Surface Segregation Assessment In Poly( ε -caprolactone)-poly(ethylene glycol) Multiblock Copolymer...

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Surface Segregation Assessment in Poly(e-caprolactone)-poly(ethylene glycol) MultiblockCopolymer Films

Stefania Cometa, Federica Chiellini, Irene Bartolozzi, Emo Chiellini,*Elvira De Giglio, Luigia Sabbatini

The ability to predict the in vivo performance of multiblock-copolymer-based biomaterials iscrucial for their applicability in the biomedical field. In this work, XPS analysis of PCL-PEGcopolymers was carried out, as well as morphological and wettability evaluations by SEM andCA measurements, respectively. XPS analysis onfilms equilibrated in PBS demonstrated a furtherenrichment in the PEG component on the surface.Copolymer films obtained by casting using differentsolvents showed a dependence in segregationaccording to the solvent employed. Cell adhesiontests demonstrated the importance of copolymersegregation and rearrangement in a wet environ-ment, with a dependence of these phenomena onthe copolymer molecular weight.

Introduction

Although most of the degradable polymers proposed as

biomaterials are well established in the biomedical and

pharmaceutical fields, multiblock copolymers are often

preferredsincebiocompatibilityandefficacyof the relevant

homopolymers could be optimized by refining and/or

combining their structural and functional features. Poly-

(ethylene glycol) (PEG) has been widely recognized as a

S. Cometa, F. Chiellini, I. Bartolozzi, E. ChielliniLaboratory of Bioactive Polymeric Materials for Biomedical andEnvironmental Applications (BIOlab) UdR-INSTM, Department ofChemistry and Industrial Chemistry, University of Pisa, ViaVecchia Livornese 1291, 56010, S. Piero a Grado (Pisa), ItalyFax: þ39 50 221 0332; E-mail: [email protected]. De Giglio, L. SabbatiniDepartment of Chemistry, University of Bari, Via E. Orabona 4,70126 Bari, Italy

Macromol. Biosci. 2010, 10, 317–327

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

bioinert polymer, mainly employed for its hydrophilic

nature, which allows a poor protein adsorption and

consequent limited cell adhesion.[1–5] Poly(e-caprolactone)(PCL) is a semi-crystalline bioresorbable aliphatic polyester,

with slightly hydrophobic characteristics that make it

slowly hydro-biodegradable under physiological condi-

tions.[6–8] When PCL is copolymerized with PEG, its

hydrophilicity and biodegradability can be improved,

covering a wider field of applications formerly impeded

by the inherent hydrophobic character of PCL homopoly-

mer.[9–11]

The biological response and long term performances of

polymeric biomaterials are hugely influenced by their top

layer chemical composition, the presence of contaminants,

segregation of elements or blocks, functional group

orientation etc. Surface dynamics of polymeric biomater-

ials can be much more complex than those of other rigid

biomaterials (i.e., metals, ceramics, etc.), due to the high

mobility of macromolecules on the surface. Many block

DOI: 10.1002/mabi.200900284 317

S. Cometa, F. Chiellini, I. Bartolozzi, E. Chiellini, E. De Giglio, L. Sabbatini

318

copolymers spontaneously undergo phase separation

during processing.[12,13] Copolymer components having a

lower surface energy always tend to enrich the surface in

order to minimize the free energy of the whole system: for

instance, amphiphilic multiblock copolymers tend to

rearrange so that the hydrophilic portion will dominate

the interface in polar hydrophilic media, whereas the

hydrophobic portionwill dominate in air. In themajority of

cases, in going froma specificmedium to an antagonist one

(i.e., from a dry environment to a wet one), a structural

rearrangementof theexposed surfaceoccurs. Ina fewcases,

a locking of the surface can be achieved thanks to a

segmental self-assembly thermodynamically impervious

to restructuring.[14,15]

Surface properties of biomedical polymers should be

properly characterized and optimized. In this respect, thin

films obtained by solvent casting methods (phase immer-

sion, spin coating or solvent evaporation) are often used to

determine the effect of surface chemical composition and

morphology on cell adhesion and proliferation. Solvent

casting of polymers on a Petri dish can introduce large

differences in terms of molecular conformation,

surface chemistry and topography.[16] The instrumental

surfaceanalysis techniquesdevelopedover the last30years

are being increasingly applied for the study of copolymers

surfaces and interfaces. Great efforts have been made in

order to elucidate the surface features of polymers in terms

of their elemental/molecular composition and physical

morphology, as well as in terms of evaluation of their

cellular response, by performing in vitro and in vivo

experiments.[17–21] However, only a limited number of

research works have been carried out in order to highlight

the role of surface science in understanding the complex

dynamics of materials occurring in physiological environ-

ments.

In this work, X-ray photoelectron spectroscopy (XPS),

scanning electron microscopy (SEM) and static and

dynamic contact angle (SCA and DCA) measurements have

been employed to characterize the surface chemistry/

morphology/wettability of PCL-PEG hydro-biodegradable

copolymers (named PEGCL copolymers). In particular, XPS

representsavaluable tool forprobingsurfacesegregation in

common multi-phase amphiphilic polymer systems.[22–26]

It is widely accepted that the polymer surface is indeed

stable and the results of the analysis obtained under high

vacuum conditions can also be applied to non-vacuum

environments. Since conventional XPS probes the top

10nm, whereas cells only probe the outermost molecular

layer, angle-resolvedXPS (AR-XPS)hasbeenutilized inorder

to investigate more surface layers. In vitro cell adhesion

tests, performedonPEGCL chloroformcast copolymerfilms,

have indeedshownnodependenceonthePCL/PEGnominal

ratio, whereas different molecular weights elicited differ-

ent cell adhesion. In this respect, here we present an XPS



investigation on surface rearrangements of copolymer

films after equilibration in an aqueous environment, in

correlation to their molecular weight. XPS analysis, as well

as SEM, SCA andDCA characterizations, have substantiated

the evidence for surface segregation of PEGCL copolymers,

providing a satisfactory explanation of the biological

response to the investigated systems.

Experimental Part

Materials

All reagents were purchased from Sigma Aldrich. Two different

PCL/PEG molar ratios have been employed: PCL/PEG 50/50 (coded

as PEGCL50) and 75/25 (coded as PEGCL25). Hexamethylene 1,6-

diisocyanate (HMDI) was used as a chain extender. A polymer

obtainedusingHMDIandPCL, asauniquerepeatingblock (codedas

PCL-HMDI), was synthesized as a control. Moreover, a higher

molecularweight copolymer having PCL/PEG¼75/25was synthe-

sized (coded as high MW-PEGCL25), in order to gain information

about a possible influence of the molecular weight parameter on

surface segregation phenomena. Briefly, the synthesis was

achieved by dissolving the previously dried dihydroxylated

prepolymers (PEG and PCL diols – molecular weight 2 000) in bulk

under a dry nitrogen atmosphere. A slight excess of HMDI and

0.5wt.-% stannous octoate [Sn(Oct)2] as a catalyst were added and

the reactionmixturewas allowed to react at 70 8C and the reaction

wasstoppedwhentheviscositywastoohightoallowstirringof the

mixture. The copolymers were obtained in yields > 85% by

precipitating the reaction mixture in excess of cold methanol. The

isolatedcopolymerswerepurifiedbydissolution in chloroformand

reprecipitation indiethylether, andfinallydriedunderavacuumat

room temperature for at least 48 h. A slight excess of HMDI (1.1:1

ratio)wasused inorder tohaveaprecisecontrolover themultiblock

copolymer’s molecular weight. When higher molecular weight

copolymers were desired, the reaction was carried out under

rigorously anhydrous conditions to prevent the eventual hydro-

lysis of HMDI.

Bulk Characterization

1H NMR spectra of PEGCL copolymers were acquired on a Varian

Gemini 200 spectrometer in CDCl3. Size exclusion chromatography

(SEC)wasperformedataflowrateof 1.0mL �min�1 byusinga Jasco

PU-1580 high-performance liquid chromatograph (HPLC) con-

nected to Jasco 830-RI and Perkin-Elmer LC-75 spectrophotometric

(l¼260nm) detectors, equippedwith twoMixed-D PLgel columns

(300�7.5mm2). Chloroform was used as the eluent and the

calibration curve was established using mono-dispersed polystyr-

ene standards.

Biological Investigation

Materials

Cell line BALB/3T3 Clone A31 mouse embryo fibroblasts (CCL163)

were obtained from American Type Culture Collection (ATCC) and

DOI: 10.1002/mabi.200900284

Surface Segregation Assessment In Poly(e-caprolactone)-poly(ethylene glycol) . . .

propagatedas indicatedbythesupplier.Dulbecco’sModifiedEagles

Medium (DMEM), 0.01M pH¼7.4 phosphate-buffered saline

without Ca2þ and Mg2þ (PBS 1X), calf serum (CS), trypsin/

ethylenediamine tetraacetate (EDTA), glutamine and antibiotics

(penicillin/streptomycin) were purchased from GIBCO Brl. The cell

proliferation reagentWST-1waspurchased fromRocheDiagnostic.

Tissue culture grade disposable plastics were obtained from

Corning Costar.

Cytotoxicity Tests on PEGCL Copolymers Extracts

To assess the cytotoxicity of possible substances that could leach

from the prepared PEGCL copolymers, 0.2 g of each polymer were

immersed in 1mL of DMEM supplemented with 10% Calf Serum,

4�10�3M L-Glutamine and 100U �mL�1:100mg �mL�1 penicillin:

streptomycin for 48h at 37 8C in an enriched 5% CO2 atmosphere.

The medium containing the extract was tested undiluted and

diluted at a volume ratio of 1:1 and 1:4 using the complete culture

medium. Balb/c 3T3 Clone A31 cells were seeded at a density of

1�103/well in a 96 well plate and allowed to proliferate for 24h.

Then the culturemediumwas changedwith theDMEMcontaining

the extracts and cellswere allowed to proliferate for further 24h at

37 8C in an 5% CO2 enriched atmosphere. Cells incubated

with complete DMEM and wells containing only complete DMEM

were used as controls. At the endof the exposure time, cell viability

was measured using WST-1 tetrazolium salt as previously

described.[27] Absorbance was measured at 450nm and values

relative to a controlwere reported. Experimentswere performed in

triplicate.

Cell Adhesion and Proliferation onto PEGCL Cast Films

To investigate the ability of the prepared PEGCL cast films to

support cell adhesion and proliferation, films were placed into 12

well culture plates and sterilized for 10min under UV light. BALB/

3T3 Clone A31 mouse embryo fibroblasts cells were seeded onto

the film at a concentration of 1�104 per well and allowed to

proliferate for 5 d at 37 8C in a 5% CO2 enriched atmosphere. The

complete culture media was renewed every 48h. At the end of the

experiment, cells were washed with PBS 1X, fixed with 3.8%

paraformaldehyde in PBS 1X for 1 h at room temperature and

stained with 1% solution of toluidine blue in PBS 1X. The cell

morphologywas investigatedbymeansof lightmicroscopyusinga

Nikon Eclipse TE2000 inverted microscope.

Surface Characterization

SCA measurements were performed on films spin coated from

chloroform solutions using HPLC-grade water and a sessile drop

method at 25�0.1 8C with a DSA 10 drop shape analysis system

(Kruss, Hamburg, Germany). In particular, 1wt.-% copolymer

solution in chloroform was filtered on a 0.45mm nylon filter,

dropped on the surface of amicroscope glass plate and spin coated

at 6 200 rpm for 60 s. The film was kept in a dry atmosphere for

further analysis. Data were averaged over at least nine measure-

ments.

Dynamic contact angles (DCA) were measured using a DCAT11

instrument (Dataphysics, Filderstadt, Germany) onglass slides dip-



coated in chloroform polymeric solution. Three cycles of immer-

sion/withdrawal andHPLC-gradewaterwere used.Measurements

were averaged over six samples.

Copolymer films for XPS and SEM analysis were prepared by a

solution-castingmethod from a 5% chloroform (in some cases also

inacetoneor ethyl acetate) solution. The solutionwas stirred for 3h

at room temperature and then filtered with a 0.2mm poly(tetra-

fluoroethylene) (PTFE) filter. The filtratewas cast into a Petri dish to

formapurifiedpolymerfilm. The solventwas allowed to evaporate

slowly at room temperature for approximately 1week and

subsequently the filmswere dried in a vacuum for 48h. In general,

all the films were easily removed from the Petri dishes since

detaching from the glass occurred consequently to the solvent

evaporation.

SEM analysis was performed using a JEOL LSM5600LV (Tokyo,

Japan) scanning electron microscope. Before analysis, samples

were gold sputtered under a high vacuum.

XPS spectra of dry films and PBS-equilibrated films

were obtained using a ThermoVG Theta Probe spectrometer

(Thermo Fisher Scientific, Inc., Waltham, MA, USA), equipped with

a microspot monochromatized Al Ka source and a flood

gun combined with an argon gun for compensation of sample

electrostatic charging. The Al Ka line (1 486.6 eV) was used

throughout the work and the base pressure of the

instrument was 10�9mbar. Survey scans (binding energy

range 0–1200 eV, FAT mode, pass energy¼150eV) and detailed

spectra (FAT mode, pass energy¼50 eV) were recorded for each

sample. The stability of the samples during X-ray exposure

was examined, and no degradation was observed within the

acquisition time. Data analysiswas performed using the Avantage

software package, which consists of a non-linear least

squares fitting program. The surface composition was determined

using the manufacturer’s sensitivity factors. The fractional

concentration of a particular element X (% X) was computed using

Equation (1):

X% ¼ Ix=fxð ÞP

Ii=fið Þ � 100 (1)

where Ii and fi are the integrated peak areas of each of the n

detected elements and their sensitivity factors, respectively. The

values of binding energies (BE, eV) were taken relative to the

binding energy of the C1s electrons of hydrocarbon contaminants

on the sample surface (from an adventitious carbon), which is

accepted to be equal to 285.0 eV. The curve fitting process was

done by imposing the peak full width at half maximum (FWHM)

of the most resolved peak (i.e., the peak at the highest BE in C1s

spectrum) to the other peaks. For AR-XPS, the Theta Probe

spectrometer was equipped with a radian lens and a two-

dimensional detector that collects the electrons, which are

discriminated for signal intensities in both photoelectron energy

and photoemission angle. Three simultaneous emission angles

were acquired ranging from 30.5 to 60.58. The higher the emission

angle, the smaller the sampling depth, resulting in a more surface

sensitive measurement. All data were averaged over at least three

replicates (i.e., analyzing three randomly selected portions of the

polymeric surface) and the mean� standard deviation was

reported for each value.

www.mbs-journal.de 319


Figure 1. 3T3/balb Clone A31 mouse embryo fibroblasts cell linegrown onto a) tissue culture polystyrene (control), b) PEGCL50,c) PEGCL25 and d) high MW-PEGCL25.

Table 1.Molecular weights of copolymers with different PCL/PEGratios, obtained using SEC analysis.

Copolymer PCL2000/PEG2000 Mn Mw=Mn

PCL-HMDI 100/0 42 000 1.7

PEGCL50 50/50 72 000 3.3

PEGCL25 75/25 62 000 2.6

highMW-PEGCL25 75/25 331 000 1.9

320

Results and Discussion

Bulk Characterization

Examination of copolymers by 1H NMR spectroscopy

confirmed all the peak characteristics of the two compo-

nents. The ratio of the PEG and PCL blocks was calculated

from the relative intensities of the peak at d¼ 3.6 for the

(CH2CH2O) of PEG and from the peak at d¼ 4.1 for the

(CH2�OC¼O) of PCL,which is in agreementwith the ratio of

the feed mixture.

Molecular weights and molecular weight distributions,

obtained by SEC analysis, are reported in Table 1.

Biological Evaluation

Solvent cast films could not be representative of the

ultimate manufactured biomaterial, since the copolymers

have to be processed in different manners to obtain three-

dimensional (3D) polymeric scaffolds (electrospinning,

rapid prototyping, etc.). On the other hand, biocompat-

ibility evaluation of novel synthesized materials is

imperative in biomaterial research, therefore cast films

are generally employed for this purpose.

Materials intended to be used in biomedical applications

should not release any cytotoxic agent susceptible to being

harmful for the cells. As reported in the literature,[28–29] PCL

homopolymer has been widely investigated in vitro as

material forpotentialbiomedicalapplications,demonstrat-

ing an interesting capacity to sustain cell adhesion and

proliferation. To know whether the prepared PEGCL

copolymers extracts might be harmful to cells, the

fibroblast cell line balb/c 3T3 Clone A31 was cultured in

the presence of the extractables of these materials for 24h

at 37 8C. A WST-1 assay was carried out to evaluate the

potential cytotoxicity of the PEGCL extracts. Aqueous

extracts of the investigated samples, undiluted, anddiluted

1:1 and 1:4were used. The result of this study revealed that

no significant amount of cytotoxic compounds were

released from the prepared copolymers. Moreover, cells

not only remainedviable but alsoproliferated similar to the

control, even in the case of the undiluted extract.



Investigations regarding PEGCL50 and 25 cast film

bioactivity in terms of cell adhesion and proliferation

revealeda limited cell adhesion, as shown in Figure1(b) and

1(c), probably correlated to the PEG and PCL component

phase segregation (higher surface PEG content on both the

chloroform cast films). Interestingly, in the case of high

MW-PEGCL25, a significantly higher cell adhesion was

observed, as reported in Figure 1(d), suggesting that, at least

in the investigated specimens, an improvement in the

biological response seems to be attained by the employ-

mentofhighermolecularweight copolymers. Cell adhesion

onto tissue culture polystyrene (TCPS) as a control is shown

in Figure 1(a). No significant morphological differences

were found between cells grown onto TCPS [Figure 1(a)],

whichwas used as a control and cells cultured onhighMW-

PEGCL25 [Figure 1(d)].

These biological tests pointed out that cell adhesion on

these samples cannot be predicted on the basis of the

copolymer’s composition, but appears to be related to the

molecular weight of the copolymers (i.e., PEGCL50 and

PEGCL25, despite their different bulk composition, gave a

similar biological response while PEGCL25 and high MW-

PEGCL25, despite having the same bulk composition,

provided very different biological responses). These unex-

pected results could be explained by investigating the

surface features of the copolymers, in terms of surface

wettability, morphology and chemical composition.

Morphological Investigation

PEGCLfilmsforbiological andsurface characterizationwere

cast from the solutions onto glass Petri dishes. Copolymer

solutionswere dried slowly in an enclosed space to provide

DOI: 10.1002/mabi.200900284


Figure 2. SEM images of air- (on the left) and glass- (on the right) exposed sides ofPEGCL25 film cast by chloroform. The scale bar shown in each micrograph is 10mm inlength.

an environment saturated with solvent vapor and the

resulting cast films appeared as whitish films having

thickness of about 150–170mm, with an opaque air-

exposed side and a translucent glass-exposed side.

The surface morphology of PEGCL50, PEGCL25 and high

MW-PEGCL25 chloroform cast films was investigated by

SEM. In particular, SEM micrographs of PEGCL25 - air and

glass sides - are illustrated in Figure 2 (PEGCL50, having a

morphology very similar to PEGCL25, is not reported).

The air side, on the left side of Figure 2, appeared rough

with visible pores and was characterized by discrete,

spherule-like aggregations (diameters ranging between 85

and 100mm) partially fused together. In contrast, the glass

side of PEGCL25 (on the right side of Figure 2) appeared

much smoother than the air side. SEM analysis of chloro-

form cast high MW-PEGCL25 film, reported in Figure 3,

evidenced less porous and elongated filamentous aggre-

gates on the air side, and a less porous and smoother glass

side. The filamentous structure could be due to the lower

solubility observed for high MW-PEGCL25 with respect to

that of its homologue with lower molecular weight.

On the basis of the observed morphologies, we have

hypothesized that, when the solution is cast from chloro-

form into a Petri dish, PEG blocks tend to assemble

according to the hydrophilic properties of the glass surface,

forming a laminated surface structure, while a substantial

Figure 3. SEM images of air- (on the left) and glass-exposed sides (on the right) of highMW-PEGCL25 film cast by chloroform. The scale bar shown in each micrograph is 10mmin length.



number of round and irregular aggre-

gateswith significant gaps in the form of

voids are predominant on the air-

exposed surface. Regarding high MW-

PEGCL25, it was hypothesized that the

filamentous structure observed on the air

side of the copolymer, characterized by a

relatively high viscosity, (on the left of

Figure 3) was a consequence of a faster

nucleation taking place with poorly

soluble systems. Therefore, we suggest

that the different copolymer domains

could havemore freedom to aggregate in

visible nucleated structures in PEGCL25

than in high MW-PEGCL25, probably due to the higher

solubility and lower viscosity of the former. In conclusion,

no evidence of a relationship between the morphological

characteristics and copolymer bulk composition was

observed, whereas significant differences between cast

films having the same bulk composition and different

molecular weights were noticed.

Static and Dynamic Contact Angle Measurements

Static contact angle measurements, reported in Table 2,

pointedout a similarhydrophilic character of both the spin-

coated copolymers with different PCL/PEG ratios (compar-

ing PEGCL50 and 25) and different molecular weights

(PEGCL25 and high MW-PEGCL25). These values, obtained

by measuring the right and left angles of deionized water

droppedonto the surface of thefilms, could be related to the

hydrophilic nature of PEG chains present on the material

surfaces. Interestingly, surfaces containing different nom-

inal PEG contents resulted in a similar contact angle value,

suggesting no relevant surface differences between

PEGCL50 and 25 spin-coated films, irrespectively of their

different bulk composition. For a comparison, contact angle

measurements were performed on PCL-HMDI. The value

resulted was quite similar to the typical PCL contact

angle reported in the literature.

In order to obtain information about

surface roughness and homogeneity (i.e.,

phase separation), dynamic contact

angle measurements were performed.

For PEGCL50, hysteresis decreased

after the first immersion/withdrawal

cycle, indicating high chain mobility

with a dramatic rearrangement of the

surface after the first contact withwater,

which results into a more homogeneous

hydrophilic surface. In fact, while Q1adv

was about 748,Q2adv andQ3

adv were about

298 and the Qrec angles were always

around238. Therefore, hysteresiswas 458



Table 2. Static contact angles of PEGCL50, PEGCL25 and PCL-HMDIspin-coated films (data averaged over nine measurements).

Copolymer Contact angle

-

PEGCL50 72.0� 2.0

PEGCL25 71.2� 1.2

high MW-PEGCL25 74.0� 3.0

PCL-HMDI 82.0� 3.0

322

in the first cycle and only 68 in the second and third cycles.

Similar observations were reported by Crisci et al.[30] This

phenomenoncouldbe related to several contributions, such

as the surface rearrangement and the swelling of the PEG

component.

PEGCL25 exhibited a slightly more stable and hydro-

phobic surface thanPEGCL50. In fact,Q1adv resulted758,Q

2adv

and Q3adv were about 478, while receding angles were

constant around208and thereforehysteresiswasabout558for the first cycle and 278 for the second and third ones. As

far as high MW-PEGCL25 is concerned, the film exhibited a

fairly stable and more hydrophobic surface respect to

PEGCL25: Q1adv resulted equal to 818, while Q2

adv and Q3adv

were around 758, while receding angles resulted of about

208 for all the cycles. Therefore hysteresis ranged between

61 and 558, thus indicating lower chain mobility than for

PEGCL50 and 25.

PCL-HMDI exhibited constant values for both the

advancing contact angles (958) and the receding contact

angles (268). Thus, the surface was very hydrophobic and

hysteresis was constant at around 708. This similar

behavior can be explained by considering that PCL itself

can possibly have fast rearrangements which result in the

exposition of either the hydrophobic or hydrophilic part of

the molecules.

DCA results clearly showed a surface rearrangement of

PEGCL50 and PEGCL25, with enrichment of the hydrophilic

moieties, while this phenomenon was significantly less

pronounced in high MW-PEGCL25. This molecular weight-

dependent surface rearrangement was

investigated by XPS analysis of water-

equilibrated films in order to have a

better insight and correlation with

results obtained from the biological

characterization.

Figure 4. Survey spectra of PEGCL50 (a) and PEGCL25 (b) cast films from chloroform(air side).

Surface Chemistry Assessmentby XPS

Surface segregation phenomena can be

theoretically understood in terms of sur-



face energy and chemical potential balance arguments: the

driving force for surface segregation is the difference in

surface tension between different domains of a copolymer

system. Domains with low surface energywill move to the

surface to minimize the overall surface energy of the

system. At the same time, this surface migration could

increase the chemical potential due to a demixing process.

The balance of these two factors supplies the final surface

composition.

XPSanalysiswas carriedout inorder to assess the surface

chemistry of PEGCL50 and 25 cast films, devoting particular

attention to eventual differences existing between the

surface exposed to air and that exposed to the Petri dish

glass. Survey spectra of PEGCL50 and 25 films, cast from

chloroform (air side), are reported in Figure 4(a) and 4(b)

respectively. Typical elements (C1s andO1s) of PEG and PCL

samples were detected. In addition, Si2p signals were

recorded, probably due to the silicon grease contamination,

as well as a N1s signal, due to HMDI. It is important to note

the absence of an Sn2p signal, related to the catalyst used,

thus demonstrating the suitability of the purification steps

employed.

In order to evaluate the surface composition of the two

copolymers, curve fittings of high resolution C1s spectra

were performed. For comparison, the C1s signal curve

fitting of the air/polymer interface of PCL-HMDI is reported

in Figure 5.

Theobtainedpeaksdeconvolution is inagreementwitha

typical PCL film reported in the literature,[31,32] with the

exceptionofpeakD.This contribution isdue to theCH2�NH

groups relevant toHMDImolecules, and its peak’s areawas

well comparable to the N1s corrected signal area. The

C1s spectrum of PCL-HMDI showed an A:B:C:E ratio

[i.e., CH2: CH2C(¼O)O:CH2OC(¼O):C(¼O)O ratio] equal

to (5.0� 0.2):(1.61� 0.05):(1.58� 0.09):(1.04� 0.01) (data

averaged over three sampled points) with respect to the

expected 3:1:1:1 one. This finding is in keepingwith a slight

enrichment of the non-polar segments of PCL repeating

units on the air-exposed surface, as reported by Tang et al.

for PCL cast films.[33] However, peak A resulted larger than

expected from the caprolactone formula, also due to some

CHx -type surface contamination; hence this peak was not

DOI: 10.1002/mabi.200900284


Figure 5. C1s high-resolution spectrum of air-exposed surface ofPCL-HMDI film cast from chloroform. Peaks attributions arereported in the text.

used in further calculations. As far as PEG-based polymers

are concerned, XPS literature data[31,34] report a single C1s

contribution falling at 286.6� 0.1 eV (in addition a possible

minor component at 285.0 eV, due to adventitious con-

tamination, could be present). Therefore, it can be observed

that, in PEGCL copolymers, the presence of PEG moieties

does not add peculiar signals to the XPS spectra but simply

adds contributions to carbon and oxygen spectral regions.

In Figure 6, curve-fittings of C1s high resolution spectra

relevant to PEGCL50 and PEGCL25 – air and glass sides – are

reported. Peak attributions are illustrated in Scheme 1,

which allowed us to provide a detailed analysis of

quantitative information obtainable by relating the

normalized peak areas. Peaks binding energies, as well as

their atomic percentage, are reported in Table 3.

In particular, in the C1s signal of PEGCL copolymers can

be noticed a significant increase of peak C (relevant to the

PEG COH functionality, falling at the same binding energy

as thatof theCH2OC(¼O)ofPCLmoieties)withrespect to the

Figure 6. C1s high-resolution spectra of chloroform cast films: PEGCLexposed surfaces (a and b, respectively) and PEGCL25 glass- and air-eand d, respectively).



C1s signal ofPCLalone (seeFigure5), due to the introduction

of ethylene glycol components.

The equations used for the quantitative evaluation of

surface PEG and PCL content are reported in the following

system of Equation (2):

50 glaxposed

Area peak E� Area N1s ¼ 16xArea peak C ¼ 20xþ 90y

(2)

where x is the number of PCL2000 macromers in which 20

C�O and 16 C¼O functionalities were present, and y

represents the number of PEG2000 macromers, which

contain 90 C�O groups each.

It should be underlined that, when peak E was used to

estimate PCL content in PEGCL copolymers, a slight

underestimation of this value was implicitly linked to

the lower surface presence of polar C(¼O)O groups (see

peaks area ratio in C1s PCL/HMDI curve fitting). In any case,

using the reported equations, a surface PEGenrichment can

be detected on both the air-polymer and glass-polymer

interfaceswith respect to thebulk composition: 63.3� 1.6%

and 55.9� 1.1% of PEG content in PEGCL50 glass and air

sides, respectively, vs. 50% nominal value, and 58.1� 0.6%

and56.3� 1.4% in PEGCL25 glass and air sides, respectively,

vs. 25%. These findings suggest a PEG component enrich-

ment in both PEGCL50 and 25 glass-exposed copolymer

films, as expected, but an almost similar PEGCL50 and 25

surface composition, in disagreementwith their significant

difference in terms of bulk composition. Indeed, the PEG

surface content which is higher than the nominal one,

observed on both air and glass sides of the cast films, could

be responsible for the low serumprotein adhesion onto the

surface of the films resulting in poor cell attachment.[35]

ss- and air-surfaces (c

Moreover, results of XPS analysis on dry

chloroform high MW-PEGCL25 cast film

revealed a surface composition very

similar to the homologue copolymer

having lower molecular weight. This

result did not allow us to understand

the different biological performances of

higher and lower molecular weight

PEGCL25 copolymer films, thus convin-

cing us to exploit othermodalities of XPS

analysis.

In-Depth Composition AnalysisUsing AR-XPS

In order to better assess the real surface

chemistry experimented by cells, we

have carried out AR-XPS measurements,

which can supply information about the

outermost molecular layers.



Scheme 1. Peak attributions of C1s curve fittings reported in Figure 6. Circles are used to highlight the carbon(s) whose capital letter refers topeak labels in Figure 6 and in Table 3.

Table 3. Binding energies and atomic percentages of the C1s fitting components of spectra reported in Figure 6.

Peak Peak BE Carbon content

eV at.-%

PEGCL50 (glass) PEGCL50 (air) PEGCL25 (glass) PEGCL25 (air)

A 285.0� 0.1 33.7� 0.4 55.29� 0.09 39.7� 0.4 48.9� 1.2

B 285.5� 0.1 10.8� 1.1 8.4� 0.5 8.9� 0.8 10.21� 0.02

C 286.7� 0.1 47.2� 1.9 28.8� 0.2 43.0� 0.6 33.0� 0.9

D 287.3� 0.1 2.3� 0.9 2.8� 0.3 2.16� 0.17 2.87� 0.03

E 289.4� 0.1 5.9� 0.3 4.64� 0.11 6.12� 0.03 5.0� 0.3

Table 4. PEG content on PEGCL50 and PEGCL25 (air and glass sides), at different takeoff angles, calculated from Equation (2) on C1s signalscurve fittings.

Takeoff angle PEG content

- %

PEGCL50 air PEGCL50 glass PEGCL25 air PEGCL25 glass

30.5 54� 2 60.7� 1.5 53� 2 59� 2

45.5 53.7� 1.2 62.0� 1.7 50.7� 1.5 59� 3

60.5 53� 3 62.8� 1.1 53� 3 60.0� 1.7

324

AR-XPS data for different takeoff angles showed that no

significant variations in PEG and PCL percentages were

observedbyvarying the samplingdepth (seeTable 4). These

results clearly show that surface analysis of the topmost



10nm of PEGCL50 and 25 chloroform cast films resulted in

quite homogeneously distributed layers, thus conventional

XPS analysis seems to be adequate to give information

about the real surface compositionofmultiblock copolymer

DOI: 10.1002/mabi.200900284


films experimented by cells. Similar

results have been obtained by perform-

ing AR-XPS on high MW-PEGCL25. In this

respect, after verifying this quite homo-

geneous in-depth composition, further

analyses have been acquired in the

conventional XPS mode.

Figure 7. C1s signals relevant to PEGCL25 (on the left) and high MW-PEGCL25 (on theright), before (solid line) and after (dotted line) 48h incubation in PBS.

XPS Analysis of PBS-EquilibratedFilms and Interpretation ofBiological Results

The analysis of polymer films after interaction with an

aqueous environment canbevery important tounderstand

if contactwithwater canmodify their surface composition.

In this respect, PEGCL chloroform cast films were analyzed

after immersion in PBS for 48hand successive dryingunder

a vacuum, in order to ascertain if the copolymer films

underwent reconstruction of their block composition. A

similarprocedurehasalreadybeenadopted in the literature

byother authors inorder to characterizewater-equilibrated

polymer films.[36–38] Since XPS data on ‘‘wet’’ samples was

in good agreement with DCA and cell adhesion results, we

have assumed that the ultrahigh vacuum used in the XPS

analysis did not substantially alter the copolymer surface

assembly. Therefore, XPS data on ‘‘wet’’ surfaces could be

considered indicative of the real chemical composition

experimented by the cells in aqueous environment.

Relative peak abundances, obtained by performing C1s

curve fitting on PEGCL50 and25 ‘‘wet’’ films, are reported in

Table 5.

These results demonstrated that, independently of the

glass and air sides, a further enrichment in PEG moieties

occurred, both for PEGCL50 and 25 samples; the PEG

percentage, calculated using Equation (2), reached

88.0� 0.9% and 84.3� 0.6% for PEGCL50 and 25, respec-

tively.

Table 5. Binding energies and atomic percentages of C1s peakscurve fitting relevant to PEGCL50 and 25 films after 48h immer-sion in PBS.

Peak Peak BE Carbon content

eV at.-%

PEGCL50 ‘‘wet’’ PEGCL25 ‘‘wet’’

A 285.0� 0.1 28.0� 0.6 38.3� 0.6

B 285.5� 0.1 15.2� 0.5 12.79� 0.16

C 286.7� 0.1 49.7� 0.9 41.6� 0.3

D 287.3� 0.1 3.1� 0.5 2.9� 0.3

E 289.4� 0.1 4.0� 0.3 4.7� 0.3



In Figure 7(a), a comparison between the PEGCL25 C1s

shape (glass side) before and after incubation in PBS has

beenreported, inorder tobetter clarify the incrementofPEG

contribution, as obviously expected by incubating in an

aqueousmedium a copolymer containing both hydrophilic

and hydrophobic components.

On the other hand, comparison between the high MW-

PEGCL25 C1s shape (glass side) before and after incubation

in PBS, reported in Figure 7(b), shows that quite similar C1s

shapeswere recorded. We hypothesized that themigration

of PEG domains and phase separation that resulted was

hindered by the high molecular weight of the copolymer.

Therefore, a surface rearrangement of high MW-PEGCL25

may be highly influenced by its low freedom in motion.

These results are in good agreement with DCA measure-

ments, nevertheless in XPS solvent cast films whereas in

DCA dip-coated glass slides have been analyzed. Anyway,

both XPS and DCA analysis results were useful to conclude

that the less pronounced phase segregation after immer-

sion in wet environment observed in high MW-PEGCL25

film allowed for a significant cell adhesion and prolifera-

tion, as shown in Figure 1(d). Additional detailed research is

necessary to fully delineate the complex mechanisms that

govern the surface segregation in these systems. In

particular, further investigations are in progress by

synthesizing a series of polymers with different molecular

weights and blockiness distribution. However, our main

interest is to analyze possible surface segregation phenom-

ena related to the processing methods chosen to obtain 3D

scaffolds by using these copolymers. In this respect, the

ongoing morphological, chemical and biological character-

ization of PEGCL 3D scaffolds obtained by electrospinning

and/or rapid prototyping appears of great interest for a

better understanding of their effective potential utilization

in biomedical fields.

XPS Analysis of Films Cast from Different SolventSystems

Finally, PEGCL50and25havebeenalso cast using twoother

different solvent systems, in order to investigate to what



Figure 8. C1s high-resolution spectra of air-exposed PEGCL50 films cast from acetone (a)and ethyl acetate (b) and PEGCL25 films cast from acetone (c) and ethyl acetate (d).

326

extent their surface composition could be

influenced by solvent properties. The

solvents used were: acetone (hydrophilic,

Teb 56 8C) and ethyl acetate (hydrophobic,

Teb 77 8C), compared to previously ana-

lyzed chloroform (hydrophobic, Teb 61 8C)cast films. In this respect, we report

only XPS results about the air-exposed

surface, since, during the casting process,

itwas in contactwith thegasphase,made

by a mixture of air and residual solvent

vapors. When the solution was cast on

a Petri dish, indeed, the copolymer

systems assemble according to the high

hydrophilic glass properties (contact

angle� 258), forming a laminated surface

enriched with PEG hydrophilic domains,

while the air/polymer interface will

depend, in turn, on the hydrophilicity of

the solvent used. In our investigations,

XPS analysis pointed out a clear relationship

between surface copolymer composition and solvents

used, in particular with their relative hydrophilicity and

volatility.

In Figure 8, C1s curve fittings relevant to air-exposed

PEGCL50 and 25, fromacetone and ethyl acetate, have been

reported for comparison with air-exposed PEGCL50 and 25

cast from chloroform [see Figure 6(b) and 6(d)]. In Table 6,

binding energies and relative peak abundances, the

attribution of which is based on Scheme 1, have been

summarized.

Using Equation (2), the following PEG percentages in

films cast from the examined solvents resulted:

1. 8

Ma

� 2

2.3� 1.3% PEG in PEGCL50 and 83.1� 1.1% PEG in

PEGCL25 films cast from acetone, denoting higher

surface PEG enrichment than in chloroform cast films,

Table 6. Binding energies and atomic percentages of the C1s peaks in s

Peak Peak BE

eV

PEGCL50

(acetone)

PE

(ethy

A 285.0� 0.1 31.8� 1.8 34.

B 285.5� 0.1 10.5� 0.9 16.

C 286.7� 0.1 50.3� 0.9 37.

D 287.3� 0.1 3.7� 0.6 2.

E 289.4� 0.1 3.7� 0.3 8.3

cromol. Biosci. 2010, 10, 317–327

010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

due to the high hydrophilic character and the low

boiling point of acetone;

2. 4
3.2� 0.5% PEG in PEGCL50 and 26.3� 0.9% PEG in
PEGCL25 films cast from ethyl acetate, resulting in a

surface PEG percentage more similar to the nominal

one, indicating that this more highly boiling and

hydrophobic solvent enabled PCL to reach the surface.

In conclusion, by comparing the surface PEG percentage

estimated by XPS investigation on films cast by different

solvents, the trend was that surface composition was

enriched by PCL when: (1) the casting solvent was more

hydrophobic and/or (2) it evaporated slowly.

Work is in progress to gain information about the

morphological features and biological performances of

acetone and ethyl acetate cast films, in order to correlate

these results to the obtained XPS data.

pectra reported in Figure 8.

Carbon content

at.-%

GCL50

l acetate)

PEGCL25

(acetone)

PEGCL25

(ethyl acetate)

8� 0.7 22.7� 0.6 40.0� 1.5

3� 1.8 8.0� 0.8 20.8� 1.4

9� 0.4 59.1� 0.5 27.4� 0.6

5� 0.5 5.1� 0.8 2.2� 0.5

8� 0.08 5.0� 0.4 9.6� 0.2

DOI: 10.1002/mabi.200900284


Conclusion

We focused our attention on the assessment of the

influence of multiblock copolymer film surface composi-

tion on their biological performance.

XPS investigation, supported by morphological and

wettability analysis, evidenced that cast films were

strongly affected by phase segregation phenomena, which

were found to depend on the solvent used in the casting

procedure, as well as on the molecular weight of the

copolymer. These segregation effects were enhanced after

equilibration in aqueous media and were also reflected in

cell behavior, observed in cell adhesion and proliferation

assays, using the balb/c 3T3 clone A31 mouse embryo

fibroblast cell line.

These results suggest that a systematic surface char-

acterization of random multiblock or triblock copolymers,

paying particular attention to their possible phase segrega-

tion as bound to primary structural parameters (molecular

weight, blockiness length and distribution) and to proces-

sing methods (i.e., solvent casting, spin coating, electro-

spinning, rapid prototyping)might provide useful hints for

thedevelopmentofpolymeric scaffoldswith successful and

optimized biocompatibility.

Acknowledgements: This work was performed within the frame-work of the projects FIRB-2005 - prot. RBIN043BCP, PRIN-2006 -prot. 2006038548 t and NoE Expertissues CT - 2004 - 500328. Theauthors would like to thank Mr. P. Narducci for the recording ofSEM images.

Received: August 3, 2009; Revised: September 25, 2009; Publishedonline: December 2, 2009; DOI: 10.1002/mabi.200900284

Keywords: biocompatibility; block copolymers; supports; sur-faces; X-ray

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