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Chemie
As the ROS generation of the photosensitizers under
PyTPE-CRP and light irradiation were significantly de-
white light irradiation has been known to play a pivotal role
for photodynamic antimicrobial therapy, we next studied the
ROS generation of the probe inside the infected macrophages
upon irradiation with white light by using dichlorofluorescein
creased compared to the groups without light irradiation.
Finally, to demonstrate the impact of early detection of
bacterial infection in macrophage cells, S. aureus infected
macrophages were treated with vancomycin at different
infection time points. As displayed in Figure 5A, the MIC
of vancomycin towards intracellular bacteria with 24 h
infection is much higher than that with 1 h infection (the
[19]
diacetate (DCF-DA) as the ROS indicator, which can be
rapidly oxidized in the presence of ROS to yield the green
fluorescent molecule of dichlorofluorescein (DCF). As shown
in Figure 4C, strong green fluorescent signals emerged on the
bacterial phagosomes owing to the accumulation of the probe
residues on the phagosomes, which could generate a high
level of ROS under light irradiation. The intensity profile
across the bacterial phagosomes was plotted in Figure 4D. As
noted, the average ROS indicator fluorescent intensity per
unit area in the phagosomes is about 2.7-fold higher than that
in the cytoplasm.
To determine whether the probe could eradicate the
bacteria surviving in macrophages under white light irradi-
ation, Raw 264.7 macrophages were infected with S. aureus in
the presence of the probe at different concentrations, either in
the dark or under light irradiation. The surviving intracellular
bacteria were examined by counting the colony forming units
Figure 5. A) MIC values of vancomycin towards the bacteria survived
in Raw 264.7 cells with different time infection. Raw 264.7 cells were
infected with S. aureus with a MOI of 20. After 1 h of infection,
samples were washed with PBS for three times and incubated with
(
CFU) (Figure 4E, Figure S16). As noted, the probe showed
concentration-dependent inhibition of intracellular S. aureus
under light irradiation. A significant reduction of the intra-
cellular S. aureus recovered in number after light irradiation
compared to that in dark. Meanwhile, the antimicrobial
activity of the probe towards the extracellular S. aureus was
examined by using broth dilution method to determine the
minimal inhibition concentration (MIC). The probe displayed
À1
fresh medium containing 50 mgmL of gentamycin to inhibit the
growth of S. aureus outside of Raw 264.7 and different concentrations
of vancomycin were added to the medium at 1 h and 24 h after
infection. The survival of intracellular S. aureus was assessed by lysing
the macrophages. B) Cytotoxicity of intracellular S. aureus towards Raw
2
64.7 cells. After 1 h of infection, samples were washed with PBS three
À1
times and incubated with fresh medium containing 50 mgmL of
gentamycin to inhibit the growth of extracellular S. aureus. At specific
time points, the viability of macrophages was determined by MTT
assay.
an MIC of 15 mm (Figure 4F) under white light irradiation for
À2
1
2
0 min at a power of 40 mWcm . Finally, the viability of Raw
64.7 macrophages was evaluated with different concentra-
tions of PyTPE-CRP in the dark or under light irradiation
using MTT assay (Figure 4G). In the dark, the probe
exhibited negligible dark toxicity on macrophages. Similarly,
upon light irradiation, minimal cytotoxicity for macrophages
was observed for up to 20 mm of the PyTPE-CRP, implying its
good biocompatibility.
time needed for casp-1 to give a clear signal for bacterial
infection in macrophage). This is because once engulfed by
macrophages, S. aureus was not only able to survive in the
macrophages, but also replicate in the macrophages, leading
to intracellular bacteria increase during long-term infec-
[
20]
To explore the feasibility of PyTPE-CRP for intracellular
bacteria detection and photoinactivation in vivo, we built
a simple bacterial infection model using nude mouse. In order
to elucidate that PyTPE-CRP can reach the intracellular
infection and realize molecular cleavage induced aggregation,
we built the infection model by subcutaneous orthotopic
tion. Meanwhile, we also analyzed whether the long-term
intracellular bacterial infection exerts cytotoxic effect on
macrophages. For that, the viability of S. aureus infected
macrophages was examined by MTT method. As noted in
Figure 5B, intracellular S. aureus induced a slight macrophage
death from 12 h post infection and reached approximately
30% death 24 h post infection. These results indicate that
early-stage bacterial detection and elimination could help
reduce the death of macrophages and decrease inappropriate
use of antibiotics, which are often given empirically.
7
injection of S. aureus infected Raw 264.7 cells (10 cells) on
the top of mouse right leg and injected Raw 264.7 cells using
the same quantity as the control. The fluorescence imaging
was performed after 4 h PyTPE-CRP administration through
i.v. injection. As shown in Figure S17A, the fluorescent signal
distribution suggests that the PyTPE-CRP probes are well
cleaved. However, there was no fluorescent signal observed Conclusion
for the control. Moreover, we also evaluated whether the
intracellular bacteria could be eliminated by PyTPE-CRP
through PDT. The S. aureus-infected Raw 264.7 cells treated
regions were exposed to white light irradiation for 10 min.
After 24 h, the infected regions were collected, and the
survival bacteria were measured by CFU counting (Fig-
ure S17B). The CFU of the infected regions treated with
In summary, we have developed a new molecular probe
(PyTPE-CRP) with two moieties, an AIE molecule (PyTPE)
and an enzyme-responsive peptide linker (NEAYVHDAP),
for macrophage-mediated bacterial infection detection and
intracellular bacteria elimination. We have shown that the
designed PyTPE-CRP could be specifically cleaved by casp-
Angew. Chem. Int. Ed. 2019, 58, 2 – 9
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