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1.#Introduc,on�
6.#Conclusions �
Surface6ini,ated#atom#transfer#radical#polymeriza,on#[1]�
High6density#polymer#brush#surfaces#with#controlled#physicochemical#proper,es#were#prepared,#as##model#surfaces#to#understand#the#rela,onship#between#protein#adsorp,on#and#interac,on#force. �
5.#Protein#Adsorp,on#Behavior �
Electrosta,c#interac,on#forces#and#hydrophobic#interac,on#forces#genera,ng#on#surfaces#were#clearly#separated#using#systema,cally#prepared#polymer#brush#surfaces.#
�$Long)range$strong$repulsion$in$pure$water$��Weakening$with$increase$of$ionic$strength$��→$Electrosta;c$interac;on$
Weak$repulsion$independent$of$ionic$strength$�→$No$specific$interac;on$
Electrosta,c#and#hydrophobic#interac,on#forces#hinder#the#reversible#detachment#of#proteins#from#the#surfaces,#which#would#lead#to#high#protein#adsorp,on.#
No$interac;on$force$when$retrac;ng$a?er$contact$$
4.#Surface#Interac,on#Forces �
3.#Polymer#Brush#Surfaces �
•$Gra?$density:$High$enough$to$form$the$high)density$polymer$brush$surface$[2]$•$Hydrophilicity:$High$in$wet$condi;on$[except$for$poly(BMA)]$$
����������$$Constant$regardless$of$ionic$strength$in$medium$•$Surface$poten;al:$Different$by$charge$proper;es$of$monomer$units$
[3]$Y.$Inoue$et#al.,$React.#Funct.#Polym.,$71,$350)355,$2011.�
Protein$ adsorp;on$ forces$ on$ well)defined$ polymer$ surfaces$ were$ analyzed.$ The$ electrosta;c$ or$ hydrophobic$interac;on$genera;ng$on$ the$ vicinity$of$ protein$ adsorp;ve$ surfaces$plays$ a$ role$ as$ the$ force$which$ inhibit$ the$detachment$of$proteins$from$the$surface.$�
The# fabrica,on# of# surface# which# enables# proteins# to# easily#detach# from# the# surface# would# be# important# to# suppress#protein#adsorp,on#on#biomaterials#surfaces.#
Analysis$of$$Protein$Adsorp;on$Force$Generated$at$Polymer$Surfaces$for$Designing$Non)biofouling$Materials$
Sho$SAKATA,$Yuuki$INOUE,$and$Kazuhiko$ISHIHARA�Department$of$Materials$Engineering,$School$of$Engineering,$The$University$of$Tokyo$
e)mail:[email protected])tokyo.ac.jp$$$$hep://www.mpc.t.u)tokyo.ac.jp$
$Polymer$Gra?$density$ Contact$angle$(°)$ ζ)Poten;al$(chains/nm2)$ in$water$ in$PBS$ (mV)$
$Poly(MPC)$ 0.33$ $$9$ $$9$ $$)5.9$$Poly(TMAEMA)$ 0.45$ 17$$ 14$$$$$ $64.9$$Poly(SPMA)$ 0.55$ 13$ 11$ )74.0$$Poly(BMA)$ 0.75$ 73$ 73$ )37.2$
Penetra;on$of$proteins$into$polymer$brush$layers$is$negligible.$�
Poly(TMAEMA)$$(Ca;onic)�
CH3�
C=O�CH3�
CH3�
O�O)R
SiO
2/Si$ )OH�
)OH�
)O)Si)(CH2)10)O)C)C)(CH2)C)100)Br�
Substrate � Ini;ator$ Polymer$chain �
R$:$Side$chains$with$various$chemical$structures�
Poly(MPC)$$(Zwieerionic)�
Poly(BMA)$$(Hydrophobic)�
Poly(SPMA)$$(Anionic)�
(CH2)3CH3�
(CH2)2N+(CH3)3$��Cl)�
(CH2)2OPO(CH2)2N+(CH3)3�O�
O)�
(CH2)3SO3)$�$K+�
[1]$S.$Sakata$et#al.,$Langmuir$30,$2745)2751,$2014.$
MPC$TMAEMA$SPMA$BMA�
:$2)Methacryloyloxyethyl$phosphorylcholine$:$2)Trimethylammoniumethyl$methacrylate$:$3)Sulfopropyl$methacrylate$:$n)Butyl$methacrylate�
Degree$of$polymeriza;on$(DP)$was$controlled$by$the$ra;o$of$monomer$and$free$ini;ator$in$feed.$
Development$of$regenera;ve$medicine,$cell$engineering,$and$biomaterials$engineering$→$Biological$responses$at$interfaces$should$be$precisely$understood$and$controlled.$��$
It$is$important$to$understand$each$process$of$protein$adsorp;on$phenomena$based$on$interac;on$forces.$
Cell � Tissue �Protein �Biological$responses$on$materials$surface �
Ini;al$event�
Mul;layer$adsorp;on�
Approach$
Detachment �
Conforma;on$change$
Protein)protein$interac;on�
�$Polymer$brush$layer$probe$�$Protein)immobilized$probe$
→$Forces$on$each$surface$→$Direct$interac;on$force$with$protein$
(Thickness:$ca.10$nm)$
Force$(nN)�
Distance$(nm)�
Aerac;ve�
Repu
lsive�
0�
0�
Indirect#long6ranged##Interac,on#force�
Direct#interac,on#force�
Poly(MPC)! Poly(TMAEMA)! Poly(SPMA)!
!50$ 0$ 50$ 100$ 150$ 200$ 250$Distance)(nm)�
Force)(nN)�
―)0)mmol/L)―)1.5)mmol/L)―)150)mmol/L)
!2.0$
0.0$
2.0$
4.0$
6.0$
8.0$
10.0$
12.0$
!2.0%
0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
12.0%
!50% 0% 50% 100% 150% 200% 250%Distance)(nm)�
Force)(nN)�
―)0)mmol/L)―)1.5)mmol/L)―)150)mmol/L)
!2.0%
0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
12.0%
!50% 0% 50% 100% 150% 200% 250%Distance)(nm)�
Force)(nN)�
―)0)mmol/L)―)1.5)mmol/L)―)150)mmol/L)
+�+� +�+�+�+� +�+�
−�−� −�−�−�−� −�−�
−�+� −�+� −�+�
−�+� −�+� −�+�
!4.0%
!2.0%
0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
!50% 0% 50% 100% 150% 200% 250%Distance)(nm)�
Force)(nN)�
―)Approach)―)Retract)
MPC,$TMAEMA,$SPMA$$$(Retract$curve)$
!4.0%
!2.0%
0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
!50% 0% 50% 100% 150% 200% 250%Distance)(nm)�
Force)(nN)�
―)Poly(MPC))―)Poly(TMAEMA))―)Poly(SPMA))�(150)mmol/L))
Poly(BMA)!
�$No$interac;on$force$on$approach$��Strong$aerac;on$on$retrac;on$��→$Hydrophobic$interac;on$
0.0!
1.0!
2.0!
3.0!
4.0!
Poly(MPC)$Poly(T
MAEMA)$
Poly(SPMA)$
Poly(BMA)$
Interac;on
$force$(nN)�
0.0!
1.0!
3.0!
5.0!
7.0!
Poly(MPC)$Poly(T
MAEMA)$
Poly(SPMA)$
Poly(BMA)$
Interac;on
$force$(nN)� 6.0!
4.0!
2.0!
0.0!
1.0!
2.0!
3.0!
4.0!
Interac;on
$force$(nN)�
0.0!
2.0!
4.0!
5.0!
7.0!
Interac;on
$force$(nN)� 6.0!
3.0!
1.0!
Polymer$brush$surfaces$�Surface)ini;ated$atom$transfer$radical$polymeriza;on$�→$Uniform$extension$of$polymer$chains$from$surface)$��$$immobilized$ini;ators$
Refined$understanding$of$various$molecular$interac;on$by$combina;on$of$polymer$brush$surfaces$and$AFM$
Understanding#protein#adsorp,on#based#on#analysis#of#molecular#interac,on#opera,ng#on#the#surfaces#2.#Objec,ve�
Poly(MPC)�Poly(T
MAEMA)�Poly(B
MA)�Poly(S
PMA)�
Adsorbe
d$am
ount$(n
g/cm
2 )�
700�
600�
500�
400�
300�
200�
0�
100�
N.D.�
N.D.�
N.D.�
Albumin$())�
Poly(MPC)�Poly(T
MAEMA)�Poly(B
MA)�Poly(S
PMA)�
N.D.�
N.D.�
1200�
Adsorbe
d$am
ount$(n
g/cm
2 )�
1000�
800�
600�
400�
200�
0�
Lysozyme$(+)�
��10$mmol/L$��150$mmol/L�
��10$mmol/L$��150$mmol/L�
→$Various$interac;on$forces$on$surfaces �
Poly(MPC)$Poly(TMAEMA)!Poly(SPMA)$Poly(BMA)$
→$Liele$interac;on!→$Strong$interac;on$with$albumin!→$Strong$interac;on$with$lysozyme!→$Strong$Interac;on$with$both$proteins!
Poly(MPC)$Poly(TMAEMA)!Poly(SPMA)$Poly(BMA)$
→$Liele$adsorp;on$(<$5.0$ng/cm2)!→$High$adsorp;on$of$albumin!→$High$adsorp;on$of$lysozyme!→$Independent$of$charge$proper;es!
AFM#f6d#curves#on#retrac,ng�AFM#f6d#curves#on#approaching �
Amount#of#adsorbed#proteins � Protein6protein#interac,on# �Surface6protein#interac,on �Surface$plasmon$resonance$measurement$�)$1.0$mg/mL$(PBS;$I$=$10,$150$mmol/L,$pH$7.4)$�)$37°C,$500$μL/min,$30$min$
AFM$f6d$curve$measurement$[3]$�)$Polymer$brush$surface$�)$PBS$(I$=$150$mmol/L,$pH$7.4)$
Zwieerionic$
Albumin$())� Lysozyme$(+)�
Physicochemical#surface#proper,es �
AFM$f6d$curve$measurement$�)$Protein$pre)adsorbed$surface$�)$PBS$(I$=$150$mmol/L,$pH$7.4)$
[2]$Y.$Tsujii$et#al.,$Adv.#Polym.#Sci.,$197,$1)45,$2006.�
Interac;on$force$with$proteins$drama;cally$decreased$on$the$high$protein$adsorp;on$surfaces.$�→$Protein$would$not$adsorb$on$the$surfaces$with$$$$$$$$$enough$amount$protein$layer.$
Difficult$to$detach$
Albumin$())� Lysozyme$(+)�
Zwieerionic$surface$�→$No$specific$interac;on$$Ca;onic$Surface$Anionic$Surface$�→$Electrosta;c$interac;on$$Hydrophobic$Surface$�→$Hydrophobic$interac;on$$$Single$or$No$interac;on$force$operated$on$the$surfaces.$
Dissolved$protein$ Easy$detachment$
Ca;onic$ Anionic$ Hydrophobic$ Zwieerionic$ Ca;onic$ Anionic$ Hydrophobic$
No$adsorp;on$
+� +� +� +�+� +� +�
−� −� −� −�−� −� −�−� −� +� +�
App
roach�
Retract�
Polymer$brush$surface$
AFM$probe�
Ini;ator �
Monomer �
Substrate �
Atomic$force$microscopy$(AFM)$��$Acquisi;on$of$force)versus)distance$(f6d)$curve$��$Evalua;on$of$various$interac;on$by$modified$probe��$
��$Polymer$chains$composed$of$single$monomer$unit$��$Controlled$surface$property$by$chemical$structure$��→$Clarifica;on$of$surface$interac;on$force$
Poly(MPC)$$ Poly(TMAEMA)$$
Poly(BMA)$
Poly(SPMA)$
Albumin� Lysozyme�
1$nm�
��Albumin$from$bovine$serum$(pI$4.8)$��Lysozyme$from$chicken$egg$white$(pI$11.1)$
→$Nega;ve$net$charge$at$pH$7.4$→$Posi;ve$net$charge$at$pH$7.4$
��Before$/$��A?er$immersion$in$protein$solu;on�
Enough$amount$protein$layer�
No$interac;on�Equilibrium$
state�