The combination of Diels-Alder reaction and redox polymerization for preparation of functionalized CNTs for intracellular controlled drug delivery

Journal Pre-proof The combination of Diels-Alder reaction and redox polymerization for preparation of functionalized CNTs for intracellular controlled drug delivery

Ziyang He, Ruming Jiang, Wei Long, Hongye Huang, Meiying Liu, Junyu Chen, Fengjie Deng, Naigen Zhou, Xiaoyong Zhang, Yen Wei PII:

S0928-4931(19)33472-1

DOI:

https://doi.org/10.1016/j.msec.2019.110442

Reference:

MSC 110442

To appear in:

Materials Science & Engineering C

Received date:

17 September 2019

Revised date:

13 November 2019

Accepted date:

15 November 2019

Please cite this article as: Z. He, R. Jiang, W. Long, et al., The combination of Diels-Alder reaction and redox polymerization for preparation of functionalized CNTs for intracellular controlled drug delivery, Materials Science & Engineering C (2018), https://doi.org/ 10.1016/j.msec.2019.110442

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© 2018 Published by Elsevier.

Journal Pre-proof The combination of Diels-Alder reaction and redox polymerization for 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 Zhou a,*, Xiaoyong Zhang b,*, Yen Wei c,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

Department of Chemistry and Center for Nanotechnology and Institute of Biomedical Technology,

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d

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University, Beijing, 100084, P. R. China.

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Chung-Yuan Christian University, Chung-Li 32023, Taiwan Naigen Zhou

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[email protected]

Yen Wei

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[email protected]

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[email protected]

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Xiaoyong Zhang

1

Journal Pre-proof Abstract Carbon nanotubes (CNTs) are a novel type of one-dimensional carbon nanomaterials that have been widely utilized for biomedical applications such as drug delivery, cancer photothermal treatment owing to their high surface area and unique interaction with cell membranes. However, their biomedical applications are still impeded by some drawbacks, including poor water dispersibility, lack of functional groups and toxicity. Therefore, 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

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polymerization, this strategy shows the advantages of mild reaction conditions, water tolerance, low

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temperature and hydroxyl-surfaced initiator. In this modification procedure, the hydroxyl groups were

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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

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biocompatible poly(ethylene glycol) methyl ether methacrylate (PEGMA) and carboxyl-rich acrylic

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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

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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

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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.

Key words: Carbon nanotubes, Diels-Alder reaction, Ce(IV)/HNO3 redox polymerization, intracellular controlled drug delivery 2

Journal Pre-proof 1 Introduction 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 photothermal treatment[3-7]. For the biomedical applications, CNTs could be utilized over existing drug carriers owing to their ability to be internalized by cells easily and high specific surface area provided plentiful active adsorption sites for loading drugs by covalent or non-covalent interaction methods[8]. Besides, the CNTs-drugs conjugates possess a lower potential

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cytotoxicity than bare drugs and also their internalization efficiency could be improved further[9-11].

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Although the CNTs possess many advantages in biological applications, as we know, CNTs possess

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huge specific surface areas and strong hydrophobicity, which make pristine CNTs agglomerate easily in solution, cells, tissues or organs with damages. The low dispersibility hinders seriously their

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applications in biomedicine. In addition, the toxicity of pristine CNTs still remains a controversial topic,

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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

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functionalization approaches have shown great potential in improving dispersibility, biocompatibility. Some effective strategies have been developed to decorate the CNTs to improve the water

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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 [15-18], on some level, it could increase the water dispersibility slightly. However, the process is hard to control, involvement of hazardous agents and low efficiency. Moreover, the products are difficult to separate or purify and the by-products are not friendly to the environment. 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 procedures 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 feasible polymerization methods have been developed [40-45]. For example, Ao et al. developed a versatile strategy to modify CNTs. Firstly, the surface of CNTs was covered with a thin layer of by self-polymerization of dopamine and next the reversible addition-fragmentation chain transfer (RAFT) 3

Journal Pre-proof 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 hydrophilic 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 excellent biocompatibility and suggest the potential of

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these CNTs polymeric composites for biomedical applications [48-51]. However, those approach

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require high reaction temperature, strict waterless and nitrogen gas protection, therefore, the

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development of novel surface polymerization methods that could overcome the above issues should be of utmost importance.

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In this work, we developed a novel and facile approach to functionalize CNTs that combine the D-A

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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 materials and the simple, hydrous,

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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

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CNTs were surface modified with hydroxyl groups through the reaction of furfuralcohol with CNTs via D-A reaction. Then, the monomers (poly(ethylene glycol) methyl ether methacrylate (PEGMA) and acrylic acid (AA)) were grafted on the surface of CNTs-OH through the redox polymerization, which was initiated 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, physicochemical 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.

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Scheme 1 The surface modification of CNTs with carboxyl groups and PEGMA through the

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combination of D-A reaction and Ce(IV)/HNO3 initiated redox polymerization.

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2 Materials and methods

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2.1 Materials and measurements

Multiwalled carbon nanotubes (>95%, diameter of 30–50 nm) were purchased from Chengdu

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Organic Chemicals Co., Ltd. (Chinese Academy Of Sciences), dimethyl sulfoxide (DMSO, Mw: 78.13

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Da, 67-68-5, AR), nitric acid (Mw: 63.01 Da, 7697-37-2, AR) were purchased from Tianjin Damao Chemical Reagent Co. Ltd. Furfuralcohol (Mw: 98.10 Da, 98-00-0, 99%), Ammonium ceric nitrate

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(Mw: 548.22 Da, 16774-21-3, 99.99%), Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mw: 950 Da, 26915-72-0), acrylic acid (AA, Mw: 72.06 Da, 79-10-7, 99.7%) were purchased from

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the Aladdin Industrial Co., Ltd. (Shanghai, China), cis-platinum (CDDP) was supplied 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 characterization was listed in the ESI. 2.2 Synthesis of CNTs-OH CNTs-OH was synthesized through the D-A reaction in DMSO solution containing CNTs and furfuralcohol. In brief, 300 mg CNTs were dispersed in 30 mL DMSO solution and treated with ultrasonic-processing for 10 min. Then, 500 mg furfuralcohol was dissolved in solution and the mixed solution was stirred in 40 ℃ 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 technology. Then, we obtain the dry CNTs-OH. 2.3 Preparation of CNTs-OH-PAA@PEGMA 5

Journal Pre-proof 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

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h and water was removed through vacuum drying. Finally, pure CNTs-OH-PAA@PEGMA composites

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were obtained.

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2.4 Loading and release behavior of CDDP

Plentiful carboxyl groups were introduced on the surface of CNTs through the redox polymerization

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that make CNTs-OH-PAA@PEGMA composites great potential for highly efficient drug loading and

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pH-controlled release behavior. The ability for the loading and release of CDDP on CNTs-OH-PAA@PEGMA was evaluated. First, 10 mg CDDP and 20 mg CNTs-OH-PAA@PEGMA

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were dispersed in phosphate buffer saline (PBS, 100 mL, pH = 7.4) by ultrasonic treatment. The mixed solution was kept in dark environment with electromagnetic stirring at 37 ℃ for 48 h. After that, black

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products (CNTs-OH-PAA@PEGMA@CDDP complexes) were separated by centrifugal separation and the centrifugal liquid protected from light. The concentration of dissociative CDDP existed in the centrifugal liquid was measured by UV–Vis spectrometer at 705 nm and the CDDP loading capability of

CNTs-OH-PAA@PEGMA

was

calculated.

Afterwards,

the

preparative

CNTs-OH-PAA@PEGMA@CDDP complexes were dialyzed at PBS (100 mL, pH = 7.4 and 5.2) for 48 h without light and 1 mL dialysate was sampled with time for measuring by UV–Vis spectroscopy. 2.5 Cytotoxicity evaluation Biocompatibility is crucial for the functional composite applied in biomedical field. In this work, MTT assay was adopted to evaluate the 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 6

Journal Pre-proof 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 experiment. the experiment was repeated for three times and the cell viability or standard deviation was calculated by absorbance.

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3 Results and discussion

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The successful surface modification of CNTs through the combination of D-A reaction and

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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 CDCl3. The

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peak with chemical shifts at 3.61 ppm can be attributed to the hydrogen from the polyethylene glycol,

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which demonstrated the PEGMA have been successfully grafted in the surface of CNTs. Besides, the two peaks showed at 1.22 and 0.81 ppm, which resulted from the hydrogen on the alkane backbone

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such as methylene and methyl. The results authenticated the successful surface modification of CNTs

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through surface-initiated redox polymerization based on Ce(IV)/HNO3 redox system.

Fig. 1 The 1H NMR spectrum of CNTs-OH-PAA@PEGMA composites in CDCl3.

In

order

to

confirm

the

successful

7

synthesis

of

CNTs-OH-PAA@PEGMA,

Journal Pre-proof Fourier-transform infrared (FT-IR) spectroscopy was adopted to characterize the functional groups and chemical information. The FT-IR spectra of CNTs, CNTs-OH and CNTs-OH-PAA@PEGMA were shown in Fig. 2. Apparently, no significant infrared absorption peak appeared in the FT-IR spectra of pristine CNTs, which illuminated that pristine CNTs are lack of functional groups. On the other hand, the O-H stretching vibration was found in 3446 cm-1 and C=C stretching vibration 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 of CNTs by the D-A reaction. Besides, the successful synthesis of CNTs-OH-PAA@PEGMA composites was evaluated by FT-IR spectra. As shown in Fig. 2, a series of

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characteristic infrared absorption peaks were also found in the sample of CNTs-OH-PAA@PEGMA,

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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

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vibration of C=O. A series of peaks from 1465 to 1269 cm-1 could be ascribed to the asymmetric

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stretching vibration and in-plane bending vibration of alkyl. Besides, the signal of 1099 cm-1 arise from

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the stretching vibration of -O- and the signal of 947 cm-1 could be attributed to the out-of-plane bending vibration of C-H. The -OH and C=O infrared absorption peaks were resulting from the success of AA

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grafted on CNTs-OH-PAA@PEGMA and -O- signal was found on account of the successful polymerization of PEGMA. The above results implied that CNTs-OH-PAA@PEGMA composites have

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been successfully synthesized via the Ce(IV)/HNO3 redox polymerization.

Fig. 2 The FT-IR spectra of unadorned CNTs, CNTs-OH and CNTs-OH-PAA@PEGMA samples.

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Journal Pre-proof 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 indicated 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 ℃. The weight loss should be attributed to the introduction of furfuralcohol, 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 ℃ should be ascribed to the decomposition of polyacrylic acid. Besides, the weight loss of

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CNTs-OH-PAA@PEGMA rapidly increased to 35.5% at 450 ℃, which should be attributed to the

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disintegration of polyethylene glycol. Compared with the curve of CNTs-OH, the weight loss occurred

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at 500-700 ℃ 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

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calculated to be 54.5%. The obvious weight loss could be attributed to the decomposition of

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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].

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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

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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 1s and the O 1s. 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 percentage in CNTs-OH should be ascribed to the introduction of furan ring and hydroxyl groups. For the CNTs-OH-PAA@PEGMA, it could be attributed to the successful introduction of copolymers by redox polymerization. The results also confirmed the successful preparation of CNTs-OH-PAA@PEGMA. Besides, the C 1s (Fig. 3C) and O 1s (Fig. 3D) deconvoluted scan exhibited respectively four representative domains and two representative domains. The C 1s deconvoluted scan could be ascribed to C-C/C=C (284.4 eV), C-O- (285.35 eV), C=O (286.55 eV) and C=O-O (288.5 eV), respectively. Moreover, the O 1s deconvoluted scan could be attributed to C=O (531.7 eV) and C-O (532.9 eV). All mentioned above could further confirm the successful preparation of CNTs-OH-PAA@PEGMA. 9

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Fig. 3 (A) TGA curves of CNTs-OH-PAA@PEGMA, (B) the survey XPS spectra of

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CNTs-OH-PAA@PEGMA, (C) the region of deconvoluted scan C 1s, (D) the region of deconvoluted

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scan O 1s.

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Table 1 The element contents of CNTs, CNTs-OH and CNTs-OH-PAA@PEGMA.

CNTs CNTs-OH

O (%)

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C (%)

CNTs-OH-PAA@PEGMA

99.3

0.7

97.9

2.1

86.6

13.4

Based on the representation results above, the successful 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

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Journal Pre-proof CNTs, the CNTs-OH-PAA@PEGMA composites show different 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 displayed in Fig. S1. The result showed the particle size of CNTs-OH-PAA@PEGMA has a wide distribution and the most particle sizes are

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distributed around 800 nm with PDI of 0.260.

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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.

Great water dispersibility is a pre-requisite for application in biomedical 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

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Journal Pre-proof deposited even more than 12 hours. 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. Moreover, 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 interaction 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

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combination of D-A reaction and redox polymerization. The great improvement of water dispersibility

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was also found. On the other hand, owing to the introduction of carboxyl groups, the

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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 sections, the loading of

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CDDP on CNTs-OH-PAA@PEGMA composites and the drug release behavior was investigated. The

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biocompatibility of CNTs-OH-PAA@PEGMA composites and the therapeutic effects of the

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CNTs-OH-PAA@PEGMA-drug complexes were also evaluated.

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.

It has been reported that CDDP can be captured by carboxylic groups via substitution of the chloride ion ligands, which can be designed 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 12

Journal Pre-proof CDDP was released from the CNTs-OH-PAA@PEGMA at different pH solutions (pH = 7.4 and 5.2). The whole loading and release process must be maintained in dark environment. As illustrated in Fig. 6, in the first 12 hours, 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@PEGMA@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

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CNTs-OH-PAA@PEGMA@CDDP, it can be ascribed to the protonation of carboxylic groups at

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the acidic pH, which weakens the drug and nanoparticle coupling and makes the drug more

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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

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in lower pH solution is much higher than in high pH solution. In the acid solution (pH = 5.2), the ratio

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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

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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

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composites, the drug will release fast in intracellular after cell internalization. Therefore, the CNTs-OH-PAA@PEGMA composites could be potentially used as carriers for targeted drug delivery and reduce the side effects of drug to normal cells and tissues.

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Journal Pre-proof Fig. 6 The release behavior of CDDP from CNTs-OH-PAA@PEGMA complexes at pH values of 5.2 and 7.4.

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

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cytotoxicity and good biocompatibility. Our previous reports [56-58] demonstrated that the CNTs could

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be internalized by tumor cells and exhibit strong toxicity towards different types of cells and the

this

work,

we

demonstrated

that

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surface properties of CNTs will also influence the final toxic results of CNTs and related materials. In CNTs-OH-PAA@PEGMA composites possess excellent

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biocompatibility, which is possibly attributed to the surface functionalization with the hydrophilic and

CNTs-OH-PAA@PEGMA@CDDP

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biocompatible copolymers, which could mask the toxicity of CNTs. For the cell viability of complexes,

we

can

discover

the

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CNTs-OH-PAA@PEGMA@CDDP complexes into cells and the cell viability was decreased with the increasing of concentration of CNTs-OH-PAA@PEGMA@CDDP complexes. The cell viability

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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.

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7 The

viability of

HepG2

cells after

treatment

with different concentration of

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Fig.

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CNTs-OH-PAA@PEGMA, CNTs-OH-PAA@PEGMA@CDDP and pure CDDP for 24 h.

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4 Conclusions

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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

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polymeric composites has been confirmed by a variety of characterization methods. Some necessary properties of biomaterials also have been measured and demonstrated. Firstly, essential

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biocompatibility and low cytotoxicity have been measured by MTT assay and the cell viability keep above 90%. Besides, owing to the introduction of hydrophilic 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 demonstrated 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. Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 21788102, 21865016, 51363016, 21474057, 21564006, 21561022, 21644014).

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Journal Pre-proof Conceptualization: Xiaoyong Zhang Methodology: Ziyang He, Ruming Jiang, Wei Long, Hongye Huang Resources: Hongye Huang, Junyu Chen, Fengjie Deng Writing - Original Draft: Ziyang He, Ruming Jiang Writing - Review & Editing: Xiaoyong Zhang, Naigen Zhou, Yen Wei Supervision: Xiaoyong Zhang, Naigen Zhou, Yen Wei

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Funding acquisition: Xiaoyong Zhang, Naigen Zhou, Yen Wei, Meiying Liu, Fengjie Deng

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Journal Pre-proof Declaration of competing interest

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The authors declared that there is no conflict of interest

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Graphical abstract

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Journal Pre-proof Highlights ► The CNTs were surface functionalized with copolymers ► The Diels-Alder reaction and redox polymerization is useful for surface modification of CNTs ► The CNTs-OH-PAA@PEGMA composites show excellent physicochemical properties

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► The potential drug delivery applications of CNTs-OH-PAA@PEGMA composites were examined

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