Accepted Manuscript

Title: Synthesis and characteristics of biodegradable and

temperature responsive polymeric micelles based on
poly(aspartic acid)-g-poly(N-isopropylacrylamide-co-N,N-
dimethylacrylamide)
Authors: Jih-Chao Yeh, Huei-Hung Yang, Ya-Ting Hsu,
Chao-Ming Su, Tsong-Hai Lee, Shyh-Liang Lou
PII: S0927-7757(12)00874-6
DOI: doi:10.1016/j.colsurfa.2012.12.014
Reference: COLSUA 18077
To appear in: Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: 13-5-2012
Revised date: 11-12-2012
Accepted date: 12-12-2012
Please cite this article as: Jih-Chao Yeh, Huei-Hung Yang, Ya-
Ting Hsu, Chao-Ming Su, Tsong-Hai Lee, Shyh-Liang Lou,
Synthesisandcharacteristicsofbiodegradableandtemperatureresponsivepolymericmicellesbasedonpoly(asparticacid)-
g-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide), Colloids and Surfaces
A: Physicochemical and Engineering Aspects doi:10.1016/j.colsurfa.2012.12.014
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*Graphical Abstract (for review)
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We synthesized PASp-g-PND possessing temperature response and
biodegradability
The micelles phase transition temperature is about 41.9
o
C
The micelles degraded within three days is about 25%
The micelles contains free amino groups to be able to conjugate with
bio-molecules
The micelles has potential to be used as a drug carrier for targeting treatments

*Highlights (for review)
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Synthesis and characteristics of biodegradable and temperature
responsive polymeric micelles based on poly(aspartic acid)-g-
poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)
Jih-Chao Yeh
a,b
, Huei-Hung Yang
a
, Ya-Ting Hsu
a
, Chao-Ming Su
a
, Tsong-Hai Lee
b,*
, Shyh-
Liang Lou
a,**

a
Department of Biomedical Engineering, Chung-Yuan Christian University, Chung Li 32023,
Taiwan, ROC
b
Department of Neurology and Stroke center, Chang Gung Memorial Hospital, Linkou Medical
Center and College of Medicine, Chang Gung University, Taoyuan, Taiwan, ROC.

Both Tsong-Hai Lee and Shyh-Liang Lou act as co-corresponding authors to this manuscript.

Acknowledgment and reprint request to
Shyh-Liang Lou
Tel.: +886 3 2654517fax: +886 3 2654599.
E-mail addresses: lou@cycu.edu.tw
200, Chung Pei Road, Chung Li 32023, Taiwan, R.O.C

*Manuscript
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Abstract
Temperature responsive polymeric micelles can release drug at controlled rate and
minimize the side effects, and have been widely used for drug delivery system. In this study,
biodegradable and temperature responsive micelles comprised of poly(aspartic acid)-g-poly(N-
isopropylacryamide-co-N,N-dimethylacrylamide) (PASp-g-poly(NIPAAm-co-DMAAm)) were
successfully synthesized by grafting poly(NIPAAm-co-DMAAm) onto poly(succinimide) and
followed by aminolysis with ammonia hydroxide. The micelles had free amine group and
exhibited a phase transition temperature above normal body temperature, which was suitable for
targeting drug delivery. In addition, PASp-g-poly(NIPAAm-co-DMAAm) was stable in
phosphate buffer solution at 37
o
C, and 25% of micelles could degrade within 3 days. Based on
these results, the present study demonstrated that PASp-g-poly(NIPAAm-co-DMAAm) can be
used as a drug carrier for targeting treatment.

Keywords: biodegradable, temperature responsive polymeric micelles, phase transition
temperature, drug carrier

1. Introduction
Polymeric micelles have become an interesting system for delivery of poorly water-soluble
drugs and are increasingly used in different medical applications, e.g., drug carriers for cancer
therapy[1], gene[2], protein[3] and imaging agents[4, 5]. These micelles are block or graft
copolymers that are made up of hydrophobic core and hydrophilic shell and can self-assemble in
aqueous solution by hydrophobic/hydrophilic interaction[6, 7], hydrogen bonding[8], and
electrostatic interactions[9]. Hydrophobic inner core can accommodate hydrophobic drugs, and
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hydrophilic outer shell makes particles stable in the blood stream to avoid being recognized by
reticuloendothelial system (RES) to increase treatment efficiency[10].
The stimuli-responsive micelles can sense the surrounding environment change, such as pH,
temperature, enzyme, and ion change, and response as a phase transition to control drug release.
In addition, stimuli-responsive micelles enable drugs to accumulate at the desired site either
passively by enhanced permeation and retention effect or actively through the conjugation of
recognition signal onto the surface of the micelles[11, 12]. Therefore, stimuli-responsive
polymeric micelles have been paid much attention in drug delivery system, because these
micelles can release drug at controlled rate and minimize the side effects[13].
Poly(N-isopropylacryamide), PNIPAAm, is one of the most commonly investigated
temperature responsive amphiphilic polymer, which exhibits lower critical solution temperature
(LCST) at about 32
o
C in aqueous media. Below the LCST, PNIPAAm is hydrophilic and
extended in the solution, which forms hydrogen bonds between the water and the amide side
chain. When above the LCST, they become water-insoluble and undergo volume transition to
aggregate in the solution. In addition, the LCST of PNIPAAm can be easily modified by
introducing hydrophilic or hydrophobic segments. Using temperature responsive micelles for
drug delivery at specific sites, the LCST of micelles needs modification to become slightly
higher than the body temperature by incorporating hydrophilic co-monomers, such as N,N-
(dimethyl amino)ethyl methacrylate[14], methyacrylic acid[15], acrylic acid[16] and
polyethylene glycol[17]. The release profile of doxorubicin in temperature responsive micelles
made from poly(N-isopropylacryamide-co-N,N-dimethylacrylamide-co-10-undecenoic acid)
with various compositions has been investigated. The micelles in phosphate buffer solution
exhibited LCST from 33 to 43
o
C. Also, the micelles can be stable in the blood stream at 37
o
C,
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but can be deformed to release the encapsulated drugs above the LCST by breaking the
hydrophilic/hydrophobic balance of micelles[18].
Although PNIPAAm based polymeric micelles offer the advantage of temperature
responsive behavior and can act as a potential candidate for drug delivery system, they are not
biodegradable. Recently, there is methodology to improve the effectiveness of PNIPAAm based
copolymers by grafting onto biodegradable polymers. The biodegradable and temperature
responsive polymer micelles made from dextran-g-poly(NIPAAm-co-DMAAm) and chitosan-g-
poly(NIPAAm-co-DMAAm) have been studied[19-21]. The LCST of micelles can be increased
to 38
o
C by grafting poly(NIPAAm-co-DMAAm) onto the main chain of dextran and chitosan.
Chitosan has reactive amine groups which serve to conjugate with specific antibody for targeting
delivery. However, antidextran antibodies in human limit the cell uptake and reduce the
therapeutic effectiveness for targeting treatment.
Poly(amino acid) and its derivatives have high biocompatibility, biodegradability and
diversity in the various side chain groups for conjugating to antibodies, such as poly(aspartic
acid)[22], poly(glutamic acid)[23] and polylysine[6], which have been used for drug delivery
system. Poly(aspartic acid), PASp, is a typical poly(amino acid) derivative and is a
biodegradable, nontoxic, nonantigenic material. It can be synthesized by thermal
polycondensation of L-aspartic acid and is followed by aminolysis with amino hydroxide. The
synthesis and characteristics of amphiphilic biodegradable poly(aspartic acid-co-lactic acid) has
been investigated[24]. The degradation rate of copolymer can be increased with higher aspartic
acid content.
This study thus consists of three parts: (1) In order to increase the LCST of temperature
responsive micelles, DMAAm was used to adjust the LCST of poly(NIPAAm-co-DMAAm).
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poly(NIPAAm-co-DMAAm) with three different compositions including PN35D5 (the feed ratio
of NIPAAm and DMAAm =35:5), PN35D10 (35:10), and PN35D15 (35:15) which was
synthesized by radical copolymerization. (2) The biodegradable and temperature responsive
micelles were prepared by grafting the poly(NIPAAm-co-DMAAm) onto the main chain of
poly(aspartic acid) via aminolysis of polysuccinimide (PSI) through amino-terminated
poly(NIPAAm-co-DMAAm), followed with aminolysis of residual PSI by ammonium
hydroxide. (3) The structure analysis, LCST, biodegradable ratio in PBS and cytotoxicity of
PASp-g-poly(NIPAAm-co-DMAAm) were also discussed.

2. Materials and methods
2.1 Materials
N-Isopropylacrylamide was received from Tokyo chemical industry (TCI, Tokyo, Japan).
N,N-dimethylacrylamide and hydrazine (98%) were purchased from Alfa Aesar (MA, USA).
Methyl 3-mercaptopropionate (98%), L-aspartic acid (98%), thiazolyl blue tetrazolium bromide
(97.5%), dimethyl sulfoxide, and 1,6-diphenyl-1,3,5-hexatriene (98%) were supplied by Sigma
(MO, USA). Potassium peroxydisulfate, N,N-dimethyl methanamide (99.9%), and ammonium
hydroxide (30%) were obtained from J. T. Baker (NJ, USA). N-methylprollidone (99.9%) was
received from Showa Denko (Tokyo, Japan). Dimethyl sulfoxide-d
6
(99.9%) was purchased from
Merck (Darmstadt, Germany). Orthophosphoric acid (80%) was supplied by Riedel-de Haen
(USA). Potassium bromide was obtained from Scharlan (Barcelona, Spain). Modified eagles
medium was received from GIBCO (Life Technology, NY, USA). All chemicals, reagents and
solvents were used without further purification.

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2.2 Synthesis of amino-terminated poly(N-isopropylacrylamide-co-N,N-dimethylmethanamide)
poly(NIPAAm-co-DMAAm)
Briefly, 0.41 g of NIPAAm, 0.096 mL of DMAAm and 0.02764 mL of methyl 3-mercapto
propionate were dissolved in 40 mL of deionized water and degassed with nitrogen gas for 30
min. The mixture solution was heated to 50
o
C under nitrogen gas. Then, 0.03 g of potassium
peroxydisulfate was dissolved in 10 mL of deionized water. The solution was added into the
monomer solution and reacted at 50
o
C for 2 h with magnetic stirring. Upon completion, the
resulted solution was dialyzed against deionized water using a dialysis membrane (MWCO <
1200) for 24 h and was freeze-dried. Subsequently, an appropriate amount of poly(NIPAAm-co-
DMAAm)-COOCH
3
was dissolved in 40 mL of methanol solution. Hydrazine monohydrate was
dropwised into the solution and refluxed for 5 h with magnetic stirring. Upon completion, the
solution was dialyzed against deionized water using MWCO <1200 dialysis membrane and then
freeze-dried to obtain amino-terminated poly(NIPAAm-co-DMAAm).

2.3 Synthesis of poly (succinimide) (PSI)
10 g of L-aspartic acid and 1 g of orthophosphoric acid were mixed and heated to 200
o
C in an
oil bath under nitrogen gas for 24 h. The resulted off-white fine powder was filtered and washed
with deionized water for five times to remove orthophosphoric acid. The obtained fine powder
was dried at 50
o
C for 12 h to get PSI powder.

2.4 Synthesis of PASp-g-poly(NIPAAm-co-DMAAm)
Amino-terminated poly(NIPAAm-co-DMAAm) and PSI were separately dissolved in 10 mL
of N,N-dimethylmethanamide. Then, amino-terminated poly(NIPAAm-co-DMAAm) solution
was added dropwise into the PSI solution and heated to 60
o
C for 24 h with magnetic stirring.
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After 24 h, the mixture solution was cooled down to room temperature; the ammonium
hydroxide solution was added into the reaction solution and reacted at room temperature for 5 h
with stirring. The resulting solution was dialyzed against deionized water using a dialysis
membrane (MWCO <1200) for 48 h. Finally, the solution was freeze-dried to obtain PASp-g-
poly(NIPAAm-co-DMAAm) powder.

2.5 Characterization of polymer
Amino-terminated poly(NIPAAm-co-DMAAm), PSI and PASp-g-(NIPAAm-co-DMAAm)
were analyzed by Fourier transform infrared spectroscopy (FTIR, IR-4200, Jasco, Japan) in the
region of 400-4000cm
-1
. All samples were mixed with potassium bromide and pressed into
pellets for analysis. The morphological properties of polymer were observed by a transmission
electron microscope (TEM, H7500, Hitachi, Japan) at 80 kV. A drop of nanoparticle suspension
was placed on a copper grid and dried at room temperature overnight. The composition of
P(NIPAAm-co-DMAAm) was analyzed by a 300 MHz spectrometer (NMR, DRX300, Bruker,
Germany) and the polymer was dissolved in DMSO-D
6
for analysis. The molecular weights and
polydispersity index (PDI) of polymer were characterized using gel permeation chromatography
(GPC, 150C, Waters, USA). The flow rate of mobile phase was 1 mL/min and N-
methylprollidone was used as an eluent.

2.6 Lower critical solution temperature (LCST) measurement
The LCST of polymer was measured for the transmittance of polymer solution at 500 nm
using a UV-Vis spectrophotometer (V-530, Jasco, Japan) and fiber optic thermometry (Luxtron,
ROC). The solution was first heated to 50
o
C and then measured for the absorbance at different
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temperature from 25 to 50
o
C. The LCST of the polymer was defined as the temperature
decreasing a half of absorbance of polymer solution at 50
o
C.
2.7 Biodegration evaluation
25 mg of PASp-g-poly(NIPAAm-co-DMAAm) was accurately weighted and dissolved in
10 mL of 0.1 M phosphate buffer saline (pH 7.4). The solution was placed in a dialysis
membrane (MWCO <12000-14000) against 500 mL of PBS at 37
o
C in an incubator for 3, 7, 14,
21 and 28 days. Then, the solution was dialyzed against deionized water using a dialysis
membrane (MWCO <1200) for 24 h. Finally, the solution was freeze-dried and weighted. The
biodegradable ratio of the polymer was calculated by equation (1) :

, where W
0
is the weight of the polymer before degration and W
a
is the weight of the polymer
after degration.

2.8 Cytotoxicity testing
The biocompatibility of the polymer was evaluated by MTT cell viability assay using L929
fibroblast cells. 410
-6
L929 cells were cultured in a 24-well culture plate with 1 mL of modified
eagle medium (MEM, 10% horse serum, 1% penicillin-streptomycin) at 37
o
C, under 5% CO
2
.
After 24 h, the culture medium was replaced and 5 mg of polymer was added into the fresh
medium. The cells were incubated for another 24, 48 and 72 h. Then, medium with polymer was
substituted by fresh medium and 100 L of MTT solution was added. After incubation for 3 h at
37
o
C, the medium was removed. In order to dissolve the internalized purple formazan crystals, 1
mL of DMSO was added. 100 L of resulting solution was transferred to a 96-well plate, and an
ELISA reader (1500-490, Thermo, USA) was used to determine the absorbance at 570 nm. The
cell viability was calculated according to equation (2) :
(1)
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, where OD
polymer
is the absorbance obtained in the presence of polymer and OD
control
is the
absorbance obtained without polymer.

3. Results and discussion
3.1 Synthesis and characteristics of PASp-g-poly(NIPAAm-co-DMAAm)
First, PSI could be prepared by bulk polymerization of aspartic acid using phosphoric acid
as a catalyst. Then, amino-terminated poly(NIPAAm-co-DMAAm) could be synthesized by
surfactant-free radical polymerization using KPS as an initiator and methyl 3-meracptopropinate
as a chain transfer agent. Finally, the imide cyclic structure of PSI could be easily opened by an
amiable reaction. Therefore, PASp-g-poly(NIPAAm-co-DMAAm) could be prepared by
nucleophilic opening of PSI using amino-terminated poly(NIPAAm-co-DMAAm) and followed
by hydrolysis with ammonium hydroxide. The scheme of PASp-g-poly(NIPAAm-co-DMAAm)
is shown in Figure 1.

3.1.1
1
H NMR analysis
The chemical structures of amino-terminated poly(NIPAAm-co-DMAAm) and PSI were
investigated by
1
H NMR spectroscopy. The
1
H NMR of amino-terminated poly(NIPAAm-co-
DMAAm) is shown in Figure 2(a). The characteristic peaks at 1.04 and 3.83 ppm were assigned
to the isopropyl group of NIPAAm. The peaks at 2.80 to 2.88 ppm were assigned to methyl
group on the DMAAm. In addition, the broad peak at 1.23 to 2.08 ppm was assigned to -CH
2
-
CH- on the polymer [25, 26]. This indicated successful copolymerization of amino-terminated
poly(NIPAAm-co-DMAAm)-HNHN
2
. Based on the integration of the peak area of signal a and
signal f, the actual molar ratio of NIPAAm to DMAAm could be calculated. Table 1 shows the
(2)
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actual molar ratios of NIPAAm: DMAAm were approximately equaled to the feed ratio. The
1
H
NMR spectra of PSI is shown in Figure 2(b), the characteristic peaks at 2.7 to 3.21 ppm and 5.26
ppm was assigned to the protons of the methylene and methyne group on the succinimide units
of PSI [27].
The feed molar ratio of amino-terminated poly(NIPAAm-co-DMAAm) and PSI was 1:1.
The chemical structure of PASp-g-poly(NIPAAm-co-DMAAm) was analyzed by
1
H NMR. As
shown in Figure 2 (c), the NMR spectra of PASp-g-poly(NIPAAm-co-DMAAm) is similar to
that of poly(NIPAAm-co-DMAAm). Upon comparison with PSI, the disappearance of the peak
at 5.26 ppm and appearance of the peak at 4.52 ppm indicated the rings in PSI were totally
opened by the amino-terminated poly(NIPAAm-co-DMAAm) and ammonium hydroxide.
According to the results of NMR analysis, the poly(NIPAAm-co-DMAAm) was successfully
grafted to the polyaspartic acid backbone chain.

3.1.2 FT-IR analysis
The FT-IR spectrum of amino-terminated poly(NIPAAm-co-DMAAm) is shown in Figure
3(a). The characteristic absorption bands at 1645 and 1550 cm
-1
were assigned to the absorbance
of bending frequency of amide N-H bond, and at 3444 and 3311cm
-1
were assigned to the
absorbance of N-H bond in poly(NIPAAm-co-DMAAm)[19,28,29]. The characteristic
absorption bands from the methyl 3-mercaptopropionate at 663 cm
-1
was assigned to the
absorption bands of C-S bond. These results suggested there was successful polymerization of
poly(NIPAAm-co-DMAAm) and the methyl end group could be replaced by hydrazine
monohydrate.
The FT-IR spectrum of PSI is shown in Figure 3(b). The characteristic absorption bands at
1397, 1718, and 1792 cm
-1
were assigned to the absorbance of methylene group, carbonyl group
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and cyclic imide of succinimide group of PSI [30,31]. Grafting of amino-terminated
poly(NIPAAm-co-DMAAm) onto the backbone of PSI followed by hydrolysis with ammonium
hydroxide was also confirmed by FT-IR spectrum of PASp-g-poly(NIPAAm-co-DMAAm)
(Figure 3(c)). The spectrum of PASp-g-poly(NIPAAm-co-DMAAm) was similar to that of
poly(NIPAAm-co-DMAAm). The characteristic peak at 1792 cm
-1
assigned to the succinimide
group of PSI was disappeared. In addition, the peak at 1718 cm
-1
was significantly smaller than
PSI. These results indicated there was full ring-opening of PSI by amino-terminated
poly(NIPAAm-co-DMAAm) and hydrolysis with ammonium hydroxide.

3.1.3 Molecular weight
High molecular weight and conversion of PSI was prepared by bulk polycondensation of
aspartic acid with o-phosphoric acid as a catalyst [27]. The molecular weight of PSI determined
by GPC was 30,000 Da. The conversion of aspartic acid was around 90% [32]. The LCST of
PNIPAAm was around 32
o
C and could be wildly used in medical applications. In addition,
DMAAm is more hydrophilic than PNIPAAm and can be used to increase the LCST of the
copolymer [17]. In this study, DMAAm is used to adjust the LCST of PNIPAAm with three
different compositions. The molecular weight of various composition poly(NIPAAm-co-
DMAAm) is summarized in Table 1. The molecular weights of PN35D5, PN35D10, and
PN35D15 determined by GPC were 7946, 10582, and 12317 Da, respectively. Using PN35D10
to conjugate with PSI, the molecular weights of PASp-g-poly(NIPAAm-co-DMAAm) was
32,130 Da. The entire polymer has good polydispersity.

3.1.4 Thermal property and miceller morphology
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The thermal weight loss curve of poly(NIPAAm-co-DMAAm), pure poly(aspartic acid),
and PASp-g-poly(NIPAAm-co-DMAAm) are shown in Figure 4. The onset of thermal
degradation of poly(NIPAAm-co-DMAAm) occurred at about 298 C and the pure poly(aspartic
acid) had multi-decomposition steps [33]. As shown in figure 4(b), PASp-g-poly(NIPAAm-co-
DMAAm) also had multi-decomposition steps. In addition, the mass loss of approximately 43%
on heating from 298 to 458
o
C implied that PASp-g-poly(NIPAAm-co-DMAAm) consisted of
43% poly(NIPAAm-co-DMAAm) and 48% PASp. Based on the thermal decomposition
temperature of PASp-g-poly(NIPAAm-co-DMAAm) obtained from TGA, these micelles can be
sterilized by autoclaving.
The particle size and morphology of PASp-g-poly(NIPAAm-co-DMAAm) was observed by
DLS and TEM microscopy. TEM image of PASp-g-poly(NIPAAm-co-DMAAm) micelles is
shown in Figure 5. All PASp-g-poly(NIPAAm-co-DMAAm) micelles showed spherical shape
and the average particle size of micelles was approximately 89.1 nm. However, the particle size
analyzed by DLS was 195 nm, which was due to the dehydration of the polymer for the
measurement of TEM microscopy.
Based on the above results of FTIR, NMR, thermal property and morphology, the
biodegradable and temperature responsive polymer PASp-g-P(NIPAAm-co-DMAAm), could be
successfully synthesized and sterilized by autoclave.

3.2 Lower critical solution temperature (LCST) of polymer
The LCST of different composition of P(NIPAAm-co-DMAAm) in phosphate buffered
saline (pH 7.4) including PN35D5, PN35D10 and PN35D15 was 37.2, 40.7 and 45.7
o
C,
respectively (Table 1). The LCST of poly(NIPAAm-co-DMAAm) could be increased with
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increasing DMAAm monomer. In addition, the LCST of PN35D10 and PN35D15 was above
normal body temperature, which is more suitable to be used for drug releasing. After grafting of
PN35D10 onto the PSI, as shown in Figure 6, the LCST of PASp-g-PN35D10 is slightly
increased to 41.9
o
C. This can be explained as due to the ring-opening of PSI by PN35D10 and
ammonium hydroxide resulting in increase of hydrogen bonding between the hydrophilic
segment of PASp and water molecular, therefore, increasing the LCST. However, the LCST of
PN35D15 without grafting onto the PSI was 45.7
o
C, which is much higher than body
temperature. Therefore, PASp-g-PN35D10 is more suitable for drug releasing in living bodies. In
pursuance of the observations, we focused on PASp-g-PN35D10 for following investigations.

3.3 Biodegradable rate of polymer
The biodegradable rate of PASp-g-poly(NIPAAm-co-DMAAm) was studied in Phosphate
buffered saline (pH 7.4) at 37
o
C. As shown in Figure 7, approximately 25% of micelle weight
disappeared in the PBS within 3 days. The copolymer composition of poly(NIPAAm-co-
DMAAm)/PSI was 1. After 7 days, about 40% of micelle weight disappeared in the PBS. The
structure of degraded micelles determined by FT-IR was poly(NIPAAm-co-DMAAm). These
results suggested that the temperature responsive micelles were degraded causing chain scission
at the primary amine site in PASp-g-poly(NIPAAm-co-DMAAm). In addition, we used
PN35D10 conjugated to low molecular weight PSI (MW=10,000, poly(NIPAAAm-co-
DMAAm)/PSI=1) and investigated the biodegradable rate in PBS at 37
o
C (data not shown).
Approximately 40 % of micelles disappeared in the PBS within 24 h suggesting a reduced
molecular weight of PSI could be used to increase the degradable rate. Based on these research
results, it is suggested that the kidney cells can filtrate the particles with molecular weight
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smaller than 40k Da [34], which means that PASp-g-poly(NIPAAm-co-DMAAm) can be
filtrated through blood stream by kidney.
3.4 Cytotoxicity of polymer
For the ultimate of PASp-g-poly(NIPAAm-co-DMAAm) micelles as drug carrier for drug
delivery, it is important that these micelles and its degradation products retain low toxicity. L929
celles were incubated with poly(NIPAAm-co-DMAAm)(PN35D10), polyaspartic acid (PASp)
and PASp-g-poly(NIPAAm-co-DMAAm) in cultured medium containing 5mg of polymer for
24, 48, and 72 h, respectively. Figure 8 shows that the variability was above 95% and there was
no significant difference from that in the control experiment. After 72 h, PASp-g-poly(NIPAAm-
co-DMAAm) was degraded about 20% and its degradation products could be dissolved in the
cultured medium. There is no significant difference between the PASp-g-poly(NIPAAm-co-
DMAAm) and control experiment. Thus, it can be concluded that both PASp-g-poly(NIPAAm-
co-DMAAm) and its degradation products have very low or no cytotoxicity for using as drug
carriers.

4. Conclusions
The present study demonstrated that a biodegradable and temperature responsive micelle,
polyaspartic acid-g-poly(N-isopropylacryamide-co-N,N-dimethylacrylamide) (PASp-g-
poly(NIPAAm-co-DMAAm)), with a LCST at 41.6
o
C could be synthesized for the use as drug
delivery system. These micelles were stable in the phosphate buffered saline (pH 7.4) at 37
o
C,
but could be deformed and aggregated above its LCST. In addition, the micelles contained free
amine groups, which allowed further conjugation with specific antibodies for targeting treatment
and could be degraded after drug delivery. Thus, these micelles may contribute to the selective
accumulation and release of drugs in the desired site.
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15


Acknowledgement
The study was supported by the National Science Council, Taiwan (Contract No. NSC 98-
2314-B-182-054-MY2, NSC 99-2628-B-182-027-MY3) and Chang Gung Memorial Hospital
under the Medical Research Project (Contract No. Animal Molecular Imaging Center
CMRPG340203). And we thank Microscopy Core Laboratory of Chang Gung Memorial
Hospital at Linkou for the use of transmission electron microscopy.
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Table 1
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Figure 1 Synthetic scheme
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Figure 2 NMR
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Figure 3 FTIR
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Figure 4 TGA
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Figure 5 TEM of PASPPND
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Figure 6 LCST
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Figure 7 percent degrgradation
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Figure 8 survial ratio