Journal of Biomaterials and Nanobiotechnology, 2011, 2, 337-346
doi:10.4236/jbnb.2011.24042 Published Online October 2011 (http://www.SciRP.org/journal/jbnb)
Copyright © 2011 SciRes. JBNB
337
Blood Compatibility of Amphiphilic
Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane)
Block Copolymers
Kazuo Sugiyama, Nobuyuki Tanigawa, Kohei Shiraishi
Cluster of Biotechnology and Chemistry Systems, Program in Systems Engineering, Graduate School of Systems Engineering, Kinki
University, Higashihiroshima-City, Japan.
Email: sugiyama@hiro.kindai.ac.jp
Received June 21st, 2011; revised July 22nd, 2011; accepted August 20th, 2011.
ABSTRACT
Amphiphilic block copolymers poly(LysAA-b-DMS) consisting of a hydrophilic poly(N-α-acrylamide-L-lysine) [poly(LysAA)]
segment with different molecular weights and a hydrophobic polydimethylsiloxane (PDMS ) segment were prepared as
follows. The precursor copolymer poly(Bo c-L ys AA- Ot Bu-b -PD MS) was obtained from radical polymerization of
N-α-acrylamide-N-ε-tert-butoxycarbonyl-L-lysine-tert-butylester (Boc-LysAA-OtBu) initiated with 4,4’-azobis(polydi-
methylsiloxane 4-cyanopentanoate) (azo-PDMS) with the molecular weight of PDMS Mw = 4.3 × 103 in the presence of
2-mercaptoethanol (2-ME) as a chain-transfer agent. Removal of the protecting groups of the precursor copolymer was
carried out in 80% trifluoroacetic acid aqueous solution to give poly(LysAA-b-DMS)-1-3. The weight average molecu-
lar weight of poly(LysAA-b-DMS)-1-3 was Mw = 1.02 × 104 - 2.52 × 104. From the 1H-NMR and fluorescence spectra
measurements, poly(LysAA-b-DMS)-1-3 was determined to self-organize and form core-shell micelles in water. The
critical micelle concentration (CMC) increased to 1000 - 4000 mg·L–1 with increasing molar ratio of the poly(LysAA)
segment from 0.42 to 0.65. From morphological analysis with a scanning probe microscope (SPM), poly(LysAA-b-DMS)
has microphase-separated structures made up of hydrophilic and hydrophobic regions with the domain size ranging
from several tens to several hundreds of nanometers. Inhibition of thrombin activity of poly(LysAA-b-DMS) was evalu-
ated from the Michaelis constant (KM) and catalytic activity (kcat) for the enzymatic reaction of thrombin and synthetic
substrate S-2238 in the presence of poly(LysAA-b-DMS). The KM and kcat were 0.10 - 0.11 mM and 4.04 × 105 - 4.26 ×
105 min–1, respectively. Fibrinolytic activity was also verified from the transformation of plasminogen to plasmin by
tissue plasminogen activator (t-PA) using synthetic substrate S-2251 in the presence of poly(LysAA-b-DMS). The KM
and kcat were 0.07 mM and 5.73 × 106 - 5.95 × 106 min1, respectively.
Keywords: Poly(N-α-acrylamide-L-lysine), Polydimethylsiloxane, Block Copolymer, Molecular Assembly, Blood
Compatibility, S-2238/S-2251, Biomedical Polymer Material
1. Introduction
Some graft and block copolymers containing the polydi-
methylsiloxane (PDMS) segment have been synthesized
in order to improve the mechanical properties and bio-
compatibility of silicone rubber as a useful biomedical
material [1-3]. In poly(etherurethaneurea)s including va-
rious molar ratios of the tetramethyldisiloxane moiety in
the main chain, the siloxane moiety was located on the
surface. As the hydrophobicity was increased with in-
creasing siloxane content, the surface was able to adsorb
bovine serum albumin [4]. On the other hand, a series of
block copolymers consisting of PDMS and hydrophilic
polymethacrylates, such as poly(2-hydroxyethyl metha-
crylate), poly(2,3-dihydroxypropyl methacrylate), and poly
(2,3,4,5,6-pentahydroxyhexyl methacrylate) suppressed the
adsorption of albumin and -globulin as well as platelets,
to a level less than PDMS [5].
The microphase-separated structure of a polymer is
considered to exhibit effective suppression of the adsorp-
tion of fibrinogen, an important blood coagulation factor,
as well as inhibition of adhesion and activation of human
platelets. Plasma protein adsorption, which is the initial
event in blood–material interaction, influences subse-
quent platelet adhesion and activation. The amphiphilic
poly(MPC-b-PDMS) obtained from introduction of the
Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers
338
2-(methacryloyloxy)ethyl phosphorylcholine (MPC) mo-
iety into PDMS has a microphase-separated structure and
shows no adhesion or activation of human platelets [6,7].
In our work on biocompatible polymeric materials [4-
9], we found that incorporation of L-serine or L-lysine
residues into polymers as side groups, for example, poly-
(O-methacryloyl-L-serine) [poly(SerMA)] and poly(N-
α-methacrylamide-L-lysine) [poly(LysMA)] [10], was
useful for creating blood compatible materials. The am-
phiphilic block copolymer poly(LysMA-b-DMS) con-
sisting of poly(LysMA) as the hydrophilic segment and a
PDMS segment with almost monodispersed molecular
weight as the hydrophobic segment has also been re-
ported [11]. The poly(LysMA-b-DMS) also exhibits an-
tithrombotic properties. Blood compatibility of poly(Lys-
MA) and poly(LysMA-b-DMS) may arise from the
strong interaction of the L-lysine group in the polymer
and the L-lysine-binding sites (LBS) of plasminogen, the
precursor of plasmin [12]. Plasminogen is converted to
the fibrinolytic active plasmin by the tissue plasminogen
activator (t-PA), thus showing increased fibrinolytic ac-
tivities.
It is well known that differences in the glass transition
temperatures (Tg) of polymers reflect the flexibility of
the main chain and side chain. For instance, the micro-
brownian motion of poly(methyl acrylate) and poly(N-
tert-butylacrylamide) begins at Tg = 10˚C and Tg =
128˚C, respectively, whereas that of poly(methyl metha-
crylate) and poly(N-tert-butylmethacrylamide) begins at
Tg = 105˚C and Tg = 160˚C, respectively [13]. The for-
mer polymers are more flexible due to the difference in
the substituent group (H or methyl) at the α-position of
the monomer units in the vinyl polymers. The more
flexible main chain is therefore expected to be the origin
of the stronger interaction between the polymer L-lysine
moiety and the LBS of plasminogen. In the course of our
study on biomedical polymeric materials, our interest has
been focused on the preparation of amphiphilic block
copolymers poly(LysAA-b-DMS) consisting of the hy-
drophilic poly(N-α-acrylamide-L-lysine) [poly(LysAA)]
segment with different molecular weights and a hydro-
phobic PDMS segment. Poly (LysAA-b-DMS) is ex-
pected to exhibit stronger fibrinolytic activity than poly
(LysMA-b-DMS).
There has also been growing interest in the self-orga-
nization phenomena of water soluble polymers contain-
ing a few hydrophobic substituents per chain and hydro-
phobic groups attached to the polymer end [8,9], as well
as amphiphilic block copolymers consisting of hy- dro-
philic and hydrophobic segments [11,14-17]. These am-
phiphilic polymers and copolymers are expected to play
a role as carrier molecules for drug delivery systems
(DDS), because they form a core-shell polymer micelle
composed of a hydrophobic core and a hydrophilic shell,
making it possible to stably hold hydrophobic medicines
in the core when placed in water.
In this article, it is confirmed that poly(LysAA-b-DMS)
can self-assemble to form a core-shell polymer micelle
composed of a hydrophobic core and a hydrophilic shell
in water, which can be used for the intravenous injection
of DDS carrier macromolecules. It is also confirmed that
its polymer film has a microphase-separated structure
useful for biomedical materials such as artificial veins.
The blood compatibility of poly (LysAA-b-DMS) was
evaluated by the Michaelis constant (KM) and catalytic
activity (kcat) for the enzymatic reaction of thrombin and
a synthetic substrate S-2238, as well as by the transfor-
mation of plasminogen to plasmin by tissue plasminogen
activator (t-PA) using synthetic substrate S-2251 in the
presence of poly(LysAA-b-DMS).
2. Materials and Methods
2.1. Materials
4,4’-Azobis (4-cyanopentanoic acid) was kindly supplied
by Wako Pure Chemical Co. (Japan) and purified by re-
crystallization from methanol. 4,4’-Azobis(4-cyano-pen-
tanoic acid chloride) was prepared according to the me-
thod of Smith [18]. Reactive silicone oil (PDMS-OH; X-
22-170BX, OH value: 21.0 KOH mg·g–1, Mw = 4.3 × 103,
Mn = 3.9 × 103, Mw/Mn = 1.09) was used as obtained
from Shinetsu Chemicals Ind. (Tokyo, Japan). 1-(6-Di-
methylamino-2-naphthyl)-1-dodecanone (DMAND; Mo-
lecular ProbesTM, invitrogen) was used as obtained. Hu-
man thrombin was purchased from Mochida Pharmaceu-
tical CO., LTD. (Tokyo, Japan), whereas the synthetic
chromogenic substrates S-2238 and S-2251 were pur-
chased from Sekisui medical CO. LTD. (Tokyo, Japan),
and used without any purification. Distilled and deio-
nized water was used for all experiments.
2.2. Synthesis of
4,4’-Azobis(polydimethyl-siloxane
4-cyanopentanoate) (azo-PDMS)
To a CHCl3 solution (150 mL) of reactive silicone oil
(PDMS-OH: 19 mmol) and triethylamine (10 mmol) was
added dropwise a dichloromethane solution (30 mL) of
4,4’-azobis(4-cyanopentanoic acid chloride) (9.5 mmol)
at 25˚C. The reaction mixture was washed with water,
and then the solution was evaporated under reduced
pressure. The crude azo-PDMS was purified by frac-
tional precipitation with a solvent system of tetrahydro-
furan-methanol. The molecular weight distribution was
found to be Mw/Mn = 1.12 by means of gel permeation
chromatography analysis conducted in CHCl3 solution
with TSK gel (column: TOSOH G4000HXL, G3000HXL,
C
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Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers339
G2000HXL), using TOSOH LC-8020 GPC. Yield: 68.3%.
1H-NMR (CDCl3, TMS): δ = 0.14 [s, 420H, -Si(CH3)2],
0.79 - 0.82 [m, 6H, -CH3], 1.23 - 1.72 [m, 12H, -CH2-],
2.32 - 2.52 [m, 4H, -CH2-], 3.34 - 3.39 [m, 4H, -OCH2-],
3.55 - 3.58 [m, 4H, -OCH2-], 4.18 - 4.20 [t, 4H, -COOCH2-].
IR(KBr)ν(cm–1): 1255 (Si-CH3), 1735 (-COOR), 2250
(-CN).
2.3. Synthesis of N-α-Acrylamide-N-ε-tert-
butoxycarbonyl-L-lysine-tert-butylester
(Boc-LysAA-OtBu)
To a CHCl3 solution (150 mL) of N-ε-tert-butoxycar-
bonyl-L-lysine-tert-butyl ester (15 mmol) and triethyl-
amine (23 mmol) was added dropwise a CHCl3 solution
(30 mL) of acrylic acid chloride (18 mmol) at 4˚C. The
reaction mixture was washed with 1 M HCl and water,
and then the solution was evaporated under reduced
pressure. The crude Boc-LysAA-OtBu was purified by
passage through a basic alumina column with the mixed
solvent of dichloromethane and hexane (50/50 vol%).
Yield: 64.1%; mp: 62.0˚C - 62.5˚C. [α]D
20 = +20.1°.
1H-NMR (CDCl3, TMS): δ = 1.24 - 1.47(m, 2H, >CH-
CH2-CH2-CH2-CH2-), 1.43(s, 9H, -C(CH3)3), 1.47(s, 9H,
-C(CH3)3), 1.63 - 1.82 (m, 2H, >CH-CH2-CH2-CH2-CH2-),
1.81 - 2.05 (m, 1H, >CH-CH2-CH2-CH2-CH2-), 2.99 -
3.24 (m, 2H, >CH-CH2-CH2-CH2-CH2-), 4.56 - 4.60 (m,
2H, >CH-CH2-CH2-CH2-CH2-), 5.63 - 5.79(m, 1H, H-
CH=CH-), 4.58 (br, 1H, -NH-), 6.12 - 6.29 (m, 1H, H-
CH=CH-), 6.24 (br, 1H, -NH-), 6.28 - 6.47 (m, 1H, CH2
=CH-). 13C-NMR (CDCl3, TMS): δ = 22.27 [-CH2-],
28.01 [-(CH3)3], 28.43 [-(CH3)3], 29.59 [-CH2-], 32.34
[-CH2], 40.12 [-CH2-], 52.44 [-CH], 79.02 [>C<], 82.21
[>C<], 126.77 [=CH2], 130.43 [=CH], 155.94 [-CONH],
164.95 [-COO-], 171.51 [-COO-]. Elemental Anal.;
Calcd for C18H32N2 356.461: C, 60.65%; H, 9.05%; N,
7.86%, Found: C, 60.46%; H, 9.06%; N, 8.08%.
2.4. Synthesis of Poly(LysAA-b-DMS)
A mixed solution of tetrahydrofuran and ethanol (40:60
vol%, 15 mL) containing Boc-LysAA-OtBu (10 mM),
azo-PDMS (10 mM) as an initiator, and 2-mercapto-
ethanol (2-ME: 0.10 - 1.0 mM) as a chain transfer agent
in a glass tube was degassed by the freeze-thaw tech-
nique with a liquid nitrogen bath and was sealed in a
vacuum. After polymerization at 60˚C for 20 h, the con-
tents of the tube were poured into a large quantity of
hexane to precipitate the precursor block copolymer,
poly (Bo c - Ly sAA- O t Bu- b-DMS)-1-3. The number-aver-
age molecular weight (Mn) was calculated from gel per-
meation chromatography (GPC) with a polystyrene stan-
dard. GPC analysis was conducted in a dimethylfor-
mamide solution with a TSK gel column (TOSOH HHR
6000, 5000, 4000, 3000, and 2000) with a TOSOH LC-
8020 GPC apparatus. The composition ratio of the Boc-
LysAA-OtBu polymer segment (p) and PDMS segment
(q) of poly(Boc-LysAA-OtBu-b-DMS) was calculated
using the N content of elemental analysis performed with
a Silber Hegner elemental analysensysteme GmbH. The
removal of the tert-butoxycarbonyl (Boc) and tert-Bu
(t-Bu) protecting groups in the Boc-LysAA-OtBu poly-
mer segment was carried out as follows. Poly (Boc-Lys-
AA-OtBu-b-DMS)-1-3 was dissolved in tetrahydrofuran,
80% trifluoroacetic acid was added, and the mixture was
stirred at 25˚C for 48 h. After removing the solvent under
reduced pressure and washing with diethyl ether, the
resulting copolymer was neutralized with 0.1 M sodium
hydroxide, and then purified by dialysis against water
using a seamless cellulose membrane (Cell-SepT1TM,
Spectra/PorTM Biotech, USA; MWCO 3500). Poly (Ly-
sAA-Zb-DMS)-1-3 was obtained from the aqueous solu-
tion by freeze drying.
2.5. Measurements of Critical Micelle
Concentration (CMC)
The fluorescence spectra of an artificial blood plasma
solution of poly(LysAA-b-DMS) were recorded with a
Shimadzu RF-1500 fluorophotometer [19]. DMAND
with a 364 nm excitation wavelength (λEX) was used as a
fluorescent probe for hydrophobicity. The concentration
of DMAND was adjusted to 1 μM by the addition of 20
μL of a solution containing 500 μM DMAND in metha-
nol to 10 mL of each amphiphilic poly(LysAA-b-DMS)
solution. The concentration of poly(LysAA-b-DMS) was
varied in the range of 34 - 1.0 × 104 mg·L–1. The CMC
was determined by the break point in the plot of the
fluorescence intensity and poly(LysAA-b-DMS) concen-
tration.
2.6. Measurements with Scanning Probe
Microscope (SPM)
SPM images of poly(LysAA-b-DMS) film were taken by
means of a Shimadzu SPM-9500J3 with Budget sensors
BS-Tap 300AI (Innovative Solutions Bulgaria, Ltd.,
USA). The copolymer film for SPM measurements was
prepared by casting 20 mL of an aqueous solution con-
taining a known amount of poly(LysAA-b-DMS) on a
mercury bath of 6.0 cm diameter. The solvent was eva-
porated by the freeze-dry method and a film with a thick-
ness of 0.06 - 0.08 mm was obtained.
2.7. Measurements of Degree of Thrombin
Activity Inhibition
Twenty microliters of thrombin (10 unit mL–1) dissolved
in a Tris buffer solution (pH 7.4), and 70 μL of poly
(LysAA-b-DMS) (2 mg·mL–1) were placed in a 96-well
microplate of diameter φ = 6.4 mm (Iwaki brand, Scitech
Copyright © 2011 SciRes. JBNB
Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers
Copyright © 2011 SciRes. JBNB
340
division, Asahi Techno Glass Corporation, Japan) and
shaken for 10 min. Next, 10 μL of calcium chloride
aqueous solution (300 mg·L–1) and 100 μL of S-2238
(0.1 - 1.0 mM), which specifically reacts to thrombin,
were added, and the mixture was shaken at 37˚C for 2 h.
The degree of inhibition of thrombin activity was evalu-
ated from the changes in the maximum absorption
strength (λmax) of 405 nm of p-nitroaniline, which was
derived from S-2238, with a microplate reader (BIO-
TEC Instruments, Inc., Elx808, USA). Kinetics parame-
ters, Michaelis constant (KM) and catalyst rate constant
(kcat) for the reaction of thrombin and S-2238 in the
presence of poly(LysAA-b-DMS) were calculated from a
Lineweaver-Burk plot.
10 min. Next, 70 μL of S-2251 (0.1 - 1.0 mM) solution
and 40 μL of t-PA (0.25μg·mL–1) solution were added
and the mixture was shaken at 37˚C for 2 h. The degree
of plasminogen activity by t-PA in the presence of poly
(LysAA-b-DMS) was evaluated based on the amount of
p-nitroaniline, which was derived from S-2251, with a
microplate reader (BIO-TEC Instruments, Inc., Elx808,
USA). KM and kcat for the enzymatic reaction in the
presence of poly(LysAA-b-DMS) were determined ac-
cording to the method mentioned above.
3. Results and Discussion
3.1. Characterization of Block Copolymers
Amphiphilic block copolymers consisting of hydrophilic
and hydrophobic segments can be prepared by the po-
lymerization of hydrophilic monomers initiated with a
polymer initiator such as azo-PDMS. A series of amphi-
philic block copolymers poly(LysAA-b-DMS)-1-3 with
different molar ratios of poly(LysAA) to PDMS segment
(p:q) were synthesized according to Scheme 1. First, pre-
2.8. Measurements of Degree of Plasminogen
Activation
Twenty microliters of plasminogen (34 μg·mL–1) dis-
solved in a Tris buffer solution (pH 7.4), and 70 μL of
poly(LysAA-b-DMS) (2 mg·mL–1) were placed in a 96-
well microplate of diameter φ = 6.4 mm, and shaken for
Scheme 1. Preparation of amphiphilic block polymers.
Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers341
cursor block copolymers, poly(Boc-LysAA-OtBu-b-
DMS)-1-3, were obtained by radical polymerization of
Boc-LysAA-OtBu, in which amino and carbonyl groups
of the L-lysine moieties were protected with tert-butoxy-
carbonyl (Boc) and tert-butyl (t-Bu) groups, respectively,
initiated with azo-PDMS in the presence of chain trans-
fer agent 2-ME, and varying the molar ratio of 2-ME to
Boc-LysAA-OtBu in the feed from 10:0.1, 10:0.5, and
10:1. For the higher concentration of 2-ME, the smaller
molecular weight poly(Boc-LysAA-OtBu) segment was
obtained based on the larger chain-transfer constant of
2-ME. Second, the removal of the protecting groups of
poly (Bo c - Ly sAA- O t Bu- b-DMS)-1-3 was carried out in
80% trifluoroacetic acid aqueous solution, and then puri-
fication by dialysis against water to give poly(LysAA-
b-DMS)-1-3. Block copolymer, poly(Boc-LysAA-OtBu-
b-DMS) with differing chain lengths of the LysAA
polymer segment, was obtained as tabulated in Table 1,
along with the results for poly(Boc-LysMA-OtBu- b-
DMS) [11], where Boc-LysMA-OtBu represents N-α-
methacrylamide-N-ε-tert-butoxycarbonyl-L-lysine-tert-
butyl ester. The composition ratio of poly(Boc-LysAA-
OtBu) (p) : PDMS (q) was then altered to p:q = 0.35:0.65
- 0.58:0.42. With an increase in the concentration of
2-ME, the number-average molecular weight (Mn) of the
poly(Boc-LysAA-OtBu) segment decreased from Mn =
1.56 104 to 0.88 104 and the weight-average molecu-
lar weight (Mw) decreased to Mw = 2.52 104 – 1.02
104, whereas the molecular weight of the PDMS seg-
ment derived from azo-PDMS had a constant molecular
weight of Mn = 3.9 103. Comparing the molecular
weight of poly(Boc-LysAA-OtBu-b-DMS) and poly-
(Boc-LysMA-OtBu-b-DMS), it can be seen that Boc-
LysAA-OtBu without the methyl moiety at the α-posi-
tion of the monomer causes a somewhat smaller molecu-
lar weight and larger degree of polydispersion (Mw/Mn)
than those of Boc-LysMA-OtBu, because the chain
transfer constant of Boc-LysAA-OtBu to 2-ME is ex-
pected to be larger than that of Boc-LysMA-OtBu based
on the higher monomer reactivity of Boc-LysAA-OtBu.
The propagating rate constant (kp) of Boc-LysAA-OtBu
is also expected to be higher than that of Boc-LysMA-
OtBu, though the kp is not known yet. For example, the
kp of methyl acrylate and methyl methacrylate are 2090
and 734 L·mol–1·sec–1, respectively [13]. The protecting
groups in the Boc-LysAA-OtBu polymer segment were
then hydrolyzed in 80% trifluoroacetic acid, converting it
to a hydrophilic LysAA polymer segment, and poly-
(LysAA-b-DMS)-1-3 was obtained. The removal of the
protecting groups (δ = 1.42 ppm, 1.46 ppm) was con-
firmed by 1H-NMR spectroscopy. The removal of the
protecting group of Boc-LysAA-OtBu resulted in a
change in its solubility. Poly(Boc-LysAA-OtBu-b-DMS)
is insoluble in water but soluble in acetone, diethyl ether
and tetrahydrofuran, yet poly(LysAA-b-DMS) is soluble
in water but insoluble in the organic solvents mentioned
above.
3.2. Self-Organization
The self-organization of poly(LysAA-b-DMS) was first
confirmed by 1H-NMR measurements. The spectra of
poly(LysAA-b-DMS)-1 in CD3OD and D2O, used as ty-
pical examples, are shown in Figure 1. The proton signal
(HB) of the methyl moiety in PDMS that appeared in
CD3OD weakened gradually in D2O, whereas the signal
of HA in the LysAA moiety sharpened gradually with
increasing water content. The half-width of the two in-
dependent peaks as a function of the D2O content in
CD3OD is shown in Figure 2. The half-width of HB (δ =
0 ppm) broadened gradually with an increase in the D2O
content.
However, the half-width of HA (δ = 2.8 ppm) became
narrower with an increase in the water content. The line
broadening of the proton signals of the methyl group in
PDMS in aqueous medium is ascribed to the restricted
molecular motion of the PDMS chains upon self-organ-
ization [20,21]. Nevertheless, the mobility of the hydro-
philic segments increased when the solvent polarity in-
Table 1. Preparationa) and Charactarization of poly(BocLysAA-OtBu-b-DMS).
Copolymer 2-ME Yield Elemental analysis P:qb Mnc
mmol % C(%) H(%) N(%) ×10–4 ×10–4 Mwc Mw/Mn
poly(Boc LysAA-OtBu-b-DMS) 1 0.10 60.1 58.21 8.56 7.07 0.65:0.35 1.56 2.52 1.62
2 0.50 58.7 55.33 8.48 6.54 0.51:0.49 1.29 1.69 1.31
3 1.00 50.2 54.28 8.41 6.12 0.42:0.58 0.88 1.02 1.16
poly(Boc LysAA-OtBu-b-DMS) 1d 0.10 66.4 58.52 9.02 6.63 0.59:0.41 2.32 3.41 1.47
2 0.50 63.0 56.84 8.95 6.27 0.49:0.51 1.52 1.91 1.26
3 1.00 58.7 54.83 8.63 5.66 0.38:0.62 1.25 1.39 1.11
a) Reaction condition: Boc LysAA-OtBu or Boc LysMA-OtBu: 10.0 mmol. DMS: 100mmol, 2-ME: 0.10 - 1.0 mmol, Mixed solvent of tetrahydrofuran-etha-
nol(40:60 vol %) 15 Ml, 60˚C, 20 hr; b) Molar ratio (p:q) in block copolymer was calculated from nitrogen content obtained by elemental analysis; c) Mn and
Mw of block copolymers were estimated from GPC; d) Ref.12.
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Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers
342
Figure 1. 1H-NMR spectra of poly(LysAA-b-DMS)-1 in a
mixed solvent of D2O and CD3OD, [poly(LysAA-b-DMS)-1]
= 1.0 × 10-4 mg·L–1.
Figure 2. Effect of D2O on the half-width of the 1H-NMR
proton signal of poly(LysAA-b-DMS)-1, :HA, :HB, see
Scheme 1.
creased. These data suggest that poly(LysAA-b-DMS)
self-aggregates have microdomains provided by both a
core of hydrophobic PDMS segments and a mobile shell
of hydrophilic poly(LysAA) segments. The self-organi-
zation ability of poly(LysAA-b-DMS-1-3 was also veri-
fied in a solution of artificial blood plasma by a fluores-
cent probe method using DMAND at 20˚C [8,17]. The
concentration of poly(LysAA-b-DMS)-3 increased, as
did the fluorescent intensity from DMAND, thus show-
ing that the self-organization of the PDMS segment due
to hydrophobic interaction created a hydrophobic core
that absorbed DMAND. Note that poly(LysAA-b-DMS)-3
is not itself fluorescent. By plotting the largest value of
fluorescent intensity and concentration of poly(LysAA-
b-DMS)-3, we can see the breaking point at which the
fluorescent intensity begins to suddenly increase (1000
mg·L-1), as shown in Figure 3, and we take this as the
critical micelle concentration (CMC) for self-organiza-
tion. The CMCs of poly(LysAA-b-DMS)-1-3 with diffe-
rent composition ratios of the copolymer are summarized
in Table 2. While the molecular weight of the PDMS
segment is a constant, CMC decreases from 4000 to 1000
mg·L–1 as the amount of the hydrophilic LysAA polymer
segment decreases.
Figure 3. Relationship between the fluorescensity of DMAND
and concentration of poly(lysAA-b-DMS)-3, [poly(LysAA-b-
DMS)-3] was varied: 34 to 1.0 × 104 mg·L–1 [DMAND] = 1.0
μmol·L–1, λEX = 364 nm, CMC = 1000 mg·L–1.
While the particle sizes of the poly(LysAA-b-DMS)-1-3
micelles have not yet been measured, it has been re-
ported that in the case of poly(LysMA-b-DMS)-1-3, the
diameters of micelles were from 220 to 270 nm as mea-
sured by light scattering [11]. It is presumed that poly
(LysAA-b-DMS) self-assembles to form nanometersi-
zed polymer micelles in water, similar to particles of
poly(LysMA-b-DMS). Considering the core-shell mice-
lle, this means that hydrophobic interactions of the PD-
MS segment in water play a dominant role with the de-
crease in the length of the LysAA polymer segment in
the shell, allowing a hydrophobic core to form even
when the concentration of poly(LysAA-b-DMS)-3 is low
[13]. The self-assembled poly(LysAA-b-DMS) is ex-
pected to play a role as a carrier molecule for DDS,
making it possible to stably hold hydrophobic medicines
in the core when placed in water.
3.3. Microphase-Separated Structure
As amphiphilic poly(LysAA-b-DMS) is expected to be
applied in biomedical materials such as artificial veins,
its film manufactured by freeze drying was morphologi-
cally analyzed with a scanning probe microscope (SPM)
in order to confirm its microphase-separated structure.
The poly(LysAA-b-DMS) was found to have a micro-
phase-separated structure with a domain size ranging
from several tens to several hundreds of nanometers, as
shown in Figure 4. The red and yellow areas of the
phase image represent the soft domain from PDMS,
whereas the blue areas represent the hard domain from
poly(LysAA). In the case of poly(LysAA-b-DMS)-1,
based on the composition ratio p:q = 0.65:0.35, the blue
areas of the poly(LysAA) segment are more numerous
than the yellow areas representing PDMS. On the other
hand, with poly(LysAA-b-DMS)-3, based on the compo-
sition ratio p:q = 0.42:0.58, the red and yellow areas
C
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Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers343
Table 2. Kinetics Parameters for the Reaction of Thrombin and S-2238 and for the Reaction of t-PA and S-2251 in the Pres-
ence of poly(LysAA-b-DMS).
S-2238a S-2238a
CMC Vmax K
M k
cat k
cat/KM V
max KM kcat kcat/KM
Copolymer p:qc
mg·L–1 ×10–2
mM·min–1 mM ×10–5
min–1
×10–5
mM·min–1
×10–3
mM·min–1 mM ×10–6
min–1
×10–5
mM·min–1
poly(LysAA-b-DMS) 1 0.65:0.35 4000 2.13 0.11 4.26 3.87 8.63 0.07 5.94 8.50
2 0.51:0.49 3000 2.11 0.11 4.22 3.84 8.49 0.07 5.83 8.37
3 0.42:0.58 1000 2.02 0.10 4.04 4.04 8.31 0.07 5.71 8.19
poly(LysAA-b-DMS) 1 0.59:0.41 2000 1.96 0.17 3.92 2.31 6.86 0.09 4.93 5.26
2 0.49:0.51 1000 1.86 0.16 3.72 2.33 6.31 0.09 4.35 4.83
3 0.38:0.62 500 1.82 0.15 3.64 2.43 6.03 0.09 4.02 4.62
Control - - 0.14 1.62 2.68 1.91 0.09 4.01 1.28 1.72
a) [Thrombin] = 10 unit·mL–1, [poly(LysAA-b-DMS)] = 2 mg·mL–1, [S-2238] = 0.1 - 1.0 mM, Ph 7.4, 37˚C, 2 h; b) [plasminogen] = 34 μg·mL–1, [t-PA] = 0.25
μg·mL–1 [poly(LysAA-b-DMS)] = 2 mg·mL–1, [S-2251] = 0.1~1.0 mM pH 7.4, 37˚C, 2 h; c) p:q represents the molar ratio of poly(LysAA) and PDMS seg-
ments; d) Ref.12.
Figure 4. SPM images of poly(LysAA-b-DMS) films.
outnumber the blue. The ratio of poly(LysAA) segment
(p) in the copolymer decreased with increasing amounts
of 2-ME due to lowering of the molecular weight of
poly(LysAA) based on the chain transfer reaction. In this
manner, we can see that poly(LysAA-b-DMS) possesses a
sea-island structure, made up of domains corresponding
to the composition ratio.
3.4. Evaluation of Inhibition of Thrombin
Activity
In order to use poly(LysAA-b-DMS) in biomedical ma-
terials such as DDS carrier molecules for intravenous
injection and artificial blood vessels, it must have a high
blood compatibility. To confirm this, the inhibition of
thrombin activity was evaluated. Thrombus formation in-
cludes a process whereby fibrinogen is converted to fi-
brin by thrombin. Furthermore, the fibrin forms a cross-
linked structure by transglutaminase XIIIa and completes
the blood clotting. The level of antithrombotic activity
can be verified by the enzymatic reaction of thrombin
and a synthetic substrate S-2238 in the presence of
poly(LysAA-b-DMS)-1-3 as shown in Scheme 2 [22].
The reaction in the absence of poly(LysAA-b-DMS) was
used as a control. The reaction rate [V] is monitored by
the absorption change at 405 nm of p-nitroaniline de-
rived from S-2238 over time when thrombin causes the
C-terminal of the residual arginine to hydrolyze [16].
A Lineweaver-Burk plot of this reaction is shown in
Figure 5. [V] is the rate in terms of the amount of
p-nitroaniline produced by one unit of thrombin in one
minute, while [S] represents the initial concentration of
S-2238. By the slope of the line (KM/Vmax) and the in-
tercept (1/Vmax), kinetics parameters such as the maxi-
mum rate of enzymatic reaction (Vmax), Michaelis con-
stant (KM) and the catalyst constant of enzyme (kcat) in
the presence of poly(LysAA-b-DMS)-3 were calculated
as summarized in Table 2, together with those data ob-
tained in the presence of poly(LysMA-b-DMS). The
Vmax was 2.02 102 - 2.13 102 mM·min–1 and that of
the control was 0.14 102 mM·min–1. With increasing
molar ratio of the poly(LysAA) segment, kcat increased
from 4.04 105 - 4.26 105 min–1, while KM remained
almost constant. kcat/KM decreased from 4.04 105 -
3.87 105 mM–1·min–1 with increasing molar ratio of the
poly(LysAA) segment. KM, kcat, and kcat/KM were 0.06 -
0.07, 1.51 - 1.59, and 2.12 - 2.03 times those of the con-
trol, respectively. The effect of the molecular weight of
the poly(LysAA) segment on the kinetics data cannot be
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Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers
344
Scheme 2. Enzymatic reaction of thrombin/S-2238 and plasminogen /S-2251 in the presence of poly(LysAA-b-DMS).
[V]
–1
/min·mM
–1
Figure 5. Lineweaver-Burk plots for the reaction of throm-
bin and S-2238 in the absence (:control) and in the pre-
sence of poly (LysAA-b-DMS)-3 () in a Tris buffer solution
(pH = 7.4) at 37˚C, [Thrombin] = 1 unit, [S-2238] was varied
from 0.05 to 0.5 mM. [poly(LysAA-b-DMS)-3] = 0.7 mg·L–1.
regarded as significant. Comparing poly(LysAA-b-DMS)
and poly(LysMA-b-DMS), the former shows larger kcat
and kcat/KM than the latter with kcat (3.64 105 - 3.92
105 min–1) and kcat/KM (2.43 106 - 2.31 106 mM–1·min–1).
From the difference in kcat/KM of the enzymatic reaction
in the presence of the two different copolymers, poly
(LysAA-b-DMS) shows a somewhat stronger tendency to
promote thrombin activity than poly(LysMA-b-DMS).
The poly(LysAA) segment can interact with thrombin
and promote the enzymatic reaction more efficiently than
the poly(LysMA) segment, due to the differences in their
flexibility. From the kinetics parameters of the enzymatic
reaction, it was found that neither of the two copolymers
could inhibit the thrombin activity.
3.5. Evaluation of Activation of Tissue
Plasminogen Activator (t-PA)
Plasmin, which converts fibrinogen to a fibrin net, exists
as a precursor called plasminogen in the blood. Plasmi-
nogen is activated by t-PA, becoming the fibrinolytic
agent plasmin, and dissolving the fibrin net. At this point,
the L-lysine-binding sites (LBS) of the plasminogen or
t-PA interact with the L-lysine group of fibrin. The de-
gree of activation can be verified using the enzymatic
reaction of plasmin and a synthetic substrate S-2251 [23].
It is known that polymeric materials with surfaces modi-
fied by L-lysine groups exhibit strong interaction with
plasminogen, increasing the fibrinolytic activity [24-26].
We have also reported that plasminogen activation by
t-PA using S-2251 in the presence of poly(LysMA) in-
creases fibrinolytic characteristics compared to a control
in the absence of poly(LysMA) [12].
Based on this finding, fibrinolytic activity was verified
by enzymatic reaction of plasminogen and t-PA using
S-2251 in the presence of poly(LysMA-b-DMS) as
shown in Scheme 2. A Lineweaver-Burk plot of this en-
zymatic reaction is shown in Figure 6. Kinetic pa- rame-
ters in the presence of poly(LysAA-b-DMS)-3 were cal-
culated as summarized in Table 2, together with those
parameters obtained in the presence of poly(LysMA-b-
DMS). Vmax, KM, and kcat increased with increasing mo-
lar ratio of the poly(LysAA) segment. The Vmax of the
transformation of plasminogen to plasmin by t-PA in
C
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Blood Compatibility of Amphiphilic Poly(N-α-acrylamide-L-lysine-b-dimethylsiloxane) Block Copolymers345
[V]
–1
/min·mM
–1
Figure 6. Lineweaver-Burk plots for the reaction of plas-
minogen by t-PA and S-2251 in the absence (: control) and
in the presence of poly (LysAA-b-DMS)-3 () in a Tris buf-
fer solution (pH=7.4) at 37˚C, [Plg] = 34 μL–1, [S-2251] was
varied from 0.035 to 3.5 mM. [poly(LysAA-b-DMS)-3] = 0.7
mg·mL–1, [t-PA] = 0.2 μg·mL–1.
the presence of poly(LysAA-b-DMS)-1-3 using S-2251
was found to be 92 - 96 times faster than the control,
proving its increased fibrinolytic activities. KM was 0.017
times that of the control, while kcat was 4.46 - 4.64 times
that of the control. It is suggested that the blood com-
patibility of the L-lysine group in poly(LysAA-b-DMS) is
due to the strong interaction of the L-lysine residue and
the L-lysine-binding sites (LBS) of the plasminogen [12].
With increasing molar ratio of the poly(LysAA) segment,
Vmax, kcat, and kcat/KM increased from 8.31 103 - 8.63
103 mM·min–1, 5.71 106 - 5.95 106 min–1, and 8.19
105 - 8.50 105 M–1·min–1, respectively, while KM was
constant. Comparing poly(LysAA-b-DMS) and poly
(LysMA-b-DMS), the former shows larger kcat and
kcat/KM than the latter with kcat (4.02 106 - 4.93 106
min–1) and kcat/KM (4.62 105 - 5.26 105 mM–1·min–1).
These data indicate that poly(LysAA-b-DMS) shows a
stronger tendency to promote fibrinolytic activity than
poly(LysMA-b-DMS). It is explained that the L-lysine
residue of poly(LysAA) segment can interact with t-PA
easier and accelerate the fibrinolysis better than the poly
(LysMA) segment can based on the difference in their
flexibilities as mentioned above. It should be emphasized
from the difference in kcat/KM of the enzymatic reaction
in the presence of different block copolymers that poly
(LysAA-b-DMS) exhibits much more tendency to pro-
mote fibrinolysis than poly(LysMA-b-DMS) does.
3.6. Surface Plasmon Resonance Measurements
From the surface plasmon resonance measurements on
the polymer immobilized sensor surface, the association
equilibrium constants (KA) for the enzymatic reaction of
plasminogen with poly(α-LysAA) and poly(α-LysMA)
were determined to be 389 109 and 0.281 109, re-
spectively. KA for the reaction of t-PA with poly(α-Lys-
AA) and poly(α-LysMA) were found to be 155 109 and
6.24 109, respectively [27]. This means that poly(α-
LysAA) binds more strongly to plasminogen and t-PA
than poly(α-LysMA). Based on these results, it was found
that poly(LysAA-b-DMS) promotes thrombin activity
and is a better enhancer of fibrinolytic activity than poly
(LysMA-b-DMS) in promoting the conversion of plas-
minogen into plasmin.
4. Conclusions
From the results obtained here, the following conclusions
can be drawn:
1) Because the nanometer-sized micelles derived from
self-assembly of poly(LysAA-b-DMS) had microdomains
consisting of a hydrophobic core of PDMS segments and
a mobile shell of hydrophilic poly(LysAA) segments,
lipophilic drugs could be included in the hydrophobic
core to form a novel intravenous injection DDS.
2) A film of amphiphilic copolymer poly(LysAA-b-
DMS) adopts a microphase-separated structure. The co-
polymer is expected to be applied in biomedical poly-
meric materials for artificial organs such as blood ves-
sels.
3) The inhibition of thrombin activity is promoted by
the presence of poly(LysAA-b-DMS) in the enzymatic
reaction system more than poly(LysMA-b-DMS).
4) Because it can promote the conversion of plasmi-
nogen into plasmin by t-PA, poly(LysAA-b-DMS) en-
hances the fibrinolytic activity of t-PA, thus increasing
blood compatibility.
5) Both activities of the inhibition of thrombin and fi-
brinolysis are influenced by the differences in the molar
ratio of poly(LysAA) and the flexibility of the main chain
hydrophilic polymer segment. The kinetic parameters of
the enzymatic reaction in the presence of poly (LysAA-
b-DMS) containing the flexible poly(LysAA) segment
showed larger KM and kcat than poly(LysMA-b-DMS).
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