Synthesis of amphiphilic polyaspartamide derivatives and construction of reverse micelles

Article abstract of DOI:10.1039/c4ra04432k

A series of amphiphilic graft copolymers based on biodegradable and biocompatible poly(aspartic acid)s were synthesized by a successive aminolysis reaction of polysuccinimide using octadecylamine/dodecylamine, and ethylenediamine-β-cyclodextrin/ethanediamine. The chemical structures of the copolymers were confirmed by FT-IR and ¹H NMR spectroscopy. Large compound reverse micelles consisting of numerous small reverse micelles with polar cores and hydrophobic shells were formed in octanol solution. The reverse micelles showed various particle sizes based on the different length of hydrophobic alkyl chains and molecular weight of polysuccinimide, as determined by dynamic light scattering. Interestingly, the particle size of micelles showed temperature dependence, the diameter decreased continuously with increasing temperature. Their morphology and assembly properties were characterized using scanning electron microscopy, transmission electron microscopy and fluorescence spectroscopy. The reverse micelles were extremely efficient in extracting Congo red from water into octanol, exhibiting a potential application as delivery vehicles in the pharmaceutical and cosmetic fields, and as nanocontainers for separation of inorganic molecules as well. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra04432k

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Synthesis of amphiphilic polyaspartamide
derivatives and construction of reverse micelles
Cite this: RSC Adv., 2014, 4, 37130
De-E Liu,a Hui Han,a Hongguang Lu,a Guolin Wu,b Yinong Wang,b Jianbiao Maa
a
*
and Hui Gao
A series of amphiphilic graft copolymers based on biodegradable and biocompatible poly(aspartic acid)s
were synthesized by successive aminolysis reaction of polysuccinimide using octadecylamine/
a
dodecylamine, and ethylenediamine-b-cyclodextrin/ethanediamine. The chemical structures of the
copolymers were confirmed by FT-IR and 1H NMR spectroscopy. Large compound reverse micelles
consisting of numerous small reverse micelles with polar cores and hydrophobic shells were formed in
octanol solution. The reverse micelles showed various particle sizes based on the different length of
hydrophobic alkyl chains and molecular weight of polysuccinimide, as determined by dynamic light
scattering. Interestingly, the particle size of micelles showed temperature dependence, the diameter
decreased continuously with increasing temperature. Their morphology and assembly properties were
characterized using scanning electron microscopy, transmission electron microscopy and fluorescence
spectroscopy. The reverse micelles were extremely efficient in extracting Congo red from water into
octanol, exhibiting a potential application as delivery vehicles in the pharmaceutical and cosmetic fields,
and as nanocontainers for separation of inorganic molecules as well.
Received 12th May 2014
Accepted 4th August 2014
DOI: 10.1039/c4ra04432k
www.rsc.org/advances
hydrophilic biodegradable poly(amino acid) derivatives such as
poly(aspartic acid), poly(aspartamide)s, poly(hydroxyethyl
aspartamide)s, etc.22–26 The self-assembly behaviour of the
Introduction
The self-assembly behaviour of amphiphilic copolymers in
aqueous media has been extensively investigated during the
past decades due to their special physicochemical properties
and potential applications in target and transdermal drug
delivery systems, catalysis, and microelectronics.1–5 Amphi-
philic gra copolymers, in which the hydrophilic and hydro-
phobic moieties are tunable, have been widely studied because
of their various applications and relatively easy preparation
methods.6–9 Gra copolymers possess additional complexity in
self-assembly due to their complicated and conned struc-
tures,10–12 which can provide more information about the
control of micellar morphologies and the design of new
nanomaterials.13,14
Poly(amino acid)s and their derivatives have been known to
exhibit interesting biochemical properties on account of their
superior biocompatible, biodegradability and non-toxicity.15–17
They are used in various elds including biomedicine, phar-
maceuticals, cosmetics and agrochemicals.18–21 Poly-
(succinimide) (PSI) is a reactive precursor for synthetic poly-
(amino acid) derivatives because PSI can be converted easily to
amphiphilic gra poly(amino acid) derivatives have been
investigated to form regular micelles in water.27–32 However, to
the best of our knowledge, investigations about reverse micel-
lization of them have not been reported. RMs has been widely
used as nanospace microreactors and so template for
preparing inorganic nanoparticles,33,34 carriers for drugs,35–37
and they can be used to selectively enrich biomarker peptides
and protein fragments from human serum and selectively
extract/fractionate peptides based on their isoelectric points.38,39
In this paper, the amphiphilic gra copolymers of poly(aspartic
acid) (PASP) derivatives were synthesized by a successive ami-
nolysis reaction of PSI using octadecylamine (ODA)/dodecyl-
amine (DDA) as hydrophobic moieties, ethanediamine-b-
cyclodextrin (CDen)/ethanediamine (EDA) as hydrophilic
moieties. Spherical large compound reverse micelles (RMs),
which consist of numerous small reverse micelles with polar
cores and hydrophobic shells in octanol were prepared by using
the single-solvent dissolving method. The reverse micellization
behaviour was explored effectively by using the polar uores-
cence compound N-(1-naphthyl)ethylenediamine dihydro-
chloride (NEAH) as a polar uorescence probe.40 The effect of
microstructure of PASP derivatives in octanol on the reverse
micellization behaviour, temperature-responsive behaviour, as
well as on the extraction efficiency for a hydrophilic dye, were
investigated.
aSchool of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic
Solar Cells and Photochemical Conversion, Tianjin University of Technology, Tianjin
300384, China. E-mail: ghhigher@hotmail.com; hgao@tjut.edu.cn; Fax: +86
2260214251; Tel: +86 2260214259
bKey Laboratory of Functional Polymer Materials (Ministry of Education), Institute of
Polymer Chemistry, Nankai University, Tianjin 300071, China
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(molecular weight cut-off ¼ 10–12 kDa) for three days to remove
the residual ODA and CDen, and then freeze-dried (yield: 74–
85%). PASP-DDA-CDen was prepared similarly. The reaction
was performed at 100 ꢀC for 7 h to obtain PASP-DDA, according
to the reference,22 then excess CDen was added and the reaction
continued as above (yield: 80–85%).
Synthesis of octadecylamine/dodecylamine and EDA-graf-
ted–polysuccinimide (PASP-ODA/DDA-EDA). To obtain PASP-
ODA/DDA-EDA, excess EDA was then added to the PASP-ODA/
DDA solution to react with the remaining succinimide units.
Aer stirring at 75 ꢀC for 6 h, the product was puried as
described above in “Synthesis of PASP-ODA/DDA-CDen” (yield:
80–85%).
Experimental sections
Material
L-aspartic acid (L-Asp, 99%) was purchased from Alfa Co., Ltd.
(Tianjin, China). Octadecylamine (ODA) and dodecylamine
(DDA) were purchased from Heowns biochem technologies Co.,
Ltd. (Tianjin, China). Tetramethylene sulfone was purchased
from J&K Scientic Co., Ltd. (Beijing, China). Mesitylene, b-
cyclodextrin (b-CD, 98%), ethanediamine (EDA), dime-
thylformamide (DMF) and NEAH were purchased from Tianjin
Chemical Reagent Co. Ltd. (Tianjin, China). b-CD was used aer
recrystallization from water and drying under vacuum. DMF
and EDA were distilled before use.
Synthesis of mono(6-(2-aminoethyl)amino-6-deoxy)-b-cyclo-
dextrin (CDen). Mono-6-deoxy-6-(p-tolylsulfonyl)-b-cyclodextrin
(Mono-6-OTs-b-CD) was synthesized according to the method
reported in the literature.41 CDen was synthesized as described
in the previously reported method.42 Briey, 5.0 g of mono-6-
OTs-b-CD was reacted with excess amount of EDA at 75 ꢀC for 4
h. Aer the reaction was completed, the mixture was allowed to
cool to room temperature, followed by addition of 30 mL of cold
acetone, resulting in immediate formation of white precipitate.
Aer the precipitate was collected by suction ltration, the
crude product was repeatedly dissolved in 30 mL of water–
methanol mixture, and poured into a large amount of cold
acetone several times for the removal of unreacted EDA. The
Characterization of polymers
The FT-IR spectra were recorded using KBr discs on a Bio-Rod
6000 spectrometer (Thermo Electron, USA) at room tempera-
1
ture. The H NMR spectra were recorded on a Bruker AV-400
spectrometer (400 MHz, Bruker, Freemont, CA) at room
temperature with D2O or DMSO-d6 as solvents, respectively. The
molecular weight of PSI was estimated in DMF containing LiBr
(20 mmol Lꢁ1) by gel permeation chromatography [GPC;
column, PL gel 5 mm MIXED-C X 2 (Polymer Laboratories Ltd.,
U.K.); detector, refractive index; standard, polystyrene].
Preparation of RMs
ꢀ
sample obtained was dried at 50 C for 3 d in a vacuum oven,
RMs of PASP-ODA/DDA-CDen/EDA in octanol were prepared by
using the single-solvent dissolving method. The polymer (5 mg)
was dissolved in octanol (5 mL), sonicated at 100 W for 4 min in
an ice bath, and agitated until visibly clear solutions were
obtained.
and 4.3 g of CDen were obtained (yield: 80%).
Preparation of poly(succinimide) (PSI). PSI was synthesized
according to the method reported in the literature.43 Briey, L-
aspartic acid (25 g, 0.188 mol) was suspended in the mixed
solution (mesitylene/sulfolane ¼ 56 g/24 g) in the presence of o-
ꢀ
phosphoric acid (2 g, 17.34 mmol) and stirred at 180 C under
N2 atmosphere. Water formed in the reaction process was
continuously removed using the Dean–Stark trap. The reaction
was processed for 4.5 h. Aer the reaction mixture was cooled
down, the solvent was removed and DMF was added to dissolve
the polymer, and precipitated into excess methanol twice. Aer
ltration, the precipitate was washed several times with
distilled water to remove the phosphoric acid. The product was
Particle size analysis and morphology examination
The mean particle size and size distribution of the polymer
aggregates were investigated in octanol at 20 ꢀC by dynamic
light scattering measurements (DLS) on a Zetasizer Nano ZS90
(Malvern, Worcestershire, U.K.).
Transmission electron microscopy (TEM, JEOL, Japan) was
conducted on a Jeol 1400 instrument with an accelerating
voltage of 100 kV. Samples were prepared by dropping two drops
of the RM solutions (0.1 mg mLꢁ1) onto a carbon coated copper
grid and drying for 1 week in the fume cupboards.
Scanning electron microscope (SEM, JEOL, Japan) was con-
ducted on a JSM-6700F type instrument with an accelerated
voltage of 10 kV. Samples were prepared by dropping two drops
of the RM solutions (0.5 mg mLꢁ1) on coverslip. The samples
were dried and coated with a thin layer of gold.
ꢀ
nally dried at 80 C in vacuum for three days to obtain PSI in
white powder (yield: 80%).
Synthesis of octadecylamine/dodecylamine and CDen-graf-
ted-polysuccinimide (PASP-ODA/DDA-CDen). The preparation
of PASP-ODA-CDen was according to the synthesis method of
PASP-ODA and PASP-CDen.6,44 Briey, PSI (97 mg, 1 mmol) was
dissolved in DMF (10 mL), followed by addition of ODA (135 mg,
0.5 mmol) dissolved in DMF (5 mL) rapidly. The reaction
mixture was stirred continuously under a N2 atmosphere at 70
ꢀC for 24 h. Excess CDen dissolved in DMF was then added to
the solution to react with the remaining succinimide units.
Aer stirring at 60 ꢀC for another 48 h, the product was
precipitated in excess ether twice to remove the residual ODA.
The precipitate was ltered and dissolved in DMF, aer which
the product solution was dialyzed against ethanol, then dia-
lyzed against distilled water using a dialysis membrane
Aggregation behaviour and critical aggregation concentration
(CAC)
The self-assembly behaviour of PASP derivatives in octanol was
explored by using the polar uorescence compound NEAH as a
polar uorescence probe. The prepared copolymer micellar
solution was diluted step by step in the range of 1 ꢂ 10ꢁ1 to 5 ꢂ
10ꢁ6 mg mLꢁ1 to obtain octanol solutions of PASP derivatives
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with continuously varying concentrations. Aer stirring for half Subsequently, the copolymer suspensions with the concentra-
an hour, 10 mL of the solutions was taken and added into a tions of 2.5, 2.0, 1.5, 1.0, and 0.5 mg mLꢁ1 were added to 96-well
conical ask of 50 mL, respectively, and 6 mL of methanol plates at 10 mL per well and incubated for 24 h, respectively. The
solution of NEAH (2 mg mLꢁ1) was added into these conical solution was then removed and replaced with 100 mL of RPMI-
ask using a microinjector to obtain a solution with NEAH 1640. Then, CCK-8 solution was added to 96-well plates at 10 mL
concentration of 4.6 ꢂ 10ꢁ6 mmol mLꢁ1. Aer stirring for 15 per well and incubated for 24 h, and the resulting solution was
min, the uorescence emission of these solutions was analyzed at 450 nm by means of a plate reader with a back-
measured by the excitation at 320 nm using a spectro- ground correction using
a Bio-Tek FLx800 Fluorescence
uorophotometer (F-7000, Hitachi, Japan). The CACs were Microplate Reader. This process was repeated for eight times in
determined as the concentration where the uorescence parallel. The results are expressed as the relative cell viability
intensity substantially changed.
(%) with respect to control wells containing culture medium.
Temperature sensitivity of RMs
Results and discussion
Synthesis and characterization of the polymers
The temperature responsibility of the RMs was investigated
using DLS performed from 20 ꢀC to 75 ꢀC in octanol solution at
ꢀ
Scheme 1 shows the reaction scheme for the synthesis of PASP-
DDA/ODA-CDen/EDA. These copolymers were synthesized from
PSI through a successive aminolysis reaction with quantitative
a temperature interval of 5 C.
Thermodynamic investigation of RMs
5 mL of deionized water was added into 1.5 mL (0.5 mg mLꢁ1) of
RMs each time under stirring. Aer stirring for 1 h, DLS was
used to measure the size of the RMs. The volume was recorded
as the critical volume when micro-phase separation occurred.
Extraction of Congo red from water
The aqueous solution of Congo red with a concentration of 0.25
mg mLꢁ1 was equilibrated with equal volume of polymer solu-
tions in octanol (1 mg mLꢁ1) for 2 h. The amount of dye
extracted into the organic layer was determined by the nal dye
concentrations in water, which can be estimated by monitoring
the absorbance changes at the wavelength of maximum
absorption (496 nm) using a UV-vis spectrophotometer (TU-
1900, Persee, Beijing).
Cell viability assays
Cell viability was detected using Cell Counting Kit-8 (CCK8)
assay. Mouse broblast cells (L929) were cultivated in a
humidied 5% carbon dioxide atmosphere at 37 ꢀC on a 96-well
microplate, with 5000 cells immersed in complete growth
medium per well. The cells were allowed to attach for 24 h.
Fig. 1 1H NMR spectra of PSI (A), PASP-ODA-EDA (B) in DMSO-d6,
CDen (C) in D2O and PASP-ODA-CDen (D) in DMSO-d6.
Scheme 1 Synthesis of PSI (A), PASP-DDA/ODA-CDen (B) and PASP-
DDA/ODA-EDA (C).
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DDA/ODA, and CDen/EDA. The weight average molecular
weight of PSI was determined by GPC as 25 kDa and 45 kDa
respectively. Their chemical structures were characterized by 1H
NMR spectroscopy (Fig. 1). The signals at 5.0–5.4 ppm and 2.5–
3.3 ppm were assigned to methine proton (b) and methylene
proton (a) of PSI. While in the 1H NMR spectrum of PASP-DDA/
ODA-CDen, new signals from the N-dodecyl/octadecyl asparti-
mide units were observed at 0.8 ppm for methyl protons (c), at
1.2–1.50 ppm for methylene protons (d, e) and at 3.0 ppm for
methylene protons (f) adjacent to amino group of the dodecyl/
octadecyl chain. In addition, the ring opening of succinimide
units caused the appearance of a new signal at 4.5 ppm assigned
to the methine proton of the N-dodecyl/octadecyl aspartamide
units. The absence of the peak at 5.0–5.4 ppm related to the
methine proton in succinimide units clearly indicated that the
ring opening reaction of PSI completed. The peaks “1” at 4.80
ppm and “2–6” at 3.20–3.80 ppm could be assigned to the
protons on C1 and on C2–C6 of the CDen unit. The peaks “a”
and “b” at 2.5–2.8 ppm arose from the methylene protons of
ethanediamine groups in CDens. In addition, the peaks located
at 5.5–6.0 ppm assigned to active hydrogens (–NH, –OH) was
observed. The 1H NMR results indicated that the PASP-DDA/
ODA-CDen was synthesized successfully via aminolysis of PSI.
The 1H NMR spectrum of PASP-DDA/ODA-EDA was shown in
Fig. 1B. The peaks “g” and “h” were methylene protons of EDA.
Other peaks could be well assigned as above.
Fig. 2 FT-IR spectra of PSI (A), CDen (B), PASP-DDA-CDen (C), and
PASP-DDA-EDA (D).
Especially, the carbonyl C]O from cyclic imides stretching
located at 1710 cmꢁ1 was absence in Fig. 2C and D. A new peak
located at 1670 cmꢁ1, assigned to the stretching vibration of
carbonyl in the amido group on the PASP-DDA-CDen/EDA
appeared, indicating that the aminolysis of PSI completed.
Characterization of RMs
The aminolysis degree (AD) is the mole percent of the
attached unit with alkyl group per total succinimide unit. Table
1 shows AD of DDA/ODA and CDen according to feed molar
ratio. The AD of each side chain was determined by comparing
the integral ratio of peaks assigned to the methyl protons (c) of
N-dodecyl/octadecyl aspartimide units, the protons on C1 (1) of
the CD unit with the integral peak assigned to the methine
proton (b) of the polyaspartamide backbone in 1H NMR. The AD
of CDen-contained polymers was much lower than that of ODA/
DDA-contained ones due to the steric hindrance of CDen unit.
As shown in Fig. 2, the FT-IR spectra of PSI showed the
stretching vibration of –NH– located at about 3430 cmꢁ1, and
the carbonyl C]O from cyclic imides stretching located at 1710
cmꢁ1. The peaks located at 3380 and 1030 cmꢁ1 were attributed
to multihydroxy of the CDen moiety in PASP-DDA-CDen.
The amphiphilic PASP derivatives were then employed to
prepare RMs. The DLS graph of the PASP(45K)-ODA-CDen RMs
with narrow particle size distribution was shown in Fig. 3. Their
particle size and distribution were measured by DLS (Table 1).
The average diameters of nanoparticles were around 300–400
nm for the PASP derivatives, and increased with increasing alkyl
length of the alkyl chain and the molecular weight of PSI. The
longer alkyl chain led to the lower ratio of the hydrophilic and
hydrophobic segments, thus the size of PASP(45K)-ODA-CDen
was larger than that of PASP(45K)-DDA-CDen. On the other
hand, a larger space volume produced from the copolymers
with higher molecular weight, thus the PASP(45K)-based poly-
mers exhibited larger size than PASP(25K)-based ones. Most of
PDI of these assemblies was lower than 0.2, indicating the
Table 1 Characteristics of PASP derivatives
Sample
ADa (%)
ADb (%)
Diameter (nm)
PDI
Extraction of Congo redc (%)
PASP(25K)-DDA-CDen
PASP(45K)-DDA-CDen
PASP(25K)-ODA-CDen
PASP(45K)-ODA-CDen
PASP(25K)-DDA-EDA
PASP(45K)-DDA-EDA
PASP(25K)-ODA-EDA
PASP(45K)-ODA-EDA
45.7
48.3
43.0
47.3
43.0
46.0
40.0
36.3
19.7
15.1
21.4
20.6
—
—
—
—
305
331
310
362
315
376
350
423
0.163
0.171
0.145
0.143
0.148
0.103
0.149
0.252
53.5
63.0
59.8
66.3
92.3
95.0
93.0
97.0
a
The aminolysis degree of the N-dodecyl/octadecyl aspartimide units, dened as the molar ratio of the N-dodecyl/octadecyl aspartimide units to the
succinimide unit in original polysuccinimide, and was determined by 1H NMR spectra (mol%). b The aminolysis degree of the CDen units, dened
as the molar ratio of the CDen units to the succinimide unit in original polysuccinimide, and was determined by 1H NMR spectra (mol%). c The
initial concentration of Congo red in water is 0.25 mg mLꢁ1
.
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Scheme 2 Formation of large compound reverse micelles by PASP
derivatives.
numerous small reverse micelles with polar cores and hydro-
phobic shells as shown in Scheme 2. We then examined the
assembly properties of these micelles using a polar uorescence
probe, and found that reverse micelles with hydrophilic cores
and hydrophobic shells were formed (Fig. 5).
Fig. 3 DLS graph of the PASP(45K)-ODA-CDen RMs.
narrow distribution of the RMs. Only PASP(45K)-ODA-EDA
exhibited a relative high PDI. Its larger diameter probably led to
a wider dispersion.
Determination of the CAC
Fig. 5 shows the varied uorescence emission of NEAH at
different copolymer concentrations in octanol. The variation
in uorescence intensity was similar for these four polymers.
When the concentration of copolymers was lower, the uo-
rescence emissions of NEAH decreased slightly with the
increased concentrations of copolymers. While as the
concentrations of copolymers increased to a certain value, the
uorescence emission of NEAH decreased sharply, and then
the emissions remained constant. The abrupt changes of the
uorescence emissions of NEAH evidenced the formations of
RMs, indicating that NEAH can be used as a polar uores-
cence probe, and it can be used to explore macroscopically the
reverse micellation behaviour of the amphiphilic gra
copolymers of PASP derivatives in octanol. PASP derivatives
could self-assemble into RMs at a certain concentration in
octanol, consisting of hydrophilic PASP-CDen/EDA units as
core and hydrophobic alkyl chain as shell. The CACs of the
four copolymer samples were 5 mg mLꢁ1 for PASP(25K)-ODA-
CDen (Fig. 5A), 1 mg mLꢁ1 for PASP(25K)-DDA-CDen (Fig. 5B),
5 mg mLꢁ1 for PASP(45K)-ODA-CDen (Fig. 5C) and 10 mg mLꢁ1
for PASP(45K)-DDA-EDA (Fig. 5D). By comparing these data, it
could be found that for the copolymer samples with the same
length of PASP unit but different length of alkyl chain (ODA,
DDA), the longer the alkyl chain is, the lower ratio of the
hydrophilic and hydrophobic segments is, and the higher the
CAC is. Therefore, the CAC was 5 mg mLꢁ1 for PASP(25K)-ODA-
CDen and 1 mg mLꢁ1 for PASP(25K)-DDA-CDen. For polymer
PASP(45K)-DDA-EDA with EDA as the hydrophilic chain,
which is shorter than CDen units, had the highest CAC (10 mg
mLꢁ1). PASP(25K)-ODA-CDen and PASP(45K)-ODA-CDen had
the same CAC (5 mg mLꢁ1), indicating that the molecular
weight of the polymers did not inuence on the CACs. The
experimental results are in agreement with general rule that
the CAC of the amphiphilic copolymer in the selective solvent
depends greatly on the length of the insoluble segments,
and the copolymer with longer insoluble segments has a
lower CAC.40
Fig. 4A shows a typical SEM image of PASP(45K)-DDA-CDen
RMs formed in octanol solution (0.1 mg mLꢁ1) at 25 ꢀC.
Spherical particles with a diameter of approximately 200 nm
were observed. The TEM image (Fig. 4B) also shows the pres-
ence of spherical micelles with a diameter of 200 nm. The size
observed from TEM and SEM was smaller than that measured
by DLS (331 nm) because the samples were in the dried state
when observed using microscopy. Here, the size of spherical
micelles (300–400 nm) from DLS was much larger than the
maximum length of the repeating unit of copolymers estimated
by ChemOffice soware. The enlarged TEM image (Fig. 4C and
D) also showed multi-cores of the micelle. This kind of micelle
was named as a large compound reverse micelle consisting of
Fig.
4 SEM image (A), TEM image (B) of PASP-DDA-CDen and
enlarged TEM of PASP(45K)-DDA-CDen (C) and PASP(45K)-DDA-EDA
(D) in octanol solution at 25 ꢀC.
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Fig. 5 Varying fluorescence emission of NEAH in copolymer octanol solution with copolymer concentration (mg mLꢁ1) for different copoly-
mers: a, 5 ꢂ 10ꢁ6; b, 1 ꢂ 10ꢁ5; c, 5 ꢂ 10ꢁ5; d, 1 ꢂ 10ꢁ4; e, 5 ꢂ 10ꢁ4; f, 1 ꢂ 10ꢁ3; g, 5 ꢂ 10ꢁ3; h, 1 ꢂ 10ꢁ2; i, 5 ꢂ 10ꢁ2; j, 1 ꢂ 10ꢁ1
.
Fig. 6 Effects of temperature on average diameter (A–D) and relative scattering intensity (C and D) of different RMs.
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Table 2 The maximum water content in RMs
copolymers with higher molecular weight. Therefore, The crit-
ical water volume of PASP(45K)-ODA-CDen/EDA was higher
than that of PASP(25K)-ODA-CDen/EDA.
Sample
Vwater (mL)
Vwater/Voctanol
PASP(25K)-ODA-CDen
PASP(45K)-ODA-CDen
PASP(25K)-ODA-EDA
PASP(45K)-ODA-EDA
20
30
40
60
0.013
0.020
0.026
0.040
Encapsulation of Congo red in RMs
Fig. 7 shows the differences in color intensity of dye solution
when different polymers were used for the transfer, exhibiting
the difference in loading capacity of polymeric micelles. We
distinguished the differences between these copolymers by
comparing an initial Congo red concentration with the nal dye
concentration in water aer the transfer, measured by optical
spectroscopy. The extraction rate of Congo red by different PASP
derivatives was shown in Table 2. The extraction rate of PASP
derivatives with CDen unites was around 60%, and 90% for the
polymers without CDen unites. Analysis of the dye concentra-
tion in aqueous revealed that vast majority of the dye molecules
was transferred to the octanol. The polymers without CDen
units were more efficient to transfer the Congo red. The larger
size of PASP-ODA/DDA-EDA may be suitable to accommodate
more Congo red molecules. However, the RMs of PASP-ODA/
DDA-EDA was less stable during storage than PASP-ODA/DDA-
CDen probably due to their larger sizes.
Temperature sensitivity of RMs
Fig. 6 shows temperature dependence of the diameter of
different RMs. The size of the polymers decreased upon
increasing temperature at a uniform speed. The polymer chain
shrinked with increasing temperature, which probably led to
the decrease in particle size. The size variation upon tempera-
ture changes was reversible for PASP-ODA/DDA-CDen (Fig. 6A
and B). However, the particle size variation was irreversible for
PASP(25K)-ODA/DDA-EDA (Fig. 6C and D) with EDA as the
hydrophilic chains and the RMs could not be reconstructed
upon decreasing temperature. This is probably because that the
relative shorter hydrophilic EDA chain, compared with CDen
unit, as the driving force to form hydrophilic core is lower. To
check the existence or dissolution of micelles, we measured the
relative scattering intensity. As for PASP(25K)-ODA-EDA, when
the temperature was higher than 55 ꢀC, the intensity decreased
sharply to zero. While for PASP(25K)-DDA-EDA the transition
temperature was 65 ꢀC, which is higher than that of PASP(25K)-
ODA-EDA. The longer alkyl chain (ODA) made it easier dissolve
in octanol solution, leading to a disaggregation.
Cell viability assays
The in vitro cytotoxicity of the polymers is an extremely impor-
tant factor for their potential application as delivery vehicles in
the pharmaceutical and cosmetic elds. In this work, the in vitro
cytotoxicity of the polymers of PASP(45K)-ODA-CDen and
PASP(45K)-ODA-EDA were evaluated using CCK8 assays against
L929 cells as a function of the concentrations (Fig. 8). All the
samples retained high cell viability (>70%) aer 24 h of incu-
bation at all tested concentrations up to 0.25 mg mLꢁ1. Some
average cell viabilities were a little bit larger than 100% (about
110%) in Fig. 8, probably due to the experimental error of the
data, with standard deviation of ꢃ10%. Their low cytotoxicity
further highlighted the polymers could be used as biomaterials.
Thermodynamic investigation of RMs
A similar method was employed to explore the thermodynamic
characteristic of the RMs as reported by Eisenberg45 and
Cheng.46 Table 2 showed the critical water volume in 1.5 mL
RMs before generating micro-phase separation. The polymer
solutions of PASP(25K)-ODA-EDA and PASP(45K)-ODA-EDA
containing EDA unit displayed higher water content than
PASP(25K)-ODA-CDen and PASP(45K)-ODA-CDen The larger
sizes of EDA-contained polymers (Table 1) endowed them with
larger inside water cores to accommodate more water. Addi-
tionally, a larger space volume probably produced from the
Fig. 7 The transfer of Congo red by PASP(45K)-ODA-CDen (A) and Fig. 8 Cell viabilities of polymers against L929 cells. The cells were
PASP(45K)-ODA-EDA (B) micellar solutions. The upper is the octanol treated with increasing concentrations of polymers, incubated for 24 h
layer, the lower is the water layer.
before analysis by CCK8 assay.
37136 | RSC Adv., 2014, 4, 37130–37137
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Paper
RSC Advances
19 S. R. Yang, H. J. Lee and J. D. Kim, J. Controlled Release, 2006,
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Conclusions
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In this study, a series of amphiphilic PASP derivatives were
synthesized and characterized. All copolymers could form RMs
in octanol solution using the single-solvent method. The size of
the RMs decreased upon heating and it can be reversible when
cooling for copolymers containing CDen units. The micro-
structures and CACs of the RMs were explored using NEAH as a
polar uorescence probe. Their low cytotoxicity and the ability
to efficient extraction of Congo red from water into octanol may
be particularly interesting for the application of sustained drug
delivery systems and nanocontainers.
´
´
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Acknowledgements
Financial support from National Natural Science Foundation of
China (21374079), Program for New Century Excellent Talents
in University (NCET-11-1063), Program of for Prominent Young
College Teachers of Tianjin Educational Committee is highly
acknowledged.
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RSC Adv., 2014, 4, 37130–37137 | 37137