Interactions of new synthesized fluorescent cationic amphiphiles bearing pyrene hydrophobe with plasmid DNA: Binding affinities, aggregation and intracellular uptake

Article abstract of DOI:10.1002/mabi.201000047

In this work, we prepared a novel series of cationic amphiphiles denoted as the Py-cations (Py-Gly, Py-Ala, Py-Cap, Py-G₁-Lys and Py-G ₂-Lys) bearing fluorescent pyrene and various hydrocarbon linkers between the pyrene hydrophobe and cationic block. Employing these new cationic amphiphiles with pyrene as the fluorescent probe, the interactions between these Py-cations and plasmid DNA (pDNA) in distilled water and 0.1M PBS buffer solution have been explored by means of UV-vis and fluorescent spectrometers, and ethidium bromide dye displacement and agarose-gel retardant assays were also implemented to evaluate their pDNA binding affinities in aqueous solution. Furthermore, the average sizes and morphologies of self-assembled Py-cation/ pDNA lipoplex aggregates were examined by dynamic laser light scattering (DLS) and atomic force microscopy (AFM). It was found that these fluorescent cationic amphiphiles showed blue fluorescence emission of pyrene probe at λ=340 nm in distilled water while their interactions with pDNA led to new strong green emission at λ=490 nm, and this may be due to the stacking of pyrene and new formation of excimers via the rigid pDNA templated self-assembly. It was also revealed that the binding between new Py-cations and pDNA in aqueous solution was strongly influenced by the Py-cation hydrophobicity, charges of the cation and the presence of electrolytes.With respect to the Py-cation/pDNA aggregate morphologies, very interesting 1-D hybrid nanofibers were predominantly observed by AFM for the Py-Cap/pDNA aggregates. In addition, utilizing a COS-7 cell-line, in-vitro cellular uptakes of new cationic amphiphiles with pyrene probe were studied and visualized by fluorescent microscopy. As a result, this may provide a new approach to investigate the interactions between synthetic cationic lipids and nucleic acids, and pave an alternative clue to design new organic gene delivery carriers.

Full text of DOI:10.1002/mabi.201000047

Full Paper
Interactions of New Synthesized Fluorescent
Cationic Amphiphiles Bearing Pyrene
Hydrophobe with Plasmid DNA: Binding
Affinities, Aggregation and Intracellular
Uptakea
Ruilong Sheng, Ting Luo, Yingdan Zhu, Hui Li, Amin Cao*
In this work, we prepared a novel series of cationic amphiphiles denoted as the Py-cations (Py-Gly, Py-Ala, Py-
Cap, Py-G1-Lys and Py-G2-Lys) bearing fluorescent pyrene and various hydrocarbon linkers between the pyrene
hydrophobe and cationic block. Employing these new cationic amphiphiles with pyrene as the fluorescent
probe, the interactions between these Py-cations and plasmid DNA (pDNA) in distilled water and 0.1 M PBS
buffer solution have been explored by means of UV-vis and fluorescent spectrometers, and ethidium bromide
dye displacement and agarose-gel retardant assays were also implemented to evaluate their pDNA binding
affinities in aqueous solution. Furthermore, the average sizes and morphologies of self-assembled Py-cation/
pDNA lipoplex aggregates were examined by dynamic laser light scattering (DLS) and atomic force microscopy
(AFM). It was found that these fluorescent cationic amphiphiles showed blue fluorescence emission of pyrene
probe at l ¼ 340 nm in distilled water while their interactions with pDNA led to new strong green emission at
l ¼ 490 nm, and this may be due to the stacking of pyrene and new formation of excimers via the rigid pDNA
templated self-assembly. It was also revealed that the binding between new Py-cations and pDNA in aqueous
solution was strongly influenced by the Py-cation hydrophobicity, charges of the cation and the presence of
electrolytes. With respect to the Py-cation/pDNA aggregate
morphologies, very interesting 1-D hybrid nanofibers were
predominantly observed by AFM for the Py-Cap/pDNA
aggregates. In addition, utilizing a COS-7 cell-line, in-vitro
cellular uptakes of new cationic amphiphiles with pyrene
probe were studied and visualized by fluorescent micro-
scopy. As a result, this may provide a new approach to
investigate the interactions between synthetic cationic
lipids and nucleic acids, and pave an alternative clue to
design new organic gene delivery carriers.
R. Sheng, T. Luo, Y. Zhu, H. Li, A. Cao
Laboratory of Polymer Materials, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 345 Lingling Road,
Shanghai 200032, China
Introduction
Fax: þ86-21-54925303.; E-mail: acao@mail.sioc.ac.cn
Design and synthesis of amphiphilic cationic molecules
towards biomedical applications have been attracting
increasing attention over the past decade, and numerous
amphiphilic cationic compounds such as organic
cationic lipids,[1–5] cationic amphiphilic polymers,[6–11]
a : Supporting information for this article is available at the bottom
of the article’s abstract page, which can be accessed from the
journal’s homepage at http://www.mbs-journal.de, or from the
author.
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Interactions of New Synthesized Fluorescent Cationic Amphiphiles Bearing Pyrene Hydrophobe . . .
and modified cationic nanoparticles[12] have been devel-
opedandexploited toconstructnon-viral genedrugcarriers
for the delivery of therapeutic DNA, small interfering RNA
(siRNA) and oligomer DNA (ODNA). Since the efficacy of
DNA delivery has already been found to be greatly
influenced by the DNA binding affinity of a cationic
amphiphilic vector and stability of their lipoplex aggre-
gates,[13,14] it would be of great importance to make clear
the interactions between functional cationic amphiphilic
gene delivery vector and DNA.
probes and their corresponding in-vitro/in-vivo transgene
mechanism.[31] Understanding the important interactions
between pDNA and cationic amphiphiles was essential for
new rational design offunctional genecarriers withdesired
low cell toxicity, high pDNA loading efficiency and
transgene efficacy.
In this work, we have synthesized and characterized a
novel series of fluorescent cationic amphiphiles with well-
defined structures, in which the pyrene block played dual
functionsas afluorescentlabel andhydrophobe, which was
attached to a variety of hydrophilic cationic head-groups
through different hydrocarbon linkers. Subsequently, these
new fluorescent cationic amphiphiles were examined to
reveal their structure dependence of pDNA binding affinity,
aggregation and the organized complex particle morphol-
ogies by means of UV-vis and fluorescent spectrometers,
ethidium bromide dye displacement and agarose gel
retardant assays, dynamic laser light scattering (DLS) and
atomic force microscopy (AFM). Finally, to further explore
their potential possibility for biomedical application, the
synthesized cationic amphiphiles were cultivated with
COS-7 cell-line in vitro, and their intracellular uptakes were
imaged by fluorescence microscopy, and were discussed.
So far, with regard to amphiphilic gene delivery carriers,
there have been a few works reported to disclose that the
plasmidDNA(pDNA)bindingaffinitystronglydependedon
molecular structures of the cationic amphiphiles.[15,16] For
example, Chaudhuri et al.[17] found that the head-group
orientation of cationic amphiphilic lipids greatly affected
their pDNA binding affinities. Alternatively, Stupp et al.[18]
reported a peptide amphiphile conjugated with hydro-
phobicalkylgroupcouldinterestinglyshowextremelyhigh
DNA affinity. Recently, Smith et al. disclosed the hydro-
phobicity[19] of their hyper-branched cationic amphiphiles
and an interesting synergistic effect[20] would play
important roles in pDNA binding as well as successive cell
transgene efficacy. In the previous works,[21,22] we also
revealed the binding of pDNA could be enhanced by
tailoring molecular hydrophobicity of a dendritic poly-
(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) sys-
tem. However, in a general view, the factors which regulate
the important interactions between amphiphilic gene
delivery vector and pDNA, are now still kept not so clear
yet. In order to further shed new light on the interactions
between pDNA and a cationic amphiphile, an organic
cationic amphiphile bearing a less complicated structure
and architecture may be more favorable as an ideal model.
For this purpose, efforts to create new cationic amphiphiles
with well-defined structures and new integrated functions
are demanded for exploring their crucial interactions with
pDNA.
Experimental Part
Materials
Pyrene (97.5%) was purchased from Sigma & Aldrich Chemical, and
utilized as-received. Glysine (98.5%), alanine(98.5%), 6-amino
caproic acid (97.0%), (L-)-lysine (99.0%) and Boc2O anhydride
(99.0%) were commercially supplied by GL Biochem Co. Ltd.
(Shanghai, China) Dicyclohexylcarbodiimide (DCC, 99.0%),
4-dimethylaminopyridine (DMAP, 98.0%) and trifluoroacetic acid
(TFA, 97.0%) were purchased from Shanghai Sinopharm Chemical
Reagent Co. Ltd (Shanghai, China), and were utilized as-received.
Plasmid DNA (PCS DNA, 1.0 mg ꢀ mLꢁ1) and Cos-7 cell-line were
generously gifted by Prof. Yuhong Xu of the School of Pharmacy,
Shanghai Jiaotong University. 96-well microplates and 50 mL cell
cultivation flasks were purchased from Corning Co. Ltd. 0.1 M
Phosphate buffer solution (PBS), Dulbecco’s Modified Eagle Media
(DMEM)and 10%FBSweresuppliedbyHangzhouGenomCo. Ltd. In
this study, all the other reagents and organic solvents were of
analytical grade, and were utilized without further purification.
Additionally, chemical synthesis and molecular structural char-
acterization of the new cationic amphiphile Py-cations were
described in detail in the Supporting Information.
On the other hand, fluorescent labeling techniques had
recently been developed as an efficient strategy for probing
inter/intramolecular interactions. Taking the advantages
of its high sensitivity and simplicity into consideration,
many new fluorescent molecule probes have been devel-
oped and examined to show great potential in labeling the
biologically important molecules and their detection.[24–27]
For instance, pyrene has frequently been explored for the
construction of fluorescent probe due to its possible
formation of pyrene excimer via intermolecular p-p
stacking/aggregation,[28–30] which is extremely sensitive
to the microenvironment. Regarding the investigation on
supramolecular interaction between the cationic amphi-
phile and pDNA toward the fundamental studies of gene
therapy, there have been only very few studies concerning
cationic amphiphile delivery carriers with fluorescent
Analytical Methods
NMR measurements: 1H NMR spectra were recorded at room
temperatureon aVarianVXR300FT-NMRinstrument, operatingat
300.0 MHz for 1H nuclei. Measurements of 13C NMR spectra were
implemented on a Bruker Avance 300 NMR spectrometer at room
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R. Sheng, T. Luo, Y. Zhu, H. Li, A. Cao
temperature. Tetramethylsilane (TMS) was applied as the internal
chemical shift reference.
various Py-cation/pDNA charge ratios and a fixed 20 mg of pDNA in
1.0 mL of distilled water.
MS characterization: ESI mass spectra were routinely measured
on a Varian SATURN 2000 spectrometer for new synthesized
cationic amphiphiles.
Atomic force microscopy: Morphologies of the Py-cation/pDNA
lipoplex aggregates were characterized at room temperature on a
NanoScope IVa atomic force microscopy (Veeco Instrument Co. Ltd)
intappingmode.Here,theAFMsampleswerepreparedbyplacinga
drop of the diluted mixture solution comprising new cationic
amphiphile and 6.0 mg pDNA under a Py-cation/pDNA charge ratio
equalto 24 onto a fresh mica surface, and then wereair-dried under
room temperature prior to measurement.
UV-vis and fluorescence spectra measurements: A predeter-
mined amount of the synthesized cationic amphiphile was
preliminarily well-dissolved in distilled water or 0.1 M PBS buffer
solution (pH ¼ 7.4) to prepare stock solution with
a molar
concentration equal to 1.0 ꢂ 10ꢁ2 M, and then this stock solution
was further diluted into 1.0 ꢂ 10ꢁ4 M with corresponding distilled
water or 0.1 M PBS buffer solution (pH ¼ 7.4), and was hereby
denoted as the dilute solution. Spectral characterization of the
In vitro intracellular uptake assay and imaging:COS-7cells were
seededintoa96-wellmicroplatewithadensityof3 ꢂ 103 cells/well
and100 mLDMEMmediumwith10%FBS.Aftera24-hincubationat
37 8C with 5% CO2, the medium was removed, and was replaced
with 100 mL of fresh DMEM medium containing new cationic
amphiphile under 1.0 ꢂ 10ꢁ4 M, and continuously kept incubation
at 37 8C for 10 min. After repeated washing of the harvested cells
with PBS buffer solution, intracellular uptakes of new fluorescent
interaction between pDNA and
a new fluorescent cationic
amphiphile was separately implemented in a 1 cm-length quartz
cuvettewithgraduallydroppingapredeterminedamountofpDNA
stock solution (pDNA Tris-buffer solution under 1.0 mg ꢀ mLꢁ1 and
pH ¼ 7.4) into each prepared dilute solution, and their UV-vis and
fluorescence spectra were thus recorded at ambient temperature
on a Hitachi UV-2800 UV-vis spectrometer and Hitachi F-7000
fluorescence spectrometer, respectively.
cationic amphiphiles were studied by
a Nikon Ti-S invert
fluorescence microscopy, and were further imaged under a bright
light mode and a fluorescent mode.
Ethidium-bromide (EB) dye displacement assay: At first, the EB
dye (5.0 mg) and pDNA (PCS plasmid DNA, 5.0 mg) were separately
mixed in 1 mL of distilled water and 0.1 M PBS buffer solution
(pH ¼ 7.4). Then, the mixture solution was allowed to incubate for
2 min at room temperature, and new prepared cationic amphi-
phile under a calculated N/P charge ratio (N/P ꢃ 3.0–24.0) was
placed into the prepared pDNA/EB mixture solution, and
continuously incubated at ambient temperature for 2 min.
Thereafter, the fluorescence spectra were measured on a Hitachi
F-7000 fluorescence spectrometer under an excitation at
l ¼ 510 nm and an emission at l ¼ 590 nm, respectively. Here,
the fluorescence emission intensities at l ¼ 590 nm of the initial
pDNA/EB mixture solution (5.0 mg/5.0 mg) (I100) and the pDNA-
free EB solution (I0) were taken as the references of relative
intensity 100% (F100) and 0% (F0), respectively. Hence, the
fluorescence intensities at l ¼ 590 nm for the pDNA/EB mixture
solution with various amounts of new cationic amphiphile were
measured, and their relative intensities F was further evaluated in
accordance to F ¼ (I ꢁ I0)/(I100 ꢁ I0).
Results and Discussion
Synthesis of New Fluorescent Cationic Amphiphiles
For the sake of preparing new fluorescent cationic
amphiphiles in a way as shown in Scheme S1 (Supporting
Information), 1-aminopyrene was at first synthesized via
the reduction of 1-nitropyrene in the presence of 10% Pd/C
as the catalyst and hydrazine anhydride as the proton
donor, respectively.[32] Furthermore, with a combination of
DCC and DMAP, Boc-protected intermediates as the
synthetic precursor of new fluorescent cationic amphiphile
were prepared through coupling the amino group of as-
synthesized 1-aminopyrene and carboxylic acid group of
amino functional group Boc-protected amino acid or poly-
(L-lysine) dendrimer.[21] Continuously, the amino deprotec-
tion was conducted with TFA to finally prepare a new series
of fluorescent cationic amphiphiles with the yields of
65ꢄ80% (Supporting Information). Figure 1 depicts the
1H NMR spectra for new Py-cations, and the evidences of
structural elucidation as seen in the Supporting Informa-
tion demonstrated successful preparation of new fluor-
escent cationic amphiphile Py-cations (Py-Gly, Py-Ala, Py-
Cap, Py-G1-Lys and Py-G2-Lys) bearing different cationic
groups and hydrocarbon linkers, and fluorescent pyrene as
seen in Scheme 1.
Agarose-gel retardant assay: As an alternative way to char-
acterize the interaction between a new cationic amphiphile and
pDNA, agarose-gel retardant assay was also implemented. The
cationic amphiphile/pDNA complex solution were preliminarily
prepared under a variety of Py-cation/pDNA charge ratios in the
presence of 150 ꢂ 10ꢁ3 M NaCl and a fixed 2.0 mg pDNA, and were
further incubated at ambient temperature for 15 min. Then, each
preparedsolutionwasloadedonto2%agarosegel(w/v)withEBdye
(0.5 mg ꢀ mLꢁ1 in the gel) in TAE buffer solution (pH ¼ 7.4). Finally,
pDNA molecular migration and retardant were recorded with
pDNA as the control under UV excitation on UVP benchtop 2UV
transilluminator system.
Dynamic laser light scattering: The average aggregate sizes of
new Py-cation/pDNA lipoplexes were characterized at ambient
temperature on a dynamic laser light scattering instrument (Nano
ZS-90, Malvern, UK) at l ¼ 633 nm and a fixed scattering angle of
908. The aggregate solutions were prepared at the room tempera-
ture by simply mixing new fluorescent cationic amphiphile under
UV-Vis and Fluorescence Spectroscopic Study on
Interactions between Fluorescent Cationic
Amphiphiles and pDNA
Employing the new prepared cationic amphiphiles bearing
thepyreneasfluorescentprobeandfunctionalhydrophobe,
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Interactions of New Synthesized Fluorescent Cationic Amphiphiles Bearing Pyrene Hydrophobe . . .
was fixed to 1.0 ꢂ 10ꢁ4 M. It could be seen that the addition
of pDNA into the Py-Ala aqueous solution apparently gave
rise to the decrease of UV absorption at l ¼ 340 nm for the
Py-Ala while the UV absorption at l ¼ 370 nm concurrently
increased in a gradual manner with increasing pDNA
concentration from 0 to 1.25 ꢂ 10ꢁ4 M. Further, an isobestic
point occurring at l ¼ 348 nm in Figure 2 (a) might imply
new formation of lipoplex by the Py-Ala and pDNA.[30,33]
Meanwhile, fluorescence emission intensities at
l ¼ 380 nm for the Py-Ala in Figure 2 (b) tended to decrease
with gradual increase in fluorescence emission at
l ¼ 490 nm.Thiscouldaccordinglysuggestanewformation
of the pyrene excimers.[30,33] Similar phenomena were also
observed in the UV-vis and fluorescence spectra once
mixing pDNA and a synthesized Py-cation like Py-Gly, Py-
Cap, Py-G1-Lys and Py-G2-Lys, regardless of their different
hydrophilic cationic moieties and architectures. On these
evidences, it could be concluded that the self-assembly of
pyrene excimers occurred upon mixing pDNA and new Py-
cation in aqueous solution, and this could further be
accounted for the electrostatic interactions between the
feature negatively charged nucleic acid phosphate anions
along the pDNA long chain and positively charged nitrogen
counterparts in each new Py-cation. As illustrated in
Scheme 1. The lipoplex self-assembly may be reasonably
interpreted by the electrostatic interaction and successive
p-p stacking of the condensed hydrophobic pyrene moieties
at the vicinity of pDNA chains, and the rigid- rod structural
pDNAwithrodlengthofseveraltensofnanometers[31] may
work as an important template for assembling these Py-
cation/pDNA hybrid aggregates.
Figure 1. 1H NMR spectra for new fluorescent cationic amphiphiles
in DMSO-d6 solution.
UV-vis and fluorescence spectra were applied to explore the
interactions between these synthetic organic lipids and
pDNA in dilute aqueous solution and PBS buffer solution,
respectively. Figure 2 shows the UV-vis (a) and fluorescence
(b) spectra as a function of pDNA molar concentration for
the Py-Ala in aqueous solution. The Py-Ala concentration
cationic block
Pyrene
linker
O
HN
O
HN
O
H
N
HN
A
TF
H3N
TFA
H3N
O
-Ala
Py
Py-Gly
NH3
HN
O
NH3
NH3
O
O
HN
HN
NH3
NH3
-
4 TFA
2 TFA
TFA
Py-G2-Lys
Py-G1-Lys
H3N
Py-Cap
H3N
Scheme 1. Schematic diagram of molecular structures of new fluorescent cationic amphiphile Py-cations and their interactions with plasmid
DNA.
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R. Sheng, T. Luo, Y. Zhu, H. Li, A. Cao
a)1.6
b) 4500
Alternatively, thefluorescentemission
of pyrene excimer was visualized under
the UV excitation. Only blue emission
was observed in Figure S1a (see Support-
ing Information) for the Py-cation aqu-
eous solution under 1.0 ꢂ 10ꢁ4 M, respec-
tively. Upon adding pDNA into the Py-
cation aqueous solution, new green
emission stemming from the pyrene
excimer could be observed as shown in
Figure S1b (see Supporting Information).
Among the Py-cations, it intriguingly
seems that the mixed solution with
pDNA and Py-Cap or Py-G1-Lys exhibited
green fluorescence emission much stron-
ger than those solutions with pDNA and
the Py-cations of Py-Gly, Py-Ala and Py-
G2-Lys. In a view of the structures of new
Py-cations, it is noteworthy that the
molecules of Py-Gly, Py-Ala and Py-Cap
which have the same one positively-
charged amino functional group, tended
to exhibit distinct excimer emission
behavior, and the green emission inten-
sities increased with an increase in the
length of hydrocarbon linker for the Py-
cation. This result demonstrated the
lipoplex aggregation of pDNA/Py-cation
and pyrene p-p stacking strongly
depended on the hydrophobicity of the
Py-cation, and an increase in the hydro-
phobicity of new amphiphiles would
gave rise to stronger interaction with
pDNA and formation of more pyrene
excimers.
1.4
4000
3500
3000
2500
2000
1500
1000
500
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.25
1.0
x
10-4
M
1.25 x10-4 M
10-4
10-5
10-5
10-5
-4
x
1.0 x10
-5
7.5
x
7.5 x 10
-5
5.0
2.5
0
x
x
5.0 x 10
-5
2.5 x 10
0
0
300
320
340
360
380
400
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)
Figure 2. UV-vis (a) and fluorescence (b) spectra of the Py-Ala (1.0 ꢂ 10ꢁ4 M) upon
addition of pDNA under various concentration (0, 2.5 ꢂ 10ꢁ5, 5.0 ꢂ 10ꢁ5, 7.5 ꢂ 10ꢁ5
1.0 ꢂ 10ꢁ4, 1.2 ꢂ 10ꢁ4 M) in aqueous solution.
,
a)
b)400
1800
1600
1400
1200
1000
800
600
400
200
0
Py-Gly
Py-Ala
Py-Cap
Py-G1-Lys
Py-G2-Lys
Py-Gly
Py-Ala
Py-Cap
Py-G1-Lys
Py-G2-Lys
300
200
100
0.0
0.5
1.0
1.5
2.0
2.5
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
DNA/Py-cation
DNA/Py-Cation
Figure 3. Fluorescence emissions at 490 nm for the fluorescent cationic amphiphiles
under 1.0 ꢂ 10ꢁ4 M of each fluorescent cationic amphiphile and various ratios of the
pDNA/Py-cation in aqueous solution (a) and 0.1 M PBS buffer solution (b), respectively.
Figure 3 depicts the pDNA/Py-cation
charge ratio dependence of new green
fluorescence emission at l ¼ 490 nm in
solution. It could be seen that in aqueous
solution (Figure 3a), the green emission
intensities tended to gradually increase
upon increasing the DNA/Py-cation
charge ratio, and reached different max-
imum emission intensities, strongly
depending on the their distinct Py-cation
structures. Meanwhile, it was also found
that the addition of excessive pDNA
apparently resulted in a remarkable
decrease of the green emission intensi-
ties. This may imply that the new green
fluorescent emission was initially
enhanced due to the more and well
self-assembled pyrene excimers with
increasing the pDNA/Py-cation charge
a)
b)
110
110
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
Py-Gly
Py-Ala
Py-Cap
Py-G1-Lys
Py-G2-lys
Py-Gly
Py-Ala
Py-Cap
Py-G1-Lys
Py-G2-lys
-10
-10
0
5
10
15
20
0
5
10
15
20
25
N/P charge ratio
N/P charge ratio
Figure 4. Ethidium bromide displacement assay of plasmid DNA binding affinity for the
synthesized fluorescent cationic amphiphiles under different Py-cation/pDNA charge
ratios in aqueous solution (a) and in 0.1 M PBS buffer solution (b), respectively.
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Interactions of New Synthesized Fluorescent Cationic Amphiphiles Bearing Pyrene Hydrophobe . . .
ratio. On the contrary, if mixing the Py-cation with
excessive amount of pDNA, possible competitive electro-
static interactions between pDNA and Py-cation might
happen, and would finally increase water-solubility of as-
assembled insoluble pDNA/Py-cation lipoplex aggregates
and lead to the disassembly of pyrene excimers and
decreased green fluorescence emission. In a view of
Figure 3a, the category of Py-Gly, Py-Ala and Py-Cap with
only one positive charge, was found to strongly interact
withpDNAunder aDNA/Py-cation chargeratioof1/1.25. In
comparison, the Py-G1-Lys and Py-G2-Lys with correspond-
ing 2 and 4 positive charges exhibited strong interactions
with pDNA under pDNA/Py-cation charge ratios of 1/1.8
and 1/1.2, respectively. This could demonstrate that the
multiple cationic binding sites and total molecular hydro-
phobicity of an organic cationic amphiphile would
simultaneously regulate apparent interactions with pDNA
in solution, and a similar phenomenon has already been
revealed by Smith et al.[19,20] for their hyper-branched
molecules with much more surface cationic termini and
higher molecular weights.
electrolytes and ionic strength of the mixture solution,[35]
and the presence of electrolytes would decrease the driving
forces demanded to be strong enough for organizing pyrene
excimers as formed in distilled water.
Ethidium Bromide (EB) Dye Displacement Assay
As for a candidate of cationic molecule toward the
application as non-viral gene delivery carrier, its high
pDNA binding affinity seems to be very important for good
geneloadingefficiency,andEB-dyedisplacementassaywas
hereby utilized to evaluate the pDNA binding affinity for
the new synthesized fluorescent Py-cations in distilled
water solution and 0.1 M PBS buffer solution, respec-
tively.[22] In Figure 4a, the relative fluorescence intensities
F at l ¼ 590 nm seemed to decrease to a value of 40% when
the N/P charge ratio increased to 4 for the Py-Ala and Py-Gly
in distilled water, indicating their relatively lower pDNA
binding affinities. In comparison, the relative fluorescence
intensities F decreased to about 15% when the N/P charge
On the other hand, the presence of an
electrolyte has also been known to affect
electrostatic interactions in solution,[34]
new lights were here shed on the
interactions between the Py-cation and
pDNA in 0.1 M PBS buffer solution under a
fixed
Py-cation
concentration
of
1.0 ꢂ 10ꢁ4 M. Figure 3b presents the
pDNA/Py-cation charge ratio depen-
dence of fluorescence emission at
l ¼ 490 nm of the assembled pyrene
excimers for the Py-cations of Py-Gly,
Py-Ala, Py-Cap, Py-G1-Lys and Py-G2-Lys,
and negligible changes in emission
intensities at l ¼ 490 nm could be
observed, apparently independent of
different Py-cation/pDNA charge ratios
and distinct Py-cation structures. Addi-
tionally, Figures S1c and S1d (see Sup-
porting Information) also show fluores-
cence emission of the quartz-cuvettes
with the pDNA/Py-cation mixture solu-
tion prepared by 0.1 M PBS buffer solution
under UV excitation, and green emission
attributable to the self-assembled inso-
luble pyrene excimers could hardly be
detected. The remarkable difference in
apparent green emission at l ¼ 490 nm of
the Py-cation/pDNA mixture in indivi-
dual aqueous solution and 0.1 M PBS
buffer solution may imply the Py-
cation/pDNA electrostatic interaction
were significantly influenced by the
Figure 5. Agarose-gel retardant assay of pDNA binding affinity for new synthesized
fluorescent cationic amphiphiles under various Py-cation/pDNA charge ratios. (Lane: 1.
pDNA; 2. N/P ¼ 3; 3. N/P ¼ 6; 4. N/P ¼ 9; 5. N/P ¼ 12; 6. N/P ¼ 15; 7. N/P ¼ 18).
Macromol. Biosci. 2010, 10, 974–982
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R. Sheng, T. Luo, Y. Zhu, H. Li, A. Cao
1000
900
800
700
600
500
400
300
200
100
ratios were 6 for the Py-Cap and 2 for the Py-G1-Lys and Py-
G2-Lys, respectively. This demonstrated high pDNA binding
affinities of new synthesized Py-cations, and the higher
hydrophobicity and/or the more cationic binding sites of a
Py-cation would lead to its better pDNA binding affinity in
distilled water solution, and this phenomenon of hydro-
phobic block association enhanced DNA binding is very
similar to that we recently reported for a triblock
amphiphilic copolymer system.[21,22] With respect to the
pDNA binding in 0.1 M PBS buffer solution in the presence of
possible electrolytes, the N/P charge ratio dependence of F
values is shown in Figure 4b. It could be seen that the F
values decreased to be 40 and 15% with the N/P charge ratio
of 6 for the Py-Ala and Py-Gly and 11 for the Py-Cap,
respectively, implying their decreased pDNA binding
affinities. In particular, remarkable decrease in the pDNA
binding affinity was detected for the Py-G1-Lys and Py-G2-
Lys with multiple cationic termini. As a result, the presence
of electrolyte in buffer solution could be concluded to
decrease pDNA binding affinities for new organic Py-
cations.
Py-Gly
Py-Ala
Py-Cap
Py-G1-Lys
Py-G2-Lys
0
5
10
15
20
25
N/P charge ratio
Figure 6. DLS measurements of the aggregate sizes for the Py-
cation/pDNA lipoplexes under varies N/P charge ratio in aqueous
solution.
Morphologies of the Self-assembled Py-Cation/pDNA
Lipoplex Aggregates
Agarose-gel Retardant Assay
In addition, agarose-gel retardant assay was also employed
to examine the interaction between new Py-cations and
pDNA in the presence of 150 ꢂ 10ꢁ3 M NaCl in TAE buffer
solution,[22] and the results are shown in Figure 5. The
Py-cationsofPy-Gly,Py-AlaandPy-Capbearingonecationic
charge but distinct flexible hydrophobic hydrocarbon
linkers between the fluorescent pyrene and cationic
head-groups exhibited remarkably different retardant
effects in pDNA migration along the electrophoresis
lanes, and the pDNA migration could not be entirely
inhibited even at a high N/P charge ratio of 18 for either
Py-Gly or Py-Ala. In contrast, the effective retardant of
pDNA migration could be observed at N/P charge ratio of
12 for the more hydrophobic Py-Cap,
Particle sizes and distribution have been recognized to be
important for cellular uptakes of the DNA/carrier complex
aggregates[36] and their particle distribution in different
organs as well as successive transgene efficiency in vivo for
agenecarrier.[22,37] Regardingthesizesandmorphologiesof
new Py-cation/pDNA aggregates, DLS and AFM were
employedtocharacterizetheirN/Pchargeratiodependence
of organized aggregate sizes and morphologies under a
fixed 20.0 mg of pDNA in distilled water. Figure 6 depicts
averageaggregatesizesasafunctionoftheN/Pchargeratio,
and the organized aggregate sizes of pDNA with Py-Cap, Py-
G1-LysandPy-G2-Lystendedtoincreasewiththeincreasein
theN/Pchargeratio. Incontrast, theaverageaggregatesizes
demonstrating the pDNA binding
affinity of an organic lipid would be
readily enhanced simply by improving
its molecular hydrophobicity.[21] Con-
cerning the Py-G1-Lys and Py-G2-Lys,
the pDNA migration could not be effec-
tively inhibited even at a N/P charge ratio
of 15, and this may be due to the presence
of multiple cationic charges and much
enhanced molecular hydrophilicity as
ever reported.[21,22] Hence, in buffer
solution, the overall molecular hydro-
phobicity and cationic charge number
were important factors, and may regu-
Figure 7. AFM morphologies recorded in the tapping mode for the Py-Gly/pDNA (a) and
Py-Cap/pDNA (b) lipoplex aggregates formed under N/P charge ratio equal to 24,
respectively.
late the DNA migration along the electro-
phoresis lanes.
Macromol. Biosci. 2010, 10, 974–982
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ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.201000047

Interactions of New Synthesized Fluorescent Cationic Amphiphiles Bearing Pyrene Hydrophobe . . .
of the pDNA with Py-Gly and Py-Ala were observed to
initially decrease when the N/P charge ratio increased up to
20, indicating obvious collapse and condensation of the Py-
Gly/pDNA and Py-Ala/pDNA aggregates.[38,39] The differ-
ence inaggregate sizesforthese Py-cation/pDNAlipoplexes
may further be accounted for their distinct morphologies.
demonstrate higher intracellular uptake capability for
new Py-Ala, Py-Cap and Py-G1-Lys. The fluorescent Py-
Cap with simple structure, high cellular uptake capability
and pDNA binding affinity might be interesting for further
study on cell transgenetic behavior and lightening the gene
transfection pathway, and continuous works are now
under investigation, and are to be appeared in a forth-
coming work.
Figure
7 shows morphologies of the air-dried self-
assembled Py-Gly/pDNA and Py-Cap/pDNA lipoplex aggre-
gates prepared under N/P ¼ 24, respectively. It is very
interestingly to note that sphere-like aggregates (Py-Gly/
pDNA) and well-organized nanofibers with very large
aspect ratios (Py-Cap/pDNA) could be clearly observed, and
the significantly distinct morphologies may be one of
possible reasons for their different particle sizes measured
by DLS. It is also noteworthy that to the best of our
knowledge, the lipoplex and polyplex aggregates have
generally been known to organize spherical particles, and
till now very few literatures reported the self-assembly of
lipoplex aggregates with fine nanofiber morphologies, and
this 1-D self-assembling mechanism needs to continuously
be explored in future works.
Conclusion
In summary, we have designed and successfully prepared a
new series of water-soluble organic cationic amphiphilic
Py-cations bearing dual-functional pyrene as fluorescent
probe and hydrophobe. UV-vis and fluorescence spectral
studies of new cationic amphiphile and pDNA in dilute
aqueous solution demonstrated strong Py-Cap/pDNA and
Py-G1-Lys/pDNA interactions withnew occurrence of green
fluorescence emission due to the self-assembly of pyrene
excimers via pꢁp stacking, while negligible interactions
Intracellular Uptake of Fluorescent
Cationic Amphiphiles
To further explore the potentiality for
these new Py-cations as functional
organic gene delivery carriers, COS-7
cell-line was employed for their intracel-
lular uptake assay in vitro. Here, the COS-
7 cell-line was first incubated at 37 8C and
5% CO2 for 10 min under 1.0 ꢂ 10ꢁ4 M for
each synthesized new Py-cation, and
then washed repeatedly with 0.1 M PBS
buffersolutiontocompletelyremovefree
fluorescent cationic amphiphiles possi-
bly absorbed on the harvested cell sur-
face. Figure 8 depicts the fluorescence
images of COS-7 cells incubated with Py-
Ala and Py-Cap under a bright mode (left)
and a fluorescent mode (right), and much
stronger blue fluorescence emission
could be seen for the cells incubated with
thePy-Ala, Py-Cap andPy-G1-Lysthanthe
Py-Gly and Py-G2-Lys (Figure S2 in
Supporting Information). Since the blue
fluorescence emission stemmed from the
pyrene probe attached to fluorescent Py-
cations, a stronger blue pyrene fluores-
cence emission could imply the more
cellular uptakes of corresponding fluor-
escent Py-cation. Therefore, the stronger
Figure 8. Images of Py-Ala(top), Py-G1-Lys(middle) and Py-Cap (bottom) intracellular
uptake by the COS-7 cells recorded under bright mode (left) and fluorescent mode
(right).
blue fluorescence emission of the har-
vested cells could unambiguously
Macromol. Biosci. 2010, 10, 974–982
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981

R. Sheng, T. Luo, Y. Zhu, H. Li, A. Cao
could be observed for the same mixture systems in PBS
buffer solution. It was intriguingly found that the inter-
actions between a new Py-cation and rod structural pDNA
significantly depended on the Py-cation molecular hydro-
phobicity, cationic charges and the presence of electrolyte
in solution. With regard to the pDNA binding affinity, EB-
dye displacement assay conducted in individual aqueous
solution and PBS buffer solution indicated stronger pDNA
binding affinities for new Py-Cap, Py-G1-Lys and Py-G2-Lys
in aqueous solution than those results in PBS buffer
solution, and this agrees well with the tendency as revealed
by agarose-gel retardant assay. Moreover, the sizes and
morphologies of Py-cation/pDNA lipoplex aggregates self-
assembled in aqueous solution demonstrated Py-cation
structure dependence of the Py-cation/pDNA lipoplex
aggregate sizes, and besides well-reported lipoplex sphere
structural morphologies, new 1-D nanofiber morphology
was interestingly observed for the Py-Cap/pDNA aggre-
gates self-assembled under a N/P charge ratio of 24 in
solution. Moreover, employing a COS-7 cell-line, intracel-
lular uptake assay of these fluorescent Py-cations indicated
relatively higher cellular uptake capabilities for the Py-Ala,
Py-Cap and Py-G1-Lys.
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Keywords: amphiphiles; cellular uptakes; DNA binding; gene
deliveries; self-assemblies
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DOI: 10.1002/mabi.201000047