Particles 2011
Stimuli‐Responsive Particles and Particle Assemblies
9‐12 July 2011 Berlin, Germany
y g
Takao Aoyagi
National Institute for Materials Science
International Center for Materials Nanoarchitectonics(MANA)
1‐1, Namiki, Tsukuba Ibaraki, 305‐0044 JAPAN
1
1. Brief introduction of nano‐associates formation of thermo‐
responsive block copolymer
2. Concept of this study
3. Double thermo‐responsive block copolymers
4. Multi‐functional nano‐associates by simple mixing of block copolymers
2
Thermo‐responsive Amphiphilic Block Copolymer with PNIPAAm Block
Amphiphilic block copolymer
Hydrophilic
block
Hydrophobic
block
Amphiphilic block copolymers are expected to apply for drug delivery system because of forming nanoparticles such as micelle and
forming nanoparticles such as micelle and vesicle in aqueous solution.
Block copolymer consisting of PNIPAAm block
Block copolymer consisting of PNIPAAm block
PNIPAAm‐b‐P(Hydrophobic monomer)
Drug release by increasing temperature
J.E. Chung et al., J. Control. Rel. 1999, 62, 115
1999, 62, 115
J.E. Chung et al., J. Control. Rel.
PNIPAAm‐b‐P(Hydrophilic monomer)
Forming nanoparticle and drug capsule by increasing temperature
capsule by increasing temperature
S. Qin et al., Adv. Mater. 2006, 18, 2905
3
Double Thermo‐Responsive Block Copolymer
p
p y
Double thermo‐responsive b oc copo y e
block copolymer
Thermo‐responsive Thermo‐responsive
block 1 (LCST 1)
block 1 (LCST 1)
block 2 (LCST 2)
block 2 (LCST 2)
Drug
T < LCST1
Self‐assembly
and Drug loading
LCST1 < T < LCST2
Drug release
LCST2 < T
D
Drug Carrier
C i
4
co
CH2 CH
C
n
O
C
Monomer 1
NH
n
O
Monomer 2
NH
CH
CH3
co
CH2 CH
CH
CH3
Copolymer
block 1
CH3
CH3
Copolymer
block 2
Tran
nsmittance
e / %
Control of Responsive Temperature by Changing Copolymer
l
Contents
in water
T1
T2
p
/ oC
Temperature / By copolymerization with hydrophobic or hydrophilic monomer, the LCST of PNIPAAm can be controlled. Therefore, a block copolymer with different copolymerized block show double thermo‐response which can be tuned easily.
5
Development of Multi‐Stimuli‐Responsive Block Copolymers as Nano‐associates for Drug Delivery Applications
N
i t f D
D li
A li ti
Stimuli‐responsive
block
Selected stimuli‐responsive block
Thermo response
Drug release
Biocompatibility
Common thermo‐responsive
block
Molecular recognition
Targeting
g
g
Mixture
Heating
Heating
Target
Drug
Drug loading
Drug release
6
Double Thermo‐Responsive Block Copolymers with Controlled Assembly Temperature in Aqueous Solution
Controlled Assembly Temperature in Aqueous Solution
HN
Me6TREN
CuBr
DMF/Water
20 deg.
O
O
HN
O
b
HN
LCST control by copolymerization y p y
O
HN
HN
O
O
EBB
HN
O
O
PNIPAAm
(N) block
(N) block
C
O
NH
CH2O
P(NIPAAm‐co‐BMAAm)
(NB) block
CH2
CH
CH3
CH3
N‐(isobutoxymethyl)
acrylamide (BMAAm)
Traansmittance / %
100
CH2 CH
80
60
40
20
0
5
15
25
35
45
Temperature / oC
7
Characterization of PNIPAAm‐b‐P(NIPAAm‐co‐BMAAm) Block Copolymers
Block Copolymers
Table. Characteristic data of PNIPAAm‐b‐P(NIPAAm‐co‐BMAAm) block copolymer
BMAAm content of
the block copolymera
(mol%)
Mn b
Mw/Mn b
N
0
50600
1 19
1.19
NbNB_5
2.4
73400
1.51
NbNB_10
5.4
74700
1.52
NbNB_15
7.3
73800
1.46
Code
aEstimated by 1H‐NMR. bMeasured by GPC using DMF with 10 mM LiBr.
8
Controlled Double Thermo‐Responsive Behavior by DLS and Transmittance Measurement
Transmittance Measurement
200
100
80
150
60
100
40
50
20
0
15
Diameterr / nm
Tra
ansmittance / %
(A)
0
20
25
30
35
40
Temperature / oC
Figure . (A) Temperature‐dependent behavior of 0.5 w/v% aqueous solution of NbNB_10 as a function of temperature, as followed by transmittance and DLS. (B) Photographs of obtained for 0.5 w/v% aqueous solution of NbNB_10 at different temperatures.
9
Controlled Double Thermo‐Responsive Behavior by DLS and Transmittance Measurement
Transmittance Measurement
(B)
200
150
60
100
40
20
50
NbNB 5
NbNB_5
0
15
0
20
25
30
35
Temperature / oC
40
Transmitttance / %
80
200
100
Diametter / nm
Transmitttance / %
100
80
150
60
100
40
50
20
Diametter / nm
(A)
NbNB 15
NbNB_15
0
10
0
15
20
25
30
35
40
Temperature / oC
Figure . Temperature‐dependence behavior of 0.5 w/v% aqueous solution of NbNB_5 (A), NbNB_15 (B) as a function of temperature, as followed by transmittance and DLS.
By changig the BMAAm content on NB‐block, the responsive temperature B
h
i th BMAA
t t
NB bl k th
i t
t
could be controlled easily.
10
Controlled Double Thermo‐Responsive Behavior by 1H NMR Measurement
D2O
(A) 22.5 (A)
22.5 oC
BMAAm
(B) 27.5 oC
(C) 37.5 (C)
37 5 oC
6
5
4
3
2
1
0 ppm
Figure. Temperature‐dependent 1H‐NMR spectra of 1 w/v% solution of NbNB_10 in D2O at 22.5 oC oC (B) and 37.5 oC (C)
((A), 27.5 ),
( )
( ).
11
Reversible Self‐Assembly Formation by DLS and Maximum Wavelength of ANS
Wavelength of ANS
520
Diameter // nm
80
500
60
480
40
460
Temp. / oC
20
0
27.5
27.5 27
5 oC
Max. Wavelenggth / nm
100
17.5 oC
440
17.5
0
2
4
6
8
Step
Figure Reversible self‐assembly formation of NbNB_10 in response to temperature cycle between 17 5 and 27 5 oC, as followed by DLS and maximum wavelength of ANS.
17.5 and 27.5 C as followed by DLS and maximum wavelength of ANS
The assembly behavior was completely reversible depending on
solution temperature.
12
CAC Measurement by Fluorescence Emission Spectra of Pyrene
fP
(A)
(B)
1.8
I1 / I3
In
ntensity
5 х 10‐1 g/l
1 х 10‐1 g/l
5 х 10‐2 g/l
5 х 10‐3 g/l
5 х 10‐4 g/l
2.0
1.6
1.4
360
380
400
420
Wavelength / nm
440
460
1.2
-5
-4
-3
-2
-1
0
1
Log C / g/l
Figure (A) Fluorescence emission spectra of pyrene (6 * 10
Figure
(A) Fluorescence emission spectra of pyrene (6 * 10‐77 M) in aqueous solution of NbNB_15 at M) in aqueous solution of NbNB 15 at
27.5 oC, λex = 337 nm. (B) Change in fluorescence intensity ratio of I1/I3 of pyrene as a function of increasing concentration of NbNB_15.
By increasing polymer concentration, the intensity and I
B
i
i
l
t ti
th i t it
d I1/13 were dramatically changed because d
ti ll h
db
of changing the surrounding environment on pyrene. The critical association concentration(CAC) of NbNB_15 was approximately 10 mg/l.
13
Double Thermo Responsive Block Copolymers
Double Thermo‐Responsive Block Copolymers
Y. Kotsuchibashi et al.,
J. Polym. Sci. Part A : Polym. Chem., 2010, 48, 4393
Double thermo‐responsive block copolymers which form nanoassemblies were synthesized. The D
bl h
i bl k
l
hi h f
bli
h i d Th
nanoasemblies consisting of NB‐block into core and N‐block into shell showed completely reversible assembly behavior by cycling solution temperature.
14
Development of Multi‐Stimuli‐Responsive Block Copolymers as nano‐associates for Drug Delivery Applications
i t f D
D li
A li ti
Common Thermo‐responsive
Block
Stimuli‐Responsive
Block 1
Block 1
Stimuli‐Responsive
Block 2
Mixture
Solution
ΔT
Multi‐Stimuli‐Responsive Nanoassembly
15
Characterization of PNIPAAm‐b‐P(NIPAAm‐co‐HMAAm) and PNIPAAm‐b‐P(NIPAAm‐co‐AMPS) Block Copolymers
d PNIPAA b P(NIPAA
AMPS) Bl k C
l
HN
O
Me6TREN
CuBr
DMF/Water
20 deg.
HN
HN
O
O
b
SO3Na
N
HN
O
HN
O
HN
O
EBB
SO3Na
Figure. Synthesis of the PNIPAAm‐b‐P(NIPAAm‐co‐HMAAm) and PNIPAAm‐b‐P(NIPAAm‐co‐
AMPS).
16
Characterization of PNIPAAm‐b‐P(NIPAAm‐co‐HMAAm) and PNIPAAm‐b‐P(NIPAAm‐co‐AMPS)
PNIPAAm
b P(NIPAAm co AMPS) Block Copolymers
Block Copolymers
Table . Characteristic data of PNIPAAm
Table
. Characteristic data of PNIPAAm‐b‐P(NIPAAm‐co‐HMAAm)
b P(NIPAAm co HMAAm) and PNIPAAm
and PNIPAAm‐b‐
b
P(NIPAAm‐co‐AMPS)
HMAAm or AMPS
content of the
copolymer block
HMAAm or AMPS
content of the
copolymer block
in feed
in block copolymer b
(mol%)
N248
b
Mn,theo.
n theo
Mn c
Mw /Mnc
(mol%)
(-)
(-)
(-)
0
0
28300
21700
1.14
N248bN419H113
20
21
87100
75100
1.58
N248bN461A108
20
19
105100
118900
1.54
Code
a
aPNIPAAm‐b‐P(NIPAAm‐co‐HMAAm) and PNIPAAm‐b‐P(NIPAAm‐co‐AMPS) were abbreviated as PNIPAA b P(NIPAA
HMAA ) d PNIPAA b P(NIPAA
AMPS)
bb i d
N248bN419H113 and N248bN461A108 respectively, where bottom right numbers are monomer units. bEstimated by 1H‐NMR. cDetermined by GPC using DMF with 10 mM LiBr.
17
Double Thermo‐Responsive Behavior by Transmittance Measurement
Transmittance / %
%
100
b
(□) PNbP(NcoA)
HN
80
O
HN
O
HN
O
SO3Na
60
40
(○) PNbP(NcoH)
20
0
20
30
40
50
T
Temperature / / oC
60
Figure. Temperature‐dependent transmittance change of 0.5 w/v% block copolymers in 100 mM NaClaq..
The common thermo‐responsive block which are PNIPAAm blocks showed similar responsive temperature in both block copolymers.
18
Size Distribution Histogram of nano
Size
Distribution Histogram of nano‐
assemblies from each block copolymers
PNbP(NcoA)
D = 240 ± 59 nm
20
15
10
5
0
1
37
134 491
Di
t /
Diameter / nm
D = 148 ± 36 nm
25
1000
Light scaattering inttensity / %
%
25
Light scaattering inttensity / %
%
PNbP(NcoH)
20
15
10
5
0
1
6
32
178
Diameter / nm
Diameter / nm
1000
Figure . Size distribution histogram for block copolymers in 100 mM NaClaq at 40 oC.
At 40 oC, the stability assemblies were formed successfully. The diameter were 240 ±
C, the stability assemblies were formed successfully. The diameter were 240 ± 59 nm and 59 nm and
At 40 148 ± 36 nm on PNbP(NcoH) and PNbP(NcoA) respectively.
19
Size Distribution Histogram of nano‐assemblies
from mixed block copolymers
from mixed block copolymers
250
Diam
meter / nm
200
150
100
50
(A)
0
0
20
40
60
80 100
content / v/v%
N248bN419H113 content / v/v%
Lightt scatteringg intensityy / %
25
D =
20 178 ± 53 nm
15
10
5
(B)
0
1
6
37
229
1000
Diameter / nm
Figure . (A) Diameter for 0.1 w/v% of mixted block copoly‐mers in 100 mM NaClaq at 40 oC, (B) Size distribution histogram of N248bN419H113 : N248bN461A108 = 50 : 50 v/v%.
By changing the mixture ration of block copolymers, the diameter of assemblies could be controlled easily.
20
Nanoassembly with Thermo‐Response and Negative Ch
Charge into Shell
i t Sh ll
N248bN461A108
Mixture
Aggregation
N248bN419H113
Temperature
Precipitation
20 oC
40 oC
45 oC
A nanoassembly with thermo‐response and negative charge into shell were prepared by only mixing two block copolymers and increasing solution temperature to above LCST.
21
Synthesis of Block Copolymers by Reversible Addition‐
Fragmentation Chain Transfer (RAFT) Polymerization
Fragmentation Chain Transfer (RAFT) Polymerization
P(NIPAAm‐co‐HMAAm)‐b‐P(NIPAAm‐co‐BMAAm)
Thermo response
Biocompatibility
Common thermo‐responsive
block
PEG‐b‐P(NIPAAm‐co‐BMAAm)
22
Characterization of P(NIPAAm‐co‐HMAAm)‐b‐
P(NIPAAm co BMAAm) Block Copolymer
P(NIPAAm‐co‐BMAAm) Block Copolymer
Table. Characteristic data of P(NIPAAm‐co‐HMAAm)‐b‐P(NIPAAm‐co‐BMAAm)
HMAA
HMAAm
BMAA
BMAAm
HMAA
HMAAm
BMAA
BMAAm
in feed
in feed
in copolymera
in copolymera
(mol%)
(mol%)
(mol%)
P(NcoH)
11
-
(
) (
)
P(NcoH)bP(NcoB)
11
17
Code
Mn b
Mw/Mn b
(mol%)
(-)
(-)
11
-
14400
1.32
11
18
22400
1.54
aEstimated by 1H‐NMR. bMeasured by GPC using DMF with 10 mM LiBr.
23
Double Thermo‐responsive Behavior Observed f
from Transmittance Changes
T
i
Ch
□ P(N H)
□: P(NcoH)
Transm
mittance / %
%
100
80
60
○ (N H)b (N )
○: P(NcoH)bP(NcoB) 40
20
0
5
15
25
35
Temperature / oC
Temperature / 45
Figure . Temperature‐dependent behavior of 0.5 w/v% aqueous solution of P(NcoH)bP(NcoB) (○), and P(NcoH) (□), as a function of temperature.
24
Diameter of P(NIPAAm‐co‐HMAAm)‐b‐P(NIPAAm‐co‐
BMAA ) Bl k C
BMAAm) Block Copolymer at Room Temperature
l
tR
T
t
Light scatte
ering intenssity / %
25
Transm
mittance / %
%
100
80
60
40
Dave. = 105 ± 23 nm
20
15
10
5
0
4
20
0
5
15
25
35
Temperature / oC
45
19
91
Diameter / nm
/
426
Figure. Size distribution histogram for 0.5 w/v% aqueous solution of P(NcoH)bP(NcoB) at 25 oC. At 25 oC, the block copolymers formed nanoassemblies. Moreover, at 40 oC, the assemblies aggregated each other due to dehydration of P(NcoH) block in shell.
25
Characterization of PEG‐b‐P(NIPAAm‐co‐
BMAA ) Bl k C
BMAAm) Block Copolymer
l
HN
HN
O
O
O
HO
O
O
O
O
n
S
S
AIBN
O
HO
O
O
n
S
O
S
b
O
HN
O
O
HN
O
O
PEG‐b‐P(NIPAAm‐co‐BMAAm)
PEG
b P(NIPAAm co BMAAm)
(PEGbP(NcoB))
Table. Characteristic data of PEG‐b‐P(NIPAAm‐co‐BMAAm)
HMAAm
BMAAm
HMAAm
BMAAm
a
a
Mn b
Mw/Mn b
in feed
in feed
in copolymer
in copolymer
(mol%)
(mol%)
(mol%)
(mol%)
(-)
(-)
P(NcoH)bP(NcoB)
11
17
11
18
22400
1.54
(
)
PEGbP(NcoB)
-
17
-
18
17800
1.52
Code
aEstimated by 1H NMR. bMeasured by GPC using DMF with 10 mM LiBr.
26
Multiple Stimuli‐Responsive Assembly by Dehydration of P(NIPAAm‐co‐BMAAm) Copolymer Block
P(NIPAAm‐co‐BMAAm) Copolymer Block
○: P(NcoH)bP(NcoB)
○: P(NcoH)bP(NcoB) 100
0
Transm
mittance / %
%
Macro CTA
80
0
b
HN
O
60
0
HN
O
HN
O
OH
HN
O
O
□: PEGbP(NcoB)
40
0
20
0
Similar LCST
0
5
15
25
35
45
o
Temperature / C
Temperature / Figure . Temperature‐dependent behavior of 0.1 w/v% aqueous solution of P(NcoH)bP(NcoB) (○), and PEGbP(NcoB) (□), as a function of temperature.
27
Diameter of The Block Copolymer at Room Temperature
15 Dave. = 99 ± 39 nm
(A)
P(NcoH)bP(NcoB)
(
) (
)
20
10
0
Light scatterring intensity / %
Light scatterring intensity / %
30 Dave. = 93 ± 19 nm
(B)
PEGbP(NcoB)
10
5
0
1
4
14
50
185 1000
Di
Diameter / nm
t /
1
4
16
66
266 1000
Diameter / nm
Diameter / nm
Figure. Size distribution histogram for 0.1 w/v% aqueous solution of P(NcoH)bP(NcoB) (A), and PEGbP(NcoB) (B) at 25 oC. The diameter were similar on both block copolymers.
28
P(NcoH)bP(NcoB)
0.1 w/v%
PEGbP(NcoB)
0.1 w/v%
Mixture ratio = 1:1 v/v%
Mixture solution
i
l i
Total concentration
= 0.1 w/v%
Ligght scatterring intensiity / %
Diameter of Mixed Block Copolymers Solution at Room Temperature
R
T
25 Dave. = 106 ± 27 nm
20 Mixture
15
10
5
0
1
4
15
60
232 1000
Diameter / nm
i
/
Figure. Size distribution histogram for 0.1 w/v% (total concentration) aqueous solution of mixture solution of P(NcoH)bP(NcoB) and PEGbP(NcoB) (1:1 v/v%) at 25 oC. By increasing temperature at 25 oC, the nanoassemblies were prepared from mixture solution consisting of two block copolymers.
29
Multiple Stimuli‐Responsive Assembly by Dehydration of P(NIPAA
P(NIPAAm‐co‐BMAAm) Copolymer Block
BMAA ) C
l
Bl k
?
Transmittance change of P(NcoH)bP‐ (NcoB) were not affected by only PEG.
t ff t d b
l PEG
80
80
Transmitttance / %
100
Transmitttance / %
100
60
P(NcoH)bP(NcoB) PEGbP(NcoB)
= 0.05 w/v%
respectively
40
20
0
60
40
P(NcoH)bP(NcoB) PEG
= 0.05 w/v%
p
y
20 respectively
0
5
15
25
35
T
/ oC
Temperature / 45
5
15
25
35
45
T
t
/ oC
Temperature / These results suggested that the shell on nanoassemblies are composed of PEG block and P(NcoH) block.
30
Thermo‐Responsive Behavior of P(NcoH) Block Shell
P(NcoH)bP(NcoB) : PEGbP(NcoB) = 1:1 (v/v%), total concentration = 1 wt/v%
25 oC
PEG
D2O
NIPAAm
40 oC
D2O
BMAAm peaks: disappeared
NIPAAm
and
d
HMAAm NIPAAm and HMAAm peaks: disappeared
4
2
0 ppm
6
Figure Temperature‐dependent 1H‐NMR spectra of 1 w/v% mixture solution of P(NcoH)bP(NcoB) and PEGbP(NcoB) in D2O at 25 O at 25 oC and 40 C and 40 oC.
C
and PEGbP(NcoB) in D
P(NcoH) block in shell showed thermo‐responsive property even as the shell are composed of mixture with PEG block.
31
Nanoassembly with Thermo‐Response and PEG block into Shell at Room Temperature
into Shell at Room Temperature
Stimuli‐responsive
Sti
li
i
block
S l t d ti li
Selected stimuli‐responsive block
i bl k
Thermo response
= P(NIPAAm‐co‐HMAAm) block
P(NIPAAm co HMAAm) block
(Drug release)
Common thermo‐responsive
block
Biocompatibility
Mixture
= PEG block
((Biocompatibility)
p
y)
Room
temperature
Above body Above
body
temperature
At room temperature, a mixed nanoassembly with P(NIPAAm‐co‐HMAAm) block and PEG block into the shell were prepared. The nanoassemblies kept high stability even as the shell showed thermo‐
responsive property due to P(NIPAAm‐co‐HMAAm) block due to hydrophilic PEG block.
32
Conclusion Stimuli‐responsive
block
Selected stimuli‐responsive block
Thermo response
Drug release
Drug release
Biocompatibility
Common thermo responsive
thermo‐responsive
block
Molecular recognition
g
Targeting
Mixture
Heating
eat g
Heating
Target
Drug
D
Drug loading
l di
Drug release
Drug release
By only changing the mixture ratio of block copolymers, nanoassemblies which were tuned the kind and amount of function into shell were prepared. These novel ‘smart’ material will apply to Drug Delivery System as drug carrier.
l
d
1) Y. Kotsuchibashi, et al., J. Polym. Sci., Polym. Chem., 46, 6142(2008)
2) Y. Kotsuchibashi, et al., J. Colloid Interface Sci., 336, 67(2009)
3) Y. Kotsuchibashi, et al., J. Polym. Sci., Polym. Chem., 48, 4393(2010).
33
4) Y. Kotsuchibashi, et al., Polym. Chem., 2, 1362(2011)
Acknowledgment
Dr. M. Ebara
Dr. Giancarlo Forte
Dr. John Hoffman
Dr. N. Idota (JSPS)
Dr K Uto
Dr. K.
Dr. Ewelina Zawadzak
Dr. Monika Bil
Dr. Janice Tam
Dr. Janice Tam
Dr. Y.Kotsuchibashi (JSPS)
Univ. Tsukuba
D3 T. Prapatsorn
D2 Y‐J. Kim
M2 T. Takiguchi
M1 S. Yasuda
M1 T. Okada
B4 T. Sato
・Bioconjates
Bioconjates
・Function control of stem cell by cellmaterial interaction
・stimuli responsive Nanofibers
・stimuli-responsive
・Shape memory surfaces
Secretary Dr. A. Ebara
Mrs. Hiroyama
34
35