Hypoxia and temperature dual-stimuli-responsive random copolymers: Facile synthesis, self-assembly and controlled release of drug

Article abstract of DOI:10.1039/d0nj02114h

Amphiphilic random copolymers poly(isopropylacryamide-co-acrylic acid-co-2-nitroimidazole acrylate) (P(NIPAM-co-AA-co-NIA)) with different contents of the 2-nitroimidazole (NI) group were facilely synthesized through RAFT and EDC reactions. These random c

Full text of DOI:10.1039/d0nj02114h

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Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
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DOI: 10.1039/D0NJ02114H
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Hypoxia and temperature dually responsive random copolymers:
facile synthesis, self-assembly and controlled release of drug
Chenming Jia, Yinlu Denga, Hua Yuana*, Yongzhen Wub* and Weizhong Yuana*
Received 00th January 20xx,
Accepted 00th January 20xx
Amphiphilic random copolymers poly(isopropylacryamide-co-acrylic acid-co-2-nitroimidazole acrylate) (P(NIPAM-co-AA-co-
NIA)) with different contents of 2-nitroimidazole (NI) group were facilely synthesized through RAFT and EDC reactions. These
random copolymers self-assembled into spherical micelles at room temperature through dialysis method. NI groups
exhibited stimulus responsiveness under hypoxia condition. The hydrophobicity-hydrophilicity transformation could be
realized through the change of NI groups into aminoimidazole (AI) groups. Moreover, NIPAM segments endowed the
copolymers with thermo-responsive property. In addition, the content of NI group in the copolymers would influence the
hydrophilic-hydrophobic equilibrium of the copolymers, and resultingly generated an obvious effect on the self-assembly
behaviour of the copolymers and the morphologies of the self-assemblies. The micelles or vesicles can be used as the drug
carriers to realize the controlled release of doxorubicin (DOX). The in vitro cell viability study of the DOX-loaded self-
assemblies indicated the cytotoxicities under hypoxic condition were much higher than those under normoxic condition.
Moreover, the drug release behaviors also could be adjusted by changing the temperatures. Therefore, these random
copolymers are expected to be the multifunctional and smart carrier for the elimination of cancer cells.
DOI: 10.1039/x0xx00000x
www.rsc.org/
aminated NI groups onto amphiphilic polymers, and achieved
controllable drug release under hypoxic conditions.22
Introduction
Recently, there are significant interests focused on the dual-
As the novel and smart polymeric materials, hypoxia-responsive
polymers have attracted considerable interests owing to their
promising applications in the therapic fields of hypoxia-
associated diseases including cancer, vascular diseases, anemia,
cardiomyopathy and rheumatoid arthritis and so on.1-10 The
nitroaromatic and azobenzene derivatives are two commonly
used hypoxia-responsive substances which could be introduced
to polymeric chains to construct hypoxia-responsive
copolymers.11-16 As for the nitroaromatic derivatives, the
hydrophobic-to-hydrophilic transformation can be completed
resulting from the convertion of nitro groups to amino groups
under hypoxic conditions. Moreover, the nitroaromatic groups
could be facilely linked to the backbones of polymers to
construct hypoxia-responsive polymeric materials under mild
conditions.17, 18 Among them, 2-nitroimidazole (NI) groups are
important and typical hypoxia-sensitive groups and can present
obvious sensitivity to hypoxia conditions.19-22 For example,
Thambi et al. grafted hydrophobic modified NI groups onto
carboxymethyl dextran (CM-DeX) framework to prepare
hypoxia-responsive nanoparticles (HR-NPs).19 Deng et al.
reported the copolymer self-assembled micelles by grafting the
stimuli-responsive polymers with a distinct opportunity to well
adjust their responses to dual stimuli independently.23-33 Dually
responsive copolymers including temperature-pH dual
responses34-36
,
temperature-glucose dual responses37-40
,
temperature-redox responses41-43 had been reported, and the
controlled release properties of these responsive polymeric
systems were also proved. For example, Yuba et al. reported the
pH-temperature dually responsive copolymer which could be
modified on liposome surfaces for controlled cargo release.44
Wang et al. reported the micelles assembled by using photo-
temperature responsive copolymer for controlled-release
under compound stimulation.45 However, up to now, the dual-
stimuli responsive polymers containing hypoxic response have
rarely been reported. Therefore, it is necessary and worthwhile
to investigate dual-response polymers with hypoxic response
and other responses.
The change of temperature would lead to the morphological
transformation of thermoresponsive polymeric self-
assembles.46-50 In fact, temperature is one of the most
controllable stimuli.51-55 Among the temperature responsive
polymers, poly(isopropylacryamide) (PNIPAM) has been widely
used as thermoresponsive materials in biomedical fields
because its lower critical solution temperature (LCST) is close to
human body temperature.56-60 The PNIPAM segments can
undergo a reversible phase transition accompanying with the
swelling or shrinkage of structures, which endowed these
polymers with potential application value in releasing drugs.61-
aDepartment of Interventional and Vascular surgery, Shanghai Tenth People’s
Hospital, School of Materials Science and Engineering, Tongji University, Shanghai
201804, People’s Republic of China. E-mail: yuanwz@tongji.edu.cn (W. Yuan),
Tel.: +86 21 69580234, Fax: +86 21 69584723.
bEYE & ENT Hospital of Fudan University, Shanghai 200031, People’s Republic of
China. E-mail: wuyongzhen_fd@sina.com (Y. Wu)
66
In addition, acrylic acid has been widely used in the field of
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Fig. 1 Schematic illustration of the hypoxia and temperature responsive properties of
biomedicine because of its good water solubility,
biocompatibility and active chemical properties, which can be
used for RAFT and EDC reactions.67, 68
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P(NIPAM-co-AA-co-NIA) random copolymer micelles or vesicles and their controlled DOX
DOI: 10.1039/D0NJ02114H
release.
Herein, the random copolymers of poly(isopropylacrylamide-
co-acrylic acid-co-2-nitroimidazole acrylate) (P(NIPAM-co-AA-
co-NIA)) with containing different amounts of NI group were
synthesized by RAFT and EDC reactions (Scheme 1). The random
copolymers could self-assemble in aqueous solution to form
micelles. Under hypoxic conditions, the hydrophobic NI groups
in P(NIPAM-co-AA-co-NIA) would be converted to hydrophilic 2-
aminoimidazole (AI) groups through the catalytic action of
reduced nicotinamide adenine dinucleotide phosphate
(NADPH). Changes in hydrophilic properties of copolymers
further induced the expansion of micelles or transformation
into vesicles. In addition, the change of temperature also would
alter the morphology of micelles. As a result, the P(NIPAM-co-
AA-co-NIA) copolymer micelles could be used as a promising
drug carrier to achieve the controlled release of doxorubicin
(DOX), as shown in Fig. 1.
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Experimental
Materials
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2,2'-Azobisisobutyronitrile (AIBN, Aldrich, 98%) was
recrystallized from ethanol. Acrylic acid (AA, Acros, 99.5%) was
used directly without further purification. 2-nitroimidazole (NI,
Adamas, 98%), Carbon disulfide (CS2, Adamas, 99.9%), 1-
Dodecanethiol (C12H25SH, Adamas, 98%), Triethylamine (TEA,
Adamas, 99.5%), N-hydroxysuccinimide (NHS, Adamas, 98%), N-
boc-6-bromohexylamine (Energy Chemical, 98%) and 3-(3-
dimethylaminopropyl)-1-ethylcarbodiimide
hydrochloride
(EDC∙HCl, Adamas, 97%) were used as received. Aminated 2-
nitroimidazole (NH2-NI) was synthesized by the reaction of NI,
K2CO3 and 6-(boc-amino)ethyl bromide according to the
literature.19 NIPAM was recrystallized from the toluene/hexane
mixture (1:3).
Characterizations
Attenuated total reflection fourier transform infrared
spectroscopy (ATR FT-IR). An EQUINOSS/HYPERRION 2000
spectrometer (Bruker, Germany) was applied to collect ATR FT-
IR spectra with a resolution of 1 cm-1 from 650 to 4000 cm-1 for
30 scans. Perkin Elmer Spectrum software was used for data
acquisition and analysis.
Proton nuclear magnetic resonance spectroscopy (1H NMR).
Bruker AVANCE-400 MHz NMR was used to obtain the H NMR
spectra of the synthesized polymers and modified hypoxic
sensitive substance with CDCl3 or DMSO-d6 as the solvent.
Gel permeation chromatography (GPC). HLC-8320 (Tosoh,
Japan) analysis system equipped with two columns (TSK gel
super AWM-H×2, R0091+R0093) was used to obtain GPC data
1
Scheme 1 Synthesis of P(NIPAM-co-AA-co-NIA) amphiphilic random copolymer by
combining RAFT and EDC reactions.
under a flow rate of 0.6 mL min-1 at 40 C by using DMF
containing 10 mM LiBr as the eluent. The PMMA calibration kit
was used as the calibration standard.
o
UV-vis absorption spectroscopy. The UV-vis absorption spectra
were recorded on a UV-vis spectrophotometer (Lambda 35,
PerkinElmer) with scanning rate of wavelength at 1 nm/s from
260 nm to 400 nm. Under hypoxic and normoxic conditions, the
sample of the micelle solutions in a quartz cuvette was placed
vertically under a high-pressure mercury lamp (wavelength 365
nm, power 200 W).
Transmission electron microscopy (TEM). The morphologies of
the assembled micelles were observed on a JEOL JEM-2010 TEM
at an acceleration voltage of 80 kV. The samples for observation
were prepared by dropping a drop of assembled micellar
solution on carbon coated copper grids and drying overnight
before testing.
Dynamic light scattering (DLS). DLS were measured by using a
Malven Autosizer 4700 DLS spectrometer to obtain the
hydrodynamic radius (Rh) value of the copolymer micelles. The
scattering light was recorded at a fixed angle of 90o and the Rh
values were obtained by a cumulant analysis.
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Zeta potential. ZETASIZER Nano series instrument (Malvern N), 1420 (νN=O), 1381 (νO-H). H NMR (DMSO-d6, δV,iepwpAmrti)c:le7O.n1li6ne-
DOI: 10.1039/D0NJ02114H
Instruments) was used to record the Zeta potential of self- 7.67 (NCH=CHN), 3.82 (NHCH and NCH2C), 3.40 (CH in PNIPAM,
assembled micelles under normoxic and hypoxic conditions.
NHCH2), 1.16-2.23 (CH2 in PNIPAM, CH2 and C
C(CH3)2CO, C(CH2)4CNH), 1.06 (NHCH(CH3 2 and C12H25 in
DDMAT).
H in PAA,
)
Synthsis of DDMAT
The chain transfer agent DDMAT was synthesized by using CS2,
CHCl3, acetone, C12H25SH and NaOH. The specific procudures
Micellization of P(NIPAM-co-AA-co-NIA) random copolymers
are as follows. Firstly, C12H25SH (48.46 g, 0.24 mol), (CH3)2CO Firstly, 50 mg of P(NIPAM-co-AA-co-NIA) copolymer was
(146.5 mL) and Aliquot 336 (3.90 g, 0.0096 mol) were added into dissolved in 10 mL of DMF. Then, 20 ml deionized water was
a three-necked flask. After the system was cooled to 10 oC and injected into the resulting solution by using a microinjection
filled with nitrogen. 50% NaOH solution (13.19 ml) was added pump. Finally, the self-assembled micelles were formed by
dropwise into the mixed system and stirred for 20 min. Secondly, dialyzing the mixed solution against deionized water for 48 h
CS2 (18.24 g, 0.24 mol) in acetone (30.74 mL) were added using a dialysis membrane (molecular weight cut-off: 3500 Da),
dropwise into the flask within 12 min, followed by the addition and the dialysis water was changed every 6 h.
of CHCl3 (28.8 mL). Add 50% NaOH solution (28.03 ml) dropwise
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DOX loading and controlled release from micelles
into the system to conduct the reaction overnight. Thirdly,
deionized water (360 mL) and hydrochloric acid (60 mL) were The process for DOX loading was implemented as fellows. Firstly,
added into the mixture, followed by the rotary evaporation to DOX∙HCl (6 mg) and P(NIPAM-co-AA-co-NIA) (20 mg) were
remove acetone. Finally, the obtained mixture was filtered, dissolved in 4 mL of DMF, then 0.6 mL of TEA was added into
recrystallized from n-hexane and dried for 24 h to obtain the the mixture, followed by the continuous stirring for several
orange solid.
1H NMR (CDCl3, δ, ppm): 11.0 (COO
1.28 (CH3(CH2)10), 0.82 (CCH3).
minutes to form a homogeneous crimson solution. Then, the
), 1.62 (CH2CS and C(CH3)2), resulting solution was slowly added to 8 ml deionized water
dropwise under ultrasonic treatment. After that, the system
H
was sonicated for addtional 30 min to obtain a purple-blue
homogeneous solution. The solution was dialyzed against
Synthesis of P(NIPAM-co-AA)
P(NIPAM-co-AA) was synthesized by RAFT of AA and NIPAM deionized water for 24 h using a dialysis membrane (molecular
with DDMAT as the chain transfer agent. Typically, NIPAM (5 g, weight cut-off: 3500 Da), and the deionized water need to be
44.2 mmol), AA (1.6 g, 22.2 mmol), DDMAT (0.268 g, ), AIBN refreshed every 2 h in the first 8 h and then every 6 h in later to
(12.1 mg, 0.074 mmol) and anhydrous DMF (8 mL) were added remove the unloaded DOX molecules and organic solvents.
into the flask. After, repeating freeze-vacuum-charge argon for Finally the dry micelles/vesicles loading DOX were obtained by
three times, the reaction was carried out at 70
℃ under argon freeze drying. The drug loading efficiency (DLE) and drug loading
for 6-8 h. Thereafter, the resulting solution was added dropwise content (DLC) were calculated according to the following
into ice-bathed n-hexane and then stayed overnight. formulae:
Afterwards, the supernatant solution was removed and the final DLC(wt%)= (weight of loading DOX/weight of DOX-loaded
product was obtained after vacuum drying (yield: 93%).
polymeric micelles )×100%
(1)
Mn, NMR=7826 g/mol, Mn,GPC=7480 g/mol, Mw/Mn=1.21. ATR FT- DLE(wt%)=(weight of loading DOX/weight of DOX in feed)×100%
IR (cm-1): 3160-3700 (νN-H), 1724 (νC=O), 1647 (νC=O), 1540 (νC-N),
(2)
1
1381 (νO-H). H NMR (CDCl3, δ, ppm): 11.00 (-COO
H
), 8.00 In the DOX release investigation, the freeze-dried DOX-loaded
in PAA, micelles were dispersed in PBS (pH=7.4) and transferred into a
H2 in PNIPAM and CH2 in PAA, C(CH3)2CO in DDMAT), 1.06 dialysis membrane (molecular weight cut-off: 3500 Da). For
(CNHC), 3.82 (NHCH), 3.38 (CH in PNIPAM), 1.28-2.20 (CH
C
(NHCH(CH3)2 and C12H25 in DDMAT).
DOX release under hypoxic conditions, the dialysis membrane
was immersed in the erlenmeyer flask filled with deoxidized PBS
(pH=7.4) containing 100 mM NADPH which was continuously
Synthesis of P(NIPAM-co-AA-co-NIA)
P(NIPAM-co-AA-co-NIA) random copolymers were synthesized purged by using argon to maintain anaerobic condition. Then
by EDC reaction of P(NIPAM-co-AA) with different amounts of the flask was sealed and transferred into a 37 oC shaker with
NH2-NI. The typical procedure is described as follows. P(NIPAM- shaking at 150 rpm. 2.0 mL of release solution in the flask was
co-AA) (0.5 g, 0.06 mmol) was dissolved in 12 mL of DMF, then collected for measurements and the equal volume of fresh PBS
EDC∙HCl (3.21 g, 16.7 mmol) and NHS (1.93 g, 16.7 mmol) were was replenished and the DOX concentration was obtained by
added to obtain the solution via continuous stirring for 15 min. measuring the extracted solution at 485 nm on a UV-vis
Afterthat, 8 mL DMF solution of NH2-NI (0.36 g, 1.7 mmol) was spectrophotometer.
slowly added into the mixture to perform the reaction at room
temperature for 24 h. The pure copolymers were obtained by
In vitro cytotoxicity assay of copolymer micelles
dialyzing against methano/water mixing solvents (v1/v2=1/1) The 293T cells were used for investigating the cytotoxicities of
and deionized water respectively for 24 h and 48 h. The purified the micelles or vesicles. In brief, the logarithmic 293T cells were
and dry product was obtained after freeze drying (yield: 89%).
seeded into a 96-well plate with the density of 5×103 cells per
Mn, NMR=11516 g/mol, Mn,GPC=10650 g/mol, Mw/Mn=1.24. ATR well, and the wells at edges were filled with sterile PBS. After
FT-IR (cm-1): 3136-3706 (νN-H), 1724 (νC=O), 1641 (νC=O), 1533 (νC- that, the subject was incubated overnight at 37 oC humidified 5%
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group of PAA segment could be detected at 8.00 ppm and 11.00
DOI: 10.1039/D0NJ02114H
ppm respectively, which confirmed the successful synthesis of
CO2 condition with DMEM supplemental medium (containing
10% FBS) to ensure that the monolayer cells were covered the
bottom of the well. Then, the cells were treated with different
concentrations of pure and DOX loaded micelles/vesicles for 24
h. After removing the medium, MTT stock solution (20 μL, 5
mg/mL in PBS) was added into each well, followed by the
incubation for another 4 h. Afterwards, the medium was sucked
our carefully in each well and 150 μL of DMSO was added
successively. The 96-well plate was transferred in a shaker with
slight shaking for 10 min to ensure the complete dissolution of
the intracellular MTT formazan crystals. Finally, a Multiscan
MK3 plate reader was used to record the absorbance at 570 nm
of the resulting solution. The relative cell viability can be
calculated by the following formula:
View Article Online
P(NIPAM-co-AA). Mn.NMR of P(NIPAM-co-AA) copolymer was
calculated according to the integral ratio of corresponding
peaks and the known molecular weight of PNIPAM and PAA
blocks. The result was 7826 g/mol.
1H NMR spectrum of P(NIPAM-co-AA-co-NIA) shown in Fig. 2S(c)
revealed the characteristic peaks of NI groups, which means the
successful grafting of P(NIPAM-co-AA) and NH2-NI. Besides, the
grafting number of NI group could be calculated from the
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integral ratio of the proton peak (k+1) in NI to the methyl proton
풂
peak (
) in PNIPAM segment. The results indicated that the
풂+풎
number of NI groups in copolymer was 19. Based on this result
and Mn,NMR of P(NIPAM-co-AA), the molecular weight of
P(NIPAM-co-AA-co-NIA) was further calculated to be 11516
Relative cell viability %=(ODtreated/ODcontrol)×100
where ODtreated was obtained from the presence of the
micelles/vesicles and ODcontrol was obtained from the absence g/mol.
of the micelles/vesicles.
By controlling the feed ratio of NI and AA in copolymer, three
kinds of P(NIPAM-co-AA-co-NIA) with different grafting
numbers of NI groups were prepared. As shown in Table 1, the
numbers of NI groups grafted to PAA segment are 8, 17 and 19
respectively. Namely, P(NIPAM48-co-AA18-co-NIA8), P(NIPAM48-
co-AA9-co-NIA17) and P(NIPAM48-co-AA7-co-NIA19) have been
successful synthesized.
Results and discussion
Synthesis of P(NIPAM-co-AA-co-NIA) random copolymers
The ATR FT-IR spectra of P(NIPAM-co-AA) and P(NIPAM-co-AA-co-
NIA) were shown in Fig. S1. As shown in Fig. S1(a), the strong
absorption peaks at 3160-3700 cm-1 correspond to the N-H
stretching vibration in the PNIPAM segment, while the strong
absorption peak at 1647 cm-1 is attributed to the C=O stretching
vibration in the PNIPAM segment, and C-N stretching vibration peak
appear at 1540 cm-1. These signals fully proved the successful
polymerization of N-isopropylacrylamide monomer. Moreover, the
characteristic group signal of PAA segment could also be detected by
IR spectrum, the stretching vibration peaks of C=O and bending
vibration peaks of O-H appeared at 1724 cm-1 and 1381 cm-1
respectively, indicating that acrylic monomers were successfully
polymerized. Fig. S1(b) shows the ATR FT-IR spectrum of P(NIPAM-
co-AA-co-NIA), in addition to the characteristic group signals in
PNIPAM and PAA segments, the absorption peak at 1420 cm-1
corresponded to N=O group, and the strengths of the stretching
vibration peaks of C=O and bending vibration peaks of O-H in PAA
segment decreased, which indicated that NI groups were successfully
Table 1 Results of the random copolymers with different amount of NI groups
b
Sample
Mn,NMR
(g/mol)a
9221
10967
11516
Mn,GPC
(g/mol)b
9012
9985
10650
Mw/Mn
P(NIPAM48-co-AA18-co-NIA8)
P(NIPAM48-co-AA9-co-NIA17)
P(NIPAM48-co-AA7-co-NIA19)
1.12
1.23
1.24
a Determined by 1H NMR spectroscopy of the random copolymers.
b Determined by GPC analysis with polystyrene standards. Use THF as eluent.
Fig. S3 showed the GPC curves of random copolymer P(NIPAM-
co-AA-co-NIA), these curves are unimodal and symmetrical,
indicating that the synthesized polymers are pure substances.
The molecular weight and its distribution are shown in Table 1.
Micellization of amphiphilic P(NIPAM-co-AA-co-NIA) random
copolymers
grafted onto the side chains of random copolymer P(NIPAM-co-AA). To further explore the morphology and hydrodynamic radius of
1H NMR spectra of DDMAT, P(NIPAM-co-AA) and P(NIPAM-co-
the micelles obtained by self-assembled amphiphilic copolymer
P(NIPAM-co-AA-co-NIA), three polymer micelle samples with
different grafting ratios of NI group were characterized by TEM
and DLS respectively. The results are shown in Fig. 2.
1
AA-NIA) are shown in Fig. S2. H NMR spectrum of DDMAT is
shown in Fig. 2S(a). The proton of methyl-terminal can be
detected at 0.82 ppm, the absorption peak at 1.28 ppm
belonged to ten methylene’s protons. Meanwhile, the proton
peaks of methylene linked to C=S and two methyl linked to C-S
appeared at 1.62 ppm, and the proton signal of carboxyl group
appeared at 11.0 ppm. All these results proved the successful
The results of TEM characterization are shown in Fig. 2(a-c). The
morphologies of micelles obtained by self-assembled
P(NIPAM48-co-AA18-co-NIA8), P(NIPAM48-co-AA9-co-NIA17) and
P(NIPAM48-co-AA7-co-NIA19) were regular spheres, which
conformed the core-shell structure of amphipathic molecule. In
addition, the comparison among (a), (b) and (c) in Fig. 2
indicated that the size of micelles increased with the increasing
graft number of NI groups in the copolymer. Fig. 2(a’-c’) shows
the hydrodynamic radius (Rh) values of self-assembled
P(NIPAM48-co-AA18-co-NIA8), P(NIPAM48-co-AA9-co-NIA17) and
1
synthesis of DDMAT. H NMR spectrum of P(NIPAM-co-AA)
which was synthesized from N-isopropylacrylamide and acroleic
acid monomer via RAFT, by using DDMAT as chain transfer and
azodiisobutyronitrile as evocating agent was shown in Fig. S2(b).
The proton signals of -NH group of PNIPAM chains and -COOH
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P(NIPAM48-co-AA7-co-NIA19), respectively. Obviously, the solutions from 260 nm to 400 nm. Micelle solutions were
View Article Online
change tendency of the hydrodynamic radius was similar to that respectively treated under hypoxic (0.1% O2D, O5%I: 1C0O.102)3a9/nDd0nNoJ0rm21o14xiHc
displayed in TEM images. Namely, the Rh values increased from (20% O2, 5% CO2) conditions, in PBS media containing NADPH
63.7 nm, 85.1 nm to 101.8 nm with the increase of grafting (amount to the molar amount of nitro) at 37oC for 3 h. As revealed in
number of NI groups. By comparing the results of the last two Fig. 3a, the characteristic peak of NI group at 325 nm can be detected
samples, while the grafting number of NI groups increased by under normoxic conditions. Whereas, the peak of NI completely
only two, it was also caused a significant increase in Rh value, disappeared under hypoxic conditions, and a new peak appeared at
which was due to the increase of steric hindrance.
278 nm which was attributed to the characteristic peak of 2-
aminoimidazole (AI), indicating that the convertion occurred from
nitro group of NI to the amino group under hypoxic conditions. In the
convertion progress, NADPH was acted as electron donor and
transferred six electrons to catalyze the nitro groups. Furthermore,
the intensities of peak were increased with the amount increase of
AI groups in copolymer, correspondingly.
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Fig. 2 TEM images of (a) P(NIPAM48-co-AA18-co-NIA8), (b) P(NIPAM48-co-AA9-co-NIA17
)
and (c) P(NIPAM48-co-AA7-co-NIA19) and hydrodynamic radius (Rh) of copolymer self-
assemblies of (a’) P(NIPAM48-co-AA18-co-NIA8), (b’) P(NIPAM48-co-AA9-co-NIA17) and (c’)
P(NIPAM48-co-AA7-co-NIA19).
In addition, the Rh value of DLS was bigger than micellar radius
obtained by TEM image, which was due to the fact that DLS test
was for the micelle solution of polymer, including the thickness
of hydration layer, but TEM test was for the morphology of
dehydrated micelles. As a result, TEM test results were smaller
than corresponding Rh values. To sum up, the increase trend of
self-assembled micelles caused by the increased hydrophobicity
of copolymers with the increase of the grafting NI groups
number, which required more molecular to self-assembly into
micelles. By designing polymers with different number of NI
groups, more targeted biological applications can be achieved.
Fig. 3 (a) UV absorption spectra of P(NIPAM48-co-AA18-co-NIA8) (Sample 1), P(NIPAM48
-
co-AA9-co-NIA17 (Sample 2) and P(NIPAM48-co-AA7-co-NIA19 (Sample 3) micelle
)
)
solutions incubated for 3 h under normoxic and hypoxic conditions respectively.
The samples were measured in PBS buffer containing 100 mM NADPH as electron
donor. The illustration shows the conversion of NI into 2-aminoimidazole under
hypoxic condition containing NADPH and (b) Zeta potential of P(NIPAM48-co-AA18-co-
NIA8) (Sample 1), P(NIPAM48-co-AA9-co-NIA17) (Sample 2) and P(NIPAM48-co-AA7-co-NIA19
(Sample 3) micelle solutions incubated with 100 mM NADPH as electron donor
under normoxic and hypoxic conditions.
)
Hypoxia-responsive property of the random copolymer micelles
The performance of P(NIPAM-co-AA-co-NIA) random copolymer
micelles in terms of hypoxic response was characterized and
revealed by ultraviolet absorption measurement of the micelle
The Zeta potential images of the P(NIPAM48-co-AA18-co-NIA8),
P(NIPAM48-co-AA9-co-NIA17) and P(NIPAM48-co-AA7-co-NIA19) micelle
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solutions are shown in Fig. 3b. The Zeta potentials of samples
incubated with NADPH under normoxic conditions were -16.03 mV,
-23.73 mV and -24.2 mV, respectively. After hypoxia treatment, the
Zeta potentials of three copolymer micelles decreased rapidly to -
7.90 mV, -10.59 mV and -8.80 mV, which was due to the change that
the negatively charged nitro group of the NI was converted to
positively charged amino group under anoxic circumstance with
NADPH as electron donor. In addition, the absolute values of the
decreased Zeta potentials were 8.13 mV, 13.14 mV and 15.4 mV
respectively, which indicated that the increased content of NI group
could enhance the hypoxic sensitivity of micelles.
respectively. And the particle sizes obtained by DLS and TEM
displayed the same change tendency, which can be attributed
View Article Online
DOI: 10.1039/D0NJ02114H
to the convertion from hydrophobic NI groups to hydrophilic AI
groups catalyzed by NADPH under hypoxic conditions, leading
to the expansion of micelles and even the structural change into
vesicles like sample P(NIPAM48-co-AA7-co-NIA19). The
morphological changes of micelles caused by their
responsiveness indicated that it was possible to use them as
smart carriers for drug targeting and controlled release.
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Temperature-responsive property of the random copolymer
micelles
The ultraviolet transmittance tests of P(NIPAM-co-AA-NIA) self-
assembled micelles were carried out under different
temperatures as shown in Fig. 5. The micelle solution was
irradiated by ultraviolet light with wavelength of 500 nm from
20oC to 56oC, and the amplitude of variation was 0.5 oC per time.
The ultraviolet transmittance of micelle solution was 50.2% at
20 oC. After that, with the increase of temperature, the
ultraviolet transmittance decreased slightly in the temperatures
o
ranging from 20 oC to 30 C. When the temperatures ranged
o
o
from 30 C to 40 C, the decrease in UV transmittance was
o
accelerated. When the temperature increased up to 56 C, the
ultraviolet transmittance decreased slightly to the lowest value
of 6.3%. The above results verified the temperature response
property of copolymer P(NIPAM-co-AA-co-NIA), with the
o
o
sensitive temperature ranges from 30 C to 40 C, which was
similar to that of PNIPAM chain segment. The realization of
temperature response property can be attributed to the
morphological variation of the PNIPAM chain segment at the
response temperature.
Fig. 4 TEM images of (a) P(NIPAM48-co-AA18-co-NIA8), (b) P(NIPAM48-co-AA9-co-NIA17
)
and (c) P(NIPAM48-co-AA7-co-NIA19) micellar and hydrodynamic radius (Rh) of copolymer
self-assemblies of (a’) P(NIPAM48-co-AA18-co-NIA8), (b’) P(NIPAM48-co-AA9-co-NIA17) and
(c’) P(NIPAM48-co-AA7-co-NIA19) after anoxic treatment.
The TEM and DLS characterizations of P(NIPAM48-co-AA18-co-
NIA8), P(NIPAM48-co-AA9-co-NIA17) and P(NIPAM48-co-AA7-co-
NIA19) after anoxic treatment are shown in Fig. 4. Compared
with the image of micelles under normoxic conditions in Fig. 2,
the copolymer micelles of P(NIPAM48-co-AA18-co-NIA8) and
P(NIPAM48-co-AA9-co-NIA17) were still spherical but the radius
increased obviously. For P(NIPAM48-co-AA7-co-NIA19), the
micelles were transformed into vesicles with the increase of size,
which can be attributed to the varying proportions of
hydrophobic segments of the copolymer under hypoxic
conditions. Fig. 4(a’-c’) shows the variation in the Rh values of
samples after anoxic treatment. Among them, the Rh values of
copolymer micelles were 141.3 nm, 198.3 nm and 222.1 nm,
Fig. 5 Trend Diagram of UV transmittance of P(NIPAM48-co-AA7-co-NIA19) copolymer
micelle with temperatures ranging from 20 oC to 56 oC.
The TEM images of P(NIPAM48-co-AA18-co-NIA8), P(NIPAM48-co-
AA9-co-NIA17) and P(NIPAM48-co-AA7-co-NIA19) copolymer
micelles after heating treatment under human body
o
temperature (37 C) and the Rh value of P(NIPAM48-co-AA7-co-
NIA19) copolymer micelles with temperatures ranging from 25
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oC to 50 C have been recorded in Fig. 6. Obviously, the normoxia conditions, which was due to the acceleVriaewteAdrtircleelOenalisnee
DOI: 10.1039/D0NJ02114H
morphologies of micelle samples observed by TEM after heating of DOX under hypoxic conditions.
treatment were no longer regular spherical, but were irregular Moreover, the IC50 of DOX-loaded copolymer micelles can also
small particles. Compared with Fig. 2, the size of each particle be evaluated by the in vitro cell assay and the IC50 for three
decreased, but the particles were aggregated together. As samples were 4.16, 1.96 and 1.75 μg/L respectively, which can
shown in Fig. 6d, the Rh value of self-assembled micelles was be seen in the Table 2.
recorded at a scattering angle of 90° for each 1 oC. At
o
o
temperature range from 25 C to 33 C, Rh decreased slightly.
When the temperature exceeded above 33 oC, the Rh increased
rapidly and continuously, which should be attributed to
temperature responsiveness of the PNIPAM chain segment
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o
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(LCST is 33 C). With the increase of temperature below 33 C,
PNIPAM segments gradually became hydrophobic but not
obviously, thus leading to the shrinkage of particle size. When
the temperature was higher than LCST, the hydrophobic
transformation occurred and led to aggregation of micelles into
larger particle size.
Fig. 6 TEM images of (a) P(NIPAM48-co-AA18-co-NIA8), (b) P(NIPAM48-co-AA9-co-NIA17
and (c) P(NIPAM48-co-AA7-co-NIA19) micellar after heating treatment under 37 oC and (d)
trend diagram of hydrodynamic radius (Rh) of P(NIPAM48-co-AA7-co-NIA19) with
temperatures ranging from 25 oC to 50 oC.
)
Fig. 7 (a) In vitro cytotoxicities of pure P(NIPAM48-co-AA18-co-NIA8) (Sample 1),
P(NIPAM48-co-AA9-co-NIA17) (Sample 2) and P(NIPAM48-co-AA7-co-NIA19) (Sample 3)
assembled micelles incubated with 293T cells for 24 h and (b) The cytotoxicities of
DOX-loaded Sample 3 and free DOX incubated with 293T cells for 24 h under
normoxic and hypoxic conditions with different concentration of DOX. Asterisks
(*) denote statistically significant differences (p<0.05 compared with DOX-loaded
In vitro cell assay and the controlled release of DOX
The in vitro cytotoxicity assays of micelles were carried out by
using 293T cells after incubated at 37 oC for 24 h with copolymer
micelles solution, and the cell viabilities were measured by MTT
method. As shown in Fig. 7a, the cell viability was still up to 80%
as the concentration of the copolymer micelles reached 100
mg/L, which indicated that the copolymer micelles had low
cytotoxicity and good biocompatibility. In addition, Fig. 7b
shows the cytotoxicities of Sample 3 loading DOX and free DOX
under hypoxic and normoxic conditions. The free DOX exhibited
the similarly strong cytotoxicities in both conditions. However,
the cytotoxicity of DOX loaded copolymer micelles under
hypoxic conditions was significantly higher than that under
sample incubated under normoxic condition).
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Table 2 The IC50 of three DOX-loaded polymeric micelles with different amount of NI
groupsa
View Article Online
DOI: 10.1039/D0NJ02114H
5
6
Sample
IC50 Micelle concentration
(μg/L)
7
8
P(NIPAM48-co-AA18-co-
NIMA8)
4.16
9
P(NIPAM48-co-AA9-co-
NIMA17)
P(NIPAM48-co-AA7-co-
NIMA19)
1.96
1.75
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a The results determined by the in vitro cell assay experiments.
The drug-loading efficiency (DLE) of self-assembled micelles was
64.3% and the drug-loading content (DLC) was 8.39% which
were calculated by formula (1) and (2) respectively.
In order to further study the effect of drug-loaded micelles on
the controlled performance of drug release, the release
experiments of DOX-loaded micelles were carried out under
normoxic room temperature (23 oC), normoxic 37 oC and
hypoxia 37 oC respectively. 2 mL of external PBS was taken out
to determine the ultraviolet absorbance at 485 nm, and the
cumulative drug release was calculated, as shown in Fig. 8.
Fig. 8 In vitro DOX release curves of P(NIPAM48-co-AA7-co-NIA19) (Sample 3) micelles
under normal, hypoxic and heat conditions. Measurements were performed in PBS
buffer (pH 7.4) containing 100 mM NADPH as reduction catalytic medium
Conclusions
o
Obviously, under normoxic 23 C conditions, the drug-loaded
The amphiphilic copolymer P(NIPAM-co-AA-co-NIA) with both
temperature and hypoxia responsiveness was successfully
synthesized by RAFT and EDC reactions. By TEM, DLS and other
characterizations, the copolymer assembled micelles exhibited
a regular spherical shape with different sizes that closely related
with the grafting rate of NI groups in copolymer. Meanwhile, by
UV, DLS and Zeta etc., the P(NIPAM-co-AA-co-NIA) copolymer
micelles under hypoxia conditions displayed the obviously
hypoxic sensitivity, which was due to the hydrophilic
transformation of NI groups. In addition, through the studies of
TEM, UV transmittance and DLS in different temperatures, the
morphology and hydrodynamic radius of micelles changed,
indicating that the micelles possessed good temperature
responsiveness. Furthermore, through in vitro cytotoxicity and
cumulative release of drug-loaded micelles, it was found that
the copolymer with biocompatible property could be used as a
smart drug carrier to achieve the encapsulation and controlled
release of hydrophobic drugs. In the double stimulation
conditions of hypoxia and high temperature (37 oC), the
cumulative release of drug was further enhanced and the
release process was more controllable, thus verifying the
potential for future applications in the biomedical field.
micelles could only release 24.61% of DOX after 24 h, and the
release rate was slow. When the temperature was raised (37 oC),
the rate of drug release in the first four hours was significantly
increased, and the final drug release level was 35.49%.
Therefore, it could be concluded that the temperature
responsiveness of the copolymer micelles would lead to an
increase in release rate and enhanced the drug-release capacity,
that is, the temperature responsiveness facilitated the
intelligent and controlled release of the copolymer micelles as
o
drug carrier. Besides, under hypoxic conditions at 37 C, the
cumulative release level of DOX was significantly higher than
that under normoxic conditions. After 10 h, the release rate was
still fast, and the release level could reach 49.74 %. The final
cumulative release level of drug-loaded micelles was 53.24 %,
which was significantly higher than that under normoxic
condition. The result further revealed that the conversion from
the NI derivative to the AI derivative under hypoxic conditions
could further promote DOX release. Compared with the single
responsive self-assembled micelles in previous literature, the
hypoxia and temperature dually responsive copolymer revealed
stronger targeted drug delivery and more controllable release
efficiency.17, 22, 69
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The project was funded by the National Natural Science
Foundation of China (no. 81771942).
Notes and references
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View Article Online
DOI: 10.1039/D0NJ02114H
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DOI: 10.1039/D0NJ02114H
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The micelles self-assembled from P(NIPAM-co-AA-co-NIA) copolymers presented
hypoxia and temperature dual responsive properties and achieve the controlled release
of drug.
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