A highly fluorescent cationic bifunctional conjugate

Article abstract of DOI:10.1039/c2jm16166d

It was of crucial importance to modify perylene-3,4,9,10-tetracarboxylic acid bisimides (PBIs) for the design of new perylene-based bio-dye agents with strong fluorescence. Recently, we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (or PGEA) can produce good transfection efficiency, while exhibiting very low toxicity. Herein, the low-toxic PGEA was proposed to be conjugated with PBIs via facile atom transfer radical polymerization for the well-defined highly fluorescent cationic bifunctional conjugate (PBI-PGEA). The obtained PBI-PGEA exhibited good water-solubility properties, characteristic spectroscopic patterns of PBIs, and excellent photostability. The PBI-PGEA conjugate can be used as an efficient cell bio-dye for rapid (2-5 min) cell labeling at low concentrations (0.06-0.12 mg mL^-1). Such a fast labeling process did not induce obvious cytotoxicity, avoiding possible side-effects to the cells. In addition, the PBI-PGEA still possessed good gene transfection efficiency in different cell lines. With the strong fluorescence in water and good transfection properties, the developed bifunctional PBI-PGEA should possess more potential in bioimaging and gene delivery.

Full text of DOI:10.1039/c2jm16166d

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PAPER
A highly fluorescent cationic bifunctional conjugate
*
X. C. Yang, M. Y. Chai, Y. Zhu, W. T. Yang and F. J. Xu
Received 25th November 2011, Accepted 23rd February 2012
DOI: 10.1039/c2jm16166d
It was of crucial importance to modify perylene-3,4,9,10-tetracarboxylic acid bisimides (PBIs) for the
design of new perylene-based bio-dye agents with strong fluorescence. Recently, we reported that
ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (or PGEA) can produce good transfection
efficiency, while exhibiting very low toxicity. Herein, the low-toxic PGEA was proposed to be conjugated
with PBIs via facile atom transfer radical polymerization for the well-defined highly fluorescent cationic
bifunctional conjugate (PBI–PGEA). The obtained PBI–PGEA exhibited good water-solubility
properties, characteristic spectroscopic patterns of PBIs, and excellent photostability. The PBI–PGEA
conjugate can be used as an efficient cell bio-dye for rapid (2–5 min) cell labeling at low concentrations
(0.06–0.12 mg mLꢀ1). Such a fast labeling process did not induce obvious cytotoxicity, avoiding possible
side-effects to the cells. In addition, the PBI–PGEA still possessed good gene transfection efficiency in
different cell lines. With the strong fluorescence in water and good transfection properties, the developed
bifunctional PBI–PGEA should possess more potential in bioimaging and gene delivery.
by modifying PBIs with low-toxicity cationic PGEA. The novel
1. Introduction
fluorescent PBI–PGEA can be used as an efficient bio-dye for
rapid (2–5 min) cell labeling. Such a fast labeling process did not
induce cytotoxicity, avoiding possible side-effects to the cells. In
addition, the PBI–PGEA also possessed good gene transfection
efficiency in different cell lines. The present bifunctional PBI–
PGEA conjugate should possess more potential applications in
biomedical fields.
Because of their outstanding thermal and photophysical stabili-
ties, perylene-3,4,9,10-tetracarboxylic acid bisimides (PBIs) have
been widely used to develop perylene-based bio-dye agents with
high fluorescence.1–3 PBIs possess extremely low solubility with
strong aggregation in water. Great efforts have been made to
increase the water solubility of PBIs by modifying the perylene
core with bulky substituents4 or by conjugating hydrophilic
species (including cyclodextrin,5 poly(ethylene glycol) (PEG),3,6,7
poly(vinyl alcohol)8 and polyglycerol-dendron2) at the imide
positions.Itshouldbepointedoutthatthebulkysubstituentsofthe
perylene core would shift the fluorescence color of the fluorophore
and did not preserve the green–yellow emission of the parent core-
unsubstituted derivative. For the fluorescent cell labeling applica-
tion, the uncharged hydrophilic species-functionalized PBIs
mainly depend on the cellular uptake process. The hydrophilic
nature, especially for PEG, does not benefit rapid cellular uptake.9
It generally takes several hours to complete the cell labeling
process.3 Further improvement of the cell labeling process of PBIs
is still required to extend their biomedical applications.
2. Experimental section
Materials
Branched polyethylenimine (PEI, Mw ꢁ25 000 Da), glycidyl
methacrylate (GMA, 98%), ethanolamine (EA, 98%),
N,N,N0,N00,N00-pentamethyldiethylenetriamine (PMDETA,
99%), 2-bromoisobutyryl bromide (BIBB, 98%), and copper(I)
bromide (CuBr, 99%) were obtained from Sigma-Aldrich
Chemical Co., St. Louis, MO. GMA was used after removal of
the inhibitors in a ready-to-use disposable inhibitor-removal
column (Sigma-Aldrich). N,N-bis(2-[2-hydroxyethoxy]ethyl)
perylene-3,4,9,10-tetracarboxylic acid bisimide (PBI–OH) was
synthesized according to the literature.12 3-(4,5-Dimethylthiazol-
2yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin, and
streptomycin were purchased from Sigma Chemical Co., St.
Louis, MO. C6 and HEK293 cell lines were purchased from the
American Type Culture Collection (ATCC, Rockville, MD).
We recently reported that ethanolamine (EA)-functionalized
poly(glycidyl methacrylate) (PGMA), or PGEA with plentiful
flanking secondary amine and hydroxyl groups, can produce
good transfection efficiency in different cell lines, while exhibit-
ing very low toxicity.10,11 In this present work, we report the
highly fluorescent cationic bifunctional conjugate (PBI–PGEA)
Synthesis of PBI–PGEA
State Key Laboratory of Chemical Resource Engineering, Key Laboratory
of Carbon Fiber and Functional Polymers, Ministry of Education, College
of Materials Science & Engineering, Beijing University of Chemical
Technology, Beijing, China 100029. E-mail: xufj@mail.buct.edu.cn
As shown in Fig.1, PBI–OH was first modified with PGMA
based on facile atom transfer radical polymerization (ATRP).
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The starting bromoisobutyryl-terminated PBI (PBI–Br) was
synthesized according to the following procedures. About 570
mg (about 1 mmol) of PBI–OH was suspended in 100 mL of
anhydrous methylene chloride with stirring and then kept in an
ice bath. About 2.1 g (about 9 mmol) of BIBB in 10 mL of
methylene chloride was added drop-wise into the ꢂflask through
an equalizing funnel for a period of 10 min at 0 C. After this
addition, the flask was sealed with continuous stirring for 2 h.
The reaction was allowed to proceed at room temperature for
another 24 h to produce the crude PBI–Br initiator. The resultant
(C2H5)3N$HBr and unreacted PBI–OH were removed by
centrifugation. After removal of the solvent by rotary evapora-
tion, the crude polymer was purified by passing through a silica
gel column using CHCl3 as eluent.
The PBI–PGMA polymers (Fig.1) were synthesized using
a molar feed ratio [GMA (4 mL)] : [CuBr (34.6 mg, 0.24
mol)] : [PMDETA (62.4 mL, 0.36 mmol)] of 120 : 1 : 1.5 in 10 mL
of THF containing 0.1 g (0.24 mol) of PBI–Br at 50 ꢂC. The used
THF solvent was purified by distillation. The reaction was per-
formed in a 25 mL flask equipped with a magnetic stirrer and
under the typical conditions for ATRP. GMA, PBI–Br, and
PMDETA were introduced into the flask containing 10 mL of
THF. After PBI–Br had dissolved completely, the reaction
mixture was degassed by bubbling argon for 30 min. Then, CuBr
was added into the mixture under an argon atmosphere. The
reaction mixture was purged with argon for another 10 min. The
flask was then sealed with a rubber stopper under an argon
atmosphere. The polymerization was allowed to proceed under
continuous stirring at 50 ꢂC for 12 (PBI–PGMA1 from 12 h
of ATRP, Mn ¼ 1.06 ꢃ 104 g molꢀ1, PDI ¼ 1.2) to 24 h
Fig. 2 Typical 1H NMR spectra of (a) PBI–Br (in CDCl3), (b) PBI–
PGMA (in CDCl3), and (c) PBI–PGEA (in D2O).
(PBI–PGMA2 from 24 h of ATRP, Mn ¼ 1.48 ꢃ 104 g molꢀ1
,
PDI ¼ 1.3). The reaction was stopped by exposing to air. The
PBI–PGMA was precipitated in excess methanol to remove
the catalyst complex. The crude polymer was purified by re-
precipitation cycles into methanol to remove the reactant resi-
dues, prior to being dried under reduced pressure.
For the preparation of PBI–PGEA, 0.4 g of PBI–PGMA was
dissolved in 7 mL of DMF (HPLC grade). 4 mL of EA and 2 mL
of triethyleneamine was then added. The reaction mixture was
stirred at 50 ꢂC for 72 h to produce PBI–PGEA. The final
reaction mixture was precipitated with excess diethyl ether. The
crude produce was purified by 24 h dialysis against DDW using
a dialysis membrane (MWCO 3500) prior to lyophilization.
Polymer characterization
The polymers were characterized by gel permeation chroma-
tography (GPC), nuclear magnetic resonance (NMR) spectros-
copy, UV-Vis absorption spectroscopy and fluorescence
emission spectroscopy. GPC measurements were performed on
a Waters GPC system equipped with Waters Styragel columns,
a Waters-2487 dual wavelength (l) UV detector, and a Waters-
2414 refractive index detector. THF was used as the eluent at
a low flow rate of 1.0 mL minꢀ1. Monodispersed PEG standards
1
were used to generate the calibration curve. H NMR spectra
were measured on a Bruker ARX 300 MHz spectrometer, using
d-chloroform (CCl3D) as the solvent with 1000 scans at a relax-
ation time of 2 s. UV-Vis absorption spectra in the wavelength
range of 200 to 900 nm were obtained from a Shimadzu UV-
3101PC spectrophotometer. Fluorescence emission spectra in the
wavelength range from 510 to 700 nm were measured on
a HITACHI F4600 fluorescence spectrophotometer.
Fig. 1 Preparation processes of fluorescent ethanolamine-functionalized
poly(glycidyl methacrylate) (PGEA).
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and incubated in 100 mL of DMEM/well for 24 h. The culture
media were replaced with fresh culture media containing serial
dilutions of polymers (0.06–0.24 mg mLꢀ1), and the cells were
incubated for 5–10 min. Then, 10 mL of sterile-filtered MTT
stock solution in PBS (5 mg mLꢀ1) was added to each well,
reaching a final MTT concentration of 0.5 mg mLꢀ1. After 5 h,
the unreacted dye was removed by aspiration. The produced
formazan crystals were dissolved in DMSO (100 mL/well). The
absorbance was measured using a Bio-Rad Model 680 Micro-
plate Reader (UK) at a wavelength of 570 nm. The cell viability
(%) relative to control cells cultured in media without polymers
was calculated from [A]test/[A]control ꢃ 100%, where [A]test and
[A]control are the absorbance values of the wells (with the poly-
mers) and control wells (without the polymers), respectively. For
each sample, the final absorbance was the average of those
measured from six wells in parallel.
Characterization of PBI–PGEA–pDNA complexes
The plasmid (encoding Renilla luciferase) used in this work was
pRL-CMV (Promega Co., Cergy Pontoise, France).10 The puri-
fied pDNA was resuspended in Tris-EDTA (TE) buffer, pH 7.4,
and kept in aliquots of 0.5 mg mLꢀ1 in concentration. All poly-
mer stock solutions were prepared at a nitrogen concentration of
10 mM in distilled water. Polymers to DNA ratios are expressed
as molar ratios of nitrogen (N) in PGEAs to phosphate (P) in
DNA (or as N : P ratios). The average mass weight of 325 per
phosphate group of DNA was assumed. All polymer–pDNA
complexes were formed by mixing equal volumes of polymer and
pDNA solutions to achieve the desired N : P ratio. Each mixture
was vortexed and incubated for 30 min at room temperature.
Each cationic polymer was examined for its ability to bind
pDNA through agarose gel electrophoresis using the similar
procedures to those described earlier.10 The particle sizes and zeta
potentials of the polymer–pDNA complexes were measured in
triplicate using a Zetasizer Nano ZS (Malvern Instruments,
Southborough, MA) and procedures similar to those described
earlier.10
Fig. 3 UV-Vis absorption spectra (a) and fluorescence spectra (b,b0) of
aqueous solutions of PBI–PGEA polymers.
Cell labeling
Prior to labeling, C6 and HEK293 cell lines were seeded into the
24 wells at a density of 5 ꢃ 104 cells/well and incubated for 24 h at
37 ꢂC under a humidified 5% CO2 atmosphere in 1 mL Dulbec-
co’s modified eagle media (DMEM) supplemented with 10% fetal
bovine serum, 100 units mLꢀ1 of penicillin and 100 mg mLꢀ1 of
streptomycin. The culture media were replaced with fresh culture
media containing serial dilutions of PBI–PGEA polymers (0.06–
0.24 mg mLꢀ1), and the cells were dyed for 2–10 min. The cells
were then washed with phosphate buffered saline (PBS; pH ¼
7.4) thoroughly to remove loosely attached polymer in the
medium. The cells were then fixed in 4% formaldehyde in PBS for
1 h, and dehydration in a series of ethanol aqueous solutions (50–
100%) was carried out. The fixed cells were imaged by using
a Leica DMIL Fluorescence Microscope with a FITC filter.
Transfection assay
Transfection assays were performed first using plasmid pRL-
CMV as the reporter gene in HEK293 and C6 cell lines in the
presence of serum. In brief, the cells were seeded in 24-well plates
at a density of 5 ꢃ 104 cells with 500 mL of medium/well and in-
cubated for 24 h. The PBI–PGEA–pDNA complexes (20 mL/well
containing 1.0 mg of pDNA) at various N : P ratios were
prepared by adding the polymer into the DNA solutions, fol-
lowed by vortexing and incubation for 30 min at room temper-
ature. At the time of transfection, the medium in each well was
replaced with 300 mL of fresh normal medium (supplemented
with 10% FBS). The complexes were added into the transfection
medium and incubated with the cells for 4 h under standard
incubator conditions. Then, the medium was replaced with
500 mL of the fresh normal medium. The cells were further
incubated for an additional 20 h under the same conditions,
resulting in a total transfection time of 24 h. The cultured cells
were washed with PBS twice, and lysed in 100 mL of the cell
culture lysis reagent (Promega Co., Cergy Pontoise, France).
Cytotoxicity assay
The cell viability under the cell labeling conditions was evaluated
using the MTT assay in HEK293 and C6 cell lines. The cells were
seeded in a 96-well microtiter plate at a density of 104 cells/well
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Fig. 4 Fluorescent microscopy images of C6 cells after culturing in the medium containing different concentrations of PBI–PGEA polymers for 2
(a,b,c,d,e), 5 (a0,b0,c0,d0,e0), and 10 (a00,b00,c00,d00,e00) min.
Luciferase gene expression was quantified using a commercial kit
(Promega Co., Cergy Pontoise, France) and a luminometer
(Berthold Lumat LB 9507, Berthold Technologies GmbH. KG,
Bad Wildbad, Germany) using similar procedures to those
described earlier.10 Gene expression results were expressed as
relative light units (RLUs) per milligram of cell protein lysate
(RLU mgꢀ1 protein).
structure of PBI–Br was characterized by 1H NMR spectroscopy
in CDCl3 (Fig. 2(a)). The signals at 8.19 and 8.41 ppm (a) were
the contribution from the aromatic structure of the PBI initiator.
The chemical shifts at 4.46 and 4.34 ppm were mainly attribut-
able to the (b) CH2–N–C]O and (c) CH2–O–C]O methylene
protons, respectively. The signals at 3.86 and 3.92 ppm were
associated with the (d) CH2–O methylene protons. The chemical
shift at d ¼ 1.90 ppm was associated with the methyl protons
(e, C(Br)–CH3) of the 2-bromoisobutyryl groups. The area ratio
of peak e and peak b indicated that both hydroxyl groups of
PBI–OH were successfully converted into the corresponding
initiation sites.
3. Results and discussion
Synthesis of PBI–PGEA
As shown in Fig. 1, the PBI–PGEA conjugate was prepared
based on ATRP. ATRP is a well-known facile ‘controlled’
radical polymerization method, which has been widely used in
the biological field.13 The ATRP was readily initiated from the
aromatic ring or imide group of perylene.3,4,14 The starting bro-
moisobutyryl-terminated PBI (PBI–Br) initiator was synthesized
via the direct reaction of hydroxyl groups of PBI–OH with
2-bromoisobutyryl bromide (BIBB). Unlike PBI–OH, the PBI–
Br initiator can dissolve in CH2Cl2 and THF. The chemical
Well-defined PBI–PGMA conjugates were subsequently
synthesized from PBI–Br (Fig.1). The PBI–PGMA with different
lengths of PGMA can be synthesized by varying the ATRP time.
In this work, two PBI–PGMA conjugates (PBI–PGMA1 from
12 h of ATRP and PBI–PGMA2 from 24 h of ATRP) were
obtained. The corresponding number average molecular weights
(Mn) (and polydispersity index (PDI)) were 1.06 ꢃ 104 (and 1.2)
for PBI–PGMA1 and 1.48 ꢃ 104 g molꢀ1 (and 1.3) for PBI–
PGMA2. The pendant epoxide groups of PGMA were readily
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Fig. 7 Electrophoretic mobility of pDNA in the complexes of the
cationic polymers ((a) PBI–PGEA1, (b) PBI–PGEA2, and (c) PEI) at
various N : P ratios.
Fig. 5 Fluorescent microscopy images of HEK293 cells after culturing
in the media containing different concentrations of PBI–PGEA polymers
for 2 (a,b,c,d) and 5 (a0,b0,c0,d0) min.
Fig. 8 Particle size (a) and zeta potential (b) of the complexes between
the cationic polymers (PEI (25 kDa), PBI–PGEA1, and PBI–PGEA2)
and pDNA at various N : P ratios.
1
PGMA and PBI–PGEA were characterized by H NMR spec-
troscopy as shown in Fig. 2(b) and Fig. 2(c), respectively. For
PBI–PGMA, the signals at d ¼ 3.8 and 4.3 ppm correspond to
the methylene protons adjacent to the oxygen moieties of the
ester linkages (c0, CH2–O–C]O). The peaks at d ¼ 3.3 ppm (d0)
and d ¼ 2.65 and 2.87 ppm (d00) can be assigned to the protons of
the epoxide ring. The ratio of peak areas of c0, d0 and d00 was
about 2 : 1 : 2, indicating that the epoxy groups in the PGMA
remained intact throughout ATRP. After the ring-opening
reactions of PGMA with EA, the peaks (d0,d00) associated with
the epoxide ring had disappeared completely (Fig. 2(c)). The
peaks (c0, CH2–O–C]O) shifted to one position (c00) at d ¼ 3.95
ppm. The new peaks at d ¼ 3.7 and d ¼ 2.7 ppm were
mainly attributable to the CH–OH methylidyne and CH2–OH
Fig. 6 Cell viability assay in (a) C6 and (b) HEK293 cells after culturing
in the media containing different concentrations (0.24–0.12 mg mLꢀ1) of
PBI–PGEA polymers for 5 and 10 min. Cell viability was determined by
the MTT assay and expressed as a percentage of the control cell culture.
reacted with the amine moieties of ethanolamine (EA).10,11 The
PBI–PGEA conjugates were derived from the corresponding
PBI–PGMA counterparts. The representative structures of PBI–
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present cationic PBI–PGEA conjugates probably can be used as
efficient cell labels with a short labeling time. To investigate the
possibility of the PBI–PGEA conjugates as rapid cell labeling
agents, the fluorescent microscopy images of C6 cells were first
obtained after culturing the cells in media containing different
concentrations (0.24–0.06 mg mLꢀ1) of PBI–PGEA for 2–10 min
(Fig. 4). The significant green–yellow fluorescence derived from
the parent PBI parts was observed from the images of the labeled
cells. The whole cells were labeled and their morphologies could
be clearly seen. At the same polymer concentration, the fluo-
rescence from the cells labeled for 5 and 10 min seems slightly
more intense than that from 2 min. No obvious changes were
observed among the fluorescence intensities of the cells labeled
for 5 and 10 min. About the effects of dye concentrations, at the
same labeling time the fluorescence from the cells labeled from
0.12 and 0.24 mg mLꢀ1 was slightly more intense than that from
0.06 mg mLꢀ1. There was no obvious difference among the
fluorescence intensities of the cells labeled from 0.12 and 0.24 mg
mLꢀ1. The above results indicated that the dye concentration and
labeling time for the PBI–PGEA conjugates were 0.06–0.12 mg
mLꢀ1 and 2–5 min, respectively.
Fig. 9 In vitro gene transfection efficiency of the cationic polymers (PBI–
PGEA1, PBI–PGEA2, and PGEA)–pDNA complexes at various N : P
ratios in comparison with that of PEI (25 kDa) at the optimal N : P ratio
of 10 in (a) C6 and (b) HEK293 cells, where the PGEA was derived from
the PGMA homopolymer (Mn ¼ 1.28 ꢃ 104 g molꢀ1).
To further investigate the applicability of the PBI–PGEA
conjugates as rapid cell labeling agents, another type of cell,
HEK293, was used also in this work. Fig. 5 shows fluorescent
microscopy images of HEK293 cells after culturing the cells in
the media containing different concentrations (0.12–0.06 mg
mLꢀ1) for 2 and 5 min. The bright green–yellow fluorescence was
also observed from the images of the labeled HEK293 cells. The
morphologies of the HEK293 cells, different from those of the
C6 cells (Fig. 4), were clearly exhibited out (Fig. 5). The above
rapid labeling was probably attributed to the fast interaction
between the PBI–PGEA conjugates and cells. It is well known
that cell membranes are generally negatively charged. It was
quite easy for the cationic PBI–PGEA conjugates to complex the
negative-charged cells and rapidly cover the cell membranes,
making the labeled cells exhibit bright fluorescence. For the
cellular uptake-dependent labeling process of the uncharged
hydrophilic species-functionalized PBIs, such PBI-based dyes
had to be internalized and accumulated within the cytoplasm.3
The involved hydrophilic species, especially PEG, does not
benefit rapid cellular uptake,9 which would take quite a long time
to complete the cell labeling process.
methylene protons (g) and methylene protons (f, NH–CH2),
respectively. The area ratio (about 4 : 5) of peak f and peaks g,c00
indicated that almost all oxirane rings of PGMAs were opened
by EA under the present reaction conditions.
Spectroscopic properties of PBI–PGEA
Fig. 3 shows the UV-Vis absorption spectra (a) and fluorescence
spectra (b,b0) of different PBI–PGEA polymers. All the spectra
possessed well-resolved vibronic patterns which are characteris-
tics of monomeric PBIs.15,16 At the same mass concentration, the
spectroscopic intensities of PBI–PGEA2 were slightly lower than
those of PBI–PGEA1, which was consistent with the fact that
PBI–PGEA2 possessed a relatively low PBI content. For the UV-
Vis absorption spectra, the PBI–PGEA conjugates possessed the
typical absorption peaks of PBIs at 470 and 520 nm. For the UV-
Vis fluorescence spectra, the PBI–PGEA conjugates demon-
strated the characteristic fluorescence emission peaks of PBIs at
540 and 580 nm. As shown in Fig. 3(b,b0), the PBI–PGEA
conjugates exhibited the concentration-dependent fluorescent
behavior. As expected, the fluorescence became stronger, as the
polymer concentration increased. In addition, the PBI–PGEA
polymers still possessed excellent photostability. After exposure
to natural light for one month, the given PBI–PGEA solution did
not produce obvious changes in the vibronic patterns and
intensities of the UV-Vis absorption and fluorescence spectra.
Cytotoxicity is one of the most important factors to be
considered in selecting cell-labeling dyes. Our recently studies
indicated that PGEA exhibited very low toxicity in different cell
lines.10,11 Fig. 6 shows the cell viabilities of C6 and HEK293 cells
under culture conditions the same as those used for cell labeling.
Under the mass concentrations (0.24–0.06 mg mLꢀ1) of PBI–
PGEA and culture time (5–10 min), no obvious cytotoxicity was
observed. The cell viability assay indicated that at the low
concentration of PBI–PGEA used in this present work, the above
fast labeling process did not induce cytotoxicity, which could
avoid possible side-effect to the cells.
Cell labeling
For the fluorescent cell labeling application, the water soluble
uncharged PBI-based species mainly depend on the cellular
uptake process, which generally needs several hours to complete
the cell labeling process.3 The mass content of the hydrophobic
PBI molecule in PBI–PGEA was very low (<4%), and the PBI–
PGEA readily dissolved in water due to the high water solubility
of the PGEA species. With their high fluorescence in water, the
Gene transfection
For cellular transfection, DNA has to be condensed by cationic
polymers into polymer–plasmid nanoparticles suitable for
cellular uptake. The ability of PBI–PGEAs to condense plasmid
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DNA (pDNA) into particulate structures was confirmed by
agarose gel electrophoresis, particle size and zeta potential
measurements. Fig. 7 shows the gel retardation results of cationic
PBI–PGEAs–pDNA complexes with increasing N : P ratios in
comparison with that of PEI (25 kDa). PBI–PGEA could
compact pDNA completely at a N : P ratio of above 2. No
obvious difference in their condensation capability was observed.
As shown in Fig. 8(a), all the cationic PBI–PGEA can efficiently
compact pDNA into small nanoparticles. PBI–PGEA can
condense pDNA into nanoparticles of around 200 nm in diam-
eter above the a N : P ratio of 10, which was similar to those of
PGEA.10 Zeta potential is an indicator of surface charges on
the polymer–pDNA nanoparticles. The PBI–PGEA–pDNA
complexes possessed positively charged surfaces above a N : P
ratio of 2 (Fig. 8(b)). A positively charged surface allows elec-
trostatic interaction with anionic cell surfaces and facilitates
cellular uptake. In addition, it was found that the PBI–PGEA
conjugates possessed similar cytotoxicity to that of PGEA (data
not shown here).10
photostability. The present cationic PBI–PGEA conjugates can
be used as efficient cell bio-dyes at low concentrations with
a short labeling time (2–5 min). Such a fast labeling process did
not induce cytotoxicity, avoiding possible side-effects to the cells.
In addition, the PBI–PGEA also possessed a good gene trans-
fection efficiency comparable to that mediated by the ‘gold-
standard’ PEI (25 kDa) in different cell lines. The present
bifunctional PBI–PGEA conjugate should possess more poten-
tial applications in bioimaging and gene delivery.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (grant numbers 21074007 and 51173014),
the Research Fund for the Doctoral Program of Higher
Education of China (project no. 20090010120007), the Program
for New Century Excellent Talents in University (NCET-10-
0203), SRF for ROCS, SEM, and the National High Technology
Development Program of China (863 Program 2011AA030102).
The in vitro gene transfection efficiency of the cationic PBI–
PGEA–pDNA nanoparticles was assessed using luciferase as
a gene reporter. Fig. 9 shows the gene transfection efficiency of
PBI–PGEA at various N : P ratios in comparison to that of PEI
(25 kDa) at its optimal N : P ratio of 10 in C6 and HEK293
cells. The optimal N : P ratios for PBI–PGEA were 15–20, in
comparison with the optimal N : P ratio (10)10 of PEI. The lower
optimal N : P ratio for PEI was consistent with its higher DNA
condensation capability (Fig. (8)). It was reported that the
incorporation of hydrophobic segments into gene carriers could
enhance gene transfection efficiency.17,18 In comparison with the
PGEA derived from the PGMA homopolymer (Mn ¼ 1.28 ꢃ 104
g molꢀ110) (Fig. 9), the PBI–PGEA1 (derived from the PBI–
PGMA with Mn of 1.06 ꢃ 104 g molꢀ1) and PBI–PGEA2
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(derived from the PBI–PGMA with Mn of 1.48 ꢃ 104 g molꢀ1
)
exhibited slightly lower transfection efficiencies in both cell lines
at most N : P ratios. The above results indicated that in this
present work the hydrophobic PBI species did not enhance the
gene transfection efficiency, probably because the incorporated
PBI could not facilitate the interaction between the cationic
carrier and pDNA. The hydrophobic PBI species did not
significantly deteriorate transfection efficiency either. As shown
in Fig. 9, the transfection efficiency mediated by the PBI–PGEA
conjugates at their optimal N : P ratios was comparable to that
mediated by the ‘gold-standard’ PEI (25 kDa).
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4. Conclusions
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The well-defined highly fluorescent cationic bifunctional conju-
gates (PBI–PGEA) were successfully prepared by modifying
PBIs with low-toxicity cationic PGEA. The resultant PBI–PGEA
conjugates exhibited good water-solubility properties, charac-
teristic spectroscopic pattern of PBIs, and excellent
18 H. Tian, W. Xiong, J. Wei, Y. Wang, X. Chen, X. Jing and Q. Zhu,
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7812 | J. Mater. Chem., 2012, 22, 7806–7812
This journal is ª The Royal Society of Chemistry 2012