Manganese porphyrin-incorporated conjugated polymer nanoparticles for T1-enhanced magnetic resonance and fluorescent imaging

Article abstract of DOI:10.1016/j.ica.2017.06.014

Conjugated polymer nanoparticles (CPNs) possess many useful and fascinating properties, including high brightness, excellent photostability, good water-dispersibility, low cytotoxicity, and easy functionalization, showing promising application in bioimaging. In this work, one kind of optical/magnetic conjugated polyelectrolyte has been designed and synthesized by introducing Mn(III) porphyrin (T₁-weighted relaxivity) into a fluorescent fluorene based polymer backbone. Fluorescent/magnetic conjugated polymer nanoparticles (FM-CPNs) were prepared by self-assembly in the phosphate buffer solution caused by their amphiphilic structures with hydrophobic backbones and hydrophilic side chains. Their photophysical properties have been investigated in details via UV–vis absorption and fluorescent emission spectra. Investigation of its magnetic properties has shown that the FM-CPNs exhibit high T₁-weighted relaxivity value, making them promising candidates for T₁-enhanced magnetic resonance imaging agent. Further cell imaging has been realized successfully using FM-CPNs as staining label, and cytotoxicity was evaluated by the methyl thiazolyl tetrazolium (MTT) assay.

Full text of DOI:10.1016/j.ica.2017.06.014

Accepted Manuscript
Research paper
Manganese Porphyrin-Incorporated Conjugated Polymer Nanoparticles for T1-
Enhanced Magnetic Resonance and Fluorescent Imaging
Tianshe Yang, Wenguo Feng, Changyong Hu, Zhuang Lv, Huanjie Wei, Jiayang
Jiang, Shujuan Liu, Qiang Zhao
PII:
S0020-1693(17)30579-0
http://dx.doi.org/10.1016/j.ica.2017.06.014
ICA 17662
DOI:
Reference:
Inorganica Chimica Acta
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15 April 2017
1 June 2017
7 June 2017
Please cite this article as: T. Yang, W. Feng, C. Hu, Z. Lv, H. Wei, J. Jiang, S. Liu, Q. Zhao, Manganese Porphyrin-
Incorporated Conjugated Polymer Nanoparticles for T1-Enhanced Magnetic Resonance and Fluorescent Imaging,
Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.06.014
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Manganese Porphyrin-Incorporated Conjugated Polymer Nanoparticles for T1-
Enhanced Magnetic Resonance and Fluorescent Imaging
Tianshe Yang,‡ Wenguo Feng,‡ Changyong Hu,‡ Zhuang Lv, Huanjie Wei, Jiayang
Jiang, Shujuan Liu, Qiang Zhao*
Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced
Materials (IAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing
210023, China. Email: iamqzhao@njupt.edu.cn (Q. Zhao), Fax: (+86) 25-85866396.
Abstract:
Conjugated polymer nanoparticles (CPNs) possess many useful and fascinating properties,
including high brightness, excellent photostability, good water-dispersibility, low cytotoxicity, and
easy functionalization, showing promising application in bioimaging. In this work, one kind of
optical/magnetic conjugated polyelectrolyte has been designed and synthesized by introducing
Mn(III) porphyrin (T1-weighted relaxivity) into a fluorescent fluorene based polymer backbone.
Fluorescent/magnetic conjugated polymer nanoparticles (FM-CPNs) were prepared by self-
assembly in the phosphate buffer solution caused by their amphiphilic structures with
hydrophobic backbones and hydrophilic side chains. Their photophysical properties have been
investigated in details via UV-vis absorption and fluorescenct emission spectra. Investigation of
its magnetic properties has shown that the FM-CPNs exhibit high T1-weighted relaxivity value,
making them promising candidates for T1-enhanced magnetic resonance imaging agent. Further
cell imaging has been realized successfully using FM-CPNs as staining label, and cytotoxicity
was evaluated by the methyl thiazolyl tetrazolium (MTT) assay.
Keywords: Conjugated polyelectrolytes; Cell imaging; Magnetic resonance imaging; Polymer
nanoparticles
1

1. Introduction
Conjugated polymers, regarded as promising materials for next-generation optoelectronic
devices, have gained wide interest for applications in organic field-effect transistors, organic
light-emitting diodes, organic solar cells, and so on [1-7]. Recently, the applications of
conjugated polymer have been extensively developed to chemical/biological detections and
bioimaging fields [8-15], due to their large absorption cross sections and good photostability [16-
18]. In particular, conjugated polymer nanoparticles (CPNs) have attracted considerable
attention in bioapplication owing to their intriguing chemical and optical features, such as strong
absorbance, high fluorescent efficiency, excellent photostability, good biocompatibility and
amplified signal output [6-8,12,19-26]. Thus, CPNs as promising nanosized agents have
practical applications in biomedical research. Besides, CPNs can be designed to possess
multifunction by introducing some special moiety to endow multifunctional capacity sensor,
imaging, therapeutics, and so on [8,27-33].
Magnetic resonance imaging (MRI) has been successfully applied to the clinical diagnosis of
many diseases, because of its advantages such as noninvasiveness, high lateral and depth
resolution [34-36]. Paramagnetic metals (e.g., FeIII, MnII, MnIII, and GdIII) are typical MR contrast
agents, increasing the longitudinal (1/T1) and transverse (1/T2) relaxation rates [37-43].
Paramagnetic gadolinium complexes are the most widespread MRI contrast agents in clinical
applications. However, recent research has reported that Gd(III)-based contrast agents can
induce patients with renal dysfunction to nephrogenic systemic fibrosis [44-47]. Manganese,
one of the first examples of MRI paramagnetic contrast material due to its efficient T1
enhancement [48-51], is a good candidate as MRI agent. Manganese-porphyrin complexes
have merged as a promising MRI contrast agents because of their increasing nuclear spin
relaxation rate [37,44,52-53].
Herein, we reported a kind of polyfluorene-based cationic conjugated polyelectrolytes (P2)
including Mn(III) porphyrin for fabricating water-soluble FM-CPNs (Fig. 1) with fluorescent/MR
features. The charged side chain with triglycol moiety improved their aqueous solubility of the
FM-CPNs [54], which was obtained from P2 self-assembly in phosphate buffer solution (PBS).
2

We introduced Mn(III) porphyrin into the conjugated polymer to endow T1-weighted enhanced
MR character. The optical and T1-weighted MR properties have been investigated in detail. The
FM-CPNs were further applied in cell imaging as staining agent. Moreover, the cell cytotoxicity
of the FM-CPNs was also investigated. These experiments have shown that this kind of FM-
CPNs can be used as an excellent contrast agent for multifunctional imaging.
Fig. 1. The chemical structures of conjugated polymers P1, P2, and a diagrammatic sketch of
the self-assembly behavior of P2 into the FM-CPNs.
2. Experimental
2.1 Instruments and methods
All starting chemicals and solvents were commercially purchased and were used without any
1
purification unless otherwise noted. The H and 13C NMR spectra were recorded on a Bruker
Ultra Shield Plus 400 MHz NMR spectrometer. Mass spectra were recorded on a Bruker
autoflex matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-
3

TOF/MS). UV-visible absorption spectra were obtained with an HP-8453 spectrophotometer.
Emission spectra were measured on an Edinburgh FL920 fluorescence spectrometer. The
molecular weight of polymer was determined by gel permeation chromatography (GPC) in THF
at 35 °C using polystyrene as the standard. The zeta-potentials and particle sizes were studied
by dynamic light scattering (DLS) with Zetasizer Nanoseries (Nano ZS90). The particle
morphology was investigated by a JEOL JEM-2100 transmission electron microscope (TEM) at
an acceleration voltage of 100 kV.
2.2 Cell culture
Human cervical cancer cells HeLa were obtained from the Institute of Biochemistry and Cell
Biology, SIBS, CAS (China). The HeLa cells were seeded in DMEM medium containing 10%
FBS (fetal bovine serum) under an atmosphere of at 37 ºC 5% CO2. The HeLa cells were
precultured and allowed to adhere for overnight before experiments.
2.3 Confocal luminescence imaging
Confocal luminescence imaging and Z-scaning were taken by using an Olympus IX81 laser
scanning confocal microscope and a 40 oil-immersion objective lens. A pulse diode laser of 405
nm was used as excitation of the HeLa cells incubated with FM-CPNs. The HeLa cells were
incubated at 37 ºC for 2 h with 10 μg mL-1 FM-CPNs in DMEM solution. The emission channel
was collected from 410 nm to 460 nm.
2.4 Cell viability assay
In vitro cell cytotoxicity was investigated by MTT assays on HeLa cells. Briefly, cells were
grown in a 96-well plates at 1×104/well. Various concentrations of FM-CPNs (0, 5, 10, 20, 40
and 60 μg mL−1, diluted in DMEM) were then added into the wells. The cells were incubated for
24 h under 5 % CO2 atmosphere at 37 ºC. Then, the MTT (5 mg mL-1)/PBS solution was added
to each well of the 96-well assay plate and treated as above for an additional 4 h. Each well was
added with 150 μL DMSO after cleaning off the culture dish, then the OD570 of each well was
monitored with reference at 490 nm using an enzyme-linked immunosorbent assay (ELISA)
reader. The formula of calculating the inhibition of cell growth is as following:
Viability (%) = (mean of Abs. value of treatment group/mean Abs. value of control) × 100%.
2.5 ICP-AES analysis
4

The manganese content in aqueous samples was determined by inductively coupled plasma-
atomic emission spectroscopy (ICP-AES) (Optima 5300DV, PerkinElmer, USA). The samples
were digested by microwave oven after adding nitric acid solution (HNO3 for trace analysis).
2.6 T1-weighted MRI phantom images
The MRI measurement of FM-CPNs was carried out on a 0.55 T NMR analyzer (NMI20,
China). Different concentrations of Mn3+ solution were prepared before MRI scanning, which
varied from 0.0066 to 0.121 mM with a volume of 500 μL in 1.5 mL centrifuge tube. The
parameters of data acquisition were set as follows: echo time (TE) = 18.2 ms, repetition time
(TR) = 460 ms, field of view (FOV) = 7.5 cm × 7.5 cm, matrix = 256 × 192, slice thickness = 5.0
mm, and spacing = 1.0 mm.
2.7 T1 measurement and r1 relaxivity in vitro
The longitudinal relaxation time T1 and relaxivitiy r1 of the FM-CPNs were measured by a
standard inversion−recovery pulse sequence on a small MRI instrument whit 0.55 T magnetic
field. Dilutions of FM-CPNs in water as contrast agent were added into five 1.0 mL centrifuge
tubes according to different proportions to performing T1 assay. The T1 values were obtained at
various concentration and plotted as 1/T1 (R1) vs. molar concentration of Mn3+. The molar
relaxivity r1 was obtained by the slope of this line.
2.8 Synthesis
5

Scheme 1. The synthetic routes of monomers.
Scheme 1 and Fig. 1 showed the chemical structures and synthesis route of monomer 1, 2, 3
and polymer P1 and P2, and preparation of the polymer nanoparticles (FM-CPNs). The
synthesis procedure is as the following.
1,8-Dibromo-3,6-dioxaoctane: The mixture of triglycol (20 mL, 0.15 mol) and 50 mL dry
dichloromethane in ice water bath was dropped with PBr3 (14.2 mL, 0.15 mol) with constant
pressure funnel about 40 min. The solution was stirred at room temperature for 12 h. Then the
reaction was quenched with 20 mL water. The organic phase after extracting three times with
chloroform was collected and evaporated to dryness under vacuum. The crude product was
purified by a silica gel column and eluted with petroleum ether/dichloromethane (2 : 1, v/v) to
1
get an oily liquid in 65% yield. H NMR (400 MHz, CDCl3, δ): 3.83 (t, J = 6.4 Hz, 4H), 3.69 (s,
4H), 3.48 (t, J = 6.0 Hz, 4H). GC-MS: calcd for C6H12Br2O2 275.97, Found 276.24.
Synthesis of monomer 1: The mixture of 2,7-dibromofluorene (3.0 g, 9 mmol), Bu4NBr (996
6

mg, 3 mmol), 1,8-dibromo-3,6-dioxaoctane (10.0 g, 36 mmol) and KOH (50 g) in distilled water
(50 mL) was stirred at 75 ºC under air for 6 h. Then the mixture was washed three times with
distilled water, and extracted with dichloromethane. The organic phase was collected and
condensed under reduced pressure. The crude product was purified by a silica gel column and
eluted with petroleum ether/dichloromethane (1 : 2, v/v) to obtain a white solid. Yields: 4.3 g
(72%). 1H NMR (400 MHz, CDCl3, δ) 7.54 (d, J = 1.6 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.47 (dd,
J = 8.0 Hz, 2H), 3.68 (t, J = 6.4 Hz, 4H), 3.38 - 3.42 (m, 8H), 3.18 - 3.22 (m, 4H), 2.79 (t, J = 6.8
Hz, 4H), 2.34 (t, J = 7.6 Hz, 4H). 13C NMR (100 MHz, CDCl3, δ): 150.95, 138.48, 130.69, 126.76,
121.65, 121.25, 71.15, 70.36, 70.01, 66.90, 51.97, 39.50, 30.24. MS (MALDI-TOF) m/z: calcd
for C25H30Br4O4 714.12, Found: 735.858 [M+Na]+.
Synthesis of monomer 2: The compound 1 (2.86 g, 4 mmol), Pd(dppf)2Cl2 (0.16 g, 0.2
mmol), potassium acetate (2.70 g, 28 mmol), bis(pinacolato)diboron (2.54 g, 10 mmol) were
added to a flask. The flask was vacuumized for 5 minute and subsequently filled with nitrogen
for 3 minute, and the process was repeated for three times. 50 mL anhydrous dioxane was
injected into the system with a syringe. The solution was heated to 85 ºC and maintained for 12
h under nitrogen protection. After cooled down to room temperature, the mixture was diluted
with dichloromethane, extracted with water several times. The solvent was removed by rotation
evaporation.
The
crude
products
were purified by column chromatography
(hexane/dichloromethane = 1/1) to obtain a white solid 2 (2.50 g, yield: 77%).
1H NMR (400 MHz, CDCl3, δ) 7.84 (s, 2H), 7.80 (d, J = 7.6 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H),
3.66 (t, J = 6.4 Hz, 4H), 3.35 - 3.41 (m, 8H), 3.15 - 3.19 (m, 4H), 2.65 - 2.72 (m, 4H), 2.44 (t, J =
8.4 Hz, 4H), 1.39 (s, 24H). 13C NMR (100 MHz, CDCl3, δ): 148.56, 143.14, 134.09, 129.25,
128.44, 119.56, 83.87, 71.11, 70.34, 69.92, 67.04, 51.10, 39.50, 30.15, 24.93. MS (MALDI-TOF)
m/z: calcd for C37H54B2Br2O8 808.25, Found: 830.132 [M+Na]+.
5,15-Bis(4-bromophenyl)-10,20-bis(pentafluorophenyl)-porphyrin: The mixture of
2,3,4,5,6-pentafluorobenzaldehyde (490 mg, 2.5 mmol), 4-bromo-benzaldehyde (463 mg, 2.5
mmol) and pyrrole (0.35 mL, 5.0 mmol) in dried dichloromethane (500 mL) was stirred for 10
min under nitrogen at room temperature, and then BF3·O(Et)2 (0.15 mL) was added. After
stirring for 1 h, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 860 mg, 3.76 mmol) was
7

added and stirred for further 3 h. The mixture was added triethylamine (0.35 mL) and purified
through a silica column with petroleum ether/CH2Cl2 (1:1) as eluent. The crude products were
purified by a second silica column with petroleum ether/CH2Cl2 (5:1) to obtain a purple powder
(115 mg, yield: 12%). 1H NMR (400 MHz, CDCl3, δ): 8.93 (d, J = 4.8 Hz, 4H), 8.83 (d, J = 4.8 Hz,
4H), 8.06 - 8.11 (m, 4H), 7.90 - 7.98 (m, 4H), - 2.89 (s, 2H). MS (MALDI-TOF) m/z: calcd for
C44H18N4F10Br2 952.43, Found: 951.112.
Synthesis of monomer 3: The procedure was modified by previous literature [55].
Mn(OAc)2·4H2O
(245
mg,
1.0
mmol)
and
5,15-bis(4-bromophenyl)-10,20-
bis(pentafluorophenyl)-porphyrin (95 mg, 0.1 mmol) in DMF (8 mL) was heated to reflux for 6 h.
After cooling down to room temperature, the mixture was extracted with H2O and
dichloromethane. Then the organic phase was removed under vacuum. The residue was
purified by column chromatography using ethyl acetate as eluent to afford a gray green powder
(85 mg, yield: 85%). MS (MALDI-TOF) m/z: calcd for C46H19N4O2F10Br2Mn 1064.40, Found:
1005.994 [M−AcO]−. UV−vis (CH2Cl2): λabs = 370, 398, 446, 475, 576, 610 nm, λmax = 475 nm; IR
(KBr): ν, cm-1 473 (ν Mn−N), 705, 745, 770 (γ C−H, Ph), 805 (γ C−H, Pyr), 930, 957, 990,1007
(ν C−C, δ C−Н, ν C−N, Pyr), 1074 (ν C−Br), 1071, 1162 (ν C−H, Ph), 1336 (ν C−N), 1427 (ν
C=N), 1493, 1587 (ν C=C, Ph), 1520 (vibrations of the pyrrol ring), 1650 (O−C=O) 2925, 3080
( C−H, Ph).
P1: The mixture of monomer 1 (57 mg, 0.08 mmol), monomer 2 (81 mg, 0.1 mmol) and
monomer 3 (20 mg, 0.02 mmol) was dissolved in toluene (2.5 mL), then Bu4NBr (a granule) and
K2CO3 (2 M, 1.5 mL) were added. The system was degassed and refilled with N2 (repeated
several times) before and after addition of Pd(PPh3)4 (0.002 mmol, 2.3 mg). The mixture was
stirred at 85 ºC under N2 for 48 h, and then extracted three times with chloroform and water.
The organic phase was collected and the solvent was removed by rotation evaporation. The
residue was dissolved in CH2Cl2 (0.5 mL) and then precipitated in CH3OH / H2O (20:1). The
precipitate re-dissolved in THF after being collected by filtration, then repeated one time
previous operation. These floccules were filtered and thoroughly washed by Soxhlet extractions
1
for 48 with acetone to afford target polymer (110 mg, 73%) as a gray solid. H NMR (400 MHz,
CDCl3, δ): 7.55 - 7.94 (m, 6H, Ar H of fluorene), 3.59 - 3.74 (m, 4H −CH2Br of fluorene), 3.22 -
8

3.56 (m, 12H −CH2 of fluorene), 2.80 - 3.08 (m, 4H −CH2 of fluorene), 2.38 - 2.72 (m, 4H −CH2
of fluorene). GPC (THF, polystyrene standard): GPC: Mw 70063 g/mol, Mn 36522 g/mol, PDI
1.91.
P2: The neutral polymer P1 (25 mg) was dissolved in THF (2.0 mL). Then 2 mL
trimethylamine (2 M in THF solutions) was added. The solution was stirred under hermetic
environment at 25 ºC about 24 h until the large amount of solid precipitated. After removing the
supernatant, the precipitates were re-dissolved by supplementary methanol (2 mL) and extral
trimethylamine (2 mL) to make further quaternary ammonium. The solution was stirred overnight.
After evaporating most the solvent, acetone was added to give P2 (26 mg) as a gray green solid.
1H NMR (400 MHz, CD3OD, δ): 7.84 - 8.06 (m, 6H, Ar H of fluorene), 3.74 - 3.82 (m, 4H), 3.48 -
3.55 (br, 4H) 3.39 - 3.45 (br, 4H), 3.10 - 3.18 (m, 17H), 2.91 - 3.03 (br, 4H), 2.50 - 2.72 (br, 4H),
1.18 - 1.36 (m, 4H); Quarternization degree: 96.5%.
Preparation of FM-CPNs [56]: P2 (4.0 mg) was dissolved in methanol (4.0 mL). The solution
was injected rapidly into 8 mL phosphate buffered saline in a vigorous bath sonicator for 10 min.
Then, the nanoparticle suspension was evaporated under vacuum at 38 °C to remove methanol.
The resulting FM-CPNs were gained via filtration with a 0.22 micron filter.
3. Results and Discussion
3.1 Design, synthesis, and characterization
We chose conjugated polyelectrolytes of polyfluorene and Mn(III) complex as π-conjugated
polymer backbone and charged side chains with triglycol moiety for improving solubility. The
chemical structure of the conjugated polymer and their synthetic routes are shown in Fig. 1. The
polymer P1 containing Mn(III) complex was synthesized via Suzuki polycondensation reaction,
1
1
and then its structure was confirmed by H NMR, GPC. H NMR spectrum only shows the
chemical shift of the polyfluorene segments, because paramagnetism of Mn(III) porphyrins
leads to NMR signal absent, which proved that, on the other hand, Mn(III) porphyrins segments
have successfully been incorporated into the polymer backbone. The number average
molecular weight of P1 was determined by gel permeation chromatography (GPC), using THF
9

as eluent and polystyrene as standard, and its value was 36522 with a polydispersity index (PDI)
of 1.91. The Mn(III) complex–polymer P2 was obtained by the quarternization of the
intermediate P1 (Fig. 1). The molar feed ratios of Mn(III) porphyrins to the total monomers was
10% and the actual Mn(III) porphyrins content in the obtained sample was calculated to be
about 6.8%, by inductively coupled plasma atomic emission spectrometry (ICP-AES). The
decrease of actual Mn(III) porphyrins content in the obtained polymer, comparing with the initial
feed ratio, may be due to the steric hindrance or reaction activity.
The FM-CPNs was obtained from P2 injecting in PBS under sonicate condition. The
morphology of the as-prepared FM-CPNs was observed by transmission electron microscopy.
Fig. 2A and 2B showed the TEM images of FM-CPNs at low and high magnification,
respectively, which confirm that the FM-CPNs are spheres with uniform particle size of ~100 nm,
via self-assembly owing to their amphiphilic structures with hydrophobic backbones and
hydrophilic side chains. Dynamic light scattering (DLS) measurements showed a dominant
diameter of 110 nm (Fig. 2C), which is slight bigger than the result observed from TEM images,
indicating the as-prepared FM-CPNs were well dispersed in aqueous phase. This may be
caused by the fact that DLS monitored the sizes of FM-CPNs in solution where they were water-
swelling state, while TEM revealed the sizes of the FM-CPNs when they had been dried on the
surface [57]. One week later, the DLS data of the FM-CPNs was monitored again and no
significant change was observed (Fig. S1). All results showed the FM-CPNs good stability in
aqueous phase. Moreover, the zeta-potential of FM-CPNs was recorded to be 59.7 mV. The
FM-CPNs dispersions were clear for weeks without tendencies of aggregation, further showing
the good dispersability and stability of FM-CPNs in PBS, which may be related to well-
hydrophilic side-chains [58].
10

Fig. 2. TEM image (A: scale bar = 500 nm; B: scale bar = 100 nm) and DLS (C) of FM-CPNs in
aqueous solution.
3.2 Photophysical properties
The absorption spectrum of Mn(III) porphyrin monomer 3 has firstly been measured. As
shown in Fig. S2, the dominant absorption band at 475 nm was attributed to the Soret bands of
Mn(III) porphyrin, and two weak peaks at 576 nm and 610 nm are originated from Q(1,0) and
Q(0,0) of the Mn(III) porphyrin [59]. The absorption spectra of P1 in dichloromethane (15μg mL-
1), P2 (15 μg mL-1) in methanol solution; and FM-CPNs in aqueous solution (15 μg mL-1) were
shown in Fig. 3A. P1 and P2 exhibit a dominant absorption band at 377 nm which can be
attributed to the π–π* transition of fluorene units. The weak bands at 475 nm and 550 nm can
be attributed to the Soret band and Q band of Mn(III) porphyrins moieties, which also confirmed
that Mn(III) porphyrin have been incorporated into the polymer backbone. The absorption peak
of P2 on the π–π* transition of fluorene units is as same as that of P1. Meanwhile, the Soret
and Q band absorptions of P2 and FM-CPNs are almost same, indicating that the FM-CPNs
retain the intrinsic absorption property of P2. Their subtle discrepancies of absorption spectra
on P1, P2 and FM-CPNs may be caused by solvent effect. The fluorescence emission spectra
of P1, P2 and FM-CPNs were shown in Fig. 3B. Under excitation at 375 nm, P1 in
dichloromethane solution and P2 in methanol solution emitted intense blue fluorescence light
with emission peak at 418 nm and a shoulder peak at 440 nm (see in Fig. 3B and inset),
originating from the π–π* emission of the polyfluorene segments. The luminescence quantum
efficiencies were measured to be 47.7% and 49.9%, for P1 in dichloromethane and P2 in
methanol, respectively, using an integrating sphere for measuring the efficiency data. FM-
CPNs also revealed blue fluorescence emission at 437 nm and a shoulder peak at 475 nm in
aqueous solution at 375 nm excitation (Fig. 3B and inset), which display a slight red shift
comparing with P1. There is no evident difference in emission wavelength and peak shape. The
luminescence quantum efficiencies of FM-CPNs in aqueous phase were measured to be 31.0%.
11

Fig. 3. Normalized absorption (A) and emission (B) spectra: P1 (15 μg mL-1) in dichloromethane
solution; P2 (15 μg mL-1) in methanol solution; FM-CPNs (15 μg mL-1) in aqueous solution; λex
= 375 nm. The insets of A and B shows the pictures of FM-CPNs and P1, P2 solution under
daylight and 365 nm UV light illumination, respectively.
3.3 T1 relaxivity of FM-CPNs MR imaging
MRI can provide a depth for in vivo imaging with excellent spatial resolution, as well as
exceptional anatomical information. Manganese (Mn3+) can be used as a positive contrast agent
for MRI. Owing to the presence of Mn3+ on the FM-CPNs, these FM-CPNs could also act as a
T1 MRI contrast agent. The T1 relaxivity coefficient of the FM-CPNs was measured by a small
MRI instrument with a 0.55 T magnetic field. Fig. 4A displays the T1-weighted relaxivity of FM-
CPNs in the range 0.0066-0.121 mM of Mn3+ based on ICP measurement, which shows a signal
that was enhanced with increasing Mn3+ concentrations. A good linear relationship was obtained
along with the molar concentration of the Mn3+ being plotted versus 1/T1 (R1). The relaxivity
parameter (r1) was calculated from the slope of the concentration-dependent relaxation 1/T1 (R1).
The r1 value of the FM-CPNs was estimated to be 13.15 mM–1 s–1, which was three times than
that of the Gd-DTPA (r1 = 4.08 mM–1 s–1) [60]. To examine the MR properties of FM-CPNs, an in
vitro T1-weighted MR imaging for FM-CPNs was conducted on a 0.55 T NMR analyzer. Tubes
containing the FM-CPNs solution were arrayed with the order of Mn(III) concentrations. Fig. 4B
displays T1-weight and color-mapped images in the range 0.0066-0.121 mM, and the T1-
12

weighted MR images were brightened with an increase in the Mn(III) concentration. This results
has suggested that the FM-CPNs containing Mn(III)-porphyrin complexes could be indeed used
as an effective T1 MRI contrast agent.
Fig. 4. (A) Longitudinal relaxation rate (1/T1) of FM-CPNs at different Mn3+ concentrations. (B)
T1-weighted phantom MR images for FM-CPNs; these experiments were performed on a 0.55 T
NMR analyzer at 25 °C.
3.4 Cell cytotoxicity
Generally, to apply in biological imaging, the fluorescent materials prefer to be hydrophilic (or
amphiphilic). Based on the above results, the FM-CPNs showed the good dispersability and
stability in PBS. Furthermore, the cytotoxicity and cell permeability features of FM-CPNs are
essential in cell imaging. To study the cytotoxicity of the FM-CPNs, a MTT viability assay with
HeLa cells was used to investigate the effect of the FM-CPNs at various concentrations on cell
proliferation at 24 h. Fig. 5 showed the viability of cells at FM-CPNs concentrations of 0, 5, 10,
20, 40 and 60 μg mL–1 after 24 h incubation, respectively. When the HeLa cells have been
incubated with the FM-CPNs for 24 h under the concentration of 60 μg mL-1, the cell viability still
remained up 90%. The trivial concentration-dependent viability was likely to be caused by
aggregation of CPNs in cells or heavy metal ion effect. The results indicated that the FM-CPNs
can be considered non-cytotoxic to HeLa cells and can be used in luminescence cell imaging as
contrast agent.
13

Fig. 5. In vitro viability of HeLa cells incubated with FM-CPNs at different concentrations for 24
h.
3.5 Cell imaging
To investigate the interaction of living cells with the FM-CPNs, confocal luminescence
imaging has been performed. After HeLa cells cultured at 37 °C for 24 h under air atmosphere,
the FM-CPNs (10 μg mL–1, in PBS) were placed into the medium for excess 3 h. We chose a
410–460 nm band pass emission filter, with 405 nm excitation. Significant intracellular signal
was observed for the living HeLa cells, as shown in Fig. 6A. Overlays of the confocal
luminescence and bright-field images (Fig. 6C) showed that the luminescence was evident in
the intracellular region, indicating that the fluorescence FM-CPNs were internalized into the
HeLa cells rather than merely adhering the membrane surface. The Z-scan confocal experiment
has also been taken, as shown in Fig. 6D. The results of Z-scan imaging also demonstrated that
the FM-CPNs have stained the cytoplasm region of HeLa cells. Fig. 6E showed the signal
intensity profile of special region of HeLa cell (Fig. 6D arrow indicating), suggesting strong
fluorescence emission of FM-CPNs from the intracellular region. Further quantization of
fluorescence signal from the FM-CPNs in the cells was shown in Fig. S3. The fluorescence
intensity profile (across the line shown in the insert cell image) of the HeLa cell revealed an
appreciable signal-to-noise ratio with high intensity at cytoplasm regions (counts >3000) and low
intensity at nucleus region (counts ~1000), further revealing that the FM-CPNs were internalized
14

into the cells. The above results suggested that the FM-CPNs based on conjugated polymer
containing polyfluorene are promising as specific cell-imaging agents.
Fig. 6. Confocal luminescence (A), brightfield (B) and overlay images (C) of HeLa cells
incubated with FM-CPNs for 3 h at 37 °C, (D) Z-scan images of HeLa cells, (E) Fluorescence
intensity profile of special region (arrow in Fig. 6D) of HeLa cell. λex = 405 nm, the emission
channel at wavelengths of 410–460 nm.
4. Conclusions
In conclusion, we have demonstrated a water-soluble multifunctional FM-CPNs with
fluorenscent/MR features, which was prepared from conjugated polyelectrolyte via self-
assembly in aqueous phase by using polyfluorene as fluorophore and Mn(III) complex providing
T1-weighted relaxivity property. The FM-CPNs exhibit a blue emission, low cytotoxicity, and
excellent staining ability of living cells. All of these results showed them potential candidates as
living-cell imaging agents. Moreover, Mn(III) complex in the conjugated polymers makes the
FM-CPNs possess high r1 relaxivity and suitable for T1 enhanced MR imaging. Our results,
incorporating fluorophore and metal complex or other functional unit into one conjugated
polyelectrolyte, provide a potential design strategy to fabricate dual/multi-modality imaging
agents.
15

Acknowledgements
This work was supported by the National Natural Science Foundation of China (51473078,
21401108 and 21671108), National Program for Support of Top-Notch Young Professionals, the
Ministry of Education of China (20133223110006), Natural Science Foundation of Jiangsu
Province of China (BK20140866), Scientific and Technological Innovation Teams of Colleges
and Universities in Jiangsu Province (TJ215006), Natural Science Fund for Colleges and
Universities in Jiangsu Province (14KJB150016), and Priority Academic Program Development
of Jiangsu Higher Education Institutions (YX03001).
‡ T. S. Yang and W. G. Feng, C. Y. Hu contributed equally to this work.
Conflict of interest
The authors declare no competing financial interest.
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Supplementary Information
Manganese Porphyrin-Incorporated Conjugated Polymer Nanoparticles for T1-
Enhanced Magnetic Resonance and Fluorescent Imaging
Tianshe Yang,‡ Wenguo Feng,‡ ChangYong Hu,‡ Zhuang Lv, Huanjie Wei,
Jiayang Jiang, Shujuan Liu, Qiang Zhao*
Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced
Materials (IAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing
210023, China. Email: iamqzhao@njupt.edu.cn (Q. Zhao), Fax: (+86) 25-85866396.
Contents
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Diameter / nm
Fig. S1 Diameter distribution of FM-CPNs dispersed in water for one week, determined by
dynamic light scattering.
22

Fig. S2. Absorption spectra of free-base porphyrin (red) and Mn(III) porphyrin (blue).
Fig. S3. Luminescence intensity profile (across the line shown in the insert cell image) of the
HeLa cell.
23

Fig. S4. 1H NMR spectrum of 1,8-dibromo-3,6-dioxaoctane in CDCl3.
24

Fig. S5. 1H NMR spectrum of monomer 1 in CDCl3.
25

Fig. S6. 13C NMR spectrum of monomer 1 in CDCl3.
26

Fig. S7. MALIDI-TOF spectrum of monomer 1.
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Fig. S8. 1H NMR spectrum of monomer 2 in CDCl3.
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Fig. S9. 13C NMR spectrum of monomer 2 in CDCl3.
29