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Tuning the enzymatic hydrolysis of biodegradable

polyesters and its application to surface patterning†
Cite this: J. Mater. Chem. A, 2013, 1,
4667
Yoshihiro Kikkawa,*a Masato Fukuda,b Nobuhiro Ichikawa,b Ayumi Kashiwada,b
Received 28th December 2012 Kiyomi Matsuda,b Masatoshi Kanesatoa and Tomohiro Hiraishic
Accepted 28th February 2013

DOI: 10.1039/c3ta01670f

www.rsc.org/MaterialsA

Biodegradable polyester surfaces with controlled enzymatic important and attractive challenges to realize site-selective
degradability were fabricated by simple UV–ozone and UV treat- patterning based on the substrate specicity of the enzymes and
ments. The surface modification was applied for the development of to create so interfaces with various surface morphologies.8 In
an enzymatic lithographic technique to produce a patterned soft order to develop a wide range of applications, such enzymatic
interface with various architecture designs. lithography would require versatility, precision and ability for
large-area patterning. Even in the eld of nanotechnology,
biocompatibility and low environmental impact of the by-
Biodegradable polyesters (BPs) have attracted signicant
products are desired because of the increasing awareness of
attention as environmentally friendly, sustainable and
global environmental issues.
biocompatible materials produced from renewable biomass,
In this communication, we prepare BP surfaces with
and controlling the biodegradation rate in nature is one of the
different enzymatic degradability by the simple surface modi-
fundamental issues for the effective use of such green mate-
cations of UV–ozone treatment/UV irradiation. The controlled
rials.1 For example, BPs for agricultural and marine applications
enzymatic degradation rate of BPs allows the regular surface
should not lose their functions as polymeric materials during
patterning. UV irradiation alone provides the opposite patterns
the usage, but it is better to start the biodegradation just aer
when compared to UV–ozone treatment aer the enzymatic
their disposal. Therefore, methods for both enhancement and
degradation.
retardation of the biodegradation are required from the view-
Generally, the UV–ozone treatment has been exploited for
point of practical applications. Copolymerization, chemical
the cleaning of the metal surfaces due to the strong oxidation of
modication, and blending of BPs have been applied to control
the organic materials.9 However, we found that the UV–ozone
the degradation rate.2 However, these techniques require
treatment retards the enzymatic degradation of the polyesters.
complicated synthesis and blending procedures prior to the
Fig. 1A shows the frequency change of pristine PLLA thin lms
molding and processing of BPs.
measured by QCM. The control experiment using 50 mM Tris–
Patterning of polymeric and organic surfaces has been
HCl buffer without enzyme showed almost no frequency
widely investigated for the fabrication of so interfaces with
change, suggesting that the hydrolytic degradation can be
various properties,3 and self-assembly of block copolymers4 and
negligible within the present time scale.10 In the Tris–HCl buffer
colloidal particles5 has been used in lithographic techniques. In
containing 1 mg mL 1 proteinase K, the frequency gradually
addition, micro-contact printing6 and scanning probe micros-
increased due to the weight loss of the thin lm during enzy-
copy-based lithography7 have also been used as the surface
matic degradation, and then plateaued. The PLLA lm of
patterning methods for so materials. Recently, development of
200 nm thickness was degraded within ca. 20 min, whereas that
enzyme-catalyzed surface patterning has been one of the most
treated by UV–ozone needed ca. 40 min for the complete
degradation. The enzymatic erosion rates were calculated from
a
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba the slope of the time-course of frequency change. The erosion
Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. E-mail: y.kikkkawa@ rates of pristine and UV–ozone treated PLLA were 0.93  0.06 mg
aist.go.jp; Fax: +81-29-861-3029; Tel: +81-29-861-2955 cm 2 min 1 and 0.49  0.04 mg cm 2 min 1 respectively, sug-
b
Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon
gesting that UV–ozone treatment retards the enzymatic degra-
University, 1-2-1 Izumi-cho, Narashino, Chiba 275-8575, Japan
c
dation of PLLA. The retardation of enzymatic degradation could
Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
† Electronic supplementary information (ESI) available: Experimental details and
also be conrmed by the erosion depth measurement from the
additional data. See DOI: 10.1039/c3ta01670f time-dependent AFM observations during the enzymatic

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UV–ozone treated PLLA was more hydrophilic than the pristine

one (see Fig. S1 in the ESI†). These results suggest that the
adsorption of proteinase K is disturbed possibly due to the
hydrophilic surface formed by the UV–ozone treatment, result-
ing in the retardation of the enzymatic hydrolysis rate.
As shown in Fig. 1, UV–ozone treatment provided the
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differences in the enzymatic degradation rate. Therefore, we

tried to apply the combinations of UV–ozone treatment and the
following enzymatic degradation for the surface patterning of
PLLA (Fig. 2A). By using the TEM grid as a mask, micrometer
sized patterning could be accomplished by enzymatic lithog-
raphy (Fig. 2B and C). Since the UV–ozone treatment gives the
less adsorbable PLLA surface by proteinase K, the region
masked by the TEM grid was preferentially eroded, resulting in
the formation of square shaped matrices. The patterns were
dependent on the mask shape and size, and therefore creation
of various morphologies is possible by the UV–ozone treatment
(see Fig. S2 in the ESI†).
Thus, erosion rate control supplied the biodegradable and
biocompatible PLLA surface with various regular patterns. To
Fig. 1 Time course of frequency change (DF) observed during enzymatic
fabricate the opposite patterns compared to the UV–ozone
degradation by proteinase K at 25  C (A), depth profile plotted as a function of treated materials, UV irradiation without ozone was examined
degradation time (B) for the pristine (blue) and UV–ozone treated PLLA thin films because reduction of the molecular weight for the polymeric
(red). AFM images of pristine (C) and UV–ozone treated PLLA thin films (D) after materials can be caused by the UV irradiation, resulting in the
the enzymatic degradation by proteinase K (50 mg mL 1) for 7 min. The average
enhancement of the enzymatic degradation.11 Fig. 2D and E
number of the adsorbed enzyme molecules per 100  100 nm2 on the thin film
was counted, and plotted as a function of the concentration of proteinase K (E).
respectively show the AFM images of PLLA thin lms irradiated
The blue and red curves indicate the pristine10 and UV–ozone treated PLLA thin by UV light masked with TEM grid and followed by enzymatic
films, respectively. degradation by proteinase K. In Fig. 2D and E, PLLA under the
masked region remained on the Si substrate, whereas the other
open region was completely eroded. Thus, it is demonstrated
degradation by proteinase K (Fig. 1B). 30 mL of proteinase K
solution was dropped on the PLLA surface, and the erosion
depth was measured by AFM. The erosion rate in the vertical
direction for pristine PLLA was ca. 10.0 nm min 1, whereas that
for the UV–ozone treated one was ca. 5.2 nm min 1. These QCM
and AFM results indicate that the UV–ozone treatment retards
the enzymatic degradation by proteinase K, and that the erosion
rate can be almost half of that of pristine PLLA. Note that such
retardation of the erosion rate could be maintained for at least
10 days aer the UV–ozone treatment.
Since the adsorption of proteinase K is the initial step for the
enzymatic hydrolysis reaction, the enzymes on PLLA were
directly visualized by AFM to compare the apparent number of
enzymes participating in the hydrolysis reaction.10 The PLLA
thin lms aer the enzymatic degradation for 7 min were
observed by AFM. The thin lms were gently washed with Milli
Q water and dried. On the pristine (Fig. 1B) and UV–ozone
treated PLLA thin lms (Fig. 1C), enzyme molecules were
recognized as dark “dots” in the AFM phase images. More
enzymes were found on the surface of the pristine PLLA thin
lm (Fig. 1B) than on the UV–ozone treated one (Fig. 1C). Such
observation was repeated aer the enzymatic degradation by
proteinase K with different concentrations. Then, the enzymes
Fig. 2 (A) Schematic illustration of the UV–ozone treatment and enzymatic
per 100  100 nm2 on both PLLA thin lms were counted and
patterning by proteinase K. Optical micrographs, AFM height images and cross-
the data are plotted in Fig. 1D. The number of proteinase K on sectional data of patterned surface for UV–ozone (B and C) and UV treated PLLA
UV–ozone treated PLLA was almost half of that on bare PLLA.10 thin films (D and E) after the enzymatic degradation by proteinase K. Insets in
In addition, contact angle measurement revealed that panels (B) and (D) show cartoons of the surface pattern.

4668 | J. Mater. Chem. A, 2013, 1, 4667–4670 This journal is ª The Royal Society of Chemistry 2013

Communication
that opposite patterns were created in comparison to the
UV–ozone treated PLLA samples.
Towards the polyester surface with nanometer-sized arrays,
colloidal lithography5 was combined with the UV–ozone treat-
ment (Fig. 3). The hexagonally packed colloidal monolayer
composed of PS particles with 500 nm diameter was placed on
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the PLLA surface, and UV–ozone treatment was carried out.
Aer the removal of colloidal particles by sonication, enzymatic
degradation was performed using proteinase K. As a result,
small disks of 30 nm height and 100 nm diameter were regularly
aligned in 500 nm periodicities over 20  20 mm2, and displayed
hexagonal patterns, which were traced from the self-assembled
PS particle alignments. Accordingly, the nanometer-sized arrays
could be achieved by the combinations of colloidal mask,
UV–ozone treatment, and enzymatic degradation.
The versatility of the enzymatic degradation combined with
UV–ozone treatment was secured for the other biodegradable
aliphatic polyesters of poly(3-caprolactone) (PCL) and poly(3-
hydroxybutyrate) (PHB). Similar to the PLLA case, the erosion
rate of PCL by lipase and that of PHB by PHB depolymerase
could also be retarded as revealed by QCM results (see Fig. S3 in
the ESI†), namely, the erosion rate of PCL and PHB aer the UV–
ozone treatment was almost half of those of the pristine mate-
rials. Especially for PCL, regular patterns based on the mask
were demonstrated aer the enzymatic degradation by lipase,
and the concave and convex were dependent on the method of
surface modication (see Fig. S4 in the ESI†). Thus, we could
fabricate the negative and positive patterns of biodegradable
polyesters without special photo-sensitized reagents, regardless
of the surface modication by either UV–ozone treatment or UV
irradiation. The detailed characterizations aer the UV–ozone
treatment and creation of smaller patterns are now in progress.
In conclusion, control over the enzymatic degradation rate
was successfully accomplished by the surface modication
through UV–ozone treatment/UV irradiation, which could be
applied to the representative BPs such as PLLA, PCL, and PHB.
QCM and AFM analyses revealed that the UV–ozone and UV
Fig. 3 AFM images of the colloidal mask on a glass support (A and B), and disk
structures of PLLA formed after the UV–ozone treatment and following enzymatic
degradation by proteinase K (C and D). The graphs at the right of panels (B) and
(D) are the cross-sectional data at the white lined region in each image.
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treatments respectively retard and enhance the enzymatic
degradation. The facile UV–ozone/UV treatments combined
with enzymatic degradation were available as the enzymatic
lithography of BP surfaces. Thus, so interfaces composed of
BPs with nanometer- to micrometer-sized architectures could
be fabricated by the environmentally friendly processing
method.
Acknowledgements
This work has been partly supported by JSPS (23750137) and
MEXT KAKENHI (23106722), Japan.
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