Multi-colored fibers by self-assembly of DNA, histone proteins, and cationic conjugated polymers

Article abstract of DOI:10.1002/anie.201308795

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color-encoded IPC fibers were also obtained based on the co-assembly of DNA, histone proteins, and blue-, green-, or red- (RGB-) emissive CCPs by tuning the fluorescence resonance energy-transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP-coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi-colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering. Artificial spider silk: Cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers (see picture) with DNA and histone proteins. The color of the fibers could be fine-tuned by changing the molar ratio of the three different CCPs.

Full text of DOI:10.1002/anie.201308795

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DOI: 10.1002/anie.201308795
Self-Assembled Fibers
Multi-Colored Fibers by Self-Assembly of DNA, Histone Proteins, and
Cationic Conjugated Polymers**
Fengyan Wang, Zhang Liu, Bing Wang, Liheng Feng, Libing Liu,* Fengting Lv, Yilin Wang,* and
Shu Wang*
Abstract: The development of biomolecular fiber materials
with imaging ability has become more and more useful for
biological applications. In this work, cationic conjugated
polymers (CCPs) were used to construct inherent fluorescent
microfibers with natural biological macromolecules (DNA
and histone proteins) through the interfacial polyelectrolyte
complexation (IPC) procedure. Isothermal titration micro-
calorimetry results show that the driving forces for fiber
formation are electrostatic and hydrophobic interactions, as
well as the release of counterions and bound water molecules.
Color-encoded IPC fibers were also obtained based on the co-
assembly of DNA, histone proteins, and blue-, green-, or red-
(RGB-) emissive CCPs by tuning the fluorescence resonance
energy-transfer among the CCPs at a single excitation wave-
length. The fibers could encapsulate GFP-coded Escherichia
coli BL21, and the expression of GFP proteins was successfully
regulated by the external environment of the fibers. These
multi-colored fibers show a great potential in biomedical
applications, such as biosensor, delivery, and release of
biological molecules and tissue engineering.
fibers constructed by natural biological macromolecules
have been rarely reported.[8] IPC fibers possess good biocom-
patibility and are attractive materials for biomedical applica-
tions, such as biosensors,[9] delivery of biological mole-
cules,[8,10] encapsulation of cells, and tissue engineering.[7a,11]
Recently, biomaterials with imaging ability have become
more and more useful because one can monitor biological
processes and visualize the medical imaging for diagnosis and
therapy. Until now, the reported way to make fluorescent IPC
fibers has been to incorporate or stain them with organic dyes
or quantum dots (QDs).[7a,8] However, fluorescent dyes
usually suffer from photobleaching,[12] and QDs are cytotoxic
because of the leaking of heavy metals from their nanocrystal
core.[13] These facts provide the motivation for designing new
inherent fluorescent IPC fibers.
Cationic conjugated polymers (CCPs) can form com-
plexes with negatively charged DNA through electrostatic
interactions, which have been widely used for highly sensitive
DNA detection.[14,15] They exhibit high fluorescence bright-
ness, excellent photostability, and lower toxicity,[16] thus
fabrication of fluorescent IPC fibers with CCPs, DNA, and
histone proteins are expected. Herein we synthesized four
CCPs (PFP, PPE, PT, and PBF) that exhibit blue, green,
orange, and red fluorescent emissions, respectively. These
CCPs could respectively form inherent fluorescent micro-
fibers with natural salmon sperm DNA and histone proteins
through the IPC procedure. Color-encoded IPC fibers could
also be prepared through co-assembly of DNA, histone
proteins, and RGB-emissive CCPs. We further encapsulated
GFP-coded Escherichia coli BL21 into the DNA/histone/
CCPs IPC fibers, and the expression of GFP proteins was
successfully regulated in the external environment of the
fibers.
Four CCPs (PFP, PPE, PT, and PBF) with different
fluorescent colors (blue, green, orange, and red) were used.
Their chemical structures are shown in Scheme 1A. PFP[17]
and PBF[18] were prepared according to the previous proce-
dures. The synthesis of PPE is illustrated in Scheme 1B. Boc-
PPE was prepared by copolymerization of 1 and 2 through
a Sonogashira coupling reaction. Then, deprotection of the
tert-butoxycarbonyl group affords PPE. The synthesis of PT is
outlined in Scheme 1C. Treatment of 2-(thiophen-3-yl)ethyl
4-methylbenzenesulfonate (3) with NaH and triethylene
glycol in anhydrous THF affords 3-(2-(2-(2-hydroxyethoxy)e-
thoxy)ethoxy)ethylthiophene (4) in 23% yield. Reaction of 4
and 4-methylbenzene-1-sulfonyl chloride in the presence of
pyridine in CH2Cl2 provides 2-(2-(2-(2-(thiophen-3-yl)ethox-
y)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (5) in 92%
yield. Treatment of 5 with LiBr·H2O in acetone gives 3-(2-(2-
P
olyion complexes are formed by self-assembly of two
oppositely charged polyelectrolytes in aqueous solution. They
have been widely used in constructing membranes, antistatic
coatings, surfactants, and microcapsules.[1–4] The polyion
complexes could form fibers at an interface through an
interfacial polyelectrolyte complexation (IPC) process.[5,6]
Chromatin is one perfect example of natural IPC fibers
formed through self-assembly of oppositely charged DNA
and histone proteins. Although IPC fibers composed of many
different polyelectrolytes have been extensively studied,[7]
[*] F. Wang, B. Wang, Dr. L. Feng, Prof. L. Liu, Prof. F. Lv, Prof. S. Wang
Beijing National Laboratory for Molecular Sciences
Key Laboratory of Organic Solids
Chinese Academy of Sciences, Beijing 100190 (P. R. China)
E-mail: liulibing@iccas.ac.cn
wangshu@iccas.ac.cn
Z. Liu, Prof. Y. Wang
Beijing National Laboratory for Molecular Sciences
Key Laboratory of Colloid, Interface
and Chemical Thermodynamics, Institute of Chemistry
Chinese Academy of Sciences, Beijing 100190 (P. R. China)
E-mail: yilinwang@iccas.ac.cn
[**] The authors are grateful to the National Natural Science Foundation
of China (grant numbers 21033010 and TRR61) and the Major
Research Plan of China (grant numbers 2011CB932302 and
2012CB932600).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201308795.
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emissive polymers. Therefore, if the distance between
these CCPs is close enough, intermolecular multi-stepped
fluorescence resonance energy transfer (FRET) will occur
between them upon excitation of the blue-emissive
polymer with a short excitation wavelength.[19]
Composed mainly of negatively charged DNA and
positively charged histone proteins, chromatin can be
regarded as an example of a natural IPC fiber. To obtain
IPC fibers with good biocompatibility like chromatin,
DNA and histone proteins were chosen as two primary
materials. As shown in Figure 1A, using a pair of tweezers
in a continuous upward movement, we could draw straight
IPC fibers from the interface of positively charged histone
proteins (H) and negatively charged DNA solutions.
Using 5 mL of droplets of polyelectrolyte solution, fibers
could be up to a length of 30 cm. IPC fibers constructed
with DNA/H/PT and DNA/PT were also fabricated using
the same method as that of DNA/H. Light microscope
images in Figure 1B show the morphology of these fibers.
The most obvious feature of these fibers is that beadlike
structures are distributed periodically along the fiber axis.
When freshly drawn these beads took the form of viscous
fluid droplets, but then became protuberances upon
drying. These beads probably have been formed because
of accumulation of water droplets, as discovered in spider
silk and artificial fibers which mimic the natural structure
of spider silk.[20] Diameters of these fibers are in the range
from several to ten micrometers, which probably depends
on both components and drawing rate. Field-emission
scanning electron microscope (FSEM) images in Fig-
ure 1C demonstrate the surface morphology of these
fibers. On the surface of these fibers, parallel ridges and
valleys were observed, as noticed by others.[8] However,
the roughness extent of these surfaces is different from
them. The phenomenon may be attributed to the surface
tension of the composing polyelectrolyte solutions. Further-
more, we used the other three CCPs (PFP, PPE and PBF) to
make DNA/H/CCPs fibers with blue, green, and red emission
colors, respectively. Confocal laser scanning microscopy
(CLSM) images show that the fluorescence of CCPs is
uniformly distributed in the fibers (Figure 1D).
Scheme 1. A) Chemical structures of four CCPs (PFP, PPE, PT, and PBF)
that exhibit blue, green, orange, and red fluorescent emissions, respectively.
B–C) Synthetic routes of PPE and PT.
(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)thiophene (6) in
88% yield. Then, treatment of 6 with trimethylamine (33%
in methanol) in THF offers N,N,N-trimethyl-2-(2-(2-(2-(thio-
phen-3-yl)ethoxy)ethoxy)ethoxy) ethanaminium bromide (7)
in 97% yield. Oxidative copolymerization of 7 in nitrogen in
the presence of FeCl3 gives PT in 12% yield. Because of the
quaternized amine-terminated groups in the side chains, the
four CCPs are all soluble in water. Their photophysical
properties were measured in aqueous solution and are
summarized in Table S1 (see the Supporting Information).
PPE exhibits a maximum absorption at 436 nm with a molar
extinction coefficient of 3.58 ꢀ 104 mÀ1 cmÀ1, corresponding to
the p–p* transition of the back bone. Its emission displays
a maximum peak at 514 nm with a fluorescence quantum
yield (QY) of 8.8%. The maximum absorption wavelength of
PT is 410 nm with a molar extinction coefficient of 0.32 ꢀ
104 mÀ1 cmÀ1, and the emission maximum is 565 nm with a QY
of 3.5%. From the normalized UV/Vis absorption and
fluorescent emission spectra (Figure S1), we could see that
the blue-emissive polymer acts as the donor for green-
emissive and red-emissive polymers, the green-emissive
polymer acts as the acceptor for the blue-emissive one and
the donor for the red-emissive one, while the red-emissive
polymer acts as the acceptor for the green-emissive and red-
To study the formation mechanism of DNA/H/CCP fibers
at a microlevel, we used isothermal titration microcalorimetry
(ITC) technique[21] to study the interactions among different
components of these fibers. We selected PFP and PT as two
examples of the four CCPs in the following research. Firstly,
we studied the interactions between DNA and CCPs. Fig-
ure S2A shows the changing of the observed enthalpy (DHobs
)
when DNA (1.52 mm) is titrated into the solution of PT
(0.34 mm in RU (repeat unit)). When the molar ratio of DNA
to PT is less than 0.8, the DHobs values is negative and displays
a platform, which indicates that the interaction between DNA
and PT is an exothermic process, and electrostatic binding
between DNA and PT is the dominant force accompanied
with the release of counterions and dehydration. When the
DNA/PT molar ratio goes beyond 0.8, phase separation
occurs. The exothermic DHobs increases with further addition
of DNA, and it reaches a maximum when the molar ratio
reaches to 1.2. At this point, the precipitation process is over,
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sponding to the charge ratio of the two systems respectively,
so electrostatic interaction is the major interaction. The
binding constant is 1.36 ꢀ 106 mÀ1 for the DNA/PT system,
3.74 ꢀ 1011mÀ2 for the DNA/PFP system, so it is obvious that
the binding ability of DNAwith PFP is stronger than that with
PT. Moreover, the binding of DNA and PT is an enthalpy–
entropy double driven process, because DH is negative and
TDS is positive in this system. However, because of the
positive DH and TDS, the binding of DNA and PFP is
entropy-driven but opposed by the enthalpy. Furthermore,
interactions between DNA and H or H and CCP mixtures
were studied. As shown in Figures S4 and S5, the DNA/H
system also undergoes a three-step progress, an endothermic
platform, a strong exothermic process, and a dilution process,
similar to the DNA/PT system. But when DNA is added into
the mixed solution of H and PT, the system exhibits an
exothermic process (Figure S4A). The phenomenon suggests
the interaction of DNA and PT is a strong exothermic process.
The variation of the DHobs curves means that the interaction
between DNA and the mixture of H and PT solution may
contain two binding processes. The first process shows
a turning point at the molar ratio of 0.015, corresponding to
the molar ratio of DNA and PT mixed in H solution. Thus,
this binding process is primarily aroused by the interaction
between DNA and PT. The second process beyond the molar
ratio of 0.015 and before the phase separation indicates the
interactions among DNA, H, and PT. Similar with the DNA/
PT system, as the molar ratio increases, the DNA/H/PT
system also goes through a three-step process and phase
separation arises as well. Figure S4B shows that interactions
in DNA/H/PFP system are similar to that in the DNA/H/PT
system. However, the interaction between DNA and PFP is
an endothermic process, so the endothermic enthalpy of DNA
and the mixture of H and PFP is larger than that of DNA and
H alone. In summary, the interactions in the DNA/H/CCP
system may contain electrostatic and hydrophobic interac-
tions, and the release of counterions and bound water
molecules. Using the fitting method mentioned above, we
obtained the binding ratio (N), the binding constant (K), the
binding enthalpy (DH), the Gibbs free-energy (DG), and the
entropy change (DS) of the two systems (Table S3). The fitting
results indicate that the interactions in both DNA/H/PT and
DNA/H/PFP systems contain two binding processes. From
this table we could see that the binding ratio of DNA and H is
0.11 and the binding constant of them is much less than that of
DNA with PT or PFP (Table S2), in other words the binding
strength of them is far smaller than that of DNA with PT or
PFP. Compared with the binding constant of DNA/PT and
DNA/H, the K1 and K2 values decline for both DNA/H/PT
and DNA/H/PFP systems. But in the DNA/H/PFP system, K1
declines and K2 is enhanced. The disparity may relate to the
hydrophobicity difference of PFP and PT. The aforemen-
tioned ITC data analysis indicates the occurrence of binding
and phase separation processes.[6]
Figure 1. A) Photographs of the IPC fiber drawing process. polyanion:
DNA; polycation: H. Light microscope images (B; scale bars 500 mm)
and FSEM images (C; scale bars 10 mm) of the IPC fibers: DNA/H,
DNA/H/PT ([PT]=0.2 mgmLÀ1) and DNA/PT ([PT]=8 mgmLÀ1).
D) Confocal laser scanning microscopy images of the IPC fibers of
DNA/H/CCP (PFP, scale bar 10 mm; PPE, scale bar 30 mm; PT scale
bar 10 mm; PBF, scale bar 10 mm; each 0.2 mgmLÀ1).
[DNA]=[H]=8 mgmLÀ1. All colors are false colors in these images.
which suggests that PT is fully bound with DNA and the point
signifies the saturation of the interaction between them.
According to this consideration, we identified the fitting line
(Figure S3A) of the interaction between DNA and PT by
assuming that the enthalpy change for the binding became
zero at this point. After this molar ratio, further addition of
DNA only generates dilution enthalpy. In brief, with the
gradual addition of DNA into the system, the DHobs curves
experience a three-step process: an exothermic platform,
a strong exothermic process and a dilution process, which
correspond to a gradual combination of DNA with PT, phase
separation of DNA/PT complexes, and the dilution of DNA,
respectively. Figure S2B shows the DHobs change when DNA
(1.52 mm) is titrated into the solution of PFP (0.17 mm in
RU). It also experienced a three-step process similar to the
DNA/PT system. However, different from the DNA/PT
system, DHobs is positive as DNA was added progressively.
This is because the heat released when DNA/PFP is formed
by electrostatic binding is overcome by the heat absorbed
when the counterions and bound water molecules are
released. By fitting the DHobs curves using the method
described in the Supporting Information, the binding model,
binding constant (K), and binding enthalpy (DH) are
obtained as shown in Table S2. Moreover, the Gibbs free-
energy (DG) is calculated from DG =-RTlnK and the entropy
change (DS) from TDS = DHÀDG. The binding ratio is 1:1 for
the DNA/PT system, and 1:2 for DNA/PFP system, corre-
As the CCPs could be incorporated into the IPC fibers
successfully, and intermolecular multi-stepped FRET
between them can occur upon the excitation of blue-emissive
polymer, we prepared color-encoded IPC fibers based on the
co-assembly of DNA, histone proteins, and CCPs. To simplify
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names, a new nomenclature is introduced; DNA/H/CCPs
fibers with different molar ratios of the CCPs are defined as
mBnGkR, where the B, G, and R stand for PFP, PPE, and
PBF, respectively, and m, n, and k stand for the molar ratio of
them. Red (R), green (G), and blue (B) are three primary
colors of light, and mixing them in different proportion could
produce a wide variety of emission colors. As shown in
Figure 2, we can easily prepare various color-barcoded fibers
Figure 3. Fluorescent, bright-field, and merged images of IPC fibers
encapsulating GFP-coded Escherichia coli BL21 by CLSM. These fibers
were incubated in the liquid LB culture medium with 50 mgmLÀ1
Kanamycin without (A) and with (B) 25 mgmLÀ1 of IPTG for 4.5 h at
378C. GFP emission was collected from 500 to 520 nm. The false color
Figure 2. Images of multi-colored fibers fabricated with different con-
of GFP is green. [DNA]=[H]=8 mgmLÀ1, [PT]=0.4 mgmLÀ1
.
centrations of the four CCPs were taken at 365 nm UV-light irradiation.
They were constructed by DNA/H/CCPs. [DNA]=[H]=8 mgmLÀ1
,
[CCPs]=0.4 mgmLÀ1. B, G, O, and R stand for PFP, PPE, PT, and PBF,
respectively. Parameters of these images: (B) exposure time (1/4 s),
aperture (F/5.6), sensitivity (ISO 1600); (R) exposure time (7 s),
aperture (F/5.6), sensitivity (ISO 6400); (the others) exposure time (1/
4 s), aperture (F/5.6), sensitivity (ISO 6400).
The control experiment without IPTG exhibits that no GFP
was expressed in the bacteria (Figure 3A). After incubation
in the liquid LB culture medium with IPTG, the bacteria-
occupied region gives a green color as shown in Figure 3B,
which indicates that GFP-coded Escherichia coli BL21 encap-
sulated in the fibers is alive and meanwhile keeps its
biological activity. Even after incubation in culture medium
for 24 h, the IPC fibers were not broken, which exhibits that
they possess good stability in the biological environment.
These results display that the fiber has good permeability, and
the nutrients required for bacteria growth would diffuse into
the fibers. Also, the IPC progress is moderate enough to
prevent the encapsulated bacteria from exposing to the
external environment of the fibers.
In conclusion, four CCPs have been synthesized with four
emission colors (blue, green, orange, and red). These CCPs
can respectively form inherent fluorescent microfibers with
natural salmon sperm DNA and histone proteins through the
interfacial polyelectrolyte complexation procedure. We also
obtained color-encoded IPC fibers based on the co-assembly
of DNA, histone proteins, and RGB-emissive CCPs. The
DNA/histone/CCPs IPC fibers show multi-colored emissions
by tuning the FRET efficiencies among CCPs under a single
excitation wavelength with large Stokes shifts. The color
could be adjusted through fine-tuning the molar ratios of the
three CCPs (FRET occurs among them) and twelve color-
encoded IPC fibers were obtained. Isothermal titration
microcalorimetry (ITC) shows that the driving forces for
fiber formation are electrostatic and hydrophobic interac-
by just incorporating different molar ratios of the three CCPs
into the fibers at a single excitation wavelength (365 nm). The
emission spectra of the color-barcoded fibers were measured
to evaluate the occurrence of multi-stepped FRET. As shown
in Figure S6, for the color-barcoded fibers, when the donor is
excited, the efficient FRET from donor to acceptor polymers
causes a remarkable quenching of the donor and the
appearance of acceptor emission. Taking B/G/R fibers as
examples, when they are excited at 380 nm (maximum
excitation wavelength of PFP), multi-stepped FRET occurs,
FRET from PFP to PPE, then FRET from PPE to PBF and
direct FRET from PFP to PBF, respectively. These results also
show that the fluorescence intensities of PFP, PPE, and PBF
components in the fibers are different by varying the ratios of
the three CCPs. In other words, the colors can be regulated
through fine-tuning the molar ratios of CCPs.
The IPC procedure is carried out at room temperature
and in aqueous solutions, thus the resulted fibers possess good
biocompatibility. In order to detect the biocompatibility of
the newly constructed multi-colored IPC fibers, we encapsu-
lated GFP-coded Escherichia coli BL21 cells into DNA/H/PT
fibers, and used IPTG (isopropyl-d-1-thiogalactopyranoside)
which can induce the GFP gene to express the GFP protein to
confirm whether the bacteria in the fiber were alive or not.
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Fiber formation: Salmon sperm DNA, histone proteins (H) and PT
were dissolved in water (8 mgmLÀ1 for each polyelectrolyte).
Droplets of two oppositely charged polyelectrolyte solutions (5 mL
each) were placed adjacent to each other on a plastic Petri dish. The
two droplets were then brought in contact with each other by using
a pair of tweezers to form a stable interface. The fiber was fabricated
by continuously drawing up the interface.
Incorporation of cationic conjugated polymers (CCPs) into the
IPC fibers: PFP (1 mm, 0.7 mgmLÀ1, 5% DMSO aqueous solution),
PPE (1 mm, 0.9 mgmLÀ1, THF), PT (20.9 mm, 8 mgmLÀ1, H2O), PBF
(5.7 mm, 8 mgmLÀ1
, 20% DMSO aqueous solution) and H
(100 mgmLÀ1, H2O) were diluted to sterilized water according to
the required concentration. The acquired mixtures were used as the
positively charged substances to make a fiber.
Incapsulation of Escherichia coli in the IPC fibers: GFP-coded
Escherichia coli BL21 (6 mL) that was just recovered was transferred
to 10 mL liquid LB (Luria-Bertani) culture medium and grown at
a shaker (378C, 180 rpm) overnight. The obtained bacterial suspen-
sion (2mL) was centrifuged and the residue was washed with 1 ꢀ PBS
for three times. Then the supernatant was discarded and the
remaining GFP-coded Escherichia coli BL21 were resuspended in
sterilized water (50 mL). After that, 8 mL of the resuspended bacterial
suspension was added to 42 mL DNA solution (8 mgmLÀ1), the
obtained suspension was used as the negatively charged substance to
make a fiber.
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Keywords: conjugation · DNA · fibers · polymers · proteins
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