Hierarchical Acceleration of Wound Healing through Intelligent

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Biological and Medical Applications of Materials and Interfaces

Hierarchical Acceleration of Wound Healing through Intelligent Nanosystem to Promote Multiple Stages Yan Cheng, Yun Chang, Yanlin Feng, Hui Jian, Xiaqing Wu, Runxiao Zheng, Li Wang, Xiaomin Ma, Keqiang Xu, Panpan Song, Yanjing Wang, and Haiyuan Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b13267 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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ACS Applied Materials & Interfaces

Hierarchical Acceleration of Wound Healing through Intelligent Nanosystem to Promote Multiple Stages Yan Cheng1, Yun Chang1, Yanlin Feng1, 2, Hui Jian1, Xiaqing Wu1, 2, Runxiao Zheng1, 2, Li Wang1, 3,

Xiaomin Ma1, 3, Keqiang Xu1, 2, Panpan Song1, 2, Yanjing Wang1, 2, and Haiyuan Zhang1, 2, *

1

Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Changchun, 130022, Jilin, China. 2

3

University of Science and Technology of China, Hefei, 230026, Anhui, China. School of Chemistry and Life Science, Changchun University of Technology, Changchun,

130022, Jilin, China. KEYWORDS: Wound healing; Antibacterial; Proliferation; Charge transfer; Intelligent nanosystem

ABSTRACT. A Wound healing is a dynamic, interactive, and complex process, including multiple stages. Although various nanomaterials are applied to accelerate the wound healing process through exhibiting antibacterial activity or promoting cell proliferation, only single stage is promoted during the process, lowering healing efficacy. It is necessary to develop programmable nanosystems for promoting multiple wound healing stages in sequence. Herein,

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arginine loaded and detachable ceria-graphene nanocomposites (ACG NCs) were designed to achieve this purpose. Ceria NPs and graphene were linked by base cleavable Nhydroxysuccinimide (NHS) ester. At inflammation stage, ACG NCs could effectively generate ROS and kill bacteria under white light irradiation due to their efficient electron-hole separation between ceria NPs and graphene. At proliferation stage, ceria NPs could be detached from ACG NCs and taken up by cells to scarify intracellular ROS and promote cell proliferation, while the separated graphene could act as a scaffold to promote fibroblast migration to wound site. A series of in vitro and in vivo assessments demonstrated that

ACG

NCs could effectively

accelerate wound healing process.

Introduction Wound healing is a dynamic, interactive, and complex process, involving hemostasis, inflammation, proliferation and remodeling stages.1-2 At inflammation stage, bacterial infection can delay the healing and cause serious tissue damages, compromising the overall health of an individual, because the bacteria can compete with the host immune system and subsequently invade viable tissue.3 At proliferation stage, fibroblast migration to and proliferation within the wound site are prerequisites for wound granulation.4 Currently, various nanomaterials (NMs) such as graphene quantum dots,5 molybdenum disulfide,6 silver,7 and copper metal–organic framework hydrogel,8 have been widely investigated to accelerate the wound healing process, however, most of them only can promote the individual stage, such as either killing bacteria or promoting cell migration and proliferation. Therefore, it will be necessary to develop programmable nanosystems for hierarchically promoting multiple wound healing stages, achieving accelerated wounding healing process.


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Ceria nanoparticles (NPs), as a semiconductor with bandgap of ~3.1 eV, have been used for bacteria inactivation under white light irradiation based on their photo-induced reactive oxygen species (ROS) oxidative attacks.9-10 However, the fast recombination of photoinduced electrons and holes can dramatically lower ROS production efficiency, as a result of a low antibacterial efficacy. Graphene can be incorporated into semiconductors to separate photo-induced electrons and holes, leading to the more efficient ROS generation.11 Therefore, the formation of ceriagraphene nanocomposites (CG NCs) potentially can enhance white light triggered antibacterial efficacy. Under white light irradiation, the electrons of ceria can be excited from valence band (VB) to conduction band (CB) and further transfer to graphene, leaving holes in VB of ceria. This efficient electron-hole spatial separation can facilitate the free electrons in graphene and free holes in ceria to react with oxygen (O2) and water (H2O), respectively, forming superoxide radical anion (O2•-) and hydroxyl radical (•OH), respectively, as a result more significant antibacterial activity of CG NCs compared with ceria NPs. Moreover, ceria NPs have been demonstrated to exhibit cell proliferation feature12 due to their cellular ROS scarifying ability originating from their superoxide dismutase (SOD) and catalase activities,13-14 while graphene has also been utilized as scaffolds for wound healing15 based on their cell adhesion and migration abilities.16-18 Thus, integrating ceria and graphene into a sophisticated nanosystem potentially can significantly accelerate the wound healing process through programmable promotion of inflammation and proliferation stages. In the present study, ceria NP-detachable graphene nanocomposites (denoted as ACG NCs) were designed to enhance the wound healing efficacy through promoting both inflammation and proliferation stages. In ACG NCs (Figure 1A), hollow ceria NPs were attached to graphene through a base-cleavable linker (N-hydroxysuccinimide (NHS) ester),19-20 and arginine (Arg)


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was loaded into hollow ceria NPs and capped with folded i-motif DNA,21 and a matrix metalloproteinase

(MMP)-

cleavable

stealth

peptide

(GGGPLGLAGGNYTCEVTELTREGETIIELK)22 was further engineered on graphene. Once applied on wound site, ACG NCs can work as follows: (1) At inflammation stage that remains an acidic pH (~6.0),23 ACG NCs are subjected to white light irradiation that has been reported to deliver less pain to skin,24-25 generating a large magnitude of ROS to get rid of bacteria due to the efficient electron-hole spatial separation between ceria and graphene (Figure 1B); (2) With the wound healing process proceeding to proliferation stage whose pH level is naturally raised to a neutral level,23 the folded i-motif DNA is allosterically changed to unfolded single-stranded DNA form,21 facilitating the pore of hollow ceria NPs opening to release Arg that can be decomposed into urea with the help of arginase overexpressed at wound site;26-27 (3) The produced urea could be further decomposed into ammonia (NH3) due to urease-mimicking activity of ceria NPs,28 which can further elevate the pH to a more basic level, leading to breakage of NHS ester and detachment of ceria NPs from graphene (Figure 1C); (4) The detached ceria NPs can be freely taken up into fibroblasts to scarify intracellular ROS and promote the cell proliferation, while the stealth peptide prevents graphene from uptake by macrophage and prolongs the retention time of graphene as a scaffold at wound site to promote fibroblast migration (Figure 1C). Finally, at remodeling stage, the high expressed MMP in extracellular matrix could cleave GPLGLAG peptide and facilitate the macrophage to internalize the graphene through endocytosis,

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leading to the biodegradation of ACG NCs in wound site.

Thus, these compact ACG NCs can programmably promote the inflammation and proliferation stages, accelerating the wound healing process. Results and discussions


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Synthesis and characterization of CG NCs. Hollow ceria NPs were prepared by silica template-assisted synthesis approach (Figure S1A).30 Silica templates with diameters of 70 ± 5.1 nm (Figure S1B) were firstly fabricated through a modified Stöber method,31 and then ceria shell were grown on silica cores (Figure S1C). After etching the silica cores using sodium hydroxide aqueous solution, hollow ceria NPs with diameters of 90 ± 6.4 nm (Figure S1D) were obtained. And graphene was prepared through Hummers method.32 CG NCs was synthesized as descripted in Figure S2. Amino groups (NH2) were introduced into both ceria NPs and graphene through modifying their surface with 3-aminopropyltriethoxysilane (APTES), as confirmed by zeta potentials and Fourier transform infrared (FTIR) spectra (Figure S3). Then, NH2 modified ceria NPs were connected with NHS ester-poly (ethylene glycol) (PEG)-carboxyl acid (COOH) through the reaction between NH2 of ceria NPs and NHS of PEG, and then fabricated with NH2 modified i-motif DNA to form ceriaDNA NPs through NHS/1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling. Finally, CG NCs were obtained by coupling the amino group of graphene with the COOH groups of both ceriaDNA NPs and MMP-cleavable stealth peptide (COOH-GGGPLGLAGGNYTCEVTELTREGETIIELK) through NHS/EDC coupling. The content of ceria in CG NCs was 90.31 % as determined by inductively coupled plasma optical emission spectrometer (ICP-OES). Transmission electron microscopy (TEM) images of CG NCs (Figure 2A) showed that hollow ceria NPs were successfully linked to graphene. The existence of ceria NPs in CG NCs was confirmed by the cubic phase fluorite structure of ceria (JCPDS 340394) and F2g vibrational mode (464 cmK ) in cubic fluorite lattice of ceria, as evidenced by Xray diffraction (XRD) patterns (Figure 2B) and Raman spectra (Figure 2C). The peaks at 1324 and 1598 cmK in Raman spectra (Figure 2C) were related to the D band and G band of graphene, confirming the existence of graphene in CG NCs. And the ultraviolet-visible (UV-Vis) spectra


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of superoxide binds to oxygen vacancy sites around Ce3+ and Ce4+ to oxidize a second molecule of Ce3+ into Ce4+ and release a second of H2O2; then, one molecule of the released H2O2 can reduce two molecule of Ce4+ into Ce3+ and generate one molecule of O2, leading to the fully reduced oxygen vacancy site returning to the initial state. For catalase mimetic activity, one molecule of H2O2 firstly reacts with Ce4+, reducing it to Ce3+ and releasing protons and O2; a second H2O2 molecule can bind to the oxygen vacancy site around two Ce3+, oxidizing the Ce3+ back to the initial Ce4+ state and releasing H2O. Antibacterial activity of CG NCs. To investigate the antibacterial activities of CG NCs, the ROS generation of CG NCs was firstly accessed by 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. With white light irradiation, CG NCs could generate more ROS than ceria NPs or graphene alone, as indicated by their more intense DCF fluorescence intensity (Figure 3A), whereas without white light irradiation, CG NCs, ceria NPs, or graphene could not generate ROS (Figure 3B). Furthermore, 2,3-bis-(2-methoxy-4-nitro-5-sulfophehyl)-2H-tetrazolium-5carboxanilide (XTT) and 3O-(p-aminophenyl) fluorescein (APF) assay were used to detect the ROS types generated by CG NCs. As shown in Figure 3C-D, under white light irradiation, CG NCs and ceria NPs could enhance the APF fluorescence, while no obvious XTT absorption enhancement was induced by all NPs, meaning that the main ROS type is the hydroxyl radical. Meanwhile, CG NCs could generate stronger APF fluorescence than ceria NPs alone, further proving the more potent ROS generation ability of CG NCs. The antibacterial activities of CG NCs, ceria and graphene NPs were examined in gram-negative E. coli and gram-positive S. aureus bacteria by investigating their growth kinetics and colony-forming capabilities. As shown in Figure 3E-G, under white light irradiation, CG NCs could more dramatically delay the growth rates and reduce the colony numbers of both types of bacteria than ceria NPs and graphene,


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E. coli

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Figure 3. Antibacterial activity of CG NCs and the underlying mechanism. (A-B) ROS generation ability of graphene (10 P mL-1), ceria NPs (90 P mL-1) ,and CG NCs (100 P mL-1) with (A) or without (B) white light irradiation; (C-D) Superoxide (C) and hydroxyl radical (D) generation ability of graphene (10 P mL-1), ceria NPs (90 P mL-1) ,and CG NCs (100 P mL-1) with white light irradiation; (E-F) The 0-6 h growth curves of E. coli (E) and S. aureus (F) incubated with graphene (50 P mL-1), ceria NPs (450 P mL-1) and CG NCs (500 P mL-1) with white light irradiation; *p < 0.05; (G) The colony-forming capability of E. coli and S. aureus incubated with graphene (50 P mL-1), ceria (450 P mL-1) and CG NCs (500 P

mL-1) for 24 h with white light irradiation. (H-J) Cellular ROS generation (H),

malondialdehyde level (I), and GSH level (J) induced by graphene (10 P mL-1), ceria NPs (90 P mL-1), and CG NCs (100 P mL-1) with white light irradiation.

showing their pronounced antibacterial activity, which originated from the excellent photoinduced electrons and holes separation ability of CG NCs. In comparison, all these NMs showed no antibacterial activity if not subjected to white light irradiation (Figure S6). In order to


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investigate the molecular mechanism of bacterial inactivation, the intracellular ROS generation was accessed by H2DCFDA assay, in which CG NCs could induce more ROS under white light irradiation than ceria NPs (Figure 3H), indicating ROS-mediated bacterial inactivation mechanism. Moreover, the increased malondialdehyde level (MDA) and decreased glutathione (GSH) level induced by CG NCs (Figure 3I-J) further prove that the bacteria are inactivated through oxidative stress injury.34 Base-responsive detachment of ceria NPs from ACG NCs. Arg was loaded into CG NCs through incubation of Arg and CG NCs in phosphate buffered saline (PBS, pH 6.0) for 6 h to form ACG NCs. The Arg content was detected through Sakaguchi reaction between guanidine group of Arg and S/

&35 The loading efficacy of Arg in ACG NCs was determined to be

211.8 µg Arg per mg CG NCs. The Arg release profile of ACG NCs was examined in the buffer of pH 7.4 and 6.0. It was found 40.6 % of Arg could be released after 12 h of incubation at pH 7.4 (Figure 4A). However, during the same time period, only 11.6 % of Arg was released at pH 6.0, demonstrating the base-responsive Arg release profile. The urease activity of ACG NCs was testified by a phenol red method, where the absorption at 560 nm of phenol red will be enhanced with the pH value increasing.28 As shown in Figure 4B, after mixing ACG NCs, phenol red, and urea into PBS (pH 7.4), the absorption of the mixture at 560 nm gradually increased with the reaction proceeding, and their pH value could reach up to 8.5 within 1 h, revealing the excellent urease activity of ACG in Arg decomposition into NH3. In order to testify the NHS ester broken ability of ACG NCs at proliferation stage, fluorescein isothiocyanate (FITC) labeled ceria NPs were used in CG NPs to form ACFG NCs. When ACFG NCs were dispersed in PBS at pH 6.0, they showed weak fluorescence intensity due to the fluorescence resonance energy transfer (FRET) between FITC and graphene within a short distance (Figure 4C-D). When the pH of PBS


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was adjusted to 7.4, the fluorescence intensity of the mixture was significantly enhanced after addition of arginase, indicating NHS ester can be broken at pH 8.5 and FITC is far away from graphene (Figure 4D). Taken all together, at proliferation stage, the neutral pH value could unfold i-motif DNA and release Arg, which can be catalyzed into urea by arginase. Urea could be further decomposed into NH3, leading to the basic environment and ceria NP detaching from ACG

NCs as schemed in Figure 1C.

ROS scarifying ability of ACG NCs. The ROS scarifying ability of ACG NCs intensely correlates with their SOD and catalase activities originating from regenerative redox switching between Ce3+ and Ce4+ ions.14, 36 As shown in Figure 4E, ACG NCs showed the excellent SOD and catalase activities, and the superoxide anion and H2O2 inhibition rates of ACG NCs (200 µg mL-1) were determined as 84.13 % and 25.9 % through SOD and H2O2 assays, respectively, indicating their excellent ROS scarifying ability. Considering the different processes of ACG NCs, the valence of Ce ions and ROS scarifying ability of ACG NCs after NHS ester broken or after white light irradiation was also examined compared with those of untreated conditions. Figure S7 reveals the similar Ce ion distribution of ACG NCs after these treatments compared with untreated samples (Figure S4), while Figure S8 demonstrates their similar SOD and catalase activities, demonstrating the stability of SOD activity of ACG NCs. All these results suggest the excellent ROS-scavenging activity of ACG NCs, which potentially can protect healthy cells and tissues against deleterious ROS injury.


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Cell migration and proliferation abilities of ACG NCs. The detachment of ceria NPs from ACG

NCs can facilitate the cellular uptake of ceria NPs, executing the excellent ROS scarifying

ability inside cells at proliferation stage. Firstly, the cell viability in basic environment was examined by MTT assay. As shown in Figure S9, no obvious dead cells were observed from pH 7.4 to pH 9.0 after 24 h incubation, similar with the reported result.37 Therefore, the basic environment had no effect on the cell viability. Then, detachment and cellular uptake of detached ceria NPs were investigated by flow cytometry using FITC labeled ACG (ACFG) and CG (CFG) NCs, where FITC was only labeled on ceria NPs rather than graphene. To mimic wound site microenvironment, Transwell system was used to culture fibroblasts (3T3 cells) in the upper chamber and macrophages (Raw 264.7 cells) in the lower chamber in acidic culture medium (pH 6.0). IL-4 was used to induce M2 polarization in Raw 264.7 cells. Then, the cells were cultured in neutral culture medium (pH 7.4) containing ACFG or CFG NCs for 6 h (Figure 5A). As shown in Figure 5B, ACFG NC treatment could result in the higher fluorescence intensity in 3T3 cells as detected by flow cytometry, while CFG NC treatment could only induce weak fluorescence, similar to that of untreated cells, meaning the Arg loading in CG NPs facilitates the detachment and cellular uptake of ceria NPs. The intracellular ROS scarifying ability of ACG NCs was assessed in H2O2-treated 3T3 cells of above 3T3-Raw 264.7 Transwell system using H2DCFDA assay. Both fluorescence microscopy and flow cytometry analysis revealed

ACG

NC or ceria NP treatment could

significantly weaken the DCF fluorescence intensity of H2O2-treated 3T3 cells while CG NCs treatment could not (Figure 5C and Figure S10), indicating the excellent ROS scarifying ability of ACG NCs after internalization of cells. Simultaneously, the cell proliferation ability of ACG NCs was evaluated in 3T3 cells by EdU assay. As shown in Figure 5D and Figure S11, the


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proliferation of 3T3 cells could be more significantly increased by ACG NCs and ceria NPs than CG NCs. MTT assay (Figure S12) also confirmed this increased proliferation of 3T3 cells when exposed to ACG NCs. All above results indicate that ceria NPs can be detached from ACG NCs and enter into fibroblasts, further scarifying cellular ROS and promoting cell proliferation. Moreover, the graphene once after ceria NP separation is expected to promote the cell migration based on their cell scaffold property at the proliferation stage of wound healing,38 however, the prerequisite is that graphene cannot be removed by macrophage from the wound site, where the stealth peptide of ACG NCs plays a critical role. Therefore, FITC labeled ACG (ACGF) NCs where FITC was only labeled on graphene rather than ceria NPs were used to investigate the cellular uptake of ACG NCs in macrophage cells using above Transwell system. Figure S13 indicated Raw 264.7 cells showed low fluorescence intensity after treatment with ACGF

NCs, but high intensity after treatment with

ACGF

without peptide. These results

demonstrate the stealth peptide in ACG NCs can prevent macrophage cells from internalizing the graphene. Meanwhile, when MMP was added into the cell culture, the enhanced fluorescence intensity indicated that graphene could enter into macrophage again due to the detachment of stealth peptide, implying the excellent biodegradable ability of ACG NCs. Scratch wound healing assay was further executed to investigate the cell migration ability of ACG NCs. Figure 5E and Figure S14 indicated that 3T3 cell migration into the cell-free region (outlined) was dramatically accelerated by ACG NCs. Taken all together, ACG NCs can significantly promote cell migration and proliferation at the proliferation stage, potentially capable of accelerating the wound healing process.


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**

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* *

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* 6 3 0

Figure 6. In vivo wound healing effect of ACG NCs. (A) Wound closure rates expressed by the percentage of closed wound area. *p < 0.05; (B) The representative photographs of wound site of mice before and after 14-day treatment; H&E staining (C) and hydroxyproline content (D) of wound tissues after 14-day healing. For (A) to (D), the wounds of mice were treated with PBS, CG NCs, CG NCs (light), ACG NCs, and ACG NCs (light) for 14 days; *p < 0.05, **p < 0.01.

In vivo wound healing effect. Encouraged by the excellent bacteria inactivation, cell migration and proliferation abilities of ACG NCs, ICR mice with wounds on their backs were utilized as in vivo models to investigate the wound healing capability of ACG NCs through hierarchically promoting multiple stages. The wound were treated with PBS, CG NCs, and ACG NCs, respectively, under white light irradiation or not, and the wound sizes were measured every two days after treatment. Figure 6A shows the wound closure rates within 14 days of treatment. All the wound areas were gradually decreased, and the wound closure rates of mice at the end of


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treatment was 61.5 ± 5.1 % for PBS, 69.9 ± 2.9 % for CG NCs, 79.1 ± 0.6 % for ACG NCs, 74.5 ± 0.5 % for CG NCs (light), and 95.7 ± 0.3 % for ACG NCs (light), respectively, revealing the most promising healing ability of ACG NCs with white light irradiation. Figure 6B further shows the photographs of wound site before and after 14 days of treatment, in which ACG NCs (light) also showed the best wound healing capability. The wound healing ability of ACG NCs was also evaluated by histological analysis. Hematoxylin & eosin (H&E) stained wound tissues showed that the wound boundary between wound and normal tissue was obvious in wounds treated with PBS, CG NCs, ACG NCs, and CG NCs (light), while complete and thickened epidermis was formed after treatment with ACG NCs (light) (Figure 6C). Moreover, the hydroxyproline content of wounds was measured to evaluate collagen formation in granulation tissue. The most significant increase was observed in ACG NCs (light)-treated mice, followed by those of ACG NCs-, CG NCs (light)-, CG NCs-, and PBS-treated mice (Figure 6D), confirming the excellent wound healing ability of ACG NCs with white light irradiation. Conclusion In summary, ACG NCs were designed to exhibit hierarchical wound healing ability through promoting inflammation and proliferation stages. At inflammation stage,

ACG

NCs could

effectively kill bacteria due to their efficient electron-hole separation ability. At proliferation stage, ceria NPs could be detached from ACG NCs and taken up by cells to scarify intracellular ROS and promote fibroblast proliferation, while the left grapheme as a scaffold could accelerate fibroblast migration to wound site. This design develops a new strategy for hierarchically accelerating the multiple stages of wound healing. Experimental Section


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Preparation of hollow ceria NPs. Silica NPs were first prepared through a modified Stöber method.31 A solution of 5 mL of tetraethyl orthosilicate (TEOS) in 20 mL of ethanol was added dropwise into a mixture containing 30 mL of ethanol, 4 mL of water, and 5 mL of ammonium hydroxide (4 mol L-1) under stirring at 60 ºC. After 2 h of reaction, the products were centrifuged at 10000 rpm for 5 min, washed with ethanol, and dried for 12 h at 60 °C. Hollow ceria NPs was synthesized by using the above silica NPs (~70 nm) as templates. 0.1 g of silica NPs and 1 g of polyvinyl pyrrolidone (PVP, MW = 55000) were dispersed in 40 mL of deionized water and stirred for 30 min at 95 °C, followed by addition of cerium (III) nitrate hexahydrate (0.5 mmol L1,

5 mL) and hexamethylenetetramine (0.5 mmol L-1, 5 mL) aqueous solutions in turn. After

vigorous stirring for 2 h, the mixture was cooled down to room temperature, centrifuged at 7000 rpm for 5 min, washed with water, and dried for 12 h at 60 °C. Then, 0.05 g of the dried power was heated to 600 °C and maintained for 2 h, and then dispersed into 20 mL of sodium hydroxide aqueous solution (2 mol L-1) at room temperature and stirred for 24 h. The hollow ceria NPs were obtained through centrifugation at 7000 rpm for 5 min, washing with water, and drying at 60 °C for 12 h. Preparation of amino modified hollow ceria NPs and graphene. Graphene was synthesized through a Hummer method.32 Amino groups were introduced into both ceria NPs and graphene through modification of their surface with 3-aminopropyltriethoxysilane (APTES). For amino modified hollow ceria NPs, 0.05 g of hollow ceria NPs was dispersed into 10 mL of deionized water under vigorous stirring, and then 0.2 mL of APTES was added and stirred for overnight. After centrifugation, washing and drying at 60 °C, amino modified ceria NPs were obtained. For amino modified graphene, 0.05 g of graphene and 1 mL of APTES were dispersed into a mixed solution containing 100 mL of deionized water and 100 mL of ethanol. After bubbling with


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nitrogen for 30 min, the mixture was refluxed at 70 °C for 24 h under nitrogen atmosphere, and amino modified graphene was obtained through centrifugation, washing and lyophilizing. Preparation of CG and ACG. Amino modified hollow ceria NPs were first attached with NHS ester-PEG-COOH (MW = 3400) and i-motif DNA, then coupled with amino modified graphene and stealth peptides through EDC/NHS method. Briefly, amino modified hollow ceria NPs (10 mg mL-1, 10 mL) and NHS ester-PEG3400-COOH (1 mg mL-1, 10 mL) aqueous suspensions were mixed and stirred for 12 h. After centrifugation, the above precipitate was redispersed in 10 mL of deionized water, and 1 mL of NHS (1 mg mL-1) and 1 mL of EDC (1 mg mL-1) were added, followed by the addition 1 mL of 100 µmol L-1 i-motif DNA (5’-NH2-(CH2)6CCCTAACCCTAACCCTAACCC-3’) at room temperature with continuous stirring for 6 h. Then ceriaDNA was obtain through centrifugation at 7000 rpm for 5 min, then mixed with 10 mL of stealth peptide (COOH-GGGPLGLAGGNYTCEVTELTREGETIIELK) aqueous solution (1 mg mL-1), followed by adding 1 mL of NHS (1 mg mL-1) and 1 mL of EDC (1 mg mL-1). The resulting mixture was stirred for 1 h. After dialysis (MWCO = 10000) to remove the unreacted EDC and NHS, amino modified graphene aqueous suspension (1 mg mL-1, 1 mL) was added. Finally, CG NCs were obtained through centrifugation at 7000 rpm for 5 min, washing with water, and drying at 60 °C. To obtain ACG NCs, 500 µg of CG NCs was dispersed into 2 mL of PBS (pH 6.0), followed by addition of 1 mL of Arg (500 µg mL-1 in PBS, pH 6.0) and 6 h of stirring. After centrifugation at 7000 rpm for 5 min and drying at 60 °C for 12 h, ACG NCs were obtained. Preparation of FITC labeled CG NCs (CFG NCs) and ACG NCs (ACFG NCs). FITC was firstly labeled on hollow ceria through the attachment of isothiocyanate groups of FITC to NH2 of hollow ceria NPs. In detail, a mixture of NHS ester-PEG3400-COOH (1 mg mL-1, 10 mL) and


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FITC (1 mg mL-1, 1 mL) were added into amino modified ceria (10 mg mL-1, 10 mL) and stirred for 12 h. After dialysis (MWCO = 10000) to remove the unreacted PEG and FITC, FITC labeled ceria (ceriaF) NPs were obtained through lyophilizing. CeriaF NPs were used instead of ceria NPs to prepare CFG and ACFG according to the protocol mentioned above. Fabrication of FITC labeled graphene. For FITC labeled graphene, FITC (1 mg mL-1, 1 mL) was mixed with amino modified graphene (1 mg mL-1, 1 mL) and stirred for 12 h. After centrifugation at 7000 rpm for 5 min, washing with water, and drying at 60 °C, FITC labeled graphene were obtained. For FITC and stealth peptide co-modified graphene, 10 mL of peptide (COOH-GGGPLGLAGGNYTCEVTELTREGETIIELK) aqueous solution (1 mg mL-1) were firstly added with 1 mL of NHS (1 mg mL-1) and 1 mL of EDC (1 mg mL-1) and stirred for 1 h. After dialysis (MWCO = 10000) to remove the unreacted EDC and NHS, FITC (1 mg mL-1, 1 mL) was mixed with the above peptide, then amino modified graphene (1 mg mL-1, 1 mL) was added and stirred for 12 h. Finally, FITC and peptide labeled graphene were obtained through centrifugation at 7000 rpm for 5 min, washing with water, and drying at 60 °C. Physicochemical Characterizations. TEM images were taken using a JEM-2010 transmission electron microscope (JEOL, Japan). XRD patterns were collected with a D8 Focus X-ray diffractometer (Bruker, Germany). Raman spectra were recorded with a LabRAM HR Evolution Raman spectrometer (Horiba, Japan). UV-Vis absorption spectra were collected on a UV-3600 UV-Vis spectrometer (Shimadzu, Japan). Zeta potential was measured by Zetasizer Nano ZS apparatus (Malvern, UK). XPS spectra were characterized with Escalab 250Xi X-ray photoelectron spectrometer (Thermo Scientific, USA).


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Detection of ROS. Total ROS generation was determined by H2DCFDA assay.39 To each well of a 96 multiwell black plate, we added 80 P of H2DCFDA working solution (10 µM, pH 6.0) and 20 P of hollow ceria NP (450 P mL-1), graphene (50 P mL-1, more than the content of graphene in ACG NCs due to ICP results), or ACG NC suspension (500 P mL-1), followed by irradiation with white light (Sun 2000 Solar Simulator, Abet Technologies) for 20 min or not. After another 2 h of incubation, the fluorescence emission spectra were collected in the range of 500-600 nm with an excitation wavelength of 490 nm. Similar protocols were used to detect superoxide and hydroxyl radical by XTT (100 µM, pH 6.0) and APF (10 µM, pH 6.0). Antibacterial assessments. Monocolonies of E. coli and S. aureus were grown at 37 °C for 12 h under 160 rpm rotation in liquid Luria-Bertani (LB) culture medium (pH 6.0). The bacteria concentration was determined by measuring the optical density of suspension at 600 nm (OD600). 150 P of E. coli or S. aureus bacterial suspension (OD600=0.1) containing graphene (50 P mL1),

ceria NPs (450 P mL-1) and CG NCs (500 P mL-1) was incubated in each well of a 96-well

plate. After irradiating with white light for 20 min or not, the bacteria were incubate for 6 h at 37 °C at 160 rpm rotation. The value of OD600 was recorded every hour. After 6 hours of growth, the resulting bacterial suspension was diluted for 10000 times, and 50 P of the suspension was spread on agar plate. The number of the colonies was counted after 24 h of incubation at 37 °C. ROS, MDA and GSH levels assessments in bacteria. The bacteria were incubated with graphene (10 P mL-1), ceria NPs (90 P mL-1), and CG NCs (100 P mL-1) for 6 h after white light irradiation (0.1 W cm-2, 20 min). For ROS level assessment, 0.2 mL of the above bacteria solution was collected and incubated with H2DCFDA (10 P ) for 30 min under darkness. Then the bacteria were washed with PBS twice and dropped into a slide, finally imaged by fluorescence microscope. For MDA and GSH level assessments, 1 mL of the above bacteria


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solution was collected and lysed by lysis buffer (KeyGEN BioTECH, KGP701), then MDA and GSH were detected by MDA Assay Kit (KeyGEN BioTECH, KGT004) and GSH Assay Kit (KeyGEN BioTECH, KGT006) according the manufacturer’s instructions, respectively. SOD, catalase and urease activities assessments. For SOD activity assessment, 2 mL of ACG

NCs (50, 100, 200 P mL-1) dispersions were mixed with a solution containing 3 mM

xanthine, 200 mU mL-1 xanthine oxidase, and 100 P

2,3-bis-(2-methoxy-4-nitro-5-

sulfophehyl)-2H-tetrazolium-5-carboxanilide) (XTT). After 1 h of incubation, the above suspension was centrifuged at 6000 rpm for 5 min, and the absorbance of supernatant at 465 nm was recorded. For catalase activity assessment, 1 mL of ACG NC (50, 100, 200 P mL-1) dispersions were mixed with 10 mM H2O2 and incubate for 2 h, then the above suspension was centrifuged at 6000 rpm for 5 min, and 1 mL of the supernatant was mixed with hydrogen peroxide detection kits (KeyGEN BioTECH, KGT018) and the finally H2O2 concentration was determined by the absorption at 450 nm. For urease activity assessment, phenol red (30 P mL-1, 50 P 2 and urea (3 mol L-1, 100 P 2 were mixed in PBS (pH 7.4) to form a detection solution. Then, 100 P of ACG NC (2 mg mL-1 in PBS) suspension was added, and the absorbance of this mixture at 560 nm (OD560) was monitored every minute within 60 minutes. Cell culture. 3T3 and Raw 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (10%), penicillin (100 units mL-1), and streptomycin (100 mg mL-1) at 37 °C in a humidified 5% CO2 atmosphere. The culture media was changed every two days, and the cells were passaged by trypsinization before confluence. Cell viability at different pH values. 1×104 3T3 cells in 100 P

of culture medium at

different pH values (7.4, 7.8, 8.2, 8.8, and 9.0) were seeded in each well of 96-well plate. After 24 h-incubation, the viability of 3T3 cells was accessed by MTT assay.


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Cellular uptake of ACG NCs using 3T3-Raw 264.7 co-culture Transwell system. 4×104 3T3 cells in 400 P of culture medium (pH 6.0) were seeded in lower chamber, and 1×104 Raw 264.7 cells in 100 P of culture medium were seeded in upper chamber of 24-well Transwell system. Following overnight incubation at 37 °C, the medium was replaced with fresh medium, and Raw 264.7 cells were simulated by IL-4 (5 ng mL-1) for 6 h. For 3T3 cells uptake, 3T3 cells were treated with 400 P of cell culture medium (pH 7.4) containing 100 P mL-1 (equivalent to ceria) CFG or ACFG NCs for 8 h, then 3T3 cells were collected and their FITC fluorescence was detected by flow cytometry (BD Accuri™ C6 plus). For Raw 264.7 cell uptake, Raw 264.7 cells were incubated with 100 P

cell culture medium (pH 7.4) containing ACGF (100 P mL-1

equivalent to ceria) with or without stealth peptide for 8 h. Then Raw 264.7 cells were collected and their FITC fluorescence was detected by flow cytometry. Cellular ROS scarifying ability of ACG NCs. 3T3 and Raw 264.7 cells were co-cultured in Transwell system with Raw 264.7 cells stimulated by IL-4 as described before. Then, 3T3 cells were treated with 400 P of cell culture medium containing 10 mmol L-1 H2O2 and 100 P mL-1 (equivalent to ceria) ceria, CG or ACG NCs for 10 h and stained with 10 µmol L-1 H2DCFDA in DMEM (without fetal bovine serum) for 2 h, and collected for flow cytometry analysis and fluorescence imaging. Cell proliferation ability of

ACG

NCs. 3T3 and Raw 264.7 cells were co-cultured in

Transwell system with Raw 264.7 cells stimulated by IL-4 as described before. Then, 3T3 cells were incubated with 400 P of cell culture medium containing 100 P mL-1 (equivalent to ceria) ceria, CG or ACG NCs for 24 h. The proliferation of 3T3 cells were accessed by EdU (KeyGEN BioTECH, KGA331-100) and MTT assay according the manufacturer’s instructions, respectively.


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Cell migration ability of

ACG

Page 24 of 32

NCs using 3T3 and Raw 264.7 co-culture Transwell

system. 3T3 and Raw 264.7 cells were co-cultured in Transwell system. Following overnight incubation at 37 °C, a scratch was made by a sterile pipette tip (200 P 2 on the monolayers of 3T3 cells and incubated with cell culture without fetal bovine serum, and IL-4 (5 ng mL-1) were added into Raw 264.7 cells for 6 h of stimulation. Then, 3T3 cells were treated with 400 P of cell culture medium (without fetal bovine serum, pH 7.4) containing 100 P mL-1 (equivalent to ceria) CG or ACG NCs for 24 h. The bright field images of 3T3 cells were taken at 24 h and 48 h after creating a scratch. The percentages of wound healing were analyzed using Image J software. In vivo wound healing effect assessment. Female ICR mice (18-21 g, 4-6 weeks old) were purchased from Center for Experimental Animals, Jilin University, and divided into five groups: PBS, CG NCs, CG NCs (light), ACG NCs, and ACG NCs (light). A wound (0.5 cm X 0.5 cm) was obtained by surgical procedure on the back of the mice after anesthesia and treated with 50 P of 1 mg mL-1 (equivalent to ceria) CG or ACG NC suspension in PBS after 12 h. Then, the wound site was irradiated with or without white light for 20 min at the power density of 0.1 W cm-2. The wounds were photographed after another 24 h and 14 days, and the wound sizes measured every two days. After 14 days, the wound tissues collected from the mice were used for H&E staining and hydroxyproline assay. All animal studies were performed in Center for Experimental Animals, Jilin University, and the procedures involving experimental animals were in accordance with protocols approved by the Committee for Animal Research of Jilin University, China.


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Statistical Analysis. All data were presented as mean or means ± standard deviation. Statistical significance was evaluated using Student’s one-slide t-test according to the T. TEST function in Microsoft Excel. P < 0.05 was considered to be statistically significant.

ASSOCIATED CONTENT Supporting Information. Additional figures including synthesis scheme of hollow ceria and CG NCs, TEM images of silica and ceria, zeta potential, FTIR spectra of NH2 modified ceria and graphene, Ce 4d XPS spectra of CG NCs, antibacterial activity of graphene, ceria and CG NCs without light irradiation, Ce 4d XPS spectra and superoxide anions inhibition of ACG NCs after different treatments, cell internalization of ACG NCs with or without stealth peptide in Raw 264.7 cells, in vitro ROS scarfing ability, promoting proliferation rate, and wound healing percentage of ACG NCs. AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Tel: 86-431-85262136. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT


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Hierarchical Acceleration of Wound Healing through Intelligent

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