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Graphene Nanoparticles-Based Self-Healing Hydrogel in Preventing Post-Operative Recurrence of Breast Cancer JinFeng Liao, Qiwen Li, Junru Wen, Chenlu Liu, Yanpeng Jia, Yongzhi Wu, Yue Shan, and Zhiyong Qian ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01475 • Publication Date (Web): 01 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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ACS Biomaterials Science & Engineering

Graphene Nanoparticles-Based Self-Healing Hydrogel in Preventing Post-Operative Recurrence of Breast Cancer Qiwen Li†§, Junru Wen†§, Chenlu Liu†, Yanpeng Jia‡, Yongzhi Wu†, Yue Shan†, Zhiyong Qian‡, Jinfeng Liao*,† † State

Key Laboratory of Oral Diseases, National Clinical Research Centre for Oral

Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China. ‡ State

Key Laboratory of Biotherapy/Collaborative Innovation Centre for Biotherapy,

West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.

Qiwen Li and Junru Wen contributed equally to this work.

§

*Corresponding

author:

Dr. Jinfeng Liao, State Key Laboratory of Oral Diseases, National Clinical Research Centre for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China. E-mail: [email protected]

ABSTRACT Hydrogel is an ideal scaffold in fields of regenerative medicine and tumor therapy owing to its biomimetic ability to modulate tissue microenvironment. Herein, we fabricated a new kind of self-healing hydrogel based on graphene nanoparticle and expanded its application in post-operative recurrence of breast cancer. Firstly, a facile method was used to prepare self-healing hydrogel via Schiff-base linkage, which composed of chondroitin sulfate multi-aldehyde (CSMA), branched polyethylenimine (BPEI) and BPEI conjugated graphene (BPEI-GO). BPEI-GO was doped in the network and participated in Schiff-base reaction and stabilized the structure, as well as provided sustained drug delivery, and near-infrared laser (NIR)-triggered

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photothermal effect. The hydrogels exhibited excellent self-healing (~100%) and improved mechanical properties (7,000 Pa). Further, in vitro breast cancer cell inhibition study showed enhanced cell killing efficiency with synergistic chemo-photothermal therapy. In the breast cancer post-operative recurrence prevention mice model, we found that combination of Doxorubicin (DOX) and photothermal therapy in CSMA/BPEI/BPEI-GO hydrogels group reduced tumor recurrence to 33.3%, compared with 66.7% for DOX-loaded hydrogels without NIR irradiation, 66.7% for local administration of free DOX, 100% for hydrogels with NIR irradiation, blank hydrogels and blank control. This study suggests the great potential of CSMA/BPEI/BPEI-GO hydrogels for postoperative recurrence prevention of breast cancer.

Key words: Hydrogels; Self-healing; Nanoparticles; Graphene Oxide; Cancer Recurrence


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1. INTRODUCTION The prevention of post-operative recurrence of the breast cancer is critical to patients’ long-term relief.1 Conventional adjuvant chemotherapy, endocrine therapy and radiotherapy are routinely utilized in clinic to reduce the tumor relapse risk, but they bring about high toxicity and systemic side effects.2,3 As breast cancer recurrence often starts locoregionally, recent study focuses on the localized and site-specific treatment strategy at the early stage of recurrence. To achieve this goal, one of the approaches is adopting scaffolds (e.g. hydrogels, spinning membranes, etc.) loaded with functional molecules into the tumor site.4,5 Hydrogel is an ideal candidate in the fields of tumor therapy.6,7 However, hydrogel is susceptible to external stimuli when used in sophisticated in vivo environment. The integrity of hydrogel structure can be easily disrupted, consequently resulting in the impairment of functional treatment effect, for instance, sustained drug release and stimuli-responsiveness.6,7 Hence, hydrogel able to resist damage or recover from damage to achieve its long-term function is expected for tumor therapy compared with conventional hydrogels or other materials like spinning membranes. Self-healing hydrogel is defined as the hydrogel with intrinsic ability to recover to its initial set of properties once exposed to damaging factors. The design of it adopted the idea of dynamic chemistry, in which the mobile phase generated by dynamic and reversible dissociation and recombination of bonding confers the self-healing ability. Various design strategies have emerged, which can be divided into two categories: the non-covalent bonding (e.g. hydrophobic interaction, hydrogen bonding, host-guest interactions) and the dynamic covalent bonding (e.g. imine bonding, boronate ester bonding, disulfide bonding).8-10 Imine bonding, also known as Schiff base linkage, is the dynamic covalent interaction between amine group and aldehyde group. Compared with other strategies which required complicated reaction system, studies have shown that imine-based hydrogels could form in a facile way.11-13 Various natural polymers can be easily tailored with amine and aldehyde groups and reaction can occur in physiologic environment, making the hydrogels biocompatible.12,14,15 Chondroitin sulfate (CS) is an ideal natural polymer for construction of self-healing



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hydrogels. Several studies have successfully modified it with aldehyde groups using periodate, which breaks down the vicinal hydroxyls of the glucuronic acid ring of CS to form chondroitin sulfate multi-aldehyde (CSMA).16 CSMA-based hydrogels have been utilized for cell encapsulation and cartilage regeneration.12,17 However, its application in tumor therapy has not been explored. Furthermore,

the

introduction

of

nanoparticles

such

as

carbon-based

nanoparticles into self-healing hydrogels have been shown to improve mechanical property.18 Thus, in our study, we aimed to apply functional nanoparticles as an additional component to participate in the Schiff-base reaction, in order to improve the hydrogel performance. Graphene oxide (GO) is one of the outstanding candidates to improve mechanical strength as well as enhance tumor therapy. It has remarkable properties

such

as

good

colloidal

stability,

surface

modifiability

and

biocompatibility.19-22 Specifically, GO displays near infrared (NIR) absorption, which converts absorbed light into heat. Numerous studies have successfully harnessed this photothermal effect into cancer therapy without apparent toxicity to normal tissue.19-22 GO is also an ideal candidate for drug delivery, considering its large surface area and facile functionalization.20 Therefore, chemo-phototherapy, which combines heat and anti-cancer drugs, shows great potential in future clinical application and is now under intensive investigation for tumor therapy. However, GO nanoparticles lack stability in vivo. They disperse well in solutions but are prone to aggregate when exposed to physiological environment. Surface modification is a way to improve stability. Here we try to modify GO with amine terminated polymer branched polyethylenimine (BPEI). We speculate that BPEI can break GO into smaller nanoparticles (BPEI-GO) with better aqueous dispersion, as well as help GO participate in hydrogel formation via Schiff base reaction by grafting enough amine groups on surface. As far as we know, several studies have developed GO-based “tough” self-healing hydrogels via non-covalent bonding (e.g. hydrophobic interactions).23-25 Incorporating GO nanoparticles into hydrogels via dynamic covalent bonding and with self-healing ability for expanded application in tumor therapy has not been explored yet.


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In this work, we integrated BPEI-GO with CSMA and BPEI to develop a self-healing hydrogel and explored its potential to prevent recurrence of locoregional breast cancer with combined chemo-photothermal therapy. The structural analysis, rheological characterization, drug delivery, photothermal effect and biocompatibility of the hydrogels were systemically studied. In vitro inhibition of cancer cells and in vivo therapeutic application as anti-tumor scaffold were exploited.

2. MATERIALS AND METHODS 2.1 Materials Chondroitin sulfate A sodium salt (mixture of isomers, main component: chondroitin 4-sulfate, 499.38 Da) and branched polyethylenimine (BPEI, 1.8 kDa) were

purchased

from

Aladdin

(Shanghai,

1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide

China).

hydrochloride

Graphite, (EDC)

and

N-Hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Periodate (NaIO4), sulphuric acid (H2SO4), phosphoric acid (H3PO4), potassium permanganate (KMnO4), anhydrous ethanol and anhydrous diethyl ether were purchased from Chengdu KeLong chemicals Co. Ltd (Chengdu, China). Hydrogen peroxide (H2O2, 30%) was supplied by Tianjin RuiJinTe chemicals Co. Ltd. (Tianjin, China). Doxorubicin chloride (Doxorubicin, DOX) was obtained from Zhejiang Hisun Pharmaceutical Company (Zhejiang, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from GibcoTM (United States). All the reagents were of analytical grade. 2.2 Synthetic Procedure 2.2.1. Synthesis of Chondroitin Sulfate Multiple Aldehyde (CSMA) Periodate can break the vicinal hydroxyls of the glucuronic acid ring of chondroitin sulfate (CS), leading to the formation of multiple aldehyde groups. Briefly, chondroitin sulfate (5 g) was dissolved in deionized water (100 mL), and then stoichiometric amount of sodium periodate (1.35 g) was added. The mixture was stirred in the dark for 6 h at 20 °C. Then, the resulted product was dialyzed against



deionized water for 3 days with several changes of water. Finally, lyophilize to obtain CSMA. FT-IR was used to characterize CSMA. 2.2.2. Synthesis of Graphene Oxide (GO) and BPEI-conjugated GO Graphene oxide was prepared according to modified Hummers method with some modification.26 Briefly, a mixture of 0.3 g of graphite powder and 1.8 g of KMnO4 was added to 40 mL of concentrated H2SO4/H3PO4 (9:1) in a 250 mL flask. The mixture was then stirred for 12 h at 50 °C. Afterward, the mixture was poured down onto ice. 0.3 mL H2O2 was added dropwise and reacted for 30 min with stirring. For purification, the mixture was centrifuged at 8,000 rpm for 30 min and the supernatant was decanted away. The precipitate was washed with deionized water, 0.2 M HCl, and ethyl alcohol in sequence for 3 times, and anhydrous diethyl ether. The GO was vacuum dried at 37 °C. Conjugating amino groups of BPEI to carboxyl groups of GO was achieved via EDC/NHS coupling. Before conjugation, the amount of carboxyl groups of GO was measured by acid-base titration. To conjugate BPEI (Mw = 1.8 kDa) to GO, 20 mg dried GO was dispersed in phosphate buffered saline (PBS, pH = 7.4) solution by sonication for 1 h. Stoichiometric amount of EDC (85.8 mg) and NHS (102 mg) were added to the dispersion. After sonication for 15 min, BPEI solution (200 mg) was added to the GO dispersion. The mixture was sonicated in ice water for 2 h and stirred overnight at room temperature. The resulting BPEI-GO dispersion was washed several times by centrifugal ultrafiltration (10 kDa, 3,000 rpm) to remove the unreacted BPEI. The UV-vis absorption curves of GO, BPEI-GO were measured by UV-vis spectrophotometer (UV-2600, SHIMADZU, Japan). Samples were diluted to 0.05 mg/mL for analysis. The particle sizes of GO and BPEI-GO were measured by a Zetasizer Nano-ZS from Malvern Instruments (Zetasizer nano ZSP, Malven, England). Samples were diluted to 0.05-0.1 mg/mL for measurement three times at 25 °C. Atomic force microscopy (AFM) of GO and BPEI-GO was conducted on a SPA-400 microscope (Seiko Instruments Inc., Japan) operating in the tapping mode with standard silicon nitride tips. 2.2.3. Preparation and Characterization of Hydrogels


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The desired amount of CSMA was dissolved in deionized water to get pre-set concentration of 30 wt%. BPEI was diluted with deionized water and adjusted pH to 7.4 to get a final concentration of 10, 20, 30, 40 or 60 wt%. BPEI-GO was dispersed in deionized water to 0.2, 0.4 or 0.6 wt%. The hydrogels were instantaneously formed upon mixing the three components (300 μL CSMA: 150 μL BPEI: 50 μL BPEI-GO) (Table S1, ESI). The Fourier transform infrared (FT-IR) spectra were performed using Nicolet 6700 spectrophotometer (Nicolet, Thermo Scientific, Waltham, MA, USA). Dried samples were completely ground with KBr and compressed into films under vacuum for measurement. For scanning electron microscopy, the prepared hydrogels were frozen in liquid nitrogen and lyophilized (SCIENTZ-10N, NINGBO SCIENTZ BIOTECHNOLOGY Co., Ltd., China) for 2 days. Hydrogels were then transversely cut off with a blade. A JEOL SEM (JSM-5900LV, JEOL, Tokyo, Japan) was used to observe the cross-sectional hydrogel after gold was sputtered on them. Rheological tests were carried out on HAAKE RheoStress 6000 (Thermo Scientific, United States). The storage modulus (G’) and loss modulus (G’’) were recorded in response to dynamic frequency sweep and dynamic strain sweep test. Continuous strain sweep test was used to characterize the self-healing behavior of hydrogels. Briefly, 1) a small amplitude of shear strain (1% strain, frequency = 1.0 Hz) was applied and the hydrogels remained in gel state (G’ > G”); 2) a large amplitude of shear strain (400% strain, frequency = 1.0 Hz, 30 s) was implemented, leading to gel fracture (G” < G’), and 3) returned to the initial state (1% strain, frequency = 1.0 Hz, 200 s). This circulating procedure was repeated several times. 2.4 Photothermal Effect BPEI-GO dispersions with different concentrations (0.2, 0.4, 0.6 wt %), CSMA/BPEI/BPEI-GO hydrogels (0.6 wt % BPEI-GO) and CSMA/BPEI hydrogels were irradiated with an 808 nm NIR laser at a power density of 2.5 W·cm-2 for 6 min. This process was simultaneously recorded by a Fluke Ti32 Infrared (IR) thermal camera (Infrared Cameras, Fluke, Avery, WA, USA). 2.5 In vitro Drug Release Behavior



Drug loading was achieved by simply mixing desired amount of DOX with BPEI-GO overnight. The unbound DOX was removed by centrifugal ultrafiltration (10 kDa, 3,000 rpm). The BPEI-GO/DOX complex was re-suspended. The amount of DOX loading onto BPEI-GO was measured by UV-vis (Y = 0.01266X - 0.02562; X: DOX concentration, mg/mL; Y: Abs). The release profile of DOX-loaded BPEI-GO dispersion, CSMA/BPEI hydrogels and CSMA/BPEI/BPEI-GO hydrogels were measured with dialysis method. Samples were placed into dialysis bags (molecular weight cut off = 8,000-14,000 Da) and immersed in release media which composed of 30 mL PBS (PH = 6.5, 7.4, 10.0) with shaking at 80 rpm at 37 °C. At predetermined time intervals, the buffer was removed and replaced with fresh PBS. 2.6 In vitro Cytotoxicity Assay of CSMA/BPEI/BPEI-GO Hydrogel. The cytotoxicity assay of CSMA/BPEI/BPEI-GO hydrogel was measured with 3T3 cells and human periodontal ligament cells (hPDLCs). The hydrogel (100 μL) leachate was extracted using DMEM (1 mL) for 24 h and sequentially diluted to different concentrations. Cells were seeded in 96-well plates and incubated for 2.5 h. Then, 100 μL hydrogel leachates with varied concentrations (0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/mL, n=6) were added. At predetermined time, 20 μL of MTT (5 mg/mL) was added to each well and the cells were further incubated for 4 h. The formed formazan was dissolved in 150 μL DMSO for each well and the absorbance was measured at 570 nm. 2.7 In vitro Inhibition of Breast Cancer Cell MCF-7 cells (human breast adenocarcinoma cell line) were incubated on Transwell (Corning Incorporated, USA) plate for adhesion. After 24 h, hydrogels were put into upper chamber of the plate and co-cultured with hydrogels without DOX, free DOX, DOX-loaded hydrogels (hydrogels-DOX) without NIR laser irradiation (2.5 W·cm-2 for 5 min/day), hydrogels without DOX and hydrogels-DOX with NIR laser irradiation. The killing efficiency of hydrogel in tumor cell was tested with MTT. 2.8 In vivo Inhibition of Breast Cancer Recurrence All animal procedures were carried out under the guidance of State Key laboratory of Oral Diseases at Sichuan University, China. Balb/c female mice (4-6 weeks old)


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were purchased from Beijing HFK Bioscience Co. Ltd, Beijing, China. Mice were randomly divided into 6 groups. After the mice grow to eight weeks, they were injected with 1.0×106 4T1 cells in the right thoracic mammary fat pad. When the tumor grew to 200 mm3, it was removed but left with a layer of surrounding skin to allow the post-operative recurrence with follow-up. Mice were then administered with A) normal saline (200 μL), B) free DOX (5mg/kg, 200 μL), C&D) CSMA/BPEI/BPEI-GO hydrogels (80mm3) without DOX, E&F) DOX-loaded CSMA/BPEI/BPEI-GO hydrogels (5mg/kg). Group D and Group F were subjected to NIR laser irradiation once (2.5W cm-2, 5 min) 24 h post-operation. Body weight and tumor recurrence were monitored every two days. In conformity with animal ethics, mice were sacrificed when tumors reached 14-16 mm. When treatment finished, the heart, lung, liver, spleen and kidney of all groups were collected for hematoxylin and eosin (H&E) histopathological analysis. 2.9 Statistical Analysis. Mean values and standard deviations were calculated for the drug release, cytotoxicity, tumor cell inhibition and mice body weight. Kaplan-Meier survival curve of mice was plotted using Origin 2015 (OriginLab Corp., Massachusetts, USA). Statistical analysis was performed using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA) and differences were considered significant if P values < 0.05.

3. RESULTS AND DISCUSSION 3.1 Synthesis of CSMA The CSMA/BPEI/BPEI-GO self-healing hydrogel was prepared by mixing of CSMA, BPEI and BPEI-GO at room temperature, as is shown in schematics of hydrogel preparation (Figure 1 & 2). In this system, aldehyde modified biopolymers chondroitin sulfate multi-aldehyde (CSMA) functioned as the backbone of the network, which actively crosslinked with amine terminated polymers branched polyethylenimine (BPEI). Meanwhile, BPEI conjugated graphene (BPEI-GO) participated in the cross-linking to stabilize the network.



Specifically, chondroitin sulfate (CS), a subset of glycosaminoglycan enriched in extracellular matrix, was chosen as the backbone material in our hydrogel for its good hydrophilicity, biocompatibility and biodegradability. To facilitate its usage, sodium periodate was used to oxidize CS.12 The FT-IR spectra of CSMA (Figure S1, Supporting Information) showed a peak appeared at ~1745 cm-1, which denoted the aldehyde symmetric vibration. This peak was inconspicuous because of the formation of hemiacetals between aldehyde group and unoxidized hydroxyl group.

Figure 1. Schematic illustration of (A) conjugation of BPEI to graphene oxide (GO), (B) oxidation of chondroitin sulfate (CS), and (C) formation of CSMA/ BPEI /BPEI-GO hydrogels.


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Figure 2. Schematic illustration of CSMA/BPEI/BPEI hydrogel and its application in prevention of locoregional recurrence of breast cancer.

3.2 Synthesis of BPEI-GO Graphene oxide was synthesized by previous method with minor modification,26 and subsequently conjugated with BPEI (Mw = 1.8 kDa) by EDC/NHS coupling.27 After GO modification with BPEI, the color changed from yellow-brown to black. UV-vis test showed that the peaks of GO were at ~230 nm and ~300 nm, indicating π → π* transition of C=C band and n → π* transition of C=O band. When BPEI was grafted to GO, the absorption peaks red-shifted from ~230 nm to ~260 nm, indicating the restoration of electronic conjugation (Figure 3A). The mean zeta potential also increased from negative (-30.8 mV) to positive (+20.0 mV) after BPEI conjugation. Meanwhile, the peak of n → π* transition of C=O band weakened. Additionally,



FT-IR spectra showed the appearance of amide band (1630-1695 cm-1) and the absence of carboxylic group (~1726 cm-1) and epoxide group (~1050 cm-1) in BPEI-GO (Figure 3B). The morphology of GO (Figure 3C) and BPEI-GO (Figure 3D) was characterized by atomic force microscopy. It was clearly seen that wrinkled paper-like GO was broken into smaller pieces after BPEI conjugation. The thickness of GO also increased from ~ 1 nm to ~1.5 nm after BPEI modification (Figure S2, Supporting Information) due to the attachment of BPEI on different carboxylic groups on single of other GO sheet. Accurate size distributions of BPEI-GO and GO were measured by nano-sizer instrument. The results showed the average diameter of GO and BPEI-GO was 1101.5 nm (PDI = 0.339) and 133.8 nm (PDI = 0.194), respectively (Figure 3E).

Figure 3. (A) UV-vis absorption spectra of GO and BPEI-GO aqueous dispersions. (B) FT-IR spectra of GO and BPEI-GO. (C) AFM image of GO, inset: the photo of GO dispersion (2 mg/mL). (D) AFM image of BPEI-GO, inset: the photo of BPEI-GO dispersion (2 mg/mL). (E) The size distribution curves of GO and BPEI-GO.

3.3 Characterization of Hydrogel The CSMA/BPEI/BPEI-GO self-healing hydrogel formed within 30 seconds upon mixing the three ingredients at room temperature. To uncover the critical role of BPEI-GO nanoparticles in this system, hydrogel without BPEI-GO was prepared


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(CSMA/BPEI hydrogel) as comparison. FT-IR spectra showed the formation of imine bond in both hydrogels (Figure S3, Supporting Information). The morphology of the hydrogel was characterized with scanning electron microscope (Figure 4). Both hydrogels exhibited three-dimensional porous structure. The average pore size of CSMA/BPEI/BPEI-GO hydrogel (~13.85 μm) was smaller than that of CSMA/BPEI hydrogel (~22.57 μm). We ascribed this difference to BPEI-GO nanoparticles that covalently bond with CSMA backbone. The more cross-linkage forms, the smaller pore size is.28

Figure 4. The cross-sectional SEM images of (A) CSMA/BPEI hydrogel (30 wt % CSMA, 30 wt % BPEI), and (B) CSMA/BPEI/BPEI-GO hydrogel (30 wt % CSMA, 30 wt % BPEI, 0.6 wt % BPEI-GO). Scale bar: 100 μm. (C) Dynamic frequency sweep test of CSMA/BPEI hydrogels with 300 mg/mL CSMA and different BPEI concentration (100, 200, 300, 400 and 600 mg/mL); (D) Dynamic frequency sweep test of CSMA/BPEI/BPEI-GO hydrogels with 300 mg/mL CSMA, 300 mg/mL BPEI and 2, 4, 6 mg/mL BPEI-GO. (E & F) Strain amplitude sweep test of CSMA/BPEI hydrogels and CSMA/BPEI/BPEI-GO hydrogels (6 mg/mL BPEI-GO) respectively.

The mechanical property affects hydrogel applied range. The rheological test was performed. Storage modulus (G’) of CSMA/BPEI hydrogel exhibited a concentration dependent behavior within the range of 10-60 wt % BPEI and 20-30 wt % CSMA in dynamic frequency sweep test, with a maximum G’ of ~ 1,700 Pa. Introducing BPEI-GO (0.2-0.6 wt %) into the system significantly improved the storage modulus,



with a maximum G’ of ~ 7,000 Pa (Figure 4C & 4D). The elastic response of the CSMA/BPEI hydrogels and CSMA/BPEI/BPEI-GO hydrogels were also measured by strain amplitude sweep test (Figure 4E & 4F). The G’ and G’’ curve intersects at the strain of ~ 200% both for hydrogels with or without BPEI-GO. As the strain further increased, the G’ decreased dramatically, indicating the collapse of the network. In the network, the imine bond disassociated and regenerated autonomously and continuously, reaching a dynamic equilibrium that enables the self-healing property. To test the self-healing ability of the hydrogel, several blocks of CSMA/BPEI hydrogels were put side by side without further intervention (Figure 5A). After 10 min, the hydrogels merged into a single piece, which could be lifted by a tweezer and withstood its own weight. As BPEI-GO participated into the dynamic reaction of hydrogel, it remained to concern if introduction of BPEI-GO compromised the flowability and self-healing property of hydrogels. To test it, the same experiment was performed on CSMA/BPEI/BPEI-GO hydrogels (Figure 5B). After 10 min, the hydrogels also merged into a single piece and was lifted by a tweezer. Moreover, after 2-hour healing, the interface of different blocks of the hydrogels fused together and the hydrogels can be further stretched. Besides, we tested if CSMA/BPEI/BPEI-GO hydrogel can be injected through a syringe and self-heal (Figure S4, Supporting Information). The hydrogel was squeezed through the syringe and quickly fused together and could be lifted within 1 minute. After autonomous healing for 10 min, the rough surface of the hydrogel turned smooth, with the disappearance of minor sags and crests. Furthermore, continuous strain sweep test was conducted (Figure 5C&D). Both hydrogels stayed in the gel state when subjected to 1% strain (frequency = 1.0 Hz, time = 200 s). When a large amplitude oscillatory force of 400% strain (frequency = 1.0 Hz, time = 30 s) was implemented, storage modulus of hydrogels dropped sharply (G’ < G”), indicating the collapse of the network. Subsequent reversal of amplitude oscillatory force strain to 1% led to G’ quick recovery, demonstrating the restoration of the network. By comparing the G’ value, the healing ratio of CMSA/BPEI hydrogel was calculated to be ~78.4% after the initial cycle. It is worth noting that the healing ratio of CSMA/BPEI/BPEI-GO hydrogel was ~100%


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even after 3 cycles. This effect can be explained as the stabilizing role of BPEI-GO in hydrogel. Due to the fluidity of BPEI,29,30 it failed to assist in network recovery once the network collapsed. However, BPEI-GO stabilized the structure by providing accessible bonding sites for rapid gel formation.

Figure 5. (A) Self-healing photos of CSMA/BPEI hydrogels. Rhodamine B and trypan blue were

added

for

distinguishable

observation.

(B)

Self-healing

photos

of

CSMA/BPEI/BPEI-GO. Alcian blue and Alizarin Red were added for distinguishable observation. (C) Continuous strain sweep test of CSMA/BPEI hydrogels subjected to a small oscillatory force (1%, 200 s) and a large oscillatory force (400%, 30 s). (D) Continuous strain sweep test of CSMA/BPEI/BPEI-GO hydrogels subjected to a small oscillatory force (1%, 200 s) and a large oscillatory force (400%, 30 s).

3.4 Photothermal Effect in vitro



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After that, we explored the photothermal effect of CSMA/BPEI/BPEI-GO hydrogel. Grafting BPEI to GO partially restored the disrupted π conjugation of GO, resulting in the increase of optical absorption in near-infrared (NIR) region.31 Figure 6A showed the temperature changes of BPEI-GO dispersion under NIR laser irradiation at 808 nm with 2.5 W·cm-2 for 6 min. Along with the increased concentrations (2, 4, 6 mg/mL), the temperature increased to 55.5 °C, 59.5 °C and 65.4 °C respectively. Similar result was also observed in CSMA/BPEI/BPEI-GO hydrogel (Figure 6B). The temperature of CSMA/BPEI/BPEI-GO hydrogel increased to 50.9 °C, while no obvious changes were observed on CSMA/BPEI hydrogel. The photothermal effect of BPEI-GO nanoparticles and CSMA/BPEI/BPEI-GO hydrogel still remained stable after five cycles. Previous studies have shown that cancer cells are thermo-sensitive and

cannot

survive

over

42

°C,32

herein

the

photothermal

effect

CSMA/BPEI/BPEI-GO hydrogel can be further harnessed for tumor treatment.


of

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Figure 6. Photothermal effect and drug releasing behavior of BPEI-GO dispersions and hydrogels. (A) The temperature change in BPEI-GO dispersions at different concentrations with NIR laser irradiation. (B) The temperature change of CSMA/BPEI hydrogel and CSMA/BPEI/BPEI-GO hydrogel with NIR laser irradiation (6 mg/mL BPEI-GO). (C) Near infrared imaging of CSMA/BPEI/BPEI-GO hydrogel (6 mg/mL BPEI-GO) under 2.5 W·cm-2 irradiation at 808 nm for 0 min, 2 min, 4min and 6 min. (D) DOX release profile of BPEI-GO dispersion at pH of 6.5, 7.4 and 10.0. (E) DOX release profile of BPEI-GO dispersion, CSMA/BPEI hydrogel and CSMA/BPEI/BPEI-GO hydrogel at pH of 6.5. Data are represented as mean ± SD (n = 3).



3.5 Sustainable Drug Release from Hydrogel To investigate its therapeutic application, we further explored the drug-loading and sustained drug-release behavior of CSMA/BPEI/BPEI-GO hydrogel. Doxorubicin (DOX), an aromatic drug widely used for chemotherapy, was selected as the model drug.33 We firstly studied the drug loading capacity of BPEI-GO. The result showed that the loading efficiency was 60.1%. Next, drug release from BPEI-GO was observed at different pH conditions (Figure 6D). More DOX was released from BPEI-GO at pH = 6.5 (57.05%) than at pH = 7.4 (33.89%) and at pH = 10.0 (36.73%) in 24 h. As tumor micro-environment was acidic, the increased release of DOX at lower pH showed a better acid-induced tumor killing effect. We then explored the DOX-release profile of hydrogels at pH = 6.5. As is shown in Figure 6E, CSMA/BPEI/BPEI-GO hydrogel exhibited more sustainable drug release behavior compared with CSMA/BPEI hydrogel loaded with stoichiometric free DOX, and BPEI-GO nanoparticle alone. A cumulative release of 35.05% DOX in CSMA/BPEI/BPEI-GO hydrogel was observed within 24 h, but 62.02% and 53.50% drug were respectively released from CSMA/BPEI hydrogel and BPEI-GO dispersion. After an initial burst, the release of the hydrogels remained steady in the following time. Although the initial burst release of DOX appeared in BPEI-GO dispersion and hydrogels, the results still revealed an enhanced drug sustainable release effect by combining hydrogel and nanoparticle into one system. Furthermore, we studied the effect NIR laser irradiation on drug release profile of BPEI-GO nanoparticles and CSMA/BEPI/BPEI-GO hydrogels, and no apparent difference was observed. Therefore, the CSMA/BPEI/BPEI-GO hydrogel serves as an ideal scaffold for sustained drug release.

3.6 In vitro Cancer Cell Inhibition The photothermal effect and sustainable drug release ability of the self-healing hydrogel motivated us to exploit the potential application in tumor therapy. First of all, the cytotoxicity of CSMA/BPEI/BPEI-GO hydrogel was tested. The hydrogel leachates with different concentrations incubated with primary mouse embryonic


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fibroblast (3T3 cells) (Figure 7A) and human periodontal ligament cells (Figure S5, Supporting Information).34 No obvious toxicity was observed in all ranges of leachates in 24 h. Cells cultured in 10 mg/mL leachate showed ~ 80% viability after 72 h. The result indicated that the cytotoxicity of polycationic polymer BPEI can be greatly

reduced

by

crosslinking.

Therefore,

the

hydrogel

showed

good

biocompatibility. Next, the killing efficiency of hydrogel for cancer cell was tested in vitro. Human breast adenocarcinoma cells (MCF-7 cells) were incubated for 24 h for adhesion. Then, cells were co-cultured with DOX-free hydrogels, free DOX, DOX-loaded hydrogels without NIR laser irradiation, DOX-free hydrogels and DOX-loaded hydrogels with NIR laser irradiation. Figure 7B. displayed the relative viability of MCF-7 cells after treatment. Free DOX exerted direct toxicity, with 44.58%, 5.20% and 2.92% of cell survival at day 1, 2 and 3. DOX-loaded hydrogels exhibited decreased but prolonged cytotoxicity due to the delayed drug release from hydrogel. NIR laser irradiation (2.5 W·cm-2 for 5 min/treatment/day) of DOX-free hydrogels led to 58.71%, 43.67% and 36.60% of cell survival at day 1, 2, and 3, which demonstrated the killing effect of photothermal treatment. Importantly, the synergetic chemo-photothermal therapy achieved improved killing efficiency, with 37.8%, 22.8% and 9.2% of cell survival at day 1, 2 and 3. These results demonstrate the ideal cancer cell killing effect of CSMA/BPEI/BPEI-GO in vitro, which encouraged us to further apply it to locoregional inhibition of post-operative breast cancer recurrence in vivo.



Figure 7. (A) Cytotoxicity at different concentrations (mg/mL) of CSMA/BPEI/BPEI-GO hydrogel leachates on 3T3 cells at 24 h, 48 h and 72 h. (B) Relative viability of MCF-7 cells after anti-tumor treatment after 24 h, 48 h and 72 h (2.5 W·cm-2 for 5 min per treatment, one treatment per day for photothermal irradiation). Data are represented as mean ± SD (n = 3). *P < 0.05 by two-sample student’s t-test.

3.7 In vivo Prevention of Post-operative Breast Cancer Recurrence A period of 32-day in vivo study was conducted. When the implanted tumor volume reached 200 mm3, it was removed but left with a layer of surrounding skin to allow tumor recurrence (Figure 8A). Hydrogel was implanted in situ and mice were irradiated with NIR laser for 5 min. The temperature quickly reached 44.5 °C within 1 min and peaked 53.1 °C at 2 min, and then remained stable from 2 min to 5 min. This result indicated hydrogel could respond quickly and stably to NIR laser in vivo (Figure 8B). After 32-day continuous observation, the outcome varied with different treatment strategies (Figure 8C). All six mice developed cancer in groups of control, DOX-free hydrogel and DOX-free hydrogel with laser. Unfortunately, DOX-free hydrogels with laser failed to inhibit cancer recurrence, which suggests photothermal treatment alone might not be effective enough to cure tumor and should combine with other therapeutic options. Previous study harnessing phototherapy to prevent breast cancer recurrence also yielded similar results, where phototherapy alone is less effective than chemotherapy.34 In groups of DOX-loaded hydrogels without NIR laser irradiation, and mice receiving free DOX treatment, four of six mice did re-grow cancer (66.7%) in the end. Nevertheless, in DOX-loaded hydrogel group, the cancer recurred about 7 days later than the free DOX treatment, indicating that sustainable release and a prolonged cytotoxicity were achieved by DOX loaded into hydrogels. As expected, combined chemo-photothermal treatment strategy yield better result, with only two out of six mice developing cancer recurrence (33.3%) in this group. A 32-day follow-up showed apparent rodent weight loss with free DOX treatment subcutaneously (Figure 8D). This side effect was alleviated when DOX was loaded into hydrogels.


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Figure 8. (A) Surgical process of locoregional breast cancer removal and treatment. When the tumor volume reached 200 mm3 (A1), mice were anaesthetized and the tumor was removed but left with a layer of surrounding skin (A2); hydrogel was implanted in situ (A3) and incision was sutured (A4); mice were irradiated with NIR laser at 808 nm for 5 min (2.5 W·cm-2) after 24 h (A5). (B) Near infrared imaging of mice implanted with CSMA/BPEI/BPEI-GO hydrogels. (C) Kaplan-Meier survival curve plotting breast cancer recurrence. (D) Body weight variation during treatment.

The late stage of breast cancer could couple with lung metastasis.35 At the end of follow-up, lung samples of all groups were collected and sliced for H&E staining. As Figure 9 shows, numerous tumor nodules can be observed in control and DOX-free hydrogel groups. In groups of free DOX, DOX-loaded hydrogel without laser and DOX-free hydrogel with laser, the nodules were reduced. In combined DOX and NIR laser irradiation group, no visible cancer metastasis can be detected. This result was in accordance with survival data.



Figure 9. Lung metastasis of breast cancer after the treatment. (A) control, (B) blank hydrogel, (C) Free DOX, (D) DOX-free hydrogel with laser, (E) DOX-loaded hydrogel without laser, (F) DOX-loaded hydrogel with laser.

Pathohistological analysis by H&E staining showed severe cardiotoxicity of free DOX (Figure 10C). When DOX-free hydrogel was implanted in the tumor site, no visible lesions were found in heart, liver, spleen, lung and kidney, which further demonstrated the biocompatibility of the hydrogel (Figure 10B). Combined DOX and NIR laser irradiation did not lead to systemic lesions. More importantly, compared with free DOX, DOX-loaded hydrogel displayed reduced cardiotoxicity (Figure 10D).


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Figure 10. H&E staining of heart, liver, spleen, lung and kidney. (A) control, (B) blank hydrogel, (C) free DOX, (D) DOX-loaded hydrogel with NIR laser irradiation. No visible cytotoxicity in heart, liver, spleen, lung and kidney can be observed in blank hydrogel (B). Apparent cardiotoxicity is present in free DOX treated group (C), but cardiotoxicity was greatly reduced in DOX-loaded hydrogel with NIR laser irradiation group (D).

Prevention of post-operative recurrence of breast cancer is the long-term goal in both basic and clinical study. Recent years, various drug delivery systems using hydrogels as carriers are developed. One is thermosensitive hydrogel system which responds well to body temperature or near infrared laser irradiation. Another strategy is “injectable” hydrogel system. The hydrogels are delivered in situ, not only enhancing the cure effect but also minimizing systemic side effect. For instance, one study shows paclitaxel-loaded PECE converts into gel state in body temperature and can prevent breast cancer recurrence when injected.36 Besides, pH responsive hydrogels sustainably releasing doxorubicin are reported.37 It is also reported that co-assemble of tailor-made hexapeptide and DOX can prevent recurrence in tumor



site.38 Finally, hydrogels that combine natural or synthetic polymers with nanoparticles have emerged. One study reports hydrogel prepared from chitosan (CS) functionalized GO can release DTX to inhibit tumor.39 In our study, the improved inhibition of breast cancer recurrence demonstrated that CSMA/BPEI/BPEI-GO hydrogel is a suitable scaffold for inhibition of post-operative recurrence of breast cancer through combined chemo-photothermal therapy. Moreover, added with an advanced self-healing and enhanced mechanical properties, the application of CSMA/BPEI/BPEI-GO hydrogel is more flexible, owing to its resilience to destructive factors and a more stable structure in vivo when encountered with external or internal force. Besides, hydrogels with an extensive range of mechanical strength can be obtained by tuning the amount of BPEI-GO nanoparticle, which could match with different stiffness accurately.40-42 CSMA/BPEI/BPEI-GO self-healing hydrogel could therefore be utilized for the prevention of cancer postoperative recurrence, whose self-healing and chemo-photothermal effect are believed to exhibit enhanced treatment outcomes compared with conventional hydrogels. Finally, the self-healing property of the hydrogel makes it attractive for future application in regeneration of tissues, especially those bearing mechanical strength such as bone, cartilage and muscle. However, direct evidence that self-healing precedes non-self-healing is absent in vivo. Therefore, setting up a standard to evaluate the contribution of self-healing property to the whole hydrogel system in tissue repair and tumor therapy are warranted in future study.

4. CONCLUSIONS In summary, a graphene-based self-healing hydrogel was developed via Schiff-base reaction. The CSMA/BPEI/BPEI-GO hydrogel exhibited enhanced mechanical and self-healing properties, as well as sustained drug delivery and biocompatibility. Exploitation of the hydrogel for in vitro cancer cell inhibition and in vivo prevention of post-operative breast cancer recurrence via combined chemo-photothermal therapy yielded an effective result. This novel hydrogel showed great promise in the field of biomedical application.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ASC Publications website at DOI: Table S1, Components of CSMA/BPEI/BPEI-GO self-healing hydrogels. Figure S1, FT-IR spectra of CS and CSMA. Some characteristic peaks appeared both in CS and CSMA, such as ~1630 cm-1 for amide group, ~1260 cm-1 for S=O bond, etc. Specially, a new peak at ~1745 cm-1 appeared in spectrum of CSMA, demonstrating the aldehyde symmetric vibration. Figure S2, thickness of two-dimensional GO and BPEI-GO characterized by AFM. Figure S3, FT-IR spectra of CSMA/BPEI hydrogel and CSMA/BPEI/BPEI-GO hydrogel. Figure S4, Self-healing test of CSMA/BPEI/BPEI-GO hydrogels. Figure S5, Cytotoxicity of CSMA/BPEI/BPEI-GO hydrogel leachates at different concentrations on hPDLCs cells.

ACKNOWLEDGEMENTS The authors declare no conflicts of interest. The authors thank the financial support from the National Natural Science Foundation of China (31600778) and Sichuan University (2018SCUH0029). REFERENCES (1) Kottke, T.; Boisgerault, N.; Diaz, R.; Donnelly, O.; Rommelfanger-Konkol, D.; Pulido, J.; Thompson, J.; Mukhopadhyay, D.; Kaspar, R.; Coffey, M.; Pandha, H.; Melcher, A.; Harrington, K.; Selby, P.; Vile, R. Detecting and targeting tumor relapse by its resistance to innate effectors at early recurrence. Nat. Med. 19(2013): 1625–1630. DOI: 10.1038/nm.3397. (2) Wöckel, A.; Wolters, R.; Wiegel, T.; Novopashenny, I.; Janni, W.; Kreienberg, R.; Wischnewsky, M.; Schwentner, L. The impact of adjuvant radiotherapy on the survival of primary breast cancer patients: a retrospective multicenter cohort study of 8935 subjects. Ann. Oncol. 25(2014): 628–632. DOI: 10.1093/annonc/mdt584. (3) Aebi, S.; Gelber, S.; Anderson, S.J.; Láng, I.; Robidoux, A.; Martín, M.; Nortier,



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For Table of Contents Use Only Graphene Nanoparticles-Based Self-Healing Hydrogel in Preventing Post-Operative Recurrence of Breast Cancer Qiwen Li, Junru Wen, Chenlu Liu, Yanpeng Jia, Yongzhi Wu, Yue Shan, Zhiyong Qian, Jinfeng Liao


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