Journal Pre-proof

Ultrafast discrimination of Gram-positive bacteria and highly efficient photodynamic

antibacterial therapy using near-infrared photosensitizer with aggregation-induced
emission characteristics

Michelle M.S. Lee, Wenhan Xu, Liang Zheng, Bingran Yu, Anakin C.S. Leung, Ryan
T.K. Kwok, Jacky W.Y. Lam, Fu-Jian Xu, Dong Wang, Ben Zhong Tang
PII: S0142-9612(19)30681-7
DOI: https://doi.org/10.1016/j.biomaterials.2019.119582
Reference: JBMT 119582

To appear in: Biomaterials

Received Date: 12 May 2019

Revised Date: 25 September 2019
Accepted Date: 25 October 2019

Please cite this article as: Lee MMS, Xu W, Zheng L, Yu B, Leung ACS, Kwok RTK, Lam JWY,
Xu F-J, Wang D, Tang BZ, Ultrafast discrimination of Gram-positive bacteria and highly efficient
photodynamic antibacterial therapy using near-infrared photosensitizer with aggregation-induced
emission characteristics, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119582.

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Ultrafast Discrimination of Gram-Positive Bacteria and Highly Efficient

Photodynamic Antibacterial Therapy Using Near-Infrared Photosensitizer with

Aggregation-Induced Emission Characteristics

Michelle M. S. Leea,b,1, Wenhan Xub,1, Liang Zhengc,1, Bingran Yuc, Anakin C. S.

Leungb, Ryan T. K. Kwokb, Jacky W. Y. Lamb, Fu-Jian Xuc, Dong Wanga,*, Ben Zhong

Tangb,*

a: Center for AIE Research, College of Materials Science and Engineering, Shenzhen

University, Shenzhen 518060, China

b: Department of Chemistry, Hong Kong Branch of Chinese National Engineering

Research, Center for Tissue Restoration and Reconstruction, Institute of Molecular

Functional Materials, State Key Laboratory of Neuroscience, and Division of

Biomedical Engineering and Division of Life Science, The Hong Kong University of

Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077, China.

c: Beijing Advanced Innovation Center for Soft Matter Science and Engineering,

Beijing University of Chemical Technology, Beijing 100029, China

*Corresponding author.

Email addresses: wangd@szu.edu.cn (D.W.), tangbenz@ust.hk (B. Z. T)

Keywords: bacterial discrimination, photodynamic antibacterial therapy,

anti-infection, aggregation-induced emission

Abstract

With the increase of bacterial infections in clinical practice, it becomes a public

health problem which has aroused worldwide attention. Fluorescence imaging-guided

photodynamic antibiosis has recently emerged as a promising protocol to solve this

problem. However, developing a super powerful fluorescent material allowing facile

preparation, long emission wavelength, rapid bacterial discrimination, washing-free

staining, and high photodynamic antibacterial efficiency in a single entity, is highly

desirable but remains challenging. In this study, we utilize for the first time a

water-soluble near-infrared (NIR) emissive luminogen with aggregation-induced

emission (AIE) characteristics, namely TTVP, for simultaneous dual applications of

Gram-positive bacteria discrimination and photodynamic antibiosis. TTVP is able to

selectively target Gram-positive bacteria over Gram-negative bacteria through a

washing-free procedure after only 3 s incubation period, which is at least 100-fold

shorter than those of previously reported protocols, implying ultrafast bacterial

discrimination features. Meanwhile, TTVP exhibits extremely high reactive oxygen

species generation efficiency, which is far superior to that of most popularly used

photosensitizers, representing one of the best candidates for photodynamic antibiosis.

In vitro and in vivo results demonstrate that TTVP provides extraordinary

performance on photodynamic antibacterial therapy. This study thus offers a blueprint

for the next generation of antibacterial materials.

1. Introduction

The population of bacteria on earth is approximately 5 x 1030, which largely

exceeds the sum of animals and plants [1,2]. Bacteria are very adaptive to

environmental changes, which can survive and reproduce in harsh environment.

Although most bacteria are harmless or even beneficial to human, considerable

species are infectious and can even cause fatal diseases [3,4], which has become a

global health issue and provoked an urgent need to discover effective bacterial

discrimination and anti-bacterial strategies.

Rapid and reliable discrimination of bacteria is crucial for anti-bacterial

application. Conventional approaches for discriminating bacteria include

Gram-staining test, plate-culture, polymerase chain reaction, gene-related techniques

and immunological methods [5-7]. Indeed, these methods are generally

time-consuming, costly and laborious, as well as require sophisticated instruments

[8,9]. In the meantime, antibiotics have become the most commonly used practice to

treat bacterial infections in recent years [10-13]. Despite the intrinsic bacterial killing

power and easy access, the overuse and misuse of antibiotics have largely suppressed

their effectiveness and thus antibiotic resistance to certain bacteria has evolved

[14,15]. According to the World Health Organization (WHO), antimicrobial

resistance is becoming a serious threat now happening in every region of the world

[16-18]. Therefore, developing alternative effective bacterial diagnosis and killing

methods without causing antimicrobial resistance remains an important and

challenging task with very limited success achieved.

 Fluorescence imaging technology has recently captivated much interest in

bacterial detection by virtue of its non-invasiveness, superb sensitivity, low cost and

simple operation [19-23]. Moreover, as a non-invasive therapy modality with high

spatiotemporal precision, photodynamic inactivation that is driven by activating

photosensitizer (PS) to generate cytotoxic reactive oxygen species (ROS) to induce

bacterial inactivation upon light irradiation [24-26], has been developed to serve as a

promising alternative for antibacterial practice, mainly because photodynamic

antibiosis does not induce antimicrobial resistance [27,28]. In this context,

fluorescence imaging-guided photodynamic antibiosis involving the use of PSs could

be a promising alternative protocol [29-31]. However, conventional PSs suffer from

inherent fluorescence quenching with reduced ROS generation upon aggregates

formation, which seriously impedes their practical applications [32]. Fortunately, the

emergence of luminogens with aggregation-induced emission (AIE) features has

provided a powerful solution to solve this problem [33]. AIE refers to a unique

phenomenon that luminogens are non-emissive or weakly emissive in the

molecularly dissolved state but are induced to emit intensely in the aggregates

resulting from the restriction of the intramolecular motions (RIM), revealing intrinsic

capability of AIE luminogens (AIEgens) to perfectly work at high concentrations with

high fluorescence efficiency and extraordinary photo-stability [34,35]. In particular,

water-soluble AIEgens perform outstandingly in aqueous biological system, which do

not require washing procedures and give high signal-to-noise ratio in bioimaging

[36-38]. Furthermore, AIEgens also usually exhibit efficient photosensitization ability

in aggregate state, simultaneously allowing efficient ROS generation. Therefore, AIE

has opened a venue to effective bacterial discrimination and anti-bacteria.

Notwithstanding a handful of AIEgens have been successfully utilized in

bacterial imaging and inactivation in the recent few years, current situation is still far

from ideal [39,40]. All previously reported AIEgens for bacterial study possess these

collective properties: (1) water-insoluble feature, which causes aggregation of

AIEgens in bacterial culture medium, and thus tedious washing procedures are

required for removing non-internalized AIE aggregates; (2) short emission

wavelengths, which exhibit low penetration, severe photo-damage to biological

structure and high background autofluorescence; (3) relatively long incubation period

(>5 min), which is time-consuming and often causes inaccurate detection output

owing to the change of bacterial status; (4) insufficient efficiency for bacterial

inactivation, in particular for in vivo anti-bacterial application [41-44].

In this contribution, we used an AIE-active molecule sharing good

water-solubility, near-infrared (NIR) emission and extremely high ROS generation

efficiency for bacterial discrimination and antibacterial study. This AIEgen can

discriminate Gram-positive bacteria through a washing-free and ultrafast staining

procedure, in addition, it provides significantly prominent performance in

photodynamic antibacterial application in vivo and in vitro towards Gram-positive

bacteria (Fig. 1).

Fig. 1. Schematic illustration of using AIEgen TTVP for ultrafast discrimination of
Gram-positive bacteria and highly efficient photodynamic antibacterial therapy.

2. Experimental Section

2.1 Materials and methods

Ultra-pure water was supplied by Milli-Q Plus System (Millipore Corporation,

United States). Phosphate buffered saline (PBS) was purchased from Thermo Fisher

Scientific. 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA), Ce6 and

Rose Bengal were purchased from Sigma-Aldrich and used as received. TTVP was

synthesized according to the literature method [45].

2.2 Bacterial imaging

A single colony on solid culture medium [Luria broth (LB) for E. coli, S. aureus

and S. epidermidis was transferred to 5 mL of liquid culture medium and grown at

37 °C for 10 h. The concentrations of bacteria were determined by measuring the

optical density at 600 nm (OD 600) and 109 colony forming unit (CFU) of bacteria

was transferred to 1.5 mL EP tube. Bacteria were harvested by centrifugation at 7000

rpm for 3 min, followed by removal of supernatant. 1 mL of TTVP solution in PBS at

a certain concentration was then added into the EP tube. After dispersing with vortex,

the bacteria were incubated at room temperature for a certain time. To take confocal

images, 2 µL of stained bacteria solution was transferred to glass slide and then

covered by a coverslip. The image was collected using 63x objective. The bacteria

were imaged using Zeiss laser scanning confocal microscope (LSM7 DUO) under the

condition: Excitation: 488 nm, Emission range: 600-740 nm.

2.3 Reactive oxygen species measurement

ABDA was used to detect the reactive oxygen species generation of TTVP, rose

Bengal and Ce6 upon light irradiation. The absorbance of each TTVP, rose Bengal

and Ce6 solution (1 x 10-6 M) was firstly set as blank. Afterwards, 1 x 10-5 M of

ABDA was mixed to each solution (DMSO/water (v:v) = 1/100) in a dark room, and

the absorbance of solution was measured immediately. The solution mixture was then

irradiated using white light lamp at intervals of 1 min until 6 min. After each

irradiation interval, the absorbance of solution was recorded immediately. The

absorbance change of ABDA alone in 6 min light irradiation time was also checked as

control.

2.4 Photodynamic bacterial killing study

108 CFU bacteria were dispersed in 500 µL of PBS. After incubation with certain

concentration of TTVP for 10 min. After that, the bacteria were dispersed in PBS and

exposed to white light lamp for 30 min, while the control groups were put in the dark

for 30 min. The viability of bacteria was quantified by plate-count method. The
bacteria were diluted with a dilution factor of 105. Then 100 µL of the bacteria

solution was evenly spread on a LB agar plate, which was then subjected to culturing

at 37 °C for 18 h before quantification and photo taking.

2.5 SEM imaging

The concentration of S. epidermidis was diluted to an optical density of 0.2 at

OD600. Then the bacteria were incubated with 2 µM TTVP for 30 min and

illuminated with white light for 30 min, followed by drying, and collection of SEM

images. Bacteria without treatment were also imaged under SEM for comparison.

2.6 Zeta potential measurement

Bacteria were incubated with TTVP for 3 s, 20 min and 40 min at room

temperature, respectively. The samples were harvested by centrifugation, followed by

resuspension in H2O for Zeta potential measurements with Nano ZS (ZEN3600). The

bacteria without TTVP treatment were also measurement under the same condition as

control.

2.7 In vivo antibacterial test on infected skin wound

16 of Wistar mice were divided into four groups: (1) control group without

bacterial infection and treatment, (2) S. aureus-infected wounds without treatment, (3)

S. aureus-infected wounds treated by TTVP only, and (4) phototherapy group of

TTVP plus white light irradiation. The mice were anesthetized by intraperitoneal

injection of 10% chloral hydrate. Then, two 1.5 × 1.5 cm2 open excision wounds were

cut on the both sides of the spine. 50 µL of S. aureus suspension (2×108 cfus) and 50

µL of TTVP (2 µM) was inoculated over each wound and covered with sterile
nonwoven fabrics. Then the wound was irradiated by white light with a power density

of 60 mW cm−2 for 30 min. On the 1st, 3rd and the 7th day after surgery, the entire

wound with adjacent normal skin were harvested. The infectious tissues were

separated and homogenized in normal saline. The homogenates were diluted 1000

times with normal saline. 20 µL of the bacteria solution was sprayed onto LB agar

plate and subjected to culturing at 37 °C. After 24 h, the bacterial colonies on the

plate were counted for analysis. The other tissues were fixed in 4% paraformaldehyde

for the histological analysis.

2.8 Histological examination

The collected tissues were fixed in 4% paraformaldehyde for the histological

analysis, and then embedded in paraffin. The fixed tissues were cut into slices with a

thickness of 4 mm. Next, H&E staining was carried out according to the standard

protocols as described in the previous work [30].

3. Results and discussion

3.1 Ultrafast discrimination to Gram-positive bacteria

TTVP having good water-solubility was facilely synthesized by two-step

reaction [45], and displays a maximum absorption at 480 nm, as well as AIE features

with maximum emission at 704 nm in NIR region (Fig. S1). In the preliminary

bacterial study, bacterial imaging experiment was conducted by using S. epidermidis

as Gram-positive bacteria model, which was incubated with 10 µM of TTVP for 10

min. As illustrated in Fig. S2, S. epidermidis can be clearly visualized with high

signal-to-noise ratio regardless of washing or non-washing the bacteria after staining,

indicating a washing-free bacterial staining protocol, which can be reasonably

attributed to both the water-soluble and AIE-active characteristics of TTVP. TTVP

remains at molecular state in aqueous buffer solution and shows no emission, so it

gives ultra-low background signal in bio-imaging even without undergoing washing

procedures. The concentration influence involving 10, 5, 2 and 1 µM of TTVP was

then investigated, as shown in Fig. S3, the brightness of TTVP emission in S.

epidermidis did not obviously decline even when the concentration was down to 1 µM.

Moreover, the evaluation of incubation period was carried out by treating S.

epidermidis with 1 µM of TTVP with different incubation time period from 5 min to a

few seconds at room temperature (Fig. 2A and 2B). It was observed that the red

emission of TTVP-stained S. epidermidis can be clearly detected showing excellent

image contrast to the background after very fast incubation (approximately 3 s). As

shown in Fig. S4, the Zeta potential of S.epidermidis is -19.18 mV, the surface of

Gram-positive bacteria is negatively charged. Therefore, the ultrafast bacterial

imaging could result from excellent monodispersity of TTVP in the culture media,

and strong electrostatic interaction between positively charged TTVP and negatively

charged S. epidermidis, as well as extremely sensitive fluorescence enhancement of

AIEgen upon the restrictions of intramolecular motions. Noteworthily, “halo” patterns

around the exterior of the bacteria from the emission of TTVP was observed (Fig. 2B

and S5), suggesting that TTVP preferentially accumulates on the outer portion of S.

epidermidis. Furthermore, this ultrafast washing-free bacterial imaging protocol was

utilized to target Gram-positive S. aureus and Gram-negative E. coli. The imaging

outcome shows that TTVP efficiently “light up” S. aureus with a very high
signal-to-noise ratio (Fig. 2B, S6), on the contrary, the emission of E. coli incubated

with TTVP was barely detected (Fig. 2B), implying excellent staining-specificity of

TTVP towards Gram-positive bacteria.

40 min, and only partial E. coli was stained even incubation time is up to 40 min (Fig.

S7). These results solidly demonstrate the extraordinary performance of TTVP for

accurate and ultrafast discrimination of Gram-positive bacteria through fluorescence

technique. It seems reasonable to infer that the effective discrimination benefits from

the significant structural difference of outer envelopes between Gram-positive and

Gram-negative bacteria. The outer envelope of Gram-negative bacteria that consists

of an outer membrane, crosslinked peptidoglycan network and a cytoplasm membrane,

is much more complicated than that of Gram-positive bacteria having only

peptidoglycan network and a cytoplasm membrane [43,46-50]. Therefore,

Gram-positive bacteria are short of efficient barriers to prevent the insertion of

molecules, consequently achieving ultrafast differentiation of Gram-positive bacteria

over Gram-negative bacteria. Aiming to further clarify the efficient bacterial

discrimination outcomes, Zeta potentials of both Gram-positive bacteria S.

epidermidis and Gram-negative bacteria E. coli with TTVP incubation for different

time period (3 s, 20 min and 40 min) were measured. As illustrated in Fig. S4, before

and after TTVP incubation for different time period, the Zeta potential of S.

epidermidis maintained almost constant. Considering that TTVP is able to efficiently

“light up” S. epidermidis after only 3 s incubation period, the almost constant Zeta

potential of S. epidermidis strongly indicates that TTVP can efficiently insert into the

cell wall of S. epidermidis in 3 s and prevent the positive charges exposure to the

bacterial surface. In the case of E. coli., the Zeta potential increased significantly from

-52.92 to -34.32 mV after 3 s TTVP incubation, suggesting that positively charged

TTVP could be effectively attracted to the surface of E. coli. due to electrostatic

interaction, but cannot efficiently insert into the cell wall of E. coli. in such short

period owing to its much more complicated outer envelope of E. coli. TTVP that was

attracted to the surface of E. coli. can still undergo free intramolecular motions as

their motions are not fully restricted compared to the cell wall insertion, thus no

emission could be observed from confocal images. Furthermore, as the incubation

time prolonged from 3 s to 40 min, the Zeta potential of E. coli. gradually recovered

to -53.30 mV. This can be resulted from the gradual insertion of TTVP to the cell wall

of E. coli. with longer incubation time. After 40 min incubation time, almost all

TTVP are inserted to the cell wall of Gram-negative bacteria, leaving the bacteria

surface a negatively charged environment similar to the bacteria without any

treatment. These results are in good accordance with the imaging outcomes shown in

Fig. S7. Apart from discrimination of Gram-positive bacteria over Gram-negative

bacteria, TTVP can achieve selective staining to Gram-positive bacteria over normal

cells. Fig. S8 shows the in vitro staining result of TTVP towards the mix culture of

HaCaT cell (a normal human keratinocyte cell line that covers the majority of

epidermis in the outermost layer of skin), and S.aureus. It was observed that 2 µM of

TTVP could selectively target Gram-positive bacteria giving bright red emission, and

no HaCaT cells were stained by TTVP under the conditions. This could be attributed

to the less negatively charged surface of HaCaT cells in comparison to S.aureus,

which was determined by the less negative Zeta potential value of HaCaT cells than

that of S.aureus (-8.07 mV versus -29.34 mV). The result provides a solid evidence

for the selectivity of TTVP to Gram-positive bacteria over normal skin cells, which
suggests potential application of TTVP to in vivo photodynamic skin wound bacterial

inactivation therapy.

3.2 Photodynamic antibacterial study

Inspiring by the remarkable performance for ultrafast discrimination of

Gram-positive bacteria, TTVP was further employed as PS for photodynamic

antibacterial evaluation using S. epidermidis, S. aureus and E. coli. as bacteria models.

Considering the critical role of ROS in photodynamic inactivation, in the preliminary

experiment, the ROS generation efficiency of TTVP was assessed by the use of

ABDA as indicator. ABDA is a commercially available probe that can be

decomposed in the presence of ROS and show decrease in absorbance. It was

observed that absorption intensity of ABDA rapidly declined upon white light

irradiation at a power density of 60 mW/cm2 in the presence of 1 µM of TTVP, and

almost all the ABDA were consumed after 6 min light exposure (Fig. 3A and S9).

Furthermore, as two of the most widely used and reputable PSs for photodynamic

therapy, Ce6 and Rose Bengal were tested under the same conditions for comparisons.

As depicted in Fig. 3A and S9, about 30% and 70% of ABDA were decomposed by

Ce6 and Rose Bengal respectively, strongly revealing the superior property of TTVP

comparing with both Ce6 and Rose Bengal. To the best of our knowledge, TTVP with

80.16% of 1O2 quantum yield represents one of the best PSs in terms of ROS

production comparing with various previously reported ones [45]. The

dose-dependent photodynamic antibacterial study was then performed upon treatment

with various TTVP concentrations (0, 0.125, 0.25, 0.5, 1 µM) in the presence or
absence of light illumination. As shown in Fig. 3B and 4, TTVP exerted negligible

toxicity to both Gram-positive and Gram-negative bacteria without light irradiation

process in all tested concentrations. After 30 min exposure to white light, the viability

of TTVP-treated Gram-positive S. aureus and S. epidermidis rapidly dropped, and

almost complete bacterial inactivation was realized by utilizing 0.125 µM and 0.25

µM of TTVP, respectively (Fig. 3B and 4). Comparing with other PSs used for

photodynamic antibacterial study, TTVP holds the lowest working concentrations for

completely inactivating Gram-positive bacteria. Moreover, scanning electron

microscopy (SEM) of S. epidermidis stained with 1 µM of TTVP reveals that the

bacteria without light treatment consisted of well-defined and clear membrane borders

with smooth morphology, as comparison, morphology of the bacteria treated with

light became irregular and was greatly distorted with blurred bacterial membrane

borders, which unambiguously verify that the combination of TTVP and light

illumination can powerfully deconstruct Gram-positive S. epidermidis (Fig. 3C). In

contrast, E.coli remained over 80% viability when 0.5 µM of TTVP was used. The

results solidly demonstrate that TTVP is remarkably prominent for selective

inactivation of Gram-positive bacteria through photodynamic antibacterial pathway,

implying its promising practice as effective in-vivo bactericide.

(A) 100 (B) S. epidermidis w/o light (C) Without treatment

140 S. epidermidis + light
S. aureus w/o light S. aureus + light
80 120 E.coli w/o light E.coli + light
100
A/A0 (%)

60
Viability (%)

80

40 60 After treatment
Without PS 40
20 Ce6
Rose Bengal 20
TTVP
0 0
0 60 120 180 240 300 360 0 0.125 0.25 0.5 1 1 M
Time (s) Concentration (µM)
Fig. 3. ROS generation and photodynamic antibacterial study. (A) Decomposition
rates of ABDA in the presence of different PSs under light irradiation, where A0 and
A are the absorbance of ABDA at 378 nm. (B) Statistical analysis of photodynamic
antibacterial data in Fig. 4. (C) SEM images of S. epidermidis without and with 1 µM
TTVP and 30 min light irradiation treatment.

Fig. 4. Bacterial killing ability of TTVP. Plates of S. epidermidis, S. aureus and E.

coli after incubated with different concentrations of TTVP for 10 min without or with
light irradiation (60 mW/cm2) for 30 min.

3.3 In vivo photodynamic bacterial killing in wound infection

To evaluate photodynamic antibacterial efficiency of TTVP in combating skin

wound infection, S. aureus-infected wound model of Wistar rats was established. The

rats bearing infected full-thickness skin wounds were randomly assigned to 3 groups,

i.e., (1) TTVP plus light group treated by both 2 µM of TTVP and white light
irradiation at 60 mW/cm2 for 30 min, (2) TTVP group treated by only 2 µM of TTVP,

and (3) the group without any treatment. The photos of the wounds at different time

points (1st, 3rd and 7th day) were captured (Fig. 5A). Certain degree of pyosis in

wounds was observed on day 1, owing to the inoculation of S. aureus, and both TTVP

only and nontreatment groups exhibited severer pyosis than TTVP plus light group.

On day 3, the pyosis around the wounds almost disappeared in the TTVP plus light

group, while pyosis can be clearly observed in the other two infection groups. On day

7 post-treatment, TTVP plus light group showed the best healing status in the wounds,

which is even comparable to the control group where no bacteria were inoculated. To

quantitatively evaluate the photodynamic antibacterial activity of TTVP, the bacteria

in the pus around the wounds in each group were cultured and counted at different

time points, respectively. As displayed in Fig. 5B and 5C, the bacterial amount in

wounds of TTVP plus light group was significantly low on day 1, and almost no

bacterial colonies were determined on days 3 and 7. For the TTVP only group, the

amount of bacteria gradually decreased from day 1 to 7, and bacteria could be still

detected in the wounds on day 7. On the contrary, a large number of bacteria in the

wounds of the group without any treatment was observed on day 1, 3 and 7. The

results show that TTVP is capable of inactivating S. aureus in vivo under dark

conditions but the efficiency is much lower than light-driven therapy, solidly

revealing the high photodynamic antibacterial efficiency of TTVP. After the 7 days

treatment, the wounds of the rats were harvested and subjected to hematoxylin and

eosin (H&E) staining. Neutrophils, generated in infected tissues, were stained in blue.

As illustrated in Fig. 6, both TTVP only group and the group without treatment
possessed much higher amount of neutrophils, especially for day 3 post-treatment,

indicating more serious infection of wound in these two groups. In addition, newly

formed vessels and fibroblasts were obviously observed for the wounds of both

control and TTVP plus light groups, suggesting the success of wound healing.

Fig. 5. In vivo photodynamic antibacterial study towards S. aureus. (A) Wound

healing images of mice on day 1, 3 and 7. Scale: 1 cm. (B) Images of bacterial
colony-forming units obtained from different tissues of mice treated under various
conditions. (C) Number of bacterial colony-forming units obtained from different
tissues of mice treated under various conditions.
Fig. 6. H&E staining of the wound tissues harvested from different rats on 1, 3, 7th
day post-treatment. (blue arrows: inflammatory cells; red arrows: new blood vessels;
scale: 100 µm).

4. Conclusions

In summary, we have developed an extremely simple and effective protocol for

simultaneous dual applications of Gram-positive bacteria discrimination and

photodynamic antibiosis by using a water-soluble NIR emissive AIEgen (TTVP).

Benefiting from its excellent monodispersity in the culture media, strong electrostatic

interaction with Gram-positive bacteria and AIE features, TTVP can rapidly

discriminate Gram-positive bacteria over Gram-negative bacteria through

fluorescence technique, after ultrafast incubation as short as a few seconds and a

washing-free staining procedure. Remarkably, the presented incubation period for

bacteria discrimination is at least two orders of magnitude shorter than previously

reported fluorescent probes. TTVP can also efficiently function to generate ROS,

even is far superior to both Rose Bengal and Ce6, which are two of the most effective

and popularly used PSs. Along with both its strong absorption in visible light region
and rapid membrane insertion ability, TTVP with a concentration down to 0.125 µM

is capable of completely inactivating Gram-positive bacteria upon white light

irradiation, making it a super powerful optically mediated antimicrobial agent. More

importantly, TTVP exhibits prominent performance for in vivo photodynamic

antibacterial therapy on a skin wound infected rat model. This successful example

will provide an efficient strategy for the rational design of easy-to-perform and

time-saving agents for bacterial discrimination in potential clinical applications, and

stimulate the development of high-performance antibacterial materials to defeat

increasingly serious bacterial infection problem.

Acknowledgements

M.M.S.L., W.X. and L.Z. contributed equally to this work. This work was

partially supported by the Natural Science Foundation of China (Grant No. 21801169),

the President Fund of Shenzhen University Foundation (848-0000106), the National

Basic Research Program of China (973 Program; 2013CB834701 and

2013CB834702), the University Grants Committee of Hong Kong (AoE/P-03/08).

Animal experiments were approved by the China Committee for Research and

Animals Ethics in compliance with the law on experimental animals.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

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