Phage-Guided Targeting, Discriminative Imaging, and Synergistic Killing of Bacteria by AIE Bioconjugates

Article abstract of DOI:10.1021/jacs.9b12936

New agents with particular specificity toward targeted bacteria and superefficacy in antibacterial activity are urgently needed in facing the crisis of worldwide antibiotic resistance. Herein, a novel strategy by equipping bacteriophage (PAP) with photody

Full text of DOI:10.1021/jacs.9b12936

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Article
Phage-Guided Targeting, Discriminated Imaging and
Synergistic Killing of Bacteria by AIE Bioconjugates
Xuewen He, Yujun Yang, Yongcan Guo, Shuguang Lu, Yao Du, Jun-Jie Li, XUEPENG
ZHANG, Nelson L. C. Leung, Zheng Zhao, Guangle Niu, Shuangshuang Yang, Zhi
Weng, Ryan T. K. Kwok, Jacky W. Y. Lam, Guoming Xie, and Ben Zhong Tang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12936 • Publication Date (Web): 30 Jan 2020
Downloaded from pubs.acs.org on January 30, 2020
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Phage-Guided Targeting, Discriminated Imaging and Synergistic
Killing of Bacteria by AIE Bioconjugates
Xuewen He,†,‖,# Yujun Yang,*,‡,# Yongcan Guo,¶ Shuguang Lu,§ Yao Du,‡ Jun-Jie Li,‡ Xuepeng
Zhang,†,‖ Nelson L. C. Leung,†,‖ Zheng Zhao,†,‖ Guangle Niu,†,‖ Shuangshuang Yang,‡ Zhi Weng,‡ Ryan
T. K. Kwok,†,‖ Jacky W. Y. Lam,†,‖ Guoming Xie,*,‡ and Ben Zhong Tang*,†,‖,$
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†Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Centre for Tissue Restoration
and Reconstruction, Institute for Advanced Study, Division of Life Science, Department of Chemical and Biomedical
‡
Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Key
Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing
Medical Laboratory Microfluidics and SPRi Engineering Research Center, Chongqing Medical University, Chongqing
400016, China. ¶Clinical Laboratory, Traditional Chinese Medicine Hospital Affiliated to Southwest Medical University,
§
Luzhou 646000, China. Department of Microbiology, College of Basic Medical Science, Army Medical University,
Chongqing 400038, China. ‖HKUST-Shenzhen Research Institute, Shenzhen 518057, China. $NSFC Center for
Luminescence from Molecular Aggregates, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of
Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China.
ABSTRACT: New agents with particular specificity toward targeted bacteria and super efficacy in antibacterial activity, are
urgently needed in facing the crisis of worldwide antibiotic resistance. Herein, a novel strategy by equipping bacteriophage
(PAP) with photodynamic inactivation (PDI)-active AIEgens (luminogens with aggregation-induced emission property) was
presented to generate a type of AIE-PAP bioconjugates with superior capability for both targeted imaging and synergistic
killing of certain species of bacteria. The targeting ability inherited from the bacteriophage enabled the bioconjugates to
specifically recognize the host bacteria with preserved infection activity of phage itself. Meanwhile, the AIE characteristic
empowered them a monitoring functionality, and the real-time tracking of their interactions with targets was therefore
realized via convenient fluorescence imaging. More importantly, the PDI-active AIEgens could serve as powerful in-situ
photosensitizers producing high-efficiency reactive oxygen species (ROS) under white light irradiation. As a result, selective
targeting and synergistic killing of both antibiotic-sensitive and multidrug resistant (MDR) bacteria were successfully
achieved in in vitro and in vivo antibacterial tests with excellent biocompatibility. This novel AIE-phage integrated strategy
would diversify the existing pool of antibacterial agents and inspire the development of promising drug candidates in the
future.
INTRODUCTION
resistance.11,12 The situation is worsening with the rapidly
increased difficulty and cost in producing new robust
antibacterial agents.13 Thus, it is highly desirable to develop
pinpoint accurate drugs to kill certain species of harmful
pathogens. Fortunately, bacteriophage had been verified
with particular specificity to their hosts,14 and could evolve
synchronously to adapt to infect resistant bacteria.15,16
Phagotherapy, which was used before antibiotics
discovered, is now going through a revival driven by the
crisis of antibiotic resistance.17 However, the bacteriophage
alone normally shows moderate antibacterial effect,
hindering the curing of infectious diseases especially the
acute infections.18 Due to the lack of imaging moieties, the
processes of phagotherapy including target recognition,
binding and infection could not be easily monitored,
resulting in difficult real-time evaluation of therapeutic
performance. The introduction of sensitively detectable
“radar” with powerful “weaponry”, will therefore promote
phagotherapy as a deluxe ensemble for both visualization of
its infection process and their subsequent discriminative
eradication.
Bacterial infectious diseases have become one of the
greatest global challenges, which seriously threaten the
health and civilization of human beings.1,2 And increasing
evidences revealed that bacteria can also indirectly
promote the occurrence and progression of other diseases,
such as cancer.3-6 On the other hand, bacterial communities
are indispensable to human health, such as intestinal
microbes whose balances determined the nutrient
absorption from digested food in our bodies and should be
always protected from unintentional damage.7,8 Therefore,
in the antibacterial battle, promoting antibacterial agent in
terms of targeting and killing efficiency is persistently
pursued. Antibiotics have been predominately employed in
the treatment of bacterial infections since the discovery of
penicillin. However, using antibiotics, even narrow-
spectrum antibiotics, both harmful bacteria and beneficial
microbes could be killed, resulting side effects such as
diarrhea, constipation and inflammatory bowel.9,10 The
global abuse or overuse of antibiotics has promoted
bacterial evolution and intensified the antibiotic
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Fluorescence imaging technology has emerged as a function of real-time monitoring of phage-bacterium
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rapid, facile and powerful tool for visualization and tracking
of bioanalytes with merits of high sensitivity, low cost and
fast responsiveness.19-21 Surpassing traditional organic
fluorophores that have usually suffered severe
photobleaching and moderate signal-to-noise ratio, the
newly developed luminogens with aggregation-induced
emission characteristics (AIEgens) are becoming attractive
candidates for advanced biosensing and imaging
application.22-25 AIEgens display weak emission in solution,
but intensified fluorescence upon aggregation via the
restriction of intramolecular motion.26 The unique light-up
fluorescence with negligible background noise and
remarkable photostability, qualify AIEgens to be a superior
choice in long-lasting fluorescence imaging and tracking for
dynamic bioprocesses.27-29 What’s more, with intelligent
molecular designing, AIE-based materials display
exceptional photodynamic activity for the treatments of
malignant cancer and the inactivation of bacteria.30-34
However, the specific discrimination of a certain species of
bacterium, has not yet been previously demonstrated by
AIEgens, making them unsuitable for accurately removing
certain targeted bacterium.35,36 Although the bioorthogonal
method showed improvement in targeted labeling, the
incorporation of exogenous reactive groups was demanding
with challenges for large-scale applications.37,38 These
limitations propelled us to engineer AIEgens with pinpoint
specificity in order to realize the discriminative imaging and
killing of a certain species of bacterium.
interaction and particular specificity in bacterial targeting.
What’s more, the synergistic antibacterial effect with
efficiency that significantly surpassing both individuals in
the bioconjugates was achieved by integrating the PDI
activity of AIEgen and the infection ability of phage itself.
Superior performance of the bioconjugate was also
demonstrated in in vivo treatment of infectious wounds
caused by both antibiotic-sensitive and MDR P. aeruginosa.
This novel strategy by integrating specific targeting,
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discriminative
imaging,
and
synergistic
killing
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functionalities together into an AIE-phage bioconjugate, is a
promising methodology for the design of next-generation
antibacterial agents.
RESULTS AND DISCUSSION
AIEgen Synthesis, Property Characterizations, and
Bioconjugation with Bacteriophage. AIE compound TVP-
S
was designed and synthesized through three-step
reactions (Figure 1a and Figure S1). Its structure was
determined by nuclear magnetic resonance (NMR), high
resolution mass spectroscopy and X-ray diffraction (XRD,
Figure S2) as shown in the Supporting Information (SI).
Attributed to the existence of positively charged pyridinium
group and the hydrophilic carboxyl group in its structure,
TVP-S could be well dissolved in high polarity solvent
including water. More importantly, the activated carboxyl
group in TVP-S structure endows it capability to
straightforwardly react the amino group that is widespread
in the bioorganism. The obvious absorbance of TVP-S in the
range between 400 and 600 nm with a maximum at 457 nm
showed its strong capability of visible light absorption
(Figure 1b). As TVP-S could be well dissolved in solvent
with large polarity, we investigated its AIE features in
dimethyl sulfoxide (DMSO)/chloroform (CHCl3) mixtures
via varying the fractions of CHCl3. As shown in Figure 1c,
TVP-S exhibited an apparent AIE property. Fluorescence
could hardly be observed when it was dissolved in a good
solvent, DMSO, whereas strong aggregated-state emission
with 47 times enhancement in intensity appeared when
Herein, a novel strategy was presented by integrating
AIEgens with bacteriophage to form a new class of
antimicrobial bioconjugates (TVP-PAP) for the imaging and
killing of a certain species of bacterium (Scheme 1). Phage
targeting P. aeruginosa was chosen as a prototype. The AIE
molecule, TVP-S, was designed with excellent fluorescence
properties and efficient PDI activity. Through a simple
amino-carboxyl reaction, the AIEgens could be facilely
modified onto the surface of phage entity. The generated
TVP-PAP perfectly preserved the properties of both AIEgen
and phage, with intrinsic AIE fluorescence serving the
Scheme 1. Phage-guided targeting, discriminative imaging and synergistic killing of bacteria by AIE bioconjugatesa
aThe processes including: (Ⅰ) the particular recognition of bacteria by AIEgen conjugated phage; (Ⅱ) the bacterial lighting
up imaging by AIE fluorescence and the phage infections as well as the ROS generation of AIEgens; and (Ⅲ) the synergistic
killing of the target bacteria by phage infections and AIE-base photodynamic inactivation.
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the fraction of chloroform reached 99% (Figure 1d). Their
which could emit fluorescence with a “turn on” process
triggered by ROS. As depicted in Figure S6, DCFH alone was
non-emissive and remained almost constant during 10 min
white irradiation (~ 4.2 mW·cm-2). In contrast, the
emission intensity of DCFH gradually enhanced and
reached 23-fold within the same period exposure to white
light irradiation in the presence of TVP-S. In addition, up to
72.3% of singlet oxygen (1O2) quantum yield for TVP-S was
determined using 9,10-anthracenediyl-bis(methylene)
dimalonic acid (ABDA) as indicator and Rose Bengal (RB)
as standard with ~ 75% quantum yield for 1O239,40 (Figure
1e,f and Figure S7). Sodium azide (NaN3) as a 1O2
quencher41 was further added to the ABDA solution in the
presence of TVP-S. As shown in Figure S8, the
decomposition rate of ABDA slowed down gradually with
increase of the concentration of NaN3, and 2.0 mg/mL NaN3
could completely inhibited the decomposition of ABDA,
maximum emissions in the aggregation state are located at
630 nm, indicating its long-wavelength emission properties
and large Stokes shifts. The significant increase in the
absolute fluorescence quantum yield of TVP-S in
chloroform (33.3%) versus in DMSO (1.6%), further
verified its typical AIE feature. BSA and bacteria could also
induce the aggregation of TVP-S in aqueous solution with
significant emission enhancements (Figure S3 and S4).
Moreover, dynamic light scattering (DLS) analysis was
performed to confirm the aggregation process by
decreasing the solvent polarity, with obvious
increasements in the average hydrodynamic diameters
(Figure S5).
The strong absorption of TVP-S in the visible light
region could lead to the utilization of white light as the
source for PDI-based antibacterial application. The ROS
generation efficiency of TVP-S was initially determined by
using of 2′,7′-dichlorofluorescin diacetate (DCFH) as
indicator,
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implying O2 was indeed generated by TVP-S. Meanwhile,
no obvious enhancements could be observed in the signal
from hydroxyphenyl fluorescein (HBF) indicator for
hydroxyl radical (OH•) and dihydrorhodamine (DHR)
1
indicator for superoxide radical (O2•−), indicating the O2
accounted for nearly all of the ROS generated by TVP-S
(Figure S9 and S10). As the 1O2 should be stemmed from the
energy transfer process between the triplet oxygen and
triplet excited-state photosensitizer, we further checked
the existence of triplet excitons in TVP-S. As shown in
Figure S11, TVP-S exhibited strong phosphorescence
emission with a lifetime of up to 31.3 μs at 77 K, clearly
demonstrated its efficient intersystem crossing to the
triplet excited state. Theoretically, this is also quite rational
given TVP-S contain Br atom which usually boost ISC
process in organic molecular systems (the well-known
heavy atom effect42).
With the activated carboxyl group in its molecular
structure, TVP-S could facilely react with the amino group
in the outside proteins of bacteriophage and was ready to
form the AIE-featured phage bioconjugate (TVP-PAP). As
illustrated in Figure 2a, the intensified absorbance of PAP
phage in the range of 400-500 nm after AIEgen
modification, confirmed the successful bioconjugation
reaction between TVP-S and PVP. According to
concentration titrations, the molar extinction coefficients
of TVP-S and PAP were calculated to be 1.94 × 104 and 3.41
× 1010 M-1·cm-1, respectively (Figure 2b,c and Figure S12).
And the molar ratio of TVP-S to PAP in the TVP-PAP
bioconjugate was determined to be averagely 8200 AIEgen
molecules bound on one PAP entity, according to the
enhanced absorbance at 457 nm of TVP-PAP comparing to
nude PAP. Due to the robust ROS generation capability of
prepared AIE-phage bioconjugates (Figure S13), the
particular bacterial recognition, real-time tracking, and
synergistic bacterial killing were highly expectable (Figure
2d).
Figure 1. (a) Molecular structure of AIEgen (TVP-S). (b)
Normalized UV–vis spectra of TVP-S dissolved in water,
DMSO and chloroform, respectively. (c) AIE curve of TVP-S
in the mixture of DMSO and CHCl3 with varying volume
fraction. (d) Plotting of the emission maximum of TVP-S
versus the fraction of the DMSO/CHCl3 mixture. (e)
Photodegradation of ABDA with TVP-S under white light
irradiation in water. [TVP-S] = [Rose Bengal] = 5 × 10-6 M,
and [ABDA] = 2 × [TVP-S]. (f) Decomposition rates of ABDA
with light irradiation at 379 nm absorbance.
Particularly Specific Bacteria Imaging by TVP-PAP.
Taking advantage of the specific recognition feature of
phage and the unique AIE property from TVP-S, the
efficiency of
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Figure 2. (a) UV–vis spectra of the aqueous solution of TVP-PAP (9.375 × 109 PFU·mL-1), PAP (9.375 × 109 PFU·mL-1) and
TVP-S (4.26 × 10-6 mol·L-1). The inner plotting was from 16-fold concentrated TVP-PAP (1.5 × 1011 PFU·mL-1) and PAP (1.5 ×
1011 PFU·mL-1). (b) and (c) The standard absorbance curve of TVP-S and PAP, respectively. Abs457 nm and Abs256 nm could be
fitted linearly with their concentrations. Through subtraction of Abs457 nm of TVP-PAP by Abs457nm of PAP, the concentration of
modified AIEgens in the TVP-PAP conjugates could be calculated. And averagely 8200 AIEgens were bound on one PAP entity.
(d) Cartoon illumination of the synergistic effect of AIEgens modified phage for particularly specific bacterial recognition,
real-time fluorescent tracking, phage infection and AIE PDI activity integrated synergistic bacterial killing.
bacterial imaging using TVP-PAP was first examined on its
target bacteria (P. aeruginosa, abbreviated as P. a.). As
shown in Figure 3a, P. aeruginosa could be stained with
bright fluorescence upon incubating with TVP-PAP for 30
min, and the staining efficiency could reach 100% efficiency
(also shown in Figure S14). Comparing to the commercial
cell membrane staining dye, DiO, P. aeruginosa could be
light-up stained by the TVP-PAP probe with a much higher
staining efficiency (Figure S15). To test the particular
specificity of this TVP-PAP probe stemming from the
inherent recognition capability of bacteriophage, we
employed two types of bacteria, G A. baumannii
(abbreviated as A. b., Figure S16) and G+ S. aureus (Figure
S17), as control. The results showed that even the process
extending to 60 min, neither bacteria could be stained by
TVP-PAP probes. And no apparent fluorescence signals
emitted out from neither of them in the AIE fluorescence
channel.
Encouraged by this exciting result, we further
conducted real-time tracking of the bacterial staining
process in the mixed microbes including A. baumannii (G)
and host bacteria P. aeruginosa (G ) to verify the particular
selectivity of the TVP-PAP probe to its target P. aeruginosa.
Clearly, the arrow indicated A. baumannii could never be
stained throughout the whole incubation process by
monitoring both the bright-field pictures and the
fluorescence images as showed in Figure 3b. In contrast, the
recognition and attachment between TVP-PAP and P.
aeruginosa started after 10 min’s coincubation to emit out
fluorescence signal. With the time elapsing to 30 min, the
fluorescence signal became gradually stronger. And more
detailed tracking of TVP-PAP was also carried out, in which
the fluorescence signal of bacteria intensified to a platform
after 30 min, consistent with the necessary time for phage
targeted binding and infection to the host (Figure S18).
These results of nearly all P. aeruginosa being successfully
stained while no fluorescence being observed from A.
baumannii, demonstrated the particular specificity of TVP-
PAP probe to its target bacteria. The use of AIEgen alone for
bacterial imaging via non-specific electrostatic or
hydrophobicity-hydrophily interaction indicated that it is
impossible to realize the absolute discrimination of a
certain species of bacterium.31 And the stable and efficient
staining of G bacteria was always challenged due to the
blocking of their complicated cell walls.43,44 The
introduction of bacteriophage could guide AIEgen to realize
discriminative light-up imaging likely equipped with a
sensing “radar”. And the TVP-PAP carried AIEgen to anchor
and aggregate on the surface of target bacteria, resulting in
the enrichment of a large number of AIEgens at local site
with enhancement in their fluorescence emission for light-
up imaging of bacteria. Therefore, significant advancement
in targeting specificity could be achieved by TVP-PAP
bioconjugate comparing to the previous AIEgens that
suffered moderate selectivity.35
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influence on bacterial survival rate. Similarly, TVP-S alone
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could neither inhibit the bacterial growth crossing the
tested concentration range (4.33 ~ 21.65 pM, according to
3.18:1 ~ 15.9:1 molar ratio), no matter with or without light
irradiation. It was possibly that the non-specific AIEgens
and their relative low concentration on the surface of
bacteria weakened the destructive effect. In contrast, upon
PAP treatment, antibacterial effect initiated when its
concentration rising to 9.54 × 105 PFU·mL-1 (according to
9.54:1 molar ratio) under darkness (PAP I-60). And the
bacterial survival rate further decreased to 48.3% when the
concentration increasing to 15.9 × 105 PFU·mL-1 (according
to 15.9:1 molar ratio) with a dose-dependent manner. This
result indicated the inherited infection capability of the
TVP-PAP bioconjugate from PAP moiety. With assistance
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from AIEgen, TVP-PAP showed
a little bit higher
antibacterial efficiency in the darkness comparing to PAP
alone (TVP-PAP I-30 vs PAP I-30) crossing the tested
concentration range, possibly contributed by the dark
toxicity of the AIEgens enriched on the surface of P.
aeruginosa. With elongating the interaction time for
another 30 min (TVP-PAP I-60), this dark toxicity became
much stronger and could cause another 20% decrease in
the bacterial survival rate at the concentration of 9.54 × 105
PFU·mL-1. What’s more, by harnessing the extra PDI activity
under white light irradiation, significant enhancements in
the antibacterial efficiency could be observed (TVP-PAP I-
30 & L-30). With the concentration of TVP-PAP increasing
to 1.59 × 106 PFU·mL-1, the bacteria survival rate rapidly
decreased to 6.9%. The cloning plate pictures also verified
the excellent antibacterial effect from TVP-PAP
bioconjugates which was much superior to TVP-S and PAP
alone (Figure S21). And complete inhibition could be
achieved by increasing TVP-PAP to 2.544 × 106 PFU·mL-1
(Figure S22 and S23). PAP could keep infectious capability
after TVP-S modification for at least 1 week (Figure S24 and
S25). Extending to the clinical isolated MDR P. aeruginosa, a
similar dose-dependent antibacterial profile was presented
upon TVP-PAP treatment. When the concentration of TVP-
PAP rising to 6.36 × 107 PFU·mL-1, the eradication efficiency
could reach 64.8% (TVP-PAP I-60). And the efficiency could
even rise up to 99.5% under white light irradiation (TVP-
PAP I-30 & L-30). In contrast, at the same concentration, the
individual AIEgen (TVP-S I30 & L-30) and PAP (PAP I30 &
L-30) could only kill 7.0% and 46.5% bacteria, respectively,
definitely verified the significantly synergistic antibacterial
effect boosted by the PDI activity of AIEgens.
Figure 3. Targeted bacterial imaging. (a) Fluorescence
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imaging of P. aeruginosa incubated with TVP-PAP for 30 min.
All the scale bars were 10 μm. (b) Specificity test of TVP-PAP
by fluorescence imaging of P. aeruginosa and A. baumanni
co-incubated with TVP-PAP. Arrow indicated A. baumannii
without staining by TVP-PAP. TVP-PAP was exited at 488
nm, and the emission was collected at 580-680 nm.
Targeted Antibacterial Effect via Phage-Guided
Photodynamic Inactivation. Inspired by the exceptional
bacteria-targeting ability of PAP and high 1O2 generation
efficiency of AIEgens, we next checked their synergistic
antibacterial performances in facing antibiotic-sensitive
and MDR P. aeruginosa. The times for incubation and white
light illumination were first optimized by examining the
survival rate of P. aeruginosa. In order to avoid the instant
decrease of bacterial survival rate, a small amount of TVP-
PAP (9.54 × 105 PFU·mL-1) was added in each group. As
shown in Figure S19, with elongation of the incubation time
from 0 to 30 min, the bacterial survival rate decreased from
98.3% to 74.7%, consistent with the saturation time in the
above imaging process (Figure S18). Next, we fixed the
incubation time at 30 min and then irradiated the bacterial
suspensions by white light (4.2 mW·cm-2) for 2, 10, 30, 60
min, respectively. Nearly 79% of P. aeruginosa could be
eradicated after 30 min’s illumination and the efficacy
remained unchanged even irradiation prolonged to 60 min,
indicating the saturation of ROS generation at this condition.
What’s more, the bacterial survival rate significantly
increased after reversing the sequence of incubation and
illumination, suggesting the critical role of phage targeting
and infection before TVP-PAP-guided ROS generation on
the bacterial surface (Figure S20). Thus, light illumination
following 30 minutes’ coincubation was applied in the
subsequent antibacterial tests.
The targeted killing is another critical consideration in
antibacterial test. Herein, the character of PAP was
introduced for the bacterial discrimination and their
following targeted killing. This capability of TVP-PAP was
checked in two-component systems by culturing host
bacteria, P. aeruginosa (G), with non-host bacteria, A.
baumannii (G) or S. aureus (G+), respectively. As shown in
Figure 4b,c, in the presence of light irradiation, P.
aeruginosa were killed effectively and few colonies formed
upon TVP-PAP treatment. Whereas the colony quantity of A.
baumannii or S. aureus was nearly unaffected, consistent
with the above selective staining result of TVP-PAP to its
target. The phage-guided
The synergistic antibacterial effect of TVP-PAP was next
tested by comparing with the same molar concentrations of
individual AIEgen and PAP entity. As shown in Figure 4a, for
antibiotic-sensitive P. aeruginosa, neither incubation in
darkness (control I-30, control I-60) nor with further white
light irradiation (control I-30 & L-30) could pose apparent
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exposure to white light starting from time point at 30 min,
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bacterial morphologies became gradually blurred with
illumination lasting, with obvious appearance of fragments
in the bright field. In the fluorescence field, signals
disappeared in a large fraction of bacteria after 10 min’s
irradiation, along with the apparent agglomeration of
bacterial cells. These change in morphology and fluidity
indicated destruction happened on the targeted bacteria. To
MDR P. aeruginosa, the real-time tracking was performed
upon interaction with a higher concentration of TVP-PAP
(6.36 × 107 PFU·mL-1) (Figure S27). Similarly, after 30 min’s
coincubation, it was easy to observe the bacterial cell
fragment whose amount obviously increased with the
elapsing of white light irradiation time, indicating the
aggravating damage to the MDR bacteria.
In order to gain more insights into the antibacterial
effect of TVP-PAP, SEM imaging was employed to monitor
the morphological changes on antibiotic-sensitive and MDR
P. aeruginosa. As shown in Figure 6a, without TVP-PAP
treatment, P. aeruginosa possessed smooth surface with
intact cell wall and well stereo-shape body. In contrast, after
treatment with TVP-PAP and white light irradiation, the cell
walls of P. aeruginosa apparently became rough and even
ruptured with the disappearance of its original stereo shape.
targeting was proposed to play a critical role in achieving
this super antibacterial performance. PAP first specifically
recognized the antibiotic-sensitive or MDR P. aeruginosa
through its tail protein binding to lipopolysaccharide of the
host bacteria.14 Then the AIEgens (low to 21.65 pM in 1.59
× 106 PFU·mL-1 TVP-PAP) were enriched on the bacterial
surface. Following the infection process of phage itself, the
AIEgens generated efficient 1O2 in situ under white light
irradiation could further promote the destruction on the
targeted bacteria. Comparing to the previous PDI-based
antibacterial tests demanding up to several μM of
AIEgens,36,45-47 our phage-guided method showed pretty
higher efficacy and would remarkably mitigate the toxic
concerns regarding the random interaction of traditional
photosensitizers with surrounding biomolecules.
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Tracking of the Phage-Guided Targeting and Killing
Process. The unique fluorescence property of AIEgen was
also harnessed for real-time monitoring this targeting and
killing process. As shown in Figure 5, the successful staining
of P. aeruginosa was first achieved by incubating with TVP-
PAP bioconjugates, with emitting out robust fluorescence
signal under imaging (also quantitated in Figure S26). After
Figure 4. Antibacterial evaluation. (a) Survival rate of antibiotic-sensitive (left) and MDR P. aeruginosa (right) upon various
treatments (bacteria were fixed at 1.0 × 105 CFU·mL-1 in each group). (b) P. aeruginosa and A. baumannii (each with 1.0 × 105
CFU·mL-1) incubated together with TVP-PAP (1.59 × 106 PFU·mL-1). (c) Same concentrations of P. aeruginosa and S. aureus
incubated together with TVP-PAP. Inner pictures in (b) and (c) were the corresponding CFU count assays with I-30 & L-30
treatment. The arrow indicated P. aeruginosa identified via colony morphology and colony color for another 36h incubation
(I: incubation; L: light illumination. I-30: co-incubated for 30 min at 37 ℃ in darkness; I-60: co-incubated at 37 ℃ for 30 min
and then at 25 ℃ for 30 min in darkness; I-30 & L-30: co-incubated at 37 ℃ for 30 min in darkness and then at 25 ℃ for 30
min under white light). The concentration of TVP-PAP was determined using PAP as standard and the concentration of TVP-
S was calculated according to the molar ratio of TVP-S to PAP in TVP-PAP bioconjugate. **P < 0.01, ***P < 0.001.
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Figure 5. Real-time monitoring of TVP-PAP infecting P. aeruginosa and PDI effect. Bacteria were first co-incubated with TVP-
PAP (molar ratio of TVP-PAP to P. aeruginosa was 15.9:1) for 30 min, and irradiated under white light for another 50 min.
further damages to P. aeruginosa (Figure 6b). Similar result
with coarse cell surfaces and collapsed morphologies, could
also be observed in the case of MDR P. aeruginosa, verifying
the tremendous destruction from the synergistic effect48,49
of TVP-PAP bioconjugates (Figure S28).
In Vivo Synergistic Eradication of Skin Infections.
Inspired by the outstanding bacteria targeting and
elimination activity of TVP-PAP in vitro, we further
extended antibacterial examinations to in vivo using a
mouse model with antibiotic-sensitive or MDR P.
aeruginosa infectious skin wounds. The biocompatibility of
TVP-PAP on normal cells was first checked. As illustrated in
Figure S29, for both dark and illumination groups, the
viability of HaCaT cells still remained up to 83% even the
TVP-PAP concentration went up to 8.45 × 1010 PFU·mL-1.
The imaging result of HaCaT cell incubated with TVP-PAP
further verified its low toxicity as no obvious fluorescence
Figure 6. SEM imaging of bacterial killing effect. (a) P.
aeruginosa without any treatment. (b) P. aeruginosa
incubated with TVP-PAP and under white light irradiation.
signal could be detected (Figure S30). Compared to the TVP-
S molecules, the improved biocompatibility of TVP-PAP
provided a basis for their in vivo antibacterial application
(Figure S31).
Also numerous holes appeared on the bacterial surface.
These holes punched by phage would accelerate the
1
permeation of O2 into the bacteria cytoplasm and caused
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After model building, the antibiotic-sensitive and MDR P. collected and bacterial colony numbers were counted to
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aeruginosa infected mice were randomly separated to three
groups with various treatments (as depicted in Figure 7a).
Through monitoring the healing processes of the wounds, it
was clear that the mice treated with TVP-PAP nearly
recovered at 8 day posttreatment (Figure 7b,d). And both of
the relative wound sizes infected by antibiotic-sensitive and
MDR P. aeruginosa decreasing to below 10%, significantly
smaller comparing to the PBS control or TVP-PAP alone
group (Figure 7c,e). The wound healing was pretty faster
than the previous report using AIE polymer32 and AIEgen-
antibiotic conjugate in case of G bacterial infection,45
demonstrating the super efficiency of the phage-guided
bacterial killing. The infected skin tissues were then
quantify the antibacterial efficacy of TVP-PAP. It turned out
that both numbers of bacteria in the groups treated with
TVP-PAP plus white light irradiation were much smaller
comparing to the PBS control and TVP-PAP alone treated
group (Figure S32). The hematoxylin and eosin (H&E)
staining also proved the superior outcome of TVP-PAP
treatment plus white light irradiation by showing intact
histological characteristics. Whereas in control and TVP-
PAP treated alone, invaded lymphocytes could be obviously
observed (Figure 8). Collectively, these outstanding
performances in vivo suggested the great potential of TVP-
PAP bioconjugates in the application of target bacterial
killing and infectious disease therapeutics.
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Figure 7. In vivo evaluation of TVP-PAP in treatment of antibiotic-sensitive and MDR P. aeruginosa infected wounds on mice.
(a) The treatment process illustrated with time elapsing. (b) Photographs of wounds treated by TVP-PAP plus white light
irradiation for different time periods. (c) Relative wound size analysis after diverse treatments for 8 days. The Initial P.
aeruginosa was 2.0 × 107 CFU. (d) Photographs of wounds treated by TVP-PAP plus white light irradiation for different time
periods. (e) Relative wound size analysis after diverse treatments for 8 days. The initial MDR P. aeruginosa was 2.0 × 106 CFU.
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Figure 8. Histologic analysis of the bacterial infected injury tissue. (a) P. aeruginosa and (b) MDR P. aeruginosa infected injury
tissue by H&E staining for different treatment groups, respectively. All scale bars were equal to 10 μm.
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CONCLUSIONS
In summary, we proposed a novel antibacterial strategy by
integrating the fluorescence monitoring, phage targeting
and AIE-based photodynamic inactivation together toward
ORCID
Xuewen He: 0000-0002-8414-5164
Yujun Yang: 0000-0002-5637-7304
Xuepeng Zhang: 0000-0002-2608-6098
Guangle Niu: 0000-0002-5403-6880
a certain species of bacterium. Benefiting from the unique
1
fluorescence property, effective O2 generation capability,
Ryan T. K. Kwok: 0000-0002-6866-3877
particular targeting specificity, TVP-PAP had been
Guoming Xie: 0000-0002-7497-1964
successfully utilized in the discriminative imaging, selective
Ben Zhong Tang: 0000-0002-0293-964X
and efficient killing of targeted bacteria, without apparent
Author Contributions
#X. He and Y. Yang contributed equally to this work.
influence in the non-targeting bacteria or the normal
mammalian cells. The in vitro test demonstrated that even
MDR P. aeruginosa could be killed with nearly 100%
efficiency attributing to the synergistic effect within TVP-
PAP. Additionally, in vivo study verified that the healing
rates of both antibiotic-sensitive and MDR bacteria infected
wound could be significantly accelerated. These remarkable
performances validated that the superior potential of TVP-
PAP bioconjugate in synergistic bacterial recognizing and
killing that surpassing each individual component within
the bioconjugate. Through changing to other phages, this
AIE-phage conjugated strategy could be extended to be a
universal approaching for producing particular specific
antibacterial agents, holding great potential to the
treatment of bacterial infections as well as diseases
indirectly influenced by bacteria, such as the chemo- or
immuno-therapy resistant cancer with critical bacterial
participation.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors acknowledge funding to B.Z.T. from the Research
Grants Council of Hong Kong (C6009-17G), the Innovation and
Technology Commission (ITC-CNERC14SC01), the National
Natural Science Foundation of China (21788102), the Natural
Science
Foundation
of
Guangdong
Province
(2019B030301003), and the National Key Research and
Development program of China (2018YFE0190200). B.Z.T. is
also grateful for the support from the Science and Technology
Plan of Shenzhen (JCYJ20160229205601482). Funding to G. X.
from the National Natural Science Foundation of China
(81672112, 81972025), the Chongqing Technology Innovation
and Application Demonstration Project (cstc2018jscx-
msybX0010). And funding to Y. Y. from the Chongqing Special
Postdoctoral Science Foundation (Xm2017090), the Science
and Technology Research Program of Chongqing Municipal
Education Commission (Kjqn201900416). Funding to R.T.K.K.
from the Science and Technology Plan of Shenzhen
(JCYJ20180507183832744). The authors also thank Dr. Ling-
Hong Xiong in Shenzhen Center for Disease Control and
Prevention, and her funding from the National Natural Science
Foundation of China (21705111). And thanks to Dr. Weijun
Zhao in the measurements of phosphorescence spectra and
lifetime decay, to Dr. Li Zhang for help in drawing the schematic
figures, to Dr. Lei Xu for help in building of the animal models.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website. Materials, methods, supporting
figures (Figures S1−S32) (PDF)
AUTHOR INFORMATION
Corresponding Authors
* B. Z. T. tangbenz@ust.hk
* G. X. guomingxie@cqmu.edu.cn
* Y. Y. yangyujun@cqmu.edu.cn
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(18) Zhang, Q.-G.; Buckling, A. Phages Limit the Evolution of
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