Nano adamantane-conjugated BODIPY for lipase affinity and light driven antibacterial

Article abstract of DOI:10.1016/j.saa.2020.118252

The increasing number of resistant bacterial strains has raised efforts in developing alternative treatment strategies. Lipase is highly expressed in most bacteria and lipase targeting dyes will be non-sacrificed materials for a sustainable method against

Full text of DOI:10.1016/j.saa.2020.118252

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Nano adamantane-conjugated BODIPY for lipase affinity and
light driven antibacterial
Jian Shao, Pu-Zhen Huang, Qiu-Yun Chen, Qing-Lin Zheng
PII:
S1386-1425(20)30230-4
https://doi.org/10.1016/j.saa.2020.118252
SAA 118252
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 17 February 2020
Revised date:
7 March 2020
10 March 2020
Accepted
date:
Please cite this article as: J. Shao, P.-Z. Huang, Q.-Y. Chen, et al., Nano adamantane-
conjugated BODIPY for lipase affinity and light driven antibacterial, Spectrochimica
Acta Part A: Molecular and Biomolecular Spectroscopy (2020), https://doi.org/10.1016/
j.saa.2020.118252
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Nano adamantane-conjugated BODIPY for lipase affinity and light driven antibacterial
Jian Shaoɑ, Pu-Zhen Huangɑ, Qiu-Yun Chen*ɑ, Qing-Lin Zhenga,b
a School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, 212013, China.
b THOR Specialty Chemical (Zhenjiang) Company Limited, No. 182 Jingang Avenue, New District,
Zhenjiang, Jiangsu, 212132, China.
* Corresponding Author, E-mail: chenqy@ujs.edu.cn.
ABSTRACT
The increasing number of resistant bacterial strains has raised efforts in developing alternative
treatment strategies. Lipase is highly expressed in most bacteria and lipase targeting dyes will be
non-sacrificed materials for a sustainable method against microorganism. The combination of
chemotherapy and antimicrobial photodynamic inactivation (aPDI) method will be an effective
method due to enhanced antibacterial activity. Here we reported the spectroscopic features of five
boron dipyrrolylmethene (BODIPY) derivatives with different functional groups for lipase affinity
and antibacterial activity. Lipase affinity tests and antibacterial assays were conducted by
spectroscopic methods. Adamantane-conjugated BODIPY (BDP-2) was found to be the active
compound against E. coli. Next, BDP-2 was brominated, and then assembled with PEG resulting
biocompatible BDP2-Br2@mPEG nanoparticles. The MTT assay indicated that BDP2-Br2@mPEG
was less toxicity on BGC-823 cancer cells without irradiation. The BDP2-Br2@mPEG can inhibit
the proliferation of E. coli and damage the membrane of bacterial cell under green LED light
irradiation. The results proved BDP2-Br2@mPEG can be a very promising green LED light driven
antibacterial material.
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Keywords: BODIPY, adamantane, antibacterial, photodynamic therapy, single oxygen
1. Introduction
The severity of bacterial infectious diseases has caused worldwide concern. The abuse of
antibiotics has caused bacterial resistance mutations, making people commit to find more
suitable antibacterial methods [1-3]. Antimicrobial photodynamic inactivation (aPDI) has
been introduced into bacteriostatic treatment [3-4]. The aPDI utilizes a photosensitizer (PS) to
produce reactive oxygen species (ROS) including singlet oxygen (1O2) or free radicals when
PS is illuminated with visible or near-infrared light. Singlet oxygen can destruct bacterial
reactivity with most biomolecules, and it can be turned into harmless oxygen [5-8]. More
1
importantly, due to non-specific damage caused by O2, its killing effect does not cause any
drug resistance of pathogenic bacteria [9].
Boron dipyrrolylmethene (BODIPY) is a kind of tunable fluorescent dyes for
photodynamic therapy (PDT) and aPDI [10]. Functional groups conjugated BODIPY
derivatives can convert the O2 into singlet oxygen (1O2) against cancer cells or bacteria under
irradiation [11-14]. Specific structures in BODIPY derivatives can also increase the bacteria
targeting affinity [15]. Nitazoxanide and benzamide derivatives were found to be useful class
of functional groups in those reported antibacterial agents [16-17]. Moreover, adamantane
derivatives can interfere with a variety of enzymes, and have good therapeutic activities such
as anti-viral, anti-Parkinson, antibacterial and anti-cancer [18-19]. Therefore, multifunctional
photodynamic therapy may have the advantages of both photodynamic therapy and
chemotherapy. BODIPYs are recently studied in biological areas because of its good
absorption to visible light. Lipases are the components of the outer membrane of
Gram-negative bacteria. Compounds with aromatic amides groups, such as 1.2.3-triazole
ureas and 1,5-diphenylpyrazole-3-carboxamide derivatives were as selective lipase inhibitors
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[20. 21]. Therefore, the conjugates of functional groups (such as benzamide, adamantane,
etc.) with BODIPY would have the potential properties of both lipase recognition and visible
light-driven photodynamic inactivation. Over-dose antibacterial agents have been
demonstrated to be harmful for both environments and human being. The lipase targeting
PDT agent could be an effective and green antibacterial agent due to its selectivity and visible
light-driven photodynamic inactivation, which would have a smaller impact on normal lives.
In this work, we conceived a design that employed BODIPY part as a photosensitizer,
bromine as heavy atom to generate singlet oxygen, and functional groups (such as benzamide,
adamantane, etc.) as lipase targeting and anti-bacterial functional group (Scheme 1). To
enhance the biocompatible property of dyes, a nano-dye (BDP2-Br2@mPEG) was produced.
Porcine pancreatic lipase (PL) was used to screen the affinity of compounds with lipase.
Activities against E. coli were studied with and without illumination.
2. Materials and Methods
2.1 Reagents
All chemicals were purchased from commercial suppliers and were used without further
purification. 2, 4-Dimethylpyrrole (98%), p-chloromethylbenzoyl chloride, adamantane, methyl
bromoacetate and N-bromosuccinimide (NBS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[methoxy(polyethyleneglycol)-5000] (DSPE-mPEG5000)and other chemicals were purchased from
Energy Chemical Reagent Co., Ltd. LB broth and agar were purchased from Sangon Biotech
(Shanghai, China) Co., Ltd. The bacterial strain Escherichia coli (E. coli, ATCC 25922) were
obtained
from
Luwei
Technology
(Shanghai,
China)
Co.,
Ltd.
(3s,5s,7s)-N-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin
-10-yl)benzyl)adamantan-1-amine
(BDP-Ad)
and
8-(4-chlorobenzyl)-4,4-difluoro-1,3,5,7-
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tetramethyl-4-bora-3a,4a-diaza-sindacen (BODIPY-Cl) were synthesized according to literature
procedures [22, 23].
2.2 Characterization
The electronic absorption spectra were measured at room temperature by a UV-2450 UV–vis
spectrophotometer. Fluorescence measurements were carried out on
a
fluorescence
spectrofluorometer Model CARY Eclipse (VARIAN, USA), a 1.0 cm quartz cell (slit width = 5 nm).
13
The 1H NMR (400 MHz) and C NMR (100 MHz) data was recorded on a Bruker AVANCE II 400
MHz spectrometer using CDCl3 as a solvent. The chemical shifts (δ) were reported in ppm and
coupling constants (J) in Hz. The electrospray mass spectra (ES-MS) were measured on a Finnigan
LCQ mass spectrograph. The fluorescence lifetime was determined using a time-dependent single
photon counting setup (TCSPC) (Fluo Time 200, Picoquant GmbH). The size distribution of the
nanoparticle was measured on a Brookhaven BI-9000 (Brookhaven Instruments, US). The SEM
images of the samples were obtained by using a scanning electron microscope (SEM, Shimadzu
SS-550).
2.3
Synthesis
of
1-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo
[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)-N-(5-nitrothiazol-2-yl)pyrrolidine-2-carboxamid
e (BDP-1)
BODIPY-Cl (500 mg, 1.34 mmol) and L-proline (154 mg, 1.34 mmol) were dissolved in THF
(20 mL) at room temperature. After mixing evenly, Et3N (186 μL, 1.34 mmol), KI (80 mg, 0.48
mmol) and three drops of 18-crown-6 were added. After the reaction mixture was heated to 65 °C in
an oil bath and stirred for 10 h, the solution was concentrated and recrystallized with ethyl acetate to
get the crude intermediate. The final product was synthesized according to literature procedures with
slight modification [24]. The crude intermediate (540 mg, 1.2 mmol) was dissolved in DCM (10 mL),
2-amino-5-nitro-thiazol (173 mg, 1.2 mmol), DCC (375 mg, 1.8 mmol) and DMAP (61 mg, 0.5
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mmol) were then added under N2 atmosphere. The reaction mixture was stirred at room temperature
overnight. The solvent was removed in vacuo and residue was dissolved in DCM. The organic layer
was washed with saturated sodium chloride (3×10 mL), dried with sodium sulfate, and concentrated
in vacuo. The residue was loaded onto a column of silica gel to give BDP-1 (245 mg, 42% yield) as
an orange solid. 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.49 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 9.0
Hz, 2H), 5.98 (s, 2H), 3.91 (dd, J = 9.6, 12.6 Hz, 2H), 3.21 (t, J = 7.4 Hz, 1H), 2.65 (d, J = 5.8 Hz,
1H), 2.56 (s, 6H), 2.47 – 2.28 (m, 1H), 2.14 – 1.70 (m, 4H), 1.30 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 160.30, 155.69, 143.69, 142.79, 140.77, 134.83, 131.32, 129.91, 128.62, 121.36, 65.89,
59.93, 54.51, 31.44, 31.00, 30.20, 29.69, 24.55, 14.57, 14.32. ES-MS(ESI-, MeOH): Calcd for
C28H29BF2N6O3S [M-H]- m/z=577.45, found m/z=577.31.
2.4
Synthesis
of
methyl
N-((3s,5s,7s)-adamantan-1-yl)-N-(4-(5,5-difluoro-1,3,7,9-
tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)glycinate
(BDP-2)
BODIPY-Ad (487 mg, 1 mmol) was dissolved in MeCN (20 mL) at room temperature, then
methyl bromoacetate (306 mg, 2 mmol) and Et3N (202 mg, 2 mmol) were added. After the reaction
mixture was heated to 80 °C in an oil bath and stirred for 10 h, orange solution was obtained. The
solvent was removed in vacuo and residue was dissolved in DCM. The organic layer was washed
with saturated sodium chloride (3×10 mL), dried with sodium sulfate, and then concentrated in
vacuo. The residue was loaded onto a column of silica gel to give BDP-2 (352 mg, 63% yield) as an
1
orange solid. H NMR (400 MHz, CDCl3) δ: 7.56 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 5.98
(s, 2H), 4.00 (s, 2H), 3.64 (s, 3H), 3.39 (s, 2H), 2.57 (s, 6H), 2.12 (s, 3H), 1.81 (d, J = 2.1 Hz, 6H),
1.66 (q, J = 12.2 Hz, 6H), 1.39 (s, 6H). 13C NMR (100 MHz, CDCl3) δ: 174.03, 155.22, 143.13,
142.19, 133.14, 131.56, 129.27, 127.62, 121.10, 55.19, 51.34, 51.20, 48.93, 40.15, 36.63, 29.73,
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14.55, 14.32. ES-MS (ESI+, CH3CN): Calcd for C33H40BF2N3O2 [M+H]+ m/z=560.51, found
m/z=560.96.
2.5
Synthesis
of
methyl
N-((3s,5s,7s)-adamantan-1-yl)-N-(4-(2,8-dibromo-5,5-
difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)
glycinate (BDP2-Br2)
BDP-2 (280 mg, 0.5 mmol) and NBS (334 mg, 2 mmol) were dissolved in dry CH2Cl2 (20 mL)
at room temperature. After stirred at room temperature for 30 min, the solvent was removed in vacuo.
The residue was loaded onto a column of silica gel to give BDP2-Br2 (308 mg, 86% yield) as a red
solid. 1H NMR (400 MHz, CDCl3) δ: 7.61 (d, J = 7.9 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 4.01 (s, 2H),
3.65 (s, 3H), 3.40 (s, 2H), 2.61 (s, 6H), 2.12 (s, 3H), 1.80 (s, 6H), 1.66 (q, J = 12.2 Hz, 6H), 1.38
(s,6H). 13C NMR (100 MHz, CDCl3) δ: 173.90, 153.70, 143.77, 142.63, 140.62, 132.46, 130.53,
129.53, 127.43, 111.67, 111.64, 55.32, 51.40, 51.30, 49.05, 40.14, 36.62, 29.72, 13.66. ES-MS (ESI+,
CH3CN): Calcd for C33H38BBr2F2N3O2 [M+H]+ m/z=718.30, found m/z=718.47.
2.6 General Procedure for the Synthesis of Benzoyl Piperazine BODIPYs
Benzoic acid (366 mg, 3 mmol), 1-boc-piperazine (560 mg, 3 mmol), DCC (927 mg, 4.5 mmol)
and DMAP (122 mg, 1 mmol) were dissolved in DCM (10 mL) at room temperature under N2
atmosphere. The reaction mixture was stirred for 10 h before it was filtered. The mixture solution
was then concentrated in vacuo and the residue was loaded onto a column of silica gel to give crude
piperazine benzoic acylamide as a white solid. Piperazine benzoic acylamide (830 mg, 2.8 mmol)
was dissolved in DCM (5 mL) and TFA (1 mL) was added. The reaction mixture was stirred for 10 h
before it was concentrated in vacuo. The residue was dissolved in MeCN (5 mL) and the pH value of
the mixture was adjusted to 7~8 with NaHCO3 before filtered. After that, BDP-Cl (100 mg) and Et3N
(200 μL) were added. After the reaction mixture was heated to 80 °C and stirred for 10 h, the solvent
was removed in vacuo and residue was dissolved in DCM. The organic layer was washed with
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saturated sodium chloride (3×10 mL), dried with sodium sulfate, and concentrated in vacuo. The
residue was loaded onto a column of silica gel to give piperazine-substituent BODIPY (122 mg, 83%
yield) as an orange solid.
(4-(4-(5,5-Difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-1
0-yl)benzyl)piperazin-1-yl)(3-hydroxypyridin-2-yl)methanone (BDP-3). Orange solid (183 mg, 36 %
1
yield); H NMR (400 MHz, CDCl3) δ 11.81 (s, 1H), 8.11 (dd, J = 4.2, 1.6 Hz, 1H), 7.49 (d, J = 7.9
Hz, 2H), 7.38 – 7.25 (m, 4H), 4.46 (s, 2H), 3.88 (s, 2H), 3.66 (s, 2H), 2.58 (d, J = 9.1 Hz, 10H), 1.40
(s, 6H). 13C NMR (100 MHz, CDCl3) δ 168.00, 158.47, 155.46, 142.99, 141.59, 138.57, 134.62,
134.05, 131.47, 129.78, 128.06, 127.43, 126.06, 121.24, 62.46, 53.67, 47.87, 43.15, 14.58,
14.36.ES-MS (ESI+, CH3CN): Calcd for C30H32BF2N5O2 [M+H]+ m/z=544.43, found m/z=544.39.
(4-(4-(5,5-Difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-1
0-yl)benzyl)piperazin-1-yl)(2-hydroxyphenyl)methanone (BDP-4). Orange solid (85 mg, 63% yield);
1H NMR (400 MHz, CDCl3) δ 9.63 (s, 1H), 7.48 (d, J = 6.7 Hz, 2H), 7.34 (d, J = 7.0 Hz, 1H), 7.27
(d, J = 6.7 Hz, 3H), 7.03 (d, J = 7.8 Hz, 1H), 6.88 (d, J = 6.7 Hz, 1H), 6.00 (s, 2H), 3.79 (s, 4H), 3.66
(s, 2H), 2.56 (d, J = 13.0 Hz, 10H), 1.39 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 170.82, 159.12,
155.49, 142.95, 141.52, 138.42, 134.12, 132.71, 131.45, 129.74, 128.33, 128.10, 121.25, 118.57,
118.14, 116.90, 62.42, 53.03, 29.70, 14.58, 14.35. ES-MS (ESI+, CH3CN): Calcd for C31H33BF2N4O2
[M+H]+ m/z=543.44, found m/z=543.35.
(4-(4-(5,5-Difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-1
1
0-yl)benzyl)piperazin-1-yl)(phenyl)methanone (BDP-5). Orange solid (122 mg, 83% yield); H
NMR (400 MHz, CDCl3) δ 7.45 (d, J = 7.9 Hz, 2H), 7.41 (s, 5H), 7.23 (d, J = 7.9 Hz, 2H), 5.98 (s,
13
2H), 3.83 (s, 2H), 3.64 (s, 2H), 3.46 (s, 2H), 2.55 (s, 8H), 2.39 (s, 2H), 1.37 (s, 6H). C NMR (100
MHz, CDCl3) δ 170.30, 155.42, 142.97, 141.60, 138.61, 135.85, 133.98, 131.46, 129.73, 128.49,
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128.02, 127.04, 121.24, 62.47, 53.12, 47.78, 42.16, 14.57, 14.33. ES-MS (ESI+, CH3CN): Calcd for
C31H33BF2N4O [M+H]+ m/z=527.44, found m/z=527.45.
2.7 Preparation of BDP2-Br2@mPEG nanoparticles
The nanoparticles were prepared using the same method as the literature reported [25].
DSPE-mPEG5000 (20 mg) was added, followed by the dissolution of BDP2-Br2 (13 mg) in CHCl3
(10 mL) at normal atmospheric temperature. The solution was stirred at room temperature for 10 min,
and the solvent was removed in a vacuum. The residue was dissolved in PBS. Particles in the
solution were then isolated by centrifugation at 8000 rpm for 20 min and dried at 40 °C to generate
BDP2-Br2@mPEG nanoparticles as red-purple solids. BODIPY content was up to 12.6 ± 1.0 wt %
calculated on the basis of UV-vis absorption at 522 nm of BDP2-Br2 in the supernatant (mean value
based on three parallel results).
2.8 Determination of fluorescence quantum yield
The relative fluorescence quantum yields of the BODIPY derivatives were determined by
comparison methods. The fluorescence quantum yield was calculated by the following equations:
Φ=ΦS × F/FS × AS/A
Φ and ΦS are the fluorescence quantum yields of the sample and the reference, F/Fs is the
integral fluorescence intensity ratio of the sample and the reference, and AS/A is the absorbance ratio
of the reference and sample at the same excitation wavelength. The methoxyfluoroborazole
(BDPOMe, ΦS = 0.30/MeCN) reported in the literature was used as the reference [26]. The excitation
wavelength was 475 nm and the integrated area was from 480 nm to 700 nm when BODIPYs were
tested.
2.9 Determination of transient fluorescence lifetime
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The fluorescence lifetime of the compounds was determined using a time-dependent single
photon counting setup (TCSPC) (Fluo Time 200, Picoquant GmbH) and a diode laser (LDH-P-670,
PDL 800-B, Picoquant GmbH, 670 nm, 20 MHz, 44 ps), and fluorescence lifetime values and error
analysis were calculated using FluorFit software.
2.10 Interaction with lipase
The affinity interaction of BODIPY derivatives to ligase were assayed using a fluorescence
spectrofluorometer Model CARY Eclipse. The tryptophan present in pancreatic lipase (PL) was used
as an intrinsic fluorophore (λex = 290 nm; λem = 340 nm). The stock solution of PL was 0.5 mg/mL
with distilled water. In several microtubes, the BODIPY derivatives (0 μM to 60 μM) in phosphate
buffer (50 mM, pH 7.0), were mixed with PL (0.5 mg/mL) and allowed to react for 5 min. The
emission spectrum of the mixture was measured. Since the BODIPY derivatives may possess
intrinsic fluorescence at the λex (290 nm), their spectra were measured and subtracted in all
fluorescence experiments.
2.11 Singlet oxygen measurement
To assess whether the nanoparticle could generate singlet oxygen under LED light irradiation,
following tests were carried out [25]. A 30 mL of constantly aerated acetonitrile solution containing
BDP-2 (1 μM) or BDP2-Br2 (1 μM), or BDP2-Br2@mPEG (6 μg/mL, its BDP2-Br2 content: 1 μM),
or Rose Bengal (1 μM) and 1, 3-diphenylisobenzofuran (DPBF) (50 μM), was irradiated with a 4 W
green LED light at 25°C for 10 min. Aliquots of 1 mL were taken from the solution every 1 min for
10 min, and then a UV-visible spectrum was recorded. 1,3-Diphenylisobenzofuran (DPBF) is a
singlet oxygen quencher. The singlet oxygen produced by the nanoparticles under irradiation was
confirmed by the decrease in UV absorbance of DPBF at 414 nm.
2.12 Cytotoxicity testing
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The cytotoxicity assays were measured with BGC-823 cells in normal culture conditions.
BGC-823 cells were seeded at a density of 5×103 cells per mL into sterile 96-well plates. Samples
were dispersed in H2O and diluted with culture media. The cytostatic effects of different
concentration of BDP2-Br2@mPEG nanoparticles (0 to 96 μg/mL) in the dark conditions or green
LED light irradiation (4 W) for 120 min were measured. Cell viability was determined with the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenpyltetrazoliumbromide (MTT) assay by measuring the
absorbance at 570 nm. Each test was performed in triplicate.
2.13 Bacterial culture
All the equipments used in this experiment were sterilized before used. E. coli were chosen as
the bacterial model to explore the antimicrobial activity of compounds and BDP2-Br2@mPEG
nanoparticles. A single colony of E. coli was used to inoculate 3 mL of Luria-Bertani (LB) medium
and cells were grown at 37 °C under aerobic conditions on a shaker incubator (180 rpm) until an
OD600 nm of approximately 0.8 was reached. Cells were harvested by centrifugation (10 min, ~2000
g) and washed twice with PBS. The cells were then resuspended in PBS and diluted to ~106
CFU·mL−1 for antibacterial assays and photodynamic inactivation assay.
2.14 Antibacterial assays
Gram-negative Escherichia coli (E. coli) strain was purchased from Shanghai Luwei Technology
Co., Ltd. The bacteria were cultured in LB broth medium. The bacterial concentration was monitored
by measuring the OD value of the bacteria at 600 nm. All assays were conducted in 96-well, flat
bottom, sterile plates. Bacterial suspension was adjusted to contain 1 × 106 CFU/mL in fresh sterile
PBS solution. Five compounds (1 μM to 16 μM) and BDP2-Br2@mPEG (6 μg/mL to 96 μg/mL, its
BDP2-Br2 content: 1 μM to 16 μM) were transferred into the assay plate, respectively. Next, the
plates were serially twofold diluted in triplicate, and 100 μL of the bacterial solution was added (final
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incolum 5 × 105 CFU/mL). The final volume for the assay was 200 μL. The control groups were set
without compounds for each assay. Each test was performed in triplicate. Plates were incubated in a
plate shaker (200 rpm) at 37 °C for 24 h. The above operation is carried out in a clean bench. Then
the absorbance at 600 nm of bacterial suspension was measured. The inhibition rate on E. coli was
calculated by the following formula:
Inhibition Rate (%) = (COD600 –TOD600) COD600 / × 100 %
where COD600 and TOD600 represent the absorbance at 600 nm in the control group and the
treatment group, respectively.
2.15 Photodynamic inactivation assay
Photodynamic inactivation assay was carried out as previously reported [26]. The
BDP2-Br2@mPEG solutions (96, 48, 24, 12, 6 and 0 μg/mL) were mixed with the same volume of
bacterial suspension (1 × 106 CFU/mL). After 10 min of static incubation at 37 °C in the dark, the
cultures were irradiated with the green LED device (4 W) for 1 h. Survivors were quantified using
the viable count technique. Plates were then incubated for 24 h at 37 °C. For each experiment the
following controls were set: samples exposed to the photosensitizer and not irradiated (+PS, -light),
samples without the photosensitizer and irradiated (-PS, +light), samples without the photosensitizer
and not irradiated (-PS, -light), samples exposed to DSPE-mPEG and irradiated (+DSPE-mPEG,
+light) and samples exposed to DSPE-mPEG and not irradiated (+DSPE-mPEG, -light). For each test
three independent experiments were carried out.
2.16 Release of cytoplasmaic constituents
The cytoplasmaic constituents such as DNA and RNA have strong absorption at 260 nm,
therefore the release of the cytoplasmaic constituents from the bacteria can be monitored by their
strong absorption at 260 nm [27]. The concentration of E. coli was adjusted to OD600=1.0,
centrifugated and washed with PBS three times. Then, the bacteria were incubated with the
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BDP2-Br2@mPEG under green LED light (5 W) for 60 min, the samples in the dark conditions were
used as control. Finally, the bacterial suspensions were filtered with 220 nm syringe filters
immediately to remove the bacteria, and optical density at 260 nm was recorded. Each test was
performed in triplicate.
2.17 Preparation of bacterial samples for SEM
E. coli suspension and E. coli suspension treated with BDP2-Br2@mPEG under irradiation (1
mL, 1 × 106 CFU/mL) were collected. All bacterial suspensions were placed on the slide, fixed with
glutaraldehyde for 12 h, dried in a freeze-dryer (Flexi-Dry TM MP), gold sputter-coated, and imaged
using a scanning electron microscope (SEM, Shimadzu SS-550).
3. Results and discussion
3.1 Syntheses and characterizations
Five functional groups BODIPY conjugates were synthesized by electrophilic substitution or
amide condensation respectively (Scheme 1a and Scheme S1). BDP2-Br2 was synthesized by
1
bromination with NBS. The H NMR, 13C NMR and MS spectra of the compounds confirm the
compounds with exact structures and high purity (Fig. S1-S18). To enhance solubility of BDP2-Br2
in water, DSPE-mPEG5000 was used to produce nanoself-assembly of BDP2-Br2@mPEG by
hydrophilic and hydrophobic interactions (Scheme 1b). BDP2-Br2@mPEG has a cuboid structure
(Figure 1a), which can be dispersed uniformly with an average diameter of 262.1 nm (Fig. 1b),
confirming that the BDP2-Br2 self-assembled into nanoscaled cuboids with DSPE-mPEG in aqueous
solutions. The UV-vis absorption of BDP2-Br2@mPEG is in the range of 500-600 nm, which red
shifted compared to BDP2-Br2. This indicates that the nanoparticles can absorb visible light (Fig. 1c,
Fig. S19). The maximum emission wavelength of BDP2-Br2@mPEG shifted to the infrared region
compared to BDP-2 and BDP2-Br2 (Fig. 1d, Fig. S20).
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Scheme 1. (a) Chemical structures of the five BODIPYs and BDP2-Br2 and (b) Schematic
illustration of the synthesis and antibacterial mechanism of BDP2-Br2@mPEG nanoparticles.
a
Fig. 1. Characterization of BDP2-Br2 and BDP2-Br2@mPEG nanoparticles. (a) SEM image of
BDP2-Br2@mPEG. (b) Diameter sizes of the BDP2-Br2@mPEG nanoparticles as measured by
dynamic light scattering. (c) UV−vis spectra of BDP2-Br2 (10 μM, MeCN) and BDP2-Br2@mPEG
(50 μg/mL, H2O).(d) Excitation and emission spectra of BDP2-Br2@mPEG (25 μg/mL, H2O).
3.2 Spectroscopic properties of BODIPYs
The fluorescence emission and fluorescence quantum yield of BDP derivatives were measured (Table
1). The emission wavelength of the BDP2-Br2 is 535 nm and its stock shift (Δλ = λem -λex) is 9 nm
(Table 1). What’s more, the maximum emission of BDP2-Br2@mPEG is 545 nm, and its stock shift
(18 nm), which is two times higher than BDP2-Br2, indicating the existence of π-π interaction and
intramolecular charge transfer in BDP2-Br2@mPEG [30]. The methoxy-substituted BODIPY
(BDPOMe, ΦS = 0.30/MeCN) reported in the literature was used as a reference for the determination
of fluorescence quantum yield [26]. The results show that the fluorescence quantum yields of the five
non-brominated derivatives except BDP-1 are similar to that of BDPOMe, with values ranging from
0.30 to 0.31, indicating less effect of substitutes on the emission of BODIPY core. The fluorescence
quantum yield of BDP-1 is lower than that of other BODIPY derivatives because of the nitro group
quenching effect. Also, the fluorescence quantum yield of BDP2-Br2 was reduced because of the
heavy atom effect. The fluorescence lifetime of compounds was determined using a time-dependent
single photon technique to investigate the transient changes in fluorescence (Fig. S21). The
attenuation curve integral was obtained by FluorFit software simulation under normal temperature
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test conditions. As shown in Table 1, the fluorescence lifetimes of the compounds BDP-2, BDP-3,
BDP-4 and BDP-5 are longer than that of BDP-1. The life time (3.93 ns) of BDP-3 is longer than
others, indicating that the chain in the body of BODIPY has effects on its life time.
Table 1. Spectroscopic properties of BODIPYs at room temperature
3.3 Affinity to lipase
Porcine pancreatic lipase (PL) consists of more than 400 amino acid residues in a single
polypeptide of globular shape which contains seven tryptophans with intrinsic fluorescence. The
addition of BODIPY derivatives to lipase caused a reduction of protein fluorescence intensity
(λem=362 nm) known as quenching effect (Fig. 2a). It was assumed that the interaction between the
enzyme and BODIPY derivatives did not significantly change the environment of the tryptophan
residues since the shape of the protein spectra did not change [31]. The decrease in fluorescence
intensity indicates the strong interaction between the compounds and the lipase. As shown in Fig. 2b,
BDP-2 is the one that decreased the fluorescence intensity of tryptophan residues in lipase
significantly, which means that BDP-2 has a good affinity to lipase. It was reported that lipase was
widely found in the out membrane of E. coli [32], which was used as a standard model for
lipase-targeting compounds to achieve an easier way of bacteriostasis with good effects [33]. Based
on this feature, we chose BDP-2 as the delicate compound for bromination in order to enhance its
1
ability of generating O2. DSPE-mPEG was then used to produce water dispersible nanoparticles
with BDP-2 by hydrophobic interactions.
Fig. 2. Emission spectra of PL (0.5 mg/mL, H2O) in the presence of various amount of BODIPYs; (a)
BDP-2 (a to g: 0, 10, 20, 30, 40, 50, 60 μM); The exciting wavelength was 290 nm. (b) Fluoresce
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intensity changes of PL at 334 nm in the presence of BODIPYs (60 μM) (A, BDP-1; B. BDP-2; C.
BDP-3; D. BDP-4; E. BDP-5)
3.4 Generation of singlet oxygen
The rate of singlet oxygen generation is an important parameter for an aPDI reagent. Rose
Bengal was used as a positive control, which is considered to be one of the powerful singlet oxygen
generators under irradiation [34]. Singlet oxygen was measured by monitoring the absorption
decrease of DPBF at 414 nm. As was shown in Fig. 3, the BDP-2 had little effect on DPBF under
green LED light irradiation, while the BDP2-Br2 and BDP2-Br2@mPEG caused obvious absorption
decrease of DPBF at 414 nm. This means the generation of 1O2 because of the heavy atom effect. The
relative rate of DPBF degradation under given conditions is in a ratio of 1:0.80:0.66:0.04 for Rose
Bengal:BDP2-Br2:BDP2-Br2@mPEG:BDP-2, where Rose Bengal is generally considered to be a
1
powerful substance that produces O2 fast. The results indicate that the BDP2-Br2@mPEG can
generate ROS and has the potential to be an excellent aPDI agent.
Fig. 3. (a) DPBF degradation profiles in 8 min by BDP2-Br2@mPEG (Insert: Changes of absorbance
at 414 nm); (b) Comparative reactive oxygen species generation rates plot for BDP-2 (A),
BDP2-Br2@mPEG (B), BDP2-Br2 (C) and Rose Bengal (D). The concentration of all
photosensitizers was at 1×10-6 M, and DPBF was initially at 5×10-5 M in acetonitrile. The slopes (m)
and the R2 coefficients of the lines are determined by linear regression.
3.5 Cytotoxicity testing
The inhibitory effect of BDP2-Br2@mPEG on the growth of BGC-823 cells was determined by
MTT reduction method. Experimental results demonstrate that BDP2-Br2@mPEG does not show
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significant cytostatic effects without irradiation (Fig. 4). Conversely, BDP2-Br2@mPEG exhibits an
obvious cytotoxicity under illumination and its IC50 is 18.3 μg/mL. This indicates that the
cytotoxicity of BDP2-Br2@mPEG is initiated by green LED light irradiation. The results indicate
that the BDP2-Br2@mPEG is low-toxicity without irradiation.
Fig 4. Cytotoxicity of different concentrations of BDP2-Br2@mPEG to BGC-823 cells in dark
conditions and green LED light for 120 min. The results are represented as mean ± SD (n = 3). The
t-test revealed the statistical significance of samples in the dark and samples illuminated with green
LED light with respect to control sample. Statistical significance is denoted by * (p < 0.05), ** (p <
0.005), *** (p < 0.001), and **** (p < 0.0001).
3.6 Antibacterial assays
To compare the antimicrobial properties of five compounds, the following antibacterial assays
were conducted. As shown in Fig. 5, the inhibition rates of all non-brominated compounds on E. coli
is below 5% at low concentrations (1 μM to 4 μM), and as the concentration of compounds increases,
BDP-2, BDP-3 and BDP2-Br2@mPEG show the relatively higher inhibition rate on E. coli, in which
the BDP-2 shows the highest inhibition rate of 48.2 ± 7.9 %. The antibacterial activity of
BDP2-Br2@mPEG nanoparticles was slightly reduced, which may be caused by DSPE-mPEG
encapsulation. Therefore, we believe that BDP2-Br2@mPEG will have a good antibacterial effect
among these compounds and can be a promising choice for antimicrobial dyes because
BDP2-Br2@mPEG nanoparticles has better water solubility and can also generate 1O2.
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Fig. 5. Inhibition rates of five compounds (1 μM to 16 μM) and BDP2-Br2@mPEG (6 μg/mL to 96
μg/mL, its BDP2-Br2 content: 1 μM to 16 μM) on E. coli. Data are expressed as the mean from three
independent experiments performed in triplicate.
The photo-induced antibacterial activity of the nanoparticles was evaluated. The light dose was
fixed (green LED, 500 nm). Various concentration of the BDP2-Br2@mPEG nanoparticles (0, 6, 12,
24, 48, 96 μg/mL) were investigated and controls including samples exposed to the pohotosensitizer
and not irradiated (+PS, -light), samples without the pohotosensitizer and irradiated (-PS, +light),
samples without the pohotosensitizer and not irradiated (-PS, -light), samples exposed to
DSPE-mPEG and irradiated (+DSPE-mPEG, +light) and samples exposed to DSPE-mPEG and not
irradiated (+DSPE-mPEG, -light), were set (Figure S22). Results show that untreated E. coli could
grow normally and its growth was slightly influenced by the green LED light irradiation. The
number of bacteria decreased a lot when the BDP2-Br2@mPEG nanoparticles were added. The
adamantane derivatives were reported to be active against drug-susceptible and drug-resistant TB
strains [35]. Experimental results indicate that the toxicity of the nanoparticles was not caused by
DSPE-mPEG (Fig. S22e and S22f). We propose that the antibacterial property of BDP2-Br2@mPEG
was due to the dark-cytotoxicity from the adamantane group. Furthermore, the concentration effect
of BDP2-Br2@mPEG on the photodynamic inactivation of E. coli was investigated (Fig. 6). As
expected, the inhibition of BDP2-Br2@mPEG to E. coli is dose-dependent. The number of survivors
was reduced by 5 log units when the concentration of BDP2-Br2@mPEG was increased from 0 to 24
μg/mL. When the concentration of the nanoparticles continued to double, there were no visible
colonies.
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Fig. 6. Effects of increasing concentrations of BDP2-Br2@mPEG on the photodynamic inactivation
of E. coli. Light dose: 5 J/cm2. Data are expressed as the means of three independent experiments ±
SD.
Previous studies reported that the bacterial apoptosis is associated with bacterial membrane
damage [36, 37]. In this work, absorption study at 260 nm was used to confirm whether the
membrane of E. coli was broken. Absorption at 260 nm of the bacteria incubated with
BDP2-Br2@mPEG under illumination is higher than the bacteria under dark condition (Fig. 7). The
result indicates that the membrane of more bacteria was broken and the cytoplasmaic constituents
inside had leaked out under irradiation.
Fig. 7. Absorption at 260 nm of E. coli after incubated with BDP2-Br2@mPEG under dark and LED
light. The results are represented as mean ± SD (n = 3).
SEM images were used to illustrate the antibacterial effect of the BDP2-Br2@mPEG
nanoparticles. The morphology of the bacteria treated with nanoparticles shows significant changes
after illumination compared to the control group (without nanoparticles) (Fig. 23). Distorted bacterial
walls can be clearly observed after illumination compared to the smooth surface of control cells. The
membrane of bacterial cell was severely damaged by 1O2 so that most of them were broken. This led
to an irreversible destruction of the membrane structure of the bacteria, indicating that the bacteria
were dead.
4. Conclusions
In this work, five functional groups (such as benzamide, adamantane, etc.) conjugated BODIPY
were synthesized and characterized. Lipase affinity and antibacterial assays were conducted by
spectroscopic methods. The spectroscopic features of BODIPY derivatives for lipases affinity and
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antibacterial activity were reported. Lipase affinity assay results indicate that BDP-2 was the most
active compound among the five compounds. Therefore, BDP-2 was used to synthesize
photoresponsive nanoparticle (BDP2-Br2@mPEG). BDP2-Br2@mPEG was very active against E.
coli under illumination, making it a very promising aPDI agent. Our results confirm that functional
group conjugated BODIPY would be potential visible light driven antibacterial dyes. Visible light is
healthy to human. Bacterial targeting photodriven antibacterial materials will be new green materials
for the sustainable treatment of bacteria.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Science Foundation of China
(21571085).
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Scheme 1. (a) Chemical structures of the five BODIPYs and BDP2-Br2 and (b) Schematic
illustration of the synthesis and antibacterial mechanism of BDP2-Br2@mPEG nanoparticles.
Fig. 1. Characterization of BDP2-Br2 and BDP2-Br2@mPEG nanoparticles. (a) SEM image of
BDP2-Br2@mPEG. (b) Diameter sizes of the BDP2-Br2@mPEG nanoparticles as measured by
dynamic light scattering. (c) UV−vis spectra of BDP2-Br2 (10 μM, MeCN) and BDP2-Br2@mPEG
(50 μg/mL, H2O).(d) Excitation and emission spectra of BDP2-Br2@mPEG (25 μg/mL, H2O).
Fig. 2. Emission spectra of PL (0.5 mg/mL, H2O) in the presence of various amount of BODIPYs;
(a) BDP-2 (a to g: 0, 10, 20, 30, 40, 50, 60 μM); The exciting wavelength was 290 nm. (b) Fluoresce
intensity changes of PL at 334 nm in the presence of BODIPYs (60 μM) (A, BDP-1; B. BDP-2; C.
BDP-3; D. BDP-4; E. BDP-5).
Fig. 3. (a) DPBF degradation profiles in 8 min by BDP2-Br2@mPEG (Insert: Changes of
absorbance at 414 nm); (b) Comparative reactive oxygen species generation rates plot for BDP-2 (A),
BDP2-Br2@mPEG (B), BDP2-Br2 (C) and Rose Bengal (D). The concentration of all
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photosensitizers was at 1×10-6 M, and DPBF was initially at 5×10-5 M in acetonitrile. The slopes (m)
and the R2 coefficients of the lines are determined by linear regression.
Fig 4. Cytotoxicity of different concentrations of BDP2-Br2@mPEG to BGC-823 cells in dark
conditions and green LED light for 120 min. The results are represented as mean ± SD (n = 3). The
t-test revealed the statistical significance of samples in the dark and samples illuminated with green
LED light with respect to control sample. Statistical significance is denoted by * (p < 0.05), ** (p <
0.005), *** (p < 0.001), and **** (p < 0.0001).
Fig. 5. Inhibition rates of five compounds (1 μM to 16 μM) and BDP2-Br2@mPEG (6 μg/mL to 96
μg/mL, its BDP2-Br2 content: 1 μM to 16 μM) on E. coli. Data are expressed as the mean from three
independent experiments.
Fig. 6. Effects of increasing concentrations of BDP2-Br2@mPEG on the photodynamic
inactivation of E. coli. Light dose: 5 J/cm2. Data are expressed as the means of three independent
experiments ± SD.
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Fig. 7. Absorption at 260 nm of E. coli after incubated with BDP2-Br2@mPEG under dark and
LED light. The results are represented as mean ± SD (n = 3).
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Scheme 1.
(a) Chemical structures of the five BODIPYs and BDP2-Br2 and (b) Schematic illustration of the
synthesis and antibacterial mechanism of BDP2-Br2@mPEG nanoparticles.
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b
a
25
20
15
10
5
0
1
10
100
1000
10000
Diameter (nm)
1.0
BDP2-Br2 (10 M, MeCN)
d
c
Ex
Em
BDP2-Br2@mPEG (50 g/mL, MeCN)
120
80
40
0
0.8
0.6
0.4
0.2
0.0
450
500
550
600
650
700
250 300 350 400 450 500 550 600 650 700
Wavelength(nm)
Wavelength (nm)
Fig. 1. Characterization of BDP2-Br2 and BDP2-Br2@mPEG nanoparticles. (a) SEM image of
BDP2-Br2@mPEG. (b) Diameter sizes of the BDP2-Br2@mPEG nanoparticles as measured by
dynamic light scattering. (c) UV−vis spectra of BDP2-Br2 (10 μM, MeCN) and BDP2-Br2@mPEG
(50 μg/mL, H2O).(d) Excitation and emission spectra of BDP2-Br2@mPEG (25 μg/mL, H2O).
29