Vancomycin-conjugated polythiophene for the detection and imaging of Gram-positive bacteria

Article abstract of DOI:10.1039/c7tb02061a

Bacterial infections can cause serious health problems. The rapid identification of bacteria plays a vital role in the treatment of bacterial infection at an early stage of the disease. In this work, an active polythiophene derivative containing reactive pentafluorophenyl (PFP) ester pendant groups was prepared via Fe³⁺-catalyzed oxidative polymerization. As far as we know, this is the first report of active polythiophene with reactive PFP ester moieties. The active polythiophene derivative was conjugated with vancomycin and α-methoxy-ω-amino poly(ethylene glycol) (mPEG-NH₂) via a reactive ester-amine reaction, resulting in the formation of water-soluble and fluorescent vancomycin-containing polythiophene (PTPVan). Since vancomycin can selectively interact with Gram-positive bacteria and kill them, the antibacterial properties of PTPVan were evaluated. The detection of Gram-positive bacteria was carried out by observing the color change and the fluorescence response of bacteria upon incubation with PTPVan through the naked eye and a fluorescence spectroscope, respectively. The staining of Gram-positive bacteria was observed using a confocal laser scanning microscope (CLSM).

Full text of DOI:10.1039/c7tb02061a

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Vancomycin-conjugated polythiophene for the
detection and imaging of Gram-positive bacteria†
Cite this:DOI: 10.1039/c7tb02061a
a
a
Ling Gui Ning,a Shuai Wang,a Xue Feng Hu,*b Chang Ming Li
and Li Qun Xu
*
Bacterial infections can cause serious health problems. The rapid identification of bacteria plays a vital
role in the treatment of bacterial infection at an early stage of the disease. In this work, an active
polythiophene derivative containing reactive pentafluorophenyl (PFP) ester pendant groups was prepared
via Fe3+-catalyzed oxidative polymerization. As far as we know, this is the first report of active
polythiophene with reactive PFP ester moieties. The active polythiophene derivative was conjugated with
vancomycin and a-methoxy-o-amino poly(ethylene glycol) (mPEG-NH2) via a reactive ester–amine
reaction, resulting in the formation of water-soluble and fluorescent vancomycin-containing
polythiophene (PTPVan). Since vancomycin can selectively interact with Gram-positive bacteria and kill
them, the antibacterial properties of PTPVan were evaluated. The detection of Gram-positive bacteria
was carried out by observing the color change and the fluorescence response of bacteria upon
incubation with PTPVan through the naked eye and a fluorescence spectroscope, respectively. The
staining of Gram-positive bacteria was observed using a confocal laser scanning microscope (CLSM).
Received 1st August 2017,
Accepted 16th October 2017
DOI: 10.1039/c7tb02061a
rsc.li/materials-b
for biological optical imaging, due to their reduced cytotoxicity
and improved photostability in comparison to small molecular
Introduction
Bacterial infections are some of the most serious health problems dyes.23–27 Functional moieties on the side chains of conjugated
and have led to the increased morbidity, mortality and cost of polymers play an important role as they are indispensable for
health care for patients.1,2 In particular, infections resulting from the conjugation of bioactive molecules.28 In addition, the post-
Gram-positive bacteria can cause a variety of serious diseases functionalization step is critical to impart water solubility/
including sepsis, bacteremia, pneumonia, osteomyelitis and dispersibility to the conjugated polymers. The reactive penta-
endocarditis.3,4 Bacterial identifications play vital roles in clinical fluorophenyl (PFP) ester-based ester–amine reaction is an
diagnosis to determine the origins of infections and provide efficient and versatile method for the post-functionalization
information for the selection of appropriate courses of treatment. of polymers, substrates, particles and graphene sheets.29–35 The
Conventional methods are mainly based on recovering and culturing introduction of reactive PFP esters on the side chains of the
of bacteria from patients.5–7 Advanced detection methods, including conjugated polymer could offer an elegant approach for attaching
the polymerase chain reaction (PCR), NMR spectrometry, surface bioactive molecules and improving its water solubility/dispersibility.
plasmon resonance (SPR) and immunoassays,5,8–11 are also employed
Herein, PFP thiophene-3-acetate (PFPTA) was synthesized
to detect the pathogenic bacteria. Although these detection techno- and polymerized using FeCl3 as an oxidant. As far as we know,
logies can meet the requirements for clinical diagnosis and have this is the first report of an active thiophene monomer and
desirable sensitivity and selectivity, they are time-consuming or polymer with PFP ester moieties. The resulting poly(PFPTA)
require sophisticated equipment.12–16 Thus, it is still interesting to (PPFPTA) polymer was then conjugated with vancomycin and
develop rapid and simple methods for the identification of microbes, a-methoxy-o-amino poly(ethylene glycol) (mPEG-NH2) via a
especially Gram-positive bacteria.
reactive ester–amine reaction, leading to the formation of
Staining that can differentiate bacteria by the naked eye vancomycin-conjugated polythiophene (PTPVan) (Scheme 1).
or fluorescence techniques is a powerful tool for bacterial Vancomycin is a glycopeptide antibiotic that can bind to D-alanyl–
detection.17–22 Conjugated polymers are promising candidates D-alanine (D-Ala–D-Ala) moieties on the cell wall of Gram-positive
bacteria.4,11,36–44 This binding could interfere the transglycosylase
a Institute for Clean Energy and Advanced Materials, Faculty of Materials and
step of peptidoglycan biosynthesis, lower the rigidity of the cell wall,
and ultimately cause bacterial death.3,45,46 PEG is a non-toxic and
Energy, Southwest University, Chongqing, 400715, P. R. China.
E-mail: xulq@swu.edu.cn
b National Engineering Research Center for Biomaterials, Sichuan University,
non-immunogenic polymer, and PEGylation could increase the
solubility and stability of conjugated polymers under physiological
Chengdu, 610064, P. R. China. E-mail: huxuefeng@scu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7tb02061a conditions.47,48 The attachment of vancomycin and PEG to the side
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Scheme 1 Synthetic route of PPFPTA and PTPVan.
chains of polythiophene could endow it with good solubility as Preparation of poly(PFPTA) (PPFPTA)
well as selective interaction with Gram-positive bacteria. The
Dry FeCl3 (1.94 g, 12.0 mmol) and anhydrous chloroform
antibacterial properties of PTPVan against both Gram-positive
and Gram-negative bacteria were studied. The identification of
Gram-positive bacteria was carried out by staining the bacteria
with PTPVan and observing the phenomenon with the naked
eye, a fluorescence spectroscope and a confocal laser scanning
microscope (CLSM).
(30 mL) were added to a 100 mL three-neck round bottom
flask. The mixture was stirred at room temperature under an
argon atmosphere for 30 min, giving a dark brown solution.
To this suspension, PFPTA (0.92 g, 3.0 mmol) dissolved in
anhydrous chloroform (10 mL) was added dropwise within
15 min. After stirring at room temperature under argon for
24 h, the solution was poured into 500 mL of methanol. The
precipitate was collected by filtration, soxhlet extracted with
methanol for 3 days and dried under reduced pressure for 24 h.
Experimental section
Materials
Preparation of vancomycin-conjugated polythiophene
(PTPVan)
Pentafluorophenyl trifluoroacetate (98%) and 3-thiopheneacetic
acid (98%) were purchased from Sigma-Aldrich Chemical Co.
(Shanghai, China). Methoxypolyethylene glycol amine (mPEG-
amine, M.W. = 1000) was purchased from Alfa Aesar Co. Ltd
(Tianjin, China). Vancomycin hydrochloride (USP grade)
was purchased from Aladdin Reagent Co. (Shanghai, China).
Escherichia coli (E. coli, ATCC 25922), Pseudomonas aeruginosa
(P. aeruginosa, ATCC 27853), Staphylococcus aureus (S. aureus,
ATCC 25923 and 43300), methicillin-resistant S. aureus (MRSA,
ATCC 33592) and Staphylococcus epidermidis (S. epidermidis,
ATCC 12228) were obtained from the American Type Culture
Collection. All other regents were purchased from J&K Scientific
or Aladdin Reagent Co., and were used without further
purification.
PPFPTA (50.0 mg, 0.16 mmol of repeat units) was dissolved in
20 mL of N,N-dimethylformamide. The mixture was stirred at
50 1C for 30 min, followed by the successive addition of
vancomycin hydrochloride (89.1 mg, 0.06 mmol) and triethyl-
amine (22.3 mL, 0.16 mmol). After stirring at 50 1C for 24 h,
mPEG-amine (160 mg, 0.16 mmol) in 5 mL of deionized water
was added dropwise to the solution through a syringe needle
within 30 min. The reaction mixture was stirred at 50 1C for
another 24 h, followed by the dialysis with deionized water
(molecular weight cutoff = 5000 Da) for 3 days. The solution
was further passed through a 0.22 mm filter membrane and
lyophilized. The resulting yellow brown powder was referred to
as PTPVan1. The PTPVan sample prepared by the addition of
vancomycin hydrochloride (133.7 mg, 0.09 mmol) was referred
to as PTPVan2.
Synthesis of PFPTA
Tetrahydrofuran (150 mL) and 3-thiopheneacetic acid (5.0 g,
35.2 mmol) were added to a 250 mL round bottom flask. The
solution was cooled to 0 1C in an ice bath, followed by the
Determination of the antimicrobial properties of PTPVan
successive addition of triethylamine (19.6 mL, 140.6 mmol) and E. coli, P. aeruginosa, S. aureus and S. epidermidis were cultured
pentafluorophenyl trifluoroacetate (12.1 mL, 70.4 mmol). After according to the ATCC protocols/specifications. The bacterial
that, the reaction mixture was stirred at room temperature for suspensions were then dispersed in phosphate-buffered saline
1.5 h. The solvent was removed by rotary evaporation, and the (PBS, pH 7.4) solution to give rise to a final concentration of
residue was diluted with 200 mL of ethyl acetate. The solution 1 Â 105 bacterial cells per mL. Stock solution of PTPVan1 (or
was washed with 200 mL of saturated NaHCO3 aqueous PTPVan2) was prepared in PBS solution at concentrations of
solution twice. The separated organic phase was dried with 0.5 and 1.0 mg mLÀ1. The bacterial suspension was then mixed
anhydrous Na2SO4 overnight, and the solvent was removed by with the PTPVan stock solution at a volume ratio of 1 : 2. After
rotary evaporation. The obtained crude product was purified culturing at 37 1C for 2 h, 100 mL of the solution was introduced
by column chromatography using ethyl acetate and hexane into 2 mL of tryptic soy broth (TSB). The mixture was vortexed
1
(v/v = 1 : 10) as eluents. H NMR (400 MHz, CDCl3-d, d, ppm): for 10 s and 100 mL of the mixture was added to each well of a
7.34 (thiophene–H, 1H), 7.25 (thiophene–H, 1H), 7.10 (thio- 96-well plate. The plate was incubated at 37 1C, and the time-
phene–H, 1H) and 4.00 (thiophene–CH2–, 2H). ESI-MS: m/z dependent optical density (OD) of each well at 600 nm was
309.29 ([M + H+], calcd 309.23).
recorded on a microplate reader (BioTek Instruments ELX800, USA).
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Controlled experiment was carried out by using PBS solution to The crude product was purified by soxhlet extraction. The
culture with the bacteria for 2 h.
as-obtained PPFPTA has a number-average molecular weight
(Mn) of 23 000 g molÀ1 and a polydispersity index (PDI) of 1.68.
The degree of polymerization of PPFPTA is about 74. PPFPTA
was reacted with vancomycin and mPEG-NH2 via a reactive ester–
amine reaction in the presence of triethylamine. The FT-IR
spectrum of PPFPTA (Fig. 1a) exhibits two absorption bands
at 1785 cmÀ1 and 1514 cmÀ1, attributable to the stretches of
the activated carbonyl (CQO) group and aromatic fluorine,
respectively.49 After substitution with vancomycin and PEG, the
strong absorption bands at 1785 cmÀ1 and 1514 cmÀ1 are barely
discernible and the amide CQO stretch is appeared in the FT-IR
spectra of PTPVan1 and PTPVan2 (Fig. 1b and c). This result
indicates that the reactive PFP ester moieties have been success-
fully converted into amide groups. Fig. S3 (ESI†) shows the
1H NMR spectra of PTPVan1 and PTPVan2 in D2O. The appearance
of characteristic proton signals of PEG and vancomycin indicates
that this post-functionalization step has been successfully carried
out. The molar ratios of PEG to vancomycin are calculated based on
the integrated area ratios of methylene protons in PEG and methyl
(CH3–CH(R)–) protons in vancomycin. The PEG to vancomycin
molar ratios are determined to be 2.1 : 1 and 1.4 : 1, respectively,
for PTPVan1 and PTPVan2. Since the degree of polymerization
of PPFPTA is about 74, the respective degrees of substitution of
vancomycin in PTPVan1 and PTPVan2 are around 23 and 30.
The presence of vancomycin in the side chains of polythiophene
could endow it with antimicrobial properties. This hypothesis
was verified using the growth inhibition assay. PTPVan (or free
vancomycin) and bacteria (E. coli, MRSA, P. aeruginosa, S. aureus or
S. epidermidis) were cultured for 2 h, followed by mixing with TSB
and measuring OD at 600 nm. The growth curves of E. coli, MRSA,
P. aeruginosa, S. aureus and S. epidermidis treated with PTPVan1
and free vancomycin are shown in Fig. 2. Both Gram-negative
and Gram-positive bacteria grow in similar manners but with
differences in turbidity in the controlled experiments (incubated
with PBS solution). The growths of Gram-negative bacteria
(E. coli and P. aeruginosa strains) are not influenced in the presence
of PTPVan1 and free vancomycin. Upon the addition of
PTPVan1 (0.5 mg mLÀ1), the growth rates of Gram-positive bacteria
Conjugation of PTPVan with bacteria measured by fluorescence
spectroscopy
The bacteria were suspended in PBS solution to reach a
concentration of 1 Â 109 bacterial cells per mL. Four hundred
microliter of the PTPVan1 stock solution (1 mg mLÀ1) was
mixed with 800 mL of the bacterial suspension. After culturing
at 37 1C for 30 min, the bacterial suspension was centrifuged at
11 000 rpm for 5 min. The bacterial cakes were re-dispersed in 1 mL
of PBS solution and the bacterial suspension was centrifuged again.
Three hundred microliter of Buffer BS (TaKaRa Bio Inc., Catalog
No. 9763-4) and 50 mL of lysozyme solution (TaKaRa Bio Inc.,
Catalog No. 9763-2) were added to the bacterial cake in a 2 mL PCR
tube. The mixture was vortexed and incubated at 37 1C for 1 h.
During incubation, the PCR tube was taken out from the water bath
and vortexed for 10 s after every 20 min. After lysis, the sample was
further sonicated for 10 min and measured using a fluorescence
spectrophotometer via excitation at 458 nm. Controlled experiment
was carried out using PBS solution to culture with the bacteria for
30 min.
Conjugation of PTPVan with bacteria measured by confocal
laser scanning microscopy (CLSM)
The bacteria were dispersed in PBS solution to achieve a concen-
tration of 1 Â 107 bacterial cells per mL, and 1.5 mL of the
bacterial suspension was added into a glass bottom cell culture
dish (Titan Technology, Catalog No. 02036560) at 37 1C for 4 h. The
solution was then aspirated and replaced with 0.5 mL of the
PTPVan2 stock solution (1 mg mLÀ1). The cells were cultured for
30 min, and then washed with deionized water exhaustively to
remove the free and loosely attached PTPVan2. The bacteria were
then observed on a LSM800 confocal microscope (Carl Zeiss,
Germany). Controlled experiment was carried out by using PBS
solution to culture with the bacteria for 30 min.
Characterization
The chemical structures were characterized using 1H NMR spectro-
scopy on a Bruker DRX 400 MHz spectrometer. The FT-IR spectro-
scopy measurements were carried out on a Thermo Scientific
Nicolet 6700 FT-IR spectrometer. The UV-visible absorption spectra
were obtained from a Shimadzu UV-2550 spectrophotometer. The
fluorescence spectra were measured on a Shimadzu RF-5301PC
spectrophotometer. Gel permeation chromatography (GPC) was
performed on a Waters GPC system, equipped with a Waters 1515
isocratic HPLC pump, a Waters 717 plus Autosampler injector and
a Waters 2414 refractive index detector, using tetrahydrofuran as
an eluent at 35 1C and at a flow rate of 1.0 mL minÀ1
.
Results and discussion
PFPTA was polymerized by chemical oxidation with anhydrous
FeCl3 in chloroform, resulting in the formation of an active
polythiophene derivative with reactive PFP ester groups (PPFPTA).
Fig. 1 FT-IR spectra of (a) PPFPTA, (b) PTPVan1 and (c) PTPVan2.
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Fig. 2 Growth curves of (a) E. coli, (b) MRSA, (c) P. aeruginosa, (d) S. aureus 25923, (e) S. aureus 43300 and (f) S. epidermidis treated with PTPVan1
(0.5 mg mLÀ1) and free vancomycin (0.2 mg mLÀ1).
Fig. 3 (a) UV-visible absorption and (b) fluorescence spectra of PTPVan1 and PTPVan2 in deionized water. Inset: Photograph of PTPVan2 aqueous
solution under ambient light (left) and a 365 nm UV lamp (right).
(MRSA, S. aureus and S. epidermidis strains) are affected. However,
the growths of Gram-positive bacteria treated with free vancomycin
(0.2 mg mLÀ1) can be completely inhibited within the incubation
time. These results indicate that vancomycin conjugated on the
side chains of polythiophene via amidation has weaken its anti-
bacterial activity against Gram-positive bacteria. Free vancomycin
has MIC values of 250, 41000, 0.98, 0.98, 0.98 and 0.49 mg mLÀ1
,
respectively, for E. coli, P. aeruginosa, MRSA, S. aureus 25923,
S. aureus 43300 and S. epidermidis strains. Unfortunately, the MIC
values of PTPVan against all of the bacterial strains are larger than
1000 mg mLÀ1. The significantly reduced antibacterial properties of
PTPVan suggest that they could not be used as antibacterial agents.
The optical properties of PTPVan were then investigated
using UV-visible absorption and fluorescence spectroscopies.
The UV-visible absorption spectra of PTPVan1 and PTPVan2
(Fig. 3a) in deionized water show absorption maxima at 403
and 413 nm, respectively. The absorption peak originates from
the p–p* transition of the polythiophene backbone.50,51 The
photoluminescence (PL) spectra of PTPVan1 and PTPVan2 in
Fig. 4 Photographs of the blank (left) and stained (right) bacterial cakes
after incubation with PBS and PTPVan1 solutions, respectively.
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deionized water (Fig. 3b) exhibit emission peaks at about
The identification of bacteria was carried out by staining
558 nm with an excitation wavelength of 458 nm. Under 365 nm the bacteria with PTPVan. After incubation with PTPVan1 for
UV illumination, the PTPVan2 aqueous solution can emit fluores- 30 min at 37 1C, the bacteria were centrifuged, washed with PBS
cence (inset of Fig. 3b). The fluorescence quantum yields of solution and centrifuged again. The blank bacterial samples
PTPVan1 and PTPVan2 in deionized water are determined to be were obtained by incubation of the bacteria with PBS solution. The
0.076 and 0.078, respectively, as measured using rhodamine 6G collected bacterial cakes are shown in Fig. 4. Except P. aeruginosa,
as the standard. These results suggest that the polythiophene all other blank bacteria cakes are white in color. After incubation
derivatives still exhibit their intrinsic optical properties after the with PTPVan1, the color of Gram-positive bacteria (MRSA, S. aureus
post-functionalization process and the fluorescent PTPVan can be 25923, S. aureus 43300 and S. epidermidis strains) changes to brown.
used to detect bacteria via fluorescence techniques.
However, there is no obvious color change for Gram-negative
Fig. 5 Fluorescence spectra of blank and stained bacteria after lysis with lysosome via excitation at 458 nm.
Fig. 6 Merged fluorescent and bright-field CLSM images of E. coli, MRSA, P. aeruginosa, S. aureus 25923, S. aureus 43300 and S. epidermidis before and
after incubation with PTPVan2 for 30 min.
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bacteria (E. coli and P. aeruginosa strains) after exposure to Conflicts of interest
PTPVan1. Thus, Gram-positive bacteria could be distinguished
through observing the color change of bacterial cakes by the
There are no conflicts to declare.
naked eye.
To verify the feasibility of identification of the Gram-positive
bacteria using fluorescence techniques, the PL spectra of bacteria
Acknowledgements
This research was supported by the National Natural Scientific
Foundation of China (21504072, 51741304) and the Program
for the Fundamental Research Funds for the Central Univer-
sities (SWU115005). The authors thank Prof. Li Li from School
of Life Sciences, Southwest University, for providing access and
measure time at his CLSM.
before and after incubation with PTPVan were examined. Fig. 5
show the fluorescence responses of E. coli, MRSA, P. aeruginosa,
S. aureus 25923, S. aureus 43300 and S. epidermidis. Upon incuba-
tion with PTPVan1, MRSA and S. aureus strains show significantly
enhanced fluorescence intensities centred at about 535 nm, and
S. epidermidis strain also shows increased fluorescence intensity.
However, the Gram-negative bacteria (E. coli and P. aeruginosa
strains) exhibit a negligible increase in the fluorescence intensity
after interaction with PTPVan1. The increase in the fluorescence
intensity of Gram-positive bacteria may be due to its binding
capacity toward vancomycin moieties in the side chains of
PTPVan1 and the subsequent enrichment of fluorescent polythio-
phene derivatives on the bacterial membranes. These results
indicate that the fluorescence spectroscopy could be used to
identify the Gram-positive bacteria with the aid of fluorescent
targeting molecules.
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