Novel Aliphatic Poly(ester-carbonate) with Pendant Allyl Ester Groups and its Folic Acid...

Novel Aliphatic Poly(ester-carbonate) with Pendant AllylEster Groups and its Folic Acid Functionalization

XIULI HU,1,2 XUESI CHEN,1 SHI LIU,1 QUAN SHI,1,2 XIABIN JING1

1State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry,Chinese Academy of Sciences, Changchun 130022, People’s Republic of China

2Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China

Received 26 September 2007; accepted 4 November 2007DOI: 10.1002/pola.22530Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: A series of novel poly(ester-carbonate)s bearing pendant allyl ester groupsP(LA-co-MAC)s were prepared by ring-opening copolymerization of L-lactide (LA) and5-methyl-5-allyloxycarbonyl-1,3-dioxan-2-one (MAC) with diethyl zinc (ZnEt2) as ini-tiator. NMR analysis investigated the microstructure of the copolymer. DSC resultsindicated that the copolymers displayed a single glass-transition temperature (Tg),which was indicative of a random copolymer, and the Tg decreased with increasingcarbonate content in the copolymer. Then NHS-activated folic acid (FA) first reactedwith 2-aminoethanethiol to yield FA-SH; grafting FA-SH to P(LA-co-MAC) in thepresence of TEA produced P(LA-co-MAC)/FA. The structure of P(LA-co-MAC)/FA andits precursor were confirmed by 1H NMR and XPS analysis. Cell experiments showedthat FA-grafted P(LA-co-MAC) had improved adhesion and proliferation behaviorof vero cells on the polymer films. Therefore, the novel FA-grafted block copolymeris expected to find application in drug delivery or tissue engineering. VVC 2008 Wiley

Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 1852–1861, 2008

Keywords: allyl; biocompatible; biodegradable; functionalization of polymers; poly-carbonates

INTRODUCTION

Aliphatic polyesters based on e-caprolactone, lac-tide, and glycolide are of interest for medicaland pharmaceutical applications because oftheir low immunogenicity, good biocompatibility,and excellent mechanical properties.1–3 Currenttargets for these materials include biodegrad-able sutures, resorbable implant materials, tis-sue engineering scaffolds, and drug deliveryvehicles.4–7 While suitable for many applica-tions, such polyesters are limited in scopebecause of their hydrophobic and semicrystalline

properties and their inadequate interactionbetween the polymers and cells, leading toin vivo foreign body reactions, such as inflam-mation, infections, local tissue necrosis, andthrombosis.8,9 Furthermore, because of the lackof functional groups, they cannot be modifiedeasily with biologically active moieties. Thepreparation of functionalized aliphatic polyest-ers is a synthetic challenge.10,11

Numerous examples of chain-end functional-ized aliphatic polyesters have been reported andprepared most commonly by the use of func-tional nucleophiles to initiate ring-opening poly-merization (ROP) of lactone.12,13 However, pend-ent functionalization provides a unique opportu-nity to alter physical and chemical properties bydistributing functionality along the polymer back-

Correspondence to: X. Jing (E-mail: [email protected])

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 46, 1852–1861 (2008)VVC 2008 Wiley Periodicals, Inc.

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bone. The functionality is introduced at the mono-mer stage, or by postpolymerization chemistry, aswell as a combination of these two approaches. Inthe former case, such functionality must be com-patible with the polymerization conditions, whilein the postpolymerization chemistry the desiredtransformations must be achieved without degra-dation of the polyester backbone.

Recently, more and more attention has beenpaid to the preparation of biodegradable poly-

mers possessing functional or reactive groups bycopolymerization of functionalized cyclic mono-

mers, such as NCA’s of amino acids,14 morpho-line-2,5-dione derivatives, including those con-

taining carboxyl,15 amine, thiol, and hydroxylgroups,16,17 and e-caprolactones bearing carboxylor hydroxyl18,19 groups. But some of these mono-

mers are difficult to synthesize and need protec-tion and deprotection procedure. Functionalized

cyclic carbonate monomers and their polymer-ization have recently been attracting special

attention because their synthesis is easier andtheir polymerization activity is higher. So poly-mers or copolymers with higher molecular

weights can be easily obtained in comparisonwith other functional monomers reported in the

literature.20,21 William and Grinstaff22 reportedthe synthesis and characterization of poly(car-bonate-ester)s of glycerol and L-lactic acid. These

polymers possess a hydrolyzable backbone,tunable hydrophobic/hydrophilic properties, and

functionalizable heteroatom pendant groups.Recently, they reported new copolymers based

on 6-hydroxyhexanoic acid and glycerol whichcontained pendant side chains with varyingcommon reactive groups, such as hydroxyl, car-

boxylic acid, and amine groups by postpolymeri-zation of the hydroxyl-carrying copolymer.23

Gross and coworkers24 reported polycarbonatesbearing vicinal diol pendant groups by the ROPof trimethylene carbonate (TMC) with 1,2-O-iso-

propylidene-D-xylofuranose-3,5-cyclic carbonateand by the subsequent deprotection of the ketal

protecting groups. Zhou and coworkers25 de-scribed the synthesis of two kinds of aliphatic

polycarbonates with pendant ester groups: poly(5-methyl-5-methoxycarbonyl-1,3-dioxan-2-one)and poly(5-methyl-5-ethoxycarbonyl-1,3-dioxan-

2-one). Lee et al.26 reported novel, biodegrad-able, functional poly(ester carbonate)s by the

copolymerization of trans-4-hydroxy-L-prolinewith cyclic carbonate bearing a pendent carbox-

ylic group. Our group has reported several kindsof novel carbonate monomer successfully, 2-methyl-2-benzyloxycarbonyl-propylene carbonate(MBC), and 5-benzyloxy-trimethylene carbonate,5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-onewith protected carboxyl, hydroxyl, and acetylenegroups, respectively.27–29

Folate receptors (FR) have been known to bevastly overexpressed in several human tumorsand are overexpressed in many of the humancancerous cells, including the malignancies ofthe ovary, mammary gland, lung, kidney, brain,colon, prostate, nose, and throat; however, theyare only minimally distributed in normal tis-sues.30 Folic acid (FA), a high-affinity ligand toFR is efficiently internalized into the cellthrough the receptor mediated endocytosis evenwhen conjugated with a wide variety of mole-cules.31 Moreover, due to FA’s high stability,compatibility with both organic and aqueous sol-vent, low-cost, nonimmunogenic character, thecovalent attachment of FA to a wide range ofbiomedically relevant molecules is an estab-lished methodology that has been exploited forthe selective delivery of imaging agents, genecarriers, therapeutic agents, enzymes, lipo-somes, block copolymer micelles,32–34 and othermacromolecular complexes35,36 to tumor tissues.

Application-specific tailoring of the chemical,physical, and mechanical properties of the poly-mer for medical uses by varying monomer units,composition is highly advantageous given thevaried design requirements of a specific applica-tion.37,38 In this article, therefore, based on thenovel cyclic carbonate 5-methyl-5-allyloxycar-bonyl-1,3-dioxan-2-one (MAC) that we reportedrecently,39 the random copolymerization of MACand LA was reported in detail. ZnEt2 was usedas the catalyst/initiator. Polymers containing themonomer MAC and LA were synthesized tointroduce reactive double bond functionality intothe polymer backbone for crosslinking or furtherfunctionalization. To graft FA onto the copoly-mer, a two steps procedure was introduced: (1)activation of FA with NHS and reaction with 2-aminoethanethiol to yield FA-SH; (2) grafting ofFA-SH with P(LA-co-MAC) to produce P(LA-co-MAC)/FA. Preliminary study for the medical useshowed that this FA-grafted P(LA-co-MAC) hadimproved adhesion and proliferation behavior ofvero cells. Continued research will focus on theunique application of FA and double bond func-tionality for targeted drug delivery or polymernetworks for tissue engineering.

NOVEL ALIPHATIC POLY(ESTER-CARBONATE) 1853

Journal of Polymer Science: Part A: Polymer ChemistryDOI 10.1002/pola

EXPERIMENTAL

Materials

L-Lactide (LA) was prepared in our own labora-tory and recrystallized from ethyl acetate forthree times before use. Monomer MAC was pre-pared and purified according to the literature.39

ZnEt2 was kindly supplied by Prof. XianhongWang in Changchun Institute of Applied Chem-istry. 2-Aminoethanethiol and folic acid (FA)were purchased from Sigma. Toluene wasrefluxed over sodium for 24 h and distilledunder nitrogen before use. Other reagents werecommercially available and used as received.

Characterization of the Block Copolymer

13C NMR and 1H NMR spectra were recordedon a Bruker AV 300M in CDCl3 at 25 8C. Chemi-cal shifts were given in parts per million fromtetramethylsilane. Gel permeation chromatogra-phy (GPC) measurements were conducted witha Waters 410 GPC equipped with Waters Styra-gel column (HT3) with CHCl3 as eluent (flowrate: 1 mL/min, at 35 8C). The molecularweights were calibrated with polystyrene stand-ards (molecular weight range: 500 – 3 3 104).Differential scanning calorimetry (DSC) analy-ses were carried out at a heating rate of 10 8C/min on a Perkin Elmer Pyris 1. Surface elemen-tal compositions were analyzed on an EscalabMKII photoelectron spectrometer (VG Scientific)equipped with a standard Mg anode X-raysource (Mg Ka X-rays at 1253.6 eV) and a 150-mm hemispherical electron energy analyzer. Thespectra were obtained for each sample using atakeoff angle of 908.

Polymerization

A mixture of monomer MAC and LA with a spe-cific molar ratio was placed in a thoroughlydried glass flask. The reaction vessel was sealed,evacuated, and purged with nitrogen threetimes. Next, the reaction vessel was partiallyimmersed in a thermostated oil bath, preheatedto 100 8C. After 5 min, the monomers were wellmixed. A certain amount of the catalyst (ZnEt2in toluene) was added via a syringe. The reac-tion vessel was placed in an oil bath at 110 8Cfor 12 h, and the reaction was terminated bycooling the vessel to room temperature. The co-polymer P(LA-co-MAC) was dissolved in

dichloromethane and precipitated into anamount of cold methanol, isolated by filtration,and dried under vacuum at room temperature.

Homopolymerization of LA or MAC was car-ried out in a similar manner.

Synthesis of Folic Acid-Grafted Copolymer

N-Hydroxysuccinimide ester of folic acid (NHS-FA) was prepared in accordance to a reportedprocedure40 as depicted in Scheme 2(A). In brief,FA (0.5 g) was added into a mixture of anhy-drous dimethyl sulfoxide (DMSO, 20 mL) andtriethylamine (TEA, 0.3 mL) and dissolved inthe mixture with stirring. Then dicyclohexylcar-bodiimide (DCC, 0.25 g) and NHS (0.26 g) wereadded, and stirred in the dark for 24 h. The sideproduct dicyclohexylurea (DCU) precipitatedwas removed by filtration. DMSO and triethyl-amine were evaporated under vacuum. Vacuumdried FA-NHS was then dissolved into 2 mL ofthe mixture of DMSO and TEA with a volumeratio of 2:1. An equal molar amount of 2-aminoe-thanethiol was added to the mixture, and thereaction was conducted under an anhydrouscondition overnight. Therefore, a thiol groupwas introduced into FA to form FA-SH by nucle-ophilic substitution.

The conjugation of FA-SH to P(LA-co-MAC)was performed as depicted in Scheme 2(B). Inbrief, FA-SH was added slowly to P(LA-co-MAC)solution in DMSO with stirring and the reactionwas conducted at room temperature in the darkfor 48 h. The synthesized product was dialyzedagainst DMSO using a dialysis tubing with Mw

cut-off of 3000 Da (Spectrum Laboratories) for48 h, followed by removing the solvent by evapo-rating under vacuum. FA-g-P(LA-MAC) withvarious degrees of substitution (DS) of FA weresynthesized by changing the feed ratio of FA-SHto P(LA-co-MAC).

Cell Experiments

Test Cells

Vero cells were chosen to investigate the biocom-patibility of P(LA-co-MAC) and its FA-graftedcopolymer P(LA-co-MAC)/FA. Vero cells werefirst cultured in Dulbecco’s modified Eagle’s me-dium (DMEM, GIBCO) supplemented with 10%heat-inactivated fetal bovine serum (FBS,GIBCO), 100 U/mL penicillin, and 100 mg/L

1854 HU ET AL.


streptomycin (Sigma). And the culture mediumwas replaced once every day.

Test Films

The copolymers P(LA-co-MAC) and P(LA-co-MAC)/FA were dissolved in DMSO (1 wt %), andthe solution was cast onto glass slides. Thecoated slides were treated under vacuum for 48 hto remove the trace of DMSO, followed by steri-lizing by ultraviolet radiation for 2 h. The purePLA films were prepared in a similar way andused as a control.

Cell Adhesion and Spreading

The cover slides coated with the polymer filmswere placed in each well of 6-well tissue cultureplates (NUNC). The cells were seeded on thecover slides at a density of 1 3 105 cells/well,incubated in a humidified incubator at 37 8Cand 5% CO2, and examined after 24 and 48 h.Following incubation, the cover slides werewashed three times with PBS and fixed with 3%glutaraldehyde in PBS at room temperature for30 min. After thorough washing with PBS, thecells were dyed with one drop of Giemsa stain(Sigma) for 30 min, washed with deionizedwater, dried in air, and then enveloped withneutral balsam. The stained cells were observedunder an inverted microscope (TE2000-U,Nikon). Pictures of each cover slide were takenwith a digital camera (DXM1200F, Nikon) andanalyzed with NIH Image J.

RESULTS AND DISCUSSION

Copolymerization

The copolymerization of cyclic carbonates withlactides, glycolide, and caprolactone has beenaccomplished with a variety of polymerizationsystems, and copolymerization is a commonmethod for creating novel materials with tailora-ble properties, such as chemical, physical, andthermal properties, and so forth.41,42 The ROPof six-membered cyclic carbonates has beenreported with various catalyst/initiators.43–45

Among which, ZnEt2 is an efficient initiator forthe synthesis of biodegradable polycarbonateswith high molecular weight and narrow polydis-persity.24,29 Therefore, in this article, it wasused as catalyst/initiator to prepare the copoly-

mer P(LA-co-MAC) through ROP of monomerMAC and LA in bulk, and Scheme 1 showed thesynthesis procedure. Polymerizations weremaintained at or below 115 8C to minimizetrans-esterification and trans-carbonation events.P(LA-co-MAC)s with various molar ratios wereprepared in an analogous manner. Copolymerswith moderate molecule weight and polydisper-sity were obtained at 110 8C. The molecularweight was in the range of 1.9–8.8 3 104 andthe polydispersity (Mw/Mn) was 1.4–1.7. Table 1summarizes the results of the copolymerization.With 1H NMR, the copolymer composition wascalculated from the relative peak areas of theallyl (��CH2��CH¼¼CH2) protons at 4.6 ppm ofthe MAC repeat units and the methyl[��CH(CH3)O��] protons at 1.57 ppm of the LArepeat units. The MAC content in the polymerwas lower than that in the monomer feed, andthis might be attributed to a little bit lower ac-tivity of MAC compared with LA as reported byROP with Sn(OEt)2.

46 However, the good agree-ment between the monomer feed and polymercompositions indicated the successful polymer-ization. Therefore, the sequence length and thecomposition of the copolymer could be adjustedby changing the feed ratio. The GPC curves ofthe copolymer with different content of MAC

Scheme 1. Synthesis of (A) MAC monomer and (B)P(LA-co-MAC) copolymer.



showed a unimodal peak. These data alsoimplied that the copolymerization was carriedout successfully, and there was no homopolyme-rization of LA or MAC.

Structural Characterization

1H NMR and 13C NMR spectra were used tocharacterize the copolymer of MAC and LA. Fig-ure 1 shows the typical 1H NMR spectra ofP(LA-co-MAC) and peak assignments. Compari-son with previously published spectra of homo-polymer PLA, the signals at 5.1 and 1.56 ppmare due to the LA repeat unit CH and CH3 pro-tons, respectively.47 Signals c from 4.2 to4.5 ppm were assigned to ��CO��O��CH2 pro-tons of repeat units of MAC. Peak e at 5.8 ppmand f at 5.3 ppm were assigned to the allyl(��CH2��CH¼¼CH2) and (��CH2��CH¼¼CH2)protons of repeat units of MAC on the basis ofthe spectra of PMAC (Sample 7 in Table 1),respectively. Two sets of quartets at 5.1 and 5.0

ppm appeared. Possible dyad structures thatcould give rise to these signals are LL, LM (Ldesignates LA and M designates MAC), and ML.These assignments are consistent with thosemade previously.48,49

To examine the detailed sequence distribu-tion, the 1H NMR spectra of the copolymerswith different composition ratios were studied.As shown in Figure 2, the peaks of 5.0 ppmincreased with increasing incorporation of MACin the final copolymer. This was because thechemical shift of the methine protons of the LA[��OCH(CH3)C¼¼O��] units was sensitive to therepeat unit sequence distribution. It displayed achemical shift of 5.1 ppm when adjacent to twoester linkages; however, it was shifted upfield toa chemical shift of 5.0 ppm when adjacent to anester linkage on one side and a carbonate link-

Table 1. Related Data on the Bulk Copolymerization of LA and MACa

Entry

Mol % of MAC

Mnc Mw/Mn

c Yield (%) Tgd (8C)Feeding Productb

1 0 0 88,000 1.4 97 592 10 10 37,500 1.51 95 46.23 20 20 31,300 1.6 92 34.84 30 29 24,700 1.7 90 28.15 50 47 28,100 1.5 84 16.56 70 65 22,000 1.7 82 4.67 100 100 19,000 1.61 76 �12

a Copolymerization was carried out at 110 8C for 12 h. The molar ratio of ZnEt2 to monomer (LAþMAC) was 1/200.b Determined by 1H NMR.c Determined by GPC (CHCl3 as eluent).d Determined by DSC.

Figure 1. 1H NMR spectrum of copolymer P(LA-co-MAC) in CDCl3.

Figure 2. Expanded 1H NMR spectrum of P(LA-co-MAC) with different molar compositions: 10:90 MAC-LA (entry 2, Table 1); 30:70 MAC-LA (entry 4, Table1); 40:60 MAC-LA; PMAC (entry 7, Table 1).

1856 HU ET AL.


age on the other side. So as expected for randomcopolymers, the peaks at 5.0 ppm increased withincreasing MAC content in the copolymer. How-ever, the corresponding methylene protons ofMAC yielded no sequence information, appear-ing as one broad peak only at 4.2–4.5 ppm.

The sequence distribution of the copolymerwas also examined by 13C NMR spectra analy-sis. The full 13C NMR spectra of the copolymer(Table 1, entry 3) are displayed in Figure 3(A).Assignments of the major resonances werebased on the 13C NMR spectra of the homopoly-mers PLLA and PMAC in Figure 3(B). Asreported in previous articles,20 the 13C NMRspectra may be correlated with the followingeight possible triads for the two repeating units,LA (L) and MAC (M), that is, LLL, LLM, MLL,LML, LMM, MLM, MML, MMM. Expansions ofsignals in the carbonyl regions of the spectrumshow considerable complexity [Fig. 3(B)]. On thebasis of a comparison with the homopolymerPLA (b) and PMAC (c) in Figure 3(B), four reso-nance signals were found. The signal at 169.4ppm was assigned to the triad LLL, which wasthe most prevalent sequence in the copolymer.22

In addition there was a signal just downfield at169.6 ppm, which was attributed to the centralcarbonyl in the triad LLM. The carbonate car-

bon in the homopolymer PMAC displayed achemical shift at 154.6 ppm, while that corre-sponding to the MMM dyad in the copolymerdisplayed a chemical shift at 154.2 ppm, and thecarbonate carbon corresponding to the ML dyaddisplayed an upfield chemical shift at 153.9ppm. As expected for random copolymers, thecarbonate carbon corresponding to the ML dyadincreased with increasing incorporation of LA inthe final copolymer.

Thermal Properties

DSC was used to study how the thermal proper-ties were effected by the content of MAC repeatunits in the P(LA-co-MAC) copolymers. Study ofthe second heating scans showed that LA andMAC homopolymers have glass-transition tem-perature (Tg) values of 59 and �128C, respec-tively. The homopolymer PLA is a highly crys-talline material and has a melting temperature(Tm) at 174 8C. For the copolymers synthesized,no melting peaks were observed. Only a singleTg was detected for every sample in the exam-ined temperature range of 0–180 8C, indicativeof a random copolymer (Fig. 4). The Tg of thecopolymers with different carbonate contentsare listed in Table 1. With increasing content ofMAC incorporated into the copolymer, a Tg de-crease was observed. Specifically, the Tg valuesfor copolymers with 10, 20, 30, 50, and 70 mol %MAC were 46.2, 34.8, 28.1, 16.5, and 4.6 8C,respectively. Comparison of the experimentaland calculated (Fox equation) values of Tg, wefound that they were in excellent agreement

Figure 3. (A) 13C NMR spectrum of P(LA-co-MAC)copolymer in CDCl3 (Table 1, entry 3), (B) theexpanded carbonyl regions for (a) P(LA-co-MAC), (b)PLA, and (c) PAMC.

Figure 4. DSC thermograms of P(LA-co-MAC) withdifferent composition.



over the full range of copolymers studied (Fig.5). These results further indicated that LA wasstatistically copolymerized with MAC and wasconsistent with a copolymer microstructure thattrends toward a random distribution.

Conjugation with Folic Acid

The next point of interest was to introduce bio-logically active groups that elicit positive cellu-lar responses to biomaterial surfaces. Because ofFA’s high stability, compatibility with both or-ganic and aqueous solvent, low-cost, nonimmu-nogenic character, FA was chosen and FA-grafted copolymer P(LA-co-MAC)/FA was pre-pared with a two steps procedure: (1) activationof FA with NHS and reaction with 2-aminoetha-nethiol to yield FA-SH; (2) grafting of FA-SHwith P(LA-co-MAC) to produce P(LA-co-MAC)/FA. Heterobiofunctional 2-aminoethanethiol,which contains a thiol group and a primaryamino group was chosen as a crosslinker forconjugating FA onto P(LA-co-MAC). First, FAwas reacted with N-hydroxylsuccinimide. Thecarbodiimide-activated FA can couple either viathe a or c carboxyl group of the glutamate resi-due. Because of the steric hindrance constraintsimposed by the FA chain, the folate derivativeswere expected to consist mostly of the c-linked iso-mer.40,50 Then, the NHS activated carboxyl groupwas reacted with the NH2 group of 2-aminoetha-nethiol to obtain the amide linkage between FAand 2-aminoethanethiol as shown in Scheme 2(A),and finally, the remained thiol group was coupledto the allyl group of P(LA-co-MAC) via a radicaladdition as shown in Scheme 2(B). Coupling of

thiol to ally group was a useful preparationmethod, as the reaction was highly site-specificand convenient to control, and the reaction withthiol moiety generated a stable linkage.

Successful synthesis of P(LA-co-MAC)/FAwas confirmed by 1H NMR spectra. Typical 1Hspectra of FA and P(LA-co-MAC)/FA are shownin Figure 6. Coupling of the folate residue tothe copolymer P(LA-co-MAC) was confirmed bythe appearance of signals at 6.0–8.1 ppm inthe 1H NMR spectrum of P(LA-co-MAC)/FA [Fig.6(A)], which corresponded to the aromatic pro-tons of FA.

For more evaluation, the coupling of FA wasassessed from XPS spectroscopy. The XPS wasused to determine the surface elemental compo-

Scheme 2. Activation of folic acid (A) and graft ontopoly(ester-carbonate) (B).

Figure 5. Plot of 1/Tg (1/K) versus weight fractionof PLA.

1858 HU ET AL.


sition of the polymers synthesized. According tothe molecular formulas shown in Scheme 2,nitrogen is only present in the P(LA-co-MAC)/FA blocks and in the grafted FA. As shown inFigure 7, nitrogen appears in the spectroscopyof P(LA-co-MAC)/FA comparing with that ofP(LA-co-MAC), indicating the successful cou-pling of FA. The atom nitrogen to carbon canalso provide a qualitative measure of theamount of grafted FA on the material surface.Winnik and coworkers51 have determined thecontent of folate in the conjugates by quantita-tive UV spectrophotometry using the molarextinction coefficient value of 6197 mol/cm atk ¼ 363 nm.

Cytotoxicity of P(LA-co-MAC)

In order to be used as biomedical materials, thebiocompatibility of the poly(ester-carbonate)sshould be investigated. In vitro cytotoxicitywith vero cells was evaluated for P(LA-co-MAC)and its FA-functionalized copolymer as repre-sentative samples. Firstly, they were dissolvedin DMSO and the solution was cast onto glassslides. The cover slides coated with the polymerfilms were placed in each well of 6-well tissueculture plates. Secondly, the vero cells wereseeded onto these polymer films. Thirdly, theadhesion and growth behaviors of the cellswere examined by optical microscope. Figure 8presents the morphology of vero cells culturedon the copolymer films for 24 and 48 h, respec-tively. The same experiments were carried outon PLA films for comparison. After 48 h, cellsadhesion and spreading on PLA, P(LA-co-MAC)

films were similar, with some cells remainingrounded. After 24 h, the cells continued to prolif-erate, and their density increased gradually andfinally occupied the whole polymer surface. Thecell adhesion and growth was better and fasteron the FA-containing films than that on P(LA-co-MAC) and PLA films. The area fractions occupiedby the cells were measured for semi-quantitativecomparison. As shown in Figure 8(b), for both 24and 48 h, the order of the three films was P(LA-co-MAC)/FA > P(LA-co-MAC) > PLA. This meansthat copolymerization of MAC monomers causesimprovement in cell affinity of PLA film, andgrafting of FA onto the copolymer results in fur-ther improvement in cell affinity. Therefore, FA-grafted copolymer P(LA-co-MAC)/FA are expectedto be used in biomedical applications such asdrug delivery, tissue engineering, and so forth.

CONCLUSIONS

A series of novel poly(ester-carbonate)s bearingpendant allyl ester groups P(LA-co-MAC)s wereprepared by ROP of LA and MAC with ZnEt2 ascatalyst/initiator. The structure of P(LA-co-MAC)was examined in detail by NMR analysis. DSCresults indicated that the copolymers displayed asingle Tg, and the Tg decreased with increasingcarbonate content in the copolymer, following aFox relation, which was indicative of a randomcopolymer. The FA was grafted onto the copoly-mer P(LA-co-MAC) through a 2-aminoethanethiol

Figure 6. 1H NMR spectrum of folic acid-functional-ized copolymer P(LA-co-MAC)/FA (A) (Table 1, entry2) and folic acid (B).

Figure 7. XPS spectrum of P(LA-co-MAC) before (a)and after FA-graft (b). The insert shows the nitrogensignal at high resolution. [Color figure can be viewedin the online issue, which is available at www.interscience.wiley.com.]



bridge, on the basis of the reaction of its NH2

group with the carboxy end-group of FA and ofits SH group with the allyl group on P(LA-co-MAC). This FA-grafted copolymer P(LA-co-MAC)/FA showed improved adhesion and proliferationbehavior of vero cells than that of P(LA-co-MAC)and PLA. Therefore, the novel FA-grafted copoly-

mer was expected to find application in drugdelivery and tissue engineering.

Financial support was provided by the National Natu-ral Science Foundation of China (Project No.20674084 and 50373043), and by the National Fundfor the Distinguished Young Scholars (No: 50425309).

Figure 8. (A) Microscopic images of adhered and spread vero cells. Film sample:PLA (a and d), P(LA-co-MAC) (b and e), and P(LA-co-MAC)/FA (c and f). Incubationtime: a, b, and c, 24 h; d, e, and f, 48 h. (B) Attached fractions of vero cells adheredand proliferated on the surface of the following substrates: (a) PLA, (b), P(LA-co-MAC), (c) P(LA-co-MAC)/FA.

1860 HU ET AL.


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Novel Aliphatic Poly(ester-carbonate) with Pendant Allyl Ester Groups and its Folic Acid...

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