Proceedings of the 22ndAnnual EMBS International Conference, July 23-28,2000, Chicago IL.
Functionalization of Polylactide(PLA) surface using
Heterobifunctional PEGPLA Block Copolymers for
the Control of Cell Behavior at Surfaces
Hidenon Otsuka', Yukio Nagasaki2,Teruo Okano3,and Kaeunori Kataoka"
Abstract-This paper deals with novel approaches
established by our group for the construction of a
functionalized poly(ethy1ene glycol) (PEG) layer,
PEG-brushed layer possessing a reactive group a t
the free end of tethered PEG chain, on substrates.
An AB-type block copolymer composed of a-
acetal-poly(ethy1ene glycol) (PEG) as the
hydrophilic segment and polylactide (PLA) as the
hydrophobic segment was synthesized, and utilized
to construct the functionalized PEG layer on the
biodegradable polylactide surface by simple
coating. In this way, a PEG-brushed layer with a
terminal aldehyde group was readily prepared
which may have both non-fouling and ligand-
binding properties. Non-fouling property of PEG
strands eliminates nonspecific and uncontrolled
interactions of the surface with biological
components, including cells and proteins, while
presentation of tethered ligands attached to the
chain end of PEG brush allows cell behavior a t
the surface to be modulated in a specific manner
via receptor-mediated signaling. Based on the
characterization of these PEGylated surfaces from
a physicochemical (contact angle, atomic force
microscopy, electron spin resonance) as well a s
biological (protein adsorptionlcell adhesion) point
of view, our strategy to construct a functionalized
PEG layer was confirmed. Active functional
groups were present at the tethered PEG-chain end,
these materials will provide new insights i n t o
controlling cell behavior at surfaces for tissue
engineering and biomedical applications.
Key words- poly(ethy1ene glycol)(PEG),
polylactide(PLA), aldehyde, hepatocyte
I. INTRODUCTION
Modification with hydrophilic polymers is the most common way
to improve the surface properties of devices used for biological
and biomedical applications'. In particular, a poly(ethy1ene
glycol) (PEG) coating has most widely been used to minimize non-
specific fouling of the device surface with biocomponents,
including plasma proteins'. '. Although the PEG-coating can be
performed by a variety of methods, most of the PEG-coated
surfaces so far reported possess no reactive group on the PEG
chain end. To provide the further functionality on the PEG-coated
surface, we designed a block copolymer having end-functionalized
ffias a hydrophilic segment. Polylactide (PLA) was chosen as
the hydrophobic segment because it is biodegradable and non-
toxic, and is widely utilized as implant materials. Further, acetal
group is installed at the a-chain end of poly(ethy1ene
glycol)/polylactide block copolymers (PEGPL.4) which,
0-7803-6465-1/00/$10.0002000 IEEE
apparently, is able to be delivatized to aldehyde group by the
moderate acid treatment.
Our strategy is to construct a functionalized PEG layer on a
biodegradable PLA surface through a simple coating of a reactive
block copolymer of a-acetal-PEGPLA". After the construction
of the polymer layer composed of a-acetal-PEGPLA on a PL4
surface, acetal groups at the fire end of PEG chains are converted
into aldehyde groups. This surface-engineering is shown
schematically in Figure 1.
f Protein or
Peptide SchiffBase
Reduction
INaBHXN)
ZCWNH
I
Ligand-Receptor
Conjugation
Aldehyde group -hteraction n
Acid treatment
Functional Group Coating on
plylactide surface
(acetal group)
Figure 1. Schematic representation of the application to the
construction of a PEG layer using a PEGPLA block copolymer.
The hydrophobic PLA segments of the block copolymers anchor
into the PLA surface, while the water-soluble PEG chains should
extend into the bulk aqueous medium. Various ligands including
proteins, peptides, and sugars can be covalently immobilized to a
functionalized-PEG coating on a PLA surface. An aldehyde group
is very useful for this purpose due to its stability in water and its
high reactivity with primary amino groups. The focus of this
study was the accurate analysis of the surface properties of a-
acetal- (or aldehyde-) PEGPLA covered on a PLA matrix, as a basis
for the further application of this strategy in the biomedical field,
including functionalization of PLA-based devices. Particularly,
the effect of varying the PEG molecular weight (MW) of the block
copolymers was investigated from a physicochemical (5
potential, static and dynamic wetting) as well as a biological
(protein adsorptiodcell adhesion) point of view.
II. EXPERIMENTAL
II-1. Synthesis of Acetal-PEGIPLA Block Copolymers".
2936
 Proceedings of the 22"dAnnual EMBS International Conference, July 23-28, 2000, Chicago IL.

a-Acetal-PEGPLA block copolymers were synthesized by a For this purpose, numerous acetal-PEGPLAs with different
one-pot anionic ring-opening polymerization of EO followed by lengths of both PEG and PLA were synthesized. Molecular
LA using potassium 3.3-diethoxypropanolate (PDP) as an initiator weights (MW) of PEGPLA segments were abbreviated as follows
at room temperature under argon. The molecular weight of the PEG (Table 1): PEGPLA(0.65/11.0, 1.817.0, 3.3/5.4, 5.014.6,
and PLA segments were determined by GPC, MALDI-TOF-MS, and 8.7/6.9) where the numbers in parenthesis denote the M W of the
NMR measurements. PEG segments and PLA segments in kg/mol, respectively.
11-2. Preparation of PEGylated surface and conversion of the acetal 111-2. Dynamic contact angle measurements.
group into aldehyde group. The dynamic contact angle was measured to estimate the
The glass substrates, which were cleaned by a Piranha etch dynamics of the uppermost surface when the surface contacts with
(boiling mixture of 50% (v/v) sulfuric acid and 50 % (vlv) water(Figure 2).
hydrogen peroxide), were placed in 2 % (v/v) solution of 3- 90
(trimethoxysily1)propyl methacrylate/ethanol. The glass
I
substrates were dried at 160 k for 24 h under vacuum. The PEG-
brushed layer was constructed on this silanized glass surface by the
spin coating of toluene solution of PLA(4 % (w/v)). followed by
the acetal-PEGPLA(2 % (w/v)). The PEGylated glass string was
immersed into aqueous media adjusted to pH 2 to transform an 60
acetal group at the PEG-chain-end into an aldehyde end group. The Q)
resulting string was immersed into PBS solution of the ESR probe, 2 50
WJ
then subjected to sodium cyanoborohydride (NaBH,CN). The
electron spin resonance (ESR) was measured on an FSR 40
spectrometer using Mn" as the standard signal.
11-3. Characterization of acetal-PEGPLA surface. 30
The dynamic advancing (&J and receding (0,) angles were
obtained by extending and then contracting the volume of the drop
20
at the rate of 0.13 ml/sec. 10
Surface topography and roughness of sample surfaces were
examined by AFM, oprating in tapping mode witha Nanoscope 0
III(Digital Instruments, St. Barbara, CA). An area of 4pmx4pm P L A a b c d e
was scanned. Figure 2. Dynamic water contact angles in air on PLA and
Protein adsorption on the film samples was measured using
PEWLA block copolymers. a:PEGPLA(0.65/11 .O), b:
bovine serum albumin (BSA) at concentration of 0.01 to 4.5
PEGPLA( 1.8/7.0), c: PEG/PLA(3.3/5.4), d: PEG/PLA(5.0/4.6),
mg/ml in phosphate-buffered saline (Dulbecco PBS (-)) solution
e: PEGPLA(8.7/6.9) in Table 1.
(0.15 M, pH7.4). and the adsorbed amount was determined using
the protein analysis kit (micro BCA protein assay reagent kit,
Comparing the dynamic wetting behavior of the PEGPLA surfaces
Pierce, Rockford, IL,USA) based on the bicinchoninic acid OCA)
with different PEG MWs, the most striking finding is the marked
method'.
decrease in receding contact angle on a PEGPLA (3.3/5.0) surface
Primary hepatocytes were obtained by collagenase perfusion
resulting in the maximum hysteresis. Hysteresis in the dynamic
and purified by centrifugation. Cells were seeded and cultured in
contact angle may be caused by the hydration of PEG segments".
DMEM containing 10% fetal bovine serum and appropriate
In the dry state, the PEG chain should assume a conformation flat
chemicals.
to the surface experienced by the advancing contact line. Upon
III. RESULTS AND DISCUSSION hydration, however, the PEG chain should extend from the surface
III- 1. Synthesis of Acetal-PEGIPLA. due to the hydration of PEG chains. As a result, the receding
One of the objectives in this study is to investigate the effect contact line experiences a more hydrophilic surface than the
of the variation in PEG chain length on surface properties. This advancine contact line.
includes assays on protein adsorption and cellular attachment to
get a biochemical insight into the behavior of tethered PEG under 6- -o- V=O.I3(ml/sec)
biological conditions. E
s0.8
Table 1. Molecular weights of PEGPLA block copolymers E
v
PEG PLA U
a 0.6
sample1 Mn
GPC MS
MW
GPC MS
MwM
GPC MS
Mn g
v)

U
a 0.4
*
a 685 650 770 720 1.12 1.10 11470 3
b 1920 1880 2110 1930 1.09 1.03 7020 m
'i5 0.2
c 3570 3340 3750 3470 1.05 1.04 5410 c
c
d 5100 5050 5670 5210 1.11 1.03 4640 a
e 8910 8730 9080 8810 1.02 1.01 6940 E o
a 0 2000 4000 6000 8000 10000
a; PEGPLA(0.65/11S), b; PEGPLA( 1.8n.O), PEG molecular weight
c; PEGPLA(3.3/5.4), d; PEGPLA(5.0/4.6), Figure 3. Reduced hysteresis and the adsorbed amount of BSA on
e; PEGRLA(8.7l6.9). PEGPLA coated substrates as a function of PEG M.W.

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 Proceedings of the 22"dAnnual EMBS International Conference, July 23-28, 2000, Chicago IL.

In Figure 3, the dimensionless form, referred to as reduced

hysteresis H , was plotted as a function of PEG MW to investigate
the surface reorganization. The H value increases with increasing
PEG MW and then shows a maximum at a medium PEG chain
length (PEG/PLA(3.3/5.0)), followed by an appreciable decrease.
This surface rearrangement and interfacial orientation of PEGPLA
at the water interface occurs due to the high surface tension of
water, which provides a strong driving force for polymer strands
to orient so as to minimize the interfacial tension". It is worth
noting that the maximum value of H observed in
PEGPLA(3.3/5.0) is significantly attributed to the minimum
value of 8,. due to the highest hydration power. A OJ . 10.0
conformational reorientation of the hydrophilic segment, PEG, is 0.0 1.0 2.0 3.0 4.0
greatly influenced by the hydration power in water environment, a
and the surface property significantly affect the protein adsorption
on the surface.
111-3. AFM analysis of the PEG/PLA surfaces.
Figure 4(a) illustrates PLA surface in water, which has the
uniform appearance with considerably small roughness. On the
other hand, the representative surfaces of PEGPLA layers(Figs 4.
(b)-(d)) gave the images of densely packed clumps or islands where
size is considerably uniform, and systematically changes with
PEG MW, the topographycal features(both in horizontal and
vertical(Ra) distances) grow larger and more prominent as the PEG
MW is increased.
111-4. Protein adsorption.
Protein adsorption on PEGIPLA surfaces was measured using Ra Inm; 3.49 o.o
0
bovine serum albumin @SA) as a model protein. Figure 3 shows
the BSA adsorption from Dulbecco PBS (-) solution on various b 0.0 1.0 2.0 3.0 4.0
€"LA surfaces. On a PLA surface, BSA was significantly
adsorbed, while on PEG-coated surfaces BSA adsorption clearly
decreased. As the PEG MW was increased, the amount of BSA
adsorbed on the surface significantly decreased up to a PEG MW of
ca. 3300. A further increase in PEG MW resulted in a slight
increase in BSA adsorption. Thus, minimum adsorption was
obtained at a medium PEG chain length, i.e., PEGIPLA(3.36.4).
note that this surface revealed the maximum hysteresis and H value
in dynamic contact angle measurement. Generally, PEG-coated
substrates have shown reduced adsorption of proteins with
increasing Puj M W . However, reduction in protein adsorption
onto the present surfaces may not depend upon PEG MW but rather
upon the high levels of water that bind to the PEG component to Ra Inm; 7.09 0.0
increase PEG-hydration. 0
III-5. Conversion of an acetal group to an aldehyde group. 0.0 1.0 2.0 3.0 4.0
C
A functionalization of the PEG chain end provides the means
for attaching ligand molecules for further chemical modulation of
the surface. After the construction of the PEGIPLA surface, the
acetal groups at the PEG-chain-end were successfully transformed
into aldehyde end groups. An aldehyde group reacts smoothly
with amino groups forming a Schiff base, a chemical path which
can be employed for conjugation of proteins and peptides. To
confirm the presence as well as to examine the reactivity of
aldehyde group on the surface, model reactions with 2, 2, 6, 6-
tetramethyl-l-piperidinyloxy (TEMPO) derivatives as label agents
were performed and ESR specta of TEMF'O derivatized surfaces were
successively recorded (Figure 5)'. When the acetal surface was
treated with Camino-TEMPO, only a slight signal was observed Ra Inm; 4.12 o.o
probably due to the physical adsorption of Camino-TEMPO on 0
the surface (Fig. 5(b)). When the aldehyde surface was treated with d 0.0 1.0 2.0 3.0 4.0
TEMPO having no functional (amino) group, no ESR signal was
observed (Fig. 5(c)). Contrary to these control treatments, three
Figure 4. AFM studies of silicon wafers coated with PLA
typical signals were clearly observed when Camino-TEMPO was
homopolynier and PEGPLA block copolymers in water; a::PLA,
used as the surface modification reagent, indicating that the
b:PEGPLPi(1.8/7.0), c:PEG/PLA(5.0/4.6), d:PEGPLA(8.7/C1.9).
effective covalent-conjugation of 4-amino-TEMPO with the

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 Proceedings of the 22ndAnnual EMBS International Conference, July 23-28,2000, Chicago IL.

8. Nagasaki, Y.; Okada. T.; Scholz, C.; Iijima, M.; Kato, M.; Kataoka K.
Macromolecules 1998.31, 1473.
aldehyde group at the end of PEG on the surface took place (Fig. 9. Smith, P. K. Anal. Biochem. 1985,150.76.
5(a)). These results strongly suggest the high utility of these 10. Good.R. J. In Surface and Colloid Science; Good, R. J.. Stromberg,R. R.,
surfaces in the field of biomaterials; as proteins, sugars, and Eds.; Plenum Press: New York, 1979; Vol. 11.
11. Teraya, T.; Talyhara, A.; Kajiyama. T. Polymer 1990.31, 1149.
peptides can be immobilized at the end of PEG moiety by end- 12. Kuhl. P. R.; Gnfi,th-Cima,L. G. Nat. Med. 1996.2, 1022.
group activating PEG chains, the surface will show to support the
growth of anchorage dependent cells. Much current work in tissue
engineering and other areas of biomedical engineering points t o
the need for materials which can control cell behavior in a specific NaCNBH3 .
manner". Control over the migration, growth, and differentiation
of cells at surfaces might be obtained by combining protein
resistance with tethered ligands. Carbohydrate-based cell
recognition has been applied in tissue engineering. The most
extensively studied example is the use of monosaccharide binding
to the asialoglycoprotein receptor on hepatocytes. Our surfaces
were covalently modified with p-aminophenyl-p-D-
lactopyranoside, and the behavior of hepatocytes on these
surfaces were investigated. Primary rat hepatocytes attached to
and spread on the lactose-PEGPLA surfaces but did not attach to
control surfaces coated with PEGmLA without lactose. The
lactose-immobilized PEGlPLA surface was also characterized by
staining the surface with a fluorescent affinity label specific for
galactose and quantifying the fluorescence. The quantity of
fluorescein-labeled galactose-specific lectin, Ricinis communis
(RCA 120, vector Labs), binding to surfaces was dependent on
PEG molecular weight and therefore lactose surface concentration
andor lactose mobility at the PEG chain end. Polymers without
lactose were also investigated and showed negligible RCA Mn2+ Field: 328.9f5mT fi2+
binding, proving the specificity of the ligand-recepter Figure 5. ESR spectra after the reaction between the PEGPl-4
interaction. surface and TEMPO derivertives; a: aldehyde surface-4-amino-
TEMPO, b acetal surface-4-amino-TEMP0, c: aldehyde surface-
IV.CONCLUSION TEMPO systems.
Heterobifunctional block copolymers, a-acetal-o-hydroxy-
PEGPLA, were synthesized and coated on a polylactide surface,
followed by the conversion of the acetal group into a aldehyde I Department of Materials Science, Graduate School of Engineering,
group. In this way, a PEG-brushed layer with a terminal aldehyde The University of Tokyo,
group was readily prepared which has both non-fouling and ligand- Hongo 7-3-1, Tokyo 113-8656. Japan
otsuka@bmw.mm.t.u-tokyo.ac.jp~o~@bmw.mm.t.u-tokyo.ac.jp
binding properties. Furthermore, aldehyde groups were confirmed
to be present at the tethered PEG-chain end and can be derivertized MDepartment of Materials Science and Technology,
with bioactive proteins and peptides with amino or hydrazide Science University of Tokyo,
Noda. Chiba 278-85 10, Japan
functionality. These results highlight the potential of this system nagasaki@rs.noda.sut.ac.jp
to act as an engineered biomaterial as well as tissue engineering
scaffold because these systems will provide new insights into Institute of Biomedical Engineering,
Tokyo Women's Medical University
controlling cell behavior at surfaces for tissue engineering and tokano@lab.twmu.ac.jp
biomedical applications.

V.A C K " T S
This study was supported by a JSPS, The Japan Society for the
Promotion of Science, "Research for the Future'' Program (JSPS-
RFTF96100201).

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Macromolecules 1997.30,6489.65.

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