An enzyme-responsive and photoactivatable carbon-monoxide releasing molecule for bacterial infection theranostics

Article abstract of DOI:10.1039/d0tb01761b

Infections caused by pathogenic bacteria, especially the drug-resistant bacteria, are posing a devastating threat to public health, which underscores the urgent needs for advanced strategies to effectively prevent and treat these intractable issues. Here

Full text of DOI:10.1039/d0tb01761b

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ISSN 2050-750X
PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
particles
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DOI: 10.1039/D0TB01761B
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An Enzyme-Responsive and Photoactivatable Carbon-Monoxide
Releasing Molecule for Bacterial Infection Theranostic
Xianghong Wang, †ab Xin Chen, †bc Lingjie Song,﹡a, Rongtao Zhou, a and Shifang Luan,
Received 00th January 20xx,
Accepted 00th January 20xx
a
﹡
DOI: 10.1039/x0xx00000x
Infections caused by pathogenic bacteria, especially the drug-resistance bacteria, are posing a devastating
threat to public health, which underscores the urgent needs for advanced strategies to effectively prevent and
treat the intractable issues. Here we report a feasible and effective theranostic platform based on an enzyme-
sensitive and photoactivatable carbon monoxide releasing molecule (CORM-Ac) for successive detection and
elimination of bacterial infection. The extracellular bacterial lipase can trigger the excited state intramolecular
proton transfer (ESIPT) via elimination of ester group in CORM-Ac, thus providing fluorescence switch for an
early warning of infection. Subsequently, the potent bactericidal therapy against the model bacterial strains,
Staphylococcus aureus (S. aureus) and notorious methicillin-resistant Staphylococcus aureus (MRSA), were
readily realized via photoinduced release of CO. In addition, the CORM-Ac and CORM showed good
biocompatibility within a wide range of concentrations. The results of infected animal wound test also
demonstrated that CORM-Ac-loaded gauze was effective in indicating the wound infection and accelerating
the wound healing via the photoinduced CO release. The simplicity, functional integration, good
biocompatibility and broad adaptability make CORM-Ac very attractive for bacterial theranostic applications.
molecules (CORMs) based on transition-metal carbonyl complexes
(Ru, Mn, Co, Re and Mo) have been developed. Although effective,
several drawbacks related to those CORMs, e.g., potential
cytotoxicity, spontaneous diffusion, and lack of on-demand CO
release, heavily limit their practical applications.15 Alternatively,
photoactivatable organic CORMs are more attractive because they
can deliver CO in a more controlled manner, largely decreasing the
likelihood of toxic effects to healthy cells.16-18
Introduction
Bacterial infections with high mortalities are a major global threat
to public health.1 The prevalence of drug-resistance caused by
indiscriminate use of antibiotics is further dramatically aggravating
the global infection situations.2 To overcome these dilemmas, many
novel therapeutic strategies relying on polycationic polymers,3,
antimicrobial peptides,5,
4
6
photodynamic/photothermal therapy
Besides, sensitive bacterial detection as a warning of the early-
stage infection is of great significance, as it facilitates the timely
interventions before local manifestations induced either through
biofilm formation or a systemic spread of the infection.19, 20 To date,
several standard technologies, e.g., plate-culturing method and
polymerase chain reaction (PCR), have been widely used for
pathogen detection. However, they are generally time-consuming,
complicated and highly apparatus/skill-dependent, largely confining
their applications in real-time and nondestructive bacterial
detection.21-24 Alternatively, detecting bacterial-related biomarkers,
especially enzymes, is a more promising method because they are
directly related to the pathological progress. Fluorescence imaging
based on small-molecular probes is an efficient tool for detecting
biological targets due to their noninvasive nature, high sensitivity
and rapid response capacities.25-27 In this regard, diversified
fluorescence probes have been designed based on various
fluorescence switching mechanisms, such as excited state
agents7-9 and gaseous drugs (nitric oxide, NO and carbon monoxide,
CO)10, 11, have been widely explored. Owing to the unique
antibacterial mechanism, CO holds great promise as a novel
antimicrobial chemotherapy agent to kill bacteria without causing
potential drug-resistance. The reason is that CO can target
preferentially to transition-metal-containing proteins (e.g., heme-
containing terminal oxidase), deteriorating their biological
functions.12, 13 However, the practical application of CO is severely
limited by the systemic toxicity at high concentrations and
difficulties in targeted delivery.14 Till now, traditional CO releasing
a. State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China.
Emails: songlj@ciac.ac.cn, sfluan@ciac.ac.cn
b. School of Materials Science and Engineering, Zhengzhou University, No. 100
Science Avenue, Zhengzhou 450001, China
c. Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun 130022, China
29
intramolecular proton transfer phenomenon (ESIPT),28,
†
These authors contributed equally to this work.
fluorescence resonance energy transfer (FRET),30-32 and
aggregation-induced emission (AIE).33-36 In particular, ESIPT probes
Electronic
Supplementary
Information
(ESI)
available.
See
DOI: 10.1039/x0xx00000x
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featured with high quantum yield, large Stokes shift and dual
fluorescence emission are highly suitable for fluorescence
biosensing.37-39 Despite these unique photophysical properties, only
a few ESIPT probes have been designed for real-time detection of
bacterial infection.
Results and discussion
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DOI: 10.1039/D0TB01761B
Preparation, characteristic and properties of CORM-Ac
As a starting point of our study, CORM was synthesized based on
the Alger-Flynn-Oyamada reaction45 and CORM-Ac was
synthesized by acylation of CORM with acetic anhydride (Fig. 1A).
The successful preparation of CORM and CORM-Ac were
confirmed by 1H NMR and ESI-MS (Fig. S1 and S2). The
spectroscopic properties of CORM-Ac and CORM under mimetic
physiological condition (PBS, pH 7.4) were studied first. CORM-Ac
had a maximum adsorption peak centered at ~ 280 nm, and CORM
exhibited two absorbance peaks at ~ 280 nm and ~ 350 nm (Fig. 1B).
Due to the spontaneous intramolecular hydrogen bonding known as
ESIPT process, CORM exhibited a faint blue emission peak at ~ 410
nm and a strong green fluorescent emission at ~ 525 nm when
irradiated at 360 nm, which corresponded to the excited-state normal
(N*) and tautomeric (T*) forms, respectively (Fig. 1C and 1D).
CORM-Ac only displayed an intensive emissive peak centered at ~
450 nm, indicating that modification of hydroxyl group in CORM
can block the ESIPT process, resulting in the quenching of T*
emission. Further incubation of CORM-Ac with lipase solution gave
rise to two separate emission peaks that was similar with CORM,
except that one peak red-shifted from ~ 410 nm to ~ 450 nm,
suggesting the effective liberation of free hydroxyl group in CORM-
Ac by lipase and restore the dual emission property associated with
N*–T* tautomerism (Fig. 1D).
Nowadays, pathogen diagnosis and therapy are independent
progresses in clinic, inevitably leading to the compromised
therapeutic effects and increased economic and mental burdens.
Theranostic integrates accurate diagnosis and therapeutic capacities
in a single system can generate real-time diagnostic signals and
facilitate the in-situ therapeutic processes in time, representing a
potential strategy for precise infection treatments.40-42 Nevertheless,
it is difficult to simultaneously integrate bacterial sensing and killing
capacities into a single system without complex and tedious
procedures.43 Moreover, the theranostic capacities towards broad-
spectrum bacteria strains, as well as the validity on treating the
booming multi-resistant bacteria without causing antimicrobial
resistance, are not easily implemented.44
Herein, we report a simple and feasible unimolecular theranostic
probe, O-acetyl group protected CORM (CORM-Ac), for real-time
detection of bacterial infection and subsequent treatment. As
illustrated in Scheme 1, by utilizing the exogenous bacterial lipases
as targets, the CORM-Ac could undergo enzymatic cleavage of O-
acetyl group and transform into CORM, readily activating the ESIPT
progress and giving an early warning on infection via visualized
fluorescence signal. The image-guided bactericidal therapies could
then be administrated by releasing CO from CORM upon
Two photolysis compounds, CO and inactivated CORM
photoexcitation. This “sense-and-treat” molecular formulation with (iCORM), were supposed to be formed during the illumination
largely simplified preparation procedures shows great potentials in process according to the previous report (as shown in scheme 1).45
sensitive warning and treatment of bacterial infection.
To confirm the photoinduced CO release, a CO fluorescent probe
(COfp) was synthesized (Fig. S3). The detection mechanism is based
Scheme 1. Schematic concept of bacterial sensing of CORM-Ac process and subsequent bacterial killing in situ.
on that CO can reduce Pd2+ to Pd0 in-situ, which could induce
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range of 0 to 140 μg mL-1, with a good linear relationship (R2=0.987)
nonfluorescent COfp to release the fluorescent product via Pd0-
mediated Tsuji–Trost reaction (Fig. 1E).46, 47 As shown in Fig. 1F,
compared with COfp/CORM/dark, COfp/CORM-Ac/light and
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DOI: 10.1039/D0TB01761B
and a regression equation of 0.9113+0.04997[Lipase] (Fig. 2B).
Also, a linear relationship (R2 = 0.997) was also observed between
the ratio of fluorescence intensity (I525/I450) and incubation time with
lipase (Fig. 2C and 2D). The selectivity of CORM-Ac towards lipase
was then evaluated by measuring its fluorescence response in the
presence of different interference substances, including proteins
(alkaline phosphatase (ALP), fibrinogen (Fib)) and inorganic salts in
PBS. As displayed in Fig. 2E, the lipase can induce a significant
increase in the ratio of fluorescence intensity over other components,
suggesting the high selectivity of CORM-Ac over lipase.
Collectively, these results indicated that CORM-Ac can be a
sensitive ratiometric fluorescent sensor for quantitative detection of
lipase in an aqueous environment.
COfp/CORM-Ac/lipase/Dark groups,
a remarkable change in
fluorescent emission at 470 nm was monitored in simultaneous
presence of CORM and light or CORM-Ac, lipase and light,
accompanied by the prominent color change from colorless to bright
blue. A commercially available CO detector was also used for CO
detection (Fig. S4). Upon illumination of the oxygen-filled CORM
solution under a simulated sunlamp for 12 h, the CO at a
concentration of ~ 220 ppm was readily detected. Subsequently, the
illuminated solution was analyzed by ESI-MS to confirm the
iCORM. A discriminable mass signal at m/z 275.2 (M-OH)
originated from inactivated photolysis CORM (iCORM) was found
(Fig. S2C). Besides, both of the fluorescence emissions at 525 nm of
CORM and CORM-Ac/lipase solutions significantly attenuated,
implying the formation of non-emissive iCORM (Fig. 1D, pink and
black curves). These results collectively suggested that CORM could
release CO via photo-activated manner, while CORM-Ac is
photostable under the same condition.
A number of Gram positive pathogenic bacterial species have
been proved to produce lipases, which have been an important
49
virulence factor for many infection diseases.48,
Herein, to
demonstrate the bacterial detection ability of CORM-Ac, S. aureus,
the well-studied lipase-secretion bacteria, was used as the
representative bacterial stain. As shown in Fig. 2F, significant
changes in fluorescence spectra were seen within 30 min after
incubation of CORM-Ac with bacterial suspension. The ratio of
fluorescence intensity (I525/I450) was linearly dependent upon the
logarithm of bacterial suspension concentrations ranged from 0 to
106 cells mL-1 (Fig. 2G). More importantly, the prominent
fluorescence change of CORM-Ac was also observed in the presence
of methicillin-resistant S. aureus (MRSA) (Fig. 2H), suggesting the
valid detection ability of CORM-Ac towards lipase-secretion
bacteria.
Fig. 1. (A) Synthetic route of CORM-Ac. Reagents and conditions:
i) EtOH, KOH, RT, 12 h; 30% H2O2, RT, 12 h. ii) THF, DMAP,
0 ℃, 12 h. (B) UV-Vis absorption spectra of CORM-Ac and CORM
in H2O/DMSO mixture (1:1, v/v). (C) The ESIPT mechanism of
CORM. (D) Fluorescence emission spectra of CORM-Ac, CORM-
Ac incubated with lipase, CORM-Ac with lipase and illuminated
with a sunlamp (37,500 lx) for 12 h, CORM, and CORM
illuminated with a sunlamp (37,500 lx) for 12 h. (E) The detection
mechanism of COfp towards CO and (F) the changes in spectrum and
fluorescence of COfp after different treatments.
Fig. 2. (A) Change of fluorescence spectra of CORM-Ac in DMSO (10
μM) with incremental addition of lipase (0, 10, 20, 40, 60, 80, 100,
120, 140 μg mL-1 in PBS, pH 7.4) and (B) the corresponding linear
ratio of fluorescence intensity (I525/I450) versus lipase concentration.
(C) Change of fluorescence spectra of CORM-Ac (10 μM) under
different incubation time (0, 10, 20, 30, 40, 50, 60 min) in the presence
of lipase in PBS solution (100 μg mL-1) and (D) the corresponding
linear relationship of fluorescence intensity ratio (I525/I450) versus
incubation time. (E) Specificity evaluation of CORM-Ac towards
lipase over other interfering substances. (F) Spectrum changes of
CORM-Ac towards S. aureus at different concentrations and (G) the
corresponding linear ratio of fluorescence intensity (I525/I450) versus the
concentrations of S. aureus suspensions. (H) Changes in fluorescence
Lipase and bacterial detection
The fluorescence kinetic curves of CORM-Ac upon incubation with
lipase at different concentrations and incubation times were also
investigated. As shown in Fig. 2A, the increment in lipase
concentration resulted in the gradual increased fluorescence intensity
at 525 nm. The changes of ratio in fluorescence intensity (I525 nm/I450
nm) was linearly proportional to the lipase concentrations within the
color of CORM-Ac in the presence of MRSA (+) or not (-).
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S6D). More importantly, we confirmed that the releaseVdieCwOArtwiclaesOanllisnoe
Bactericidal activity in vitro
Prior the assay, the CORM-Ac concentration of 30 μg mL-1 and remarkably effective in killing the methiciDllOinI:-r1e0s.1is0t3a9n/tD0ST.Ba0u17r6e1uBs
illumination time of 30 min were adopted according to the optimized (MRSA), one of the main dreaded clinical superbug, as verified by
antibacterial effects of CORM against S. aureus strains (Fig. S5 and the significant reduction in colony formation and damaged cell
S6). In the bactericidal test in vitro, CORM-Ac solutions were morphology (Fig. 3B and 3D).
incubated with S. aureus suspensions for 30 min to release CORM,
Compared with planktonic cells, the bacteria in biofilms are hard
followed by illuminating the mixture for bactericidal treatment. to eradicate as a consequence of the existence of protected
Compared with control group (PBS/dark), exposure CORM-Ac to extracellular polymeric substances.50 Herein, we testified the
bacterial suspension alone did not lead to a conspicuous reduction in bactericidal activity of CORM-Ac towards the bacteria cells within
the number of bacterial colonies, manifesting no essential biofilms. As shown in CLSM images (Fig. 4A), the CORM-Ac and
bactericidal activity of CORM-Ac in the dark. However, shortly after light treated biofilms exhibited intensive red fluorescence, signifying
the exposure to CORM-Ac and light irradiation, the bacterial severe cell necrosis and death. This was in sharp contrast to the
viability was significantly impaired, as indicated by the agar plate strong green fluorescence presented in control groups (untreated
culturing results (Fig. 3A and 3B). To further confirm the bacterial biofilm, CORM-Ac/dark and antibiotic ciprofloxacin (CIP) treated
viability, the bacteria cells after CORM-Ac and light treatments were biofilms). Simultaneously, as confirmed by SEM, the visible
immediately labeled with live/dead staining dyes. As clearly seen reduction of biofilm adherence and loosely aggregated bacterial
from CLSM images (Fig. 3C), the bacteria cells presented in red clusters were observed (Fig. 4B). The membrane integrity of
fluorescence upon CORM-Ac/light treatment, indicative of cell bacterial cells also dramatically altered after the CORM-Ac/light
death. In addition, SEM results revealed that severely impaired cells treatment (Fig. 4B). The surface-associated biofilm stained by
with conspicuous cytoplasm leakage were presented after bacteria crystal violet revealed that ~ 62 % biofilm biomass was moved after
cells were treated with CORM-Ac and light, which was sharply CORM-Ac/light treatment (Fig. 4C and 4D). These results
contrast to the healthy cell morphology in control groups (Fig. 3C). collectively indicated that CORM-Ac showed the prominent
These results suggested that released CO rather than iCORM was properties in killing bacteria in biofilm and eradicating the
responsible for the potent bactericidal action. This point was also established biofilm via releasing CO after tandem lipase and light
supported by the fact that light-illuminated CORM solution (iCORM stimuli.
solution) did not decrease the viability of S. aureus cells in dark, but
can recover the bactericidal efficacy upon light irradiation (Fig.
Fig. 4. (A) The representative 3D CLSM images of S. aureus
biofilms stained by live/dead staining technique after different
treatments. B) SEM images of biofilm and cellular morphology of
bacterial cell in biofilm after various different treatments (scale: 10
μm and 2 μm, respectively). (C) Images of the crystal violet-stained
biofilms and (D) the corresponding quantitative analysis of
Fig. 3. The antibacterial activities of bacterial lipase-activated
CORM-Ac in vitro. (A) Photographs for S. aureus and MRSA
colonies grown on LB agar plate under different treatments and
(B) corresponding colony-forming unit (CFU) counting, XX
indicates almost undetectable number of bacterial cells
(mean±SD, n=3). (C) Representative CLSM images for
evaluating the activities of S. aureus via live/dead BacLight Kit
(scale: 50 μm) and the SEM images detailing cell morphology
(scale: 50 μm) (scale: 1 μm). (D) The SEM images for MRSA
remaining biomass by measuring OD value at 590 nm. (*p < 0.05,
**p < 0.01, ***p < 0.001). (Error bars: standard deviation, n = 3).
Biocompatibility in vitro
Next, to gain insight into the cytotoxicity and hemocompatibility
of CORM-Ac and CORM, the cell viability of murine fibroblasts
was studied via the CCK-8 assay and the hemolysis assay were
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performed on erythrocytes. Fig. 5A demonstrates cell viability after
the cells were incubated with two compounds at different
concentrations for 24 h. Notably, more than 80% of cells could
survive at the concentration of 250 μM for each compound (82.50 μg
mL-1 for CORM-Ac and 71.98 μg mL-1 for CORM). In the
hemolysis results, the two compounds showed considerably lower
hemolysis (< 1.5%) even when their concentrations reached up to
1000 μM (Fig. 5B and S7). At the antibacterial dose of CORM-Ac
and CORM (30 μg mL-1), the cell survival rates were higher than
90%, and the hemolysis ratios were lower than 0.7%, indicating their
good biocompatibility (Fig. 5C).
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DOI: 10.1039/D0TB01761B
Fig. 5. The biocompatibility of CORM-Ac and CORM in vitro. (A)
Plot of L929 fibroblasts percent viability and (B) hemolysis ratios
versus various concentrations of CORM-Ac and CORM. (C)The cell
viability and hemolysis ratio of CORM-Ac and CORM at a
concentration of 30 μg mL-1. (Error bars: standard deviation, n = 3)
Potential in infection theranostic application in vivo
As encouraged by the bacterial sensing and bactericidal
performances of CORM-Ac in vitro, S. aureus-infected mouse
models were established to demonstrate its potential utility in
clinical theranostics. As a proof-of-concept, medical-grade gauze
was selected as a supportive material for CORM-Ac loading prior to
the assay (CORM-Ac-Gauze for short). The application of this
wound dressing followed the steps outlined in Fig. 6A. Briefly, after
two symmetrical and full-thickness excisional wounds were created,
107 cells mL-1 of S. aureus suspension (50 μL) was injected into the
right wound for bacterial infection, while the left wound was
injected with PBS as a control. After 1 h infection, the CORM-Ac-
Gauze was covered onto the wounds for infection imaging. As
demonstrated in Fig. 6B, the bright green fluorescence of CORM-
Ac-loaded gauze clearly emerged only at the infected wound within
30 min under the assistance of a portable UV lamp, indicating the
rapid response of CORM-Ac to bacterial infection. After infection
indication, the antibacterial therapy of infected wounds was
performed by illuminating the CORM-Ac-Gauze covered wounds
for 30 min with a simulated sunlight source, while the similarly
treated wounds that kept in dark were set as controls. The traumas
were photographed on days 0, 2, 4, 6, 8, and 10, respectively. As can
be seen from macroscopic images in Fig. 6C, either in the absence of
CORM-Ac or illumination treatment, the wounds showed certain
degree of pyosis on days 2, and were still erythematous and
edematous after healing for 10 days because of severe infection. On
the contrary, CORM-Ac/light treated wound showed better skin
regeneration with smoother epidermis state over 10 days of healing.
Meanwhile, the rate of wound closure was measured over the time
course of healing and revealed that the difference in wound closure
between CORM-Ac/light and other control groups was seen at day 4
(p<0.05). This difference became more evident on day 6 (p<0.001)
(Fig. 6D). We also examined the residual number of bacteria around
Fig. 6. In vivo assessment of theranostic CORM-Ac on S. aureus-
infected mouse models (107 cells mL-1). A) The procedures of
theranostic CORM-Ac-Gauze for S. aureus-infected wound
imaging. The backs of the mice were slashed and injected with 50
µL bacteria suspension on the right side. After 1 h infection, the
CORM-Ac-Gauze were covered onto the wounds for infection
imaging. Then, the wounds were exposed to sunlight source for 30
min to kill bacteria. B) Representative photographs of fluorescence
response of CORM-Ac towards infection wound. C) Photographs of
wound closure in the time course of healing (0, 2, 4, 6, 8 and 10
days) and D) corresponding rate of wound closure. E) Remaining
bacteria of infected wound tissue evaluted by plate-counting method.
F) Representative histochemistry of the impaired skin sections on
days 10 via H&E staining. (*p < 0.05, **p < 0.01, ***p < 0.001).
(Error bars: standard deviation)
the infected tissue on days 5, relative to the controls, only a few
bacterial colonies were visible on culture plate in the CORM-
Ac/light treated group (Fig. 6E and S8).These results collectively
proved that the wound healing was appreciably accelerated, owing to
the timely infection indication and subsequent antibacterial therapy
via photo-induced CO release.
The bacterial infection in vivo can stimulate the immune system to
produce
a large number of white blood cells (WBCs) for
bacteria clearance. Therefore, H&E staining of infected wound
sections was performed after healing for 10 days (Fig. 6F). CORM-
Ac/light treated wound had remarkable fewer inflammatory cells
than other control tissues, clearly confirming that released CO could
alleviate inflammatory responses by killing bacteria. In addition, the
in vivo biocompatibility assays ascertained that uninfected wounds
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treated with CORM-Ac in the presence of light or not gave no signs
Journal Name
Benzaldehyde (0.53 g，5 mmol), 1′-hydroxy-2′-aVcieetwoAnratipclhetOhnolinnee
DOI: 10.1039/D0TB01761B
of abnormal tissue response during healing, indicating that CORM- (0.93 g，5 mmol) and potassium hydroxide (25 %, 10 mL) were
Ac was a potential safe molecule for infected tissue therapy (Fig. S9). added into ethanol (30 mL) and stirred for 12 h at room temperature.
These results collectively show that CORM-Ac could be integrated Upon addition of 5 mL of H2O2 solution (30 %), the mixture was
with bacterial sensing and therapy capacities, which might be a stirred for another 12 h at room temperature. After the reaction, the
better candidate for personalized theranostic applications.
mixture was poured into ice-water and acidified with diluted HCl.
The resulting yellow-brown precipitate was filtered and washed with
water. The crude product was recrystallized from ethanol (~ 52 %
yield) to give the CORM. 1H NMR (DMSO-d6, 300MHz): δ 9.88 (s,
1H), 8.68 (dd, J = 6.1, 3.3 Hz, 1H), 8.35 (d, J = 7.5 Hz, 2H), 8.14
(dd, J = 6.1, 3.1 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.93 (d, J = 8.8
Hz 1H), 7.84 (dd, J = 6.2, 3.2 Hz, 2H), 7.64 (t, J = 7.5 Hz, 2H), 7.55
(t, J = 7.3 Hz, 1H). ESI-MS (relative intensity) calcd. for C19H13O3
[MH]+: 289.1; found: 289.3.
Conclusion
In summary, we have presented that a single metal-free organic
molecule, CORM-Ac, as a new and powerful theranostic formulation
for effectively diagnosis and treatment of bacterial infections. In the
presence of bacteria-secreted lipase, CORM-Ac could be rapidly
hydrolyzed to CORM and activate the ESIPT progress for real-time
and sensitive monitor of bacterial infection at early stage. After the
feedback of changed fluorescence signal, antibacterial therapy could
be employed via photo-induced release of CO. The CO showed
significant bactericidal abilities on Gram-positive S. aureus and the
tracable drug-resistance bacteria, MRSA. Meanwhile, the validity of
CO on eradication of bacteria in biofilms and biofilm dispersion
were also demonstrated. In addition, both CORM-Ac and CORM
showed good biocompatibility within a wide range of concentrations,
indicative of the potential biosafety in bioapplications. By simply
loading CORM-Ac on Gauze, its theranostic application on infected
wounds were employed. The results demonstrated that CORM-Ac-
gauze was effective in indicating the wound infection and
accerlating the wound healing via the photoindcued CO release. This
smart molecule could be with easily integrated with various medical
devices, showing fascinating possibilities for the applications in real-
world infection resistance.
Synthesis of 3-Methoxy-2-phenyl-4H-benzo[h]chromen-4-One
(CORM-Ac)
CORM (0.445 g, 5 mmol) and acetic anhydride (20 mmol) was
dissolved in THF (25 mL) with adding DMAP (0.006 g, 0.05 mmol)
as the catalyst. After reaction at room temperature for 12 h, the
resulting mixture was poured into water, and the white precipitate
1
was obtained by filtration (~ 58 % yield). H NMR (DMSO-d6, 300
MHz)： δ 8.63 (d, J = 8.7 Hz, 1H), 8.18 (d, J = 7.5 Hz, 1H), 8.08-
8.02 (m, 4H), 7.89-7.82 (m, 2H), 7.68 (m, 3H), 2.38 (s, 3H). ESI-MS
(relative intensity) calcd. for C21H15O4 [MH]+: 331.1; found: 331.4.
Synthesis of CO fluorescent probe (COfp)
COfp was synthesized according to the literature.47 Briefly, 2, 4-
Dihydroxybenzaldehyde (2.50 g, 18 mmol) and ethyl acetoacetate
(2.36 g, 18 mmol) were dissolved in absolute EtOH (15 mL) with
addition of piperidine (0.10 g, 1 mmol) as the catalyst. The mixture
was heated to reflux for 12 h and then cooled to room temperature to
obtain a yellow precipitate. The solid was collected by suction
filtration and washed with cold EtOH, affording 3-acetyl-7-hydroxy-
2H-chromen-2-one as yellow powder (～ 61% yield). Then 3-acetyl-
Experimental
Materials
7-hydroxy-2H-chromen-2-one (0.64 g,
3
mmol) and allyl
Benzaldehyde, 1ʹ-hydroxy-2ʹ-acetonaphthone, acetic anhydride, 4-
(dimethylamino) pyridine (DMAP), 2, 4-dihydroxybenzaldehyde,
ethyl acetoacetate, piperidine, dimethyl sulfoxide (DMSO), crystal
violet and lipase from pseudomonas cepacia were purchased from
Sigma-Aldrich (USA). Gram-positive Staphylococcus aureus (S.
aureus; ATCC 6538) and methicillin-resistant S. aureus (MRSA,
ATCC 43300) were obtained from Nanjing Lezhen Biotechnology
Co., Ltd. (Nanjing, China). Phosphate buffered solution (PBS, 0.1
mol L-1, pH = 7.4) and Luria-Bertani (LB) were obtained from
Dingguo Biotechnology (China). L929 murine fibroblasts cell line
was purchasedprocured from American Type Culture Collection
(ATCC, USA). Dulbecco’s modified Eagle’s medium (DMEM),
0.25 wt.% trypsin, sterile filtered fetal bovine serum (FBS), and Cell
Counting Kit-8 (CCK-8) were purchased from Wuhan Boster
Biological Technology Co., Ltd. (Wuhan, China). LIVE/DEAD
Backlight Bacterial Viability Kit L7012 was purchased from
Molecular Probe Inc (USA). Unless otherwise specified, all
chemicals and reagents were obtained from commercial sources and
used as received without further purification.
chloroformate (1.13 g, 9 mmol) were mixed in 10 mL of DMF,
followed by adding triethylamine (0.95 g, 9 mmol) under ice bath
and reacting for 24 h. Afterward, 20 mL of ethyl acetate was added,
followed by washing the mixture by water (10 mL) for three times
and dried over Na2SO4. The crude product was purified by silica
chromatography eluted with ethyl acetate/n-hexane (1:1, v/v) to
afford COfp (~ 76% yield). 1H NMR (DMSO-d6, 300MHz) of 3-
acetyl-7-hydroxy-2H-chromen-2-one δ 11.18 (s, 1H), 8.59 (s, 1H),
1
7.77 (d, 1H), 6.82 (dd, 1H), 6.73 (d, 1H), 2.54 (s, 3H). H NMR
(DMSO-d6, 300MHz) of CO fluorescent probe δ 8.67 (s, 1H), 8.01
(d, 1H), 7.50 (s, 1H), 7.37 (d, 1H), 6.00 (m, 1H), 5.45 (dd, 2H), 4.78
(d, 2H), 2.58 (s, 3H)
Measurement of CO release
An Agilent headspace vial containing CORM in acetonitrile (1 mg
mL-1, acetonitrile) was filled with oxygen. After the illumination of
the vial with a sunlamp (37,500 lx) for 12 h, the released CO was
detected by a commercial CO detector. In addition, COfp was used to
sense released CO. Similarly, Briefly, O2 filled CORM solution (30
μg mL-1 in DMSO) in an Agilent headspace was illuminated under a
sunlamp for 30 min. Then, the equimolar Pb(OAc)2 and 10% H2O
Synthesis of 3-Hydroxy-2-phenyl-4H-benzo[h]chromen-4-One
(CORM)
6 | J. Name., 2012, 00, 1-3
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Journal of Materials Chemistry B
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Journal Name
ARTICLE
was added quickly into the mixture and reacted for 15 min at 37 °C. Additionally, the bacterial suspensions with various trVeiaetwmAertnictlse Ownelinree
Equimolar COfp was then added to react for another 15 min. Finally, fixed with 4 vol. % paraformaldehyde for 4DhOaI:n1d0.1d0e3h9y/dDr0aTtBed01w76i1tBh
the fluorescence images under a portable UV-lamp (365 nm) were sequential ethanol aqueous solutions (10, 30, 50, 70, 90 and 100
immediately taken by a digital camera and the fluorescent emission vol %). Then, the bacteria cells were harvested via centrifugation at
spectrum was measured. The vial that kept in the dark, vial 3000 rpm for 10 min. 50 μL of bacterial resuspension was dropped
containing CORM without illumination and vial containing CORM- onto clean silicon slices and dried for SEM measurement (XL 30
Ac upon illumination were set as controls.
FESEM FEG, FEI Co).
Detection of lipase and bacteria by CORM-Ac
The bactericidal activities of CORM-Ac against bacteria in
biofilm
Fluorescent response of CORM-Ac towards different lipase
concentrations (0-140 μg mL-1) was investigated by incubating
Biofilms were established by incubating bacterial suspension (106
CORM-Ac in DMSO (10 μM) with various lipase PBS solutions for cells mL-1) at 37 °C for 24 h without shaking in a 96-well plate.
1 h. Fluorescent response of CORM-Ac towards lipase at different Afterwards, biofilms were rinsed with PBS solution twice to remove
enzymolysis times was investigated by incubating CORM-Ac in the floating bacteria. 30 μL of CORM-Ac solution (1 mg mL-1 in
DMSO (10 μM) with determined lipase concentration (100 µg mL-1) DMSO) diluted with 1 mL of glucose-peptone-water medium was
at 37 °C for different reaction times (0, 10, 20, 30, 40, 50 and 60 added into each well. After incubation at 37 °C for 30 min, the S.
min). The ratios of fluorescent intensity (I525 450
/
) were calculated.
aureus biofilms were illuminated for 50 min. The untreated biofilm,
As for the bacteria detection, S. aureus was used as representative illumination treated biofilm, CORM-Ac/light treated biofilm and
bacteria stain, while methicillin-resistant S. aureus (MRSA) was antibiotic ciprofloxacin (CIP, 1 μg mL-1) treated biofilm were used
chosen as representative multidrug-resistant (MDR) bacteria. Briefly, as references.
bacteria were grown aerobically at 37 °C overnight in sterile LB
For CLSM observation, the biofilms were stained with
broth. The concentration of bacterial suspension was calculated by LIVE/DEAD BacLight Viability Kit for 15 min in the dark, followed
testing the absorbance of cell dispersions at OD600nm using a by washing with sterile water and vacuum freeze dehydration.
microplate reader. An optical density (OD) of 1.0 at 600 nm was Biofilm structure and bacterial morphology were observed by SEM
equivalent to ~ 109 cells mL-1. The diluted bacterial LB suspensions according to the method mentioned above. For biofilm quantitation,
(108 cells mL-1) were centrifuged and washed with PBS, followed by the biofilms after various treatments were rinsed with PBS and
resuspending in glucose-peptone-water medium to obtain the stained with 0.2% Crystal Violet (CV) solutions for 20 min, followed
suspensions with 106 cells mL-1. Sebsequently, 30 μL of CORM-Ac by rinsing with PBS. Then, acetic acid (33%) was added and
solution (1 mg mL-1 in DMSO) was then incubated with 1 mL of incubated for 10 min. The absorbance of each well at OD590 nm was
bacterial suspensions (106 cells mL-1) at 37 °C for 30 min. The measured and the corresponding color image was visualized by
fluorescence images were taken under a portable UV-lamp (365 nm) taking a photo with a digital camera.
and fluorescence emission spectra were measured.
Cytotoxicity assay in vitro
Bactericidal activity of CORM and CORM-Ac
The murine fibroblasts cell line L929 was used to explore the
Initially, the influences of CORM concentrations and illumination cytotoxicity of CORM-Ac and CORM via a standard CCK-8 method
time on antibacterial capacity was studied by the microdilution and (Cell Counting Kit-8). CORM-Ac or CORM solutions (4%
colony-forming unit (CFU) counting methods, respectively, in which DMSO/PBS, v/v) were diluted with DEME to get the final solutions
S. aureus was used as a representative bacterial strain. Specifically, with different concentrations (0.1 μM - 1000 μM). L929 cells were
different concentrations of CORM solutions were incubated with S. cultured at a density of 2 × 104 cells per well in a 96-well plate and
aureus LB suspensions (1 mL, 106 cells mL-1), followed by supplemented with 100 µL of DMEM. The well plate was then
illumination for 30 min under a sunlamp (37,500 lx). The placed in a humidified atmosphere containing 5% CO2 at 37° C for
bactericidal activity was reflected by monitoring the bacterial 24 h. Then, CORM-Ac or CORM solutions were added into each
proliferation by measuring the OD600nm (TECAN SUNRISE, Swiss). well and incubated with the cells for 24 h in the dark. After remove
The influence of illumination time was investigated by inoculating the culture medium in each well, CCK-8 solutions (10%, v/v in
100 μL of CORM/bacteria suspensions after different light DMEM) were added and co-incubated for another 1 h at 37 °C.
treatments (10 min, 20 min and 30 min) on gelatinous LB agar plate Finally, the optical absorbance at OD450
was measured on a
nm
for colony formation. The bactericidal efficiency was calculated via microplate reader. The results were expressed as percentages relative
counting the number of bacterial colonies.
to the optical absorbance obtained in the untreated cells.
As for the bactericidal activity of CORM-Ac, the CORM-Ac
solution (30 μg mL-1 in DMSO) was incubated with 1 mL of
bacterial suspensions (106 cells mL-1) for 30 min, which could give
Hemolysis assay
Fresh blood was extracted from a healthy rabbit in accordance
the significant change in fluorescence color. Afterwards, mixed with the guidelines issued by the ethical committee of the Chinese
bacterial suspension was illuminated for 30 min. The bactericidal Academy of Sciences. The blood was centrifuged at 1000 rpm for 15
efficacy was investigated by the colony-forming unit (CFU) min to obtain erythrocyte concentrates. The erythrocyte concentrates
counting method as described above. The live/dead state of bacterial were washed three times with PBS. Afterward, the erythrocytes were
was also evaluated by confocal laser scanning microscopy (CLSM, resuspended in PBS to obtain an erythrocyte suspension with 20%
LSM 700, Carl Zeiss) via LIVE/DEAD BacLight Viability Kit. hematocrit (v/v). CORM-Ac or CORM solution (1mg mL-1 in
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DMSO) was diluted with PBS to a graded concentration series (1
Journal Name
The data were expressed as mean ± standard deviation (SD).
View Article Online
DOI: 10.1039/D0TB01761B
μM - 1000 μM). Then 50 μL of diluted solutions was mixed with 50 Statistical difference between various experimental and control
μL of fresh erythrocyte suspensions (final erythrocyte concentration: groups was determined by Student's t-test. Differences were
10%, v/v) and incubated for 2 h at 37 °C in the dark. Subsequently, 2 considered statistically significant at a level of *p < 0.05; **p <
mL of PBS solution was added into each sample and incubation for 0.01; ***p< 0.001.
another 1 h at 37 °C to stop the hemolysis. Positive and negative
controls were produced by adding erythrocyte suspension into 2 mL
Conflicts of interest
There are no conflicts to declare.
ODtest − ODneg
(
)
HR % =
× 100%
(1)
ODPOS−ODneg
of distilled water and PBS solution, respectively. After incubation,
the erythrocyte cells were removed by centrifugation (3000 rpm, 3
min) and the supernatant was transferred to a 96-well plate. Optical
density (OD) of the supernatant was measured using the microplate
Acknowledgements
This study was supported by the National Natural Science
reader operating at OD540 nm. The hemolysis ratio was calculated Foundation of China (Grant Nos. 51873213; 51803212), Chinese
according to the following formula:
Academy of Sciences-Wego Group Hightech Research (2017-2020)
and National Key Research and Development Program of China
(Grant No. 2020YFC1106900).
Where ODtest, ODpos and ODneg represented the absorbance values
of test sample, the positive (water) and negative (PBS) control,
respectively.
Assessment of theranostic property of CORM-Ac on S. aureus-
infected mouse models
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DOI: 10.1039/D0TB01761B
Table of Content
A lipase-sensitive and photoactivatable carbon-monoxide releasing molecule for
successive detection and elimination of bacteria infection.