9 % hydrolyses of the susceptible urea linkages by chymotrypsin and subtilisin, respec tively. Since the polyurea chains contain 18 repeating units on the aver age, it is reasoned that i n addition to the hydrolysis of urea linkages, ester-linkage cleavage must have occurred i n order to give water-soluble products. A poly (ester-urea) of M 1,930 prepared from L-phenylalanine gly col ester was hydrolyzed by chymotrypsin up to 22.9% i n 10 days (26). Most of the hydrolysis was found to be cleavage of the ester linkages i n keeping of the known specificity of chymotrypsin ( acyl cleavage adjacent to the phenylalanine benzyl group). The corresponding poly (ester-urea) prepared from the glycine glycol ester was not degraded by chymo trypsin. n

- E - NHCHCOOCH CH OOCCHNHCONH(CH ) NHCO 2

2

2

I

I

R

R

6

P o l y (ester-ureas) R = H , not degraded R — C H P h , degraded Polyurethanes. Polyester base polyurethanes were reported to sup port fungal growth better than polyether base polyurethanes (15). Poly urethanes derived from cellulose hydrolysates were degraded by cellusin (27) . W e found that the polyurethane obtained by reacting poly(dodecamethylene D-tartrate) with 1,6-di-isocyanatohexane supported the growth of A . niger with an A S T M rating of 4 (16). A similar polyure thane derived from poly(hexamethylene tartrate) was degraded i n vivo (28) . These reports suggest that biodégradation of polyurethanes does not necessarily involve the cleavage of urethane linkages. Although sev eral polyurethanes have been used as surgical implants (29), there is very little known about their degradation in vivo. I n order to find out if the urethane linkages are biodegradable, we studied the degradation of two simple polyurethanes containing only urethane linkages. Although we have not found a purified enzyme that 2


212

STABILIZATION A N D DEGRADATION OF POLYMERS

Table IV. Degradation of Polyurethanes by Axion in 10 Days at 3 0 ° C OCH2CH2OOCNH—R—NHCO •

% R

hi

1,6-hexamethy lene 2,4-tolylene

0.20 0.21

Hydrolysis

Buffer"

Axion"

e,d

0,4

10.2 (1.0) 14.4 (1.3)

15.1 (6.2) 24.0 (7.1)

Phosphate buffer pH 8.0 + sodium dodecylsulfate. Axion solution pH 8.0. • Weight loss of solid samples, ± 1%. "Ninhydrin analysis of amino groups increase in solution based on the total amount of susceptible urethane linkages, ± 0 . 4 % . β


*

h

w i l l effectively degrade the polyurethanes the enzyme containing deter gent Axion degraded the polyurethanes, Table I V . I n addition to the hydrolysis caused b y the basic buffer, degradations were caused also by the enzyme and/or other additives i n Axion. One of the major differences between natural proteins and synthetic polymers is that proteins generally do not have repeating units along the polypeptide chains. This irregularity provides the protein chains with conformational flexibility which allows them to fit into the enzyme active sites. It is very likely that this contributes to the biodegradabilities of proteins. The synthetic polymers, on the other hand, generally have short repeating units and this regularity results i n conformational rigidity which inhibits a close fit between the polymer chains and the enzyme active sites. Thus no effective enzyme catalysis w i l l occur. W e reasoned that synthetic polymers with long repeating units might be conformationally flexible and thus biodegradable. W e prepared several poly (amide urethanes) with rather long repeating units to test this hypothesis (30). The degradation by subtilisin results are encouraging (Table V ) . Table V . Degradation of Poly (amide urethanes) by Subtilisin in 10 Days at 3 0 ° C - f - NHCH CH200CHN(CH2)eNHCOOCH2CH NHCO(CH2),CO - J — 2

2

% Wt Loss

1

X

M

mp, °C

Buffer'

Buffered Enzyme'

2 4 8

6,200 9,100 8,800

190-213 195-205 172-192

8 8 2

25 22 7

n

Phosphate buffer, pH 7.2.


17.

HUANG ET A L .


213


Conclusion Many new step-growth polymers were biodegradable. Most of the biodégradation of synthetic polymer systems are complex multicompo*nent and multiphase systems. The surface area, morphology, and the molecular weights of the polymers should have significant effects on the biodegradability of the polymer samples. Information i n these areas still awaits future research. Although it is premature to draw final con clusions on all of the factors affecting the biodegradability of polymers, several points can be made on the existing information. Although amide, ester, urea, and urethane linkages are biodegrad able, the flexible ester-containing polymers are generally more degradable than polymers containing the more rigid amide, urea, and urethane groups. Replacement of the flexible alkylene segments w i t h the more rigid olefinic and aromatic systems retards or inhibits degradation. Changing the hydrophilic-hydrophobic characteristics of the poly mer samples alters the biodegradability. In general, the presence of both hydrophilic and hydrophobic segments gives the best results of degrada tion. Proper stereoisomers of substituted polymers are more degradable thanthe corresponding unsubstituted analogs. Introduction of substituents increases the conformational flexibility of polymer chains and provides favorable hydrophobic or hydrophilic interaction between the polymer chains and the active sites of enzymes, thus improving the catalysis by en zymes. Increasing the repeating unit length has a similar effect. Copolymers, both i n terms of substituents and linkages, are generally more degradable than the corresponding homopolymers. Again, this is probably caused by the fact that copolymers are more flexible than the more regular homopolymers with the exception of some polyesters. In addition to the presence of hydrolyzable linkages, it seems that conformational flexibility is one of the most important requirements. Acknowledgment W e thank the National Science Foundation (Grant D M R 75-16912) and the University of Connecticut Research Foundation for financial support. Literature Cited

1. Huang, S. J., Roby, M., Knox, J. R., EnzymeTechnol.(1976) 5, 135. 2. Guillet, J., Ed., "Polymer and Ecological Problems," Plenum, New York, 1973. 3. Potts, J. E., Clendinning, R. Α., Ackart, U.S., E.P.A. Contract No. CPE70-124, 1972, p. 22. Allara and Hawkins; Stabilization and Degradation of Polymers Advances in Chemistry; American Chemical Society: Washington, DC, 1978.


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STABILIZATION AND DEGRADATION OF POLYMERS

4. Guillet, J., Ed., "Polymer and Ecological Problems," pp. 61-79, Plenum, New York, 1973. 5. 1970 Annual of ASTM Standards, Part 27, ASTM-D-1924, p. 593. 6. 1970 Annual of ASTM Standards, Part 26, ASTM-D-2676, p. 758. 7. Bailey, W. J., Okamoto, Y., Kuo, W.-C., Narita, T., Proc. Int. Biodegradation Symp., 3rd (1976) 765. 8. Nykvist, Ν. B., Proc. Conf. Degrad. Polym. Plast. (1973) 1. 9. Tokiwa, Y., Suzuki, T.,J.Ferment.Technol.(1974) 52, 393. 10. Fields, R. D., Rodriquez, F., Proc. Int. Biodegradation Symp., 3rd (1976) 775-784. 11. Huang, S. J., et al., Proc. Int. Bidegradation Symp., 3rd (1976) 731-741. 12. Tokiwa, Y., Suzuki, T., Agric. Biol. Chem. (1977) 41, 265. 13. Tokiwa, Y., Ando, T., Suzuki, T.,J.Ferment.Technol.(1976) 54, 603. 14. Kinoshita, S., Bisaria, V. S., Sawada, S., Okada, H., Abst. Annu. Meet. Soc. Ferment.Technol.(1974) 110. 15. Darby, R. T., Kaplan, A. M., Appl. Microbiol. (1968) 16, 900. 16. Bitritto, M. M., Bell, J. P., Brinkle, G. M., Huang, S. J., Knox, J. R., J. Appl. Polym. Sci., in press. 17. Bell, J. P., Huang, S. J., Knox, J. R., U.S. NTIS, AD-A Rep. No. 009577 (1974). 18. Tabushi, I., Yamada, H., Matsuzaki, H., Furukawa, J., J. Polym. Sci. Polym. Lett. (1975) 13, 447. 19. Rodriquez, F., Chem.Technol.(1971) 409. 20. Fukumura, T., Plant Cell Physiol. (1966) 7, 93. 21. Fukumura, T.,J.Biochem. (1966) 59, 537. 22. Kinoshita, S., Kageyama, S., Iba, K., Yamada, Y., Okada, H., Agr. Biol. Chem. (1975) 39, 1219. 23. Huang, S. J., Leong, K. W., Knox, J. R., unpublished data. 24. Leong, K. W., Ph.D. Dissertation, University of Connecticut, 1976. 25. Huang, S. J., Pavlisko, J., unpublished results. 26. Huang, S. J., Bansleben, D. Α., Knox, J. R., J. Appl. Polym. Sci., in press. 27. Kim, S., Stannett, V. T., Gilbert, R. D., J. Macromol. Sci., Chem. (1976) A10, 671. 28. Wang, P. Y., Arlitt, Β. B., Polym. Sci.Technol.(1975) 1, 173. 29. Wilkes, G. L., "Polymers in Medicine and Surgery," R. L. Kronenthal, Z. Oser, Ε. Martin, Eds., pp. 45-76, Plenum, New York, 1976. 30. Huang, S. J., Roby, M., Knox, J. R., unpublished data. RECEIVED May 12, 1977. Publication 944 from Institute of Materials Science.


The Effects of Some Structural Variations on the ... - ACS Publications

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