Lipopolysaccharide neutralizing peptide-porphyrin conjugates for effective photoinactivation and intracellular imaging of gram-negative bacteria strains

Article abstract of DOI:10.1021/bc300203d

A simple and specific strategy based on the bioconjugation of a photosensitizer protophophyrin IX (PpIX) with a lipopolysaccharide (LPS) binding antimicrobial peptide YI13WF (YVLWKRKRKFCFI-Amide) has been developed for the effective fluorescent imaging an

Full text of DOI:10.1021/bc300203d

Article
pubs.acs.org/bc
Lipopolysaccharide Neutralizing Peptide−Porphyrin Conjugates for
Effective Photoinactivation and Intracellular Imaging of Gram-
Negative Bacteria Strains
Fang Liu,† Annie Soh Yan Ni,† Yingjie Lim,† Harini Mohanram,‡ S. Bhattacharjya,*,‡ and Bengang Xing*,†
‡
†Division of Chemistry & Biological Chemistry, School of Physical & Mathematical Sciences, and Division of Structural and
Computational Biology, School of Biological Physical Sciences, Nanyang Technological University, Singapore 637371
S
* Supporting Information
ABSTRACT: A simple and specific strategy based on the
bioconjugation of a photosensitizer protophophyrin IX (PpIX)
with a lipopolysaccharide (LPS) binding antimicrobial peptide
YI13WF (YVLWKRKRKFCFI-Amide) has been developed for
the effective fluorescent imaging and photodynamic inactiva-
tion of Gram-negative bacterial strains. The intracellular
fluorescent imaging and photodynamic antimicrobial chemo-
therapy (PACT) studies supported our hypothesis that the
PpIX-YI13WF conjugates could serve as efficient probes to
image the bacterial strains and meanwhile indicated the potent
activities against Gram-negative bacterial pathogens especially
for those with antibiotics resistance when exposed to the white
light irradiation. Compared to the monomeric PpIX-YI13WF
conjugate, the dimeric conjugate indicated the stronger fluorescent imaging signals and higher photoinactivation toward the
Gram-negative bacterial pathogens throughout the whole concentration range. In addition, the photodynamic bacterial
inactivation also demonstrated more potent activity than the minimum inhibitory concentration (MIC) values of dimeric PpIX-
YI13WF conjugate itself observed for E. coli DH5a (∼4 times), S. enterica (∼8 times), and other Gram-negative strains including
antibiotic-resistant E. coli BL21 (∼8 times) and K. pneumoniae (∼16 times). Moreover, both fluorescent imaging and
photoinactivation measurements also demonstrated that the dimeric PpIX-YI13WF conjugate could selectively recognize
bacterial strains over mammalian cells and generate less photo damage to mammalian cells. We believed that the enhanced
fluorescence and bacterial inactivation were probably attributed to the higher binding affinity between dimeric photosensitizer
peptide conjugate and LPS components on the surface of bacterial strains, which were the results of efficient multivalent
interactions.
PACT, the effective affinity of the photosensitizers on the
surface of target bacterial cells would be crucial to enable the
potent photoinactivation against bacterial strains. One possible
antimicrobial strategy to minimize the side effects and enhance
the potency of bacterial PACT inactivation is the utility of
affinity ligands, which can selectively deliver the PSs to the
targeted bacterial infection sites and lead to bacterial killing
with minimum nonspecific drug accumulation.14−16 So far,
several affinity ligand conjugated photosensitizer complexes
have been used to successfully inactivate a variety of antibiotic-
resistant pathogens, especially for those methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant enter-
ococci (VRE).17−25 However, most of the existing photo-
sensitizer systems based on affinity ligands only exhibit efficient
antimicrobial activity against Gram-positive strains rather than
Gram-negative ones mostly due to the presence of a highly
INTRODUCTION
■
Since penicillin was first introduced into medical practice in the
1940ʼs, a large number of various antibiotics have been
developed to successfully inhibit bacterial infection diseases in
clinics. However, increasing bacteria resistance to most of
antibiotics has been extensively reported in medical treatment
and has currently emerged as a serious threat to public
health.1−6 As such, there is an emergent need to design new
types of antimicrobial agents and alternative treatment
strategies that can effectively kill the bacterial strains with
potent drug-resistant properties. One promising approach for
microbiological inactivation is based on photodynamic
antimicrobial chemotherapy (PACT), by which porphyrins or
related compounds such as photofrin and foscan have been
employed as photosensitizers (PS) to activate reactive oxygen
species (ROS, e.g., singlet oxygen (1O2)) when exposed to
room light with a suitable wavelength. The activated singlet
oxygen species can be used as highly reactive oxidants and are
capable of destroying the cell walls and membranes in close
proximity, thus resulting in cell death.7−13 In the process of
Received: April 13, 2012
Revised: June 19, 2012
Published: July 9, 2012
© 2012 American Chemical Society
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1
organized outer bacterial membrane structure with a highly
negatively charged lipid portion, which blocks the cellular
attachment between PS and bacteria and thus intercepts O2.
(ATCC), USA. H NMR spectra were recorded using either a
Bruker 300 or 400 MHz spectrometer. Mass spectra (MS) were
measured with a Thermo LCQ Deca XP Max for ESI.
Analytical reverse-phase HPLC analysis was performed on a
Shimadzu HPLC system using an Alltima C-18 (250 × 10 mm)
column at a flow rate of 2.5 mL/min for preparation and a C-18
(250 × 4.6 mm) for analysis. Fluorescence emission spectra
were performed on a Varian Cary eclipse Fluorescence
spectrometer. UV absorption spectra were recorded in a 5
mm path quartz cell on a Beckman coulter DU 800
spectrometer. Photoirradiation experiments were performed
at a fluence rate of 50 mW/cm2 with a cool-light fiber optic
illuminator provided by a 150 W OSRAM Quartz Halogen bulb
(400−900 nm).
26
1
Although several affinity ligands based on nanoparticles18 and
water-soluble conjugated polymers19,25 and polypeptides20 have
been recently developed to achieve close association of PSs to
the Gram-negative bacterial strains and have indicated effective
photoinactivation toward these strains, the development of
simple and specific affinity ligand molecules that can selectively
direct photosensitizers to the surface of Gram-negative bacteria
pathogens is of great importance and remains the challenge in
the antimicrobial studies, since the current approaches may
indicate potential disadvantages including tedious sample
treatment, nonspecific aggregation, possible immune response,
or less cellular penetration.
Synthesis. N-Boc-Ethylenediamine 1. Di-tert-butyldicar-
bonate (Boc2O) (10.0 g, 46 mmol) was added into a mixture of
ethylenediamine (10.0 mL, 150 mmol) and triethylamine (2
mL) in 20 mL of ethanol at 0 °C. The solution was stirred for 2
h at room temperature. The solvent was then removed using a
rotator evaporator and the contents were redissolved in 20 mL
of dichloromethane (DCM). The solution was subsequently
extracted with 1.0 M acetic acid, and the pH of the aqueous
layer was adjusted to pH 9−10 using 2.0 M sodium hydroxide
(NaOH). Extraction was carried out with DCM (3 × 10 mL). 1
was collected by removing the solvent and used in the next step
Generally, Gram-negative bacteria are characterized by two
distinct lipid membrane structures, one is an inner membrane
consisted of glycerophospholipids and the other is an
asymmetric bilayer outer membrane predominantly composed
of a highly conserved and chemically unique lipopolysaccharide
(LPS) moiety. The amphiphilic LPS contains one lipid A, a
lipid fragment with six or seven fatty-acid-acylated chains, and a
few hydrophilic polysaccharides components with negatively
charged phosphate and carboxyl groups, which have been
considered an efficient barrier to prevent bacterial pathogen
strains from the permeability of antimicrobial molecules
including hydrophobic antibiotics, surfactants, host-defense
proteins, and peptides.27−29 Apart from the function as the
major constituent of the outer membrane of Gram-negative
bacteria, LPS has also been considered as one of the most
potent bacterial signal molecules that can strongly stimulate the
host defenses to release proinflammatory mediators and often
result in endotoxic shock or sepsis in intensive care units.30−32
One promising strategy to inhibit septic shock will be the
rational development of functional antimicrobial peptides, the
ubiquitous arsenals of all living organisms, which would bind
with LPS with a high affinity and significantly block the
interactions between LPS and its recognition protein receptors.
So far, extensive studies have been recently carried out to
investigate the interactions of antimicrobial peptides toward
LPS for their important roles in antibacterial activation, outer
membrane perturbation, and LPS neutralization.33−37
In this study, we report a simple and effective dimeric peptide
porphyrin binding system, by which a cationic antimicrobial
peptide with the sequence of YVLWKRKRKFCFI-Amide
(YI13WF) was chosen as a bacterial affinity ligand to link
with a most commonly used porphyrin photosensitizer (PpIX).
This new dimeric antimicrobial PpIX−peptide conjugate may
indicate a unique advantage to selectively adhere to the surface
of Gram-negative bacterial strains based on the enhanced
multivalent/polyvalent interactions between disubstituted
peptides and LPS moieties on bacterial strains, and thus serve
as a promising fluorescent probe and potent photosensitizer for
specific imaging and photodynamic inactivation of drug-
resistant Gram-negative bacterial strains.
1
of the synthesis without further purification (9.6 g, 40%). H
NMR (300 MHz, CDCl3): δ 1.40 (9H, s), 2.76 (2H, t, J = 5.85
Hz), 3.12 (2H, t, J= 5.70 Hz), 5.07 (1H, s). C7H16N2O2, ESI-
MS: m/z 160.12 (calcd); 160.84 [M+H]+, 320.78 [2M+H]+
(found).
N-(2-[(t-Boc)amino]ethyl Maleimide 2. Maleic anhydride
(400 mg, 1.66 mmol) in 35 mL of ethanol was added dropwise
into a solution of 1 (1.2 equiv) and triethylamine in 30 mL of
ethanol at 0 °C and stirred for 4 h. The solvent was removed
and the contents were redissolved in 8 mL of acetic anhydride.
Sodium acetate (1.0 equiv) was added and the solution was
heated to 60−70 °C for 30 min then cooled to room
temperature. Water was added to the solution and subsequent
extraction was carried out with ethyl acetate. The organic layer
was collected and washed with saturated sodium bicarbonate
solution. After drying with anhydrous sodium sulfate, the
organic layer was concentrated and purified with flash
chromatography (EA/hexanes, 1:3) to give a white solid 2
1
(1.2 g, 82%). H NMR (300 MHz, CDCl3): δ 1.40 (9H, s),
3.32 (2H, d, J = 5.4 Hz), 3.65 (2H, t, J = 5.6 Hz), 4.73 (1H, s),
6.70 (2H, s). C11H16N2O4, ESI-MS: m/z 240.11 (calcd); 240.83
[M+H]+ (found).
N-(2-Aminoethyl)maleimide Trifluoroacetate Salt 3. Com-
pound 2 (1.0 g, 4.17 mmol) dissolved in 10 mL of DCM was
added with trifluoroacetic acid (TFA) (5 mL) at 0 °C and
stirred for 1 h. DCM was removed by evaporation and contents
were redissolved in 3 mL of methanol. The desired product was
precipitated with 50 mL of diethyl ether and used without
further purification.
Monomeric PpIX-Maleimide Derivative 4. Protoporphyrin
IX (PpIX) (50 mg, 0.09 mmol) and N-hydroxysuccinimide
(HOSu) (9.2 mg, 0.08 mmol, 0.9 equiv) was dissolved in 8 mL
of dimethyl formamide (DMF) at 0 °C and stirred for 10 min
before the addition of 1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide hydrochloride (EDC.HCl) (20.4 mg, 0.117
mmol, 1.3 equiv). The reaction mixture was stirred at room
temperature for 4 h. Compound 3 (20 mg, 0.08 mmol, 0.9
equiv) was then added and the system was left to stir for a
EXPERIMENTAL PROCEDURES
■
General. All the chemical reagents were purchased from
Sigma Aldrich or Fluka. Commercially available reagents were
used without further purification. The antimicrobial peptides
were synthesized commercially by GL Biochem (Shanghai,
China) and further purified by reverse-phase HPLC. All
bacterial strains were from American Type Culture Collection
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further 5 h at room temperature. The compound was purified
by flash chromatography with eluent methanol/DCM = 1:20 to
give a red solid product as mixture of two regioisomers (26.2
mg, 43%) . 1H NMR (400 MHz, DMSO-d6): δ 2.92 (2H, t, J =
7.6 Hz), 3.16−3.20 (4H, m), 3.44 (2H, t, J = 5.8 Hz), 3.61 (6H,
m), 3.71 (6H, m), 4.27 (2H, t, J = 7.6 Hz), 4.35 (2H, t, J = 7.2
Hz), 6.23 (2H, d, J = 11.2 Hz), 6.47 (2H, dd, J1 = 18.0 Hz, J2 =
2.0 Hz), 6.86 (2H, s), 8.06 (1H, s), 8.48 (2H, dd, J1 = 17.6 Hz,
J2 = 11.6 Hz), 10.20−10.25 (4H, m). C40H40N6O5, ESI-MS: m/
z 684.31 (calcd); 685.48 [M+H]+, 1368.80 [2M+H]+ (found).
Dimeric PpIX-Maleimide Derivative 5. PpIX (20 mg, 0.036
mmol) and HOSu (9.2 mg, 0.079 mmol) was dissolved in 4 mL
of DMF at 0 °C, EDC.HCl (15 mg, 0.079 mmol) was then
added to the reaction mixture and allowed to stirred at room
temperature for 4 h. Following the addition of compound 3 (20
mg, 0.079 mmol), the system was left to stir for another 5 h at
room temperature. The compound was purified by flash
chromatography with eluent methanol/DCM 1:50 to give a red
(d, J = 10.8 Hz), 3.71 (d, J = 3.6 Hz), 3.86−3.95 (m), 4.12−
4.31 (overlapped), 4.35−4.55 (overlapped), 6.17 (dd, J1 = 11.8
Hz, J2 = 6.0 Hz), 6.42 (dd, J1 = 18.5 Hz, J2 = 5.4 Hz), 6.85−7.35
(m), 7.52 (d, J = 8.0 Hz), 7.60 (d, J = 4.8 Hz), 7.80 (br s), 8.10
(br s), 8.05−8.15 (m), 8.20 (br s), 8.40−8.55 (m), 9.16 (s),
10.17 (s), 10.24 (s), 10.34 (s), 10.75 (s). C222H316N54O36S2,
ESI-MS: m/z 4380.41(calcd); m/3z 1460.97, m/4z 1096.29, m/
5z 877.34, m/6z 731.43, m/7z 627.18, m/8z 549.09 (found).
General Spectroscopic Measurements. 1.0 mM stock
solution (in DMSO) of PpIX, conjugate 6 and 7, was diluted to
5.0 μM in DMSO. The UV−vis spectra were recorded using a
Beckman coulter DU800 spectrometer. Wavelength Interval:
1.0 nm. Scan Speed: 1200 nm/min. Fluorescence spectroscopic
studies were also performed using a Varian Cary Eclipse
Fluorescence Spectrophotometer at the excitation wavelength
of 504 nm.
Bacterial and Cell Culture. Four Gram-negative bacteria
strains: E. coli DH5a (ATCC 53868), S. enterica (ATCC
14028), and ampicillin-resistant bacterial strains E. coli BL21
(Ampr E. coli) and K. pneumoniae (ATCC 700603) were used
in this study. Luria−Bertani (LB) medium was used for all four
bacterial strains tested in this study. A single colony from the
stock LB agar plate was added to 3 mL of liquid medium, then
was grown at 37 °C on a shaker incubator (200 rpm) overnight
followed by a subculture until an OD600 of approximately 0.5−
0.7 was reached.
1
solid product (20.9 mg, 73%). H NMR (400 MHz, DMSO-
d6): δ 2.95 (4H, t, J =7.6 Hz), 3.19 (4H, t, J = 6.0 Hz), 3.43
(4H, t, J =5.6 Hz), 3.61 (3H, s), 3.64 (3H, s), 3.76 (3H, s), 3.77
(3H, s), 4.29 (4H, t, J =7.2 Hz), 6.24 (2H, d, J = 11.6 Hz), 6.47
(2H, dd, J1 = 18.0 Hz, J2 = 4.0 Hz,), 6.80 (4H, s), 8.14 (2H, t, J
= 6.0 Hz,), 8.54 (3H, m), 10.21 (1H, s), 10.29 (2H, s), 10.38
(1H, s). C46H46N8O6, ESI-MS: m/z 806.35 (calcd); 807.49 [M
+H]+, 1613.92 [2M+H]+ (found).
Monomeric PpIX−Peptide Conjugate 6. Compound 4 (1.1
mg, 1.6 μmol) and peptide YI13WF (2.8 mg, 1.6 μmol) in 200
μL of dimethylsulfoxide (DMSO) were added with 5 μL
DMSO solution containing 10% diisopropylethylamine
(DIPEA). The mixture was allowed to stir at room temperature
overnight and then was added to 10 mL diethyl ether to give a
red precipitate. This crude solid was purified by reverse-phase
high performance liquid chromatography (HPLC) with eluting
system consisting of A (water with 0.1% TFA) and B
(acetonitrile with 0.1% TFA) under a linear gradient,
monitored by UV absorbance at 280 and 400 nm. The linear
gradient stretched over 43 min from t = 2 min at 70% solution
A and 30% solution B to t = 45 min at 20% solution A and 80%
solution B. The reaction yielded a deep red solid monomeric
PpIX−peptide conjugate 6 (2.3 mg 56%) after lyophilization.
1H NMR (400 MHz, DMSO-d6): δ 0.66−0.80 (m), 0.96 (br s),
1.12 (s), 1.21−1.71 (overlapped), 1.84−1.92 (m), 2.25−2.36
(m), 2.65−2.75 (overlapped), 2.76−2.92 (m), 2.81−3.15 (m),
3.57 (dd, J1 = 10.6 Hz, J2 = 7.2 Hz), 3.69−3.71 (m), 3.89−3.96
(m), 4.12−4.30 (m), 4.47−4.55 (m), 6.15−6.19 (m), 6.41 (d, J
= 17.6 Hz), 6.56 (d, J = 8.4 Hz), 6.87−6.98 (m), 7.00−7.20
(m), 7.24 (d, J = 8.0 Hz), 7.52−7.63 (m), 7.68−7.75 (br s),
7.82−7.94 (br s), 7.96−8.21 (m), 8.42−8.52 (m), 9.15 (br s),
10.19 (s), 10.23 (s), 10.25 (s), 10.32 (s),10.70 (s).
Minimum Inhibitory Concentration (MIC) Test. Anti-
microbial activities of the designed peptides were determined
following a previously reported method with a light
modification.38 Briefly, 1.0 mL aliquots of bacterial strains
cultured in LB solution were collected and centrifuged. The cell
pellets were washed twice and resuspended in PBS (pH 7.2) at
OD600 of 0.5, then further diluted to a final concentration of 2
× 105 cfu/mL. Aliquots of this suspension (25 μL) were placed
into a 96-well plate. PpIX and its synthesized conjugates were
diluted in 25 μL PBS and then added into the bacteria
suspensions to give the desired concentration. After treating
with compounds at 37 °C in the dark for 2 h, the cultures were
then added to 50 μL of twice-LB solution and further incubated
at 37 °C for 16 h. The wells containing the same number of
cells but no compounds and the wells containing the same
culture solution but without bacterial cells were set as control
groups. The plate was then read using a 96-well plate reader at
600 nm. The MIC value was the lowest concentration of
compound that prevented the growth of bacteria.
Bacterial Cell Imaging. Bacterial strains cultured overnight
in Luria−Bertani (LB) solution were harvested and washed
twice with sterile phosphate-buffered saline (PBS, pH 7.2). The
washed cells were resuspended in PBS with an OD600 of 0.7,
Then 100 μL aliquots were treated with 2 μM of compounds
PpIX and these two PpIX−peptide conjugates. After incubation
for 20 min in the dark at 37 °C, the cells were washed with PBS
by centrifugation to remove the unbound compounds and then
a drop of the suspension was immobilized on poly(L-lysine)-
treated coverslips followed by covering with another coverslip.
Fluorescent images were acquired with confocal laser micro-
scope (Nikon Eclipse TE2000-E, CFI VC 100× oil immersed
optics), using an excitation wavelength of 488 nm and a LP
filter at 650 nm.
C
128H175N29O20S, ESI-MS: m/z 2471.33 (calcd); m/2z,
1236.53, m/3z 825.11, m/4z 619.18, m/5z, 495.69 (found).
Dimeric PpIX−Peptide Conjugate 7. Compound 5 (2.0 mg,
2.5 μmol) and peptide YI13WF (10 mg) (10.0 mg, 5.6 μmol)
in 200 μL of dimethylsulfoxide (DMSO) was added with 5 μL
of DMSO solution containing 10% diisopropylethylamine
(DIPEA). The reaction was treated as described above and
the crude products were purified by reverse-phase HPLC with
the same eluting system as purification of the monomeric
conjugate 6. The reaction yielded a deep red solid 7 (5.3 mg,
Quantification of bacterial cell staining was performed with
Image J software v 1.44 (NIH) according to its standard
manual. Statistic analysis was carried out using Origin v 8.0
software. Error bars reflect the standard error of the mean.
1
48%) after lyophilization. H NMR (400 MHz, DMSO-d6): δ
0.65−0.80 (m), 0.95 (br s), 1.12−1.74 (overlapped), 1.84−1.92
(m), 2.25−2.36 (m), 2.70 (br s), 2.83−3.15 (overlapped), 3.60
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a
Scheme 1. Synthesis and Characterization of PpIX−Peptide Conjugates
a
Reagents and conditions: (a) Boc2O, Et3N, EtOH, 0 °C; (b) 1. Maleic anhydride, Et3N, Et2O, 0 °C; 2. Ac2O, Na2OAc, 60−70 °C; (c) TFA, DCM,
0 °C−rt; (d) and (e) HOSu, EDC·HCl, DMF, 0 °C−rt; (f) DIPEA, DMSO, rt.
Statistical significance (set at P < 0.05) was determined by
Student’s t test.
then were incubated with the dimeric PpIX−peptide conjugate
(7) with varying concentrations at 37 °C for 20 min. After
incubation, the cells were exposed to a white light source for 10
min (30 J/cm2), and then incubated for a further 24 h in the
dark. As for the dark control assays, the plate was kept in the
dark during the whole process. After removing the conjugate 7
containing medium by centrifugation, the cells were incubated
with the Tox-8 reagent. After two hours’ incubation, the
absorbance was measured at the wavelength of 600 nm by a
Tecan Infinite M200 microplate reader. The measurement at
the wavelength of 690 nm was set as reference, which was
subtracted from the 600 nm measurement later. The wells
without cells but with Tox-8 reagent were used as controls.
Each experiment was performed in quintuplicate.
Selective Bacterial Cell Staining over Mammalian
Cells. A mixture of live Jurkat T cells [(0.5−1) × 105 cfu/mL]
and bacteria (108 cfu/mL) in PBS (pH = 7.2) was incubated
with 4 μM of dimeric PpIX−peptide conjugate 7 at 37 °C for
10 min. After being harvested by centrifugation (5 min at 500 g
for Jurkat T cells and then 5 min at 3000 g for bacterial cells),
the pellets of both mammalian and bacterial cells were mixed
again in PBS, and 8 μL of the suspensions was added to a
poly(L-lysine)-coated coverslip followed by slightly covering
with another coverslip for immobilization. The images were
taken as described above.
Photodynamic Inactivation against Bacterial Cells.
Bacteria were grown and washed as imaging and MIC tests.
From OD600 of 0.7 in PBS, the bacteria suspension was diluted
to a final concentration of 2 × 108 cfu/mL. 60 μL aliquots of
this suspension were mixed with the compounds (in 60 μL
PBS) and then added into the wells of the 96-well plate made
up the calculated concentration of compound (0, 0.25, 0.5, 1.0,
or 2.0 μM). The plate was incubated in the dark at 37 °C for 20
min. Before irradiation, 20 μL aliquots from each well were
pipeted out and further diluted to a series of sample
concentrations from 107 cfu/mL to 104 cfu/mL. The remaining
was exposed to a white light source for 2, 5, and 10 min, and
then again, 20 μL aliquots from each well were diluted to the
same series of concentrations after the specific light exposure
time. Twenty-five microliter aliquots of each suspension of
bacteria were subsequently plated onto LB agar plates then
incubated at 37 °C overnight. The number of colony-forming
units was counted after incubation and plotted onto a log scale.
Phototoxicity of Conjugate 7 toward Jurkat T Cells.
The cell toxicity assay was performed using the Tox-8 assay
(Sigma Aldrich) according to the manufacturer’s instructions
and the reported method.39 In brief, Jurkat T cells were
cultured in 96-well plates at a density of 2 × 104 cells/well, and
RESULTS AND DISCUSSION
■
Chemistry and Spectroscopic Studies. Scheme 1
illustrated the synthetic pathway for the preparation of
porphyrin and peptide derivatives. Typically, unlike the
Gram-positive organisms, the cell wall of Gram-negative strains
contains LPS in the outer membrane, which generally acts as a
protective permeability barrier. The LPS component increases
the negative charge in the cell wall, thus reducing the binding
effect of many neutral and anion drugs. In our previous work,
we described a series of de novo designed antimicrobial and
anti-endotoxic peptides. These peptides selectively interacted
with negatively charged lipids including LPS and adopted β-
boomerang structures in LPS micelles.40 As a mode of action,
these peptides destabilized LPS structures and penetrated into
the outer membrane of Gram-negative bacterial strains. In this
study, we chose an analogue of the β-boomerang antimicrobial
peptide YI13WF with the sequence of YVLWKRKRKFCFI-
Amide as target ligand due to the higher affinity to neutralize
LPS in Gram-negative bacterial membrane. The designed
peptide sequence was prepared based on the standard solid-
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Figure 1. UV−vis absorption and fluorescent emission spectrum. (A) Absorption at concentration of 5.0 μM in DMSO; (B) emission spectrum
excited at 504 nm at same concentration.
phase preparation through a commercial source. Considering a
single cysteine residue in the peptide sequence, a thiol-reactive
maleimide group was first introduced into the PpIX structure
through ethylenediamine as the linkage to yield the maleimide-
modified monomeric PpIX intermediates 4, which could further
react with the cysteine residue in the peptide sequence to afford
the PpIX−peptide conjugate through the specific interaction
between free thiol and maleimide group. A simple reverse-
phase HPLC purification produced the monomeric PpIX-
peptide derivative 6 with the yield of 56%. Similarly, the
dimeric peptide adduct with PpIX moiety was also prepared in
48% yield by conjugating the bimaleimide PpIX intermediate 5
with excess YI13WF peptide sequence. The final products 6
and 7 were purified to >95% purity (Supporting Information
Figure S1) by reverse-phase HPLC and further characterized by
NMR and mass spectrometry (ESI-MS).
After obtaining the porphyrin−peptide derivatives, their
photochemical properties were investigated by UV−vis
absorption and fluorescence spectra. The spectroscopic
characterization demonstrated that both the monomeric and
dimeric PpIX-conjugates exhibited similar absorptions from 330
to 650 nm with an intense soret band near 400 nm and four Q-
band peaks between 500 and 620 nm (Figure 1A). The
emission spectra of monomeric and dimeric peptide PpIX−
peptide conjugates are similar and showed no difference from
those of PpIX molecule (Figure 1B). The results indicated that
the peptide conjugation has no significant effect on the
fluorescent properties of PpIX molecule. Moreover, the PpIX−
peptide conjugates together with PpIX could also produce
reactive oxygen species (e.g., singlet oxygen, etc.) when excited
by white light irradiation, which were determined by the
standard assay based on the specific probe of 9,10-
anthracennediyl-bis(methylene) dimalonic acid (ABDA) re-
ported previously (Supporting Information Figure S2).19,21,22
MIC Tests. The antibacterial studies were first carried out by
investigating the minimum inhibitory concentration (MIC) of
PpIX-peptide derivatives. In order to avoid the potential
interferences of photoexcitation during the MIC tests, the MIC
experiments were performed in the dark. In a typical study, four
standard Gram-negative bacteria including E. coli DH5a
(ATCC 53868), S. enterica (ATCC 14028), and ampicillin-
resistant bacterial strains E. coli BL21 (Ampr E. coli) and K.
pneumoniae (ATCC 700603) were used as model organisms. As
expected, the antimicrobial peptide sequence, YI13WF,
demonstrated potent MIC activity against these Gram-negative
bacterial strains mostly attributed to its stronger binding effect
toward lipids exists at the outer membrane of Gram-negative
bacteria.29,40 Compared to the parent peptide sequence,
monomeric PpIX-peptide derivative showed a similar trend to
inhibit bacterial growth (Table 1), suggesting that conjugation
of PpIX to the peptide would not affect the binding affinity
Table 1. MIC Values of PpIX, Free Peptide YI13WF, and
PpIX-YI13WF Conjugates
MIC (μM)
E. coli DH5a E. coli BL-21 K. pneumoniae S. enterica
Conjugate 7
Conjugate 6
YI13WF
2
4
8
4
8
32
32
>64
32
32
>64
32
8
∼ 24
>64
PpIX
>64
between the antimicrobial peptide and LPS moieties on the
bacterial membranes. Under the same assay conditions, the
MIC value of dimeric PpIX-peptide against bacterial strains was
found to be 4- to 8-fold less than that of YI13WF peptide or
monomeric PpIX-YI13WF derivative, indicating that the greater
potency of bacterial inactivation could be caused by the dimeric
derivative. As control, similar antimicrobial activity was also
evaluated by using the PpIX molecule alone (Table 1). The
higher MIC value of PpIX clearly demonstrated the limited
activity to inhibit the bacterial growth, which was consistent
with the bacterial inactivation results reported previously.17−26
Bacterial Imaging Studies. In order to verify the binding
affinity of PpIX-peptide derivatives toward various Gram-
negative bacterial strains, living cell imaging was conducted
under a confocal microscope upon laser excitation of the Q-
band absorbance of PpIX. In this study, the bacterial strains
were incubated with PpIX−peptide conjugates as described in
the experiment section. As show in Figure 2A, the bacterial
strains incubated with PpIX itself did not show any obvious
fluorescence. However, after incubation of the bacterial strains
with PpIX−peptide conjugates, the confocal microscope
indicated a significant fluorescent imaging of the bacterial
cells stained with both dimeric and monomeric PpIX−peptide
conjugates. In contrast to the monomeric conjugate 6 and free
PpIX, the dimeric conjugate 7 exhibited stronger fluorescence,
suggesting a higher binding ability of dimeric peptide conjugate
toward the surface of bacterial strains. The different staining
effects of the tested compounds were further quantified by
measuring the fluorescence imaging intensity.41 As shown in
Figure 2B, fluorescence intensity from the monomeric PpIX-
peptide stained cells is about 8 times greater than that of free
PpIX stained cells, and such a difference would even increase
up to about 20 times when the same bacterial strains were
stained with dimeric PpIX−peptide conjugate, suggesting an
even higher binding association between the dimeric peptide
conjugate and Gram-negative bacteria. By contrast, the
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Figure 3. Fluorescent images of Jurkat T cells and two antibiotic-
resistant Gram-negative bacteria: (A) E. coli BL21 and (B) K.
pneumoniae incubated with conjugate 7 (4 μM). Left: bright field
images, right: fluorescent images. The fluorescence of PpIX is
highlighted in red.
tidylserine (PS) are commonly found lipid components located
at the exoplasmic leaflet and inner leaflet of the plasma
membrane of mammalian cells, respectively. Compared to the
different phospholipids in the mammalian cell membranes, the
major components of LPS located at the surface of Gram-
negative bacterial strains would be the main reason to greatly
contribute the selective recognition of dimeric PpIX−peptide
conjugate to bacterial strains instead of mammalian cells.37
Photodynamic Bacterial Inactivation Studies. To
demonstrate that the PpIX-peptide derivatives are effective
for the photodynamic inactivation of bacterial growth, the
PACT treatment was conducted by exposure of Gram-negative
strains with white light illumination in the presence of PpIX−
peptide conjugates. Upon white light irradiation, the bacteria
lethality was evaluated by counting the number of colony
forming units (cfu) on the LB agar plate. It is found that both
of the monomeric and dimeric PpIX−peptide conjugates
displayed effective photoinactivation to the tested bacterial
strains including those strains (e.g., ampicillin-resistant E. coli
BL2l and K. pneumoniae) with drug resistance properties. In
contrast to the monomeric PpIX−peptide conjugate, the
dimeric one showed that higher antibacterial activity and
more than 99% bacterial lethality (>2.0 log10 reduction) could
be observed at only 0.5 μM after 30 J/cm2 of light irradiation
(Figure 4A,C), whereas the control studies without light
irradiation only showed limited bacterial lethality. Under a fixed
concentration (0.5 μM) of photosensitizers, these two PpIX−
peptide conjugates also demonstrated even more significant
bacterial reduction when higher doses of light irradiation were
employed (Figure 4A,B). This photodynamic bacterial
inactivation indicated more potent activity than the MIC
values of dimeric PpIX−peptide conjugate observed for E. coli
DH5a (∼4 times) and S. enterica (∼8 times) including other
Gram-negative strains such as ampicillin-resistant E. coli BL21
(∼8 times) and K. pneumoniae (∼16 times). Moreover, the
efficacy of bacterial photoinactivation was also found to be
concentration-dependent, where the significant bacterial killing
with more than 99.9% bacterial lethality (>3.0 log10 reduction)
could still be easily obtained when photodynamic inactivation
was conducted in the presence of higher concentrations of
dimeric PpIX−peptide conjugate (Figure 4 and Supporting
Information Figure S3). In contrast, the presence of free PpIX
alone would not produce any detectable bacterial inactivation
when the bacterial strains were treated with identical white light
Figure 2. Fluorescent imaging of bacterial staining with PpIX and its
derivatives. (A) The bacterial cells were treated with 2 μM compounds
at room temperature for 10 min. Excitation wavelength was 488 nm,
and fluorescence was detected using a long-pass filter at 650 nm. Scale
bar = 10 μm. (B) Quantitative analysis of bacterial staining, mean
S.E. * P < 0.05 versus the monomeric conjugate.
incubation of porphyrin itself with bacterial strains would not
result in obvious fluorescence signals, mostly due to the weak
binding affinity of porphyrin molecules with LPS moieties on
the surface of Gram-negative bacteria. The living bacterial
imaging and fluorescent intensity measurements clearly
confirmed the hypothesis that antimicrobial peptide sequence
YI13WF may act as affinity ligands for specific targeting the
surface of Gram-negative bacterial strains. The enhanced
bacterial imaging and bacterial MIC inactivation properties
observed in the living cells treated with dimeric porphyrin−
peptide conjugate may be attributable to the multivalent
effects.18,42−46
Encouraged by the effective bacterial imaging results, the
dimeric PpIX−peptide conjugate was further applied to
investigate the possibility of selective recognition and imaging
of bacterial strains over mammalian cells. As proof-of-concept,
the Jurkat T cells were chosen and co-cultured with different
Gram-negative bacteria such as Ampr E. coli and K. pneumonia,
respectively, and then incubated with dimeric PpIX−peptide
conjugate at 37 °C for 10 min. The bacterial and cellular
imaging measurements were acquired by using a confocal
fluorescence microscope. As shown in Figure 3, for both
mixtures of Jurkat T cells with Ampr E. coli and Jurkat T cells
with K. pneumonia, the significant red fluorescence could only
be observed in bacteria, and there was no obvious fluorescence
detected in Jurkat T cells (Figure 3 and Supporting Information
Figure S4). The higher fluorescence observed in bacteria clearly
indicated the ability of the dimeric PpIX−peptide conjugate to
selectively recognize and image bacterial strains over mamma-
lian cells. Generally, phosphatidylcholine (PC) and phospha-
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Figure 4. Bacterial photoinactivation with PpIX−peptide conjugates. (A and B) Dose effects in light-induced toxicity against bacterial cells at the
fixed concentarion (0.5 μM) of PpIX−peptide conjugates 7 and 6. (C and D) Survival curves of the bacterial cells treated with several concentrations
2
■
●
▲
of PpIX−peptide conjugates 7 and 6 at the fixed energy of irradiation (30 J/cm ). E. coli DH5a ( ), E. coli BL21 ( ), S. enterica ( ), and K.
▼
pneumoniae ( ).
irradiation (Supporting Information Figure S3). A similar
mammalian cell viability analysis toward Jurkat T cells with and
without white light irradiation was also conducted by using
Tox-8 assay. There were no significant mammalian cell
viabilities changed with the increase of dimeric PpIX-peptide
concentration and no obvious cytotoxicity detected for both the
light irradiation and dark conditions (Supporting Information
Figure S5). These results clearly supported our hypothesis that
PpIX-conjugated dimeric peptide derivative could serve as an
effective reagent to inactivate Gram-negative bacteria over
mammalian cells, mostly owing to the higher binding affinity of
the dimeric peptide sequence toward the LPS moieties on the
surface of bacterial strains, which may lead to the locally high
concentration of photosensitizers caused by the efficient
polyvalent/multivalent interactions. The high concentration of
dimeric porphyrin peptide conjugate accumulated at close
proximity to the bacterial surface could thus constitute
significant bacterial lethality upon exposure to some dose of
light illumination.
ties, which is more active than the MIC values of YI13WF
peptide and PpIX alone. Apart from the higher photodynamic
antimicrobial application, the dimeric PpIX−peptide conjugate
also displayed the promising function in real-time fluorescent
imaging of living bacterial strains. Moreover, both fluorescent
imaging and photoinactivation experiments also demonstrated
that the dimeric PpIX-YI13WF conjugate could selectively
target bacterial strains over mammalian cells and generate less
damage to mammalian cells. The demonstrated strategy based
on the dimeric lipids affinity peptide sequence derivative may
provide the great possibility for further study of biological
functions in Gram-negative bacterial strains under living
settings. It may also supply the potential alternative for the
effective inactivation of bacterial pathogens especially for those
with drug-resistant properties.
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC analysis of peptide conjugates, measurement of singlet
oxygen generation, controlled bacterial and cellular photo-
inactivation, controlled bacterial and cellular fluorescent
imaging, bacterial imaging and fluorescent intensity quantifica-
tion, H NMR for compounds 1, 2, 4−7, and ESI-MS for
compounds 6 and 7. This material is available free of charge via
CONCLUSIONS
■
In this work, we have successfully constructed a simple and
specific strategy for effective fluorescent imaging and photo-
dynamic inactivation of Gram-negative bacterial strains through
the conjugation of photosensitizer protoporphyrin IX (PpIX)
with two lipopolysaccharide (LPS) neutralizing peptide
sequences: YI13WF (YVLWKRKRKFCFI-Amide). By taking
advantage of the higher binding affinity of the peptide sequence
toward the LPS components, the dimeric PpIX peptide
conjugate can effectively deliver the photosensitizer to the
surface of Gram-negative bacterial strains and thus enhance the
local concentration through the effects of multivalency. Upon
the white light illumination, the dimeric PpIX−peptide
derivative indicated the potent activity against Gram-negative
bacterial pathogens including those with drug-resistant proper-
1
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Xing Bengang: Phone 65-63168758, e-mail bengang@ntu.edu.
sg. S. Bhattacharjya: Phone 65-63167997, e-mail surajit@ntu.
edu.sg.
Notes
The authors declare no competing financial interest.
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Bioconjugate Chemistry
Article
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Chem. Soc. 131, 13117−13124.
ACKNOWLEDGMENTS
■
This work was supported in part by Nanyang Technological
University (NTU) Start-Up Grant and MOE/NTU Tier 1
Grant (RG 64/10) in Singapore.
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ABBREVIATIONS:
■
PACT, photodynamic antimicrobial chemotherapy; HPLC,
high-performance liquid chromatography; LPS, lipopolysac-
charide; PpIX, protoporphyrin IX; MIC, minimum inhibitory
concentration.
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