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:$sakata@mpc.t.u)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�