Copolyesteramides, 10a

Enzymatic degradation of 6-iminohexanoyl/12-oxydodecanoylcopolyesteramides

Isaac Goodman*, Maria Teresa Rodrı´guezb

Department of Chemistry and Chemical Technology, University of Bradford, Bradford, BD7 1DP, WestYorkshire, U.K.

(Received: May 4, 1998; revised: June 17, 1998)

SUMMARY: Films of several 6-iminohexanoyl/12-oxydodecanoyl random copolymers containing from 19to 46 wt.-% (29–60 mol-%) of 6-iminohexanoyl units, together with comparison films of nylon 6 homopoly-mer, were incubated at pH 7,4in vitro for 336 h at 378 with solutions of protease, collagenase,a-chymotryp-sin and pancreatin. The first three mentioned enzymes were without visible effect upon any of the polymerfilms, and there was no significant evidence with any enzyme or substrate of the formation of 6-aminohexa-noic acid as a product of amide-bond splitting. By contrast, incubation with pancreatin caused the topochemi-cal surface erosion of each copolyesteramide (but not of nylon 6), with the depletion of ester group content inthe surface layer and the development of an amide-richer characteristic striated surface morphology. Randomincorporation of amide groups into polyesters, in the form of oligoamide sequences, is suggested as a meansof protecting the ester component against esterolytic attack.

IntroductionThe degradation of synthetic polymers in real or simu-lated biological environments has been investigatedintensively for many years, impelled by widely diversemedical, agricultural and industrial interests ranging atone extreme from requirements for long-term durabilityin the relevant conditions of use, and at the other formaterials which will undergo planned breakdown in theshorter term.

Depending upon the use concerned, the time span overwhich polymers may be required to undergo (or conver-sely to resist) decomposition to simple molecular frag-ments may be anything from a few days to many monthsor longer. The selection of such materials must thereforebe influenced by the (often unknown) identity of thepotential attacking species, by the conditions to whichthey will be exposed, and by their own molecular and tex-tural parameters, as well as by their physical capabilityfor fabrication to the required format. The multiple andoften conflicting factors involved, and the dilemmas ofestablishing satisfactory laboratory criteria for their eva-luation are discussed in several major reviews2–8).

Biodegradation most often occurs in water-containingsystems, and when required to take place within a limitedtime span, it has been generally accepted that the polymersshould contain main-chain groups such as ester, amide,

urethane or others that are potentially susceptible to hydro-lysis, whether simple in aqueous media, or by body fluids,cellular enzymes or micro-organisms, depending on thetopic of interest. With regard to synthetic polymers pro-posed for resorbable surgical and drug delivery devices,some workers have emphasized biomimetic considera-tions, arguing that the incorporation of enantiomericgroupings having the same chirality as their natural conge-ners would facilitatein vivo enzymatic attack9–16). How-ever, such positive results as have been reported mayreflect also the effects of differences in polymer phasestates, hydrophilicity and diffusion characteristics as com-pared with ‘non-natural’ reference materials.

Studies with different polymer classes have confirmedthat aliphatic homo- and copolyesters, albeit with consid-erable individual differences, are usually susceptible tohydrolytic breakdown in physiological conditions,whether simulated or actively biotic16–21). There are fewercomparative studies with polyamides, but it is clear thatthe linear aliphatic members (nylons) are considerablylonger-lived in physiological environments (althougheventual disintegration occurs after lengthyin vivoimplantation)2, 6, 9, 10, 21–24)and that chemical modificationwould be needed to encourage shorter-term breakdown.The combination of both types of characteristic groups,as polyesteramides, has therefore been envisaged as a

Macromol. Chem. Phys.200, No. 1 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 1999 1022-1352/99/0101–0044$17.50+.50/0

a Part 9: cf. ref.1)

b Present address: Universidad de Burgos, Facultad de CyTA y Ciencias Quı´mica, Departamento de Quı´mica, E-09001 Burgos,Spain.

44 Macromol. Chem. Phys.200,44–50 (1999)

Copolyesteramides,10 45

meansof affording wider ranges,andhencemorespecificchoices, of biodegradation behaviour11–14,25–33). Moreover,themechanicalandthermalpropertiesof polyesteramidesareopento wide variation by structuralchoice1,34–37)soasto makepotentially availablematerialscombining desiredphysical character with the chemical responserequiredfor particular biotic conditions.

Part 9 of this series1) described random copolyester-amidescomposedof NH(CH2)5CO (6A) andO(CH2)11CO(12E) unitsasmaterials which,whencontaining from ca.20 to 55 mol-% of 6A units, displayedan unusualrela-tively homogeneousphasecharacter, free of the distinc-tive crystalline phasescommonly found in polyester-amides but retaining physical solidity and mechanicaltoughnessto temperatureswell abovethoseexpected foramorphous copolymers of this class.Sinceseveralreportsdescribethebiodegradation of crystalline(i. e.polyphase)polyestersandpolyesteramidesasbeingfocussedinitial lywithin the amorphousregions,leadingto an increaseincrystallinity of the residual material20,29,30,38), it was ofinterestto examine theresponseof the6A/12E polymersto some enzymes ordinarily displaying proteolytic orlipolytic specificity which might act, respectively, uponthe amideor ester unionspresent.The latter in particularmight besusceptible sinceit is known that the12-oxydo-decanoyl(12E) unit participatesin natural synthetic aswell as laboratory enzyme-mediated polymerization pro-cessesyielding oligo-12E products39–41); that 12-hydroxydodecanoic acid is involved in theenzymaticmetabolismof dodecanoic (lauric) acid in both animals andplants42–46); andthat 12-dodecanolactone(the cyclic formof the 12E unit) canbe homo- andcopolymerized undertheaction of lipase47).

Resultsand discussionMelt-pressed films of 6A/12E copolymers containingfrom 29 to 60 mol-% of 6A units (53–81 weight-% of12E) wereincubatedat 378C for 336h, eachwith oneoffour enzymesin a pH 7,4 buffer solution. Parallelsimilarrunswere madewith nylon 6 homopolymer film, andofall five materials in anenzyme-freebuffer solution.Sincethe enzymeswere likely to undergo autolysis during theexperiments, separatecomparisonruns were made withthesein buffer but without polymer substrates. The sys-temswere unsterilisedand,althoughbacterial growth didoccurin somecases,this wasnot disadvantageoussinceageneralobject wasto note whetheranybiotic breakdownwould takeplace; in theoutcome,no effect dueto bacter-ial growth wasfound.

Essential characteristics of the copolymersstudied aregiven in Tab. 1. Their preparation and propertiesweredescribedin Part91) but it maybenotedthatall hadbeenmadehydroxyl-ended in order to inhibit carboxyl auto-

catalysis of hydrolysis. R6 and R7, although similar inco-unit contents,resulted from differentpreparative con-ditions, the former having a highercontentof single 6Aunits andthe latter of multi-6A sequencescombinedran-domly in the polymer chains. The enzymesemployedwere (a) protease(bacterial,ex. Subtilisin BPN1), (b) col-lagenase(ex. Clostridium sp.), (c) a-chymotrypsin (ex.bovine pancreas), and (d) pancreatin (ex. porcine pan-creas). Of these,(a)–(c) haveproteolytic specificity, andhencea formal potentialfor CO1NH bond scissionin thecopolymers (note: chymotrypsin also cleaves CO1Obonds in someester-containing polymers15,16)), whilst (d)hasa broaderspectrumof action, thoughmainly esteroly-tic. The lettersa–d, asabove,togetherwith the copoly-mer identifiers will be used to denoteindividual runs;thosewith nylon 6 homopolymer will beshownasN6-a,etc., andenzyme-freecontrol runswith thesuffix f.

Generalobservations

At the outset,all the treating media were clear or onlyslightly hazy, andfor a–c andf remainedsofor ca.140hafter which small amounts of filamentary or gelatinousparticleswereseensuspendedin theliquids.With d, how-ever, flocculentprecipitatesappearedafter 24 h andper-sisted to the endwhenthey separatedleaving clearsolu-tions.All a andf systems,aswell at R7-c, R8-c andR11-c remainedcolourless,whereasall b andd films andR6-c becameyellow. No pH changehadoccurred in any ofthe incubation liquids at theendof theexperiment. Filmsof R6-d, R7-d andf, andR11-b, d andf crackedor brokeduring the period of treatment,and a subsequent simplefolding test showedfailure with all recoveredR11 sam-plesandwith R7-d andR8-a. Becauseof the breakagesthe gravimetric dataareincomplete,but thosespecimensrecoveredintact (including the N6 andthe f series films)showed weight lossesof between5 and 11%, averagingabout 2,6 g N m–2 of film surfacewith no obvious speciescorrelation.Thesevalues aresomewhat larger than havebeen reported for other copolyesteramides of similarweight-%estergroupcontent28,31,33), but certain of thelat-

Tab.1. General characteristicsof the6A/12E copolymers

Copoly-mer

ginh

dL=ga�mol-%

6 A unitsWeight-%6 A units

Weight-%12E units

Tm �Kofler��C

b�

R6 0,89 29,1 19,0 81,0 125R7 0,90 29,7 19,4 80,6 122R8 0,93 37,7 25,7 74,3 151R11 0,75 60,2 46,3 53,7 169

a) In m-cresol(c = 0,5g/100 mL) at 308C.b) Apparentmelting temperatures(for detailsseeref.1)): films

weremelt-pressedat ca.208C abovethesetemperatures.

46 I. Goodman,M. T. Rodrıguez

ter were examined in static conditionsin contrast to thesustained agitationusedherewhich entailedan extentofmechanical attrition of thefilms.

Examinationof thesupernatantliquids

Hydrolytic breakdownof the copolymers by amidolysiswould be expectedto yield 6-aminohexanoic acid (6-AHA) as a water-soluble degradation product. Evidencefor its presencehasthereforebeensought by aminoacidanalysis of the incubation fluids where, if present, itwould haveto be distinguishedfrom otherproducts thatmight have resulted from enzymeautolysis during theperiodof theexperiments.

With the chromatographicsystememployed (Fig. 1),referencedagainst standard aminoacid mixtures,6-AHAwas resolved at a characteristic elution time of ca.113min, approximatedotherwiseonly by 3-methylhisti-dine (ca. 112,8 min) which is not expected as a productfrom enzymeprotein though it might occurasa bacterialmetabolite. However, this did not preclude thepossibility(asprovedto bethecase with a-chymotrypsin) thatotherunidentified enzymederivatives might appear at elution

timessimilar to thatof 6-AHA, or that,with thecomplexsystems present(or the total degradation product load-ing), theremight be interferencewith the lineardetectionvs. concentrationrelationship of 6-AHA alone. Conse-quently, the quantifiedresults in Tab. 2 areexpressedasapparent6-AHA values,representing the maximum pos-sible contentsof 6-AHA in the incubates.The figureswould only bea meaningful measureof real6-AHA con-tent if theapparentvalues were significantly greaterthanthose found for the enzyme-autolysis (substrate-free)experiments. This was the case only for the pancreatinrunsR6-d andR8-d, whosevaluesin relationto all othersareof uncertain validity. With thesepossible exceptions,therefore,therewasno evidencefor thereleaseof signifi-cant quantities of 6-AHA from any of the copolymers,indicating that no extensivescissionof amidebondshadoccurredin theconditionsemployed.

Polymersurfacetexture andcomposition

Scanning electronmicrographs (SEM) of the surfacesofR6 films following various incubationsareshown in Fig.2. Thoserecovered from treatmentswith protease,col-lagenase and a-chymotrypsin showed no significantchange of surfacemorphology from that of the enzyme-free experiment R6-f. However, pancreatin incubation(R6-d) gave rise to a very different pattern, formed ofparallel longitudinal striations spaced approximately3 lm apartandseparatedtransversely by groupsof shortnodular structures.Similar striated patternswere foundwith eachof the other copolyesteramide-pancreatincom-binations (with no striations in the a, b and c series),whilst the N6 specimensshowed essentially clean pat-tern-free surfaces from all incubations, including thatwith pancreatin.

Fig. 3 comparesthe surfaceSEM of pancreatin-treatedcopolymer R7 with those following treatment of thesame copolymer with dilute solutions of sodium hydro-xide and ethanolamineas chemical etching agents. Thedistinctive morphology of the first-mentioned(R7-d) isagain apparent.

Thefindingsarethus consistentwith a form of specificsurface erosion of the copolymers by pancreatin, andsinceno similar effect occurredwith nylon 6 it wasprob-able that the surfacetexture changesas compared withthe enzyme-free runs, involved the estercomponentofthe copolyesteramides.This wasconfirmed by the FT-IRATR (reflectance)spectraof the film surfaces (Fig. 4)which showed,for pancreatin, a marked reduction in theintensityof the ester-CO stretching band(ca.1725 cm–1)relative to the Amide I and II bands(ca. 1635, 1533cm–1), correspondingto a diminution of 12E unit contentin the surfacelayer. Since in this spectralrange the IRradiation penetratesto a depthof ca.1 lm, the chemical

Fig. 1. Representativeelution chromatograms of supernatantincubation liquids: (i) and (iii) runs R11-a and R8-c, respec-tively; (ii) and(iv) autolysispatternsof proteaseanda-chymo-trypsin, respectively. The asterisk(*) at ca. 113 min indicatestheelutionpositionexpectedfor 6-aminohexanoicacid

Copolyesteramides,10 47

Tab.2. Apparent6-AHA contentsin incubationsupernatant liquids (valuesin nanomolespermLa))

System Code N6 R6 R7 R8 R11 Enzymeautolysis(substrate-free)

Protease a nil 299,7 140,1 425,4 230,4 436,9Collagenase b nil 74,7 66,7 72,4 46,5 not determineda-Chymotrypsin c 4657,2 6616,7 4894,5 5179,6 4826,2 7688,9Pancreatin d 488,7 (2487,4)b) 641,4 (2009,9)b) 143,2 453,2Blank (enzyme-free) f nil nil nil nil nil –

a) 1 nmol/mL of 6-AHA is equivalent to 1,312610–7 g/mL.b) Valuesof uncertainvalidity.

Fig. 2. SurfaceSEMsof incubatedR6 films: (i)–(v) respectivelyfrom runsf, a, b, c andd.Original magnification 10006

48 I. Goodman,M. T. Rodrıguez

change(i. e. lossof 12E units) hasoccurred in a depthofsimilar dimensions to that of the striae in the SEMs,theorganized upper features of which must therefore beamide-richer.

Generaldiscussion

Thesignificant findingsof this work arethat: (1) with thequestionable exception of casesR6-d andR8-d, 6-AHA(an indicator of amide-bond fission) did not result fromincubation of the 6A/12E copolymersor of nylon 6 withany of the enzymesemployed, and (2) all four copoly-mers,but not nylon 6, underwentsurfaceerosionby pan-creatinalone of thoseenzymes,with the development ofa distinctive striolate surfacemorphology. Otherthantheuncertain 6-AHA figure in the caseof R6, no significantdifference attributable to their qualitatively differentmicrostructureswasobservedin the responseof R6 andR7 to pancreatin.

Since positive effects occurred with pancreatin, it isclear that the non-formation of 6-AHA is not an effectsimply of polymer surfaceinertness.The 6A units in thecopolymersarecombinedwith theestercomponenteitherisolated (l12E-6A-12El) or in oligomeric sequences(l12E-(6A)x-12El), requiring in eithercasethescissionof two successive hydrolysable linkages (1 amide + 1ester, or 2 amide)for 6-AHA to result.Evidently none ofthe enzymesusedpossessedthe structuralspecificity forsuchpairedreactions to takeplace,andtheresults concurwith other reports that only free low-molecular-weightnylon 6 oligomers, and thosehavingunsubstituted term-inal groups,aremetabolisedby bacteria48) or degraded bya bacterial hydrolase49).

The action of pancreatin on R6–R11 was selectiveboth with regard to the entity (the 12E group) attacked,andtheresulting surfacemorphology. IR evidenceof pre-ferential ester attack in polyesteramideshasbeengivenpreviously for the in vitro non-enzymatic breakdown of

Fig. 3. SurfaceSEMsof R7 films: (i) and (ii) following treatmentwith dilute NaOH and ethanolaminesolutions, respectively; (iii) following incubation with pancreatin(run R7-d). Original magnification25006

Copolyesteramides,10 49

L-lactide/nylon 6,10 copolymers12), and for someunsub-stituted linear aliphatic copolymers incubated in livingfungalcultures28). Neitheraccountreportedon thesurfacetexture,but anSEM studyof alternating poly(L-alanyl-L-lactyl) recovered after lengthy in vivo implantationshowedextensive formation of a porous reticular struc-tureextendinginto thedepthof thematerial,anddifferentfrom thesurfaceeffectsdescribedabove14).

The extent of degradation of the presentmaterials bypancreatin is relatively slight and limited to the surfacelayer. This is seen as a consequence of the hydrophobiccharacter conferredby the12E segmentswhich, togetherwith thehomogeneouspolymer texture,hinders thepene-tration of the attacking agentinto the depth of the poly-mers.Further, sincethe surface erosionoccurspreferen-tially by ester-group loss, the residualsurfacebecomesricher in amidewhich serves asa physicalbarrier to con-tinued esterolytic attack.Whether the organisedamide-richer features seenin the SEMs reflect a pre-existingsupramolecular pattern or are an artefact of pancreatintreatmentremainsanopen question.

The abovefindings refer, of course,to aqueouscondi-tions but are paralleled by the different results whichoccur on non-aqueousaminolysis of alternating and ofrandom6A/12E copolymers50). The sequential order ofsub-units in copolyesteramidesis thus a further structuralfactorto beconsideredin designing polymersof this classfor specificresponses to biodegradation.

Experimental partThe incubationswereperformedin a thermostatted shakingapparatus,with the polymers in the form of melt-pressedfilm strips of approximatedimensions1062560,1 mm(eachweightingca. 25 mg), placedtogetherwith 10 mL ofincubation liquid in glass vials of 15 mL capacity, withPTFE-lined screwcapclosures.Theactivitiesof theenzymes(Sigma)andtheconcentrationsusedwereasfollows:

The solutions were made up in deionised water withHydrion (Aldrich) pH 7,40buffer, the latter alsobeingusedfor the enzyme-freeruns.The films recoveredafter incuba-tion wererinsedanddried.

Examinationof thesupernatantliquids for 6-AHA contentwas performed after filtration through Gelman SciencesNylon Acro discs of 0,45 lm pore size, using an LKBAlpha-Plusaminoacidanalyzerwith stepwiselithium citrateelution and ninhydrin detection.Except for the initial cali-brationwith authentic6-AHA, 80 lL of samplewasloadedpertest,peakareasbeingdeterminedautomatically.

Fig. 4. PartialIR-ATR spectra (1800–1400cm–1) of polyesteramidefilm surfaces:(i) and(iii) from R8-f andR11-f enzyme-freeruns,respectively; (ii) and(iv) fromcorresponding R8-d andR11-d pancreatinruns.A, B, C andD aretheC2O stretch,Amide I, Amide II andC1H bandsat ca.1725,1635,1533and1460cm–1, respec-tively

Activity(suppliersdata)

Concentrationin mg/mL–1

(a) ProteaseTypeXXVII (Nagase)

7–14 units per mgsolid

0,4

(b) CollagenaseType1A

A125 collagen diges-tion unitspermg

0,5

(c) a-Chymotrypsin 40–60unitspermg 3,0TypeII protein

(d) Pancreatin 86USPspecification 3,0

50 I. Goodman,M. T. Rodrıguez

The film surfaceswereexaminedby FT-IR ATR spectro-scopy using a Perkin Elmer Model 1720 instrumentfittedwith a SpectrotechATR, ZnSe crystal attachment,and bySEM using a JEOL 6400 instrumentwith gold sputtering.Fig. 2 and 3 are printed with the conventionthat the whiteareasare the topologicaluplandsand the dark portionsarethevalleys.

Acknowledgement:We gratefully acknowledgethe provisionof adviceand facilities by ProfessorT. G. Baker and Dr. (nowProfessor)M. L. G. Gardnerof theBiomedicalSciencesDepart-ment of this University. We also thank Mrs. K. M. Illingworthfor the amino acid analyses, Mr. S. C. Mitchell for the SEMwork, andtheSpanishMinistry of EducationandSciencefor theawardof aScholarshipto M.T.R.

1) I. Goodman,M. T. Rodrıguez,Macromol. Chem.Phys. 197,881(1996)

2) R. Langer, N. Pappas,J. Macromol. Sci., Rev. Macromol.Chem.Phys. C23(1), 61 (1983)

3) G. E. Zaikov, J. Macromol. Sci., Rev. Macromol. Chem.Phys. C25(4), 551(1985)

4) S. J. Holland,B. J. Tighe,P. L. Gould,J. Controlled Release4, 155(1986)

5) J. P. Singhal, H. Singh, A. R. Ray, J. Macromol. Sci., Rev.Macromol. Chem.Phys. C28 (3,4),475(1988)

6) S. J. Huang(Ch.21,pp. 597–606) and D. F. Williams (Ch.22, pp. 607–619), in: ComprehensivePolymerScience, Vol.6, G. Allen, J. C. Bevington andG. C. Eastmond,Eds.,Per-gamonPress,Oxford 1989

7) G. E. Zaikov, V. S. Livshits, “Biodegradable Polymers”, in:Polymer Yearbook-4, R. A. Pethrick, Ed., Harwood Aca-demicPublishers,London 1990,pp.177–201

8) T. Tsuruta,“ContemporaryTopicsin Polymeric MaterialsforBiomedicalApplications”, in: Advancesin Polymer Science,Vol. 126,SpringerVerlag,Berlin 1996,pp.1–51

9) W. J.Bailey, Y. Okamoto,W.-C. Kuo,T. Narita,in: Proc.3rdInter. Biodegradation Symp., J. M. Sharpley and A. M.Kaplan, Eds., Applied Science Publishers,London 1976,pp.765–773

10) R. D. Katsarava,D. P. Kharadze, L. I. Kirmelashvili, T. M.Bendiashvili, M. M. Zaalishvili, Soobshch. Akad.NaukGruz.SSR114 (2), 321(1984);Chem. Abstr. 102, 62640s(1985)

11) S. Andini, L. Ferrara,G. Maglio, R. Palumbo,Makromol.Chem.,RapidCommun. 9, 119 (1988)

12) V. de Simone, G. Maglio, R. Palumbo,V. Scardi,J. Appl.Polym.Sci. 46, 1813(1992)

13) L. Castaldo,P. Corbo, G. Maglio, R. Palumbo,Polym.Bull.(Berlin) 28, 301(1992)

14) M. Yoshida, M. Asano, M. Kumakura, Makromol. Chem.,RapidCommun. 11, 337(1990)

15) S. J. Huang,D. A. Bansleben,J. R. Knox, J. Appl. Polym.Sci. 23, 429 (1979)

16) I. Tabushi,H. Yamada, H. Matsuzaki, J.Furukawa,J. Polym.Sci.,Polym.Lett.Ed. 13, 447(1975)

17) R. D. Fields,F. Rodriguezin: Proc.3rd Internat. Biodegrada-tion Symp.,J. M. Sharpley andA. M. Kaplan,Eds.,AppliedSciencePublishers,London1976,pp.775–784

18) Y. Tokiwa,T. Suzuki,Nature 270, 76 (1977)

19) K. Imasaka,T. Nagai,M. Yoshida,H. Fukuzaki,M. Asano,M. Kumakura,Makromol.Chem. 192, 1067(1991)

20) Y. Doi, “Microbial Polyesters”, VCH PublishersInc., NewYork 1990

21) S. J. Huang,M. Bitritto, K.W. Leong,J. Pavlisko,M. Roby,J. R. Knox, in: Stabilizationand Degradation of Polymers,D. L. Allara andW. L. Hawkins,Eds.,Advancesin Chemis-try Series169, AmericanChemical Society, WashingtonD.C.1978,pp.205–214

22) O. S.Voronkova,T. T. Daurova,M. V. Shablygin,L. P. Mil’-kova,N.M. Mikhailov,Vysokomol.Soedin.A13(3),677(1971)

23) K. Z. Gumargalieva,Yu. V. Moiseev, T. T. Daurova,O. S.Voronkova,I. B. Rozanova,Biomaterials1, 214(1980)

24) K. E. Gonsalves,P. M. Mungara,TrendsPolym.Sci.4 (1), 25(1996)

25) Y. Tokiwa, T. Suzuki, T. Ando, J. Appl. Polym.Sci. 24, 1701(1979)

26) T. H. Barrows,J. D. Johnson, S. J. Gibson,D. M. Grussing,in: Polym.Sci. Technol., Vol. 34 (Polymers in Medicine II),PlenumPress,New York 1986,pp.85–90

27) T. H. Barrows,G. J.Quarforth,P. E. Blegen,R. L. McQuinn,Polym. Mater. Sci. Eng. 59, 376 (1988); Chem.Abstr. 110,63672f(1989)

28) K. E. Gonsalves,X. Chen,J. A. Cameron,Macromolecules25, 3309(1992)

29) P. J. A. In’t Veld, P. J. Dijkstra, J. Feijen, Adv. Biomat. 10(Biomaterial-TissueInterfaces) 421(1992)

30) P. J.A. In’t Veld,P. J.Dijkstra,J.Feijen,Clin. Mater. 13, 143(1993)

31) M. Nagata,T. Kiyotsukuri,Eur. Polym.J. 30, 1277(1994)32) P. J. A. In’ t Veld, Z.-R. Shen, G. A. J. Takens,P. J. Dijkstra,

J. Feijen, J. Polym. Sci., Part. A: Polym. Chem.32, 1063(1994)

33) M. Nagata,Macromol.RapidCommun. 17, 583(1996)34) B. Jasse,Bull. Soc.Chim.France1640(1965)35) P. Akcatel, B. Jasse,J. Polym. Sci., Polym. Chem.Ed. 16,

1401(1978)36) F. deCandia,G. Maglio, R. Palumbo,Polym.Bull (Berlin) 8,

109 (1982)37) L. Castaldo, F. de Candia, G. Maglio, R. Palumbo, G.

Strazza,J. Appl.Polym.Sci. 27, 1809(1982)38) C. G. Pitt, F. I. Chasalow, Y. M. Hibionada, D. M. Klimas,A.

Schindler, J. Appl.Polym.Sci. 26, 3779(1981)39) J.Bougault,L. Bourdier, J. Pharm.Chim. [6] 29, 561;Chem.

Abstr. 3, 2445(1909)40) S. Matsumura,J. Takahashi,Makromol. Chem.,RapidCom-

mun. 7, 369(1986)41) K. Pavel, H. Ritter, Makromol.Chem. 192, 1941(1991)42) M. Kusunose,E. Kusunose,M. J. Coon,J. Biol. Chem. 239

(5), 1374(1964)43) I. Bjorkhem,Biochim.Biophys.Acta260(2), 178(1972)44) R. M. Laetham, D. R. Koop, Mol. Pharmacol. 42 (6), 958

(1992)45) P. Lemaire,M. Lafavrie, D. Weissbart,F. Durst, P. Pflieger,

C. Mioskowski,J.P. Salaun,Lipids 27 (3), 187(1992)46) D. E. Moreland,F. T. Corbin,T. J.Fleischmann,J.E. McFar-

land,Pestic. Biochem.Physiol. 52 (2), 98 (1995)47) H. Uyama, S. Kobayashi,Front. Biomed.Biotechnol. 3, 5

(1996)48) T. Fukumara,Plant Cell. Physiol. 7, 93 (1996)49) S. Kinoshita, T. Terada,T. Taniguchi,Y. Takene,S. Masuda,

N. Matsunaga,H. Okada, Eur. J. Biochem. 116, 547(1981)50) I. Goodman,M. T. Rodrıguez,Macromol. Chem. Phys. 195,

1075(1994)