One Stone with Two Birds: Functional Gold Nanostar for Targeted

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Biological and Medical Applications of Materials and Interfaces

One stone with two birds: functional gold nanostar for targeted combination therapy of drug resistant Staphylococcus aureus infection Huajuan Wang, Zhiyong Song, Shuojun Li, Yang Wu, and Heyou Han ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09824 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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ACS Applied Materials & Interfaces

One stone with two birds: functional gold nanostar for targeted

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combination therapy of drug resistant Staphylococcus aureus infection

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Huajuan Wang,a Zhiyong Song,b Shuojun Li, b Yang Wu c and Heyou Han * a,b,c

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a

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Technology, Huazhong Agricultural University, Wuhan, 430070, PR China.

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b

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Agricultural University, Wuhan, 430070, PR China.

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c

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Technology, Huazhong Agricultural University, Wuhan, 430070, PR China.

State Key Laboratory of Agricultural Microbiology, College of Food Science and

State Key Laboratory of Agricultural Microbiology, College of Science, Huazhong

State Key Laboratory of Agricultural Microbiology, College of Life Science and

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Corresponding author: Tel.: 86-27-87288505

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E-mail addresses: [email protected]

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ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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The development of new antibacterial agents to deal with the emergence and spread of

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antibiotic resistance in Gram-positive bacterial pathogens has become an increasing

4

problem. Here, a new strategy is developed for the effective targeting and killing of

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Gram-positive bacteria based on Vancomycin (Van) modified gold nanostars (AuNSs).

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Our work has demonstrated that Van-modified AuNSs (AuNSs@Van ) can not only

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selectively recognize MRSA, but also kill MRSA under NIR laser irradiation in vitro.

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Additionally, the AuNSs@Van shows satisfactory biocompatibility and antibacterial

9

activity in treating bacterial infection in vivo. The attractive trait of AuNSs@Van is

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attributed to the physical effect of its antibacterial activity, with less potential for

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resistance development. The aforementioned advantages indicate the potential of

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AuNSs@Van as a photothermal antibacterial agent for effectively combating

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Gram-positive bacteria in the field of healthcare.

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KEYWORDS: Gold nanostars, Recognition, MRSA, Infections, Photothermal

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therapy

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1 INTRODUCTION

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Bacterial infections have posed a serious threat to public health.1-4 The most widely

3

employed strategies to handle bacterial infections are therapies with conventional

4

antibiotics.5 However, conventional antibiotics are becoming less efficient as a result

5

of the development of drug-resistant (DR) or multi-drug-resistant (MDR) bacterial

6

strains.6 Therefore, it is of great urgency to develop new antibiotics to relieve the

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serious situation. However, the sad news is that new antibiotics is far behind in the

8

development rate than pathogens resistance.7 Actually, the discovery of new drugs has

9

greatly decreased and even essentially stagnated in the past few years, which implies

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that we are now approaching a post-antibiotic era.8,9 The resistance of DR or MDR

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bacteria to drugs is mainly attributed to the structure transformation, the gene

12

mutation of planktonic bacteria and the formation of biofilms.10-12 Therefore, the

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development of new approaches based on novel treatment programs and with less

14

potential to induce resistance is becoming more and more urgent. To combat

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microbial drug resistance, nanoparticle (NP)-based antibiotics have been extensively

16

investigated in recent years.13 For instance, Sabo-Attwood and co-workers discovered

17

the use of functional gold nanorods for targeted photothermal lysis of the pathogenic

18

bacteria;14 Chen and co-workers developed multifunctional Fe3O4@Au nanoeggs as

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photothermal agents for selective killing of nosocomial and antibiotic-resistant

20

bacteria;15 Rotello and co-workers demonstrated the use of functionalized Au NPs to

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combat MDR pathogenic bacteria, with the bacterial resistance not observed even

22

after 20 generations.16 Overall, NPs with unique optical and magnetic properties can 3



1

provide excellent platforms for the selective non-invasive photothermal therapeutic

2

applications for MDR and other pathogens.17 One promising means, using a

3

nanomaterial-based photothermal process for selective treatment of bacterial

4

infections, is still in its infant stage.

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Photothermal therapy (PTT) has been widely developed, which including pulsed laser

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and plasmon absorption nanomaterials (grapheme oxide, gold nanoparticles) in the

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NIR region.18,19 near-infrared (NIR) light (700-1100 nm) has a relatively high

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transmission rate through biological tissue, which is very suitable for biological

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applications.20-23 Compared with other metal nanoparticles, gold nanoparticles have

10

the properties of good photostability, low toxicity and easy to surface modification.24

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Moreover, gold nanoparticles can be tuned to strongly absorb NIR radiation

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depending on their shape,25 and can ultimately transfer this light energy into the

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surrounding environment as heat.26,27 If the nanoparticles are directly attached to

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bacterial surfaces, the local high temperature that occurs during NIR irradiation can

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lead to irreparable cellular damage.28,29 A variety of gold nanoconstructs, including

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gold naonrods,30 nanoshells,31 nanocages,32 nanocrossess33 and branched gold

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nanoparticles,34 have been demonstrated to display plasmon resonance in the NIR

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region and produce hyperthermia upon NIR laser irradiation. Branched or star-shaped

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Au nanostructures consisting of a core and protruding arms have attracted particular

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interest recently due to their unique morphology and optical properties.35 The

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presence of sharp tips and high surface to volume ratios make branched Au

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nanostructures more effective than those with smooth surfaces in photothermal 4


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conversion and drug loading.36 However, in many collaborative antibacterial studies

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that combined PTT with other therapeutic strategies, plasmon absorption

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nanomaterials were not specifically dispersed to the bacteria surface, thus the high

4

temperature were not fully delivered to bacteria.37 If the nanomaterials are specifically

5

bound to the cell surface, the high temperature will produced on the bacterial surface

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directly, thus a better antibacterial effect will be achieved.

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Herein, we designed a strategy through covalent conjugation of AuNSs to Van for

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specific recognition and killing the Gram-positive pathogen, Methicillin-resistant

9

Staphylococcus aureus (MRSA) (Scheme 1). Specifically, vancomycin-modified

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AuNSs can actively target Gram-positive bacteria by binding to the D-Ala-D-Ala

11

moiety of the cell wall of Gram-positive bacteria and anchor on the surface of

12

bacteria.38,39 Subsequently, the irradiation of NIR laser generate hyperthermia in situ,

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leading to bacterial death. In short, this antibacterial platform aimed at killing MRSA

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efficiently and precisely by photothermal effect and with less possibility to cause

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bacterial resistance.

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Scheme 1. Schematic illustration of the preparation and application of AuNSs@Van: (ⅰ)

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Preparation of AuNSs@Van; and (ⅰⅰ) AuNSs@Van for distinguishing and killing MRSA using

4

808 nm light.

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2 EXPERIMENTAL SECTION

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2.1 Materials

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HAuCl4, N-hydroxysuccinimide (NHS), was obtained from Shanghai Reagent

8

Chemical Co. (China). 3-(4, 5Dimethylthiazol- yl)-2, 5Dimethylthiazol-2-yl)-2,

9

5diphenyltetrazolium

bromide

(MTT),

vancomycin

(Van),

and

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1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from

11

Aladdin. 808 nm Laser (MDL-N-808nm-8W-14050284), was purchased from

12

Changchun New Industries Optoelectronics Tech. Co., Ltd. PI (propidium iodide) and

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DAPI (4′-6-diamidino-2-phenylindole) were acquired from Beyotime Institute of

14

Biotechology (Shanghai, China). Dulbecco’s modifed Eagle’s medium (DMEM), fetal 6


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bovine serum (FBS), were purchased from GIBCO Invitrogen Corp. SDS-PAGE Gel

2

Parparation Kit (ZD304A-2) was provided by ZOMANBIO. Agarose B was

3

purchased from BBI Life Sciences.

4

2.2 Synthesis of AuNSs

5

AuNSs were prepared according to previous literature.40 Briefly, the seed solution was

6

fabricated by adding 15 mL of citrate solution into 100 mL of boiling HAuCl4

7

solution, and after boiling for 15 min, the solution was cooled to room temperature

8

and then kept at 4℃ for further use. Next, AuNSs were synthesized by mixing 0.5 mL

9

of the above-prepared seed solution with 25 mL water, 260 µL HAuCl4 (2%) solution

10

and 50 µL HCl (1 M) solution in a 50 flask under moderate stirring. Subsequently, 0.5

11

mL AgNO3 solution (2 mM) and 0.25 mL ascorbic acid (100 mM) were added into

12

the flask at the same time, followed by stirring the solution until the color changed

13

from light red to blue or greenish black. Finally, the obtained solution was kept at 4

14

°C for further use.

15

2.3 Synthesis of and Characterization of AuNSs@Van

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First, Van was activated by adding EDC (50 μL, 50 mg/mL) and NHS (50 μL, 25

17

mg/mL) into the Van solution (10 mg/mL, 1 mL), followed by gentle shaking for 1 h

18

at 4 °C. Next, Van was thiolated by mixing the activated Van with NH2-PEG-SH

19

(M.W.: 2000 Da) for 8 h with slowly shaking at room temperature. Subsequently, the

20

modified Van solution was supplemented with AuNSs (75 mL) solution under stirring

21

for 8 h to form Au-S bonds between Van and AuNSs. In order to purify the

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AuNSs@Van and remove the free Van, the obtained solution was transferred into 7



1

dialysis bag (MWCO, 35 kDa) and then washed with phosphate buffered saline (PBS)

2

several times under centrifugation at 6500 rpm for 6 min. The final solution was

3

stored at 4 °C for further use. The concentration of Au in the solution was determined

4

by atomic absorption spectroscopy (AAS, iCE 3500, Thermo Fisher Scientific,

5

Germany): the solution (30 µL) treated with aqua regia (volume ratio,

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HNO3/HCL=1:3) for 2 h. Then, the obtained solution was diluted with water to 3 mL

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before determining Au concentration by AAS. The size of the nanomaterial were

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determined by transmission electron microscopy (TEM, JEM-2010 TEM) and the

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software of Nano Measurer 1.2, respectively. Additionally, the dynamic light

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scattering (DLS, Malvern zetasizer Nano ZS90) was also used to measure the size and

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the dispersibility of the particles. The loading amount of Van on AuNSs could be

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calculated according to the calibration absorption curves of Van (the value of its

13

absorption at 280 nm).41 Besides, zeta potential and TEM elemental mappings were

14

also measured to confirm the successful loading of Van on the particles of AuNSs.

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2.4 Bacterial Culture

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MRSA (011P6B5A), Gram-positive S. aureus (1213P46B) and ampicillin-resistant E.

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coli (PCN033), were isolated from pigs with clinical symptoms. Gram-positive S.

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aureus (AB91093) and Gram-negative E. coli (AB 93154) were obtained from China

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Center for Type Culture Collection (CCTCC). The above strains were used as

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experimental models in the following experiments and were stored at -80 ℃. The

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strains were cultured in the Luria Bertani (LB) broth medium at 37 ℃. The

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concentration of bacterial cells was determined by measuring the absorbance of cell 8


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suspensions at 600 nm with a ultraviolet–visible (UV-vis) spectrometer (PerkinElmer,

2

Lambda 25). The bacterial suspensions were adjusted to the desired concentrations

3

with LB medium before use.

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2.5 Photothermal Effect

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The obtained AuNSs@Van (0.5 mL) with different concentrations (40, 80, 120, and

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150 µg/mL) in EP tubes were exposed to 808 nm light for 10 min (2.5 W/cm2) at

7

room temperature, using PBS as the control. The temperature was measured by a

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thermometer.

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2.6 In vitro Antibacterial Test of AuNSs@Van irradiated by NIR light

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The activity of AuNSs@Van against MRSA was determined by the plate count

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method. Briefly, 200 µL bacterial cells (1×107 CFU/mL) was centrifuged (5000 r/min,

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3 min) at room temperature, followed by the addition of 200 µL AuNSs@Van at

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different concentrations (80, 100, and 120 µg/mL) and incubation for 1 h. Next, 808

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nm (2.5 W/cm2) light was used to illuminate the samples for 10 min, followed by

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spreading 20 µL diluted (1000 folds) bacterial suspensions onto the LB agar plates

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and incubation at 37 ℃ for 12 h. Besides, the bacteria treated with PBS as control.

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Finally, the colony-forming units (CFUs) grew on the plates were counted to calculate

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the antibacterial activity of AuNSs@Van. The antibacterial activity was presented in

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two forms: the reduction in the CFU number per and survival rate. The survival rate

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was calculated according to the following formula:

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group)/CFU(control group)

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Survival rate (%) = CFU(experimental

× 100%, where CFU

2

(experimental group)

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with and without samples, respectively.

4

In addition, the minimal inhibitory concentration (MIC) was carried out according to

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the microdilution method.42

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2.7 Live/Dead Staining Assay

and CFU(control group) were the numbers of colonies in the groups treated

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After NIR irradiation, the antibacterial activity of AuNSs@Van against bacteria was

8

further determined via live/dead viability assay. Briefly, the bacterial suspension

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(200 µL) was centrifuged (5000 r/min, 3 min), then, treated with 200 µL

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AuNSs@Van with different concentrations (80, 100, and 120 μg/mL) for 1 h, after

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that, the suspension was illuminated by NIR light for 10 min, then, the suspension

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centrifuged (5000 r/min, 3 min) before dyed by PI (30 µL) and DAPI (30 µL) in

13

dark for 30 min. Subsequently, the bacteria were washed with PBS (added 200

14

µL PBS to resuspend the bacteria and then centrifuged at 5000 r/min for 3 min).

15

Finally, the solutions were dropped on the slides and observed by a fluorescence

16

microscope (DM6000 B fluorescence microscope, Leica Microsystems,

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Germany). Besides, the permeability of cells treated with/without AuNSs@Van

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under 808 nm NIR light could also be observed by PI staining. Briefly, bacterial

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suspension (107 CFU/mL 200 µL) was centrifuged (5000 r/min, 3 min), then 200

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μL AuNSs@Van (80 μg/mL) was added and co-cultured for 1 h, after that, the

21

bacterial suspension was illuminated with 808 nm light (2.5W/cm2) for 10 min.

22

Then, the suspension was centrifuged before dyed with PI, finally, the 10


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suspension was washed with PBS before observed with a fluorescence

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microscope. Additionally, bacteria treated with AuNSs@Van and PBS were as

3

control.

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2.8 Morphology Study of Bacterial Cells

5

Morphological changes of bacterial cells treated with AuNSs@Van were observed by

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scanning electron microscopy (SEM, SIGMA-500) and TEM. Briefly, MRSA and

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E.coli suspension (107 CFU/mL, 200µL) centrifuged (5000 r/min, 3 min) before

8

treated with AuNSs@Van (80 μg/mL, 200µL) for 1 h, and then irradiated with 808

9

nm (2.5 W/cm2) NIR light for 10 min, after that, the suspension was washed with PBS

10

before fixed with glutaraldehyde (2.5%, 4 h) at room temperature, then, the obtained

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bacteria were dehydrated with different concentrations of ethanol (30, 50, 70, 90, and

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100 %, each for 15 min). Finally, the bacterial suspension was dropped on copper

13

grids and sputtered with gold for SEM; the bacterial suspension was dropped on

14

copper grids for TEM.

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2.9 Cell Cytotoxicity assay

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MTT assay was carried out to determine the AuNSs@Van cytotoxicity. Briefly,

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COS7 cells (monkey kidney cells) (100 µL, 1.0 × 104 cells per well) were first seeded

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in 96-well plates and cultured for 24 h at 37 ℃ containing 5% CO2. Then, the culture

19

medium in each well was substituted by AuNSs@Van solution with different

20

concentrations (50, 80, 100, 120, and 150 µg/mL) or control DMEM (10% FBS) for

21

12 h. Next, DMEM (100 µL, 10% FBS) was added replaced AuNSs@Van in each

22

well and cultured for 8 h, then the solution was supplemented with 20 μL MTT 11



1

reagent in each well and cultured for 4 h at 37 ℃, followed by discarding the medium

2

and adding DMSO (150 µL) to dissolve the formazan in each well. Finally, the

3

absorbance at 490 nm was measured on a microplate reader (Wallac 1420). The

4

relative cell viability was calculated via the formular: cell viability (%) = OD(experimental

5

group)/OD(control group)

6

with AuNSs@Van and without AuNSs@Van, respectively.

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2.10 Leakage of Intracellular Components

8

For the purpose of further confirming the destruction of the bacterial cell membrane,

9

related experiments were carried out on the leakage of protein and nucleic acid.

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Briefly, 600 µL (109 CFU/ mL) bacterial suspension was centrifuged (3000 r/min, 8

11

min) and then washed with PBS twice (added PBS to resuspended the bacteria, then

12

centrifuged 3000 r/min for 8 min, repeated this step once) before treatment with 50

13

µL different concentrations of AuNSs@Van (80, 100, and 120 µg/mL,) under 808 nm

14

light irradiation (2.5 W/cm2, 10 min). The bacterial suspension treated with PBS was

15

used as the control group. After treatment, the suspension was centrifuged at 3000

16

r/min for 10 min at 4 ℃ to obtain the supernatant. Finally, the supernatant was

17

denatured (95 ℃，3 min), followed by added 50 µL denatured supernatant into the

18

SDS-PAGE gel (10%) to do the denaturing polyacrylamide gel electrophoresis

19

(stacking gel, 120 V; separating gel, 180 V). After that, the gel was stained by

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Coomassie Brilliant Blue G-250, and then used a mobile phone to take photos. In

21

addition, Enhanced BCA Protein Assay Kit (Beyotime Biotechnology) was used to

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quantitative the protein content. Besides, using the same method to obtain the

× 100%, where OD(experimental

group)

12


were the OD values treated

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supernatant. Then the supernatant was demonstrated the presence of the nucleic acid

2

by agarose gel (2%) electrophoresis and stained with Ethidium bromide (EB). Finally,

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the picture was taken by gel imaging system (GelDoc XR+, BIO-RAD).

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2.11 In vivo Animal Model Studies

5

In order to investigate the antibacterial effect of AuNSs@Van in vivo, the animal

6

infection model was established. Briefly, healthy female BALB/c mice (6-8 weeks)

7

were shaved on the back, followed by excising a round wound about 1 cm in diameter,

8

removing the skin, and injection of MRSA suspension (107 CFU/mL, 100 μL). After

9

24 h infection, all the mice were randomly divided into 4 groups. Then, the 4 groups

10

accepted 4 treatments (3 times, once a day): AuNSs@Van (808 nm, 2.5 W/cm2, 10

11

min), Dark treatment, Van, and PBS. Specifically, 20 µL AuNSs@Van (80 µg/mL)

12

was smeared over the surface of wounds, followed by 808 nm light irradiation. For

13

the other 3 groups, 20 µL AuNSs@Van (80 µg/mL), 20 µL Van solutions (1.1

14

µg/mL), and PBS were dropped on the wounds, respectively. During the irradiation

15

treatment, the mice were under anesthesia. Besides, on days 0, 1, 3, 5, and 7 of the

16

treatment, the mice were photographed and the bacteria were sampled from the

17

wounds, respectively. Additionally, after 7 days of treatment, the mice were sacrificed

18

to extract the wounded tissues for histology analysis by Hematoxylin and Eosin (H&E)

19

staining and Masson’s trichrome staining. In addition, major organs (liver, spleen,

20

kidney, heart and lung) were also collected for H&E staining to analyze the toxicity of

21

AuNSs@Van in vivo. The samples (collected tissues and organs) were tested in

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Wuhan Google Biotechnology Co., Ltd. 13



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3 RESULTS AND DISCUSSION

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3.1 Characterization of AuNSs@Van

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In the work, the AuNSs@Van was synthesized as described in the Experimental

4

Section. As displayed by the TEM photograph in Figure 1A, the average diameter

5

(104.4 ± 13.3 nm) of AuNSs@Van was calculated

6

particles from the TEM images, which was in line with the DLS result (Figure 1B).

7

Further, the polydiseperse index (PDI) of AuNSs@Van nanoparticles were measured

8

by DLS, the results showed that the nanoparticles have a good dispersibility

9

(PDI=0.232). The Van have carboxyl groups (-COOH), which was able to be

10

covalently coupled with amino groups (-NH2) of NH2-PEG-SH by EDC and NHS,

11

then the Van was given sulfhydryl groups (-SH), when added the AuNSs solution,

12

Au-S bonds was formed between Van and AuNSs, thus AuNSs@Van was

13

synthesized.43 The loading amounts of Van on AuNSs (0.014 mg Van per milligram

14

of AuNSs) was calculated according to the calibration absorption curves of Van

15

(Figure S1). The successful loading of Van was verified by DLS, UV-vis spectra,

16

zeta potential, and TEM elemental mappings. As displayed in Figure 1B, the size of

17

AuNSs@Van was larger than that of AuNSs as a whole, for the Van was modified on

18

the AuNSs. Figure 1C exhibited the results of UV-vis spectra, it is known that

19

AuNSs had the surface plasmon resonance (SPR) peak near 800 nm and Van had a

20

peak at 280 nm, compared with AuNSs, AuNSs@Van not only possessed an SPR

21

absorption peak at 800 nm, but also had an absorption peak at 280 nm, which

22

indicated the successful loading of Van onto AuNSs. Figure 1D showed the zeta 14


by randomly selecting 100

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1

potential of AuNSs, Van, and AuNSs@Van, which was -39.3 ± 1.5 mV, -9.3 ± 1.3

2

mV and -3.1 ±0.4 mV, respectively, the changes of zeta potential suggested the Van

3

was loaded onto AuNSs. In addition, the TEM elemental mappings (Figure 1E)

4

showed the uniform distribution of Au, N, C, Cl and O elements in the same particle.

5

Because Cl is a typical element of Van, hence, the elemental mappings further

6

demonstrated the existence of Van in the nanoparticles.

7 8

Figure 1. Characterization of AuNSs@Van. (A) TEM images of AuNSs@Van. (B) Size

9

distribution of AuNSs and AuNSs@Van measured by DLS. (C) UV-vis absorption spectra AuNSs,

10

Van, and AuNSs@Van. (D) Zeta potential of AuNSs, Van, and AuNSs@Van. (E) elemental

11

mapping of a typical nanoparticle of AuNSs@Van.

12

All the aforementioned results indicated that the AuNSs@Van was successfully

13

synthesized. 15



1

3.2 Photothermal conversion efficiency of AuNSs@Van

2

Figure 2 displayed the photothermal conversion efficiency of AuNSs@Van. It could

3

be seen that the temperature of a series of concentrations of AuNSs@Van (40, 80, 120

4

and 150 µg/mL) increased from 29 to 69.8 ℃ under the same power of 808 nm light

5

(2.5 W/cm2). Figure 2B exhibited the temperature of AuNSs@Van at same

6

concentration (120 µg/mL) increased obviously (from 42.4 to 66.2 ℃) under the

7

excitation of a different NIR light power (1.5-2.5 W/cm2) for 10 min. In Figure 2C,

8

AuNSs@Van was shown to have highly stable photothermal conversion capability

9

during the three test cycles. All the results implied that AuNSs@Van possessed

10

superior photothermal conversion efficiency.

11 12

Figure 2. The photothermal property of AuNSs@Van. (A) Photothermal curves of AuNSs@Van

13

with different concentrations under 808 light (2.5 W/cm2) for 10 min. (B) Photothermal curves of

14

AuNSs@Van (120 µg/mL) under 808 nm light (different power) irradiation for 10 min. (C)

15

Temperature rising and cooling profiles of AuNSs@Van (120 µg/mL) with irradiation on/off

16

under 808 nm light (2.5 W/cm2).

17

3.3 Selective recognition of Gram-positive bacteria

18

For the purpose of verifying the recognition of AuNSs@Van to Gram-positive

19

bacteria in vitro, some related experiments were performed. The recognition of

20

AuNSs@Van to Gram-positive bacteria was demonstrated by SEM data. As exhibited 16


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1

in Figure 3, the surface of MRSA in the control group (Figure 3a,a1) was smooth.

2

When MRSA treated with AuNSs, it could be found that there were a few particles

3

(AuNSs) on the surface of MRSA (Figure 3b,b1). However, when MRSA treated

4

with AuNSs@Van under the same conditions, a lot of nanoparticles (AuNSs@Van)

5

existed on the surface of bacteria (Figure 3c,c1). This difference was mainly due to

6

the modified by the Van which could targeted binding to the cell wall of MRSA, so

7

that the nanoparticles were better enriched on the surface of the bacteria. Besides,

8

TEM data was also presented to demonstrate AuNSs@Van possessed the capability of

9

identification Gram-positive bacteria. In our experiment, Gram-positive and

10

Gram-negative strains were used as the model to evaluate the targeted recognition

11

ability of nanoparticles. Figure 3a2,a3 displayed the results of two Gram-positive

12

strains S.aureus (1213P46B) and S.aureus (AB91093) treated with AuNSs, separately.

13

It could be observed that a few nanoparticles (AuNSs) existed the surface of bacteria,

14

however, lots of nanoparticles (AuNSs@Van) were appeared around bacteria treated

15

with AuNSs@Van (Figure 3b2,b3), these results were in line with the SEM results.

16

Moreover, When the E.coli (AB 93154, PCN033) were incubated with AuNSs@Van,

17

the results showed that almost no nanoparticles could be observed around the surface

18

of the bacteria (Figure 3c2,c3). When the mixed bacterial suspensions of MRSA and

19

E.coli (PCN033) were treated with AuNSs@Van, the results (Figure 3d2,d3) showed

20

that only MRSA cells were surrounded with nanoparticles, and almost no nanoparticle

21

was found to around the E.coli cells. The above results fully demonstrated that

22

AuNSs@Van had a good ability to recognize Gram-positive bacteria, which was 17



1

mainly due to the modification of vancomycin. Previous studies have shown that

2

vancomycin can be anchored to the surface of the Gram-positive bacterial cell wall by

3

binding to the D-Ala-D-Ala molecule, thereby achieving targeted recognition of

4

Gram-positive bacteria.44

5 6

Figure 3.

7

treated with PBS as control (a, a1), treated with AuNSs (b, b1), and treated with AuNSs@Van (c,

8

c1). Typical TEM images of S.aureus (1213P46B, AB91093) treated with AuNSs (a2, a3). (b2,b3)

9

TEM images (down layer) of S.aureus (1213P46B, AB91093) treated with AuNSs@Van. (c2,c3)

10

TEM images of E.coli (AB 93154, PCN033) treated with AuNSs@Van for 1 h. (d2,d3) MRSA

11

and E.coli (PCN033) treated with AuNSs@Van for 1 h. AuNSs: 80 µg/mL, AuNSs@Van: 80

12

µg/mL.

13

3.4 The Antimicrobial Activity of AuNSs@Van in vitro

Selectivity of AuNSs@Van to MRSA. Typical SEM images (upper layer) of MRSA

18


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1

The antimicrobial activity of AuNSs@Van against Gram-positive bacteria in vitro

2

was evaluated by plate count method. Figure 4 presented the antibacterial activity of

3

AuNSs@Van against the two Gram-positive strains (MRSA: a~d, and S.aureus:

4

a1~d1) through the agar plates, which directly exhibited the antibacterial effect of

5

AuNSs@Van. Specifically, when compared with the control groups (a, a1), only a

6

few or even no colonies presented on the agar plates (b~d, b1~d1) after treatment with

7

AuNSs@Van (80, 100, and 120 µg/mL) under the irradiation of 808 nm light. Figure

8

4e, e1 showed the corresponding statistics of the antibacterial effect. It displayed that

9

AuNSs@Van with different concentrations (80, 100, and 120 µg/mL) exhibited a

10

almost 2-log reduction in the CFU number per against the two strains with 808 nm

11

NIR light irradiation, which indicated that AuNSs@Van plus NIR light irradiation

12

could effectively inhibit the growth of bacteria. Notably, in terms of survival rate,

13

nearly 99% antibacterial efficiency was observed with 80 µg/mL of AuNSs@Van

14

against both the strains under 808 nm NIR light illumination, and the antibacterial

15

efficiency was over 99.9% when the treatment concentration increased to 120 µg/mL.

16

Of course, the antibacterial efficiency could be further improved through increasing

17

the laser power, extending the illumination time, and increasing the concentration of

18

nanomaterial. Whether Van in the nanomaterials played a bactericidal role during the

19

experiment was tested by some related experiments. In Figure S2b, it could be

20

observed that no significant reduction in the number of colonies on the agar plate of

21

the free Van (1.1 µg/mL, the concentration is comparable to the amount of

22

vancomycin contained in 80 µg/mL AuNSs@Van.) treated group versus the control 19



1

group (Figure S2a), and the bacterial survival rate decreased to 81% (Figure S2c).

2

However, there were only a few colonies on the agar plate of the AuNSs@Van

3

(80µg/mL) plus NIR light group under the same conditions, and the bacterial survival

4

rate was below 1%. These observations indicated that it was the high temperature

5

generated by AuNSs@Van after absorbing 808 nm light that made a major

6

contribution to the antibacterial effect of AuNSs@Van.

7 8

Figure 4. In vitro antibacterial activity test. Photographs of the agar plates of MRSA (a~d) and

9

S.aureus (a1~d1) treated with AuNSs@Van (80, 100, and 120 µg/mL) under 808 nm light

10

irradiation (808 nm: 2.5 W/cm2, 10 min.). (a, a1) Treatment with PBS was set up as control. Effect

11

of AuNSs@Van on bacterial counts of MRSA (e) and S.aureus (e1). The error bars indicate means

12

± SD (n = 3).

13

The antibacterial activity of AuNSs@Van was further verified by the live/dead

14

viability assay using PI and DAPI staining. The dye of PI can enter the damaged cells,

15

stain the nucleus, and emit red fluorescence, while the dye of DAPI can pass through 20


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1

bacterial cells with intact membrane, combine with DNA and emit blue fluorescence.

2

Hence, the above two dyes can be used to indicate the state of the bacteria, dead (red

3

fluorescence) or alive (blue fluorescence).45-47 In this study, MRSA (b~d) and

4

S.aureus (b1~d1) were treated with different concentrations (80, 100, and 120 µg/mL)

5

of AuNSs@Van under 808 nm NIR light irradiation (2.5 W/cm2) for 10 min. In

6

Figure 5, obvious red fluorescence (dead bacteria) could be observed in (b~d) and

7

(b1~d1), especially at the treatment concentration of 100 and 120 µg/mL (c~d, c1~d1),

8

which showed almost no blue fluorescence (live bacteria). In contrast, the control

9

group (a, a1) displayed a large area of blue fluorescence, with almost no detectable

10

red fluorescence. The above results implied that AuNSs@Van plus 808 nm light

11

irradiation did result in the death of bacteria. In addition, the corresponding

12

quantitatibve flourescence data were measured by image pro plus software, and the

13

results were displayed in Figure 5e, e1, which were consistent with the bacterial

14

survival rates shown in Figure 4.

15

21



1

Figure 5. Fluorescence microscope images of MRSA (a~d) and S.aureus (a1~d1) with PI and

2

DAPI staining after treatment with different concentrations of AuNSs@Van under 808 nm light

3

(2.5 W/cm2, 10 min). (a, a1) Treatment with PBS was set up as control, (b, b1) 80 g/mL, (c, c1)

4

100 µg/mL, and (d, d1) 120 µg/mL. The corresponding measurement of live/dead fluorescence of

5

MRSA (e) and S.aureus (e1). The error bars indicate means ± SD (n = 3).

6

3.5 Cell Integrity Damage of MRSA Induced by AuNSs@Van

7

The integrity of MRSA membrane under different treatments was analyzed by SEM

8

techniques. From the SEM image in Figure 6Aa (the control group), it could be

9

clearly observed the integrity membrane/wall of MRSA. In Figure 6Ab, when MRSA

10

was treated with AuNSs@Van but without 808 nm light, a number of AuNSs@Van

11

particles on the surface of MRSA were found, and the MRSA also possessed

12

complete cell membrane/wall. In Figure 6Ac, it was shown that after treating MRSA

13

with AuNSs@Van plus 808 nm light irradiation (2.5 W/cm2, 10 min), the bacterial

14

membrane became collapsed, which indicated the bacterial structure was destroyed.

15

The disruption of bacterial membrane treated with AuNSs@Van was further verified

16

by using PI staining.42 In Figure 6B, it could be observed that only a few MRSA cells

17

were stained with PI in the control and treated with AuNSs@Van (without NIR light

18

irradiation). In contrast, a large number of MRSA cells showed red fluorescence when

19

treated with AuNSs@Van under NIR laser, which implied that the cell

20

membranes/walls were destroyed and lost integrity. Furthermore, the leakage of

21

cytoplasmatic content, including nucleic acid and protein, was detected. Figure S3

22

presented the results of released nucleic acid and protein of MRSA treated with

23

different concentrations of AuNSs@Van under NIR light irradiation. In Figure S3A, 22


Page 22 of 38

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1

it could be seen that after treatment with different concentrations of AuNSs@Van (80,

2

100, and 120 µg/mL) under NIR light irradiation, the protein strips appeared, besides,

3

the corresponding data shown in Figure S3B. In Figure S3C, it illustrated that

4

treatment with AuNSs@Van plus NIR light irradiation led to the occurrence of

5

nucleic acid strips. The above results indicated that AuNSs@Van plus NIR light

6

treatment caused the destruction of bacterial cell membranes, thereby releasing

7

bacterial contents and ultimately leading to bacterial death.

8 9

Figure 6. (A) SEM images of MRSA treated with AuNSs@Van with or without NIR light

10

irradiation. (a) The untreated group is used as the control. (b) A representative SEM image of

11

MRSA treated with AuNSs@Van but without 808 nm light, on the surface of MRSA. (c) A

12

typical SEM image of MRSA treated with AuNSs@Van (80 µg/mL) plus 808 nm light irradiation

13

(2.5 W/cm2, 10 min), indicating the destroyed bacterial structure. (B) Fluorescent and bright field

14

images of MRSA stained by PI following various treatments, bar=20 µm. 23



1

The superior bacteria capturing ability of AuNSs@Van synergistically improved its

2

photothermal efficiency, and the antibacterial rate was close to 99% after the NIR

3

light irradiation for 10 min at 80 µg/mL. A possible explanation for this as follows:

4

the capturing ablity of Van toward the wall of MRSA cells and the photothermal

5

effects of AuNSs after absorption of 808 nm light (the release of the energy as heat in

6

situ),

7

moderate temperature (for instance, 37 ℃) is suitable for the growth of bacteria;

8

however, if the temperature increased to ~ 50℃ or > 50 ℃, the growth of bacteria

9

would be affected, even leading to their death. The heat generated by AuNSs@Van

10

under 808 nm light irradiation was able to increase the solution temperature to ~60 ℃.

11

At this high temperature, the enzymes necessary for bacterial metabolism were

12

denatured, which hindered intracellular reactions, destroyed the cell membrane,

13

prevented the bacteria from performing normal life activities, and finally resulted in

14

bacterial death.50

15

3.6 In Vivo antibacterial activity of AuNSs@Van irradiated by NIR light

16

The biocompatibility of AuNSs@Van was examined by MTT cytotoxicity assay.51 In

17

Figure S4, the viability of Hela cells was observed to be >90% even at 150 µg/mL of

18

AuNSs@Van, which indicated AuNSs@Van had good biocompatibility. Considering

19

the antibacterial activity in vitro and the MTT results, 80 µg/mL (AuNSs@Van) was

20

selected for the experiment in vivo. In order to demonstrate the potential clinical

21

application of the obtained antibacterial nanoparticles in vivo, MRSA-infected mice

22

models were established. Figure 7A showed the process of wound repair.

48,49

besides, the Van also showed a weak antibacterial effect. Generally, a

24


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1

Specifically, the AuNSs@Van solutions (80 µg/mL) were applied to the infected

2

wound, followed by irradiation of 808 nm NIR light; Van (a conventional antibiotic,

3

1.1 µg/mL) was used as a control. The circular wounds of mice were generated by

4

knife injury and infected with 100 µL MRSA (107 CFU/ mL), followed by different

5

treatments: AuNSs@Van (808 nm, 2.5 W/cm2, 10 min), Dark treatment, Van, and

6

PBS.52 Figure 7B displayed the healing process of wounds, including the wound

7

shape at 0, 1, 3, 5, and 7 d of treatment. After 7 days, compared with the control

8

groups (PBS, Van or Dark treatment), the wound area in the group of AuNSs@Van

9

obviously reduced (11.98 %) in contrast to over 45% in the other groups (Figure 7C).

10

This proved that the treatment of AuNSs@Van promoted the healing of wound. The

11

antibacterial effect was assessed by collecting the bacteria at the wounds and the

12

results were shown in Figure 7D. It could be seen that, in the AuNSs@Van group, the

13

number of colonies grew on the agar plates became less and less with time increased,

14

in contrast to no significant decline in the number of colonies in the other groups. The

15

relative bacterial survival rate was quantified and shown in Figure 7E, with the rate

16

(54.5%, 28.94%, and 5.26%) in the group of AuNSs@Van being obviously lower

17

than that in the control groups at 3, 5, and 7 d of treatment. Overall, AuNSs@Van

18

plus NIR light expressed satisfactory antibacterial effect in vivo. Moreover,

19

histological analysis of the infected area was conducted during the treatment to

20

evaluate the healing ability of mice in infected wound. Optical micrographs were

21

taken after H&E staining and Massosn trichrome staining of histological sections,

22

respectively. Infected tissues are prone to generate neutrophils, which can be stained 25



1

in blue.53 As exhibited in Figure 7F, after 7-day, there were still some blue spots in

2

the wounds in the control groups (PBS, Van, and Dark treatment). In contrast, the

3

blue area was obviously reduced in the wound treated with AuNSs@Van under NIR

4

light irradiation. Hence, the treatment with AuNSs@Van plus NIR light alleviated the

5

inflammation of infected area. In addition, Masson’s trichrome staining was employed

6

to evaluate the formation of collagen fiber (blue) during the wound healing process.

7

As displayed in Figure 7F, it could be observed that there were many blue spots

8

(established collagen fbers) presented in the group of AuNSs@Van, but very few blue

9

spots could be found in the control groups (PBS, Van, and Dark treatment). As blue

10

represents collagen fibers, the results suggested that the group treated with

11

AuNSs@Van formed more connective tissues than the other three control groups,

12

which was very beneficial to the healing of the wounds. Furthermore, the main organs

13

collected from AuNSs@Van-treated (80 mg/mL) mice (after 7-day treatment),

14

including the heart, liver, spleen, lungs, and kidneys, were stained with H&E to

15

evaluate the toxicity of AuNSs@Van in vivo.

16

26


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1 2

Figure 7. In vivo therapeutic effect of AuNSs@Van against MRSA-infected mouse model. (A)

3

Schematic illustration for the construction of mouse model and the treating process. (B)

4

Photographs of wounds and (C) corresponding wound area from the four groups with different

5

treatments after 0, 1, 3, 5, and 7 d. (D) LB-agar plate images of surviving bacteria and bacterial

6

inhibition rate (E) of wounds from the four groups at different times during the therapeutic process.

7

(F) Histologic analysis of wound tissue samples obtained from the MRSA-infected mice by H&E

8

and Masson’s trichrome staining after 7 days of treatment. (Ⅰ-1~Ⅰ-4) H&E staining, (Ⅱ-1~Ⅱ-4)

9

Masson’s trichrome staining, (1) Control, (2) Dark, (3) Van, and (4) AuNSs@Van. Error bars are 27



1

taken from three mice per group.

2

As exhibited in Figure 8, no obvious inflammatory lesions and damages were

3

observed in them, which implied that very weak toxicity of AuNSs@Van in vivo. The

4

overall results indicated that AuNSs@Van could serve as a good antibacterial agent to

5

inhibit the growth of bacteria with minimal toxicity.

6 7

Figure 8. In vivo toxicity of AuNSs@Van. H&E staining images of the major organs, including

8

heart, liver, spleen, lung, and kidney of MRSA-infected mice obtained by slicing for H&E staining

9

at 7 d of experiment.

10

4. CONCLUSIONS

11

In summary, we had successfully synthesized a nanocomposite: vancomycin-modified

12

gold nanostars (AuNSs@Van). This work had demonstrated that AuNSs@Van with

13

absorption ability in the NIR region could be employed as a photothermal agent,

14

which was able to recognize MRSA (Gram-positive bacteria) and further effectively 28


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1

inhibit the growth of MRSA under NIR light irradiation. Additionally, the low

2

concentration (less than MIC: 2 µg/mL) of Van (1.1 µg/mL) in AuNSs@Van (80

3

µg/mL) not only had the capacity of targeting MRSA, but also presented antibacterial

4

effect to some extent. Our strategy of designing an antibacterial agent with high

5

selectivity and low cytotoxicity made AuNSs@Van a promising agent for combating

6

MRSA infection, and with less potential to induce bacterial resistance. But,

7

AuNSs@Van also have some limitations, such as, AuNSs@Van may be less effective

8

against

9

modification is made to AuNSs@Van, more widely application would be available

vancomycin-resistant

Gram-positive

bacteria,

hence,

if

appropriate

10

for AuNSs@Van in antibacterial field.

11

Supporting Information

12

The Supporting Information is available free of charge on the ACS Publications

13

website at DOI: 10.1021/acsami.XXXXXXX

14

Quantification of vancomycin, antibacterial effects of free Van, the leakage of

15

intracellular substance, cell viability.

16

Corresponding Author

17

* Corresponding Author E-mail: [email protected]

18

Notes

19

The authors declare no competing financial interest.

20

ACKNOWLEDGMENTS

21

The authors are grateful to the financial support by the National Natural Science

22

Foundation of China (21807036, 21778020), Sci-tech Innovation Foundation of 29



1

Huazhong Agriculture University (2662017PY042, 2662018PY024), Science and

2

Technology Major Project of Guangxi (Gui Ke AA18118046). We are also thankful

3

to Prof. Hanchang Zhu for editing of the language.

4

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Table of Contents One stone with two birds: functional gold nanostar for targeted combination therapy of drug resistant Staphylococcus aureus infection Huajuan Wang,a Zhiyong Song,b Shuojun Li, b Yang Wu c and Heyou Han * a,b,c

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One Stone with Two Birds: Functional Gold Nanostar for Targeted

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