Photoresponsive micelles enabling codelivery of nitric oxide and formaldehyde for combinatorial antibacterial applications

Article abstract of DOI:10.1021/acs.biomac.1c00251

It is of particular interest to develop new antibacterial agents with low risk of drug resistance development and low toxicity toward mammalian cells to combat pathogen infections. Although gaseous signaling molecules (GSMs) such as nitric oxide (NO) and formaldehyde (FA) have broad-spectrum antibacterial performance and the low propensity of drug resistance development, many previous studies heavily focused on nanocarriers capable of delivering only one GSM. Herein, we developed a micellar nanoparticle platform that can simultaneously deliver NO and FA under visible light irradiation. An amphiphilic diblock copolymer of poly(ethylene oxide)-b-poly(4-((2-nitro-5-(((2-nitrobenzyl)oxy)methoxy)benzyl)-(nitroso)amino)benzyl methacrylate) (PEO-b-PNNBM) was successfully synthesized through atom transfer radical polymerization (ATRP). The resulting diblock copolymer self-assembled into micellar nanoparticles without premature NO and FA leakage, whereas they underwent phototriggered disassembly with the corelease of NO and FA. We showed that the NO- and FA-releasing micellar nanoparticles exhibited a combinatorial antibacterial performance, efficiently killing both Gram-negative (e.g., Escherichia coli) and Gram-positive (e.g., Staphylococcus aureus) bacteria with low toxicity to mammalian cells and low hemolytic property. This work provides new insights into the development of GSM-based antibacterial agents.

Full text of DOI:10.1021/acs.biomac.1c00251

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Photoresponsive Micelles Enabling Codelivery of Nitric Oxide and
Formaldehyde for Combinatorial Antibacterial Applications
Yutian Duan,§ Kewu He,§ Guoying Zhang, and Jinming Hu*
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ABSTRACT: It is of particular interest to develop new antibacterial agents with low
risk of drug resistance development and low toxicity toward mammalian cells to
combat pathogen infections. Although gaseous signaling molecules (GSMs) such as
nitric oxide (NO) and formaldehyde (FA) have broad-spectrum antibacterial
performance and the low propensity of drug resistance development, many previous
studies heavily focused on nanocarriers capable of delivering only one GSM. Herein,
we developed a micellar nanoparticle platform that can simultaneously deliver NO
and FA under visible light irradiation. An amphiphilic diblock copolymer of
poly(ethylene oxide)-b-poly(4-((2-nitro-5-(((2-nitrobenzyl)oxy)methoxy)benzyl)-
(nitroso)amino)benzyl methacrylate) (PEO-b-PNNBM) was successfully synthesized
through atom transfer radical polymerization (ATRP). The resulting diblock
copolymer self-assembled into micellar nanoparticles without premature NO and
FA leakage, whereas they underwent phototriggered disassembly with the corelease of
NO and FA. We showed that the NO- and FA-releasing micellar nanoparticles exhibited a combinatorial antibacterial performance,
efficiently killing both Gram-negative (e.g., Escherichia coli) and Gram-positive (e.g., Staphylococcus aureus) bacteria with low toxicity
to mammalian cells and low hemolytic property. This work provides new insights into the development of GSM-based antibacterial
agents.
antibacterial activity with a low risk of resistance develop-
ment.6,7,36,37 It should be mentioned that the antibacterial
performance of NO is highly concentration-dependent. That is,
NO can not only efficiently disperse bacterial biofilms at low
concentrations but also kill planktonic bacteria at elevated
doses.38−40 Note that the formation of bacterial biofilms
invalidated conventional antibiotics, making it rather challeng-
ing to treat biofilm-related infections.41 The unique biofilm
dispersal and bacterial killing capacity of NO thus made it a
promising candidate for new antibiotics.42
On the other hand, like NO, formaldehyde (FA) is not only
an air pollutant but also an endogenous reactive carbonyl
species (RCS).43 Recent studies also suggested that FA could
be an endogenous signaling molecule,44 and abnormal FA
concentrations were closely related to Alzheimer’s disease,45
diabetes,46 and so on.47 As such, much effort has been devoted
to developing FA probes to disentangle the physiological and
pathological roles of FA.48−51 Notably, the antibacterial
capacity of an FA solution (i.e., formalin) has been known
INTRODUCTION
■
The advent of a postantibiotic era necessitates the develop-
ment of new antibiotic agents.1,2 Unfortunately, microbes can
develop antibiotic resistance much faster than that of the
discovery of new antibiotics.3,4 To resolve this challenge, much
attention has been paid to develop antibacterial agents with a
low propensity to evolve drug resistance.5−7 In sharp contrast
to conventional antibiotics having specific targets, antibacterial
agents such as cationic polymers8 and antibacterial peptides9
that can interact with negatively charged cell walls having no
defined targets remarkably prevented bacteria from developing
resistance. However, the cationic nature of positively charged
polymers was generally associated with high cytotoxicity (e.g.,
hemolysis),10 while the insufficient in vivo stability and high
costs of antibacterial peptides remained a major concern.11
Therefore, it is rather challenging to develop new antibacterial
agents that can selectively kill microbes without affecting the
viability of normal cells.12,13
Nitric oxide (NO) is endogenously produced by the
conversion of L-arginine to L-citrulline,14,15 exhibiting multiple
physiological roles such as vasodilation,16,17 wound heal-
ing,18,19 and immune regulation.20,21 Notably, NO also plays a
dominant role in living organisms to combat infections,22,23
which can kill invading pathogens through nitrosative and
oxidative stress.6 As such, a plethora of NO-releasing materials
have been developed24−31 and their antibacterial performance
was systematically evaluated,32−35 revealing a board-spectrum
Received: February 24, 2021
Revised: April 7, 2021
Published: April 22, 2021
https://doi.org/10.1021/acs.biomac.1c00251
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© 2021 American Chemical Society
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Scheme 1. Schematic Illustration of the Photomediated Conversion of PEO-b-PNNBM (PNOFA) to PEO-b-PMAA Diblock
Copolymers with the Corelease of NO and FA Signaling Molecules, Showing a Broad-Spectrum Antibacterial Activity toward
Both Gram-Positive and Gram-Negative Bacteria
for years,52 and it has been widely used as a preservative in
cosmetics and medicinal creams. However, although NO and
FA are both endogenously produced and show antibacterial
performance, the development of antibacterial agents that can
simultaneously release NO and FA has yet been achieved.
Herein, NO-/FA-releasing diblock copolymers were success-
fully synthesized through atom transfer radical polymerization
(ATRP)53 of the photoresponsive 4-((2-nitro-5-(((2-
nitrobenzyl)oxy)methoxy)benzyl)(nitroso)amino)benzyl
methacrylate (abbreviated as NNBM). The as-synthesized
amphiphilic diblock copolymers of PEO-b-PNNBM (PNOFA;
Scheme 1) self-assembled into micellar nanoparticles in an
aqueous solution. Under visible light irradiation, the micellar
nanoparticles underwent the photolysis reaction and simulta-
neously released NO and FA, killing both Gram-negative (e.g.,
Escherichia coli) and Gram-positive (e.g., Staphylococcus aureus)
bacteria in a combinatorial manner without incurring obvious
cytotoxicity toward mammalian cells (Scheme 1). This work
opens an avenue toward new antibacterial agents by engineer-
ing endogenous gaseous signaling molecules as broad-spectrum
antibiotics.
amine (DIPEA) were purchased from Sinopharm Co., Ltd. 2,2′-
Bipyridyl (bpy) was purchased from Thermo Fisher. All other
chemicals were purchased from commercially available sources and
were used as received unless otherwise indicated. Solvents were
purified through a Pure SolvTM system. Water was deionized through
a Milli-Q apparatus (Millipore) with a resistivity of 18.2 MΩ·cm. 1-
((Chloromethoxy)methyl)-2-nitrobenzene (Scheme 2),54 poly-
(ethylene oxide) (PEO)-based macroinitiator (PEG-Br),55 and
PEO45-b-PNBM21 (PNO) diblock copolymer were synthesized
according to reported procedures.
Sample Synthesis. The synthetic scheme employed for the
preparation of the NNBM monomer and the PEO-b-PNNBM
(PNOFA) amphiphilic diblock copolymer through atom transfer
radical polymerization (ATRP) is shown in Scheme 2.
Synthesis of 2-Nitro-5-(((2-nitrobenzyl)oxy)methoxy)-
benzaldehyde (Compound 1, Scheme 2a). 5-Hydroxy-2-nitro-
benzaldehyde (2.39 g, 14.29 mmol, 1.0 equiv), 1-((chloromethoxy)-
methyl)-2-nitrobenzene (4.03 g, 20 mmol, 1.4 equiv), and anhydrous
DIPEA (5.54 g, 42.87 mmol, 3.0 equiv) were dissolved in dry N,N-
dimethylformamide (70 mL) and were stirred at room temperature
overnight under a nitrogen atmosphere. After removing the organic
solvent under reduced pressure, the solid residue was purified by
column chromatography, yielding compound 1 as a yellowish solid
(3.89 g, yield: 81.9%; Figure S1). 1H NMR (400 MHz, chloroform-d,
δ): 10.47 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.07 (dd, J = 8.2, 1.2 Hz,
1H), 7.71 (dd, J = 7.7, 1.4 Hz, 1H), 7.65 (td, J = 7.6, 1.2 Hz, 1H),
7.53 (d, J = 2.8 Hz, 1H), 7.50−7.42 (m, 1H), 7.32 (dd, J = 9.0, 2.8
Hz, 1H), 5.52 (s, 2H), 5.15 (s, 2H); 13C NMR (101 MHz,
chloroform-d, δ): 188.25, 161.35, 147.18, 143.10, 134.18, 133.86,
133.09, 128.78, 128.62, 127.20, 124.92, 120.11, 115.71, 93.01, 68.09;
EXPERIMENTAL SECTION
■
Materials. 5-Hydroxy-2-nitrobenzaldehyde, 2-nitrobenzyl alcohol,
4-nitrobenzyl alcohol, acetic anhydride, acetic acid (AcOH), sodium
nitrite (NaNO2), sodium borohydride (NaBH4), copper(I) bromide,
anhydrous sodium sulfate (Na2SO4), 2-bromoisobutyryl bromide,
methacryloyl chloride, sulfuryl chloride, and N,N-diisopropylethyl-
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Scheme 2. (a) Synthetic Routes of a NO- and FA-Releasing NNBM Monomer and a PNOFA Amphiphilic Diblock Copolymer;
(b) Proposed Photolysis Mechanism of an NNBM Monomer under Visible Light Irradiation
ESI-MS m/z: [M + Na]+ calcd for C15H12N2O7, 355.0537; found,
355.0545.
solvent, the solid residue was purified by column chromatography,
yielding compound 2 as a yellowish solid (3.62 g, yield: 56.3%; Figure
S2). H NMR (400 MHz, DMSO-d6, δ): 8.18 (d, J = 9.1 Hz, 1H),
1
Synthesis of N-(4-(Hydroxymethyl)phenyl)-N-(2-nitro-5-(((2-
nitrobenzyl)oxy) methoxy)benzyl)nitrous Amide (Compound 2,
Scheme 2). Compound 1 (4.56 g, 13.72 mmol, 1.0 equiv) and 4-
aminobenzyl alcohol (1.69 g, 13.72 mmol, 1.0 equiv) were dissolved
in a mixed solvent (EtOH/tetrahydrofuran (THF), v/v = 1/2; 50
mL) and were stirred at room temperature overnight. After removing
the organic solvent, the solid residue was dissolved in a mixed solvent
(CH3OH/THF, v/v = 1/1; 50 mL) and NaBH4 (0.25 g, 13.72 mmol,
1.0 equiv) was added dropwise. The mixture solution was stirred at
room temperature for 3 h. Then, water (10 mL) was added to the
reaction solution and the solvents were removed. The residue was
extracted with dichloromethane, and the organic phase was dried over
anhydrous Na2SO4. After that, dichloromethane (DCM) was removed
under reduced pressure, the resulting mixture was dissolved in AcOH
(67.65 mL), and NaNO2 (4.03 g, 15.09 mmol, 1.1 equiv) aqueous
solution (150 mL) was added. The mixture was stirred at room
temperature for 30 min. Afterward, the resultant solution was slowly
poured into the NaHCO3 aqueous solution. The resulting mixture
was extracted with dichloromethane (DCM) and the organic phase
was dried with anhydrous Na2SO4. After removing the organic
8.04 (d, J = 7.9 Hz, 1H), 7.72−7.65 (m, 1H), 7.66−7.48 (m, 4H),
7.41 (d, J = 8.3 Hz, 2H), 7.16 (dd, J = 9.2, 2.7 Hz, 1H), 6.45 (d, J =
2.7 Hz, 1H), 5.59 (s, 2H), 5.42 (s, 2H), 5.30 (t, J = 5.7 Hz, 1H), 4.98
(s, 2H), 4.52 (d, J = 5.7 Hz, 2H); 13C NMR (101 MHz, DMSO-d6,
δ): 161.23, 147.51, 142.71, 141.63, 139.82, 134.35, 133.37, 132.82,
129.32, 129.31, 128.74, 127.94, 125.06, 120.09, 115.35, 93.40, 67.79,
62.68, 46.01; ESI-MS m/z: [M + Na]+ calcd for C22H20N4O8,
491.1173; found, 491.1181.
Synthesis of 4-((2-Nitro-5-(((2-nitrobenzyl)oxy)methoxy)benzyl)-
(nitroso)amino)benzyl Methacrylate (NNBM, Scheme 2). Com-
pound 2 (2.41 g, 5.14 mmol, 1.0 equiv), TEA (1.04 g, 10.28 mmol,
2.0 equiv), and methacryloyl chloride (0.64 g, 6.17 mmol, 1.2 equiv)
were dissolved in dry dichloromethane (50 mL) and were stirred at
room temperature overnight. After removing the organic solvent, the
solid residue was purified by column chromatography, yielding
NNBM as a yellowish solid (2.03 g, yield: 73.6%; Figure 1). 1H NMR
(400 MHz, chloroform-d, δ): 8.24 (d, J = 9.1 Hz, 1H), 8.06 (d, J = 8.2
Hz, 1H), 7.64−7.40 (m, 7H), 7.07 (dd, J = 9.1, 2.6 Hz, 1H), 6.48 (d, J
= 2.5 Hz, 1H), 6.17 (s, 1H), 5.63−5.60 (m, 1H), 5.59 (s, 2H), 5.33
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1
Figure 1. (a) H and (b) 13C NMR spectra recorded in CDCl3 for an NNBM monomer. (c) HPLC profile recorded at 312 nm for an NNBM
monomer (0.5 mM; CH3CN/H2O, v/v = 7/3 as the eluent). (d) ESI mass spectrum recorded for an NNBM monomer.
Figure 2. (a, b) TEM images of aqueous dispersions (0.1 g/L) of PNOFA micelles (a) without and (b) with irradiation for 1 h. (c) Irradiation
time-dependent changes in scattering intensity and number-average hydrodynamic diameters of PNOFA micelles. (d) Number-average
hydrodynamic distributions of PNOFA micelles without and with light irradiation. In all cases, the irradiation wavelength was 410 nm and the
intensity was 28 mW/cm2.
(s, 2H), 5.22 (s, 2H), 5.05 (s, 2H), 1.97 (s, 3H); 13C NMR (101
MHz, chloroform-d, δ): 167.11, 161.60, 147.05, 141.29, 141.20,
136.02, 135.71, 133.87, 133.21, 132.63, 129.39, 128.71, 128.58,
128.48, 126.20, 124.86, 118.96, 114.97, 114.54, 92.95, 67.93, 65.51,
46.55, 18.37; ESI-MS m/z: [M + Na]+ calcd for C26H24N4O9,
559.1435; found, 559.1429.
Synthesis of PEO45-b-PNNBM21 (PNOFA) Diblock Copolymers
(Scheme 2). Typically, an NNBM monomer (268.3 mg, 0.5 mmol,
25.0 equiv), PEO-based macroinitiator (43.2 mg, 0.02 mmol, 1.0
equiv), bpy (6.3 mg, 0.04 mmol, 2.0 equiv), and CuBr (2.9 mg, 0.02
mmol, 1.0 equiv) were dissolved in dimethyl sulfoxide (DMSO, 1
mL) and were added into a sealed tube. The tube was degassed by
three freeze−pump−thaw cycles and then sealed under vacuum. After
stirring at 40 °C for 24 h, the polymerization reaction was quenched
by freezing the reaction solution in liquid nitrogen. The reaction tube
was opened and diluted with DCM and precipitated into an excess of
ether. The above dissolution−precipitation cycle was repeated three
times. The precipitate was collected by filtration and was dried in a
vacuum oven overnight at room temperature, affording a yellowish
solid (80 mg, yield: 29.8%). The degree of polymerization (DP) of
1
the PNNBM block was determined to be 21 by H NMR analysis
(Figure S3). As such, the resulting diblock copolymer was denoted as
PEO45-b-PNNBM21. GPC analysis of the PEO-b-PNNBM diblock
copolymer revealed an Mn of 5.4 kDa and a polydispersity index (Mw/
Mn) of 1.16 (Figure S4).
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Self-Assembly of the PNOFA Diblock Copolymer. Two milligrams
of a PNOFA diblock copolymer was dissolved in THF (1 mL), and
the solution was stirred at 25 °C for 30 min. Then, the THF solution
of the PNOFA diblock copolymer was injected into deionized water
(4 mL). After stirring at room temperature for 30 min, the organic
solvent was removed by dialysis (MWCO 14 kDa) against pure water.
Visible Light-Triggered Degradation of PNOFA Micelles. Photo-
triggered degradation of PNOFA micelles was monitored by UV−vis
spectroscopy, ALV, transmission electron microscopy (TEM), and
NanoSight. Briefly, absorbance spectra of PNOFA micelles (0.1 g/L)
under 410 nm irradiation (28 mW/cm2) for varying time (0−150
min) were monitored by UV−vis spectroscopy (Figure 3a). The size
changes and morphological transitions of PNOFA micelles (0.1 g/L)
after visible light irradiation (410 nm, 28 mW/cm2) were analyzed by
ALV, TEM, and NanoSight (Figures 2 and S7).
NO Release of PNOFA Micelles by Griess Assay. PNOFA micelles
were irradiated with a hand-held LED lamp (410 nm, 28 mW/cm2),
and the irradiated dispersion (200 μL) was mixed with a sulfanilamide
solution (200 μL; 1% sulfanilamide in 5% phosphoric acid). The
mixture was cultured in the dark for 5 min, N-(1-naphthyl)-
ethylenediamine dihydrochloride solution (200 μL; 0.1% NED in
water) was then added, and the resulting mixture was incubated for 10
min under a dark condition. The NO release contents were quantified
through the standard curve established by absorbance intensity
changes at 530 nm of a sodium nitrite solution (Figures 3b and S10).
HCHO Release of PNOFA Micelles by a Formaldehyde Test Kit.
PNOFA micelles were irradiated with a hand-held LED lamp (410
nm, 28 mW/cm2), and the irradiated dispersion (200 μL) was mixed
with a formaldehyde test kit according to the manufacturer’s
instructions. Briefly, 3-methyl-2-benzothiazolone hydrazone hydro-
chloride (200 μL) was added to the irradiated PNOFA dispersion
(200 μL). The mixture was cultured in a dark condition for 20 min at
30 °C. Then, 200 μL of an aqueous ammonium iron(III) sulfate
solution was added. After 10 min, the HCHO release contents were
quantified according to the standard curve established by absorbance
intensity at 622 nm (Figures 3c and S10).56
Antibacterial Assay of PNOFA Micelles against E. coli and S.
aureus Bacteria. The antibacterial activity of PNOFA micelles was
tested against E. coli (ATCC 25922) and S. aureus (ATCC 25923). In
all assays, two colonies of E. coli were cultured in a Luria Bertani
medium (2.5 mL) at 37 °C with shaking at 200 rpm for 16 h and then
40 μL of the resulting bacterial suspension was added into 4 mL of the
Luria Bertani medium for 1.5 h. In all assays, two colonies of S. aureus
were cultured in a Trypticase soy broth (TSB) medium (2.5 mL) at
37 °C with shaking at 200 rpm for 16 h and then 40 μL of the
resulting bacterial suspension was added into 4 mL of the Trypticase
soy broth medium for 1.5 h. The initial E. coli and S. aureus bacteria
were diluted to 5 × 105 cfu/mL with phosphate-buffered saline (PBS).
The E. coli and S. aureus suspensions (50 μL, 5 × 105 CFU/mL)
were added into 96-well plates, respectively, and they were incubated
at 37 °C for 20 min with 100 μL of PNOFA micelles (0.128, 0.064,
0.032, 0.016, and 0.008 mg/mL), preirradiated PNOFA dispersions
(0.128, 0.064, 0.032, 0.016, and 0.008 mg/mL) that released NO and
HCHO were removed by N2 flash, PNOFA vesicles (0.128, 0.064,
0.032, 0.016, and 0.008 mg/mL), and HCHO aqueous solutions (50,
25, 12.5, 6.25, and 3.125 μM), respectively. Then, all groups were
irradiated with or without 410 nm LED lamp (28 mW/cm2) for 10
min. After 30 min of incubation, bacterial suspensions were diluted to
1% and plated onto LB agar for further culture at 37 °C. Bacteria
colonies were counted. All assays included at least three replicates and
were repeated in three independent experiments.
Antibiofilm Assay of PNOFA Micelles against a S. aureus Biofilm.
Two colonies of S. aureus were cultured in a Trypticase soy broth
medium (2.5 mL) at 37 °C with shaking at 200 rpm for 12 h; a
bacterial suspension (10 μL) was mixed with 990 μL of TSB and 1
mL of diluted bacteria was added to each well of a 24-well plate. After
24 h culture at 37 °C, a S. aureus biofilm was formed. Biofilm dispersal
was quantified by crystal violet (CV) staining. The preformed biofilms
were incubated with PNOFA micelles (0.128 mg/mL), PNO vesicles
(0.128 mg/mL), and FA solution (50 μM) for 20 min, respectively.
Then, all groups were irradiated with or without 410 nm LED lamp
(28 mW/cm2) for 10 min. After 30 min of incubation, the resultant
biofilm was rinsed once with PBS (1 mL) and then a CV staining
agent (500 μL) was added. The biofilm was further cultured for 20
min. After that, the CV staining agent was removed and the biofilm
was washed with PBS (1 mL) twice. Afterward, the remaining CV-
stained biofilm was dissolved in ethanol (1 mL) and was quantified by
measuring the OD550 of the homogenized suspension using a
microtiter plate reader (Thermo Fisher). All of the reported results
were repeated in at least three independent experiments (Figure S17).
In Vitro Cytotoxicity Assay of PNOFA Micelles. Cell viability was
examined by MTT assay. Mouse fibroblast (L929) cells were seeded
in a 96-well plate at an initial density of 10 000 cells/well in 100 μL of
the Dulbecco’s modified Eagle’s medium (DMEM) complete
medium. After overnight culture, L929 cells were cocultured with
PNOFA micelles (0.128, 0.064, 0.032, 0.016, and 0.008 mg/mL) for
24 h before 10 μL of an MTT solution was added for further culture
of 4 h. Then, the suspension was removed and 100 μL of DMSO was
added to each well of 96-well plates. Cell viability was determined by
the absorbance intensity at 490 nm that was recorded on a microplate
reader.
Hemolysis of PNOFA Micelles. The hemolytic activity of PNOFA
micelles with or without 410 nm light irradiation (28 mW/cm2, 10
min) was tested with sheep erythrocytes (2%). The sheep
erythrocytes were washed with PBS (10 mM, pH 7.4) and centrifuged
at 2000 rpm (10 min) three times. Then, the collected erythrocytes
were diluted to 2%, following the treatment with PNOFA micelles,
preirradiated PNOFA dispersions, Triton X-100 (1%; positive
Figure 3. (a) Evolution of absorbance spectra of PNOFA micelles
(0.1 g/L) under 410 nm light irradiation (28 mW/cm2). (b) NO and
(c) FA release profiles of PNOFA micelles without (black squares)
and with (red squares) 410 nm light irradiation (28 mW/cm2). Data
are presented as mean s.d. (n = 3).
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polymerized through conventional ATRP53 with the formation
of an amphiphilic diblock copolymer of PEO-b-PNNBM
(PNOFA) using a PEO-based macroinitiator55 (Scheme 2a).
In addition, a NO-releasing diblock copolymer of PEO-b-
PNBM (PNO)38 was synthesized according to our previous
report as a control sample (Scheme S1).
control), and PBS (negative control), respectively. The mixture was
incubated at 37 °C for 1 h. After that, the resulting mixture was
centrifuged at 2000 rpm for 10 min. The supernatants (100 μL) of
each group were added to a 96-well plate and the OD values at 576
nm were recorded to calculate the hemolytic values.
Statistical Analysis. Data are presented as mean
standard
deviations (mean s.d.) and were analyzed using Prism 8.0 software
(GraphPad, San Diego, California) and student’s t-test. The level of
significance was set at p < 0.05.
The chemical structures of all of the intermediates and the
NNBM monomer were carefully characterized by nuclear
magnetic resonance (NMR), high-performance liquid chroma-
tography (HPLC), and high-resolution mass spectroscopy
(HRMS, Figures 1 and S1−S2). The resulting degrees of
polymerization of the PNNBM block in PNOFA and the
NBM block in PNO diblock copolymers were both
determined to be 21 by NMR spectroscopy (Figure S3).
The molecular weights of PNOFA and PNO were determined
to be 5.4 kDa (Mw/Mn = 1.16) and 4.6 kDa (Mw/Mn = 1.16)
by gel permeation chromatography (GPC), respectively
(Figure S4). The critical micellization concentration (CMC)
of the PNOFA diblock copolymer was calculated to be 8.1
mg/L (Figure S5). Transmission electron microscopy (TEM)
observation revealed that PNOFA diblock copolymers self-
assembled into micellar nanoparticles with PEO coronas in an
aqueous solution with a number-average hydrodynamic
diameter of ∼52 nm as determined by dynamic light scattering
(Figure 2a,d). We found that the micellar nanoparticles were
relatively stable in an aqueous solution, and the sizes of
micellar nanoparticles showed negligible changes and no
macroscopic precipitates were observed within at least one
week, as monitored by DLS (Figure S6). Moreover, our
previous results suggested that PNO diblock copolymers had a
CMC value of ∼32 mg/L, which self-assembled into vesicular
nanoparticles in an aqueous solution.38
Visible Light-Triggered Corelease of NO and FA.
Irradiation of the PNOFA micellar nanoparticles with a hand-
held LED lamp (410 nm, 28 mW/cm2) for 1 h led to a
significant decrease of the micellar nanoparticles, as observed
by TEM (Figure 2b). This result was further corroborated by
NanoSight nanoparticle tracking analysis (NTA), revealing
that the micellar nanoparticles dropped from 3.32 × 109/mL to
5.53 × 108/mL at a micelle concentration of 0.1 g/L (Figure
S7). Moreover, the scattering intensities and sizes of micelles
gradually decreased under light irradiation (Figure 2c).
Specifically, the size of micelle nanoparticles decreased from
52 to 16 nm after 1 h irradiation (Figure 2d). These results
suggested that PNOFA micellar nanoparticles were responsive
to 410 nm light irradiation and underwent photomediated
disassembly under irradiation.
Characterization. Nuclear magnetic resonance (NMR) spectra
were recorded on a 400 MHz Bruker NMR spectrometer operated in
the Fourier transform mode. Deuterated chloroform (CDCl3) was
used as a solvent. A high-resolution electrospray ionization mass
spectrometry (HR-ESI-MS) experiment was performed on a Waters
XEVOG2-XS-TOF Mass Spectrometer equipped with an electrospray
interface. High-performance liquid chromatography (HPLC) analysis
was performed with a Shimadzu HPLC system, equipped with an LC-
20AP binary pump, an SPD-20A UV−vis detector, and a Symmetry
C18 column. Molecular weights and molecular-weight distributions
were determined by gel permeation chromatography (GPC) equipped
with a Waters 1515 pump and a Waters 2414 differential refractive
index detector (set at 30 °C). It used a series of two linear Styragel
columns (HR2 and HR4) at an oven temperature of 45 °C. The
eluent was THF at a flow rate of 1.0 mL/min. A series of polystyrene
standards with varying molecular weights and low polydispersities
were employed for calibration. UV/vis spectra were recorded on a
TU-1910 double-beam UV−vis spectrophotometer (Puxi General
Instrumental Company, China). Fluorescence spectra were obtained
on an F-4600spectrofluorometer (Hitachi). Transmission electron
microscopy (TEM) was conducted on a JEOL JEM 1400 Flash
electron microscope at an acceleration voltage of 120 kV. Dynamic
light scattering (DLS) measurements were conducted using an ALV/
CGS3 dynamic light scattering with a 632.8 nm laser light set at a
fixed scattering angle of 90°. The nanoparticle concentrations of
micellar nanoparticles with or without visible light irradiation (410
nm, 28 mW/cm2) were analyzed using a NanoSight NS 300
(Malvern). Electron paramagnetic resonance (EPR) spectra were
recorded on a JEOL JES FA200 ESR spectrometer (300 K, 9.063
GHz, X-band) at room temperature. The following parameters were
applied, microwave power: 1 mW; sweep width ranged from 319.3 to
329.3 mT; modulation frequency: 100 kHz; and modulation
amplitude: 0.35 mT.
RESULTS AND DISCUSSION
■
Synthesis and Self-Assembly of Photoresponsive NO-
and FA-Releasing Diblock Copolymers. Recently, we have
successfully synthesized NO-releasing diblock copolymers
based on photoresponsive N-nitrosamine moieties.38,57 These
NO-releasing diblock copolymers self-assembled into vesicles
in an aqueous solution without premature NO leakage.
However, phototriggered NO release was achieved under
ultraviolet (UV) or visible light irradiation, showing potential
applications in corneal wound healing and biofilm dispersal.
Building on the previous work, to introduce photoresponsive
FA-releasing moieties, we started from 5-hydroxy-2-nitro-
benzaldehyde and functionalized the phenolic group with 1-
((chloromethoxy)methyl)-2-nitrobenzene with the formation
of compound 1 (Scheme 2a). Compound 1 was reacted with
4-aminobenzyl alcohol with the formation of the correspond-
ing Schiff base intermediate, which was further treated with
sodium borohydride (NaBH4) and sodium nitrite (NaNO2).
The three-step reactions were performed in a successive
manner, generating the NO- and FA-releasing precursor
(compound 2) with an overall yield of 56.3%. After that, the
hydroxyl group of compound 2 was then esterified with
methacryloyl chloride with the formation of an NNBM
monomer. The resulting NNBM monomer could be easily
To further demonstrate the PNOFA micellar nanoparticles
being responsive to visible light irradiation, we first followed
UV−vis spectroscopy of the NNBM monomer under 410 nm
light irradiation. The absorbance peak at 282 nm gradually
decreased subjected to irradiation, whilst the absorbance
intensities at 245 and 410 nm gradually intensified. Specifically,
the absorbance intensity at 410 nm reached a plateau after ∼60
min irradiation (Figure S8). Using 2-phenyl-4,4,5,5-tetrame-
thylimidazoline-1-oxyl-3-oxide (PTIO) as a spin-trapping
agent, phototriggered NO release could be readily identified
by the appearance of the characteristic signals of 2-phenyl-
4,4,5,5-tetramethylimidazoline-1-oxyl (PTI) in the electron
paramagnetic spectra (EPR),58 which unequivocally suggested
the release of a NO radical under visible light irradiation
(Figure S9).
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Figure 4. Bacterial viability of (a, b) E. coli and (c, d) S. aureus treated with (a, c) PNO and (b, d) PNOFA micelles with or without 410 nm light
irradiation (28 mW/cm2, 10 min). Data are presented as mean s.d. (n = 3); n.s., not significant; p values were calculated in comparison with the
PBS control or nonirradiated groups. *p < 0.05; **p < 0.01; *** p < 0.001; and ****p < 0.0001.
Quantitative analysis using standard Griess assay and the FA
test kit56 revealed that a 1 mol NNBM monomer could
simultaneously release ∼0.686 mol NO and ∼0.657 mol FA
after 1 h irradiation (410 nm, 28 mW/cm2), respectively
(Figures S10 and S11). This result potently demonstrated that
NNBM could be potentially used as a photoresponsive donor
of NO and FA signaling molecules, which could concurrently
release NO and FA under visible light irradiation. Intuitionally,
the photoresponsive property of the NNBM monomer
underpinned the observed results of photomediated disintegra-
tion of PNOFA micellar nanoparticles (Figure 2). Indeed,
PNOFA micellar nanoparticles displayed similar absorbance
changes under identical light irradiation compared to that of
the NNBM monomer (Figure 3a), indicating that the
formation of micellar nanoparticles did not affect the
photoresponsive property.32 Specifically, at a micelle concen-
tration of 0.1 g/L, the released NO and FA contents were
determined to be ∼68 and ∼107 μM after 1 h irradiation,
corresponding to ∼43.6 and ∼68.6% relative to that of NNBM
moieties. By striking contrast, no released NO and FA were
detected without light irradiation (Figure 3b,c). This result
demonstrated not only the excellent stability of PNOFA
micelles in an aqueous solution but also the great potential for
photoactivated codelivery of NO and FA.
S12). As such, we proposed the following mechanism for the
photolysis of the NNBM monomer: the N-nitrosoamine (N−
NO) bond of the NNBM monomer was cleaved first with the
release of NO, which has been known as a photoresponsive
weak bond.17,59 The resulting intermediate underwent a
spontaneous 1,6-elimination reaction with the release of
methacrylic acid and the formation of compound 3.
Compound 3 was subjected to further photolysis, generating
4-aminobenzyl alcohol (compound 4), o-nitrosobenzaldehyde,
and hemiacetal intermediate (compound 5). FA was then
spontaneously released from unstable compound 5.44 Notably,
the photomediated corelease of NO and FA led to the
disintegration of micellar nanoparticles that transformed the
initial diblock copolymer of PEO-b-PNNBM to hydrophilic
PEO-b-PMAA (Scheme 1), in agreement with the remarkable
decrease of micellar nanoparticles after irradiation (Figure 2).
The residual nanoparticles after irradiation were likely ascribed
to the aggregates of small-molecule fractions of degraded
micellar nanoparticles (Scheme 2b).
Antibacterial Assessment of Photoresponsive PNOFA
Micellar Nanoparticles. As detailed above, PNOFA micellar
particles can simultaneously release NO and FA, both of which
exhibited well-documented antibacterial capacity. We envi-
sioned that the corelease of NO and FA may exert a
combinatorial antibacterial performance. We first examined
whether the photoirradiation alone affected bacterial viability.
As shown in Figure S13, we found that 10 min irradiation (410
nm, 28 mW/cm2) did not incur any statistical difference in the
To unravel the phototriggered degradation mechanism of
the NNBM monomer, we monitored the photolyzed products
of NNBM using HRMS and two main intermediates
(compounds 3 and 4, Scheme 2b) were identified (Figure
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viability for both E. coli and S.aureus bacteria. However, the
bacterial viability gradually decreased subjected to longer
irradiation periods, which could be ascribed to the innate
phototoxicity of light irradiation.60 To alleviate the bacterial
killing effect of photoirradiation, it will be necessary to develop
photoresponsive NO/FA donors responding to near-infrared
(NIR) light, which was known to have better penetration
depths in biological samples and lower toxicity. In this study,
to minimize the toxicity of photoirradiation, in the next phase
of work, we chose 10 min irradiation for further antibacterial
assessment.
Bacterial viability assay revealed that both PNOFA and the
control sample, PNO, did not show any bacterial killing
capacity without light irradiation at all tested concentrations
(0−0.128 mg/mL; Figure 4). By striking contrast, both
PNOFA and PNO showed concentration-dependent anti-
bacterial performance under irradiation for 10 min (410 nm,
28 mW/cm2). Moreover, PNOFA micelles revealed a better
bacterial killing performance than that of NO-releasing PNO
nanoparticles at the same concentrations. Specifically, at a
micelle concentration of 0.128 mg/mL, PNOFA micelles killed
almost all of the bacteria (>99.99%) and no colonies were
observed on the plate for both E. coli and S. aureus bacteria,
whereas PNO nanoparticles at the same concentration only led
to 66.14 and 89.98% bacterial death for E. coli and S. aureus,
respectively (Figure 4).
cm2, 10 min) (Figure S17). Taken together, PNOFA micelles
capable of releasing both NO and FA exert antibacterial and
antibiofilm effects.
Ideal antibacterial materials were expected to selectively kill
bacterial pathogens without affecting mammalian cells.
Unfortunately, many cationic polymer-based antibacterial
materials showed unfavorable hemolytic behavior that
significantly hampered their practical applications.62 In sharp
contrast to previous antibacterial materials, PNOFA micelles
did not have cationic components and were negatively charged
in an aqueous solution with a ζ-potential of −13 mV (Figure
S18), showing neglectable toxicity toward mammalian cells
(e.g., L929) after 24 h incubation regardless of light irradiation
(Figure 5a). Importantly, PNOFA micellar nanoparticles did
It is worth noting that the released NO concentrations were
calculated to be 28.7 and 19.3 μM for PNO and PNOFA at the
concentration of 0.128 mg/mL with 10 min irradiation (Figure
S14), whilst the released FA concentration from PNOFA was
estimated to be 38.3 μM. Although the released NO
concentration from PNOFA was lower than that of the
PNO control at the same micelle concentrations (e.g., 0.128
mg/mL), PNOFA micellar nanoparticles showed much higher
antibacterial capacity (Figure 4). On the other hand, the
control experiments revealed that at the FA concentration of
50 μM with 10 min light irradiation, the bacterial viability of E.
coli and S. aureus was determined to be 76.8 and 87.7%,
respectively (Figure S15), in line with the antiseptic property
of FA. Therefore, either NO-releasing PNO or an FA solution
with even higher concentrations showed lower antibacterial
performance than that of NO- and FA-releasing PNOFA
micelles. The combination index (CI) between NO and FA on
E. coli and S. aureus elimination was calculated to be 0.43 and
0.80, respectively, which confirmed a synergistic effect of NO
and FA on bacterial elimination.61 Moreover, we found that
the preirradiated PNOFA nanoparticles following the removal
of released NO and FA exhibited negligible antibacterial
capacity at otherwise identical conditions (Figure S16),
suggesting the bacterial killing capacity originating from the
corelease of NO and FA. Taken together, we can conclude that
the incorporation of both NO- and FA-releasing moieties into
PNOFA micellar nanoparticles exerted a combinatorial
antibacterial performance, showing better bacterial killing
performance than that of any single ingredients of NO and
FA. We further evaluated the antibiofilm performance of
PNOFA micelles as well. At the same concentration of 0.128
mg/mL, PNO released 28.7 μM NO, while PNOFA micelles
released 19.3 μM NO and 38.3 μM FA under 410 nm
irradiation (28 mW/cm2, 10 min). Crystal violet staining
showed that PNOFA micelles led to ∼42% S. aureus biofilm
decrease and ∼33.5% biofilm decrease was observed in the
presence of PNO micelles under 410 nm irradiation (28 mW/
Figure 5. (a) Cytotoxicity assay of L929 cells against PNOFA
micelles with or without 410 nm light irradiation (28 mW/cm2, 10
min). Data are presented as mean s.d. (n = 3); n.s., not significant;
p values were calculated in comparison with the PBS control or
nonirradiated groups, *p < 0.05. (b) Macroscopic images and (c)
quantification of hemolysis of sheep erythrocytes (2%) treated with
Triton X-100 (1%; positive control), PBS (negative control), and
PNOFA micelles without or with 410 nm light irradiation (28 mW/
cm2, 10 min).
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not show evident hemolysis at micelle concentrations up to
0.128 mg/mL either (Figure 5b,c). Therefore, the NO- and
FA-releasing micellar nanoparticles may be useful for practical
antibacterial applications with low toxicity toward normal cells.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge financial support by the
National Key R&D Program of China (2020YFA0710700), the
National Natural Scientific Foundation of China (NNSFC)
Projects (51690150, 51690154, 52073270, 51722307, and
51973206), the Fundamental Research Funds for the Central
Universities (WK2060190102), and the Hefei Independent
Innovation Policy Project (J2020Y06).
CONCLUSIONS
■
In summary, we have successfully developed a micellar
nanoparticle-based platform for the simultaneous release of
NO and FA signaling molecules. We showed that the micellar
nanoparticles were relatively stable in an aqueous solution
without premature NO and FA leakage but could release NO
and FA under visible light irradiation. The photomediated NO
and FA release led to the disassembly of the micellar
nanoparticles, exhibiting a combinatorial antibacterial perform-
ance against both Gram-positive and Gram-negative bacterial
pathogens. More importantly, the micellar nanoparticles with
negative potential did not incur obvious hemolysis and showed
negligible toxicity toward normal mammalian cells. The
current strategy may provide a feasible way to develop gaseous
signaling molecule-based antibacterial materials by rational
molecular engineering.
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AUTHOR INFORMATION
■
Corresponding Author
Jinming Hu − CAS Key Laboratory of Soft Matter Chemistry,
Hefei National Laboratory for Physical Science at the
Microscale, Department of Polymer Science and Engineering,
University of Science and Technology of China, Hefei, Anhui
230026, P. R. China; orcid.org/0000-0002-6969-1343;
Email: jmhu@ustc.edu.cn
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Authors
Yutian Duan − CAS Key Laboratory of Soft Matter Chemistry,
Hefei National Laboratory for Physical Science at the
Microscale, Department of Polymer Science and Engineering,
University of Science and Technology of China, Hefei, Anhui
230026, P. R. China
Kewu He − Imaging Center of the Third Affiliated Hospital of
Anhui Medical University, Hefei 230031, Anhui, China
Guoying Zhang − CAS Key Laboratory of Soft Matter
Chemistry, Hefei National Laboratory for Physical Science at
the Microscale, Department of Polymer Science and
Engineering, University of Science and Technology of China,
Hefei, Anhui 230026, P. R. China
Complete contact information is available at:
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