Materials Science & Engineering C 109 (2020) 110442

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

The combination of Diels-Alder reaction and redox polymerization for T

preparation of functionalized CNTs for intracellular controlled drug delivery
Ziyang Hea,b, Ruming Jiangb, Wei Longa,b, Hongye Huanga,b, Meiying Liub, Junyu Chenb,
⁎ ⁎ ⁎⁎
Fengjie Dengb, Naigen Zhoua, , Xiaoyong Zhangb, , Yen Weic,d,
a
School of Materials Science and Engineering, Nanchang University, Nanchang, Jiangxi 330031, China
b
Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China
c
Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084, China
d
Department of Chemistry and Center for Nanotechnology and Institute of Biomedical Technology, Chung-Yuan Christian University, Chung-Li 32023, Taiwan

A R T I C LE I N FO A B S T R A C T

Keywords: Carbon nanotubes (CNTs) are a novel type of one-dimensional carbon nanomaterials that have been widely
Carbon nanotubes utilized for biomedical applications such as drug delivery, cancer photothermal treatment owing to their high
Diels-Alder reaction surface area and unique interaction with cell membranes. However, their biomedical applications are still im-
Ce(IV)/HNO3 redox polymerization peded by some drawbacks, including poor water dispersibility, lack of functional groups and toxicity. Therefore,
intracellular controlled drug delivery
surface modification of CNTs to overcome these issues should be importance and of great interest. In this work,
we reported for the first time that CNTs could be surface modification through the combination of Diels-Alder
(D-A) reaction and redox polymerization, this strategy shows the advantages of mild reaction conditions, water
tolerance, low temperature and hydroxyl-surfaced initiator. In this modification procedure, the hydroxyl groups
were introduced on the surface of CNTs through the D-A reaction that was adopted for grafting the copolymers,
which were initiated by the Ce(IV)/HNO3 redox system using the hydrophilic and biocompatible poly(ethylene
glycol) methyl ether methacrylate (PEGMA) and carboxyl-rich acrylic acid (AA) as monomers. The final CNTs-
OH-PAA@PEGMA composites were characterized by a series of characterization techniques. The drug loading
and release results suggested that anticancer agent cis‑platinum (CDDP) could be loaded on CNTs-OH-PAA@
PEGMA composites through coordination with carboxyl groups and drug release behavior could be controlled by
pH. More importantly, the cell viability results clearly demonstrated that CNTs-OH-PAA@PEGMA composites
displayed low toxicity and the drug could be transported in cells and still maintain their therapeutic effects.

1. Introduction the CNTs possess many advantages in biological applications, as we

For the past few years, carbon nanotubes (CNTs) are getting more
and more attention owing to their special one-dimensional structure
and physicochemical properties [1,2]. Therefore, CNTs have been
widely applied in biomedical fields such as medical devices and sensors,
tissue engineering, drug delivery, antibacterial and cancer photo-
thermal treatment [3–7]. For the biomedical applications, CNTs could
be utilized over existing drug carriers owing to their ability to be in-
ternalized by cells easily and high specific surface area provided plen-
tiful active adsorption sites for loading drugs by covalent or non-
covalent interaction methods [8]. Besides, the CNTs-drugs conjugates
possess a lower potential cytotoxicity than bare drugs and also their
internalization efficiency could be improved further [9–11]. Although
know, CNTs possess huge specific surface areas and strong hydro-
phobicity, which make pristine CNTs agglomerate easily in solution,
cells, tissues or organs with damages. The low dispersibility hinders
seriously their applications in biomedicine. In addition, the toxicity of
pristine CNTs still remains a controversial topic, different preparation
and purification procedures could influence their toxicity [11–14].
Therefore, developing an efficient, low cytotoxic and high dispersibility
of drug carrier is needed. Surface functionalization approaches have
shown great potential in improving dispersibility, biocompatibility.
Some effective strategies have been developed to decorate the CNTs
to improve the water dispersibility. For example, strong oxidizing acid
can oxidize the surface of CNTs and some oxygen functional groups
could be introduced in surface such as hydroxyl and carboxyl groups

⁎
Corresponding authors.
⁎⁎
Correspondence to: Yen Wei, Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084, China.
E-mail addresses: ngzhou@ncu.edu.cn (N. Zhou), zhangxiaoyong@ncu.edu.cn (X. Zhang), weiyen@tsinghua.edu.cn (Y. Wei).

https://doi.org/10.1016/j.msec.2019.110442
Received 17 September 2019; Received in revised form 13 November 2019; Accepted 15 November 2019
Available online 18 November 2019
0928-4931/ © 2019 Published by Elsevier B.V.
Z. He, et al.
[15–18], on some level, it could increase the water dispersibility
slightly. However, the process is hard to control, involvement of ha-
zardous agents and low efficiency. Moreover, the products are difficult
to separate or purify and the by-products are not friendly to the en-
vironment. Therefore, Diels-Alder (D-A) reaction [19–23] was adopted
to endow CNTs with functional groups, which process advantages of
low reaction temperature, mild experimental conditions, facile proce-
dures and absent of hazardous agents. Through the functional groups,
we can further modify CNTs with polymerization reaction for further
functionalization. Besides, the surface modification of CNTs with
functional polymers can also endow their other functions such as cell
imaging, drug delivery and probes [24–39]. In recent years, some fea-
sible polymerization methods have been developed [40–45]. For ex-
ample, Ao et al. developed a versatile strategy to modify CNTs. Firstly,
the surface of CNTs was covered with a thin layer of by self-poly-
merization of dopamine and next the reversible addition-fragmentation
chain transfer (RAFT) agent was combined with dopamine. Finally, the
polymer is grafted onto the surface of the CNTs by RAFT polymerization
[46]. Song et al. introduced 2-bromoisobutyryl bromide on the surface
of CNTs by mussel biochemistry to form initiators and decorated the
surface via surface-initiated polymerization by electron transfer atom
transfer radical polymerization (ATRP) [47]. Our recent report has
demonstrated that the CNTs could be facilely functionalized with hy-
drophilic copolymers containing PEGMA through the combination of
mussel-inspired chemistry and surface-initiated single-electron transfer
living radical polymerization (SET-LRP). The resultant CNTs polymeric
composites show enhanced dispersibility in aqueous solution and ex-
cellent biocompatibility and suggest the potential of these CNTs poly-
meric composites for biomedical applications [48–51]. However, those
approaches require high reaction temperature, strict waterless and ni-
trogen gas protection, therefore, the development of novel surface
polymerization methods that could overcome the above issues should
be of utmost importance.
In this work, we developed a novel and facile approach to functio-
nalize CNTs that combine the D-A reaction and Ce(IV)/HNO3 redox
polymerization for the first time. Compared with the other methods, the
mild, simple D-A reaction possess extensive reliability for carbon ma-
terials and the simple, hydrous, low temperature and hydroxyl-surfaced
initiated Ce(IV)/HNO3 redox polymerization make it become more
easier for the material surface modification with polymer. As displayed
in Scheme 1, the pristine CNTs were surface modified with hydroxyl
groups through the reaction of furfuralcohol with CNTs via D-A reac-
tion. Then, the monomers (poly(ethylene glycol) methyl ether
Scheme 1. The surface modification of CNTs with carboxyl groups and PEGMA through the combination of D-A reaction and Ce(IV)/HNO3 initiated redox poly-
merization.
2
Materials Science & Engineering C 109 (2020) 110442
methacrylate (PEGMA) and acrylic acid (AA)) were grafted on the
surface of CNTs-OH through the redox polymerization, which was in-
itiated by Ce(IV)/HNO3 redox system. The polymerization could take
place under low reaction temperature and organic solvent, absent of
expensive and hazardous agents. The size and morphology, physico-
chemical properties and drug delivery performance were detailed
characterized and examined. Owing to the introduction of carboxyl
groups and PEGMA, we could expect that the resultant CNTs-OH-PAA@
PEGMA composites could possess the ability for drug loading and
controlled drug release as well as for biomedical applications.
2. Materials and methods
2.1. Materials and measurements
Multiwalled carbon nanotubes (> 95%, diameter of 30–50 nm)
were purchased from Chengdu Organic Chemicals Co., Ltd. (Chinese
Academy Of Sciences), dimethyl sulfoxide (DMSO, Mw: 78.13 Da,
67–68-5, AR), nitric acid (Mw: 63.01 Da, 7697-37-2, AR) were pur-
chased from Tianjin Damao Chemical Reagent Co. Ltd. Furfuralcohol
(Mw: 98.10 Da, 98–00-0, 99%), Ammonium ceric nitrate (Mw:
548.22 Da, 16,774–21-3, 99.99%), Poly(ethylene glycol) methyl ether
methacrylate (PEGMA, Mw: 950 Da, 26,915–72-0), acrylic acid (AA,
Mw: 72.06 Da, 79–10-7, 99.7%) were purchased from the Aladdin
Industrial Co., Ltd. (Shanghai, China), cis‑platinum (CDDP) was sup-
plied by Huaxia Co., Ltd., (Chengdu, China), cell lines HepG2 were
obtained from the Cell Bank of the Chinese Academy of Sciences
(Shanghai, China) and all above chemicals were used directly without
any further purification. The samples were characterized by a series of
characterization equipment and the detailed information for char-
acterization was listed in the ESI.
2.2. Synthesis of CNTs-OH
CNTs-OH was synthesized through the D-A reaction in DMSO so-
lution containing CNTs and furfuralcohol. In brief, 300 mg CNTs were
dispersed in 30 mL DMSO solution and treated with ultrasonic-pro-
cessing for 10 min. Then, 500 mg furfuralcohol was dissolved in solu-
tion and the mixed solution was stirred in 40 °C for 24 h. After that, the
mixture was separated through centrifugation and the solid particles
were dialyzed against distilled water for 24 h to remove the unreacted
impurities. Afterwards, we remove the water by freeze-drying tech-
nology. Then, we obtain the dry CNTs-OH.
Z. He, et al. Materials Science & Engineering C 109 (2020) 110442

2.3. Preparation of CNTs-OH-PAA@PEGMA

For preparation of CNTs-OH-PAA@PEGMA, 300 mg CNTs-OH were

well dispersed in deionized water by ultrasonic treatment for 10 min.
Then, 1 g PEGMA and 200 mg AA were dissolved in mixed solution and
mixed liquid was transferred into reaction flask, removing the air by
repeated blowing N2 flow and vacuum supply for 10 min. A nitrogen
atmosphere should be guaranteed throughout the process. After that,
1 mL of 0.2 M solution of ceric ammonium nitrate in 1 M nitric acid was
injected into the reaction flask. The polymerization reaction was
maintained at 50 °C for 5 h. Finally, black solid were separated and
purified by centrifugal treatment (7000 rpm) with deionized water for 5
times. Besides, in order to further eliminate impurities, black solid was
dialyzed against distilled water for 24 h and water was removed
through vacuum drying. Finally, pure CNTs-OH-PAA@PEGMA com-
posites were obtained.

2.4. Loading and release behavior of CDDP

Plentiful carboxyl groups were introduced on the surface of CNTs

through the redox polymerization that makes CNTs-OH-PAA@PEGMA Fig. 1. The 1H NMR spectrum of CNTs-OH-PAA@PEGMA composites in CDCl3.
composites great potential for highly efficient drug loading and pH-
controlled release behavior. The ability for the loading and release of
the hydrogen from the polyethylene glycol, which demonstrated the
CDDP on CNTs-OH-PAA@PEGMA was evaluated. First, 10 mg CDDP
PEGMA have been successfully grafted in the surface of CNTs. Besides,
and 20 mg CNTs-OH-PAA@PEGMA were dispersed in phosphate buffer
the two peaks showed at 1.22 and 0.81 ppm, which resulted from the
saline (PBS, 100 mL, pH = 7.4) by ultrasonic treatment. The mixed
hydrogen on the alkane backbone such as methylene and methyl. The
solution was kept in dark environment with electromagnetic stirring at
results authenticated the successful surface modification of CNTs
37 °C for 48 h. After that, black products (CNTs-OH-PAA@
through surface-initiated redox polymerization based on Ce(IV)/HNO3
PEGMA@CDDP complexes) were separated by centrifugal separation
redox system.
and the centrifugal liquid protected from light. The concentration of
In order to confirm the successful synthesis of CNTs-OH-PAA@
dissociative CDDP existed in the centrifugal liquid was measured by
PEGMA, Fourier-transform infrared (FT-IR) spectroscopy was adopted
UV–Vis spectrometer at 705 nm and the CDDP loading capability of
to characterize the functional groups and chemical information. The FT-
CNTs-OH-PAA@PEGMA was calculated. Afterwards, the preparative
IR spectra of CNTs, CNTs-OH and CNTs-OH-PAA@PEGMA were shown
CNTs-OH-PAA@PEGMA@CDDP complexes were dialyzed at PBS
in Fig. 2. Apparently, no significant infrared absorption peak appeared
(100 mL, pH = 7.4 and 5.2) for 48 h without light and 1 mL dialysate
in the FT-IR spectra of pristine CNTs, which illuminated that pristine
was sampled with time for measuring by UV–Vis spectroscopy.
CNTs are lack of functional groups. On the other hand, the OeH
stretching vibration was found in 3446 cm−1 and C]C stretching vi-
2.5. Cytotoxicity evaluation
bration occurred in 1616 cm−1 in the FT-IR spectra of CNTs-OH. It can
prove that the furfuralcohol was introduced successfully on the surface
Biocompatibility is crucial for the functional composite applied in
of CNTs by the D-A reaction. Besides, the successful synthesis of CNTs-
biomedical field. In this work, MTT assay was adopted to evaluate the
OH-PAA@PEGMA composites was evaluated by FT-IR spectra. As
cytotoxicity of CNTs-OH-PAA@PEGMA [52]. Firstly, the cultured
HepG2 cells were inoculated into 96-well plates at a density of 5 × 103
cells well and supplemented with DMEM medium containing 10% fetal
bovine serum to provide nutrition and the cells were cultivated for 24 h
in cell incubator and until the cell fusion rate is over 80% and adheres
completely. Then, the prepared CNTs-OH-PAA@PEGMA, CNTs-OH-
PAA@PEGMA@CDDP and pure CDDP were added to 96-well plate
(100 μL/well), and reserve two rows of empty space. Moreover, equal
volume phosphate buffer saline (PBS) was applied to seal the outer edge
and two empty holes were set as control group by adding pure DMEM
medium with same amount. After cultivated for 12 or 24 h, 5 mg/mL
MTT solution was added to each orifice plate until reach a dose of 50 μL
for each well. After three hours, the added liquid was absorbed and
150 μL DMSO was added for each hole with shaking for 10 min. Finally,
the absorbance was recorded with 490 nm laser to accomplish the ex-
periment. The experiment was repeated for three times and the cell
viability or standard deviation was calculated by absorbance.

3. Results and discussion

The successful surface modification of CNTs through the combina-

tion of D-A reaction and surface-initiated redox polymerization was
affirmed by 1H nuclear magnetic resonance (NMR) spectrum. As shown
in Fig. 1, the signal appeared at 7.23 ppm, it can be ascribed to the Fig. 2. The FT-IR spectra of unadorned CNTs, CNTs-OH and CNTs-OH-PAA@
CDCl3. The peak with chemical shifts at 3.61 ppm can be attributed to PEGMA samples.

3
Z. He, et al.
Fig. 3. (A) TGA curves of CNTs-OH-PAA@PEGMA, (B) the survey XPS spectra of CNTs-OH-PAA@PEGMA, (C) the region of deconvoluted scan C 1 s, (D) the region of
deconvoluted scan O 1 s.
shown in Fig. 2, a series of characteristic infrared absorption peaks were
also found in the sample of CNTs-OH-PAA@PEGMA, the signal of
3438 cm−1 results from the stretching vibration of –OH, the signal of
2904 cm−1 results from the stretching vibration of –CH2, the peaks of
1729 and 1641 cm−1 root from the stretching vibration of C]O. A
series of peaks from 1465 to 1269 cm−1 could be ascribed to the
asymmetric stretching vibration and in-plane bending vibration of
alkyl. Besides, the signal of 1099 cm−1 arise from the stretching vi-
bration of eOe and the signal of 947 cm−1 could be attributed to the
out-of-plane bending vibration of CeH. The –OH and C]O infrared
absorption peaks were resulting from the success of AA grafted on
CNTs-OH-PAA@PEGMA and eOe signal was found on account of the
successful polymerization of PEGMA. The above results implied that
CNTs-OH-PAA@PEGMA composites have been successfully synthesized
via the Ce(IV)/HNO3 redox polymerization.
The successful surface modification of CNTs also confirmed by the
thermogravimetric analyzer (TGA). As shown in Fig. 3A, there is no
significant changes occurred the TGA curve pristine CNTs, which in-
dicated pristine CNTs possess great thermodynamic stability. Besides, it
clearly shows in the TGA curve of CNTs-OH that the weight loss of
CNTs-OH was larger than pristine CNTs at the temperature 400–700 °C.
The weight loss should be attributed to the introduction of furfur-
alcohol, which could be detached from CNTs when the temperature was
elevated. Furthermore, more apparent weight loss was observed in the
curve of CNTs-OH-PAA@PEGMA. The obvious weight loss occurred at
100–350 °C should be ascribed to the decomposition of polyacrylic acid.
Besides, the weight loss of CNTs-OH-PAA@PEGMA rapidly increased to
35.5% at 450 °C, which should be attributed to the disintegration of
polyethylene glycol. Compared with the curve of CNTs-OH, the weight
loss occurred at 500–700 °C in the curve of CNTs-OH-PAA@PEGMA
should be blamed to the introduction of furan rings. The final weight
loss of CNTs-OH-PAA@PEGMA composites based on TGA curve was
calculated to be 54.5%. The obvious weight loss could be attributed to
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Materials Science & Engineering C 109 (2020) 110442
the decomposition of copolymers on the surface of CNTs. Therefore,
based on the TGA curves, the successful preparation of CNTs-OH-PAA@
PEGMA could be further confirmed [53].
X-ray photoelectron spectroscopy (XPS) was conducted to testify the
successful modification in surface of CNTs. The full survey XPS spectra
of CNTs, CNTs-OH and CNTs-OH-PAA@PEGMA were demonstrated in
Fig. 3B. From the spectrum, we could judge there are two elements
(carbon and oxygen) existed in sample and the detailed signal of C 1 s
and the O 1 s. The atomic percentage of elements present of samples
was shown in the Table 1. Apparently, the oxygen atomic percentage of
CNTs (0.7%), CNTs-OH (2.1%) and CNTs-OH-PAA@PEGMA (13.4%)
was gradually improved, the improvement of the oxygen atomic per-
centage in CNTs-OH should be ascribed to the introduction of furan ring
and hydroxyl groups. For the CNTs-OH-PAA@PEGMA, it could be at-
tributed to the successful introduction of copolymers by redox poly-
merization. The results also confirmed the successful preparation of
CNTs-OH-PAA@PEGMA. Besides, the C 1 s (Fig. 3C) and O 1 s (Fig. 3D)
deconvoluted scan exhibited respectively four representative domains
and two representative domains. The C 1 s deconvoluted scan could be
ascribed to C-C/C=C (284.4 eV), CeOe (285.35 eV), C]O (286.55 eV)
and C=O-O (288.5 eV), respectively. Moreover, the O 1 s deconvoluted
scan could be attributed to C]O (531.7 eV) and CeO (532.9 eV). All
mentioned above could further confirm the successful preparation of
CNTs-OH-PAA@PEGMA.
Based on the representation results above, the successful
Table 1
The element contents of CNTs, CNTs-OH and CNTs-OH-PAA@PEGMA.
C (%) O (%)
CNTs 99.3 0.7
CNTs-OH 97.9 2.1
CNTs-OH-PAA@PEGMA 86.6 13.4
Z. He, et al. Materials Science & Engineering C 109 (2020) 110442

Fig. 4. Representative TEM images of pristine CNTs and CNTs-OH-PAA@PEGMA composites. (A) pristine CNTs, (B) CNTs-OH-PAA@PEGMA composites, the scale
bar of A and B is 50 nm. (C) A partial enlarged view of pristine CNTs, (D) a partial enlarged view of CNTs-OH-PAA@PEGMA composites, the scale bar of C and D is
20 nm.

preparation of CNTs-OH-PAA@PEGMA composites could be confirmed.
The TEM images further provided the evidence of size and morphology.
As shown in Fig. 4, the clear TEM images of pristine CNTs and CNTs-
OH-PAA@PEGMA composites was exhibited. The photographs of
Fig. 4A and Fig. 4C have displayed the size and morphology pristine
CNTs, we could find the surface of pristine CNTs is very smooth, which
was decided by the stable physical structure. However, compared with
the pristine CNTs, the CNTs-OH-PAA@PEGMA composites show dif-
ferent surface morphologies in Fig. 4D. From the Fig. 4D, we could
discover the surface of CNTs-OH-PAA@PEGMA is rough. Moreover,
combined with the characterization results of XPS, the rough surface of
CNTs-OH-PAA@PEGMA could be judged as the macromolecules, which
were grafted on the surface of CNTs through the redox polymerization
induced by cerium ions and resulted in the surface of CNTs-OH-PAA@
PEGMA rough. The particle sizes of CNTs-OH-PAA@PEGMA was dis-
played in Fig. S1. The result showed the particle size of CNTs-OH-
PAA@PEGMA has a wide distribution and the most particle sizes are
distributed around 800 nm with PDI of 0.260.
Great water dispersibility is a pre-requisite for application in bio-
medical fields. In order to improve the hydrophilicity of CNTs, PEGMA
was grafted onto surface of CNTs and the dispersion ability of CNTs and
CNTs-OH-PAA@PEGMA was also investigated. As shown in Fig. 5, most
of pristine CNTs were rapidly accumulated and deposited in water
within 2 min. And after 1 h, the pristine CNTs completely deposited on
the bottom. On the contrary, the CNTs-OH-PAA@PEGMA composites
displayed excellent dispersion. The water suspension of CNTs-OH-
PAA@PEGMA is homogeneous and no aggregation was observed when
the water suspension of CNTs-OH-PAA@PEGMA was deposited
even > 12 h. Furthermore, the results further attested the successful Ce
(IV)/HNO3 redox polymerization occurred on the surface of CNTs. The
admirable water dispersibility makes the CNTs-OH-PAA@PEGMA
composites great potential in further drug deliver applications. More-
over, PEG is a hydrophilic and biocompatible macromolecule that has
been extensively utilized for surface modification of materials. It has
been demonstrated that the surface modification of materials could not
only improve their water dispersibility, but also influence the interac-
tion of materials and biological systems even the toxic outcome. In this
work, we demonstrated that PEG and carboxyl groups could be grafted
on the surface of CNTs successfully through the combination of D-A
reaction and redox polymerization. The great improvement of water

Fig. 5. Photographs of CNTs (bottle A) and CNTs-OH-PAA@PEGMA (bottle B) in water for different deposition time points 2 min, 1 h and 12 h.

5
Z. He, et al.
dispersibility was also found. On the other hand, owing to the in-
troduction of carboxyl groups, the CNTs-OH-PAA@PEGMA composites
could also potentially utilized for carrying the ionic drug such as CDDP
through the electrostatic interaction. Therefore, in the following sec-
tions, the loading of CDDP on CNTs-OH-PAA@PEGMA composites and
the drug release behavior was investigated. The biocompatibility of
CNTs-OH-PAA@PEGMA composites and the therapeutic effects of the
CNTs-OH-PAA@PEGMA-drug complexes were also evaluated.
It has been reported that CDDP can be captured by carboxylic
groups via substitution of the chloride ion ligands, which can be de-
signed for the preparation of drug carriers. Therefore, the drug loading
and release behaviors of CNTs-OH-PAA@PEGMA were also evaluated.
Based on coordination complexation formed by o-phenylenediamine
and CDDP, the concentrations of CDDP could be measures by UV
spectrophotometer (maximum absorption wavelength located at
705 nm). In this work, we demonstrated that the loading capacity of
CNTs-OH-PAA@PEGMA is 189.22 mg/g. Besides, we further assessed
pH responsive release behavior of CNTs-OH-PAA@PEGMA loaded
CDDP. The CDDP was released from the CNTs-OH-PAA@PEGMA at
different pH solutions (pH = 7.4 and 5.2). The whole loading and re-
lease process must be maintained in dark environment. As illustrated in
Fig. 6, in the first 12 h, CDDP was rapidly released from CNTs-OH-
PAA@PEGMA and the ratio of release gradually decreases. And after
12 h, the quantity of CDDP released from CNTs-OH-PAA@PEG-
MA@CPPD almost no changes over time. More importantly, the release
capacity of CNTs-OH-PAA@PEGMA@CPPD is sensitive to pH value.
The coordinate bonds between CNTs-OH-PAA@PEGMA and CDDP can
be destroyed in pH 5.2 solution than in pH 7.4 solution, which decided
the pH-controlled drug release behavior of CNTs-OH-PAA@PEG-
MA@CDDP, it can be ascribed to the protonation of carboxylic groups
at the acidic pH, which weakens the drug and nanoparticle coupling
and makes the drug more replaceable by chloride [54,55]. The higher
solubility of CDDP under pH 5.2 than pH 7.4 can further promote the
release. The results indicated that the release ratio of CNTs-OH-PAA@
PEGMA in lower pH solution is much higher than in high pH solution.
In the acid solution (pH = 5.2), the ratio of release could reach 59.78%
while in basic solution (pH = 7.4), the ratio of release merely reached
17.62%. The results demonstrated the great potential of CNTs-OH-
PAA@PEGMA for intracellular controlled drug delivery owing to the
acidic environment after internalization. The drug release behavior
indicates that the CDDP could be effectively loaded on the CNTs-OH-
PAA@PEGMA composites, the drug will release fast in intracellular
Fig. 6. The release behavior of CDDP from CNTs-OH-PAA@PEGMA complexes
at pH values of 5.2 and 7.4.
6
Materials Science & Engineering C 109 (2020) 110442
Fig. 7. The viability of HepG2 cells after treatment with different concentration
of CNTs-OH-PAA@PEGMA, CNTs-OH-PAA@PEGMA@CDDP and pure CDDP
for 24 h.
after cell internalization. Therefore, the CNTs-OH-PAA@PEGMA com-
posites could be potentially used as carriers for targeted drug delivery
and reduce the side effects of drug to normal cells and tissues.
Low cytotoxicity is crucial factor for the biomaterials applied in
biomedical fields. In this work, the cytotoxicity studies were conducted
by MTT assay. As shown in Fig. 7, the figure displays the viability of
HepG2 cells after treatment with different concentrations of CNTs-OH-
PAA@PEGMA, CNTs-OH-PAA@PEGMA@CPPD and pure CDDP for
24 h. The cell viability still can stay around 100% after cultivated with
CNTs-OH-PAA@PEGMA for 24 h, which demonstrated their low cyto-
toxicity and good biocompatibility. Our previous reports [56–58] de-
monstrated that the CNTs could be internalized by tumor cells and
exhibit strong toxicity towards different types of cells and the surface
properties of CNTs will also influence the final toxic results of CNTs and
related materials. In this work, we demonstrated that CNTs-OH-PAA@
PEGMA composites possess excellent biocompatibility, which is pos-
sibly attributed to the surface functionalization with the hydrophilic
and biocompatible copolymers, which could mask the toxicity of CNTs.
For the cell viability of CNTs-OH-PAA@PEGMA@CDDP complexes, we
can discover the CNTs-OH-PAA@PEGMA@CDDP complexes into cells
and the cell viability was decreased with the increasing of concentra-
tion of CNTs-OH-PAA@PEGMA@CDDP complexes. The cell viability
reduced to 64.6% when the concentration reached to 200 μg mL−1, the
result represents the CDDP has been successfully loaded in the CNTs-
OH-PAA@PEGMA and released in the cells. The great potential in drug
delivery application has been further confirmed. The cytotoxicity of
pure CDDP also has been evaluated by MTT assays. In the Fig. 7, we can
find the cells are very sensitive to the pure CDDP, the cell viability was
decreased to 34.98% when the concentration of pure CDDP reached to
200 μg mL−1. The result revealed the high cytotoxicity of pure CDDP
and also showed the necessity of drug carrier to weaken the damage for
normal cells or tissues. In consequence, the CNTs-OH-PAA@PEGMA
composites possess great potential in drug delivery application and
show good biocompatibility, which makes them more suitable for
biomedical applications.
4. Conclusions
In summary, we have successfully established a mild, valid and
aqueous polymerization system to modify surface of CNTs for anti-
cancer drug delivery. The successful preparation of these CNTs-based
Z. He, et al.
polymeric composites has been confirmed by a variety of character-
ization methods. Some necessary properties of biomaterials also have
been measured and demonstrated. Firstly, essential biocompatibility
and low cytotoxicity have been measured by MTT assay and the cell
viability keep above 90%. Besides, owing to the introduction of hy-
drophilic copolymers, the resultant CNTs-OH-PAA@PEGMA composites
displayed greater improvement of water dispersibility. Moreover, high
drug loading and pH-controlled drug release behavior have been de-
monstrated in a buffer solution thanks to the successful introducing of
carboxyl groups. Therefore, the advantages mentioned above provided
CNTs-OH-PAA@PEGMA composites a huge potential in drug delivery.
More importantly, this modification method that combined the D-A
reaction and Ce(IV)/HNO3 redox polymerization provided a mild,
aqueous and valid way to prepare CNTs-based functional polymeric
composites for biomedicine applications.
CRediT authorship contribution statement
Ziyang He: Methodology, Writing - original draft. Ruming Jiang:
Methodology, Writing - original draft. Wei Long: Methodology.
Hongye Huang: Methodology, Resources. Meiying Liu: Funding
acquisition. Junyu Chen: Resources. Fengjie Deng: Resources,
Funding acquisition. Naigen Zhou: Writing - review & editing,
Supervision, Funding acquisition. Xiaoyong Zhang:
Conceptualization, Writing - review & editing, Supervision,
Funding acquisition. Yen Wei: Writing - review & editing,
Supervision, Funding acquisition.
Declaration of competing interest
The authors declared that there is no conflict of interest.
Acknowledgments
This research was supported by the National Natural Science
Foundation of China (Nos. 21788102, 21865016, 51363016,
21474057, 21564006, 21561022, 21644014).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.msec.2019.110442.
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