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ARTICLE
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Cross-linking strategy provides a new generation of biodegradable

Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
and biocompatible cyanoacrylate medical adhesives
Liang Xua, Tao Zhanga, Huajin Donga, Dazheng Caiab, Han Hana, Qingbin Menga, Yongjia Tanga,
Qingguo Mengb, Zehui Gonga, Tianhong Zhanga, Zhenqing Zhanga, Husheng Yanc*, Keliang Liua*
Addition polymerization usually results in polymers with long carbon-carbon main chains. Cyanoacrylate (CA) is arguably
an important example of such polymerization and has obtained widespread acceptance as an all-purpose adhesive.
However, CA-based medical adhesives have never been approved by the U.S. Federal Drug Administration for use below
the skin, mainly due to the low biodegradability and biocompatibility of their solid glue after polymerization. In this
research, a cross-linking strategy involving the combination of alkyl-CA and the cross-linking agent poly(ethylene glycol)-
di(cyanoacrylate) (CA-PEG-CA) to form a copolymeric network was used to synthesize a new generation of biodegradable
CA medical adhesives. The degradability could be modulated by adjusting the ratio of CA-PEG-CA to alkyl-CA and the
length of PEG. An optimal composite adhesive, LKJ11, was shown to have excellent biodegradability, adhesive capability,
and biocompatibility. Importantly, the molecular weight of the polycyanoacrylate chains in the polymerized LKJ11 was
greatly reduced than those polymerized from pure butyl-CA. Thus, the degradation product could be readily extracted. The
results showed that LKJ11 represents a new generation of CA-based biodegradable medical adhesives to date. This
advance also provides a general strategy to facilitate the conversion of other polymers with long carbon-carbon main
chains to a biodegradable form, thereby expanding the novel applications available for traditional polymeric materials.

biodegradable. In addition, the polymers always have huge

Introduction
Addition polymerization usually results in polymers with long
carbon-carbon main chains, which are not very biodegradable
under most normal conditions. Cyanoacrylate (CA) is an important
example of such polymerization and has obtained widespread
acceptance as an all-purpose adhesive since it was first synthesized
[1-7]
in 1949. The liquid monomers can undergo an almost
instantaneous polymerization when applied to amine or water-
containing substances such as skin and tissues, resulting in a stable
material with water-resistant adhesive strength; these properties
thus facilitate medical applications such as closing surgical incisions
[8,9]
or trauma lacerations. However, commercialized CA medical
adhesives (mainly butyl-CA, octyl-CA, and 2-octyl-CA) have never
been approved by the U.S. Federal Drug Administration for use
below the skin, mainly due to the low biodegradability of their solid
[10,11]
glue after polymerization. Polycyanoacrylates (PCAs) usually
contain long carbon-carbon main chains and are almost non-
molecular weights (hundreds of thousands of Daltons), far beyond
[12]
the extent that the human kidney can filter. A long-time
persistence in vivo may cause inflammation, tissue necrosis, or even
[13,14]
potential carcinogenicity. Furthermore, the persistence will
cause continuous generation and accumulation of formaldehyde in
treated sites by a reverse-Knoevenagel reaction pathway (although
[15]
with low efficiency), which is also responsible for the
[16,17]
cytotoxicity. Chemically modifying the alkyl chain with moieties
[18,19]
including etheric oxygen or ester groups and physically
blending with other components such as a plasticizer have been
[20]
reported to improve the elasticity and toxicity. However, these
modifications do not affect the chemical characteristics of CA-based
polymerization, and most of them do not shorten the long carbon-
carbon main chains in PCA. Therefore, the drawback of poor
biodegradability of CA-based medical adhesives remains to be
resolved.
In this research, a cross-linking strategy was used to build up a
network structure for developing a new generation of CA-based
biodegradable medical adhesives (Figure 1). For this purpose,
poly(ethylene glycol)-di(cyanoacrylate) (CA-PEG-CA) was designed
as the cross-linking agent in the CA-based copolymer. The two
hydrolytically labile ester bonds and the flexible and water-soluble
PEG segment in the agent provide the potential for the polymer to
be biodegradable and biocompatible when it is copolymerized with
the alkyl-CA monomer. Specifically, during the copolymerization,
the steric hindrance of the PEG segments in the cross-linking agents
would hinder the diffusion of monomers and lower the efficiency of

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H, I, and J: 100.0%, 61.2%, 61.6%, 36.1%, and 14.1, respectively)

than that of the control (Group K, 4.3%). It was also observed that

Table 1.Degradability of composite adhesives

Journal of Materials Chemistry B Accepted Manuscript

a)
Group Composite ratio Apparent degradation rate(%)

A 1:4 BCA:CA-TEG-CAb) 4.0±0.4

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B 1:1 BCA:CA-TEG-CA 1.4±0.4

C 4:1 BCA:CA-TEG-CA 1.4±1.2

Figure 1.Graphical scheme of a biodegradable CA-based copolymer.
D 1:4 BCA:CA-PEG600-CA 100.0±0.0

linear polymerization; as a result, the PCA chains would become
much shorter than those polymerized from pure alkyl-CA. Since
these short PCA chains are cross-linked by PEG, the PCA-PEG
network structure would still possess a huge molecular weight,
which is important for a strong adhesive capability. First, the cross-
linked copolymer would be degraded in the interface into small
pieces at the ester sites between the PEG and PCA chains. Because
the PEG segments are water-soluble and would interrupt the tight
alignment of pure PCAs, the copolymer would be partially swollen
near the interface so that molecules around the copolymer would
exchange in and out in vivo. As a result, the copolymer degradation
would be further promoted into deep sites of the material. The
degradation products, PEG and short PCA segments, both have
[21-23]
been approved for safety in the clinic and would be readily
excreted via the renal filtration process once their molecular
weights were small enough. Moreover, this copolymer would elicit
improved biocompatibility with tissues over pure PCA because the
PEG segments inside of the network would change the
physicochemical and metabolic properties. For the reasons stated
above, it was reasonable to hypothesize that this cross-linked
copolymer would be biodegradable and biocompatible so that
employing CA-based materials in vivo might become feasible.
Results and discussion
Three monomers, CA-tetra(ethylene glycol)-CA (CA-TEG-CA), CA-
PEG600-CA, and CA-PEG2000-CA, were designed and synthesized
with different PEG lengths (supporting information). Butyl-CA (BCA)
and CA-PEG-CA combined in appropriate weight ratios (Table 1,
except group G) could mix uniformly and retain the transparent
liquid state at room temperature. All these composite adhesives
could polymerize to solid glue within 5–15 s once they made
contact with skin or tissue.
Apparent degradation rates in vitro
A degradation test was carried out in vitro to evaluate the
degradability of each polymer. As shown in Table 1, different
apparent degradation rates were observed for various composite
ratios of BCA to CA-PEG-CA. The composite adhesives containing
CA-TEG-CA all showed low apparent degradation rates (Groups A, B,
and C: 4.0%, 1.4%, and 1.4%, respectively). While the composite
adhesives containing longer PEG chains (CA-PEG600-CA and CA-
PEG2000-CA) showed much higher degradation rates (Groups D, E,
E 1:1 BCA:CA-PEG600-CA 61.2±1.6
4:1 BCA:CA-PEG600-CA 1.8±1.7
F
G 1:4 BCA:CA-PEG2000-CA --C)
H 1:1 BCA:CA-PEG2000-CA 61.6±1.9
I 2:1 BCA:CA-PEG2000-CA 36.1±10.1
J 4:1 BCA:CA-PEG2000-CA 14.1±3.8
100% BCA(control) 4.3±1.0
K
a)
Apparent degradation rate (100%) = (W0 – W1) / W0. W0 is the weight of the
polymerized composite adhesive before degradation, and W1 is the weight of the
residual glue after degradation. Data were collected after degradation in PBS
buffer (pH 7.0) for 14 days and are given as mean ± standard deviation; b)BCA,
butyl-cyanoacrylate; CA, cyanoacrylate; TEG, tetra(ethylene glycol); PEG,
poly(ethylene glycol); C)
the two components could not mix uniformly and retain
transparent liquid state at room temperature.
as the weight ratio of CA-PEG600-CA and CA-PEG2000-CA
increased, the apparent degradation rate increased as well. For
example, the apparent degradation rate of Group H (61.6%; 1:1 BCA
to CA-PEG2000-CA) was higher than that of Group I and J (36.1%
and 14.1%, 2:1 and 4:1 of BCA to CA-PEG2000-CA, respectively).
A copolymer with a higher CA-PEG-CA content means that it
contains more ester groups or longer PEG chains. The former would
have more hydrolytically labile sites, while the latter would result in
a looser and more hydrophilic network; therefore, both would
favour network degradation. These results indicated that the
degradability could be modulated both by adjusting the ratio of CA-
PEG-CA to BCA and the length of the PEG chain. In addition, the
adhesive capabilities were also found to be modulated by the
composite ratios (Table S1, supporting information).
Group H (named as LKJ11 below) was selected to clarify our
proposed strategy due to its excellent general properties as a
medical adhesive, including high monomer purity, proper
biodegradability, and adhesive capability.
Biodegradability of LKJ11
Molecular weight of PCA chains in the copolymer
To determine in vitro degradation, polymerized LKJ11 (P-LKJ11)
was detected by gel permeation chromatography (GPC) analysis. P-
LKJ11 could swell but not dissolve in tetrahydrofuran (THF) before

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degradation due to its cross-linked structure; as a result, no obvious
peak was observed in the GPC chromatogram (Figure 2, line 1).
After incubation in phosphate-buffered saline (PBS) for 7 days, the
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Figure 2. GPC chromatogram results of the degradation test in vitro. Lines1,
2, and 3 designate P-LKJ11 before incubation in PBS, after incubation in PBS
for 7 days, and after incubation in PBS for 14 days, respectively; lines 4, 5,
and 6 designate PBCA before incubation in PBS, after incubation in PBS for 7
days, and after incubation in PBS for 14 days, respectively.
Figure 3. Percentages of residual P-LKJ11 and PBCA in the long-term
degradation test.
residual solid glue could dissolve in THF; thus, polymers with a
molecular weight less than 4 kDa were detected in the GPC
chromatogram (Figure 2, line 2). 1H-NMR analysis of the residue
further demonstrated the typical spectra of polybutylcyanoacrylate
(PBCA) and PEG (Figure S1, supporting information). Moreover,
polymers with a molecular weight of 2.3 kDa were detected in the
PBS buffer, which was revealed to be mainly PEG by 1H-NMR (Figure
S2, supporting information). This result indicated that the cross-
linked structure degraded into linear or branched segments. In
addition, the molecular weight of the residual glue was further
reduced when the incubation time was extended to 14 days (Figure
2, line 3). Subsequently, the molecular weights of these degradation
products were determined to be much less than the limit of the
human renal system (approximately 50–70 kDa). Therefore, they
would have the potential to be excreted in vivo. In contrast, the
control PBCA had a molecular weight of 367 kDa before
degradation, as shown in the chromatogram (Figure 2, line 4),
indicating its huge linear structure after polymerization. After
incubation for 7 days or 14 days in PBS, the molecular weight of the
residual polymers was still huge, 288 kDa and 242 kDa (lines 5 and
6), respectively. The decrease in molecular weight can be mainly
explained by hydrolysis of the butyl ester groups because the long
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carbon-carbon main chains should not be degraded under these
conditions.
The residues of adhesives with good apparent degradation rates
Journal of Materials Chemistry B Accepted Manuscript
(Table 1, Groups E and I) were also tested by GPC. Similarly, the
molecular weights were found to be lower than that of PBCA
Figure 4. Photographs of muscle injected with LKJ11 or BCA at 1 day, 14
days, and 3 months after injection; the arrows and the dashed lines indicate
the polymers.
Figure 5. Pathological analysis of muscle applied with LKJ11 or BCA. Pictures
were taken at 1 month, 3 months, 6 months, and 15 months after injection.
(Figure S3, supporting information). Together, these GPC results
indicated that copolymerization of alkyl-CA with a proper cross-
linking agent did result in a low degree of linear polymerization. In
other words, the PCA chains in the copolymer were much shorter
than those polymerized from pure alkyl-CA.
Long-term in vitro degradation test
The long-term in vitro degradation test was carried out in PBS
containing 50% bovine serum. As shown in Figure 3. The
percentages of P-LKJ11 residue at 7, 14, and 21 days were
36.7±3.2%, 3.3±2.5%, and 1.2±0.4%, respectively. After incubation
for 28 days, the solid glue was completely dissolved in PBS solution,
indicating that P-LKJ11 could be completely degraded in vitro. In
contrast, the percentages of PBCA residue at 7, 14, 21, and 28 days
were 91.5±2.4%, 83.7±3.5%, 76.4±4.6%, and 74.0±5.7%,
respectively. In addition, the higher degradation rate of either P-
LKJ11 or PBCA in serum-containing PBS than in pure PBS indicated
that enzymatic hydrolysis might take place (the percentages of P-
LKJ11 residue in pure PBS at 7, 14, 21, and 28 days were 51.5±0.5%,
38.4±1.9%, 30.1±3.4%, and 24.3±4.1%, respectively; the PBCA group
were 96.8±0.5%, 95.7±1.0%, 94.1±2.1%, and 91.7±2.4%,
respectively).
Degradation tests in rabbit models and in rat models
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An intramuscular degradation test was carried out in a rabbit
model. LKJ11 or BCA was injected into the legs of rabbits, and the
injected muscle was cut open at 1 day, 14 days, and 3 months after
injection to observe the in vivo degradability of P-LKJ11 and PBCA.
As shown in Figure 4, both LKJ11 and BCA polymerized quickly into
solid glue at the injection sites and looked the same after 1 day. P-
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LKJ11 was enlarged at 14 days, and the solid glue finally
Figure 6. Rate of radioactivity in urine, feces, stomach residue, and blood.
disappeared at 3 months. In contrast, PBCA still remained at the
injection site after 3 months, and no significant changes were
observed under the same conditions.
A degradation test in a rat model was also carried out, and
sections were pathologically analyzed. As shown in Figure 5, for the
LKJ11 group, only a small amount of gel remained visible after 3
months. The muscles on both sides of the original gel film almost
completely reconnected and regenerated, with only a few
inflammatory cells. As time progressed, the inflammatory cells
gradually disappeared and the muscle was able to recover
completely. In contrast, the solid glue of PBCA was still detectable
even after 15 months. As a result, the muscle tissue in the control
BCA group did not recover, and inflammatory cell accumulation and
granulation tissue growth were detected.
Excretion test of radioactively labeled LKJ11
A radioactive LKJ11 biodegradation test was carried out to
further demonstrate that the partially or completely hydrolyzed
PCA could be excreted from the rat body. One month after applying
the radioactive LKJ11 onto the stomach, 70.1% of the radioactivity
was detected in the urine and feces collected daily (62.1% and 8%,
respectively), with only 0.06% in the blood and 13.7% in the
stomach residue (Figure 6). In contrast, in the control BCA group,
only 20.2% of radioactive PBCA was excreted, and 70.3% remained
on the stomach (Figure S4, supporting information). As
hypothesized above, the degradation products would be readily
excreted once the molecular weights were small enough. This result
was in line with the GPC analysis as the small molecular weight of
the degradation products facilitated the excretion. As most of the
radioactivity was detected in the urine and little was noticed in the
blood, it might also be concluded that the degradation products
were mainly excreted by the glomerular filtration process.
Scanning electron microscopy test and water-vapor permeability
test
In addition, a scanning electron microscopy test (Figure S5,
supporting information) showed that the P-LKJ11 membrane
presented a rough and loose morphology, compared to the smooth
and compact morphology for PBCA. This physical characteristic
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increased the specific surface area and might facilitate the
approach of solvent and enzyme molecules during degradation of P-
LKJ11. A water-vapor permeability test (Table S2, supporting
information) did demonstrate that P-LKJ11 possessed higher water
Journal of Materials Chemistry B Accepted Manuscript
permeability than PBCA. These results provided further explanation
to the better biodegradability of P-LKJ11compared to PBCA.
Table 2. The adhesive capability of LKJ11 and BCAa
Group shear tension T-peel Tension Wound
strength (kPa) strength strength closure
(N/cm) (kPa) strength(N)
LKJ11 190.8±31.4 1.21±0.36 418.1±39.3 9.75±3.2
BCA 310.4±26.2 1.36±0.25 511.5±67.8 13.1±5.9
a
For each sample, six parallel experiments were performed to derive the
means ± standard deviations (SD).
Together, these in vitro and in vivo results revealed that our
cross-linking strategy did endow LKJ11 with biodegradability. The
copolymeric material could be degraded into small fragments, then
absorbed, and finally excreted. Key factors that enabled this
copolymeric material to be degraded included both the small
molecular weight of the PCAs and the hydrolytic ester bonds in the
copolymer network. However, it should be noted that difunctional
bis(cyanoacrylate) monomers have been used previously to
increase the solvent resistance and mechanical strength of CA-
based materials.[26-28] These results can be mainly attributed to the
different structures of the cross-linking agents used. In addition,
different ratios of cross-linking agents might also provide discrepant
biodegradabilities.
Adhesive capability of LKJ11
The adhesive capability of LKJ11 was tested both in vivo and in vitro.
In vitro adhesive intensity test
The adhesive intensity of LKJ11 was tested according to national
standards of the medicine industry (YY/T 0729-2009). The results of
shear tension strength, T-peel strength, tension strength, and
wound closure strength are given in Table 2. The bond strength of
LKJ11 was a little lower than that of BCA. However, it should be
pointed out that when used as tissue sealant or for small incision
adhesion, an effective adhesive capability should be interpreted as
stronger than the essential physiological requirement; as a result,
LKJ11 was further tested in vivo. As shown in the animal models
below, LKJ11 did provide enough adhesive capability when
used as a tissue sealant or for small incision adhesion.
Burst pressure of the rat intestinal wound test
Because the adhesive effectivity might be affected by the
degradation of LKJ11 in vivo, a burst pressure test in a rat intestinal
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wound model was carried out to assay the mechanical support Acute toxicity test
whether being maintained until the wound is cicatrized. Quickly
after application, the polymeric film of P-LKJ11 could firmly No acute toxicity was observed in a rat model. As shown in Figure
agglutinate the tissue wound. As shown in Figure 7, the burst 8, the body weight of the rats in the LKJ11 group increased as time

Journal of Materials Chemistry B Accepted Manuscript

pressure of the LKJ11 group at 0 h was 118.6±37.4 mmHg. As the
physiological intestinal peristaltic pressure of rats in vivo is 15
mmHg, this result indicated that P-LKJ11 allowed the intestinal
wound to sustain the pressure to which it would be exposed. The
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progressed, similar to those in the control group fed a normal diet.
After 14 days, the rats were killed for pathological examination, and
no abnormalities were observed.

Figure7. The maximum tolerated pressure in the rat intestinal wound test.

burst pressure remained at a high level (more than 130mmHg)

during the wound recovery period (7-10 days), meaning that P-
LKJ11 maintained its efficacy throughout this timeframe. These
results also indicated that the degradation of P-LKJ11 during the
Figure8. Body weights of rats in the acute toxicity test.
wound recovery period did not influence the mechanical support
and that the degradation rate met the clinical requirements of
wound cicatrization. In the control group, animals injured but
without further treatment showed no burst pressure after
operation (0 h), and the burst pressure only increased gradually as
time progressed. Furthermore, intestinal fluid leakage was
observed before the wound fully recovered.

Guinea pig skin wound healing test

The effectiveness of LKJ11 in maintaining adhesion was also
demonstrated in a guinea pig surface wound model. As shown in
Figure S6 (supporting information), both LKJ11 and BCA
polymerized within 15s after application, generating a solid
polymeric film that adhered the incision together. Daily movement
of the guinea pigs did not cause wound dehiscence or secondary
hemorrhage. The wounds treated with LKJ11 or BCA healed well
within 5–7 days, and the solid films gradually sloughed off
spontaneously in approximately 5–10 days. Wounds without
treatment recovered slowly, and dehiscence was still detected after
7 days.
In addition, it was observed that the gel film of P-LKJ11 had
better flexibility and plasticity than that of PBCA in this test. The
polymeric film of P-LKJ11 felt soft when touched by hand. By
comparison, obvious shrinking (a hard lump) of PBCA was observed
on the skin surface, which even remained after 7 days. Moreover,
as time progressed, partial loss of the gel films was more frequent
in the PBCA group due to its frangibility and hardness. This result
might be due to the PEG2000 fragments introduced by our cross-
linking strategy. This advantage is especially beneficial for use on
soft tissue and will likely reduce the surface tension on wound sites.
Safety of LKJ11
Figure9. Skin histopathology. A, normal rabbit skin; B, rabbit skin treated
with LKJ11.
Skin irritation test
No symptoms of erythema or edema in the treated area were
observed after the skin irritation test in rabbits. The tissue section
results showed that the epidermal structure of the skin of the LKJ11
group was intact, and an obvious cutinized layer was visible. Cells of
epidermal appendages, such as dermal follicles and sebaceous
glands, were arranged in an orderly manner; and vascular tissue,
collagen fibers, and other structures did not present proliferation,
which was in line with the normal tissue results (Figure9).
Intracutaneous irritation test
No symptoms of erythema or edema in the test area were
observed after the intracutaneous irritation test in rabbits. The
results did not depend on whether sodium chloride solution or
vegetable oil was used for the extraction, indicating that no
intracutaneous irritation occurred with the application of LKJ11.

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Cytotoxicity
The Hs27 cells that came into contact with medium exposed to P-
LKJ11 were observed to be normal, with no obvious cell death
under microscopic examination (FigureS7, supporting information).
The cell counting kit-8 assay further indicated that the cell growth
inhibition of LKJ11 was 16.42±9.26% (for PBCA, and positive control,
this data was 22.57±16.76%, and 93.01±2.22% respectively). This
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result indicated that LKJ11 was cytocompatible under the evaluated
conditions.
Conclusions
In conclusion, a cross-linking strategy was utilized to develop
biodegradable CA-based medical adhesives. The biodegradation
rate of the copolymers could be modulated by changing the length
of the PEG chain and the ratio of CA-PEG-CA to BCA. An optimal
composite adhesive, LKJ11, was shown to have excellent
biodegradability, adhesive capability, and biocompatibility.
Importantly, the molecular weight of the PCA main chains in the
copolymer of P-LKJ11 was greatly reduced than those polymerized
from pure butyl-CA. Thus, the degradation product could be readily
extracted in vitro and excreted in vivo. LKJ11 represents a new
generation of CA-based biodegradable materials to date, and it has
the potential to be used in vivo as a sealant or tissue adhesive. For
example, its application may reduce the risk of anastomotic leakage
in most general surgeries. To the best of our knowledge, this is the
first detailed report of safely employing a nonbiodegradable
carbon-carbon polymeric material, in this case PCA, in vivo.
Moreover, this advance provides a general strategy to facilitate the
conversion of other polymers with long carbon-carbon main chains
to a biodegradable form, thereby expanding the novel applications
available for traditional macromolecule materials that are
polymerized from addition polymerization.
Materials and methods
Chemicals
PEG was obtained from Sigma. Benzene and dimethylbenzene
were used after refluxing with sodium. Other commercially
available reagents were used without further purification. Silica gel
TLC was carried out on HS GF254 plates (Yantai Institute of
Chemical Industry, China) and visualized under a UV light. Silica gel
(200–300 mesh, Qingdaohaiyang Chemical. Co., China) was used for
1
flash column chromatography. The H-NMR spectra were recorded
on a JNM-ECA-400 spectrometer (JEOL, Japan). Chemical shifts are
reported in ppm downfield from an internal TMS standard.
Synthesis of BCA
Butyl-CA (BCA) was synthesized according to a conventional
[24]
method and was purified by decompression distillation.
Synthesis of CA-PEG-CA
CA-TEG-BCA (5a), CA-PEG600-CA (5b), and CA-PEG2000-CA (5c)
[25]
were synthesized according to Scheme 1.
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Anthracene/ethyl α-cyanoacrylate adduct (A/ECA); ethyl 9,10-
dihydro-9,10-endoethanoanthracene-11-cyano-11-carboxylate (2)
To a 250-mL three-neck flask fitted with a magnetic stirrer and
condenser that was protected with the drying agent SO2 was added
Journal of Materials Chemistry B Accepted Manuscript
150 mL of anhydrous toluene, 60 g (0.48 mol) of ethyl 2-
cyanoacrylate (ECA), and 87 g(0.48 mol) of anthracene. The
suspension was refluxed with stirring for 48 h, and the solvent was
evaporated. After recrystallization of the residue twice with 200 mL
of 95% ethanol, 130 g of the crude A/ECA adduct (2) was obtained
in 88% yield and was used directly without further purification in
1
the next step.m.p.120–122 °C; H-NMR (400 MHz, CDCl3, 25 °C,
TMS): δ 7.48 ppm (m, 1H; Ar-H), 7.30 ppm (m, 2H; Ar-H), 7.21 ppm
(m, 5 H; Ar-H), 4.87ppm (s, 1H;Ar-CH-C-CN), 4.43ppm (d, 3J(H,H)
Scheme 1. The synthesis of CA-PEG-CA. Conditions: (i) anthracene, toluene,
reflux, 48h; (ii) KOH; (iii)HCl; (iv)EDCI, DMAP; (v) maleic anhydride, xylene,
reflux, 6h.
=2.4Hz,1H;Ar-CH-CH2), 4.15ppm (m, 2H;OCH2CH3), 2.80ppm (dd,
2 2
J(H,H)=-12.8Hz, 1H;CH2CCN), 2.20ppm (dd, J(H,H)=-
3
12.8Hz,1H;CH2CCN), 1.27ppm(t, J(H,H)=14.0Hz,3H;CH3); MS (50
+
eV):m/z(%):321.4 [M+NH4 ].
Anthracene/α-cyanoacrylic acid adduct (A/CAA); 9,10-dihydro-9,10-
endoethanoanthracene-11-cyano-11-carboxylaic acid (3)
To a 1-L flask fitted with a stirrer and condenser was added 130 g
of the crude A/ECA product (2) and 400 mL of 95% ethanol. The
suspension was refluxed, 200 mL of KOH solution (3.6 M) was
added slowly, and the solution was heated at reflux for 3 h. The
solution was quenched into 1500 mL of water and filtered, and the
filtrate was washed with 600 mL of dichloromethane (DCM) to
remove free anthracene. Next, the aqueous phase was acidified
with 6N HCl to pH 2 to precipitate the adduct. The solids were
filtered, washed with water, and dried to afford 104 g of the A/CAA
1
adduct (3) in 78.8% yield. m.p.199–201 °C; H-NMR (400 MHz,
CDCl3, 25 °C, TMS): δ 7.48 ppm (m, 1H; Ar-H), 7.30 ppm (m, 2H; Ar-
H), 7.21 ppm (m, 4 H; Ar-H), 7.11 ppm (m,1H; Ar-H), 4.87 ppm (s,
3
1H;Ar-CH-C-CN), 4.43 ppm (d, J(H,H) =2.4Hz,1H; Ar-CH-CH2), 2.80
2
ppm (dd, J(H,H)=-12.8Hz, 1H;CH2CCN), 2.20 ppm (dd, J(H,H)=-
2
12.8Hz,1H;CH2CCN); MS (50 eV): m/z(%):274.3 [M+H ].
+
Bis-anthracene/tetraethyene glycol bis(α-cyanoacrylate) adduct
(bis-A/tetraethyene glycol BCA); tetraethyene glycol bis(9,10-
dihydro-9,10-endoethanoanthracene-11-cyano-11-carboxylate) (4a)
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To 600 mL of DCM in a 1000-mLround-bottom flask was added
10.5 g (0.0382 mol) of A/CAA (3), 2.96 g (0.0152 mol) of
tetraethyene glycol, 7.37 g (0.0384 mol) of EDC, and 0.49 g (0.004
mol) of DMAP. The solution was stirred at room temperature for 7 h
Compound (5b) was obtained similar to compound 5a (74.2%
1
yield). H-NMR (400 MHz, CDCl3, 25 °C, TMS): δ 7.08 ppm
(s,2H,H2C=C), 6.66 ppm (s,2H,H2C=C), 4.43 ppm (m,4H,COOCH2CH2),
3.68 ppm (m, 54H; OCH2CH2).

Journal of Materials Chemistry B Accepted Manuscript

and washed with 200 mL of saturated NaHCO3 solution followed by
Polyethylene glycol 2000 bis(α-cyanoacrylate) (5c, CA-PEG2000-CA)
200 mL of saturated NaCl solution. The organic phase was dried
with anhydrous sodium sulfate overnight. The organic phase was Compound (5c) was obtained similar to compound 5a (89.0%
1
concentrated for flash column chromatography (3:1 petroleum yield). H-NMR (400 MHz, CDCl3, 25 °C, TMS): δ 7.09 (s,2H,H2C=C),
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3
ether/ethyl acetate as the eluent). The product was obtained as a 6.66 ppm (s,2H,H2C=C), 4.43 ppm (t, J(H,H)=8.3Hz,4H; COOCH2),
1
white solid (8.02g, 74.3% yield). m.p.51–54 °C; H-NMR (400 MHz, 3.68 ppm (m, 232H; OCH2CH2).
CDCl3, 25 °C, TMS): δ 7.48 (m, 2H; Ar-H), 7.1–7.4 ppm (m, 14H; Ar-
In vitro apparent degradation rate test
H), 4.92 ppm (s, 2H, Ar-CH-C-CN), 4.42 ppm (s, 2H, Ar-CH-CH2), 4.87
3
ppm (s, 1H;Ar-CH-C-CN), 4.43 ppm (d, J(H,H) =2.4Hz, 1H;Ar-CH- A total of 100 mg of composite adhesive was polymerized into a
2
CH2), 4.22 ppm (m, 4H, COOCH2), 3.63 ppm (m, 12H; Ar-H), 2.80 piece of solid glue (2.5×3cm ), dried, and weighed. The solid glue
2 2
ppm (dd, J(H,H)=-12.8Hz, 1H;CH2CCN), 2.20 ppm (dd, J(H,H)=- was immersed into a continuously shaking conical flask containing
+
12.8Hz,1H;CH2CCN); MS (50 eV): m/z(%):731.6 [M+Na ]. 100mL of phosphate-buffered saline (PBS). After certain days at 37
°C, the residue was filtered, washed, dried, and weighed to
Bis-anthracene/polyethylene glycol 600 bis(α-cyanoacrylate)adduct
calculate the apparent degradation rate. Long term degradation
(bis-A/PEG600BCA); polyethylene glycol 600 bis(9,10-dihydro-9,10-
test was carried out in PBS containing 50% bovine serum that was
endoethanoanthracene-11-cyano-11-carboxylate) (4b)
changed every 2 days. For each sample, 6 parallel experiments were
Compound (4b) was obtained similar to compound 4a. The performed to derive the means ± standard deviations (SD) of
1
product was obtained as a yellowish oily liquid (57.3% yield). H- apparent degradation rate. 1.5 GPC analysis
NMR (400 MHz, CDCl3, 25 °C, TMS): δ 7.49 ppm (m, 2H; Ar-H), 7.1– R
GPC was performed on Waters 1515，Styragel HT2~4. The solid
7.4 ppm (m, 14H; Ar-H), 4.92 ppm (s, 2H, Ar-CH-C-CN), 4.42 ppm (s,
glue (25 mg) before and after incubation in PBS in the in vitro
2H, Ar-CH-CH2), 4.22 ppm (m, 4H, COOCH2), 3.63 ppm (m, 86H;
2 apparent degradation rate test was dissolved in dry THF (0.5 mL) for
CH2OCH2CH2), 2.80 ppm (dd, J(H,H)=-12.8Hz, 1H; CH2-C-CN), 2.20
2 testing, respectively. The mother liquor was evaporated and then
ppm (dd, J(H,H)=-12.8Hz,1H; CH2CCN); MS (50 eV):
extracted by THF for the test. At least 3 repeated experiments were
m/z(%):1166.76±44n[M+].
preformed.
Bis-anthracene/polyethylene glycol 2000 bis(α-cyanoacrylate)
Intramuscular degradation test in a rabbit model
adduct (bis-A/PEG2000BCA); polyethylene glycol 2000 bis(9,10-
dihydro-9,10-endoethanoanthracene-11-cyano-11-carboxylate) (4c) Healthy rabbits were anesthetized with Nembutal (30mg/kg, i.v.),
and a 10-mm-long incision on the lateral skin of the rear leg was cut
Compound (4c) was obtained similar to compound 4a (87.67%
1 to expose the muscles, without damaging the dermis.
yield. m.p. 28–31 °C). H-NMR (400 MHz, CDCl3, 25 °C, TMS): δ 7.50
Subsequently, 30 μL of adhesive was administered by intramuscular
ppm (m, 2H; Ar-H), 7.1–7.4 ppm (m, 14H; Ar-H), 4.93 ppm (s, 2H, Ar-
injection. LKJ11 and BCA (as the control) were injected,
CH-C-CN), 4.43 ppm (s, 2H, Ar-CH-CH2), 4.22 ppm (m, 4H, COOCH2),
2 respectively, into the left and right legs of the same rabbit. Next,
3.63 ppm (m, 172H; CH2OCH2CH2), 2.79 ppm (dd, J(H,H)=-12.8Hz,
2 the skins were sutured, and the rabbits were fed normally. The
1H; CN-C-CH2), 2.22 ppm (dd, J(H,H)=-12.8Hz,1H;CH2CCN); MS (50
injected muscle was cut open to observe the degradability at 1d,
eV): m/z(%):2225.98 [M+].
14d and 3 months, respectively. In total, 18 animals were used and
Tetraethyene glycol bis(α-cyanoacrylate) (5a, CA-TEG-CA) 6 animals were cut each time.
A solution consisting of 4.00 g (5.65 mmol) of bis-A/tetraethyene In vivo biodegradation test in a rat model
glycol BCA (4a), 3.32 g (33.8 mmol) of maleic anhydride, 5mg
Sprague Dawley rats were anesthetized with Nembutal (45
(0.0454mmol) of hydroquinone, and 15 mg (0.106mmol) of
mg/kg, i.p.), and a 2–3 cm incision in the skin was made on the back
phosphorous pentoxide in 50 mL of anhydrous xylene was stirred at
to expose the psoas major. An incision of 5–8 mm in length and 3–5
reflux for 6h. The solvent was evaporated in vacuo, and 30 mL of
mm deep on the psoas was made. A total of 10 μL of adhesive was
dry benzene was added to the residue; this process was repeated
applied on the inner sites of the incision, which bound the wound
three times. After another 30mL of dry benzene was added, the
together. LKJ11 and BCA (as the control) were used on each side of
slurry was filtered and the solvent was removed in vacuo again.
the same rat, respectively. Next, the skins were sutured, and the
DCM and anhydrous diethyl ether were used to precipitate the
rats were fed normally. The skins were cut open at certain time
product 5a (1.71 g, 75.3% yield).
points(1 month, 3 months, 6 months and 15 months), and the
1
H-NMR (400 MHz, CDCl3, 25 °C, TMS): δ 7.08 ppm (s, 2H,H2C=C), tissues at the incision sites were prepared into pathological sections
3
6.66 ppm (s, 2H,H2C=C), 4.43 ppm (t, J(H,H)=9.6Hz,4H; for observation of both the solid glue degradation and the tissue
3
COOCH2CH2), 3.80 ppm (t, J(H,H)=6.0Hz, 4H;COOCH2), 3.68 ppm regeneration status. In total, 28 animals were used and 7 animals
(m, 8H; OCH2CH2). were cut each time.

Polyethylene glycol 600 bis(α-cyanoacrylate) (5b, CA-PEG600-CA) Excretion test of radioactively labeled LKJ11

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ARTICLE
14 14
Radioactive C-labelled BCA was synthesized from C-
[25]
formaldehyde according to methods published previously. The
-1
product with specific activity 33 MBq·ml was used to prepare
radioactive LKJ11. 5 rats (male 190-210 g) were anesthetized with
Nembutal (50 mg/kg, i.v.). Following abdominal incision, the
stomach was separated. A total of 5 μL of radioactive LKJ11 (about
88 kBq) was applied onto the stomach surface. Next, the skin was
sutured, and the rats were individually housed in motabolic cages.
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The urine and feces were collected every 24 hours for 30 days.
Finally, the rats were sacrificed, and the stomach and blood were
collected. The radioactivity of the standard, blank or unknown
sample (urine, blood, feces or stomach) was measured by LSC in a
Tri-Carb 2910TR liquid scintillation counter (PerkinElmer Inc., USA).
Aliquots of urine (50 μl) were directly mixed with scintillation fluid
cocktails and counted. Feces and stomach were homogenized with
addition of ethanol/water (50/50) at approximately 3 times of
original sample weight. Aliquots of fecal and stomach homogenates
(approximately 0.3 g) and whole blood (approximately 0.2g) were
14
combusted in a Packard Sample Oxidizer. The resulting CO2 was
trapped with scintillation fluid cocktails and assayed by LSC. The
radioactive recovery in each sample was calculated by dividing the
total radioactivity administrated.
Burst pressure of the rat intestinal wound test
Sprague Dawley rats were anesthetized (50 mg/kg, i.v.), and their
abdominal cavities were opened to separate a segment of the small
intestine (45 mm) for use. A hole of 2 mm in diameter on the small
intestine was made using a syringe needle. A total of 5 μL of LKJ11
was applied to seal the wound, after drying with cotton. The rabbits
were then sutured and were fed normally. At certain time points,
the rat was opened again, one end of the operated small intestine
was ligated by silk sutures, and a small incision was made at the
other end. A three-way tube, a pressure gauge, and a
tonotransducer were connected. The pressure was gradually
increased until the small intestine was damaged to cause gas
leakage, namely the burst pressure value. Animals in the control
group had a hole created but not sealed. In total, 72 animals were
used and each group contained 36 animals. 6 animals in each group
were cut each time.
Guinea pig skin wound healing test
6 adult guinea pigs were sampled, and their back hair was
removed. The animals were anesthetized with Nembutal (50mg/kg,
i.v.). After the back was disinfected, two incisions were made on
each side of the back along the animal ridge; each incision was20
mm in length and reached the muscular layer. After hemostasis, the
skin incisions were aligned closely, and LKJ11 and BCA (as the
control) were uniformly applied onto each wound surface,
respectively. The wounds were kept aligned manually for
approximately 30s until the adhesive fully solidified. The guinea pigs
were fed normally, and the wound healing was observed over time.
T-peel strength test, tension strength test and wound closure
strength test
8 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6TB00235H
Journal Name
These tests were carried out according to national standard of
medicine industry (YY/T 0729-2009). For each sample, 10 parallel
experiments were performed to derive the means ± SD.
Acute toxicity test
Journal of Materials Chemistry B Accepted Manuscript
12 male mice were used, including 6 in the LKJ11 group and 6 in
the control group. All mice fasted for 24h before the test. P-LKJ11
was ground to a powder and suspended in 0.5%
carboxymethylcellulose aqueous solution to make a final
concentration of 20%. A dose of 10g/kg (maximum tolerated dose)
was administered intragastrically to each mouse in the LKJ11 group.
The control group received only 0.5% carboxymethylcellulose
aqueous solution. The mice were fed normally, and dynamic
changes of animal body weight were recorded.
Skin irritation test
6 healthy rabbits were selected. At 24h before the test, hair at
both sides of the midline of the back was removed by sodium
2
sulfide to prepare an area of approximately 8×8 cm . It was
required that the skin was intact. A total of 0.1mL of LKJ11 was
coated onto the skin within a circular area with a diameter of 2.5
cm. The skin was covered with two layers of gauze and fixed with
nonirritant tape and a bandage. After 4h, the polymer was removed
with warm water. Next, skin reactions were observed at 1h, 24h,
48h, and 72h.
Intracutaneous irritation test
6 rabbits used in the intracutaneous irritation test were prepared
in the same manner as described in 2.12. P-LKJ11 was extracted
with 0.9% sodium chloride solution or vegetable oil (0.2 g in 6 mL,
37 °C, 72 h). The extract was injected intracutaneously into the back
of a rabbit, and the injected area was observed at 24 h, 48 h, and
72 h post injection.
Cytotoxicity
Hs27 cells, obtained from Zhongyuan Union Cell and Gene
Engineering Corp., Ltd, were cultured in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
under standard conditions (37°C, 5% CO2). Cells were digested with
5
0.25% typsin-EDTA and were seeded at 1 × 10 cells/well in a 96-
well plate overnight. Compound Treatment: LKJ11 (100 μL) was
polymerized in a 24-well plate. Then, 1.0 mL of DMEM containing
10% FBS was added to each well, and the plate was incubated at
37°C for 24 h. Next, the extract was added to Hs27 cells to test the
cytotoxicity of LKJ11 (100μL/well). After incubation for 72 h, a CCK-8
assay was used to determine the cell number. CCK-8 Assay: The old
culture medium was discarded, the Hs27 cells were washed with
fresh PBS three times, 100 μL of new culture medium and 10μL of
CCK-8 reagent were added, and the plate was incubated at 37 °C for
1.5 h before the optical density at 450 nm was measured. All
experiments were done in triplicate and repeated three times.
Negative control was DMEM containing 10% FBS. Positive control
was DMEM containing 5% DMSO.
Animals
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Animals were supplied by Beijing Experimental Animal Center 19 China Patent: CN 101180085A.
(Beijing China). All of the procedures were carried out in accordance 20 P. Fadaie, M. Atai, M. Imani, A. Karkhaneh, S. Ghasaban.
with the standards established in the Guide for the Care and Use of Dent. Mater. 2013, 29, 61-69.
Laboratory Animals published by the Institute of Laboratory Animal 21 J. J. Verhoef, J. F. Carpenter, T. J. Anchordoguy, H.

Journal of Materials Chemistry B Accepted Manuscript

Resources of the National Research Council (United States), and
also approved by the Animal Care and Use Committee of the Beijing
Institute of Pharmacology and Toxicology. All the efforts were made
to minimize the number of animals used and their suffering.
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Nanomedicine, 2009, 5, 419-423.

Acknowledgements
Financial support from the National Science and Technology Major
Project of China (2012ZX09301003) and the National Natural
Science Foundation of China (81573345) are gratefully appreciated.
24 F. Leonard, R. K. Kulkarni, G. Brandes, J. Nelson and J. J.
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67x29mm (300 x 300 DPI)

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