Designing an Amino-Fullerene Derivative C70â(EDA)8 to Fight

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Biological and Medical Applications of Materials and Interfaces

Designing an Amino-Fullerene Derivative C70-(EDA)8 to Fight Super Bacteria Junfang Zhang, Jiachao Xu, Haijun Ma, Haotian Bai, Libing Liu, Chunying Shu, Hui Li, Shu Wang, and Chunru Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01483 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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ACS Applied Materials & Interfaces

Designing an Amino-Fullerene Derivative C70(EDA)8 to Fight Super Bacteria Junfang Zhang, §, † Jiachao Xu, §Haijun Ma, §Haotian Bai, ‡ Libing Liu, ‡Chunying Shu,§ Hui Li,*,§ Shu Wang,*,‡ and Chunru Wang*,§ §Beijing

National Laboratory for Molecular Sciences, Key Laboratory of Molecular

Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences Beijing 100190, China ‡Beijing

National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids,

Institute of Chemistry, Chinese Academy of Sciences Beijing 100190, China

KEYWORDS: amino fullerene, super bacteria, cytoprotection, sterilization mechanism, selectivity, wound healing

Email: *[email protected], *[email protected], *[email protected]

ACS Paragon Plus Environment

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ABSTRACT: Along with the rapid appearance of super bacteria with multidrug resistance (MDR), it is a challenge to develop new antibacterial materials to address this big issue. Herein, we report a novel amine group modified fullerene derivative (C70-(ethylenediamine)8 abrr. C70(EDA)8), which reveals a high performance in killing super bacteria, and most importantly, it shows negligible toxicity to the mammalian cells. The strong antibacterial ability of this material was attributed to its unique molecular structure. On the one hand, the amino groups on the EDA part make it easy to affix onto the outer membrane of multidrug resistance E. coli by electrostatic interactions. On the other hand, the hydrophobic surface on the C70 part makes it to form a strong hydrophobic interaction with the inner membrane of bacteria. Finally, C70-(EDA)8 leads to the cytoplast leakage of super bacteria. In contrast, the C70-(EDA)8 is nontoxic for mammalian cells due to different distribution of the negative charges in the cell membrane. In vivo studies indicated that C70-(EDA)8 mitigated bacterial infection and accelerated wound healing by regulating the immune response and the secretion of growth factors. Our amine group-based fullerene derivatives are promising for use in clinical treatment of wound infection and offer a new way to fight against the super bacteria.

1. INTRODUCTION Since the invention of antibiotics, billions of people have been saved from the pathogen infections.1 However, due to the continuously extensive use and abuse of antibiotics in the last 70 years, some super bacteria with multidrug resistance (MDR) appear rapidly.2-3 Thus, it is a big challenge to develop new and highly efficient antibacterial materials to protect us from these dangerous super bacteria. Although many potential materials with reasonable antimicrobial activity have been investigated, e.g., silver nanoparticles,4-6 antibacterial peptides7-10, quaternary ammonium salt compounds 11-12 and so on13-16, these materials still have more or less toxicity to


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mammalian cells which limits their extensive applications. It is really urgent to develop new antibacterial materials without toxicity. Recently, fullerene has attracted extensive attention due to its excellent biocompatibility and high yields of ROS even under lower oxygen conditions.17-19 It has been widely applied in biomedical research for sterilization or tumor therapy as a new type of photosensitive agents in PDT,20-23 for example, it is reported that nC60 (aggregate of pristine C60 in the aqueous phase) and C60 derivatives (C60-OH, C60-COOH, C60-NH2, C60-polymer etc.) could kill bacteria under light irradiation.24-27 However, the preparation of these fullerene materials are always tedious or time-consuming, and the antibacterial ability depends on the light irradiation. Herein, we reported a novel amino group-modified fullerene derivative C70-(ethylenediamine)8 (C70-(EDA)8) that could selectively kill antibiotic-resistant bacteria over mammalian cells without light irradiation, and the sterilization mechanism was attributed to its unique molecular structure. In vivo, C70-(EDA)8 mitigated bacterial infection and accelerated wound healing by regulating the immune response and the secretion of growth factors. 2. RESULTS AND DISCUSSIONS 2.1. Preparation and Characterization. The C70-EDA complex was prepared by a facile liquid-liquid reaction under room temperature (Scheme 1), and the Fourier translation infrared spectroscopy (FT-IR) was employed to determine its molecular structure. In Figure. 1a, the absorption bands located at ca. 3350 and 1650 cm−1 are separately assigned as the N-H stretching and bending vibrations, revealing the presence of amino groups on the molecules. Figure. 1b shows the X-ray photoelectron spectroscopy (XPS) of this molecule, in which the C1s peaks centering at the binding energy of 284.8 eV and 286.2 eV are assigned to C–C and C–N, respectively, further confirming the connection of amino group and C70. Finally, the elemental


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analysis was employed to reveal the chemical composition of this derivative as C70-(EDA)8 considering the relative contents of carbon and nitrogen elements (Table S1). The hydrodynamic diameter in aqueous solution was 116.2 nm (Fig. S1). This was a reflection of the aggregation behavior.

Scheme 1. Synthetic procedure of C70-(ethylenediamine)8 (C70-(EDA)8)complex by liquid−liquid reaction method.

Figure 1. (a) Fourier translation infrared spectroscopy (FT-IR) of C70-(EDA)8, (b) X-ray photoelectron spectroscopy (XPS) of C70-(EDA)8 2.2. Antibacterial capacity of C70-(EDA)8 in vitro. Here the Escherichia coli top 10 with MDR E. coli (gram-negative) and Staphylococcus aureus ATCC6538 (S. aureus, gram-positive)


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were chosen as typical bacteria, and a traditional surface plating method was performed to analyze the antibacterial activity of C70 derivatives. C70-(EDA)8 with a concentration of 40 μM could kill 94.9% MDR E. coli and 99.6% S. aureus, respectively (Fig. 2). By comparison, penicillin G potassium salt and the streptomycin as the two contrast medicines killed only a small part of the MDR E. coli even with a very high concentration (40 mM) (Figure. S3 and S4). Therefore, this result indicates the high-efficiency of C70-(EDA)8 in killing both Gram-negative and Gram-positive bacteria, even for the multidrug resistant bacteria. It should be noted that not all fullerene derivatives own the antibacterial capacity. By comparison, we synthesized two other amino modified fullerene derivatives, namely C70(propylenediamine)5 (C70-(PDA)5) and C70-(butanediamine)3 (C70-(DAB)3) (Fig. S2), as well as other two fullerene complexes without amino groups, i.e., the hydroxyl group-containing fullerene derivative C70-(OH)1828 and the fullerene and poly-(vinylpyrrolidone) complex (C70PVP)29, and their antibacterial abilities were evaluated by the minimum inhibitory concentration (MIC), which was figured out by counting the number of visible colonies on the plates and fitting the growth inhibition curve of the materials to the bacteria30 (Figure. S5 and S6). Since C70 is insolvable in water and there is no functional group on the surface of C70-PVP, C70-PVP was chose as control for other factionalized C70 derivatives. As revealed in Table 1, all the amine modified C70 derivatives effectively killed MDR E. coli and S. aureus, while those fullerene derivatives without amine groups such as C70-(OH)18 and C70-PVP have no obvious antibacterial activity. Moreover, the C70-(EDA)8 molecule possessed the lowest MIC in comparison to C70(PDA)5 and C70-(DAB)6, suggesting that the antibacterial effect of the fullerene derivatives depend on the number of amine groups on a single molecule. According to pervious work,17-19


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fullerene derivatives can generate high yields of ROS under light irradiation. So this could definitely enhance the antimicrobial efficacy of C70-(EDA)8 when we need.

Figure 2. (a) Photographs of visible colony forming units (CFUs) of multidrug resistance (MDR) E. coli treated with different concentrations of C70-(EDA)8 (5-80 μM). (b) CFU reduction of MDR E. coli treated with C70-(EDA)8. (c) Photographs of CFUs of S. aureus treated with different concentrations of C70-(EDA)8 (5-80 μM). (d) CFU reduction of S. aureus treated with C70-(EDA)8.

Table 1. Minimum inhibitory concentration (MIC) values of C70 derivatives


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C70 derivatives

Multidrug Resistance E. coli (μM)

S. aureus (μM)

C70-(OH)18

>400

>400

C70-PVP

>400

>400

C70-(ethylenediamine)8

39.15

14.08

C70-(propylenediamine)5

127.74

41.43

C70-(butanediamine)3

79.79

32.24

2.3. Selective sterilization effect of C70-(EDA)8 over mammalian cells. The human epidermal keratinocytes-adult (HEK-a) cells were used as a representative to further studied the selective sterilization effect of C70-(EDA)8 over mammalian cells. Three groups of cells with (1) HEK-a cells only, (2) HEK-a cells and MDR E. coli (Control group), and (3) HEK-a cells and MDR E. coli and C70-(EDA)8 (Experimental group) were set as a comparing cell culturing study. As shown in Figure 3, it was observed that in the control group the color of the Dulbecco’s Modified Eagle Medium (DMEM) with phenol red changed from purple to yellow after incubation with MDR E. coli for 8 h, which was resulted from the metabolites produced by the fast bacteria proliferation to alter the pH of DMEM (Figure 3a). Moreover, the DMEM became somewhat turbid in this process for the flourish of this bacteria, while the solution was still clear after incubation with MDR E. coli and C70-(EDA)8 (40 μM) for 8 h, indicating MDR E. coli had been efficiently inhibited by the C70-(EDA)8. When the HEK-a cells were treated with S. aureus and C70-(EDA)8, similar phenomena were observed (Fig. S7). In Figure 3b, the optical microscopy images of the three groups were taken to show the status of the HEK-a cells. The HEK-a cells in both group (1) and (3) emerged as a healthy status, indicating that the MDR E. coli bacteria in experimental group had been inhibited efficiently by


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C70-(EDA)8, whereas for the group 2, the HEK-a cells were scarce due to the lots of MDR E. coli in the cell culture dish. MDR E.coli proliferates fast in DMEM because of adequate nutrients. This means HEK-a cells have a competition with bacteria and they lack of nutrients in comparison to the untreated cells. Although C70-(EDA)8 can effectively kill MDR E.coli in a short time, the metabolic waste of MDR E.coli is still in the DMEM solution. This metabolic waste influences the HEK-a cells in a negative way. As a result, the morphology of HEK-a cells changed after treated with MDR E.coli and C70-(EDA)8. Flow cytometry was employed to further investigate the status of HEK-a cells in the three groups31-32. As shown in Figure 3c, only 47.5% of the HEK-a cells in group (2) showed Annexin V-negative/PI-negative staining pattern, indicating that 47.5% of the HEK-a cells were alive. The others with Annexin V-negative/PI-positive staining represented the debris of those apoptosis cells owing to the lack of nutrients and toxins produced by metabolism of bacteria. By comparison, 95.6% of HEK-a cells in the experimental group showed Annexin V-negative/ PInegative staining, reconfirming the fact that the C70-(EDA)8 could selectively kill bacteria and has negligible cytotoxicity to the mammalian cells.


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Figure 3. (a) Photographs of cell culture dishes. (b) Optical microscopy images of human epidermal keratinocytes-adult (HEK-a) cells. The Scale bar is 100 µm. (c) Flow cytometry analysis of HEK-a cells. Untreated HEK-a cells as blank. HEK-a cells incubated with 1 ml multidrug resistance (MDR) E. coli (OD600=1) for 8 h as control. HEK-a cells incubated with 1 ml MDR E. coli (OD600=1) and treated with C70-(EDA)8 (40 μM) as experimental group.

In fact, due to the strong radical scavenging property of fullerene materials,33-37 they not only show negligible toxicity to mammalian cells, but also could promote the cell growth efficiently.17 With the increase of the C70-(EDA)8 concentration, the viability of HEK-a cells had no


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significant change at low concentrations from 10, 20, to 40 μM, but along with the C70-(EDA)8 concentration increasing to above 40 μM (p

Designing an Amino-Fullerene Derivative C70â(EDA)8 to Fight

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