Journal of Industrial and Engineering Chemistry 118 (2023) 101–108

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Journal of Industrial and Engineering Chemistry

journal homepage: www.elsevier.com/locate/jiec

Effect of thrombin conjugation on hemostatic efficacy of PLGA mesh

through reagent free surface modification
In Kyu Lee a,1, Su Jung You b,1, Young Jin Yun b,1, Jae Kwang Kim c, Dae Hyeok Yang b, Heung Jae Chun b,d,⇑,
Jaehoon Ko e, Youngjoo Koh f
a
Department of Surgery, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
b
Institute of Cell & Tissue Engineering, The Catholic University of Korea, Seoul 06591, Republic of Korea
c
Department of Internal Medicine, Bucheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Bucheon 14647, Republic of Korea
d
Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
e
Smart Textile R&D Group, Korea Institute of Industrial Technology, Ansan 15588, Republic of Korea
f
Pharmaceuticals Research R&D Center, Samyang Biopharmaceuticals Corp., Seongnam 13488, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: In this study, thrombin-conjugated PLGA (THR-PLGA) electrospun meshes were prepared through
Received 5 September 2022 reagent free surface modification based on Schiff base reaction between amine groups in thrombin and
Revised 17 October 2022 carbonyls on the surface of PLGA meshes, and their feasibilities on the improvement of hemostasis were
Accepted 27 October 2022
evaluated. In vitro studies were performed with chromogenic substrate and transglutaminase activity
Available online 2 November 2022
assays to evaluate the thrombin activity and FXIIIa activity of the THR-PLGA samples, respectively. A
rat tail injury model was employed for measuring hemostatic time of the samples. The results showed
Keywords:
that the hemostatic efficacy of the samples increased with increase in the content of THR on the surface
Thrombin
PLGA
of the samples. In addition, partial splenectomy was carried out in rat model to investigate the potential
Reagent free surface modification management of hemorrhage of THR-PLGA, as compared to VicrylÒ and TachoSilÒ. The results showed that
Schiff base reaction the inflammatory response was significantly reduced compared to VicrylÒ and TachoSilÒ, and a dense
Hemostasis fibrous layer was produced to protect the inside from laceration at 2 weeks of treatment. Moreover,
neo pulps were appeared right below the fibrous capsule. These findings suggest that THR-PLGA samples
prepared through the reagent free immobilization method deserves further advanced investigations for
future clinical applications.
Ó 2022 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
reserved.

Introduction
The treatment of the lacerations following both blunt and pen-
etrating trauma varies depending on the severity of the injury [1–
4]. Major vascular lacerations of internal organs often require the
immediate interventions [2]. The primary concern of intervention
is hemostasis control, but suturing with conventional technique
cannot be suitable for the severely lacerated organs such as liver,
spleen and kidney consist of fragile parenchyma and capsules [5–
7]. The fragility of capsule and parenchyma may not hold suture,
in turn, results in the additional loss of parenchyma. Moreover,
the macerated wound edges in traumatized parenchyma hardly
⇑ Corresponding author at: Institute of Cell & Tissue Engineering, College of
Medicine, The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, Seoul
06591, Republic of Korea.
E-mail address: chunhj@catholic.ac.kr (H.J. Chun).
1
These authors have equal contribution.
provides sufficient anchorage for the tying of knots which is asso-
ciated with a risk of incomplete hemostasis.
For decades, therefore, packing or wrapping using bio-
absorbable polymeric mesh has been used as alternatives [3,4].
The representatives of bio-absorbable polymeric meshes com-
monly used for repair of laceration are Dexon and VicrylÒ (polyglac-
tin 910), made of poly (glycolic acid) (PGA) and copolymers of
glycolic and lactic acid (PLGA), respectively [8]. Both products, as
the United States Food and Drug Administration approved synthetic
polyesters [9], provide soft and flexible but tear-proof properties
enough to allow compression with packing or wrapping of lacer-
ated organs. However, biodegradable aliphatic polyesters, PGA,
PLA and PLGA, similar to other synthetic polyesters, are of a
hydrophobic nature [10], which make difficulties to achieve
hemostasis through tamponade effect. In addition, since these
materials are lack of biochemical hemostatic elements, the use of
additional hemostatic agent during intervention is required. There-
fore, given the crucial role of hemostasis in the treatment of

https://doi.org/10.1016/j.jiec.2022.10.049
1226-086X/Ó 2022 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
I.K. Lee, S.J. You, Y.J. Yun et al. Journal of Industrial and Engineering Chemistry 118 (2023) 101–108

lacerated organs, modification with hydrophilic hemostatic factors
may increase the potential of PLGA mesh as the advanced wrapping
and packing material.
In this study, we selected thrombin as hemostatic agent and
coupled to PLGA mesh (THR-PLGA) using reagent-free surface
modification using plasma treatment (scheme 1); the carbonyl
species were produced on the surface of PLGA mesh due to oxida-
tion by plasma treatment and covalently coupled to primary amine
groups of thrombin through Schiff base reaction [11]. The amounts
of thrombin coupled onto the surface of THR-PLGAs were quanti-
fied using a chromogenic substrate assay, and the changes in the
surface chemical composition and the wettability of the samples
were characterized by ATR-FTIR, XPS and contact angle measure-
ment, respectively. In addition, the efficacies on hemostasis were
systemically evaluated in vitro and in vivo.
Materials and method
Materials
to characterize the modified PLGA samples. The spectra were mon-
itored from 600 to 4000 cm 1. The changes in elemental composi-
tions of the PLGA samples were analyzed by X-ray photoelectron
spectroscopy (PHI 5000 Versa Probe, ULVAC PHI, Japan). The source
was a 15 kV, 24.5 W monochromated X-ray beam (photoelectron
energy = 1486.6 eV) obtained from an aluminum anode. Pass
energy of wide scan was recorded at 117.4 eV at 2.0  10-7 Pa.
The wettability of the sample was also confirmed by measuring
water contact angle using OCA 40 (Optical contact angle measuring
and contour analysis systems, Dataphysics, Germany).
Specific activity [15]
Thrombin-specific activity on the surface of the samples was
measured using a chromogenic substrate assay. 1.0  1.0 cm2 of
the sample meshes were placed in a 12-well plate and treated with
1 mL of tris-HCL buffer (20 mM, pH 7.4). N-(p-Tosyl)-Gly-Pro-Arg
p-nitroanilide acetate salt solution was added into the well plate
and the absorbance was measured at 405 nm using a microplate
reader.

PLGA, Neosorb (10/90, Mw: 200 kDa) was gifted from Samyang
Animals
Biopharmaceuticals Co. ltd. (Seongnam, Korea). 1,1,1,3,3,3-Hexa
fluoro-2-propanol (HFIP) was supplied by Acros Organics (Pitts-
Animals used in this study were adult male Sprague-Dawley
burgh, PA, USA). Thrombin from bovine plasma was purchased from
rats aged 9 weeks and weighing 150 to 200 g. All animal proce-
Sigma-Aldrich (referring to units as quoted by the supplier; Catalog
dures were performed in accordance with the Laboratory Animals
Number; T4648, Batch Number; SLBH3919V; Saint Louis, Mo, USA).
Welfare Act, the Guide for the Care and Use of Laboratory Animals
N-(p-Tosyl)-Gly-Pro-Arg p-nitroanilide acetate salt (T1637, Sigma-
experiment of Yeouido St. Mary’s Hospital of the Catholic Univer-
Aldrich, Saint Louis, Mo, USA) and Factor XIIIa Activity Assay Kit,
sity of Korea with the animal ethics approval (YEO20153401FA)
Catalog # K522-100, BioVision, USA) were purchased and used.
issued by IACUC (Institutional Animal Care and Use Committee)
VicrylÒ mesh, (Ethicon, NJ, USA) and TachoSilÒ sealant matrix,
of the Catholic University of Korea.
(Takeda Austria, Linz, Austria) were purchased and used.
Conjugation of thrombin (THR) to the electrospun PLGA fibrous
Fxiiia activity assay [16]
mesh (THR-PLGA).
PLGA fibrous mesh was fabricated by spun 10 wt% of PLGA solu-
The rat was deeply anesthetized with ZoletilÒ (Virbac, Carros,
tion dissolved in HFIP through a 21 G needle at 25 kV of power, and
France) and RompunÒ (Bayer, Toronto, Canada). 3 ml of blood in
4 mL/h of flow rate as described in previous studies [12–13].
each rat was obtained by cardiac puncture using a 5 ml syringe
Thrombin was conjugated onto PLGA samples using the induc-
after the abdomen of rat was disinfected with iodine. The collected
tively coupled plasma-assisted chemical vapor deposition (ICP
blood was transferred to vacuum tubes containing sodium citrate,
CVD) apparatus [14]. Prior to plasma treatment, PLGA fibrous
and gently inverted 10 times. The blood was transferred to a 1.5 mL
meshes were washed with MilliQ water and dried at room temper-
tube within 1 hour. Plasma was isolated by centrifugation at 1300
ature. The samples were placed on the bottom of the chamber and
rcf for 10 minutes. The FXIIIa activities in rat plasma were mea-
subjected to argon plasma treatment with 13.56 MHz of radio fre-
sured an enzyme-linked immunosorbent assay (ELISA) according
quency. Argon gas was introduced into the chamber at a flow rate
to the manufacturer’s instructions. The THR-PLGA FSs were placed
of 5 cm3/min. Plasma was generated at an electric power of 30 W
to the bottom of a 96-well plate with a diameter of 6 mm. A mix-
for 30 seconds. After plasma treatment, the samples were immedi-
ture of 25 lL FXIIIa activation buffer, 25 lL reaction buffer, 48 lL
ately exposed to the oxygen atmosphere by supplying oxygen gas
detection buffer, 2 lL probe and 10 lL of rat plasma were placed
to the reaction chamber to promote the oxidation of the surface.
in a well containing the samples. The absorbance of solution was
The samples were cut into 1  1 cm2 pieces and placed on the bot-
measured using an ELISA plate reader at 340 nm.
tom of 24 well plates for thrombin conjugation.
Thrombin solutions were prepared by dissolving 0.29 mg of
Rat tail snip model [17]
thrombin in 2 ml of 20 mM tris buffer, (pH 7.4) which is equivalent
to 80 IU/ml. Through serial dilution, 100 lL of THR solutions that
Twelve rats were used for coagulation time test. Prior to test,
correspond to 1 to 8 IU/cm2 with respect to the samples were
the rats were anesthetized by ZoletilÒ and RompunÒ at recom-
added dropwise to each well plate, and incubated at 4 °C. The cou-
mended dosage. The tail tip was depilated and disinfected with
pling reaction between thrombin and PLGA sample was allowed to
70 % of ethyl alcohol. The tail of the rat was cut 1 cm from its
continue for 24 hours, then the samples were rinsed with PBS and
tip. After 5 seconds of bleeding, THR-PLGA was covered the wound.
lyophilized at 80 ° C and 5 mTorr for 24 hours. The samples were
The blood coagulation time was measured when the THR-PLGA did
designated in accordance with the content of thrombin feed (THR1
not fall from the wound site. The samples after hemostasis was
–PLGA, THR2-PLGA, THR3-PLGA etc.). Prior to the experiments, all
lyophilized and observed using field emission scanning electron
samples were sterilized using ethylene oxide gas.
microscopy (FE-SEM; Inspect F, FEI, USA).

Surface characterizations Spleen wrapping

Fourier transform infrared spectroscopy-attenuated total reflec- The hemostatic capacity of the THR-PLGA samples was further
tion (FTIR-ATR; Nicolet iS50, Fisher Scientific, USA) was employed evaluated through a rat splenic laceration model compared to
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I.K. Lee, S.J. You, Y.J. Yun et al. Journal of Industrial and Engineering Chemistry 118 (2023) 101–108

Scheme 1. Schematic representation of THR-PLGA through reagent free surface modification.

VicrylÒ and TachoSilÒ. The laceration model of rat spleen was made
through partial splenectomy [18]. Briefly, considering the size of
the rat spleen, less than 0.5 cm from its distal tip is transected
sharply. Hemostasis was made by wrapping the transected portion
with the samples. In the case of THR-PLGA and TachoSilÒ, creating
compression on the parenchyma was enough to prevent further
bleeding. While VcrylÒ need the additional application of fibrin
glue to accomplish stable wrapping.

Results

The FTIR/ATR spectra of control PLGA and THR-PLGA samples

are shown in Fig. 1. PLGA shows the IR characteristic absorption
bands of CAH group at 2960 cm 1, CAH carbonyl (C@O) peak at
1750 cm 1 and CAO peak at 1150 cm 1 [19,20]. The conjugation
of THR to PLGA produces new peaks at 1600 cm 1 –1700 cm 1
and 3000–3500 cm 1 corresponding to the amide I, amide II and
NH2 groups, respectively [21]. The transmittance of these peaks
Fig. 1. FTIR/ATR spectra of control PLGA and THR-PLGA samples. PLGA and THR-
increased with increase in THR feed content. Fig. 2 shows the PLGA samples show their characteristic absorption bands: CAH (2960 cm 1),
XPS survey scan spectra of control and THR- PLGA samples [21]. carbonyl C@O (1750 cm 1), CAO (1150 cm 1), amide I (1600 cm 1), amide II
In all THR-PLGA samples, conjugation of THR generated similar (1700 cm 1), and NH2 groups (3000–3500 cm 1).
patterns of N1s spectra at 400 eV, whose intensities increased with
the increase in the content of THR. The C1s core spectrum of PLGA
(Fig. 3A-1) exhibits peaks at 284.5, 286.8, and 289.1 eV, corre-
sponding to CAC, CAO, and O-C@O bonds, respectively [22]. The
coupling of thrombin to PLGA reveals a new c1s spectrum
(Fig. 3A-2). The N-C@O of the peptide bond in the main chain of
thrombin appeared at 287.48 eV, and the CAO peak of 286.8 eV
was slightly shifted to 286.24 eV and its intensity increased due
to the incorporation of the imine bond. PLGA displays O1s core
spectra at 533.4 and 532.12 eV in Fig. 3B-1 assigned to CAOAC
and O@C groups in PLGA. The conjugation of thrombin to PLGA
results in the peak at 530.95 eV corresponds to N-C@O group
(Fig. 3B-2). In addition, the N1s core spectrum of THR-PLGA
(Fig. 3C-2) shows the peaks at 398.25, 399.71 and 401.47 eV that
may convince the successful condensation of thrombin to the sur-
face of PLGA sample through imine group formation.Fig. 3.
The yield of thrombin grafted onto the PLGA surface was deter-
mined by measuring thrombin specific activity of the feed solution
and the surface. The specific activities of thrombin on the surface of
the samples increased with increase in those of the feed solutions
(Fig. 4). Fig. 5 shows the contact angles of the samples as a function
of graft efficiency of thrombin. The control PLGA sample had a Fig. 2. The XPS survey scan spectra of control and THR- PLGA samples.

water contact angle of 126.2°. In the case of thrombin conjugated

samples exhibited the remarkable decrease in water contact
angles. The contact angle of water drop on the surface of THR2-
PLGA was 27.5°, and the contact angle of the samples showed a
tendency to decrease to 16.5° as the concentration of thrombin
increased.
Thrombin has a number of well-studied biological activities,
which serve to achieve coagulation and hemostasis [23]. One of
the important activity of thrombin is to activate Factor XIII to FXIIIa
that cross-link soluble fibrin to insoluble fibrin network and stabi-

103
I.K. Lee, S.J. You, Y.J. Yun et al.
Fig. 3. C1s, O1s and N1s core spectra of PLGA and THR- PLGA.
Fig. 4. Immobilized thrombin activities on PLGA. The yield of thrombin grafted onto
the PLGA surface was determined by chromogenic substrate assay.
lize blood clots. Therefore, the activation of FXIIIa in blood plasma
upon contact with the sample surface provides strong evidence
that the immobilized thrombin is involved in blood coagulation.
Fig. 6 shows the FXIIIa activities with respect to the samples. The
activity proportionally increased as the concentration of thrombin
increased, and attenuated at 5 IU of thrombin feed (THR5-PLGA)
Fig. 5. Water contact angle measurement of PLGA and THR-PLGA samples.
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Journal of Industrial and Engineering Chemistry 118 (2023) 101–108
then reached a plateau. Compared to Fig. 4, the difference in the
pattern of increase can be attributed to the fact that blood coagu-
lation is a threshold-limited intertwined set of processes. FXIII acti-
vation takes place slightly prior to the release of fibrinopeptides A
by another function of thrombin in blood coagulation process. As
fibrinopeptides A has a threshold level in the amount of release,
FXIIIa activation reaches the maximum levels around 60 % of con-
centration, therefore, a graph that is directly proportional to
thrombin feed cannot be expected.
Animal test using rat tail snip model was designed with four
groups of samples including control PLGA, THR2-PLGA, THR4-
PLGA and THR8-PLGA in order to narrow down the number of cases
(Fig. 7). Similar to Fig. 5 in which the control PLGA showed strong
hydrophobicity, the control PLGA could not absorb bleeding from
the tail tip of the rat, and could not retain the additional blood that
flows continuously over time, eventually failed the formation of
hemostatic plug. On the other hand, thrombin-conjugated samples
show an instantaneous absorption of bleeding and thereby
strongly stuck to tail tip, in turn, led the formation of an initial pla-
telet plug with the aid of conjugated thrombin. The hemostatic
effect of the samples was also investigated by electron microscopic
observation.
In Fig. 8A, a small number of round and biconcave shaved red
blood cells are attached to the surface of the PLGA fiber. In the case
of thrombin conjugated samples, thrombin promoted the activa-
tion and aggregation of platelets. The activated platelets became
adhered to the surface of the samples and serve as the site for coag-
ulation (Fig. 8B). The adhered platelets changed their shape and
I.K. Lee, S.J. You, Y.J. Yun et al.
Fig. 6. FXIIIa activities of rat blood plasma against the samples.
release the contents of their granules to attract more platelets.
Thrombin also cleaves soluble fibrinogen to produce insoluble fib-
rin. Fibrin mesh trapped a greater number of platelets and red
blood cells (Fig. 8C). Eventually, bunches of well grown blood clots
made up of the activated platelets, red blood cells and fibrin were
Fig. 7. Animal test using rat tail snip model; (a) PLGA, (b) THR2-PLGA (c) THR4-PLGA and (d) THR8-PLGA.
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Journal of Industrial and Engineering Chemistry 118 (2023) 101–108
produced that tightly hold the clot together (Fig. 8D). As shown in
the rat tail model and electron microscopic examination, the
thrombin conjugation resulted in a significant decrease in blood
coagulation time. In the case of the control sample, it took more
than 4 minutes to stop bleeding, whereas in the case of THR-
PLGA samples, it did not exceed 3 minutes. In addition, the hemo-
static time of PLGA, THR2-PLGA, THR4-PLGA and THR8-PLGA were
found to be 248, 168, 116 and 103 seconds, respectively, therefore,
the hemostatic effect increased with increase in the content of
thrombin at the surface of samples (Fig. 9).
Fig. 10 shows that VicrylÒ, TachoSilÒ and THR-PLGA wrap the
incision part of the spleen. VicrylÒ and THR-PLGA absorbed bleed-
ing instantaneously upon contact with the wound bed, and wrap-
ping was accomplished without difficulties. However, in the case of
hydrophobic VicrylÒ, fibrin glue was topically applied for
wrapping.
Fig. 11A-1 shows the optical microscopy of H&E stained normal
spleen. The fibrous capsule surrounds the spleen parenchyma that
consists white pulps between red pulps areas. Spleen is the largest
lymphoid organ in the body that receives blood from the splenic
artery and primarily filters blood by destroying old red blood cells
and platelets. Therefore, laceration of grade III injuries involving
trabecular vessels results in ongoing bleeding appearing as splenic
hematoma (Fig. 11A-2). Compared to other samples, the spleen
treated with VicrylÒ for 48 hours shows some voids at the inter-
face, presumably the result of poor absorbing capacity of the bleed-
ing because VicrylÒ is hydrophobic. A number of macrophages
I.K. Lee, S.J. You, Y.J. Yun et al.
Fig. 8. SEM images of blood clot on PLGA and THR-PLGA samples. (a) PLGA, (b) THR2-PLGA (c) THR4-PLGA and (d) THR8-PLGA.
Fig. 9. Comparison of coagulation time of PLGA and THR-PLGA samples in rat tail
snip model (*p < 0.05).
exist at the interface, and fibrosis is progressing by the reticulum
cells (Fig. 11B-1). After 2 weeks (Fig. 11B-2), the number of macro-
phages was greatly decreased and a fibrous capsule was found at
the interface. Because TachoSilÒ is hydrophilic, it absorbed a large
amount of bleeding with the infiltration of macrophages to some
extent (Fig. 11C-1). An immature fibrous layers are found at the
interface, but a number of inflammatory reactions are in progress
inside TachoSilÒ (Fig. 11C-2). THR-PLGA also showed the strong
capacity to absorb bleeding. While a large number of macrophages
exist at the interface, there is a strong tendency to form fibrosis by
a large number of reticular cells (Fig. 11D-1). Compared with
TachoSilÒ, the inflammatory response was significantly reduced,
and a dense fibrous layer was produced to protect the inside from
laceration at 2 weeks of treatment (Fig. 11D-2). Moreover, pulps
Fig. 10. In vivo application of THR-PLGA using rat spleen injury model compared with VicrylÒ and TachoSilÒ. (A) VicrylÒ, (B) TachoSilÒ and (C) THR-PLGA.
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Journal of Industrial and Engineering Chemistry 118 (2023) 101–108
are seen right below the fibrous capsule, although they are not
grown up enough.
Discussion
Biomaterials, regardless of whether used inside or outside the
human body, the surface comes in contact with body components;
proteins, cells and tissues, therefore, the characteristics of the sur-
face are responsible for the subsequent events of bioreactions to
materials. In this regard, a number of studies have been conducted
on surface modification of biomaterials for several decades [24,25].
In the case of the biodegradable aliphatic polyesters; PLA, PGA and
PLGA, new attempts to surface treatment with proteins so called
protein-polymer conjugates have made, because proteins feature
a unique bio-recognition and binding depending on their particular
function, while the synthetic counterparts lack this ability [26].
In order to covalently bind proteins to a polymer, it is necessary
to introduce the functional groups on the surface of the polymer
capable of condensation with the proteins. Conventional methods
for introduction of functional groups were constrained for com-
mercialization because a number of wet processes using toxic
chemicals were involved [27]. Therefore, the surface modification
using plasma treatment through a simple dry method has attracted
many attentions in the area of protein-polymer conjugate. How-
ever, the toxicity problems cannot be completely overcome
through this method because most of the monomers for surface
deposition have been clinically proven to be toxic [28,29]. More-
over, the coupling agent linking one end, and another still has to
be used in a solution state. In the meantime, there have been
attempts to use a simple method using the generation of functional
groups by oxidation of the polymer surface, so called reagent free
immobilization or modification instead of using a coupling agent
for condensation [27]. When the polymer is exposed to oxygen
after plasma treatment, peroxide is formed at the surface [15].
I.K. Lee, S.J. You, Y.J. Yun et al.
Fig. 11. H & E stained histological sections of normal spleen and sample treated groups: (A-1) normal spleen, (A-2) lacerated spleen, (B-1) VicrylÒ treated 48 hrs, (B-2) VicrylÒ
treated 2 weeks, (C-1) TachoSilÒ treated 48 hrs, (C-2) TachoSilÒ treated 2 weeks, (D-1) THR-PLGA treated 48 hrs and (D-2) THR-PLGA treated 2 weeks; white circles represent
white pulps and dashed lines indicate the interface between sample and host tissue (Scale bar = 100 lm).
Unstable peroxide is immediately decomposed and oxidized to
hydroxyl, carboxyl and carbonyl groups [30]. Most of the carbonyls
are present in the form of ketone and aldehyde. These carbonyls
can be easily condensed with free amines of the protein and form
imines through Schiff base reaction [11]. This method does not
seem to be innovative in terms of chemistry, especially stoichiom-
etry. However, this method can be very useful because it does not
couple a single or the small molecules on the surface of the mate-
rial, but because it binds the proteins that have a large number of
functional groups. Thrombin, in particular, contains a number of
basic amino acids such as lysine, which is capable of covalent cou-
pling with a carbonyl group formed on the surface of the material
by plasma treatment [31]. In addition, since platelets respond with
a very high sensitivity to thrombin even in the range of picomolar
to nanomolar scale [32–34], the reagent free immobilization
method was proven to be sufficient to improve the hydrophobicity
of the material surface and to increase IU as well as FXIII activity of
the samples. The positive aspects from in vitro tests were also lar-
gely reflected in the results of animal studies. THR-PLGA exhibited
moderate inflammatory responses and showed the tissue regener-
ation capacity that white and red pulp, the most important compo-
nents of spleen, were formed within a short period of time.
Therefore, the series of the extensive surface treatment by conven-
tional methods as performed in our previous studies seemed to be
unnecessary, and this technology can take a step closer to commer-
cialization. Consequently, THR-PLGA samples prepared through
the reagent free immobilization method are enough to warrants
further in vivo investigations for future clinical applications.
Conclusions
In this study, the reagent free surface modified PLGA-THR elec-
trospun meshes in which amine groups in thrombin were directly
bound to carbonyl groups PLGA mesh through Schiff base reaction
were prepared. The studies on FTIR together with XPS confirmed
the successful condensation of thrombin to the surface of PLGA
sample through imine group formation. The specific activities of
thrombin on the surface of the samples increased with increase
in those of the feed solutions. The increase of the hydrophilic prop-
erty and FXIIIa activity of the surface of PLGA-THR samples due to
the conjugation of thrombin resulted in a marked hemostatic effect
compared to the control PLGA as demonstrated in animal studies.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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Journal of Industrial and Engineering Chemistry 118 (2023) 101–108
Acknowledgment
This research was supported by the grant of ministry of Trade,
Industry and Energy (MOTIE) (Grant no. 10047811).
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