Selective and Efficient Photoinactivation of Intracellular Staphylococcus aureus and MRSA with Little Accumulation of Drug Resistance: Application of a Ru(II) Complex with Photolabile Ligands

Article abstract of DOI:10.1021/acs.jmedchem.0c02257

Novel antibacterial agents capable of efficiently sterilizing intracellular Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) but with low cytotoxicity and low resistance development are quite appealing. In this work, three Ru(II) complexes with photolabile ligands were explored to realize such a goal. Complex 3 (5 μM) can inhibit more than 90% growth of S. aureus/MRSA that has invaded in J774A.1 cells upon visible light irradiation, being much more efficient than vancomycin. In similar conditions, negligible dark- and phototoxicity were found toward the host cells. The bactericidal activity is highly correlated with DNA covalent binding by the Ru(II) fractions generated after ligand photodissociation. Moreover, S. aureus quickly developed resistance toward vancomycin, while negligible resistance toward complex 3 even after 700 generations was obtained. These appealing results may pave a new way for fighting against intracellular antibiotic-resistant pathogens.

Full text of DOI:10.1021/acs.jmedchem.0c02257

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Article
Selective and Efficient Photoinactivation of Intracellular
Staphylococcus aureus and MRSA with Little Accumulation of Drug
Resistance: Application of a Ru(II) Complex with Photolabile Ligands
Weize Sun,# Yao Jian,# Mengxue Zhou, Yishan Yao, Na Tian, Chao Li, Jun Chen, Xuesong Wang,*
and Qianxiong Zhou*
Cite This: J. Med. Chem. 2021, 64, 7359−7370
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ABSTRACT: Novel antibacterial agents capable of efficiently
sterilizing intracellular Staphylococcus aureus and methicillin-resistant
S. aureus (MRSA) but with low cytotoxicity and low resistance
development are quite appealing. In this work, three Ru(II) complexes
with photolabile ligands were explored to realize such a goal. Complex
3 (5 μM) can inhibit more than 90% growth of S. aureus/MRSA that
has invaded in J774A.1 cells upon visible light irradiation, being much
more efficient than vancomycin. In similar conditions, negligible dark-
and phototoxicity were found toward the host cells. The bactericidal
activity is highly correlated with DNA covalent binding by the Ru(II)
fractions generated after ligand photodissociation. Moreover, S. aureus
quickly developed resistance toward vancomycin, while negligible resistance toward complex 3 even after 700 generations was
obtained. These appealing results may pave a new way for fighting against intracellular antibiotic-resistant pathogens.
Though significant improvements have been achieved, these
methods have suffered from instability in biological fluids and
poor drug loading efficiency of nanoparticles as well as
complex synthesis of conjugates. Moreover, the long-
established antibiotics gradually become incompetent to fight
against rapidly evolving MRSA, necessitating the development
of novel antibacterial agents with a different mechanism of
action. Particularly, a strategy that does not easily elicit
resistant responses is more appealing but challenging.
While most clinical antibiotics exert their activity via either
impairing the function of membranes or restricting the
biosynthesis of cell walls and intracellular proteins,17 DNA-
targeting antibiotics are rare though bacterial DNA differs from
cellular DNA remarkably. Cellular DNA is mainly located in
nuclei enveloped with a tight nuclear membrane, while
bacterial DNA is exposed in the cytoplasm to allow for free
access of internalized drugs. Such a disparity may open an
avenue for developing novel and highly selective antibiotics to
damage intracellular bacterial DNA rather than host cell DNA;
INTRODUCTION
■
Staphylococcus aureus (S. aureus) is one of the most common
causes of nosocomial and community-acquired infections,
closely related to pneumonia, endocarditis, osteomyelitis,
arthritis, and sepsis.1,2 Treatment of such infections is getting
more and more difficult due to the emergence and wide spread
of methicillin-resistant S. aureus (MRSA), which so far has
evolved to be resistant to nearly all clinically approved
antibiotics, including vancomycin,3 clindamycin,4 mupirocin,5
and linezolid.6 Moreover, substantial reports have revealed that
S. aureus is able to invade and survive within mammalian cells,
primarily macrophages and neutrophils.7−9 The infected
immune cells may serve as “Trojan horses” to disseminate
the bacteria away from the initial site, causing larger areas of
infections. The intracellular location protects S. aureus from the
host immune system and common antibiotics. It has been
reported that the minimum inhibitory concentration (MIC)
value of vancomycin against intracellular MRSA is more than
100 times larger than that against extracellular one, and similar
results were also observed for other antibiotics.10 As a result,
large antibiotic doses and a long treatment time are generally
required for intracellular bacteria, placing a heavy burden on
the patients as well as on society.
Received: December 29, 2020
Published: May 25, 2021
Current strategies against intracellular S. aureus are mainly to
enhance the mammalian cell penetration capability of common
antibiotics by either nanoparticle encapsulation11−13 or
covalent conjugation to antibodies,10 cell surface reporters,14
and peptides with unique sequences and functions.15,16
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Scheme 1. Chemical Structures of Complexes 1−3 and an Illustration of Selective Photo Damage of Intracellular S. aureus/
MRSA DNA by Complex 3
however, no such exploration has been reported for treating
the intractable intracellular bacteria.
best of our knowledge, complex 3 represents the first example
of leveraging a transition metal complex to selectively kill
intracellular S. aureus and MRSA without causing damage to
the host cells and showing low drug resistance after repeated
treatment. Our results may provide a new direction for
developing novel weapons against intractable intracellular
antibiotic-resistant bacteria.
Ru(II) complexes with photolabile ligands may have such a
potential. They can undergo photoinduced ligand dissociation,
and the resultant Ru(II) aqua compounds may coordinately
bind to DNA in a manner similar to cisplatin.18 Over the past
decade, many groups, including us, have focused on their
application as novel anticancer agents.19−25 Since DNA is the
potential target of these Ru(II) complexes, it occurs to us that
if rendering them with good cellular uptake but poor nucleus
penetration, these complexes may effectively kill intracellular S.
aureus, even MRSA, but have low toxicity toward mammalian
cells by virtue of their selective access to bacterial DNA rather
than nuclear DNA. Additionally, such a photoactivation
strategy can provide a spatial and temporal control of drug
activity, which will confine their action within the infected
sites, avoiding possible side effects further.26−28
In this work, three dppz (dppz = dipyrido[3,2-a:2′,3′-
c]phenazine) or 7-OCH3-dppz (7-OCH3-dppz = 7-methoxyl-
dipyrido[3,2-a:2′,3′-c]phenazine) coordinated Ru(II) polypyr-
idine complexes containing four monodentate pyridine or 4-
methoxyl-pyridine (4-OCH3-py) ligands (Scheme 1) were
designed and synthesized. Dppz ligand is well-known for its
DNA intercalation ability,29 and -OCH3 is a common
functional group in antibiotics, such as methicillin, cefotaxime,
cefmetazole, and clarithromycin.30,31 By changing the number
of -OCH3 groups and sites, the cellular uptake and nucleus
localization abilities of the resulting complexes are finely tuned.
As a result, complex 3 shows good uptake by both mammalian
and bacterial cells but poor nucleus accumulation and displays
negligible cytotoxicity toward mammalian cells upon irradi-
ation but efficiently photoinactivates intracellular S. aureus and
MRSA. More interestingly, little resistance occurred by
complex 3-treated S. aureus after 700 generations. To the
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Complexes 1−3 were
synthesized based on our reported method.32 Taking complex
2 as an example, the 7-OCH3-dppz ligand reacted with
benzeneruthenium(II) chloride dimer in CH3OH for 3 h at
room temperature, and then pyridine was added and the
solution was refluxed under a N2 atmosphere for another 4 h.
After purification by silica gel chromatography, the desired
−
complex was obtained as PF6 salts by precipitation with
1
NH4PF6. All the complexes were characterized by H NMR,
13C NMR, HR ESI-MS, and HPLC (see Supporting
Information, Figures S4−S13).
Absorption and Emission. All complexes show typical
absorption and emission spectra of Ru(II) polypyridine
complexes (Figure 1), with ligand centered (LC) transitions
in the UV region and metal to ligand charge transfer (MLCT)
transitions in the visible region. Though nonluminescent at
room temperature, all complexes are emissive at 77 K in a
EtOH/MeOH (4:1, v/v) glass, a typical character of the
Ru(II) complexes with weak ligand fields.33 Introduction of
-OCH3 groups onto the pyridine ligands decreases the energy
gap between the highest occupied molecular orbital (HOMO)
and lowest unoccupied molecular orbital (LUMO), leading to
a redshift of both absorption and emission spectra of complex
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DNA Binding. To evaluate the DNA-targeting capability of
complexes 1−3, their interactions with calf thymus DNA (CT-
DNA) and plasmid DNA were examined first. The DNA
binding constant was measured by CT-DNA titration (Figure
S14), and all the complexes show strong CT-DNA binding in
the dark with binding constants of around 5× 105 M−1 (Table
1). Their similar binding constants suggest that the -OCH3
substitution on the dppz ligand does not change its
intercalation behavior significantly.
Table 1. DNA Binding Constants, Oil/Water Partition
Coefficients, and Hemolysis Percentage of 1−3 towards
Rabbit RBCs
1
2
3
a
Kapp (× 105 M−1
Log PO/W
)
4.59 0.11
−0.15 0.01
0.64 0.01
4.67 0.08
0.08 0.01
1.12 0.03
5.17 0.15
0.28 0.01
1.40 0.02
b
Figure 1. Absorption (in H2O) and normalized emission (77 K, in
ethanol/methanol glass (4:1 in volume ratio)) spectra of complexes
1−3.
hemolysis percentage
c
(%)
a
b
DNA binding constants acquired by DNA titration. Logarithmic
c
3, consistent with other electron-donating group modified
Ru(II) complexes.34
values of n-octanol/water partition coefficients. The hemolysis
percentage toward rabbit red blood cells at 20 μM of 1−3.
Photoinduced Ligand Dissociation. The photostability
1
of complexes 1−3 was examined by UV/vis, H NMR, and
Further, complexes 1−3 had no effects on plasmid DNA
migration, which is in good agreement with their excellent dark
stability (Figure S15). Upon light irradiation (470 nm, 22.5
mW cm−2), complexes 1−3 slowed down the DNA mobility
and bleached the DNA bands (Figure 3), which is the typical
mass spectra. Our previous study showed that complex 1
dissociated one pyridine ligand upon irradiation (470 nm, 22.5
mW cm−2).32 Similarly, complexes 2 and 3 also underwent
photoinduced monodentate ligand dissociation. The absorp-
tion of complex 2 at around 500 nm increased upon 470 nm
LED irradiation (22.5 mW cm−2, Figures S1−S3), accompany-
ing an absorbance decrease at about 350 nm. Two isosbestic
points appeared at 281 and 476 nm, indicating the formation
of one single photoproduct. The absorption spectra changes of
complex 3 were indistinctive, probably due to the similar
1
absorption of complex 3 and the photoproducts. H NMR
Figure 3. Agarose gel electrophoresis pattern of pBR322 DNA (100
mM in base pairs) in Tris-EDTA (5 mM, pH = 7.5) upon irradiation
(470 nm, 22.5 mW cm−2) for 5 min in the presence of complexes 1−3
with varied concentrations. Lanes 1, 13: DNA marker; lanes 2, 12:
DNA + light; lanes 3−5: complex 1 + light with a concentration of 2,
5, and 10 μM, respectively; lanes 6−8: complex 2 + light with a
concentration of 2, 5, and 10 μM, respectively; lanes 9−11: complex 3
+ light with a concentration of 2, 5, and 10 μM, respectively.
spectra present more confident evidence. As shown in Figure 2,
two groups of peaks centered at 6.93 and 8.53 ppm appeared
upon irradiation (470 nm, 22.5 mW cm−2) for complex 3,
which can be ascribed to the meta and ortho protons of free 4-
OCH3-py, respectively. The chemical shift comparison with
the reported systems and peak integral indicates that one
equatorial 4-OCH3-py ligand dissociated from the Ru center
(Figure S5). This is further confirmed by the HR ESI-MS in
physiological saline where only one 4-OCH3-py ligand
exchanged with Cl− upon irradiation (470 nm, 22.5 mW
cm−2, Figure S7), in line with the previous reports.32,35 Similar
results were obtained for complex 2 (Figures S4 and S6). In
contrast, all complexes are stable in aqueous solutions in the
dark, a prerequisite for photoactivated processes.
phenomenon of DNA coordinate binding with a coordination-
unsaturated Ru(II) complex or a Ru(II) aqua complex,18
indicative of the photoinduced ligand dissociation of
complexes 1−3. No obvious DNA photocleavage was
observed, which is consistent with their weak singlet oxygen
generation ability. As shown in Figure S16 using 3,3′-
1
Figure 2. H NMR spectra of 3 in mixed CD3COCD3/D2O (4:1 in volume ratio) before and after irradiation (470 nm LED) with different
▼
times. indicates the peaks of free pyridine.
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(anthracene-9,10- diyl)-dipropanoic acid (9,10-ADPA) as an
1O2 trapping agent,36 [Ru(bpy)3]2+ (bpy = 2,2′-bipyridine) can
continuously bleach the absorbance of 9,10-ADPA upon
irradiation (470 nm, 22.5 mW cm−2), while complexes 1−3
quickly lost the bleaching ability probably due to the
photoinduced ligand dissociation.
Photoinactivation of Planktonic S. aureus and MRSA.
Antibacterial performances of complexes 1−3 against S. aureus
and MRSA are displayed in Figure 4. Almost no effects on the
of 1 (Figures S17 and S18). On the other hand, −OCH3 may
also favor interaction with biomolecules and partially accounts
for its prevalence in antibiotics, though the reasons behind it
are not clear yet. In the cases of complexes 2 and 3, the
presence of −OCH3 gives rise to somewhat binding enhance-
ment toward DNA (Table 1), a potential target for their
antibacterial properties.
The minimum inhibitory concentrations (MIC) and
minimum bactericidal concentrations (MBC) of 1−3 upon
470 nm irradiation (22.5 mW cm−2) were also measured,
taking methicillin and vancomycin as controls (Table S1). The
MIC and MBC values of 2 and 3 against planktonic S. aureus
and MRSA are 1−2 μg/mL, much lower than that of
methicillin, and comparable with that of vancomycin (2−4
μg/mL), an antibiotic regarded currently as the last resort
against MRSA.
Antibacterial Mechanism. To ensure the Ru(II) aqua
compounds is the real active species, we checked the
antibacterial properties of pyridine/4-OCH3-py alone and the
photoproducts of complexes 2 and 3 in the dark. As expected,
comparable bactericidal activities (Figure S19 vs Figure 4)
were observed for the photoproducts, while pyridine/4-OCH3-
py had no effect. Similar effects on DNA electrophoresis were
observed for the photoproducts of complex 3 (Figure S20 vs
Figure 3). The Ru(II) aqua compounds may coordinately bind
to the bacterial DNA, which may impede the DNA replication
and transcription in a nonreversible manner. Such a
mechanism is an oxygen-independent process, which is further
convinced by the similar antibacterial activities of 2 and 3 in
both normal and hypoxic conditions (3% O2, Figure S21).
To evaluate the DNA-targeting ability of complexes 1−3 in
bacteria, the genomes of S. aureus and MRSA were isolated
using commercial kits, and the bound Ru(II) complexes were
examined by ICP-MS (Figures S17 and S18). Before
irradiation, about 20% of 1−3 taken in the bacteria was
found to associate with DNA. After irradiation for 20 min with
470 nm LED (22.5 mW cm−2), the percentage of the bound
complexes escalated to about 30%. The DNA damage of S.
aureus was also examined by qPCR. Two genes (autolysin (alt)
and penicillin-binding protein 2a (mecA)) were selected, and
corresponding genomic segments are listed in Table 2. As
Table 2. Primers Used for Genes Amplification in qPCR
Figure 4. Antibacterial activity of complexes 1−3 against S. aureus
(A) and MRSA (B) in the dark or under light irradiation (470 nm,
LED, 22.5 mW cm−2, 20 min).
gene
atl
forward
reverse
5′-TTCGTATCCAT-
TGCATGCTTAAC-3′
5′-GTATGACATTAA-
ACCAAGTTGCTC-3′
mecA 5′-CAATTTGTCT-
5′-CTGATGGTATGC-
GCCAGTTTCTCC-3′
AACAAGTCG-3′
growth of S. aureus and MRSA were observed for 1−3 in the
dark. Upon 470 nm irradiation (22.5 mW cm−2) for 20 min,
complexes 2 and 3 (10 μM) can result in colony-forming unit
(CFU) reduction of about 5 log units against S. aureus,
suggesting the bactericidal activity is highly associated with
their photoinduced ligand dissociation. Encouragingly, 2 and 3
are also active toward MRSA with a CFU reduction of about 4
log units at 10 μM. The antibacterial activity of complex 1 is
much weaker than that of 2 and 3, hinting at the important
role of −OCH3 groups. On one hand, the introduction of
−OCH3 groups increases the lipophilicity of 2 and 3 as
characterized by oil/water partition coefficients (Table 1),
which may facilitate cell membrane penetration. The uptake
levels of 2 and 3 measured by inductively coupled plasma mass
spectrometry (ICP-MS) were about 3−4 times larger than that
shown in Figure 5, the control groups, including irradiation
(470 nm, 22.5 mW cm−2) only, complex 3 in the dark, and
vancomycin, did not cause obvious changes on DNA
amplification. In contrast, complex 3 upon irradiation can
significantly inhibit it with the Ct (cycle threshold) values
increased from about 18 to 21, indicating DNA damage had
occurred. So far, the clinical antibiotics that directly cause
DNA damage are scarce. The different antibacterial mecha-
nisms of complexes 2−3, i.e., formation of Ru-DNA coordinate
binding, are of great importance in fighting against multidrug-
resistant pathogens, including MRSA. Thomas and coauthors
also reported that Ru(II) complexes with DNA intercalating
ligand can target the nucleoid of S. aureus efficiently in a
noncoordinate way.37
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Figure 5. qPCR amplification lines (A: alt gene; C: mecA gene) and corresponding Ct values (B: alt gene; D: mecA gene) of S. aureus upon various
treatments. Each experiment was carried out three times (*p < 0.05). (E) Outer membrane permeation of S. aureus investigated by ANS. (F) Inner
membrane depolarization of S. aureus examined by diSC3(5).
To rule out other possibilities, such as membrane damage,
fluorescence staining experiments were carried out.38 8-
Anilino-1-naphthalenesulfonic acid (ANS), which displays
increased fluorescence upon binding to hydrophobic mem-
brane, is usually used as an indicator of outer membrane
permeation of bacteria. As shown in Figure 5E, the
fluorescence intensity of ANS did not show an obvious change
for the S. aureus treated by complex 3 and irradiation (470 nm,
22.5 mW cm−2), while that of the positive control (1% Triton-
X-100) was greatly enhanced. Similar results were also
obtained for 3,3′-dipropylthiadicarbocyanine iodide
(diSC3(5)), a common probe for the inner membrane
depolarization (Figure 5F). The transmission electron micro-
scope (TEM) images of S. aureus treated by complex 3 and
Figure 6. Resistance evolvement of S. aureus toward vancomycin and
irradiation (470 nm, 22.5 mW cm−2, 20 min) did not show
complex 3 upon irradiation (470 nm, 22.5 mW cm−2).
obvious membrane damage either (Figure S22). These results
suggest that bacterial membrane may not be the target of
complex 3.
generations. For complex 3, only a 2-fold enhancement in
MIC was observed after 700 generations. The following
characteristics of complex 3 may account for this interesting
result. First, bacteria may show resistance through enhanced
efflux and decreased uptake of antibiotics, but a pro-drug
strategy may bypass these traps,41 which may also be the case
for photoactivatable complex 3. Second, target modification
associated resistance is prone to take place for the single-target
antibiotics. A DNA strand consists of lots of nucleotides that
can coordinate with complex 3 through photoinduced ligand
exchange. Such a multitarget feature can significantly slow
down the resistance development through the target
modification pathway.42 Vancomycin which binds to lipid II,
is generally regarded as a single-target antibiotic; therefore, it is
very easy to suffer from high resistance development.42
Though more detailed exploration is needed, the small
Resistance Evolvement. Though drug resistance is
unavoidable for any type of antibacterial agents, there are
really some agents that can make it difficult for bacteria to
develop a resistance mechanism. Antibacterial peptides and
antibacterial photodynamic agents are two examples. While the
antibacterial peptides mainly disrupt the integrity of the
bacterial membranes,39 the multitarget feature of the
antibacterial photodynamic agents is believed to be the reason
for their low drug-resistance tendency.40 To examine the drug-
resistance development of complex 3 and irradiation-treated S.
aureus (470 nm, 22.5 mW cm−2, 20 min), a half MIC
concentration was repeatedly used (see Supporting Informa-
tion, Table S1), taking vancomycin as a control. S. aureus
quickly developed resistance toward vancomycin (Figure 6),
showing a 256 times enhancement in MIC after 320
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accumulation of drug resistance makes complex 3 quite
appealing in fighting against the quick emergence of resistance.
Hemolysis and Cytotoxicity toward Mammalian
Cells. To be clinically useful, an antibacterial agent also
needs to meet both prerequisites, i.e., low cytotoxicity toward
mammalian cells and negligible hemolysis. The hemolysis rates
of complexes 1−3 (20 μM) toward rabbit red blood cells were
measured to be less than 1.40% (Table 1 and Figure S23),
indicating their good biocompatibility. The cytotoxicity of
complexes 1−3 was evaluated toward human cervical cancer
(HeLa), and human normal liver (L-02) cells through MTT
assays. As shown in Table 3 and Figures S24 and S25,
Table 3. IC50 Values (μM) of 1−3 towards HeLa and L-O2
Cells
HeLa
L-02
a
b
L
D
L
D
1
2
3
75.5 2.3
19.3 0.4
181.0 4.8
148.3 2.6
>200
>200
53.8 1.6
2.4 0.1
174.1 4.9
93.7 0.9
170.6 4.2
>200
a
b
470 nm LED, 22.5 mW cm−2, 20 min. In the dark.
complexes 1−3 displayed little cytotoxicity in the dark,
especially for complex 3 with half maximal inhibitory
concentration (IC50) values above 200 μM. Upon 470 nm
irradiation (22.5 mW cm−2, 20 min), complex 1 exhibited a
medium cytotoxicity, and complex 2 became highly toxic with
IC50 values of 19.8 and 2.6 μM toward HeLa and L-O2 cells,
respectively. Interestingly, complex 3 still showed low toxicity
with IC50 values of about 180 μM, confirming the good
biocompatibility of complex 3.
Figure 7. L-02 (A) and HeLa (B) cellular uptake and subcellular
distribution of 1−3 (5 μM).
To better understand the cytotoxicity of 1−3, their cellular
uptake and subcellular localization were assessed using ICP-
MS after isolation of the cell nucleus from the cytoplasm. As
shown in Figure 7, the cellular uptake levels increased from 1
to 3 in both HeLa and L-O2 cells, which is consistent with
their gradually enhanced lipophilicity (Table 1). Though it has
the highest uptake level, complex 3 is nearly nontoxic
irrespective of irradiation, which may be ascribed to the
lowest nucleus localization of 3. The possible structure−
function relationship of nucleus accumulation is not clear for
Ru(II) polypyridine complexes. It has also been reported that
minor modifications can totally change the nucleus-targeting
ability of Ru(II) complexes.43 The above results imply that the
targets of complexes 1−3 in mammalian cells are most likely
nucleus DNA. As a result, the poor nucleus localization makes
nucleus DNA unavailable to complex 3. This is in sharp
contrast to the cases of bacteria. Because of no nucleus
membrane protection, both complex 2 and 3 can access
bacterial DNA freely once internalized and therefore have
similar antibacterial abilities.
Photoinactivation of Intracellular S. aureus and
MRSA. The high uptake levels of complex 3 in both
mammalian and bacterial cells, coupled with the immense
difference in its phototoxicity toward both types of cells,
prompt us to examine its potential to photoinactivate
intracellular bacteria. S. aureus and MRSA invading in
J774A.1 (mouse mononuclear macrophage) cells were used
as a model of the intracellular bacteria. Vancomycin was also
studied as a control. Vancomycin displayed a very poor activity
against intracellular S. aureus and MRSA, with an inhibition
rate of only around 10% at 20 μM (Figure 8A,B). Upon
irradiation (470 nm, 22.5 mW cm−2), complex 3 exhibited a
much better performance than vancomycin, with a more than
90% inhibition at 5 μM toward S. aureus and MRSA.
Additionally, we also examined the antibacterial activity toward
intracellular vancomycin-resistant S. aureus obtained by
repeated culture as mentioned before. Complex 3 did not
display obvious cross-resistance toward vancomycin-resistant S.
aureus (Figure 8C), probably owing to its unique DNA
coordinate binding mechanism different from most of the
current antibiotics. In similar conditions, complex 3 also
displayed little cytotoxicity toward the host J774A.1 cells
(Figure S26). Obviously, the high activity and specificity of
complex 3 benefit from its good mammalian cell uptake in
combination with its main cytoplasmic localization, which
enables it to selectively target bacterial DNA rather than
mammalian cells, leading to efficient inactivation of intra-
cellular bacteria but leaving mammalian cells largely unaffected.
Photoinactivation of Intracellular S. aureus in Vivo.
To examine the photoinactivation activity of complex 3 against
intracellular S. aureus in vivo, an intracellular S. aureus infection
nude mouse model was built as a proof of concept. Thirty
microliters of J774A.1 cells (107/mL) infected with S. aureus
were subcutaneously injected on the back of the mice. After 12
h, the mice were randomly divided into four groups with three
mice of each group. Drugs (50 μL complex 3 or vancomycin (1
mM)) or control samples (50 μL of PBS (phosphate buffer
solution, pH = 7.4)) were injected subcutaneously at the
infection region. One hour after drug injection, the light groups
were irradiated by a 520 nm LED for 10 min (350 mW cm−2).
As shown in Figure 9A, complex 3 can photoinactivate 91.2%
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Figure 8. Intracellular antibacterial activities of complex 3 (0−20 μM) in J774A.1 cells infected with S. aureus (A), MRSA (B), and vancomycin-
resistant S. aureus (C) upon irradiation (470 nm LED, 22.5 mW cm−2, 30 min). Vancomycin without irradiation was studied as a control.
Figure 9. (A) In vivo antibacterial activities of complex 3 against intracellular S. aureus in the dark or under 520 nm irradiation (350 mW cm−2, 10
min). Vancomycin without irradiation and PBS with irradiation were studied as controls; (B−F) H&E images of heart, liver, spleen, lung, and
kidney, respectively, of the mice treated with complex 3 and 520 nm irradiation. Scale bar: 100 μm.
of intracellular S. aureus, which is also much better than that of
vancomycin (63.2%). The hematoxylin and eosin (H&E)
images of the organs did not show any abnormalities,
consistent with the little cytotoxicity of complex 3 (Figure 9
and Figure S27).
remarkably. In contrast, the nucleus localization is improved
from complex 1 to complex 2 but experiences a dramatic
decrease to complex 3. Once internalized into a bacteria-
infected mammalian cell, complex 3 can access the DNA of the
intracellular bacteria but is blocked away from nucleus DNA of
the mammalian cell. Consequently, Ru(II) fractions generated
upon photodissociation of one monodentate ligand may
coordinately bind to bacterial DNA and lead to efficient
inactivation of intracellular S. aureus and drug-resistant ones
but leaving mammalian cells viable. More interestingly, S.
aureus hardly develop resistance against complex 3. These
novel results may stimulate further exploration of such types of
transition metal complexes in the battle between humans and
pathogenic microbes.
CONCLUSION
■
In summary, intracellular drug-resistant S. aureus represents
one of the most difficult to treat pathogens. In this work Ru(II)
complexes with photolabile ligands were explored for the first
time to treat such intractable bacteria. By introducing −OCH3
groups from complex 1 to complexes 2 and 3, their uptake
levels by both mammalian and bacterial cells are enhanced
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[Ru(7-OCH3-dppz)(4-OCH3-py)4]2+ calculated: 425.1124, found:
425.1082. HPLC purity >95%.
Synthesis and Characterization of [Ru(dppz)(py)4](PF6)2 (Com-
plex 1). Complex 1 was obtained similarly as complex 3. Yield: 64%.
1H NMR (400 MHz, CD3COCD3) δ 9.82 (d, J = 7.9 Hz, 2H), 9.34
(d, J = 5.3 Hz, 2H), 8.74 (dd, J = 5.3 Hz, 2H), 8.74 (d, J = 5.3 Hz,
4H), 8.53 (d, J = 6.4 Hz, 2H), 8.31 (d, J = 5.5 Hz, 2H), 8.23 (d, J =
5.3 Hz, 4H), 8.09 (d, J = 5.1 Hz, 4H), 7.86 (t, J = 7.3 Hz, 2H), 7.80−
7.72 (d, J = 4.8 Hz, 4H), 7.81−7.71 (t, 4H), 7.25 (t, J = 7.4, 6.4 Hz,
4H). 13C NMR (101 MHz, CD3CN) δ 157.09, 156.20, 155.70,
152.85, 143.92, 141.04, 139.42, 139.16, 134.73, 133.60, 132.11,
130.71, 128.27, 127.57, 127.02. HR ESI-MS of [Ru(dppz)(py)4]2+
calculated: 350.0818, found: 350.0820. HPLC purity >95%.
EXPERIMENTAL SECTION
■
Materials. Benzeneruthenium(II) chloride dimer, 1,10-phenan-
throline-5,6-dione, thiazolyl blue tetrazolium bromide, 4-methoxyl-
1,2-phenylenediamine, 9,10-anthracenedipropanoicacid (9,10-
ADPA), 8-anilino-1-naphthalenesulfonic acid (ANS), 3,3′-dipropylth-
iadicarbocyanine iodide (diSC3(5)), and 4-methoxyl-1,2-phenyl-
enediamine were purchased from Acros or Alfa Aesar. Solvents
were purchased from Beijing Chemical Works, except for acetonitrile,
which was provided by China National Medicines Corporation.
Supercoiled pBR322 plasmid DNA was obtained from TaKaRa
Biotechology. Phosphate buffer saline (PBS) (1 mM, pH = 7.4),
Dulbecco’s modification of Eagle’s medium (DMEM), penicillin,
streptomycin, and fetal bovine serum (FBS) were purchased from
Corning. Hoechst, propidium iodide (PI), and DNA isolation kits for
Gram-positive bacteria were purchased from Solarbio. Nucleus
isolation kits for mammalian cells were produced by Invent
Biotechnologies. The genomic DNA of S. aureus was isolated using
a TIANamp bacteria DNA kit (TiangenBiotec, Beijing, China). All
primers were purchased from GENEWIZ (Suzhou, China).
Methicillin and vancomycin were obtained from Inalco. Transparent
plastic centrifugal tubes were purchased from Axygen. Gram-(+) S.
aureus (ATCC6538), and MRSA (ATCC43300) were purchased
from Antibacterial Center of Technical Institute of Physics and
Chemistry and China General Microbiological Culture Collection
Center.
Synthesis and Characterization of [Ru(7-OCH3-dppz)(py)4](PF6)2
(Complex 2). Complex 2 was obtained similarly as complex 3. Yield:
1
65%. H NMR (400 MHz, CD3CN) δ 9.82 (dd, J = 14.0, 8.2 Hz,
2H), 9.34 (dd, J = 11.6, 5.4 Hz, 2H), 8.74 (d, J = 5.3 Hz, 4H), 8.53
(d, J = 6.4 Hz, 2H), 8.31 (d, J = 5.5 Hz, 2H), 8.23 (d, J = 5.3 Hz, 4H),
8.09 (d, J = 5.1 Hz, 4H), 7.86 (t, J = 7.3 Hz, 2H), 7.81−7.71 (m, 4H),
7.80−7.72 (m, 4H), 7.25 (t, J = 6.4 Hz, 4H), 4.09 (s, 3H). 13C NMR
(101 MHz, CD3CN) δ 163.88, 157.01, 155.95, 155.53, 154.68,
152.50, 151.86, 145.87, 140.78, 140.57, 139.27, 139.04, 138.47,
134.49, 134.07, 132.06, 131.73, 128.01, 127.95, 127.43, 126.90,
106.83, 57.27.HR ESI-MS of [Ru(7-OCH3-dppz)(py)4]2+ calculated:
365.0872, found: 365.0874. HPLC purity >95%.
Oil/Water Partition Coefficient (Log PO/W) Measurements.
Complex 1, 2, or 3 (10 μM) was added to a mixed solution of 10 mL
of H2O/n-octanol (v/v = 1:1), and the mixtures were sonicated in the
dark for 30 min. The oil and water phases were measured on a UV−
vis spectrophotometer to obtain the concentrations of the complexes
in each phase after centrifugation. The oil/water partition coefficients
were calculated based on the following equation:
Instruments. 1H NMR spectra were recorded on a Bruker DMX-
400 MHz using tetramethylsilane (TMS) as the chemical shift
reference. High-resolution ESI mass (HR ESI-MS) spectra were
recorded on a Bruker APEX IV (7.0T) FT_MS. UV−vis absorption
spectra were obtained using a Shimadzu UV-4100 spectrophotometer.
An LED lamp (470
10 nm) was purchased from Suzhou D&R
Instruments Co., Ltd. MTT assays were carried out on a Thermo
MK3Multiscan microplate reader at 570 nm.
Log P
= Log CO/CW
(1)
O/W
where CO and CW represent the concentrations of the complexes in
octanol and H2O, respectively. Each measurement was repeated three
times.
HPLC Analysis. The purities of complexes 1−3 were analyzed by
high-performance liquid chromatography (HPLC), which was carried
out on a HITACHI chromaster using a WH-C-18 column (5 μm, 4
mm × 150 mm) under the following conditions: detection at 254 nm,
1 mL/min flow rate with 100% CH3CN in 10 min. The HPLC purity
of complexes 1−3 were more than 95%. The HPLC spectra are
shown in Figures S11, S12, and S13.
DNA Binding Constant Measurement. CT-DNA was dissolved
in PBS (5 mM, pH 7.4) buffer solution, stirred overnight in ice bath,
followed by measuring the absorbance at 260 nm (ε = 6600 M−1
cm−1) to determine its concentration.
The DNA binding constant was determined by titrating CT-DNA
to a studied complex solution and fitting the following equation with
the data:
ICP-MS was tested on a PerkinElmer ELAN DRC-e inductively
coupled plasma mass spectrometer. Measurements were acquired in
KED or kinetic energy discrimination mode at 1200 W plasma RF
power and 3 V KED voltage. The nebulizer, auxiliary, and collision gas
flow rates were 15, 0.7, and 1.0 L/min, respectively. Ru was the target
isotope monitored. Calibration curves were obtained along with each
run on the ICP-MS using the Ru standard from Sigma-Aldrich. Each
measurement was carried out three times.
HPLC analysis was done on a HITACHI chromaster (pump: 5110,
auto sampler: 5210, column oven: 5310, UV−vis detector: 5420).
Synthesis and Characterization of [Ru(7-OCH3-dppz)(4-OCH3-
py)4](PF6)2 (Complex 3).32 First, 7-methoxyl-dipyrido[3,2-a:2′,3′-
c]phenazine (7-OCH3-dppz) was synthesized by refluxing 1,10-
phenanthroline-5,6-dione (210 mg, 1 mmol) and 4-methoxyl-1,2-
phenylenediamine (140 mg, 1 mmol) for 3 h in ethanol and
recrystallizing in methanol. Afterward, 94 mg (0.3 mmol) of 7-OCH3-
dppz and 75 mg (0.15 mmol) of benzeneruthenium(II)chloride dimer
were mixed with 20 mL of methanol and stirred for 2 h until it
became a homogeneous brownish solution. Then the solvent was
removed by rotary evaporation and redissolved in 40 mL of water.
After adding 500 μL of excess 4-methoxyl-pyridine, the solution was
refluxed for 4 h in N2. After cooling to room temperature, the product
was precipitated by NH4PF6 and purified on silica gel using CH3CN/
saturated KNO3 aqueous solution (v/v = 10/1) as eluent. The excess
KNO3 was removed by dissolving the product in ethanol and
filtration. Finally, the product was washed with acetone, ether, and
hexane to gain pure complex 3 as an orange-red powder. Yield: 65%.
1H NMR (400 MHz, CD3COCD3) δ 9.64 (dd, J = 18.9, 8.2 Hz, 2H),
0.5
2
i
j
j
j
y
z
z
z
z
z
z
i
j
j
j
y
z
z
z
z
z
2K C [DNA]
2
t
(ε − ε )(ε − ε ) = b − b −
/2KCt
j
j
j
j
j
a
f
b
f
s
(2)
(3)
k
{
k
{
b = 1 + KCt + K[DNA]/2s
where εf and εb are the extinction coefficients of the free and bound
complex, respectively. εa is the apparent extinction coefficient of the
complex in the presence of DNA. [DNA] denotes the concentration
of DNA in nuclear phosphate, while Ct is the concentration of the
complex, K is the DNA binding constant, and s is the binding site
size.44
9.22 (dd, J = 12.7, 5.5 Hz, 2H), 8.40 (d, J = 6.3 Hz, 4H), 8.32 (d, J =
9.3 Hz, 1H), 8.19 (dd, J = 13.9, 5.9 Hz, 2H), 7.77−7.69 (m, 6H), 7.21
(t, J = 6.9 Hz, 4H), 6.69 (d, J = 6.5 Hz, 4H), 4.09 (s, 3H), 3.97 (s,
6H), 3.68 (s, 6H). 13C NMR (101 MHz, CD3OCD3 δ 168.09, 167.87,
163.59, 157.46, 155.93, 155.15, 152.96, 152.32, 145.54, 140.53,
140.23, 138.24, 131.54, 131.45, 131.27, 127.77, 127.70, 127.62,
113.73, 113.22, 112.93, 106.51, 56.76, 56.68, 56.36.HR ESI-MS of
Agarose Gel Electrophoresis. Complexes 1, 2, and 3 of different
concentrations were added to supercoiled pBR322 DNA solutions
(Tris-acetic acid-EDTA buffer) and irradiated for 5 min (470 nm,
22.5 mW cm−2). Loading buffer was added to the samples before they
were loaded on the agarose gel, and the electrophoresis lasted for 60
min at 80 V. Then, the gel was soaked in an ethidium bromide (EB)
solution for 15 min and photographed by a Gel Doc XR system (Bio-
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Rad). Dark experiments were conducted similarly except for no
irradiation.
Cell Culture. The cells were cultured in DMEM with 10% fetal
bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at
37 °C and 5% CO2.
MTT Assay. 5000−8000 cells were seeded to each well in a 96-well
plate and cultured for 24 h. Complexes (dissolved in DMSO) with
gradient concentrations were added to the wells (with final DMSO
volume ratio less than 1% for all samples) and replaced by fresh media
after 4 h incubation. After 20 min irradiation (470 nm, 22.5 mW
cm−2), the cells were incubated for another 20 h. Then, the cell media
were refreshed with the one containing MTT (1 mg/mL). Four hours
later, the media with MTT were discarded, and DMSO was added. A
microplate reader was employed to measure the results.
was replaced by 0.4 μM diSC3 (3,3′-dipropylthiadicarbocyanine
iodide), and fluorescence signal was monitored between 650 and 700
nm with excitation at 622 nm.
TEM Characterization. S. aureus cells were harvested at the
exponential phase, washed using PBS, and divided into three groups.
The control group was left untreated all along. The light group was
incubated with 5 μM of complex 3 for 30 min, followed by 20 min of
irradiation (470 nm, 22.5 mW cm−2). The dark group was cultured
with 5 μM of complex 3 without irradiation. The cell pellets were
obtained and subjected to fixation with 2% glutaraldehyde at 4 °C for
12 h. After washing with PBS, samples were dehydrated for 15 min in
a graded ethanol series (30%, 50%, 70%, 90%, and 95%) and another
15 min in 100% ethanol. Finally, the dried specimens were
investigated by TEM.
Bacterial DNA Isolation and qPCR. The S. aureus cells
(including five groups: (1) control; (2) S. aureus + light irradiation
(470 nm, 22.5 mW cm−2 for 20 min); (3) S. aureus + 5 μM complex 3
in the dark; (4) S. aureus + 5 μM vancomycin in the dark; (5) S.
aureus + 5 μM complex 3 upon irradiation (470 nm, 22.5 mW cm−2
for 20 min)) were collected using centrifugation and washed with
PBS. The genomic DNA was isolated by the TIANamp bacteria DNA
kit (TiangenBiotec, Beijing, China) and quantified with Nano-
dropOne UV−vis spectrophotometers (Thermo Fisher Scientific,
Waltham, MA, USA). The amplification efficiency of the selected atl
(autolysin) and mecA (penicillin-binding protein 2a) genes was
investigated by qPCR. Reactions were performed in triplicate in three
independent experiments. The primer sequences were designed based
on the sequence data from NCBI and are listed in Table 2.
MIC/MBC Measurement against S. aureus and MRSA. Serial
2-fold dilutions of each drug (128−0.5 μg/mL or 0.25−0.002 μg/mL)
were added to the LB media containing ∼105 bacteria per 1 mL. After
30 min of incubation, the ones with complexes 1−3 were irradiated
(470 nm, 22.5 mW cm−2) for 20 min, while the other groups
remained in the dark. MIC and MBC were obtained after 24 h
incubation by measuring the optical density at 570 nm (OD570) on a
microplate reader or resazurin tests, respectively.
Evaluation of Antimicrobial Resistance. The antimicrobial
resistance was evaluated by following the literature method.45 S.
aureus was inoculated and cultured in LB medium at 37 °C for 18 h,
and the bacteria were diluted to ∼105 CFU/mL in MH medium as
the working suspension. A mother solution of complex 3 (0.5 mg
mL−1 in DMSO) was used. One microliter mother solution was added
to 1 mL of LB media containing ∼105 bacteria, yielding a solution of
complex 3 with a concentration of 0.5 μg mL−1 (equal to 0.5 × MIC).
After complex 3 was added (0.5 × MIC) for 30 min, the mixture was
irradiated (470 nm, 22.5 mW cm−2) for 20 min and then was
incubated at 37 °C for another 23 h and 10 min. Vancomycin at a
concentration of 0.5 × MIC without irradiation was also studied as
the control. Twenty-four hours later, the cell suspension was diluted
1000-fold with MH medium in a new container for another cycle of
bacteria-drug incubation. This cycle was repeated every 24 h (about
10 generations of growth per serial passage). The MIC values were
examined every 2 days. The concentrations of the complex 3 or
antibiotics were adjusted accordingly to remain at 0.5 × MIC. MIC
tests were repeated at least twice at different times.
Cell Uptake and Subcellular Distribution Measured by ICP-
MS. A total of 105 cells were seeded in a 25 cm2 bottle and cultured
for 24 h to reach 80% confluence. Then, the cells were incubated for
another 4 h with 1 μM of a certain complex. After being washed by
PBS for at least three times, the cells were harvested, and the nucleus
was separated from the cytoplasm with a toolkit purchased from the
company of Invent Biotechnologies. The samples were digested in a
solution of nitric acid and hydrochloric acid (1:3) and finally made
into a 2% HNO3 solutions for further ICP-MS detections.
Bacterial Culture. Gram-(+) S. aureus and MRSA were cultured
in either an LB medium or an agar plate medium at 37 °C for 16 h in
an incubator with a shaking rate at 100 rpm. The bacteria suspension
was centrifuged and washed by PBS, the concentration of which was
estimated based on the optical density at 600 nm.
Antibacterial Studies. Suspensions (1 mL) of bacteria (∼108
mL−1 in PBS) at the exponential phase in a transparent plastic
centrifugal tube were added with gradient concentrations of
complexes (dissolved in DMSO, and the final DMSO volume ratio
is less than 1% for all the samples). The control experiments
containing 1% DMSO (in volume ratio) were also carried out. The
mixtures were cultured in the dark in an incubator (37 °C, 100 rpm)
for 30 min, and the light groups were irradiated (470 nm, 22.5 mW
cm−2) for 20 min; meanwhile, the dark group remained in the dark.
Then, the samples were diluted with PBS, added on 3M Petrifilm
aerobic count plates, and incubated for another 24 h at 37 °C before
the number of colony-forming units (CFUs) was counted. Each
experiment was conducted at least three times.
Antibacterial Studies of the Photoproducts of 2 and 3.
Complexes 2 and 3 in PBS were irradiated by 470 nm LED (22.5 mW
cm−2) for 20 min, and the resultant mixtures were not purified and
were directly used as the “photoproducts”. Irradiation by 470 nm LED
(22.5 mW cm−2) for 20 min can lead to almost total photolysis of
these two complexes, and thus the concentrations of photoproducts
were estimated by using the initial concentrations of complexes 2 or 3.
The antibacterial activity of the photoproducts was examined against
S. aureus without further irradiation.
Antibacterial Studies in Anaerobic Conditions. The studied
complexes were added to the bacteria suspensions in plastic
centrifugal tubes, and then the tubes were sealed with AnaeroGenTM
2.5 L biobags produced by Mitsubishi Gas Chemical Co. to generate
anaerobic conditions (O2% < 0.1%). After 30 min, the centrifugal
tubes in the transparent bag were exposed to irradiation (470 nm,
22.5 mW cm−2) for 20 min, followed by CFU counting in the above-
mentioned manner.
Hemolysis Assay. To 20 μL of rabbit erythrocytes, varied
concentrations of Ru(II) complexes dissolved in PBS were added, and
the final volumes of the solutions were adjusted to 1 mL with PBS. A
total of 980 μL of H2O with 20 μL of erythrocytes and 980 μL of PBS
with 20 μL of erythrocytes were employed as the positive and negative
controls, respectively. All samples were incubated at 37 °C on a shake
bed for 2 h before centrifugation. The supernatants were transferred
to a 96-well plate with 200 μL for each well, and the OD570 values
were measured on a microplate reader. The hemolysis was calculated
using the following equation
Outer Membrane Permeability Assay.38 Measurements were
performed using the 8-anilinonapthalene-1-sulfonic acid (ANS)
staining assay. S. aureus cells were grown overnight in LB media
and washed with PBS (pH 7.4) and resuspended in the same buffer
with 5 μM complex 3. Thirty minutes later, the mixture was irradiated
(470 nm, 22.5 mW cm−2) for 30 min and incubated for another 2 h.
1% Triton-X-100 without irradiation was used as a positive control.
Ten micromolar ANS was added to each group of samples, and the
fluorescence intensity was measured between 450−600 nm with
excitation at 380 nm.
hemolysis (%) = (As − An)/(Ap − An) × 100%
(4)
Inner Membrane Depolarization Assay.38 The method was
similar to the outer membrane permeability assay. Only 10 μM ANS
where As, An, and Ap stand for the OD570 of the samples, negative
controls, and positive controls, respectively.
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Bacterial Uptake and Subcellular Distribution. Suspensions of
bacterial cells (108 CFU/mL) in PBS (1 mM, pH = 7.4) were
incubated with complexes 1−3 (5 μM) at 37 °C for 40 min in the
dark. The light groups were irradiated with 470 nm LED (22.5 mW
cm−2) for 20 min, while the dark groups remained in the dark. After
irradiation, the cells were collected immediately by centrifugation and
washed with PBS three times. The DNA was isolated by a kit
provided by Solarbio, and the residuals were also collected. Both were
made into 2% nitric acid solutions for ICP-MS measurements to
determine the ruthenium concentrations.
Beijing 100190, P. R. China; orcid.org/0000-0001-7472-
0164; Email: zhouqianxiong@mail.ipc.ac.cn
Authors
Weize Sun − Key Laboratory of Photochemical Conversion
and Optoelectronic Materials, Technical Institute of Physics
and Chemistry, Chinese Academy of Sciences, Beijing 100190,
P. R. China; University of Chinese Academy of Sciences,
Beijing 100049, P. R. China
Yao Jian − Key Laboratory of Photochemical Conversion and
Optoelectronic Materials, Technical Institute of Physics and
Chemistry, Chinese Academy of Sciences, Beijing 100190, P.
R. China; University of Chinese Academy of Sciences, Beijing
100049, P. R. China
Mengxue Zhou − University of Chinese Academy of Sciences,
Beijing 100049, P. R. China; ACAS Key Laboratory for
Biomedical Effects of Nanomaterials and Nanosafety, Multi-
disciplinary Research Division, Institute of High Energy
Physics, Chinese Academy of Sciences, Beijing 100190, P. R.
China
Determination of Intracellular Survival Index.16 J774A.1 cells
were plated at a density of 4 × 105 cells and infected with S. aureus or
MRSA at a ratio of 10−20 bacteria per cell. Then J774A.1 cells were
cultured in DMEM with 50 μg/mL gentamicin to inhibit the growth
of extracellular bacteria. Complex 3 or the examined antibiotics were
added to the media 1 day after infection and incubated for 4 h. The
plates with complex 3 were irradiated for 30 min, and the survival of
intracellular bacteria was assayed after another 4 h by the following
method. The plates with the antibiotics were handled in parallel
except for no irradiation.
J774A.1 cells were lysed with Hanks buffered saline solution
supplemented with 0.1% bovine serum albumin and 0.1% Triton-X,
and the resulting solution was diluted with PBS containing 0.05%
Tween-20. The survival intracellular bacteria were quantified by CFU
counting after spreading and culturing the lysate on 3 M Petrifilm
aerobic count plates. Each experiment was performed at least three
independent times.
Yishan Yao − Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, P. R. China
Na Tian − Key Laboratory of Photochemical Conversion and
Optoelectronic Materials, Technical Institute of Physics and
Chemistry, Chinese Academy of Sciences, Beijing 100190, P.
R. China; University of Chinese Academy of Sciences, Beijing
100049, P. R. China
Chao Li − Key Laboratory of Photochemical Conversion and
Optoelectronic Materials, Technical Institute of Physics and
Chemistry, Chinese Academy of Sciences, Beijing 100190, P.
R. China
Jun Chen − University of Chinese Academy of Sciences, Beijing
100049, P. R. China; ACAS Key Laboratory for Biomedical
Effects of Nanomaterials and Nanosafety, Multi-disciplinary
Research Division, Institute of High Energy Physics, Chinese
Academy of Sciences, Beijing 100190, P. R. China
In Vivo Photoinactivation of Intracellular Bacteria.28 Experi-
ments involving animals were performed following guidelines
approved by the Animal Care and Use Committee of CAS Key
Laboratory for Biomedical Effects of Nanomaterials and Nanosafety.
Female BALB/c mice (4−5 weeks, 13 2 g) were obtained from
Beijing WeiTongLiHua Animal Co. Ltd. S. aureus-infected J774A.1
cells (30 μL, about 107 cells) was subcutaneously injected into right
back of mice to establish the intracellular bacterial infection model.
Twelve hours later, complex 3 or vancomycin (50 μL, 1 mM in PBS)
was subcutaneously injected into the infection region. One hour later,
the infected sites of mice in PBS control and “complex 3 + light”
group were exposed to light (520 nm, 350 mW cm−2) for 10 min.
After 24 h, all the mice were sacrificed, and the infected skin was
collected and then homogenized in a tissue grinder with 2 mL of PBS
and then spread onto 3M Petrifilm aerobic count plates.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02257
Statistical Analysis. All data are expressed as the mean SD of
at least three independent experiments. p-Values below 0.05 were
considered as statistically significant, which were analyzed using a
two-tailed Student’s t test.
Author Contributions
#W.S. and Y.J. contributed equally.
Funding
ASSOCIATED CONTENT
This work was financially supported by National Key R&D
Program of China (2018YFC1602204), NSFC (21390400,
21571181 and 21773277) and the Strategic Priority Research
Program of Chinese Academy of Sciences (XDB17000000).
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02257.
1
UV−vis absorption, H NMR, 13C NMR, HPLC trace,
Notes
The authors declare no competing financial interest.
antibacterial activity, and MTT assays of complexes 1−3
(PDF)
ACKNOWLEDGMENTS
Molecular formula strings (CSV)
■
The authors thank Prof. Yong Chen, Ye Tian, Zhongwei Niu
(Technical Institute of Physics and Chemistry, CAS) and Dr.
Chuanchao Du (Peking University Third Hospital) for helpful
discussion.
AUTHOR INFORMATION
■
Corresponding Authors
Xuesong Wang − Key Laboratory of Photochemical
Conversion and Optoelectronic Materials, Technical Institute
of Physics and Chemistry, Chinese Academy of Sciences,
Beijing 100190, P. R. China; University of Chinese Academy
of Sciences, Beijing 100049, P. R. China; orcid.org/0000-
0002-4552-5762; Email: xswang@mail.ipc.ac.cn
Qianxiong Zhou − Key Laboratory of Photochemical
Conversion and Optoelectronic Materials, Technical Institute
of Physics and Chemistry, Chinese Academy of Sciences,
ABBREVIATIONS USED
■
9,10-ADPA, 3,3′-(anthracene-9,10-diyl)-dipropanoic acid; alt,
autolysin; ANS, 8-anilino-1-naphthalenesulfonic acid; bpy,
2,2′-bipyridine; CFU, colony-forming unit; Ct, cycle threshold;
CT-DNA, calf thymus DNA; diSC3(5), 3,3′-dipropylthiadi-
carbocyanine iodide; DMEM, Dulbecco’s modification of
Eagle’s medium; DMSO, dimethyl sulfoxide; DNA, deoxy-
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ribonucleic acid; dppz, dipyrido[3,2-a:2′,3′-c]phenazine; EB,
ethidium bromide; EDTA, ethylene diamine tetraacetic acid;
FBS, fetal bovine serum; H&E, hematoxylin and eosin; HeLa,
human cervical cancer cell; HOMO, highest occupied
molecular orbital; HPLC, high-performance liquid chromatog-
raphy; HR ESI-MS, high-resolution ESI mass; IC50, half
maximal inhibitory concentration values; ICP-MS, inductively
coupled plasma mass spectrometry; J, coupling constant (in
NMR spectrometry); L-02, human normal liver cell; LB,
Luria−Bertani broth; LC, ligand centered; Log P, logarithm of
partition coefficient; LUMO, lowest unoccupied molecular
orbital; M, molar (moles per liter); MBC, minimum
bactericidal concentrations; mecA, penicillin-binding protein
2a; MH, Mueller−Hinton Broth; MHz, megahertz; MIC,
minimum inhibitory concentrations; mL, milliliter; MLCT,
metal to ligand charge transfer; mM, millimolar (millimoles per
liter); MRSA, methicillin-resistant S. aureus; MS, mass
spectrometry; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl
tetrazolium bromide; PBS, phosphate buffer solution; PI,
propidium iodide; py, pyridine; S. aureus, Staphylococcus
aureus; TEM, transmission electron microscope; TMS,
tetramethylsilane
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