Carbon quantum dots embedded electrospun nanofibers for efficient antibacterial photodynamic inactivation

Journal Pre-proof Carbon quantum dots embedded electrospun nanofibers for efficient antibacterial photodynamic inactivation Xiaolin Nie, Shuanglin Wu, Alfred Mensah, Keyu Lu, Qufu Wei PII:

S0928-4931(19)33141-8

DOI:

https://doi.org/10.1016/j.msec.2019.110377

Reference:

MSC 110377

To appear in:

Materials Science & Engineering C

Received Date: 24 August 2019 Revised Date:

27 October 2019

Accepted Date: 29 October 2019

Please cite this article as: X. Nie, S. Wu, A. Mensah, K. Lu, Q. Wei, Carbon quantum dots embedded electrospun nanofibers for efficient antibacterial photodynamic inactivation, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/j.msec.2019.110377. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Carbon Quantum Dots Embedded Electrospun Nanofibers for Efficient Antibacterial Photodynamic Inactivation

Xiaolin Nie1, Shuanglin Wu1, Alfred Mensah1, Keyu Lu3 and Qufu Wei1, 2*

1

Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi 214122,

China 2

Fujian Key Laboratory of Novel Functional Textile Fibers and Materials, Minjiang

University, Fuzhou, Fujian, 350108, China 3

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122,

China

*Manuscript Correspondence: Prof. Qufu Wei Professor of Textile Science & Engineering Key Laboratory of Eco-textiles Jiangnan University 1800 Lihu Avenue, Wuxi 214122, China E-mail: [email protected]

ABSTRACT Faced with the emergence and proliferation of antibiotic resistant pathogens, novel nonspecific materials and approaches are required. Herein, we employed electrospinning technology to fabricate nanofibers with antibacterial photodynamic inactivation. This material combines polyacrylonitrile, as a photostable polymer, and biocompatible carbon quantum dots. The resulted nanofibers were successfully characterized by physical and spectroscopic methods. The microbicidal reactive oxygen species (i.e., singlet oxygen) upon illumination was confirmed, and cytotoxicity assay demonstrated that the nanofibers had low cytotoxicity and good biocompatibility. Antibacterial photodynamic inactivation studies demonstrated broad antibacterial efficacy of Gram-negative Escherichia coli ATCC-8099 (99.9999+%, 6 log units inactivation), Gram-negative Pseudomonas aeruginosa CMCC (B) 10104 (99.9999+%, 6 log units inactivation), and Gram-positive Bacillus subtilis CMCC (B) 63501 (99.9999+%, 6 log units inactivation) upon illumination with visible light (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h). However modest inactivation results were observed against Gram-positive Staphylococcus aureus ATCC-6538 (98.3%, 1.8 log units inactivation). Owing to the prepared nanofibers exhibiting efficient antibacterial activity against Gram-negative and Gram-positive bacteria, such materials could be potentially used in anti-infective therapy. KEYWORD antibacterial, carbon quantum dots, nanofiber, photodynamic inactivation, electrospinning

1. INTRODUCTION Since the 20th century, the global incidence of infectious diseases has had a series of ups and downs, infectious diseases have become more frequent in recent decades [1-3]. Although the global mortality caused by infectious diseases decreased from 23% to 17% since 2009 to 2012 [4, 5], the World Health Organization forecasts 13 million deaths from infectious diseases by 2050 [6], which seems to be a long way to go in the fight against infectious diseases. Even more concerning is the emergence of drug resistance, i.e., multidrug-resistant pathogens menacing the human ability to resist usual infections, generating great deal of attention [7, 8]. It is reported that 23,000 died in a year from drug-resistant pathogens in the United States [9] and upwards of 700,000 people die of resistant infections worldwide annually [10]. Faced with the threat of the “post-antibiotic era” [11], novel approaches are urgently needed for prevention and treatment of infectious diseases. One potential approach for various types of infectious diseases is antimicrobial photodynamic inactivation (aPDI) [12, 13], which is a promising technology for cancer treatment and infectious diseases. It differs from other medical treatment (Surgery, chemotherapy and radiotherapy), and remains non-toxic in the dark as well as having no resistance [14-16]. aPDI consists of three key components: a non-toxic photosensitizer (PS), uninjurious visible light illumination and oxygen molecules. The therapeutic principle of aPDI is the production of reactive oxygen species (ROS) with bacterial inactivation under the illumination of PS via one or two mechanisms [17]: Type I mechanism generates hydrogen peroxide (H2O2), superoxide anion radical (O2•‒) or hydroxyl radicals (•OH); Type II

mechanism generates singlet oxygen (1O2). ROS are non-specific and can cause serious damage to pathogens, including bacteria, fungi, viruses or even natural parasites [18, 19]. Based on the statement above, aPDI has two novel and important advantages: i) as an alternative strategy for the treatment of microbial infections, aPDI is equally effective against both drug-susceptible and drug-resistant pathogens [20, 21]; ii) resistance to aPDI is unlikely because ROS could cause non-specific damage to pathogens [22, 23]. Given the emergence of antibiotic resistance, these two attributes make aPDI particularly attractive. Although aPDI is promising for the treatment of pathogenic infections, there are limitations.

Current

PSs,

including

natural

and

synthetic

tetrapyrroles

[24-26],

phthalocyanines [27], phenothiazines [28, 29] and xanthene dyes [30, 31] have been utilized in antimicrobial photodynamic inactivation, anti-cancer photodynamic therapy and even simultaneous cancer imaging [32]. However, many drawbacks of these classical PSs, including poor water dispersibility, complex synthesis steps, difficulty to purification, undesirable biodegradability, poor biocompatibility in tissue engineering, and unsatisfying photostability, greatly limit their further applications in photodynamic therapy (PDT) and photothermal therapy (PTT) [33-36]. To overcome these shortcomings, a lot of efforts have been made to modify PSs, including synthesis method modifications [25, 37, 38], conjugation with water-soluble and targeting agents [39-41], and the utilization of carriers [42-45]. Most recently, applications of nanoscale materials have been potential nanotechnologies for therapeutic applications [46], including quantum dots, nanocages, doped nanoparticles, nanosheets, and polymer dots [47-51]. Among these developments, carbon quantum dots has emerged as a novel promising

zero-dimensional nanomaterial with some unique properties of ease-of-synthesis, favorable dispersibility in water, nontoxicity, good biocompatibility and widely controllable optical properties [52-55]. Carbon quantum dots are reported to produce ROS by absorbing light for PDT [56] and PTT [57], and as such, carbon quantum dots have been utilized widely for applications in bio-imaging [58], nanomedicine [59], photocatalysis [60, 61] sensors [62, 63] and electret-transducer materials [64]. Compared with traditional PSs, carbon quantum dots do not contain any heavy-metal elements, it has the advantage of environmental friendliness. In the past few years, various methods have been tried to prepare carbon quantum dots, such as top-down and bottom-up approaches. Top-down approaches include breaking down larger precursors such as graphite powder or carbon nanotube [65, 66]. Bottom-up approaches include the carbonization of certain precursors [67]. The precursors and synthesis steps may affect

the

physicochemical

properties

of

carbon

quantum

dot,

such

as

hydrophilicity/hydrophobicity, particle size, optical property and surface functional groups. Among them, solvothermal method using some certain green precursors and solvent to produce carbon quantum dots has been widely used because of its wide source of precursors, green chemistry nature, simple procedure and high purity product [68]. Although carbon quantum dots have prominent PDT capacities due to some unique properties, a number of outstanding questions on the utility of PDT materials remain. For instance, is there a way to recycle the carbon quantum dots to avoid the potential threat posed by residues in vivo? Is there a support that could load carbon quantum dots during the inactivation of bacteria? If so, will the support guarantee that the carbon quantum dots will not be released? Will the materials become fragile after illumination?

To address these issues, we employed electrospinning technology [6, 69-74]; a facile, green and versatile route to fabricate carbon quantum dots embedded polyacrylonitrile nanofibers (PAN-CQDs NFs) (Figure 1) with high antibacterial photodynamic inactivation (aPDI) efficacy. Our experimental design takes advantage of the solvothermal method as a bottom-up approach to synthesize carbon quantum dots using environmentally-friendly precursors and solvents, and the versatile electrospinning technology to produce PAN-CQDs NFs. On one hand, nanofibers have a large specific surface area, providing more space for bacteria to attach [75]. On another hand, PAN nanofibers have prominent photostability, high hydrophilicity and spinnability [76-78]. In addition, bactericidal carbon quantum dots with PAN NFs as support could be recycled after bacterial inactivation. The results show that the composite fluorescent PAN-CQDs NFs presented ultra-efficient broad-spectrum antibacterial properties against four strains of bacteria, including Gram-negative and Gram-positive ones. Mechanistic study presented that 1O2 was responsible for pathogen inactivation, and the aPDI process was performed through Type II mechanism. Our findings suggest that in addition to being highly efficient aPDI nanomaterials for the photodynamic inactivation of bacteria, PAN-CQDs NFs could be produced in a simple approach and facile methods represent a next generation, nanoscale antibacterial materials to combat the transmission of pathogens that lead to healthcare associated infections.

Figure 1. Fabrication of PAN-CQDs NFs and the photodynamic inactivation of bacteria upon visible light illumination. 2. EXPERIMENTAL 2.1 Materials and reagents Polyacrylonitrile powder (PAN: Mw=150,000, 99%) was purchased from Shaoxing Gimel Advanced Materials Technology Co., Ltd. 1,5-diaminonaphthalene (1,5-DAN), and 1,3-diphenylisobenzofuran (DPBF) were purchased from Shanghai Vita Chemical Reagent Co. Ltd. N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethanol, methanol, glutaric dialdehyde (25 %), citric acid (CA), potassium chloride, sodium chloride, yeast extract, plate count agar, tween-80, disodium hydrogen phosphate, monopotassium phosphate, tryptone and soy peptone were purchased from Sinopharm Chemical Reagent Co., Ltd. Chemicals were of analytical grade and all reagents were used without any further purification. Sterilized deionized water was used through all experiments.

2.2 Synthesis of carbon quantum dots (CQDs) CQDs was synthesized by a facile one-pot solvothermal method with the mass ratio of

CA to 1,5-DAN of 1:2. In brief, CA (130 mg, 0.68 mmol) and 1,5-DAN (260 mg, 1.64 mmol) were added to absolute ethanol (130 mL). After ultrasound dispersion treatment for 10 min, the above solution was transferred into a poly(tetrafluoroethylene) (Teflon)-lined autoclave (250 mL) and heated at 200 ℃ for 9 h. After cooling down to room temperature slowly, ethanol was removed by rotary evaporation and then resuspended into water. The resulted solution was filtered, dialyzed and freeze-dried to obtain the brown CQD particles.

2.3 Electrospinning of NFs First, 3.0 g PAN was dispersed in DMF 25 (mL), then the mixture was stirred at room temperature for 24 h to fully dissolve the PAN. After polyacrylonitrile was completely dissolved, 0 mg, 30 mg and 158 mg of as synthesized CQD particles were added to the PAN solution under vigorous stirring. The resulted clear homogenous solution was used for electrospinning. The percentages of CQDs in PAN were 0 wt %, 1 wt % and 5 wt %. The electrospinning parameters were as follows: the feeding speed of the solution in the syringe was set at 0.7 mL/h by a syringe pump, the implemented voltage of 11.7 kV was applied and the distance between the tip of the needle and the collector was 15 cm.

2.4 Determination of CQDs loading The stability of the CQDs loaded in PAN NFs in phosphate buffered saline (PBS, aqueous solution of 170 mM NaCl, 3.4 mM KCl, 10.0 mM Na2HPO4, 1.8 mM KH2PO4, pH=7.2, 0.05 % Tween-80) was determined as follows: PAN-CQDs NF membrane (Diameter: 14 mm, thickness: 30.8±1.25 µm) were immersed in PBS (10 mL, 37 ℃, 100 rpm) for 120 h.

The UV-visible spectrum of the PBS was measured to monitor leaching of the CQDs from the NFs into the surrounding solution by using an ultraviolet-visible spectrophotometer (UV-2600, China). For the determination of CQDs loading, dry and washed NFs was dissolved in 5 mL DMF, thereby fully solubilizing the NFs. The concentration of CQDs was determined by UV-visible spectroscopy.

2.5 Characterization The morphology and structure of the NFs were observed by scanning electron microscopy (SEM, Hitachi SU1510, Japan) and transmission electron microscopy (TEM, JEM-2100, Japan). The diameters of the fibers are calculated by the scale bar, 50 number of nanofibers were randomly selected for the average diameter calculation. The Fourier transform infrared spectroscopy spectra (FTIR)were obtained using a Nicolet Nexus spectrometer (USA). The thermal degradation behaviors of NFs were examined in an inert nitrogen atmosphere using a thermogravimetric instrument (TGA, SDTA 851, Beijing). The static water contact angle tests were performed using surface contact angle measuring system (DCAT-21, Data Physics Company, Germany). Crystal structure (X-ray diffraction pattern) was obtained using an X-ray diffractometer (XRD, D2 PHASER, German). The fluorescence spectra

with

different

excitation

wavelength

was

obtained

by

using

a

fluoro-spectrophotometer (F-4600, Japan) with excitation of Xenon lamp. The confocal laser scanning microscopy was performed on a TCS SP8 instrument (Leica Microsystems GmbH, Germany) to visualize CQDs localization on the NFs, with the excitation wavelength of 405

nm and an emission wavelength range of 440-500 nm. An electronic fabrics strength tester from Ningbo Textile Instrument Factory (YG028) was employed to study the mechanical strength of the NF membrane in the presence or absence of CQDs both pre- and post-illumination (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h). The samples were prepared with a width of 5 mm and length of 15 mm. In order to better fasten the sample to the clamps and prevent the membrane from breaking near both ends, the samples prepared had a trapezoid area in the middle. The clamping distance was set 100 mm and the tensile speed was 50 mm/min.

2.6 Antibacterial assays 2.6.1 Culturing bacteria As the most widely distributed bacterial species in nature, Escherichia coli and Staphylococcus aureus were selected as experimental strains. In order to demonstrate the universality of antibacterial properties of the nanofibers, Pseudomonas aeruginosa and Bacillus subtilis were also selected. Escherichia coli ATCC-8099 was cultured overnight at 37 °C in lysogeny broth (LB) in an orbital shaker (120 rpm). Pseudomonas aeruginosa CMCC (B) 10104, Staphylococcus aureus ATCC-6538 and Bacillus subtilis CMCC (B) 63501 followed the same procedure, substituting LB with tryptic soy broth (TSB). Bacterial strains were grown to an initial concentration of 1-5×108 colony forming units (CFU)/mL; the concentration of cultures was monitored by optical density measurements at 600 nm (OD600). Cultures were pelleted by centrifugation for 10 min (10,000 rpm), and then resuspended into PBS for further experiments. All broth and PBS solution used in the study were sterilized.

2.6.2. Photodynamic inactivation instrumentation For aPDI studies of all the bacteria strains, illumination was provided by a Xenon arc lamp (500 W), with a vertical distance of 12 cm, and equipped with a long-pass filter (λ≥420 nm).

2.6.3 Antibacterial photodynamic inactivation assay Inactivation studies employing the above four bacteria stains were performed in sterile flat-bottom 24-well plate. In brief, PAN-CQDs NF membrane (Diameter: 14 mm, thickness: 30.8±1.25 µm) was first added into adjacent wells of 24-well plate, and then 0.1 mL PBS containing 1-5×108 CFU/mL of bacteria was added. The 24-well plate was kept in dark for 60 min and then further subjected to illumination (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h). All bacterial illumination studies were conducted in triplicate, with two dark control samples, two PAN NFs light control samples and two NFs-free dark control samples taken prior to cell pelleting to verify the final growth concentration in colony forming units (CFU). Upon the completion of illumination, 0.9 mL of sterile PBS was added to each well in both the illuminated and the control plate, then 100 µL aliquots were taken from each of the wells, then diluted by a factor of 10 in 1.5 mL Eppendorf tubes containing 0.9 mL of PBS. A 1:10 serial dilution was conducted a total of six times for each sample. Then each series was plated (LB-agar culture plate for E. coli, TSB-agar culture plate for P. aeruginosa, S. aureus and B. subtilis). The plates were incubated in the dark at 37 °C for a period of 15-24 h. The

highest degree inactivation detectable was limited by the plating technique employed, 6 log units of detection in CFU/mL were possible with a starting concentration of 108 CFU/mL. Survivability values limited to ≥0.0001 % were recorded, with above value representing the detection limit. Statistical significance was assessed using an unpaired Student’s two-tailed t-test.

2.7 Morphological characterization of bacteria. After antibacterial photodynamic inactivation, the morphology of bacteria was observed by scanning electron microscopy on a Hitachi SU1510. The bacteria were centrifuged (10,000 rpm), the supernatant was decanted, the cell pellet was resuspended in 1 mL PBS solution containing 2.5% glutaraldehyde, and transferred to a 24-well plate containing a glass slide (10 × 10 mm). The solution was subsequently incubated for 12 h at 5 °C to fix the morphology of the bacteria, followed by dehydration in an ethanol series of 25%, 50%, 75%, 95% and 100% (v/v) for 10 min each, air-dried in the dark, then finally gold sputtered prior to visualization.

2.8 Detection of ROS (1O2) The generation of 1O2 of the PAN-CQDs NFs was identified by the oxidative bleaching 1,3-diphenylisobenzofuran (DPBF) as a 1O2 quencher [79]. Briefly, a piece of PAN-CQDs NFs membrane (14 mm) was placed on the bottom in a 10 mL beaker, then 3 mL methanol solution containing 18.5 µM DPBF was added, illuminated by a handheld laser (532 nm, 85±1 mW/cm2; HTPOW Laser Limited) for 90 min, and the UV-visible spectrum of the

solution was recorded every 10 min with the absorbance at 410 nm.

2.9 Cell viability assay The

in

vitro

cytotoxicity

of

the

PAN-CQD

NFs

was

evaluated

by

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) (MedChemExpress LLC) assay. Three small pieces of PAN-CQDs-2.5% membranes with a diameter of 7 mm were sterilized by ultraviolet irradiation. Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) was used for the growth of L929 cells (Supplemented with 10% fetal bovine serun (FBS), 100 µg/mL penicillin as well as 100 µg/mL streptomycin). L929 cells were seeded at 20,000 cells/well and treated with the sterilized NF membranes. After 24 h, the medium was removed, then 100 µL of MTT solution (In complete growth medium, 0.5 mg/mL) was added into each well. The plate was incubated in a humidified incubator containing 5% CO2 at 37 °C for 4 h. Then, 100 µL of the solution in each well was removed carefully and 100 µL of DMSO was added followed by incubation in a shaker with a low shaking speed for 10 min. Subsequently, 100 µL of solution was transferred into a new 96-well plate. The absorbance of each well at 570 nm was measured using a microplate spectrophotometer (EPOCH2T, BioTek Instruments, Inc. Highland Park). Cells seeded on TCP without treatment acted as control group. Each group was tested in triplicate.

3. RESULTS AND DISCUSSION 3.1 Characterization Washed NFs, yielding PAN, PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs were used for

further characterization and antibacterial photodynamic inactivation. SEM images, diameter distribution and TEM images of electrospun PAN NFs alone and in the presence of CQDs (Represented as PAN, PAN-CQDs-0.6% and PAN-CQDs-2.5%) are shown in Figure 2. Compared with the smooth surface of PAN NFs, CQDs seems to aggregate slightly on the surface of PAN-CQDs NFs (Figure 2 panel A, B and C). It can also be seen that PAN NFs in the presence or absence of CQDs have a nearly uniform distribution of the diameter size. The average diameters of PAN, PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs are about 179 nm (Figure 2 panel D), 305 nm (Figure 2 panel E) and 433 nm (Figure 2 panel F), respectively. An increase in fiber diameter is obtained in the presence of the CQDs. The polymer solution properties such viscosity, conductivity and surface tension may affect the fiber formation, hence the diameter of the fibers [80-82]. The presence of CQDs in the composite NFs was confirmed by the TEM images. The corresponding TEM images are demonstrated in Figure 2 panel G, H and I. The presence of CQDs in composite NFs is identified inside the NFs. The CQDs are found to be uniformly distributed inside the fiber structure as shown without any aggregation. Herein, we conclude that the final NF diameter could be tuned by controlling the mass ration of PAN and CQDs, the introduced uniformly distributed CQDs in the NFs affect the surface of the fibers slightly.

Figure 2. SEM, diameter distribution and TEM images of PAN (panel A, D and G), PAN-CQDs-0.6% (panel B, E and H) and PAN-CQDs-2.5% NFs (panel C, F and I). The scale bar is 600 nm for the insets in SEM images. The FT-IR spectra in Figure 3 panel A reveals the functional groups of PAN, PAN-CQDs-0.6%, PAN-CQDs-2.5% NFs and CQDs. For CQDs, the stretching vibration bands of -OH, N-H, C=C, C=N and C-N are observed at 3373, 3250, 1631, 1520 and 1370 cm-1, respectively [83, 84]. The peak at 1584 cm-1 is attributed to N-H bending [85]. For

pristine PAN NFs, the peak at 2243 cm-1 confirms the nitrile (C≡N) position, same with PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs [86]. PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs show new peaks at 1631, 1520 and 1584 cm −1 compared with PAN NFs, which correspond to C=C, C=N stretching vibration band and N-H bending from the CQDs. Figure 3 panel B presents TGA curves of PAN, PAN-CQDs-0.6%, PAN-CQDs-2.5% NFs and CQDs. For CQDs, there are three stepwise weight-loss cycles at 120, 262 and 497 ℃. The first stage weight loss of ~ 4.34% from 30 ℃ to 120 ℃ is due to evaporation of moisture, then the second stage weight loss of ~ 55.76% from 120 ℃ to 262 ℃ is attributed to some species attached through weak hydrogen interactions present within the CQDs [87]. The final stage weight loss of ~ 26.88% after 262 ℃ is probably due to the decomposition of organic functional groups of CQDs [88]. PAN-CQDs-0.6%, PAN-CQDs-2.5% NFs present good thermal stability below 295 ℃, however, they suffer three sharp mass loss stage over this temperature like PAN NFs, which might be due to the cyclization (From 295 to 310 ℃), decomposition-carbonization

(From 310 to 480 ℃) and pyrolytic reaction (After 480 ℃)

[89]. The residual weight of PAN-CQDs-0.6% NFs is lower than that of PAN-CQDs-2.5% NFs by nearly 2%, which indicates the difference of mixing ratio of CQDs loaded. Compared with CQDs, the thermal stability of PAN-CQDs NFs is greatly improved, which further justifies PAN NFs as a suitable support. The hydrophilicity of the NF surfaces was determined by contact angle measurements (Figure 3 panel C). PBS aqueous solution droplet was deposited on the NF surfaces successfully. Because of CQDs hydrophilicity, the contact angles of PAN-CQDs-0.6% (44.7°) and PAN-CQDs-2.5% (30.6°) NFs are decreased compared with that of PAN NFs (52.3°).

This decrease in contact angle from 52.3° to 30.6° proves the presence of CQDs in the PAN matrix. Moreover, the improved wettability of photobactericidal NFs plays a very important role in photodynamic inactivation. With the improvement of surface wettability, the photodynamic ability and antibacterial activity of nanofibers in aqueous environment were significantly improved. The poor wettability may be effected by the tendency of water droplets to immerse into pores. This effect allows diffusion of pathogens into porous structure of the materials, where they could be more vulnerable to 1O2 than on the surface of the materials. [90]. The X-ray diffraction pattern (XRD) shows a single broad diffraction peak at 2θ=26° (Figure S2) that corresponded to the crystal lattice distance of (002), in accordance with previous studies of CQDs [91, 92]. From Figure 3 panel D it could be found that PAN, PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs have the same diffraction peak at 2θ=17°, corresponding to the (200) plane of PAN. Compared with PAN NFs, the PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs show a new diffraction peak appearing at 2θ=26°. The broad peaks for the PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs show that the CQDs are introduced successfully. To evaluate how the CQDs affect the mechanical strength of the NF membrane, tensile strength tests were conducted on the PAN and PAN-CQDs NF fabrics before and after illumination (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h) (Figure 2 panel E and Table S1). All stress-strain curves contain two stages: the elastic deformation stage and the plastic deformation stage. Prior to illumination, the PAN-CQDs-2.5% NF membrane showed the best mechanical properties, with a breaking stress at 48.04 MPa and a breaking

elongation of 96.49%, in contrast to 32.67 MPa and 46.57% for the PAN NF membrane (Table S1). We inferred that the increase in stress and gain in elongation at break might be attributed to the enhanced segment interactions by hydrogen bond. After illumination, the light-aged PAN-CQDs NF membrane do not show a significant decrease in breaking stress and elongation. The elongation at break for PAN-CQDs-0.6% NF membrane is decreased by 13.44% after illumination, while for 2.5% loaded membrane, the essentially elongation at break remain essentially unchanged. These results prove that PAN NFs could be an outstanding support for CQDs.

Figure 3. Characterization of PAN, PAN-CQDs-0.6%, PAN-CQDs-2.5% NFs and CQDs. Panel A and B: FTIR spectra and TGA curve of PAN, PAN-CQDs-0.6%, PAN-CQDs-2.5% NFs and CQDs. Panel C and D: Contact angle and XRD pattern of PAN, PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs. Panel E: Strain-stress curves of PAN and PAN-CQDs NF membrane before and after illumination (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h). Curves before illumination: a (PAN NFs), c (PAN-CQDs-0.6% NFs) and e (PAN-CQDs-2.5% NFs); Curves after illumination: b (PAN NFs), d (PAN-CQDs-0.6% NFs) and f (PAN-CQDs-2.5% NFs).

To explore the optical properties of the photoluminescent NFs, photoluminescence spectra was obtained at different excitation wavelengths (From 280 to 350 nm). The CQDs exhibit an emission maximum at 402 nm, and the maximal fluorescence intensity was

obtained with an excitation at 320 nm, as indicated in Figure 4 panel A. While for PAN-CQDs NFs (0.6% and 2.5% loading), the emission maximum is at 385 nm. Interestingly, the FL emission of photoluminescent NFs shows visibly blue-shift behavior (About 17 nm). The reason of emission wavelength shift is due to the changes of the environment around CQDs [93]. It was noted that the excitation wavelength of photoluminescent NFs was 310 nm (PAN-CQDs-0.6%) and 330 nm (PAN-CQDs-2.5%), which is consistent with that of UV-visible spectra of an aqueous solution of CQDs (Figure S3), the absorbance wavelength of CQDs varied from 300 nm to 350 nm.

Figure 4. Fluorescence spectra of the CQDs solution (panel A), PAN (panel B), PAN-CQDs-0.6% (panel C) and PAN-CQDs-2.5% (panel D) NFs. Excitation wavelength: from 280 nm to 350 nm. Insets: aqueous CQDs and NFs under UV light (λex=365 nm) (left) and visible light (right). The confocal microscopic image of electrospun PAN-CQDs NFs was visualized via confocal laser scanning microscopy (Figure 5). Fluorescence emission was identified when it was excited at 405 nm laser. As expected, the CLSM images of individual PAN-CQDs NFs

do exhibit some slight, discontinuous fluorescence, which is attributed to the embedded CQDs. Importantly, the uniformity of the fluorescence color emission throughout the electrospun PAN-CQDs NFs confirms the uniform distribution of CQDs without aggregation in the polymer matrix. The characteristic photoluminescent behavior of CQDs is well preserved inside the NFs.

Figure. 5 CLSM images. Panel A and D: Bright-feld images, Panel B and E: merged image, and C and F: luminescent images of the PAN-CQDs NFs (0.6% and 2.5%), respectively. 3.2 Antibacterial activity In vitro antibacterial photodynamic inactivation (aPDI) studies employing the electrospun NFs as a CQDs embedded antibacterial materials were performed against the model bacteria Gram-negative bacteria E. coli (ATCC-8099) and P. aeruginosa CMCC (B) 10104, as well as Gram-positive bacteria S. aureus (ATCC-6538) and B. subtilis CMCC (B)

63501, the results are presented in Figure 6. Unless otherwise marked, fixed illumination intensity (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h) was employed for the essays, and a starting concentration of 1-5×108 CFU/mL as determined by colony counting. The Gram-negative bacteria E. coli (ATCC-8099) and P. aeruginosa CMCC (B) 10104 are found to be vastly susceptible to photodynamic inactivation by the PAN-CQDs NFs. With 60 min dark pre-incubation followed by 90 min illumination, PAN-CQDs-2.5% and PAN-CQDs-0.6% NFs inactivate E. coli and P. aeruginosa to the detect limit of 6 log units (99.9999+%; E. coli: P < 0.005, P. aeruginosa: P < 0.01; Figure 6 panel A). No obvious inactivation is observed in the presence of the PAN NFs with illumination (PAN NFs light control) and in the presence of the PAN-CQDs-2.5% NFs but absence of illumination (Dark control), which both further confirm a photodynamic inactivation effect towards Gram-negative bacteria and illustrates that there is no dark toxicity. Gram-positive bacteria S. aureus (ATCC-6538) and B. subtilis CMCC (B) 63501 are also found to be susceptible to photodynamic inactivation by the PAN-CQDs NFs, while inactivation result of S. aureus was modest. After 60 min dark pre-incubation and 90 min illumination as described before, PAN-CQDs-0.6% and PAN-CQDs-2.5% NFs inactivate S. aureus by 98.3% (1.8 log units; P=0.00018) and 99.4% (2.2 log units; P=0.00016, Figure 6 panel B). While for B. subtilis, the result is satisfying, PAN-CQDs-2.5% and PAN-CQDs-0.6% NFs inactivates B. subtilis to the detect limit of 6 log units (99.9999+%; P < 0.005, Figure 6 panel B). As was found for S. aureus and B. subtilis, the PAN NFs light controls and dark controls reveal no significant inactivation of the Gram-positive bacteria.

Figure 6. Photodynamic inactivation studies employing PAN-CQDs NFs against Gram-negative bacteria E. coli (ATCC-8099) and P. aeruginosa CMCC (B) 10104, as well as Gram-positive bacteria S. aureus (ATCC-6538) and B. subtilis CMCC (B) 63501. Displayed is the % survival for the PAN-CQDs-2.5% NFs dark control (dark grey bar) and illuminated PAN NFs light control (red bar) conditions, blue and green bars represent the PAN-CQDs NFs (0.6% and 2.5%) against bacteria, respectively. Studies were performed with a 60 min dark pre-incubation followed by 90 min illumination. It is generally believed that photodynamic inactivation of Gram-negative bacteria is more difficult than that of Gram-positive strains because of an outer cell membrane [94], while we found that E. coli was more likely to be inactivated by the CQDs-loaded NFs than S. aureus. This result is similar to a previous aPDI studies employing Mn-doped Zn/S quantum dots as antimicrobial photosensitizer [95]. It is accepted that physical interaction between photosensitizers and bacterias plays an important role in the process of photodynamic antimicrobial inactivation. The diffusion distance of 1O2 in aqueous phase is less than 200 nm [96, 97]. This may localize the antibacterial effect to within a fraction of a bacterium

(Bacterium size: about 1 µm) from the surface of the NFs. In addition, it is easy for S. aureus itself to form grape-like cluster [98]. Given the limited diffusion capacity of 1O2 and formation of S. aureus, one probable reason of the discrepant inactivation noted here between Gram-negative E. coli and Gram-positive S. aureus is the inability of the 1O2 generated from the CQDs embedded the NFs.

3.3 Scanning Electron Microscopy of Inactivated Bacteria To further investigate the effect of photodynamic inactivation on typical Gram-negative E. coli and Gram-positive S. aureus, scanning electron microscopy was used to observe the bacterial morphologies in the presence of the NF materials, both prior to and post illumination (Figure 7). In the absence of NFs and illumination, the size and morphology of native bacteria are typical and normal (Figure 7 panel A and E): E. coli shapes like a rod, S. aureus appears spherical. The surfaces of both are smooth and intact. When incubated with PAN NFs after illumination (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h) (Figure 7 panel B and F) and incubated with PAN-CQDs-2.5% NFs prior illumination (Figure 7 panel C and G), the size and morphology of both bacteria presented the same morphologies as the native cells, and no damage could be observed on the cell surface. However, when incubated with PAN-CQDs-2.5% NFs after illumination (Figure 7 panel D and H), both bacteria are found to be damaged seriously on the surface, including morphology deformation and volume loss. All of these are strongly in favor of membrane leakage [99]. These results are consistent with a previous study of E. coli and S. aureus destroyed by oxidative material RC-TETA-PPIX-Zn [100], an anti-infective nanoscale

material consisted of regenerated cellulose as scaffold and covalently appended protoporphyrin IX. Consistent with our previous assumption that E. coli is more susceptible than that of S. aureus to photodynamic inactivation, undamaged S. aureus is still discernible inside and out of cell death residue (Figure 7 panel H), suggesting the limited diffusion capacity of 1O2 and the formation of S. aureus cluster. As a whole, the SEM observation results are consistent with bacterial cell damage by 1O2 generated photodynamically by the illumination of the PAN-CQDs NFs.

Figure 7. SEM images of E. coli (A-D) and S. aureus (E-F). Panel A and E were the cells without any treatment, panel B and F were the cells after incubation with the PAN NFs followed by illumination, panel C and G were the cells after incubation with the PAN-CQDs-2.5% NFs without illumination, and panel D and H were the cells after incubation with the PAN-CQDs-2.5% NFs followed by photodynamic inactivation (Xe lamp, 500 W, 12 cm sample distance, λ≥420 nm, 1.5 h). 3.4 Detection of ROS (1O2) To further investigate the mechanism of antibacterial photodynamic inactivation by the PAN-CQDs NFs, we explored if 1O2 was generated upon illumination. As a colorimetric chemical trapping agent, 1,3-diphenylisobenzofuran (DPBF) possesses a highly specific reactivity towards 1O2, undergoing oxidative bleaching to form colorless 1,2-dibenzoylbenzene [101]. As shown in Figure 8, the absorbance of DPBF at 410 nm

decreases gradually in the presence of the PAN-CQDs NFs (0.6% and 2.5%) under a 532 nm laser illumination. When the studies were performed in pure PAN NFs or in the absence of illumination (PAN-CQDs-2.5% present dark control), no obvious decrease in DPBF absorption is noted (Figure S4). Taken together, these results provide unequivocal evidence for the formation of 1O2 upon visible light illumination of the PAN-CQDs NFs. In summary, the mechanism of antibacterial photodynamic inactivation by the CQDs prepared here is consistent with the photodynamic generation of biocidal singlet 1O2, and confirms the PAN-CQDs NFs as a Type II photodynamic materials.

Figure 8. A): The single wavelength (410 nm) data from panel B (red) and panel C (blue), in comparison to the study performed in the absence of light (dark control; green, Figure S4 panel A), and in the absence of the CQDs (PAN NFs light control; dark grey, Figure S4 panel B). B and C UV-visible spectrum of the photooxidation of 18.5 µM 1,3-diphenylisobenzofuran (DPBF) to colorless 1,2-dibenzoylbenzene of the PAN-CQDs NFs (0.6% and 2.5%) as a function of illumination time (532 nm laser, 85±1 mW/cm2). 3.5 Cell Viability Assay To test the cytotoxicity and biocompatibility of as-prepared NFs, the in vitro cytotoxicity upon L929 cells were performed. As depicted in Figure 9, the cell survival rate of L929 cells still remains greater than 95% even after PAN-CQDs-2.5% were co-cultured cells for 24 h.

This results demonstrate that the synthesized NFs had low cytotoxicity and good biocompatibility.

Figure 9. MTT assay by L929 on the surface of PAN-CQDs-2.5% NFs after 24 h.

4. CONCLUSIONS In conclusion, carbon quantum dots synthesized by solvothermal method were successfully embedded in polyacrylonitrile nanofibers by a versatile electrospinning method. It was found that the diameter of the NFs varied with the load of the CQDs and CQDs were nearly uniformly distributed without any aggregation in the structure. Also, illumination did not affect the mechanical property of the nanofiber membrane. Finally, their ability to mediate the antibacterial photodynamic inactivation of Gram-positive and Gram-negative bacteria was demonstrated. Although the PAN-CQDs nanofibers were modestly effective against Gram-positive S. aureus, it showed excellent antibacterial activity against other three bacteria, including E. coli, P. aeruginosa and B. subtilis, by upwards of ~ 6 log units inactivation. With the mechanism of photodynamic inactivation attributed to singlet oxygen, the higher activity against E. coli compared to S. aureus was related to the propensity for S.

aureus to form clusters that may limit CQDs-bacteria interaction. Taken together, the bacteria inactivation results demonstrate the promising nanofiber-based aPDI composite materials could be applied as a next-generation photodynamic materials for anti-infection.

Acknowledgement This research was financially supported by National Key R&D Program of China (2017YFB0309100), Key Laboratory of Eco-textiles, Ministry of Education-supported by the Fundamental Research Funds for the Central Universities (JUSRP51907A), the national first-class discipline program of Light Industry Technology and Engineering (LITE2018-21), the Natural Science Foundation of Jiangsu Province (BK20180628), the National Natural Science Foundation of China (51803078), the 111 Project (B17021) and the International Science and Technology Center (BZ2018032).

Supplementary information Transmission electron microscopy image, XRD pattern of the CQDs. UV-visible spectra of an aqueous solution of CQDs. Control studies for ROS formation of dark control and PAN NFs light control. Breaking stress and breaking elongation of nanofiber membranes.

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Highlights Carbon quantum dots was synthesized by a facile one-pot solvothermal method. A PAN nanofiber containing carbon quantum dots was fabricated. The PAN nanofibers could be applied as a suitable support for carbon quantum dots. Resulted nanofibers exhibited broad photodynamic antibacterial inactivation. The inactivation possessed singlet oxygen was the bactericidal agent.

Conflict of interest The authors declared that they have no conflicts of interest to this work.