Journal of Biomaterials Science, Polymer Edition

ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsp20

In vivo imaging/detection of MRSA bacterial

infections in mice using fluorescence labelled
polymeric nanoparticles carrying vancomycin as
the targeting agent

Araz Norouz Dizaji, Dan Ding, Tulin Kutsal, Mustafa Turk, Deling Kong &
Erhan Piskin

To cite this article: Araz Norouz Dizaji, Dan Ding, Tulin Kutsal, Mustafa Turk, Deling Kong
& Erhan Piskin (2019): In�vivo imaging/detection of MRSA bacterial infections in mice using
fluorescence labelled polymeric nanoparticles carrying vancomycin as the targeting agent, Journal
of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2019.1692631

To link to this article: https://doi.org/10.1080/09205063.2019.1692631

Published online: 25 Nov 2019.

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JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION
https://doi.org/10.1080/09205063.2019.1692631

In vivo imaging/detection of MRSA bacterial infections

in mice using fluorescence labelled polymeric
nanoparticles carrying vancomycin as the targeting agent
Araz Norouz Dizajia, Dan Dingb, Tulin Kutsalc, Mustafa Turkd, Deling Kongb and
Erhan Piskina,e
a
Bioengineering Division, Institute of Graduate Studies, Hacettepe University, Beytepe, Ankara,
Turkey; bState Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials,
Ministry of Education, and College of Life Sciences, Nankai University, Tianjin, China; cFaculty of
Engineering, Chemical Engineering Department, Hacettepe University, Beytepe, Ankara, Turkey;
d
Faculty of Engineering, Department of Bioengineering, Kirikkale University, Yahsihan, Kirikkale,
Turkey; eNanoBMT: Nanobiyomedtek Biyomedikal ve Biyoteknoloji San.Tic.Ltd.Şti, Bilkent,
Ankara, Turkey

ABSTRACT ARTICLE HISTORY

This study aims to develop fluorescence labelled polymeric nano- Received 16 August 2019
particle (NP) carrying vancomycin as the targeting agent for Accepted 11 November 2019
in vivo imaging of Methicillin-resistant Staphylococcus aureus
KEYWORDS
bacterial infections in animal models. Maleimide functionalized
Fluorescence probes/dyes;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide in vivo detection of
(polyethylene glycol)-2000] as the main was carrier matrix to bacterial infections;
prepare the NPs. A fluorescence probe, namely, poly[9,90 -bis Methicillin-resistant
(600 -N,N,N-trimethylammonium) hexyl) fluorene-co-alt-4,7-(2,1,3- Staphylococcus aureus;
benzothiadiazole) dibromide] was encapsulated within these NPs polymeric nanoparticles;
by ultrasonication successfully. UV-Vis spectro- photometry of the targeting; vancomycin
NPs showed the characteristic shifting on the peak of conjugated
polymers indicating successful packaging of this compound with
lipid bilayers in nanoscales. Zeta-sizer and TEM analysis showed
that the prepared NPs have a diameter of 80-100 nm in a narrow
size distribution. Thiolated vancomycin was synthesized and
attached to the NPs as the targeting agent. FTIR and MALDI-TOF
spectroscopy analysis confirmed the immobilization. The specific
targeting properties of the vancomycin conjugated NPs to the tar-
get bacteria were first confirmed in in vitro bacterial cultures in
which Escherichia coli was the non-target bacteria – using confocal
microscopy and TEM. Imaging of bacterial infections in vivo was
investigated in mice model using a non-invasive live animal fluor-
escence imaging technique. The results confirmed that bacterial
infections can be detected using these novel polymeric NPs carry-
ing fluorescence probes for imaging and vancomycin as the tar-
geting agent – in vivo successfully.

CONTACT Erhan Piskin erhanpiskin@icloud.com NanoBMT: Nanobiyomedtek Biyomedikal ve Biyoteknoloji

San.Tic.Ltd.Şti, Bilkent, Ankara, Turkey
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/tbsp.
Supplemental data for this article is available online at https://doi.org/10.1080/09205063.2019.1692631.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 A. NOROUZ DIZAJI ET AL.

1. Introduction
Imaging of the diseased cells and tissues in the body has a great value/contribution
not only for effective diagnosis but also allows selection of the most appropriate treat-
ment protocols in therapy [1–3]. Therefore, live/non-invasive imaging of biological
structures has become the focus of many research groups [4, 5]. Fluorescent dyes are
among the most popular molecules for in vivo imaging and have been widely applied
[6–8]. Note that these optical probes exhibit a certain wavelength excitation property
and emit light at a different wavelength [9]. Many of them can be used as qualitative
or quantitative diagnostic probes with unique properties/advantages including rapid
diagnosis, high sensitivity in low quantities, non-radiation and low toxicity [10–13].
Conjugated polymers (CPs) are macromolecules exhibiting extraordinary/diverse
properties such as excellent photoluminescence, light absorption and conductivity –
as a result of the unsaturated delocalized p-conjugated electrons on their main chains
[14, 15]. Due to these unique properties, CPs have attracted a great attention for
imaging [16, 17]. CPs exhibiting fluorescence properties have recently become the
focus of biological imaging, especially for non-invasive in vivo imaging [16, 18–21].
The CP used in this study – that have been utilized for in vivo imaging of
Tumour cells previously – was poly[9,90 -bis(600 -N,N,N-trimethylammonium)hexyl)-
fluorene-co-alt-4,7-(2,1,3-benzot hiadiazole)-dibromide] (PFBT) [14, 22].
These optical probes are used with carriers – usually in/on nano and microparticles/
capsules or structures made of a variety of polymeric materials (both synthetic and
natural polymares [22–26]. Following the previous study – here, we have also used a
maleimide functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimi-
de(polyethyleneglycol)-2000] (DSPE-PEG2000-Mal) molecule for encapsulation of PFBT,
which is highly biocompatibility and forms capsule wall similar to cell membranes
[27, 28].
Targeting of active agents to the desired cells/tissues in the body is one of the
main rational both for successful/selective imaging/diagnosis and more efficient local-
ized therapy [29–31]. There is a huge literature about targeted delivery of drugs and
other medicinal agents related to therapeutical applications [32–34]. There are several
reasons for targeted delivery including: (i) the active agents can be quite sensitive and
lose their activity and/or eliminated by several mechanisms before they do reach their
target sites with desired doses/concentrations; (ii) however they may have quite sig-
nificant side effects [35, 36]. For imaging the same concerns are valid – but targeting
is a must not an option.
For targeting, usually carriers (nanoparticles, nanocapsules, etc.) are used to deliver
the active agents in which their external surfaces are decorated by attachment/immo-
bilization of the targeting agents. Several molecules – mainly antibodies against the
target cell surface receptors – have been studied as targeting agents extensively but
unfortunately with limited successes [31, 37, 38]. Not only their targeting abilities but
also other positive or negative properties have been discussed in the related litera-
ture [39–42].
The main aim of this study is imaging of bacterial infections within the body – the
targets are not the cells but bacteria themselves. Antibiotics are known as antibacterial
compounds that have a lethal or inhibitory effect against bacteria [43, 44]. Depending
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 3

on the mechanism of action, antibiotics can bind to the sites/receptors on/in different
specific targets (molecules and/or structures) such as DNA/RNA, DNA/RNA poly-
merases, peptidoglycans and lipopolysaccharides, ribosome and cell membrane
[45–50]. By utilizing these usual properties of antibiotics, it is feasible to assign them
as targeting agents.
Methicillin-resistant Staphylococcus aureus (MRSA) refers to a group of Gram-
positive bacteria that are genetically distinct from other strains of Staphylococcus aur-
eus. MRSA is responsible for several difficult-to-treat infections in humans, therefore
has been selected as the target in this study [51–53]. Escherichia coli (E. coli) is
another important pathogen – but in a group of Gram-negative bacteria – with very
different bacterial wall structure comparing the ones in the Gram-positive bacteria –
that was selected as the non-target in this study [53–55]. Vancomycin is one of the
rare antibiotics that are effective against MRSA but not E. coli [56]. Therefore, it has
been proposed not only to treat the MRSA infection but also as specific targeting
agent as we have investigated in this study. Note that our focus here not to treat the
MRSA infections but only imaging/detection of these infected areas. In addition, it
should be pointed out that the presence of the carbocyclic groups allowed us chemical
modification (thiolization) of vancomycin molecules which let easier and more reli-
able immobilization it onto our carriers described above [57].
This article describes preparation protocols of the polymeric nanocapsules carrying
a CP – as a fluorescence dye and vancomycin on their surface as the targeting agent
for selective imaging/detection of MRSA both in vitro bacterial cell cultures and
in vivo (an animal – mice model) infected with both MRSA (the target bacteria – at
one side) and E. coli (a non-target bacteria – on the other side).

2. Material and methods

2.1. Materials
The main component of the nanoparticles, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-
N-[maleimide(polyethylene glycol)-2000] Maleimide (DSPE-PEG2000-Mal) was a com-
mercial product of Avanti Polar Lipids, Inc., USA. The photoactive (fluorescent)
component/label – PFBT was purchased from Sigma-Aldrich, St. Louis, MO, USA.
Vancomycin dihydrochloride (Van), cystamine dihydrochloride, dimethyl sulfoxide
(DMSO), dimethylformamide (DMF), N,N,N0 ,N0 -Tetramethyl-O-(1H-benzotriazole-1-yl)
uronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIEA), glycerol,
H2O2 and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) that were used for the
synthesis of the thiolated-vancomycin (HS-vancomycin) and also in other treatments
were also purchased from Sigma-Aldrich, USA. Tetrahydrofuran (THF) was distilled
from sodium benzophenone ketyl under dry nitrogen immediately prior to use.
Dulbecco’s Modified Eagle’s Medium (DMEM) and Fetal Bovine Serum (FBS), which
were used for cell culture studies, were obtained from Sigma-Aldrich, USA. L929 fibro-
blast cell line (derivative of strain L mice fibroblast cells), Water Soluble Tetrazolium
Salts-1 (WST-1) and horseradish peroxidase (HRP) antibody labeling kit (Abcam,
Cambridge, MA, USA), and Caspas-3 assay kits (AB3623, Millipore, Billerica, MA,
USA) were provided by the Kirikkale University Central Laboratories, Turkey.
4 A. NOROUZ DIZAJI ET AL.

In the specific targeting studies two bacteria MRSA (ATCC 33591, Manassas, VA,
USA) – ‘as the target bacteria’ and Escherichia coli (E. coli), (ATCC 25922, USA) –
‘as the non-target bacteria for comparison’ were used. Male ICR mice (6-8 weeks old)
provided by the animal center of the Drum-Tower Hospital (Nanjing, China) were
used in the animal model studies.

2.2. Methods
2.2.1. Multifunctional nanoparticles
The ‘Fluorescence Probe Loaded Nanoparticles’ (CPDP-Mal NPs) were prepared as
follows: 1 ml THF solution containing 0.5 mg of PFBT and 2 mg of DSPE-PEG2000-
Mal was added to 9 ml of distilled water. This mixture was sonicated via 12 W micro-
tip probe sonicator (12 W output, XL2000, Misonix Incorporated, NY) for 60 s. After
evaporation of THF overnight at the room temperature, the nanoparticles were puri-
fied by centrifugation at 3000g for 30 min which was followed ultrafiltration through
a 0.2 lm syringe driven filter. The obtained nanoparticles were characterized using
dynamic light scattering (DLS; BIC-ZetaPALS, USA), zeta potential (BIC-ZetaPALS,
USA) and TEM (Hitachi TEM System, Tachikawa, Tokyo, Japan).
To demonstrate the successful encapsulation of PFBT molecules into CPDP-Mal
NPs, PFBT CPs and their encapsulated forms were characterized using UV-Vis spec-
trophotometer (Jena Analytik, Germany) to observe the possible shifting in the excita-
tion spectra due to the packaging process.
In order to produce the nanoparticles carrying the targeting agent (i.e. vanco-
mycin) the following protocols were applied. First, the thiol ended-vancomycin
(HS-Van) was prepared according to the previous protocols described in the related
literature [57, 58]. Briefly, 100 mg vancomycin was dissolved in 1 ml of DMSO by
sonication until the solution become clear. Cystamine dihydrochloride of 6.8 mg con-
taining 1 ml DMF solution was prepared and added to the vancomycin solution and
stirred in an ice bath. HBTU of 34 mg was dissolved in 60 ml DIEA solution which
was then added into the previous mixture. After treating in ice bath 30 min, the
reaction was completed by stirring overnight – at room temperature. The resultant
mixture was purified first treating in an HPLC (Shimadzu, Japan). The thiol ended-
vancomycin (HS-Van) formed was separated, then dried with air pump overnight
that was followed freeze-drying for 3 days. The cleavage of disulfide bonds was
achieved using TCEP. The synthesized HS-Van was characterized using MALDI-TOF
Spectroscopy (Applied Biosystem, Foster City, CA, USA).
The bacterial targeting agent ‘HS-Van’ was attached on the surfaces of the nano-
particles as follows: 1 ml of the CPDP-Mal NPs (3 nM) was added to the 3 mM HS-
Van solution (3 mM) by shaking for 1 h. According to the mechanism reported by
Greg T. Hermanson et al, the maleimide functional group can react specifically with
any biomolecules that have a sulfhydryl functional groups [59]. This reaction was car-
ried out in a pH range between 6.5 and 7.5. At the end of this reaction, stable thio-
ether bonds were formed as described in Figure 1. The free and unconjugated
vancomycin molecules were separated the nanoparticle in a dialysis tube (Cut-
off:14000, Sigma-Aldrich, USA) within 12 h. PFBT, DSPE-PEG2000-Mal, the
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 5

Figure 1. HS-Van conjugation onto the DSPE-PEG2000-Mal NPs.

synthesized HS-Van and the CPDP@Van NPs obtained were characterized using
ATR–FTIR spectroscopy (Nicolet iS10 FT-IR Spectrometer, Thermo ScientificTM,
Waltham, MA, USA).

2.2.2. In vitro cytotoxicity and apoptosis tests

Cytotoxicity of the synthesized CPDP@Van NPs were investigated against L929
mouse fibroblast cell line via WST-1 assay test. L929 mouse fibroblast cells were cul-
tured in 48-well plates at a density of 2  104 cells/ml with in a culture medium
(DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin. After 24 h
post-incubation of cultured cells at the 37  C in the presence of 5% CO2 atmosphere,
the CPDP@Van NPs solutions with different concentrations (0.37–3 nM) were added
to the culture medium and incubated for 24 h at the same conditions. After incuba-
tion, the media within the wells were discarded, and replaced with 100 ll of phenol
red-free medium (Sigma-aldrich, USA) and 10 ll of WST-1 solution and further incu-
bated for 4 h. The absorbances were measured/obtained at 440 nm using a microplate
Reader (BIO RAD-i MarkTM-Microplate Reader, Hercules, CA, USA).
The apoptotic effects of the CPDP@Van NPs synthesized in this study were
investigated by Caspase-3 antibody staining of the L929 mouse fibroblast cells.
2  104 cells/ml of L929 mouse fibroblast cells were cultured within 48-well plate in
the DMEM media supplemented 10% FBS and 1% penicillin-streptomycin. The cul-
tured cells were incubated at 37  C in a 5% CO2 atmospheric conditions for 24 h.
The CPDP@Van NPs solutions with different concentrations (0.37–3 nM) was
added to the cells and re-incubated for 24 h. Then, the medium was discarded and
the cells were treated with 3% methanolic H2O2 for 15 min and lastly washed with
PBS and the HRP antibody labeling kit was used according to the protocol [60].
Finally, the treated cells were imaged at 20 magnification on a microscope to
observe the apoptotic effects of the CPDP@Van NPs. It should be noted that only
6 A. NOROUZ DIZAJI ET AL.

the multifunctional nanoparticles carrying both optical probes/labels and targeting

agent (vancomycin) were included in the cytotoxicity and apoptosis cell culture tests
here in this study.

2.2.3. In vitro targeting tests

Specific targeting properties of the CPDP@Van NPs synthesized in this study were
investigated on MRSA (the target bacteria) and E. coli (the non-target bacteria) in
bacterial cultures. The proper amount of bacterial population (3  106 cfu/ml) were
obtained after culturing in bacteria in LB broth at 37  C by shaking 200 rpm for 18 h.
The cultured bacteria were washed twice with physiological serum after centrifugation
at 3000 rpm for 3 min. The bacteria were treated with the CPDP@Van NPs at 37  C
for 1 h. Non-specifically attached/adhered nanoparticles were removed by centrifuga-
tion and washing with physiological serum.
The specific targeting properties of the CPDP@Van NPs against MRSA bacteria
was investigated using Confocal Laser Scanning Microscopy (CLSM; Zeiss LSM 410,
Jena, Germany). Quantitative analyses of the CLSM images were determined using
Image Pro Plus software. The bacteria were centrifuged at 3000 rpm and the super-
natant was removed. Then, 50 ml of glycerol were added and the bacteria for fixation
purpose. Ten microliter of each sample was dropped on microscope slides images
were taken with the confocal microscope upon excitation at 494 nm with the absorp-
tion of fluorescent signals around 531 nm.
In parallel, targeting of the CPDP@Van NPs to MRSA were investigated by TEM
(FEI/Tecnai G2 Spirit Biotwin, Hillsboro, OR, USA) analysis. For this purpose, the
CPDP NPs containing no vancomycin and/or vancomycin conjugated were treated
with MRSA separately. Ten microliter of each sample was dropped to the electron
microscopy grids and – after drying – images were taken with TEM.

2.2.4. In vivo studies

The in vivo performances of the CPDP@Van NPs were investigated in the animal
model studies. The mice were infected with the MRSA and E. coli by subcutaneous
injection of 50 ml (3  106 cfu/ml) of the bacterial dispersions to the left and right
back side of ICR mice, respectively. All animal studies were performed under the
guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals, and
the overall project protocols were approved by the Animal Ethics Committee of
Nankai University. Intravenous injection of 200 ml (3 nM) CPDP@Van NPs were car-
ried out two days after bacterial infections of the test animals. The in vivo live tracing
studies were performed on these animals by a ‘non-invasive live animal fluorescence
imaging technique’ using Maestro EX Fluorescence Imaging System (CRi, Inc.,
Waltham, MA, USA). The mouse auto-fluorescence was removed by spectral unmix-
ing with the Maestro software. The infected mice were imaged by receiving 1 s to
5 min scans. The bioluminescence signals were quantified in units of maximum pho-
tons per second per square centimeter per steradian.
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 7

3. Results and discussion

3.1. Nanoparticles
The excitation properties of PFBT and the CPDP-Mal NPs – prepared in this study
-were determined by UV-vis spectrophotometer – characteristic peaks are shown in
Figure 2. UV-Vis spectra indicated that the characteristic excitation peak of PFBT
shifted from 460 to 480 nm after encapsulation. This is well-known that encapsulation
of CPs causes to red-shifting of emission and excitation peaks. Moreover, this should
be noted that this is a preferred event for the reception of higher sensitivity in the
application of biological imaging [61].
The diameter average and potential charge of the synthetized CPDP-Mal NPs and
CPDP@Van NPs were determined by DLS and Zeta potential. The zeta potential
value showed an increase from –32.65 to –21.86 which is referred to the positive
charge nature of vancomycin [62]. There was also an increase in the size the nano-
particles (from 70 to 80 nm) after conjugation of vancomycin onto the CPDP-Mal
NPs according to the DLS analysis (Supporting Information, Figure S1). This increase
was most probably due to both the increase molecular weight increase and change of
the spatial 3D structure of the immobilized vancomycin [63]. These results were
accepted as an indication of conjugation of vancomycin molecules to the CPDP-Mal
NPs. According to TEM results, the nanoparticles were on spherical shape with distri-
bution around 70–100 nm (Figure 3) that can be supported DLS results.
The MALDI-TOF has the potential to accurately measure the mass changes that
occur after the interaction between the molecules [64]. It has also become a preferred
technique for the analysis of molecules [65]. Therefore, one of the most reliable ways
to prove that the HS-Van molecule was successfully synthesized is to use the
MALDI-TOF technique – as we have done in this study. In order to bind the vanco-
mycin to the nanoparticles, a thiol functional group was initially created on the
vancomycin molecule. The successful synthesis of HS-Van was confirmed using
MALDI-TOF spectral analysis – as shown in Figure 4. Note that the molecular weight

Figure 2. UV-Vis spectra of PFBT and the PFBT encapsulated within CPDP-Mal NPs. As a result of
encapsulation, a red shift was observed in the characteristic excitation PFBT peak – shifted from
460 to 480 nm.
8 A. NOROUZ DIZAJI ET AL.

Figure 3. TEM imaging of CPDP-Mal NPs.

Figure 4. A representative MALDI-TOF spectra of the thiolated-vancomycin molecules prepared in

this study.

of HS-Van is 1507 g mol1. The peak at 1508 m z1 indicates that the molecule is
ionized with Hþ (M ¼ 1507 þ 1) with a molecular weight of 1 g mol1 and the peak
at 1530 m z1 indicates that the molecule was ionized with Naþ (M ¼ 1507 þ 23),
which has a molecular weight of 23 g mol1. The peak with relatively low intensity at
3013 m z1 belongs to bis-vancomycin, which does not hydrolyze with TCEP and
remains in solution. Bis-vancomycin has a molecular weight of 3012 g mol1 and ion-
ization with Hþ during laser application in the MALDI-TOF device (M ¼ 3012 þ 1).
The FTIR analysis was also applied to understand/prove the binding of HS-
vancomycin to the nanoparticles. Representative spectra of PFBT, DSPE-PEG2000-
Mal and HS-Van and CPDP@Van NPs were shown in Figure 5(a–c). The peaks at
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 9

Figure 5. Representative FTIR spectra: (a) PFBT; (b) DSPE-PEG2000-Mal; (c) HS-Van and (d)
CPDP@Van NPs. The OH peaks that correspond to the vancomycin molecule were observed
3100–3600 cm–1.

2849, 2918 and 2954 cm1 belong to the -CH3, -CH2 and -CH stretching vibrations
that from PFBT and DSPE-PEG2000-Mal, respectively. The peaks at 1100 and
1115 cm1 were assigned to the etheric C-O tensile vibration from DSPE-PEG2000-
Mal and the peak observed at 1731 cm1 corresponds to the carbonyl tensile vibra-
tion in the strong ester group resulting from DSPE-PEG2000-Mal. The -OH peaks
corresponding to the vancomycin molecules were observed in the range of
3100–3600 cm1. All of these peaks that belongs to PFBT, DSPE-PEG2000-Mal and
HS-Van were appeared in the spectrum of the CPDP-Mal NPs too. In order to
prove successful binding of a molecule or ligand to nanoparticles, the results of
FTIR analysis are indisputably known to provide a great contribution [66–68]. In
the light of FTIR analysis, we can conclude that the PFBT molecules were encapsu-
lated by DSPE-PEG2000-Mal molecules. The most striking result is that HS-Van
molecules were successfully synthesized and immobilized onto CPDP-Mal NPs.

3.2. Cytotoxicity and apoptosis

In vitro cytotoxicity and apoptosis properties of materials can almost mimic the limi-
tations of in vivo applications [69]. Therefore, we have designed and conducted the
cytotoxicity and apoptosis tests as explained in Section 2 given above. The WST-1
proliferation assay technique was performed, and the relevant results obtained via the
microplate reader which are illustrated in Figure 6. As shown here, the lowest level of
the cell viability was observed at the maximum concentration (3 nM) of the CPDP-
Mal NPs – which was in quite acceptable level. Therefore, we have concluded that
10 A. NOROUZ DIZAJI ET AL.

Figure 6. Cytotoxicity of the CPDP@Van NPs on L929 cell line in different concentrations.

the synthesized CPDP@Van NPs has low cytotoxicity and it is reliable carry them to
in vivo studies that we have done and demonstrated below.
The apoptosis effects of the NPs at different doses were investigated in the related
cell culture studies in which L929 mouse fibroblast cell line was used by applying a
Caspas-3 antibody staining protocol. Even at the highest dose (3 nM) of the
CPDP@Van NPs, there was no adverse observations related to cell deformation,
nuclear breakdown, chromatin distribution, cell membrane orientation and mem-
brane blebbing (see also Supporting Information, given Figure S2). This results clearly
proved that the prepared NPs can be used in in vivo studies without adverse apop-
tosis effects.

3.3. In vitro targeting

As shown in Figure 7(a–c and e), in the presence of nanoparticle system significant
fluorescence signals were observed for MRSA which localized around the cell wall.
However, no signal was detected for E. coli (Figure 7(d,e)). Gram-positive bacteria
have a receptor against vancomycin on peptidoglycan layers. On the contrary, Gram-
negative bacteria’s cell wall is surrounded by the outer membrane with low perme-
ability abilities to molecules as large as vancomycin [70]. Therefore, the CPDP@Van
NPs could not bind to Gram-negative bacteria which was the main initiative to select
and use vancomycin as the targeting agent on the NPs that we have synthesized in
this study.
Demonstration of successful targeting of the CPDP@Van NPs to MRSA bacteria
was investigated using TEM as a validator analysis. Figure 8(c,b) show the MRSA
bacteria in diplococcus form and the CPDP-Mal NPs (vancomycin free) present in
the distance, respectively. the CPDP-Mal NPs are thought not to have binding ability
to MRSA bacteria. Therefore, – as expected – the vancomycin free NPs do not have
any specific binding properties to MRSA. The specific interaction of the CPDP@Van
NPs carrying the targeting agent vancomycin with MRSA was shown in Figure 9. The
CPDP@Van NPs that accumulate on the MRSA is evidence of specific binding
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 11

Figure 7. Representative confocal microscopy images: (a) Single MRSA bacteria; (b) another single
MRSA bacteria; (c) triple MRSA bacteria; (d) E. coli bacteria and (e) MRSA and E. coli in the same
environment.

Figure 8. Representative TEM images: (a) Interaction of CPDP-Mal NPs (vancomycin free) and
MRSA; (b) CPDP-Mal NPs with no binding properties against MRSA and (c) MRSA bacteria without
any bonded CPDP-Mal NPs.
12 A. NOROUZ DIZAJI ET AL.

Figure 9. Representative TEM images: (a) Specific interaction of MRSA with CPDP@Van NPs carry-
ing vancomycin as the targeting agent; (b); (c) and (d) Magnifications to different regions of
same image.

properties of these nanoparticles. Since each CPDP@Van NP has an alternative ability

to bind to more than one bacteria (due to more than one vancomycin were conju-
gated on nanoparticles surface), it caused to bacterial aggregation/coagulation.

3.4. In vivo studies

The fluorescence imaging of spectral of infected mice was obtained with the Maestro
software to obliterate of the mouse background auto-fluorescence. Figure 10 shows
time-dependent (1, 3 and 24 h post-injection) fluorescence distribution profile of the
CPDP@Van NPs in living mice. One hour after intravenous injection of CPDP@Van
NPs to the infected mice, the nanoparticles distributed in blood stream/cycle and low
amount of them were targeted successfully to the infected part of the body. For the
case of 3 h post-injection, more nanoparticles were absorbed and after 24 h significant
concentration of the CPDP@Van NPs were targeted/accumulated to the MRSA
infected part (shown in the left). Due to the high blood flow on infected tissues and
enhanced permeability and retention effect [71], both infected sides of the mice indi-
cated the higher fluorescence signals. Since MRSA has a receptor against vancomycin,
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 13

Figure 10. Representative live images: (a) Live imaging of infected mice model without applying
fluorescence effect. Time depended distribution of CPDP@Van NPs in the infected mice model: (b)
after 1 h; (C) after 3 h and (D) after 24 h post intravenous injection of CPDP@Van NPs.

the intensity of the fluorescence signal is higher than E. coli’s side (shown in the
right; Figure 10).

4. Conclusion
In this study, novel multifunctional nanoparticles carrying fluorescence labels for
imaging and vancomycin as the targeting agent were prepared for the imaging/
detection of MRSA bacterial infections in vivo. The characterization techniques
used (e.g. UV-Vis, MALDI-TOF, FTIR, TEM, Zeta Size) approved the protocols
that have been applied to prepare NPs within the desired particle size around
70–100 nm and also carrying optical (i.e. PFBT) and targeting (i.e. vancomycin)
agents successfully. First, the specific targeting properties of the NPs carrying
vancomycin were illustrated in the in vitro bacterial cultures. TEM and confocal
microscopy images demonstrate that CPDP-Van NPs have specific targeting proper-
ties against MRSA comparing to the non-target bacteria, i.e. E. coli. Cytotoxicity
and apoptotic assays demonstrated that these nanoparticles are safe to use in
in vivo. Therefore, the animal model studies described above have designed and
performed. The selected animals, i.e. mice were infected with both MRSA and
E. coli, two different sides. The invasive live imaging clearly demonstrated that these
novel nanoparticles having both optical and specific targeting agents can success-
fully use to image/detect MRSA bacterial infections in vivo – which could be trans-
lated/applied to other targets with specific targeting agents.

Acknowledgments
The studies included in this paper are a part of the Ph.D. thesis of Araz Norouz Dizaji which
were mainly performed in the Chinese partner’s labs in Tianjin and in the Chemical
Engineering Department of Hacettepe University. The authors specially thanks to Chao Chen
from the Nankai University and Matin Yazdani Kohneshahri from Hacettepe University for
helping in in vivo experiments and MALDI-TOF analysis, respectively. Erhan Piskin also
acknowledges support from the Turkish Academy of Science as an honorary member.
14 A. NOROUZ DIZAJI ET AL.

Disclosure statement
No potential conflict of interest of interest was reported by the authors.

Funding
This study was conducted in the frame of a EU project between Europe and China – the
shortly called ‘ABREM’, ‘FP7-PEOPLE-2009-IRSES’ and also supported by Turkish Scientific
and Technological Council (Project Number: 1130864).

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