Construction and DNA condensation of cyclodextrin-coated gold nanoparticles with anthryl grafts

Article abstract of DOI:10.1002/asia.201402078

The condensation of DNA in a controlled manner is one of the key steps in gene delivery and gene therapy. For this purpose, a water-soluble supramolecular nanostructure is constructed by coating 14 β-cyclodextrins onto the surface of a gold nanoparticle, followed by the noncovalent association of different amounts of anthryl-modified adamantanes with coated β-cyclodextrins. The strong binding of β-cyclodextrins with anthryl adamantanes (K_S=8.61×10⁴ M^-1) efficiently stabilizes the supramolecular nanostructure. Spectrophotometric fluorescence spectra and microscopic studies demonstrated that, with many anthryl grafts that can intercalate in the outer space of the DNA double helix, this supramolecular nanostructure showed good condensation abilities to calf thymus DNA. Significantly, the condensation efficiency of supramolecular nanostructure towards DNA could be conveniently controlled by adjusting the ratio between gold nanoparticles and anthryl adamantane grafts, leading to the formation of DNA condensates of a size that are suitable for the endocytosis of hepatoma cells, which will make it potentially applicable in many fields of medicinal science and biotechnology. Congenial host: A supramolecular DNA condenser containing gold nanoparticles (AuNPs; see picture), β-cyclodextrins (CDs), and anthryl adamantanes exhibits good DNA condensation efficiency and low toxicity; thus making it potentially applicable in medicinal science and biotechnology.

Full text of DOI:10.1002/asia.201402078

FULL PAPER
DOI: 10.1002/asia.201402078
Construction and DNA Condensation of Cyclodextrin-Coated Gold
Nanoparticles with Anthryl Grafts
[
a]
Di Zhao, Yong Chen, and Yu Liu*
Abstract: The condensation of DNA in
the supramolecular nanostructure.
Spectrophotometric fluorescence spec-
tra and microscopic studies demon-
strated that, with many anthryl grafts
that can intercalate in the outer space
of the DNA double helix, this supra-
molecular nanostructure showed good
condensation abilities to calf thymus
DNA. Significantly, the condensation
efficiency of supramolecular nanostruc-
ture towards DNA could be conven-
iently controlled by adjusting the ratio
between gold nanoparticles and anthryl
adamantane grafts, leading to the for-
mation of DNA condensates of a size
that are suitable for the endocytosis of
hepatoma cells, which will make it po-
tentially applicable in many fields of
medicinal science and biotechnology.
a controlled manner is one of the key
steps in gene delivery and gene thera-
py. For this purpose, a water-soluble
supramolecular nanostructure is con-
structed by coating 14 b-cyclodextrins
onto the surface of a gold nanoparticle,
followed by the noncovalent associa-
tion of different amounts of anthryl-
modified adamantanes with coated b-
cyclodextrins. The strong binding of b-
cyclodextrins with anthryl adamantanes
Keywords: cyclodextrins · cytotox-
icity · DNA · gene technology ·
host–guest systems
4
ꢀ1
(
K =8.61ꢀ10 m ) efficiently stabilizes
S
[
12]
Introduction
linker.
Klibanov and Thomas demonstrated that AuNPs
functionalized with branched polyethylenimine (PEI) chains
were 12 times more efficient than unmodified PEI.
[13]
Gene therapy is the use of genes (DNA and RNA) instead
of conventional drugs to treat diseases. After the first suc-
cessful case of gene therapy occurred in 1990, significant
progress has been made in this area over past two decades.
Considering that naked genes are not effectively endocy-
Al-
though AuNP-based gene-delivery systems have been inves-
tigated extensively, most of these systems involve the cova-
lent linkage of functional molecules to the AuNP surface.
As a result, the preparation processes for these kinds of vec-
tors are usually complex and it becomes difficult to adjust
the DNA condensing capabilities of vectors without chang-
ing the ratio of the reactants when preparing AuNPs.
Supramolecular chemistry, which is the chemistry of non-
covalent interactions, provides an easy way to construct
complicated and controllable molecular assemblies for appli-
cations in biomedical science. A number of host–guest
supramolecular systems have been reported for gene and
drug delivery. Of all macrocyclic host molecules, cyclodex-
trins (CDs), which are cyclic oligosaccharides composed of
six to eight d-glucopyranoside units, have attracted great in-
terest for their benign water solubility, low toxicity, biologi-
cal compatibility, and unique inclusion ability for various or-
ganic or biological molecules that fit their hydrophobic
cavity. Therefore, CDs are extensively applied in biochem-
istry and pharmaceuticals, such as enhancing cell membrane
[
1]
[2]
tosed by cells and are easily degraded by serum nucleases,
it is important for scientists to find efficient and safe gene-
[3]
delivery systems. Nowadays, several kinds of functional
[4]
[5]
[6]
systems, such as viruses, lipoplexes, polyplexes, den-
[
7]
[8]
drimers, and inorganic metal nanoparticles, have been
widely used as vectors for gene therapy. Among them, gold
nanoparticles (AuNPs) have attracted increasing attention
because of their unique chemical and physical properties,
such as high surface-to-volume ratios, being essentially inert,
[14]
[
9]
nontoxicity, ease of preparation, high transfection efficien-
[10]
cy, and high drug transportation ability. Rotello et al. re-
ported that tetraalkylammonium ligand modified AuNPs
could interact with the DNA backbone through charge com-
They also developed light-triggered DNA
release by AuNP, which was modified with a quaternary am-
[
11]
plementarity.
monium salt, through a photocleavable o-nitrobenzyl ester
[15]
absorption, increasing the nuclease resistance of oligonu-
cleotides,
and compacting and carrying DNA into cells.
[
16]
[17]
reducing the immunostimulatory response,
[
a] D. Zhao, Dr. Y. Chen, Prof. Dr. Y. Liu
Department of Chemistry
[8, 18]
In prelimi-
State Key Laboratory of Elemento-Organic Chemistry
Collaborative Innovation Center of Chemical
Science and Engineering (Tianjin)
Nankai University, Tianjin, 300071 (P.R. China)
E-mail: yuliu@nankai.edu.cn
nary research, we found that oligo(ethylenediamine)-b-CD-
modified AuNP can concentrate and carry DNA into cells.
We also found that b-CD based supramolecular architec-
[8]
tures with aryl grafts could act as promising DNA concen-
[8,18]
trators.
These findings led us to the design and construc-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201402078.
tion of supramolecular architectures based on CDs and
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Yu Liu et al.
AuNPs, and one could hypothesize that the combination of
CDs and AuNPs, especially nanometer-scaled CD-AuNP
systems, might lead to a breakthrough in many fields of
chemistry and materials science. Herein, we report a soluble
nanometer-scaled AuNPs system noncovalently grafted by
anthryl groups (Scheme 1). Considering the controllable as-
Figure 1. TGA results for 1 and 2.
Table 1. Elemental analysis of 2.
Element
Analysis method
Content [%]
Au
C
H
N
ICP-AES
25.7
9.73
2.73
0.30
[
[
[
a]
a]
a]
Vario EL Cube
Vario EL Cube
Vario EL Cube
Scheme 1. Construction of the CD-AuNP (2)/anthryl adamantine (3)
host–guest system.
[
a] See the Experimental Section for more details about the Vario EL
Cube analyzer.
sociation between the CD-modified AuNP core (2) and an-
thryl grafts through host–guest binding of the b-CD cavity
with anthryl adamantine (3), the DNA condensation capa-
bilities of the desired nanostructure can be conveniently
tuned by adjusting the 2/3 ratio.
average number of gold atoms per AuNP and the average
number of CD units around one nanoparticle (NCD) could
[19]
be calculated by means of Equations (1)–(4).
ꢀ
ꢁ
3
2
3
D
a
Results and Discussion
Characterization of 2
U ¼
p
ð1Þ
b-CD-coated AuNPs were obtained by the reaction of
M
AuU
Au
WNP ¼ m
ð2Þ
ð3Þ
ð4Þ
1
with gold chloride tetrahydrate and were characterized by
1
13
H and C NMR spectroscopy, elemental analysis, and in-
ductively coupled plasma atomic emission spectroscopy
(
ICP-AES). FTIR spectra showed that the shape of the
M
C
WCD ¼ m
FTIR spectrum of b-CD-coated AuNPs (2) was similar to
that of free 1, accompanied by the clear broadening of sev-
eral characteristic peaks (Figure S1 in the Supporting Infor-
mation).
In addition, thermogravimetric analysis (TGA) showed
that 1 rapidly decomposed at approximately 3308C, accom-
panied by 68.5% weight loss up to 8008C (Figure 1); howev-
er, 2 presented a smooth and slow weight loss and only gave
n
CD
WCD M mAuU
WNP mCDMAu
C
NCD
¼
¼
n
in which U is the number of gold atoms per AuNP; D is the
average diameter of AuNPs; a refers to the edge of a gold
14.5% weight loss up to 8008C. Through a calculation based
unit cell, which has a value of 4.0786 ꢂ; MAu and M are the
C
on the weight losses of 1 and 2 within 8008C, the content of
units of 1 in 2 was measured to be 21.2%, which corre-
sponded to a WCD value of 0.159 mmolg . This result is in
gold and carbon contents, respectively; mAu and mCD are the
atomic weight of gold and the molecular weight of host 1,
respectively; WNP and WCD are contents of AuNPs and host
1, respectively; and n is the number of carbon atoms per
host 1. The results of the calculations are as follows: U=
ꢀ
1
accordance with the result calculated from elemental analy-
ꢀ
1
sis and ICP-AES data (WCD =0.162 mmolg ; Table 1)
Table 1 shows the elemental analysis of 2. By assuming
that the core shape of the nanoparticles is spherical, the
ꢀ
6
ꢀ1
ꢀ4
ꢀ1
1109, WNP =1.18ꢀ10 molg , W =1.62ꢀ10 molg , and
CD
NCD =14.
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Yu Liu et al.
tration experiments into water. The net reaction heat was
given by subtracting the dilution heat from the apparent re-
action heat.
Generally, the first point of the titration curve was disre-
garded because some liquid mixing near the tip of the injec-
tion needle was known to occur at the beginning of each
ITC run. Two independent titration experiments were per-
formed to afford self-consistent parameters and to give aver-
Binding of the b-CD Cavity with 3
To investigate quantitatively the inclusion complexation be-
havior of the b-CD cavity with 3 and their thermodynamic
origins, isothermal titration calorimetry (ITC) experiments
were performed in water. The VP-ITC instrument was cali-
brated chemically by measurement of the complexation re-
action of b-CD with cyclohexanol, and the obtained thermo-
dynamic data were shown to be in good agreement (error<
aged values. The binding stoichiometry (N), complex stabili-
[20]
o
2
%) with the literature data. All microcalorimetric titra-
ty constant (K ), standard molar reaction enthalpy (DH ),
S
tions between b-CD and 3 were performed in aqueous solu-
tion at atmospheric pressure and 298.15 K to give the com-
and standard deviation were obtained by using the “one set
of binding sites” model (Origin software, Microcal Inc.).
The standard free energy (DG ) and entropy changes (DS )
o
o
plex stability constants (K ) and thermodynamic parameters.
S
Twenty-five successive injections were made for each titra-
tion experiment. In each run, an aqueous solution of host in
a 250 mL syringe was sequentially injected with stirring at
were calculated according to Equation (5)
o
o
o
DG ¼ ꢀRTlnK
¼ DH ꢀTDS
ð5Þ
S
300 rpm into an aqueous solution of guest in the sample cell
(
1.4227 mL volume). Figure 2 shows a representative titra-
in which R is the gas constant and T is the absolute tempera-
ture. Figure 3 shows a typical curve fitting result for the
complexation of 3 with b-CD in water. From the ITC results,
Figure 2. Microcalorimetric titration of 3 with b-CD in aqueous solution
at 298.15 K. a) Raw ITC data for 25 sequential injections (10 mL per in-
jection) of a solution of b-CD (2.02 mm) into a solution of 3 (0.097 mm).
b) Heat effects of the dilution and complexation reaction of 3 with b-CD
for each injection during the microcalorimetric titration experiment.
Figure 3. “Net” heat effects of complexation of b-CD with 3 for each in-
jection, obtained by subtracting the dilution heat from the reaction heat,
which was fitted by computer simulation with the “one set of binding
sites” model.
o
tion curve; each titration of b-CD into the sample cell gave
an apparent reaction heat caused by the formation of an in-
clusion complex between b-CD and 3. A control experiment
to determine the heat of dilution was performed for each
run by performing the same number of injections with the
same concentration of host compound as that used in the ti-
complex stability constants (K ), enthalpic changes (DH ),
S
o
and entropic changes (TDS ) for the complexation of b-CD
4
ꢀ1
and
3
were determined to be [(8.61ꢁ0.05)ꢀ10 ]m ,
ꢀ1 ꢀ1
(ꢀ31.99ꢁ0.36) kJmol , and (ꢀ3.85ꢁ0.37) kJmol , respec-
tively.
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Yu Liu et al.
corded (Figure 5). The b-CD was used as a competitive re-
agent to exclude probable interactions between the adaman-
tane group and ct-DNA. Binding of the anthryl group to the
2
D ROESY NMR Spectroscopy
2
D NMR spectroscopy has become a powerful method to
study the conformations of host–guest complexes. NOE cor-
relation signals in the NOESY or ROESY spectrum means
that two protons are closely located in space (0.4 nm apart
[21]
at most). The inclusion structure of 3 and b-CD was iden-
tified by 2D ROESY spectroscopy (Figure 4). It is known
Figure 5. The fluorescence intensity of b-CD/3 (0.10 mm/0.01 mm) with in-
creasing concentrations of ct-DNA (0–13.9 mm) in 5 mm Tris-HCl and
5
0 mm NaCl buffer (pH 7.1), with excitation at l=366 nm. Inset: Stern–
Volmer quenching plot of 3 with increasing concentrations of ct-DNA,
with excitation at l=366 nm and monitored at l=415 nm.
DNA helix quenched the fluorescence very strongly. The
fluorescence quenching data were plotted according to
[22]
Equation (6), in which I and I are the fluorescence inten-
o
sities in the absence and presence of DNA, respectively;
and KSV is the Stern–Volmer quenching constant, which is
a measure of the efficiency of quenching by DNA.
Io=I ¼ 1 þ KSV½DNAꢂ
ð6Þ
2
Figure 4. The 2D ROESY spectrum of 3 and b-CD in D O with a mixing
time of 250 ms.
As shown in Figure 5, the fluorescence intensity of 3 de-
creased gradually with increasing concentration of ct-DNA.
The fluorescence quenching constant evaluated by using
4
ꢀ1
Equation (6) was 2.2ꢀ10 m of DNA phosphates.
that only the H3, H5, and H6 protons of CDs can give the
NOE cross-peaks for analyzing the conformation of host–
guest complexes because the H2 and H4 protons face the
outer cavity. As shown in Figure 4, all 15 protons of the ada-
mantane group present clear cross-peaks with the H3 and
H5 protons. Moreover, no NOE correlation signal was de-
tected between the protons of the anthryl group and b-CD.
These results unambiguously showed that the adamantane
group of 3 was included in the b-CD cavity through the
wide opening and the anthyl group was located out of the b-
CD cavity and had a chance to react with the DNA back-
bone.
The DNA binding behavior of 3 was also investigated by
H NMR spectroscopy. As shown in Figure 7b below, the
1
signals of anthryl protons gradually broadened, accompa-
nied by slight chemical shift changes with the addition of ct-
DNA. This phenomenon also demonstrated the intercalation
of the anthryl groups into the hydrophobic DNA double
helix.
Absorption Spectra
To investigate the aggregation behavior of 2/3 with DNA,
the absorption spectra of 2 and 2/3 were recorded in the ab-
sence and presence of ct-DNA at varying concentrations.
Because the absorption band of 2 at l=260 nm overlapped
with that of ct-DNA, we only analyzed the absorption maxi-
mum of 2 beyond l=450 nm, which was assigned to the sur-
face plasmon resonance (SPR) band of AuNP. Generally,
because of electric dipole–dipole interactions and coupling
DNA Binding Behavior of 3
The DNA binding behavior of 3 was investigated by the
fluorescence titrations, in which calf thymus DNA (ct-DNA)
of different concentrations was gradually added to a solution
of b-CD/3 complex, and the fluorescence emissions were re-
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Yu Liu et al.
between the neighboring particles in the aggregates of gold
particles, the SPR band of gold particles will show an appre-
ciate bathochromic shift in the UV absorption spectrum.
increased intensity of the SPR band; this indicated that the
increased surface coverage of 3 promoted the reactivity of 2/
3 with DNA.
[23]
As shown in Figure 6a, the addition of ct-DNA to an aque-
ous solution of the 2/3 system produced a bathochromic
shift of the SPR maximum from l=508 to 519 nm, accom-
panied by the increased intensity of the SPR band. This phe-
nomenon indicated an effective aggregation of AuNPs. For
comparative purposes, the absorption spectra of 2 in the
presence of ct-DNA at varying concentrations were also re-
corded (Figure S8 in the Supporting Information). The UV
absorption band of 2 was almost unchanged upon the gradu-
al addition of DNA. This phenomenon means that there is
no appreciable aggregation of 2 without 3. In addition, the
absorption changes of 2 in the presence of a constant con-
centration of ct-DNA and different concentrations of 3 were
also recorded (Figure 6b) to compare the effect of the sur-
face coverage of 3 on the interactions of 2/3 with DNA. As
shown in Figure 6b, the addition of 3 to an aqueous solution
of 2+ct-DNA also led to a bathochromic shift (7 nm) and
1
H NMR Spectroscopy
The aggregation behavior of 2/3 with DNA was also investi-
gated by H NMR spectroscopy. Figure 7a shows the partial
H NMR spectra of 2 (5.0ꢀ10 m) and a mixture of 2 (5.0ꢀ
10 m) and ct-DNA (1.0ꢀ10 m) in D O by adding 0, 5.0ꢀ
10 , 1.0ꢀ10 , 2.0ꢀ10 , 2.5ꢀ10 , or 5.0ꢀ10 m of 3. The
signals of 2 gradually broadened and shift upfield with the
increasing guest/host ratio in the presence of ct-DNA. This
phenomenon may indicate the formation of tight supra-
molecular complexes between DNA and 2/3,
condensation efficiency of 2/3 could be controlled by chang-
1
1
ꢀ4
ꢀ
4
ꢀ3
2
ꢀ
5
ꢀ4
ꢀ4
ꢀ4
ꢀ4
[
8,18]
and the
ing the guest/host ratio.
1
ꢀ4
Figure 7. a) Partial H NMR spectra of 2 (1; 5.0ꢀ10 m) and the mixture
ꢀ
1
ꢀ4
ꢀ3
Figure 6. a) UV/Vis absorption spectra of 2/3 (0.62 gL /0.2 mm) in the
of 2 (5.0ꢀ10 m) and ct-DNA (1.0ꢀ10 m) in D
10 , 1.0ꢀ10 , 2.0ꢀ10 , 2.5ꢀ10 , or 5.0ꢀ10 m of 3 (2 to 7). b) Partial
H NMR spectra of 3 (5.0ꢀ10 m) in D
10 , 2.5ꢀ10 , 5.0ꢀ10 , or 1.0ꢀ10 m of ct-DNA (1 to 6).
2
O by adding 0, 5.0ꢀ
ꢀ
5
ꢀ4
ꢀ4
ꢀ4
ꢀ4
presence of ct-DNA at different concentrations in water (pH 7.0). b) UV/
ꢀ
1
ꢀ1
1
ꢀ4
ꢀ5
Vis absorption spectra of 2/ct-DNA (0.62 gL /0.18 gL ) in the presence
of 3 at different concentrations in water (pH 7.0).
2
O by adding 0, 5.0ꢀ10 , 1.0ꢀ
ꢀ
4
ꢀ4
ꢀ4
ꢀ3
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a height of approximately 30.3 nm (Figure 8d). The 3D view
of supramolecular aggregates clearly shows that the large
aggregates are composed of smaller tight particles (Fig-
ure 8e). In contrast, the AMF images of 2 and 2/3 (Figure S9
in the Supporting Information) indicated that free 2 and 2/3
existed as dispersed solid particles with heights of approxi-
mately 3.5 and 4.1 nm, respectively. A similar phenomenon
also was observed by TEM. Under our experimental condi-
tions, host 2 existed as dispersed solid particles with an aver-
age core diameter of (3.3ꢁ0.5) nm (based on individual
measurements on at least 100 particles; Figure 9a). When
Microscopy
Direct information about the size, shape, and distribution of
/3 and its aggregates with DNA came from AFM and TEM
results. Figure 8 shows typical AMF images of ct-DNA in
the absence and presence of 2/3. As shown in Figure 8a,
2
ꢀ
1
ꢀ1
Figure 9. TEM images of a) 2 (0.62 gL ) and b) 2/3 (0.62 gL , 0.10 mm)
with DNA (0.37 gL ).
ꢀ1
only 3 (Figure S10a in the Supporting Information) or ct-
DNA (Figure S10b in the Supporting Information) were
added individually, no appreciable aggregation of AuNPs
was observed. Comparatively, when 3 and ct-DNA were
added, host 2 tended to gather together and large aggregates
of AuNPs with an average diameter of (33.3ꢁ7.4) nm were
observed (Figure 9b); this indicated that small tight particles
tended to form larger fluffy structures. This phenomenon
demonstrated that complexes of 2/3 could act as promising
DNA concentrators and gave good binding abilities toward
ct-DNA and the condensation efficiency could be conven-
iently controlled by adjusting the ratio between the AuNPs
and anthryl adamantane grafts. From the viewpoint of biol-
ogy, the larger size of the DNA condensates is favorable for
their intracellular uptake and the smaller size of free 2/3
complexes means that the complexes can be eliminated
[25]
faster from the cell after completing the delivery mission.
ꢀ1
Specifically, DNA condensates of this size (~40 nm) could
Figure 8. AFM images of ct-DNA (0.37 gL ) in the absence (a) and
presence of 2/3 with different guest/host ratios (1:20 (b), 1:5 (c), and 1:1
be preferentially internalized by the Hepa 1–6 hepatoma
cell line through clathrin-mediated endocytosis.
[26]
(
d)). e) Partial 3D view of d).
Zeta Potential
free ct-DNA was loose and silklike with a height of approxi-
[24]
mately 2.3 nm (typical diameter of DNA). When 3 and 2
were added in different guest/host ratios (1:20, 1:5, and 1:1),
host 2 tended to aggregate. As shown in Figure 8b, at a low
guest/host ratio (1:20), most of the AuNPs still existed as
discrete particles and some of them began to come into con-
tact with DNA strands. When the guest/host ratio increased
to 1:5, loose DNA strands could not be observed, and small
aggregates formed with a height of approximately 16.7 nm
As shown in Table 2, the zeta potential of 2 slightly in-
creased after being mixed with 3, probably because the sur-
face charge of nanoparticles was partly shielded by un-
charged guest. The zeta potential of 2/3 decreased after
being mixed with ct-DNA because the surface of the nano-
particles was covered by negatively charged DNA. General-
ly, electrostatic interactions are considered to be the main
driving force for the condensation of DNA by complexation
[27]
(
Figure 8c). When the guest/host ratio further increased to
:1, discrete AuNPs tended to form large aggregates with
with condensing agents, such as multivalent cations, pep-
[28]
[29]
1
tides, cationic polymers, and dendrimers. On the other
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Yu Liu et al.
Table 2. Zeta potential data for the nanoparticles.
Nanoparticle
amounts of 3 by 2 showed different condensation abilities
and DNA was totally retained in the gel well at a guest/host
molar ratio of 2:5; this means the condensation efficiency of
2/3 could be conveniently controlled by changing the guest/
host ratio. In addition, the transfection experiments were
also performed in HGC-27 human gastric carcinoma cells,
which were tested with pCMV-AC-GFP DNA, and no visi-
ble green fluorescent protein (GFP) expression was ob-
served. We think that improvements may be achieved by the
further chemical modification of carriers, the appropriate se-
lection of cells and DNA, and the optimization of the ratio
of carriers and DNA in the mixture.
Zeta potential [mV]
[
a]
2
2
2
2
ꢀ18.19
ꢀ15.64
ꢀ18.04
ꢀ20.69
[
a]
/3
[
a]
[b]
+ct-DNA
[
a]
[b]
/3 +ct-DNA
[
a] [CD]=[3]=0.10 mm. [b] [ct-DNA]=0.05 mm.
hand, the intercalation of aryl grafts into DNA also contrib-
utes to the condensation of DNA in cooperation with cat-
[8, 18]
ionic compounds.
Considering the negative zeta poten-
tial of the 2/3 complex, intercalation should be the only driv-
ing force to condense DNA in the present system. The bind-
ing constant of aryl grafts with DNA is commonly in the
order of 10 m in DNA base pairs. Therefore, intercala-
tion may be not very strong, which is consist with the results
of microscopy that large aggregates formed by adjusting the
guest/host ratio, but not very compact, and that very high
nanoparticle and ct-DNA concentrations should be used for
spectroscopy and microscopy studies.
Cytotoxicity Assay
4
ꢀ1
[22]
Cytotoxicity assay results of aggregates of 2/3 with different
concentrations are presented in Figure 11. We can see that
aggregates of 2/3 exhibit no measurable cytotoxicity up to
a concentration of 1 mm. The low cytotoxicity of aggregates
of 2/3 probably arose from extrusive CD cavities on the sur-
face of aggregates of 2/3 protecting the plasma membranes
[8]
of cells from deposition by gold clusters. The low cytotox-
icity can also be attributed to its negative zeta potential
Agarose Gel Electrophoresis
(
Table 2).
The formation of 2/3/DNA complexes was analyzed by their
electrophoretic mobility on an agarose gel at various w/w/w
ratios. As shown in Figure 10a, the interaction of the 2/3
complex ([CD]/[3]=1:1) with DNA is molar ratio depen-
dent. As the concentration of 2/3 increased, the gel mobility
of DNA decreased, and DNA was totally retained in the gel
well at a wCD-AuNP/w /w
ratio of 43:3:1. Figure 10b shows
DNA
3
the agarose gel assay of DNA in the presence of complex 2/
at different guest/host ratios. The grafting of different
3
Figure 11. Cytotoxicity assay results for aggregates of 2/3 with different
concentrations.
Conclusion
We constructed a water-soluble, nontoxic, nanometer-sized
conjugate containing AuNPs, b-CDs, and anthryl adaman-
tanes through a supramolecular assembly approach. Owing
to the good DNA reactivity of anthracene moieties, this
supramolecular conjugate exhibited good condensation abil-
ities toward ct-DNA, and the condensation efficiency could
be conveniently controlled through the binding equilibrium
of CD cavities attached to the surfaces of AuNPs with an-
thryl adamantine grafts. The larger size of the DNA supra-
molecular aggregates was beneficial to their intracellular
uptake and the smaller size of free complexes of 2/3 meant
Figure 10. Agarose gel electrophoresis assay of pBR322 DNA
ꢀ
1
(
10 ngmL ). a) [CD]=0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, and
0
0
.10 mm from lanes 1 to 10, respectively. [3]=0.10, 0.01, 0.02, 0.03, 0.04,
.05, 0.06, 0.07, 0.08, and 0.10 mm from lanes 1 to 10, respectively.
b) [CD]=0.10 mm from lanes 1 to 10. [3]=0, 0.01, 0.02, 0.03, 0.04, 0.05,
.06, 0.07, 0.08, and 0.10 mm from lanes 1 to 10, respectively.
0
Chem. Asian J. 2014, 9, 1895 – 1903
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www.chemasianj.org
Yu Liu et al.
the complexes could be eliminated faster from the cell after
Cell Culture
completion of the delivery mission. Owing to these findings,
this supramolecular nanostructure is expected to have excit-
ing applications in gene therapy with the promising poten-
tial to control gene expression and delivery.
The human breast cancer MCF-7 cell line was cultured in Dulbeccoꢃs
modified eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) at 378C in a humidified atmosphere of 5% of CO .
2
Agarose Gel Electrophoresis
Agarose gels of 1% were prepared by heating agarose (250 mg) in TAE
ꢀ
2
ꢀ1
ꢀ2
ꢀ1
buffer (25 mL; 4.0ꢀ10 molL Tris, 2.0ꢀ10 molL acetic acid, 2ꢀ
ꢀ
3
ꢀ1
Experimental Section
10 molL ethylenediaminetetraacetic acid (EDTA); Dingguo Chang-
sheng Biotechnology Co. Ltd.). Sample solutions containing pBR322
DNA, 2, and 3 with different w/w/w ratios were prepared by adding an
appropriate volume of solutions of 2, 3, and DNA into Eppendorf tubes,
which were then diluted to a total volume of 10 mL. After incubation at
Materials
All solvents and reagents were commercially available and used without
further purification, unless otherwise noted. Anhydrous N,N-dimethylfor-
mamide (DMF) was dried and distilled over CaH under reduced pres-
2
4
6
8C for 30 min, the sample solutions were subjected to electrophoresis at
0 V for 1 h (current 120 mA) and visualized by ethidium bromide stain-
sure. All aqueous solutions were prepared from distilled water. b-CD of
reagent grade (Shanghai Reagent Factory) was recrystallized twice from
water and dried in vacuo at 958C for 24 h prior to use. 6-Amino-6-deoxy-
ing. The DNA bands were visualized and photographed by means of
a UV transilluminator and WD-9413B gel documentation system (Beijing
Liuyi Instrument Factory, P.R. China).
[
30]
b-CD was synthesized according to published procedures.
Cytotoxicity Studies
Instruments
1
13
The cytotoxicity of 2/3 was investigated by means of a 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO,
USA) viability test on the MCF-7 cell line. Briefly, cells were plated at
a density of 1.0ꢀ10 cells per well in 96 wells. After 24 h of incubation,
cells were treated with 2/3 at indicated concentrations for 48 h, then the
medium was removed, and fresh medium (200 mL) plus MTT reagent
2
H, C, and 2D NMR spectra were recorded in D O on a Bruker AV 400
spectrometer. TGA was performed with a RIGAKU Standard type TG
analyzer. Carefully weighed quantities of every sample were subjected at
4
ꢀ
1
a heating rate of 10.0 Kmin under a nitrogen atmosphere from 25 to
008C. FTIR spectra were recorded on a Bio-Rad FTS6000 spectrometer.
Mass spectra were recorded on a Varian 7.0T FTICR mass spectrometer
MALDI). All microcalorimetric experiments were performed on a ther-
8
(
20 mL; 2.5 mg dissolved in 50 mL of dimethylsulfoxide (DMSO)) were
(
added to each well. After incubation for 4 h at 378C, the culture medium
containing MTT was withdrawn and DMSO (200 mL) was added, fol-
lowed by shaking for 10 min until the crystals dissolved. Viable cells were
detected by measuring the absorbance at l=490 nm by using an MRX II
absorbance reader (DYNEX Technologies, Chantilly, VA, USA). Cell
growth was expressed as a percentage of absorbance in cells treated with
mostated and fully computer-operated isothermal calorimetry (VP-ITC)
instrument (Microcal Inc., Northampton, MA). UV/Vis spectra were re-
corded in a quartz cell (light path 5 mm) on a Shimadzu UV-3600 spec-
trophotometer equipped with a PTC-384WI temperature controller. The
ICP-AES data were measured by means of an ICP-9000 (N+M) instru-
ment (USA, Thermo Jarrell-Ash Corp.). Elemental analysis (C, H, and
N) was performed by using a Vario EL Cube elemental analyzer (Ele-
mentar Ltd. Corp., Germany). AFM images were examined by means of
a Nanoscope IIIa Multimode 8 AFM (Bruker). TEM images were exam-
ined by means of a high-resolution transmission electron microscope
2
/3 to that in cells without 2/3 treatment (100%). The survival rate (IR)
was calculated as follows: IR=(A value of 2/3 well/A value of control
well)ꢀ100%.
Synthesis of 1
(
8
Tecnai G2 F20 microscope, FEI) equipped with a CCD camera (Orius
32, Gatan) operating at an acceleration voltage of 200 kV. The zeta po-
dl-Lipoic acid (227.0 mg, 1.1 mmol), O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU; 418.3 mg,
1.1 mmol), hydroxybenzotriazole (HOBt; 675.2 mg, 5.0 mmol), and 4-di-
methylaminopyridine (DMAP; 61.09 mg, 0.5 mmol) were dissolved in
DMF (10 mL) at 08C. The mixture was stirred for 1 h at 08C under an
argon atmosphere, and then a solution of DMF (10 mL) containing 6-
amino-6-deoxy-b-CD (1.1 g, 1.0 mmol) was added. The reaction mixture
was stirred for 6 h at 08C and then for 8 h at room temperature under an
argon atmosphere. The mixture was poured into acetone (300 mL). The
precipitate was collected by filtration to obtain a yellow powder, which
was purified by column chromatography on silica gel by using propanol/
tential of the nanoparticles was recorded by means of a Zeta-Plus z po-
tential analyzer (Zetapals/BI-200SM, Brookhaven, USA).
Absorption Spectroscopy
Samples for absorption spectroscopic measurements were prepared by
adding an aqueous solution of ct-DNA (Beijing Dingguo Changsheng
Biotechnology Co. Ltd.) to an aqueous solution of 2 (or 2/3). The ct-
DNA concentration per nucleotide was determined by measuring the
ꢀ
1
ꢀ1 [31]
UV absorbance at 260 nm (e=6600m cm ). The stock solution of ct-
DNA was stored at 48C and kept at room temperature for 1 h before
use.
water/ammonia (v/v/v 6:3:1) as the eluent to obtain 1 as a white solid
1
(
501.2 mg, 37.9%). H NMR ([D
6
]DMSO, 400 MHz): d=1.22–1.38 (m,
Fluorescence Measurements
2H), 1.42–1.68 (m, 4H), 1.82–1.93 (m, 1H), 2.05–2.17 (m, 2H), 2.32–2.45,
m, 1H), 3.06–3.19 (m, 3H), 3.26–3.32 (m, 14H), 3.53–3.75 (m, 28H),
.37–4.61 (m, 6H), 4.77–4.90 (m, 7H), 5.59–5.86 (m, 14H), 7.55–7.69 ppm
(
4
Fluorescence experiments were performed in a conventional quartz cell
(
light path 10 mm) on a Varian Cary Eclipse equipped with a Varian
+
(
1
C
s, 1H); HRMS (MALDAI): m/z calcd for C50
H
83NO35
S
2
[M+H] :
322.4265; found: 1322.4220; elemental analysis calcd (%) for
·7H O: C 41.46, H 6.75, N 0.97; found: C 41.66, H 7.02, N
Cary single-cell Peltier accessory to control the temperature (lex
=
3
5
66.0 nm, bandwidth(ex)=2.5 nm, bandwidth(em)=10.0 nm) at 258C in
mm Tris-HCl 50 mm NaCl buffer (pH 7.1).
50
H
83NO35
S
2
2
0
.92.
Synthesis of 2
A solution of DMSO (20 mL) containing gold chloride tetrahydrate
AFM Measurements
Samples were prepared by dropping the solution on mica. The mica sam-
ples were then air-dried and the samples were examined in tapping
mode.
(50.0 mg, 0.1 mmol) was quickly mixed with another solution of DMSO
(20 mL) containing 1 (20.0 mg, 0.02 mmol) and sodium borohydride
(75.5 mg, 2.0 mmol). The reaction mixture was stirred for 24 h at room
TEM Measurements
temperature, and then acetonitrile (40 mL) was added. The precipitate
was collected by centrifugation and washed with DMSO/CH CN
Each sample for TEM measurements was prepared by dropping sample
solution (50 mL) on a copper grid. The grids were then air-dried and the
samples were examined by means of a high-resolution TEM instrument.
3
(100 mL; v/v 1:1) and ethanol (100 mL). Subsequently, the product was
purified by dialysis (molecular weight cutoff 3500) in distilled water sev-
Chem. Asian J. 2014, 9, 1895 – 1903
1902
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemasianj.org
Yu Liu et al.
eral times and dried under vacuum for 12 h at 808C. From the elemental
analysis and ICP-AES data, the content of CD units (WCD) was calculat-
ed to be 0.162 mmolg ; the average number of b-CD units around one
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ꢀ
1
nanoparticle was calculated to be 14. Elemental analysis (%) found: C
9
.73, H 2.73, N 0.30; ICP-AES (%): Au 25.7.
Synthesis of 3
-Anthracene carboxaldehyde (1.1 g, 5.0 mmol) and diethylenetriamine
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11] G. Han, C. T. Martin, V. M. Rotello, Chem. Biol. Drug Des. 2006,
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(
(
2.7 mL, 25 mmol) were dissolved in a mixture of anhydrous ethanol
125 mL) and dichloromethane (75 mL), and stirred for 24 h at room
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13] M. Thomas, A. M. Klibanov, Proc. Natl. Acad. Sci. USA 2003, 100,
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water (50 mL) and extracted with dichloromethane (200 mL) three times.
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9
138–9143.
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2
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1
768, 1311–1324.
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3
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20 mL) and added dropwise to a solution of triethylamine (2.1 mL,
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(
1
[
33]
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column chromatography on silica gel by using dichloromethane/methanol
2
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(
v/v 10:1) as the eluent to give 3 as a white powder ( 1.27 g, 55.7%).
1
H NMR (D
2
O, 400 MHz): d=1.47–1.72 (m, 12H), 1.84–1.93 (s, 3H),
.00–3.09 (t, 2H), 3.27–3.50 (m, 6H), 5.08–5.15 (s, 2H), 7.53–7.61 (t, 2H),
.63–7.72 (t, 2H), 8.04–8.13 (d, 2H), 8.18–8.26 (d, 2H), 8.56–8.62 ppm (s,
[
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7
1
1
(
4
1
3
H); C NMR (D
2
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25.5, 122.7, 47.7, 43.5, 40.4, 38.2, 36.2, 35.6, 27.5 ppm; HRMS
+
MALDAI): m/z calcd for
56.3013; elemental analysis calcd (%) for C30
C
30
H
37
N
3
O
[M+H] : 456.3015; found:
O·5H O: C 66.03, H
[
[
[
H
37
N
3
2
8
553.
8
.68, N 7.70; found: C 66.29, H 8.64, N 8.00.
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Received: February 18, 2014
Published online: May 26, 2014
Chem. Asian J. 2014, 9, 1895 – 1903
1903
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim