Well-defined biodegradable amphiphilic conetworks

Article abstract of DOI:10.1039/c3sm27853k

A series of amphiphilic conetworks (APCNs) with well-defined molecular structures were prepared via a copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) of tetrakis(2-propynyloxymethyl)methane (TMOP), diazide end-functionalized triblock copolymers of poly(ε-caprolactone) with poly(ethylene glycol) (N₃-PCL-PEG-PCL-N₃). The so-prepared APCNs exhibit unique properties of ordered nanophase separation of hydrophilic (HI) and hydrophobic (HO) phases, and a variable swelling capacity both in water and organic solvent. The morphology, surface properties and thermal behavior of the APCNs were investigated by scanning electron microscopy (SEM), water contact angle (WCA), and differential scanning calorimetry (DSC), respectively. The physical properties of APCNs depended on the ratio of HI-HO, which can be regulated via precise synthesis of N₃-PCL-PEG-PCL-N₃. The analysis of an in vitro cell viability assay suggests that the APCNs have excellent biocompatibility. The prepared APCNs are excellent carriers for controlled drug release. The hydrophilic choline theophyllinate and hydrophobic 5-fluorouracil (5-FU) were loaded into the APCNs simultaneously as model drugs to study the release from APCNs. The well-controlled drug release is attributable to the well-defined molecular structure and tunable HI/HO composition of the APCNs.

Full text of DOI:10.1039/c3sm27853k

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PAPER
Well-defined biodegradable amphiphilic conetworks†
a
a
b
c
c
a
Yang Yuan, Ao-Kai Zhang, Jun Ling,* Li-Hong Yin, Yue Chen and Guo-Dong Fu*
Cite this: DOI: 10.1039/c3sm27853k
A series of amphiphilic conetworks (APCNs) with well-defined molecular structures were prepared via a
copper-catalyzed 1,3-dipolar azide–alkyne cycloaddition (CuAAC) of tetrakis(2-propynyloxymethyl)-
methane (TMOP), diazide end-functionalized triblock copolymers of poly(3-caprolactone) with
3 3
poly(ethylene glycol) (N -PCL-PEG-PCL-N ). The so-prepared APCNs exhibit unique properties of ordered
nanophase separation of hydrophilic (HI) and hydrophobic (HO) phases, and a variable swelling capacity
both in water and organic solvent. The morphology, surface properties and thermal behavior of the
APCNs were investigated by scanning electron microscopy (SEM), water contact angle (WCA), and
differential scanning calorimetry (DSC), respectively. The physical properties of APCNs depended on the
3 3
ratio of HI–HO, which can be regulated via precise synthesis of N -PCL-PEG-PCL-N . The analysis of an
in vitro cell viability assay suggests that the APCNs have excellent biocompatibility. The prepared APCNs
are excellent carriers for controlled drug release. The hydrophilic choline theophyllinate and
hydrophobic 5-fluorouracil (5-FU) were loaded into the APCNs simultaneously as model drugs to study
the release from APCNs. The well-controlled drug release is attributable to the well-defined molecular
structure and tunable HI/HO composition of the APCNs.
Received 13th December 2012
Accepted 18th April 2013
DOI: 10.1039/c3sm27853k
www.rsc.org/softmatter
32,33
response.
Chemical combination of HI and HO pre-poly-
1
Introduction
mers renders another plausible synthetic approach of APCNs,
34
Amphiphilic conetworks (APCNs), composed of covalently for instance, Diels–Alder reaction, and Michael-type additions
interconnected hydrophilic and hydrophobic (HI/HO) chain reactions.35–38 However, high defects and low strength in
segments, have attracted increasing attention owing to their
the prepared APCN are their major limitations due to low
reaction yield.
1–3
unique structures and properties.
The incompatibility of
39–48
HI/HO segments results in a phase separated morphology on
“Click chemistry”,
especially copper(I)-catalyzed azide–
the nanoscale and renders APCNs capable of swelling in both alkyne cycloaddition (CuAAC), has been approved as a highly
aqueous and organic media without encountering macroscopic effective approach for the preparation of three-dimensional
phase separation or polymer leaching. APCNs have found well-dened polymeric networks due to its reaction specicity,
4
–12
13–15
49–52
versatile applications as drug carriers,
contact lenses,
quantitative yields and good functional group tolerance.
16–20
biomaterials for tissue engineering,
membranes for ltra- CuAAC has exhibited outstanding capability to prepare hydro-
21
22
23–26
tion and pervaporation, and catalysis supports.
APCNs can be synthesized by random free radical copoly-
merization of multifunctional crosslinkers.
gels with well-dened molecular structures and good physical
53–57
and mechanical properties.
CuAAC was also applied to
27–31
41
48
However, the
´ ˆ
prepare APCNs, as reported by Jerome et al. and Dubois et al.
uncontrollable polymerization and the low monomer conver- However, these APCNs are prepared from the macromonomer
sion lead to defects and disorder in the molecular structure of with cross-linking points randomly distributed on the back-
the APCN. Additionally, the un-reacted monomers in the APCNs bones, resulting in a random HO/HI distribution in the
limit their application in biomedical elds, because the alkenes conetworks.
may cause a local inammatory reaction or systematic immune
Polyethylene glycol (PEG) is a well-known uncharged, water-
soluble, nontoxic and nonimmunogenic biomaterial. Poly-
(3-caprolactone) (PCL) is another FDA-approved biocompatible
a
School of Chemistry and Chemical Engineering, Southeast University, Jiangning
District, Nanjing, Jiangsu Province, P.R. China 211189. E-mail: fu7352@seu.edu.cn; thermoplastic polyester with adjustable degradation time,
5
8–60
Fax: +86-25-52090625; Tel: +86-25-52090625
which can fulll targeted therapeutic applications.
Herein,
b
MOE Key Laboratory of Macromolecular Synthesis and Functionalization,
we prepare PEG/PCL APCNs with a well-dened molecular
structure and precisely controlled composition of HO/HI by
CuAAC. The special swelling properties and low cytotoxicity of
prepared APCNs, as well as the interesting drug release proles,
suggest that the prepared APCNs could be a kind of excellent
drug carrier for controlled release.
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou,
Zhejiang Province, P.R. China 310027. E-mail: lingjun@zju.edu.cn
c
School of Public Health, Southeast University, Nanjing, Jiangsu Province, P.R. China
2
†
1
11189
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3sm27853k
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mixture was stirred for another 24 h at room temperature. The
solvent was evaporated and the residue was extracted with
2
Experimental section
2.1 Materials
7
0 mL methylene chloride. Aer ltration, the organic layer was
ꢁ
1
ꢁ1
Propargyl bromide (80%), pentamethyldiethylenetriamine washed with water, 1 mol L HCl, and 1 mol L NaHCO3
PMDETA, 99%), stannous octoate (Sn(Oct) , 95%), 2-bromo- aqueous solution successively, and dried over anhydrous
(
2
propionyl bromide (97%), 5-uorouracil (5-Fu), choline MgSO . White solid was precipitated by pouring the solution
4
theophillinate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- into cold diethyl ether. Aer being ltered and dried in vacuum,
trazolium bromide were purchased from Aldrich Chemical Co. the white solid was dissolved in 70 mL DMF. Aer sodium azide
and used as received. Sodium azide (99%), copper(I) bromide (Warning: sodium azide may cause explosion. 3.8 g, 60 mmol)
(CuBr, 98%) and pentaerythritol were purchased from Shanghai was added, the reaction mixture was stirred for 72 h at room
Chemical Reagent Plant. L929 cells were obtained from the temperature, and then was ltered. DMF was removed by
Chinese Academy of Sciences. Copper(I) bromide was puried distillation under reduced pressure. Methylene chloride (80 mL)
by stirring in acetic acid, washing with methanol, and drying was added and washed with water 3 times. The organic layer
under vacuum. RPMI-1640 medium, a nutrient solution for cell was dried over anhydrous MgSO . N -PCL -PEG -PCL -N was
4
3
10
23
10
3
culture, and phosphate buffered saline (PBS) were purchased obtained aer evaporation and dried in vacuum (1.87 g, yield ¼
from Gibco. Dimethyl sulfoxide (DMSO) was purchased from 18.7%). N -PCL -PEG -PCL -N and N -PCL -PEG -PCL -N
3
3
10
45
10
3
3
5
91
5
the Chemical Reagent Company of National Pharmaceutica were synthesized with yields of 24.9% and 22.1%, respectively.
Group. 3-Caprolactone (CL, 99%, Acros) was distilled under
2.2.4 Synthesis of APCNs via CuAAC. About 0.066 g
reduced pressure aer being stirred over CaH for 24 h. Toluene (0.02 mmol) N -PCL -PEG -PCL -N , 2.88 mg (0.01 mmol) of
2
3
10
23
10
3
and tetrahydrofuran (THF) were dried by reuxing over sodium tetrakis(2-propynyloxymethyl)methane and 1 mL DMF was
benzophenone ketyl and distilled directly into reaction asks. added into a small vial, then the vial was put under ultrasonic
Triethylamine was distilled before use. Poly(ethylene glycol)s agitation to make the reactants dissolve in DMF completely.
(PEGs with degrees of polymerization of 23, 45 and 91) were When the solution turned clear, 2.87 mg (0.02 mmol) CuBr was
carefully dried by azeotropic distillation with toluene.
added into the vial and degassed with N for about 15 min. Aer
2
that, 3.46 mg (0.02 mmol) PMDETA was added into the vial with
a syringe. Then the vial was put under ultrasonic agitation at
room temperature. Gelation of the APCN was obtained in about
2.2 Chemical synthesis
2
.2.1 Synthesis of tetrakis(2-propynyloxymethyl)methane 1 min. The reaction was allowed to react at room temperature
TPOM). Tetrakis(2-propynyloxymethyl)methane (TPOM) was for another 12 h to obtain a uniform solid structure. Aer
synthesized according to the method reported in the litera- complete gelation, the gel was rstly immersed into an EDTA
(
54
ture. About 2 g (0.014 mmol) pentaerythritol and 12.5 g (5%) solution and then DMF containing PMDETA to remove the
(
(
0.22 mmol) KOH was dissolved in 25 mL dimethylformamide copper ions. At last, the gel was immersed in deionized water to
ꢀ
DMF). The mixture was stirred for 30 min at 5 C. Aer that, achieve maximum water absorption. Two other gels from N
3
-
2
0 g (0.17 mmol) propargyl bromide was slowly added into the PCL
reaction mixture and reacted at 40 C for 24 h. Aer cooling, prepared in the same way in 1.5 mL DMF at room temperature.
5
-PEG91-PCL
5
-N
3
and N
3
-PCL10-PEG45-PCL10-N
3
were also
ꢀ
water (c.a. 100 mL) was added into the mixture and the mixture (Molar
ratio:
[N -PCL -PEG -PCL -N ]–[TPOM]–[CuBr]–
3 y x y 3
was extracted with ether. The organic layers were washed with [PMDETA] ¼ 0.5 : 0.25 : 1 : 1.)
water and brine, and then dried over Na SO . Aer removal of
2
4
ether, the residue was puried by a silica gel column using ethyl
2
.3 Characterization
acetate–hexane (v/v ¼ 2 : 8) as the eluent, giving about 2.9 g
1
yellow solid. Yield ¼ 78%; H NMR (CDCl
3
, ppm): 4.13 (8H, The chemical structures of Br-PCL
y
-PEG
x
-PCL
y
-Br and N
3 y
-PCL -
ꢁ
1
1
–
OCH
2
), 3.54 (8H, C(CH
2
)
4
) and 2.43 (4H, CH); FTIR (cm ): PEG
exclusion chromatography (SEC). H NMR spectra were recor-
-OH. 5.0 g PEG ded on a Bruker Avance DMX500 spectrometer in CDCl with
x y 3
-PCL -N were characterized by H-NMR, FTIR and size
1
n
(^CH) ¼ 3297.
2
.2.2 Synthesis of HO-PCL
y
-PEG
x
-PCL
y
3
(
(
M
n
¼ 1000) was dissolved in toluene (15 mL). 3-Caprolactone tetramethylsilane as the internal standard. FT-IR spectra were
10.0 g, 87.7 mmol) and stannous octoate (16 mL, 0.04 mmol) recorded on a Bruker Vector 22 IR spectrometer. Number-
ꢀ
were added to the reaction mixture and stirred at 110 C for average molecular weights (M ) were measured by size exclusion
n
24 h. White product was isolated by precipitation from n-hexane chromatography (SEC) using a Waters 1515 system equipped
with a yield of 99%. When PEG-2000 and PEG-4000 were used, with two PL Mixed-C columns using THF as the eluent at a ow
ꢁ1
ꢀ
the yields of the corresponding HO-PCL -PEG -PCL -OH block rate of 1.0 mL min at 40 C against commercial polystyrene
y x y
copolymers were 99% and 76%, respectively. M
calculated by H NMR analyses.
n
and DP were (PSt) standards (Waters ShodexVR). The morphology of the
APCNs was studied on a scanning electron microscope (XL30
1
2.2.3 Synthesis of
N
3
-PCL
y
-PEG
x
-PCL
y
-N
3
.
HO-PCL10
-
SEM) at an accelerating voltage of 25 kV. The swollen gels were
PEG -PCL -OH (10.0 g, 3.0 mmol) was mixed with triethyl- dried in a LGJ-10C freeze-dryer. The DSC measurements were
2
3
10
ꢀ
amine (1.8 g, 18.0 mmol) in 50 mL THF and cooled to 0 C. A performed under a nitrogen atmosphere on a Perkin-Elmer
ꢀ
solution of 2-bromopropionyl bromide (5.2 g, 24.0 mmol) in DSC7 calorimeter. All samples were rstly heated to 100 C to
ꢀ
1
0 mL THF was added dropwise over 15 min. The reaction erase the thermal history, then cooled to ꢁ100 C at a rate of
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ꢀ
ꢁ1
ꢀ
ꢀ
1
0 C min , and nally heated to 100 C again by a rate of 10 C cells grew to the expected concentration, they were trypsinized
ꢁ
1
min . For cytotoxicity evaluation, the optical densities (OD with 0.05% trypsin/EDTA and then incubated for 5 min to
70) were measured at 570 nm with a Rayto RT-6000 ELISA obtain complete cell detachment. The cell suspension was
analyzer. The transmittance spectra of the prepared APCNs were centrifuged at 1300 r min for 10 min followed by resus-
measured on a Hitachi U-4100 Ultraviolet-visible (UV-Vis) pension of the cell pellets with complete medium. The prepared
5
ꢁ
1
6
spectrophotometer. The drug release concentration was deter- cell suspension was seeded into 96-well plates at 1 ꢂ 10 viable
mined by measuring the absorbance at 264.5 nm and 273 nm. cells per well and incubated with conditioned media for 24 h,
ꢀ
4
8 h and 72 h, respectively, at 37 C in 5% CO
2
before MTT
2
.4 Swelling of APCNs
The swelling of the networks was studied by immersing dry gels
Weightdry gel) in various solvents at room temperature. They
assays. Then, cell survival was measured using an MTT assay.
Samples, both negative control group and positive control
group were lled with treated samples, RPMI-1640 medium,
and phenol solution, respectively. Aer 24 h, 48 h and 72 h
(
were weighed (Weightswollen gel) at intervals until the swelling
equilibration was reached. Each value was the average of three
measurements. The swelling ratio (SR) (g/g) of a gel was deter-
mined using eqn (1).
ꢁ
1
treatment, 10 mL MTT (5 mg mL ) was added to each well and
ꢀ
the plates were incubated for another 4 h at 37 C. Then 150 mL
DMSO was added to each cell to dissolve intracellular MTT
formazan crystals. The absorbance was determined at 570 nm
using a multifunctional microplate reader. Experiments were
done in triplicate and were repeated at least twice. The cell
weight
SR
¼ [(Weightswollen gel/Weightdry gel) ꢁ 1] ꢂ 100% (1)
Additionally, taking the differences of solvent density into relative growth rate (RGR) was calculated as eqn (4).
consideration, the equation from a gravimetric approach was
revised as eqn (2).
RGR (%) ¼ [ODtest ꢁ ODblank]/[ODcontrol ꢁ ODblank] ꢂ 100%(4)
volume
where ODtest is the experiment group optical density; ODcontrol is
the negative group optical density (OD); ODblank is the blank
group optical density (OD). The cell MTT viability in the three
SR
¼ [(Weightswollen gel ꢁ Weightdry gel)/
solvent ꢂ Volumedry gel)] ꢂ 100%
(
r
(2)
where rsolvent is the density of the solvent and Volumedry gel is experiment group extracts was compared with that of the
the volume of the dry gel. negative group and positive group.
2.5 In vitro degradation studies
2.7 Control and release of drugs
The degradation tests were carried out by immersing the APCNs
specimens in the phosphate-buffered saline solution (pH ¼ 7.4)
APCN partly swells in aqueous solvent. In that case, the drug can
not be encapsulated in the HO phase. In order to load the drugs
both in the HO and HI phase, a mixture of solvents was used for
drug encapsulation. The dried APCNs were soaked in a THF–
water (50 : 50 wt/wt) mixture with 0.5 wt% choline theophylli-
nate and 0.5 wt% 5-uorouracil (5-Fu), respectively. Aer
swelling with a mixture of solvents at room temperature for
ꢀ
at 37 C. These specimens were periodically removed from the
degradation medium at specied time points. Their nal mass
was obtained aer completely drying in a vacuum oven. The
61
percentage of the mass loss was calculated from eqn (3).
Mass loss (%) ¼ (m ꢁ m )/m ꢂ 100%
(3)
i
d
i
7
2 h, they were taken out and washed with the mixture of
i d
where m is the initial dry gel mass and m is the gel mass aer
degradation at certain time. The mass loss was measured as a
function of time in the buffer solution for each APCN system.
solvents to remove the model drugs on the surface. Then, the
APCNs loaded with drugs were dried under ambient conditions
for 1 day, and in a vacuum oven at 40 C for 4 days. These
ꢀ
samples were available for drug release experiments without
any further treatment.
The amount of drug loading in the gel samples were esti-
mated in an indirect way. Choline theophyllinate and 5-Fu in
the gels exhibited the same lmax and intensities in the UV
spectra as the free drugs, since the matrix and the mixed solvent
2
.6 MTT viability assay
The relative cytotoxicity of the prepared APCNs was estimated
by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
a
bromide (MTT)) viability assay. Prior to cell culture, all gels
samples were sterilized by immersing them in 70% (v/v) ethanol
for 1 h, and then rinsing 3 times with sterile phosphate buffer
saline (PBS) (pH 7.4) to remove residual alcohol. Three samples
were extracted with RPMI-1640 medium (0.1 g mL ) for 24 h,
respectively. These extracts were collected as a conditioned
medium for subsequent cell viability tests and determined by
an MTT-dye reduction assay using L929 cells. L929 cells were
did not react with the model drugs or affect the absorbance of
62,63
the drugs.
The difference between the initial and nal
ꢁ
1
amount of drugs in the media was tested by UV spectroscopy at
max values of 273 nm and 264.5 nm for choline theophyllinate
l
and 5-FU, respectively, and used to calculate the amount of
drugs loading in the APCN. The amount of drug loading (ADL)
and encapsulation efficiency (EE) were calculated as eqn (5) and
2
cultured in a 25 cm culture ask in RPMI-1640 medium sup-
plemented with 10% fetal bovine serum (FBS), 1% L-glutamine,
and 1% penicillin/streptomycin and then incubated at 37 C in
(6), respectively.
ꢀ
5
% CO
2
, 95% humidied atmosphere, for 2 days. Aer L929
ADL (wt%) ¼ (mass of loaded drug/mass of gel) ꢂ 100% (5)
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EE (wt%) ¼ (mass of loaded drug/mass of drug in feed) ꢂ 100%(6)
ꢀ
In vitro release studies were performed in PBS at 37.4 C. An
APCN loaded with the drug was immersed in 100 mL buffer
solution of pH 7.4. In a special interval, 8 mL of the solution was
withdrawn from the release medium and replaced with 8 mL of
fresh solution. The concentrations of the choline theophyllinate
and 5-Fu solutions were measured by UV spectroscopy at
273 nm for choline theophyllinate and 264.5 nm for 5-Fu. Each
measurement was repeated at least three times and the average
values were plotted.
3
Results and discussion
3.1 Synthesis
1
Some research teams have reported several feasible methods Fig. 1 H NMR spectra of Br-PCL10-PEG23-PCL10-Br and N -PCL10-PEG23-PCL10-N3
3
to synthesize multi-block copolymers of PCL via poly- polymers.
condensation, anionic polymerization, and ring opening
64–68
polymerization (ROP).
PEG -PCL -N
triblock copolymer was synthesized aer three The chemical shi of the methyl group at the chain ends
steps. HO-PCL -PEG -PCL
-OH was synthesized by ROP of CL changes from 1.83 ppm of Br-PCL10-PEG23-PCL10-Br to 1.47 ppm
initiated by PEG with Sn(Oct)
as the catalyst. Then, they were of N -PCL10-PEG23-PCL10-N , indicating the successful replace-
esteried with 2-bromopropionyl bromide to generate Br-PCLy- ment of bromide by azide groups. The conversion of N
PEG -PCL -Br. Finally, N -PCL -PEG -PCL -N triblock polymers substitution from Br is 88% according to the peak intensities in
3 y
As shown in Scheme 1, a N -PCL -
x
y
3
y
x
y
2
3
3
3
x
y
3
y
x
y
3
1
were obtained aer the substitution of Br by NaN in dry DMF.
H NMR. The chemical composition of N
can be precisely regulated by controlled ROP and calculated
copolymers are from the integral ratio of signals at 4.09 ppm, 3.65 ppm and
summarized with the characteristic data in Table S1 in the ESI. 1.83 ppm. The mono-dispersion and precisely controlled
Their SEC traces are all symmetrical monomodal (Fig. S1) and chemical composition of N -PCL -PEG -PCL -N enable the
3 y x y 3
-PCL -PEG -PCL -N
3
Well-dened
PCL -N and
N
3
-PCL10-PEG23-PCL10-N
-PCL10-PEG45-PCL10-N
3
3
,
3 5
N -PCL -PEG91-
5
3
,
N
3
3
y
x
y
3
their polydispersity indices (PDIs) are less than 1.22 (Table S1), preparation of APCNs with well-controlled HI–HO ratios and
which indicates that no transesterication or backbiting side well-dened molecular structures.
6
9
reactions occurred during the polymerizations. N -PCL -PEG -
As shown in Scheme 1, APCNs were prepared from a reaction
PCL -N was also studied by FT-IR (Fig. S2). The appearance of a mixture of N -PCL -PEG -PCL -N , TPOM, CuBr and PMDETA
molar ratio of [N -PCL -PEG -PCL -N ] : [TPOM] :
[CuBr] : [PMDETA] at 0.5 : 0.25 : 1 : 1. It is worth noting that it
3
y
x
y
3
3
y
x
y
3
ꢁ1
characteristic absorption peak at 2098 cm assigned to azide with
a
3
y
x
y
3
groups conrms the successful preparation of N
PCL10-N
3
-PCL10-PEG23
1
-
. The copolymers were further conrmed by H NMR only shows a basic connection of the polymer chain connection.
3
analyses, as shown in Fig. 1. The signals at 1.42, 1.62, 2.34 and In fact, triangle, quadrangle, pentagon, hexagon and even
.09 ppm are assigned to the protons of the PCL backbone, and heptagon morphology may exist in the polymer network. The gel
4
the peak at 3.65 ppm is the methylene protons of the PEG block. fraction of the APCN(PCLy-PEGx-PCLy) is one of the major concerns,
which is obtained from eqn (7):
Gel fraction ¼ Massdried gel/Masspolymer precursors
.
(7)
APCN(PCL10-PEG23-PCL10) prepared from N -PCL -PEG -
3
10
23
3
PCL10-N has a gel fraction of about 89.8%, slightly lower
than 92.3% for APCN(PCL10-PEG45-PCL10) and 93.3% for
APCN(PCL5-PEG91-PCL5) (Table 1).
3.2 Physical properties
One of the most attractive features of APCNs is their various
physical properties in different solvents due to the existence of
both HO and HI phases in polymer networks. Scheme 2 illustrates
the behavior of the APCNs in various mediums via morphological
isomerization. In a good solvent for both the HI and HO, the
polymer chains in the network are highly extended, while in a
selective solvent for either HI or HO, only the corresponding part of
the polymer chains adopts extension. The characteristics result in
Scheme 1 Synthesis of N
3
-PCL
y
-PEG
x
-PCL
y
-N
3
and preparation of APCNs by
¼ sodium azide, PMDETA ¼
“
click chemistry”. DMF ¼ dimethylformamide, NaN
3
pentamethyldiethylenetriamine, APCNs ¼ amphiphilic conetworks.
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Table 1 Physical properties of APCNs
Kinetic of swelling
b
c
d
(W )
ADL (%)
EE (%)
Gel fraction
(%)
a
e
0
ꢀ
e
ꢀ
Sample
In Water
In DMF
5-FU
Choline theophyllinate
5-FU
Choline theophyllinate
WCA
( )
WCA ( )
3
Gel-1
Gel-2
Gel-3
89.8
92.3
93.3
45 ꢃ 11
88 ꢃ 17
690 ꢃ 9
1419 ꢃ 12
1935 ꢃ 16
1.75
4.46
7.20
2.00
3.43
7.70
0.673
2.00
3.86
0.801
1.62
4.61
84.5
68
51
43
24
0
866 ꢃ 20
a
b
Gel-1: Gel (PCL10-PEG23-PCL10); Gel-2: Gel (PCL10-PEG45-PCL10); Gel-3: Gel (PCLe
5
-PEG91-PCL
5
). W is the equilibrium solvent uptake in percentage
c
d
(see Experimental section). Amount of drug loaded. Encapsulation efficiency. WCA
0 3
and WCA are the relative water contact angle values with a
residence time of 0 and 3 min, respectively.
interesting swelling properties and unique physical properties. PCL10) (45%) are comparable to the theoretical SRmaximum
DMF is a good solvent for both PEG and PCL, in which values, which are 970%, 144% and 27%, respectively, revealing
APCNs(PCLy-PEGx-PCLy) can fully swell as shown in Fig. 2(a). All that the APCNs have well-dened molecular structures.
samples swelled quickly in 10 h. APCN(PCL5-PEG91-PCL5) has a
The unique properties of APCNs in different mediums are
maximum swelling ratio of 1935%, which is larger than 1420% for also observed in optic studies. Fig. 3(b) shows the photos of
APCN(PCL10-PEG45-PCL10) and 690% for APCN(PCL10-PEG23-PCL10). The APCN(PCL10-PEG45-PCL10) in DMF, ether and H O. In DMF,
2
swelling ratio fully agreed with the fact that longer polymer chains APCN(PCL10-PEG45-PCL10) presents the highest transparency and
result in larger space among the polymer network lattice. largest SR, while in H O and ether, the opacity and small SR of
2
Fig. 2(b) shows the swelling ratios of the APCNs in water, which is APCN(PCL10-PEG45-PCL10) account for the partial dissolution of
a good solvent for PEG but poor for PCL. The SR value of PCL and PEG, which causes hydrophobic/hydrophilic (HI/HO)
APCN(PCL5-PEG91-PCL5) in water is 866%; smaller than that in DMF phase separation in APCNs. The optic property was character-
(1935%). The reduced SR in water is consistent with the fact that ized by UV-Vis spectroscopy (Fig. 3(a)). In DMF, the trans-
only the PEG segments can extend in water. The theoretical mittance of APCN(PCL10-PEG45-PCL10) is as high as 91.2% in the
maximum SR of APCN in water can be calculated based on the PEG range 300–800 nm. In H O and ether, the transmittances of
2
content with eqn (8).
APCN(PCL10-PEG45-PCL10) are only 63.0% and 55.0%. The SR and
optical properties of the APCN can be regulated by changing
the HI and HO composition. Fig. 3(c) shows the photos
3
SRmaximum ¼ (LPEG/(LPEG + LPCL)) ꢂ SRDMF
(8)
of
APCN(PCL10-PEG23-PCL10)
,
APCN(PCL10-PEG45-PCL10)
and
APCN(PCL5-PEG91-PCL5) in water. With the increase of PEG
concentrations, the transmittances of the APCNs in water
increase correspondingly.
The experimental SR values in water for APCN(PCL5-PEG91-PCL5)
866%), APCN(PCL10-PEG45-PCL10) (88%) and APCN(PCL10-PEG23
(
-
Scheme 2 Illustration of swelling behavior of APCN in different solvents.
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Fig. 3 (a) UV-Vis transmittance spectra of APCN(PCL10-PEG45-PCL10) films swollen
Fig. 2 Weight–swelling ratio of (I) APCN(PCL5-PEG91-PCL5), (II) APCN(PCL10-PEG45-
with DMF (curve I), H
APCN(PCL10-PEG45-PCL10) swollen with H
of APCN(PCL10-PEG23-PCL10), APCN(PCL5-PEG91-PCL5) and APCN(PCL10-PEG45-PCL10) swollen
2
O (curve II) and diethyl ether (curve III). (b) Photographs of
2
PCL10) and (III) APCN(PCL10-PEG23-PCL10) in (a) DMF and (b) H O.
2
O, DMF and diethyl ether. (c) Photographs
2
with H O.
The water contact angle (WCA) was measured to study
the surface property of APCN. In Fig. 4(a), the WCA
of
APCN(PCL5-PEG91-PCL5)
,
APCN(PCL10-PEG45-PCL10)
and
of the polymers may affect both the drug permeability and
biodegradability. Fig. 6 shows the differential scanning
calorimetry (DSC) heating and cooling curves of APCNs. The
ꢀ
ꢀ
ꢀ
APCN(PCL10-PEG23-PCL10) is 51 , 68 and 84.5 at 0 min, respec-
tively. The WCA results indicate that, with the increase of HI
content, the hydrophilicity of the APCN increases correspond-
ingly. Dynamic WCAs of APCNs were also applied to investigate
the water absorbing rate. Fig. 4(b) shows the WCAs of APCNs
70
crystallization behavior of the APCNs is largely affected by the
ꢀ
length of PEG and PCL blocks. One exothermic peak at 2.59 C
ꢀ
in the cooling process and one endothermic at 30.2 C in the
aer 3 min residence time. The WCAs of APCN(PCL10-PEG23-PCL10)
,
heating process, assigned to T and T of PCL, are detected in
c
m
APCN(PCL10-PEG45-PCL10) and APCN(PCL5-PEG91-PCL5) reduced from
APCN(PCL10-PEG23-PCL10). The PEG length is too short to be
crystallized in APCN(PCL10-PEG23-PCL10). There are two
ꢀ ꢀ ꢀ ꢀ ꢀ ꢀ
4.5 , 68 and 51 to 43 , 24 and 0 , respectively. Water can be
8
completely absorbed on the surface of APCN(PCL5-PEG91-PCL5)
within 3 min. This is consistent with the fact that it has the
highest PEG content among the three studied APCNs, resulting
in the best hydrophilicity.
ꢀ
ꢀ
exothermic peaks at ꢁ18.9 C and 20.35 C in the cooling
ꢀ
ꢀ
process and endothermic peaks at 41.4 C and 47.9 C in the
heating process appear in APCN(PCL10-PEG45-PCL10). These
c m
peaks are assigned to T and T of PCL and PEG segments.
The morphology of APCN was further investigated by scan-
ning electron microscopy (SEM). Fig. 5 shows the SEM cross-
section view of freeze-dried samples of APCNs and PEG gel
without PCL blocks as a control. All of the APCN samples show a
denser morphology than that of pure PEG gel (Fig. 5(d)), which
is highly porous with pore sizes in hundreds of micrometers. A
higher concentration of the PCL in APCN leads to a denser
morphology of the APCN.
The increase of the PEG length not only allows the appear-
ance of T of PEG, but also affects T and T of the PCL block.
c
c
m
ꢀ
Tc of PCL in APCN
shis to 20.4 C in
(PCL10-PEG45-PCL10)
ꢀ
comparison with 2.6 C in APCN(PCL10-PEG23-PCL10). For
APCN(PCL5-PEG91-PCL5), the exothermic peaks appeared at
8.1 C and endothermic peaks at 45.7 C attributed to T of
ꢀ
ꢀ
2
c
the PEG block. In this case, the PCL block is too short
to crystallize.
3.3 Thermal properties
3.4 Degradation and cytocompatibility
The thermal property is another major concern for APCNs The hydrolytically labile PCL segments provide the APCN
aiming for biomedical applications because the crystallinity biodegradability. Fig. 7 illustrates the mass loss of APCNs in
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Fig. 4 Water contact angles of (1) APCN(PCL5-PEG91-PCL5), (2) APCN(PCL10-PEG45-PCL10) and (3) APCN(PCL10-PEG23-PCL10) films with different residence times of (a) 0 min and
b) 3 min.
(
Fig. 7 Mass loss profiles of (a) APCN(PCL5-PEG91-PCL5), (b) APCN(PCL10-PEG45-PCL10)
,
and (c) APCN(PCL10-PEG23-PCL10) as a function of degradation time.
PBS solution with a pH value of 7.4. APCN(PCL5-PEG91-PCL5) reaches
00% decomposition at 410 h, which is faster than that of
1
APCN(PCL10-PEG45-PCL10) (790 h) and APCN(PCL10-PEG23-PCL10) (960 h).
The degradation of the APCNs is controlled by diffusion of water
and cleavage of ester bonds in the PCL blocks. Because the well-
dened molecular structure in APCN affords a well-ordered
HI/HO nanophase separation, PCL domains are in the nanoscale
and surrounded by HI phase with PBS, which provides a large
surface area and fast degradation rate. Therefore, all APCNs
degrade much faster than pure PCL polymers (in years).
APCN(PCL10-PEG45-PCL10) exhibits the fastest degradation due to its
high up-take of water content and amorphous PCL domain as
mentioned above by DSC.
Fig. 5 SEM images (cross-section view) of freeze-dried (a) APCN(PCL10-PEG23
-
PCL10), (b) APCN(PCL10-PEG45-PCL10), (c) APCN(PCL
PEG4k gel.
5 5
-PEG91-PCL ), and (d) pristine
To evaluate the biocompatibility of APCNs, an MTT assay was
conducted on L929 cells exposed to APCNs. Fig. 8 shows
the MTT assay for cytotoxicity of APCN(PCL5-PEG91-PCL5)
,
APCN(PCL10-PEG23-PCL10)
,
APCN(PCL10-PEG45-PCL10) and control
experiments in L929 cells aer cultivation for 24, 48 and 72 h,
respectively. All cell viabilities of APCNs are greater than 75%. It
is noteworthy that the cell viability of APCN(PCL5-PEG91-PCL5)
reaches 93.85%, 97.42%, and 102.58% aer treatment for 24 h,
48 h and 72 h, respectively, which is comparable to that of the
negative control experiment. Therefore, the prepared APCNs
Fig. 6 Heating and cooling curves in DSC measurements of (a) APCN(PCL10-PEG23-PCL10)
,
(
b) APCN(PCL10-PEG45-PCL10), and (c) APCN(PCL5-PEG91-PCL5)
.
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Fig. 8 MTT assay for cytotoxicity of APCN(PCL10-PEG23-PCL10), APCN(PCL5-PEG91-PCL5)
and APCN(PCL10-PEG45-PCL10) in L929 cell culture.
show excellent biocompatibility with L929 cells and have nearly
no effect on cell growth and reproduction.
3.5 Control and release as drug carrier
The coexistence of HO and HI domains in APCN makes it
71–73
possible to be used as a dual-release drug carrier.
Herein,
hydrophilic choline theophyllinate and hydrophobic 5-FU were Fig. 9 In vitro release profiles of 5-FU (a) and choline theophyllinate (b) from
APCN(PCL10-PEG23-PCL10) (I), APCN(PCL5-PEG91-PCL5) (II) and APCN(PCL10-PEG45-PCL10) (III)
used at the same time for controlled release from the prepared
APCN. The drug encapsulation was conducted by immersion of
loaded with 5-Fu and choline theophyllinate at the same time.
dried APCNs into a mixture of choline theophyllinate and 5-FU
(
(
(
2
1 : 1, molar ratio) with a concentration of 1 wt% in THF–H O
50 : 50 wt/wt). Table 1 shows the amount of drug loading
ADL) and encapsulation efficiency (EE) of the APCNs. The and 38% 5-Fu, 63% and 45% choline theophyllinate are released
APCN(PCL5-PEG91-PCL5) has an ADL of 5-Fu of 7.20% and choline from APCN(PCL10-PEG45-PCL10) and APCN(PCL10-PEG23-PCL10) in
theophyllinate of 7.70%, which are higher than those of 200 h, respectively. The time of completed release of 5-Fu and
APCN(PCL10-PEG45-PCL10) (5-FU: 4.46%; choline theophyllinate: choline theophyllinate from APCN(PCL10-PEG23-PCL10) and
3.43%) and APCN(PCL10-PEG23-PCL10) (5-FU: 1.75%; choline the- APCN(PCL10-PEG45-PCL10) is nearly 800 h, which is close to their
ophyllinate: 2.00%). The EE of APCN(PCL5-PEG91-PCL5) is degradation time. All the APCNs present a relatively faster
also higher than those of APCN(PCL10-PEG23-PCL10) and release of hydrophilic choline theophyllinate than 5-FU.
APCN(PCL10-PEG45-PCL10). The ADLs of 5-FU and choline theo- Scheme 3 proposes a plausible mechanism of the release of
phyllinate are comparable to each other in all APCNs, indi- 5-FU and choline theophyllinate for APCN. Both the drug
cating that the ADL of the hydrophilic and hydrophobic drug diffusion and the APCN degradation serve as the driving force
is dominated by the SR of the APCN in THF/H O rather for the release of hydrophilic choline theophyllinate, while only
2
than the HO/HI composition. The APCN(PCL5-PEG91-PCL5) has the degradation is responsible for the release of hydrophobic 5-
a SR of about 1510%, which is larger than 960% for FU. Because APCN(PCL5-PEG91-PCL5) has a high HI content, high
APCN(PCL10-PEG45-PCL10) and 420% for APCN(PCL10-PEG23-PCL10)
.
SR in water and a short degradation time, it presents a fast drug
The larger SR of APCN allows more choline theophyllinate and release prole. For the hydrophilic choline theophyllinate
5-FU solution encapsulation, and thus a higher ADL.
stored in the HI phase, they can diffuse quickly out from
Figure 9(a) and (b) present the in vitro release proles of free the APCN. In contrast, most of the hydrophobic 5-FU attaches to
5
L
-FU and choline theophyllinate with a concentration of 8 g the HO phase and releases slowly until the decomposition
, as well as 5-FU and choline theophyllinate from APCN of HO. Therefore, diffusion from the HI phase and
ꢁ
1
samples in PBS (pH ¼ 7.4), respectively. The free 5-Fu and degradation of the HO phase afford faster release of
choline theophyllinate reach a maximal release in PBS in 10 hydrophilic choline theophyllinate than that of hydrophobic
minutes, which accounts for the dissolution procedure of the 5-FU from APCN(PCL5-PEG91-PCL5) in PBS solution. Since
drugs. APCN(PCL5-PEG91-PCL5) exhibits a relatively fast drug APCN(PCL10-PEG23-PCL10) and APCN(PCL10-PEG45-PCL10) have a high HO
release. About 60% 5-FU and 80% choline theophyllinate are content, the HO component forms a continuous shell, preventing
released from the APCN in 200 h, and about 90% 5-FU and 95% water diffusion in the APCN. Therefore, degradation of the APCNs
choline theophyllinate aer 400 h. APCN(PCL10-PEG23-PCL23) and plays a very important role in drug release. The degradation times
APCN(PCL10-PEG45-PCL10) present a slow drug release. About 45% of APCN(PCL10-PEG23-PCL10) and APCN(PCL10-PEG45-PCL10) are longer
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Scheme 3 Illustration of drug release procedures. (Note: cubic is not the true structure of the polymer networks.)
than that of APCN(PCL5-PEG91-PCL5). Thus, these two APCNs present
slower drug release proles than APCN(PCL5-PEG91-PCL5)
7 C. Lin and I. Gitsov, Macromolecules, 2010, 43, 10017–10030.
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J. P. Kennedy, G. Fenyvesi, S. Na, B. Keszler and
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4
Conclusion
The combination of controlled ROP and CuAAC demonstrates a
feasible approach to prepare PCL-involved APCNs with a well-
dened molecular structure and precisely controlled HO/HI 10 K. Podual, F. Doyle III and N. Peppas, Polymer, 2000, 41,
composition. The APCNs exhibit unique swelling behavior,
3975–3983.
optical properties, surface properties and thermal properties as 11 I. Gitsov and C. Zhu, Macromolecules, 2002, 35, 8418–8427.
well as phase separation of HI and HO domains. The APCNs 12 I. Gitsov and C. Zhu, J. Am. Chem. Soc., 2003, 125, 11228–
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1089.
11234.
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3509–3516.
18 R. Haigh, S. Rimmer and N. J. Fullwood, Biomaterials, 2000,
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1
9 Y. Sun, J. Collett, N. J. Fullwood, S. Mac Neil and S. Rimmer,
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Acknowledgements
This work was supported by National Natural Science Founda- 20 S. Rimmer, M. J. German, J. Maughan, Y. Sun, N. Fullwood,
tion of China under the grants 21274020 and 21074022. This
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371.
Soft Matter
This journal is ª The Royal Society of Chemistry 2013