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Hindawi Publishing CorporationInternational Journal of Polymer ScienceVolume 2013 Article ID 476748 10 pageshttpdxdoiorg1011552013476748

Research ArticleEnzyme-Mediated Ring-Opening Polymerization ofPentadecalactone to Obtain Biodegradable Polymer forFabrication of Scaffolds for Bone Tissue Engineering

V A Korzhikov12 K V Gusevskaya1 E N Litvinchuk1

E G Vlakh12 and T B Tennikova12

1 Institute of Macromolecular Compounds RAS Bolshoy Prospect 31 Saint Petersburg 199004 Russia2 Chemistry Department Saint-Petersburg State University Peterhof Universitetskii Prospect 26 Saint Petersburg 198504 Russia

Correspondence should be addressed to V A Korzhikov v korzhikovmailru

Received 16 April 2013 Accepted 20 August 2013

Academic Editor Marek Cypryk

Copyright copy 2013 V A Korzhikov et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The optimization of enzyme-mediated polymerization of pentadecalactone (PDL) was performed to obtain macromolecularproducts suitable for generation of 3D cell supports (scaffolds) for bone tissue engineering Such parameters as temperaturemonomerenzyme ratio and monomer concentration were studied The maximummolecular weight of synthesized polymers wasabout 90000 Methods allowing the introduction of reactive double bonds into polypentadecalactone (polyPDL) structure weredeveloped The macroporous matrices were obtained by modification of thermoinduced phase separation method

1 Introduction

The development of synthesis and application of biodegrad-able polymers represent great achievement due to ecological[1] and medical problems [2] associated with utilization ofcarbon-chained nondegradable polymers One of the mostemerging classes of synthetic biodegradable polymers isaliphatic poly(hydroxy acids) (PHAs) [3] Their physicalproperties are close to most plastics [4] but possessing ofester bonds in the main chain allows polymer degradationvia chemical or enzymatic hydrolysis [5] It is known that thetime of degradation can be controlled by varying the mono-mer units in a polymer chain [6]

In spite of the fact that PHAs can be obtained via polycon-densation process the polymers with fine and controllableproperties are usually derived using ring-opening polymer-ization (ROP) of lactides or lactones of corresponding HAs[7] Generally such type of polymerization is initiated andcatalyzed by organometallic substances like stannous octoate(Sn[Oct]

2) [8 9] zinc lactate (Zn[Lac]

2) [10] and many oth-

ers [7 9 11 12] These initiators allow obtaining the polymerswith various molecular weights (MWs) and quite narrow

molecular weight distribution (MWD) The main drawbackof the mentioned method is that the resulting polymers con-tain metal ions that further become part of the polymermaterials [1]

Relatively recently the novel approach to synthesize PHAsvia ROP namely enzyme-mediated ring-opening polymer-ization (EMROP) was developed and thoroughly studied bynumerous scientific groups [1 13ndash22] Usually such polymer-ization is performed in the presence of various lipases [18ndash22] for example the enzymes catalyzing the ester bond cleav-age via its hydrolysis However in nonpolar organic mediaand in the absence of water the same enzymes enable theopening of the lactone cycle followed by new ester bond for-mation [13 14] Very detailed and numerous data on EMROPproduced at different conditions can be found in literature[23ndash26] However the polymers with low or mediate MWmostly reported in cited papers cannot be used for materialconstruction In our study we optimized EMROP conditionsin order to synthesize themacromolecular productswith highMW allowing the formation of biodegradable supraporousscaffolds for bone tissue engineering using thermally inducedphase separation (TIPS) method [27]

2 International Journal of Polymer Science

The presented paper demonstrates the results on thedevelopment of optimized experimental strategy to obtainthe biodegradable polymers suitable for supraporous scaffoldconstruction using enzyme-mediated synthesis in non-polarmediumThedevelopment of easy laboratory-scale techniqueto synthesize polypentadecalactone (polyPDL) of high MWneeded for material construction represents a supplementaryobjective of presented research

2 Experimental

21 Materials and Instruments The monomer 120596-pentadeca-lactone (PDL 98) was purchased from Aldrich and usedwithout additional purification Before polymerization thereagent was characterized by proton nuclear magnetic reso-nance (1H NMR) 415 (2H t) 235 (2H t) 164 (4H m) and085 (22H br s) ppm The enzymes Candida rugosa lipase(CRL) and acrylic resin immobilized Candida antarcticalipase B (CALB Novozyme-435) possessing specific activityge40 000Ug and ge10 000Ug respectively 2-hydroxyethylmethacrylate (HEMA) and stannous octoate were purchasedfrom Sigma-Aldrich Toluene chloroform and methanolwere the products of ZAO ldquoVectonrdquo Toluene was driedover calcium hydride and distilled HPLC grade THF waspurchased fromMerck

Syntheses of polymers as well as drying of enzymesand products were carried out in Schlenck tubes (Aldrich)connected to a vacuum-argon line provided with oil vacuumpump 2B-11 (Gidromekh) To seal the reaction tubes and bot-tles with solvents Precision Seal rubber septa caps (Aldrich)were used

The precise heating of reaction mixture was reached bythe application of magnetic stirrer MR Hei-Standard (Hei-dolph) equipped with temperature controller as well as usinga glycerol bath while the stirring of polymerization mediumwas provided using an IKA T-16 basic overhead stirrer

For concentration of polymer solutions IR-1 rotary evap-orator (Khimlaborpribor) was used The intrinsic viscositymeasurements were performed in Ostwald type viscometer(capillary diameter 037mm ZAO ldquoNeva-Reactivrdquo) suppliedby LOIP LT-912 cryothermostat (ZAO ldquoLOIPrdquo) that wasalso applied for cryotropic phase separation at macroporousmaterials preparation

Themolecular weight (MW) andmolecular weight distri-bution (MWD) analysis was produced using Shimadzu liquidchromatography consisting of LC-10AD pump and RID-10Arefractometric detector and supplied with Waters StyragelHMW 6E analytical column To calculate MW and MWDLC Solution 124SP1 GPC software was used Polystyrenestandards synthesized and characterized at the Institute ofMacromolecular Compounds of RAS by Dr Ludmila Vino-gradova were used to build a calibration curve

The 1H NMR spectra were recorded with BrukerAVANCE-400 instrument

22 Methods221 Enzyme-Mediated Polymerization of PDL The typicalprocedure to obtain polyPDL was followed 093mL of

Ar

Figure 1 Scheme of reaction installation for EMROP of PDL

toluene was dropped under intensive stirring and dry argonflow to the mixture of 400mg of monomer and 40mg ofenzyme (CRL or CALB [M][E] = 101wtwt other ratioswere also tested see Figure 5) in the reaction tube (Figure 1)The temperature was raised up to predetermined value (from35 to 115∘C see Table 4) and reaction was carried out until asolidification of reaction mixture (usually 1ndash3 hours) If thatdid not occur the reaction time was prolonged for 6 hoursmaximum To stop the polymerization the reaction tubewas quenched into ice-cold water The content of reactiontube was dissolved in the excess of hot chloroform Thenpolymer solution was separated from granules of solid-phaseenzyme by filtration (Schott glass filter number 3)The filtratewas concentrated in a rotary evaporator and the polymerwas drop-wise precipitated to cold methanol The precipitateobtained was isolated by filtration on glass Schott filternumber 1 and dried in vacuum

222 Enzyme-Mediated Polymerization of PDL in a Presenceof 2-Hydroxymethacrylate (HEMA) The synthetic procedureto obtain polyPDL-HEMA was the same as that applied forsynthesis of polyPDL The only difference was the additionof HEMA to the reaction mixture ([M][HEMA] = 20 and40mol ratio)The derived polymer was dried by sublimationsealed and stored in a freezer at minus20∘C

223 Radical Reaction of PolyPDL-HEMA A sample of150mg of polyPDL-HEMA and 75mg of radical initia-tor 221015840-azo-bis-isobutyronitrile (AIBN) ([polyPDL-HEMA][AIBN] = 201 wtwt) was inserted into the ampule and dis-solved in 1mL of dry toluene ([polyPDL-HEMA][toluene] =17 wtwt) The ampule was sealed and placed into preheatedup to 70∘C thermostat and kept there for 24 hours Then theampule was cooled down and the polymer was precipitatedin cold methanol filtered and dried over calcium chloride

International Journal of Polymer Science 3

224 Polymer Analysis

(1) 1119867 NMR Spectroscopy The structures of synthesizedpolymerswere confirmed by 1HNMRspectra recorded in 6d-chloroform (CDCI

3)

(2) Intrinsic Viscosity Measurement To get the intrinsic vis-cosity ([120578]) of synthesized polymers dissolved in chloroformOstwald capillary viscometer was used The flow time mea-surements were performed at 25∘C for the following polymerconcentrations 150 133 119 105 and 094plusmn001 gdL then120578sp119888 parameter was calculated and extrapolated to the zeroconcentration to establish [120578] No Mark-Houwink equationwas found for polyPDL in the literature

(3) Gel Permeation Chromatography (GPC) To evaluate MWof synthesized samples their solutions in THF with concen-trations of 015 wt and 025wtwere injected into Styragelcolumn via 50 120583L valve loop and eluted at 03mLmin usingTHF as a mobile phase The elution curves obtained wereconverted into MWD ones using a preliminary built calibra-tion curve (polystyrene standards)

225 Macroporous Matrices Fabrication

Formation of Macroporous Matrices via Thermally InducedPhase Separation (TIPS) Technique In a glass tube 1 wtsolution of amixture of polyPDL and polyPDL-HEMA (from85 to 15wt resp) in 14-dioxane containing 15 wt ofwater and 1 wt of AIBNwas prepared To realize simultane-ous homogenization of a prepared solution and free-radicalinitiation the mixture was heated up to 65∘C and sustainedduring 2 hours After that the reaction medium was slowlycooled down up to occurrence of solid-liquid phase sepa-ration and quenched in liquid nitrogen Then the tube wasplaced into a fuming liquid nitrogen bath at minus40∘C and 10ndash15mL of ethanol was added in order to extract frozen dioxanefrom the formed pores The bath together with the tube wasshook for 3 hours and every 30min ethanol was exchangedon a fresh portion Then the matrix was left in the excess ofethanol in a freezer (minus20∘C) for 2 days and further dried atroom temperature

3 Results and Discussion

As it was noticed the final goal of the presented research wasto obtain biodegradable polypentadecalactone (polyPDL)with molecular weight sufficient for supraporous materialpreparation Enzyme-mediated synthesis which allows for-mation of polymers with controlled molecular characteris-tics was chosen to reach the goal Nevertheless the firstexperiments were directed on a comparison of chemical andenzymatic methods to synthesize the same macromolecularproduct

31 Comparison of Enzyme-Mediated and Chemically Cat-alyzed Ring-Opening Polymerization Preliminary EMROPOptimization According to our experiments the ring-opening polymerization of lactones can be performed at

Table 1 Reaction yield and polymer intrinsic viscosity at stannousoctoate initiated ROP of PDL

Sample number T ∘C Yield [120578] dLg1 120 5 Not determined2 160 9 Not determined3 200 64 0384 220 61 033Conditions bulk process reaction timemdash70 hours [PDL][Sn(Oct)2] molarratiomdash50 1

high temperature either in a sealed ampoule or in an evac-uated Schlenk tube Also the process can be carried out ata relatively lower temperature using double-neck flask underargon atmosphere While the first two methods give similarand good results (meaning the reaction yield and MW of aproduct) the latter demonstrates definite advantage becauseit is much less volatile

In the present experiments the chemically catalyzed ROPof PDL was initiated by stannous octoate and performed in abulk in vacuum-sealed ampoule

From the data presented in Table 1 one can see that syn-thesis of a real macromolecular product requires applicationof quite high temperatures In comparison the applicationof similar conditions to another monomer namely L-lactideresulted in a product with [120578] = 07 dLg at 150∘C Thus itis obvious that reactivity of PDL in polymerization initiatedby stannous octoate seems to be relatively low this is inaccordance with previously published data [9 14]

The attempts to realize bulk EMROP of PDL in ampouleusing two commercial enzymes Candida rugosa lipase (CRL)and Candida antarctica lipase B (CALB Novozyme 435) ledto formation of oligomers with very lowmolecular weight Inmost cases the product appeared as a fine dispersed dust innon-solvating (cold methanol) media Additionally the yieldof polymerization never exceeded 2-3 The reason for sucheffect can be explained by a difference of mechanisms ofenzyme-mediated and chemically catalyzed reactions [8 13]When the polymerization process is initiated by stannousoctoate the lactone cycle is cleaved to form active specie andthen the propagation occurs via nucleophilic attack of neigh-boring monomer molecules On the contrary in the case ofEMROP the repeating cycles of enzymatic catalysis followedby formation and dissociation of enzyme-monomer complexare necessary for polymerization to proceed Hence thesynthesis of the discussed polymer via chemically initiatedROP looks favorable being performed by reaction in a bulk ofmonomers with well-dispersed initiators while for enzyme-mediated ROP of the same monomer the sufficient diffu-sionalmobility of enzyme in reactionmixture is requiredTherealization of such kind of polymerization in toluene solutionat intensive mixing should improve the diffusion of substratemolecules to enzyme active sites and product withdrawingas well as minimizing a probability of side reactions Theuse of toluene as a solvent was dictated by good solubility ofPDL high boiling point and high hydrophobicity Finally thereactionwas performed in a double-necked flask under argonatmosphere and at intensive mixing of reaction medium(Figure 1)


Table 2 EMROP of pentadecalactone Dependence of reactionyield and polymer intrinsic viscosity on amount of water added tothe enzyme before the reaction

Sample number H2O wt Yield [120578] dLg5 15 1 mdash6 25 mdash mdash7 0 88 028Conditions CALB 75∘C [M][E] = 101 wtwt [M] concentrationmdash50wt reaction timemdash6 hours

Table 3 EMROPof pentadecalactone Dependence of product yieldand polymer intrinsic viscosity on reaction time

Sample number Time h Yield [120578] dLg9 28 93 0218 4 92 038Conditions CALB 65∘C [M][E] = 101 wtwt [M] concentrationmdash50wt

The application of the suggested experimental installationresulted in high reaction yields and intrinsic viscosity valuesof polymers obtained usingCALB while for theCRL case theyield never exceeded 10 Therefore all further experimentswere carried out using first commercial lipaseThe advantageof CALB can be also explained by its immobilization onacrylic resin that leads to additional stability of enzymemacromolecule Moreover some definite contents of waterwithin relatively hydrophilic resin can be a key factor for suc-cessful catalysis because it is known that enzyme activity innonpolar organic media still depends on water presence [28ndash31] The small amount of water is necessary to preserve theactive conformation of enzymemolecule and plays the role ofthe additional initiator of the polymerization process [7 31]According to that the hydrophobicity and hygroscopicity ofthe chosen organic solvent is of a great importanceThus thesolvent has to be hydrophobic enough and non-hydroscopicto avoid removal of water from surrounding enzyme Fromthis point of view it was interesting to evaluate the impact ofsmall amounts of water on the activation of polymerizationprocess For this purpose the commercial CALB (immobi-lized form) was dried over P

2O5 and then different portions

of water were added just before synthesisThe results of theseexperiments are presented in Table 2

It is obvious that even very small amounts of wateradded to the enzyme before mixing with monomer solutioncompletely stopped the polymerization while the applicationof enzyme in its initial form led to polymer formation Itmeans that the content of water provided by swollen resin isenough for the catalyzing process

The very curious effect concerning the reaction timewas observed Thus the reaction mixture was solidified afterseveral hours (Table 3 sample 8) whereas the continuationof a process till 24 hours led to its total clarification followedby a fall of intrinsic viscosity (Table 3 sample 9) This phe-nomenon might be explained by postpolymerization chaincleavage (inverse enzymatic process) as well as by interchain

Table 4 EMROP of pentadecalactone Dependence of reactiontime yield and intrinsic viscosity of resulting products on reactiontemperature

Samplenumber T ∘C Time h Yield [120578] dLg 119872119908times10

minus3

(GPC)lowast 119872119908119872lowast

119899

10 35 60 3 mdash mdash mdash11 45 20 87 030 mdash mdash12 55 10 87 054 393 17813 65 15 85 056 291 15814 75 30 89 059 mdash mdash15 85 30 90 075 mdash mdash16 95 50 86 079 mdash mdash17 105 25 91 102 880 16518 115 20 84 089 mdash mdashlowastHere and further the value corresponds to MW of a major elutedpeak related to preliminary built calibration curve (polystyrene standards)Conditions [M][E] = 101 wtwt [PDL] = 50wt

Table 5 Dependences of polyPDL yield reaction time (visualfixation of polymerization mixture solidification) and productintrinsic viscosity on monomer concentration

Sample number Time h [M] conc wt Yield [120578] dLg19 15 50 85 05620 6 25 90 07321 6 17 84 056Reaction conditions T = 65∘C [M][E] = 101 wtwt

side reactions leading to an increasing number of short frag-ments

According to this result the time of a process when thereaction mixture became solid has been chosen for furtherexperiments The maximal MW of the polymer obtainedthat was reached at chosen conditions was also taken intoconsideration

From preliminary investigations it was also found thatthe temperature of enzymatic reaction is equal to 65∘C and[M][E] ratio is equal to 10wtwt and monomer concentra-tion of 50wt led to a maximum yield and final productintrinsic viscosity Therefore these conditions were taken asa reference for further experiments

32 Optimization of PDL EMROP Conditions321 Effect of Reaction Temperature on Yield and Intrinsic Vis-cosity of Synthesized PolyPDL To evaluate the effect of reac-tion temperature on the mentioned parameters a series ofsyntheses at identical conditions with only temperature vari-ation was performed The results obtained are presented inTable 4

According to results presented in Table 4 at temperaturesbelow 40∘C the polymerization proceeds only to a negligibleextent with very low yield of a product Increasing thetemperature above 45∘C leads to formation of polymers withquite high yieldsThese values (86ndash91) remain constant in awhole studied temperature interval At the same time thegrowth of intrinsic viscosity takes placewhen the temperature


Table 6 Effects of polymer yield reaction time and product intrinsic viscosity on [M][HEMA]

Sample number Time h [M][HEMA] molmol Yield [120578] dLg 119872119908times 10minus3 (GPC) 119872

119908119872lowast

119899

22 5 Control 87 058 267 14323 5 20 85 021 164 15624 5 40 88 032 220 163Reaction conditions T = 55∘C [M][E]mdash30wtwt [M]mdash25wtlowastThe ratio is given for macromolecular peak only while for the whole sample the PDI is much higher

is increased from45∘C to 105∘C Further temperature increas-ing does not lead to [120578] growth This result can be caused bypartial loss of enzyme activity because of the removal of someamount of bound water Also the high temperature can be areason of conformational changes in protein macromolecule

Figure 3 demonstrates the complex graphs related tothe dependences of reaction time and intrinsic viscosity onapplied temperature Obviously two local intervals with opti-mal temperature can be registered

First optimum is within the temperature range 55ndash75∘CAt this temperature range the product with quite high viscos-ity is obtained at a minimum reaction time The presence ofsuch optimal zones means that the product with high viscos-ity (high MW) can be obtained at quite short reaction timesThe second optimum is detected in a narrow range of tem-peratures for example between 100∘C and 110∘C Here thepolymers with maximum viscosity are formed

The first optimum represents the special interest becauseit allows synthesis of really macromolecular products at verylow temperaturesThe polymerization at similar temperaturevalues is also possible at chemical initiation by alkali-earthmetals and lanthanide alkoxides [7 32 33] Though suchmethods necessarily require application of special experi-mental techniques namely inert atmosphere (glove-box) andhigh vacuum (vacuum line) because the air moisture easilyhydrolyzes the counted initiators Contrary to that EMROPcatalyzed by CALB can be easily performed at mild heatingintensive mixing and under argon flow in order to excludethe effect of airmoisture on enzyme activationThus EMROPdemonstrates significant experimental advantages especiallyregarding laboratory scaled-up synthesis Moreover the mildreaction conditions should allow introduction of reactiveterminal groups such as double bonds which is not possibleat high temperature polymerization

As to the second optimum it is interesting that theenzyme keeps its activity at the temperatures above 100∘Cwhich is much higher than physiological conditions In factthe reaction proceeds at nearly boiling point of tolueneThis phenomenon can be related to the fact that the poly-merization is performed in hydrophobic organic solvent(dry toluene) From classic enzymology it is known thatthermoinactivation of biocatalyst requires the participationof water which promotes conformational mobility and acti-vates a decomposition of protein molecule Dry toluenecontains no water and thus the enzyme molecules seemto be stabilized by organic solvent This effect can be areason for remaining enzyme activity and makes EMROPthe prefered method to obtain the polymer of high MW in alarge scale Additionally this method allows synthesis of

macromolecular products free of metal admixtures andenzyme could be easily separated by filtration of polymersolution in chloroform

322 Effects of Reaction Time Product Yield and IntrinsicViscosity onMonomerEnzyme Ratio The effect of monomerto enzyme ratio on reaction time poly(PDL) yield andintrinsic viscosity was evaluated (Figure 4)

The presented data show that at monomerenzyme ratioequals 2 the reaction is fast but results in the polymer withcomparatively lowMWand low yieldThe increase of relativeenzyme concentration leads to the formation of biggeramounts of activated monomer species which initiate theelevated number propagating chains This in turn leads topredominating synthesis of short polymer chainsThe latter isin a good agreement with low viscosity of the resulting prod-uct Moreover relatively high enzyme content of biocatalystcan probably promote the higher extent of side reactions(both inter- and intramolecular)

Ten times increase of PDL excess over enzyme molarconcentration leads to the extension of reaction time At thesame time the yield and intrinsic viscosity of poly(PDL) aregrowing The graphs presented on Figure 4 clearly show thatmaximum reaction time causes the formation of polymerproduct with highest values of yield and viscosity ([M][E]= 30)

Further increasing the monomer to enzyme ratio didnot lead to the yield and viscosity growth This result canbe diminished by low enzyme concentration number ofmonomers (PDL) participating in the polymerization pro-cess

323 Effects of Reaction Time Product Yield and IntrinsicViscosity on Monomer Concentration The results obtainedin the study of dependence of PDL EMROP parameters onmonomer concentration are demonstrated in Table 5Despitea good solubility of PDL in toluene the application of high-concentrated solutions seems to be irrational because of theirhigh viscosityThe conclusion from Table 5 is that decreasingthe monomer concentration from 50 to 25wt prolongsthe reaction time as well as the product yield and intrinsicviscosity Similar to [M][E] effect the result obtained can beexplained by the diminishing number of activation acts andthus by the increasing of both kinetic andmaterial lengths ofpolymer chains

It is important to note that in the cases of samples 20 and21 no solidification of reaction mixture was observed andthe reaction was stopped after 6 hours according to the abovespecified protocol (see Section 321) Obviously increasing


Solvent

Polymer

Solution

Temperaturedecreasing

Phase separation Solvent removal

Porous matrix

Figure 2 General scheme of matrix formation using TIPS technique

20 40 60 80 100 1201

2

3

4

5

6

7

Reaction time

Reac

tion

time (

hour

s)

20 40 60 80 100 120

02

04

06

08

10

[120578]

[120578] (

dLg

)

T (∘C)

T (∘C)

Figure 3 Temperature dependences of reaction time (visual detec-tion of reactionmixture solidification) and polymer intrinsic viscos-ity Reaction conditions [M][E] = 101 wtwt [PDL] = 50wt

reaction time will lead to formation of products with higherviscosity values The concentration of PDL equal to 25wtseems to be optimal to obtain the polymer with sufficientmolecular characteristics within a reasonable time interval

Summarizing the results presented in this section we canconclude that two optimal temperature intervals were foundThese are 50ndash85∘C (for temperature sensitive reactions) and100ndash110∘C (high-molecular product formation) The optimalmonomerenzyme ratio and optimal monomer concentra-tion were established as 30wtwt and 25wt respectively

33 Synthesis of Poly(PDL)-HEMA EMROP Facilities forMolecular Architecture Variation As it was pointed in theintroduction the final goal of this work was to develop andoptimize the EMROPmethod to obtain the product namelypoly(PDL) which could be appropriate for fabrication ofrigid scaffolds for bone tissue engineering According to ouridea this should be a polymer bearing active double bond

76

78

80

82

84

86

88

90

Polymer yield

Yiel

d (

)

0

2

4

6

8

10

Reac

tion

time (

hour

s)

0 20 40 60 80

0 20 40 60 80

[M][E] (wtwt)

[M][E] (wtwt)

[120578] (

dLg

)

05

06

07

08

09

[120578]Reaction time

Figure 4 Graphs of dependences of polyPDL yield reaction time(visual fixation of solidification of reaction medium) and productintrinsic viscosity on [M][E] ratio Reaction conditions 119879 = 65∘Cmonomer concentration-50wt

Unsaturated groupsCross-linking via

free-radical reaction

Chemical cross-links

Figure 5 Enhancement ofmechanical properties of polymermatrixvia chemical cross-linking

in its molecular structure These groups can be used bothfor regulation of mechanical properties of created supportvia radical cross-linking of polymer chains (Figure 5) andfor grafting of hydrophilic reactive polymer intermediates forfurther biofunctionalization of scaffold surface


O

O

HO

O

OHaa 11 11

d bb

c ce

a

bcd

e

Chloroform

8 7 6 5 4 3 2 1

(ppm)

d998400

d998400

(a)

O

O O

OHO

O

Me

H

HO a a 1111

bbc c

d ef

g

j

h

i

a

bc

jChloroform

d

eg fh

h

i

i

7 6 5 4 3 2 1

(ppm)

(ppm)62 60 58 56 54

d998400

d998400

(b)

Figure 6 1H NMR spectra (a)mdashpolyPDL (b)mdashpolyPDL obtained in presence of HEMA

25 30 35 40

500

1000

1500

2000

Resp

onse

(mV

)

Elution time (min)

(a)

0

20

40

60

80

100(

)

30 35 40 45 50 55

log(MW)

(b)

Figure 7 GPC elution (a) and MWD (b) curves for sample number 17 ([120578] = 102 dLg see Table 4)

From mechanism of EMROP published elsewhere [14]it is known that participation of hydroxyl-containing com-pound is required for initiating a step It is important thatsuch a compound becomes the part of the resulting polymerstructure Initially such initiation takes place because ofwaterthat is present in acrylic resin used for CALB immobilizationThis process results in the appearance of carboxylic-endgroup on a polymer chain The addition of some amounts ofalcohol to the reaction mixture will provoke another type ofinitiation resulting in ester-end groups formation [7]

In our study we developed the original approach namely2-hydroxymethacrylate (HEMA) was used as both hydroxyl-and double-bond containing initiator HEMA possesses aquite reactive methacrylate residue which is separated fromhydroxyl by two methylene groups

Two polymerization runs were performed at mild tem-perature and different PDLHEMA ratios In parallel thecontrol synthesis at the same conditions but without HEMA

addition was produced (Table 6) It was established thatintroduction of significant amounts of HEMA to the poly-merization mixture led to decrease of the productsrsquo intrinsicviscosity while the yield remained on the same levelThis factconfirms HEMA participating in the initiation of PDL ROPand its entering into the polymer structureThemore HEMAis introduced into reaction mixture the shorter polymerchains are obtained This fact has to be taken into consid-eration because too low MW of synthesized and modifiedpolymer prevents the following porous matrix formationbased on temperature-induced phase separation

The presence of end chain methacrylate groups was alsoconfirmed by 1HNMR spectroscopy (Figure 6) In the NMRspectra of polyPDL obtained in the presence of HEMA well-identified signals of diastereotopic protons of methacrylategroup at 609 and 554 ppm are detected A signal at 194 ppmwhich confirms the presence of HEMA introduced withmethyl group was also found


30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)

Figure 8 MWD curves calculated from GP chromatograms (a)mdashsample 22 (control) (b)mdashsample 23 ([M][HEMA] = 20mol ratio) (c)mdashsample 24 ([M][HEMA] = 40mol ratio) for details see Table 6

From the spectrum on Figure 6(b) the HEMA contentwas calculated as the integral ratio of signals correspondingto the protons of HEMA residue (Figure 6(b) j (194 ppm) h(609 ppm) and i (554 ppm)) and those of methylene groupadjacent to carbonyl group in monomer units (Figure 6(b) c(228 ppm)) It was found that there is 7mol of HEMA inthe synthesized polymer productMoreover the functionalityofmacromonomer was evaluated as the relation of number ofHEMA groups (Figure 6(b) i (554 ppm)) to the number ofthe total molecules of poly(PDL) determined from terminalndashCH2CH2OH groups content (Figure 6(b) d1015840 (364 ppm))

The obtained functionality value is equal to 82 This meansthat more than a half of the poly(PDL) chains are initiatedwith HEMA and finally possess HEMA moiety in theirstructure

Free-radical polymerization with participation of intro-duced methacrylic double bonds was performed using thesample 23 (Table 6)The reactionwas carried out in toluene inthe presence of AIBN as an initiator at 60∘C for 19 hours As aresult the intrinsic viscosity of poly(PDL) increased from021for initial polymer to 030 dLg (plusmn04 dLg) This can be a

reason for concluding that introduced HEMA moietiesenable to regulate macromolecular chains length via theircross-linking which in turn influence the mechanical prop-erties of formed polymer matrix

34 GPC Studies of Poly(PDL) Obtained in Different Condi-tions Additional to intrinsic viscosity measurements GPCanalysis of some samples of specific interest was performed toevaluate the molecular weights of synthesized polymer prod-ucts For that the calibration curve plottedwith application ofpolystyrene standards was used The119872

119908and119872

119908119872119899values

were presented in the foregoing tablesFigure 7 represents the typical elution and MWD curves

are presented The chromatogram usually consisted of twopeaks that resulted in bimodal MW distribution

The presence of two elution peaks found in our experi-ments is in good correlation with previously published data[34] The authors of the cited paper discovered two types ofmolecular architectures obtained linear and cyclic macro-molecules Obviously these products will have different sizesand thus different retention characteristics in GPC


(a) (b)

Figure 9 SEM microphotographs of PDL+PDL-HEMA-based macroporous materials (a)mdashmagnification times2 000 (b)mdashmagnificationtimes10 000

Figure 8 demonstrates MWD curves related to the sam-ples obtained without HEMA (Figure 8(a)) and in the pres-ence of HEMA (Figures 8(b) and 8(c)) It is obvious that theintroduction of HEMA into polymerization medium defi-nitely affects the character of MWD While the first curve issimilar to that presented in Figure 7 the peaks ratio in the lat-ter two cases significantly differsThe observed phenomenoncan be explained by a tendency to decrease cyclic macro-molecules formation along with increasing the amount ofHEMA introduced into reaction mixture (Figures 8(a) 8(c)and 8(b)) It means that the participation of HEMA inpolymer structuring (end chain formation) dramaticallydecreases the probability of the appearance of cyclic macro-molecular form

35 Formation of Poly(PDL)-Based Macroporous Supports viaThermally-Induced Phase Separation (TIPS) and Freeze Sol-vent Extraction The experiments approving the possibilityof formation of macroporous materials based on poly(PDL)were carried out For that thermally-induced phase sepa-ration (TIPS) technique was applied (see Figure 2) On thefirst step a homogenous mixture of poly(PDL) (sample 1785wt) with poly(PDL)-HEMA (sample 24 15 wt) in 14-dioxane containing 15 wt of water and 1 wt of AIBN wasprepared and kept at 65∘C for 2 hoursThe slight opalescencewas observed after one hour which can be referred to thereaction-induced phase separation Then slowly decreasingof temperature was performed until solid-liquid phase sepa-ration was observed After a quenching of polymer matrix inliquid nitrogen the freeze solvent extraction by ethanol wascarried out within 3 days This method is based on a goodmixing of 14-dioxane with ethanol low freezing point of thelatter as well as nonsolubility of poly(PDL) in alcoholsThuscrystalline 14-dioxane was washed out from macroporousstructure of the poly(PDL) by liquid ethanol at a temperatureabout minus40∘C The SEM images of obtained materials arepresented in Figure 9

The microphotographs obtained demonstrate the uni-form morphology of formed materials which possess inter-connected pore structure with mean pore size about 10120583m(calculated from SEM images)

4 Conclusions

Both chemically catalyzed and enzyme-mediated methodswere compared regarding the possibility of synthesis ofpoly(PDL) with highmolecular weightsThe advantage of theenzymatic method was demonstrated

The optimized EMROP method was used to obtainpoly(PDL)-HEMA macromolecules which were shown tobe perspective for formation of macroporous matrices withporous characteristics sufficient for tissue engineering scaf-folds fabrication

Abbreviations

PHA Poly(hydroxy acid)EMROP Enzyme-mediated ring-opening polymerizationTIPS Thermoinduced phase separationPDL PentadecalactoneMW Molecular weightCRL Candida rugosa lipaseCALB Candida antarctica lipase BHEMA 2-Hydroxyethylmethacrylate

Acknowledgments

The work was financially supported by the Russian Ministryof Education and Science (program ldquoScientific and scientific-pedagogical personnel of innovative Russiardquo Agreements no8471 and no 14740110382) and the Russian Foundation forBasic Research (Grant no 11-03-00829-a)

References

[1] S Kobayashi and A Makino ldquoEnzymatic polymer synthesis anopportunity for green polymer chemistryrdquo Chemical Reviewsvol 109 no 11 pp 5288ndash5353 2009

[2] J Feijen ldquoBiodegradable polymers for medical purposerdquo inPolymeric Biomaterials E Piskin and A S Hoffman Eds pp62ndash77 Martinus Nijhoff Publishers Dordrecht The Nether-lands 1986


[3] A-C Albertsson and I K Varma ldquoRecent developments inring opening polymerization of lactones for biomedical appli-cationsrdquo Biomacromolecules vol 4 no 6 pp 1466ndash1486 2003

[4] T H Barrows ldquoSynthetic bioabsorbable polymersrdquo inHigh Per-formance BiomaterialsM Szycher Ed pp 243ndash257 TechnomicPublishing Lancaster Pa USA 1991

[5] M Vert ldquoBioresorbable polymers for temporary therapeuticapplicationsrdquo Angewandte Makromolekulare Chemie vol 166pp 155ndash168 1989

[6] R Y Zhang and P Ma ldquoPoly(alpha-hydroxyl acids) hydroxya-patite porous composites for bone tissue engineering Prepara-tion andmorphologyrdquo Journal of BiomedicalMaterials Researchvol 44 pp 446ndash455 1999

[7] J-P Puaux I Banu I Nagy and G Bozga ldquoA study of L-lactide ring-opening polymerization kineticsrdquoMacromolecularSymposia vol 259 pp 318ndash326 2007

[8] R F Storey and J W Sherman ldquoKinetics and mechanism of thestannous octoate-catalyzed bulk polymerization of 120576-capro-lactonerdquoMacromolecules vol 35 no 5 pp 1504ndash1512 2002

[9] H von Schenck M Ryner A-C Albertsson and M SvenssonldquoRing-opening polymerization of lactones and lactides withSn(IV) and Al(III) initiatorsrdquoMacromolecules vol 35 no 5 pp1556ndash1562 2002

[10] R R Gowda and D Chakraborty ldquoZinc acetate as a catalystfor the bulk ring opening polymerization of cyclic esters andlactiderdquo Journal of Molecular Catalysis A vol 333 no 1-2 pp167ndash172 2010

[11] X Wang K Liao D Quan and Q Wu ldquoBulk ring-openingpolymerization of lactides initiated by ferric alkoxidesrdquoMacro-molecules vol 38 no 11 pp 4611ndash4617 2005

[12] P S Umare G L Tembe K V Rao U S Satpathy and BTrivedi ldquoCatalytic ring-opening polymerization of l-lactide bytitanium biphenoxy-alkoxide initiatorsrdquo Journal of MolecularCatalysis A vol 268 no 1-2 pp 235ndash243 2007

[13] D Philippe C Olivier and J M Raquez Eds Handbookof Ring-Opening Polymerization WHILEY-VCH WeinheimGermany 2009

[14] S Matsumura ldquoEnzymatic synthesis of polyesters via ring-opening polymerizationrdquo Advances in Polymer Science vol 194no 1 pp 95ndash132 2006

[15] A-C Albertsson and R K Srivastava ldquoRecent developmentsin enzyme-catalyzed ring-opening polymerizationrdquo AdvancedDrug Delivery Reviews vol 60 no 9 pp 1077ndash1093 2008

[16] SMatsumura ldquoEnzyme-catalyzed synthesis and chemical recy-cling of polyestersrdquo Macromolecular Bioscience vol 2 pp 105ndash126 2002

[17] Y Suzuki S Taguchi T Hisano K Toshima S Matsumuraand Y Doi ldquoCorrelation between structure of the lactones andsubstance specificity in enzyme-catalyzed polymerization forthe synthesis of polyestersrdquo Biomacromolecules vol 4 no 3 pp537ndash543 2003

[18] S Kobayashi H Uyama S Namekawa and H HayakawaldquoEnzymatic ring-opening polymerization and copolymeriza-tion of 8-octanolide by lipase catalystrdquoMacromolecules vol 31no 17 pp 5655ndash5659 1998

[19] H Uyama K Takeya N Hoshi and S Kobayashi ldquoLipase-catalyzed ring-opening polymerization of 12-dodecanoliderdquoMacromolecules vol 28 no 21 pp 7046ndash7050 1995

[20] S Noda N Kamiya M Goto and F Nakashio ldquoEnzy-matic polymerization catalyzed by surfactant-coated lipases inorganicmediardquo Biotechnology Letters vol 19 no 4 pp 307ndash3091997

[21] K Kullmer H Kikuchi H Uyama and S Kobayashi ldquoLipase-catalyzed ring-opening polymerization of 120572-methyl-120575-valero-lactone and 120572-methyl-120576-caprolactonerdquo Macromolecular RapidCommunications vol 19 no 2 pp 127ndash130 1998

[22] C Hedfors E Ostmark E Malmstrom K Hult and M Mar-tinelle ldquoThiol end-functionalization of poly(isin-caprolactone)catalyzed by candida antarctica lipase Brdquo Macromolecules vol38 no 3 pp 647ndash649 2005

[23] G Broze P M Lefebvre R Jerome and Ph Teyssie ldquoBlockcopolymerization of 33-dimethyl-2-oxetanone 1 About themechanism of 120572120572-disubstituted 120573-propiolactones block copol-ymerizationrdquoMacromolecules vol 12 pp 1047ndash1051 1979

[24] P Dubois P Degee R Jerome and Ph Teyssie ldquoMacromolec-ular engineering of polylactones and polylactides 8 Ring-opening polymerization of 120576-caprolactone initiated by primaryamines and trialkylaluminumrdquoMacromolecules vol 25 no 10pp 2614ndash2618 1992

[25] H Uyama and S Kobayashi ldquoEnzyme-catalyzed polymeriza-tion to functional polymersrdquo Journal of Molecular Catalysis Bvol 19-20 pp 117ndash127 2002

[26] H Uyama H Kikuchi and S Kobayashi ldquoOne-shot synthesisof polyester macromonomer by en-zymatic ring-opening poly-merization of lactone in the presence of vinyl esterrdquo ChemistryLetters vol 11 pp 1047ndash1048 1995

[27] S N Yoon and G P Tae ldquoPorous biodegradable polymericscaffolds prepared by thermally in-duced phase separationrdquoJournal of Biomedical Materials Research vol 47 pp 8ndash17 1999

[28] Y Mei A Kumar and R A Gross ldquoProbing water-temperaturerelationships for Lipase-catalyzed lactone ring-opening poly-merizationsrdquo Macromolecules vol 35 no 14 pp 5444ndash54482002

[29] C S Lee M T Ru M Haake J S Dordick J A Reimer and DS Clark ldquoMultinuclear NMR study of enzyme hydration in anorganic solventrdquo Biotechnology and Bioengineering vol 57 pp686ndash693 1998

[30] N A Turner and E N Vulfson ldquoAt what temperature canenzymes maintain their catalytic activityrdquo Enzyme and Micro-bial Technology vol 27 no 1-2 pp 108ndash113 2000

[31] A Klibanov ldquoWhy are enzymes less active in organic solventsthan inwaterrdquoTrends in Biotechnology vol 15 no 3 pp 97ndash1011997

[32] Z Zhong S Schneiderbauer P J Dijkstra M Westerhausenand J Feijen ldquoFast and living ring-opening polymerization ofL-lactide initiated with in-situ-generated calcium alkoxidesrdquoJournal of Polymers and the Environment vol 9 no 1 pp 31ndash38 2001

[33] C Jing-Lei Y Ying-Ming L Yun-Jie Z Li-Ying Z Yongand S Qi ldquoSynthesis characterization of homoleptic guanidinolanthanide complexes and their catalytic activity for thering-opening polymerization of 120576-caprolactonerdquo Journal ofOrganometallic Chemistry vol 689 no 6 pp 1019ndash1024 2004

[34] E Pamuła P Dobrzynski M Bero and C PaluszkiewiczldquoHydrolytic degradation of porous scaffolds for tissue engineer-ing from terpolymer of L-lactide 120576-caprolactone and glycoliderdquoJournal of Molecular Structure vol 744ndash747 pp 557ndash562 2005

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


The presented paper demonstrates the results on thedevelopment of optimized experimental strategy to obtainthe biodegradable polymers suitable for supraporous scaffoldconstruction using enzyme-mediated synthesis in non-polarmediumThedevelopment of easy laboratory-scale techniqueto synthesize polypentadecalactone (polyPDL) of high MWneeded for material construction represents a supplementaryobjective of presented research

2 Experimental

21 Materials and Instruments The monomer 120596-pentadeca-lactone (PDL 98) was purchased from Aldrich and usedwithout additional purification Before polymerization thereagent was characterized by proton nuclear magnetic reso-nance (1H NMR) 415 (2H t) 235 (2H t) 164 (4H m) and085 (22H br s) ppm The enzymes Candida rugosa lipase(CRL) and acrylic resin immobilized Candida antarcticalipase B (CALB Novozyme-435) possessing specific activityge40 000Ug and ge10 000Ug respectively 2-hydroxyethylmethacrylate (HEMA) and stannous octoate were purchasedfrom Sigma-Aldrich Toluene chloroform and methanolwere the products of ZAO ldquoVectonrdquo Toluene was driedover calcium hydride and distilled HPLC grade THF waspurchased fromMerck

Syntheses of polymers as well as drying of enzymesand products were carried out in Schlenck tubes (Aldrich)connected to a vacuum-argon line provided with oil vacuumpump 2B-11 (Gidromekh) To seal the reaction tubes and bot-tles with solvents Precision Seal rubber septa caps (Aldrich)were used

The precise heating of reaction mixture was reached bythe application of magnetic stirrer MR Hei-Standard (Hei-dolph) equipped with temperature controller as well as usinga glycerol bath while the stirring of polymerization mediumwas provided using an IKA T-16 basic overhead stirrer

For concentration of polymer solutions IR-1 rotary evap-orator (Khimlaborpribor) was used The intrinsic viscositymeasurements were performed in Ostwald type viscometer(capillary diameter 037mm ZAO ldquoNeva-Reactivrdquo) suppliedby LOIP LT-912 cryothermostat (ZAO ldquoLOIPrdquo) that wasalso applied for cryotropic phase separation at macroporousmaterials preparation

Themolecular weight (MW) andmolecular weight distri-bution (MWD) analysis was produced using Shimadzu liquidchromatography consisting of LC-10AD pump and RID-10Arefractometric detector and supplied with Waters StyragelHMW 6E analytical column To calculate MW and MWDLC Solution 124SP1 GPC software was used Polystyrenestandards synthesized and characterized at the Institute ofMacromolecular Compounds of RAS by Dr Ludmila Vino-gradova were used to build a calibration curve

The 1H NMR spectra were recorded with BrukerAVANCE-400 instrument

22 Methods221 Enzyme-Mediated Polymerization of PDL The typicalprocedure to obtain polyPDL was followed 093mL of

Ar

Figure 1 Scheme of reaction installation for EMROP of PDL

toluene was dropped under intensive stirring and dry argonflow to the mixture of 400mg of monomer and 40mg ofenzyme (CRL or CALB [M][E] = 101wtwt other ratioswere also tested see Figure 5) in the reaction tube (Figure 1)The temperature was raised up to predetermined value (from35 to 115∘C see Table 4) and reaction was carried out until asolidification of reaction mixture (usually 1ndash3 hours) If thatdid not occur the reaction time was prolonged for 6 hoursmaximum To stop the polymerization the reaction tubewas quenched into ice-cold water The content of reactiontube was dissolved in the excess of hot chloroform Thenpolymer solution was separated from granules of solid-phaseenzyme by filtration (Schott glass filter number 3)The filtratewas concentrated in a rotary evaporator and the polymerwas drop-wise precipitated to cold methanol The precipitateobtained was isolated by filtration on glass Schott filternumber 1 and dried in vacuum

222 Enzyme-Mediated Polymerization of PDL in a Presenceof 2-Hydroxymethacrylate (HEMA) The synthetic procedureto obtain polyPDL-HEMA was the same as that applied forsynthesis of polyPDL The only difference was the additionof HEMA to the reaction mixture ([M][HEMA] = 20 and40mol ratio)The derived polymer was dried by sublimationsealed and stored in a freezer at minus20∘C

223 Radical Reaction of PolyPDL-HEMA A sample of150mg of polyPDL-HEMA and 75mg of radical initia-tor 221015840-azo-bis-isobutyronitrile (AIBN) ([polyPDL-HEMA][AIBN] = 201 wtwt) was inserted into the ampule and dis-solved in 1mL of dry toluene ([polyPDL-HEMA][toluene] =17 wtwt) The ampule was sealed and placed into preheatedup to 70∘C thermostat and kept there for 24 hours Then theampule was cooled down and the polymer was precipitatedin cold methanol filtered and dried over calcium chloride




3)


























minus3


119899












119908119872lowast

119899















Solvent

Polymer

Solution



Porous matrix


20 40 60 80 100 1201

2

3

4

5

6

7

Reaction time

Reac

tion

time (

hour

s)

20 40 60 80 100 120

02

04

06

08

10

[120578]

[120578] (

dLg

)

T (∘C)

T (∘C)





76

78

80

82

84

86

88

90

Polymer yield

Yiel

d (

)

0

2

4

6

8

10

Reac

tion

time (

hour

s)

0 20 40 60 80

0 20 40 60 80

[M][E] (wtwt)

[M][E] (wtwt)

[120578] (

dLg

)

05

06

07

08

09









O

O

HO

O

OHaa 11 11

d bb

c ce

a

bcd

e

Chloroform

8 7 6 5 4 3 2 1

(ppm)

d998400

d998400

(a)

O

O O

OHO

O

Me

H

HO a a 1111

bbc c

d ef

g

j

h

i

a

bc

jChloroform

d

eg fh

h

i

i

7 6 5 4 3 2 1

(ppm)

(ppm)62 60 58 56 54

d998400

d998400

(b)


25 30 35 40

500

1000

1500

2000

Resp

onse

(mV

)

Elution time (min)

(a)

0

20

40

60

80

100(

)

30 35 40 45 50 55

log(MW)

(b)








30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)







119908and119872

119908119872119899values





(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






3)


























minus3


119899












119908119872lowast

119899















Solvent

Polymer

Solution



Porous matrix


20 40 60 80 100 1201

2

3

4

5

6

7

Reaction time

Reac

tion

time (

hour

s)

20 40 60 80 100 120

02

04

06

08

10

[120578]

[120578] (

dLg

)

T (∘C)

T (∘C)





76

78

80

82

84

86

88

90

Polymer yield

Yiel

d (

)

0

2

4

6

8

10

Reac

tion

time (

hour

s)

0 20 40 60 80

0 20 40 60 80

[M][E] (wtwt)

[M][E] (wtwt)

[120578] (

dLg

)

05

06

07

08

09









O

O

HO

O

OHaa 11 11

d bb

c ce

a

bcd

e

Chloroform

8 7 6 5 4 3 2 1

(ppm)

d998400

d998400

(a)

O

O O

OHO

O

Me

H

HO a a 1111

bbc c

d ef

g

j

h

i

a

bc

jChloroform

d

eg fh

h

i

i

7 6 5 4 3 2 1

(ppm)

(ppm)62 60 58 56 54

d998400

d998400

(b)


25 30 35 40

500

1000

1500

2000

Resp

onse

(mV

)

Elution time (min)

(a)

0

20

40

60

80

100(

)

30 35 40 45 50 55

log(MW)

(b)








30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)







119908and119872

119908119872119899values





(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials















minus3


119899












119908119872lowast

119899















Solvent

Polymer

Solution



Porous matrix


20 40 60 80 100 1201

2

3

4

5

6

7

Reaction time

Reac

tion

time (

hour

s)

20 40 60 80 100 120

02

04

06

08

10

[120578]

[120578] (

dLg

)

T (∘C)

T (∘C)





76

78

80

82

84

86

88

90

Polymer yield

Yiel

d (

)

0

2

4

6

8

10

Reac

tion

time (

hour

s)

0 20 40 60 80

0 20 40 60 80

[M][E] (wtwt)

[M][E] (wtwt)

[120578] (

dLg

)

05

06

07

08

09









O

O

HO

O

OHaa 11 11

d bb

c ce

a

bcd

e

Chloroform

8 7 6 5 4 3 2 1

(ppm)

d998400

d998400

(a)

O

O O

OHO

O

Me

H

HO a a 1111

bbc c

d ef

g

j

h

i

a

bc

jChloroform

d

eg fh

h

i

i

7 6 5 4 3 2 1

(ppm)

(ppm)62 60 58 56 54

d998400

d998400

(b)


25 30 35 40

500

1000

1500

2000

Resp

onse

(mV

)

Elution time (min)

(a)

0

20

40

60

80

100(

)

30 35 40 45 50 55

log(MW)

(b)








30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)







119908and119872

119908119872119899values





(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






119908119872lowast

119899















Solvent

Polymer

Solution



Porous matrix


20 40 60 80 100 1201

2

3

4

5

6

7

Reaction time

Reac

tion

time (

hour

s)

20 40 60 80 100 120

02

04

06

08

10

[120578]

[120578] (

dLg

)

T (∘C)

T (∘C)





76

78

80

82

84

86

88

90

Polymer yield

Yiel

d (

)

0

2

4

6

8

10

Reac

tion

time (

hour

s)

0 20 40 60 80

0 20 40 60 80

[M][E] (wtwt)

[M][E] (wtwt)

[120578] (

dLg

)

05

06

07

08

09









O

O

HO

O

OHaa 11 11

d bb

c ce

a

bcd

e

Chloroform

8 7 6 5 4 3 2 1

(ppm)

d998400

d998400

(a)

O

O O

OHO

O

Me

H

HO a a 1111

bbc c

d ef

g

j

h

i

a

bc

jChloroform

d

eg fh

h

i

i

7 6 5 4 3 2 1

(ppm)

(ppm)62 60 58 56 54

d998400

d998400

(b)


25 30 35 40

500

1000

1500

2000

Resp

onse

(mV

)

Elution time (min)

(a)

0

20

40

60

80

100(

)

30 35 40 45 50 55

log(MW)

(b)








30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)







119908and119872

119908119872119899values





(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Solvent

Polymer

Solution



Porous matrix


20 40 60 80 100 1201

2

3

4

5

6

7

Reaction time

Reac

tion

time (

hour

s)

20 40 60 80 100 120

02

04

06

08

10

[120578]

[120578] (

dLg

)

T (∘C)

T (∘C)





76

78

80

82

84

86

88

90

Polymer yield

Yiel

d (

)

0

2

4

6

8

10

Reac

tion

time (

hour

s)

0 20 40 60 80

0 20 40 60 80

[M][E] (wtwt)

[M][E] (wtwt)

[120578] (

dLg

)

05

06

07

08

09









O

O

HO

O

OHaa 11 11

d bb

c ce

a

bcd

e

Chloroform

8 7 6 5 4 3 2 1

(ppm)

d998400

d998400

(a)

O

O O

OHO

O

Me

H

HO a a 1111

bbc c

d ef

g

j

h

i

a

bc

jChloroform

d

eg fh

h

i

i

7 6 5 4 3 2 1

(ppm)

(ppm)62 60 58 56 54

d998400

d998400

(b)


25 30 35 40

500

1000

1500

2000

Resp

onse

(mV

)

Elution time (min)

(a)

0

20

40

60

80

100(

)

30 35 40 45 50 55

log(MW)

(b)








30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)







119908and119872

119908119872119899values





(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




O

O

HO

O

OHaa 11 11

d bb

c ce

a

bcd

e

Chloroform

8 7 6 5 4 3 2 1

(ppm)

d998400

d998400

(a)

O

O O

OHO

O

Me

H

HO a a 1111

bbc c

d ef

g

j

h

i

a

bc

jChloroform

d

eg fh

h

i

i

7 6 5 4 3 2 1

(ppm)

(ppm)62 60 58 56 54

d998400

d998400

(b)


25 30 35 40

500

1000

1500

2000

Resp

onse

(mV

)

Elution time (min)

(a)

0

20

40

60

80

100(

)

30 35 40 45 50 55

log(MW)

(b)








30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)







119908and119872

119908119872119899values





(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




30 35 40 45

log(MW)

0

20

40

60

80

100(

)

(a)

0

20

40

60

80

100

()

30 3525 40 45

log(MW)

(b)

30 35 40 45

log(MW)

0

20

40

60

80

100

()

(c)







119908and119872

119908119872119899values





(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)





4 Conclusions



Abbreviations


Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



research article enzyme-mediated ring-opening...

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